Biology subjects

Jagannathan, N. S.

Publications and source records attributed to Jagannathan, N. S..

6 recordsLinked to original sources

bioRxiv2025

Dynamic estimation of metabolic state during CAR T cell production and the relationship of early metabolism to final therapeutic product

Adoptive cell therapies such as CAR T cells have revolutionized cancer treatment and shown successes even with refractory cases of haematological malignancies. There is burgeoning interest in the optimization and improved manufacturing of cell therapy products. For CAR T cell therapy, enrichment of certain phenotypes of T cells in the infusion product have been correlated with improved long-term treatment outcomes. While metabolic control of T cell phenotypic fates has been demonstrated in some contexts, gaps still exist in our knowledge of how T cell metabolic dynamics early during CAR T manufacturing might affect critical quality attributes (CQAs) of the final product (e.g., differentiation/exhaustion/potency). We present a modelling framework that can perform real-time estimation of per-cell metabolic rates of T cells expanded ex vivo in a reactor. We validate our estimated rates using metabolic assays, show how average rates can be deconvoluted to rates of individual T cell phenotypes, and demonstrate applicability to different reactor types. Applying our tool to the expansion of both healthy and patient-derived cells in a perfusion-based microbioreactor, we offer proof-of-principle to show that correlations exist between early metabolic rates of T cells in culture and cellular attributes related to growth, differentiation and exhaustion of the final product. Given the biological variation that exists in the growth and dynamics of patient-derived cells in culture, such modelling contributes to the overarching goal of improving the consistency of cell therapy through Adaptive Process Control (APC).

cancer biology↗

bioRxiv2023

Contributions of individual satellite cells to muscle regeneration assessed using a confetti mouse model

Insufficient regeneration is implicated in muscle pathologies, but much remains unknown about the regenerative output of individual muscle stem cells, called satellite cells (SCs). Prior work showed that individual SCs contribute to regeneration of more than one muscle fiber ("fiber-crossing") after full-muscle damage. We investigated whether fiber-crossing also occurred in peripheral regions of a localized muscle injury. To assess fiber-crossing with a minimum number of mice, we used lineage tracing with confetti fluorescence, and developed a novel stochastic modeling method to interpret the ambiguity of multi-color fluorescent lineage tags. Microscopy of the regenerated muscle showed that adjacent fibers often expressed the same-colored tags. Computational analysis concluded that the observed color patches would be extremely unlikely to occur by chance unless SCs contributed myonuclei to multiple adjacent fibers (26-33% of SCs contributing to at most 1-2 additional fibers). Interestingly, these results were similar across the different regions studied, suggesting that severe destruction is not required for fiber-crossing. Our method to assess fiber-crossing may be useful for future study of gene and cell therapies that use fiber-crossing to aid muscle regeneration.

physiology↗

bioRxiv2023

Multi-omic analysis of bat versus human fibroblasts reveals altered central metabolism

Bats have unique characteristics compared to other mammals, including increased longevity and higher resistance to cancer and infectious disease. While previous studies have analyzed the metabolic requirements for flight, it is still unclear how bat metabolism supports these unique features, and no study has integrated metabolomics, transcriptomics, and proteomics to characterize bat metabolism. In this work, we performed a multi-omics data analysis using a computational model of metabolic fluxes to identify fundamental differences in central metabolism between primary lung fibroblast cell lines from the black flying fox fruit bat (Pteropus alecto) and human. Bat cells showed higher expression levels of Complex I components of electron transport chain (ETC), but, remarkably, a lower rate of oxygen consumption (OCR). Computational modeling interpreted these results as indicating that Complex II activity may be low or reversed, similar to an ischemic state. An ischemic-like state of bats was also supported by decreased levels of central metabolites and increased ratios of succinate to fumarate in bat cells. Ischemic states tend to produce reactive oxygen species (ROS), which would be incompatible with the longevity of bats. However, bat cells had higher antioxidant reservoirs (higher total glutathione and higher ratio of NADPH to NADP) despite higher mitochondrial ROS levels. In addition, bat cells were more resistant to glucose deprivation and had increased resistance to ferroptosis, one of the characteristics of which is oxidative stress. Thus, our studies revealed distinct differences in the ETC regulation and metabolic stress responses between human and bat cells.

systems biology↗

bioRxiv2023

High-density microbioreactor process designed for automated point-of-care manufacturing of CAR T cells

While adoptive cell therapies have revolutionized cancer immunotherapy, current autologous chimeric antigen receptor (CAR) T cell manufacturing face challenges in scaling to meet patient demands. CAR T cell production still largely rely on fed-batch, manual, open processes that lack environmental monitoring and control, whereas most perfusion-based, automated, closed-system bioreactors currently suffer from large footprints and working volumes, thus hindering process development and scaling-out. Here, we present a means of conducting anti-CD19 CAR T cell culture-on-a-chip. We show that T cells can be activated, transduced, and expanded to densities exceeding 150 million cells/mL in a two-milliliter perfusion-capable microfluidic bioreactor, thus enabling the production of CAR T cells at clinical dose levels in a small footprint. Key functional attributes such as exhaustion phenotype and cytolytic function were comparable to T cells generated in a gas-permeable well. The process intensification and online analytics offered by the microbioreactor could facilitate high-throughput process optimization studies, as well as enable efficient scale-out of cell therapy manufacturing, while providing insights into the growth and metabolic state of the CAR T cells during ex vivo culture.

bioengineering↗

bioRxiv2020

BRAWNIN: A sORF-encoded Peptide Essential for Vertebrate Mitochondrial Complex III Assembly

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution1,2. Here, we show that the mitochondria proteome is enriched for proteins smaller than 100 a.a. (defined as SEPs). Using a mitochondrial prediction and validation pipeline for small open-reading-frame (sORF)-encoded peptides (SEPs), we report the discovery of 16 endogenous mitochondrial SEPs (mito-SEPs) associated with oxidative phosphorylation (OXPHOS). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN (BR), a 71 amino acid peptide encoded by the C12orf73 gene, is essential for respiratory chain complex III (CIII) assembly. In human cells, BR is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In vivo, BR is enriched in muscle tissues and its maternal zygotic deletion in zebrafish causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BR is essential for oxidative phosphorylation across vertebrate species. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

cell biology↗

bioRxiv2019

Proteins encoded by Novel ORFs have increased disorder but can be biochemically regulated and harbour pathogenic mutations

Recent evidence has suggested that protein or protein-like products can be encoded by previously uncharacterized Open Reading Frames (ORFs) that we define as Novel Open Reading Frames (nORFs)1,2. These nORFs are present in both coding and non coding regions of the human genome and the novel proteins that they encode have increased the number and complexity of cellular proteome from bacteria to humans. It is a conundrum whether these protein or protein-like products could play any significant functional biological role. But hopes have been raised to target them for anticancer and antimicrobial therapy3,4. To infer whether these novel proteins can perform biological functions, we used computational predictions to systematically investigate whether their amino acid sequences can form ordered or disordered structures. Our results indicated that that these novel proteins have significantly higher predicted disorder structure compared to all known proteins, yet we do not find any correlation between the pathogenicity of the mutations and whether they are present in the ordered and disordered regions of these novel proteins. This study reveals that we should investigate these novel proteins more systematically as they may be important to understand complex diseases.

genetics↗