Biology subjects

Kumar, S. R.

Publications and source records attributed to Kumar, S. R..

7 recordsLinked to original sources

bioRxiv2026

Human iPSC-Derived Cardiomyocytes Maladaptively Remodel to Cyclic Stretch in Hypoplastic Left Heart Syndrome

Background: Hypoplastic Left Heart Syndrome (HLHS) is a severe congenital heart defect characterized by underdevelopment of left-sided cardiac structures, requiring staged surgical palliation that establishes the right ventricle as the single ventricle maintaining systemic circulation. After surgery, patients remain highly susceptible to heart failure, possibly due to the increased hemodynamic load imposed on the single surviving ventricle. Because the etiology of HLHS remains poorly understood, elucidating the mechanisms that drive ventricular dysfunction requires model systems that capture both patient-specific genetics and the dynamic mechanical environment of the ventricle. Thus, our objective was to compare how induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) from patients with HLHS differ from control in static culture and in response to cyclic stretch. Methods: Control iPSCs from two unaffected donors and iPSCs from four HLHS patients were differentiated into iPSC-CMs, engineered into aligned tissues, and cultured statically or exposed to cyclic 20% uniaxial stretch at 1 Hz for 48h. Sarcomere organization was quantified in static tissues using images of immunostained -actinin. Calcium transients at 1 Hz pacing frequency in static and stretched tissues were evaluated using Fluo-4 calcium indicator dye. Bulk RNA sequencing was performed to evaluate stretch-induced transcriptomic changes in control and HLHS iPSC-CMs. Results: In static culture, HLHS iPSC-CMs had modest defects in sarcomere organization and calcium transient morphology compared to control. In response to cyclic stretch, control iPSC-CMs exhibited calcium transients with quicker rise time, a response observed in only one of four lines of HLHS iPSC-CMs. Transcriptomic analysis revealed that all four lines of HLHS iPSC-CMs failed to upregulate calcium handling and sarcomere-associated genes in response to stretch, unlike control iPSC-CMs. Stretch also induced HLHS iPSC-CMs to exhibit pronounced activation of inflammatory signaling pathways, a response not observed in control iPSC-CMs. Conclusions: iPSC-CMs from HLHS patients demonstrate aberrant responses to cyclic stretch, including blunted adaptation of calcium transients and heightened inflammatory signaling. These findings implicate defective mechanoadaptation as a potential contributor to single ventricle failure and identify potential candidate biomarkers and new opportunities for therapeutic interventions for HLHS patients.

physiology↗

bioRxiv2023

Spatial genomics of AAVs reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo

Integrating cell type-specific regulatory elements (e.g. enhancers) with recombinant adeno-associated viruses (AAVs) can provide broad and efficient genetic access to specific cell types. However, the packaging capacity of AAVs restricts the size of both the enhancers and the cargo that can be delivered. Transcriptional crosstalk offers a novel paradigm for cell type-specific expression of large cargo, by separating distally-acting regulatory elements into a second AAV genome. Here, we identify and profile transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. To understand transcriptional crosstalk, we develop spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue. Using these methods, we construct detailed views of the dynamics of AAV transduction and demonstrate that transcriptional crosstalk is dependent upon concatemer formation. Finally, we leverage transcriptional crosstalk to drive expression of a large Cas9 cargo in a cell type-specific manner with systemically-administered engineered AAVs and demonstrate AAV-delivered, minimally-invasive, cell type-specific gene editing in wildtype animals that recapitulates known disease phenotypes. HighlightsO_LITranscriptional crosstalk between enhancers and promoters delivered in trans by AAVs is a generalized phenomenon. C_LIO_LISpatial genomics techniques, AAV-Zombie and SpECTr, reveal that AAV genome concatemerization facilitates transcriptional crosstalk. C_LIO_LITranscriptional crosstalk can be leveraged for minimally-invasive, targeted AAV delivery of large cargo, including machinery for CRISPR-based gene editing and manipulation. C_LIO_LITranscriptional crosstalk enables cell-type specific gene disruption in wildtype animals, recapitulating behavioural phenotypes of genetic knockouts. C_LI

bioengineering↗

bioRxiv2023

The cytochrome P450 enzyme WsCYP71B35 from Withania somnifera has a role in withanolides biosynthesis and defense against bacteria

The medicinal properties of Ashwagandha (Withania somnifera L. Dunal) are attributed to withanolides, which belong to the triterpenoid steroidal lactones class of compounds. Though it is proposed that intermediates of the universal phytosterol pathway are utilized by cytochrome P450 (CYP450) enzymes to form withanolides, studies on functional characterization of these enzymes has been sparse. This study reports the functional characterization of a CYP450 candidate from W. somnifera (WsCYP71B35) that exhibited induced expression in response to methyl jasmonate treatment and showed higher expression in tissues that accumulate withanolides. Biochemical assay with yeast microsomal fraction expressing recombinant WsCYP71B35 indicated no activity when phytosterols and their intermediate 24-methylene cholesterol were used as substrates. However, WsCYP71B35 catalyzed product formation with withaferin A, withanolide A, withanolide B, and withanoside IV among the tested substrates. Moreover, virus-induced gene silencing (VIGS) and transient overexpression of WsCYP71B35 in W. somnifera leaves modulated the levels of withaferin A, withanolide A, and withanolide B, indicating the role of WsCYP71B35 in withanolides pathway. Furthermore, VIGS of WsCYP71B35 in W. somnifera reduced its tolerance to Pseudomonas syringae (DC3000) infection, whereas overexpression enhanced the tolerance to the bacterium in W. somnifera and transgenic tobacco. Overall, these results provide insights into the role of W. somnifera WsCYP71B35 in withanolides biosynthesis and bacterial defense.

plant biology↗

bioRxiv2023

Primate-conserved Carbonic Anhydrase IV and murine-restricted Ly6c1 are new targets for crossing the blood-brain barrier

The blood-brain barrier (BBB) presents a major challenge to delivering large molecules to study and treat the central nervous system (CNS). This is due in part to the scarcity of effective targets for BBB crossing, the identification of which is the crucial first step of drug development. Here, we leveraged a panel of adeno-associated viruses (AAVs) previously identified through directed evolution for improved BBB transport to reverse engineer protein targets for enhanced BBB crossing. We identify both murine-restricted Ly6c1 and primate-conserved carbonic anhydrase IV (Car4; CA4) as novel receptors for crossing the BBB. We demonstrate how these receptors can unlock new experimental and computational target-focused engineering strategies by creating the enhanced Ly6c1-binding vector AAV-PHP.eC and by applying AlphaFold2-enabled in silico methods to rank capsids against identified receptors and generate capsid-receptor binding models. Here, with Car4, we add a completely new receptor to the very short list currently available for crossing the BBB in humans and, with Ly6c1, we validate a pipeline for receptor-targeted engineering. The identification of Car4/CA4 and structural insights from computational modeling provide new paths toward human brain-penetrant chemicals (drugs) and biologicals (including gene delivery).

bioengineering↗

bioRxiv2021

ROS/RNS balancing, aerobic fermentation regulation and cell cycle control a complex early trait ('CoV-MAC-TED') for combating SARS-CoV-2-induced cell reprogramming

In a perspective entitled From plant survival under severe stress to anti-viral human defense we raised and justified the hypothesis that transcript level profiles of justified target genes established from in vitro somatic embryogenesis (SE) induction in plants as a reference compared to virus-induced profiles can identify differential virus signatures that link to harmful reprogramming. A standard profile of selected genes named ReprogVirus was proposed for in vitro-scanning of early virus-induced reprogramming in critical primary infected cells/tissues as target trait. For data collection, the ReprogVirus platform was initiated. This initiative aims to identify in a common effort across scientific boundaries critical virus footprints from diverse virus origins and variants as a basis for anti-viral strategy design. This approach is open for validation and extension. In the present study, we initiated validation by experimental transcriptome data available in public domain combined with advancing plant wet lab research. We compared plant-adapted transcriptomes according to RegroVirus complemented by alternative oxidase (AOX) genes during de novo programming under SE-inducing conditions with in vitro corona virus-induced transcriptome profiles. This approach enabled identifying a major complex trait for early de novo programming during SARS-CoV-2 infection, called CoV-MAC-TED. It consists of unbalanced ROS/RNS levels, which are connected to increased aerobic fermentation that links to alpha-tubulin-based cell restructuration and progression of cell cycle. We conclude that anti-viral/anti-SARS-CoV-2 strategies need to rigorously target CoV-MAC-TED in primary infected nose and mouth cells through prophylactic and very early therapeutic strategies. We also discuss potential strategies in the view of the beneficial role of AOX for resilient behavior in plants. Furthermore, following the general observation that ROS/RNS equilibration/redox homeostasis is of utmost importance at the very beginning of viral infection, we highlight that de-stressing disease and social handling should be seen as essential part of anti-viral/anti-SARS-CoV-2 strategies.

cell biology↗

bioRxiv2021

Adaptive reprogramming during early seed germination requires temporarily enhanced fermentation a critical role for alternative oxidase (AOX) regulation that concerns also microbiota effectiveness

Plants respond to environmental cues via adaptive cell reprogramming that can affect whole plant and ecosystem functionality. Microbiota constitutes part of plants inner and outer environment. This Umwelt underlies steady dynamics, due to complex local and global biotic and abiotic changes. Hence, adaptive plant holobiont responses are crucial for continuous metabolic adjustment at systems levels. Plants require oxygen-dependent respiration for energy-dependent adaptive morphology, such as, germination, root and shoot growth, formation of adventitious, clonal and reproductive organs, fruits and seeds. Fermentative paths can help in acclimation and, to our view the role of alternative oxidase (AOX) in coordinating complex metabolic and physiologic adjustments is underestimated. Cellular level of sucrose is an important sensor of environmental stress. We explored the role of exogenous sucrose and its interplay with AOX during early seed germination. We found that sucrose-dependent initiation of fermentation during the first 12 hours after imbibition (HAI) was beneficial to germination. However, parallel enhanced AOX expression was essential to control negative effects by prolonged sucrose treatment. Early down-regulated AOX activity until 12 HAI improved germination efficiency in the absence of sucrose, but suppressed early germination in its presence. Our results also suggest that seeds-inoculated arbuscular mycorrhizal fungi can buffer sucrose stress during germination to restore normal respiration more efficiently. Following this approach, we propose a simple method to identify organic seeds and low-cost on-farm perspectives for early selection on disease tolerance, predicting plant holobiont behavior and improving germination. Furthermore, our research strengthens the view that AOX can serve as powerful functional marker source for seed hologenomes.

cell biology↗

bioRxiv2018

Widespread and targeted gene expression by systemic AAV vectors: Production, purification, and administration

We recently developed novel AAV capsids for efficient and noninvasive gene transfer across the central and peripheral nervous systems. In this protocol, we describe how to produce and systemically administer AAV-PHP viruses to label and/or genetically manipulate cells in the mouse nervous system and organs including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans eight days, excluding the time required to assess gene expression, and can be readily adopted by laboratories with standard molecular and cell culture capabilities. We provide guidelines for experimental design and choosing the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

neuroscience↗