Biology subjects

Govindan, S.

Publications and source records attributed to Govindan, S..

6 recordsLinked to original sources

bioRxiv2022

Neuro-toxicogenomic mapping of TMT induced neurotoxicity using human minibrain reveals associated adverse outcome events

Genome-wide transcriptomic interrogation of organoids and 3D tissue models are increasingly used for characterizing drug, toxicity responses and neurodevelopmental disorders. We established here a neuro-toxicogenomic assay by utilizing "minibrain", a human in vitro 3D brain model system and a low-cost, highly multiplexable RNA-seq methodology (BRB-seq) for screening the effect of trimethyltin chloride (TMT) induced neurotoxicity. We demonstrate that transcriptomic profiling is insightful to the cellular composition and regional identity of the minibrain. Further, we characterize the transcriptomic changes associated with the dose-time neurotoxic response of minibrain upon exposure to TMT. The distinct gene expression changes and molecular candidates identified with our pipeline provides insight to map the key events involved in the adverse outcome pathway of TMT associated neurotoxicity. We identify processes such as endoplasmic reticulum stress, dysregulation of synaptic genes and downregulation of neuron-morphology associated genes upon exposure to TMT. In response to TMT, we identify activation of an early response homeostatic mechanism in minibrain and an interplay of STAT pathways correlating with the dose severity. In this study, we present a neuro-toxicogenomic assay that demonstrates the power of a low-cost transcriptomic screening to study chemical induced neurotoxicity.

neuroscience↗

bioRxiv2021

Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

Lactoferrin binding protein B (LbpB) is a lipoprotein present on the surface of Neisseria that has been postulated to serve dual functions during pathogenesis in both iron acquisition from lactoferrin, and in providing protection against the cationic antimicrobial peptide lactoferricin. Here, we present the structures of LbpB from N. meningitidis and N. gonorrhoeae in complex with human holo-lactoferrin, forming a 1:1 complex and confirmed by SEC-SAXS. LbpB consists of N- and C-lobes with the N-lobe interacting extensively with the C-lobe of lactoferrin. Our structures provides insight into LbpBs preference towards holo-lactoferrin, and our mutagenesis and binding studies show that lactoferrin and lactoferricin bind independently. Our studies provide the molecular details for how LbpB serves to capture and preserve lactoferrin in an iron-bound state for delivery to the membrane transporter LbpA for iron piracy, and as an antimicrobial peptide sink to evade host immune defenses.

biophysics↗

bioRxiv2020

Mass Generation, Neuron Labelling and 3D Imaging of Minibrains

Minibrain is a spherical in vitro 3D brain organoid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human brain organoid models, there is a lack of labelling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains. Using the pipeline, we present i. dendrite morphometric properties obtained from 3D neuron morphology reconstructions and ii. distribution and quantification of cell types in 3D across whole mount organoids.

neuroscience↗

bioRxiv2019

Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.

genomics↗

bioRxiv2018

Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions

SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. However, how temporal fluctuations in their expression levels bias lineage commitment is unknown. Here we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cell towards both neuroectodermal and mesendodermal fates. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

developmental biology↗

bioRxiv2018

In vivo pulse-labeling of isochronic cohorts of cells in the central nervous system using FlashTag

This protocol describes a fluorescence birthdating technique to label, track and isolate isochronic cohorts of newborn cells in the central nervous system in vivo. Injection of carboxyfluorescein esters into the cerebral ventricle allows pulse-labeling of M-phase progenitors in touch with the ventricle and their progeny across the central nervous system, a procedure we termed FlashTag. Labeled cells can be imaged ex vivo or in fixed tissue, targeted for electrophysiological experiments, or isolated using Fluorescence-Activated Cell Sorting (FACS) for cell culture or (single-cell) RNA-sequencing. The dye is retained for several weeks, allowing labeled cells to be identified postnatally. This protocol describes the labeling procedure using in utero injection, the isolation of live cells using FACS, as well as the processing of labeled tissue using immunohistochemistry.

neuroscience↗