Evaluation of Noninvasive Versus Invasive Techniques for the Diagnosis of Helicobacter pylori Infection (2013-06-19 23:46:54+01:00)

Background:Helicobacter pylori is one of the most common bacterial strains causing chronic infections, affecting over one half of the world’s population. There is increasing interest in noninvasive methods for diagnosing H. pylori infection. The aim of the study was to evaluate 3 different noninvasive methods of diagnosis: the stool antigen test (HpSA), the serum antibody test, and the stool-polymerase chain reaction (PCR) test as against invasive methods based on histopathologic diagnosis. Materials and Methods:Gastric biopsies were obtained during endoscopy. Sections were stained with hematoxylin and eosin and Giemsa stain. Serum samples were tested for H. pylori antibody using an enzyme-linked immnunosorbent assay kit for the semiquantitative determination of IgG antibodies; stool samples were tested for H. pylori antigen using polyclonal enzyme-linked immnunosorbent assay kits. DNA samples from stool specimens were extracted, followed by PCR for the detection of H. pylori UreA. Results:The results revealed that 18/19 (94.7%) patients were positive for H. pylori infection as detected by Giemsa stain, and 84.2% were positive on the basis of hematoxylin and eosin stain, with a sensitivity and specificity of 88.9% and 100%, respectively. Diagnosis by noninvasive methods, including the serum antibody test, revealed a sensitivity and positive predictive value of 88.9% and 94.2%, respectively, whereas the stool antigen test recorded a sensitivity and positive predictive value of 72.2% and 92.9%, respectively. The stool-PCR test recorded a sensitivity of 72.2% and specificity of 100%. Conclusions:Among the noninvasive methods for diagnosis of H. pylori infection, the 3 methods used in this study recorded promising results, including good sensitivity, which was the highest in the serum antibody test, whereas the stool-PCR test recorded excellent specificity.

Implementation of a Microwave-assisted Tissue-processing System and an Automated Embedding System for Breast Needle Core Biopsy Samples: Morphology, Immunohistochemistry, and FISH Evaluation (2013-06-19 23:46:54+01:00)

A platform composed of a microwave (MW)-assisted tissue-processing system and an automated embedding system has been recently introduced in pathology laboratories. Needle core biopsy (NCB) is an established, highly accurate method for diagnosing breast lesions and for providing important pathologic, predictive, and prognostic information such as biomarker expression in case of breast carcinoma. The aim of this study was to evaluate whether breast NCBs processed with the MW-assisted tissue-processing system and automatically embedded show good-quality histology preparations and whether they are suitable for the assessment of estrogen receptor (ER), progesterone receptor (PR), Ki-67, and HER2 in breast carcinoma. A series of 233 consecutive breast NCBs processed by both conventional and MW-assisted tissue-processing systems was included in this study. The histomorphologic and immunohistochemical quality, as well as the results of the evaluation of the biomarkers, were compared—the conventional processing method being the gold standard for comparison. The quality of hematoxylin-eosin and immunohistochemical tissue sections provided by the new system is comparable to that obtained after the conventional processing method. Moreover, in breast carcinomas, a perfect agreement between the paired tissues when evaluating ER and PR status (Cohen κ=1) and a very good agreement when evaluating Ki-67 (κ=0.91) and HER2 (κ=0.93) have been found. In conclusion, applying strict criteria in tissue-handling steps, breast NCB can be processed and automatically embedded with these platforms. The diagnosability and the evaluation of the main prognostic and predictive biomarkers have been proved to be reliable.

The Influence of Tissue Ischemia on Biomarker Expression in Colorectal Cancer (2013-06-19 23:46:54+01:00)

The aim of the present study was to investigate the influence of fixation delay and the perioperative ischemia on hypoxia inducible factor (HIF)-1α gene expression, HIF-1α protein expression, and immunohistochemical (IHC) expression of HIF-1α, GLUT-1, Bcl-2, and Ki-67 in colorectal cancer. The study included 25 surgically removed colorectal tumors. Three sets of samples were collected readily after removal and exposed to 0, 30, and 60 minutes of delay of fixation or freezing. The perioperative ischemia time was registered. In each set of the samples, HIF-1α gene expression was analyzed by quantitative real time polymerase chain reaction, protein concentration of HIF-1α was assessed by enzyme-linked immunosorbent assay, and IHC staining of HIF-1α, GLUT-1, Bcl-2, and Ki-67 was performed. Preoperative formalin-fixed paraffin-embedded biopsies and whole sections of the entire tumor were analyzed by IHC. We found that the HIF-1α gene expression, HIF-1α protein concentration, and IHC expression of HIF-1α, GLUT-1, Ki-67, and Bcl-2 were not systematically affected by either the fixation or freezing delay of the tissue, the perioperative ischemia time, or the total ischemia time (perioperative ischemia+delay of fixation or freezing) in colorectal tumors. However, the intraindividual variation was quite large, which may question the use of individually, non-standardized–handled single biopsies or small tissue samples for analysis of often rather heterogenously expressed biomarkers.

Relationship Between MDM2 and p53 Alterations in Colorectal Cancer and Their Involvement and Prognostic Value in the Tunisian Population (2013-05-18 21:25:34+01:00)

Background:The prevalence of p53 mutations in colorectal cancer could reach 90%. The most important regulator of this protein that was identified originally was the Murine Double Minute2 (MDM2) oncoprotein, by which the levels of p53 were fixed through an autoregulatory feedback loop. In cancer cases, the overexpression of MDM2 deregulates this feedback, and the signaling pathway between MDM2 and p53 is blocked. Materials and Methods:We genotyped 167 patients and 167 healthy blood donors to determinate the mutational status of MDM2 and p53. Immunohistochemical analysis was performed on tumor and normal mucosa. Results:The MDM2 polymorphism study showed a higher distribution of MDM2 SNP309 in tumors compared with healthy controls. At the same time, the majority of samples with SNP309 indicated a positive expression of MDM2 protein in the tumor. In this case, we found a first significant association between p53 expression and the single-strand conformational polymorphism analysis and a second association between the MDM2 polymorphism and p53 mutation. Moreover, the nuclear overexpression of MDM2 and SNP309 was significantly related to a higher mortality rate. Conclusions:In this work we wanted to highlight the role, which is becoming increasingly important, of MDM2. In fact, we conclude that the effects of MDM2 SNP309 may be considered a valuable prognostic marker to predict poor outcome for Tunisian patients with colorectal cancer.

Applications and Limitations of Immunohistochemical Expression of "Napsin-A" in Distinguishing Lung Adenocarcinoma From Adenocarcinomas of Other Organs (2013-05-14 18:08:44+01:00)

Background:Distinguishing between primary lung adenocarcinoma and metastatic adenocarcinoma of lung before planning patient treatment is clinically important. Immunohistochemical markers play an important role in classification of primary lung tumors and are an effective method for separating metastatic tumors from primary pulmonary carcinoma. In this study, we evaluated the expression of Napsin-A in primary pulmonary carcinoma and some cases of nonpulmonary adenocarcinoma. Materials and Methods:The Napsin-A immunohistochemical evaluation was carried out using surgical specimens from 18 cases of adenocarcinoma, 19 cases of squamous cell carcinoma, 2 cases of large cell carcinoma, 1 case of bronchoalveolar carcinoma of lung, as well as 33 cases of renal cell carcinoma, 30 cases of thyroid neoplasm, 31 cases of colonic carcinoma, 31 cases of breast carcinoma, and 30 cases of endometrial adenocarcinoma. Results:For the primary lung carcinoma cases, all 18 cases of adenocarcinoma, 2 of the large cell carcinomas, and the 1 bronchioloalveolar carcinoma case were positive for Napsin-A. For the thyroid tumors, Napsin-A was positive in 14 cases of papillary carcinoma. Napsin-A was positive for 87.5% of papillary renal cell carcinoma cases and in 29.4% of clear cell carcinoma cases and for 1 chromophobe renal cell carcinoma case. Three out of 30 endometrial adenocarcinomas showed Napsin-A reactivity. All squamous cell carcinoma cases and adenocarcinomas of colon and breast were negative for Napsin-A. Conclusions:Napsin-A is a useful marker for differentiating primary lung adenocarcinoma from squamous cell carcinoma. However, Napsin-A immunoreactivity has the potential to misguide a pathologist to conclude a metastasis from renal, thyroid, or endometrial carcinoma as a primary lung adenocarcinoma. Therefore, when there is a need to rule out lung metastasis from other organs, implementation of other biologically specific markers should be considered.

Stem Cell Properties in Cell Cultures From Different Stage of Melanoma Progression (2013-05-24 18:48:11+01:00)

Cutaneous melanoma is an extremely heterogenous human cancer. The most aggressive melanoma may contain deregulated cells with undifferentiated/stem cell-like phenotype. A critical mechanism by which melanoma cells enhance their invasive capacity is the dissolution of the intercellular adhesion and the acquisition of mesenchymal features as a part of an epithelial-to-mesenchymal transition. The aim of this study was to clarify the role of a stem cell-like population in human melanomas by means of melanocytic cell culture analysis obtained from distinct histotypes of primary and metastatic malignant melanoma. Patients with advanced melanoma >2 cm in diameter and/or >300 mm2 surface were enrolled. The melanoma cells were isolated from skin biopsies of lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and metastatic melanoma. The colony forming unit assay and alkaline phosphatase stain were evaluated. Cells were subsequently cultured and maintained in different media to evaluate their ability to differentiate into osteogenic and adipogenic lineages. Immunohistochemistry and flow cytometry analysis were performed to evaluate antigenic markers CD90, CD73, CD105, CD146, CD20, CD166, and Nestin. This study confirms that melanoma can include heterogenous cell populations with the ability both to self-renew and to a give rise to differentiated progeny. Melanoma cells displayed intratumoral heterogeneity and dynamic antigen phenotypes. Histologically, transitions from normal skin to melanoma were associated with a gradual increase in the expression of CD146, CD20, CD133, Nestin, and CD73. These molecular profiles could be further analyzed and, in the future, used for the development of novel biomolecular targeted-therapy approaches. (C) 2013 Lippincott Williams & Wilkins, Inc.

Water Bath and Pressure Cooker Antigen Retrieval in Immunohistochemistry: A Comparative Study (2013-05-24 18:48:11+01:00)

No abstract available

Expression of Topoisomerase II-[alpha] in Triple Negative Breast Cancer (2013-05-24 18:48:11+01:00)

Triple negative breast cancer (TNBC)-defined by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 negativity is a group with poor prognosis, due to aggressive tumor biology and lack of targeted therapy. Topoisomerase II-[alpha] (topoII[alpha]) protein is one of the intracellular targets for anthracycline-based therapy, and high levels of topoII[alpha] expression are recently observed in TNBC. The study included 83 patients who underwent surgery between January 2003 and December 2009. Paraffin blocks were stained immunohistochemically with CK5/6, CK14, EGFR, Ki-67, and topoII[alpha]. Basal-like (BL) immunophenotype was defined by positivity for >=1 basal cell markers: CK5/6, CK14, or EGFR. Of 83 TNBC, 66.26% were of the BL immunophenotype, which was significantly associated with higher mitotic count (P=0.023), BL morphology (P=0.005), higher histologic grade (P=0.022), and higher proliferation rate assessed by Ki-67 (P<0.001). TopoII[alpha] expression was significantly correlated with invasive ductal carcinoma NOS (P=0.010), higher mitotic count (P=0.001), higher histologic grade (P=0.007), and higher Ki-67 (P<0.001). In conclusion, due to lack of expression of ER, PR, and human epidermal growth factor receptor 2 receptor in TNBC, specific targeted therapies are not effective, and chemotherapy is currently the only modality of available systemic therapy. Due to expression of topoII[alpha], anthracyclines may be effective in treatment of TNBC. (C) 2013 Lippincott Williams & Wilkins, Inc.

Claudin-3 and Claudin-4: Distinct Prognostic Significance in Triple-Negative and Luminal Breast Cancer (2013-05-24 18:48:11+01:00)

Introduction: To investigate the immunohistochemical expression of claudin-1, claudin-3, and claudin-4 in triple-negative breast carcinomas and compare it with several clinicopathologic parameters as well as their expression in luminal cancers. Materials and Methods: A total of 128 cases of breast carcinoma were included in the study. For all these cases, immunohistochemistry for estrogen and progesterone receptors, Ki-67, and Her2 had already been performed, whereas Her2 2+ cases had been further characterized as positive or negative for Her2 amplification with the chromogenic in situ hybridization technique. Seventy-six tumors were triple negative. The remaining 52 were luminal cancers. All tumors were evaluated for the expression of claudin-1, claudin-3, and claudin-4. Results: In the triple-negative group, the positive expression of claudin-3 and claudin-4 was related to unfavorable and favorable prognostic factors, respectively. Claudin-1 was not related to any parameter under evaluation. In the luminal cancer group, claudin-4 positivity was related to a shorter disease-free survival, whereas the inverse was observed for claudin-3. Moreover, all 3 claudins increased with increase of the grade and Ki-67 value in the luminal cancers. Conclusion: A distinct prognostic significance in the expression of claudin-3 and mostly of claudin-4 between triple-negative and luminal breast carcinomas was identified. Specifically, in triple-negative carcinomas, claudin-4 positivity could probably be considered as a biomarker of favorable prognosis, whereas in luminal cancers with claudin-4-positive expression, the administration of targeted therapy should eventually be part of the patients' management in the near future. (C) 2013 Lippincott Williams & Wilkins, Inc.

Utility of BCL2, PD1, and CD25 Immunohistochemical Expression in the Diagnosis of T-cell Lymphomas (2013-05-24 18:48:11+01:00)

T-cell lymphomas (TCLs) are a heterogenous group of diseases that show histologic and immunophenotypic features overlapping with reactive lymphoid proliferations and often require the use of ancillary testing for accurate diagnosis. The oncoprotein, bcl-2, is expressed in various types of lymphoma. At present, expression of this protein is useful for distinguishing several B-cell lymphomas. Although there are some anecdotal reports that the lack of bcl-2 expression by T cells might also be a useful marker for the diagnosis of TCL, there are no focused studies to address this hypothesis. Another antigen with value in TCL diagnosis is programmed death-1 (PD-1), a marker of follicular helper T cells, which has been reported to be sensitive in the detection of angioimmunoblastic TCL and peripheral T-cell lymphoma, unclassified. However, several reports have also shown that PD-1-positive cells may be increased in a number of settings other than TCL, including reactive and atypical lymphadenopathies. Finally, lymphoma cells express a variety of cytokine receptors and signaling molecules that are current or potential targets for immunomodulatory therapy. One such target is the interleukin (IL)-2 receptor (CD25), which is acted on by denileukin diftitox/ONTAK, a recombinant diphtheria toxin-IL-2 fusion protein. Selection of suitable patients for therapy often includes pretreatment assessment of CD25 expression in tumor cells. In order to further assess the diagnostic and therapeutic utility of these antigens, we compared the expression of the CD25, PD-1, and bcl-2 in 119 cases of T-cell non-Hodgkin lymphoma using immunohistochemical techniques applied to routinely processed and paraffin-embedded tissues. We show that lack of expression of bcl-2 was observed in 52% cases of TCL and may aid in identification of neoplastic T-cell populations. In combination, bcl-2, CD25, and PD-1 provide diagnostic utility and may aid in selecting appropriate patients for immunomodulatory therapy. (C) 2013 Lippincott Williams & Wilkins, Inc.

No-cost Manual Method for Preparation of Tissue Microarrays Having High Quality Comparable to Semiautomated Methods (2013-04-25 07:28:54+01:00)

Manual tissue microarray (TMA) construction had been introduced to avoid the high cost of automated and semiautomated techniques. The cheapest and simplest technique for constructing manual TMA was that of using mechanical pencil tips. This study was carried out to modify this method, aiming to raise its quality to reach that of expensive ones. Some modifications were introduced to Shebl’s technique. Two conventional mechanical pencil tips of different diameters were used to construct the recipient blocks. A source of mild heat was used, and blocks were incubated at 38°C overnight. With our modifications, 3 high-density TMA blocks were constructed. We successfully performed immunostaining without substantial tissue loss. Our modifications increased the number of cores per block and improved the stability of the cores within the paraffin block. This new, modified technique is a good alternative for expensive machines in many laboratories.

Immunoprofile of Mucinous Non-neoplastic Cyst of the Pancreas (2013-04-25 07:28:54+01:00)

Background:A recently described mucinous non-neoplastic cyst (MNNC) of the pancreas is a benign cyst and should be distinguished from mucinous cystic neoplasm (MCN) and intraductal papillary mucinous neoplasm (IPMN) due to different management and prognosis. The immunoprofile of MNNC has not been well studied. Design:Twenty-three MNNCs diagnosed on surgical resection were retrieved. Immunohistochemical (IHC) staining was performed on surgically resected specimen. Sixteen IPMN and 15 MCN cases were also retrieved for comparison. Cyst fluid carcinoembryonic antigen and amylase concentrations were retrieved. Result:MNNCs were randomly located in the pancreas and were either unilocular or multilocular cysts that were lined by a single layer of bland columnar or cuboidal mucinous cells and supported by paucicellular stroma. The glandular epithelial cells were diffusely positive for CK7 (100%) and PDX-1 (65%); focally positive for CD10 (superficial, 65%), CD99 (basally, 100%), CDX-2 (17%), and CK20 (4%); and negative for MUC2. Only rare stromal cells in the cyst wall were weakly positive for estrogen receptor or progesterone receptor in only 6% of cases and negative for inhibin. These results were also compared with the immunoprofile of IPMN and MCN. Conclusions:Although MNNC shares some IHC markers with IPMN or MCN, an IHC panel can help distinguish MNNC from IPMN or MCN. The results suggest that MNNC is different from IPMN and MCN.

Glutamine Synthetase, Heat shock Protein-70, and Glypican-3 in Intrahepatic Cholangiocarcinoma and Tumors Metastatic to Liver (2013-04-25 07:28:54+01:00)

Introduction:Glutamine synthetase (GS), heat shock protein-70 (HSP-70), and glypican-3 (GPC-3) are markers best characterized in hepatocellular lesions, where they are useful in distinguishing hepatocellular carcinoma from dysplastic nodules. Their staining patterns in intrahepatic cholangiocarcinoma (IH-ChCa) and metastatic tumors in liver are not well described. Methods:Tissue microarrays containing 41 IH-ChCa and 24 metastatic tumors in liver were stained with commercially available antibodies to GS, HSP-70, and GPC-3. Five percent staining of tumor cells was considered positive for HSP-70 and GPC-3. For GS, 50% was the cut-off. Results:GS reactivity was present in 31 of 41 IH-ChCa (76%), with the median amount of staining being 65% of tumor cells. HSP-70 reactivity was present in 36 of 41 IH-ChCa (88%) with the median amount of staining being 75% of tumor cells. GPC-3 reactivity was absent from all IH-ChCa. Twenty-seven of 41 IH-ChCa cases were positive for both GS and HSP-70 (66%). GS reactivity was present in 17 of 24 tumors metastatic to liver (71%), with the median amount of staining being 50% of tumor cells. HSP-70 reactivity was present in 21 of 24 tumors metastatic to liver (88%) with the median amount of staining being 80% of tumor cells. GPC-3 reactivity was present in 2 of 24 tumors metastatic to liver (8%) with one showing 5% staining and the other showing 50% staining of tumor cells. Fifteen of 24 cases were positive for both GS and HSP-70 (63%), and 2 cases were positive for all 3 markers (8%). Discussion:Of the panel of immunostains currently commonly used to distinguish hepatocellular carcinoma from dysplastic hepatocytic nodules, only GPC-3 did not react frequently with metastatic tumors and IH-ChCa, although there was staining in 2 metastatic tumors. GS and HSP-70 are typically positive in IH-ChCa and metastatic tumors. Nothing should be inferred about the histogenesis of a tumor based on positive staining with either of these 2 markers, which currently have no role in tumor of unknown origin panels.

Melanin Bleaching With Dilute Hydrogen Peroxide: A Simple and Rapid Method (2013-04-25 07:28:54+01:00)

Melanins are naturally occurring pigments in both normal and pathologic tissues. Two common bleaching processes are potassium permanganate followed by oxalic acid treatment and dilute hydrogen peroxide (H2O2) process. The potassium permanganate/oxalic acid method is faster and more easily incorporated in conventional daily immunostaining protocols, whereas the dilute H2O2 method requires 24 hours. This study aimed to reduce melanin bleaching time by using a 10% H2O2 dilution. First, reaction time was reduced to 30 minutes by raising the temperature to 65°C. Second, containers with high thermal conductivity were used to improve bleaching effectiveness. Experimental comparisons of melanin treatments with H2O2 contained in an iron jar, a glass coplin jar, and a plastic steel jar obtained bleaching time of 20, 30, and 40 minutes, respectively. These modifications of the conventional bleaching method significantly improve the speed and efficiency of the procedure and are recommended when performing immunohistochemical studies.

A Simple Way to Improve Immunoreactivity of Prostate Needle Biopsies Fixed in Bouin's Solution to AMACR (2013-04-25 07:28:54+01:00)

No abstract available

Characterization of a novel angiogenic model based on stable, fluorescently labelled endothelial cell lines amenable to scale-up for high content screening (2011-12-01)

Jumping the barrier: VE-cadherin, VEGF and other angiogenic modifiers in cancer (2011-12-01)

The endothelial barrier controls the passage of fluids, nutrients and cells through the vascular wall. This physiological function is closely related to developmental and adult angiogenesis, blood pressure control, as well as immune responses. Moreover, cancer progression is frequently characterized by disorganized and leaky blood vessels. In this context, vascular permeability drives tumour-induced angiogenesis, blood flow disturbances, inflammatory cell infiltration and tumour cell extravasation. Although various molecules have been implicated, the vascular endothelial adhesion molecule, VE-cadherin (vascular endothelial cadherin), has emerged as a critical player involved in maintaining endothelial barrier integrity and homoeostasis. Indeed, VE-cadherin coordinates the endothelial cell-cell junctions through its adhesive and signalling properties. Of note, many angiogenic and inflammatory mediators released into the tumour microenvironment influence VE-cadherin behaviour. Therefore restoring VE-cadherin function could be one very promising target for vascular normalization in cancer therapies. In this review, we will mainly focus on recent discoveries concerning the molecular mechanisms involved in modulating VE-cadherin plasticity in cancer.

Defining the role of TRIP6 in cell physiology and cancer (2011-12-01)

Integrating signals from the ECM (extracellular matrix) via the cell surface into the nucleus is an essential feature of multicellular life and often malfunctions in cancer. To date many signal transducers known as shuttle proteins have been identified that act as both: a cytoskeletal and a signalling protein. Here, we highlight the interesting member of the Zyxin family TRIP6 [thyroid receptor interactor protein 6; also designated ZRP-1 (zyxin-related protein 1)] and review current literature to define its role in cell physiology and cancer. TRIP6 is a versatile scaffolding protein at FAs (focal adhesions) involved in cytoskeletal organization, coordinated cell migration and tissue invasion. Via its LIM and TDC domains TRIP6 interacts with different components of the LPA (lysophosphatidic acid), NF-κB (nuclear factor κB), glucocorticoid and AMPK (AMP-activated protein kinase) signalling pathway and thereby modulates their activity. Within the nucleus TRIP6 acts as a transcriptional cofactor regulating the transcriptional responses of these pathways. Moreover, intranuclear TRIP6 associates with proteins ensuring telomere protection and hence may contribute to genome stability. Accordingly, TRIP6 is engaged in key cellular processes such as cell proliferation, differentiation and survival. These diverse functions of TRIP6 are found to be dysregulated in various cancers and may have pleiotropic roles in tumour initiation, tumour growth and metastasis, which turn TRIP6 into an attractive candidate for cancer diagnosis and targeted therapy.

The Golgi apparatus in the endomembrane-rich gastric parietal cells exist as functional stable mini-stacks dispersed throughout the cytoplasm (2011-12-01)

Background information. Acid-secreting gastric parietal cells are polarized epithelial cells that harbour highly abundant and specialized, H⁺,K⁺ ATPase-containing, tubulovesicular membranes in the apical cytoplasm. The Golgi apparatus has been implicated in the biogenesis of the tubulovesicular membranes; however, an unanswered question is how a typical Golgi organization could regulate normal membrane transport within the membrane-dense cytoplasm of parietal cells.

Results. Here, we demonstrate that the Golgi apparatus of parietal cells is not the typical juxta-nuclear ribbon of stacks, but rather individual Golgi units are scattered throughout the cytoplasm. The Golgi membrane structures labelled with markers of both cis- and trans-Golgi membrane, indicating the presence of intact Golgi stacks. The parietal cell Golgi stacks were closely aligned with the microtubule network and were shown to participate in both anterograde and retrograde transport pathways. Dispersed Golgi stacks were also observed in parietal cells from H⁺,K⁺ ATPase-deficient mice that lack tubulovesicular membranes.

Conclusions. These results indicate that the unusual organization of individual Golgi stacks dispersed throughout the cytoplasm of these terminally differentiated cells is likely to be a developmentally regulated event.

 (1970-01-01 01:00:00+01:00)

Different responses to mechanical injury in neonatal and adult ovine articular cartilage (2013-06-17T00:00:00Z)

Background: Articular cartilage injury remains a major challenge in orthopedic surgery. This study aimed to identify differences in gene expression and molecular responses between neonatal and adult articular cartilage during the healing of an injury. Methods: An established in vitro model was used to compare the transcriptional response to cartilage injury in neonatal and adult sheep by microarray analysis of gene expression. Total RNA was isolated from tissue samples, linearly amplified, and 15,208 ovine probes were applied to cDNA microarray. Validation for selected genes was obtained by real-time quantitative polymerase chain reaction (RT-qPCR). Results: We found 1,075 (11.6%) differentially expressed probe sets in adult injured cartilage relative to normal cartilage. A total of 1,016 (11.0%) probe sets were differentially expressed in neonatal injured cartilage relative to normal cartilage. A total of 1,492 (16.1%) probe sets were differentially expressed in adult normal cartilage relative to neonatal normal cartilage. A total of 1,411 (15.3%) probe sets were differentially expressed in adult injured cartilage relative to neonatal injured cartilage. Significant functional clusters included genes associated with wound healing, articular protection, inflammation, and energy metabolism. Selected genes (PPARG, LDH, TOM, HIF1A, SMAD7, and NF-kappaB) were also found and validated by RT-qPCR. Conclusions: There are significant differences in gene expression between neonatal and adult ovine articular cartilage following acute injury. They are partly due to intrinsic differences in the process of development, and partly to different biological responses to mechanical trauma between neonatal and adult articular cartilage.

Mathematical model of the anatomy and fibre orientation field of the left ventricle of the heart (2013-06-18T00:00:00Z)

Background: One of the main factors affecting propagation of electrical waves and contraction in ventricles of the heart is anisotropy of cardiac tissue. Anisotropy is determined by orientation of myocardial fibres. Determining fibre orientation field and shape of the heart is important for anatomically accurate modelling of electrical and mechanical function of the heart. The aim of this paper is to introduce a theoretical rule-based model for anatomy and fibre orientation of the left ventricle (LV) of the heart and to compare it with experimental data. We suggest explicit analytical formulae that allow us to obtain the left ventricle form and its fibre direction field. The ventricle band concept of cardiac architecture given by Torrent-Guasp is chosen as the model postulate. Methods: In our approach, anisotropy of the heart is derived from some general principles. The LV is considered as a set of identical spiral surfaces, each of which can be produced from the other by rotation around one vertical axis. Each spiral surface is filled with non-intersecting curves which represent myocardial fibres.For model verification, we use experimental data on fibre orientation in human and canine hearts. Results: LV shape and anisotropy are represented by explicit analytical expressions in a curvilinear 3-D coordinate system. The derived fibre orientation field shows good qualitative agreement with experimental data. The model reveals the most thorough quantitative simulation of fibre angles at the LV middle zone. Conclusions: Our analysis shows that the band concept can generate realistic anisotropy of the LV. Our model shows good qualitative agreement between the simulated fibre orientation field and the experimental data on LV anisotropy, and the model can be used for various numerical simulations to study the effects of anisotropy on cardiac excitation and mechanical function.

Chromoendoscopy in magnetically guided capsule endoscopy (2013-06-11T00:00:00Z)

Background: Diagnosis of intestinal metaplasia and dysplasia via conventional endoscopy is characterized by low interobserver agreement and poor correlation with histopathologic findings. Chromoendoscopy significantly enhances the visibility of mucosa irregularities, like metaplasia and dysplasia mucosa. Magnetically guided capsule endoscopy (MGCE) offers an alternative technology for upper GI examination. We expect the difficulties of diagnosis of neoplasm in conventional endoscopy to transfer to MGCE. Thus, we aim to chart a path for the application of chromoendoscopy on MGCE via an ex-vivo animal study. Methods: We propose a modified preparation protocol which adds a staining step to the existing MGCE preparation protocol. An optimal staining concentration is quantitatively determined for different stain types and pathologies. To that end 190 pig stomach tissue samples with and without lesion imitations were stained with different dye concentrations. Quantitative visual criteria are introduced to measure the quality of the staining with respect to mucosa and lesion visibility. Thusly determined optimal concentrations are tested in an ex-vivo pig stomach experiment under magnetic guidance of an endoscopic capsule with the modified protocol. Results: We found that the proposed protocol modification does not impact the visibility in the stomach or steerability of the endoscopy capsule. An average optimal staining concentration for the proposed protocol was found at 0.4% for Methylene blue and Indigo carmine. The lesion visibility is improved using the previously obtained optimal dye concentration. Conclusions: We conclude that chromoendoscopy may be applied in MGCE and improves mucosa and lesion visibility. Systematic evaluation provides important information on appropriate staining concentration. However, further animal and human in-vivo studies are necessary.

Image analysis and processing methods in verifying the correctness of performing low-invasive esthetic medical procedures (2013-06-09T00:00:00Z)

Background: Efficacy and safety of various treatments using fractional laser or radiofrequency depend, to a large extent, on precise movement of equipment head across the patient's skin. In addition, they both depend on uniform distribution of emitted pulses throughout the treated skin area. The pulses should be closely adjacent but they should not overlap. Pulse overlapping results in amplification of irradiation dose and carries the danger of unwanted effects. Methods: Images obtained in infrared mode (Flir SC5200 thermovision camera equipped with photon detector) were entered into Matlab environment. Thermal changes in the skin were forced by CO2RE laser. Proposed image analysis and processing methods enable automatic recognition of CO2RE laser sites of action, making possible to assess the correctness of performed cosmetic procedures. Results: 80 images were acquired and analyzed. Regions of interest (ROI) for the entire treatment field were determined automatically. In accordance with the proposed algorithm, laser-irradiated Li areas (ROI) were determined for the treatment area. On this basis, error values were calculated and expressed as percentage of area not covered by any irradiation dose (deltao) and as percentage area which received double dose (deltaz). The respective values for the analyzed images were deltao=17.87+/-10.5% and deltaz=1.97+/-1.5%, respectively. Conclusions: The presented method of verifying the correctness of performing low-invasive esthetic medical (cosmetic) procedures has proved itself numerous times in practice. Advantages of the method include: automatic determination of coverage error values deltao and deltaz, non-invasive, sterile and remote-controlled thermovisual mode of measurements, and possibility of assessing dynamics of patient's skin temperature changes.

An improved level set method for vertebra CT image segmentation (2013-05-28T00:00:00Z)

Background: Clinical diagnosis and therapy for the lumbar disc herniation requires accurate vertebra segmentation. The complex anatomical structure and the degenerative deformations of the vertebrae makes its segmentation challenging. Methods: An improved level set method, namely edge- and region-based level set method (ERBLS), is proposed for vertebra CT images segmentation. By considering the gradient information and local region characteristics of images, the proposed model can efficiently segment images with intensity inhomogeneity and blurry or discontinuous boundaries. To reduce the dependency on manual initialization in many active contour models and for an automatic segmentation, a simple initialization method for the level set function is built, which utilizes the Otsu threshold. In addition, the need of the costly re-initialization procedure is completely eliminated. Results: Experimental results on both synthetic and real images demonstrated that the proposed ERBLS model is very robust and efficient. Compared with the well-known local binary fitting (LBF) model, our method is much more computationally efficient and much less sensitive to the initial contour. The proposed method has also applied to 56 patient data sets and produced very promising results. Conclusions: An improved level set method suitable for vertebra CT images segmentation is proposed. It has the flexibility of segmenting the vertebra CT images with blurry or discontinuous edges, internal inhomogeneity and no need of re-initialization.

Interview with Sabato D'Auria, Section Editor for the Chemical Biology section in BMC Biochemistry (2013-06-14T00:00:00Z)

Sabato D'Auria is currently a senior scientist with a tenured position within the National Research Council of Italy as the head of the Laboratory for Molecular Sensing at the Institute of Protein Biochemistry CNR, Naples, Italy. Dr D'Auria is also invited Professor at the INRS-Institute Armand-Frappier, Laval, Quebec, Canada.Dr D'Auria is a well known biochemist whose scientific interests are focused on the identification, isolation and structural characterization of proteins, and their application as a specific probe for the design of advanced optical biosensors. Dr. D'Auria's laboratory uses some of the most advanced biophysical techniques available in their research, including Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy for single molecule detection, time-resolved fluorescence, and near infra-red fluorescence spectroscopy.In this article we find out a little more about the field of chemical biology and discuss what Dr D'Auria would like to see submitted to his section.

Non-steroidal anti-inflammatory drugs activate NADPH oxidase in adipocytes and raise the H2O2 pool to prevent cAMP-stimulated protein kinase a activation and inhibit lipolysis (2013-05-30T00:00:00Z)

Background: Non-steroidal anti-inflammatory drugs (NSAIDs) ---aspirin, naproxen, nimesulide, and piroxicam--- lowered activation of type II cAMP-dependent protein kinase A (PKA-II) in isolated rat adipocytes, decreasing adrenaline- and dibutyryl cAMP (Bt2cAMP)-stimulated lipolysis. The molecular bases of insulin-like actions of NSAID were studied. Results: Based on the reported inhibition of lipolysis by H2O2, catalase was successfully used to block NSAID inhibitory action on Bt2cAMP-stimulated lipolysis. NSAID, at (sub)micromolar range, induced an H2O2 burst in rat adipocyte plasma membranes and in whole adipocytes. NSAID-mediated rise of H2O2 was abrogated in adipocyte plasma membranes by: diphenyleneiodonium, an inhibitor of NADPH oxidase (NOX); the NOX4 antibody; and cytochrome c, trapping the NOX-formed superoxide. These three compounds prevented the inhibition of Bt2cAMP-stimulated lipolysis by NSAIDs. Inhibition of aquaporin-mediated H2O2 transport with AgNO3 in adipocytes allowed NOX activation but prevented the lipolysis inhibition promoted by NSAID: i.e., once synthesized, H2O2 must reach the lipolytic machinery. Since insulin inhibits adrenaline-stimulated lipolysis, the effect of aspirin on isoproterenol-stimulated lipolysis in rat adipocytes was studied. As expected, isoproterenol-mediated lipolysis was blunted by both insulin and aspirin. Conclusions: NSAIDs activate NOX4 in adipocytes to produce H2O2, which impairs cAMP-dependent PKA-II activation, thus preventing isoproterenol-activated lipolysis. H2O2 signaling in adipocytes is a novel and important cyclooxygenase-independent effect of NSAID.

Cell attachment on poly(3-hydroxybutyrate)-poly(ethylene glycol) copolymer produced by Azotobacter chroococcum 7B (2013-05-21T00:00:00Z)

Background: The improvement of biomedical properties, e.g. biocompatibility, of poly(3-hydroxyalkanoates) (PHAs) by copolymerization is a promising trend in bioengineering. We used strain Azotobacter chroococcum 7B, an effective producer of PHAs, for biosynthesis of not only poly(3-hydroxybutyrate) (PHB) and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also alternative copolymer, poly(3-hydroxybutyrate)-poly(ethylene glycol) (PHB-PEG). Results: In biosynthesis we used sucrose as the primary carbon source and valeric acid or poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-PEG and PHB-HV was confirmed by 1H nuclear-magnetic resonance (1H NMR) analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) and surface morphology of films from PHB copolymers were studied. To study copolymers biocompatibility in vitro the protein adsorption and COS-1 fibroblasts growth on biopolymer films by XTT assay were analyzed. Both copolymers had changed physico-chemical properties compared to PHB homopolymer: PHB-HV and PHB-PEG had less crystallinity than PHB; PHB-HV was more hydrophobic than PHB in contrast to PHB-PEG appeared to have greater hydrophilicity than PHB; whereas the morphology of polymer films did not differ significantly. The protein adsorption to PHB-PEG was greater and more uniform than to PHB and PHB-PEG copolymer promoted better growth of COS-1 fibroblasts compared with PHB homopolymer. Conclusions: Thus, despite low EG-monomers content in bacterial origin PHB-PEG copolymer, this polymer demonstrated significant improvement in biocompatibility in contrast to PHB and PHB-HV copolymers, which may be coupled with increased protein adsorption and hydrophilicity of PEG-containing copolymer.

Construction of a chimeric thermoacidophilic beta-endoglucanase (2013-04-29T00:00:00Z)

Background: The archeaon Sulfolobus solfataricus P2 encodes a thermoacidophilic cellulase which shows an extreme acid and thermal stability with a pH optimum at 1.8 and a temperature optimum at 80[degree sign]C. This extraordinary enzyme could be useful for biotechnological exploitation but the expression and purification in expression hosts like E. coli is unsatisfactory due to the high aggregation tendency of the recombinant enzyme. The thermophilic cellulase CelA from Thermotoga maritima belongs to the same glycoside hydrolase family (GH12) but has a neutral pH optimum. In contrast to SSO1949 this enzyme is expressed partially soluble in E. coli. Results: We aimed to constructed a hybrid enzyme based on these two beta-endoglucanases which should successfully combine the advantageous properties of both cellulases, i.e. recombinant expression in E. coli, acidophily and thermophily. We constructed two hybrid proteins after bioinformatic analysis: both hybrids are expressed insoluble in E. coli, but one hybrid enzyme was successfully refolded from washed inclusion bodies. Conclusions: The refolded active chimeric enzyme shows a temperature optimum of approximately 85[degree sign]C and a pH optimum of approximately pH 3 thus retaining the advantageous properties of the Sulfolobus parent enzyme. This study suggests that the targeted construction of chimeric enzymes is an alternative to point mutational engineering efforts as long as parent enzymes with the wanted properties are available.

Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment (2013-04-08T00:00:00Z)

Background: Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464--5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor. Results: Here we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1--3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5. Conclusions: Together these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel information about this high-affinity interaction involving an antibody from the VH1-69 germline segment.

Towards a novel influenza vaccine: engineering of hemagglutinin on a platform of adenovirus dodecahedron (2013-06-16T00:00:00Z)

Background: The production process for the current influenza vaccine takes about 6 months and its antigenic composition must be modified annually. In the attempt towards developing influenza vaccine production that would be faster, safer and cheaper we engineered an influenza vaccine in which multiple copies of hemagglutinin (HA) would be delivered by a vector, adenovirus dodecahedron (Ad Dd). Dd is a virus-like particle, formed by assembly of twelve copies of pentameric penton base (Pb) proteins responsible for virus penetration. In order to attach HA to the vector, an adaptor containing WW domains was used. The WW domain is a linear peptide fragment identified as a partner of proline-proline-x-tyrosine (PPxY) motif present at the N-terminal extremity of the Pb protein, which is a building block of Dd. That tandem of three WW domains in fusion with the protein of interest enables interaction with Dd and efficient translocation to the cytoplasm of cells in culture. Results: Since HA is an oligomeric protein with complicated processing, we prepared six different constructs of HA (A/swan/Poland/467/2006(H5N1)) in fusion with the WW adaptor. Herein we report baculovirus expression and functional analysis of six HA-WW variants. The best behaving variant was successfully delivered into human cells in vitro. Conclusions: Engineering of a soluble complex of HA with Dd, a virus-like particle that serves as a vector, an adjuvant and as a multivalent presentation platform, is an important step toward a novel influenza vaccine.

Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli (2013-06-03T00:00:00Z)

Background: Activators of Nitrile hydratase (NHase) are essential for functional NHase biosynthesis. However, the activator P14K in P. putida is difficult to heterogeneously express, which retards the clarification of the mechanism of P14K involved in the maturation of NHase. Although a strep tag containing P14K (strep-P14K) was over-expressed, its low expression level and low stability affect the further analysis. Results: We successfully expressed P14K through genetic modifications according to N-end rule and analyzed the mechanism for its difficult expression. We found that mutation of the second N-terminal amino-acid of the protein from lysine to alanine or truncating the N-terminal 16 amino-acid sequence resulted in successful expression of P14K. Moreover, fusion of a pelB leader and strep tag together (pelB-strep-P14K) at the N-terminus increased P14K expression. In addition, the pelB-strep-P14K was more stable than the strep-P14K. Conclusions: Our results are not only useful for clarification of the role of P14K involved in the NHase maturation, but also helpful for heterologous expression of other difficult expression proteins.

Generation of a tumor- and tissue-specific episomal non-viral vector system (2013-06-04T00:00:00Z)

Background: A key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. Here we present the generation of non-viral, episomally replicating vectors that are able to replicate in a tissue specific manner thus allowing tissue specific transgene expression in combination with episomal replication. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR). Results: Here, we exchanged the constitutive promoter in the pEPI derivative pEPito by the tumor specific alpha fetoprotein (AFP) or the muscle specific smooth muscle 22 (SM22) promoter leading to specific transgene expression in AFP positive human hepatocellular carcinoma (HUH7) and in a SM22 positive cell line, respectively. The incorporation of the hCMV enhancer element into the expression cassette further boosted the expression levels with both promoters. Tissue specific-replication could be exemplary proven for the smooth muscle protein 22 (SM22) promoter in vitro. With the AFP promoter-driven pEPito vector hepatocellular carcinoma-specific expression could be achieved in vivo after systemic vector application together with polyethylenimine as transfection enhancer. Conclusions: In this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. The human AFP-promoter in combination with the hCMV enhancer element was demonstrated to be a valuable tissue-specific promoter for targeting hepatocellular carcinomas with non-viral gene delivery system, and tissue specific replication could be shown in vitro with the muscle specific SM22 promoter. In combination with appropriate delivery systems, the tissue specific pEPito vector system will allow higher tissue-specificity with less undesired side effects and is suitable for long term transgene expression in vivo within gene therapeutical approaches.

Expression and efficient secretion of a functional chitinase from Chromobacterium violaceum in Escherichia coli (2013-06-01T00:00:00Z)

Background: Chromobacterium violaceum is a free-living beta-proteobacterium found in tropical and subtropical regions. The genomic sequencing of C. violaceum ATCC 12472 has revealed many genes that underpin its adaptability to diverse ecosystems. Moreover, C. violaceum genes with potential applications in industry, medicine and agriculture have also been identified, such as those encoding chitinases. However, none of the chitinase genes of the ATCC 12472 strain have been subjected to experimental validation. Chitinases (EC 3.2.1.14) hydrolyze the beta-(1,4) linkages in chitin, an abundant biopolymer found in arthropods, mollusks and fungi. These enzymes are of great biotechnological interest as potential biocontrol agents against pests and pathogens. This work aimed to experimentally validate one of the chitinases from C. violaceum. Results: The open reading frame (ORF) CV2935 of C. violaceum ATCC 12472 encodes a protein (439 residues) that is composed of a signal peptide, a chitin-binding domain, a linker region, and a C-terminal catalytic domain belonging to family 18 of the glycoside hydrolases. The ORF was amplified by PCR and cloned into the expression vector pET303/CT-His. High levels of chitinolytic activity were detected in the cell-free culture supernatant of E. coli BL21(DE3) cells harboring the recombinant plasmid and induced with IPTG. The secreted recombinant protein was purified by affinity chromatography on a chitin matrix and showed an apparent molecular mass of 43.8 kDa, as estimated by denaturing polyacrylamide gel electrophoresis. N-terminal sequencing confirmed the proper removal of the native signal peptide during the secretion of the recombinant product. The enzyme was able to hydrolyze colloidal chitin and the synthetic substrates p-nitrophenyl-beta-D-N,N'-diacetylchitobiose and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose. The optimum pH for its activity was 5.0, and the enzyme retained ~32% of its activity when heated to 60[degree sign]C for 30 min. Conclusions: A C. violaceum chitinase was expressed in E. coli and purified by affinity chromatography on a chitin matrix. The secretion of the recombinant protein into the culture medium was directed by its native signal peptide. The mature enzyme was able to hydrolyze colloidal chitin and synthetic substrates. This newly identified signal peptide is a promising secretion factor that should be further investigated in future studies, aiming to demonstrate its usefulness as an alternative tool for the extracellular production of recombinant proteins in E. coli.

Metabolic evolution of Corynebacterium glutamicum for increased production of L-ornithine (2013-06-01T00:00:00Z)

Background: L-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. The commercial applications mean that efficient biotechnological production of L-ornithine has become increasingly necessary. Adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. The evolved strains can be further optimised by metabolic engineering. Thus, metabolic evolution strategy was used for engineering Corynebacterium glutamicum to enhance L-ornithine production. Results: A C. glutamicum strain was engineered by using a combination of gene deletions and adaptive evolution with 70 passages of growth-based selection. The metabolically evolved C. glutamicum strain, named DeltaAPE6937R42, produced 24.1 g/L of L-ornithine in a 5-L bioreactor. The mechanism used by C. glutamicum DeltaAPE6937R42 to produce L-ornithine was investigated by analysing transcriptional levels of select genes and NADPH contents. The upregulation of the transcription levels of genes involved in the upstream pathway of glutamate biosynthesis and the elevated NADPH concentration caused by the upregulation of the transcriptional level of the ppnK gene promoted L-ornithine production in C. glutamicum DeltaAPE6937R42. Conclusions: The availability of NADPH plays an important role in L-ornithine production in C. glutamicum. Our results demonstrated that the combination of growth-coupled evolution with analysis of transcript abundances provides a strategy to engineer microbial strains for improving production of target compounds.

Vitamin D analogs enhance the anticancer activity of 5-fluorouracil in an in vivo mouse colon cancer model (2013-06-18T00:00:00Z)

Background: Active vitamin D analogs that are less toxic than calcitriol can be useful in the combined treatment of patients suffering from colon cancer. In the present study we demonstrate, for the first time in an in vivo model system, the biological effect of combined therapy using 5-fluorouracil (5-FU) along with vitamin D analog PRI-2191 (tacalcitol, 1,24-dihydroxyvitamin D3) or PRI-2205 (5,6-trans-isomer of calcipotriol) on colon cancer. Methods: We investigated the influence of vitamin D analogs on the anticancer activity of 5-FU or capecitabine in the treatment of mice bearing MC38 mouse colon tumors implanted subcutaneously or orthotopically. The cell cycle distribution, E-cadherin expression and caspase 3/7 activity in vitro were also evaluated. Results: We observed that both PRI-2191 and PRI-2205 significantly enhanced the antitumor activity of 5-FU; but these results depend on the treatment regimen. Applying the optimal schedule of combined therapy we observed a significant decrease in tumor growth, metastasis and also a prolongation of the survival time of mice, in comparison with the administrations of 5-FU given alone. Both combinations indicated a synergistic effect and did not cause toxicity. Moreover, analogs applied after completed course of administration of 5-FU, prolonged the antitumor effect of the drug. Furthermore, when the prodrug of 5-FU, capecitabine, was used, potentiation of its activity was also observed. Conclusions: Our data suggest that vitamin D analogs (especially PRI-2191) might be potentially applied to clinical use in order to enhance the anticancer effect of 5-FU and also prolong its activity against colon cancer. The activity of PRI-2191 is realized through stopping the cells in the G0/G1 cell cycle phase and increasing the expression of E-cadherin.

Radiotherapy plus nimotuzumab or placebo in the treatment of high grade glioma patients: results from a randomized, double blind trial (2013-06-19T00:00:00Z)

Background: The prognosis of patients bearing high grade glioma remains dismal. Epidermal Growth Factor Receptor (EGFR) is well validated as a primary contributor of glioma initiation and progression. Nimotuzumab is a humanized monoclonal antibody that recognizes the EGFR extracellular domain and reaches Central Nervous System tumors, in nonclinical and clinical setting. While it has similar activity when compared to other anti-EGFR antibodies, it does not induce skin toxicity or hypomagnesemia. Methods: A randomized, double blind, multicentric clinical trial was conducted in high grade glioma patients (41 anaplastic astrocytoma and 29 glioblastoma multiforme) that received radiotherapy plus nimotuzumab or placebo. Treatment and placebo groups were well-balanced for the most important prognostic variables. Patients received 6 weekly doses of 200 mg nimotuzumab or placebo together with irradiation as induction therapy. Maintenance treatment was given for 1 year with subsequent doses administered every 3 weeks. The objectives of this study were to assess the comparative overall survival, progression free survival, response rate, immunogenicity and safety. Results: The median cumulative dose was 3200 mg of nimotuzumab given over a median number of 16 doses. The combination of nimotuzumab and RT was well-tolerated. The most prevalent related adverse reactions included nausea, fever, tremors, anorexia and hepatic test alteration. No anti-idiotypic response was detected, confirming the antibody low immunogenicity. The mean and median survival time for subjects treated with nimotuzumab was 31.06 and 17.76 vs. 21.07 and 12.63 months for the control group. Conclusions: In this randomized trial, nimotuzumab showed an excellent safety profile and significant survival benefit in combination with irradiation.Trial registration: Cuban National Register for clinical trials (No. 1745) (http://registroclinico.sld.cu/ensayos).

Association between genomic recurrence risk and well-being among breast cancer patients (2013-06-18T00:00:00Z)

Background: Gene expression profiling (GEP) is increasingly used in the rapidly evolving field of personalized medicine. We sought to evaluate the association between GEP-assessed of breast cancer recurrence risk and patients' well-being. Methods: Participants were Dutch women from 10 hospitals being treated for early stage breast cancer who were enrolled in the MINDACT trial (Microarray In Node-negative and 1 to 3 positive lymph node Disease may Avoid ChemoTherapy). As part of the trial, they received a disease recurrence risk estimate based on a 70-gene signature and on standard clinical criteria as scored via a modified version of Adjuvant! Online. \Women completed a questionnaire 6--8 weeks after surgery and after their decision regarding adjuvant chemotherapy. The questionnaire assessed perceived understanding, knowledge, risk perception, satisfaction, distress, cancer worry and health-related quality of life (HRQoL), 6--8 weeks after surgery and decision regarding adjuvant chemotherapy. Results: Women (n = 347, response rate 62%) reported high satisfaction with and a good understanding of the GEP information they received. Women with low risk estimates from both the standard and genomic tests reported the lowest distress levels. Distress was higher predominately among patients who had received high genomic risk estimates, who did not receive genomic risk estimates, or who received conflicting estimates based on genomic and clinical criteria. Cancer worry was highest for patients with higher risk perceptions and lower satisfaction. Patients with concordant high-risk profiles and those for whom such profiles were not available reported lower quality of life. Conclusion: Patients were generally satisfied with the information they received about recurrence risk based on genomic testing. Some types of genomic test results were associated with greater distress levels, but not with cancer worry or HRQoL.Trial registration: ISRCTN: ISRCTN18543567

Quantitative expression analysis and prognostic significance of the BCL2-associated X gene in nasopharyngeal carcinoma: a retrospective cohort study (2013-06-18T00:00:00Z)

Background: Nasopharyngeal carcinoma (NPC) is a highly metastatic epithelial malignancy showing high prevalence in Southeast Asia and North Africa. The BCL2-associated X (BAX) gene encodes the most important pro-apoptotic member of the BCL2 family. We have recently shown that BCL2 and BCL2L12, two other members of the same apoptosis-related family, possess significant prognostic value in NPC. The objective of the current study was to analyze BAX mRNA expression in nasopharyngeal biopsies of NPC patients, and to assess its prognostic potential in this disease. Methods: Total RNA was isolated from 88 malignant and 9 hyperplastic nasopharyngeal biopsies, resected from Tunisian patients. After cDNA synthesis by reverse transcription of polyadenylated RNA, BAX mRNA expression was analyzed using a highly sensitive quantitative real-time polymerase chain reaction (qRT-PCR) method. Results: Lower BAX mRNA levels were detected in NPC biopsies than in hyperplastic nasopharyngeal samples. BAX mRNA expression status was associated with low tumor extent, negative regional lymph node status, and absence of distant metastases. Kaplan-Meier survival analysis demonstrated that patients with BAX mRNA-positive NPC have significantly longer disease-free survival (DFS) and overall survival (OS). In accordance with these findings, Cox regression analysis revealed that BAX mRNA expression can be considered as a favorable prognostic indicator of DFS and OS in NPC, independent of their gender, age, tumor histology, tumor extent, and nodal status. Furthermore, NPC patients without distant metastases are less likely to relapse when their primary tumor is BAX mRNA-positive, compared to metastasis-free patients with a BAX-negative nasopharyngeal malignancy. Conclusion: This is the first study examining the potential clinical utility of BAX as a prognostic tumor biomarker in NPC. We provide evidence that BAX mRNA expression can be considered as an independent favorable prognostic indicator of DFS and OS in NPC.

Secular trends of salted fish consumption and nasopharyngeal carcinoma: a multi-jurisdiction ecological study in 8 regions from 3 continents (2013-06-19T00:00:00Z)

Background: Despite salted fish being a classical risk factor of Nasopharyngeal Carcinoma (NPC), whether secular trends in salted fish consumption worldwide accounted for changes in NPC rates were unknown. The relationship between vegetable and cigarette consumption to NPC risk worldwide were also largely uncertain. We investigated the longitudinal trends in standardised NPC incidence/mortality rates across 8 regions and their associations with secular trends in salted fish, vegetable and tobacco consumptions. Methods: Age standardised mortality rate (ASMR) and age standardised incidence rate (ASIR) of NPC were obtained from the WHO cancer mortality database and Hong Kong Cancer Registry. Per capita consumption of salted fish, tobacco and vegetables in Hong Kong and 7 countries (China, Finland, Japan, Portugal, Singapore, United Kingdom and United States) were obtained from the Food and Agriculture Organization of the United Nation (FAO) and Hong Kong Trade and Census Statistics. Pearson correlation and multivariate analysis were performed to examine both crude and adjusted associations. Results: There were markedly decreasing trends of NPC ASIR and ASMR in Hong Kong over the past three decades, which were correlated with corresponding secular changes in salted fish consumption per capita (Pearson r for 10 cumulative years : ASIR = 0.729 (male), 0.674 (female); ASMR = 0.943 (male), 0.622 (female), all p < 0.05 except for female ASMR). However such associations no longer correlated with adjustments for decreasing tobacco and increasing vegetable consumption per capita (Pearson r for 10 cumulative years: ASIR = 2.007 (male), 0.339 (female), ASMR = 0.289 (male), 1.992 (female), all p > 0.05). However, there were no clear or consistent patterns in relations between NPC ASIR and ASMR with salted fish consumption across 7 regions in 3 continents. Conclusions: Our results do not support the notion that changes in salted fish consumption had played an important role in explaining secular trends of NPC rates in Hong Kong and worldwide. Further studies should explore other lifestyle and genetic factors. However, our findings do support the potentially protective effects of vegetable consumption against NPC.

Glucose-induced gradual phenotypic modulation of cultured human glomerular epithelial cells may be independent of Wilms' tumor 1 (WT1) (2013-06-14T00:00:00Z)

Background: Renal podocytes form the main filtration barrier possessing a unique phenotype maintained by proteins including podocalyxin and nephrin, the expression of which is suppressed in pathological conditions. We used an in vitro model of human glomerular epithelial cells (HGEC) to investigate the role of high glucose in dysregulating the podocytic epithelial phenotype and determined the time needed for this change to occur. Results: In our in vitro podocyte system changes indicating podocyte dedifferentiation in the prolonged presence of high glucose included loss of podocalyxin, nephrin and CD10/CALLA concomitant with upregulation of mesenchymal vimentin. Our study demonstrates for the first time that podocyte-specific markers undergo changes of expression at different time intervals, since glucose-mediated podocalyxin downregulation is a progressive process that precedes downregulation of nephrin expression. Finally we demonstrate that high glucose permanently impaired WT1 binding to the podocalyxin gene promoter region but did not affect WT1 binding on the nephrin gene promoter region. Conclusion: The presence of high glucose induced a phenotypic conversion of podocytes resembling partial dedifferentiation. Our study demonstrates that dysregulation of the normal podocytic phenotype is an event differentially affecting the expression of function-specific podocytic markers, exhibiting downregulation of the epithelial marker CD10/CALLA and PC first, followed by stably downregulated nephrin. Furthermore, it is herein suggested that WT1 may not be directly involved with upregulation of previously reduced PC and nephrin expression.

Arginine68 is an essential residue for the C-terminal cleavage of human Atg8 family proteins (2013-05-30T00:00:00Z)

Background: Autophagy is a conserved cellular process that degrades and recycles cytoplasmic components via a lysosomal pathway. The phosphatidylethanolamine (PE)-conjugation of the Atg8 protein plays an important role in the yeast autophagy process. In humans, six Atg8 homologs, including MAP1LC3A, MAP1LC3B, MAP1LC3C (refer to LC3A, LC3B, and LC3C hereafter), GABARAP, GABARAPL1, and GABARAPL2 have been reported. All of them can be conjugated to PE through a ubiquitin-like conjugation system, and be located to autophagosomes. Results: In this study, we found 3 new alternative splicing isoforms in LC3B, GABARAP, and GABARAPL1, (designated as LC3B-a, GABARAP-a and GABARAPL1-a, respectively). None of them can go through the PE-conjugation process and be located to autophagosomes. Interestingly, compared with LC3B, LC3B-a has a single amino acid (Arg68) deletion due to the NAGNAG alternative splicing in intron 3. Through structural simulations, we found that the C-terminal tail of LC3B-a is less mobile than that of LC3B, thus affecting its C-terminal cleavage by human ATG4 family proteins. Furthermore, we found that Arg68 is an essential residue facilitating the interaction between human Atg8 family proteins and ATG4B by forming a salt bridge with Asp171 of ATG4B. Depletion of this salt bridge reduces autophagosomes formation and autophagic flux under both normal and nutrition starvation conditions. Conclusions: These results suggest Arg68 is an essential residue for the C-terminal cleavage of Atg8 family proteins during the autophagy process.

Syndecan-2 is upregulated in colorectal cancer cells through interactions with extracellular matrix produced by stromal fibroblasts (2013-05-25T00:00:00Z)

Background: The extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines. Results: Our results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments. Conclusions: Overall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.

Lentivirus-mediated RNA interference targeting the H19 gene inhibits cell proliferation and apoptosis in human choriocarcinoma cell line JAR (2013-05-27T00:00:00Z)

Background: H19 is a paternally imprinted gene that has been shown to be highly expressed in the trophoblast tissue. Results from previous studies have initiated a debate as to whether noncoding RNA H19 acts as a tumor suppressor or as a tumor promotor in trophoblast tissue. In the present study, we developed lentiviral vectors expressing H19-specific small interfering RNA (siRNA) to specifically block the expression of H19 in the human choriocarcinoma cell line JAR. Using this approach, we investigated the impact of the H19 gene on the proliferation, invasion and apoptosis of JAR cells. Moreover, we examined the effect of H19 knockdown on the expression of insulin-like growth factor 2 (IGF2), hairy and enhancer of split homologue-1 (HES-1) and dual-specific phosphatase 5 (DUSP5) genes. Results: H19 knockdown inhibited apoptosis and proliferation of JAR cells, but had no significant impact on cell invasion. In addition, H19 knockdown resulted in significant upregulation of HES-1 and DUSP5 expression, but not IGF2 expression in JAR cells. Conclusions: The finding that H19 downregulation could simultaneously inhibit proliferation and apoptosis of JAR cells highlights a putative dual function for H19 in choriocarcinoma and may explain the debate on whether H19 acts as a tumor suppressor or a tumor promotor in trophoblast tissue. Furthermore, upregulation of HES-1 and DUSP5 may mediate H19 downregulation-induced suppression of proliferation and apoptosis of JAR cells.

Cell metabolism sets the differences between subpopulations of satellite cells (SCs) (2013-05-03T00:00:00Z)

Background: We have recently characterized two distinct populations of Satellite Cells (SCs) that differ in proliferation, regenerative potential, and mitochondrial coupling efficiency and classified these in Low Proliferative Clones (LPC) and High Proliferative Clones (HPC). Herewith, we have investigated their cell metabolism and individuated features that remark an intrinsic difference in basal physiology but that are retrievable also at the initial phases of their cloning. Results: Indeed, LPC and HPC can be distinguished for mitochondrial membrane potential (DeltaPsim) just after isolation from the fiber. This is matched by mitochondrial redox state measured via NAD+/NADH analysis and alternative respiratory CO2 production in cloned cells. All these parameters are accountable for metabolic differences reflected indeed by alternative expression of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3). Also Ca2+ handling by mitochondria is different together with the sensitivity to apoptosis triggered via this pathway. Finally, according to the above, we were able to determine which one among the clones represents the suitable stem cell. Conclusions: These experimental observations report novel physiological features in the cell biology of SCs and refer to an intrinsic heterogeneity within which their stemness may reside.

Evaluation in an emergency department of rapid separator tubes containing thrombin for serum preparation prior to hs-cTnT and CK-MB analyses (2013-06-13T00:00:00Z)

Background: In our emergency department, we collect blood in Rapid Serum Tubes (RSTs; Becton Dickinson, Franklin Lakes, NJ), in which clotting times are reduced. We investigated the influence of RST use on cardiac troponin T (hs-cTnT) and creatine kinase-MB (CK-MB) test results, in comparison with the use of tubes featuring a separator gel containing a clotting activator (SSTs; Green-vac, Yongin, Korea). Methods: Samples from 60 patients were divided into equal aliquots and placed into RSTs and SSTs; hs-cTnT and CK-MB concentrations were determined using an autoanalyzer (Elecsys 2010) running commercial assays (Roche Diagnostics, Penzberg, Germany). Between-tube differences in CK-MB and hs-cTnT values were compared using the paired t-test, and correlations among variables were evaluated by calculation of Spearman correlation coefficients (r values). Deming regression analysis was performed and Bland-Altman plots were constructed. Results: The hs-cTnT and CK-MB test results obtained from samples placed into RSTs and SSTs did not differ (p > 0.1). The correlations between the concentrations of hs-cTnT and CK-MB in samples placed into RSTs and SSTs were good; both r values were unity (p < 0.001). Deming regression analysis yielded the equation: RST [hs-cTnT] = 0.98 SST [hs-cTnT] + 0.69 pg/ml; and RST [CK-MB] = 0.95 SST [CK-MB]--0.09 ng/ml. The biases of 1.4 pg/ml (95% CI: minus 8.1--10.7 pg/ml) for hs-cTnT levels and 0.249 ng/ml (95% CI: minus 0.682--1.681 ng/ml) for CK-MB levels assayed using either tube was acceptable. Conclusion: The hs-cTnT and CK-MB test results did not significantly differ when either tube was used. RST tube use was associated with a short clotting time; this was an advantage in an emergency laboratory setting.

Relapsed angioimmunoblastic T-cell lymphoma with acquired expression of CD20: A case report and review of the literature (2013-06-05T00:00:00Z)

Background: Angioimmunoblastic T-cell lymphoma is one of the most common types of peripheral T-cell lymphomas, usually presenting at an older age with an aggressive clinical course. Its characteristic morphological presentation and follicular helper T-cell phenotype help to distinguish it from other T-cell lymphomas.Case presentationWe recently encountered the unique case of a 63-year old patient with relapsed tumour-cell rich angioimmunoblastic T-cell lymphoma, presenting with a "classical" phenotype and, in addition, an acquired, strong, aberrant expression of CD20."Lineage infidelity" of phenotypic markers is a well-documented phenomenon in lymphomas and leukemias, a circumstance currently still poorly understood and with the potential to bring about erroneous interpretations, causing diagnostic havoc. This case represents one of the few documented angioimmunoblastic T-cell lymphomas with strong CD20 expression. Of interest, CD20 expression was only detected in the recurrent lymphoma and not upon initial diagnosis. The clinical importance of this finding lies in the potential for treatment with an anti-CD20 antibody, for instance Rituximab, in addition to standard chemotherapy protocols for angioimmunoblastic T-cell lymphoma. Conclusion: Diagnostic work-up of lymphomas to determine their lineage should therefore consider morphology, pheno- as well as genotypic characteristics, where appropriate, and in particular signs of progression and change in marker profile in relapsed cases e.g. acquisition of "non-lineage" markers such as CD20 in T-cell lymphoma.

EphA4 is a prognostic factor in gastric cancer (2013-06-05T00:00:00Z)

Background: Erythropoietin-producing hepatocellular (Eph) receptor, consisting of a family of receptor tyrosine kinases, plays critical roles in tumour development and is considered an attractive target for cancer therapy. Methods: Tumour samples were obtained from 222 patients with gastric adenocarcinoma who underwent gastrectomy. The expressions of EphA2, EphA4, and ephrinA1 were evaluated immunohistochemically. Results: High expressions of EphA2, EphA4, and ephrinA1 significantly correlated with variables related to tumour progression, including the depth of invasion, metastatic lymph nodes, pathological stage, and distant metastasis or recurrent disease. High expressions of EphA2, EphA4, and ephrinA1 were significantly associated with poorer disease-specific survival (DSS; p < 0.001, p < 0.001, p = 0.026). On multivariate analysis, EphA4 was an independent prognostic factor of DSS (hazard ratio [HR], 2.3; 95% confidence interval [CI], 1.1-4.8; p = 0.028), and EphA2 tended to be a prognostic factor (HR, 2.4; 95% CI, 1.0-5.8; p = 0.050). In stage II and III cancer, EphA4 and EphA2 were both significantly associated with shorter survival (p = 0.007 and 0.019), but only EphA2 was an independent prognostic factor (HR, 2.6; 95% CI, 1.1-6.3; p = 0.039). Conclusion: EphA4 may play important roles in tumor progression and outcomes in patients with gastric cancer.

Primary adenocarcinoma of the thymus: an immunohistochemical and molecular study with review of the literature (2013-05-31T00:00:00Z)

Background: Primary adenocarcinoma of thymus is extremely rare.Case presentationThis is a case of primary adenocarcinoma with intestinal differentiation and focal mucin production in the thymus. Thymic cyst was associated with this tumor. Intestinal differentiation was confirmed by immunohistochemical stain with positivity for CDX-2, CK20, villin, MOC31 and focal positivity of CK7. Array comperative genomic hybridization (CGH) analysis showed a complex pattern of chromosomal imbalances including homozygous deletion at the HLA locus in chromosomal region 6p21.32. Conclusion: This rare tumor shows a similar genetic aberration with other studied thymic epithelial tumors.

Predictive value of preoperative serum CCL2, CCL18, and VEGF for the patients with gastric cancer (2013-05-22T00:00:00Z)

Background: To investigate the expression of chemokine ligand 2 (CCL2), chemokine ligand 18 (CCL18), and vascular endothelial growth factor (VEGF) in peripheral blood of patients with gastric cancer and their correlation with presence of malignancy and disease progression. Methods: Sixty patients with pathological proved gastric cancer were prospectively included into study. The levels of CCL2, CCL18, and VEGF in peripheral blood were examined by enzyme-linked immunosorbentassay (ELISA). Peripheral blood from 20 healthy people was examined as control. Results: The preoperative serum levels of CCL2, CCL18 and VEGF in gastric cancer patients were significantly higher than that of controls (P <0.001, P <0.001, and P <0.001, respectively). ROC curve analysis showed that with a cut-off value of >=1272.8, the VEGF*CCL2 predicted the presence of gastric cancer with 83% sensitivity and 80% specificity. Preoperative serum CCL2 was significantly correlated to N stage (P =0.040); CCL18 associated with N stage (P =0.002), and TNM stage (P =0.002); VEGF correlated to T stage (P =0.000), N stage (P =0.015), and TNM stage (P =0.000). Conclusion: Preoperative serum levels of CCL2 and VEGF could play a crucial role in predicting the presence and progression of gastric cancer.

Computational cloning of drug target genes of a parasitic nematode, Oesophagostomum dentatum (2013-06-18T00:00:00Z)

Background: Gene identification and sequence determination are critical requirements for many biological, genomic, and bioinformatic studies. With the advent of next generation sequencing (NGS) technologies, such determinations are predominantly accomplished in silico for organisms for which the genome is known or for which there exists substantial gene sequence information. Without detailed genomic/gene information, in silico sequence determination is not straightforward, and full coding sequence determination typically involves time- and labor-intensive PCR-based amplification and cloning methods. Results: An improved method was developed with which to determine full length gene coding sequences in silico using de novo assembly of RNA-Seq data. The scheme improves upon initial contigs through contig-to-gene identification by BLAST nearest--neighbor comparison, and through single-contig refinement by iterative-binning and -assembly of reads. Application of the iterative method produced the gene identification and full coding sequence for 9 of 12 genes and improved the sequence of 3 of the 12 genes targeted by benzimidazole, macrocyclic lactone, and nicotinic agonist classes of anthelminthic drugs in the swine nodular parasite Oesophagostomum dentatum. The approach improved upon the initial optimized assembly with Velvet that only identified full coding sequences for 2 genes. Conclusions: Our reiterative methodology represents a simplified pipeline with which to determine longer gene sequences in silico from next generation sequence data for any nematode for which detailed genetic/gene information is lacking. The method significantly improved upon an initial Velvet assembly of RNA-Seq data that yielded only 2 full length sequences. The identified coding sequences for the 11 target genes enables further future examinations including: (i) the use of recombinant target protein in functional assays seeking a better understanding of the mechanism of drug resistance, and (ii) seeking comparative genomic and transcriptomic assessments between parasite isolates that exhibit varied drug sensitivities.

High-throughput sequencing of methylated cytosine enriched by modification-dependent restriction endonuclease MspJI (2013-06-18T00:00:00Z)

Background: As a well-known epigenomic modification, DNA methylation is found to be common in plants and plays an important role in many biological processes. Relying on the unique feature of methylation-dependent digestion, the family of methylation-requiring restriction-like endonuclease, such as MspJI and its homologs, was suggested for a potential usage in methylation detection. Results: In this study, we combine MspJI digestion and electrophoretic band selection with next generation high-throughput sequencing technology to detect 5-methylcytosines in Arabidopsis genome. By developing a bioinformatics workflow to attribute the CNNR sites recognized by MspJI to the reference genome, we fulfilled the systematic assessment of this method. Conclusions: According to the assessment, here we provide the method for generating a detailed map of plant methylome that could be feasible, reliable and economical in methylation investigation.

Low-frequency intermediate penetrance variants in the ROCK1 gene predispose to Tetralogy of Fallot (2013-06-19T00:00:00Z)

Background: Epidemiological studies indicate a substantial excess familial recurrence of non-syndromic Tetralogy of Fallot (TOF), implicating genetic factors that remain largely unknown. The Rho induced kinase 1 gene (ROCK1) is a key component of the planar cell polarity signalling pathway, which plays an important role in normal cardiac development. The aim of this study was to investigate the role of genetic variation in ROCK1 on the risk of TOF. Results: ROCK1 was sequenced in a discovery cohort of 93 non-syndromic TOF probands to identify rare variants. TagSNPs were selected to capture commoner variation in ROCK1. Novel variants and TagSNPs were genotyped in a discovery cohort of 458 TOF cases and 1331 healthy controls, and positive findings were replicated in a further 209 TOF cases and 1290 healthy controls. Association between genotypes and TOF was assessed using LAMP.A rare SNP (c.807C > T; rs56085230) discovered by sequencing was associated with TOF risk (p = 0.006) in the discovery cohort. The variant was also significantly associated with the risk of TOF in the replication cohort (p = 0.018). In the combined cohorts the odds ratio for TOF was 2.61 (95% CI 1.58-4.30); p < 0.0001. The minor allele frequency of rs56085230 in the cases was 0.02, and in the controls it was 0.007. The variant accounted for 1% of the population attributable risk (PAR) of TOF. We also found significant association with TOF for an uncommon TagSNP in ROCK1, rs288979 (OR 1.64 [95% CI 1.15-2.30]; p = 1.5x10-5). The minor allele frequency of rs288979 in the controls was 0.043, and the variant accounted for 11% of the PAR of TOF. These association signals were independent of each other, providing additional internal validation of our result. Conclusions: Low frequency intermediate penetrance (LFIP) variants in the ROCK1 gene predispose to the risk of TOF.

Contrasting effects of Deadend1 (Dnd1) gain and loss of function mutations on allelic inheritance, testicular cancer, and intestinal polyposis (2013-06-17T00:00:00Z)

Background: Certain mutations in the Deadend1 (Dnd1) gene are the most potent modifiers of testicular germ cell tumor (TGCT) susceptibility in mice and rats. In the 129 family of mice, the Dnd1Ter mutation significantly increases occurrence of TGCT-affected males. To test the hypothesis that he Dnd1Ter allele is a loss-of-function mutation; we characterized the consequences of a genetically-engineered loss-of-function mutation in mice, and compared these results with those for Dnd1Ter. Results: We found that intercrossing Dnd1+/KO heterozygotes to generate a complete loss-of-function led to absence of Dnd1KO/KO homozygotes and significantly reduced numbers of Dnd1+/KO heterozygotes. Further crosses showed that Dnd1Ter partially rescues loss of Dnd1KO mice. We also found that loss of a single copy of Dnd1 in Dnd1KO/+ heterozygotes did not affect baseline occurrence of TGCT-affected males and that Dnd1Ter increased TGCT risk regardless whether the alternative allele was loss-of-function (Dnd1KO) or wild-type (Dnd1+). Finally, we found that the action of Dnd1Ter was not limited to testicular cancer, but also significantly increased polyp number and burden in the Apc+/Min model of intestinal polyposis. Conclusion: These results show that Dnd1 is essential for normal allelic inheritance and that Dnd1Ter has a novel combination of functions that significantly increase risk for both testicular and intestinal cancer.

Breed, sex and anatomical location-specific gene expression profiling of the porcine skeletal muscles (2013-06-15T00:00:00Z)

Background: Skeletal muscle is one of the most important economic traits in agricultural animals, especially in pigs. In the modern pig industry, lean type pigs have undergone strong artificial selection for muscle growth, which has led to remarkable phenotypic variations compared with fatty type pigs, making these different breeds an ideal model for comparative studies. Results: Here, we present comprehensive gene expression profiling for the white (longissimus dorsi muscle) and the red (psoas major muscle) skeletal muscles among male and female fatty Rongchang, feral Tibetan and lean Landrace pigs, using a microarray approach. We identified differentially expressed genes that may be associated the phenotypic differences of porcine muscles among the breeds, between the sexes and the anatomical locations. We also used a clustering method to identify sets of functionally coexpressed genes that are linked to different muscle phenotypes. We showed that, compared with the white muscles, which primarily modulate metabolic processes, the red muscles show a tendency to be a risk factor for inflammation and immune-related disorders. Conclusions: This analysis presents breed-, sex- and anatomical location-specific gene expression profiles and further identified genes that may be associated with the phenotypic differences in porcine muscles among breeds, between the sexes and the anatomical locations.

The genesis of an exceptionally lethal venom in the timber rattlesnake (Crotalus horridus) revealed through comparative venom-gland transcriptomics (2013-06-12T00:00:00Z)

Background: Snake venoms generally show sequence and quantitative variation within and between species, but some rattlesnakes have undergone exceptionally rapid, dramatic shifts in the composition, lethality, and pharmacological effects of their venoms. Such shifts have occurred within species, most notably in Mojave (Crotalus scutulatus), South American (C. durissus), and timber (C. horridus) rattlesnakes, resulting in some populations with extremely potent, neurotoxic venoms without the hemorrhagic effects typical of rattlesnake bites. Results: To better understand the evolutionary changes that resulted in the potent venom of a population of C. horridus from northern Florida, we sequenced the venom-gland transcriptome of an animal from this population for comparison with the previously described transcriptome of the eastern diamondback rattlesnake (C. adamanteus), a congener with a more typical rattlesnake venom. Relative to the toxin transcription of C. adamanteus, which consisted primarily of snake-venom metalloproteinases, C-type lectins, snake-venom serine proteinases, and myotoxin-A, the toxin transcription of C. horridus was far simpler in composition and consisted almost entirely of snake-venom serine proteinases, phospholipases A2, and bradykinin-potentiating and C-type natriuretic peptides. Crotalus horridus lacked significant expression of the hemorrhagic snake-venom metalloproteinases and C-type lectins. Evolution of shared toxin families involved differential expansion and loss of toxin clades within each species and pronounced differences in the highly expressed toxin paralogs. Toxin genes showed significantly higher rates of nonsynonymous substitution than nontoxin genes. The expression patterns of nontoxin genes were conserved between species, despite the vast differences in toxin expression. Conclusions: Our results represent the first complete, sequence-based comparison between the venoms of closely related snake species and reveal in unprecedented detail the rapid evolution of snake venoms. We found that the difference in venom properties resulted from major changes in expression levels of toxin gene families, differential gene-family expansion and loss, changes in which paralogs within gene families were expressed at high levels, and higher nonsynonymous substitution rates in the toxin genes relative to nontoxins. These massive alterations in the genetics of the venom phenotype emphasize the evolutionary lability and flexibility of this ecologically critical trait.

Novel genomic approaches unravel genetic architecture of complex traits in apple (2013-06-12T00:00:00Z)

Background: Understanding the genetic architecture of quantitative traits is important for developing genome-based crop improvement methods. Genome-wide association study (GWAS) is a powerful technique for mining novel functional variants. Using a family-based design involving 1,200 apple (Malus x domestica Borkh.) seedlings genotyped for an 8K SNP array, we report the first systematic evaluation of the relative contributions of different genomic regions to various traits related to eating quality and susceptibility to some physiological disorders. Single-SNP analyses models that accounted for population structure, or not, were compared with models fitting all markers simultaneously. The patterns of linkage disequilibrium (LD) were also investigated. Results: A high degree of LD even at longer distances between markers was observed, and the patterns of LD decay were similar across successive generations. Genomic regions were identified, some of which coincided with known candidate genes, with significant effects on various traits. Phenotypic variation explained by the loci identified through a whole-genome scan ranged from 3% to 25% across different traits, while fitting all markers simultaneously generally provided heritability estimates close to those from pedigree-based analysis. Results from 'Q+K' and 'K' models were very similar, suggesting that the SNP-based kinship matrix captures most of the underlying population structure. Correlations between allele substitution effects obtained from single-marker and all-marker analyses were about 0.90 for all traits. Use of SNP-derived realized relationships in linear mixed models provided a better goodness-of-fit than pedigree-based expected relationships. Genomic regions with probable pleiotropic effects were supported by the corresponding higher linkage group (LG) level estimated genetic correlations. Conclusions: The accuracy of artificial selection in plants species can be increased by using more precise marker-derived estimates of realized coefficients of relationships. All-marker analyses that indirectly account for population- and pedigree structure will be a credible alternative to single-SNP analyses in GWAS. This study revealed large differences in the genetic architecture of apple fruit traits, and the marker-trait associations identified here will help develop genome-based breeding methods for apple cultivar development.

The acute transcriptional response of the coral Acropora millepora to immune challenge: expression of GiMAP/IAN genes links the innate immune responses of corals with those of mammals and plants (2013-06-14T00:00:00Z)

Background: As a step towards understanding coral immunity we present the first whole transcriptome analysis of the acute responses of Acropora millepora to challenge with the bacterial cell wall derivative MDP and the viral mimic poly I:C, defined immunogens provoking distinct but well characterised responses in higher animals. Results: These experiments reveal similarities with the responses both of arthropods and mammals, as well as coral-specific effects. The most surprising finding was that MDP specifically induced three members of the GiMAP gene family, which has been implicated in immunity in mammals but is absent from Drosophila and Caenorhabditis. Like their mammalian homologs, GiMAP genes are arranged in a tandem cluster in the coral genome. Conclusions: A phylogenomic survey of this gene family implies ancient origins, multiple independent losses and lineage-specific expansions during animal evolution. Whilst functional convergence cannot be ruled out, GiMAP expression in corals may reflect an ancestral role in immunity, perhaps in phagolysosomal processing.

Transcriptome analysis reveals unique C4-like photosynthesis and oil body formation in an arachidonic acid-rich microalga Myrmecia incisa Reisigl H4301 (2013-06-13T00:00:00Z)

Background: Arachidonic acid (ArA) is important for human health because it is one of the major components of mammalian brain membrane phospholipids. The interest in ArA inspired the search for a new sustainable source, and the green microalga Myrmecia incisa Reisigl H4301 has been found a potential ArA-producer due to a high content of intracellular ArA. To gain more molecular information about metabolism pathways, including the biosynthesis of ArA in the non-model microalga, a transcriptomic analysis was performed. Results: The 454 pyrosequencing generated 371,740 high-quality reads, which were assembled into 51,908 unique sequences consisting of 22,749 contigs and 29,159 singletons. A total of 11,873 unique sequences were annotated through BLAST analysis, and 3,733 were assigned to Gene Ontology (GO) categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered a C4-like photosynthesis pathway in M. incisa. The biosynthesis pathways of lipid particularly those of ArA and triacylglycerol (TAG) were analyzed in detail, and TAG was proposed to be accumulated in oil bodies in the cytosol with the help of caleosin or oil globule-associated proteins. In addition, the carotenoid biosynthesis pathways are discussed. Conclusion: This transcriptomic analysis of M. incisa enabled a global understanding of mechanisms involved in photosynthesis, de novo biosynthesis of ArA, metabolism of carotenoids, and accumulation of TAG in M. incisa. These findings provided a molecular basis for the research and possibly economic exploitation of this ArA-rich microalga.

A genome-wide integrative study of microRNAs in human liver (2013-06-13T00:00:00Z)

Background: Recent studies have illuminated the diversity of roles for microRNAs in cellular, developmental, and pathophysiological processes. The study of microRNAs in human liver tissue promises to clarify the therapeutic and diagnostic value of this important regulatory mechanism of gene expression. Results: We conducted genome-wide profiling of microRNA expression in liver and performed an integrative analysis with previously collected genotype and transcriptome data. We report here that the Very Important Pharmacogenes (VIP Genes), comprising of genes of particular relevance for pharmacogenomics, are under substantial microRNA regulatory effect in the liver. We set out to elucidate the genetic basis of microRNA expression variation in liver and mapped microRNA expression to genomic loci as microRNA expression quantitative trait loci (miR-eQTLs). We identified common variants that attain genome-wide significant association (p < 10-10) with microRNA expression. We also found that the miR-eQTLs are significantly more likely to predict mRNA levels at a range of p-value thresholds than a random set of allele frequency matched SNPs, showing the functional effect of these loci on the transcriptome. Finally, we show that a large number of miR-eQTLs overlap with SNPs reproducibly associated with complex traits from the NHGRI repository of published genome-wide association studies as well as variants from a comprehensive catalog of manually curated pharmacogenetic associations. Conclusion: Our study provides important insights into the genomic architecture of gene regulation in a vital human organ, with important implications for our understanding of disease pathogenesis, therapeutic outcome, and other complex human phenotypes.

Comparative analysis of the alveolar macrophage proteome in ALI/ARDS patients between the exudative phase and recovery phase (2013-06-17T00:00:00Z)

Background: Despite decades of extensive studies, the morbidity and mortality for acute lung injury/acute respiratory distress syndrome (ALI/ARDS) remained high. Particularly, biomarkers essential for its early diagnosis and prognosis are lacking. Methods: Recent studies suggest that alveolar macrophages (AMs) at the exudative phase of ALI/ARDS initiate, amplify and perpetuate inflammatory responses, while they resolve inflammation in the recovery phase to prevent further tissue injury and perpetuated inflammation in the lung. Therefore, proteins relevant to this functional switch could be valuable biomarkers for ALI/ARDS diagnosis and prognosis. We thus conducted comparative analysis of the AM proteome to assess its dynamic proteomic changes during ALI/ARDS progression and recovery. Results: 135 proteins were characterized to be differentially expressed between AMs at the exudative and recovery phase. MALDI-TOF-MS and peptide mass fingerprint (PMF) analysis characterized 27 informative proteins, in which 17 proteins were found with a marked increase at the recovery phase, while the rest of 10 proteins were manifested by the significantly higher levels of expression at the exudative phase. Conclusions: Given the role of above identified proteins played in the regulation of inflammatory responses, cell skeleton organization, oxidative stress, apoptosis and metabolism, they have the potential to serve as biomarkers for early diagnosis and prognosis in the setting of patients with ALI/ARDS.

Proton channel HVCN1 is required for effector functions of mouse eosinophils (2013-05-24T00:00:00Z)

Background: Proton currents are required for optimal respiratory burst in phagocytes. Recently, HVCN1 was identified as the molecule required for the voltage-gated proton channel activity associated with the respiratory burst in neutrophils. Although there are similarities between eosinophils and neutrophils regarding their mechanism for respiratory burst, the role of proton channels in eosinophil functions has not been fully understood. Results: In the present study, we first identified the expression of the proton channel HVCN1 in mouse eosinophils. Furthermore, using HVCN1-deficient eosinophils, we demonstrated important cell-specific effector functions for HVCN1. Similar to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils produced significantly less reactive oxygen species (ROS) upon phorbol myristate acetate (PMA) stimulation compared with WT eosinophils. In contrast to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils did not show impaired calcium mobilization or migration ability compared with wild-type (WT) cells. Uniquely, HVCN1-deficient eosinophils underwent significantly increased cell death induced by PMA stimulation compared with WT eosinophils. The increased cell death was dependent on NADPH oxidase activation, and correlated with the failure of HVCN1-deficient cells to maintain membrane polarization and intracellular pH in the physiological range upon activation. Conclusions: Eosinophils require proton channel HVCN1 for optimal ROS generation and prevention of activation-induced cell death.

Impact of hepatitis C virus co-infection on HIV patients before and after highly active antiretroviral therapy: an immunological and clinical chemistry observation, Addis Ababa, Ethiopia (2013-05-17T00:00:00Z)

Background: Hepatitis C virus (HCV) is an RNA virus which has been known to cause acute and chronic necro-inflammatory disease of the liver. It is the leading cause of end-stage liver disease and hepatocellular carcinoma. HIV is known to have a negative impact on the natural disease outcome and immune response of HCV infection, whereas the reverse remains unclear. We evaluated the impact of HCV co-infection on recovery of CD4+ and CD8+ T-cells and liver enzyme levels before and after initiation of highly active antiretroviral therapy (HAART) in HIV/HCV co-infected patients. Methods: A hospital-based, observational, prospective cohort study design was used for this study. Pre-antiretroviral treatment (Pre-ART) and under HAART HIV mono-infected and HCV/HIV co-infected individuals who are under regular follow-up were recruited for this study. 387 blood samples were collected from volunteer, known HIV positive Ethiopian patients and screened for HCV. Twenty five HCV/HIV co-infected patients were prospectively followed for four years. CD4+ and CD8+ T-cells and liver enzyme levels were determined annually for each of the participant. Results: The prevalence of HCV/HIV co-infection in this study was 6.5%. Both HCV/HIV co-infected and HIV mono-infected under HAART groups showed CD4+ recovery (343 Vs 426; P < 0.004, OR = 4.97, 95% CI = 2.41 to 10.27) respectively; but, the recovery rate was higher in mono-infected (80 Vs 426) than co-infected group (148 Vs 343). The recovery and/or decline pattern of CD8+ T-cells was the same with that of CD4+. In 75% of co-infected groups, the mean alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were above the upper limit of normal reference range. Analyses restricted to individuals who initiated HAART and pre-ART showed similar results. Conclusion: We found that CD4+ T-cell recovery was negatively affected by the presence of ongoing HCV replication in under HAART co-infected individuals and fast decline of CD4+ T-cells in pre-ART patients. It was also associated with increased ALT and AST enzyme levels in both HAART initiated and treatment naive co-infected patients.

Expression of Toll-like Receptors 2 and 9 in cells of dog jejunum and colon naturally infected with Leishmania infantum (2013-05-14T00:00:00Z)

Background: Infection with parasite protozoa is a long-term health issue in tropical and subtropical regions throughout the world. The Toll-like receptor (TLR) signaling pathway is one of the first-responding defense systems against Leishmania. The aim of this study was to investigate the expression of TLR2 and TLR9 in jejunum and colon and its correlation with CD11c, CD11b, and CD14 receptors used as markers for dendritic cells and macrophages. Methods: Twenty four dogs infected with Leishmania infantum were used in this study. Cytometry was carried out in lamina propria cells from jejunum and colon using markers for TLR2, TLR9, CD11b, CD11c and CD14. Results: Cellular inflammatory exudate was diffuse in the mucosa and submucosa, predominately comprising mononuclear cells: plasma cells, macrophages, and lymphocytes. Despite the parasite load, microscopy showed no erosion was evident in the epithelial mucosa layers. The colon harbored more parasites than the jejunum. Flow cytometry revealed higher frequency of TLR2+ and CD11c+ dendritic cells in the colon than in the jejunum. Conversely, TLR9-expressing cells were more frequent in jejunum. Moreover, frequency of macrophages (CD11b+ and CD14+) expressing simultaneity TLR9 were lower in the colon than in jejunum, while CD11c+ cells predominated in the colon. Despite of the negative ELISA serum results, IL-10 and TNF-alpha were higher in jejunum than colon of infected animals. However, IL-4 was higher in colon than jejunum of infected animals. A higher expression these cytokines were demonstrated in infected dogs compared to uninfected dogs. Conclusions: There was no correlation between clinical signs and pathological changes and immunological and parasitological findings in the gastrointestinal tract in canine visceral leishmaniasis. However, jejunum showed a lower parasite load with increased frequency and expression of CD11b, TLR9, CD14/CD11b/TLR9 receptors and IL-10 and TNF-alpha cytokines. Conversely, the colon showed a higher parasite load along with increased frequency and expression of TLR2, CD11c receptors, and IL-4 cytokine. Thus, Leishmania infantum is able to interfere in jejunum increased expression of TLR2, TLR9, CD11b, CD14, CD14/CD11b/TLR9 receptors, IL-10, and TNF-alpha; and in colon increased expression of CD11c, TLR2, TLR9, CD11b, CD14 e, CD14/CD11b/TLR9 receptors, IL-10, and TNF-alpha.

The impact of polymorphisms in STAT6 on treatment outcome in HCV infected Taiwanese Chinese (2013-05-08T00:00:00Z)

Genetic polymorphisms observed in various disease states associated with sensitivity or resistance to specific treatments have been a robust area of investigation for decades, with the potential to allow clinicians to make evidence-based decisions on the appropriate course of treatment. This study aimed to evaluate whether genetic polymorphisms of the signal transducer and activator of transcription 6 gene (STAT6) could be associated with a sustained virological response (SVR) among patients infected with hepatitis C virus genotypes 1 and 2 (HCV-1 and HCV-2) who were treated with peginterferon plus ribavirin (PEG-IFNalpha-RBV). We analyzed the associations between SVR to PEG-IFNalpha-RBV therapy and 4 single nucleotide polymorphisms (SNPs) in STAT6. This study included Taiwanese Chinese patients infected with either HCV-1 (n = 265) or HCV-2 (n = 195) in the presence or absence of an SVR. Among the STAT6 SNPs examined, the dosage effect of the A allele and allele frequency in rs1059513 were inversely correlated with SVR in patients infected with HCV-1 (P = 0.0179 and P = 0.0235, respectively). This effect was not observed in patients infected with HCV-2. The GG, GGG, and GGGC STAT6 haplotypes comprising 2, 3, and 4 SNPs (rs1059513, rs703817, rs324015, and rs3024974) were found to be associated with SVR, and their presence may increase the probability of a successful treatment outcome in patients infected with HCV-1 (P = 0.0273, 0.0352, and 0.0368, respectively). Moreover, a multivariate logistic regression model for predicting an SVR revealed that the presence of the GGGC haplotype carriers mutually affected the outcome of PEG-IFNalpha-RBV treatment. The presence of STAT6 SNPs and the association with SVR demonstrated that STAT6 polymorphisms might influence the therapeutic outcomes of patients infected with HCV-1 under standard-of-care (SOC) treatment.

Differential biodistribution of intravenously administered endothelial progenitor and cytotoxic T-cells in rat bearing orthotopic human glioma (2013-06-10T00:00:00Z)

Background: A major challenge in the development of cell based therapies for glioma is to deliver optimal number of cells (therapeutic dose) to the tumor. Imaging tools such as magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET) and single-photon emission computed tomography (SPECT) has been used in cell tracking and/or biodistribution studies. In this study, we evaluate the dynamic biodistribution of systemic injected labeled cells [human cord blood derived endothelial progenitor cells (EPCs) and cytotoxic T-cells (CTLs)] in rat glioma model with in vivo SPECT imaging. Methods: Human cord blood EPCs, T-cells and CD14+ cells (monocytes/dendritic cells) were isolated using the MidiMACS system. CD14+ cells were converted to dendritic cells (DC) and also primed with U251 tumor cell line lysate. T-cells were co-cultured with irradiated primed DCs at 10:1 ratio to make CTLs. Both EPCs and CTLs were labeled with In-111-oxine at 37[degree sign]C in normal saline. Glioma bearing animals were randomly assigned into three groups. In-111 labeled cells or In-111 oxine alone were injected through tail vein and SPECT imaging was performed on day 0, 1, and 3. In-111 oxine activity in various organs and tumor area was determined. Histochemical analysis was performed to further confirm the migration and homing of injected cells at the tumor site. Results: EPCs and CTLs showed an In-111 labeling efficiency of 87.06 +/- 7.75% and 70.8 +/- 12.9% respectively. Initially cell migration was observed in lung following inravenous administration of In-111 labeled cells and decreased on day 1 and 3, which indicate re-distribution of labeled cells from lung to other organs. Relatively higher In-111 oxine activity was observed in tumor areas at 24 hours in animals received In-111 labeled cells (EPCs or CTLs). Histiological analysis revealed iron positive cells in and around the tumor area in animals that received labeled cells (CTLs and EPCs). Conclusion: We observed differential biodistribution of In-111-oxine labeled EPCs and CTLs in different organs and intracranial glioma. This study indicates In-111 oxine based SPECT imaging is an effective tool to study the biodistribution of therapeutically important cells.

Evaluation of ultrasound Tissue Velocity Imaging: a phantom study of velocity estimation in skeletal muscle low-level contractions (2013-06-07T00:00:00Z)

Background: Tissue Velocity Imaging (TVI) is an ultrasound based technique used for quantitative analysis of the cardiac function and has earlier been evaluated according to myocardial velocities. Recent years several studies have reported applying TVI in the analysis of skeletal muscles. Skeletal tissue velocities can be very low. In particular, when performing isometric contractions or contractions of low force level the velocities may be much lower compared to the myocardial tissue velocities. Methods: In this study TVI was evaluated for estimation of tissue velocities below the typical myocardial velocities. An in-house phantom was used to see how different PRF-settings affected the accuracy of the velocity estimations. Results: With phantom peak velocity at 0.03 cm/s the error ranged from 31% up to 313% with the different PRF-settings in this study. For the peak velocities at 0.17 cm/s and 0.26 cm/s there was no difference in error with tested PFR settings, it is kept approximately around 20%. Conclusions: The results from the present study showed that the PRF setting did not seem to affect the accuracy of the velocity estimation at tissue velocities above 0.17 cm/s. However at lower velocities (0.03 cm/s) the setting was crucial for the accuracy. The PRF should therefore preferable be reduced when the method is applied in low-level muscle contraction.

Evaluation of magnetic nanoparticle samples made from biocompatible ferucarbotran by time-correlation magnetic particle imaging reconstruction method (2013-06-05T00:00:00Z)

Background: Molecular imaging using magnetic nanoparticles (MNPs)---magnetic particle imaging (MPI)---has attracted interest for the early diagnosis of cancer and cardiovascular disease. However, because a steep local magnetic field distribution is required to obtain a defined image, sophisticated hardware is required. Therefore, it is desirable to realize excellent image quality even with low-performance hardware. In this study, the spatial resolution of MPI was evaluated using an image reconstruction method based on the correlation information of the magnetization signal in a time domain and by applying MNP samples made from biocompatible ferucarbotran that have adjusted particle diameters. Methods: The magnetization characteristics and particle diameters of four types of MNP samples made from ferucarbotran were evaluated. A numerical analysis based on our proposed method that calculates the image intensity from correlation information between the magnetization signal generated from MNPs and the system function was attempted, and the obtained image quality was compared with that using the prototype in terms of image resolution and image artifacts. Results: MNP samples obtained by adjusting ferucarbotran showed superior properties to conventional ferucarbotran samples, and numerical analysis showed that the same image quality could be obtained using a gradient magnetic field generator with 0.6 times the performance. However, because image blurring was included theoretically by the proposed method, an algorithm will be required to improve performance. Conclusions: MNP samples obtained by adjusting ferucarbotran showed magnetizing properties superior to conventional ferucarbotran samples, and by using such samples, comparable image quality (spatial resolution) could be obtained with a lower gradient magnetic field intensity.

Annual acknowledgement of manuscript reviewers (2013-04-23T00:00:00Z)

Contributing reviewersThe editors of BMC Medical Imaging would like to thank all our reviewers who have contributed to the journal in Volume 12 (2012).

Adding attenuation corrected images in myocardial perfusion imaging reduces the need for a rest study (2013-04-01T00:00:00Z)

Background: The American Society of Nuclear Cardiology and the Society of Nuclear Medicine conclude that incorporation of attenuation corrected (AC) images in myocardial perfusion scintigraphy (MPS) will improve diagnostic accuracy. The aim was to investigate the value of adding AC stress-only images for the decision whether a rest study is necessary or not. Methods: 1,261 patients admitted to 99mTc MPS were studied. The stress studies were interpreted by two physicians who judged each study as "no rest study necessary" or "rest study necessary", by evaluating NC stress-only and NC + AC stress-only images. When there was disagreement between the two physicians, a third physician evaluated the studies. Thus, agreement between 2 out of 3 physicians was evaluated. Results: The physicians assessed 214 more NC + AC images than NC images as "no rest study necessary" (17% of the study population). The number of no-rest-study-required was significantly higher for NC + AC studies compared to NC studies (859 vs 645 cases (p < 0.0001). In the final report according to clinical routine, ischemia or infarction was reported in 23 patients, assessed as "no rest study necessary" (22 NC + AC cases; 8 NC cases), (no statistically significant difference). In 11 of these, the final report stated "suspected/possible ischemia or infarction in a small area". Conclusions: Adding AC stress-only images to NC stress-only images reduce the number of unnecessary rest studies substantially.

Information-based measure of disagreement for more than two observers: a useful tool to compare the degree of observer disagreement (2013-03-22T00:00:00Z)

Background: Assessment of disagreement among multiple measurements for the same subject by different observers remains an important problem in medicine. Several measures have been applied to assess observer agreement. However, problems arise when comparing the degree of observer agreement among different methods, populations or circumstances. Methods: The recently introduced information-based measure of disagreement (IBMD) is a useful tool for comparing the degree of observer disagreement. Since the proposed IBMD assesses disagreement between two observers only, we generalized this measure to include more than two observers. Results: Two examples (one with real data and the other with hypothetical data) were employed to illustrate the utility of the proposed measure in comparing the degree of disagreement. Conclusion: The IBMD allows comparison of the disagreement in non-negative ratio scales across different populations and the generalization presents a solution to evaluate data with different number of observers for different cases, an important issue in real situations.A website for online calculation of IBMD and respective 95% confidence interval was additionally developed. The website is widely available to mathematicians, epidemiologists and physicians to facilitate easy application of this statistical strategy to their own data.

Applications of functional data analysis: A systematic review (2013-03-19T00:00:00Z)

Background: Functional data analysis (FDA) is increasingly being used to better analyze, model and predict time series data. Key aspects of FDA include the choice of smoothing technique, data reduction, adjustment for clustering, functional linear modeling and forecasting methods. Methods: A systematic review using 11 electronic databases was conducted to identify FDA application studies published in the peer-review literature during 1995--2010. Papers reporting methodological considerations only were excluded, as were non-English articles. Results: In total, 84 FDA application articles were identified; 75.0% of the reviewed articles have been published since 2005. Application of FDA has appeared in a large number of publications across various fields of sciences; the majority is related to biomedicine applications (21.4%). Overall, 72 studies (85.7%) provided information about the type of smoothing techniques used, with B-spline smoothing (29.8%) being the most popular. Functional principal component analysis (FPCA) for extracting information from functional data was reported in 51 (60.7%) studies. One-quarter (25.0%) of the published studies used functional linear models to describe relationships between explanatory and outcome variables and only 8.3% used FDA for forecasting time series data. Conclusions: Despite its clear benefits for analyzing time series data, full appreciation of the key features and value of FDA have been limited to date, though the applications show its relevance to many public health and biomedical problems. Wider application of FDA to all studies involving correlated measurements should allow better modeling of, and predictions from, such data in the future especially as FDA makes no a priori age and time effects assumptions.

Can we quantify harm in general practice records? An assessment of precision and power using computer simulation (2013-03-13T00:00:00Z)

Background: Estimating harm rates for specific patient populations and detecting significant changes in them over time are essential if patient safety in general practice is to be improved. Clinical record review (CRR) is arguably the most suitable method for these purposes, but the optimal values and combinations of its parameters (such as numbers of records and practices) remain unknown. Our aims were to: 1. Determine and quantify CRR parameters; 2. Assess the precision and power of feasible CRR scenarios; and 3. Quantify the minimum requirements for adequate precision and acceptable power.MethodWe explored precision and power of CRR scenarios using Monte Carlo simulation. A range of parameter values were combined in 864 different CRR scenarios, 1000 random data sets were generated for each, and harm rates were estimated and tested for change over time by fitting a generalised linear model with a Poisson response. Results: CRR scenarios with >=100 detected harm incidents had harm rate estimates with acceptable precision. Harm reductions of 20% or >=50% were detected with adequate power by those CRR scenarios with at least 100 and 500 harm incidents respectively. The number of detected harm incidents was dependent on the baseline harm rate multiplied by: the period of time reviewed in each record; number of records reviewed per practice; number of practices who reviewed records; and the number of times each record was reviewed. Conclusion: We developed a simple formula to calculate the minimum values of CRR parameters required to achieve adequate precision and acceptable power when monitoring harm rates. Our findings have practical implications for health care decision-makers, leaders and researchers aiming to measure and reduce harm at regional or national level.

Agreement between self-reported and measured weight and height collected in general practice patients: a prospective study (2013-03-13T00:00:00Z)

Background: Self-reported weight and height is frequently used to quantify overweight and obesity. It is however, associated with limitations such as bias and poor agreement, which may be a result of social desirability or difficulties with recall. Methods to reduce these biases would improve the accuracy of assessment of overweight and obesity using patient self-report. The level of agreement between self-reported and measured weight and height has not been widely examined in general practice patients. Methods: Consenting patients, presenting for care within four hour sessions, were randomly allocated to the informed or uninformed group. Participants were notified either a) prior to (informed group), or b) after (uninformed group) reporting their weight and height using a touchscreen computer questionnaire, that they would be measured. The differences in accuracy of self-report between the groups were examined by comparing mean differences, intraclass correlations (ICCs), Bland Altman plot with limits of agreement (LOAs) and Cohen’s kappa. Overall agreement was assessed using similar statistical methods. Results: Of consenting participants, 32% were aged between 18–39 years, 42% between 40–64 years and 25% were 65 years and above. The informed group (n = 172) did not report their weight and height more accurately than the uninformed group (n = 160). Mean differences between self-reported and measured weight (p = 0.4004), height (p = 0.5342) and body mass index (BMI) (p = 0.4409) were not statistically different between the informed and uninformed group. Overall, there were small mean differences (−1.2 kg for weight, 0.8 for height and −0.6 kg/m2 for BMI) and high ICCs (>0.9) between self-reported and measured values. A substantially high kappa (0.70) was obtained when using self-reported weight and height relative to measured values to quantify the proportion underweight, normal weight, overweight or obese. While the average bias of self-reported weight and height as estimates of the measured quantities is small, the LOAs indicate that substantial discrepancies occur at the individual level. Conclusions: Informing patients that their weight and height would be measured did not improve accuracy of reporting. The use of self-reported weight and height for surveillance studies in this setting appears acceptable; however this measure needs to be interpreted with care when used for individual patients.

"Best fit" framework synthesis: refining the method (2013-03-13T00:00:00Z)

Background: Following publication of the first worked example of the "best fit" method of evidence synthesis for the systematic review of qualitative evidence in this journal, the originators of the method identified a need to specify more fully some aspects of this particular derivative of framework synthesis.Methods and results: We therefore present a second such worked example in which all techniques are defined and explained, and their appropriateness is assessed. Specified features of the method include the development of new techniques to identify theories in a systematic manner; the creation of an a priori framework for the synthesis; and the "testing" of the synthesis. An innovative combination of existing methods of quality assessment, analysis and synthesis is used to complete the process. This second worked example was a qualitative evidence synthesis of employees' views of workplace smoking cessation interventions, in which the "best fit" method was found to be practical and fit for purpose. Conclusions: The method is suited to producing context-specific conceptual models for describing or explaining the decision-making and health behaviours of patients and other groups. It offers a pragmatic means of conducting rapid qualitative evidence synthesis and generating programme theories relating to intervention effectiveness, which might be of relevance both to researchers and policy-makers.

Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element (2013-06-19T00:00:00Z)

Background: Ribosomal protein L30 belongs to the L7Ae family of RNA-binding proteins, which recognize diverse targets. L30 binds to kink-turn motifs in the 28S ribosomal RNA, L30 pre-mRNA, and mature L30 mRNA. L30 has a noncanonical function as a component of the UGA recoding machinery that incorporates selenocysteine (Sec) into selenoproteins during translation. L30 binds to a putative kink-turn motif in the Sec Insertion Sequence (SECIS) element in the 3' UTR of mammalian selenoprotein mRNAs. The SECIS also interacts with SECIS-binding protein 2 (SBP2), an essential factor for Sec incorporation. Previous studies showed that L30 and SBP2 compete for binding to the SECIS in vitro. The SBP2:SECIS interaction has been characterized but much less is known about how L30 recognizes the SECIS. Results: Here we use enzymatic RNA footprinting to define the L30 binding site on the SECIS. Like SBP2, L30 protects nucleotides in the 5' side of the internal loop, the 5' side of the lower helix, and the SECIS core, including the GA tandem base pairs that are predicted to form a kink-turn. However, L30 has additional determinants for binding as it also protects nucleotides in the 3' side of the internal loop, which are not protected by SBP2. In support of the competitive binding model, we found that purified L30 repressed UGA recoding in an in vitro translation system, and that this inhibition was rescued by SBP2. To define the amino acid requirements for SECIS-binding, site-specific mutations in L30 were generated based on published structural studies of this protein in a complex with its canonical target, the L30 pre-mRNA. We identified point mutations that selectively inhibited binding of L30 to the SECIS, to the L30 pre-mRNA, or both RNAs, suggesting that there are subtle differences in how L30 interacts with the two targets. Conclusions: This study establishes that L30 and SBP2 bind to overlapping but non-identical sites on the SECIS. The amino acid requirements for the interaction of L30 with the SECIS differ from those that mediate binding to the L30 pre-mRNA. Our results provide insight into how L7Ae family members recognize their cognate RNAs.

Characterization of Stra8 in Southern catfish (Silurus meridionalis): evidence for its role in meiotic initiation (2013-05-22T00:00:00Z)

Background: RA (retinoic acid) signal pathway has been proved to be required for germ cell meiotic initiation in mammals, aves and amphibians. Stra8 (Stimulated by retinoic acid gene 8) is an important factor in RA signal pathway. However, the role of RA and Stra8 in germ cell meiotic initiation in teleosts is poorly characterized. Results: In this study, the full length cDNA of Stra8 was cloned from Southern catfish (Silurus meridionalis), and its spatio-temporal expression profiles were analyzed. The Stra8 cDNA (1606 bp) includes 163 bp 5'-UTR (untranslated region), 456 bp 3'-UTR, and an ORF (open reading frame) of 987 bp, encoding a polypeptide of 328 aa. Phylogenetic analysis revealed its existence in some primitive teleosts, such as Siluriformes and Salmoniformes. Tissue distribution analysis by RT-PCR showed that Stra8 is specifically expressed in gonads. By real-time PCR, in situ hybridization and immunohistochemistry, the highest expression level of Stra8/Stra8 was detected in 50 and 130 dah (day after hatching), the premeiotic stage of germ cells in XX and XY gonads, respectively. Conclusions: Our results suggest that Stra8 might be involved in germ cell meiotic initiation in S. meridionalis as it did in tetrapods.

Screening and analysis of PoAkirin1 and two related genes in response to immunological stimulants in the Japanese flounder (Paralichthys olivaceus) (2013-05-07T00:00:00Z)

A member of the NF-kappaB signaling pathway, PoAkirin1, was cloned from a full-length cDNA library of Japanese flounder (Paralichthys olivaceus). The full-length cDNA comprises a 5[prime]UTR of 202 bp, an open reading frame of 564 bp encoding a 187-amino-acid polypeptide and a 521-bp 3[prime]UTR with a poly (A) tail. The putative protein has a predicted molecular mass of 21 kDa and an isoelectric point (pI) of 9.22. Amino acid sequence alignments showed that PoAkirin1 was 99% identical to the Scophthalmus maximus Akirin protein (ADK27484). Yeast two-hybrid assays identified two proteins that interact with PoAkirin1: PoHEPN and PoC1q. The cDNA sequences of PoHEPN and PoC1q are 672 bp and 528 bp, respectively. Real-time quantitative reverse-transcriptase polymerase chain reaction analysis showed that bacteria could induce the expressions of PoAkirin1, PoHEPN and PoC1q. However, the responses of PoHEPN and PoC1q to the bacterial challenge were slower than that of PoAkirin1. To further study the function of PoAkirin1, recombinant PoAkirin1 and PoHEPN were expressed in Escherichia coli and would be used to verify the PoAkirin1-PoHEPN binding activity. These results identified two proteins that potentially interact with PoAkirin1 and that bacteria could induce their expression.

Human single-stranded DNA binding proteins are essential for maintaining genomic stability (2013-04-01T00:00:00Z)

The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.

Annual acknowledgement of manuscript reviewers (2013-03-28T00:00:00Z)

Contributing reviewersThe editors of BMC Molecular Biology would like to thank all of our reviewers who have contributed to the journal in Volume 12 (2012).

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood--brain barrier and blood-spinal cord barrier (2013-06-18T00:00:00Z)

Background: Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Methods: Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Results: Using a combination of optimised handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. Conclusions: Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood--brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.

Hemopexin induces neuroprotection in the rat subjected to focal cerebral ischemia (2013-06-10T00:00:00Z)

Background: The plasma protein hemopexin (HPX) exhibits the highest binding affinity to free heme. In vitro experiments and gene-knock out technique have suggested that HPX may have a neuroprotective effect. However, the expression of HPX in the brain was not well elucidated and its expression after cerebral ischemia-reperfusion injury was also poorly studied. Furthermore, no in vivo data were available on the effect of HPX given centrally on the prognosis of focal cerebral ischemia. Results: In the present study, we systematically investigated expression of HPX in normal rat brain by immunofluorescent staining. The results showed that HPX was mainly expressed in vascular system and neurons, as well as in a small portion of astrocytes adjacent to the vessels in normal rat brain. Further, we determined the role of HPX in the process of focal cerebral ischemic injury and explored the effects of HPX treatment in a rat model of transient focal cerebral ischemia. After 2 h' middle cerebral artery occlusion (MCAO) followed by 24 h' reperfusion, the expression of HPX was increased in the neurons and astrocytes in the penumbra area, as demonstrated by immunohistochemistry and Western blot techniques. Intracerebroventricular injection of HPX at the onset of reperfusion dose-dependently reduced the infarct volumes and improved measurements of neurological function of the rat subjected to transient focal cerebral ischemia. The neuroprotective effects of HPX sustained for up to 7 days after experiments. Conclusions: Our study provides a new insight into the potential neuroprotective role of HPX as a contributing factor of endogenous protective mechanisms against focal cerebral ischemia injury, and HPX might be developed as a potential agent for treatment of ischemic stroke.

Upregulation of myeloid cell leukemia-1 potentially modulates beclin-1-dependent autophagy in ischemic stroke in rats (2013-05-20T00:00:00Z)

Background: The mechanisms that underlie autophagy in cerebral ischemia remain poorly defined. Myeloid cell leukemia-1 (Mcl1), an anti-apoptotic member of the Bcl-2 family of proteins, regulates the balance between autophagy and apoptosis. However, little is known regarding its expression profile and contribution to cell fate in the brain following ischemic stroke. Results: In this study, we investigated the expression profile and cellular distribution of Mcl1 in brains from transient middle cerebral artery occlusion (MCAO) model rats. Brain slices from sham-operated control rats showed minimal immunoreactivity for Mcl1. Mcl1 was mainly produced in neurons. Immunoreactivity for Mcl1 increased as early as 4 hours after MCAO, peaked at 24 hours, and then declined, but still remained high, at 72 hours. Mcl1 positive cells never colocalized with either cleaved caspase-3 or terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells. Both microtubule-associated protein 1 light chain 3 (LC3) and beclin-1 were evident in ischemic brain between 4 and 72 hours after MCAO. Most cells with strong LC3 staining were also labeled with beclin-1. Beclin-1 did colocalize with caspase-3 or Mcl1. Beclin-1/caspase-3 positive cells displayed the characteristic features of apoptosis including cell shrinkage and pyknotic nuclei, whereas beclin-1/Mcl1 positive cells had normal morphology. Pretreatment with 3-methyladenine attenuated autophagy without affecting the level of Mcl1 protein. Conclusions: These findings demonstrate that the expression of Mcl1 is involved in the survival of neuronal cells. In addition, the coexpression of Mcl1 with beclin-1 may attenuate beclin-1-dependent autophagy during ischemic stroke in rats.

A small cohort of FRUM and Engrailed-expressing neurons mediate successful copulation in Drosophila melanogaster (2013-05-21T00:00:00Z)

Background: In Drosophila, male flies require the expression of the male-specific Fruitless protein (FRUM) within the developing pupal and adult nervous system in order to produce male courtship and copulation behaviors. Recent evidence has shown that specific subsets of FRUM neurons are necessary for particular steps of courtship and copulation. In these neurons, FRUM function has been shown to be important for determining sex-specific neuronal characteristics, such as neurotransmitter profile and morphology. Results: We identified a small cohort of FRUM interneurons in the brain and ventral nerve cord by their co-expression with the transcription factor Engrailed (En). We used an En-GAL4 driver to express a fruM RNAi construct in order to selectively deplete FRUM in these En/FRUM co-expressing neurons. In courtship and copulation tests, these males performed male courtship at wild-type levels but were frequently sterile. Sterility was a behavioral phenotype as these En-fruMRNAi males were less able to convert a copulation attempt into a stable copulation, or did not maintain copulation for long enough to transfer sperm and/or seminal fluid. Conclusions: We have identified a population of interneurons necessary for successful copulation in Drosophila. These data confirm a model in which subsets of FRUM neurons participate in independent neuronal circuits necessary for individual steps of male behavior. In addition, we have determined that these neurons in wild-type males have homologues in females and fru mutants, with similar placement, projection patterns, and neurochemical profiles.

Basal forebrain activation controls contrast sensitivity in primary visual cortex (2013-05-16T00:00:00Z)

Background: The basal forebrain (BF) regulates cortical activity by the action of cholinergic projections to the cortex. At the same time, it also sends substantial GABAergic projections to both cortex and thalamus, whose functional role has received far less attention. We used deep brain stimulation (DBS) in the BF, which is thought to activate both types of projections, to investigate the impact of BF activation on V1 neural activity. Results: BF stimulation robustly increased V1 single and multi-unit activity, led to moderate decreases in orientation selectivity and a remarkable increase in contrast sensitivity as demonstrated by a reduced semi-saturation contrast. The spontaneous V1 local field potential often exhibited spectral peaks centered at 40 and 70Hz as well as reliably showed a broad gamma-band (30-90Hz) increase following BF stimulation, whereas effects in a low frequency band (1-10Hz) were less consistent. The broad gamma-band, rather than low frequency activity or spectral peaks was the best predictor of both the firing rate increase and contrast sensitivity increase of V1 unit activity. Conclusions: We conclude that BF activation has a strong influence on contrast sensitivity in V1. We suggest that, in addition to cholinergic modulation, the BF GABAergic projections play a crucial role in the impact of BF DBS on cortical activity.

Phytochemical analysis and in-vitro anti-African swine fever virus activity of extracts and fractions of Ancistrocladus uncinatus, Hutch and Dalziel (Ancistrocladaceae) (2013-06-19T00:00:00Z)

Background: African swine fever (ASF), a highly contagious fatal acute haemorrhagic viral disease of pigs currently has no treatment or vaccination protocol and it threatens the pig industry worldwide. Recent outbreaks were managed by farmers with ethnoveterinary preparations with various claims of effectiveness. Results: We identified 35 compounds using GC-MS protocol and ASF virus (NIG 99) was significantly reduced by some extracts and fractions of the plant. However, the plant was poorly extracted by water and cytotoxicity was found to be a major problem with the use of the plant since its extracts also reduced the primary cells used in the assay. Conclusion: It is confirmed that the plant has antiviral potentials against ASF virus and farmers' claims seem to have certain degree of veracity, but finding the best means of exploring the potential of the plant while reducing its cytotoxic effect in-vitro and in-vivo will be necessary.

Methods used to estimate the size of the owned cat and dog population: a systematic review (2013-06-19T00:00:00Z)

Background: There are a number of different methods that can be used when estimating the size of the owned cat and dog population in a region, leading to varying population estimates. The aim of this study was to conduct a systematic review to evaluate the methods that have been used for estimating the sizes of owned cat and dog populations and to assess the biases associated with those methods.A comprehensive, systematic search of seven electronic bibliographic databases and the Google search engine was carried out using a range of different search terms for cats, dogs and population. The inclusion criteria were that the studies had involved owned or pet domestic dogs and/or cats, provided an estimate of the size of the owned dog or cat population, collected raw data on dog and cat ownership, and analysed primary data. Data relating to study methodology were extracted and assessed for biases. Results: Seven papers were included in the final analysis. Collection methods used to select participants in the included studies were: mailed surveys using a commercial list of contacts, door to door surveys, random digit dialled telephone surveys, and randomised telephone surveys using a commercial list of numbers. Analytical and statistical methods used to estimate the pet population size were: mean number of dogs/cats per household multiplied by the number of households in an area, human density multiplied by number of dogs per human, and calculations using predictors of pet ownership. Conclusion: The main biases of the studies included selection bias, non-response bias, measurement bias and biases associated with length of sampling time. Careful design and planning of studies is a necessity before executing a study to estimate pet populations.

Microarray gene expression profiling of neural tissues in bovine spastic paresis (2013-06-19T00:00:00Z)

Background: Bovine Spastic Paresis (BSP) is a neuromuscular disorder which affects both male and female cattle. BSP is characterized by spastic contraction and overextension of the gastrocnemious muscle of one or both limbs and is associated with a scarce increase in body weight. This disease seems to be caused by an autosomal and recessive gene, with incomplete penetration, although no genes clearly involved with its onset have been so far identified. We employed cDNA microarrays to identify metabolic pathways affected by BSP in Romagnola cattle breed. Investigation of those pathways at the genome level can help to understand this disease. Results: Microarray analysis of control and affected individuals resulted in 268 differentially expressed genes. These genes were subjected to KEGG pathway functional clustering analysis, revealing that they are predominantly involved in Cell Communication, Signalling Molecules and Interaction and Signal Transduction, Diseases and Nervous System classes. Significantly enriched KEGG pathway's classes for the differentially expressed genes were calculated; interestingly, all those significantly under-expressed in the affected samples are included in Neurodegenerative Diseases. To identify genome locations possibly harbouring gene(s) involved in the disease, the chromosome distribution of the differentially expressed genes was also investigated. Conclusions: The cDNA microarray we used in this study contains a brain library and, even if carrying an incomplete transcriptome representation, it has proven to be a valuable tool allowing us to add useful and new information to a poorly studied disease. By using this tool, we examined nearly 15000 transcripts and analysed gene pathways affected by the disease. Particularly, our data suggest also a defective glycinergic synaptic transmission in the development of the disease and an alteration of calcium signalling proteins. We provide data to acquire knowledge of a genetic disease for which literature still presents poor results and that could be further and specifically analysed in the next future. Moreover this study, performed in livestock, may also harbour molecular information useful for understanding human diseases.

Expression and role of PGP, BCRP, MRP1 and MRP3 in multidrug resistance of canine mammary cancer cells (2013-06-17T00:00:00Z)

Background: In both women and female dogs, the most prevalent type of malignant neoplasm is the spontaneous mammary tumor. In dogs, half of these are malignant. The treatment of choice for the canine patients is surgical mastectomy. Unfortunately, it often fails in high-risk, locally invasive mammary tumors as of during the time of the surgery the micro-metastases are present. Moreover, there are neither large studies conducting to prove of the benefit from the chemotherapy in dogs nor established chemotherapy treatment protocols available. Additionally, the effectiveness of each individual chemotherapeutic agent and drug resistance of canine mammary cancer have not yet been characterized. That has become the aim of our study, to assess the expression of PGP, BCRP, MRP1 and MRP3 in canine mammary cancer cell lines and to investigate their role in cancer resistance to vinblastine, cisplatin and cyclophosphamide with using RNAi approach. Results: The results suggested that in canine mammary cancer, the vinblastine efflux was mediated by PGP and MRP1 proteins, cisplatin efflux was mediated by all four examined efflux pumps (PGP, BCRP, MRP1 and MRP3), whereas cyclophosphamide resistance was related to BCRP activity. RNAi silencing of these efflux pumps significantly decreased IC50 doses of the examined drugs in canine mammary carcinoma cells. Conclusions: Our results have indicated the treatment of cells involving use of the siRNA targeting efflux pumps could be a beneficial approach in the future.

Expression of genes controlling fat deposition in two genetically diverse beef cattle breeds fed high or low silage diets (2013-06-17T00:00:00Z)

Background: Both genetic background and finishing system can alter fat deposition, thus indicating their influence on adipogenic and lipogenic factors. However, the molecular mechanisms underlying fat deposition and fatty acid composition in beef cattle are not fully understood. This study aimed to assess the effect of breed and dietary silage level on the expression patterns of key genes controlling lipid metabolism in subcutaneous adipose tissue (SAT) and longissimus lumborum (LL) muscle of cattle. To that purpose, forty bulls from two genetically diverse Portuguese bovine breeds with distinct maturity rates, Alentejana and Barrosa, were selected and fed either low (30% maize silage/70% concentrate) or high silage (70% maize silage/30% concentrate) diets. Results: The results suggested that enhanced deposition of fatty acids in the SAT from Barrosa bulls, when compared to Alentejana, could be due to higher expression levels of lipogenesis (SCD and LPL) and beta-oxidation (CRAT) related genes. Our results also indicated that SREBF1 expression in the SAT is increased by feeding the low silage diet. Together, these results point out to a higher lipid turnover in the SAT of Barrosa bulls when compared to Alentejana. In turn, lipid deposition in the LL muscle is related to the expression of adipogenic (PPARG and FABP4) and lipogenic (ACACA and SCD) genes. The positive correlation between ACACA expression levels and total lipids, as well trans fatty acids, points to ACACA as a major player in intramuscular deposition in ruminants. Moreover, results reinforce the role of FABP4 in intramuscular fat development and the SAT as the major site for lipid metabolism in ruminants. Conclusions: Overall, the results showed that SAT and LL muscle fatty acid composition are mostly dependent on the genetic background. In addition, dietary silage level impacted on muscle lipid metabolism to a greater extent than on that of SAT, as evaluated by gene expression levels of adipogenic and lipogenic factors. Moreover, the response to diet composition evaluated through mRNA levels and fatty acid composition showed interesting differences between Alentejana and Barrosa bulls. These findings provide evidence that the genetic background should be taken into account while devising diet-based strategies to manipulate fatty acid composition of beef cattle tissues.

Circulating serum xenoestrogens and mammographic breast density (2013-05-27T00:00:00Z)

IntroductionHumans are widely exposed to estrogenically-active phthalates, parabens, and phenols, raising concerns about potential effects on breast tissue and breast cancer risk. We sought to determine the association of circulating serum levels of these chemicals (reflecting recent exposure) with mammographic breast density (a marker of breast cancer risk). Methods: We recruited postmenopausal women aged 55-70 years from mammography clinics in Madison, Wisconsin (N=264). Subjects completed a questionnaire and provided a blood sample that was analyzed for mono-ethyl phthalate, mono-butyl phthalate, mono-benzyl phthalate, butyl paraben, propyl paraben, octylphenol, nonylphenol, and bisphenol A (BPA). Percent breast density was measured from mammograms using a computer-assisted thresholding method. Results: Serum BPA was positively associated with mammographic breast density after adjusting for age, body mass index, and other potentially confounding factors. Mean percent density was 12.6% (95% CI: 11.4, 14.0) among the 193 women with non-detectable BPA levels, 13.7% (95% CI: 10.7, 17.1) among the 35 women with detectable levels below the median (<0.55 ng/mL) and 17.6% (95% CI: 14.1, 21.5) among the 34 women with detectable levels above the median (>0.55 ng/mL; Ptrend=0.01). Percent breast density was also elevated (18.2%; 95% CI: 13.4, 23.7) among the 18 women with serum mono-ethyl phthalate above the median detected level (>3.77 ng/mL) compared to women with non-detectable BPA levels (13.1%; 95% CI: 11.9, 14.3; Ptrend=0.07). No other chemicals demonstrated associations with percent breast density. Conclusions: Postmenopausal women with high serum levels of BPA and mono-ethyl phthalate had elevated breast density. Further investigation of the impact of BPA and mono-ethyl phthalate on breast cancer risk using repeated serum measurements or other markers of xenoestrogen exposure are needed.

Mammographic density and breast cancer: a comparison of related and unrelated controls in the breast cancer family registry (2013-05-25T00:00:00Z)

IntroductionPercent mammographic density (PMD) is a strong and highly heritable risk factor for breast cancer. Studies of the role of PMD in familial breast cancer may require controls, such as the sisters of cases, selected from the same "risk set" as the cases. The use of sister controls would allow control for factors that have been shown to influence risk of breast cancer such as race/ethnicity, socio-economic status and a family history of breast cancer, but may introduce "overmatching" and attenuate case-control differences in PMD. Methods: To examine the potential effects of using sister controls rather than unrelated controls in a case-control study we examined PMD in triplets, each comprised of a case with invasive breast cancer, an unaffected full sister control, and an unaffected unrelated control. Both controls were matched to cases on age at mammogram. Total breast area and dense area in the mammogram were measured in the unaffected breast of cases and a randomly selected breast in controls, and the non-dense area and PMD calculated from these measurements. Results: The mean difference in PMD between cases and controls, and the standard deviation (SD) of the difference, were slightly less for sister controls (4.2% (SD=20.0)) than for unrelated controls (4.9% (SD=25.7)). We found statistically significant correlations in PMD between cases (n=228) and sister controls (n=228) (r= 0.39 (95% CI: 0.28, 0.50; p<0.0001)), but not between cases and unrelated controls (n=228) (r= 0.04 (95% CI: -0.09, 0.17; p=0.51)). After adjusting for other risk factors, square root transformed PMD was associated with an increased risk of breast cancer when comparing cases to sister controls (adjusted odds ratio (inter-quintile odds ratio (IQOR) = 2.19, 95% CI= 1.20, 4.00) or to unrelated controls (adjusted IQOR= 2.62, 95% CI= 1.62, 4.25). Conclusions: The use of sister controls in case-control studies of PMD resulted in a modest attenuation of case-control differences and risk estimates, but showed a statistically significant association with risk and allowed control for race/ethnicity, socio-economic status and family history.

Amphiregulin mediates progesterone-induced mammary ductal development during puberty (2013-05-25T00:00:00Z)

IntroductionPuberty is a period of increased susceptibility to factors that cause increased breast cancer risk in adulthood. Mammary end buds (EB) that develop during puberty are believed to be the targets of breast cancer initiation. While the role of estrogen (E) has been extensively studied in pubertal mammary gland development, the role of progesterone (P) during puberty is less defined. Methods: Pubertal and pre-pubertal ovariectomized mice were treated with vehicle control (C), E, P or E+P. Mammary glands from these mice were analyzed for changes in morphology, proliferation, and expression of the downstream targets amphiregulin (AREG) and Receptor Activator of NF-kB Ligand (RANKL). Results: P, acting specifically through the progesterone receptor, induced increases in mammary gland proliferation and EB formation that were associated with increased AREG expression in ducts and EBs. E, acting specifically through the estrogen receptor, produced similar responses also mediated by AREG. Blocking AREG action by treatment with an epidermal growth factor receptor (EGFR) inhibitor completely abrogated the effect of P on EB formation and proliferation and significantly reduced proliferation within ducts. P also increased expression of RANKL, primarily in ducts. Treatment with RANK-Fc, an inhibitor of RANKL, reduced P-dependent proliferation in ducts and to a lesser extent in EB, but did not cause EB regression. Conclusions: These results demonstrate a novel P-specific effect through AREG to cause EB formation and proliferation in the developing mammary gland both prior to and during puberty. Thus, hormones and/or factors in addition to E that up-regulate AREG can promote mammary gland development and have the potential to affect breast cancer risk associated with pubertal mammary gland development.

Serum microRNA expression as an early marker for breast cancer risk in prospectively collected samples from the Sister Study cohort (2013-05-24T00:00:00Z)

IntroductionMicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs between 18-22 nucleotides long that regulate gene expression. Expression of miRNAs is altered in tumors compared to normal tissue; there is some evidence that these changes may be reflected in the serum of cancer cases compared to healthy individuals. This has yet to be examined in a prospective study where samples are collected before diagnosis. Methods: We used Affymetrix arrays to examine serum miRNA expression profiles in 410 participants in the Sister Study, a prospective cohort study of 50,884 women. All women in the cohort had never been diagnosed with breast cancer at the time of enrollment. We compared global miRNA expression patterns in 205 women who subsequently developed breast cancer and 205 women who remained breast cancer-free. In addition within the case group we examined the association of miRNA expression in serum with different tumor characteristics, including hormone status (Estrogen Receptor, ER; Progesterone Receptor, PR; Human Epidermal Growth Factor Receptor 2, HER-2) and lymph node status. Results: Overall, 414 of 1,105 of the human miRNAs on the chip were expressed above background levels in 50 or more women. When the average expression among controls was compared to cases using conditional logistic regression, 21 miRNAs were found to be differentially expressed (P[less than or equal to].05). Using qRT-PCR on a small, independent sample of 5 cases and 5 controls we verified overexpression of the 3 highest expressing miRNAs among cases, miR-18a, miR-181a, and miR-222; the differences were not statistically significant in this small set. The 21 differentially expressed miRNAs are known to target at least 82 genes; using the gene list for pathway analysis we found enrichment of genes involved in cancer-related processes. In a separate case-case analyses restricted to the 21 miRNAs, we found 7 miRNAs with differential expression for women whose breast tumors differed by HER-2 expression, and 10 miRNAs with differential expression by nodal status. Conclusions: miRNA levels in serum show a number of small differences between women who later develop cancer versus those who remain cancer-free.

Overdiagnosis in breast cancer screening: the importance of length of observation period and lead time (2013-05-16T00:00:00Z)

IntroductionOverdiagnosis in breast cancer screening is a controversial topic. One difficulty in estimation of overdiagnosis is the separation of overdiagnosis from lead time that is the advance in the time of diagnosis of cancers, which confers an artificial increase in incidence when a screening programme is introduced. Methods: We postulated a female population aged 50-79 with a similar age structure and age-specific breast cancer incidence as in England and Wales before the screening programme. We then imposed a two-yearly screening programme; screening women aged 50-69, to run for twenty years, with exponentially distributed lead time with an average of 40 months in screen-detected cancers. We imposed no effect of the screening on incidence other than lead time. Results: Comparison of age- and time-specific incidence between the screened and unscreened populations showed a major effect of lead time, which could only be adjusted for by follow-up for more than two decades and including ten years after the last screen. From lead time alone, twenty-year observation at ages 50-69 would confer an observed excess incidence of 37%. The excess would only fall below 10% with 25 years or more follow-up. For the excess to be nullified, we would require 30 year follow-up including observation up to 10 years above the upper age limit for screening. Conclusions: Studies using shorter observation periods will overestimate overdiagnosis by inclusion of cancers diagnosed early due to lead time among the nominally overdiagnosed tumours.

Nip the HPV encoded Evil in the cancer bud: HPV reshapes TRAILs and signaling landscapes (2013-06-17T00:00:00Z)

HPV encoded proteins can elicit ectopic protein--protein interactions that re-wire signaling pathways, in a mode that promotes malignancy. Moreover, accumulating data related to HPV is now providing compelling substantiation of a central role played by HPV in escaping immunosurveillance and impairment of apoptotic response. What emerges is an intricate network of Wnt, TGF, Notch signaling cascades that forms higher-order ligand--receptor complexes routing downstream signaling in HPV infected cells. These HPV infected cells are regulated both extracellularly by ligand receptor axis and intracellularly by HPV encoded proteins and impair TRAIL mediated apoptosis. We divide this review into different sections addressing how linear signaling pathways integrate to facilitate carcinogenesis and compounds that directly or indirectly reverse these aberrant interactions offer new possibilities for therapy in cancer. Although HPV encoded proteins mediated misrepresentation of pathways is difficult to target, improved drug-discovery platforms and new technologies have facilitated the discovery of agents that can target dysregulated pathways in HPV infected cervical cancer cells, thus setting the stage for preclinical models and clinical trials.

Expression of ALDH1 in breast invasive ductal carcinoma: an independent predictor of early tumor relapse (2013-06-15T00:00:00Z)

Background: The specific mechanism underlying the contribution of the Aldehyde dehydrogenase 1 (ALDH1) phenotype to metastatic behavior and early tumor relapse in breast cancer is currently unclear. Methods: 147 randomly selected invasive ductal carcinoma samples were assayed for expression of ALDH1A1, NOTCH1, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2), and association of the ALDH1A1 phenotype with clinic pathological features was further evaluated. Results: ALDH1A1-positive cells were detected in 63.3% (93 of 147) of tumors. 80.0% (32 of 40) of tumors with strong ALDH1A1 staining displayed early recurrence, compared with 20.0% (8 of 40) of tumors negative for ALDH1A1 expression (P = 0.027). ALDH1A1 status was significantly correlated with strong malignant proliferative marker Ki67 staining (P = 0.001), and no significantly different expression of ALDH1A1 across the subtypes of ER, PR, and HER2 expression and triple negative features of tumor tissue. Multivariate regression analysis demonstrated that elevated ALDH1A1 expression is an independent predictor of recurrence-free survival and distant metastasis-free survival. Notably, breast cancer tissue strong for ALDH1A1 expression displayed weak NOTCH1 staining compared to ALDH1A1 weak tumor tissue (P = 0.002), and the relationship between ALDH1A1 and NOTCH1 mRNA positivity was significant (Pearson correlation - 0.337, P = 0.014; Spearman's rho - 0.376, P = 0.006). Elevated NOTCH1 mRNA level (using a cut-off value based on the median ALDH1A1 2-[white up-pointing triangle][white up-pointing triangle]CT value) was associated with reduction of ALDH1A1 mRNA level (P = 0.001). Conclusions: The ALDH1A1 phenotype is an independent predictor of early tumor relapse characteristic (specifically, incidence of early local recurrence and distant metastasis) of invasive ductal carcinoma. The NOTCH1 signaling pathway is possibly involved in the negative association of the ALDH1A1 phenotype with early malignant relapse in invasive ductal carcinoma.

Single nucleotide polymorphisms of CD20 gene and their relationship with clinical efficacy of R-CHOP in patients with diffuse large B cell lymphoma (2013-06-10T00:00:00Z)

Background: R-CHOP has significantly improved survival rates of patients with diffuse large B cell lymphoma (DLBCL) by ~20% as compared to CHOP. CD20 antigen, highly expressed on more than 80% of B-cell lymphomas, is the target for rituximab. The goal of our study was to examine polymorphism in the CD20 gene in Chinese DLBCL population and whether CD20 gene polymorphism is associated with clinical response to R-CHOP.MethodCD20 gene polymorphism was detected in the entire coding regions (CDS) including 6 exons by polymerase chain reaction (PCR)-sequencing assay in 164 patients with DLBCL. Among them, 129 patients treated with R-CHOP as frontline therapy (R >= 4 cycles) were assessable for the efficacy. Results: Polymorphisms at three single nucleotides (SNP) were identified in the entire coding regions of the CD20 gene in the 164 patients. One of them, CD20 Exon2 [216] was found to be highly correlated with response to R-CHOP. Patients with homozygous C genotype showed a trend toward higher overall response rate than others with CT plus TT genotype (90.6% vs. 79.5%; P =0.166). A trend toward higher complete remission (CR) rate was observed in patients with homozygous C genotype (67.4%) compared with CT plus TT genotype (47.1%) (P = 0.091). Conclusion: These results suggest that there are 3 SNPs in CDS of the CD20 gene in Chinese DLBCL population. The CC genotype at Exon2 [216] appears to be associated with favourable response to R-CHOP.

Role of relaxin-2 in human primary osteosarcoma (2013-06-10T00:00:00Z)

Background: The aim of this study was to clarify the clinicopathological outcome of serum relaxin-2 and tissues relaxin-2 expression levels in human primary osteosarcoma (OS), and to explore the roles of relaxin-2 inhibition and determine its possibility as a therapeutic target in human osteosarcoma. Methods: Real-time quantitative RT-PCR assay was performed to detect the expression of relaxin-2 mRNA in 36 cases of human osteosarcoma tissue samples. Serum relaxin-2 levels was measured in ELISA-based method in the 36 cases of osteosarcoma and 50 cases of controls. MTT and TUNEL assay was used to detect cell proliferation and apoptosis after relaxin-2 knockdown with siRNA transfection for 48 hs in vitro. Matrigel invasion and angiogenesis formation assay was used to detect cell metastasis and angiogenesis with HMEC-1 endothelial cells after relaxin-2 knockdown with siRNA transfection for 48 hs in vitro.The effects of relaxin-2 knockdown with anti- relaxin-2 mAb treatment on growth, apoptosis angiogenesis formation and lung metastasis in vivo was analyzed. Results: The results showed the levels of relaxin-2 mRNA expression in osteosarcoma tissue samples were significantly higher than those in the corresponding non-tumor tissue samples (P < 0.01), and the serum relaxin-2 levels were significantly higher in OS patients than in healthy controls (P < 0.01). The incidence of advanced stage cancer and hematogenous metastasis cancer in the high relaxin-2 mRNA expression group and high serum relaxin-2 levels groups was significantly higher than that in the low relaxin-2 expression group and low serum relaxin-2 levels groups ,respectively. Knockdown of relaxin-2 by siRNA transfection in vitro inhibited proliferation, invasion and angiogenesis in vitro in MG-63 OS cells. In vivo, knockdown of relaxin-2 with anti- relaxin-2 mAb treatment inhibited tumor growth by 62% (P < 0.01) and the formation of lung metastases was inhibited by 72.4% (P < 0.01). Microvascular density was reduced more than 60% due to anti- relaxin-2 mAb treatment (P < 0.01). Conclusions: Our study suggests that overexpression of relaxin-2 is critical for the metastasis of human osteosarcoma. Detection of relaxin-2 mRNA expression or serum relaxin-2 levels may provide the first biological prognostic marker for OS. Furthermore, relaxin-2 is the potential molecular target for osteosarcoma therapy.

The effects of a histone deacetylase inhibitor on biological behavior of diffuse large B-cell lymphoma cell lines and insights into the underlying mechanisms (2013-06-07T00:00:00Z)

Background: Epigenetic control using histone deacetylase (HDAC) inhibitors is a promising therapy for lymphomas. Insights into the anti-proliferative effects of HDAC inhibitors on diffuse large B-cell lymphoma (DLBCL) and further understanding of the underlying mechanisms, which remain unclear to date, are of great importance. Methods: Three DLBCL cell lines (DoHH2, LY1 and LY8) were used to define the potential epigenetic targets for Trichostatin A (TSA)-mediated anti-proliferative effects via CCK-8 assay. Cell cycle distribution and apoptosis were detected by flow cytometry. We further investigated the underlying molecular mechanisms by examining expression levels of relevant proteins using western blot analysis. Results: TSA treatment inhibited the growth of all three DLBCL cell lines and enhanced cell cycle arrest and apoptosis. Molecular analysis revealed upregulated acetylation of histone H3, alpha-tubulin and p53, and dephosphorylation of pAkt with altered expression of its main downstream effectors (p21, p27, cyclin D1 and Bcl-2). HDAC profiling revealed that all three cell lines had varying HDAC1--6 expression levels, with the highest expression of all six isoforms, in DoHH2 cells, which displayed the highest sensitivity to TSA. Conclusion: Our results demonstrated that the HDAC inhibitor TSA inhibited DLBCL cell growth, and that cell lines with higher expression of HDACs tended to be more sensitive to TSA. Our data also suggested that inhibition of pAkt and activation of p53 pathway are the main molecular events involved in inhibitory effects of TSA.

DNA and the chromosome - varied targets for chemotherapy (2004-05-24T00:00:00Z)

The nucleus of the cell serves to maintain, regulate, and replicate the critical genetic information encoded by the genome. Genomic DNA is highly associated with proteins that enable simple nuclear structures such as nucleosomes to form higher-order organisation such as chromatin fibres. The temporal association of regulatory proteins with DNA creates a dynamic environment capable of quickly responding to cellular requirements and distress. The response is often mediated through alterations in the chromatin structure, resulting in changed accessibility of specific DNA sequences that are then recognized by specific proteins. Anti-cancer drugs that target cellular DNA have been used clinically for over four decades, but it is only recently that nuclease specific drugs have been developed to not only target the DNA but also other components of the nuclear structure and its regulation. In this review, we discuss some of the new drugs aimed at primary DNA sequences, DNA secondary structures, and associated proteins, keeping in mind that these agents are not only important from a clinical perspective but also as tools for understanding the nuclear environment in normal and cancer cells.

Imaging genome abnormalities in cancer research (2004-01-13T00:00:00Z)

Increasing attention is focusing on chromosomal and genome structure in cancer research due to the fact that genomic instability plays a principal role in cancer initiation, progression and response to chemotherapeutic agents. The integrity of the genome (including structural, behavioral and functional aspects) of normal and cancer cells can be monitored with direct visualization by using a variety of cutting edge molecular cytogenetic technologies that are now available in the field of cancer research. Examples are presented in this review by grouping these methodologies into four categories visualizing different yet closely related major levels of genome structures. An integrated discussion is also presented on several ongoing projects involving the illustration of mitotic and meiotic chromatin loops; the identification of defective mitotic figures (DMF), a new type of chromosomal aberration capable of monitoring condensation defects in cancer; the establishment of a method that uses Non-Clonal Chromosomal Aberrations (NCCAs) as an index to monitor genomic instability; and the characterization of apoptosis related chromosomal fragmentations caused by drug treatments.

Microarray analysis of gene expression during the cell cycle (2003-09-19T00:00:00Z)

Microarrays have been applied to the determination of genome-wide expression patterns during the cell cycle of a number of different cells. Both eukaryotic and prokaryotic cells have been studied using whole-culture and selective synchronization methods. The published microarray data on yeast, mammalian, and bacterial cells have been uniformly interpreted as indicating that a large number of genes are expressed in a cell-cycle-dependent manner. These conclusions are reconsidered using explicit criteria for synchronization and precise criteria for identifying gene expression patterns during the cell cycle. The conclusions regarding cell-cycle-dependent gene expression based on microarray analysis are weakened by arguably problematic choices for synchronization methodology (e.g., whole-culture methods that do not synchronize cells) and questionable statistical rigor for identifying cell-cycle-dependent gene expression. Because of the uncertainties in synchrony methodology, as well as uncertainties in microarray analysis, one should be somewhat skeptical of claims that there are a large number of genes expressed in a cell-cycle-dependent manner.

MG-132, an inhibitor of proteasomes and calpains, induced inhibition of oocyte maturation and aneuploidy in mouse oocytes (2002-10-08T00:00:00Z)

Background: Although chromosome missegregation during oocyte maturation (OM) is a significant contributor to human morbidity and mortality, very little is known about the causes and mechanisms of aneuploidy. Several investigators have proposed that temporal perturbations during OM predispose oocytes to aberrant chromosome segregation. One approach for testing this proposal is to temporarily inhibit the activity of protein proteolysis during OM. We used the reversible proteasome inhibitor MG-132 to transiently perturb the temporal sequence of events during OM and subsequently analyzed mouse metaphase II (MII) for cytogenetic abnormalities. The transient inhibition of proteasome activity by MG-132 resulted in elevated levels of oocytes containing extra chromatids and chromosomes. Results: The transient inhibition of proteasome-mediated proteolysis during OM by MG-132 resulted in dose-response delays during OM and elevated levels of aneuploid MII oocytes. Oocytes exposed in vitro to MG-132 exhibited greater delays during metaphase I (MI) as demonstrated by significantly (p < 0.01) higher levels of MI arrested oocytes and lower frequencies of premature sister chromatid separation in MII oocytes. Furthermore, the proportions of MII oocytes containing single chromatids and extra chromosomes significantly (p < 0.01) increased with MG-132 dosage. Conclusions: These data suggest that the MG-132-induced transient delay of proteasomal activity during mouse OM in vitro predisposed oocytes to abnormal chromosome segregation. Although these findings support a relationship between disturbed proteasomal activity and chromosome segregation, considerable additional data are needed to further investigate the roles of proteasome-mediated proteolysis and other potential molecular mechanisms on chromosome segregation during OM.

Chromosome loops arising from intrachromosomal tethering of telomeres occur at high frequency in G1 (non-cycling) mitotic cells: Implications for telomere capture (2004-09-29T00:00:00Z)

Background: To investigate potential mechanisms for telomere capture the spatial arrangement of telomeres and chromosomes was examined in G1 (non-cycling) mitotic cells with diploid or triploid genomes. This was examined firstly by directly labelling the respective short arm (p) and long arm subtelomeres (q) with different fluorophores and probing cell preparations using a number of subtelomere probe pairs, those for chromosomes 1, 3, 4, 5, 6, 7, 9, 10, 12, 17, 18, and 20. In addition some interstitial probes (CEN15, PML and SNRPN on chromosome 15) and whole chromosome paint probes (e.g. WCP12) were jointly hybridised to investigate the co-localization of interphase chromosome domains and tethered subtelomeres. Cells were prepared by omitting exposure to colcemid and hypotonic treatments. Results: In these cells a specific interphase chromosome topology was detected. It was shown that the p and q telomeres of the each chromosome associate frequently (80% pairing) in an intrachromosomal manner, i.e. looped chromosomes with homologues usually widely spaced within the nucleus. This p-q tethering of the telomeres from the one chromosome was observed with large (chromosomes 3, 4, 5), medium sized (6, 7, 9, 10, 12), or small chromosomes (17, 18, 20). When triploid nuclei were probed there were three tetherings of p-q subtelomere signals representing the three widely separated looped chromosome homologues. The separate subtelomere pairings were shown to coincide with separate chromosome domains as defined by the WCP and interstitial probes. The 20% of apparently unpaired subtelomeric signals in diploid nuclei were partially documented to be pairings with the telomeres of other chromosomes. Conclusions: A topology for telomeres was detected where looped chromosome homologues were present at G1 interphase. These homologues were spatially arranged with respect to one-another independently of other chromosomes, i.e. there was no chromosome order on different sides of the cell nuclei and no segregation into haploid sets was detected. The normal function of this high frequency of intrachromosomal loops is unknown but a potential role is likely in the genesis of telomere captures whether of the intrachromosomal type or between non-homologues. This intrachromosomal tethering of telomeres cannot be related to telomeric or subtelomeric sequences since these are shared in varying degree with other chromosomes. In our view, these intrachromosomal telomeric tetherings with the resulting looped chromosomes arranged in a regular topology must be important to normal cell function since non-cycling cells in G1 are far from quiescent, are in fact metabolically active, and these cells represent the majority status since only a small proportion of cells are normally dividing.

Hypoxia-inducible factor (HIF) network: insights from mathematical models (2013-06-10T00:00:00Z)

Oxygen is a crucial molecule for cellular function. When oxygen demand exceeds supply, the oxygen sensing pathway centred on the hypoxia inducible factor (HIF) is switched on and promotes adaptation to hypoxia by up-regulating genes involved in angiogenesis, erythropoiesis and glycolysis. The regulation of HIF is tightly modulated through intricate regulatory mechanisms. Notably, its protein stability is controlled by the oxygen sensing prolyl hydroxylase domain (PHD) enzymes and its transcriptional activity is controlled by the asparaginyl hydroxylase FIH (factor inhibiting HIF-1).To probe the complexity of hypoxia-induced HIF signalling, efforts in mathematical modelling of the pathway have been underway for around a decade. In this paper, we review the existing mathematical models developed to describe and explain specific behaviours of the HIF pathway and how they have contributed new insights into our understanding of the network. Topics for modelling included the switch-like response to decreased oxygen gradient, the role of micro environmental factors, the regulation by FIH and the temporal dynamics of the HIF response. We will also discuss the technical aspects, extent and limitations of these models. Recently, HIF pathway has been implicated in other disease contexts such as hypoxic inflammation and cancer through crosstalking with pathways like NFkappaB and mTOR. We will examine how future mathematical modelling and simulation of interlinked networks can aid in understanding HIF behaviour in complex pathophysiological situations. Ultimately this would allow the identification of new pharmacological targets in different disease settings.

Asymmetric kinase dimer formation is crucial for the activation of oncogenic EGFRvIII but not for ERBB3 phosphorylation (2013-06-10T00:00:00Z)

Background: Formation of asymmetric kinase dimers is required for wt-EGFR activation upon ligand stimulation. The role of receptor dimerization in oncogenic EGFRvIII mutant activation is not completely understood and the molecular details of EGFRvIII interactions within homo-dimers and hetero-dimers are not elucidated yet.FindingsBy employing mutations that disrupt the asymmetric kinase dimer interface in EGFRvIII, we demonstrate that the mechanism of oncogenic EGFRvIII mutant activation is similar to that of the full-length wild-type EGFR. Surprisingly, the monomeric EGFRvIII lacks autophosphorylation and the formation of asymmetric kinase dimers is indispensable for oncogenic kinase activation. In addition, we show that ERBB3 can act as an activator of EGFRvIII by forming asymmetric kinase dimer in a ligand-independent manner. Interestingly, we found that the formation of asymmetric kinase dimer is dispensable for ERBB3 phosphorylation by the activated EGFR kinase as well as the ERBB2 kinase thus revealing a novel model for receptor function. Conclusions: Lateral signaling is a novel mechanism of signal propagation via ERBB3 upon activation by EGFR/ERBB2 kinase even in the absence of their ability to form asymmetric kinase dimers.

CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity (2013-06-10T00:00:00Z)

Background: Casein kinase 2 (CK2) is a ubiquitously expressed Ser/Thr kinase with multiple functions in the regulation of cell proliferation and transformation. In targeting adherens and tight junctions (TJs), CK2 modulates the strength and dynamics of epithelial cell-cell contacts. Occludin previously was identified as a substrate of CK2, however the functional consequences of CK2-dependent occludin phosphorylation on TJ function were unknown. Results: Here, we present evidence that phosphorylation of a Thr400-XXX-Thr404-XXX-Ser408 motif in the C-terminal cytoplasmic tail of human occludin regulates assembly/disassembly and barrier properties of TJs. In contrast to wildtype and T400A/T404A/S408A-mutated occludin, a phospho-mimetic Occ-T400E/T404E/S408E construct was impaired in binding to ZO-2. Interestingly, pre-phosphorylation of a GST-Occ C-terminal domain fusion protein attenuated binding to ZO-2, however, binding to ZO-1 was not affected. Moreover, Occ-T400E/T404E/S408E showed delayed reassembly into TJs in Ca2+-switch experiments. Interestingly, stable expression of Occ-T400E/T404E/S408E in MDCK C11 cells augments barrier properties in enhancing paracellular resistance in two-path impedance spectroscopy, whereas expression of wildtype and Occ-T400A/T404A/S408A did not affect transepithelial resistance. Conclusions: These results suggest an important role of CK2 in epithelial tight junction regulation. The occludin sequence motif at amino acids 400--408 apparently represents a hotspot for Ser/Thr-kinase phosphorylation and depending on the residue(s) which are phosphorylated it differentially modulates the functional properties of the TJ.

Down-regulation of the cancer/testis antigen 45 (CT45) is associated with altered tumor cell morphology, adhesion and migration (2013-06-10T00:00:00Z)

Background: Due to their restricted expression in male germ cells and certain tumors, cancer/testis (CT) antigens are regarded as promising targets for tumor therapy. CT45 is a recently identified nuclear CT antigen that was associated with a severe disease score in Hodgkin's lymphoma and poor prognosis in multiple myeloma. As for many CT antigens, the biological function of CT45 in developing germ cells and in tumor cells is largely unknown. Methods: CT45 expression was down-regulated in CT45-positive Hodgkin's lymphoma (L428) fibrosarcoma (HT1080) and myeloma (U266B1) cells using RNA interference. An efficient CT45 knock-down was confirmed by immunofluorescence staining and/or Western blotting. These cellular systems allowed us to analyze the impact of CT45 down-regulation on proliferation, cell cycle progression, morphology, adhesion, migration and invasive capacity of tumor cells. Results: Reduced levels of CT45 did not coincide with changes in cell cycle progression or proliferation. However, we observed alterations in cell adherence, morphology and migration/invasion after CT45 down-regulation. Significant changes in the distribution of cytoskeleton-associated proteins were detected by confocal imaging. Changes in cell adherence were recorded in real-time using the xCelligence system with control and siRNA-treated cells. Altered migratory and invasive capacity of CT45 siRNA-treated cells were visualized in 3D migration and invasion assays. Moreover, we found that CT45 down-regulation altered the level of the heterogeneous nuclear ribonucleoprotein syncrip (hnRNP-Q1) which is known to be involved in the control of focal adhesion formation and cell motility. Conclusions: Providing first evidence of a cell biological function of CT45, we suggest that this cancer/testis antigen is involved in the modulation of cell morphology, cell adherence and cell motility. Enhanced motility and/or invasiveness of CT45-positive cells could contribute to the more severe disease progression that is correlated to CT45-positivity in several malignancies.

The C-terminus region of ß-arrestin1 modulates VE-cadherin expression and endothelial cell permeability (2013-05-28T00:00:00Z)

Background: The endothelial specific cell-cell adhesion molecule, VE-cadherin, modulates barrier function and vascular homeostasis. In this context, we have previously characterized that VEGF (vascular endothelial growth factor) leads to VE-cadherin phosphorylation, β-arrestin2 recruitment and VE-cadherin internalization in mouse endothelial cells. However, exactly how this VE-cadherin/β-arrestin complex contributes to VEGF-mediated permeability in human endothelial cells remains unclear. In this study, we investigated in-depth the VE-cadherin/β-arrestin interactions in human endothelial cells exposed to VEGF.FindingsFirst, we demonstrated that VEGF induces VE-cadherin internalization in a clathrin-dependent manner in human umbilical vein endothelial cells (HUVEC). In addition to the classical components of endocytic vesicles, β-arrestin1 was recruited and bound to phosphorylated VE-cadherin. Molecular mapping of this interaction uncovered that the C-terminus tail of β-arrestin1, that comprises amino acids 375 to 418, was sufficient to directly interact with the phosphorylated form of VE-cadherin. Interestingly, the expression of the C-terminus tail of β-arrestin1 induced loss of surface exposed-VE-cadherin, promoted monolayer disorganization and enhanced permeability. Finally, this effect relied on decreased VE-cadherin expression at the transcriptional level, through inhibition of its promoter activity. Conclusions: Altogether, our results demonstrate that β-arrestin1 might play multiple functions collectively contributing to endothelial barrier properties. Indeed, in addition to a direct implication in VE-cadherin endocytosis, β-arrestin1 could also control VE-cadherin transcription and expression. Ultimately, understanding the molecular mechanisms involved in VE-cadherin function might provide therapeutic tools for many human diseases where the vascular barrier is compromised.

Cell cycle-dependent localization of CHK2 at centrosomes during mitosis (2013-05-16T00:00:00Z)

Background: Centrosomes function primarily as microtubule-organizing centres and play a crucial role during mitosis by organizing the bipolar spindle. In addition to this function, centrosomes act as reaction centers where numerous key regulators meet to control cell cycle progression. One of these factors involved in genome stability, the checkpoint kinase CHK2, was shown to localize at centrosomes throughout the cell cycle. Results: Here, we show that CHK2 only localizes to centrosomes during mitosis. Using wild-type and CHK2-/- HCT116 human colon cancer cells and human osteosarcoma U2OS cells depleted for CHK2 with small hairpin RNAs we show that several CHK2 antibodies are non-specific and cross-react with an unknown centrosomal protein(s) by immunofluorescence. To characterize the localization of CHK2, we generated cells expressing inducible GFP-CHK2 and Flag-CHK2 fusion proteins. We show that CHK2 localizes to the nucleus in interphase cells but that a fraction of CHK2 associates with the centrosomes in a Polo-like kinase 1-dependent manner during mitosis, from early mitotic stages until cytokinesis. Conclusion: Our findings demonstrate that a subpopulation of CHK2 localizes at the centrosomes in mitotic cells but not in interphase. These results are consistent with previous reports supporting a role for CHK2 in the bipolar spindle formation and the timely progression of mitosis.

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis (2013-04-27T00:00:00Z)

Background: The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. Results: We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. Conclusions: "RodCell" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells.AvailabilityRodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html, (after acceptance of the publication).

Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation (2013-04-22T00:00:00Z)

Background: Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27Xic1 (Xic1) which shares sequence homology with both p21Cip1 and p27Kip1 from mammals, p16Xic2 (Xic2) which shares sequence homology with p21Cip1, and p17Xic3 (Xic3) which shares sequence homology with p27Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4Cdt2, little is known about the processes that regulate Xic2 or Xic3. Methods: We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation. Results: Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation. Conclusions: During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell division or early development in the frog.

The novel BTB-kelch protein, KBTBD8, is located in the Golgi apparatus and translocates to the spindle apparatus during mitosis (2013-04-11T00:00:00Z)

Proteins of the BTB-kelch family are known to be involved in multiple biological processes such as migration, cytoskeleton arrangement, regulation of cell morphology, protein ubiquitination and gene expression. KBTBD8 is a new member of this family. The gene was found in a comparative transcriptome analysis of pluripotent stem cells and was therefore suggested to play a role in the regulation of pluripotency. Comparative analysis of the gene and protein sequences revealed a high conservation throughout evolution especially in the characteristic domains of BTB, BACK and kelch. We identified the Golgi apparatus as the subcellular localization of the KBTBD8 protein in non-dividing cells and could show that KBTBD8 co-localizes with alpha-tubulin on the spindle apparatus of mitotic cells suggesting a role in cell proliferation. In conclusion, KBTBD8 is a new member of the BTB-kelch superfamily that is located in the Golgi apparatus and translocates to the spindle apparatus during mitosis.

Rad53 homologue forkhead-associated kinase A (FhkA) and Ca2+-binding protein 4a (CBP4a) are nucleolar proteins that differentially redistribute during mitosis in Dictyostelium (2013-04-12T00:00:00Z)

Background: During mitosis most nucleolar proteins redistribute to other locales providing an opportunity to study the relationship between nucleolar protein localization and function. Dictyostelium is a model organism for the study of several fundamental biological processes and human diseases but only two nucleolar proteins have been studied during mitosis: NumA1 and Snf12. Both of them are linked to the cell cycle. To acquire a better understanding of nucleolar protein localization and dynamics in Dictyostelium we studied the nucleolar localization of two additional proteins during mitosis: Snf12-linked forkhead-associated kinase A (FhkA), which is involved in the cell cycle, and Ca2+-binding protein 4a (CBP4a), which is a binding partner of NumA1.

Preparation of Photoswitchable Labeled Antibodies for STORM Imaging (2013-06-03T06:00:46-07:00)
Labeling Antibodies, Fluorescence, Fluorescence, general, Labeling for Imaging, Photoactivation, Imaging: A Laboratory Manual

Stochastic optical reconstruction microscopy (STORM) is a method for superresolution fluorescence imaging based on the high accuracy localization of individual fluorophores. It uses optically switchable fluorophores: molecules that can be switched between a nonfluorescent and a fluorescent state by exposure to light. Many synthetic fluorescent dye molecules show photoswitchable fluorescence emission. In particular, photoswitchable cyanine fluorophores such as Cy5, Alexa 647, and Cy7, may be paired with a second fluorophore, which serves as an activator, determining the wavelength of light that re-activates the fluorescence of the photoswitchable molecule. This protocol describes the preparation of antibodies labeled with one such pairing scheme of synthetic fluorophores, Alexa 405 and Alexa 647. It may easily be adapted for labeling with other fluorophores or for labeling other substrate molecules.

Preparation of Nuclear Extracts from HeLa Cells (2013-06-03T06:00:46-07:00)
Cell Biology, general, Subcellular Fractionation, Topic Intro 82 Protocols 5438-5445

HeLa cells are the archetypal tissue culture cell line for the preparation of mammalian cell-free systems. These cells, derived from a cervical carcinoma, have been maintained in culture since the late 1940s. They grow with a doubling time of ~24 h and can be cultured in medium containing fetal bovine serum (optimal serum for growth) or medium containing horse serum (in which they grow more slowly, but this serum is considerably less expensive). Nuclear extracts prepared from these cells have been used to determine the mechanisms of splicing and polyadenylation, and such extracts have been characterized extensively. HeLa cells are usually the cell type of choice for initiating cell-free analysis of nearly any aspect of mammalian gene expression. In some instances (e.g., analysis of tissue-specific alternative splicing), it is necessary to use nuclei from a different cell type. We have found that the protocol described here can be used successfully to prepare active nuclear extracts from a wide variety of tissue culture cells, including Drosophila S2 cells.

Analysis of Pre-mRNA Splicing Using HeLa Cell Nuclear Extracts (2013-06-03T06:00:46-07:00)
Molecular Biology, general, RNA, RNA, general, Topic Intro 82 Protocols 5438-5445

This protocol is used to determine the splicing behavior of pre-mRNAs in cell extracts that are capable of carrying out splicing (e.g., nuclear extracts from HeLa cells). ³²P-labeled RNA is incubated under splicing conditions for various times, and the resulting products are analyzed on denaturing polyacrylamide gels.

Cycling Assay for Determining Intracellular Cyclic ADP-Ribose Levels (2013-06-03T06:00:46-07:00)
Cell Biology, general, Signal Transduction, Fluorescence, general, Collections, Calcium Techniques, PROT 072777

Cyclic ADP-ribose (cADPR) is a Ca²⁺-mobilizing second messenger involved in the regulation of various physiological processes. The ability to detect changes in endogenous cADPR is a fundamental step in the identification of its role in signal transduction triggered by hormones and other stimuli. Because the intracellular concentration of cADPR can be very low, depending on the expression level of the ADP-ribosyl cyclase activity (forming cADPR and nicotinamide from NAD) in the cell type of interest, very sensitive and selective methods are required. The method presented here exploits the ability of the ADP-ribosyl cyclase to catalyze the reverse reaction (i.e., to synthesize NAD stoichiometrically starting from cADPR) in the presence of an excess of nicotinamide. The generation of NAD can be coupled to a cycling assay using the enzymes alcohol dehydrogenase and diaphorase. The former reduces NAD to NADH in the presence of ethanol and the latter oxidizes NADH to NAD in the presence of resazurin and flavin mononucleotide. The formation of the fluorescent reduced resazurin (resofurin) can be detected with a plate reader. Thus, this cycling assay for cADPR determination can be considered a high-throughput method, potentially screening cADPR concentration simultaneously in many samples.

Imaging Green Fluorescent Protein-Labeled Neurons Using Light and Electron Microscopy (2013-06-03T06:00:46-07:00)
Fluorescence, Fluorescent Proteins, Light Microscopy, Immunostaining, Electron Microscopy, Imaging for Neuroscience, Imaging in Neuroscience: A Laboratory Manual

The ability to observe axons and dendrites with transmission electron microscopy (EM) after they have been previously imaged live with laser-scanning microscopy is a useful technique to study their synaptic connectivity. This protocol provides a detailed method by which neurons that were imaged in a live brain or slice culture can be reimaged using EM. First, brain tissue expressing green fluorescent protein (GFP) is chemically fixed. Then, an immunocytochemistry process is used to render the fluorescent protein electron dense so that it can first be located using light microscopy and then serial thin-sectioned for EM so that the ultrastructure of specific parts of neurites can be analyzed in three dimensions. Patterns of blood vessels observed in the live brain are used to locate the previously imaged neurons. The method described here allows for a complete three-dimensional (3D) reconstruction to be made of the imaged structures from serial electron micrographs.

Systematic review of the diagnostic performance of serum markers of liver fibrosis in alcoholic liver disease (2012-12-28T00:00:00Z)

Background: Alcoholic liver disease (ALD) is a significant cause of death and morbidity. Detection of liver fibrosis at an early stage could provide opportunities for more optimal management. Serum markers of liver fibrosis offer an alternative to biopsy. Evidence of the performance of biomarkers in ALD is needed and a systematic review to evaluate available studies was conducted. Methods: Electronic databases were searched. Studies were included if they evaluated paired samples of biopsy and serum, and presented data as sensitivity, specificity, or ROC curves. Results: 15 studies were included- median participant number = 146 (range 44--1034). Studies differed with respect to patient populations. 6 single markers were evaluated (mostly Hyaluronic Acid), and ten combined panels. Biomarkers could discriminate between people with severe fibrosis/cirrhosis with high diagnostic accuracy- HA (median AUROC 0.79 range 0.69-0.93), panels (median AUROC 0.83 range 0.38-0.95). Significant heterogeneity precluded pooling. Performance was poorer for detecting less severe fibrosis. Conclusions: There are limited numbers of small studies evaluating the accuracy of biomarkers in identifying fibrosis on biopsy in ALD. Some showed promise (both HA alone and some panels) in the identification of cirrhosis/severe fibrosis and could be used to rule it out in heavy drinkers. Biomarkers less accurate with less severe fibrosis.

N-acetylcysteine improves antitumoural response of Interferon alpha by NF-kB downregulation in liver cancer cells (2012-12-04T00:00:00Z)

Background: Liver cancer is one of the most common malignancies in the world and at the moment, there is no drug intervention effective for the treatment of liver tumours. Investigate the effect of N-acetylcysteine (NAC), which has been studied for its antitumoural properties, on the toxicity of hepatocarcinoma (HCC) cells in vitro when used with the drug interferon alpha-2A (IFN), which is used clinically to treat HCC. Results: NAC, IFN and NAC plus IFN reduced cell viability, as determined by MTT assay. More importantly, NAC potentiates the cytotoxic effect of IFN, with the best response achieved with 10 mM of NAC and 2.5 x 104 of IFN. These results were confirmed by Annexin/PI staining through flow cytometry and morphologic analyses. Co-treatment reduced the expression of the nuclear transcription factor kappa-B (NF-kB). In a similar way to NAC, RNAi against p65 potentiated the toxic effect of IFN, suggesting that, indeed, NAC may be enhancing the effect of IFN through inhibition of NF-kB. Conclusions: Our results support the notion that NAC may be an important drug for the treatment of liver tumours as primary or adjuvant therapy. IFN has a limited clinical response, and therefore, the anti-proliferative properties of NAC in the liver should be explored further as an alternative for non-responders to IFN treatment.

The genetic regulation of the terminating phase of liver regeneration (2012-11-20T00:00:00Z)

Background: After partial hepatectomy (PHx), the liver regeneration process terminates when the normal liver-mass/body-weight ratio of 2.5% has been re-established. To investigate the genetic regulation of the terminating phase of liver regeneration, we performed a 60% PHx in a porcine model. Liver biopsies were taken at the time of resection, after three weeks and upon termination the sixth week. Gene expression profiles were obtained using porcine oligonucleotide microarrays. Our study reveals the interactions between genes regulating the cell cycle, apoptosis and angiogenesis, and the role of Transforming Growth Factor-beta (TGF-beta) signalling towards the end of liver regeneration. Results: Microarray analysis revealed a dominance of genes regulating apoptosis towards the end of regeneration. Caspase Recruitment Domain-Containing Protein 11 (CARD11) was up-regulated six weeks after PHx, suggesting the involvement of the caspase system at this time. Zinc Finger Protein (ZNF490) gene, with a potential negative effect on cell cycle progression, was only up-regulated at three and six weeks after PHx indicating a central role at this time. TGF-beta regulation was not found to be significantly affected in the terminating phase of liver regeneration. Vasohibin 2 (VASH2) was down-regulated towards the end of regeneration, and may indicate a role in preventing a continued vascularization process. Conclusions: CARD11, ZNF490 and VASH2 are differentially expressed in the termination phase of liver regeneration. The lack of TGF-beta up-regulation suggests that signalling by TGF-beta is not required for termination of liver regeneration.

Comparative histological study of hepatic architecture in the three orders amphibian livers (2012-08-20T00:00:00Z)

Background: This report presents a detailed description of hepatic architecture in 46 amphibian livers by light microscopy, and extensively discusses the phylogenetic viewpoint. Results: The 46 amphibian livers showed a variety of histological features, but anurans were the same as in mammalian livers. The hepatocyte-sinusoidal structures of the amphibian livers were classified into three different types: (I) several-cell-thick plate type, (II) two-cell-thick plate type, and (III) one-cell-thick plate type, depending on the percentage extension of sinusoidal areas per unit area, measured by morphometry. Hematopoietic tissue structures were observed in the connective tissue of both the perihepatic subcapsular regions and portal triads in the order Caudata and Gymnophiona, but were not observed in the order Anura (except for the genus Bombina and Xenopus). As phylogenetic relationships are branched from urodeles to anurans, the parenchyma arrangement progressed from the combined several- and two-cell-thick plate type to one-cell-thick plate type as seen in the mammalian liver type. In contrast, hematopoietic tissue structures were exactly the opposite and did not involve anurans. Conclusions: This study is the first to investigate amphibian livers phylogenically, and their architectural differences are shown in the route of hepatic ontogenesis. In this process, parenchymal arrangement formation is acquired phylogenically. The occurrence of hematopoietic cells may be related with the development of the systemic immune system in the spleen and bone marrow.

Severe diabetes and leptin resistance cause differential hepatic and renal transporter expression in mice (2012-04-23T00:00:00Z)

Background: Type-2 Diabetes is a major health concern in the United States and other Westernized countries, with prevalence increasing yearly. There is a need to better model and predict adverse drug reactions, drug-induced liver injury, and drug efficacy in this population. Because transporters significantly contribute to drug clearance and disposition, it is highly significant to determine whether a severe diabetes phenotype alters drug transporter expression, and whether diabetic mouse models have altered disposition of acetaminophen (APAP) metabolites. Results: Transporter mRNA and protein expression were quantified in livers and kidneys of adult C57BKS and db/db mice, which have a severe diabetes phenotype due to a lack of a functional leptin receptor. The urinary excretion of acetaminophen-glucuronide, a substrate for multidrug resistance-associated proteins transporters was also determined. The mRNA expression of major uptake transporters, such as organic anion transporting polypeptide Slco1a1 in liver and kidney, 1a4 in liver, and Slc22a7 in kidney was decreased in db/db mice. In contrast, Abcc3 and 4 mRNA and protein expression was more than 2 fold higher in db/db male mouse livers as compared to C57BKS controls. Urine levels of APAP-glucuronide, -sulfate, and N-acetyl cysteine metabolites were higher in db/db mice. Conclusion: A severe diabetes phenotype/presentation significantly altered drug transporter expression in liver and kidney, which corresponded with urinary APAP metabolite levels.

Polymorphism of C3 complement in association with myocardial infarction in a sample of central Tunisia (2013-06-13T00:00:00Z)

Background: Myocardial infarction (MI) is a major clinical problem because of its large contribution to mortality. The genetic bases of this disease have been widely studied in recent years to find a clear association with some genetic markers that increase the risk of its occurrence. In the present investigation, the correlation between MI and the C3 complement polymorphism was analyzed using a case--control study. Methods: Our study ported on one hundred seventy survived myocardial infarction patients and ninety five healthy controls. The C3 allele identification was investigated using the amplification refractory mutation system PCR to determine the C3*S and the C3*F alleles of the C3 polymorphism Results: Frequencies of C3*S and C3*F in patients are 0.59 and 0.41 respectively. Fisher test results showed a significant increase of C3*F allele in the sample of patients (0.41; odds ratio: 2.616; C.I [1.738-3.938]) compared to controls (0.21; odds ratio: 0.382; 95% CI [0.254-0.575]), p = 2.742 x 10-6. Conclusion: A strong positive correlation was found between C3 polymorphism and MI estimating that the risk of myocardial infarction is significantly increased among patients with C3*F allele of this polymorphism.Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1190484203893646

Isolated hypoplastic circumflex coronary artery: a rare cause of haemorrhagic myocardial infarction in a young athlete (2013-06-06T00:00:00Z)

Hypoplastic coronary artery disease is a rare condition that may lead to myocardial infarction and sudden death. Here we describe for the first time an isolated hypoplasia of the leftcircumflex artery (LCX). An otherwise healthy and athletically active 16-year-old boy was admitted to the intensive care unit (ICU) after out-of-hospital cardiac arrest. He died 12 hoursafter the initial event. Autopsy revealed an isolated hypoplasticLCX and acute haemorrhagic infarction in the posterolateral myocardium. The existence of isolated hypoplasia of the LCXchallenges our understanding of coronary artery development.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1558483061962648

Solitary myofibroma of the sigmoid colon: case report and review of the literature (2013-06-06T00:00:00Z)

A 58-year-old woman presented with a solitary myofibroma that arose in the sigmoid colon. Computed tomography revealed a highly enhanced intramural mass (1.3-cm maximum diameter) in the proximal sigmoid colon. Histologically, the tumor exhibited a biphasic growth pattern, which comprised haphazardly arranged, interwoven fascicles of plump, myoid-appearing spindle cells with elongated nuclei and abundant eosinophilic cytoplasm, and more cellular areas of primitive-appearing polygonal cells that were arranged in a hemangiopericytomatous pattern. The tumor cells were positive for smooth muscle actin (SMA), and negative for desmin, h-caldesmon, CD34, cytokeratin, S100 protein, and CD117. The Ki-67 labeling index was not high (up to 7%). Based on these histologic and immunohistochemical features, our patient was diagnosed with a myofibroma of the sigmoid colon. The presence of solitary myofibroma in the intestine of an adult requires attention to avoid misdiagnosis as a more aggressive mesenchymal tumor.Virtual SlidesThe virtual silde(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/2096403796957687

Analyzing huge pathology images with open source software (2013-06-06T00:00:00Z)

No description available

Expression of the epithelial-mesenchymal transition-related proteins and their clinical significance in lung adenocarcinoma (2013-05-24T00:00:00Z)

Background: Epithelial-mesenchymal transition (EMT) is defined as switching of polarized epithelial cells to a migratory fibroblastoid phenotype. EMT is known to be involved in the progression andmetastasis of various cancers. The aim was to evaluate that whether EMT-related proteins alterations are associated with clinicopathological features and prognosis in lungadenocarcinoma. Methods: The expression of EMT-related proteins including cytokeratin, E-cadherin, TTF-1, beta-catenin, vimentin, Snail, Twist, CD44 was evaluated by immunohistochemistry using a tissue arraymethod in the lung adenocarcinoma tissues of 95 patients. In addition, clinicopathological characteristics and survival were compared with the expression of EMT-related proteins. Results: Loss of epithelial proteins and/or acquisition of the expression of mesenchymal proteins were observed in lung adenocarcinoma. These proteins' alteration was associated with poor cell differentiation and poor patients' outcome, respectively. Subjects were divided into twogroups according to the number of EMT-related proteins' alteration. A higher number of EMT-related proteins' alteration was found to be significantly as sociated with unfavorableoutcome. Multivariate analysis showed that a higher number of EMT-related proteins' alteration was independently associated with poor prognosis. Conclusions: The number of EMT-related proteins' alteration is a significantprognostic marker to predict overall survival in patients with lung adenocarcinoma. The information generated will bevaluable for the prognosis of patients with lung adenocarcinoma.Virtual slidesThe virtual slides for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1007838329872974

Comprehensive genotyping of the USA national maize inbred seed bank (2013-06-11T00:00:00Z)

Background: Genotyping by sequencing, a new low-cost, high-throughput sequencing technology was used to genotype 2,815 maize inbred accessions, preserved mostly at the National Plant Germplasm System in the USA. The collection includes inbred lines from breeding programs all over the world. Results: The method produced 681,257 single-nucleotide polymorphism (SNP) markers distributed across the entire genome, with the ability to detect rare alleles at high confidence levels. More than half of the SNPs in the collection are rare. Although most rare alleles have been incorporated into public temperate breeding programs, only a modest amount of the available diversity is present in the commercial germplasm. Analysis of genetic distances shows population stratification, including a small number of large clusters centered on key lines. Nevertheless, an average fixation index of 0.06 indicates moderate differentiation between the three major maize subpopulations. Linkage disequilibrium (LD) decays very rapidly, but the extent of LD is highly dependent on the particular group of germplasm and region of the genome. The utility of these data for performing genome-wide association studies was tested with two simply inherited traits and one complex trait. We identified trait associations at SNPs very close to known candidate genes for kernel color, sweet corn, and flowering time; however, results suggest that more SNPs are needed to better explore the genetic architecture of complex traits. Conclusions: The genotypic information described here allows this publicly available panel to be exploited by researchers facing the challenges of sustainable agriculture through better knowledge of the nature of genetic diversity.

Hyperosmotic priming of Arabidopsis seedlings establishes a long-term somatic memory accompanied by specific changes of the epigenome  (2013-06-14T00:00:00Z)

Background: In arid and semi-arid environments, drought and soil salinity usually occur at the beginning and end of a plant's life cycle, offering a natural opportunity for the priming of young plants to enhance stress tolerance in mature plants. Chromatin marks, such as histone modifications, provide a potential molecular mechanism for priming plants to environmental stresses, but whether transient exposure of seedlings to hyperosmotic stress leads to chromatin changes that are maintained throughout vegetative growth remains unclear. Results: We have established an effective protocol for hyperosmotic priming in the model plant Arabidopsis, which includes a transient mild salt treatment of seedlings followed by an extensive period of growth in control conditions. Primed plants are identical to non-primed plants in growth and development, yet they display reduced salt uptake and enhanced drought tolerance after a second stress exposure. ChIP-seq analysis of four histone modifications revealed that the priming treatment altered the epigenomic landscape; the changes were small but they were specific for the treated tissue, varied in number and direction depending on the modification, and preferentially targeted transcription factors. Notably, priming leads to shortening and fractionation of H3K27me3 islands. This effect fades over time, but is still apparent after a ten day growth period in control conditions. Several genes with priming-induced differences in H3K27me3 showed altered transcriptional responsiveness to the second stress treatment. Conclusion: Experience of transient hyperosmotic stress by young plants is stored in a long-term somatic memory comprising differences of chromatin status, transcriptional responsiveness and whole plant physiology.

Identification of pathways directly regulated by SHORT VEGETATIVE PHASE during vegetative and reproductive development in Arabidopsis (2013-06-11T00:00:00Z)

Background: MADS-domain transcription factors play important roles during plant development. The Arabidopsis MADS-box gene SHORT VEGETATIVE PHASE (SVP) is a key regulator of two developmental phases. It functions as a repressor of the floral transition during the vegetative phase and later it contributes to the specification of floral meristems. How these distinct activities are conferred by a single transcription factor is unclear, but interactions with other MADS domain proteins which specify binding to different genomic regions is likely one mechanism. Results: To compare the genome-wide DNA binding profile of SVP during vegetative and reproductive development we performed ChIP-seq analyses. These ChIP-seq data were combined with tiling array expression analysis, induction experiments and qRT-PCR to identify biologically relevant binding sites. In addition, we compared genome-wide target genes of SVP with those published for the MADS domain transcription factors FLC and AP1, which interact with SVP during the vegetative and reproductive phases, respectively. Conclusions: Our analyses resulted in the identification of pathways that are regulated by SVP including those controlling meristem development during vegetative growth and flower development whereas floral transition pathways and hormonal signaling were regulated predominantly during the vegetative phase. Thus, SVP regulates many developmental pathways, some of which are common to both of its developmental roles whereas others are specific to only one of them.

Conservation and divergence of transcriptomic and epigenomic variation in maize hybrids (2013-06-12T00:00:00Z)

Background: Recent genome-wide studies have suggested that in addition to genetic variation, epigenetic variation may also be associated with differential gene expression and growth vigor in plant hybrids. Maize is an ideal model system for the study of epigenetic variation in hybrids, given the significant heterotic performance of the plant, the well-known complexity of the genome, and the rich history of epigenetic studies using this organism. However, integrated comparative transcriptomic and epigenomic analyses in different organs of maize hybrids remain largely unexplored. Methods: We generated integrated maps of transcriptomes and epigenomes from shoots and roots of two maize inbred lines and their reciprocal hybrids, and globally surveyed the epigenetic variations and their relationships with transcriptional divergence between different organs and genotypes. Results: Whereas histone modifications varied both between organs and between genotypes, DNA-methylation patterns were more distinguishable between genotypes than between organs. Histone modifications were associated with transcriptomic divergence between organs and between hybrids and parents. Further, genes that were upregulated in both shoots and roots of hybrids were significantly enriched in the nucleosome assembly pathway. Interestingly, small interfering RNAs (siRNAs) of 22 and 24 nucleotides long were shown to be derived from distinct transposable elements, and for different transposable elements in both shoots and roots, the differences in siRNA activity between hybrids and patents were primarily driven by different siRNA species. Conclusions: These results suggest that despite variation in specific genes or genomic loci, similar mechanisms may account for the genome-wide epigenetic regulation of gene activity and transposon stability in different organs of maize hybrids.

Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome (2013-06-12T00:00:00Z)

Background: There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys. Results: A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley. Conclusions: We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes.

Differential gene hypermethylation in genital lichen sclerosus and cancer: a comparative study (2013-06-17T03:02:18.810856-05:00)

Abstract

Myogenin expression in vulvovaginal spindle cell lesions: analysis of a series of cases with emphasis on diagnostic pitfalls (2013-06-17T03:02:22.687059-05:00)

Abstract

Malignant Potential of Small Oncocytic Follicular Carcinoma/Hürthle Cell Carcinoma: An Institutional Experience (2013-06-17T03:02:32.488664-05:00)

Abstract

Immunohistochemical evaluation of MYC expression in mantle cell lymphoma (2013-06-17T03:02:35.014391-05:00)

Abstract

Corrigendum (2013-06-09T22:26:21.782806-05:00)

An integrated approach to epitope analysis I: Dimensional reduction, visualization and prediction of MHC binding using amino acid principal components and regression approaches (2010-11-02T00:00:00Z)

Background: Operation of the immune system is multivariate. Reduction of the dimensionality is essential to facilitate understanding of this complex biological system. One multi-dimensional facet of the immune system is the binding of epitopes to the MHC-I and MHC-II molecules by diverse populations of individuals. Prediction of such epitope binding is critical and several immunoinformatic strategies utilizing amino acid substitution matrices have been designed to develop predictive algorithms. Contemporaneously, computational and statistical tools have evolved to handle multivariate and megavariate analysis, but these have not been systematically deployed in prediction of MHC binding. Partial least squares analysis, principal component analysis, and associated regression techniques have become the norm in handling complex datasets in many fields. Over two decades ago Wold and colleagues showed that principal components of amino acids could be used to predict peptide binding to cellular receptors. We have applied this observation to the analysis of MHC binding, and to derivation of predictive methods applicable on a whole proteome scale. Results: We show that amino acid principal components and partial least squares approaches can be utilized to visualize the underlying physicochemical properties of the MHC binding domain by using commercially available software. We further show the application of amino acid principal components to develop both linear partial least squares and non-linear neural network regression prediction algorithms for MHC-I and MHC-II molecules. Several visualization options for the output aid in understanding the underlying physicochemical properties, enable confirmation of earlier work on the relative importance of certain peptide residues to MHC binding, and also provide new insights into differences among MHC molecules. We compared both the linear and non-linear MHC binding prediction tools to several predictive tools currently available on the Internet. Conclusions: As opposed to the highly constrained user-interaction paradigms of web-server approaches, local computational approaches enable interactive analysis and visualization of complex multidimensional data using robust mathematical tools. Our work shows that prediction tools such as these can be constructed on the widely available JMP® platform, can operate in a spreadsheet environment on a desktop computer, and are capable of handling proteome-scale analysis with high throughput.

Identification of conformational B-cell Epitopes in an antigen from its primary sequence (2010-10-20T00:00:00Z)

Background: One of the major challenges in the field of vaccine design is to predict conformational B-cell epitopes in an antigen. In the past, several methods have been developed for predicting conformational B-cell epitopes in an antigen from its tertiary structure. This is the first attempt in this area to predict conformational B-cell epitope in an antigen from its amino acid sequence. Results: All Support vector machine (SVM) models were trained and tested on 187 non-redundant protein chains consisting of 2261 antibody interacting residues of B-cell epitopes. Models have been developed using binary profile of pattern (BPP) and physiochemical profile of patterns (PPP) and achieved a maximum MCC of 0.22 and 0.17 respectively. In this study, for the first time SVM model has been developed using composition profile of patterns (CPP) and achieved a maximum MCC of 0.73 with accuracy 86.59%. We compare our CPP based model with existing structure based methods and observed that our sequence based model is as good as structure based methods. Conclusion: This study demonstrates that prediction of conformational B-cell epitope in an antigen is possible from is primary sequence. This study will be very useful in predicting conformational B-cell epitopes in antigens whose tertiary structures are not available. A web server CBTOPE has been developed for predicting B-cell epitope http://www.imtech.res.in/raghava/cbtope/.

Motif prediction to distinguish LPS-stimulated pro-inflammatory vs. antibacterial macrophage genes (2010-09-21T00:00:00Z)

Background: Innate immunity is the first line of defence offered by host cells to infections. Macrophage cells involved in innate immunity are stimulated by lipopolysaccharide (LPS), found on bacterial cell surface, to express a complex array of gene products. Persistent LPS stimulation makes a macrophage tolerant to LPS with down regulation of inflammatory genes ("pro-inflammatory") while continually expressing genes to fight the bacterial infection ("antibacterial"). Interactions of transcription factors (TF) at their cognate TF binding sites (TFBS) on the expressed genes are important in transcriptional regulatory networks that control these pro-inflammatory and antibacterial expression paradigms involved in LPS stimulation. Results: We used differential expression patterns in a public domain microarray data set from LPS-stimulated macrophages to identify 228 pro-inflammatory and 18 antibacterial genes. Employing three different motif search tools, we predicted respectively four and one statistically significant TF-TFBS interactions from the pro-inflammatory and antibacterial gene sets. The biological literature was utilized to identify target genes for the four pro-inflammatory profile TFs predicted from the three tools, and 18 of these target genes were observed to follow the pro-inflammatory expression pattern in the original microarray data. Conclusions: Our analysis distinguished pro-inflammatory vs. antibacterial transcriptomic signatures that classified their respective gene expression patterns and the corresponding TF-TFBS interactions in LPS-stimulated macrophages. By doing so, this study has attempted to characterize the temporal differences in gene expression associated with LPS tolerance, a major immune phenomenon implicated in various pathological disorders.

Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage (2010-05-17T00:00:00Z)

Background: Several arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4+ T cells are needed for optimal CD8+ T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4+ T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4+ T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy. Results: By inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4+ T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4+ T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4+ T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies. Conclusions: The identification of CD4+ T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection.

An integrated approach to epitope analysis II: A system for proteomic-scale prediction of immunological characteristics (2010-11-02T00:00:00Z)

Background: Improving our understanding of the immune response is fundamental to developing strategies to combat a wide range of diseases. We describe an integrated epitope analysis system which is based on principal component analysis of sequences of amino acids, using a multilayer perceptron neural net to conduct QSAR regression predictions for peptide binding affinities to 35 MHC-I and 14 MHC-II alleles. Results: The approach described allows rapid processing of single proteins, entire proteomes or subsets thereof, as well as multiple strains of the same organism. It enables consideration of the interface of diversity of both microorganisms and of host immunogenetics. Patterns of binding affinity are linked to topological features, such as extracellular or intramembrane location, and integrated into a graphical display which facilitates conceptual understanding of the interplay of B-cell and T-cell mediated immunity.Patterns which emerge from application of this approach include the correlations between peptides showing high affinity binding to MHC-I and to MHC-II, and also with predicted B-cell epitopes. These are characterized as coincident epitope groups (CEGs). Also evident are long range patterns across proteins which identify regions of high affinity binding for a permuted population of diverse and heterozygous HLA alleles, as well as subtle differences in reactions with MHCs of individual HLA alleles, which may be important in disease susceptibility, and in vaccine and clinical trial design. Comparisons are shown of predicted epitope mapping derived from application of the QSAR approach with experimentally derived epitope maps from a diverse multi-species dataset, from Staphylococcus aureus, and from vaccinia virus. Conclusions: A desktop application with interactive graphic capability is shown to be a useful platform for development of prediction and visualization tools for epitope mapping at scales ranging from individual proteins to proteomes from multiple strains of an organism. The possible functional implications of the patterns of peptide epitopes observed are discussed, including their implications for B-cell and T-cell cooperation and cross presentation.

Top dogs: wolf domestication and wealth (2010-02-24T00:00:00Z)

A phylogeographic analysis of gene sequences important in determining body size in dogs, recently published in BMC Biology, traces the appearance of small body size to the Neolithic Middle East. This finding strengthens the association of this event with the development of sedentary societies, and perhaps even has implications for the inception of human social inequality.See research article http://www.biomedcentral.com/1741-7007/8/16/

No better time to FRET: shedding light on host pathogen interactions (2010-02-18T00:00:00Z)

Understanding the spatio-temporal subversion of host cell signaling by bacterial virulence factors is key to combating infectious diseases. Following a recent study by Buntru and co-workers published in BMC Biology, we review how fluorescence (Forster) resonance energy transfer (FRET) has been applied to studying host-pathogen interactions and consider the prospects for its future application.See research article http://www.biomedcentral.com/1741-7007/7/81.

Making progress in genetic kin recognition among vertebrates (2010-02-17T00:00:00Z)

A recent study in BMC Evolutionary Biology has shown that genetically similar individual ring-tailed lemurs are also more similar in their scent composition, suggesting a possible mechanism of kin recognition. Theoretical and experimental studies reveal challenges ahead in achieving a true systems-level understanding of this process and its outcomes.See research article http://www.biomedcentral.com/1471-2148/9/281.

Acoel and platyhelminth models for stem-cell research (2010-02-16T00:00:00Z)

Acoel and platyhelminth worms are particularly attractive invertebrate models for stem-cell research because their bodies are continually renewed from large pools of somatic stem cells. Several recent studies, including one in BMC Developmental Biology, are beginning to reveal the cellular dynamics and molecular basis of stem-cell function in these animals.See research article http://www.biomedcentral.com/1471-213X/9/69.

Scale-eating cichlids: from hand(ed) to mouth (2010-02-24T00:00:00Z)

Two recent studies in BMC Biology and Evolution raise important questions about a textbook case of frequency-dependent selection in scale-eating cichlid fishes. They also suggest a fascinating new line of research testing the effects of handed behavior on morphological asymmetry.See research article http://www.biomedcentral.com/1741-7007/8/8.

Corpus Refactoring: a Feasibility Study (2007-09-13T00:00:00Z)

Background: Most biomedical corpora have not been used outside of the lab that created them, despite the fact that the availability of the gold-standard evaluation data that they provide is one of the rate-limiting factors for the progress of biomedical text mining. Data suggest that one major factor affecting the use of a corpus outside of its home laboratory is the format in which it is distributed. This paper tests the hypothesis that corpus refactoring – changing the format of a corpus without altering its semantics – is a feasible goal, namely that it can be accomplished with a semi-automatable process and in a time-effcient way. We used simple text processing methods and limited human validation to convert the Protein Design Group corpus into two new formats: WordFreak and embedded XML. We tracked the total time expended and the success rates of the automated steps. Results: The refactored corpus is available for download at the BioNLP SourceForge website http://bionlp.sourceforge.net. The total time expended was just over three person-weeks, consisting of about 102 hours of programming time (much of which is one-time development cost) and 20 hours of manual validation of automatic outputs. Additionally, the steps required to refactor any corpus are presented. Conclusion: We conclude that refactoring of publicly available corpora is a technically and economically feasible method for increasing the usage of data already available for evaluating biomedical language processing systems.

Nano-Bio-Genesis: Tracing the rise of nanotechnology and nanobiotechnology as 'big science' (2007-07-14T00:00:00Z)

Nanotechnology research has lately been of intense interest because of its perceived potential for many diverse fields of science. Nanotechnology's tools have found application in diverse fields, from biology to device physics. By the 1990s, there was a concerted effort in the United States to develop a national initiative to promote such research. The success of this effort led to a significant influx of resources and interest in nanotechnology and nanobiotechnology and to the establishment of centralized research programs and facilities. Further government initiatives (at federal, state, and local levels) have firmly cemented these disciplines as 'big science,' with efforts increasingly concentrated at select laboratories and centers. In many respects, these trends mirror certain changes in academic science over the past twenty years, with a greater emphasis on applied science and research that can be more directly utilized for commercial applications.We also compare the National Nanotechnology Initiative and its successors to the Human Genome Project, another large-scale, government funded initiative. These precedents made acceptance of shifts in nanotechnology easier for researchers to accept, as they followed trends already established within most fields of science. Finally, these trends are examined in the design of technologies for detection and treatment of cancer, through the Alliance for Nanotechnology in Cancer initiative of the National Cancer Institute. Federal funding of these nanotechnology initiatives has allowed for expansion into diverse fields and the impetus for expanding the scope of research of several fields, especially biomedicine, though the ultimate utility and impact of all these efforts remains to be seen.

Applied information retrieval and multidisciplinary research: new mechanistic hypotheses in Complex Regional Pain Syndrome (2007-05-04T00:00:00Z)

Background: Collaborative efforts of physicians and basic scientists are often necessary in the investigation of complex disorders. Difficulties can arise, however, when large amounts of information need to reviewed. Advanced information retrieval can be beneficial in combining and reviewing data obtained from the various scientific fields. In this paper, a team of investigators with varying backgrounds has applied advanced information retrieval methods, in the form of text mining and entity relationship tools, to review the current literature, with the intention to generate new insights into the molecular mechanisms underlying a complex disorder. As an example of such a disorder the Complex Regional Pain Syndrome (CRPS) was chosen. CRPS is a painful and debilitating syndrome with a complex etiology that is still unraveled for a considerable part, resulting in suboptimal diagnosis and treatment. Results: A text mining based approach combined with a simple network analysis identified Nuclear Factor kappa B (NFκB) as a possible central mediator in both the initiation and progression of CRPS. Conclusion: The result shows the added value of a multidisciplinary approach combined with information retrieval in hypothesis discovery in biomedical research. The new hypothesis, which was derived in silico, provides a framework for further mechanistic studies into the underlying molecular mechanisms of CRPS and requires evaluation in clinical and epidemiological studies.

Biological information specialists for biological informatics (2007-02-12T00:00:00Z)

Data management and integration are complicated and ongoing problems that will require commitment of resources and expertise from the various biological science communities. Primary components of successful cross-scale integration are smooth information management and migration from one context to another. We call for a broadening of the definition of bioinformatics and bioinformatics training to span biological disciplines and biological scales. Training programs are needed that educate a new kind of informatics professional, Biological Information Specialists, to work in collaboration with various discipline-specific research personnel. Biological Information Specialists are an extension of the informationist movement that began within library and information science (LIS) over 30 years ago as a professional position to fill a gap in clinical medicine. These professionals will help advance science by improving access to scientific information and by freeing scientists who are not interested in data management to concentrate on their science.

Generalization through similarity: motif discourse in the discovery and elaboration of zinc finger proteins (2007-10-03T00:00:00Z)

Background: Biological organisms and their components are better conceived within categories based on similarity rather than on identity. Biologists routinely operate with similarity-based concepts such as "model organism" and "motif." There has been little exploration of the characteristics of the similarity-based categories that exist in biology. This study uses the case of the discovery and classification of zinc finger proteins to explore how biological categories based in similarity are represented. Results: The existence of a category of "zinc finger proteins" was based in 1) a lumpy gradient of similarity, 2) a link between function and structure, 3) establishment of a range of appearance across systems and organisms, and 4) an evolutionary locus as a historically based common-ground. Conclusion: More systematic application of the idea of similarity-based categorization might eliminate the assumption that biological characteristics can only contribute to narrow categorization of humans. It also raises possibilities for refining data-driven exploration efforts.

Ki67 and proliferation in breast cancer (2013-05-22T21:01:44-07:00)
Breast cancer

New approaches to the prognostic assessment of breast cancer have come from molecular profiling studies. A major feature of this work has been to emphasise the importance of cancer cell proliferation as a key discriminative indicator of recurrence risk for oestrogen receptor positive breast cancer in particular. Mitotic count scoring, as a component of histopathological grade, has long formed part of a routine evaluation of breast cancer biology. However, there is an increasingly compelling case to include a specific proliferation score in breast cancer pathology reports based on expression of the cell cycle regulated protein Ki67. Immunohistochemical staining for Ki67 is a widely available and economical test with good tolerance of pre-analytical variations and staining conditions. However, there is currently no evidence based protocol established to derive a reliable and informative Ki67 score for routine clinical use. In this circumstance, pathologists must establish a standardised framework for scoring Ki67 and communicating results to a multidisciplinary team.

Low-grade adenosquamous carcinoma of the breast (2013-05-22T21:01:44-07:00)

Low-grade adenosquamous carcinoma is a rare and unique form of invasive mammary carcinoma. Though it is categorised as a variant of metaplastic carcinoma, it differs from its counterparts in this heterogeneous category by its relative clinical indolence, also reflected histologically in its low-grade cytomorphology. Descriptions of such a tumour were reported as early as 1912. However, low-grade adenosquamous carcinoma was only formally recognised in 1987 with the publication of Rosen and Ernsberger's landmark paper. Since then, several case reports and larger series have reaffirmed the clinicopathological characteristics of this unusual and uncommon tumour. Due to its rarity, however, many aspects of low-grade adenosquamous carcinoma, including its immunohistochemical and genetic profiles, remain unclear. This paper reviews the literature on this entity from 1987 to date, summarising its clinical and pathological features, and highlighting the diagnostic challenges it poses.

Occurrence and significance of epithelial-mesenchymal transition in breast cancer (2013-05-22T21:01:44-07:00)
Immunology (including allergy), Breast cancer, Molecular biology

By contrast with developmental epithelial-mesenchymal transition (EMT), where epithelial characteristics undergo transformation to a mesenchymal-like phenotype in a coordinated fashion, oncogenic EMT occurs in the context of unpredictable genetic changes present in the tumour cells, as well as an abnormal tumour microenvironment. Therefore, a partial form of EMT has been proposed as variably participating in the establishment of invasive phenotype in different types of breast carcinoma, in keeping with their morphological and phenotypical diversity. A complex network of signalling pathways and transcription factors appears responding to various growth factors and cytokines released by stromal and neoplastic elements, endowing the system with abundant regulatory opportunities. The process of EMT is largely elusive in histopathological preparations, prompting doubts regarding its significance in tumour progression. This might be related to the presumed focal occurrence of EMT in the majority of tumours. Detailed topological studies might facilitate understanding of the orchestration of events taking place in vivo. Even more importantly, clinical correlations can be endeavoured and, in parallel with advancement in molecular pathology, a contribution to taxonomy refinement can be envisaged.

Therapeutic targets in triple negative breast cancer (2013-05-22T21:01:44-07:00)
Breast cancer

Outcomes have improved significantly for many women diagnosed with breast cancer. For the heterogeneous group of tumours lacking expression of the oestrogen, progesterone and HER2 receptors, ‘triple negative’ breast cancers (TNBC), the prognosis overall has remained quite poor. When TNBC recurs, there is often little response to chemotherapy, and there are a few treatment options in this setting. Thus, there is an urgent clinical need to identify new therapeutic targets in order to improve the outlook for these patients. This review highlights the most promising therapeutic targets identified through new sequencing technologies, as well as through studies of apoptosis. We also present mounting evidence that the developmental signalling pathways Wnt/β-catenin, NOTCH and Hedgehog play an important role in the pathogenesis and progression of TNBC with new therapeutic approaches inhibiting these pathways in advanced preclinical studies or early clinical trials.

Breast pathology today: morphology and molecules (2013-05-22T21:01:43-07:00)
Breast cancer, Chemical pathology, Clinical diagnostic tests

This theme issue on breast pathology is a blend of articles of relevance for today's diagnostic practice that focuses on morphological recognition and classification of breast tumours, integration with molecular features and contribution to management decisions. The molecular reviews open the window into a future where scientific investigations into pathogenesis and biology of breast disease can be potentially translated into effective therapeutics for women with breast cancer.

Classification of breast tumours remains a mainstay of pathological assessment. In light of the recent publication of the 4th edition of WHO Classification of Tumours of the Breast in 2012,1 an update is provided of specific entities of myoepithelial, epithelial–myoepithelial, mesenchymal and fibroepithelial lesions.2 Key changes concerning these conditions emanating from the prior edition, and the rationale behind some of the revisions are summarised. Apart from emergent data and information since the prior volume that drive...

Technologies for Investigating the Physiological Barriers to Efficient Lipid Nanoparticle-siRNA Delivery (2013-05-24T09:25:57-07:00)

Small interfering RNA (siRNA) therapeutics have advanced from bench to clinical trials in recent years, along with new tools developed to enable detection of siRNA delivered at the organ, cell, and subcellular levels. Preclinical models of siRNA delivery have benefitted from methodologies such as stem-loop quantitative polymerase chain reaction, histological in situ immunofluorescent staining, endosomal escape assay, and RNA-induced silencing complex loading assay. These technologies have accelerated the detection and optimization of siRNA platforms to overcome the challenges associated with delivering therapeutic oligonucleotides to the cytosol of specific target cells. This review focuses on the methodologies and their application in the biodistribution of siRNA delivered by lipid nanoparticles.

Distinct Expression and Localization of Diacylglycerol Kinase Isozymes in Rat Retina (2013-05-24T09:25:58-07:00)

Recent studies have revealed that phosphoinositide (PI) signaling molecules are expressed in mammalian retinas, suggesting their importance in its signal transduction. We previously showed that diacylglycerol kinase (DGK) isozymes are expressed in distinct patterns in rat retina at the mRNA level. However, little is known about the nature and morphological aspects of DGKs in the retina. For this study, we performed immunohistochemical analyses to investigate in the retina the expression and localization of DGK isozymes at the protein level. Here, we show that both DGKβ and DGK localize in the outer plexiform layer, within which photoreceptor cells make contact with bipolar and horizontal cells. These isozymes exhibit distinct subcellular localization patterns: DGK localizes to the synaptic area of bipolar cells in a punctate manner, whereas DGKβ distributes diffusely in the subsynaptic and dendritic regions of bipolar and horizontal cells. However, punctate labeling for DGK is evident in the outer limiting membrane. DGK and DGKα localize predominantly to the nucleus of ganglion cells. These findings show distinct expression and localization of DGK isozymes in the retina, suggesting a different role of each isozyme.

Tissue- and Cell-Specific Co-localization of Intracellular Gelatinolytic Activity and Matrix Metalloproteinase 2 (2013-05-24T09:25:58-07:00)

Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme that degrades extracellular matrix proteins. Recent studies indicate that MMP-2 also has a role in intracellular proteolysis during various pathological conditions, such as ischemic injuries in heart and brain and in tumor growth. The present study was performed to map the distribution of intracellular MMP-2 activity in various mouse tissues and cells under physiological conditions. Samples from normal brain, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, thyroid gland, gonads, oral mucosa, salivary glands, esophagus, intestines, and skin were subjected to high-resolution in situ gelatin zymography and immunohistochemical staining. In hepatocytes, cardiac myocytes, kidney tubuli cells, epithelial cells in the oral mucosa as well as in excretory ducts of salivary glands, and adrenal cortical cells, we found strong intracellular gelatinolytic activity that was significantly reduced by the metalloprotease inhibitor EDTA but not by the cysteine protease inhibitor E-64. Furthermore, the gelatinolytic activity was co-localized with MMP-2. Western blotting and electron microscopy combined with immunogold labeling revealed the presence of MMP-2 in different intracellular compartments of isolated hepatocytes. Our results indicate that MMP-2 takes part in intracellular proteolysis in specific tissues and cells during physiological conditions.

Losartan Affects Glomerular AKT and mTOR Phosphorylation in an Experimental Model of Type 1 Diabetic Nephropathy (2013-05-24T09:25:58-07:00)

The AKT-mTOR pathway is activated in diabetic nephropathy. Renin-angiotensin system modulators exert beneficial effects on the diabetic kidney. We explored the action of losartan on AKT-mTOR phosphorylation in glomeruli and podocytes. Diabetes mellitus was induced to Sprague-Dawley rats by streptozotocin. Five months later, the rats were commenced on losartan and euthanized 2 months later. Kidneys were processed for immunofluorescence studies. Glomeruli were isolated for Western blot analysis. Diabetes increased activated forms of AKT and mTOR both in glomeruli and podocytes. In diabetic rats, losartan decreased phosphorylated/activated forms of AKT (Thr308) and mTOR (Ser2448) in glomeruli but decreased only activated mTOR in podocytes. However, in both glomeruli and podocytes of healthy animals, an inverse pattern was evident. In conclusion, a new body of evidence indicates the differential activation of AKT-mTOR in glomeruli and podocytes of healthy and diabetic animals in response to losartan.

Transient Receptor Potential Vanilloid Type 1 Channel (TRPV1) Immunolocalization in the Murine Enteric Nervous System Is Affected by the Targeted C-terminal Epitope of the Applied Antibody (2013-05-24T09:25:58-07:00)

The expression of transient receptor potential vanilloid type 1 channel (TRPV1) in the enteric nervous system is still the subject of debate. Although a number of studies have reported that TRPV1 is limited to extrinsic afferent fibers, other studies argue for an intrinsic expression of TRPV1. In the present study, reverse transcriptase PCR was employed to establish the expression of TRPV1 mRNA throughout the gastrointestinal tract. Using two antibodies directed against different epitopes of TRPV1, we were able to show at the protein level that the observed distribution pattern of TRPV1 is dependent on the antibody used in the immunohistochemical staining. A first antibody indeed mainly stained neuronal fibers, whereas a second antibody exclusively stained perikarya of enteric neurons throughout the mouse gastrointestinal tract. We argue that these different distribution patterns are due to the antibodies discriminating between different modulated forms of TRPV1 that influence the recognition of the targeted immunogen and as such distinguish intracellular from plasmalemmal forms of TRPV1. Our study is the first to directly compare these two antibodies within the same species and in identical conditions. Our observations underline that detailed knowledge of the epitope that is recognized by the antibodies employed in immunohistochemical procedures is a prerequisite for correctly interpreting experimental results.

Technologies for Investigating the Physiological Barriers to Efficient Lipid Nanoparticle-siRNA Delivery (2013-05-24T09:25:57-07:00)

Small interfering RNA (siRNA) therapeutics have advanced from bench to clinical trials in recent years, along with new tools developed to enable detection of siRNA delivered at the organ, cell, and subcellular levels. Preclinical models of siRNA delivery have benefitted from methodologies such as stem-loop quantitative polymerase chain reaction, histological in situ immunofluorescent staining, endosomal escape assay, and RNA-induced silencing complex loading assay. These technologies have accelerated the detection and optimization of siRNA platforms to overcome the challenges associated with delivering therapeutic oligonucleotides to the cytosol of specific target cells. This review focuses on the methodologies and their application in the biodistribution of siRNA delivered by lipid nanoparticles.

Distinct Expression and Localization of Diacylglycerol Kinase Isozymes in Rat Retina (2013-05-24T09:25:58-07:00)

Recent studies have revealed that phosphoinositide (PI) signaling molecules are expressed in mammalian retinas, suggesting their importance in its signal transduction. We previously showed that diacylglycerol kinase (DGK) isozymes are expressed in distinct patterns in rat retina at the mRNA level. However, little is known about the nature and morphological aspects of DGKs in the retina. For this study, we performed immunohistochemical analyses to investigate in the retina the expression and localization of DGK isozymes at the protein level. Here, we show that both DGKβ and DGK localize in the outer plexiform layer, within which photoreceptor cells make contact with bipolar and horizontal cells. These isozymes exhibit distinct subcellular localization patterns: DGK localizes to the synaptic area of bipolar cells in a punctate manner, whereas DGKβ distributes diffusely in the subsynaptic and dendritic regions of bipolar and horizontal cells. However, punctate labeling for DGK is evident in the outer limiting membrane. DGK and DGKα localize predominantly to the nucleus of ganglion cells. These findings show distinct expression and localization of DGK isozymes in the retina, suggesting a different role of each isozyme.

Tissue- and Cell-Specific Co-localization of Intracellular Gelatinolytic Activity and Matrix Metalloproteinase 2 (2013-05-24T09:25:58-07:00)

Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme that degrades extracellular matrix proteins. Recent studies indicate that MMP-2 also has a role in intracellular proteolysis during various pathological conditions, such as ischemic injuries in heart and brain and in tumor growth. The present study was performed to map the distribution of intracellular MMP-2 activity in various mouse tissues and cells under physiological conditions. Samples from normal brain, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, thyroid gland, gonads, oral mucosa, salivary glands, esophagus, intestines, and skin were subjected to high-resolution in situ gelatin zymography and immunohistochemical staining. In hepatocytes, cardiac myocytes, kidney tubuli cells, epithelial cells in the oral mucosa as well as in excretory ducts of salivary glands, and adrenal cortical cells, we found strong intracellular gelatinolytic activity that was significantly reduced by the metalloprotease inhibitor EDTA but not by the cysteine protease inhibitor E-64. Furthermore, the gelatinolytic activity was co-localized with MMP-2. Western blotting and electron microscopy combined with immunogold labeling revealed the presence of MMP-2 in different intracellular compartments of isolated hepatocytes. Our results indicate that MMP-2 takes part in intracellular proteolysis in specific tissues and cells during physiological conditions.

Losartan Affects Glomerular AKT and mTOR Phosphorylation in an Experimental Model of Type 1 Diabetic Nephropathy (2013-05-24T09:25:58-07:00)

The AKT-mTOR pathway is activated in diabetic nephropathy. Renin-angiotensin system modulators exert beneficial effects on the diabetic kidney. We explored the action of losartan on AKT-mTOR phosphorylation in glomeruli and podocytes. Diabetes mellitus was induced to Sprague-Dawley rats by streptozotocin. Five months later, the rats were commenced on losartan and euthanized 2 months later. Kidneys were processed for immunofluorescence studies. Glomeruli were isolated for Western blot analysis. Diabetes increased activated forms of AKT and mTOR both in glomeruli and podocytes. In diabetic rats, losartan decreased phosphorylated/activated forms of AKT (Thr308) and mTOR (Ser2448) in glomeruli but decreased only activated mTOR in podocytes. However, in both glomeruli and podocytes of healthy animals, an inverse pattern was evident. In conclusion, a new body of evidence indicates the differential activation of AKT-mTOR in glomeruli and podocytes of healthy and diabetic animals in response to losartan.

Transient Receptor Potential Vanilloid Type 1 Channel (TRPV1) Immunolocalization in the Murine Enteric Nervous System Is Affected by the Targeted C-terminal Epitope of the Applied Antibody (2013-05-24T09:25:58-07:00)

The expression of transient receptor potential vanilloid type 1 channel (TRPV1) in the enteric nervous system is still the subject of debate. Although a number of studies have reported that TRPV1 is limited to extrinsic afferent fibers, other studies argue for an intrinsic expression of TRPV1. In the present study, reverse transcriptase PCR was employed to establish the expression of TRPV1 mRNA throughout the gastrointestinal tract. Using two antibodies directed against different epitopes of TRPV1, we were able to show at the protein level that the observed distribution pattern of TRPV1 is dependent on the antibody used in the immunohistochemical staining. A first antibody indeed mainly stained neuronal fibers, whereas a second antibody exclusively stained perikarya of enteric neurons throughout the mouse gastrointestinal tract. We argue that these different distribution patterns are due to the antibodies discriminating between different modulated forms of TRPV1 that influence the recognition of the targeted immunogen and as such distinguish intracellular from plasmalemmal forms of TRPV1. Our study is the first to directly compare these two antibodies within the same species and in identical conditions. Our observations underline that detailed knowledge of the epitope that is recognized by the antibodies employed in immunohistochemical procedures is a prerequisite for correctly interpreting experimental results.

The Successive Alleys Test of Anxiety in Mice and Rats (2013-06-19 17:56:14+01:00)

The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics (2013-06-19 17:56:14+01:00)

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates (2013-06-19 17:56:14+01:00)

We report a simple method for fabricating an ultrahigh density array of vertically ordered small-molecular organic nanowires. This method allows for synthesis of complex heterostructured hybrid nanowire geometries, which can be inexpensively grown on arbitrary substrates. These structures have potential applications in organic electronics, optoelectronics, chemical sensing, photovoltaics and spintronics.

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease (2013-06-19 17:56:14+01:00)

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Simultaneous EEG Monitoring During Transcranial Direct Current Stimulation (2013-06-19 17:56:14+01:00)

Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that has shown initial therapeutic effects in several neurological conditions. The main mechanism underlying these therapeutic effects is the modulation of cortical excitability. Therefore, online monitoring of cortical excitability would help guide stimulation parameters and optimize its therapeutic effects. In the present article we review the use of a novel device that combines simultaneous tDCS and EEG monitoring in real time.

Anthrax vaccine design: strategies to achieve comprehensive protection against spore, bacillus, and toxin (2005-03-24T00:00:00Z)

The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and postexposure vaccination to target primarily toxins. Vaccines effective against toxin have been licensed for human use, but need improvement. Vaccines against bacilli have recently been developed by us and others. Whether effective vaccines will be developed against spores is still an open question. An ideal vaccine would confer simultaneous protection against spores, bacilli, and toxins. One step towards this goal is our dually active vaccine, designed to destroy both bacilli and toxin. Existing and potential strategies towards potent and effective anthrax vaccines are discussed in this review.

The Classics of Immunology (2005-03-10T00:00:00Z)

Medical Immunology will be publishing invited Reviews and Commentaries from investigators who are at the forefront of their fields, to up-date our readers as to the current state of their art. These Reviews and Commentaries will be accompanied by Editorials that place the current work into the perspective of the first contribution in an area, which resulted in a "Classic" paper. Where possible, links will be provided to the original publication, so that the modern student of immunology can read the original and draw their own conclusions as to the value of the "Classic" contribution, and its relationship to our contemporary views as to how the immune system functions. To begin this process at the very dawn of immunology, we highlight Sir Edward Jenner's first descriptions of the use of cowpox to immunize individuals against the dread disease smallpox.

The Future of Smallpox Vaccination: is MVA the key? (2005-03-01T00:00:00Z)

Eradication of the smallpox virus through extensive global vaccination efforts has resulted in one of the most important breakthroughs in medical history, saving countless lives from the severe morbidity and mortality that is associated with this disease. Although smallpox is now extinct in nature, laboratory stocks of this virus still remain and the subject of smallpox vaccination has gained renewed attention due to the potential risk that smallpox may be used as a biological weapon by terrorists or rogue states. Despite having the longest history of any modern vaccine, there is still much to be learned about smallpox vaccination and the correlates of protection remain to be formally defined. This Commentary will discuss the strengths and weaknesses of traditional smallpox vaccination in comparison with immunization using modified vaccinia virus Ankura (MVA), a non-replicating virus with a strong safety record but weakened immunogenicity.

FADD adaptor in cancer (2005-02-17T00:00:00Z)

FADD (Fas Associated protein with Death Domain) is a key adaptor molecule transmitting the death signal mediated by death receptors. In addition, this multiple functional protein is implicated in survival/proliferation and cell cycle progression. FADD functions are regulated via cellular sublocalization, protein phosphorylation, and inhibitory molecules. In the present review, we focus on the role of the FADD adaptor in cancer. Increasing evidence shows that defects in FADD protein expression are associated with tumor progression both in mice and humans. Better knowledge of the mechanisms leading to regulation of FADD functions will improve understanding of tumor growth and the immune escape mechanisms, and could open a new field for therapeutic interventions.

Wanted, an Anthrax vaccine: Dead or Alive? (2005-04-18T00:00:00Z)

It has been more than 100 years since the realization that microbes are capable of causing disease. In that time, we have learned a great deal as to how each organism has adapted to the immune system so as to avoid elimination. As well, we have also learned an immense amount since Louis Pasteur first proposed that the solution to infectious diseases was to culture the microbes and attenuate their virulence, so as to use them as vaccines. From the optimism and promise of the 19th century and immunization as the ultimate answer to the invasion by the microbial world, to the scientific realities of the 21st century, it is of interest to retrace the steps of the earliest microbiologists cum immunologists, to realize how far we've come, as well as how far we yet have to go. This editorial focuses on the history of anthrax as a microbial disease, and the earliest efforts at producing a vaccine for its prevention.

Surface change of root canal dentin after the use of irrigation activation protocols: Electron microscopy and an energy-dispersive X-ray microanalysis (2013-06-12T13:27:01.839277-05:00)

ABSTRACT

Microstructural characterization of Ti-6Al-4V alloy subjected to the duplex SMAT/plasma nitriding (2013-06-14T00:15:40.039082-05:00)

ABSTRACT

Dentin erosion by whitening mouthwash associated to toothbrushing abrasion: A focus variation 3D scanning microscopy study (2013-06-14T00:18:33.809145-05:00)

ABSTRACT

Microscopic analysis of recycled paper effect on print quality parameters (2013-06-14T00:18:34.011676-05:00)

ABSTRACT

Gold nanorods as probes in two-photon fluorescence correlation spectroscopy: Emerging applications and potential artifacts (2013-06-08T01:26:12.498686-05:00)

ABSTRACT

The alternative lengthening of telomere phenotype is significantly associated with loss of ATRX expression in high-grade pediatric and adult astrocytomas: a multi-institutional study of 214 astrocytomas (2013-06-14)
alternative lengthening of telomeresATRXastrocytomaFISHglioblastomaIDH1PDGFRA

The alternative lengthening of telomere phenotype is significantly associated with loss of ATRX expression in high-grade pediatric and adult astrocytomas: a multi-institutional study of 214 astrocytomas

Modern Pathology advance online publication, June 14 2013. doi:10.1038/modpathol.2013.90

Authors: Malak Abedalthagafi, Joanna J Phillips, Grace E Kim, Sabine Mueller, Daphne A Haas-Kogen, Roxanne E Marshall, Sidney E Croul, Mariarita R Santi, Jing Cheng, Shengmei Zhou, Lisa M Sullivan, Maria Martinez-Lage, Alexander R Judkins & Arie Perry

Use of cervical mucus to screen for gynecological malignancies: a pilot study (2013-06-14)
DNAfallopian tubegynecologicmucusovaryscreening

Use of cervical mucus to screen for gynecological malignancies: a pilot study

Modern Pathology advance online publication, June 14 2013. doi:10.1038/modpathol.2013.92

Authors: Ihab Lamzabi, Lela Buckingham, Mezgebe Gebrekiristos, Richa Jain, Paolo Gattuso, Vijaya Reddy, Alfred Guirguis, Summer Dewdney, Jacob Rotmensch & Pincas Bitterman

Common chromosomal aberrations detected by array comparative genomic hybridization in specialized stromal tumors of the prostate (2013-06-14)
array comparative genomic hybridizationprostatic stromal tumor of uncertain malignant potentialstromal sarcoma

Common chromosomal aberrations detected by array comparative genomic hybridization in specialized stromal tumors of the prostate

Modern Pathology advance online publication, June 14 2013. doi:10.1038/modpathol.2013.99

Authors: Chin-Chen Pan & Jonathan I Epstein

Esophageal leukoplakia or epidermoid metaplasia: a clinicopathological study of 18 patients (2013-06-14)
epidermoid metaplasiaesophagusleukoplakiaorthokeratotic dysplasiasquamous cell carcinomasquamous dysplasia

Esophageal leukoplakia or epidermoid metaplasia: a clinicopathological study of 18 patients

Modern Pathology advance online publication, June 14 2013. doi:10.1038/modpathol.2013.100

Authors: Aatur D Singhi, Christina A Arnold, Clinton D Crowder, Dora M Lam-Himlin, Lysandra Voltaggio & Elizabeth A Montgomery

Myxochondroid metaplasia of the plantar foot: a distinctive pseudoneoplastic lesion resembling nuchal fibrocartilaginous pseudotumor and the equine digital cushion (2013-06-14)
chondroid metaplasiachondroma of soft partsequine digital cushionfibrocartilaginous pseudotumor

Myxochondroid metaplasia of the plantar foot: a distinctive pseudoneoplastic lesion resembling nuchal fibrocartilaginous pseudotumor and the equine digital cushion

Modern Pathology advance online publication, June 14 2013. doi:10.1038/modpathol.2013.116

Authors: Wonwoo Shon & Andrew L Folpe

Regulation of protumorigenic pathways by Insulin like growth factor binding protein2 and its association along with beta-catenin in breast cancer lymph node metastasis (2013-06-16T00:00:00Z)

Background: Insulin like growth factor binding proteins modulate the mitogenic and pro survival effects of IGF. Elevated expression of IGFBP2 is associated with progression of tumors that include prostate, ovarian, glioma among others. Though implicated in the progression of breast cancer, the molecular mechanisms involved in IGFBP2 actions are not well defined. This study investigates the molecular targets and biological pathways targeted by IGFBP2 in breast cancer. Methods: Transcriptome analysis of breast tumor cells (BT474) with stable knockdown of IGFBP2 and breast tumors having differential expression of IGFBP2 by immunohistochemistry was performed using microarray. Differential gene expression was established using R-Bioconductor package. For validation, gene expression was determined by qPCR. Inhibitors of IGF1R and integrin pathway were utilized to study the mechanism of regulation of beta-catenin. Immunohistochemical and immunocytochemical staining was performed on breast tumors and experimental cells, respectively for beta-catenin and IGFBP2 expression. Results: Knockdown of IGFBP2 resulted in differential expression of 2067 up regulated and 2002 down regulated genes in breast cancer cells. Down regulated genes principally belong to cell cycle, DNA replication, repair, p53 signaling, oxidative phosphorylation, Wnt signaling. Whole genome expression analysis of breast tumors with or without IGFBP2 expression indicated changes in genes belonging to Focal adhesion, Map kinase and Wnt signaling pathways. Interestingly, IGFBP2 knockdown clones showed reduced expression of beta- catenin compared to control cells which was restored upon IGFBP2 re-expression. The regulation of beta-catenin by IGFBP2 was found to be IGF1R and integrin pathway dependent. Furthermore, IGFBP2 and beta-catenin are co-ordinately overexpressed in breast tumors and correlate with lymph node metastasis. Conclusion: This study highlights regulation of beta-catenin by IGFBP2 in breast cancer cells and most importantly, combined expression of IGFBP2 and beta-catenin is associated with lymph node metastasis of breast tumors.

Biochemical and biological characterization of exosomes containing prominin-1/CD133 (2013-06-14T00:00:00Z)

Exosomes can be viewed as complex "messages" packaged to survive trips to other cells in the local microenvironment and, through body fluids, to distant sites. A large body of evidence indicates a pro-metastatic role for certain types of cancer exosomes. We previously reported that prominin-1 had a pro-metastatic role in melanoma cells and that microvesicles released from metastatic melanoma cells expressed high levels of prominin-1. With the goal to explore the mechanisms that govern proteo-lipidic-microRNA sorting in cancer exosomes and their potential contribution(s) to the metastatic phenotype, we here employed prominin-1-based immunomagnetic separation in combination with filtration and ultracentrifugation to purify prominin-1-expressing exosomes (prom1-exo) from melanoma and colon carcinoma cells. Prom1-exo contained 154 proteins, including all of the 14 proteins most frequently expressed in exosomes, and multiple pro-metastatic proteins, including CD44, MAPK4K, GTP-binding proteins, ADAM10 and Annexin A2. Their lipid composition resembled that of raft microdomains, with a great enrichment in lyso-phosphatidylcholine, lyso-phosphatidyl-ethanolamine and sphingomyelin. The abundance of tetraspanins and of tetraspanin-associated proteins, together with the high levels of sphingomyelin, suggests that proteolipidic assemblies, probably tetraspanin webs, might be the essential structural determinant in the release process of prominin-1 of stem and cancer stem cells. Micro-RNA profiling revealed 49 species of micro-RNA present at higher concentrations in prom1-exo than in parental cells, including 20 with cancer-related function. Extensive accumulation of prom1-exo was observed 3 h after their addition to cultures of melanoma and bone marrow-derived stromal cells (MSC). Short-term co-culture of melanoma cells and MSC resulted in heterologous prominin-1 transfer. Exposure of MSC to prom1-exo increased their invasiveness. Our study supports the concept that specific populations of cancer exosomes contain multiple determinants of the metastatic potential of the cells from which they are derived.

Global profiling of prolactin-modulated transcripts in breast cancer in vivo (2013-06-12T00:00:00Z)

Background: Prolactin (PRL) is essential for normal mammary gland development. PRL promotes mammary tumor formation in rodents and elevated serum prolactin is associated with increased risk of estrogen-receptor positive breast cancer in women. On the other hand, PRL may also exert pro-differentiation effects and act to suppress invasive features of established breast cancer. Previously published limited global transcript profiling analyses of prolactin-regulated gene expression in human breast cancer cells have exclusively been performed in vitro. The present study aimed to shed new light on how PRL modulates estrogen receptor (ER)-positive breast cancer through global transcript profiling of a human breast cancer xenograft model in vivo. Methods: The prolactin-responsive human T47D breast cancer cell line was xenotransplanted into nude mice and global transcript profiling was carried out following treatment with or without human PRL for 48 h. A subset of PRL-modulated transcripts was further validated using qRT-PCR and immunohistochemistry. Results: The in vivo analyses identified 130 PRL-modulated transcripts, 75 upregulated and 55 downregulated, based on fold change >1.6 and P-value <0.05. From this initial panel of transcripts, a subset of 18 transcripts with established breast cancer-relevance were selected and validated by qRT-PCR. Some but not all of the transcripts were also PRL-modulated in vitro. The selected PRL-modulated transcripts were tested for dependence on Stat5, Jak1 or Jak2 activation, and for co-regulation by 17beta-estradiol (E2). The protein encoded by one of the PRL-regulated transcripts, PTHrP, was examined in a panel of 92 human breast cancers and found by in situ quantitative immunofluorescence analysis to be highly positively correlated with nuclear localized and tyrosine phosphorylated Stat5. Gene Ontology analysis revealed that PRL-upregulated genes were enriched in pathways involved in differentiation. Finally, a gene signature based on PRL-upregulated genes was associated with prolonged relapse-free and metastasis-free survival in breast cancer patients. Conclusions: This global analysis identified and validated a panel of PRL-modulated transcripts in an ER-positive human breast cancer xenotransplant model, which may have value as markers of relapse-free and metastasis-free survival. Gene products identified in the present study may facilitate ongoing deciphering of the pleiotropic effects of PRL on human breast cancer.

BCL11A overexpression predicts survival and relapse in non-small cell lung cancer and is modulated by microRNA-30a and gene amplification (2013-06-12T00:00:00Z)

Background: Aberrant activation of the proto-oncogene B-cell lymphoma/leukemia 11A (BCL11A) has been implicated in the pathogenesis of leukemia and lymphoma. However, the clinical significance of BCL11A in non-small cell lung cancer (NSCLC) remains unknown. Results: We examined BCL11A expression at the protein and mRNA levels in a cohort (n = 114) of NSCLC patients and assessed the relationship between BCL11A expression and clinicopathological parameters. Data from array-based Comparative Genomic Hybridization (aCGH) and microRNA transfection experiments were integrated to explore the potential mechanisms of abnormal BCL11A activation in NSCLC. Compared to adjacent non-cancerous lung tissues, BCL11A expression levels were specifically upregulated in NSCLC tissues at both the mRNA (t = 9.81, P < 0.001) and protein levels. BCL11A protein levels were higher in patients with squamous histology (chi2 = 15.81, P = 0.001), smokers (chi2 = 8.92, P = 0.004), patients with no lymph node involvement (chi2 = 5.14, P = 0.029), and patients with early stage disease (chi2 = 3.91, P = 0.048). A multivariate analysis demonstrated that in early stage NSCLC (IA--IIB), BCL11A was not only an independent prognostic factor for disease-free survival (hazards ratio [HR] 0.24, 95% confidence interval [CI] 0.12-0.50, P < 0.001), but also for overall survival (HR = 0.23, 95% CI 0.09-0.61, P = 0.003). The average BCL11A expression level was much higher in SCC samples with amplifications than in those without amplifications (t = 3.30, P = 0.023). Assessing functionality via an in vitro luciferase reporter system and western blotting, we found that the BCL11A protein was a target of miR-30a. Conclusions: Our results demonstrated that proto-oncogene BCL11A activation induced by miR-30a and gene amplification may be a potential diagnostic and prognostic biomarker for effective management of this disease.

Nectin-2 is a potential target for antibody therapy of breast and ovarian cancers (2013-06-12T00:00:00Z)

Background: Nectin-2 is a Ca2+-independent cell-cell adhesion molecule that is one of the plasma membrane components of adherens junctions. However, little has been reported about the involvement of Nectin-2 in cancer. Methods: To determine the expression of Nectin-2 in cancer tissues and cancer cell lines, we performed gene expression profile analysis, immunohistochemistry studies, and flow cytometry analysis. We also investigated the potential of this molecule as a target for antibody therapeutics to treat cancers by generating and characterizing an anti-Nectin-2 rabbit polyclonal antibody and 256 fully human anti-Nectin-2 monoclonal antibodies (mAbs). In addition, we tested anti-Nectin-2 mAbs in several in vivo tumor growth inhibition models to investigate the primary mechanisms of action of the mAbs. Results: In the present study, we found that Nectin-2 was over-expressed in clinical breast and ovarian cancer tissues by using gene expression profile analysis and immunohistochemistry studies. Nectin-2 was over-expressed in various cancer cell lines as well. Furthermore, the polyclonal antibody specific to Nectin-2 suppressed the in vitro proliferation of OV-90 ovarian cancer cells, which express endogenous Nectin-2 on the cell surface. The anti-Nectin-2 mAbs we generated were classified into 7 epitope bins. The anti-Nectin-2 mAbs demonstrated antibody-dependent cellular cytotoxicity (ADCC) and epitope bin-dependent features such as the inhibition of Nectin-2-Nectin-2 interaction, Nectin-2-Nectin-3 interaction, and in vitro cancer cell proliferation. A representative anti-Nectin-2 mAb in epitope bin VII, Y-443, showed anti-tumor effects against OV-90 cells and MDA-MB-231 breast cancer cells in mouse therapeutic models, and its main mechanism of action appeared to be ADCC. Conclusions: We observed the over-expression of Nectin-2 in breast and ovarian cancers and anti-tumor activity of anti-Nectin-2 mAbs via strong ADCC. These findings suggest that Nectin-2 is a potential target for antibody therapy against breast and ovarian cancers.

Diurnal difference in CAR mRNA expression (2004-08-28T00:00:00Z)

Background: The constitutive androstane receptor (CAR, NR1I3) plays a key role in the transcriptional activation of genes that encode xenobiotic/steroid and drug metabolizing enzymes. Results: The expression of CAR mRNA throughout the circadian rhythm is reported for the first time in phase with the clock gene Bmal1 and in antiphase with the clock-controlled gene Rev-erbα mRNAs, with a peak at Zeitgeber time (ZT) 20 and a trough at ZT8, and a peak/trough ratio of 2.0. The diurnal difference in CAR mRNA expression might underlie the 1.7-fold difference in the magnitude of the PB-dependent induction of CYP2B1/2 mRNA. Conclusion: The circadian oscillation of xenosensor gene CAR mRNA expression is partially responsible for chronopharmacokinetics and chronopharmacology in disease.

Binding of estrogen receptor with estrogen conjugated to bovine serum albumin (BSA) (2004-08-19T00:00:00Z)

Background: The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E2-BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E2-BSA does not compete for E2 binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E2 and E2-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E2-BSA preparations with unconjugated E2 (usually between 3–5%), the latter was carefully removed by ultrafiltration. Results: As previously reported, a 10-to 1000-fold molar excess of E2-BSA was unable to compete with 3H-E2 binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E2-BSA, the binding of 3H-E2 was significantly reduced. E2-BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E2-BSA (E2-BSA-FITC). Staining was restricted to the cell membrane when E2-BSA-FITC was incubated with stable transfectants of the murine ERα within ER-negative HeLa cells and with MC7 cells that endogenously produce ERα. This staining appeared highly specific since it was competed by pre-incubation with E2 in a dose dependent manner and with the competitor ICI-182,780. Conclusions: These results demonstrate that E2-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E2-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E2. More importantly, these findings demonstrate that in intact cells that express ER, E2-BSA binding is localized to the cell membrane, strongly suggesting a membrane bound form of the ER.

Endotoxin leads to rapid subcellular re-localization of hepatic RXRalpha: A novel mechanism for reduced hepatic gene expression in inflammation (2004-08-16T00:00:00Z)

Background: Lipopolysaccharide (LPS) treatment of animals down-regulates the expression of hepatic genes involved in a broad variety of physiological processes, collectively known as the negative hepatic acute phase response (APR). Retinoid X receptor α (RXRα), the most highly expressed RXR isoform in liver, plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. Many of the genes regulated by RXRα are repressed during the negative hepatic APR, although the underlying mechanism is not known. We hypothesized that inflammation-induced alteration of the subcellular location of RXRα was a common mechanism underlying the negative hepatic APR. Results: Nuclear RXRα protein levels were significantly reduced (~50%) within 1–2 hours after low-dose LPS treatment and remained so for at least 16 hours. RXRα was never detected in cytosolic extracts from saline-treated mice, yet was rapidly and profoundly detectable in the cytosol from 1 hour, to at least 4 hours, after LPS administration. These effects were specific, since the subcellular localization of the RXRα partner, the retinoic acid receptor (RARα), was unaffected by LPS. A potential cell-signaling modulator of RXRα activity, c-Jun-N-terminal kinase (JNK) was maximally activated at 1–2 hours, coincident with maximal levels of cytoplasmic RXRα. RNA levels of RXRα were unchanged, while expression of 6 sentinel hepatic genes regulated by RXRα were all markedly repressed after LPS treatment. This is likely due to reduced nuclear binding activities of regulatory RXRα-containing heterodimer pairs. Conclusion: The subcellular localization of native RXRα rapidly changes in response to LPS administration, correlating with induction of cell signaling pathways. This provides a novel and broad-ranging molecular mechanism for the suppression of RXRα-regulated genes in inflammation.

Estrogen receptor-dependent activation of AP-1 via non-genomic signalling (2004-06-14T00:00:00Z)

Background: Ligand-bound estrogen receptor α (ERα) and estrogen receptor β (ERβ) modulate AP-1-dependent transcription via protein-protein interactions on DNA, in a manner that depends on the type of cells and the subtype of ER. We present here evidence for an additional mechanism by which ERs modulate the transcriptional activity of AP-1. Results: We show that ERs located in the cytoplasm efficiently activate transcription at AP-1 sites in response to 17β-estradiol, while ERs present in the nucleus repress transcription under the same conditions. 17β-estradiol-induced activation of the coll-73-luc reporter correlated with cytoplasmic localization of various ERα and ERβ mutant receptors, and was inhibited in the presence of the full estrogen antagonist ICI 182,780 and the MAP-kinase inhibitor UO126. We also show that the selective estrogen receptor modulator (SERM) tamoxifen is as potent as 17β-estradiol in inducing activation of AP-1 when ERα is present in the cytoplasm. Conclusions: These results suggest that non-genomic signalling is involved in the mechanism by which ERα and ERβ influence AP-1-dependent transcription. We have previously shown that Stat3 and Stat5 are targeted by non-genomic actions of ERs, and the results presented here allow us to conclude that ERs bound to 17β-estradiol mediate the transcriptional activation of promoters regulated by AP-1 and by Stat proteins via different combinations of signal transduction pathways. Our observations thereby provide new insights into the mechanisms by which ERs act at alternate response elements, and suggest a mechanism by which tamoxifen exerts its action as a tissue-selective agonist.

The evolution of drug-activated nuclear receptors: one ancestral gene diverged into two xenosensor genes in mammals (2004-10-12T00:00:00Z)

Background: Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals. Results: To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR. Conclusion: Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species.

Reconstitution of hemisomes on budding yeast centromeric DNA (2013-06-06T22:19:45-07:00)
Protein-nucleic acid interaction, Chromatin and Epigenetics

The structure of nucleosomes that contain the cenH3 histone variant has been controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer wraps the ~80-bp Centromere DNA Element II (CDE II) sequence of each centromere into a ‘hemisome’. However, attempts to reconstitute cenH3 particles in vitro have yielded exclusively ‘octasomes’, which are observed in vivo on chromosome arms only when Cse4 (yeast cenH3) is overproduced. Here, we show that Cse4 octamers remain intact under conditions of low salt and urea that dissociate H3 octamers. However, particles consisting of two DNA duplexes wrapped around a Cse4 octamer and separated by a gap efficiently split into hemisomes. Hemisome dimensions were confirmed using a calibrated gel-shift assay and atomic force microscopy, and their identity as tightly wrapped particles was demonstrated by gelFRET. Surprisingly, Cse4 hemisomes were stable in 4 M urea. Stable Cse4 hemisomes could be reconstituted using either full-length or tailless histones and with a 78-bp CDEII segment, which is predicted to be exceptionally stiff. We propose that CDEII DNA stiffness evolved to favor Cse4 hemisome over octasome formation. The precise correspondence between Cse4 hemisomes resident on CDEII in vivo and reconstituted on CDEII in vitro without any other factors implies that CDEII is sufficient for hemisome assembly.

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response (2013-06-06T22:19:45-07:00)

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the H2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G₂/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.

Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast (2013-06-06T22:19:45-07:00)

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small ^– mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-^– cells and increased deletion mutagenesis at the ori5 region in ⁺ cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

5-Aza-2'-deoxycytidine causes replication lesions that require Fanconi anemia-dependent homologous recombination for repair (2013-06-06T22:19:45-07:00)

5-Aza-2'-deoxycytidine (5-azadC) is a DNA methyltransferase (DNMT) inhibitor increasingly used in treatments of hematological diseases and works by being incorporated into DNA and trapping DNMT. It is unclear what DNA lesions are caused by 5-azadC and if such are substrates for DNA repair. Here, we identify that 5-azadC induces DNA damage as measured by -H2AX and 53BP1 foci. Furthermore, 5-azadC induces radial chromosomes and chromatid breaks that depend on active replication, which altogether suggest that trapped DNMT collapses oncoming replication forks into double-strand breaks. We demonstrate that RAD51-mediated homologous recombination (HR) is activated to repair 5-azadC collapsed replication forks. Fanconi anemia (FA) is a rare autosomal recessive disorder, and deaths are often associated with leukemia. Here, we show that FANCG-deficient cells fail to trigger HR-mediated repair of 5-azadC-induced lesions, leading to accumulation of chromatid breaks and inter-chromosomal radial fusions as well as hypersensitivity to the cytotoxic effects of 5-azadC. These data demonstrate that the FA pathway is important to protect from 5-azadC-induced toxicity. Altogether, our data demonstrate that cytotoxicity of the epigenetic drug 5-azadC can, at least in part, be explained by collapsed replication forks requiring FA-mediated HR for repair.

De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment (2013-06-06T22:19:45-07:00)

PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences—not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.

Simple extraction methods that prevent the artifactual conversion of chlorophyll to chlorophyllide during pigment isolation from leaf samples (2013-06-19T00:00:00Z)

Background: When conducting plant research, the measurement of photosynthetic pigments can provide basic information on the physiological status of a plant. High-pressure liquid chromatography (HPLC) is becoming widely used for this purpose because it provides an accurate determination of a variety of photosynthetic pigments simultaneously. This technique has a drawback compared with conventional spectroscopic techniques, however, in that it is more prone to structural modification of pigments during extraction, thus potentially generating erroneous results. During pigment extraction procedures with acetone or alcohol, the phytol side chain of chlorophyll is sometimes removed, forming chlorophyllide, which affects chlorophyll measurement using HPLC. Results: We evaluated the artifactual chlorophyllide production during chlorophyll extraction by comparing different extraction methods with wild-type and mutant Arabidopsis leaves that lack the major isoform of chlorophyllase. Several extraction methods were compared to provide alternatives to researchers who utilize HPLC for the analysis of chlorophyll levels. As a result, the following three methods are recommended. In the first method, leaves are briefly boiled prior to extraction. In the second method, grinding and homogenization of leaves are performed at sub-zero temperatures. In the third method, N, N'-dimethylformamide (DMF) is used for the extraction of pigments. When compared, the first two methods eliminated almost all chlorophyllide-forming activity in Arabidopsis thaliana, Glebionis coronaria, Pisum sativum L. and Prunus sargentii Rehd. However, DMF effectively suppressed the activity of chlorophyllase only in Arabidopsis leaves. Conclusion: Chlorophyllide production in leaf extracts is predominantly an artifact. All three methods evaluated in this study reduce the artifactual production of chlorophyllide and are thus suitable for pigment extraction for HPLC analysis. The boiling method would be a practical choice when leaves are not too thick. However, it may convert a small fraction of chlorophyll a into pheophytin a. Although extraction at sub-zero temperatures is suitable for all plant species examined in this study, this method might be complicated for a large number of samples and it requires liquid nitrogen and equipment for leaf grinding. Using DMF as an extractant is simple and suitable with Arabidopsis samples. However, this solvent cannot completely block the formation of chlorophyllide in thicker leaves.

Improved techniques for measurement of nanolitre volumes of phloem exudate from aphid stylectomy (2013-06-17T00:00:00Z)

Background: When conducting aphid stylectomy, measuring accurate rates of phloem exudation is difficult because the volumes collected are in the nanolitre (nl) range. In a new method, exudate volume was calculated from optical measurement of droplet diameter as it forms on the tip of a severed aphid stylet. Evaporation was shown to decrease the accuracy of the measurement but was countered with the addition of water-saturated mineral oil. Volume measurements by optical estimation of the volume of a sphere suspended in oil was affected by the curvature of the oil surface. In contrast, measuring the exudate volume from optical measurement of droplet-diameter as formed on the tip of a severed aphid stylet, removes any inaccuracies due to oil surface curvature. A modified technique is proposed for measuring exudate volumes without oil by estimating the flow rate from photo-sequences of the collection period; a correction for evaporation is applied later. Results: A change in oil volume of +/-1.75% from an optimum volume of 285 mul had a statistically significant effect on droplet measurement, under or over-estimating droplet volume due to optical effects caused by the oil surface. Using microscope image capture and measurement software, a modified method for measuring phloem volume in air was developed, by reducing air exposure during measurement to approximately 5 s for each measurement. Phloem volumes were measured using both techniques with measurements in air being on average 19.9 nl less (SD 18.87, p<0.001) than those made in oil, and there was a strong linear relationship (R2=0.942) between the techniques. This linear relationship enabled the development of a correction equation with no significant difference at the 5% level between corrected volumes and actual volumes measured under oil. Conclusions: This study showed that oil has a significant role in countering evaporation but oil volume must be carefully optimised for optical measurement of droplets to ensure measurement accuracy. A linear correction factor was generated to correct the volumes measured in air for loss due to evaporation and the method provides for a much simpler alternative to previous approaches for measuring exudation rates and volumes from a cut aphid stylet.

High throughput quantitative phenotyping of plant resistance using chlorophyll fluorescence image analysis (2013-06-13T00:00:00Z)

Background: In order to select for quantitative plant resistance to pathogens, high throughput approaches that can precisely quantify disease severity are needed. Automation and use of calibrated image analysis should provide more accurate, objective and faster analyses than visual assessments. In contrast to conventional visible imaging, chlorophyll fluorescence imaging is not sensitive to environmental light variations and provides single-channel images prone to a segmentation analysis by simple thresholding approaches. Among the various parameters used in chlorophyll fluorescence imaging, the maximum quantum yield of photosystem II photochemistry (Fv/Fm) is well adapted to phenotyping disease severity. Fv/Fm is an indicator of plant stress that displays a robust contrast between infected and healthy tissues. In the present paper, we aimed at the segmentation of Fv/Fm images to quantify disease severity. Results: Based on the Fv/Fm values of each pixel of the image, a thresholding approach was developed to delimit diseased areas. A first step consisted in setting up thresholds to reproduce visual observations by trained raters of symptoms caused by Xanthomonas fuscans subsp. fuscans (Xff) CFBP4834-R on Phaseolus vulgaris cv. Flavert. In order to develop a thresholding approach valuable on any cultivars or species, a second step was based on modeling pixel-wise Fv/Fm-distributions as mixtures of Gaussian distributions. Such a modeling may discriminate various stages of the symptom development but over-weights artifacts that can occur on mock-inoculated samples. Therefore, we developed a thresholding approach based on the probability of misclassification of a healthy pixel. Then, a clustering step is performed on the diseased areas to discriminate between various stages of alteration of plant tissues. Notably, the use of chlorophyll fluorescence imaging could detect pre-symptomatic area. The interest of this image analysis procedure for assessing the levels of quantitative resistance is illustrated with the quantitation of disease severity on five commercial varieties of bean inoculated with Xff CFBP4834-R. Conclusions: In this paper, we describe an image analysis procedure for quantifying the leaf area impacted by the pathogen. In a perspective of high throughput phenotyping, the procedure was automated with the software R downloadable at http://www.r-project.org/. The R script is available at http://lisa.univ-angers.fr/PHENOTIC/telechargements.html.

High-throughput sequencing of cytosine methylation in plant DNA (2013-06-07T00:00:00Z)

Cytosine methylation is a significant and widespread regulatory factor in plant systems. Methods for the high-throughput sequencing of methylation have allowed a greatly improved characterisation of the methylome. Here we discuss currently available methods for generation and analysis of high-throughput sequencing of methylation data. We also discuss the results previously acquired through sequencing plant methylomes, and highlight remaining challenges in this field.

Simultaneous molecular formula determinations of natural compounds in a plant extract using 15 T FT-ICR MS (2013-05-30T00:00:00Z)

Background: Plant extracts are a reservoir of pharmacologically active substances; however, conventional analytical methods can analyze only a small portion of an extract. Here, we report a high-throughput analytical method capable of determining most phytochemicals in a plant extract and of providing their molecular formulae from a single experiment using ultra-high-resolution electrospray ionization mass spectrometry (UHR ESI MS). UHR mass profiling was used to analyze natural compounds in a 70% ethanol ginseng extract, which was directly infused into a 15 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer for less than 10 min without a separation process. Results: The UHR FT-ICR MS yielded a mass accuracy of 0.5 ppm and a mass resolving power (m/Deltam) of 1,000,000--270,000 for the range m/z 290--1,100. The mass resolution was sufficient to resolve the isotopic fine structure (IFS) of many compounds in the extract. After noise removal from 1,552 peaks, 405 compounds were detected. The molecular formulae of 123 compounds, including 33 ginsenosides, were determined using the observed IFS, exact monoisotopic mass, and exact mass difference. Liquid chromatography (LC)/FT-ICR MS of the extract was performed to compare the high-throughput performance of UHR ESI FT-ICR MS. The LC/FT-ICR MS detected only 129 compounds, including 19 ginsenosides. The result showed that UHR ESI FT-ICR MS identified three times more compounds than LC/FT-ICR MS and in a relatively shorter time. The molecular formula determination by UHR FT-ICR MS was validated by LC and tandem MS analyses of three known ginsenosides. Conclusions: UHR mass profiling of a plant extract by 15 T FT-ICR MS showed that multiple compounds were simultaneously detected and their molecular formulae were decisively determined by a single experiment with ultra-high mass resolution and mass accuracy. Simultaneous molecular determination of multiple natural products by UHR ESI FT-ICR MS would be a powerful method to profile a wide range of natural compounds.

Performance of endoscopic ultrasound-guided fine needle aspiration in diagnosing pancreatic neuroendocrine tumors (2013-05-31 22:13:05+01:00)

Jane Bernstein, Berrin Ustun, Ahmed Alomari, Fang Bao, Harry R Aslanian, Uzma Siddiqui, David Chhieng, Guoping Cai

CytoJournal 2013 10(1):10-10

Background: Pancreatic neuroendocrine tumors (PNETs) are rare tumors of the pancreas, which are increasingly diagnosed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). In this retrospective study, we assessed the performance of EUS-FNA in diagnosing PNETs. Materials and Methods: We identified 48 cases of surgically resected PNETs in which pre-operative EUS-FNA was performed. The clinical features, cytological diagnoses, and surgical follow-up were retrospectively reviewed. The diagnostic performance of EUS-FNA was analyzed as compared to the diagnosis in the follow-up. The cases with discrepancies between cytological diagnosis and surgical follow-up were analyzed and diagnostic pitfalls in discrepant cases were discussed. Results: The patients were 20 male and 28 female with ages ranging from 15 years to 81 years (mean 57 years). The tumors were solid and cystic in 41 and 7 cases, respectively, with sizes ranging from 0.5 cm to 11 cm (mean 2.7 cm). Based on cytomorphologic features and adjunct immunocytochemistry results, when performed, 38 patients (79%) were diagnosed with PNET, while a diagnosis of "suspicious for PNET" or a diagnosis of "neoplasm with differential diagnosis including PNET" was rendered in the 3 patients (6%). One case was diagnosed as mucinous cystic neoplasm (2%). The remaining 6 patients (13%) had non-diagnostic, negative or atypical diagnosis. Conclusions: Our data demonstrated that EUS-FNA has a relatively high sensitivity for diagnosing PNETs. Lack of additional materials for immunocytochemical studies could lead to a less definite diagnosis. Non-diagnostic or false negative FNA diagnosis can be seen in a limited number of cases, especially in those small sized tumors.

Cytodiagnosis of gouty tophus (2013-05-31 22:13:05+01:00)

Vaishali Walke, Sushma Ramraje, Vinod Jadhao

CytoJournal 2013 10(1):11-11

Metastatic colorectal adenocarcinoma in cervicovaginal cytology specimens confirmed by immunocytochemical stains on liquid base specimens: Two study cases with review of the literature (2013-05-25 00:32:35+01:00)

Muhammad Zulfiqar, Susan Liu, Dongping Shi, Shashi Madan, Suzanne Jacques, Laquita King, Vinod Shidham, Tamar Giorgadze

CytoJournal 2013 10(1):9-9

Only a few cases of adenocarcinoma (ACA) metastatic to the female lower genital tract diagnosed on cervicovaginal Pap smear have been reported during the past several decades. Both conventional and liquid based cytology (LBC) have limited sensitivity and specificity in diagnosing metastatic disease and immunocytochemical (ICC) staining may be needed for confirming the diagnosis. We present two cases of metastatic colorectal ACA diagnosed on cervicovaginal ThinPrep (TP) Pap smears, with one confirmed by ICC staining method. Recognition of extra-uterine malignancy in the cervicovaginal cytology specimen is critical for the disease diagnosis, prognosis, and the treatment. ICC staining performed on the residual LBC specimen is an important methodology to confirm the diagnosis.

Cytomorphology of Boerhaave's syndrome: A critical value in cytology (2013-05-14 12:26:14+01:00)

Walid E Khalbuss, Shveta Hooda, Manon Auger

CytoJournal 2013 10(1):8-8

Spontaneous esophageal perforation into the pleural cavity (Boerhaave's syndrome) is a rare life-threatening condition, which requires early diagnosis and urgent management. The diagnosis of such critical condition in many cases is delayed because of atypical clinical presentation, resulting in increased morbidity and mortality. Cytological examination of pleural fluid can provide early, fast and accurate diagnosis of such critical condition and help in better and early management of this disease. We describe a case of an 81-year-old female with esophageal perforation who presented with a left sided pleural effusion. The correct diagnosis was established in this case by observing gastrointestinal-like fluid characteristics of the thoracic drainage upon cytological and chemical analyses and the rupture was confirmed by esophagography. The cytological examination of pleural fluid revealed benign reactive squamous cells, fungal organisms, bacterial colonies, and vegetable material consistent with a ruptured esophagus. Cytological examination of pleural fluid is a rapid and accurate technique that can help in establishing the diagnosis of this challenging entity and guide initiation proper management of this unusual entity.

Abdominopelvic washings: A comprehensive review (2013-04-25 00:31:15+01:00)

Erika F Rodriguez, Sara E Monaco, Walid Khalbuss, R Marshall Austin, Liron Pantanowtiz

CytoJournal 2013 10(1):7-7

Intraperitoneal spread may occur with gynecological epithelial neoplasms, as well as with non-gynecological malignancies, which may result in serosal involvement with or without concomitant effusion. Therefore, washings in patients with abdominopelvic tumors represent important specimens for cytologic examination. They are primarily utilized for staging ovarian cancers, although their role has decreased in staging of endometrial and cervical carcinoma. Abdominopelvic washings can be positive in a variety of pathologic conditions, including benign conditions, borderline neoplastic tumors, locally invasive tumors, or distant metastases. In a subset of cases, washings can be diagnostically challenging due to the presence of co-existing benign cells (e.g., mesothelial hyperplasia, endosalpingiosis, or endometriosis), lesions in which there is only minimal atypia (e.g., serous borderline tumors) or scant atypical cells, and the rarity of specific tumor types (e.g., mesothelioma). Ancillary studies including immunocytochemistry and fluorescence in situ hybridization may be required in difficult cases to resolve the diagnosis. This article provides a comprehensive and contemporary review of abdominopelvic washings in the evaluation of gynecologic and non-gynecologic tumors, including primary peritoneal and mesothelial entities.

Mutations in Hedgehog pathway genes in fetal rhabdomyomas (2013-06-18T10:05:59.837266-05:00)

Abstract

Distinct evolutionary trajectories of primary high-grade serous ovarian cancers revealed through spatial mutational profiling (2013-06-18T10:05:38.943033-05:00)

Abstract

The Paracrine Effect of Cancer-associated Fibroblast-induced Interleukin-33 Regulates the Invasiveness of Head and Neck Squamous Cell Carcinoma (2013-06-14T06:39:50.918025-05:00)

Abstract

Familial Rhabdoid Tumour "Avant La Lettre" - from pathology review to exome sequencing and back again (2013-06-14T06:46:18.146341-05:00)

Abstract

B-cell lymphoma and myeloma in murine Gaucher disease (2013-06-14T06:53:25.704446-05:00)

Abstract

Clinicopathological features and CCT2 and PDIA2 expression in gallbladder squamous/adenosquamous carcinoma and gallbladder adenocarcinoma (2013-06-19T00:00:00Z)

Background: Gallbladder cancer (GBC) is a relatively uncommon carcinoma among gastrointestinal cancers and usually has a rather poor prognosis. The most common subtype of GBC is adenocarcinoma (AC), which accounts for about 90% of GBC. Squamous carcinoma/adenosquamous carcinoma (SC/ASC) are comparatively rare histopathological subtypes of GBC. The clinicopathological features and biological behaviors of SC/ASC have not been well-characterized. No molecular biomarkers are currently available for predicting the progression, metastasis, and prognosis of the SC/ASC subtype of GBC. Methods: We examined the expression levels of CCT2 and PDIA3 by immunohistochemistry (IHC) staining in human GBC tissue samples collected from 46 patients with SC/ASC and evaluated the clinicopathological significance of both CCT2 and PDIA3 expression in the SC/ASC subtypes of GBC by Kaplan-Meier analysis and multivariate Cox regression analysis. For comparison, we included specimens from 80 AC patients in our study to investigate the specificity of CCT2 and PDIA3 expression in GBC subtypes. Results: We found that the positive expression of CCT2 and PDIA3 was significantly associated with clinicopathological features of both SC/ASC and AC specimens, including high TNM stage and lymph node metastasis. Univariate analysis revealed that the two-year survival rate was significantly lower for patients with positive expression of CCT2 and PDIA3 than for those with negative expression. Multivariate analysis also indicated that the positive expression of CCT2 and PDIA3 was negatively correlated with poor postoperative patient survival and positively correlated with high mortality. Conclusions: Our study suggests that positive expression of CCT2 or PDIA3 is associated with tumor progression and the clinical behavior of gallbladder carcinoma. Therefore, CCT2 and PDIA3 could be potentially important diagnostic and prognostic biomarkers for both SC/ASC and AC subtypes of GBC.

Overexpression of HSPA2 is correlated with poor prognosis in esophageal squamous cell carcinoma (2013-06-18T00:00:00Z)

Background: Heat shock-related 70 kDa protein 2 (HSPA2) has been identified as a potential cancer-promoting protein expressed at abnormal levels in a subset of cancers. However, its important role in esophageal squamous cell carcinoma (ESCC) is hardly known by people. The purpose of this study is to assess HSPA2 expression and to explore its role in ESCC. Methods: Thirty ESCC samples, paired adjacent non-cancerous tissues and normal esophageal tissues, were collected for HSPA2 detection by quantitative RT-PCR (qRT-PCR) and western blotting. Additionally, the expression of HSPA2 in ESCC and adjacent non-cancerous tissues from 120 patients was analyzed by immunohistochemistry, and correlated with clinicopathological parameters and patients' outcome. Results: HSPA2 mRNA and protein were overexpressed in ESCC tissues. Overexpression of HSPA2 was significantly associated with primary tumor, TNM stage, lymph node metastases and recurrence, respectively (all, P <0.05). Kaplan-Meier curves showed that elevated HSPA2 expression was associated with shorter disease-free survival and overall survival in ESCC patients. Cox multivariate regression analysis revealed that overexpression of HSPA2 was an independent prognostic factor in disease-free survival and overall survival for ESCC patients (hazard ratio was 2.115 and 2.210, respectively, P <0.05). Conclusions: Our findings demonstrate that overexpression of HSPA2 may contribute to the malignant progression of ESCC and present a novel prognostic indicator for ESCC patients.

Does geography influence the treatment and outcomes of colorectal cancer? A population-based analysis (2013-06-17T00:00:00Z)

Background: The Canadian province of Manitoba covers a large geographical area but only has one major urban center, Winnipeg. We sought to determine if regional differences existed in the quality of colorectal cancer care in a publicly funded health care system. Methods: This was a population-based historical cohort analysis of the treatment and outcomes of Manitobans diagnosed with colorectal cancer between 2004 and 2006. Administrative databases were utilized to assess quality of care using published quality indicators. Results: A total of 2,086 patients were diagnosed with stage I to IV colorectal cancer and 42.2% lived outside of Winnipeg. Patients from North Manitoba had a lower odds of undergoing major surgery after controlling for other confounders (odds ratio (OR): 0.48, 95% confidence interval (CI): 0.26 to 0.90). No geographic differences existed in the quality measures of 30-day operative mortality, consultations with oncologists, surveillance colonoscopy, and 5-year survival. However, there was a trend towards lower survival in North Manitoba. Conclusion: We found minimal differences by geography. However, overall compliance with quality measures is low and there are concerning trends in North Manitoba. This study is one of the few to evaluate population-based benchmarks for colorectal cancer therapy in Canada.

Ovarian granulosa cell tumors: a retrospective study of 27 cases and a review of the literature (2013-06-18T00:00:00Z)

Background: Granulosa tumors were described for the first time in 1855 by Rokitansky. These tumors are malignancies with a relatively favorable prognosis. They are characterized by a prolonged natural history and a tendency to late recurrences. The aim of this study is to investigate the epidemiological and pathological characteristics of granulosa cell tumors and to investigate the prognosis factor for recurrences. Methods: The clinical data of patients who were treated in the period from January 2003 to December 2010 at the National Institute of Oncology in Rabat, Morocco for adult granulosa cell tumors of the ovary were investigated retrospectively. Data for age, clinical manifestation, imaging, diagnosis and treatment of the patients were reviewed and analyzed. Post-operative histology was obtained for all patients. Results: Twenty-seven cases were retrieved. The median patient age was 53 years. The most common clinical manifestations at diagnosis were abdominal pain and vaginal bleeding. Mean tumor size was 14 cm.The majority of patients had stage I (63%, n = 17), while (18,5%, n = 5) had stage III, (7.4%, n = 2) had stage IV, and (11%, n = 3) of patients had an unknown stage.In the follow-up period (median = 63.44 months), five (18.51%) patients relapsed. The median time to relapse was 41.8 months, (range: 18 to 62 months). Conclusions: Granulosa cell tumor of the ovary is an uncommon neoplasm. The adult form progresses slowly and often is diagnosed in an early stage of disease. Surgery is indicated. A prolonged post-therapeutic follow-up is necessary because of the risk of recurrences, late and exceptional for the adult form.

Sequential preoperative hepatic vein embolization after portal vein embolization for extended left hepatectomy in colorectal liver metastases (2013-06-11T00:00:00Z)

Background: The role of portal vein embolization to increase future liver remnant (FLR) is well-established in the treatment of colorectal liver metastases. However, the role of hepatic vein embolization is unclear.Case report: A patient with colorectal liver metastases received neoadjuvant chemotherapy prior to attempted resection. At the time of resection his tumor appeared to invade the left and middle hepatic vein, requiring an extended left hepatectomy including segments five and eight. Post-operatively, he underwent sequential left portal vein embolization followed by left hepatic vein embolization and finally, middle hepatic vein embolization. Hepatic vein embolization was performed to increase the FLR as well as to allow collateral drainage of the FLR to develop. A left trisectionectomy was then performed and no evidence of postoperative liver congestion or morbidity was found. Conclusion: Sequential portal vein embolization and hepatic vein embolization for extended left hepatectomy may be considered to increase FLR and may prevent right hepatic congestion after sacrificing the middle vein.