Dey G.,Stanford University |
Gupta G.D.,Samuel Lunenfeld Research Institute |
Ramalingam B.,Center for Cellular and Molecular Platforms |
Sathe M.,Tata Institute of Fundamental Research |
And 2 more authors.
PLoS ONE | Year: 2014
Any single-cell-resolved measurement generates a population distribution of phenotypes, characterized by a mean, a variance, and a shape. Here we show that changes in the shape of a phenotypic distribution can signal perturbations to cellular processes, providing a way to screen for underlying molecular machinery. We analyzed images of a Drosophila S2R+ cell line perturbed by RNA interference, and tracked 27 single-cell features which report on endocytic activity, and cell and nuclear morphology. In replicate measurements feature distributions had erratic means and variances, but reproducible shapes; RNAi down-regulation reliably induced shape deviations in at least one feature for 1072 out of 7131 genes surveyed, as revealed by a Kolmogorov-Smirnov-like statistic. We were able to use these shape deviations to identify a spectrum of genes that influenced cell morphology, nuclear morphology, and multiple pathways of endocytosis. By preserving single-cell data, our method was even able to detect effects invisible to a population-averaged analysis. These results demonstrate that cell-to-cell variability contains accessible and useful biological information, which can be exploited in existing cell-based assays. © 2014 Dey et al.
Sahadevan S.,Institute for Stem Cell Biology and Regenerative Medicine |
Antonopoulos A.,Imperial College London |
Haslam S.M.,Imperial College London |
Dell A.,Imperial College London |
And 3 more authors.
ACS Chemical Biology | Year: 2014
Cell?cell communications, cell?matrix interactions, and cell migrations play a major role in regeneration. However, little is known about the molecular players involved in these critical events, especially cell surface molecules. Here, we demonstrate the role of specific glycan?receptor interactions in the regenerative process using Hydra magnipapillata as a model system. Global characterization of the N-and O-glycans expressed by H. magnipapillata using ultrasensitive mass spectrometry revealed mainly polyfucosylated LacdiNAc antennary structures. Affinity purification showed that a putative C-type lectin (accession number Q6SIX6) is a likely endogenous receptor for the novel polyfucosylated glycans. Disruption of glycan?receptor interactions led to complete shutdown of the regeneration machinery in live Hydra. A time-dependent, lack-of-regeneration phenotype observed upon incubation with exogenous fuco-lectins suggests the involvement of a polyfucose receptor-mediated signaling mechanism during regeneration. Thus, for the first time, the results presented here provide direct evidence for the role of polyfucosylated glycan?receptor interactions in the regeneration of H. magnipapillata. © 2013 American Chemical Society.
Davidson R.M.,Michigan State University |
Gowda M.,Michigan State University |
Gowda M.,Center for Cellular and Molecular Platforms |
Moghe G.,Michigan State University |
And 5 more authors.
Plant Journal | Year: 2012
The Poaceae family, also known as the grasses, includes agronomically important cereal crops such as rice, maize, sorghum, and wheat. Previous comparative studies have shown that much of the gene content is shared among the grasses; however, functional conservation of orthologous genes has yet to be explored. To gain an understanding of the genome-wide patterns of evolution of gene expression across reproductive tissues, we employed a sequence-based approach to compare analogous transcriptomes in species representing three Poaceae subgroups including the Pooideae (Brachypodium distachyon), the Panicoideae (sorghum), and the Ehrhartoideae (rice). Our transcriptome analyses reveal that only a fraction of orthologous genes exhibit conserved expression patterns. A high proportion of conserved orthologs include genes that are upregulated in physiologically similar tissues such as leaves, anther, pistil, and embryo, while orthologs that are highly expressed in seeds show the most diverged expression patterns. More generally, we show that evolution of gene expression profiles and coding sequences in the grasses may be linked. Genes that are highly and broadly expressed tend to be conserved at the coding sequence level while genes with narrow expression patterns show accelerated rates of sequence evolution. We further show that orthologs in syntenic genomic blocks are more likely to share correlated expression patterns compared with non-syntenic orthologs. These findings are important for agricultural improvement because sequence information is transferred from model species, such as Brachypodium, rice, and sorghum to crop plants without sequenced genomes. © 2012 The Authors.
Deciphering mode of action of functionally important regions in the intrinsically disordered paxillin (residues 1-313) using its interaction with FAT (focal adhesion targeting domain of focal adhesion kinase)
Neerathilingam M.,Center for Cellular and Molecular Platforms |
Neerathilingam M.,University of Oxford |
Bairy S.G.,Center for Cellular and Molecular Platforms |
Mysore S.,Center for Cellular and Molecular Platforms
PLoS ONE | Year: 2016
Intrinsically disordered proteins (IDPs) play a major role in various cellular functions ranging from transcription to cell migration. Mutations/modifications in such IDPs are shown to be associated with various diseases. Current strategies to study the mode of action and regulatory mechanisms of disordered proteins at the structural level are time consuming and challenging. Therefore, using simple and swift strategies for identifying functionally important regions in unstructured segments and understanding their underlying mechanisms is critical for many applications. Here we propose a simple strategy that employs dissection of human paxillin (residues 1-313) that comprises intrinsically disordered regions, followed by its interaction study using FAT (Focal adhesion targeting domain of focal adhesion kinase) as its binding partner to retrace structural behavior. Our findings show that the paxillin interaction with FAT exhibits a masking and unmasking effect by a putative intra-molecular regulatory region. This phenomenon suggests how cancer associated mutations in paxillin affect its interactions with Focal Adhesion Kinase (FAK). The strategy could be used to decipher the mode of regulations and identify functionally relevant constructs for other studies. © 2016 Neerathilingam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Babu P.,Center for Cellular and Molecular Platforms
ACS Chemical Biology | Year: 2014
Glycans participate in many key cellular processes during development and in physiology and disease. In this review, the functional role of various glycans in the regeneration of neurons and body parts in adult metazoans is discussed. Understanding glycosylation may facilitate research in the field of stem cell biology and regenerative medicine. © 2013 American Chemical Society.
Gandham S.H.A.,University of Texas Health Science Center at Houston |
Volk D.E.,University of Texas Health Science Center at Houston |
Lokesh G.L.R.,University of Texas Health Science Center at Houston |
Neerathilingam M.,University of Texas Health Science Center at Houston |
And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2014
Thioaptamers targeting the dengue-2 virus (DENV-2) envelope protein domain III (EDIII) were developed. EDIII, which contains epitopes for binding neutralizing antibodies, is the putative host-receptor binding domain and is thus an attractive target for development of vaccines, anti-viral therapeutic and diagnostic agents. Thioaptamer DENTA-1 bound to DENV-2 EDIII adjacent to a known neutralizing antibody binding site with a dissociation constant of 154 nM. © 2014, Elsevier Inc. All rights reserved.
Sekar N.,Center for Cellular and Molecular Platforms |
Veetil S.K.,Center for Cellular and Molecular Platforms |
Neerathilingam M.,Center for Cellular and Molecular Platforms
BMC Biotechnology | Year: 2013
Background: Escherichia coli is most widely used prokaryotic expression system for the production of recombinant proteins. Several strategies have been employed for expressing recombinant proteins in E.coli. This includes the development of novel host systems, expression vectors and cost effective media. In this study, we exploit tender coconut water (TCW) as a natural and cheaper growth medium for E.coli and Pichia pastoris.Result: E.coli and P.pastoris were cultivated in TCW and the growth rate was monitored by measuring optical density at 600 nm (OD600nm), where 1.55 for E.coli and 8.7 for P.pastoris was obtained after 12 and 60 hours, respectively. However, variation in growth rate was observed among TCW when collected from different localities (0.15-2.5 at OD600nm), which is attributed to the varying chemical profile among samples. In this regard, we attempted the supplementation of TCW with different carbon and nitrogen sources to attain consistency in growth rate. Here, supplementation of TCW with 25 mM ammonium sulphate (TCW-S) was noted efficient for the normalization of inconsistency, which further increased the biomass of E.coli by 2 to 10 folds, and 1.5 to 2 fold in P.pastoris. These results indicate that nitrogen source is the major limiting factor for growth. This was supported by total nitrogen and carbon estimation where, nitrogen varies from 20 to 60 mg/100 ml while carbohydrates showed no considerable variation (2.32 to 3.96 g/100 ml). In this study, we also employed TCW as an expression media for recombinant proteins by demonstrating successful expression of maltose binding protein (MBP), MBP-TEV protease fusion and a photo switchable fluorescent protein (mEos2) using TCW and the expression level was found to be equivalent to Luria Broth (LB).Conclusion: This study highlights the possible application of TCW-S as a media for cultivation of a variety of microorganisms and recombinant protein expression. © 2013 Sekar et al.; licensee BioMed Central Ltd.
Rangiah K.,Center for Cellular and Molecular Platforms
Analytical Methods | Year: 2014
In recent years, the use of antipsychotics like olanzapine has increased leading to potentially serious adverse metabolic effects. A sensitive method to quantify olanzapine and its metabolites is therefore highly needed. A stable isotope dilution ultrahigh performance liquid chromatography-mass spectrometry/selected reaction monitoring based quantitative assay has been developed for the simultaneous estimation of olanzapine and its metabolites. This method includes the parent drug olanzapine, its metabolites (desmethyl olanzapine and olanzapine-N-oxide) and degradation derived piperazinium chloride, lactam and cyclic amine impurities. All analytes were well resolved and showed a linear relationship across a large dynamic range (0.017-1.25 ng mL-1) for all olanzapine metabolites except the lactam, in which the linear relationship was demonstrated at concentrations five times higher (0.085-6.25 ng mL-1). All analytes had regression coefficients higher than 0.998, accuracies between 92-113% and low coefficients of variation (0.94 to 9.3%). The recovery for all of the analytes from the sera matrix was 80 to 115%. This validated method is suitable for quantifying olanzapine and its metabolites from small volumes of sera samples with great sensitivity. © 2014 The Royal Society of Chemistry.
Ramakrishnan P.,Center for Cellular and Molecular Platforms |
Nair S.,Center for Cellular and Molecular Platforms |
Rangiah K.,Center for Cellular and Molecular Platforms
Journal of Chromatography A | Year: 2016
Developing a workflow for metabolite profiling from biological fluids using mass spectrometry is imperative to extract accurate information. In this study, urine samples from smokers (n = 10) and nonsmokers (n = 10) were analyzed using an ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) system. For the analysis, two different chromatographic methods [Reversed phase chromatography (RPC) and Hydrophilic interaction liquid chromatography (HILIC)], in two ionization modes (positive and negative) were used. Spiked reserpine (positive ion mode) or taurocholate (negative ion mode) were used for data extraction and normalization. Quality controls (QCs), prepared by pooling urine samples from both smokers and non-smokers (each n = 10), were used to assess the reproducibility of the method. The final data output from SIEVE 2.2 after applying a cut-off for QC coefficient of variation (CV) <20% and p-value <0.05 showed 165, 83, 177 and 100 unique components in RP positive/negative, HILIC positive/negative modes, respectively. Statistical analysis showed clustering of the two groups and the QCs, while the variable importance in projection (VIP) scores for the top fifteen metabolites in each of the four modes indicated the metabolites most responsible for the differences. Application of the developed workflow for comparative metabolomic analysis of urine in different diseased models will be of great use in the field of clinical metabolomics. © 2016 Elsevier B.V.
Rangiah K.,Center for Cellular and Molecular Platforms |
Palakodeti D.,Institute of Stem Cell Biology and Regenarative Medicine
Rapid Communications in Mass Spectrometry | Year: 2013
Rationale Absolute quantification of neurotransmitters (NTs) from biological systems is imperative to track how changes in concentration of active neurochemicals may affect biological behavior. A sensitive method for the absolute quantification of multiple NTs in a single method is highly needed. Methods A stable-isotope dilution ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC/MS/SRM) assay has been developed for a sensitive and quantitative assessment of NTs in planaria. We used this method for the simultaneous quantification of 16 NTs. All analytes showed a linear relationship between concentrations (0.78-50 ng/mL), regression coefficients higher than 0.97, accuracy (91-109%) and low coefficients of variation (CVs). The inter-day CVs for the lowest quality controls (1.56 ng/mL) were in the range between 2-11%. RESULTS The levels of most of the NTs were similar in both sexual and asexual planarians except for glutamic acid, which was about two-fold higher in asexual compared to sexual planarians. We identified high levels of serotonin and failed to detect tryptamine suggesting that the pathway essential for the conversion of tryptophan into tryptamine is absent in planarians. Interestingly, we also found high levels of dopamine and L-DOPA in regenerating planarians suggesting their possible role in regeneration. CONCLUSIONS For the first time, we developed novel methodology based on UHPLC/MS/SRM and quantified 16 NTs with high sensitivity and specificity from sexual and asexual strains of planarian Schmidtea mediterranea. This method will also have great application in quantifying various NTs with great precision in different model systems. © 2013 John Wiley & Sons, Ltd.