Comparing multiple ChIP-sequencing experiments

New high-throughput sequencing technologies can generate millions of short sequences in a single experiment. As the size of the data increases, comparison of multiple experiments on different cell lines under different experimental conditions becomes a big challenge. In this paper, we investigate ways to compare multiple ChIP-sequencing experiments. We specifically studied epigenetic regulation of breast cancer and the effect of estrogen using 50 ChIP-sequencing data from Illumina Genome Analyzer II. First, we evaluate the correlation among different experiments focusing on the total number of reads in transcribed and promoter regions of the genome. Then, we adopt the method that is used to identify the most stable genes in RT-PCR experiments to understand background signal across all of the experiments and to identify the most variable transcribed and promoter regions of the genome. We observed that the most variable genes for transcribed regions and promoter regions are very distinct. Gene ontology and function enrichment analysis on these most variable genes demonstrate the biological relevance of the results. In this study, we present a method that can effectively select differential regions of the genome based on protein-binding profiles over multiple experiments using real data points without any normalization among the samples.     

Diversity and Antimicrobial Activity of Actinomycetes Isolated from Lut Desert: The Extremely Arid Climatic Zones of Iran

Lut desert is situated in one of the extremely arid climatic zones of Iran and is one of the hottest deserts in our plant with the extreme fluctuation of temperature over a day. The main objective of this study is to characterize the diversity of the culturable actinomycetes and preliminary evaluation of their extracts as antimicrobial components on drug resistant pathogens. Twenty-four soil samples were collected, successively diluted and inoculated into the different culture media to support the growth of most culturable bacteria including actinomycetes. Phenotypic and molecular methods were used for accurate identification of recovered isolates particularly actinomycetes at the genus and species levels. The isolates were also evaluated for their inhibitory activities against drug resistant Acinetobacter baumannii, Enterococcus faecium, Klebsiella pneumoniae and Staphylococcus aureus. A total of 56 isolates recovered from the samples. Based on phenotypic tests, 41 isolates were identified as actinomycetes, amongst them 8 isolates were active against drug resistant pathogens. Our study revealed Lut desert, as one of the hottest deserts in the world, is the habitat to diverse taxa of bacteria particularly actinomycetes which have potential novel antimicrobial components.

     

BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models

BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation.     

Structure-Activity Relationship and Mode of Action of a Frog Secreted Antibacterial Peptide B1CTcu5 Using Synthetically and Modularly Modified or Deleted (SMMD) Peptides

All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells.     

Optimization of the Emerson–Trinder enzymatic reaction by response surface methodology

The Emerson–Trinder reaction has been optimized in this work using an initial rate spectrophotometric method and response surface methodology (RSM). In this investigation, the variation range of critical variables along with the fixed parameters were selected based on a preliminary ‘one at a time’ (OVAT) procedure for the subsequent RSM chemometric analysis as follows: pH (6–10), buffer concentration (50–250?mM), 4-aminoantipyrine (4-AAP) concentration (1–5?mM), temperature (25–45°C). The optimum values of fixed parameters were: 4-fluorophenol (4-FP, 30?mM), horseradish peroxidase (HRP) enzyme activity (0.12?U?mL^?1), and the fixed concentration of the H₂O₂ in the chemometric experiments was 11.4 µM. The non-linear nature of the experimental response of the reaction system was explained by a second-order polynomial equation, which revealed the impact of the experimental factors, their interactions and also their optimum values. The results of the reported RSM analysis proved to be quite appropriate for the design and optimization of this reaction, as illustrated by the relatively high value of the determination coefficient (R²=96.7%) for the fitting of quadratic model, along with the satisfactory results generated by the analysis of variance (ANOVA). All the evaluated analytical characteristics of this method: typical reaction progress curves, resulting linear calibration curve, within-day precisions at low and at high levels, and the upper and lower detection limits were, also, reported. In addition, to check the quality of the optimization and validity of the model, the assay of H₂O₂, in pooled serum matrix and in cosmetic samples, was performed.     

Ras Proteins Have Multiple Functions in Vegetative Cells of Dictyostelium

During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.The Ras subfamily proteins are monomeric GTPases that act as molecular switches, cycling between an active GTP-bound and an inactive GDP-bound state (17). Activation is controlled by guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP, and inactivation regulated by GTPase-activating proteins (GAPs) that stimulate the hydrolysis of bound GTP to GDP (17). Activated Ras proteins stimulate numerous downstream signaling pathways that regulate a wide range of cellular processes, including proliferation, cytoskeletal function, chemotaxis, and differentiation (4). The complexity of this regulation has been emphasized by the discovery of the presence of a large number of Ras subfamily homologues in metazoan organisms (19) and elucidation of the roles played by each protein remains a formidable challenge. An important approach to this problem is an analysis of Ras protein function in organisms amenable to genetic analysis.The Dictyostelium genome encodes 14 Ras subfamily members, an unusually large number for such a relatively simple organism (6, 25). Six of these have been partially characterized and have been shown to be involved in a wide variety of processes, including cell movement, polarity, growth, cytokinesis, chemotaxis, macropinocytosis, and multicellular development (5, 15, 23, 25). They exhibit considerable functional specificity, and even the two highly related proteins, RasD and RasG, perform different functions (23, 26). RasC and RasG are the best characterized of these proteins, and both are activated in response to cyclic AMP (cAMP) during aggregation (11). Although both proteins are involved in aggregation, signaling through RasC is more important for the regulation of the cAMP relay, whereas signaling through RasG is more important for cAMP-dependent chemotaxis, but there is some overlap of function (2, 3). Disruption of both the rasC and rasG genes results in a total loss of cAMP-mediated signaling, suggesting that all cAMP signal transduction in early development is partitioned between pathways that use either RasC or RasG (2, 3).In addition to their roles in early development, both RasG and RasC have vegetative cell functions. Cells with a disrupted rasG gene were found to exhibit a reduced growth rate, which was most apparent when cells were grown in suspension, and were multinucleate, indicating a defect in cytokinesis (13, 23). In addition, rasG⁻ cells exhibited reduced motility and polarity and an altered actin distribution. Vegetative rasC⁻ cells had a less pronounced phenotype: changes in actin distribution and motility but normal growth and cytokinesis (16). Given that there was evidence for some overlap of function between RasG and RasC during early development, it was important to determine the extent of their functional overlap in vegetative cells.In the present study, we have compared the potential overlap of RasG and RasC requirements for vegetative cell function in the recently generated isogenic rasC⁻, rasG⁻, and rasC⁻/rasG⁻ strains (2, 3). In addition, the availability of stable rasG⁻ and rasC⁻/rasG⁻ strains has enabled us to determine to what extent RasD, a protein that is highly related to RasG but not present in wild-type vegetative cells, can substitute for loss of function of RasG.     

A flavone from the ethyl acetate extract of Leea rubra leaves with DNA damage protection and antineoplastic activity

Among the major constituents of Leea rubra (Family Vitaceae) leaves, phenolic and flavonoind compounds are most important for therapeutic purposes and the plant parts have been used in traditional medicine to treat several diseases for long. Thus, in order to scientifically confirm the traditional uses of the L. rubra leaves, the present study was designed to investigate the efficacy of the isolated flavones against AAPH induced oxidative damage to pUC19 DNA by gel electrophoresis and antineoplastic activity was evaluated on Ehrlich ascites carcinoma (EAC) bearing Swiss albino mice by evaluating percentage inhibition of cell growth, morphological changes of EAC cells and hematological parameters of the mice. The isolation was carried out by column chromatography and structure was revealed by ¹H-NMR and ¹³C NMR. The result shows that, the isolated compound was identified as myricetin 4'-methoxy-3-O-α-l-rhamnopyranoside based on previously reported data. The isolated flavone effectively inhibited AAPH-induced oxidative damage to DNA; because it could inhibit the formation of circular and linear forms of the DNA. In anti-proliferative assay, 76% growth inhibition of EAC cells was observed as compare to the control mice (p<0.05) at a dose 100 mg/kg body weight. Thus the isolated flavone showed great importance as a possible therapeutic agent in preventing oxidative damage to DNA and the chronic diseases caused by such DNA damage, and can also become important in cancer chemotherapy.     

Assessment of Zoonotic Transmission of Giardia and Cryptosporidium between Cattle and Humans in Rural Villages in Bangladesh

Giardia and Cryptosporidium are important causes of diarrhoea in Bangladesh. The high prevalence of both parasites in humans and cattle in rural Bangladesh and the common use of water ponds by village inhabitants and their animals suggest a potential for zoonotic transmission. Direct transmission of Giardia and Cryptosporidium between cattle and their handlers and indirect transmission through water ponds was investigated. Faecal/stool samples were collected from 623 calves and 125 calf handlers in a cross-sectional survey. In two villages, water samples were collected monthly from water ponds and faecal/stool samples were collected monthly from inhabitants and their cattle. Giardia cysts and Cryptosporidium oocysts were detected in water samples and in faecal/stool samples and positive samples were genotyped, to determine their human or animal origin. The prevalence of Giardia and Cryptosporidium in calves was 22% and 5% respectively. In calf handlers, the prevalence of Giardia and Cryptosporidium was 11.2% and 3.2% respectively. Both in the cross-sectional survey and in the longitudinal study in the villages, G. duodenalis assemblage E was most prevalent in calves, while in humans assemblage AII, BIII and BIV were found. In cattle, Cryptosporidium parvum, C. bovis and C. andersoni were identified, but no Cryptosporidium sequences were obtained from humans. Giardia and Cryptosporidium were detected in 14/24 and 12/24 water samples respectively. G. duodenalis assemblage E and BIV (-like), as well as C. andersoni and C. hominis were identified. Although the presence of Giardia and Cryptosporidium in both water ponds suggests that water-borne transmission of Giardia and Cryptosporidium is possible, the genotyping results indicate that there is no significant direct or indirect (water-borne) transmission of Giardia between cattle and people in this area of rural Bangladesh. No conclusions could be drawn for Cryptosporidium, because of the low number of sequences that were obtained from human and water samples.     

Ablation of Promyelocytic Leukemia Protein (PML) Re-patterns Energy Balance and Protects Mice from Obesity Induced by a Western Diet

The promyelocytic leukemia protein is a well known tumor suppressor, but its role in metabolism is largely unknown. Mice with a deletion in the gene for PML (KO mice) exhibit altered gene expression in liver, adipose tissue, and skeletal muscle, an accelerated rate of fatty acid metabolism, abnormal glucose metabolism, constitutive AMP-activating kinase (AMPK) activation, and insulin resistance in skeletal muscle. Last, an increased rate of energy expenditure protects PML KO mice from the effects of obesity induced by a Western diet. Collectively, our study uncovers a previously unappreciated role of PML in the regulation of metabolism and energy balance in mice.