Comprehensive Map of Molecules Implicated in Obesity

Obesity is a global epidemic affecting over 1.5 billion people and is one of the risk factors for several diseases such as type 2 diabetes mellitus and hypertension. We have constructed a comprehensive map of the molecules reported to be implicated in obesity. A deep curation strategy was complemented by a novel semi-automated text mining system in order to screen 1,000 full-length research articles and over 90,000 abstracts that are relevant to obesity. We obtain a scale free network of 804 nodes and 971 edges, composed of 510 proteins, 115 genes, 62 complexes, 23 RNA molecules, 83 simple molecules, 3 phenotype and 3 drugs in “bow-tie” architecture. We classify this network into 5 modules and identify new links between the recently discovered fat mass and obesity associated FTO gene with well studied examples such as insulin and leptin. We further built an automated docking pipeline to dock orlistat as well as other drugs against the 24,000 proteins in the human structural proteome to explain the therapeutics and side effects at a network level. Based upon our experiments, we propose that therapeutic effect comes through the binding of one drug with several molecules in target network, and the binding propensity is both statistically significant and different in comparison with any other part of human structural proteome.     

Perturbations of liver plasma membranes induced by Ca2+ are detected using a fatty acid spin label and adenylate cyclase as membrane probes

Ca2+ decreased the lipid fluidity of rat liver plasma membranes labeled with 5-nitroxide stearate, I(12,3), as indicated by the order parameter (S). These effects form a reversible, saturable process with an association constant of 1 x 10(3) M-1. Arrhenius-type plots of S indicated that the lipid phase separation, present in the external leaflet of native membranes between 28 and 19 degrees C, is perturbed by mM Ca2+ such that the high temperature onset is elevated to 32-34 degrees C. Fluoride-stimulated adenylate cyclase was similarly inhibited by Ca2+ (ID50 = 1 mM) for the enzyme in membrane-bound or solubilized states. The glucagon-stimulated activity was more sensitive to Ca2+ inhibition with an ID50 of 0.2 mM. These inhibitory effects are due neither to perturbations of glucagon binding to its receptor nor to fluidity changes, but are instead attributed to direct Ca2+-enzyme interactions. Such binding desensitizes the enzyme to fluidity alterations induced by temperature elevation or benzyl alcohol addition. With Ca2+, Arrhenius plots of glucagon-stimulated activity indicated breaks at 32 and 16 degrees C, whereas those of fluoride-stimulated activity showed one break at 17 degrees C. Without Ca2+, Arrhenius plots exhibited one break at 28 degrees C for glucagon-stimulated activity, whereas fluoride-stimulated plots were linear. We propose that Ca2+ achieves these effects through asymmetric perturbations of the membrane lipid structure.     

Relationship between BDNF expression in major striatal afferents,striatum morphology and motor behavior in the R6/2 mouse model of Huntington's disease

Patients with Huntington's disease (HD) and transgenic mouse models of HD show neuronal loss in the striatum as a major feature, which contributes to cognitive and motor manifestations. Reduced expression of the neurotrophin brain‐derived neurotrophic factor (BDNF) in striatal afferents may play a role in neuronal loss. How progressive loss of BDNF expression in different cortical or subcortical afferents contributes to striatal atrophy and behavioral dysfunction in HD is not known, and may best be determined in animal models. We compared age‐dependent alterations of BDNF mRNA expression in major striatal afferents from the cerebral cortex, thalamus and midbrain in the R6/2 transgenic mouse model of HD. Corresponding changes in striatal morphology were quantified using unbiased stereology. Changes in motor behavior were measured using an open field, grip strength monitor, limb clasping and a rotarod apparatus. BDNF expression in cortical limbic and midbrain striatal afferents is reduced by age 4 weeks, prior to onset of motor abnormalities. BDNF expression in motor cortex and thalamic afferents is reduced by 6 weeks, coinciding with early motor dysfunction and reduced striatum volume. BDNF loss in afferents progresses until death at 13–15 weeks, correlating with progressive striatal neuronal loss and motor abnormalities. Mutant huntingtin protein expression in R6/2 mice results in progressive loss of BDNF in both cortical and subcortical striatal afferents. BDNF loss in limbic and dopaminergic striatal inputs may contribute to cognitive/psychiatric dysfunction in HD. Subsequent BDNF loss in cortical motor and thalamic afferents may accelerate striatal degeneration, resulting in progressive involuntary movements.     

Genome-wide analysis of mobile genetic element insertion sites

Mobile genetic elements (MGEs) account for a significant fraction of eukaryotic genomes and are implicated in altered gene expression and disease. We present an efficient computational protocol for MGE insertion site analysis. ELAN, the suite of tools described here uses standard techniques to identify different MGEs and their distribution on the genome. One component, DNASCANNER analyses known insertion sites of MGEs for the presence of signals that are based on a combination of local physical and chemical properties. ISF (insertion site finder) is a machine-learning tool that incorporates information derived from DNASCANNER. ISF permits classification of a given DNA sequence as a potential insertion site or not, using a support vector machine. We have studied the genomes of Homo sapiens, Mus musculus, Drosophila melanogaster and Entamoeba histolytica via a protocol whereby DNASCANNER is used to identify a common set of statistically important signals flanking the insertion sites in the various genomes. These are used in ISF for insertion site prediction, and the current accuracy of the tool is over 65%. We find similar signals at gene boundaries and splice sites. Together, these data are suggestive of a common insertion mechanism that operates in a variety of eukaryotes.     

Rationally Designed Transmembrane Peptide Mimics of the Multidrug Transporter Protein Cdr1 Act as Antagonists to Selectively Block Drug Efflux and Chemosensitize Azole-resistant Clinical Isolates of Candida albicans

Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.