Biology subjects

Nandakumar, A.

Publications and source records attributed to Nandakumar, A..

4 recordsLinked to original sources

bioRxiv2026

Chronic Electrophysiology Captures Progressive Variability in DNA-Damaged Cardiac Networks

Electrophysiological recordings provide a key functional readout of cardiac health. Most in vitro studies rely on acute measurements rather than recording evolving functional changes. Here, we implement a microelectrode array (MEA) based system for chronic electrophysiological monitoring of human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) monolayers. Electrophysiology recordings were maintained for up to 40 days, enabling longitudinal assessment of field potential amplitude, field potential duration, beat frequency, conduction velocity, and waveform morphology. Untreated iPSC-CM monolayers exhibited stable electrophysiological behavior throughout chronic culture. Following exposure to the cardiotoxic chemotherapeutic doxorubicin, chronic recordings captured progressive deterioration in electrical activity, including reduced field potential amplitude, altered repolarization dynamics, increased beat-to-beat variability, and reduced conduction that persisted after drug removal. Electrode-resolved analyses further revealed pronounced progressive spatial variability, highlighting localized responses that would be obscured by population-averaged measurements. Endpoint assays indicated reduced calcium transient amplitude and altered calcium reuptake, decreased mitochondrial membrane potential, and loss of connexin 43, findings consistent with the impaired electrical activity observed in chronic recordings. Together, these results establish chronic electrophysiological monitoring as a powerful approach for resolving the spatiotemporal progression of cardiotoxicity and other dynamic cardiac processes in vitro.

bioengineering↗

bioRxiv2025

Distinct modes of redox-mediated DegP activation govern its role as a general protease

DegP, a bifunctional chaperone-protease central to maintaining envelope proteostasis in Gram-negative bacteria, serves as a paradigm for understanding protein quality control mechanisms. Decades of research, mainly using synthetic peptides/non-native substrates that do not contain cysteines, have established the allosteric mode of DegP activation, where substrate binding remodels inactive DegP into its active conformations. In enteric bacteria, as DegP typically harbours a disulfide bond (DSB) in its protease domain, its redox-mediated activation has also been proposed. However, the cellular factors driving this activation mode and its physiological relevance have remained elusive. Here, we investigated this aspect in Escherichia coli primarily using long-chain fatty acids and alkaline pH as stressors. We show that, under these conditions, DegP considerably accumulates in its thiol form (DegPred), which represents its protease-active conformation. Besides removing damaged proteins, DegPred also induces the Cpx envelope stress response in all tested conditions. Notably, two distinct mechanisms promote redox-mediated DegP activation in a temporal and stress-specific manner: a compromised DSB-forming machinery fails to oxidize DegP, and thiol-containing substrates allosterically convert DegP into DegPred. Because in vitro, the lack of DSB sensitizes DegP to lower substrate levels, we suggest that in vivo DegPred is primed for a robust response to stresses. Finally, the diverse nature of the molecular players that activate Cpx in our tested stress conditions leads us to propose that the DSB of DegP, by acting as a redox sensor, enables DegP to integrate a wide range of inducing cues and thus work as a general protease. Significance StatementThe proteolytic activity of DegP is highly implicated in bacterial survival and virulence. Notably, DegP from the Enterobacteriaceae family of Gram-negative bacteria typically harbours a disulfide bond (DSB) in its protease domain. Because pathogens face redox stress during infection and antibiotic exposure, understanding how redox perturbants modulate DegP function is necessary for exploiting it as a target for therapeutic intervention strategies. Here, using E. coli as the model bacterium, we show that the DSB of DegP acts as a redox sensor under various inducing cues, thereby positioning DegP as a versatile, broad-spectrum protease. The present study opens avenues for investigating whether a similar redox-mediated control of DegP activation influences the pathogenesis of enteric bacteria.

microbiology↗

bioRxiv2025

Genomic diversity analysis enables development of pan-Dengue Toehold RNA sensors

The Dengue virus (DENV) like many other RNA viruses exhibits high genome sequence diversity. This poses a challenge to nucleic acid-based diagnostics, rendering them inefficient at detecting the diverse DENV strains circulating in a population. In this study, we address this challenge by developing a Toehold sensor assay that despite significant genomic diversity is able to detect [~]99.4% of all strains of DENV. To this end, our workflow first identifies relatively conserved short stretches (36-nt) within all DENV genomes, which could potentially serve as triggers that activate Toehold RNA sensors. We then add a crucial step of mismatch-tolerance that allows related triggers with high sequence diversity and multiple mismatches in the sensor-binding region to be efficiently sensed by the same sensor. The sensitivity of the Toehold sensor assay is often increased by inclusion of an isothermal RNA amplification step. We show by employing multiple primer sets that diverse trigger RNAs from all four serotypes of the dengue virus can be successfully amplified and subsequently detected by the same sensor. Together, this approach has resulted in a pan-dengue Toehold sensor assay, which presents a powerful nucleic acid detection platform to detect viruses with high sequence diversity.

molecular biology↗

bioRxiv2020

IGF targeting perturbs global replication through ribonucleotide reductase dysfunction

IGF receptor (IGF-1R) inhibition delays repair of radiation-induced DNA double-strand breaks (DSBs), prompting us to investigate whether IGF-1R influences endogenous DNA damage. We demonstrate that IGF-1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. We detect delayed replication fork progression in IGF-inhibited or IGF-1R depleted cancer cells, with activation of ATR-CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflects unanticipated regulation of global replication by IGF-1, mediated via AKT, MEK/ERK and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. IGF-1R inhibition or depletion downregulate RRM2, compromising RNR function and dNTP supply. The resulting delay in fork progression and hallmarks of replication stress are rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF-1 regulates replication. Following targeted compound screens, we identify synergy between IGF inhibition and ATM loss, with evidence that IGF inhibition compromises growth of ATM null cells and xenografts. This synthetic lethal effect reflects conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. These data implicate IGF-1R in alleviating replication stress, and the identified reciprocal IGF:ATM co-dependence provides an approach to exploit this effect in ATM-deficient cancers.

cancer biology↗