Biology subjects

Ramasamy, S.

Publications and source records attributed to Ramasamy, S..

26 records · Page 2Linked to original sources

bioRxiv2021

Cell type resolved co-expression networks of core clock genes in brain development.

The circadian clock regulates vital cellular processes by adjusting the physiology of the organism to daily changes in the environment. Rhythmic transcription of core Clock Genes (CGs) and their targets regulate these processes at the cellular level. Circadian clock disruption has been observed in people with neurodegenerative disorders like Alzheimers and Parkinsons. Also, ablation of CGs during development has been shown to affect neurogenesis in both in vivo and in vitro models. Previous studies on the function of CGs in the brain have used knock-out models of a few CGs. However, a complete catalog of CGs in different cell types of the developing brain is not available and it is also tedious to obtain. Recent advancements in single-cell RNA sequencing (scRNA-seq) has revealed novel cell types and elusive dynamic cell states of the developing brain. In this study by using publicly available single-cell transcriptome datasets we systematically explored CGs-coexpressing networks (CGs-CNs) during embryonic and adult neurogenesis. Our meta-analysis reveals CGs-CNs in human embryonic radial glia, neurons and also in lesser studied non-neuronal cell types of the developing brain.

neuroscience↗

bioRxiv2020

Chemical probe-based Nanopore direct RNA sequencing method to selectively assess the pseudouridine and inosine modifications

Current methods to identify RNA modifications with short-read sequencing are laborious and direct RNA sequencing gets proclaimed as the viable alternative. Herein, we harness the selective reactivity of the acrylonitrile towards the Inosine (I) and pseudouridine ({Psi}) modifications and developed a chemical probe-based direct RNA sequencing method. We first demonstrated that the chemical probe-induced differential signature profile using nanopore sequencing could facilitate the selective assessment of I and{Psi} in the in vitro synthesized RNA. Furthermore, we verified the I and{Psi} modification with single-nucleotide resolution using RNA derived from mouse brain without the need for a null dataset using knockouts. Our chemical probe-based nanopore sequencing strategy can be extended to profile multiple RNA modifications on a single RNA and may facilitate the diagnosis of disease-associated epitranscriptome markers by generating a comparative dataset in clinical scenarios. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=39 SRC="FIGDIR/small/105338v3_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@137799aorg.highwire.dtl.DTLVardef@110764aorg.highwire.dtl.DTLVardef@1c61ddaorg.highwire.dtl.DTLVardef@1d1db4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

Towards understanding the structure of the capsid of Banana Bunchy Top Virus

Banana is the major staple food crop for approximately 400 million people. Bunchy Top disease of Banana is one of the most devastating diseases caused by Banana Bunchy Top Virus (BBTV) that results in a significant loss of yield, stunting and bunchy appearance of leaves. While many isolates of BBTV from various regions of India have been characterized by different groups, no structural study exists for this important virus. To pursue structural studies, the pET28a clone of coat protein (CP) gene from BBTV isolate of Hill Banana grown in lower Pulney Hills (Virupakshi) of Tamilnadu was expressed in BL21 (DE3) pLysS. Purification of the CP was done using Ni-NTA affinity chromatography. In vitro capsid assembly studied using sucrose density gradient centrifugation suggested that the CP did not assemble as virus like particle (VLPs) but remained as smaller oligomers. Studies using dynamic light scattering (DLS) indicates that the purified protein is poly-dispersed represented majorly as pentamers. Studies using both homology modelling and ab initio structure determination gave useful insights into the probable fold of the CP suggesting it is a {beta}-sandwich fold similar to that seen in majority of plant viruses. In silico capsid reconstruction aided understanding of the quaternary organization of subunits in the capsid and molecular interactions present between the subunits. The location of aphid binding EAG motif was identified on the surface loops close to the pentameric axis indicating their role in vector mediated transmission.

bioinformatics↗

bioRxiv2019

Expanding AHL Acylases Horizon - Insights From Structural Analysis of Choloylglycine Hydrolases From Shewanella lohica PV-4

Acyl homoserine lactone acylases are quorum quenching enzymes that degrade the Gram negative bacterial autoinducer N-acyl homoserine lactone (AHL) and belong to the Ntn-hydrolases superfamily of enzymes. Recent findings reported AHL acylase activity of pencillin V acylases (PVA) which, alongside bile salt hydrolases, are members of the cholyolglycine hydrolase (CGH) family of the Ntn-hydrolases superfamily. The present study reports the unique activity profile of two CGHs from a marine bacterium Shewanella loihica-PV4, designated here as SlCGH1 and SlCGH2, including the structural analysis of SlCGH1. Both the enzymes exhibit AHL acylase activity while unexpectedly being inactive on standard CGH substrates PenV and bile salts. SlCGH1 differs from known homotetrameric CGHs in being a homodimer displaying a reduced active site volume attributed to loop orientation, which subsequently directs the substrate specificity. Moreover a ligand bound complex structure revealed an unusual bent conformation of the saturated acyl chain bound to the active site and also predicts a single oxyanion hole forming residue during catalysis instead of the usual two residues. Phylogenetic analysis reveals SlCGH1 homologs cluster separate from reported CGHs and AHL acylases. On the whole, SlCGH1 could represent a functionally distinct new sub-class of CGH as an adaptation to the marine environment and its structure could provide the structural framework for understanding such a novel subclass. We also make a modest proposal of a probable evolutionary link between AHL acylases and {beta} lactam acylases based on the overlap in activity and structural features.\n\nSignificanceCross-reactivity between AHL acylases and b lactam acylases has been recently identified giving us a vivid glimpse of a possible evolutionary relationship between the phenomena of quorum sensing and antibiotic resistance. We report here the first AHL acylase of the CGH structural framework. SlCGH1 from Shewanella loihica PV-4 is also the first report of a marine CGH with a unique activity and a new structural subclass of CGH family with AHL acylase activity. This finding highlights the vast diversity of AHL acylases and by extension quorum quenching enzymes as adaptation to different habitats. The results from this study also bolster the link between signal molecules and antibiotics, extending our understanding of the inadequately understood physiological roles of b-lactam acylases.

biochemistry↗

bioRxiv2019

Dissection of Catalytic Site in Crucial Gut Microbiome Enzyme: Bile Salt Hydrolase

Bile Salt Hydrolases (BSHs) are enzymes from enteric bacteria that catalyze the hydrolysis of Bile Acids and consequently promote the reduction of cholesterol level in the mammalian body. Out of several reported BSHs, the Enterococcus faecalis BSH (EfBSH) has been reported to have the highest enzymatic activity. Herein, we have investigated the mechanistic details of the EfBSH activity. The study was carried out employing two mutants of EfBSH: E269A and R207A, which shows differential catalytic activity. The mutant E269A exhibits significant loss in the BSH activity with an increased affinity towards the substrate as compared to R207A mutant. Further, R207A was found to be involved in allostery with an increased EfBSH activity towards tauro-conjugated bile acids. The structural and electrostatic force analyses of the active sites of the E269A mutant and the wild type EfBSH (wt EfBSH) revealed that the interaction between Glu21 and Arg207 is the determining factor in maintaining the dynamic allostery and high activity of EfBSH.

biochemistry↗

bioRxiv2019

Baicalein suppresses Tau fibrillization by sequestering oligomers

Alzheimers disease (AD) is a neurodegenerative disorder caused by protein misfolding, aggregation and accumulation in the brain. A large number of molecules are being screened against these pathogenic proteins but the focus for therapeutics is shifting towards the natural compounds as aggregation inhibitors, mainly due to their minimum adverse effects. Baicalein is a natural compound belonging to the class of flavonoids isolated from the Chinese herb Scutellaria baicalensis. Here we applied fluorescence, absorbance, microscopy, MALDI-TOF spectrophotometry and other biochemical techniques to investigate the interaction between Tau and Baicalein in vitro. We found the aggregation inhibitory properties of Baicalein for the repeat Tau. Overall, the potential of Baicalein in dissolving the preformed Tau oligomers as well as mature fibrils can be of utmost importance in therapeutics for Alzheimers disease.

biochemistry↗

bioRxiv2018

Identification of novel circadian transcripts in the zebrafish retina

High fecundity, transparent embryos for monitoring the rapid development of organs and the availability of a well-annotated genome has made zebrafish a model organism of choice for developmental biology and neurobiology. This vertebrate model, a favourite in chronobiology studies, shows striking circadian rhythmicity in behaviour. Here, we identify novel genes in the zebrafish genome, which shows their expression in the zebrafish retina. We further resolve the expression pattern over time and assign specific novel transcripts to the retinal cell type, predominantly in the inner nuclear layer. Using chemical ablation and free run experiments we segregate the transcripts that are rhythmic when entrained by light from those that show sustained oscillations in the absence of external cues. The transcripts reported here with rigorous annotation and specific functions in circadian biology provide the groundwork for functional characterisation of novel players in the zebrafish retinal clock.

neuroscience↗

bioRxiv2017

Aminoglycoside Antibiotics Perturb Physiologically Important MicroRNA Contributing To Drug Toxicity

miRNAs are key non-protein coding regulators of gene expression in various pathophysiological conditions. Targeting miRNA with small molecules offer an unconventional approach, where clinically active compounds with RNA binding activity can be tested for their ability to modulate miRNA levels and thus for drug repositioning. Aminoglycoside antibiotics are highly effective microbicidal RNA binding molecules that bind to prokaryotic rRNA secondary structures. Here, we report that specific subsets of miRNA can be modulated by aminoglycosides. However, ototoxicity (cochlear and vestibular) and nephrotoxicity of multiple origins resulting from prolonged use are a well-known disadvantage of aminoglycosides. Mature non-coding RNAs and their precursors can present off-target sites, by forming secondary structures that resemble ribosomal RNA, thus providing an additional molecular basis for the toxicity of aminoglycosides. Using in vitro, in cellulae and physiological responses, we provide evidence for the direct functional perturbation of the miR- 96 cluster leading to selective cell death in neuromasts- the zebrafish equivalent of cochlear hair cells, by Streptomycin, a prototype aminoglycoside antibiotic, thus contributing to the observed ototoxicity. Our observations, collectively underscore the importance of re- evaluating RNA binding drugs for their off-targeting effects in the context of miRNA and other functional non-coding RNA.

cell biology↗