Biology subjects

Sarkar, G.

Publications and source records attributed to Sarkar, G..

3 recordsLinked to original sources

bioRxiv2026

THE BILE ACID RECEPTOR TGR5 MODULATES HIPPOCAMPAL NEURAL STEM CELL STATE AND AGEING

Ageing locks hippocampal neural stem cells (NSCs) into deeper quiescence and inflammation-driven senescence, which are also associated with impaired cognition. The bile-acid receptor TGR5, a member class I G-protein coupled receptors (GPCRs), is expressed in cultured hippocampal NSCs and in SOX2-positive cells of the adult sub granular zone, yet its role in NSC homeostasis is unknown. In this study we show that TGR5 selective agonist, TC-G 1005, expanded the NSC pool without forcing neuronal differentiation. Primary NSCs treatment with TC-G 1005 increased BrdU incorporation and PCNA in a concentration-dependent manner, and both responses were lost after TGR5 knockdown. TGR5 agonism in differentiating cultures increased nestin, left doublecortin (DCX) unchanged and suppressed MAP2 expression, preserving stemness rather than neuronal commitment. The agonist also released BMP4-imposed quiescence, lowering FOXO3a and restoring phospho-EGFR and ASCL1, and reopened autophagic flux, with less p62 and LC3-II, fewer LAMP2 puncta and more Beclin-1. Furthermore, activation of TGR5 suppressed IL-6 induced p53 and p16 expression, restored proliferation and contracted the inflammatory transcriptome by more than 90%, including Cdkn2a, Cdkn2b and Cdk6. In 18-month-old mice, brain-penetrant TC-G 1005 (5 mg/kg, subcutaneously, twice daily for 28 days) expanded SOX2-positive and proliferating NSCs, reduced p16, recovered ASCL1 and improved spatial memory, without changing locomotion or generating DCX-positive neurons. Dentate gyrus-restricted TGR5 knockdown abolished these cellular effects and the rescue of spatial and social discrimination. Hippocampal proteomic analysis revealed that TC-G 1005 treated aged mice occupied an intermediate, partially rejuvenated position between young and aged proteomes, with 86 proteins shared between ageing- and treatment-responsive sets, and mitochondrial and complement C1q modules shifted toward youthful levels. TGR5 activation therefore relieves both quiescence and senescence in the aged neurogenic niche without driving neuronal differentiation. Collectively, these results suggest that TGR5 could be targeted to preserve cognition in ageing by rejuvenating the hippocampal stem-cell niche.

neuroscience↗

bioRxiv2026

DNA 6mA marks transcriptionally active chromatin in malaria parasites

DNA N6-methyladenine (6mA) has emerged as a significant epigenetic modification across a broad range of eukaryotes, from unicellular protists to metazoa. However, its role in unicellular eukaryotic parasites with highly AT-rich genomes, such as malaria-causing Plasmodium falciparum, remains unclear. Using mass spectrometry, South-western blotting, and Single Molecule Real-Time sequencing (Pacific Biosciences) across four stages of P. falciparum intra-erythrocytic development (IED), we confirmed that 0.02-0.04% of genomic adenines are modified to 6mA, with over 60% of the sites being stably maintained during the IED cycle. Notably, 6mA is enriched at transcription start sites, with genes bearing 6mA marks within their 5 and 3 untranslated regions exhibiting significantly elevated steady-state transcript levels. Consistent with this, 6mA loci show a strong positive correlation with activating histone post-translational modifications, while showing no significant association with repressive histone marks. Furthermore, in contrast to unicellular ciliates such as Oxytricha and Tetrahymena - organisms that share ancestry with Plasmodium - 6mA-marked genomic regions do not occlude nucleosomes. Lastly, we identified a putative 6mA methyltransferase belonging to the METTL4 family in P. falciparum, PfN6AMT encoded by the PF3D7_1303100 gene, and demonstrate that recombinant PfN6AMT exhibits robust methyltransferase activity in vitro, with mutation of its active site residues abolishing catalytic activity. Collectively, our findings demonstrate that 6mA is a low-abundance, yet reproducible, feature of the P. falciparum epigenome that is associated with transcriptionally active chromatin, and that the molecular mechanisms governing DNA adenine methylation may have undergone substantial evolutionary divergence, even among closely related eukaryotic lineages.

genomics↗

bioRxiv2022

Clonally selected lines after CRISPR/Cas editing are not isogenic

The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (i) editing cultured cells; (ii) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, while cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, while detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and copy number alterations acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.

genomics↗