Biology subjects

Vaid, S.

Publications and source records attributed to Vaid, S..

8 recordsLinked to original sources

bioRxiv2026

Prenatal assembly of functional cortical circuits

The extent to which early brain maturation requires interactions with the outside world is a central question in neurobiology. Because much of cortical maturation occurs after birth in Mus, it has often been viewed as dependent on postnatal experience. However, studies addressing this issue have largely relied on postnatal sensory deprivation paradigms, which perturb normal development and cannot determine to what extent postnatal experience per se drives maturation. To address this question, here we compare two related rodents with markedly different gestation lengths: the precocial Acomys dimidiatus (39-day gestation) and the altricial Mus musculus (19-day gestation). By generating a novel Acomys reference genome and using histology, cellular birth dating, electrophysiology, single-nucleus transcriptomics, and quantitative behavior, we show that Acomys preserves the canonical sequence and timing of cortical development, while shifting major milestones of neuronal, circuit and behavioral maturation into prenatal life. At birth, Acomys cortex already shows advanced cytoarchitecture, neuronal physiology, thalamocortical barrels, transcriptional states, and sensorimotor behavior, with postnatal molecular programs in Mus unfolding prenatally in Acomys. Thus, birth is not a prerequisite for early cortical maturation. Instead, evolutionarily conserved developmental programs unfold across birth, with birth occurring at different stages of these programs, reflecting a species-specific balance between neonatal competence and prolonged postnatal plasticity.

neuroscience↗

bioRxiv2025

A Novel Patient-Derived Mutation in Glucocorticoid Receptor Reveals an Essential Role for DNA binding in Development and Endocrine Regulation

Glucocorticoid receptors (GRs) are vital transcription factors regulating stress responses, metabolism, and development. We report here a novel heterozygous NR3C1 c.1310C>T (p.T437I) variant within the GR DNA-binding domain found in a family exhibiting clinical and hormonal manifestations of partial glucocorticoid resistance. Patient-derived fibroblasts demonstrated an overall suppression in dexamethasone-induced transcription. To comprehensively assess the significance of the variant, we engineered a knock-in mouse model (Nr3c1+/T444I mice) using CRISPR-Cas9, providing an in vivo model of the patient-derived GR mutation at the orthologous residue. Heterozygous mice exhibited partial glucocorticoid resistance, dysregulated HPA axis activity and impaired dexamethasone suppression, closely recapitulating the patient phenotype. Homozygous Nr3c1T444I/T444I embryos were recovered at embryonic day 12.5 (E12.5) but did not survive to term, indicating mid-gestational lethality. Transcriptomic profiling of primary mouse embryonic fibroblasts revealed dosage-dependent effects with heterozygotes showing an intermediate response to dexamethasone compared to wild-type, while homozygotes showed a markedly blunted response. Our results challenge prior assumptions by demonstrating that GR DNA-binding is essential for embryogenesis, while offering a new preclinical platform to investigate glucocorticoid resistance pathophysiology and therapeutic interventions.

genetics↗

bioRxiv2025

Delayed forebrain excitatory and inhibitory neurogenesis inSTRADA-related megalencephaly via mTOR hyperactivity

Biallelic pathogenic variants in STRADA, an upstream regulator of the mechanistic target of rapamycin (mTOR) pathway, result in megalencephaly, drug-resistant epilepsy, and severe intellectual disability. This study explores how mTOR pathway hyperactivity alters cell fate specification in dorsal and ventral forebrain development using STRADA knock-out human stem cell derived brain organoids. In both dorsal and ventral forebrain STRADA knock-out organoids, neurogenesis is delayed, with a predilection for progenitor renewal and proliferation and an increase in outer radial glia. Ventrally, interneuron subtypes shift to an increase in neuropeptide-Y expressing cells. Inhibition of the mTOR pathway with rapamycin results in rescue for most phenotypes. When mTOR pathway variants are present in all cells of the developing brain, overproduction of interneurons and altered interneuron cell fate may underlie mechanisms of megalencephaly, epilepsy, and cognitive impairment. Our findings suggest mTOR inhibition during fetal brain development as a potential therapeutic strategy in STRADA deficiency.

neuroscience↗

bioRxiv2025

Cryopreservation of Human Cortical Organoids Using Vitrification.

Cryopreservation at ultra-low temperatures is a valuable tool for preserving cells and tissues used in research. However, few protocols exist for the preservation of brain organoid models. Current methods for preserving human cortical organoids (hCOs) rely on conventional slow cooling approaches with organoids suspended in a medium containing a cocktail of cryoprotectants. In contrast, we have optimized a vitrification technique previously used to cryopreserve human embryos and oocytes for application to hCOs. We have successfully cryopreserved hCOs that were generated by two different protocols. The vitrified organoids demonstrate a growth rate, cytoarchitecture, cell type composition and electrical activity comparable to non-vitrified controls. Our hCO cryopreservation method provides a useful alternative approach for bio-banking and cross-institutional collaboration using cortical organoids as their model system. Highlights- Human pluripotent stem cell (hPSC) derived hCOs can be cryopreserved using vitrification. - Vitrified hCOs develop similarly to non-vitrified controls. - Vitrified hCOs can be stored long-term and shipped to other institutions. MotivationMany methods of cryopreservation have been developed for the maintenance of cell lines, organoid models and tissues. Most organoid models have been successfully preserved using conventional slow-cooling methods; however, human cortical organoids have been difficult to preserve. Our findings demonstrate that the vitrification of cortical organoids preserves their structure, cell type diversity and function upon rewarming and continued culture.

neuroscience↗

bioRxiv2024

Open-source cell culture automation system with integrated cell counting for passaging microplate cultures

Tissue culture in 96-well microplates is conventionally a tedious, highly manual process sensitive to individual technique and experimenter error. Here, we describe the Automated Cell Culture Splitter (ACCS), a system for passaging plates of adherent or suspension cells, for routine culture maintenance or specialized applications such as seeding plates for microscopy. The system is built around the Opentrons OT-2 liquid handling robot and incorporates a novel on-deck imaging-based cell counter which allows it to compensate for density disparities across a source plate and control the number of cells seeded on a per-well basis. We find this solution can cut hands-on time by 61% and the results compare favorably to our existing manual cell culture processes in terms of both seeding density precision and bio-logical outcomes, achieving a control of seeding density with a well-to-well coefficient of variation (CV) under 11%. The system is designed to be adaptable and an accessible entry point into automation for high-throughput cell culture; to that end, all of the source code and hardware designs are released under open source licenses.

bioengineering↗

bioRxiv2023

Global organelle profiling reveals subcellular localization and remodeling at proteome scale

Defining the subcellular distribution of all human proteins and its remodeling across cellular states remains a central goal in cell biology. Here, we present a high-resolution strategy to map subcellular organization using organelle immuno-capture coupled to mass spectrometry. We apply this proteomics workflow to a cell-wide collection of membranous and membrane-less compartments. A graph-based representation of our data reveals the subcellular localization of over 7,600 proteins, defines spatial protein networks, and uncovers interconnections between cellular compartments. We demonstrate that our approach can be deployed to comprehensively profile proteome remodeling during cellular perturbation. By characterizing the cellular landscape following hCoV-OC43 viral infection, we discover that many proteins are regulated by changes in their spatial distribution rather than by changes in their total abundance. Our results establish that proteome-wide analysis of subcellular remodeling provides essential insights for the elucidation of cellular responses. Our dataset can be explored at organelles.czbiohub.org.

cell biology↗

bioRxiv2023

An open-source FACS automation system for high-throughput cell biology

Recent advances in gene editing are enabling the engineering of cells with an unprecedented level of scale. To capitalize on this opportunity, new methods are needed to accelerate the different steps required to manufacture and handle engineered cells. Here, we describe the development of an integrated software and hardware platform to automate Fluorescence-Activated Cell Sorting (FACS), a central step for the selection of cells displaying desired molecular attributes. Sorting large numbers of samples is laborious, and, to date, no automated system exists to sequentially manage FACS samples, likely owing to the need to tailor sorting conditions ("gating") to each individual sample. Our platform is built around a commercial instrument and integrates the handling and transfer of samples to and from the instrument, autonomous control of the instruments software, and the algorithmic generation of sorting gates, resulting in walkaway functionality. Automation eliminates operator errors, standardizes gating conditions by eliminating operator-to-operator variations, and reduces hands-on labor by 93%. Moreover, our strategy for automating the operation of a commercial instrument control software in the absence of an Application Program Interface (API) exemplifies a universal solution for other instruments that lack an API. Our software and hardware designs are fully open-source and include step-by-step build documentation to contribute to a growing open ecosystem of tools for high-throughput cell biology.

bioengineering↗

bioRxiv2020

Zika virus-based immunotherapy enhances long-term survival of rodents with brain tumors through upregulation of memory T-cells

Zika virus (ZIKV) exhibits a tropism for brain tumor cells and has been used as an oncolytic virus to target brain tumors in mice with modest effects on extending median survival. Recent studies have highlighted the potential for combining virotherapy and immunotherapy to target cancer. We postulated that ZIKV could be used as an adjuvant to enhance the long-term survival of mice with malignant glioblastoma and generate memory T-cells capable of providing long-term immunity against cancer remission. To test this hypothesis mice bearing malignant intracranial GL261 tumors were subcutaneously vaccinated with irradiated GL261 cells previously infected with the ZIKV. Mice also received intracranial injections of live ZIKV, irradiated ZIKV, or irradiated GL261 cells previously infected with ZIKV. Long-term survivors were rechallenged with a second intracranial tumor to examine their immune response and look for the establishment of protective memory T-cells. Mice with subcutaneous vaccination plus intracranial irradiated ZIKV or intracranial irradiated GL261 cells previously infected with ZIKV exhibited the greatest extensions to overall survival. Flow cytometry analysis of immune cells within the brains of long-term surviving mice after tumor rechallenge revealed an upregulation in the levels of T-cells, including CD4+ and tissue-resident memory CD4+ T-cells, in comparison to long-term survivors that were mock-rechallenged, and in comparison to naive untreated mice challenged with intracranial gliomas. These results suggest that ZIKV can serve as an adjuvant to subcutaneous tumor vaccines that enhance long-term survival and generate protective tissue-resident memory CD4+ T-cells.

cancer biology↗