NucTools: analysis of chromatin feature occupancy profiles from high-throughput sequencing data

Background: Biomedical applications of high-throughput sequencing methods generate a vast amount of data in which numerous chromatin features are mapped along the genome. The results are frequently analysed by creating binary data sets that link the presence/absence of a given feature to specific genomic loci. However, the nucleosome occupancy or chromatin accessibility landscape is essentially continuous. It is currently a challenge in the field to cope with continuous distributions of deep sequencing chromatin readouts and to integrate the different types of discrete chromatin features to reveal linkages between them. Results: Here we introduce the NucTools suite of Perl scripts as well as MATLAB- and R-based visualization programs for a nucleosome-centred downstream analysis of deep sequencing data. NucTools accounts for the continuous distribution of nucleosome occupancy. It allows calculations of nucleosome occupancy profiles averaged over several replicates, comparisons of nucleosome occupancy landscapes between different experimental conditions, and the estimation of the changes of integral chromatin properties such as the nucleosome repeat length. Furthermore, NucTools facilitates the annotation of nucleosome occupancy with other chromatin features like binding of transcription factors or architectural proteins, and epigenetic marks like histone modifications or DNA methylation. The applications of NucTools are demonstrated for the comparison of several datasets for nucleosome occupancy in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). Conclusions: The typical workflows of data processing and integrative analysis with NucTools reveal information on the interplay of nucleosome positioning with other features such as for example binding of a transcription factor CTCF, regions with stable and unstable nucleosomes, and domains of large organized chromatin K9me2 modifications (LOCKs). As potential limitations and problems we discuss how inter-replicate variability of MNase-seq experiments can be addressed

New technologies for examining neuronal ensembles in drug addiction and fear

Correlational data suggest that learned associations are encoded within neuronal ensembles. However, it has been difficult to prove that neuronal ensembles mediate learned behaviours because traditional pharmacological and lesion methods, and even newer cell type-specific methods, affect both activated and non-activated neurons. Additionally, previous studies on synaptic and molecular alterations induced by learning did not distinguish between behaviourally activated and non-activated neurons. Here, we describe three new approaches—Daun02 inactivation, FACS sorting of activated neurons and c-fos-GFP transgenic rats — that have been used to selectively target and study activated neuronal ensembles in models of conditioned drug effects and relapse. We also describe two new tools — c-fos-tTA mice and inactivation of CREB-overexpressing neurons — that have been used to study the role of neuronal ensembles in conditioned fear

Identification of priority health conditions for field-based screening in urban slums in Bangalore, India

BACKGROUND: Urban slums are characterised by unique challenging living conditions, which increase their inhabitants' vulnerability to specific health conditions. The identification and prioritization of the key health issues occurring in these settings is essential for the development of programmes that aim to enhance the health of local slum communities effectively. As such, the present study sought to identify and prioritise the key health issues occurring in urban slums, with a focus on the perceptions of health professionals and community workers, in the rapidly growing city of Bangalore, India. METHODS: The study followed a two-phased mixed methods design. During Phase I of the study, a total of 60 health conditions belonging to four major categories: - 1) non-communicable diseases; 2) infectious diseases; 3) maternal and women's reproductive health; and 4) child health - were identified through a systematic literature review and semi-structured interviews conducted with health professionals and other relevant stakeholders with experience working with urban slum communities in Bangalore. In Phase II, the health issues were prioritised based on four criteria through a consensus workshop conducted in Bangalore. RESULTS: The top health issues prioritized during the workshop were: diabetes and hypertension (non-communicable diseases category), dengue fever (infectious diseases category), malnutrition and anaemia (child health, and maternal and women's reproductive health categories). Diarrhoea was also selected as a top priority in children. These health issues were in line with national and international reports that listed them as top causes of mortality and major contributors to the burden of diseases in India. CONCLUSIONS: The results of this study will be used to inform the development of technologies and the design of interventions to improve the health outcomes of local communities. Identification of priority health issues in the slums of other regions of India, and in other low and lower middle-income countries, is recommended

In silico assessment of biomedical products: the conundrum of rare but not so rare events in two case studies

In silico clinical trials, defined as “The use of individualized computer simulation in the development or regulatory evaluation of a medicinal product, medical device, or medical intervention,” have been proposed as a possible strategy to reduce the regulatory costs of innovation and the time to market for biomedical products. We review some of the the literature on this topic, focusing in particular on those applications where the current practice is recognized as inadequate, as for example, the detection of unexpected severe adverse events too rare to be detected in a clinical trial, but still likely enough to be of concern. We then describe with more details two case studies, two successful applications of in silico clinical trial approaches, one relative to the University of Virginia/Padova simulator that the Food and Drug Administration has accepted as possible replacement for animal testing in the preclinical assessment of artificial pancreas technologies, and the second, an investigation of the probability of cardiac lead fracture, where a Bayesian network was used to combine in vivo and in silico observations, suggesting a whole new strategy of in silico-augmented clinical trials, to be used to increase the numerosity where recruitment is impossible, or to explore patients’ phenotypes that are unlikely to appear in the trial cohort, but are still frequent enough to be of concern

Reduced tricarboxylic acid cycle flux in type 2 diabetes mellitus?

AIMS/HYPOTHESIS: Mitochondrial dysfunction has been postulated to underlie muscular fat accumulation, leading to muscular insulin sensitivity and ultimately type 2 diabetes mellitus. Here we re-interpret previously published data on [(13)C]acetate recovery in breath gas obtained during exercise in type 2 diabetic patients and control individuals. METHODS: When infusing [(13)C]palmitate to estimate fat oxidation, part of the label is lost in exchange reactions of the tricarboxylic acid (TCA) cycle. To correct for this loss of label, an acetate recovery factor (ARF) has previously been used, assuming that 100% of the exogenously provided acetate will enter the TCA cycle. The recovery of acetate in breath gas depends on the TCA cycle activity, hence providing an indirect measure of the latter and a marker of mitochondrial function. RESULTS: Re-evaluation of the available literature reveals that the ARF during exercise is highest in lean, healthy individuals, followed by obese individuals and type 2 diabetic patients. CONCLUSIONS/INTERPRETATION: Revisiting previously published findings on the ARF during exercise in type 2 diabetic patients reveals a reduction in muscular TCA cycle flux, reflecting mitochondrial dysfunction, in these patients. How mitochondrial dysfunction is related to type 2 diabetes mellitus-cause or consequence-requires further study

Deficiency of the Mitochondrial Electron Transport Chain in Muscle Does Not Cause Insulin Resistance

It has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis.Rats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance.The results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance

Worm Burden-Dependent Disruption of the Porcine Colon Microbiota by Trichuris suis Infection

Helminth infection in pigs serves as an excellent model for the study of the interaction between human malnutrition and parasitic infection and could have important implications in human health. We had observed that pigs infected with Trichuris suis for 21 days showed significant changes in the proximal colon microbiota. In this study, interactions between worm burden and severity of disruptions to the microbial composition and metabolic potentials in the porcine proximal colon microbiota were investigated using metagenomic tools. Pigs were infected by a single dose of T. suis eggs for 53 days. Among infected pigs, two cohorts were differentiated that either had adult worms or were worm-free. Infection resulted in a significant change in the abundance of approximately 13% of genera detected in the proximal colon microbiota regardless of worm status, suggesting a relatively persistent change over time in the microbiota due to the initial infection. A significant reduction in the abundance of Fibrobacter and Ruminococcus indicated a change in the fibrolytic capacity of the colon microbiota in T. suis infected pigs. In addition, ∼10% of identified KEGG pathways were affected by infection, including ABC transporters, peptidoglycan biosynthesis, and lipopolysaccharide biosynthesis as well as α-linolenic acid metabolism. Trichuris suis infection modulated host immunity to Campylobacter because there was a 3-fold increase in the relative abundance in the colon microbiota of infected pigs with worms compared to naïve controls, but a 3-fold reduction in worm-free infected pigs compared to controls. The level of pathology observed in infected pigs with worms compared to worm-free infected pigs may relate to the local host response because expression of several Th2-related genes were enhanced in infected pigs with worms versus those worm-free. Our findings provided insight into the dynamics of the proximal colon microbiota in pigs in response to T. suis infection

Activation of nuclear factor-κB in human prostate carcinogenesis and association to biochemical relapse

Nuclear factor (NF)-κB/p65 regulates the transcription of a wide variety of genes involved in cell survival, invasion and metastasis. We characterised by immunohistochemistry the expression of NF-κB/p65 protein in six histologically normal prostate, 13 high-grade prostatic intraepithelial neoplasia (PIN) and 86 prostate adenocarcinoma specimens. Nuclear localisation of p65 was used as a measure of NF-κB active state. Nuclear localisation of NF-κB was only seen in scattered basal cells in normal prostate glands. Prostatic intraepithelial neoplasias exhibited diffuse and strong cytoplasmic staining but no nuclear staining. In prostate adenocarcinomas, cytoplasmic NF-κB was detected in 57 (66.3%) specimens, and nuclear NF-κB (activated) in 47 (54.7%). Nuclear and cytoplasmic NF-κB staining was not correlated (P=0.19). By univariate analysis, nuclear localisation of NF-κB was associated with biochemical relapse (P=0.0009; log-rank test) while cytoplasmic expression did not. On multivariate analysis, serum preoperative prostate specific antigen (P=0.02), Gleason score (P=0.03) and nuclear NF-κB (P=0.002) were independent predictors of biochemical relapse. These results provide novel evidence for NF-κB/p65 nuclear translocation in the transition from PIN to prostate cancer. Our findings also indicate that nuclear localisation of NF-κB is an independent prognostic factor of biochemical relapse in prostate cancer

A microleakage study of gutta-percha/AH Plus and Resilon/Real self-etch systems after different irrigation protocols

The development and maintenance of the sealing of the root canal system is the key to the success of root canal treatment. The resin-based adhesive material has the potential to reduce the microleakage of the root canal because of its adhesive properties and penetration into dentinal walls. Moreover, the irrigation protocols may have an influence on the adhesiveness of resin-based sealers to root dentin. Objective: The objective of the present study was to evaluate the effect of different irrigant protocols on coronal bacterial microleakage of gutta-percha/AH Plus and Resilon/Real Seal Self-etch systems. Material and Methods: One hundred ninety pre-molars were used. The teeth were divided into 18 experimental groups according to the irrigation protocols and filling materials used. The protocols used were: distilled water; sodium hypochlorite (NaOCl)+eDTA; NaOCl+H3PO4; NaOCl+eDTA+chlorhexidine (CHX); NaOCl+H3PO4+CHX; CHX+eDTA; CHX+ H3PO4; CHX+eDTA+CHX and CHX+H3PO4+CHX. Gutta-percha/AH Plus or Resilon/Real Seal Se were used as root-filling materials. The coronal microleakage was evaluated for 90 days against Enterococcus faecalis. Data were statistically analyzed using Kaplan-Meier survival test, Kruskal-Wallis and Mann-Whitney tests. Results: No significant difference was verified in the groups using chlorhexidine or sodium hypochlorite during the chemo-mechanical preparation followed by eDTA or phosphoric acid for smear layer removal. The same results were found for filling materials. However, the statistical analyses revealed that a final flush with 2% chlorhexidine reduced significantly the coronal microleakage. Conclusion: A final flush with 2% chlorhexidine after smear layer removal reduces coronal microleakage of teeth filled with gutta-percha/AH Plus or Resilon/Real Seal SE