Biology subjects

Singh, S.

Publications and source records attributed to Singh, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2018

Simulation of spontaneous G protein activation reveals a new intermediate driving GDP unbinding

Activation of heterotrimeric G proteins is a key step in many signaling cascades. However, a complete mechanism for this process, which requires allosteric communication between binding sites that are ~30 [A] apart, remains elusive. We construct an atomically-detailed model of G protein activation by combining three powerful computational methods; metadynamics, Markov state models (MSMs), and CARDS analysis of correlated motions. We uncover a mechanism that is consistent with a wide variety of structural and biochemical data. Surprisingly, the rate-limiting step for GDP release correlates with tilting rather than translation of the GPCR-binding helix 5. {beta}-Strands 1-3 and helix 1 emerge as hubs in the allosteric network that links conformational changes in the GPCR-binding site to disordering of the distal nucleotide-binding site and consequent GDP release. Our approach and insights provide foundations for understanding disease-implicated G protein mutants, illuminating slow events in allosteric networks, and examining unbinding processes with slow off-rates.

biophysics↗

bioRxiv2018

Homology Modeling And Docking Study Of Shewanella Like Protein Phosphatase Involved In Development Of Ookinetes In Plasmodium

Parasites of the genus Plasmodium cause a great deal of morbidity and mortality in worldwide, largely in regions with limited access and indication to the tools necessary to control mosquito populations and to treat human infections of Malaria. Five species of this class of eukaryotic pathogens cause different human disease, with Plasmodium falciparum alone is infecting approximately 500 million people per year and resulting in approximately 1 million deaths. The two genes encoding the Shewanella-like phosphatases of P.falciparum, SHLP-1 and SHLP-2, are conserved among members of Plasmodium family. SHLP is frequently found in asexual blood stages and expressed at all stages of the life cycle of parasite. SHLP deletion results in a reduction in microneme formation, ookinetes (zygote) development and complete ablation of oocyst formation, thereby blocking transmission of parasite.. Structure Modeling of SHLP protein can be helpful in understanding the active site and binding site information and hence can be used for drug designing for therapeutics against malaria. Effective role of Resveratrol is studied against SHLP protein using Docking method to identify protein-ligand interaction scheme and bond formation. Study suggests that resveratrol have strong interaction with SHLP protein and can be used as ligand for drug designing.

bioinformatics↗

bioRxiv2018

Weakly supervised learning of single-cell feature embeddings

We study the problem of learning representations for single cells in microscopy images to discover biological relationships between their experimental conditions. Many new applications in drug discovery and functional genomics require capturing the morphology of individual cells as comprehensively as possible. Deep convolutional neural networks (CNNs) can learn powerful visual representations, but require ground truth for training; this is rarely available in biomedical profiling experiments. While we do not know which experimental treatments produce cells that look alike, we do know that cells exposed to the same experimental treatment should generally look similar. Thus, we explore training CNNs using a weakly supervised approach that uses this information for feature learning. In addition, the training stage is regularized to control for unwanted variations using mixup or RNNs. We conduct experiments on two different datasets; the proposed approach yields single-cell embeddings that are more accurate than the widely adopted classical features, and are competitive with previously proposed transfer learning approaches.

bioinformatics↗

bioRxiv2018

Dysregulation of the engulfment pathway in the gut fuels Inflammatory Bowel Disease

BACKGROUND & AIMSLuminal dysbiosis is ubiquitous in inflammatory bowel disease (IBD), but how the microbes trigger pro-inflammatory cascades in the epithelial and phagocytic cells remains unknown. Here we investigated the role of the microbial sensor ELMO1 (Engulfment and Cell Motility Protein-1) in sensing and responding to IBD-associated microbes in the gut epithelium and in macrophages.\n\nMETHODSA stem cell-based technique is used to grow enteroids from WT and ELMO1-/-mice and from colonic biopsies of patients with IBD and subsequently differentiate them into enteroid-derived monolayers (EDMs) that mimic the gut epithelium/Gut in a dish. EDMs infected with IBD-associated invasive E. coli-LF82 were analyzed for bacterial internalization, cytokine production and monocyte-recruitment when co-cultured with monocytes.\n\nRESULTSExpression of ELMO1 is elevated in the colonic epithelium and in the inflammatory infiltrates within the lamina propria in IBD, higher expression correlated with elevated expression of pro-inflammatory cytokines, MCP-1 and TNF-. ELMO1-/-murine EDMs displayed a significant reduction of bacterial internalization through epithelial tight junctions and in MCP-1 production compared to WT mice. MCP-1 that is released from the epithelium recruited monocytes. Once recruited, macrophages required ELMO1 to engulf the bacteria and propagate a robust pro-inflammatory cytokine storm (TNF-).\n\nCONCLUSIONSELMO1 couples microbial-sensing to inflammation in both phagocytic and non-phagocytic host cells; it is required for the production of MCP-1 in the epithelium and TNF- in macrophages. Findings raise the possibility that upregulation of epithelial ELMO1 and the epithelial ELMO1[->]MCP-1 axis may serve as an early biomarker and therapeutic target, respectively, in IBD and other disorders of inflammation.

cell biology↗

bioRxiv2018

Prevalence of Antibiotic Resistance in Commensal Escherichia Coli among the Children in Rural Hill Communities of North East India

Commensal bacteria are the representative of the reservoir of antibiotic resistance genes present in a community. Merely a few community-based studies on the prevalence of antibiotic resistance in commensal bacteria have been conducted so far in Southeast Asia and other parts of India. Northeastern India is still untapped regarding the surveillance of antibiotic-resistant genes and prevalence in commensal bacteria. In the present work, the prevalence of antibiotic resistance in commensal Escherichia coli was investigated along with the associated demographic factors in pre-school and school going children in rural areas of Sikkim. A total of 550 fecal E. coli isolates were obtained from children of the age 1-14 years living in different villages at various altitudes of Sikkim from July 2015 to June 2017. Standard antibiotic susceptibility testing of these isolates was performed. A structured questionnaire was designed to study the factors associated with carriage of antibiotic resistance in commensal E. coli isolates among children. Descriptive statistics analysis and a logistic regression model were used to identify the effect of external factors on antibiotic resistance pattern. High prevalence of resistance was found against commonly used antibiotics ampicillin (92%), ceftazidime (90%), cefoxitin (88%), streptomycin (40%) and tetracycline (36%) among the samples examined in our present study. No resistance to chloramphenicol was recorded. Fifty-two percent of the isolates were resistant to the combination of penicillin and quinolone group of antibiotics. Children living in nuclear families showed higher incidence of resistance to ampicillin (63.15%, OR 0.18,95% CI:0.11 - 0.28, p<0.01) while children of mothers having education up to school level displayed higher incidence of ceftazidime (59.27% OR 0.75, 95% CI:0.55 - 1.02, p<0.02). Our study demonstrates a high prevalence of antibiotic-resistant commensal E. coli against the commonly used antibiotics among children in the study area. A close association between different demographic factors and the pattern of carriage of antibiotic-resistant isolates was observed suggesting a concern over misuse of antibiotics and warrants a future threat of emerging multidrug resistant isolates.

microbiology↗

bioRxiv2018

Genome analysis and data sharing informs timing of molecular events in pancreatic neuroendocrine tumour

Neuroendocrine tumours (NETs) are rare, slow growing cancers that present in a diversity of tissues. To understand molecular underpinnings of gastrointestinal (GINET) and pancreatic NETs (PNETs), we profiled 45 tumours combining exome, RNA, and shallow whole genome sequencing, as well as fluorescent in situ hybridization. In addition to expected somatic mutations and copy number alterations, we found that PNETs contained a highly consistent copy neutral loss-of-heterozygosity (CN-LOH) profile affecting over half of the genome; a greater percentage than any cancer analyzed to date. Our data indicates that onset of extreme autozygosity may be progressive, associated with metastasis, and initially triggered by the loss of DAXX/ATRX, and subsequent biallelic loss of MEN1. We confirmed this molecular timing model using targeted clinical sequencing data from an additional 43 NETs made available by the AACR GENIE project. Against this background of CN-LOH, several chromosomal regions consistently retained heterozygosity, suggesting selection for crucial allele-specific components specific to PNET progression and potential new therapeutic targets.\n\nStatement of significanceWe have discovered that pancreatic neuroendocrine tumours contain a characteristic pattern of copy neutral loss-of-heterozygosity affecting the majority of the genome following mutations of MEN1 and ATRX/DAXX. Against this background of loss-of-heterozygosity, specific genomic regions are consistently retained and may therefore contain vulnerable therapeutic targets for pancreatic neuroendocrine tumours.

cancer biology↗

bioRxiv2017

Structural basis for endotoxin neutralization and anti-inflammatory activity of thrombin-derived C-terminal peptides

Thrombin-derived C-terminal peptides (TCP) of about 2 kDa are present in wounds, where they exert anti-endotoxic functions. In an effort to elucidate the structural and molecular aspects of these functions, we here employ a combination of nuclear magnetic resonance spectroscopy (NMR), ellipsometry, fluorescence spectroscopy, circular dichroism (CD) measurements, and in silico multiscale modeling to define interactions and the bound conformation of a TCP generated by neutrophil elastase, HVF18 (HVFRLKKWIQKVIDQFGE) in complex with bacterial lipopolysaccharide (LPS). In contrast to the disordered state of HVF18 in aqueous solution, its binding to LPS leads to a structural transition, wherein the N- terminus of the peptide forms a unique {beta}-turn whilst the C-terminus becomes helical. In silico modelling and simulations demonstrated that HVF18, as well as related peptides, target the LPS-binding site of CD14, and this interaction was experimentally supported using microscale thermophoresis. Collectively, the results demonstrate the role of structural transitions in LPS complex formation as well as in CD 14 interaction, and provide a molecular explanation for the previously observed therapeutic effects of TCPs in experimental models of bacterial sepsis and endotoxin shock.\n\nSignificanceThrombin-derived C-terminal peptides (TCPs) of various sizes are present in human wounds, where they bind bacteria as well as \"free\" lipopolysaccharide (LPS), and thereby reduce inflammation. In this work, employing a combination of cellular, biophysical and structural studies, combined with in silico multiscale modeling, we present the molecular structure of a TCP in association with LPS, and define a previously undisclosed interaction between TCPs and CD14. Further, we show that TCPs exhibit relatively weak but specific affinities, all in the M range, to both LPS and CD14. These novel structural insights into the function of this class of host-defense molecules will facilitate rational design of novel \"dual function\" anti-infectives, which target both bacteria and inflammatory signaling.

biophysics↗

bioRxiv2017

Increased glutaminolytic flux and activation of mitochondrial metabolism by BCL2 hyperactivity in lymphoma

B-cell lymphoma 2 (BCL2) is an important apoptosis regulator during developmental and pathological states, and its overexpression is a key feature of several malignancies. Genomic data from The Cancer Genome Atlas (TCGA) reveals significant somatic copy number amplification, overexpression, and/or elevated protein activity of BCL2 in 50 % of diffuse large B-cell lymphoma (DLBC) patients. While its canonical role in mitochondria-directed apoptosis is well established, the effect of BCL2 on transcriptional and metabolic networks remains elusive. Using an established lymphocytic pro-B-cell line overexpressing BCL2, we identified dysregulated transcriptional and metabolic networks by transcriptomic profiling arrays. Elevated BCL2 levels affect transcription factor complexes and mitogenic programs of NF-{kappa}B/REL, HIF1A/ARNT, AP1, E2F, and STAT factors. Using stable isotope-assisted metabolic flux measurements we quantify that elevated BCL2 expression increases carbon utilization boosting cellular proliferation. Tumorigenic overexpression of BCL2 significantly increases glycolytic flux, glutaminolysis, and anaplerotic flux into the TCA cycle. At the same time, the mitochondrial acetyl-CoA pool is separated from the glycolytic one by inactivating the pyruvate dehydrogenase complex via transcriptional regulation of pyruvate dehydrogenase kinase (PDK3). As compensatory fuel, mitochondrial TCA cycle metabolism is supported by asparagine synthase (ASNS) and oxidative glutaminolysis creating targets for small molecule inhibition of glutaminase. Lymphoma cells overexpressing BCL2 contained more mitochondrial mass and were more sensitive to L-glutamine deprivation and glutaminase inhibition. Cells overexpressing a mutant BCL2 G145E, which is incapable of binding BH domain members, failed to increase proliferation, glycolysis, or glutaminolysis. Taken together, the oncogene BCL2 has the ability to ramp up a metabolic phenotype supporting proliferation independent of its anti-apoptotic role. The cellular model of BCL2 activation supports NF-KB-positive subtypes of DLBC and identifies metabolic bottlenecks with dependency on anaplerotic flux as an actionable BCL2 effector network in cancer.

systems biology↗

bioRxiv2017

Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma

Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of BRAF inhibitor, vemurafenib, PLX4032. We profiled transcriptomic changes using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The transcriptomic changes were validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Downregulation of tumor suppressors and negative MAPK regulators, dual specific phosphatases, reengages mitogenic signaling. Upregulation of growth factors or cytokine receptors triggers signaling pathways circumventing BRAF blockage. Changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, an upregulation of pigmentation in inhibitor resistant melanoma cells was observed. Cellular pathways utilized during inhibitor resistance promoted melanogenesis, a pathway which partially overlaps with MAPK signaling. Upstream regulator analysis suggested gene expression changes of forkhead box and hypoxia inducible factor family transcription factors. The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance. The outcome of this transcriptional plasticity is selection for a set of transcriptional master regulators, which circumvent upstream targeted kinases and provide alternative routes of mitogenic activation. A fine-woven network of redundant signals maintains similar effector genes allowing for tumor cell survival and malignant progression in therapy resistant cancer.

systems biology↗

bioRxiv2017

CytoGAN: Generative Modeling of Cell Images

We explore the application of Generative Adversarial Networks to the domain of morphological profiling of human cultured cells imaged by fluorescence microscopy. When evaluated for their ability to group cell images responding to treatment by chemicals of known classes, we find that adversarially learned representations are superior to autoencoder-based approaches. While currently inferior to classical computer vision and transfer learning, the adversarial framework enables useful visualization of the variation of cellular images due to their generative capabilities.

bioinformatics↗

bioRxiv2017

Evaluation of protein-ligand docking methods on peptide-ligand complexes for docking small ligands to peptides

In the past, many benchmarking studies have been performed on protein-protein and protein-ligand docking however there is no study on peptide-ligand docking. In this study, we evaluated the performance of seven widely used docking methods (AutoDock, AutoDock Vina, DOCK 6, PLANTS, rDock, GEMDOCK and GOLD) on a dataset of 57 peptide-ligand complexes. Though these methods have been developed for docking ligands to proteins but we evaluate their ability to dock ligands to peptides. First, we compared TOP docking pose of these methods with original complex and achieved average RMSD from 4.74[A] for AutoDock to 12.63[A] for GEMDOCK. Next we evaluated BEST docking pose of these methods and achieved average RMSD from 3.82[A] for AutoDock to 10.83[A] for rDock. It has been observed that ranking of docking poses by these methods is not suitable for peptide-ligand docking as performance of their TOP pose is much inferior to their BEST pose. AutoDock clearly shows better performance compared to the other six docking methods based on their TOP docking poses. On the other hand, difference in performance of different docking methods (AutoDock, AutoDock Vina, PLANTS and DOCK 6) was marginal when evaluation was based on their BEST docking pose. Similar trend has been observed when performance is measured in terms of success rate at different cut-off values. In order to facilitate scientific community a web server PLDbench has been developed (http://webs.iiitd.edu.in/raghava/pldbench/).

bioinformatics↗

bioRxiv2017

Induction of autophagy by trehalose limits opportunistic mycobacterial infections in HIV-infected macrophages

Opportunistic bacterial infections amongst HIV-infected individuals pose serious health challenge. While immediate control of bacterial pathogens is typically attributed to innate defense mechanisms, whether HIV-mediated modulation of innate mechanisms like autophagy promote opportunistic infections, remains obscure. Using U1.1 and U937 macrophages, we show, HIV activation or infection inhibits autophagy and helps survival of pathogenic Mycobacterium tuberculosis and non-pathogenic non-tuberculous mycobacterial strains (NTMs) like Mycobacterium avium complex and Mycobacterium fortuitum. HIV achieves this by blocking xenophagy flux, which could be reversed by the autophagy inducer trehalose that kills intracellular Mtb and NTMs. We found trehalose acts as a PI (3,5) P2 agonist and activates TRPML1 to induce autophagy. Remarkably, trehalose treatment significantly reduced p24 levels in PBMCs infected with clinical HIV strains and in PBMCs derived from treatment-naive HIV patients. Taken together, our study highlights the immense potential of autophagy modulators in the therapeutic intervention of HIV and associated opportunistic infection.

immunology↗

bioRxiv2017

Prediction of residue-residue contacts in CASP12 targets from its predicted tertiary structures

One of the challenges in the field of structural proteomics is to predict residue-residue contacts in a protein. It is an integral part of CASP competitions due to its importance in the field of structural biology. This manuscript describes RRCPred 2.0 a method participated in CASP12 and predicted residue-residue contact in targets with high precision. In this approach, firstly 150 predicted protein structures were obtained from CASP12 Stage 2 tarball and ranked using clustering-based quality assessment software. Secondly, residue-residue contacts were assigned in top 10 protein structures based on distance between residues. Finally, residue-residue contacts were predicted in target protein based on consensus/average in top 10 predicted structures. This simple approach performs better than most of CASP12 methods in the categories of TBM and TBM/FM. It ranked 1st in following categories; i) TBM domain on list size L/5, ii) TBM/FM domain on list size L/5 and iii) TBM/FM domain on Top 10. These observations indicate that predicted tertiary structure of a protein can be used for predicting residue-residue contacts in protein with high accuracy.

bioinformatics↗

bioRxiv2017

An improved method for the expression screening of membrane protein-GFP fusions in yeast

The expression and purification of membrane proteins is an extremely challenging area of work within Protein Science. Membrane proteins are required for compound screening and structure determination in industry. Here we describe some new and innovative methodology in developing the membrane protein GFP fusion primary expression screening in yeast. This methodology enables the expression of membrane proteins fused to GFP in both Saccharomyces cerevisiae and Pichia pastoris systems. This capability helps facilitate screening of constructs to establish which are suitable for membrane protein production for compound screening and structure determination\n\nIn terms of the primary screening work, we have developed both agar plate and liquid plate expression methodology in yeast. The two approaches correspond well, but the agar plate method is more rapid and we have shown it to have the advantage of allowing cells to be taken directly into confocal microscopy for immediate cell localisation data. Innovative work to extend the methanol induction time in the Pichia agar plate method established good differentiation from the background. A novel agar plate method was also developed for S.cerevisiae which is also presented. These screening methods allow triaging of constructs for either membrane protein preps for biochemical assays or progression to fluorescence size exclusion chromatography; where various detergents can be screened to determine the most appropriate for membrane protein solubilisation, the starting point for purification, crystallisation and structure determination.\n\nMembrane targets depicted to demonstrate the improved primary screening methodology are a copper transporter Ctr1p from S.cerevisiae and a water transporter Aqp4 from human origin.\n\nHighlightsAn improved method for the production of recombinant MP-GFP fusions in yeast is presented using agar plates.\n\nAn agar plate method for MP-GFP expression screening is described for Pichia pastoris, with improved induction methodology by the simple addition of methanol, allowing longer induction times for expression clarity.\n\nA new simple rapid agar plate method for MP-GFP expression screening is described for Saccharomyces cerevisiae.\n\nCells can be taken directly from agar plates into confocal microscopy studies for immediate cell localisation data and triaging.\n\nLiquid plate based screening methods are also described for both yeasts in comparison, to show there is corresponding data, helping validate the new agar plate methods.

biochemistry↗

bioRxiv2017

Common Garlic (Allium sativum) has Potent Anti-Bacillus anthracis Activity among Various Commonly Used Spices and Herbs

Ethnopharmacological RelevanceGastrointestinal anthrax, a disease caused by Bacillus anthracis, remains an important but relatively neglected endemic disease of animals and humans in remote areas of the Indian subcontinent and some parts of Africa. Its initial symptoms include diarrhea and stomachache. In the current study, several common plants indicated for diarrhea, dysentery, stomachache or as stomachic as per traditional knowledge in the Indian subcontinent, i.e., Aegle marmelos (L.) Correa (Bael), Allium cepa L. (Onion), Allium sativum L. (Garlic), Azadirachta indica A. Juss. (Neem), Berberis asiatica Roxb. ex DC. (Daruharidra), Coriandrum sativum L. (Coriander), Curcuma longa L. (Turmeric), Cynodon dactylon (L.) Pers. (Bermuda grass), Mangifera indica L. (Mango), Morus indica L. (Black mulberry), Ocimum tenuiflorum L. (Ocimum sanctum L., Holy Basil), Ocimum gratissimum L. (Ram Tulsi), Psidium guajava L. (Guava), Zingiber officinale Roscoe (Ginger), were evaluated for their anti-Bacillus anthracis property. The usage of Azadirachta indica A. Juss. and Curcuma longa L. by Santals (India), and Allium sp. by biblical people to alleviate anthrax-like symptoms is well documented, but the usage of other plants is traditionally only indicated for different gastrointestinal disturbances/conditions. Aim of the StudyEvaluate the above listed commonly available edible plants from the Indian subcontinent that are used in the traditional medicine to treat gastrointestinal diseases including those also indicated for anthrax-like symptoms for the presence of potent anti-B. anthracis activity in a form amenable to use by the general population in the endemic areas. Materials and MethodsAqueous extracts made from fourteen plants indicated above were screened for their anti-B. anthracis activity using agar-well diffusion assay (AWDA) and broth microdilution methods. The Aqueous Garlic Extract (AGE) that displayed most potent anti-B. anthracis activity was assessed for its thermostability, stability under pH extremes encountered in the gastrointestinal tract, and potential antagonistic interaction with bile salts as well as the FDA-approved antibiotics used for anthrax control. The bioactive fractions from the AGE were isolated by TLC coupled bioautography followed by their characterization using GC-MS. ResultsGarlic (Allium sativum L.) extract was identified as the most promising candidate with bactericidal activity against B. anthracis. It consistently inhibited the growth of B. anthracis in AWDA and decreased the viable colony-forming unit counts in liquid-broth cultures by 6-logs within 6-12 h. The AGE displayed acceptable thermostability (>80% anti-B. anthracis activity retained on incubation at 50{degrees}C for 12 h) and stability in gastric pH range (2-8). It did not antagonize the activity of FDA-approved antibiotics used for anthrax control. GC-MS analysis of the TLC separated bioactive fractions of AGE indicated the presence of previously unreported constituents such as phthalic acid derivatives, acid esters, phenyl group-containing compounds, steroids etc. ConclusionThe Aqueous Garlic Extract (AGE) displayed potent anti-B. anthracis activity. It was better than that displayed by Azadirachta indica A. Juss. (Neem) and Mangifera indica L. while Curcuma longa L. (Turmeric) did not show any activity under the assay conditions used. Further work should be undertaken to explore the possible application of AGE in preventing anthrax incidences in endemic areas. O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/162214v3_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@682faaorg.highwire.dtl.DTLVardef@114d64org.highwire.dtl.DTLVardef@1efe980org.highwire.dtl.DTLVardef@1caea0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2017

Rhodosporidium toruloides: A new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts

BackgroundEconomical conversion of lignocellulosic biomass into biofuels and bioproducts is central to the establishment of a robust bioeconomy. This requires a conversion host that is able to both efficiently assimilate the major lignocellulose-derived carbon sources and divert their metabolites toward specific bioproducts.\n\nResultsIn this study, the carotenogenic yeast Rhodosporidium toruloides was examined for its ability to convert lignocellulose into two non-native sesquiterpenes with biofuel (bisabolene) and pharmaceutical (amorphadiene) applications. We found that R. toruloides can efficiently convert a mixture of glucose and xylose from hydrolyzed lignocellulose into these bioproducts, and unlike many conventional production hosts, its growth and productivity were enhanced in lignocellulosic hydrolysates relative to purified substrates. This organism was demonstrated to have superior growth in corn stover hydrolysates prepared by two different pretreatment methods, one using a novel biocompatible ionic liquid (IL) choline -ketoglutarate, which produced 261 mg/L of bisabolene at bench-scale, and the other using an alkaline pretreatment, which produced 680 mg/L of bisabolene in a high gravity fed-batch bioreactor. Interestingly, R. toruloides was also observed to assimilate p-coumaric acid liberated from acylated grass lignin in the IL hydrolysate, a finding we verified with purified substrates. R. toruloides was also able to consume several additional compounds with aromatic motifs similar to lignin monomers, suggesting that this organism may have the metabolic potential to convert depolymerized lignin streams alongside lignocellulosic sugars.\n\nConclusionsThis study highlights the natural compatibility of R. toruloides with bioprocess conditions relevant to lignocellulosic biorefineries and demonstrates its ability to produce non-native terpenes.

bioengineering↗

bioRxiv2017

Transcription Factor Target Gene Network governs the Logical Abstraction Analysis of the Synthetic Circuit in Leishmaniasis

Stochastic variations in the transcription factor target gene network influences the dynamics of protein levels. The mathematical model built, here, is useful to study the cytokine response and the resulting dynamical patterns in leishmaniasis. The fluctuations produced affect the expression rate of its target in the regulatory synthetic circuit designed by means of a negative feedback loop insertion. Thus, the faster the response time, smaller is the fluctuation of the system observed and it can be justified that the TFTG network embedded can be understood with the recurring dynamics of the designed synthetic circuit.

systems biology↗

bioRxiv2017

Repurposed High-Throughput Images Enable Biological Activity Prediction For Drug Discovery

We repurpose a High-Throughput (cell) Imaging (HTI) screen of a glucocorticoid receptor assay to predict target protein activity in multiple other seemingly unrelated assays. In two ongoing drug discovery projects, our repurposing approach increased hit rates by 60- to 250-fold over that of the primary project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from available HTI screens are a rich source of information that can be reused to empower drug discovery efforts.

pharmacology and toxicology↗