ALBERT

Description: Background: The process of creating and designing Virtual Patients for teaching students of medicine is an expensive and time-consuming task. In order to explore potential methods of mitigating these costs, our group began exploring the possibility of creating Virtual Patients based on electronic health records. This review assesses the usage of electronic health records in the creation of interactive Virtual Patients for teaching clinical decision-making. Methods: The PubMed database was accessed programmatically to find papers relating to Virtual Patients. The returned citations were classified and the relevant full text articles were reviewed to find Virtual Patient systems that used electronic health records to create learning modalities. Results: A total of n = 362 citations were found on PubMed and subsequently classified, of which n = 28 full-text articles were reviewed. Few articles used unformatted electronic health records other than patient CT or MRI scans. The use of patient data, extracted from electronic health records or otherwise, is widespread. The use of unformatted electronic health records in their raw form is less frequent. Patient data use is broad and spans several areas, such as teaching, training, 3D visualisation, and assessment. Conclusions: Virtual Patients that are based on real patient data are widespread, yet the use of unformatted electronic health records, abundant in hospital information systems, is reported less often. The majority of teaching systems use reformatted patient data gathered from electronic health records, and do not use these electronic health records directly. Furthermore, many systems were found that used patient data in the form of CT or MRI scans. Much potential research exists regarding the use of unformatted electronic health records for the creation of Virtual Patients.

Description: Background: The molecular epidemiology of C. jejuni and C. coli clinical strains isolated from children with gastroenteritis, was investigated using the multilocus sequence typing method (MLST). This analysis establishes for the first time in Greece and constitutes an important tool for the epidemiological surveillance and control of Campylobacter infection in our country. Methods: The MLST genotypes were compared with those gained by other typing methods (HS-typing, PFGE and FlaA typing) and were also phylogenetically analyzed, in order to uncover genetic relationships. Results: Among 68 C. jejuni strains, 41 different MLST-Sequence Types (MLST-STs) were found. Fifty six strains or 34 MLST-STs could be sorted into 15 different MLST-Sequence Type Complexes (MLST-STCs), while twelve strains or seven MLST-STs did not match any of the MLST-STCs of the database. Twenty C. coli strains belonged to 14 different MLST-STs. Eleven MLST-STs were classified in the same MLST-STC (828), and three were unclassifiable. There was no significant association between the MLST-STs and the results of the other typing methods.Phylogenetic analysis revealed that some strains, classified to the species of C. jejuni, formed a separate, phylogenetically distinct group. In eight strains some alleles belonging to the taxonomic cluster of C. jejuni, were also detected in C. coli and vice versa, a phenomenon caused by the genetic mosaic encountered inside the genus Campylobacter. Conclusions: The MLST-ST determination proved to be a very useful tool for the typing as well as the identification of Campylobacter on the species level.

Description: Background: Human resources are an important building block of the health system. During the last decade, enormous investment has gone into the information systems to manage human resources, but due to the lack of a clear vision, policy, and strategy, the results of these efforts have not been very visible. No reliable information portal captures the actual state of human resources in Pakistan's health sector. The World Health Organization (WHO) has provided technical support for the assessment of the existing system and development of a comprehensive Human Resource Information System (HRIS) in Pakistan. Methods: The questions in the WHO-HRIS Assessment tool were distributed into five thematic groups. Purposively selected (n=65) representatives from the government, private sector, and development partners participated in this cross sectional study, based on their programmatic affiliations. Results: Fifty-five percent of organizations and departments have an independent Human Resources (HR) section managed by an establishment branch and are fully equipped with functional computers. Forty-five organizations (70%) had HR rules, regulations and coordination mechanisms, yet these are not implemented. Data reporting is mainly in paper form, on prescribed forms (51%), registers (3%) or even plain papers (20%). Data analysis does not give inputs to the decision making process and dissemination of information is quite erratic. Most of the organizations had no feedback mechanism for cross checking the HR data, rendering it unreliable. Conclusion: Pakistan is lacking appropriate HRIS management. The current HRIS indeed has a multitude of problems. In the wake of 2011 reforms within the health sector, provinces are even in a greater need for planning their respective health department services and must work on the deficiencies and inefficiencies of their HRIS so that the gaps and HR needs are better aligned for reaching the 2015 UN Millennium Development Goals (MDGs) targets.

Description: Background: Decision support systems for differential diagnosis have traditionally been evaluated on the basis of criteria how sensitively and specifically they are able to identify the correct diagnosis established by expert clinicians.DiscussionThis article questions whether evaluation criteria pertaining to identifying the correct diagnosis are most appropriate or useful. Instead it advocates evaluation of decision support systems for differential diagnosis based on the criterion of maximizing value of information.SummaryThis approach quantitatively and systematically integrates several important clinical management priorities, including avoiding serious diagnostic errors of omission and avoiding harmful or expensive tests.

Description: Background: Mutations in the PTRF gene, coding for cavin-1, cause congenital generalized lipodystrophy type 4 (CGL4) associated with myopathy. In CGL4, symptoms are variable comprising, in addition to myopathy, smooth and skeletal muscle hypertrophy, cardiac arrhythmias, and skeletal abnormalities. Secondary features are atlantoaxial instability, acanthosis nigricans, hepatomegaly, umbilical prominence and metabolic abnormalities related to insulin resistance, such as diabetes mellitus, hyperlipidemia and hepatic steatosis.Case presentationWe describe a 3 year-old child of Moroccan origin with mild muscle phenotype, mainly characterized by mounding, muscle pain, hyperCKemia and mild caveolin 3 reduction on muscle biopsy. No CAV3 gene mutation was detected; instead we found a novel mutation, a homozygous single base pair deletion, in the PTRF gene. Only after detection of this mutation a mild generalized loss of subcutaneous fat, at first underestimated, was noticed and the diagnosis of lipodystrophy inferred. Conclusions: The PTRF gene should be investigated in patients with hyperCKemia, mild myopathy associated with spontaneous or percussion-induced muscle contractions like rippling or mounding, and no CAV3 mutation. The analysis should be performed even if cardiac or metabolic alterations are absent, particularly in young patients in whom lipodystrophy may be difficult to ascertain.

Description: Background: Little is known about the Phasmatodea gut microbial community, including whether phasmids have symbiotic bacteria aiding in their digestion. While symbionts are near ubiquitous in herbivorous insects, the Phasmatodea's distinctively thin body shape precludes the gut enlargements needed for microbial fermentation. High-throughput sequencing was used to characterize the entire microbiota of the fat bodies, salivary glands, and anterior and posterior midguts of two species of walking stick. Results: Most bacterial sequences belonged to a strain of Spiroplasma (Tenericutes) found primarily in the posterior midgut of the parthenogenetic species Ramulus artemis (Phasmatidae). Beyond this, no significant differences were found between the R. artemis midgut sections or between that species and Peruphasma schultei (Pseudophasmatidae). Histological analysis further indicated a lack of bacteriocytes. Conclusions: Phasmids are unlikely to depend on bacteria for digestion, suggesting they produce enzymes endogenously that most other herbivorous insects obtain from symbionts. This conclusion matches predictions based on phasmid anatomy. The role of Spiroplasma in insects warrants further study.

Description: Background: Angiogenesis is the main therapeutic mechanism of cell therapy for cardiovascular diseases, but diabetes is reported to reduce the function and number of progenitor cells. Therefore, we studied the effect of streptozotocin-induced diabetes on the bone marrow-mesenchymal stem cell (MSC) function, and examined whether diabetes-impaired MSC could be rescued by pretreatment with oxytocin. Results: MSCs were isolated and cultured from diabetic (DM) or non-diabetic (non-DM) rat, and proliferation rate was compared. DM-MSC was pretreated with oxytocin and compared with non-DM-MSC. Angiogenic capacity was estimated by tube formation and Matrigel plug assay, and therapeutic efficacy was studied in rat myocardial infarction (MI) model.The proliferation and angiogenic activity of DM-MSC were severely impaired but significantly improved by pretreatment with oxytocin. Kruppel-like factor 2 (KLF2), a critical angiogenic factor, was dramatically reduced in DM-MSC and significantly restored by oxytocin. In the Matrigel plug assay, vessel formation of DM-BMSCs was attenuated but was recovered by oxytocin. In rat MI model, DM-MSC injection did not ameliorate cardiac injury, whereas oxytocin-pretreated DM-MSC improved cardiac function and reduced fibrosis. Conclusions: Our results show that diabetes influenced MSC by reducing angiogenic capacity and therapeutic potential. We demonstrate the striking effect of oxytocin on stem cell dysfunction and suggest the use of oxytocin as a priming reagent in autologous stem cell therapy.

Description: Background: The choice of variable selection methods to identify important variables for binary classification modeling is critical to produce stable models that are interpretable, that generate accurate predictions and have minimum bias. This work is motivated by data on clinical and laboratory features of severe dengue infections (dengue hemorrhagic fever, DHF) obtained from 51 individuals enrolled in a prospective observational study of acute human dengue infections. Results: We carry out a comprehensive performance comparison using several classification models for DHF over the dengue data set. We compared variable selection results by Multivariate Adaptive Regression Splines, Learning Ensemble, Random Forest, Bayesian Moving Averaging, Stochastic Search Variable Selection, and Generalized Regularized Logistics Regression. Model averaging methods (bagging, boosting and ensemble learners) have higher accuracy, but the generalized regularized regression model has the highest predictive power because the linearity assumptions of candidate predictors are strongly satisfied via deviance chi-square testing procedures. Bootstrapping applications for evaluating predictive regression coefficients in regularized regression model are performed. Conclusions: Feature reduction methods introduce inherent biases and therefore are data-type dependent. We propose that these limitations can be overcome using an exhaustive approach for searching feature space. Using this approach, we results suggest that IL-10, platelet and lymphocyte counts are the major features for predicting dengue DHF on the basis of blood chemistries and cytokine measurements.

Description: Background: Sjogren's syndrome is characterized by lymphocytic infiltration of the exocrine glands, together with polyclonal B-cell activation, and lung diseases are well-known complications of the disease. Therefore, in most cases associated with Sjogren's syndrome, infiltrating lymphocytes in the lung specimen exhibit the features of B-cells. We herein report an atypical case of lymphoproliferative pulmonary involvement in a patient with Sjogren's syndrome.Case presentationA 46-year-old female was admitted to our hospital because of an abnormal chest roentgenogram finding on a medical checkup. Chest computed tomography showed randomly-distributed micronodules and patchy ground-glass opacities. A surgical biopsied specimen showed an atypical pattern of interstitial pneumonia with numerous lymphoid follicles. Among the infiltrating lymphocytes in the lung, only the monoclonality of the T-cells was proven by a gene rearrangement analysis, but there was no cytological atypicality or genetic disorder revealed by testing the bone marrow aspirate. A diagnosis of Sjogren's syndrome was made based on the patient's other symptoms and these negative findings. The patient's pulmonary lesions have been successfully treated and remission has been maintained for over three years with corticosteroid treatment alone. Conclusion: The present patient was an atypical case of lymphoproliferative pulmonary involvement in a patient with Sjogren's syndrome. Although monoclonality of the infiltrating T-cells was proven, the clinical course and the findings of the imaging and laboratory examinations were inconsistent with the previously-reported cases of primary pulmonary T-cell lymphoma. This suggests that the monoclonality of lymphocytes does not always define malignancy. The diagnosis of malignant lymphoma or lymphoproliferative diseases should be made clinically, pathologically and cytogenetically to rule out other similar diseases.

Description: Bone remodeling is a natural process that enables growth and maintenance of the skeleton. It involves the deposition of mineralized matrix by osteoblasts and resorption by osteoclasts. Several cancers that metastasize to bone negatively perturb the remodeling process through a series of interactions with osteoclasts, and osteoblasts. These interactions have been described as the “vicious cycle” of cancer metastasis in bone. Due to the inaccessibility of the skeletal tissue it is difficult to study this system in vivo . In contrast, standard tissue culture lacks sufficient complexity. We have developed a specialized three-dimensional culture system that permits growth of a non-vascularized, multiple-cell-layer of mineralized osteoblastic tissue from pre-osteoblasts. In this study, the essential properties of bone remodeling were created in vitro by co-culturing the mineralized collagenous osteoblastic tissue with actively resorbing osteoclasts followed by reinfusion with proliferating pre-osteoblasts. Cell-cell and cell-matrix interactions were determined by confocal microscopy as well as by assays for cell specific cytokines and growth factors. Osteoclasts, differentiated in the presence of osteoblasts, led to degradation of the collagen-rich extracellular matrix. Further addition of metastatic breast cancer cells to the co-culture mimicked the vicious cycle; i.e. there was a further reduction in osteoblastic tissue thickness, an increase in osteoclastogenesis, chemotaxis of cancer cells to osteoclasts and formation of cancer cells into large colonies. The resulting model system permits detailed study of fundamental osteobiological and osteopathological processes in a manner that will enhance development of therapeutic interventions to skeletal diseases. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Aberrant glycosylation by N -acetylgalactosaminyl transferases (GALNTs) is a well-described pathological alteration that is widespread in hereditary diseases, prominently including human cancers, familial tumoral calcinosis and hyperostosis-hyperphosphatemia. In this study, we integrated different computational tools to perform the in silico analysis of clinically significant mutations (nsSNPs/ single amino acid change) at both functional and structural levels, found in human GALNT3, GALNT8, GALNT12 and GALNT13 genes. From function and structure based insights, mutations encoding R162Q, T359K, C574G, G359D, R297W, Y396C & D313N substitutions were concordantly predicted highly deleterious for relevant GALNTs proteins. From intriguing findings, T359K- GALNT3 was simulated with high contribution for disease susceptibility (tumor calcinosis) as compared to its partner variant T272K [Ichikawa et al., 2006]. Similarly, the prediction of high damaging behavior, evolutionary conservation and structural destabilization for C574G were proposed as major contributing factors to regulate metabolic disorder underlying tumor calcinosis and hyperostosis-hyperphosphatemia syndrome. In case of R297W- GALNT12 , prediction of highly deleterious effect and disruption in ionic interactions were anticipated with reduction in enzymatic activity, associated with bilateral breast cancer and primary colorectal cancers. The second GALNT12 mutation (D303N)-known splice variant- was predicted with disease severity as a result of decrease in charge density and buried behavior neighboring the catalytic B domain. In the lack of adequate in silico data about systematic characterization of clinically significant mutations in GALNTs genes, current study can be used as a significant tool to interpret the role of GALNTs reaction chemistry in disease-association risks in body. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Objective To investigate whether crosstalk between RUNX2 and miRNAs is involved in tooth eruption regulated by dental follicle cells(DFCs) and the possible molecular mechanism. Methods Blood samples and embedded dental follicles were collected from patients with cleidocranial dysplasia (CCD), and RUNX2 gene mutations were analyzed, then RUNX2 +/m DFCs were isolated and identified. The characteristics of RUNX2 +/m DFCs were analyzed. The differential expression of miRNAs was detected between the RUNX2 +/m DFCs and RUNX2 +/+ DFCs by microarray, and target genes were predicted by miRGen. miR-146a was chosen for further investigation, and its effects in DFCs were analyzed by transfecting its mimics and inhibitors, and expression of genes involved in tooth eruption were detected. Results A novel insertion mutation (c.309_310insTG) of RUNX2 gene was identified which had an effect on the characteristics of DFCs. Compared with the RUNX2 +/+ DFCs, there were 69 microRNAs more than 2-fold up-regulated and 54 microRNAs more than 2-fold down-regulated in the RUNX2 +/m DFCs. Among these, miR-146a decreased significantly in RUNX 2 +/m DFCs, and expression of RUNX2, CSF-1,EGFR and OPG was significantly altered when miR-146a was over-expressed or inhibited. Conclusion RUNX2 gene mutation contributes to the characteristic change of dental follicle cells, and the crosstalk between RUNX2 gene and miRNAs may be one of the key regulatory mechanisms of differentiation of dental follicle cells. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Cancer stem cells (CSC) have a central role in driving tumor growth. Since metabolism is becoming an important diagnostic and therapeutic target, characterization of CSC line energetic properties is an emerging need. Embryonic and adult stem cells, compared to differentiated cells, exhibit a reduced mitochondrial activity and a stronger dependence on aerobic glycolysis. Here, we aimed to comparatively analyze bioenergetics features of the human osteosarcoma 3AB-OS CSC-like line, and the parental osteosarcoma MG63 cells, from which 3AB-OS cells have been previously selected. Our results suggest that 3AB-OS cells depend on glycolytic metabolism more strongly than MG63 cells. Indeed, growth in glucose shortage or in presence of galactose or pyruvate -mitochondrial specific substrates- leads to a significant reduction of their proliferation compared to MG63 cells. Accordingly, 3AB-OS cells show an increased expression of lactate dehydrogenase A (LDHA) and a larger accumulation of lactate in the culture medium. In line with these findings 3AB-OS cells as compared to MG63 cells present a reduced mitochondrial respiration, a stronger sensitivity to glucose depletion or glycolysis inhibition and a lessened sensitivity to oxidative phosphorylation inhibitors. Additionally, in contrast to MG63 cells, 3AB-OS display fragmented mitochondria, which become networked as they grow in glucose-rich medium, while almost entirely loose these structures growing in low glucose. Overall, our findings suggest that 3AB-OS CSCs energy metabolism is more similar to normal stem cells and to cancer cells characterized by a glycolytic anaerobic metabolism. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Areca chewing is an important environmental risk factor for development of oral premalignant lesions and cancer. Epidemiological evidence indicates that areca chewing is tightly linked to oral carcinogenesis. However, the pathogenetic impacts of areca nut extract (ANE) on normal human oral keratinocytes (HOKs) are unclear and possibly involve oxidative stress via redox imbalance. Sirtuin 3 (SIRT3) is a member of the sirtuin family of proteins that play an important role in regulating cellular reactive oxygen species (ROS) production. Recent studies have confirmed that ANE and other areca ingredients can induce ROS. In this study, we examined the role of SIRT3 in the regulation of ANE-induced ROS in HOK cells. We examined HOK cell viability following treatment with various ANE concentrations. ANE-induced cytotoxicity increased in a dose-dependent manner and was approximately 48% at a concentration of 50 μg/ml after 24 h. SIRT3 expression and enzyme activity were up-regulated in HOK cells by ANE-induced oxidative stress. Additionally, we identified that SIRT3 controls the enzymatic activity of mitochondrial proteins, such as forkhead box O3a (Foxo3a) transcription factor and antioxidant-encoding gene superoxide dismutase 2 (SOD2), by deacetylation in HOK cells. Moreover, SIRT3-mediated deacetylation and activation of Foxo3a promotes nuclear localization in vivo . These findings suggest that SIRT3 is an endogenous negative regulator in response to ANE-induced oxidative stress and demonstrate an essential role for redox balance in HOK cells. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Collagen is the most abundant structural protein in mammals and is expressed in various tissues. In recent years, sphingosine 1-phosphate receptors (S1PRs) have been proven to play an important role in the regulation of collagen expression. Our previous studies reported that S1PRs are involved in TGF-β1-induced collagen expression via up-regulating S1PR1/3 in mouse bone marrow-derived mesenchymal stem cells (BMSCs), and result in experimental mouse liver fibrogenesis. But it remains unclear whether this process happens in human bone marrow-derived mesenchymal stem cells (hMSCs). In this study, we provide evidences that S1PR1/3, but not S1PR2, negatively regulate the expression of collagen in hMSCs using cellular and molecular approaches in vitro . We find that treatment of hMSCs with TGF-β1 up-regulated collagen expression in a dose- and time-dependent manner. Meanwhile, TGF-β1 inhibited the expression of S1PR1/3, but not S1PR2, in hMSCs in a time-dependent manner. Furthermore, either selective knock-down of S1PR1 or silencing S1PR3 induced collagen α1(I) and collagen α1(III) expression in hMSCs. In contrast, inhibition of S1PR2 by siRNA had no effects on the expression of collagen. Altogether, all these findings demonstrated that collagen expression was negatively regulated by S1PR1 and S1PR3 in hMSCs. This study highlights the differences between hMSCs and mouse BMSCs, provides a new regulation mechanism for collagen expression, and points out the risk of utilizing hMSCs in clinical applications. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Background: Hypersensitivity pneumonitis is defined as an allergic lung disease that occurs in response to inhalation of fungal antigens, bacterial antigens, chemicals, dusts, or animal proteins. The incidence of summer-type hypersensitivity pneumonitis is higher in the summer season, especially in Japan, due to the influence of the hot and humid environment and the common style of wood house or old concrete condominiums.Case presentationThe present report describes a case of a middle-aged married couple who lived in the same house and who simultaneously suffered from summer-type hypersensitivity pneumonitis. This report analyzes these two cases in terms of environmental research and its microbiological, radiological, and pathological aspects. This case report is followed by a review of family occurrences of summer-type hypersensitivity pneumonitis from 22 studies with a total of 49 patients (including the two present cases) in Japan. Conclusion: Summer-type hypersensitivity pneumonitis may be unrecognized and misdiagnosed as pneumonia or other respiratory diseases. A greater understanding of the clinical, pathologic, and environmental features of summer-type hypersensitivity pneumonitis might help improve diagnosis and delivery of appropriate management for this condition.

Description: Background: Cell migration is a fundamental biological process and has an important role in the developing brain by regulating a highly specific pattern of connections between nerve cells. Cell migration is required for axonal guidance and neurite outgrowth and involves a series of highly co-ordinated and overlapping signalling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) has an essential role in development and is the most highly expressed kinase in the developing CNS. FAK activity is essential for neuronal cell adhesion and migration. Results: The objective of this study was to optimise a protocol for the differentiation of the neuroblastoma cell line, SH-SY5Y. We determined the optimal extracellular matrix proteins and growth factor combinations required for the optimal differentiation of SH-SY5Y cells into neuronal-like cells and determined those conditions that induce the expression of FAK. It was confirmed that the cells were morphologically and biochemically differentiated when compared to undifferentiated cells. This is in direct contrast to commonly used differentiation methods that induce morphological differentiation but not biochemical differentiation. Conclusions: We conclude that we have optimised a protocol for the differentiation of SH-SY5Y cells that results in a cell population that is both morphologically and biochemically distinct from undifferentiated SH-SY5Y cells and has a distinct adhesion and spreading pattern and display extensive neurite outgrowth. This protocol will provide a neuronal model system for studying FAK activity during cell adhesion and migration events.

Description: Background: Little is known about the Phasmatodea gut microbial community, including whether phasmids have symbiotic bacteria aiding in their digestion. While symbionts are near ubiquitous in herbivorous insects, the Phasmatodea’s distinctively thin body shape precludes the gut enlargements needed for microbial fermentation. High-throughput sequencing was used to characterize the entire microbiota of the fat bodies, salivary glands, and anterior and posterior midguts of two species of walking stick. Results: Most bacterial sequences belonged to a strain of Spiroplasma (Tenericutes) found primarily in the posterior midgut of the parthenogenetic species Ramulus artemis (Phasmatidae). Beyond this, no significant differences were found between the R. artemis midgut sections or between that species and Peruphasma schultei (Pseudophasmatidae). Histological analysis further indicated a lack of bacteriocytes. Conclusions: Phasmids are unlikely to depend on bacteria for digestion, suggesting they produce enzymes endogenously that most other herbivorous insects obtain from symbionts. This conclusion matches predictions based on phasmid anatomy. The role of Spiroplasma in insects warrants further study.

Description: Background: HIV diagnosis, prognostic and treatment requires T CD4 lymphocytes' number from flow cytometry, an expensive technique often not available to people in developing countries. The aim of this work is to apply a previous developed methodology that predicts T CD4 lymphocytes' value based on total white blood cell (WBC) count and lymphocytes count applying sets theory, from information taken from the Complete Blood Count (CBC). Methods: Sets theory was used to classify into groups named A, B, C and D the number of leucocytes/mm3, lymphocytes/mm3, and CD4/muL3 subpopulation per flow cytometry of 800 HIV diagnosed patients. Union between sets A and C, and B and D were assessed, and intersection between both unions was described in order to establish the belonging percentage to these sets. Results were classified into eight ranges taken by 1000 leucocytes/mm3, calculating the belonging percentage of each range with respect to the whole sample. Results: Intersection (A [union] C) [intersection] (B [union] D) showed an effectiveness in the prediction of 81.44% for the range between 4000 and 4999 leukocytes, 91.89% for the range between 3000 and 3999, and 100% for the range below 3000. Conclusions: Usefulness and clinical applicability of a methodology based on sets theory were confirmed to predict the T CD4 lymphocytes' value, beginning with WBC and lymphocytes' count from CBC. This methodology is new, objective, and has lower costs than the flow cytometry which is currently considered as Gold Standard.

Description: Background: Acute adrenal insufficiency is a potentially lethal condition rarely caused by bilateral adrenal haemorrhage due to heparin use. Most of the times, it is difficult to establish the diagnosis, as symptoms are not specific. Few cases have been reported in the literature.Case presentationA 52-year-old Caucasian woman presented with abdominal pain, vomiting and weakness nine days after arthroplasty and heparin use. Hyperkalemia, low cortisol and high adrenocorticotropic hormone levels were found, indicating adrenal insufficiency. Magnetic resonance imaging of the upper abdomen was compatible with preceding adrenal haemorrhage. Hydrocortisone and fludrocortisone were administered. Review of the literature revealed 36 cases of postoperative adrenal haemorrhage which are presented briefly. Conclusion: Postoperative acute adrenal insufficiency due to haemorrhage is a rare condition. If patients are treated based on clinical suspicion, they have good chances to survive. Hydrocortisone is given permanently in the majority of the patients.

Description: Background: Patient Data Management Systems (PDMS) support clinical documentation at the bedside and have demonstrated effects on completeness of patient charting and the time spent on documentation. These systems are costly and raise the question if such a major investment pays off. We tried to answer the following questions: How do costs and revenues of an intensive care unit develop before and after introduction of a PDMS? Can higher revenues be obtained with improved PDMS documentation? Can we present cost savings attributable to the PDMS? Methods: Retrospective analysis of cost and reimbursement data of a 25 bed Intensive Care Unit at a German University Hospital, three years before (2004--2006) and three years after (2007--2009) PDMS implementation. Results: Costs and revenues increased continuously over the years. The profit of the investigated ICU was fluctuating over the years and seemingly depending on other factors as well. We found a small increase in profit in the year after the introduction of the PDMS, but not in the following years. Profit per case peaked at 1039 [euro sign] in 2007, but dropped subsequently to 639 [euro sign] per case. We found no clear evidence for cost savings after the PDMS introduction. Our cautious calculation did not consider additional labour costs for IT staff needed for system maintenance. Conclusions: The introduction of a PDMS has probably minimal or no effect on reimbursement. In our case the observed increase in profit was too small to amortize the total investment for PDMS implementation.This may add some counterweight to the literature, where expectations for tools such as the PDMS can be quite unreasonable.

Description: Cervical carcinoma represents the paradigm of virus-induced cancers, where virtually all cervical cancers come from previous “high-risk” HPV infection. The persistent expression of the HPV viral oncoproteins E6 and E7 is responsible for the reprogramming of fundamental cellular functions in the host cell, thus generating a noticeable, yet only partially explored, imbalance in protein molecular networks and cell signaling pathways. Eighty-eight cellular factors, identified as HPV direct or surrogate targets, were chosen and monitored in a retrospective analysis for their mRNA expression in HPV-induced cervical lesions, from dysplasia to cancer. Real-time quantitative PCR (qPCR) was performed by using formalin-fixed, paraffin embedded archival samples. Gene expression analysis identified 40 genes significantly modulated in LSIL, HSIL and squamous cervical carcinoma. Interestingly, among these, the expression level of a panel of four genes, TOP2A, CTNNB1, PFKM and GSN, was able to distinguish between normal tissues and cervical carcinomas. Immunohistochemistry was also done to assess protein expression of two genes among those up-regulated during the transition between dysplasia and carcinoma, namely E2F1 and CDC25A, and their correlation with clinical parameters. Besides the possibility of significantly enhancing the use of some of these factors in diagnostic or prognostic procedures, these data clearly outline specific pathways, and thus key biological processes, altered in cervical dysplasia and carcinoma. Deeper insight on how these molecular mechanisms work may help widen the spectrum of novel innovative approaches to these virus-induced cell pathologies. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Cellular pathways are numerous and are highly integrated in function in the control of cellular systems. They collectively regulate cell division, proliferation, survival and apoptosis of cells and mutagenesis of key genes that control these pathways can initiate neoplastic transformations. Understanding these pathways is crucial to future therapeutic and preventive strategies of the disease. Ovarian cancers are of three major types; epithelial, germ-cell and stromal. However, ovarian cancers of epithelial origin, arising from the mesothelium, are the predominant form. Of the subtypes of ovarian cancer, the high-grade serous tumors are fatal, with low survival rate due to late detection and poor response to treatments. Close examination of preserved ovarian tissues and in vitro studies have provided insights into the mechanistic changes occurring in cells mediated by a few key genes. This review will focus on pathways and key genes of the pathways that are mutated or have aberrant functions in the pathology of ovarian cancer. Non-genetic mechanisms that are gaining prominence in the pathology of ovarian cancer, miRNAs and epigenetics, will also be discussed in the review. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Normal pregnancy is associated with systemic vasodilation and decreased vascular contraction, partly due to increased release of endothelium-derived vasodilator substances. Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor acting via endothelin receptor type A (ET A R) and possibly type B (ET B R) in vascular smooth muscle cells (VSMCs), with additional vasodilator effects via endothelial ET B R. However, the role of ET-1 receptor subtypes in the regulation of vascular function during pregnancy is unclear. We investigated whether the decreased vascular contraction during pregnancy reflects changes in the expression/activity of ET A R and ET B R. Contraction was measured in single aortic VSMCs isolated from virgin, mid-pregnant (mid-Preg, day 12) and late-Preg (day 19) Sprague-Dawley rats, and the mRNA expression, protein amount, tissue and cellular distribution of ET A R and ET B R were examined using RT-PCR, Western blots, immunohistochemistry and immunofluorescence. Phenylephrine (Phe, 10 −5  M), KCl (51 mM) and ET-1 (10 −6  M) caused VSMC contraction that was in late-Preg 〈 mid-Preg and virgin rats. In VSMCs treated with ET B R antagonist BQ788, ET-1 caused significant contraction that was still in late-Preg 〈 mid-Preg and virgin rats. In VSMCs treated with the ET A R antagonist BQ123, ET-1 caused a small contraction; and the ET B R agonists IRL-1620 and sarafotoxin 6c (S6c) caused similar contraction that was in late-Preg 〈 mid-Preg and virgin rats. RT-PCR revealed similar ET A R, but greater ET B R mRNA expression in pregnant vs. virgin rats. Western blots revealed similar ET A R, and greater protein amount of ET B R in endothelium-intact vessels, but reduced ET B R in endothelium-denuded vessels of pregnant vs. virgin rats. Immunohistochemistry revealed prominent ET B R staining in the intima, but reduced ET A R and ET B R in the aortic media of pregnant rats. Immunofluorescence signal for ET A R and ET B R was less in VSMCs of pregnant vs. virgin rats. The pregnancy-associated decrease in ET A R- and ET B R-mediated VSMC contraction appears to involve downregulation of ET A R and ET B R expression/activity in VSM, and may play a role in the adaptive vasodilation during pregnancy. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Neuroregenerative medicine is an ever-growing field in which regeneration of lost cells/tissues due to a neurodegenerative disease is the ultimate goal. With the scarcity of available replacement alternatives, stem cells provide an attractive source for regenerating neural tissue. While many stem cell sources exist, including: mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), the limited cellular potency, technical difficulties, and ethical considerations associated with these make finding alternate sources a desirable goal. Periodontal ligament stem cells (PDLSCs) derived from the neural crest were induced into neural-like cells using a combination of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Morphological changes were evident in our treated group, seen under both light microscopy and scanning electron microscopy (SEM). A statistically significant increase in the expression of neuron-specific β-tubulin III and the neural stem/progenitor cell marker nestin, along with positive immunohistochemical staining for glial fibrillary acidic protein (GFAP), demonstrated the success of our treatment in inducing both neuronal and glial phenotypes. Positive staining for synaptophysin demonstrated neural connections and electrophysiological recordings indicated that when subjected to whole cell patch clamping, our treated cells displayed inward currents conducted through voltage-gated sodium (Na + ) channels. Taken together, our results indicate the success of our treatment in inducing PDLSCs to neural-like cells. The ease of sourcing and expansion, their embryologic neural crest origin, and the lack of ethical implications in their use make PDLSCs an attractive source for use in neuroregenerative medicine. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Urotensin II (UII), a vasoactive peptide modulates renal hemodynamics. However, the physiological functions of UII in glomerular cells are unclear. In particular, whether UII alters mesangial tone remains largely unknown. The present study investigates the physiological effects of UII on intracellular Ca 2+ ([Ca 2+ ] i ) and contraction in glomerular mesangial cells (GMCs). This study also tested the hypothesis that the regulator of G-protein signaling (RGS) controls UII receptor (UTR) activity in GMCs. RT-PCR, Western immunoblotting, and immunofluorescence revealed UTR expression and localization in cultured murine GMCs. Mouse UII (mUII) stimulated [Ca 2+ ] i elevation in GMCs in the absence and presence of extracellular Ca 2+ . mUII also caused a reduction in planar GMC surface area. mUII-induced [Ca 2+ ] i elevation and contraction in GMCs were attenuated by SB 657510, a UTR antagonist, araguspongin B, an inositol 1,4,5-trisphosphate receptor antagonist, thapsigargin, a sarco/endoplasmic reticulum Ca 2+ -ATPase inhibitor, and La 3+ , a store-operated Ca 2+ channel blocker, but not nimodipine, an L-type Ca 2+ channel blocker. In situ proximity ligation assay indicated molecular proximity between endogenous RGS2 and UTR in the cells. Treatment of GMCs with mUII increased plasma membrane association of RGS2 by ∼ 2-fold. mUII also increased the interaction between RGS2 and UTR in the cells. siRNA-mediated knockdown of RGS2 in murine GMCs increased mUII-induced [Ca 2+ ] i elevation and contraction by ∼ 35 and 31%, respectively. These findings indicate that mUII induces [Ca 2+ ] i elevation and contraction in murine GMCs. Data also suggest that UTR activation stimulates RGS2 recruitment to GMC plasma membrane as a negative feedback mechanism to regulate UTR signaling. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Poly-N-acetyllactosamine (PLN) is a unique glycan composed of repeating units of the common disaccharide (Galβ1,4-GlcNAcβ1,3) n . The expression of PLN on glycoprotein core structures minimally requires enzyme activities for β1,4-galactosyltransferase (β4GalT) and β1,3-N-acetylglucosminyltransferase (β3GnT). Because β4GalTs are ubiquitous in most cells, PLN expression is generally ascribed to the tissue-specific transcription of 8 known β3GnT genes in mice. In the olfactory epithelium (OE), β3GnT2 regulates expression of extended PLN chains that are essential for axon guidance and neuronal survival. N-glycan branching and core composition, however, can also modulate the extent of PLN modification. Here we show for the first time that the β1,6-branching glycosyltransferase GCNT2 (formerly known as IGnT) is expressed at high levels specifically in the OE and other sensory ganglia. Postnatally, GCNT2 is maintained in mature olfactory neurons that coexpress β3GnT2 and PLN. This highly specific coexpression suggests that GCNT2 and β3GnT2 function cooperatively in PLN synthesis. In support of this, β3GnT2 and GCNT2 cotransfection in HEK293T cells results in high levels of PLN expression on the cell surface and on adenylyl cyclase 3, a major carrier of PLN glycans in the OE. These data clearly suggest that GCNT2 functions in vivo together with β3GnT2 to determine PLN levels in olfactory neurons by regulating β1,6-branches that promote PLN extension. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Objective The aim of this study was to evaluate the effect of artemisinin on the proliferation and apoptosis of rat vascular smooth muscle cells (VSMCs). Method Primary rat VSMCs were treated with various doses of artemisinin. Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the messenger RNA and protein expressions of proliferating cell nuclear antigen were determined by reverse-transcription polymerase chain reaction and immunohistochemistry. Apoptosis was measured using annexin V and propidium iodide double staining evaluated by flow cytometry. Protein expression of Bax, Bcl2, and cyclin-dependent kinase 4 was determined by Western blot. Results After 72 h of treatment, artemisinin significantly inhibited VSMC proliferation in a dose-dependent manner. Treatment with 1 mM artemisinin for 72 h significantly reduced the expression of proliferating cell nuclear antigen messenger RNA. On the other hand, the same treatment increased the apoptosis of VSMCs, the activation of caspase-3, the Bax protein expression, and the Bax/Bcl2 ratio. Conclusion The results suggest that artemisinin can effectively inhibit VSMC proliferation and induce VSMC apoptosis. Copyright © 2013 John Wiley & Sons, Ltd.

Description: Background: A number of studies have implicated the direct involvement of the liver in dengue virus (DENV) infection, and it has been widely shown that liver cells subsequently undergo apoptosis. The mechanism by which liver cells undergo apoptosis in response to DENV infection remains unclear. To provide further information on the mechanism of apoptosis in DENV infected liver cells, HepG2 cells were infected with DENV 2 and analyzed for the induction of ER stress, apoptosis and autophagy. Results: In response to DENV infection, HepG2 cells showed the induction of both the ER resident unfolded protein response as well as the Noxa/PUMA stress response pathways. Proteolytic activation of caspases 4, 7, 8 and 9 was observed as well as changes in mitochondrial transmembrane potential. Increased monodansylcadaverine staining was observed in DENV infected cells, consistent with the previously reported induction of autophagy. Conclusions: These results are consistent with a model in which the induction of multiple ER stress pathways is coupled with the induction of multiple cell death pathways as a mechanism to ensure the removal of infected liver cells from the system.

Description: Previous studies indicate that muscle Pgc-1α expression governs the proportion of muscle fibre types. As a first step in using diet to manipulate the proportion of muscle fibre types by using Pgc-1α expression, the present study investigates the modulation of Pgc-1α expression by feedstuffs. A luciferase-based Pgc-1α reporter construct (Pgc-1α(-2553)-luc) that contains the mouse Pgc-1α promoter (−2553 to +78 bp) was prepared. A screen of ethanol extracts from 33 feedstuffs indicated that oolong tea and roasted green tea extracts decreased Pgc-1α(-2553)-luc expression in C2C12 myoblasts. The transcriptional repression of Pgc-1α by tea leaf extracts was reproduced in hepatic HepG2 cells. We further examined the effects of the alcohol extracts of tea waste and its silage on Pgc-1α transcription; the tea waste silage extract inhibited Pgc-1α transcription. Treatment with the extracts of raw tea leaves, tea waste and tea waste silage effectively decreased Pgc-1α mRNA levels during myogenesis of myosatellite cells. The present results suggest that tea leaves and their by-products could be used to modulate proportions of muscle fibre types. Copyright © 2013 John Wiley & Sons, Ltd.

Description: Lymph nodes are often the first target of metastatic cancer which can then remetastasize to distant organs. The progression of lymph node metastasis is dependent on sufficient blood supply provided by angiogenesis. In the present study, we have developed a color-coded imaging model to visualize angiogenesis of lymph nodes metastasis using green fluorescent protein (GFP) and red fluorescent protein (RFP). Transgenic mice carrying GFP under the control of the nestin second-intron enhancer (ND-GFP mice) were used as hosts. Nascent blood vessels express GFP in these mice. B16F10-RFP melanoma cells were injected into the efferent lymph vessel of the inguinal lymph node of the ND-GFP nude mice, whereby the melanoma cells trafficked to the axillary lymph node. Three days after melanoma implantation, ND-GFP-expressing nascent blood vessels were imaged in the axillary lymph nodes. Seven days after implantation, ND-GFP-expressing nascent blood vessels formed a network in the lymph nodes. ND-GFP-positive blood vessels surrounded the tumor mass by 14 days after implantation. However, by 28 days after implantation, ND-GFP expression was diminished as the blood vessels matured. Treatment with doxorubicin significantly decreased the mean nascent blood vessel length per tumor volume. These results show that the dual-color ND-GFP blood vessels/RFP-tumor model is a powerful tool to visualize and quantitate angiogenesis of metastatic lymph nodes as well as for evaluation of its inhibition. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Spermatogenesis is a special process by which spermatogonial stem cells (SSCs) divide and differentiate to male gametes called mature spermatozoa. SSCs are the unique cells because they are adult stem cells that transmit genetic information to subsequent generations. Accumulating evidence has demonstrated that SSCs can be reprogrammed to acquire pluripotency to become embryonic stem-like cells that differentiate into all cell lineages of the three germ layers, highlighting potential important applications of SSCs for regenerative medicine. Recent studies from peers and us have made great achievements on the characterization, isolation and culture of mouse and human SSCs, which could lead to better understanding the biology of SSCs and the applications of SSCs in both reproductive and regenerative medicine. In this review, we first compared the cell identity and biochemical phenotypes between mouse SSCs and human SSCs. Notably, the cell types of mouse and human SSCs are distinct, and human SSCs share some but not all phenotypes with mouse SSCs. The approaches for isolating SSCs as well as short- and long- term culture of mouse SSCs and short-period culture of human SSCs were also discussed. We further addressed the new advances on the self-renewal of SSCs with an aim to establish the long-term culture of human SSCs which has not yet been achieved. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Background: Performing multiple tests in primary research is a frequent subject of discussion. This discussion originates from the fact that when multiple tests are performed, it becomes more likely to reject one of the null hypotheses, conditional on that these hypotheses are true and thus commit a type one error. Several correction methods for multiple testing are available. The primary aim of this study was to assess the quantity of articles published in two highly esteemed orthopedic journals in which multiple testing was performed. The secondary aims were to determine in which percentage of these studies a correction was performed and to assess the risk of committing a type one error if no correction was applied. Methods: The 2010 annals of two orthopedic journals (A and B) were systematically hand searched by two independent investigators. All articles on original research in which statistics were applied were considered. Eligible publications were reviewed for the use of multiple testing with respect to predetermined criteria. Results: A total of 763 titles were screened and 127 articles were identified and included in the analysis. A median of 15 statistical inference results were reported per publication in both journal A and B. Correction for multiple testing was performed in 15% of the articles published in journal A and in 6% from journal B. The estimated median risk of obtaining at least one significant result for uncorrected studies was calculated to be 54% for both journals. Conclusion: This study shows that the risk of false significant findings is considerable and that correcting for multiple testing is only performed in a small percentage of all articles published in the orthopedic literature reviewed.

Description: Background: Ischemic preconditioning has been proposed to involve changes in mitochondrial H+ and K+ fluxes, in particular through activation of uncoupling proteins and ATP-sensitive K+ channels (MitoKATP). The objectives of the present study were to explore how increased H+ and K+ fluxes influence heart mitochondrial physiology with regard to production and scavenging of reactive oxygen species (ROS), volume changes and resistance to calcium-induced mitochondrial permeability transition (mPT). Results: Isolated rat heart mitochondria were exposed to a wide concentration range of the protonophore CCCP or the potassium ionophore valinomycin to induce increased H+ and K+ conductance, respectively. Simultaneous monitoring of mitochondrial respiration and calcium retention capacity (CRC) demonstrated that the relative increase in respiration caused by valinomycin or CCCP correlated with a decrease in CRC, and that no level of respiratory uncoupling was associated with enhanced resistance to mPT. Mitochondria suspended in hyperosmolar buffer demonstrated a dose-dependent reduction in CRC with increasing osmolarity. However, mitochondria in hypoosmolar buffer to increase matrix volume did not display increased CRC. ROS generation was reduced by both K+- and H+-mediated respiratory uncoupling. The ability of heart mitochondria to detoxify H2O2 was substantially greater than the production rate. The H2O2 detoxification was dependent on respiratory substrates and was dramatically decreased following calcium-induced mPT, but was unaffected by uncoupling via increased K+ and H+ conductance. Conclusion: It is concluded that respiratory uncoupling is not directly beneficial to rat heart mitochondrial resistance to calcium overload irrespective of whether H+ or K+ conductance is increased. The negative effects of respiratory uncoupling thus probably outweigh the reduction in ROS generation and a potential positive effect by increased matrix volume, resulting in a net sensitization of heart mitochondria to mPT activation.

Description: Background: Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor delta (PPARdelta). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes. Results: The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARdelta agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARgamma coactivator-1alpha. Conclusions: UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA.

Description: Background: Type 2 diabetes mellitus is increasing dramatically in sub-Saharan Africa, and genetic predisposition is likely involved in that. Yet, genetic variants known to confer increased susceptibility among Caucasians are far from being established in African populations. In Ghanaian adults, we examined associations of several of these polymorphisms with type 2 diabetes. Methods: A hospital-based case--control study on type 2 diabetes (and hypertension) was conducted in Kumasi, Ghana. TCF7L2 rs7903146, KCNJ11 rs5219, PPARgamma rs1801282 and CAPN10 rs3842570, rs3792267, and rs5030952 were typed and associations with type 2 diabetes and phenotypic traits examined. Results: 675 patients with type 2 diabetes and 377 controls were compared. The minor allele frequency of the TCF7L2 (T) allele was 0.33. In the multivariate model, this allele increased the risk of type 2 diabetes by 39% (95% confidence interval (CI), 1.07-1.81; p = 0.014). The minor alleles KCNJ11 (G) and PPARgamma (G) were practically absent (each, 0.001). Minor allele frequencies of CAPN10 were for -43 (A) 0.11 and for -63 (C) 0.46. These variants showed no significant associations with type 2 diabetes. Two CAPN10 haplotypes tended to protect against type 2 diabetes: 211 (aOR, 0.32; 95%CI, 0.03-1.92; p = 0.31) and 221 (aOR, 0.73; 95%CI, 0.48-1.10; p = 0.13). Conclusions: In urban Ghana, the frequency of the TCF7L2 rs7903146 (T) allele is comparable to the one in Caucasians; the association with type 2 diabetes is slightly weaker. The risk allele KCNJ11 (G) and the protective allele PPARgamma (G) are virtually absent. The potential influence of comparatively rare CAPN10 haplotypes on type 2 diabetes risk in this population requires further evaluation. Large-scale genetic studies among native Africans aiming at fine-mapping the candidate genes are needed to identify the actual factors involved in their increased susceptibility to type 2 diabetes.

Description: Background: Kinases are important signalling molecules for modulating cellular processes and major targets of drug discovery programs. However, functional information for roughly half the human kinome is lacking. We conducted three kinome wide, 〉90%, RNAi screens and epistasis testing of some identified kinases against known intramuscular signalling systems to increase the functional annotation of the C. elegans kinome and expand our understanding of kinome influence upon muscle protein degradation. Results: 96 kinases were identified as required for normal protein homeostasis, 74 for normal mitochondrial networks and 50 for normal sarcomere structure. Knockdown of kinases required only for normal protein homeostasis and/or mitochondrial structure was significantly less likely to produce a developmental or behavioural phenotype than knockdown of kinases required for normal sarcomere structure and/or other sub-cellular processes. Lastly, assessment of kinases for which knockdown produced muscle protein degradation against the known regulatory pathways in C. elegans muscle revealed that close to half of kinase knockdowns activated autophagy in a MAPK dependent fashion. Conclusions: Roughly 40% of kinases studied, 159 of 397, are important in establishing or maintaining muscle cell health, with most required for both. For kinases where decreased expression triggers protein degradation, autophagy is most commonly activated. These results increase the annotation of the C. elegans kinome to roughly 75% and enable future kinome research. As 33% of kinases identified have orthologues expressed in human muscle, our results also enable testing of whether identified kinases function similarly in maintaining human muscle homeostasis.

Description: Long non-coding RNAs (lncRNAs) have recently gained increasing attention because of their crucial roles in gene regulatory processes. Functional studies using mammalian skin as a model system have revealed their role in controlling normal tissue homeostasis as well as the transition to a diseased state. Here, we describe how lncRNAs regulate differentiation to preserve an undifferentiated epidermal progenitor compartment, and to maintain a functional skin permeability barrier. Furthermore, we will reflect on recent work analyzing the impact of lncRNAs on the progression from normal epithelium to the development of skin disorders and cancer. Long non-coding RNAs (lncRNAs) have recently been shown to control a wide variety of gene regulatory processes. In mammalian skin, lncRNAs appear to regulate the intricate balance between progenitor cells undergoing continual regeneration in the basal layer and highly differentiated cells forming the epidermal permeability barrier.

Description: Background: Asthma genome-wide association studies (GWAS) have identified several asthma susceptibility genes with confidence; however the relative contribution of these genetic variants or single nucleotide polymorphisms (SNPs) to clinical endpoints (as opposed to disease diagnosis) remains largely unknown. Thus the aim of this study was to firstly bridge this gap in knowledge and secondly investigate whether these SNPs or those that are in linkage disequilibrium are likely to be functional candidates with respect to regulation of gene expression, using reported data from the ENCODE project. Methods: Eleven of the key SNPs identified in eight loci from recent asthma GWAS were evaluated for association with asthma and clinical outcomes including percent predicted FEV1, bronchial hyperresponsiveness (BHR) to methacholine, severity defined by British Thoracic Society steps and positive response to skin prick test using the family based association test additive model in a well characterised UK cohort consisting of 370 families with at least two asthmatic children. Results: GSDMB SNP rs2305480 (Ser311Pro) was associated with asthma diagnosis (p = 8.9x10-4), BHR (p = 8.2x10-4) and severity (p = 1.5x10-4) with supporting evidence from a second GSDMB SNP rs11078927 (intronic). SNPs evaluated in IL33, IL18R1, IL1RL1, SMAD3, IL2RB, PDE4D, CRB1 and RAD50 did not show association with any phenotype tested when corrected for multiple testing. Analysis using ENCODE data provide further insight into the functional relevance of these SNPs. Conclusions: Our results provide further support for the role of GSDMB SNPs in determining multiple asthma related phenotypes in childhood asthma including associations with lung function and disease severity.

Description: Background: Patient preference is one of the main components of clinical decision making, therefore leading to the development of patient decision aids. The goal of this study was to describe physicians' and patients' viewpoints on the barriers and limitations of using patient decision aids in Iran, their proposed solutions, and, the benefits of using these tools. Methods: This qualitative study was conducted in 2011 in Iran by holding in-depth interviews with 14 physicians and 8 arthritis patient. Interviewees were selected through purposeful and maximum variation sampling. As an example, a patient decision aid on the treatment of knee arthritis was developed upon literature reviews and gathering expert opinion, and was presented at the time of interview. Thematic analysis was conducted to analyze the data by using the OpenCode software. Results: The results were summarized into three categories and ten codes. The extracted categories were the perceived benefits of using the tools, as well as the patient-related and physician-related barriers in using decision aids. The following barriers in using patient decision aids were identified in this study: lack of patients and physicians' trainings in shared decision making, lack of specialist per capita, low treatment tariffs and lack of an exact evaluation system for patient participation in decision making. Conclusions: No doubt these barriers demand the health authorities' special attention. Hence, despite patients and physicians' inclination toward using patient decision aids, these problems have hindered the practical usage of these tools in Iran - as a developing country.

Description: Background: Most Crohn's disease (CD) genes discovered in recent years are associated with biological systems critical to the development of this disease. TGFB1 and IL10 are cytokines with important roles in CD. The aim of this study was to evaluate the association between CD, its clinical features and TGFB1 and IL10 gene polymorphisms. Methods: This case--control study enrolled 91 patients and 91 controls from the state of Bahia, Brazil. Five single nucleotide polymorphisms (SNPs) were studied in the TGFB1 gene (codon 10 T 〉 C - rs1800470; codon 25 G 〉 C - rs1800471) and IL10 gene (-1082 A 〉 G - rs1800896; -819 T 〉 C - rs1800871; -592 A 〉 C - rs1800872). An analysis of the genetic polymorphisms was performed using a commercial kit. A comparison of allele frequencies and genotypes was estimated by calculating the odds ratio (OR) with a confidence interval adjusted via the Bonferroni test for a local alpha of 1%. A stratified analysis was applied for gender, race and smoking history. Patients with CD were characterized according to the Montreal classification. Results: The C allele and CC genotype of the TGFB1 gene rs1800470 were both significantly associated with CD. The stratified analysis showed no confounding factors for the co-variables of gender, race and smoking history. The IL10 gene rs1800896 G allele was significantly associated with age at diagnosis of CD, while the T allele of the IL10 gene rs1800871 was significantly associated with perianal disease. The SNPs rs1800871 and rs1800872 were in 100% linkage disequilibrium. Conclusions: TGFB1 gene polymorphisms may be associated with susceptibility to the development of CD, and IL10 gene polymorphisms appear to influence the CD phenotype in this admixed population.

Description: Background: The lectin-like domain of TNF-alpha mimicked by an inhaled TIP peptide represents a novel approach to attenuate a pulmonary edema in respiratory failure, which is on the threshold to clinical application. In extension to a previously published study, which reported an improved pulmonary function following TIP peptide inhalation in a porcine model of lavage-induced lung injury, a post-hoc comparison to additional experiments was conducted. This analysis addresses the hypothesis that oleic acid injection-induced capillary leakage and alveolar necrosis blunts the previously reported beneficial effects of TIP peptide inhalation in a porcine model.FindingsFollowing animal care committee approval lung injury was induced by oleic acid injection in six pigs with a setting strictly according to a previously published protocol that was used for lung-lavaged pigs. Ventilation/perfusion-distribution by multiple inert gas elimination, parameters of gas exchange and pulmonary edema were assessed as surrogates of the pulmonary function. A significantly improved ventilation/perfusion-distribution following TIP inhalation was recognized only in the bronchoalveolar lavage model but not following oleic acid injection. The time course after oleic acid injection yielded no comparable impact of the TIP peptide on gas exchange and edema formation. Conclusions: Reported beneficial effects of the TIP peptide on gas exchange and pulmonary edema were not reproducible in the oleic acid injection model. This analysis assumes that sustained alveolar epithelial necrosis as induced by oleic acid injection may inhibit the TIP-induced edema resolution. Regarding the on-going clinical development of the TIP peptide this approach should hardly be effective in states of severe alveolar epithelial damage.

Description: Background: Cell-free RNA (cfRNA) naturally occurs in blood and has clinical significance. Accurate quantification of these extracellular RNAs in whole blood is hindered by the simultaneous unintended release of cellular RNA and degradation of cfRNA after blood draw. An appropriate blood collection device is needed to stabilize cfRNA during blood processing, transportation and storage, which will ensure cfRNA test reliability. In this study we compared a novel blood collection device against traditional K3EDTA tubes for its ability to stabilize cfRNA in blood when subjected to conditions that can occur during sample storage and shipping.FindingsShipping blood samples drawn into K3EDTA tubes showed a significant increase in mRNA copy numbers for β-actin, c-fos, and 18S rRNA in plasma. In contrast, shipping blood drawn into Cell-Free RNA BCT™s (BCTs) showed only a slight change in mRNA copy numbers for circulating β-actin, c-fos, and 18S rRNA. Moreover, blood stored in K3EDTA tubes at 6°C, 22°C and 30°C for 3 days showed a significant increase in mRNA copy numbers for c-fos and β-actin, whereas samples stored in BCTs only showed a slight increase. Conclusion: Our results show that BCTs minimize increases in background RNA levels caused by temperature fluctuations or agitation that can occur during blood sample storage and shipping. This novel blood collection tube could provide a method for obtaining high quality stabilized cfRNA samples for rare RNA target detection and determining accurate cfRNA concentrations.

Description: Background: Recent studies have suggested that nuclear lipid droplets (LDs) are organized into domains similar to those of cytoplasmic LDs. As cytoplasmic LDs are formed at the endoplasmic reticulum (ER) membrane, which is structurally continuous with the nuclear envelope, it could be suggested however that nuclear LDs are cytoplamic LDs trapped within an invagination of the nuclear envelope. The resolution of fluorescence confocal microscopy is not sufficiently high to exclude this hypothesis.FindingsWe therefore addressed this question by electron microscopy (EM) of serial sections. In human liver tissue, we observed some cytoplamic LDs partly surrounded by the nuclear compartment, but we were also able to identify LDs residing in the nuclear compartment that were not connected to the nuclear envelope. Conclusion: These findings indicate that nuclear LDs constitute specific subdomains of the nuclear compartment probably involved in nuclear lipid homeostasis.

Description: Background: Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. A Nicotiana hybrid (Nicotiana. tabacum x Nicotiana glauca) shows limited male fertility and could be used as a bioconfined PMP platform. Effective assessment of gene flow from these plants is augmented with methods that utilize fluorescent proteins for transgenic pollen identification. Results: We report the generation of a pollen tagging system utilizing an orange fluorescent protein to monitor pollen flow and as a visual assessment of transgene zygosity of the parent plant. This system was created to generate a tagged Nicotiana hybrid that could be used for the incidence of gene flow. Nicotiana tabacum 'TN 90' and Nicotiana glauca were successfully transformed via Agrobacterium tumefaciens to express the orange fluorescent protein gene, tdTomato-ER, in pollen and a green fluorescent protein gene, mgfp5-er, was expressed in vegetative structures of the plant. Hybrids were created that utilized the fluorescent proteins as a research tool for monitoring pollen movement and gene flow. Manual greenhouse crosses were used to assess hybrid sexual compatibility with N. tabacum, resulting in seed formation from hybrid pollination in 2% of crosses, which yielded non-viable seed. Pollen transfer to the hybrid formed seed in 19% of crosses and 10 out of 12 viable progeny showed GFP expression. Conclusion: The orange fluorescent protein is visible when expressed in the pollen of N. glauca, N. tabacum, and the Nicotiana hybrid, although hybrid pollen did not appear as bright as the parent lines. The hybrid plants, which show limited ability to outcross, could provide bioconfinement with the benefit of detectable pollen using this system. Fluorescent protein-tagging could be a valuable tool for breeding and in vivo ecological monitoring.

Description: Intact intercellular junctions and cellular matrix contacts are crucial structural components for the formation and maintenance of epithelial barrier functions in humans to control the commensal flora and protect against intruding microbes. Campylobacter jejuni is one of the most important zoonotic pathogens causing food-borne gastroenteritis and potentially more severe diseases such as reactive arthritis or Guillain--Barre syndrome. Crossing the intestinal epithelial barrier and host cell invasion by C. jejuni are considered to represent the primary reasons of gut tissue damage in humans and various animal model systems including monkeys, piglets, rabbits, hamsters and ferrets. C. jejuni is also able to invade underlying tissues such as the lamina propria, can enter the bloodstream, and possibly reach distinct organs such as spleen, liver or mesenteric lymph nodes. However, the molecular mechanisms as well as major bacterial and host cell factors involved in these activities are poorly understood. Various models exist by which the pathogen can trigger its own transmigration across polarized intestinal epithelial cells in vitro, the paracellular and/or transcellular mechanism. Recent studies suggest that bacterial factors such as flagellum, serine protease HtrA and lipooligosaccharide LOS may play an active role in bacterial transmigration. Here we review our knowledge on transmigration of C. jejuni as well as some other Campylobacter species, and discuss the pros and cons for the route(s) taken to travel across polarized epithelial cell monolayers. These studies provide fresh insights into the infection strategies employed by this important pathogen.

Description: Background: Gestational diabetes (GDM) has been shown to have long-term sequelae for both the mother and infant. Women with GDM are at increased risk of macrosomia, which predisposes the infant to birth injuries. Previous studies noted increased rates of GDM in Asian and Pacific Islander (API) women; however, the rate of macrosomia in API women with GDM is unclear. The objective of this study was to examine the relationship between ethnicity, gestational diabetes (GDM), and macrosomia in Hawaii. Methods: A retrospective cohort study was performed using Hawaii Pregnancy Risk Assessment Monitoring System (PRAMS) data. Data from 2009--2011, linked with selected items from birth certificates, were used to examine GDM and macrosomia by ethnicity. SAS-callable SUDAAN 10.0 was used to generate odds ratios, point estimates and standard errors. Results: Data from 4735 respondents were weighted to represent all pregnancies resulting in live births in Hawaii from 2009--2011. The overall prevalence of GDM in Hawaii was 10.9%. The highest prevalence of GDM was in Filipina (13.1%) and Hawaiian/Pacific Islander (12.1%) women. The lowest prevalence was in white women (7.4%). Hawaiian/Pacific Islander, Filipina, and other Asian women all had an increased risk of GDM compared to white women using bivariate analysis. Adjusting for obesity, age, maternal nativity, and smoking, Asian Pacific Islander (API) women, which includes Hawaiian/Pacific Islander, Filipina, and other Asian women, had a 50% increased odds of having GDM compared to white women when compared using multivariate analysis. Among women with GDM, the highest prevalence of macrosomia was in white women (14.5%) while the lowest was in Filipina (5.3%) women. Conclusions: API women in Hawaii have increased rates of GDM compared to white women. Paradoxically, this elevated GDM risk in API women is not associated with an increased rate of macrosomia. This suggests the relationship between GDM and macrosomia is more complex in this population.

Description: Background: Clinical decision support (CDS) for electronic prescribing systems (computerized physician order entry) should help prescribers in the safe and rational use of medicines. However, the best ways to alert users to unsafe or irrational prescribing are uncertain. Specifically, CDS systems may generate too many alerts, producing unwelcome distractions for prescribers, or too few alerts running the risk of overlooking possible harms. Obtaining the right balance of alerting to adequately improve patient safety should be a priority. Methods: A workshop funded through the European Regional Development Fund was convened by the University Hospitals Birmingham NHS Foundation Trust to assess current knowledge on alerts in CDS and to reach a consensus on a future research agenda on this topic. Leading European researchers in CDS and alerts in electronic prescribing systems were invited to the workshop. Results: We identified important knowledge gaps and suggest research priorities including (1) the need to determine the optimal sensitivity and specificity of alerts; (2) whether adaptation to the environment or characteristics of the user may improve alerts; and (3) whether modifying the timing and number of alerts will lead to improvements. We have also discussed the challenges and benefits of using naturalistic or experimental studies in the evaluation of alerts and suggested appropriate outcome measures. Conclusions: We have identified critical problems in CDS, which should help to guide priorities in research to evaluate alerts. It is hoped that this will spark the next generation of novel research from which practical steps can be taken to implement changes to CDS systems that will ultimately reduce alert fatigue and improve the design of future systems.

Description: Background: Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris.FindingsAmong tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction. Conclusion: Our gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation.

Description: Background: Spontaneous closure of an idiopathic full-thickness macular hole has been reported to occasionally occur. However, the cells involved in plugging the macular hole have not been determined conclusively. We aimed to report the early structural changes that occur during a spontaneous closure of an idiopathic full-thickness macular hole determined by spectral-domain optical coherence tomography.Case presentationA 71-year-old Japanese man with an idiopathic full-thickness macular hole and subclinical posterior vitreous detachment in the left eye was followed. Three weeks after the identification of the macular hole, optical coherence tomography showed tissue that protruded from the interior wall of the macular hole at the level of the external limiting membrane toward the center of the macular hole. Five months after the first examination, he returned with improvements of his visual symptoms, and the macular hole was closed by a thin retinal tissue which included the restored external limiting membrane that bridged across the macular hole. However, the inner segment/outer segment junction line was not intact and the fovea was detached. Two months later, optical coherence tomography showed an almost normal foveal configuration with an essentially restored inner segment/outer segment junction line and foveal reattachment. Conclusion: Our results suggest that Muller cells proliferate and/or extend at the level of the end of the external limiting membrane to form a tissue bridge across the macular hole associated with the external limiting membrane restoration first of all. This leads to the adhesion of other retinal layers and resolution of the foveal detachment.

Description: Background: Coffee production in Africa represents a significant share of the total export revenues and influences the lives of millions of people, yet severe socio-economic repercussions are annually felt in result of the overall losses caused by the coffee berry disease (CBD). This quarantine disease is caused by the fungus Colletotrichum kahawae Waller and Bridge, which remains one of the most devastating threats to Coffea arabica production in Africa at high altitude, and its dispersal to Latin America and Asia represents a serious concern. Understanding the molecular genetic basis of coffee resistance to this disease is of high priority to support breeding strategies. Selection and validation of suitable reference genes presenting stable expression in the system studied is the first step to engage studies of gene expression profiling. Results: In this study, a set of ten genes (S24, 14-3-3, RPL7, GAPDH, UBQ9, VATP16, SAND, UQCC, IDE and β-Tub9) was evaluated to identify reference genes during the first hours of interaction (12, 48 and 72 hpi) between resistant and susceptible coffee genotypes and C. kahawae. Three analyses were done for the selection of these genes considering the entire dataset and the two genotypes (resistant and susceptible), separately. The three statistical methods applied GeNorm, NormFinder, and BestKeeper, allowed identifying IDE as one of the most stable genes for all datasets analysed, and in contrast GADPH and UBQ9 as the least stable ones. In addition, the expression of two defense-related transcripts, encoding for a receptor like kinase and a pathogenesis related protein 10, were used to validate the reference genes selected. Conclusion: Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the interaction between Coffea spp. and C. kahawae.

Description: DNA replication is a highly conserved process that accurately copies the genetic information from one generation to the next. The processes of chromatin disassembly and reassembly during DNA replication also have to be precisely regulated to ensure that the genetic material is compactly packaged to fit into the nucleus while also maintaining the epigenetic information that is carried by the histone proteins bound to the DNA, through cell divisions. Half of the histones that are deposited during replication are from the parental chromatin and carry the parental epigenetic information, while the other half of the histones are newly-synthesized. It has been of growing interest to understand how the parental pattern of epigenetic marks is re-established on the newly-synthesized histones, in a DNA sequence-specific manner, in order to maintain the epigenetic information through cell divisions. In this review we will discuss how histone chaperone proteins precisely coordinate the chromatin assembly process during DNA replication. We also discuss the recent evidence that histone-modifying enzymes, rather than the parental histones, are themselves epigenetic factors that remain associated with the DNA through replication to re-establish the epigenetic information on the newly-assembled chromatin.

Description: Angiogenin (ANG) undergoes nuclear translocation and promotes ribosomal RNA (rRNA) transcription thereby enhancing cell growth and proliferation. However, the mode of action of ANG in stimulating rRNA transcription is unclear. Here, we show that ANG enhances the formation of RNA polymerase I (Pol I) pre-initiation complex at the ribosomal DNA (rDNA) promoter. ANG binds at the upstream control element (UCE) of the promoter and enhances promoter occupancy of RNA Pol I as well as the selectivity factor SL1 components TAF I 48 and TAF I 110. We also show that ANG increases the number of actively transcribing rDNA by epigenetic activation through promoter methylation and histone modification. ANG binds to histone H3, inhibits H3K9 methylation, and activates H3K4 methylation as well as H4 acetylation at the rDNA promoter. These data suggest that one of the mechanisms by which ANG stimulates rRNA transcription is through an epigenetic activation of rDNA promoter. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Background: Staphylococcus aureus (SA) is a recognized cause of nosocomial infections with 8,767 SA bacteraemia (SAB) cases reported in England only in 2012. Different typing methods have been developed but they are not generally performed as a routine investigation in hospital laboratories.FindingsWe collected epidemiological data and spa-typed all SAB isolates over a 12 months period. Spa-typing was useful to detect two potential outbreaks of methicillin-sensitive SA (MSSA). In addition, the analysis of spa-types from individuals with multiple bacteriaemias helped to distinguish between relapse and re-infection. Conclusions: Spa-typing could be used as a rapid tool to understand the epidemiology of SAB, in particular the detection of hospital clusters and to distinguish relapse from re-infection, but clinicians should be aware of its possible limitations.

Description: Background: Recently studies have demonstrated improved outcomes in patients undergoing nephron-sparing surgery (NSS) for low stage renal tumors, thus NSS is widely accepted as the treatment option for these patients. With NSS, there is a risk of renal hemorrhage and thus haemostatic agents may be routinely applied to the cut surface of the kidney. Herein we compare two commercially available haemostatic agents applied intra-operatively to the cut surface of the kidney. Post-operative outcomes (oncologic and non-oncologic) are reported. Methods: The medical records of 23 patients with suspicious renal mass documented on axial imaging and who underwent open NSS via a mini-subcostal incision were extensively reviewed. One of two haemostatic agents (Floseal(R), n = 11; Arista(R), n = 12) was intra-operatively applied to the cut surface of the kidney. Chi-square and T- student test was used to compare outcomes between the cohort of 11 patients who had Floseal(R) and the 12 patients who had Arista (R). Results: Median pre-operative size of renal mass was 4.3 cm (range 1.5-7.0 cm). Final pathology revealed 3 oncocytomas and 20 renal cell carcinoma (17 clear cell, 1 chromophobe and 2 papillary), pT1a = 14 and pT1b = 6. Mean intra-operative blood loss and hospital stay between the Floseal(R) vs. Arista(R) cohorts did not significantly differ (227 mL vs. 250 mL, p = 0.68 and 4.4 days vs. 4.5 days, p = 0.76, respectively). Intra-operative and post-operative complications were not different between the two cohorts. No recurrences have been documented with a mean follow-up of 18 months. Conclusion: Along with meticulous surgical technique, the use of either haemostatic agent (Floseal(R) or Arista(R)) was not associated with high rate of intra-operative or post-operative haemorrhage. Thus either haemostatic agent may be successfully used during NSS.

Description: Background: Gestational diabetes (GDM) has been shown to have long-term sequelae for both the mother and infant. Women with GDM are at increased risk of macrosomia, which predisposes the infant to birth injuries. Previous studies noted increased rates of GDM in Asian and Pacific Islander (API) women; however, the rate of macrosomia in API women with GDM is unclear. The objective of this study was to examine the relationship between ethnicity, gestational diabetes (GDM), and macrosomia in Hawaii. Methods: A retrospective cohort study was performed using Hawaii Pregnancy Risk Assessment Monitoring System (PRAMS) data. Data from 2009–2011, linked with selected items from birth certificates, were used to examine GDM and macrosomia by ethnicity. SAS-callable SUDAAN 10.0 was used to generate odds ratios, point estimates and standard errors. Results: Data from 4735 respondents were weighted to represent all pregnancies resulting in live births in Hawaii from 2009–2011. The overall prevalence of GDM in Hawaii was 10.9%. The highest prevalence of GDM was in Filipina (13.1%) and Hawaiian/Pacific Islander (12.1%) women. The lowest prevalence was in white women (7.4%). Hawaiian/Pacific Islander, Filipina, and other Asian women all had an increased risk of GDM compared to white women using bivariate analysis. Adjusting for obesity, age, maternal nativity, and smoking, Asian Pacific Islander (API) women, which includes Hawaiian/Pacific Islander, Filipina, and other Asian women, had a 50% increased odds of having GDM compared to white women when compared using multivariate analysis. Among women with GDM, the highest prevalence of macrosomia was in white women (14.5%) while the lowest was in Filipina (5.3%) women. Conclusions: API women in Hawaii have increased rates of GDM compared to white women. Paradoxically, this elevated GDM risk in API women is not associated with an increased rate of macrosomia. This suggests the relationship between GDM and macrosomia is more complex in this population.

Description: No abstract is available for this article.

Description: Background: In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis. Results: The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis. Conclusions: The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic properties of HtrA2/Omi and UCH-L1 may prove beneficial for the treatment of patients, e.g. in kidney failure.

Description: Background: Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix components found in the stroma. The aim of this study was to investigate mechanisms involved in tumour cell-mediated regulation of extracellular matrix and adhesion molecules in co-cultured fibroblasts. To this end, microarray analysis was performed on CCD-1068SK human fibroblast cells after direct co-culture with MDA-MB-231 human breast tumour cells. Results: We found that the expression of both connective tissue growth factor (CTGF/CCN2) and type I collagen was negatively regulated in CCD-1068SK fibroblast cells under direct co-culture conditions. Further analysis revealed that Smad7, a known negative regulator of the Smad signalling pathway involved in CCN2 promoter regulation, was increased in directly co-cultured fibroblasts. Inhibition of Smad7 expression in CCD-1068SK fibroblasts resulted in increased CCN2 expression, while Smad7 overexpression had the opposite effect. Silencing CCN2 gene expression in fibroblasts led, in turn, to a decrease in type I collagen mRNA and protein levels. ERK signalling was also shown to be impaired in CCD-1068SK fibroblasts after direct co-culture with MDA-MB-231 tumour cells, with Smad7 overexpression in fibroblasts leading to a similar decrease in ERK activity. These effects were not, however, seen in fibroblasts that were indirectly co-cultured with tumour cells. Conclusion: We therefore conclude that breast cancer cells require close contact with fibroblasts in order to upregulate Smad7 which, in turn, leads to decreased ERK signalling resulting in diminished expression of the stromal proteins CCN2 and type I collagen.

Description: Background: The actin cytoskeleton is essential for many physiological processes of eukaryotic cells. The emergence of new actin fibers is initiated by actin nucleators. Whereas most of them are evolutionary old, the cordon-bleu actin nucleator is classified as vertebrate specific.FindingsUsing sensitive methods for sequence similarity detection, we identified homologs of cordon-bleu not only in non-vertebrate chordates but also in arthropods, molluscs, annelids and platyhelminthes. These genes contain only a single WH2 domain and therefore resemble more the vertebrate cordon-bleu related 1 protein than the three WH2 domain containing cordon-bleu. Furthermore, we identified a homolog of the N-terminal, ubiquitin like, cobl domain of cordon-bleu in the cnidarian Nematostella vectensis. Conclusion: Our results suggest that the ur-form of the cordon-bleu protein family evolved already with the emergence of the bilateria by the combination of existing cobl and WH2 domains. Following a vertebrate specific gene-duplication, one copy gained two additional WH2 domains leading to the actin nucleating cordon-bleu. The function of the ur-form of the cordon-bleu protein family is so far unknown. The identification of a homolog in the model organism Drosophila melanogaster could facilitate its experimental characterization.

Description: Pancreatic cancer (PC) has a high rate of mortality and a poorly understood mechanism of progression. Investigation of the molecular mechanism of PC and exploration of the specific markers for early diagnosis and specific targets of therapy are key points to prevent and treat PC effectively and to improve their prognosis. In our study, expression profiles experiment of para-carcinoma, carcinoma and relapse human PC was performed using Agilent human whole genomic oligonucleotide microarrays with 45 000 probes. Differentially expressed genes related with PC were screened and analysed further by Gene Ontology term analysis and Kyoto encyclopaedia of genes and genomes pathway analysis. Our results showed that there were 3853 differentially expressed genes associated with pancreatic carcinogenesis and relapse. In addition, our study found that PC was related to the Jak–STAT signalling pathway, PPAR signalling pathway and Calcium signalling pathway, indicating their potential roles in pancreatic carcinogenesis and progress. Copyright © 2013 John Wiley & Sons, Ltd.

Description: ABSTRACT Uchl1 encodes the protein gene product 9.5 antigen (PGP9.5) that is a widely used to identify migrating neural progenitors in the PNS, mature neurons of the central and peripheral nervous systems, as well as neuroendocrine cells. To facilitate analysis of developing peripheral neurons, we linked regulatory regions of Uchl1 carried within a 160kb bacterial artificial chromosome (BAC) to the dual fluorescent reporter H2BmCherry:GFP-gpi. The Uchl1 -H2BmCherry:GFP-gpi transgene exhibits robust expression and allows clear discrimination of individual cells and cellular processes in cranial ganglia, sympathetic chain, the enteric nervous system (ENS), and autonomic ganglia of the urogenital system. The transgene also labels subsets of cell in endocrine tissues where prior in situ hybridization (ISH) studies have previously identified expression of this deubiquinating enzyme. The Uchl1 -H2BmCherry:GFP-gpi transgene will be a powerful tool for static and live imaging as well as isolation of viable neural progenitors to investigate processes of autonomic neurogenesis. © 2013 Wiley Periodicals, Inc.

Description: Background: Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as large genomic rearrangements. However, a large number of mutations, including missense, silent, and intronic variants, are classified as variants of unknown clinical significance. Methods: Intronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing. Results: We describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c.588 + 5G 〉 A, MLH1 c.677 + 3A 〉 T, MLH1 c.1732-2A 〉 T, MSH2 c.1276 + 1G 〉 T, and MSH2 c.1662-2A 〉 C), while four mutations had no effect on splicing compared to wild type (MLH1 c.117-34A 〉 T, MLH1 c.1039-8 T 〉 A, MSH2 c.2459-18delT, and MSH6 c.3439-16C 〉 T). Conclusions: In conclusion, we classify five MLH1/MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and their family members.

Description: Background: Trastuzumab prolongs survival of human epidermal growth factor receptor 2-positive breast cancer patients in both the adjuvant and metastatic settings. Currently toxicity data are not available on retreatment of metastatic breast cancer patients who relapse after adjuvant trastuzumab. We report one patient with metastatic breast cancer who developed acute thrombocytopenia after trastuzumab infusion. This patient had trastuzumab treatment in the adjuvant setting.Case presentationA 70-year-old Caucasian woman received a diagnosis of metastatic breast cancer four years after her initial diagnosis of locally advanced, hormone receptors-positive, human epidermal growth factor receptor 2-positive breast cancer. Trastuzumab retreatment was planned. Less than 24 hours after trastuzumab infusion, the patient was admitted to the hospital for the appearance of diffuse petechial hemorrhages and ecchymosis. The patient was confirmed to have a severe trastuzumab-induced thrombocytopenia. A rapid and complete recovery was observed after high-dose intravenous corticosteroids and immunoglobulin. No trastuzumab retreatment was attempted. Conclusion: Among the reported cases of trastuzumab-induced thrombocytopenia, this is the first report in the literature occurring in a patient retreated with trastuzumab after adjuvant therapy.

Description: Background: Biomarker discovery datasets created using mass spectrum protein profiling of complex mixtures of proteins contain many peaks that represent the same protein with different charge states. Correlated variables such as these can confound the statistical analyses of proteomic data. Previously we developed an algorithm that clustered mass spectrum peaks that were biologically or technically correlated. Here we demonstrate an algorithm that clusters correlated technical aliases only. Results: In this paper, we propose a preprocessing algorithm that can be used for grouping technical aliases in mass spectrometry protein profiling data. The stringency of the variance allowed for clustering is customizable, thereby affecting the number of peaks that are clustered. Subsequent analysis of the clusters, instead of individual peaks, help reduce difficulties associated with technically-correlated data, and can aid more efficient biomarker identification. Conclusions: This software can be used to pre-process and thereby decrease the complexity of protein profiling proteomics data, thus simplifying the subsequent analysis of biomarkers by decreasing the number of tests. The software is also a practical tool for identifying which features to investigate further by purification, identification and confirmation.

Description: Background: Dextran sodium sulfate (DSS) is commonly used in mouse studies to induce a very reproducible colitis that effectively mimics the clinical and histological features of human inflammatory bowel disease (IBD) patients, especially ulcerative colitis. However, the mechanisms of action of DSS remain poorly understood, and observations by our laboratory and other groups indicate that DSS contamination of colonic tissues from DSS-treated mice potently inhibits the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) amplification of mRNA. Results: A prior study used poly-A-mediated mRNA purification to remove DSS from RNA extracts, but we herein report a second efficient and cost-effective approach to counteract this inhibition, using lithium chloride precipitation to entirely remove DSS from RNAs. We also explored how DSS interferes with qRT-PCR process, and we report for the first time that DSS can alter the binding of reverse transcriptase to previously primed RNA and specifically inhibits the enzymatic activities of reverse transcriptase and Taq polymerase in vitro. This likely explains why DSS-treated colonic RNA is not suitable to qRT-PCR amplification without a previous purification step. Conclusion: In summary, we provide a simple method to remove DSS from colonic RNAs, and we demonstrate for the first time that DSS can inhibit the activities of both polymerase and reverse transcriptase. In order to reliably analyze gene expression in the colonic mucosa of DSS-treated mice, the efficiency rate of qRT-PCR must be the same between all the different experimental groups, including the water-treated control group, suggesting that whatever the duration and the percentage of the DSS treatment, RNAs must be purified.

Description: Background: Due to the difficult geographic terrain with lack of roads and transport, the Sikkim State in India finds difficulties in contending the respiratory diseases especially during the rainy seasons.FindingsA case--control study was conducted for two months at the Central Referral Hospital of East Sikkim involving 110 individuals in the age group of 10 years and above. Due to feasibility constraints, 55 cases and 55 controls were selected by applying the non-probability sampling method with age and sex matching. The collected data were tabulated and analyzed by using the SPSS (Statistical Package for Social Sciences) version 10.0 for windows. Findings were expressed in terms of proportion, Chi Square Test and Multiple Logistic Regression Analysis. Here, p-value

Description: Background: Acute patellar tendon ruptures with poor tissue quality. Ruptures that have been neglected are difficult to repair. Several surgical techniques for the repair of the patellar tendon have been reported, however, these techniques remain difficult because of contractures, adhesions, and atrophy of the quadriceps muscle after surgery.Case presentationWe report the cases of 2 Japanese patients (Case 1: a 16-year-old male and Case 2: a 43-year-old male ) with patellar tendon ruptures who were treated by reconstruction using semitendinosus-gracilis (STG) tendons with preserved distal insertions. Retaining the original insertion of the STG appears to preserve its viability and provide the revascularization necessary to accelerate healing. Both tendons were placed in front of the patella, in a figure-of-eight fashion, providing stability to the patella. Conclusion: Both patients recovered near normal strength and stability of the patellar tendon as well as restoration of function after the operation.

Description: Background: Paediatric tuberculosis (TB) is poorly addressed in Ethiopia and information about its magnitude and the genotype distribution of the causative Mycobacterium tuberculosis strains responsible for its spread are scanty. Methods: Gastric lavage or sputum samples were collected from consecutively enrolled TB suspect children visiting Jimma University Hospital in 2011 and cultured on Middlebrook 7H11 and Löwenstein-Jensen media. Acid fast bacterial (AFB) isolates were subjected to molecular typing targeting regions of difference (RDs), 16S rDNA gene and the direct repeat (DR) region using multiplex polymerase chain reaction (mPCR), gene sequencing and spoligotyping, respectively. Molecular drug susceptibility testing of M. tuberculosis isolates was performed by Genotype®MTBDRplus line probe assay (LPA) (Hain Life Sciences, Germany). Results: Gastric lavage (n = 43) or sputum (n = 58) samples were collected from 101 children and 31.7% (32/101) of the samples were positive for AFB by microscopy, culture and/or PCR. Out of 25 AFB isolates, 60% (15/25) were identified as M. tuberculosis by PCR, and 40% isolates (10/25) were confirmed to be non-tuberculous mycobacteria (NTM) by genus typing and 16S rDNA gene sequencing. Lineage classification assigned the M. tuberculosis strains into Euro-American (EUA, 66.7%; 10/15), East-African-Indian (EAI; 2/15), East-Asian (EA; 1/15) and Indio-Oceanic (IO; 1/15) lineages. Seven M. tuberculosis strains were new to the SpolDB4 database. All of the M. tuberculosis isolates were susceptible to isoniazid (INH) and rifampicin (RIF), except for one strain (of spoligotype SIT-149 or T3_ETH family) which had a mutation at the inhA locus which often confers resistance to INH (low level) and ethionamide. Conclusions: Analysis of the genetic population structure of paediatric M. tuberculosis strains suggested similarity with that of adults, indicating an on-going and active transmission of M. tuberculosis from adults to children in Ethiopia. There were no multidrug-resistant TB (MDR-TB) strains among the isolates.

Description: Background: We aimed to describe orbital positron emission tomography/computed tomography (PET/CT) imaging findings, both structural and metabolic, in different clinical stages of Graves ophthalmopathy (GO). This prospective, observational, cross-sectional study examined 32 eyes of 16 patients with GO. Methods: Patients were assessed with a complete ophthalmological evaluation and assigned a VISA classification for GO. All patients underwent serum thyroid hormone measurement, antibody profile, and 18-fluorodeoxyglucose positron emission tomography/computed tomography (18-FDG PET/CT) of the orbits. The 18-FDG uptake on PET images was expressed in terms of maximum standard uptake value (SUVmax). CT images were analyzed, and orbital structures were measured in millimeters. Vision, inflammation, strabismus, and overall appearance were assessed according to the VISA classification system, thyroid hormone levels, antibody values, 18-FDG uptake, and thickness of orbital structures. Results: Altogether, 32 eyes of 16 patients (10 women, 6 men; mean age 44.31 ± 13 years, range 20–71 years) were included. Three patients were hypothyroid, seven were euthyroid, and six were hyperthyroid. CT measurements of extraocular muscle diameter were elevated (P   0.05). Conclusions: We demonstrated a lack of correlation between 18-FDG extraocular muscle uptake and either clinical inflammation score or muscle diameter. Although 18-FDG uptake has been used as an inflammation marker in other pathologies, inflammation in GO may be clinically detected in PET/CT-negative cases, and cases with negative clinical findings may show inflammation on PET/CT. Clinical evaluation is mandatory but may be insufficient and inaccurate for classifying GO. A larger and homogeneous sample size and further research is needed to define the role of PET/CT in detecting, grading, and follow-up of GO to optimize treatment of the inflammatory stage respect clinical methods currently used.

Description: Background: Hepatitis B virus (HBV) and Hepatitis C virus (HCV) co-infections among HIV-1 infected individuals are growing worldwide health problems characterized by lack of effective vaccines, need for expensive treatment, chronicity of morbidity and associated mortality. Their prevalence and distribution patterns continue to vary across geographical locations with high prevalence being detected among high risk populations. To determine the prevalence of HBV and HCV among HIV-1 infected individuals, blood samples were collected from consenting study subjects visiting comprehensive HIV clinics in Nairobi during the period between October and December 2009. Methods: Blood samples from volunteers were screened with ELISA tests for detecting HIV, HBV surface antigen (HBsAg) and anti-HCV antibodies. Results: In a total of three (300) hundred infected individuals consisting of 129 (43%) males and 171 (57%) females 15.3% (46/300) were HIV-1 co-infected with either HBV or HCV or both, 10.3% (31/300) with HIV-1 and HCV and 6% (18/300) with HIV-1 and HBV infections. However, only three individuals (1%) were coinfected with the three viruses (HIV/HBV/HCV). Conclusion: Though, low levels of co-infection with all three viruses were reported, there could be higher prevalence rates than reported here especially among high risk populations.

Description: Background: Tyrosinemia type 1 (TT1) is an autosomal recessive disorder caused by deficiency of the enzyme fumarylacetoacetate hydrolase (FAH). TT1 usually presents in infancy with features suggestive of liver disease or with sepsis-like symptoms.Case presentationWe report two Saudi siblings with TT1. Case 1 was a male infant who presented at 2 months old with fever, vomiting and refusal of feeding. Examination revealed a sick-looking infant with signs of severe dehydration and hypovolemic shock. He was jaundiced, and had hepatomegaly and elevated liver enzymes. Echocardiography was performed in light of a lack of response to inotropes, and revealed biventricular and interventricular septal hypertrophies. The ventricular ejection fraction was 65%. Urine organic acid analysis showed elevated succinylacetone, consistent with a diagnosis of TT1. An FAH gene study identified a c.1 A 〉 G homozygous mutation. This patient responded well to intensive cardiorespiratory therapy, tyrosine-free formula, and oral 2-nitro-4- trifluoromethylbenzyl 1, 3 cyclohexanedione (NTBC). Echocardiographic findings reverted to normal after 4 weeks. Case 2 was the younger brother of Case 1, and was born 6 months after his brother had been confirmed with tyrosinemia. Pregnancy and delivery were uneventful. Serum amino acid and organic acid analyses 4 days after birth confirmed tyrosinemia. DNA analysis identified a c.1 A 〉 G homozygous mutation, as in his brother. Echocardiography was normal. Special formula and NTBC were commenced on day 7 of life. The infant remained asymptomatic after 9 months of follow-up. Conclusions: These cases highlight TT1 as a treatable cause of cardiomyopathy in children. It also supports the idea that early diagnosis and treatment may prevent the development of cardiomyopathy associated with tyrosinemia.

Description: Background: Hepatitis B virus (HBV) and Hepatitis C virus (HCV) co-infections among HIV-1 infected individuals are growing worldwide health problems characterized by lack of effective vaccines, need for expensive treatment, chronicity of morbidity and associated mortality. Their prevalence and distribution patterns continue to vary across geographical locations with high prevalence being detected among high risk populations. To determine the prevalence of HBV and HCV among HIV-1 infected individuals, blood samples were collected from consenting study subjects visiting comprehensive HIV clinics in Nairobi during the period between October and December 2009. Methods: Blood samples from volunteers were screened with ELISA tests for detecting HIV, HBV surface antigen (HBsAg) and anti-HCV antibodies. Results: In a total of three (300) hundred infected individuals consisting of 129 (43%) males and 171 (57%) females 15.3% (46/300) were HIV-1 co-infected with either HBV or HCV or both, 10.3% (31/300) with HIV-1 and HCV and 6% (18/300) with HIV-1 and HBV infections. However, only three individuals (1%) were coinfected with the three viruses (HIV/HBV/HCV). Conclusion: Though, low levels of co-infection with all three viruses were reported, there could be higher prevalence rates than reported here especially among high risk populations.

Description: Background: Genetic studies of the Foraminifera provide valuable insights into marine speciation and biogeography, yet the discovery of vitally needed new genetic markers for this important group is being severely limited by an extreme lack of genetic data. The establishment of a laboratory culture from a single, asexually reproducing foraminifer, will be essential to provide enough pooled genetic material from these unicellular organisms, to facilitate full genome sequencing and genetic marker discovery, using next-generation sequencing techniques.FindingsThe aim of this study was to develop a simple and inexpensive method of culturing benthic foraminifera, via asexual reproduction, in a controlled laboratory environment. Individual specimens of the benthic foraminfer Cornuloculina balkwilli (MacFadyen) were placed in 7 cm plastic beakers, containing 50 ml natural seawater, filtered to 0.2 mum, and kept at 23[degree sign]C, with a 12-hour light/dark cycle, and fed weekly on a mixed algal diet of Dunaliella tertiolecta and Phaeodactylum tricornutum. Asexually derived cultures were successfully established from 4 specimens of Cornuloculina balkwilli, originally added to the culture vessels as immature specimens. Many thousands of individuals were present after 6 months. Conclusions: The method presented here demonstrates that only basic laboratory equipment is required to establish and maintain a thriving culture of the benthic foraminfer, C. balkwilli, from a single asexually reproducing specimen, providing an excellent source of genetic material for use in next generation sequencing. The method is easily reproducible and will greatly aid in the discovery of critically needed new genetic markers in the Foraminifera. It also highlights C. balkwilli as a good candidate species for use in the field of environmental micropaleontology.

Description: Background: Cell migration is a fundamental biological process and has an important role in the developing brain by regulating a highly specific pattern of connections between nerve cells. Cell migration is required for axonal guidance and neurite outgrowth and involves a series of highly co-ordinated and overlapping signalling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) has an essential role in development and is the most highly expressed kinase in the developing CNS. FAK activity is essential for neuronal cell adhesion and migration. Results: The objective of this study was to optimise a protocol for the differentiation of the neuroblastoma cell line, SH-SY5Y. We determined the optimal extracellular matrix proteins and growth factor combinations required for the optimal differentiation of SH-SY5Y cells into neuronal-like cells and determined those conditions that induce the expression of FAK. It was confirmed that the cells were morphologically and biochemically differentiated when compared to undifferentiated cells. This is in direct contrast to commonly used differentiation methods that induce morphological differentiation but not biochemical differentiation. Conclusions: We conclude that we have optimised a protocol for the differentiation of SH-SY5Y cells that results in a cell population that is both morphologically and biochemically distinct from undifferentiated SH-SY5Y cells and has a distinct adhesion and spreading pattern and display extensive neurite outgrowth. This protocol will provide a neuronal model system for studying FAK activity during cell adhesion and migration events.

Description: Background: APOAI, a member of the APOAI/CIII/IV/V gene cluster on chromosome 11q23-24, encodes a major protein component of HDL that has been associated with serum lipid levels. The aim of this study was to determine the genetic association of polymorphisms in the APOAI promoter region with plasma lipid levels in a cohort of healthy Kuwaiti volunteers. Methods: A 435 bp region of the APOAI promoter was analyzed by re-sequencing in 549 Kuwaiti samples. DNA was extracted from blood taken from 549 healthy Kuwaiti volunteers who had fasted for the previous 12 h. Univariate and multivariate analysis was used to determine allele association with serum lipid levels. Results: The target sequence included a partial segment of the promoter region, 5'UTR and exon 1 located between nucleotides -141 to +294 upstream of the APOAI gene on chromosome 11. No novel single nucleotide polymorphisms (SNPs) were observed. The sequences obtained were deposited with the NCBI GenBank with accession number [GenBank: JX438706]. The allelic frequencies for the three SNPs were as follows: APOAI rs670G = 0.807; rs5069C = 0.964; rs1799837G = 0.997 and found to be in HWE. A significant association (p 〈 0.05) was observed for the APOAI rs670 polymorphism with increased serum LDL-C. Multivariate analysis showed that APOAI rs670 was an independent predictive factor when controlling for age, sex and BMI for both LDL-C (OR: 1.66, p = 0.014) and TC (OR: 1.77, p = 0.006) levels. Conclusion: This study is the first to report sequence analysis of the APOAI promoter in an Arab population. The unexpected positive association found between the APOAI rs670 polymorphism and increased levels of LDL-C and TC may be due to linkage disequilibrium with other polymorphisms in candidate and neighboring genes known to be associated with lipid metabolism and transport.

Description: The cytoplasmic signaling protein tumor necrosis factor (TNF) receptor-associated factor 5 (TRAF5), which was identified as a signal transducer for members of the TNF receptor super-family, has been implicated in several biological functions in T/B lymphocytes and the innate immune response against viral infection. However, the role of TRAF5 in cardiac hypertrophy has not been reported. In the present study, we investigated the effect of TRAF5 on the development of pathological cardiac hypertrophy induced by transthoracic aorta constriction (TAC) and further explored the underlying molecular mechanisms. Cardiac hypertrophy and function were evaluated with echocardiography, hemodynamic measurements, pathological and molecular analyses. For the first time, we found that TRAF5 deficiency substantially aggravated cardiac hypertrophy, cardiac dysfunction and fibrosis in response to pressure overload after 4 weeks of TAC compared to wild-type (WT) mice. Moreover, the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinases 1/2 (ERK1/2) signaling pathway was more activated in TRAF5-deficient mice than WT mice. In conclusion, our results suggest that as an intrinsic cardioprotective factor, TRAF5 plays a crucial role in the development of cardiac hypertrophy through the negative regulation of the MEK-ERK1/2 pathway. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: The SON protein is a ubiquitously expressed DNA- and RNA-binding protein primarily localized to nuclear speckles. Although several early studies implicated SON in DNA-binding, tumorigenesis and apoptosis, functional significance of this protein had not been recognized until recent studies discovered SON as a novel RNA splicing co-factor. During constitutive RNA splicing, SON ensures efficient intron removal from the transcripts containing suboptimal splice sites. Importantly, SON-mediated splicing is required for proper processing of selective transcripts related to cell cycle, microtubules/centrosomes maintenance, and genome stability. Moreover, SON regulates alternative splicing of RNAs from the genes involved in apoptosis and epigenetic modification. In addition to the role in RNA splicing, SON has an ability to suppress transcriptional activation at certain promoter/enhancer DNA sequences. Considering the multiple SON target genes which are directly involved in cell proliferation, genome stability and chromatin modifications, SON is an emerging player in gene regulation during cancer development and progression. Here, we summarize available information from several early studies on SON, and highlight recent discoveries describing molecular mechanisms of SON-mediated gene regulation. We propose that our future effort on better understanding of diverse SON functions would reveal novel targets for cancer therapy. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Background: The choice of selection methods to identify important variables for binary classification modeling is critical to produce stable models that are interpretable, that generate accurate predictions and have minimum bias. This work is motivated by data on clinical and laboratory features of severe dengue infections (dengue hemorrhagic fever, DHF) obtained from 51 individuals enrolled in a prospective observational study of acute human dengue infections. Results: We carry out a comprehensive performance comparison using several classification models for DHF over the dengue data set. We compared variable selection results by Multivariate Adaptive Regression Splines, Learning Ensemble, Random Forest, Bayesian Moving Averaging, Stochastic Search Variable Selection, and Generalized Regularized Logistics Regression. Model averaging methods (bagging, boosting and ensemble learners) have higher accuracy, but the generalized regularized regression model has the highest predictive power because the linearity assumptions of candidate predictors are strongly satisfied via deviance chi-square testing procedures. Bootstrapping applications for evaluating predictive regression coefficients in regularized regression model are performed. Conclusions: Feature reduction methods introduce inherent biases and therefore are data-type dependent. We propose that these limitations can be overcome using an exhaustive approach for searching feature space. Using this approach, our results suggest that IL-10, platelet and lymphocyte counts are the major features for predicting dengue DHF on the basis of blood chemistries and cytokine measurements.

Description: In the present study, we examined the effects of methanol extracts of Picrasma quassioides (MEPQ) on apoptosis in human cervical cancer cells. The results showed that MEPQ decreased the viability and induced caspase-dependent apoptosis in HEp-2 cells. MEPQ decreased specificity protein 1 (Sp1) in HEp-2 cells, whereas Sp1 mRNA was not changed. We found that MEPQ reduced Sp1 protein through proteasome-dependent protein degradation, but not the inhibition of protein synthesis. Also, MEPQ increased the expressions of Bad and truncated Bid (t-Bid) but did not alter other Bcl-2 family members. The knock-down of Sp1 by both Sp1 interfering RNA and Mithramycin A, Sp1 specific inhibitor clearly increased Bad and t-Bid expression to decrease cell viability and induce apoptosis. In addition, MEPQ inhibited cell viability and induced apoptotic cell death through the modulation of Sp1 in KB cells. These results suggest that MEPQ may be a potential anticancer agent for human cervical cancer. Copyright © 2013 John Wiley & Sons, Ltd.

Description: General mechanism for exaggerated sexuallyselected traits. Many animals wield sexually-selected exaggerated traits. ‘Classic’ examples include the peacock's train, and the antlers observed in male deer, as well as fiddler crabs with enlarged claws, and the enlarged head-horns of rhinoceros beetles ( Trypoxylus dichotomus , cover). These traits are used for mate choice, or to deter rival males, because they act as unusually reliable signals of the condition of individual males: only the best-quality animals produce full-sized signal structures. But what keeps their expression honest? How can signal traits evolve that are resistant to invasion by cheaters who fake attractive signals? The answer may lie in the ancient insulin-like signalling pathway, which is found in all animals and directly links individual condition to growth in a dose dependent manner. Warren et al. discuss recent evidence suggesting that exaggerated sexually-selected signal traits arise when specific structures become extra-sensitive to physiological signals like insulins or insulin-like growth factors (pages 889–899 ).

Description: Background: Hypersensitivity pneumonitis is defined as an allergic lung disease that occurs in response to inhalation of fungal antigens, bacterial antigens, chemicals, dusts, or animal proteins. The incidence of summer-type hypersensitivity pneumonitis is higher in the summer season, especially in Japan, due to the influence of the hot and humid environment and the common style of wood house or old concrete condominiums.Case presentationThe present report describes a case of a middle-aged married couple who lived in the same house and who simultaneously suffered from summer-type hypersensitivity pneumonitis. This report analyzes these two cases in terms of environmental research and its microbiological, radiological, and pathological aspects. This case report is followed by a review of family occurrences of summer-type hypersensitivity pneumonitis from 22 studies with a total of 49 patients (including the two present cases) in Japan. Conclusion: Summer-type hypersensitivity pneumonitis may be unrecognized and misdiagnosed as pneumonia or other respiratory diseases. A greater understanding of the clinical, pathologic, and environmental features of summer-type hypersensitivity pneumonitis might help improve diagnosis and delivery of appropriate management for this condition.

Description: Background: Genome-wide maps of transcription factor binding sites in primary tissues can expand our understanding of genome function, transcriptional regulation, and genetic alterations that contribute to disease risk. However, almost all genome-wide studies of transcription factors have been in cell lines, and performing these experiments in tissues has been technically challenging and limited in throughput. Results: Here we outline a simple strategy for mapping transcription factor binding sites in frozen tissues that utilizes dry pulverization of samples and is scalable for high-throughput analyses. We show that the method leads to accurate and reproducible chromatin immunoprecipitation next-generation sequencing (ChIP-seq) data, and is highly sensitive, identifying high-quality transcription factor binding sites from chromatin corresponding to only 5 mg of liver tissue. Conclusions: The enhanced reproducibility, robustness, and sensitivity of the dry pulverization method, in addition to the ease of implementation and scalability, makes ChIP-seq in primary tissues a widely accessible assay.

Description: mRNA synthesis in all organisms is performed by RNA polymerases, which work as nanomachines on DNA templates. The rate at which their product is made is an important parameter in gene expression. Transcription rate encompasses two related, yet different, concepts: the nascent transcription rate, which measures the in situ mRNA production by RNA polymerase, and the rate of synthesis of mature mRNA, which measures the contribution of transcription to the mRNA concentration. Both parameters are useful for molecular biologists, but they are not interchangeable and they are expressed in different units. It is important to distinguish when and where each one should be used. We propose that for functional genomics the use of nascent transcription rates should be restricted to the evaluation of the transcriptional process itself, whereas mature mRNA synthesis rates should be employed to address the transcriptional input to mRNA concentration balance leading to variation of gene expression. Transcription rate encompasses two related, yet different, concepts: the nascent transcription rate, which measures the in situ mRNA production by RNA polymerase, and the rate of synthesis of mature mRNA, which measures the contribution of transcription to the mRNA concentration. It is important to distinguish when to use each one.

Description: CALHM1 was recently demonstrated to be a voltage-gated ATP-permeable ion channel and to serve as a bona fide conduit for ATP release from sweet-, umami-, and bitter-sensing type II taste cells. Calhm1 is expressed in taste buds exclusively in type II cells and its product has structural and functional similarities with connexins and pannexins, two families of channel protein candidates for ATP release by type II cells. Calhm1 knockout in mice leads to loss of perception of sweet, umami, and bitter compounds and to impaired gustatory nerve responses to these tastants. These new studies validate the concept of ATP as the primary neurotransmitter from type II cells to gustatory neurons. Furthermore, they identify voltage-gated ATP release through CALHM1 as an essential molecular mechanism of ATP release in taste buds. We discuss these new findings, as well as unresolved issues in peripheral taste signaling that we hope will stimulate future research. Sweetness, umami, and bitterness are transmitted to the nervous system via ion channel-mediated ATP release from taste cells. A recent study demonstrated that CALHM1 is essential for taste cell ATP release and perception of sweetness, umami, and bitterness. We discuss the new findings and unresolved issues in peripheral taste signaling.

Description: Background: B-cell translocation gene 2 (BTG2) belongs to antiproliferative (ARPO) gene family and the expression of BTG2, human ortholog of rat PC3 and mouse TIS21 gene, has been shown to render cancer cells more sensitive to doxorubicin treatment by upregulating MnSOD expression without regulating any other reactive oxygen species (ROS) scavenging enzymes. Results: In the present study, by employing exogenous and endogenous BTG2/TIS21/Pc3 expression by transfection and transduction analyses, and by knockdown of gene expression using RNA interference or using gene knockout cells, we observed that BTG2 increased the binding of activated NF-kappaB (p65/RelA) to the enhancer element of MnSOD gene in the 2nd intron, which was regulated by p-Akt1, and the induction of MnSOD by BTG2 was accompanied with subsequent downregulation of ROS level and cyclin B1 biosynthesis along with the increase of p21WAF1, resulting in the G2/M arrest independent of p53. Conclusions: These results show for the first time that BTG2 mediates crosstalk between PI3K-Akt1 and NF-kappaB pathways, which regulates p53-independent induction of G2/M phase arrest both in normal and cancer cells.

Description: The precise orchestration of two opposing protein complexes – one in the cytoplasm (β-catenin destruction complex) and the other at the plasma membrane (LRP6 signaling complex) – is critical for controlling levels of the transcriptional co-factor β-catenin, and subsequent activation of the Wnt/β-catenin signal transduction pathway. The Wnt pathway component Axin acts as an essential scaffold for the assembly of both complexes. How the β-catenin destruction and LRP6 signaling complexes are modulated following Wnt stimulation remains controversial. A recent study in Science by He and coworkers reveals an underlying logic for Wnt pathway control in which Axin phosphorylation toggles a switch between the active and inactive states. This mini-review focuses on this and two other recent studies that provide insight into the initial signaling events triggered by Wnt exposure. We emphasize regulation of the β-catenin destruction and LRP6 signaling complexes and propose a framework for future work in this area. The mechanism by which the Wnt pathway stabilizes β-catenin, a key transcriptional co-factor, remains controversial. Recent studies have revealed that the phosphorylation state of an essential regulator of the pathway, Axin, controls its conformation and, consequently, its availability to scaffold two opposing Wnt pathway protein complexes that regulate β-catenin stability.

Description: Background: We investigated a potential link between genetic polymorphisms in genes XRCC1 (Arg399Gln), OGG1 (Ser326Cys), XRCC3 (Thr241Met), and XRCC4 (Ile401Thr) with the level of DNA damage and repair, accessed by comet and micronucleus test, in 51 COPD patients and 51 controls. Methods: Peripheral blood was used to perform the alkaline and neutral comet assay; and genetic polymorphisms by PCR/RFLP. To assess the susceptibility to exogenous DNA damage, the cells were treated with methyl methanesulphonate for 1-h or 3-h. After 3-h treatment the % residual damage was calculated assuming the value of 1-h treatment as 100%. The cytogenetic damage was evaluated by buccal micronucleus cytome assay (BMCyt). Results: COPD patients with the risk allele XRCC1 (Arg399Gln) and XRCC3 (Thr241Met) showed higher DNA damage by comet assay. The residual damage was higher for COPD with risk allele in the four genes. In COPD patients was showed negative correlation between BMCyt (binucleated, nuclear bud, condensed chromatin and karyorrhexic cells) with pulmonary function and some variant genotypes. Conclusion: Our results suggest a possible association between variant genotypes in XRCC1 (Arg399Gln), OGG1 (Ser326Cys), XRCC3 (Thr241Met), and XRCC4 (Ile401Thr), DNA damage and progression of COPD.

Description: Background: Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCtheta) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2 signaling. Results: In this study, PKCtheta knockdown (PKCthetashRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 was increased in PKCthetashRNA cells, accompanied by elevated extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation, with no change in ERK5 phosphorylation, highlighting a PKCtheta-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCthetashRNA cells. Thus, with reduced PKCtheta, differentiation and fusion occur in the absence of PI3kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCthetashRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCtheta regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation. Conclusion: Cellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCtheta regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCtheta had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCtheta regulated signaling may reveal important signaling interaction regulating skeletal muscle health in an insulin resistant state.

Description: Background: Current genetic test algorithms for Charcot Marie Tooth (CMT) disease are based on family details and comprehensive clinical and neurophysiological data gathered under ideal conditions for clinical assessment. However, in a diagnostic laboratory setting relying on external test requisitions and patient samples, such conditions are not always met. Our objective was therefore to perform a retrospective evaluation of the data given in laboratory request forms and to assess their quality and applicability with regard to the recommended algorithms for CMT diagnostics. As we are the main test centre for CMT in Norway our results also provide an overview of the spectrum of gene defects in the Norwegian CMT population. Methods: Genetic testing was performed according to polyneuropathy type; demyelinating/mixed: PMP22 duplication, MPZ, EGR2, LITAF, NEFL, PMP22, GJB1, axonal: MFN2, MPZ, NEFL, and GJB1. Results: Diagnostic testing of index patients was requested in 435 of the 549 cases. Seventy-two (16.6%) positive molecular genetic findings were made. The majority (94.6%) of mutation positive cases showed disease onset before 50 years of age. PMP22 (duplication), MPZ, GJB1 and MFN2 mutations constituted 95.8% of the positive findings. Within the nerve conduction study groups, mutation detection rates were; demyelinating 33.8%; mixed 29.0%; axonal 8.8%; unspecified 16.5%. Conclusion: We suggest a simplified algorithm intended for referral centres, dealing with DNA/blood samples, which involves the assessment of age at onset and neurophysiological data followed by testing of four genes; PMP22 (duplication), MPZ, GJB1 and MFN2. Patients negative for mutations in those four genes should be subjected to evaluation at an interdisciplinary inherited neuropathy clinic with the capacity for extended molecular genetic analysis by next generation sequencing.

Description: Background Podocytes are a terminally differentiated and highly specialized cell type in the glomerulus that forms a crucial component of the glomerular filtration barrier. Recently, Myo1e was identified in the podocytes of glomeruli. Myo1e podocyte-specific knockout mice exhibit proteinuria, podocyte foot process effacement, glomerular basement membrane disorganization, signs of chronic renal injury, and kidney inflammation. Materials and Methods After overexpression of Myo1e in a conditionally immortalized mouse podocyte cell line (MPC5), podocyte migration was evaluated via transwell assay, endocytosis was evaluated using FITC-transferrin, and adhesion was evaluated using a detachment assay after puromycin aminonucleoside treatment. Results Myo1e overexpression significantly increased the adherence of podocytes. ANOVA analysis indicated significant differences for cell adhesion between the overexpression and control groups (overexpression vs. control, t = 11.3199, P = 0.005; overexpression vs. negative control, t = 12.0570, P = 0.0006). Overexpression of Myo1e inhibited puromycin aminonucleoside-induced podocyte detachment, and the number of cells remaining on the bottom of the culture plate increased. Cell migration was enhanced in Myo1e-overexpressing podocytes in the transwell migration assay. Internalization of FITC-transferrin also increased in Myo1e-overexpressing podocytes relative to control cells. Conclusions Overexpression of Myo1e can enhance podocyte migration ability, endocytosis, and attachment to the glomerular basement membrane. Restoration of Myo1e expression in podocytes may therefore strengthen their functional integrity against environmental and mechanical injury. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Background: Tumor-specific, coordinate expression of cancer-testis (CT) genes, mapping to the X chromosome, is observed in more than 60% of non-small cell lung cancer (NSCLC) patients. Although CT gene expression has been unequivocally related to DNA demethylation of promoter regions, the underlying mechanism leading to loss of promoter methylation remains elusive. Polymorphisms of enzymes within the 1-carbon pathway have been shown to affect S-adenosyl methionine (SAM) production, which is the sole methyl donor in the cell. Allelic variants of several enzymes within this pathway have been associated with altered SAM levels either directly, or indirectly as reflected by altered levels of SAH and Homocysteine levels, and altered levels of DNA methylation. We, therefore, asked whether the five most commonly occurring polymorphisms in four of the enzymes in the 1-carbon pathway associated with CT gene expression status in patients with NSCLC. Methods: Fifty patients among a cohort of 763 with NSCLC were selected based on CT gene expression status and typed for five polymorphisms in four genes known to affect SAM generation by allele specific q-PCR and RFLP. Results: We identified a significant association between CT gene expression and the MTHFR 677 CC genotype, as well as the C allele of the SNP, in this cohort of patients. Multivariate analysis revealed that the genotype and allele strongly associate with CT gene expression, independent of potential confounders. Conclusions: Although CT gene expression is associated with DNA demethylation, in NSCLC, our data suggests this is unlikely to be the result of decreased MTHFR function.

Description: Background: Both obesity and type II diabetes mellitus are associated with insulin resistance and abnormal metabolic reactions. This study was conducted to evaluate resting metabolic rate in obese diabetic patients and to assess its relation to glycaemic control. Results: This is a case control study conducted in Gabir AbuEliz centre in Khartoum, Sudan. A random sample of 40 obese diabetic patients (cases) and 40 obese non-diabetic subjects (controls) were interviewed and examined clinically to exclude presence of acute or chronic medical illness. Haemoglobin A1c was measured for each participant using the "NycoCard Haemoglobin A1c test" (Axis -Shield/ Norway). Fasting blood sugar was measured using one touch(R) glucometer (LifeScan Canada Ltd). The PowerLab 8/35 with a gas analyzer (AD Instruments, Castle Hill Australia) was used for measurement of VO2, VCO2 and Respiratory exchange ratio (RER). Resting metabolic rate was calculated using the Weir equation. VO2 (mean+/-SD) ml/min was significantly higher among cases (209.9+/-42.7) compared to the controls (192.4+/-28.1), (P = 0.034). Similarly, VCO2 (mean+/-SD) ml/min was higher among cases (191.4+/-35.0) than controls (178.3+/-22.5), (P = 0.05). Resting metabolic rate "RMR" (mean+/-SD) kcal/day was higher in obese diabetic patients (1480.7 +/- 274.2) than obese non-diabetic subjects (1362.4+/- 184.8), (P = 0.027). Participants with high glycated haemoglobin had higher RMR than those with normal glycated haemoglobin (P = 0.016). Conclusion: It is concluded that resting metabolic rate is significantly higher in obese diabetic patients compared to obese non-diabetics, especially in those with poor glycaemic control.

Description: Background: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is an oncofetal cell surface glycoprotein. Because of its high expression in cancer cells and secretion into serum, CEA has been widely used as a serum tumor marker. Although other members of CEACAM family were investigated for splice variants/variants-derived protein isoforms, few studies about the variants of CEACAM5 have been reported. In this study, we demonstrated the existence of novel CEACAM5 splice variants and splice variant-derived protein isoforms in gastrointestinal cancer cell lines. Results: We identified two novel CEACAM5 splice variants in gastrointestinal (pancreatic, gastric, and colorectal) cancer cell lines. One of the variants possessed an alternative minor splice site that allowed generation of GC-AG intron. Furthermore, CEA protein isoforms derived from the novel splice variants were expressed in cancer cell lines and those protein isoforms were secreted into the culture medium. Although CEA protein isoforms always co-existed with the full-length protein, the secretion patterns of these isoforms did not correlate with the expression patterns. Conclusions: This is the first study to identify the expression of CEA isoforms derived from the novel splice variants processed on the unique splice site. In addition, we also revealed the secretion of those isoforms from gastrointestinal cancer cell lines. Our findings suggested that discrimination between the full-length and identified protein isoforms may improve the clinical utility of CEA as a tumor marker.