Biology subjects

Singh, S. P.

Publications and source records attributed to Singh, S. P..

43 records · Page 3Linked to original sources

bioRxiv2020

An asymmetry in the frequency and position of mitosis in the epiblast precedes gastrulation and suggests a role for mitotic rounding in cell delamination during primitive streak epithelial-mesenchymal transition.

The epiblast, a pseudostratified epithelium, is the precursor for the three main germ layers required for body shape and organogenesis: ectoderm, mesoderm, and endoderm. At gastrulation, a subpopulation of epiblast cells constitutes a transient posteriorly located structure called the primitive streak, where cells that undergo epithelial-mesenchymal transition make up the mesoderm and endoderm lineages. In order to observe the behavior of individual cells, epiblast cells were labeled ubiquitously or in a mosaic fashion using fluorescent membrane reporters. The cell shapes of individual cells and the packing and behaviour of neighbouring cells during primitive streak formation were recorded through live time-lapse imaging. Posterior epiblast displayed a higher frequency of rosettes, a signature of cell rearrangements, prior to primitive streak initiation. A third of rosettes were associated with a central cell undergoing mitosis. Interestingly, cells at the primitive streak, in particular delaminating cells, underwent mitosis twice more frequently than other epiblast cells, suggesting a role for cell division in epithelial-mesenchymal transition. Pseudostratified epithelia are characterized by interkinetic nuclear migration, where mitosis occurs at the apical side of the epithelium. However, we found that exclusively on the posterior side of the epiblast, mitosis was not restricted to the apical side. Non-apical mitosis was apparent as early as E5.75, just after the establishment of the anterior-posterior axis, and prior to initiation of epithelial-mesenchymal transition. Non-apical mitosis was associated with primitive streak morphogenesis, as it occurred specifically in the streak even when ectopically located. Most non-apical mitosis resulted in one or two daughter cells leaving the epiblast layer to become mesoderm. Furthermore, in contrast to what has been described in other pseudostratified epithelia such as neuroepithelium, the majority of cells dividing apically detached completely from the basal pole in the epiblast. Cell rearrangement associated with mitotic cell rounding in the posterior epiblast during gastrulation, in particular when it occurs on the basal side, might thus facilitate cell ingression through the PS and transition to a mesenchymal phenotype. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/959080v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@18530b2org.highwire.dtl.DTLVardef@1400a63org.highwire.dtl.DTLVardef@169f688org.highwire.dtl.DTLVardef@1b87931_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2020

Consensus mutagenesis and ancestral reconstruction provide insight into the substrate specificity and evolution of the front-end Δ6-desaturase family

Marine algae are a major source of omega ({omega})-3 long-chain polyunsaturated fatty acids ({omega}3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae begins with the desaturation of fatty acids by {Delta}6-desaturases and enzymes from different species display a range of specificities towards {omega}3 and {omega}6 LCPUFAs. In the absence of a molecular structure, the structural basis for the variable substrate specificity of {Delta}6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the {Delta}6-desaturase family, focusing on the {omega}3-specific {Delta}6-desaturase from Micromonas pusilla (Mp{Delta}6des) and the bispecific ({omega}3/{omega}6) {Delta}6-desaturase from Ostreococcus tauri (Ot{Delta}6des). Our characterization of consensus amino acid substitutions in Mp{Delta}6des revealed that residues in diverse regions of the protein, such as the N-terminal cytochrome b5 domain, can make important contributions to determining substrate specificity. Ancestral protein reconstruction also suggests that some extant {Delta}6-desaturases, such as Ot{Delta}6des, could have adapted to different environmental conditions by losing specificity for {omega}3-LCPUFAs. This dataset provides a map of regions within {Delta}6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.

biochemistry↗

bioRxiv2020

Single-cell transcriptome analysis reveals cell-cell communication and thyrocyte diversity in the zebrafish thyroid gland

The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover genetic heterogeneity in the thyrocyte population, and molecularly characterize the non-thyrocyte cells surrounding the follicle, we developed a single-cell transcriptome atlas of the zebrafish thyroid gland. The 6249-cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells and fibroblasts. Further, the thyrocytes could be split into two sub-populations with unique transcriptional signature, including differential expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9-based pax2a knock-in line, which demonstrated specific pax2a expression in the thyrocytes. However, a population of pax2a-low mature thyrocytes interspersed within individual follicles could be distinguished, corroborating heterogeneity within the thyrocyte population. Our results identify and validate transcriptional differences within the nominally homogenous thyrocyte population. One-line summarySingle-cell analysis uncovers latent heterogeneity in thyroid follicular cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/891630v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@f6df3org.highwire.dtl.DTLVardef@fb8c59org.highwire.dtl.DTLVardef@bb5be1org.highwire.dtl.DTLVardef@8d174e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2018

Machine learning based classification of cells into chronological stages using single-cell transcriptomics.

Age-associated deterioration of cellular physiology leads to pathological conditions. The ability to detect premature aging could provide a window for preventive therapies against age-related diseases. However, the techniques for determining cellular age are limited, as they rely on a limited set of histological markers and lack predictive power. Here, we implement GERAS (GEnetic Reference for Age of Single-cell), a machine learning based framework capable of assigning individual cells to chronological stages based on their trans criptomes. GERAS displays greater than 90% accuracy in classifying the chronological stage of zebrafish and human pancreatic cells. The framework demonstrates robustness against biological and technical noise, as evaluated by its performance on independent samplings of single-cells. Additionally, GERAS determines the impact of differences in calorie intake and BMI on the aging of zebrafish and human pancreatic cells, respectively. We further harness the predictive power of GERAS to identify genome-wide molecular factors that correlate with aging. We show that one of these factors, junb, is necessary to maintain the proliferative state of juvenile beta-cells. Our results showcase the applicability of a machine learning framework to classify the chronological stage of heterogeneous cell populations, while enabling to detect pro-aging factors and candidate genes associated with aging.

bioinformatics↗

bioRxiv2018

The application of text mining algorithms in summarizing trends in anti-epileptic drug research

1.Content summarization is an important area of research in traditional data mining. The volume of studies published on anti-epileptic drugs (AED) has increased exponentially over the last two decades, making it an important area for the application of text mining based summarization algorithms. In the current study, we use text analytics algorithms to mine and summarize 10,000 PubMed abstracts related to anti-epileptic drugs published within the last 10 years. A Text Frequency - Inverse Document Frequency based filtering was applied to identify drugs with highest frequency of mentions within these abstracts. The US Food and Drug database was scrapped and linked to the results to quantify the most frequently mentioned modes of action and elucidate the pharmaceutical entities marketing these drugs. A sentiment analysis model was created to score the abstracts for sentiment positivity or negativity. Finally, a modified Latent Dirichlet Allocation topic model was generated to extract key topics associated with the most frequently mentioned AEDs. Results of this study provide accurate and data intensive insights on the progress of anti-epileptic drug research.

systems biology↗

bioRxiv2017

Quantitative Analysis on the role of Raffinose Synthase in Hippocampal Neurons

1A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Raffinose synthase (RFS), a protective molecule regulates gene expression/function by controlling reactive oxygen species (ROS) levels, which has shown to be involved in a number of degenerative diseases. We confirmed the ubiquitous expression of this antioxidant protein in both human hippocampal neuron (HNN) and mouse hippocampal (HHPC-43) cell lines by an immunoblot and reverse transcriptase PCR (RT-PCR). Using a construct of RFS protein linked to CXCR-4, the transduction domain from HPV-1 CXCR-4 protein, we showed that RFS was transduced into both HNN, as well as HHPC-43 by the means of a western blot analysis. Further we proved that the protein was biologically active, and was shown to actively reduce the oxidative stress produced by paraquat and serum depletion in both human and mouse neuronal cell lines, increasing the viability of the cells. The results suggest that the intracellular delivery of RFS using CXCR-4 can be used to lower increased levels of ROS inside the cells and hence can be further investigated as a therapeutic tool in various ROS related neurodegenerative disorders.

neuroscience↗

bioRxiv2017

Biochemical, serological and molecular confirmation of Salmonella isolates

Salmonellosis is one of the most frequently reported food-borne diseases world-wide commonly caused by Salmonella Typhimurium and Salmonella Enteritidis serovars. The present study was undertaken to confirm Salmonella isolates by various techniques. All the 94 isolates were confirmed to be S. Typhimurium through various morphological, biochemical, serological and molecular methods.

microbiology↗