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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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.

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 beta-actin, c-fos, and 18S rRNA in plasma. In contrast, shipping blood drawn into Cell-Free RNA BCTTMs (BCTs) showed only a slight change in mRNA copy numbers for circulating beta-actin, c-fos, and 18S rRNA. Moreover, blood stored in K3EDTA tubes at 6[degree sign]C, 22[degree sign]C and 30[degree sign]C for 3 days showed a significant increase in mRNA copy numbers for c-fos and beta-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: A few reports suggested that low levels of Wnt signaling might drive cell reprogramming, but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/beta-catenin signaling is involved in the control of pluripotency gene networks. Additionally, whether physiological beta-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of beta-catenin and wild-type expression of p53, which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells. Results: Introduction of increased beta-catenin signaling, haploid expression of beta-catenin under control by its natural regulators in transferred chromosome 3, resulted in activation of Wnt/beta-catenin networks and dedifferentiation in HONE1 hybrid cell lines, but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous beta-catenin expression. HONE1 hybrid cells displayed stem cell-like properties, including enhancement of CD24+ and CD44+ populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells, including activation of p53- and RB1-mediated tumor suppressor pathways, up-regulation of Nanog-, Oct4-, Sox2-, and Klf4-mediated pluripotency networks, and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/beta-catenin signaling with other pathways such as epithelial-mesenchymal transition, TGF-beta, Activin, BMPR, FGFR2, and LIFR- and IL6ST-mediated cell self-renewal networks. Using beta-catenin shRNA inhibitory assays, a dominant role for beta-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9, CD24, CD44, CD90, and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres. Conclusions: Wnt/beta-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes, tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/beta-catenin signaling with stemness transition networks.

Description: Background: Electrical injury may result in arrhythmias, however atrial fibrillation following low voltage electrocution is not a common occurrence.Case presentationA 70-year-old South-Asian woman with no prior history of cardiovascular disease presented following an accidental low voltage electrocution with loss of consciousness. On initial assessment she was found to be in atrial fibrillation with a moderate to rapid ventricular rate. Troponin I and 2D echo were normal. Transient rise in markers of muscle damage were noted. The arrhythmia resolved spontaneously without active intervention. Conclusion: Loss of consciousness and the path of electrical conduction involving the heart may herald cardiac involvement following electrocution. Low voltage electrocution may cause cardiac insult. Conservative management may suffice in management of atrial fibrillation without cardiovascular compromise.

Description: Background: Vitamin D deficiency rickets is common in China. Genetic factors may play an important role in the susceptibility to rickets. Our study aimed to identify the relationship between three vitamin D-related genes (group specific component [GC], cytochrome P450, family 2, subfamily R, polypeptide 1 (CYP2R1), and 7-dehydrocholesterol reductase/nicotinamide-adenine dinucleotide synthetase 1 (DHCR7/NADSYN1) and rickets in Han Chinese children from northeastern China. Methods: A total of 506 Han children from northeastern China were enrolled in the current study. Twelve SNPs in three candidate genes were genotyped using the SNaPshot assay. Linear regression was used to examine the effect of 12 single-nucleotide polymorphisms (SNPs) on the risk of rickets. Results: In our case--control cohort, six alleles of the 12 SNPs conferred a significantly increased risk of rickets in GC (rs4588 C, P = 0.003, OR: 0.583, 95% CI: 0.412-0.836; rs222020 C, P = 0.009, OR: 1.526, 95% CI:1.117-2.0985; rs2282679 A, P = 0.010, OR: 0.636, 95% CI :0.449-0.900; and rs2298849 C, P = 0.001, OR: 1.709, 95% CI:1.250-2.338) and in CYP2R1 (rs10741657 G, P = 0.019, OR: 1.467, 95% CI:1.070-2.011; and rs2060793 G, P = 0.023, OR: 0.689, 95% CI:0.502-0.944). The results remained significant after adjustment for sex and body mass index. We further analyzed the effect of genotypes under three different genetic models. After using Bonferroni's method for multiple corrections, rs4588, rs2282679, and rs2298849 of the GC gene were significantly associated with rickets under the dominant (P =0.003 for rs4588, P =0.024 for rs2282679, and P =0.005 for rs2298849) and additive models (P = 0.006 for rs4588, P = 0.024 for rs2282679, and P = 0.005 for rs2298849). Haplotype analysis showed that the CAT haplotype of the GC gene (P = 0.005) and the GAA haplotype of the CYP2R1 gene (P = 0.026) were associated with susceptibility to rickets. Conclusions: This case--control study confirmed the strong effect of GC and CYP2R1 loci on rickets in Han children from northeastern China.

Description: Background: Quality assessment and continuous quality feedback to the staff is crucial for safety and efficiency of teleconsultation and triage. This study evaluates whether it is feasible to use an already existing telephone triage protocol to assess the appropriateness of point-of-care and time-to-treat recommendations after teleconsultations. Methods: Based on electronic patient records, we retrospectively compared the point-of-care and time-to-treat recommendations of the paediatric telephone triage protocol with the actual recommendations of trained physicians for children with abdominal pain, following a teleconsultation. Results: In 59 of 96 cases (61%) these recommendations were congruent with the paediatric telephone protocol. Discrepancies were either of organizational nature, due to factors such as local referral policies or gatekeeping insurance models, or of medical origin, such as milder than usual symptoms or clear diagnosis of a minor ailment. Conclusions: A paediatric telephone triage protocol may be applicable in healthcare systems other than the one in which it has been developed, if triage rules are adapted to match the organisational aspects of the local healthcare system.

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: Background: The lectin-like domain of TNF-α 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: Praziquantel (PZQ) is the drug of choice for treatment of all human schistosomes. It is used in population based targeted or mass deworming strategies in several countries. The effect of PZQ on S.hematobium has not been studied in Ethiopia. The objective of this study was to determine the efficacy of PZQ against S.haematobium in Dulshatalo village, western Ethiopia. Methods: A prospective study was conducted from October to December, 2007. Urine samples from 341 residents were collected and screened for haematuria and proteinuria using urinalysis dipstick. S.haematobium eggs were detected and quantified using filtration techniques. The participants who were positive for haematuria were treated with a standard dose of PZQ (40 mg/kg). Data on pre and 24 hours post treatment symptoms were collected via questionnaire. Urine samples were also collected 7 weeks after treatment and examined to assess the cure and the egg reduction rates. Results: The prevalence of S.haematobium among the study participants was 57.8% (197/341). Haematuria was detected in 234(68.6%) of the study participants. For PZQ efficacy asessment, 152 of the treated participants were considered. The presence of S.haemetaobium eggs showed statistically significant association (p 〈 0.05) with haematuria and proteinuria. Seven weeks post treatment, the extent of haematuria and proteinuria decreased from 100% to 40.8% and 94.07% to 48.7%, respectively. The cure and the parasitological egg reduction rates seven weeks post treatment were 86% and 85%, respectively. Post treatment symptoms revealed a wide range of side effects including straining, abdominal pain, nausea and headache. Conclusions: There were marked cure and egg reduction rates, together with mild and short lived side effects of PZQ for treatment of S.haematobium, in this study.

Description: Background: MicroRNAs (miRNAs) have been recognized as one of the key regulatory non-coding RNAs that are involved in a number of basic cellular processes. miRNA expression profiling helps to identify miRNAs that could serve as biomarkers. Next generation sequencing (NGS) platforms provide the most effective way of miRNA profiling, particularly as expression of different isoforms of miRNA (IsomiRs) can be estimated by NGS. Therefore, it is now possible to discern the overall complexity of miRNA populations that participate in gene regulatory networks. It is thus important to consider different isoforms of miRNA as part of total profiling in order to understand all aspects of the biology of miRNAs. Results: Here next generation sequencing data of small RNAs derived from normal peripheral blood mononuclear cells (PBMC) and Chronic myeloid leukemia (CML) patients has been used to generate miRNA profiles using a computation pipeline which can identify isomiRs that are natural variants of mature miRNAs. IsomiR profiles have been generated for all the 5p and 3p miRNAs (previously known as major mature miRNA and minor or miRNA*) and the data has been presented as a composite total miRNA transcriptome. The results indicated that the most abundant isomiR sequence of about 68% miRNAs, did not match the reference miRNA sequence as entered in the miRBase and that there is a definite pattern in relative concentration of different isomiRs derived from same precursors. Finally, a total of 17 potential novel miRNA sequences were identified suggesting that there are still some new miRNAs yet to be discovered. Conclusions: Inclusion of different isoforms provides a detailed miRnome of a cell type or tissues. Availability of miRnome will be useful for finding biomarkers of different cell types and disease states. Our results also indicate that the relative expression levels of different isoforms of a miRNA are likely to be dynamic and may change with respect to changes in the cell or differentiation status.

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: 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 beta-Tub9) was evaluated to identify reference genes during the first hours of interaction (12, 48 and 72hpi) 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: Background: Polymyositis is an immune-mediated myopathy with clinical features of proximal muscle weakness. Dysphagia and neck flexor weakness can develop along with respiratory muscle weakness as the disease progresses. Kennedy disease or X-linked spinobulbar muscular atrophy is a rare X-linked recessive disorder with clinical features of slowly progressive atrophy and weakness of limb and bulbar muscles. These two disorders may have overlapping clinical manifestations.Case presentationWe present the case of a 52-year-old Filipino man with chronic weakness involving his proximal muscle groups who carried the diagnosis of polymyositis and was refractory to multiple immunomodulatory therapies. Further neurologic examination and history taking along with selective serologic and electrodiagnostic studies instead confirmed the diagnosis of Kennedy disease. Conclusions: Distinction between polymyositis and Kennedy disease may be difficult given the potential overlapping clinical manifestations. However, with careful neurological history taking, examination, and selective serologic plus electrodiagnostic investigations the correct diagnosis may be made, thus sparing the patient ineffective therapy. One must always be sure of the diagnosis of polymyositis before it's classified as refractory.

Description: Background: Irritable bowel syndrome (IBS) is a common chronic functional gastrointestinal disorder. Post-infectious IBS (PI-IBS) is a subset of IBS that accounts for a large proportion of IBS patients. The PI-IBS symptoms meet the Rome criteria for IBS with diarrhoea (IBS-D) or IBS with mixed bowel habits (IBS-M). A low-grade inflammation has been reported to occur in PI-IBS. Abnormalities in intestinal endocrine cells have been reported in both sporadic IBS and PI-IBS.Case presentationA 20-year-old female with a diagnosis of IBS with constipation (IBS-C), according to Rome III criteria, contracted Campylobacter-induced gastroenteritis, after which her symptom pattern changed to IBS-M. She showed an intestinal low-grade inflammation that was manifested by an increase in the number of intraepithelial and lamina propria leucocytes and lymphocytes and an increase in the density of mast cells in lamina propria. There was also an increase in the density of intestinal serotonin and peptide YY (PYY) cells and a decrease in the density of rectal somatostatin cells. Follow-up of the patient at 4-months post-infection revealed reduction of IBS symptoms and an improvement in her quality of life. However, 6 months following the Campylobacter infection, the patient switched back from IBS-D to IBS-C, probably due to recovery from PI-IBS. The patient was treated with prucalopride, which is serotonin 5HT4 receptor agonist. Six months later following this treatment, the symptoms were reduced and the quality of life improved in the reported patient. Conclusions: Gastroenteritis in patients with IBS-C causes a post-infectious, low-grade inflammation. Interaction between immune-cells and intestinal endocrine cells increases the density of certain endocrine cells, which in turn might be responsible for the change in the symptom pattern, the milder symptoms and the improvement in the quality of life seen in the reported patient. The findings in this case raise the question as to whether intestinal infections are responsible for the previously reported switching of IBS from one subtype to another over time.

Description: Background: Nodular lung disease is a rare presentation of sarcoidosis. Radiologically it can present as multiple pulmonary masses or solitary lung nodule.Case presentationWe report three cases of nodular sarcoidosis in young females of Asian origin who had initially presented with dry cough and worsening dyspnea non-responsive to initially administered antibiotics. Pulmonary nodules were discovered upon radiographic imaging in all three cases which raised concern for the possibility of neoplastic processes. Subsequent biopsies revealed granulomatous inflammation indicative of sarcoidosis. All cases responded very well to systemic corticosteroids. Conclusion: Sarcoidosis may present as nodular infiltrates which alerts the treating physician to other neoplastic and infectious diseases of the lungs. Appropriate workup may reveal the true nature of this disease and hence, simplify treatment.

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: Irritable bowel syndrome (IBS) is a common chronic functional gastrointestinal disorder. Post-infectious IBS (PI-IBS) is a subset of IBS that accounts for a large proportion of IBS patients. The PI-IBS symptoms meet the Rome criteria for IBS with diarrhoea (IBS-D) or IBS with mixed bowel habits (IBS-M). A low-grade inflammation has been reported to occur in PI-IBS. Abnormalities in intestinal endocrine cells have been reported in both sporadic IBS and PI-IBS.Case presentationA 20-year-old female with a diagnosis of IBS with constipation (IBS-C), according to Rome III criteria, contracted Campylobacter-induced gastroenteritis, after which her symptom pattern changed to IBS-M. She showed an intestinal low-grade inflammation that was manifested by an increase in the number of intraepithelial and lamina propria leucocytes and lymphocytes and an increase in the density of mast cells in lamina propria. There was also an increase in the density of intestinal serotonin and peptide YY (PYY) cells and a decrease in the density of rectal somatostatin cells. Follow-up of the patient at 4-months post-infection revealed reduction of IBS symptoms and an improvement in her quality of life. However, 6 months following the Campylobacter infection, the patient switched back from IBS-M to IBS-C, probably due to recovery from PI-IBS. The patient was treated with prucalopride, which is serotonin 5HT4 receptor agonist. Six months later following this treatment, the symptoms were reduced and the quality of life improved in the reported patient. Conclusions: Gastroenteritis in patients with IBS-C causes a post-infectious, low-grade inflammation. Interaction between immune-cells and intestinal endocrine cells increases the density of certain endocrine cells, which in turn might be responsible for the change in the symptom pattern, the milder symptoms and the improvement in the quality of life seen in the reported patient. The findings in this case raise the question as to whether intestinal infections are responsible for the previously reported switching of IBS from one subtype to another over time.

Description: Background: The non-steroidal anti-inflammatory drug (NSAID) sulindac has shown efficacy in preventing colorectal cancer. This potent anti-tumorigenic effect is mediated through multiple cellular pathways but is also accompanied by gastrointestinal side effects, such as colon inflammation. We have recently shown that sulindac can cause up-regulation of pro-inflammatory factors in the mouse colon mucosa. The aim of this study was to determine the signaling pathways that mediate the transcriptional activation of pro-inflammatory cytokines in colon cancer epithelial cells treated with sulindac sulfide. Results: We found that sulindac sulfide increased NF-kappaB signaling in HCT15, HCT116, SW480 and SW620 cells, although the level of induction varied between cell lines. The drug caused a decrease in IkappaBalpha levels and an increase of p65(RelA) binding to the NF-kappaB DNA response element. It induced expression of IL-8, ICAM1 and A20, which was inhibited by the NF-kappaB inhibitor PDTC. Sulindac sulfide also induced activation of the AP-1 transcription factor, which co-operated with NF-kappaB in up-regulating IL-8. Up-regulation of NF-kappaB genes was most prominent in conditions where only a subset of cells was undergoing apoptosis. In TNFalpha stimulated conditions the drug treatment inhibited phosphorylation on IkappaBalpha (Ser 32) which is consistent with previous studies and indicates that sulindac sulfide can inhibit TNFalpha-induced NF-kappaB activation. Sulindac-induced upregulation of NF-kappaB target genes occurred early in the proximal colon of mice given a diet containing sulindac for one week. Conclusions: This study shows for the first time that sulindac sulfide can induce pro-inflammatory NF-kappaB and AP-1 signaling as well as apoptosis in the same experimental conditions. Therefore, these results provide insights into the effect of sulindac on pro-inflammatory signaling pathways, as well as contribute to a better understanding of the mechanism of sulindac-induced gastrointestinal side effects.

Description: Background: Diacylglycerol kinase (DGK) is a key enzyme that regulates diacylglycerol (DG) turnover and is involved in a variety of physiological functions. The isoform DGKtheta has a unique domain structure and is the sole member of type V DGK. To reveal the spatial and temporal expression of DGKtheta we performed immunohistochemical staining on paraffin sections of mouse embryos. Results: At an early stage of development (E10.5 and 11.5), the expression of DGKtheta was prominently detected in the brain, spinal cord, dorsal root ganglion, and limb bud, and was also moderately detected in the bulbus cordis and the primordium of the liver and gut. At later stages (E12.5 and 14.5), DGKtheta expression persisted or increased in the neocortex, epithalamus, hypothalamus, medulla oblongata, and pons. DGKtheta was also evident in the epidermis, and nearly all epithelia of the oropharyngeal membrane, digestive tract, and bronchea. At prenatal developmental stages (E16.5 and E18.5), the expression pattern of DGKtheta was maintained in the central nervous system, intestine, and kidney, but was attenuated in the differentiated epidermis. Conclusion: These results suggest that DGKtheta may play important physiological roles not only in the brain, but also in diverse organs and tissues during the embryonic stages.

Description: Background: Physician notes routinely recorded during patient care represent a vast and underutilized resource for human disease studies on a population scale. Their use in research is primarily limited by the need to separate confidential patient information from clinical annotations, a process that is resource-intensive when performed manually. This study seeks to create an automated method for de-identifying physician notes that does not require large amounts of private information: in addition to training a model to recognize Protected Health Information (PHI) within private physician notes, we reverse the problem and train a model to recognize non-PHI words and phrases that appear in public medical texts. Methods: Public and private medical text sources were analyzed to distinguish common medical words and phrases from Protected Health Information. Patient identifiers are generally nouns and numbers that appear infrequently in medical literature. To quantify this relationship, term frequencies and part of speech tags were compared between journal publications and physician notes. Standard medical concepts and phrases were then examined across ten medical dictionaries. Lists and rules were included from the US census database and previously published studies. In total, 28 features were used to train decision tree classifiers. Results: The model successfully recalled 98% of PHI tokens from 220 discharge summaries. Cost sensitive classification was used to weight recall over precision (98% F10 score, 76% F1 score). More than half of the false negatives were the word "of" appearing in a hospital name. All patient names, phone numbers, and home addresses were at least partially redacted. Medical concepts such as "elevated white blood cell count" were informative for de-identification. The results exceed the previously approved criteria established by four Institutional Review Boards. Conclusions: The results indicate that distributional differences between private and public medical text can be used to accurately classify PHI. The data and algorithms reported here are made freely available for evaluation and improvement.

Description: Background: Chromosomal instability is a hallmark of human cancer caused by errors in mitotic control and chromosome segregation. STAG2 encodes a subunit of the cohesion complex that participates in mitotic chromatid separation and was recently found to show low expression and inactivating mutations in Ewing's sarcoma, melanoma and glioblastoma.In the childhood tumor neuroblastoma (NB) segmental chromosomal alterations are associated with poor prognosis whereas tumors displaying whole chromosome gains and losses have a much better prognosis.MethodAs the genetic contribution to aneuploidy is unknown in NB, we investigated the presence of STAG2 mutations through sequence analysis of all 33 coding exons in 37 primary NB tumors.Results and conclusionAs no STAG2 mutation was detected in this study, we conclude that inactivating mutation of STAG2 is not likely causative to neuroblastoma aneuploidy.

Description: Background: Praziquantel (PZQ) is the drug of choice for treatment of all human schistosomes. It is used in population based targeted or mass deworming strategies in several countries. The effect of PZQ on S. hematobium has not been studied in Ethiopia. The objective of this study was to determine the efficacy of PZQ against S. haematobium in Dulshatalo village, western Ethiopia. Methods: A prospective study was conducted from October to December, 2007. Urine samples from 341 residents were collected and screened for haematuria and proteinuria using urinalysis dipstick. S. haematobium eggs were detected and quantified using filtration techniques. The participants who were positive for haematuria were treated with a standard dose of PZQ (40 mg/kg). Data on pre and 24 hours post treatment symptoms were collected via questionnaire. Urine samples were also collected 7 weeks after treatment and examined to assess the cure and the egg reduction rates. Results: The prevalence of S. haematobium among the study participants was 57.8% (197/341). Haematuria was detected in 234 (68.6%) of the study participants. For PZQ efficacy asessment, 152 of the treated participants were considered. The presence of S. haemetaobium eggs showed statistically significant association (p 

Description: Background: Zinc, an essential trace element, inhibits osteoclast differentiation in vitro and in vivo. The molecular mechanism for the inhibitory effect of zinc, however, is poorly understood. The purpose of this study was to investigate the effect of zinc and determine its molecular mechanism on receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis in mouse bone marrow-derived monocyte cells (BMMs) and RAW264.7 cells. Results: In BMMs, zinc treatment during osteoclast differentiation decreased RANKL-induced osteoclast formation in a dose-dependent manner. We show that zinc suppressed the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Zinc also accumulated phospho-Nfatc1 (p-Nfatc1) in the cytosol in a dose-dependent manner and inhibited the translocation of Nfatc1 to the nucleus in RAW264.7 cells. Zinc suppressed the activities of Nfatc1 in the nucleus without changing the activities of NF-kappaB in RAW264.7 cells. In contrast, calcineurin activity decreased in response to zinc but its protein level was unchanged. RANKL-induced Ca2+ oscillations were inhibited by zinc treatment, but phospho-phospholipase Cgamma1 (p-PLCgamma1), the upstream signaling molecule of Ca2+ oscillations, was unaffected. Moreover, a constitutively active form of Nfatc1 obviously rescued suppression of osteoclastogenesis by zinc. Conclusions: Taken together, these results demonstrate for the first time that the inhibitory effect of zinc during osteoclastogesis is caused by suppressing the Ca2+-Calcineurin-NFATc1 signaling pathway. Thus, zinc may be a useful therapeutic candidate for the prevention of bone loss caused by NFATc1 activation in osteoclasts.

Description: Background: As adolescents with hemophilia approach adulthood, they are expected to assume responsibility for their disease management. A bilingual (English and French) Internet-based self-management program, "Teens Taking Charge: Managing Hemophilia Online," was developed to support adolescents with hemophilia in this transition. This study explored the usability of the website and resulted in refinement of the prototype. Methods: A purposive sample (n=18; age 13--18; mean age 15.5 years) was recruited from two tertiary care centers to assess the usability of the program in English and French. Qualitative observations using a "think aloud" usability testing method and semi-structured interviews were conducted in four iterative cycles, with changes to the prototype made as necessary following each cycle. This study was approved by research ethics boards at each site. Results: Teens responded positively to the content and appearance of the website and felt that it was easy to navigate and understand. The multimedia components (videos, animations, quizzes) were felt to enrich the experience. Changes to the presentation of content and the website user-interface were made after the first, second and third cycles of testing in English. Cycle four did not result in any further changes. Conclusions: Overall, teens found the website to be easy to use. Usability testing identified end-user concerns that informed improvements to the program. Usability testing is a crucial step in the development of Internet-based self-management programs to ensure information is delivered in a manner that is accessible and understood by users.

Description: Background: Methamphetamine-abuse is a worldwide health problem for which no effective therapy is available. Inhibition of methamphetamine-induced transporter-mediated dopamine (DA) release could be a useful approach to treat methamphetamine-addiction. We assessed the potencies of bupropion, methylphenidate, and 3,4-methylenedioxypyrovalerone (MDPV) to block DA uptake or to inhibit methamphetamine-induced DA release in HEK-293 cells expressing the human DA transporter.FindingsBupropion, methylphenidate, and MDPV inhibited methamphetamine-induced DA release with relative potencies corresponding to their potencies to block DA uptake (potency ranks: MDPV 〉 methylphenidate 〉 bupropion). Conclusions: Bupropion and methylphenidate antagonize the effects of methamphetamine in vitro and may be potential candidates for the treatment of stimulant addiction. However, drugs that very potently antagonize the effect of methamphetamine are likely to also exhibit considerable abuse liability (MDPV 〉 methylphenidate 〉 bupropion).

Description: Background: Studies of online database(s) showed that convincing examples of eukaryote PPKs derived from bacteria type PPK1 and PPK2 enzymes are rare and currently confined to a few simple eukaryotes. These enzymes probably represent several separate horizontal transfer events. Retention of such sequences may be an advantage for tolerance to stresses such as desiccation or nutrient depletion for simple eukaryotes that lack more sophisticated adaptations available to multicellular organisms. We propose that the acquisition of encoding sequences for these enzymes by horizontal transfer enhanced the ability of early plants to colonise the land. The improved ability to sequester and release inorganic phosphate for carbon fixation by photosynthetic algae in the ocean may have accelerated or even triggered global glaciation events. There is some evidence for DNA sequences encoding PPKs in a wider range of eukaryotes, notably some invertebrates, though it is unclear that these represent functional genes.Polyphosphate (poly P) is found in all cells, carrying out a wide range of essential roles. Studied mainly in prokaryotes, the enzymes responsible for synthesis of poly P in eukaryotes (polyphosphate kinases PPKs) are not well understood. The best characterised enzyme from bacteria known to catalyse the formation of high molecular weight polyphosphate from ATP is PPK1 which shows some structural similarity to phospholipase D. A second bacterial PPK (PPK2) resembles thymidylate kinase. Recent reports have suggested a widespread distribution of these bacteria type enzymes in eukaryotes. Results: On -- line databases show evidence for the presence of genes encoding PPK1 in only a limited number of eukaryotes. These include the photosynthetic eukaryotes Ostreococcus tauri, O. lucimarinus, Porphyra yezoensis, Cyanidioschyzon merolae and the moss Physcomitrella patens, as well as the amoeboid symbiont Capsaspora owczarzaki and the non-photosynthetic eukaryotes Dictyostelium (3 species), Polysphondylium pallidum and Thecamonas trahens. A second bacterial PPK (PPK2) is found in just two eukaryotes (O. tauri and the sea anemone Nematostella vectensis). There is some evidence for PPK1 and PPK2 encoding sequences in other eukaryotes but some of these may be artefacts of bacterial contamination of gene libraries. Conclusions: Evidence for the possible origins of these eukaryote PPK1s and PPK2s and potential prokaryote donors via horizontal gene transfer is presented. The selective advantage of acquiring and maintaining a prokaryote PPK in a eukaryote is proposed to enhance stress tolerance in a changing environment related to the capture and metabolism of inorganic phosphate compounds. Bacterial PPKs may also have enhanced the abilities of marine phytoplankton to sequester phosphate, hence accelerating global carbon fixation.

Description: Background: With the advance of post-genomic technologies, the need for tools to manage large scale data in biology becomes more pressing. This involves annotating and storing data securely, as well as granting permissions flexibly with several technologies (all array types, flow cytometry, proteomics) for collaborative work and data sharing. This task is not easily achieved with most systems available today.FindingsWe developed Djeen (Database for Joomla!'s Extensible Engine), a new Research Information Management System (RIMS) for collaborative projects. Djeen is a user-friendly application, designed to streamline data storage and annotation collaboratively. Its database model, kept simple, is compliant with most technologies and allows storing and managing of heterogeneous data with the same system. Advanced permissions are managed through different roles. Templates allow Minimum Information (MI) compliance. Conclusion: Djeen allows managing project associated with heterogeneous data types while enforcing annotation integrity and minimum information. Projects are managed within a hierarchy and user permissions are finely-grained for each project, user and group.Djeen Component source code (version 1.5.1) and installation documentation are available under CeCILL license from http://sourceforge.net/projects/djeen/files and supplementary material.

Description: Background: Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using enrichment with the methyl-binding domain protein (MBD) are restricted to large (〉=1 mug) DNA samples, limiting the analysis of small tissue samples. Here we present a technique that enables characterization of genome-wide tissue-specific methylation patterns from nanogram quantities of DNA. Results: We have developed a methodology utilizing MBD2b/MBD3L1 enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized tiling arrays, which we termed MeKL-chip. Kinase modification in combination with the addition of PEG has increased ligation-mediated PCR amplification over 20-fold, enabling 〉400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng of DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2,498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing. Conclusions: MeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs.

Description: Background: Studies of online database(s) showed that convincing examples of eukaryote PPKs derived from bacteria type PPK1 and PPK2 enzymes are rare and currently confined to a few simple eukaryotes. These enzymes probably represent several separate horizontal transfer events. Retention of such sequences may be an advantage for tolerance to stresses such as desiccation or nutrient depletion for simple eukaryotes that lack more sophisticated adaptations available to multicellular organisms. We propose that the acquisition of encoding sequences for these enzymes by horizontal transfer enhanced the ability of early plants to colonise the land. The improved ability to sequester and release inorganic phosphate for carbon fixation by photosynthetic algae in the ocean may have accelerated or even triggered global glaciation events. There is some evidence for DNA sequences encoding PPKs in a wider range of eukaryotes, notably some invertebrates, though it is unclear that these represent functional genes.Polyphosphate (poly P) is found in all cells, carrying out a wide range of essential roles. Studied mainly in prokaryotes, the enzymes responsible for synthesis of poly P in eukaryotes (polyphosphate kinases PPKs) are not well understood. The best characterised enzyme from bacteria known to catalyse the formation of high molecular weight polyphosphate from ATP is PPK1 which shows some structural similarity to phospholipase D. A second bacterial PPK (PPK2) resembles thymidylate kinase. Recent reports have suggested a widespread distribution of these bacteria type enzymes in eukaryotes. Results: On – line databases show evidence for the presence of genes encoding PPK1 in only a limited number of eukaryotes. These include the photosynthetic eukaryotes Ostreococcus tauri, O. lucimarinus, Porphyra yezoensis, Cyanidioschyzon merolae and the moss Physcomitrella patens, as well as the amoeboid symbiont Capsaspora owczarzaki and the non-photosynthetic eukaryotes Dictyostelium (3 species), Polysphondylium pallidum and Thecamonas trahens. A second bacterial PPK (PPK2) is found in just two eukaryotes (O. tauri and the sea anemone Nematostella vectensis). There is some evidence for PPK1 and PPK2 encoding sequences in other eukaryotes but some of these may be artefacts of bacterial contamination of gene libraries. Conclusions: Evidence for the possible origins of these eukaryote PPK1s and PPK2s and potential prokaryote donors via horizontal gene transfer is presented. The selective advantage of acquiring and maintaining a prokaryote PPK in a eukaryote is proposed to enhance stress tolerance in a changing environment related to the capture and metabolism of inorganic phosphate compounds. Bacterial PPKs may also have enhanced the abilities of marine phytoplankton to sequester phosphate, hence accelerating global carbon fixation.

Description: Background: Mutations within the C-terminal region of the COL6A1 gene are only detected in Ullrich/Bethlem patients on extremely rare occasions.Case presentationHerein we report two Brazilian brothers with a classic Ullrich phenotype and compound heterozygous for two truncating mutations in COL6A1 gene, expected to result in the loss of the alpha1(VI) chain C2 subdomain. Despite the reduction in COL6A1 RNA level due to nonsense RNA decay, three truncated alpha1 (VI) chains were produced as protein variants encoded by different out-of-frame transcripts. Collagen VI matrix was severely decreased and intracellular protein retention evident. Conclusion: The altered deposition of the fibronectin network highlighted abnormal interactions of the mutated collagen VI, lacking the alpha1(VI) C2 domain, within the extracellular matrix, focusing further studies on the possible role played by collagen VI in fibronectin deposition and organization.

Description: Background: Within the field of record linkage, numerous data cleaning and standardisation techniques are employed to ensure the highest quality of links. While these facilities are common in record linkage software packages and are regularly deployed across record linkage units, little work has been published demonstrating the impact of data cleaning on linkage quality. Methods: A range of cleaning techniques was applied to both a synthetically generated dataset and a large administrative dataset previously linked to a high standard. The effect of these changes on linkage quality was investigated using pairwise F-measure to determine quality. Results: Data cleaning made little difference to the overall linkage quality, with heavy cleaning leading to a decrease in quality. Further examination showed that decreases in linkage quality were due to cleaning techniques typically reducing the variability -- although correct records were now more likely to match, incorrect records were also more likely to match, and these incorrect matches outweighed the correct matches, reducing quality overall. Conclusion: Data cleaning techniques have minimal effect on linkage quality. Care should be taken during the data cleaning process.

Description: Background: Taenia solium taeniasis/cysticercosis is a zoonotic helminth infection mainly found in rural regions of Africa, Asia and Latin America. In endemic areas, diagnosis of cysticercosis largely depends on serology, but these methods have their drawbacks and require improvement. This implies better knowledge of the proteins secreted and excreted by the parasite. In a previous study, we used a custom protein database containing protein sequences from related helminths to identify T. solium metacestode excretion/secretion proteins. An alternative or complementary approach would be to use expressed sequence tags combined with BLAST and protein mapping to supercontigs of Echinococcus granulosus, a closely related cestode. In this study, we evaluate this approach and compare the results to those obtained in the previous study.FindingsWe report 297 proteins organized in 106 protein groups based on homology. Additional classification was done using Gene Ontology information on biological process and molecular function. Of the 106 protein groups, 58 groups were newly identified, while 48 groups confirmed previous findings. Blast2GO analysis revealed that the majority of the proteins were involved in catalytic activities and binding. Conclusions: In this study, we used translated expressed sequence tags combined with BLAST and mapping strategies to both confirm and complement previous research. Our findings are comparable to recent studies on other helminth genera like Echinococcus, Schistosoma and Clonorchis, indicating similarities between helminth excretion/secretion proteomes.

Description: Background: Mobility disability is a major adverse health outcome associated with aging and an impediment to older adults’ well-being and behaviors in social and leisure activities. It has been shown that lifestyle factors, including smoking and alcohol consumption, have been used as coping strategies to deal with the negative impact of disability. The aim of this study was to determine the prevalence of smoking and alcohol consumption among older Canadians with different levels of mobility disabilities and to examine factors associated with these two lifestyle patterns among those with disabilities. Methods: Secondary data analysis was performed using individuals (n = 6,038) aged 65 years and older from both the 2001 Participation and Activity Limitation Survey and the 2003 Canadian Community Health Survey. Multivariate logistic regressions examined the relationship between disability severity and smoking as well as alcohol consumption while controlling for potential confounding socioeconomic factors. Results: The proportion of current smokers among seniors with less-severe and more-severe mobility disabilities and those in the general population was comparable with 12.55%, 11.57% and 11.93%, respectively. Forty-eight percent of seniors in the general population consumed alcohol regularly, compared to only 12.85% with more-severe mobility disabilities. No significant association was shown between the severity level of mobility disabilities and smoking (odds ratio = 0.90, 95% confidence interval: 0.75, 1.08). However, seniors having more-severe disability were less likely to consume alcohol regularly (odds ratio = 0.76, 95% confidence interval: 0.65, 0.89). Other variables including age, gender, income, living status, and social participation also impacted these lifestyle patterns among the study population. Conclusions: Smoking and alcohol patterns present different associations with the severity level of mobility disabilities. Compared with the general population, elderly Canadians with mobility disabilities had similar smoking prevalence but differ significantly in terms of alcohol consumption. Results from this research will be relevant to decision makers involved in program planning, health education, and policy development as it pertains to the prevention and management of age-related disability.

Description: Background: In the current study we report on the effects of an implementation strategy in the form of a training programme on the assessed work limitations of a client with depression by insurance physicians (IPs) participating in a RCT. These assessed work limitations of a client were in the form of scores on the List of Functional Abilities (LFA).MethodWe conducted a randomised controlled trial (RCT) for IPs in which we compared the intervention of a specially developed training programme with the usual methods of implementation and training currently used. The outcome was the mean sum score and the inter-rater reliability (Intraclass Correlation Coefficient, ICC) of the LFA scores. These LFA scores were scored by the IPs participating in the RCT for the work limitations of the cases presented in different videos, two videos before the training and two after the training of the intervention group. Results: At baseline, the intervention group (IG) consisted of 21 IPs and the control group (CG) of 19. For one participant of the IG and for one of the CG the LFAs of the two case reports after training were not available. Before training the sum scores for the first case report did not differ significantly between the groups, while the mean sum score was higher in the IG than in the CG for the second case report. For both case reports after training a higher score was found in the IG than in the CG. The inter-rater reliability measured for the two case reports before training was about the same in the IG and the CG: 0.64 and 0.65, respectively. For the two case reports after training, the ICC was higher in the IG than in the CG: 0.69 and 0.54, respectively. This difference was not significant however. Conclusion: It would appear that the implementation of a specially designed training programme on guidelines for depression may lead to greater inter-rater reliability in the assessments by insurance physicians of the work limitations of clients with depression. It is, however, important to note that insurance physicians who receive training may find more work limitations than those who do not.Trial registration: Netherlands' Trial Register NTR1863

Description: Background: Visceral leishmaniasis is a disease with great variability regarding the clinical manifestations in humans and dogs. Chronically infected dogs may develop neurological disorders, however, there are few reports that characterize the lesions and make clear the pathogenesis of the canine cerebral leishmaniasis. Concomitant with Leishmania chagasi, dogs may be infected by opportunistic pathogens, such as Toxoplasma gondii and Neospora caninum, which may contribute to the occurrence of lesions in the central nervous system. Hence, we aimed to compare the T and B lymphocytes population in the brains of infected dogs with seropositivity to L. chagasi, T. gondii and N. caninum concurrently (n = 24), seropositivity only to L. chagasi (n = 31), and seropositivity to T. gondii and N. caninum (n = 16). Uninfected dogs were used as control (n = 10). Results: Inflammatory lesions, characterised by mononuclear cell accumulation, composed mainly of CD3+ T lymphocytes predominated in several encephalic regions of the dogs from all the three infected groups, with no difference among them (P = 0.0004), whereas CD79alpha+ B lymphocytes were detected in very small intensity and presented no difference among groups (P = 0.5313). Furthermore, no association among diseases was detected at the serological enquire. Conclusions: We demonstrate that the peripheral infection by L. chagasi per se can promote the influx of lymphocytes within the nervous milieu as occurs during Toxoplasma and Neospora infections, and the concomitant seropositivity against these pathogens does not exacerbate the inflammatory brain lesions. Therefore, these findings give additional support that the brain should be included in the list of organs affected by visceral leishmaniasis and that even asymptomatic infected dogs may develop brain lesions.

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