Biology subjects

Balasubramanian, P.

Publications and source records attributed to Balasubramanian, P..

15 recordsLinked to original sources

bioRxiv2025

mCytoCounter, a deployable somatic cell counting device for the assessment of mastitis in dairy farms

Bovine mastitis is a recurring disease among herds that is often caused by unhygienic farm conditions, resulting in bacterial invasion of the udder. It is a leading cause of financial losses for dairy farmers, largely due to the widespread use of antimicrobials for treatment, which in turn fuels the development of drug-resistant bacteria. Somatic cell count (SCC) is a widely used quantitative method for diagnosing sub-clinical and clinical mastitis. We have developed a rapid and quantitative microfluidic device that enables the quantification of somatic cells in milk, thereby aiding in the diagnosis of bovine mastitis. Our platform features a microfluidic cartridge for capturing somatic cells, which is inserted into an in-house device called the mCytoCounter. The device reads the fluorescence emission signals from the somatic cells captured on the membrane in 2 minutes, with a total turnaround time of 15 minutes. The mCytoCounter is an automated and deployable system that can be used in dairy farms with minimal training. We analysed the performance of mCytoCounter with milk samples and benchmarked it against the SCC using a fluorescence microscope with a correlation coefficient of 0.97. The mCytoCounter has a detection range of 103 to 106 SCC/mL, which covers the required cell number range to discriminate between healthy, subclinical, and clinical mastitis.

immunology↗

bioRxiv2025

Mutations in antimicrobial peptides differently affect sleep and plasticity

Molecules implicated in host defense can, independent of their roles as immune mediators, regulate sleep. In mammals, cytokines such as Tumor Necrosis Factor (TNF ) and Interleukin 1{beta} (IL1{beta}) promote sleep. In Drosophila, infection stimulates the systemic release of a battery of anti-microbial peptides (AMPs). These fly immune effectors have been understudied as sleep modulators. We obtained mutants in different AMPs and characterized their functions in sleep and sleep dependent outputs. AMP mutants sleep less with a longer sleep latency, and lowered arousal thresholds. Group C mutant flies, doubly mutant for the anti-fungal peptides Metchinikowin (Mtk) and Drosomycin (Drs), showed the greatest impairments. These Group C mutants also showed an exaggerated sleep rebound. Sleep rebound was unaltered in other mutants. Different sets of AMP mutants exhibited specific disruptions in socialization and rocking induced sleep. These data are a detailed characterization of sleep regulation in AMP mutants. We also evaluated sleep functions. Group C mutants uniquely exhibited normal learning and memory, and lower synapse abundance, despite sleeping the least. Group C mutants are thus able to carry out some sleep functions without sleeping very much. Glial knockdown of Metchnikowin and Drosomycin mimicked the sleep phenotypes of the null mutants, these genes thus act from glia. Sleep and memory defects in AMP mutants was reversible - enhancing sleep of Bomanin mutants pharmacologically or behaviorally improved learning and memory. Together, these data suggest that AMPs are potent sleep modulators and that different classes of AMPs differently affect sleep and sleep dependent outcomes.

neuroscience↗

bioRxiv2025

Lipid-laden endothelial cells exhibit a transcriptomic signature linked to blood-brain barrier dysfunction, metabolic reprogramming and increased inflammation in the aging brain

Dysregulation in lipid metabolism is increasingly recognized as a key contributor to age-related diseases, including neurodegeneration and cerebrovascular dysfunction. While prior studies have largely focused on glial cells, the impact of lipid dysregulation on brain endothelial aging remains poorly understood. In this study, we conducted a secondary analysis of single-cell transcriptomic data from young and aged mouse brains, with a specific focus on endothelial cells (ECs). Our analyses revealed that aging promotes lipid droplet accumulation in brain ECs. These lipid-laden brain ECs exhibit a transcriptomic signature indicative of impaired blood-brain barrier function, increased cellular senescence, and inflammation in aging. Furthermore, lipid accumulation is associated with an altered metabolic phenotype characterized by increased fatty acid oxidation and decreased glycolysis, and impaired mitochondrial electron transport chain activity in the ECs of the aging brain. We have also validated lipid accumulation in aged ECs in vivo. Collectively, our findings indicate that lipid accumulation drives structural, functional, and metabolic impairments in the brain ECs, likely contributing to cerebrovascular aging. Understanding the mechanisms underlying lipid accumulation-induced endothelial dysfunction may offer novel therapeutic strategies for mitigating microvascular dysfunction and cognitive decline in aging.

neuroscience↗

bioRxiv2025

Phylogenetic Analysis and Machine Learning Identify Signatures of Selection and Predict Deleterious Mutations in Common Bean

Mutations are continuous source of new alleles and genetic diversity in populations. Domestication and selection influence the accumulation of alleles occurring across a range of deleteriousness. Evidence suggests that mildly deleterious mutations (DelMut) can be purged out of breeding populations, increasing favorable allele accumulation. We used phylogeny-based analyses among 36 legume genomes to identify selection signatures and predict DelMut in common bean. We also developed a multiparent advanced generation intercrossed (MAGIC) population of black beans to characterize DelMut. Genes involved in nitrogen metabolism showed signs of positive selection in the Middle American genome, whereas genes related to phosphorylation were positively selected in the Andean genome. By combining conservation and protein information with machine learning (ML) for high-dimensional feature analysis, we characterized 82,442 sites in the MAGIC founders (36,558 polymorphic) and 4,753 sites evenly sequenced among RILs that could be potentially deleterious. Variation in the number of highly DelMut (high predicted deleterious scores) among lines was observed and later correlated with agronomic traits. Phenotypic analyses showed that calculated genetic load (and number of highly DelMut) was negatively correlated with flowering time, maturity, and yield. A detailed in-silico analysis of predicted mutations showed presence in highly conserved protein regions, which is likely to affect protein functionality. Our results show that variation in genetic load can be observed in breeding populations and potentially impact plant performance. These results contribute to understanding the genome-wide accumulation patterns of DelMut in breeding populations. Our study supports future development of strategies to reduce genetic load in promising germplasm and accelerate breeding programs. Key messages- Genome-wide highly deleterious mutations were predicted in conserved protein domains potentially affecting protein functionality. - Variation in the genetic load and number of highly deleterious mutations can be observed in artificial breeding populations. - Nitrogen- and phosphorous-related genes are under positive selection in Middle American and Andean beans respectively.

plant biology↗

bioRxiv2024

Chronic β3 adrenergic agonist treatment improves brain microvascular endothelial function and cognition in aged mice

Microvascular endothelial dysfunction, characterized by impaired neurovascular coupling, reduced glucose uptake, blood-brain barrier disruption, and microvascular rarefaction, plays a critical role in the pathogenesis of age-related vascular cognitive impairment (VCI). Emerging evidence points to non-cell autonomous mechanisms mediated by adverse circulating milieu (an increased ratio of pro-geronic to anti-geronic circulating factors) in the pathogenesis of endothelial dysfunction leading to impaired cerebral blood flow and cognitive decline in the aging population. In particular, age-related adipose dysfunction contributes, at least in part, to an unfavorable systemic milieu characterized by chronic hyperglycemia, hyperinsulinemia, dyslipidemia, and altered adipokine profile, which together contribute to microvascular endothelial dysfunction. Hence, in the present study, we aimed to test whether thermogenic stimulation, an intervention known to improve adipose and systemic metabolism by increasing cellular energy expenditure, could mitigate brain endothelial dysfunction and improve cognition in the aging population. Eighteen-month-old old C57BL/6J mice were treated with saline or CL ({beta}3-adrenergic agonist) for 6 weeks followed by functional analysis to assess endothelial function and cognition. CL treatment improved neurovascular coupling responses and rescued brain glucose uptake in aged animals. In addition, CL treatment also attenuated blood-brain barrier leakage and associated neuroinflammation in the cortex of aged animals. More importantly, these beneficial changes in microvascular function translated to improved cognitive performance in radial arm water maze and Y-maze tests. Our results suggest that {beta}3-adrenergic agonist treatment improves multiple aspects of brain microvascular endothelial function and can be potentially repurposed for treating age-associated cognitive decline.

neuroscience↗

bioRxiv2024

The metabolic benefits of thermogenic stimulation are preserved in aging

Adipose thermogenesis has been actively investigated as a therapeutic target for improving metabolic dysfunction in obesity. However, its applicability to middle-aged and older populations, which bear the highest obesity prevalence in the US (approximately 40%), remains uncertain due to age-related decline in thermogenic responses. In this study, we investigated the effects of chronic thermogenic stimulation using the {beta}3-adrenergic (AR) agonist CL316,243 (CL) on systemic metabolism and adipose function in aged (18-month-old) C57BL/6JN mice. Sustained {beta}3-AR treatment resulted in reduced fat mass, increased energy expenditure, increased fatty acid oxidation and mitochondrial activity in adipose depots, improved glucose homeostasis, and a favorable adipokine profile. At the cellular level, CL treatment increased uncoupling protein 1 (UCP1)-dependent thermogenesis in brown adipose tissue (BAT). However, in white adipose tissue (WAT) depots, CL treatment increased glycerol and lipid de novo lipogenesis (DNL) and turnover suggesting the activation of the futile substrate cycle of lipolysis and reesterification in a UCP1-independent manner. Increased lipid turnover was also associated with the simultaneous upregulation of proteins involved in glycerol metabolism, fatty acid oxidation, and reesterification in WAT. Further, a dose-dependent impact of CL treatment on inflammation was observed, particularly in subcutaneous WAT, suggesting a potential mismatch between fatty acid supply and oxidation. These findings indicate that chronic {beta}3-AR stimulation activates distinct cellular mechanisms that increase energy expenditure in BAT and WAT to improve systemic metabolism in aged mice. Our study provides foundational evidence for targeting adipose thermogenesis to improve age-related metabolic dysfunction.

pharmacology and toxicology↗

bioRxiv2024

Targeting Metabolic Dependencies in Acute Myeloid Leukemia: A Dual Strategy using Arsenic Trioxide and Artesunate.

Acute myeloid leukemia (AML) remains a difficult disease to cure despite recent advances. Off-target side effects of therapy, especially prolonged cytopenia, lead to significant morbidity and mortality. There is an increased recognition that in AML, there is a potentially vulnerable dependence on OXPHOS metabolism, more so in the leukemia stem cell compartment (AML-LSC) that could be exploited. Drug re-purposing screens have suggested the possible role of artesunate (ART) in inhibiting mitochondrial respiration and arsenic trioxide (ATO) in inhibiting glycolysis. Here, we explore the potential of using a combination of ART and ATO to treat AML. Through in-vitro and in-vivo studies, we demonstrate the potential of this combination along with hypo-methylating agents and other mitocans, such as BCL-2 inhibitor venetoclax, to be used in the treatment of AML with minimal off-target effect on normal hematopoietic stem cells (HSC). These observations warrant further exploration of such novel combinations in clinical trials.

cancer biology↗

bioRxiv2023

An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

Systemic Lupus Erythematosus (SLE) is characterized by autoreactive B cell activation, upregulation of Type I Interferon (IFN) and widespread inflammation. Mitochondrial nucleic acids (NAs) are increasingly recognized as triggers of IFN1. Thus, defective removal of mitochondria from mature red blood cells (Mito+ RBCs), a feature of SLE, contributes to IFN production by myeloid cells2. Here we identify blood monocytes (Mo) that have internalized RBCs and co-express IFN-stimulated genes (ISGs) and interleukin-1{beta} (IL-1{beta}) in SLE patients with active disease. We show that ISG expression requires the interaction between Mito+ RBC-derived mitochondrial DNA (mtDNA) and cGAS, while IL-1{beta} production entails Mito+ RBC-derived mitochondrial RNA (mtRNA) triggering of RIG-I-like receptors (RLRs). This leads to the cytosolic release of Mo-derived mtDNA that activates the NLRP3 inflammasome. Importantly, IL-1{beta} release depends on the IFN-inducible myxovirus resistant protein 1 (MxA), which enables the translocation of this cytokine into a trans-Golgi network (TGN)-mediated unconventional secretory pathway. Our study highlights a novel and synergistic pathway involving IFN and the NLRP3 inflammasome in SLE.

immunology↗

bioRxiv2023

Mass spectrometry-based multi-omics identifies metabolic signatures of sarcopenia in rhesus monkey skeletal muscle

Sarcopenia is a progressive disorder characterized by age-related loss of skeletal muscle mass and function. Although significant progress has been made over the years to identify the molecular determinants of sarcopenia, the precise mechanisms underlying the age-related loss of contractile function remains unclear. Advances in omics technologies, including mass spectrometry-based proteomic and metabolomic analyses, offer great opportunities to better understand sarcopenia. Herein, we performed mass spectrometry-based analyses of the vastus lateralis from young, middle-aged, and older rhesus monkeys to identify molecular signatures of sarcopenia. In our proteomic analysis, we identified numerous proteins that change with age, including those involved in adenosine triphosphate and adenosine monophosphate metabolism as well as fatty acid beta oxidation. In our untargeted metabolomic analysis, we identified multiple metabolites that changed with age largely related to energy metabolism including fatty acid beta oxidation. Pathway analysis of age-responsive proteins and metabolites revealed changes in muscle structure and contraction as well as lipid, carbohydrate, and purine metabolism. Together, this study discovers new metabolic signatures and offer new insights into the molecular mechanism underlying sarcopenia for the evaluation and monitoring of therapeutic treatment of sarcopenia. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/551315v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1dc0addorg.highwire.dtl.DTLVardef@14d73b8org.highwire.dtl.DTLVardef@1e8733forg.highwire.dtl.DTLVardef@1e65408_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Abstract

biochemistry↗

bioRxiv2023

Inhibition of BCR::ABL1 tyrosine kinase activity Aids in the Generation of Stable Chronic Myeloid Leukemia Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) generated from patients with chronic myeloid leukemia (CML) have the potential for disease modeling to study disease pathogenesis and screening therapeutic interventions. In this study, we aimed to generate iPSCs from CD34+ hematopoietic progenitors of CML patients with varying responses to tyrosine kinase inhibitor (TKI) therapy. The generated CML-CD34-iPSC colonies displayed atypical "dome-shaped" morphology and underwent spontaneous differentiation in a few days. However, supplementation with imatinib (IM), the most widely used TKI to treat CML patients, in the culture medium improved the stability and maintenance of all isolated CML-CD34-iPSC colonies, allowing them to be maintained for more than 20 passages without significant differentiation. In contrast to previous studies, our results indicate that suppressing the BCR::ABL1 oncogenic pathway is essential for efficiently generating stable CML-iPSC colonies. Furthermore, we successfully differentiated these iPSCs to CD34+ hematopoietic progenitors both in the presence and absence of IM. This robust protocol for generating CML-iPSCs provides a valuable resource for disease modelling. The generated iPSCs will be a valuable tool for investigating CML pathophysiology, drug resistance mechanisms, and drug screening to identify novel and effective therapies for this disease.

cancer biology↗

bioRxiv2022

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is the commonly adapted strategy to ameliorate {beta}-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits the HbF tetramer production affecting the therapeutic benefits. Here, we tested various deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified a 11 kb sequence, encompassing Putative Repressor Region (PRR) to {beta}-globin Exon-1 ({beta}E1), as the core deletion that ablates HbA and exhibit superior production of HbF compared to HPFH or other well-established targets. The PRR-{beta}E1 edited hematopoietic stem and progenitor cells (HSPCs) retained engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice generating HbF+ cells in vivo. Importantly, the editing induces therapeutically relevant levels of HbF to reverse the phenotypes of both sickle cell disease and {beta}-thalassemia major. These results indicate that the PRR-{beta}E1 gene editing in patient HSPCs can potentially lead to superior therapeutic outcomes for {beta}-hemoglobinopathies gene therapy.

biochemistry↗

bioRxiv2022

Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors

{beta}-thalassemia and HbE result from mutations in the {beta}-globin locus that impedes the production of functional {beta}-hemoglobin and represents one of the most common genetic disorders worldwide. Recent advances in genome engineering have opened up new therapeutic opportunities to directly correct these pathogenic mutations using base editors that install transition mutations (A>G and C>T) in the target region with minimal generation of indels. Herein, for the first time, we demonstrate the usage of base editor in the correction of point mutations spanning multiple regions of the HBB gene, including promoter, intron and exon. To this end, we have engineered human erythroid cells harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+ HSPCs with desired mutations for the primary screening by base editors. We further performed precise creation and correction of individual HBB point mutations in human erythroid cells using base editors, which were effectively corrected in the HBB-engineered erythroid model. Intriguingly, most bystander effects produced by the base editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our study provides the proof-of-concept for the precise, efficient and scarless creation and correction of various pathogenic mutations at the coding and non-coding regions of HBB gene in human erythroid cells using base editors and establishes a novel therapeutic platform for the treatment of {beta}-thalassemia/HbE patients. This study can be further explored in correcting the other monogenic disorders caused due to single base substitutions.

genomics↗

bioRxiv2021

Adiponectin receptor agonist AdipoRon improves skeletal muscle function in aged mice

The loss of skeletal muscle function with age, known as sarcopenia, significantly reduces independence and quality of life and can have significant metabolic consequences. Although exercise is effective in treating sarcopenia it is not always a viable option clinically, and currently there are no pharmacological therapeutic interventions for sarcopenia. Here we show that chronic treatment with pan-adiponectin receptor agonist AdipoRon improved muscle function in male mice by a mechanism linked to skeletal muscle metabolism and tissue remodeling. In aged mice, 6 weeks of AdipoRon treatment improved skeletal muscle functional measures in vivo and ex vivo. Improvements were linked to changes in fiber type, including an enrichment of oxidative fibers, and an increase in mitochondrial activity. In young mice, 6 weeks of AdipoRon treatment improved contractile force and activated the energy sensing kinase AMPK and the mitochondrial regulator PGC-1a (peroxisome proliferator activated receptor gamma coactivator 1 alpha). In cultured cells, the AdipoRon induced stimulation of AMPK and PGC-1a was associated with increased mitochondrial membrane potential, reorganization of mitochondrial architecture, increased respiration, and increased ATP production. Furthermore, the ability of AdipoRon to stimulate AMPK and PGC1a was conserved in nonhuman primate cultured cells. These data show that AdipoRon is an effective agent for the prevention of sarcopenia in mice and indicate that its effects translate to primates, suggesting it may also be a suitable therapeutic for sarcopenia in clinical application.

physiology↗

bioRxiv2020

Identification of novel HPFH-like mutations by CRISPR base editing that elevates the expression of fetal hemoglobin

Switching hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin is an effective strategy for the treatment of beta-hemoglobinopathies. Fetal hemoglobin expression is down-regulated in the postnatal period due to the interplay of transcription regulators with the HBG promoters. However, in the hereditary persistence of fetal hemoglobin (HPFH) condition, naturally occurring point mutations in the HBG promoter causes continued expression of fetal globin even during adulthood. Inspired by this natural phenomenon, we screened the proximal promoter of human HBG genes using adenine and cytosine base editors to identify other nucleotide substitutions that could potentially lead to elevated levels of fetal globin. Both the base editors efficiently and precisely edited at the target sites with a minimal generation of indels and no deletion of one of the duplicated HBG genes. Through systematic tiling across the HBG proximal promoter, we identified multiple novel target sites that resulted in a significant increase in fetal globin levels. Further, we individually validated the top eight potential target sites from both the base editors and observed robust elevation in the fetal globin levels up to 47 %, without any detrimental effects on erythroid differentiation. Our screening strategy resulted in the identification of multiple novel point mutations and also validated the known non-deletional HPFH mutations that could elevate the fetal globin expression at therapeutically relevant levels. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter that normally mediates fetal globin silencing and identify additional targets for therapeutic upregulation of fetal hemoglobin.

genomics↗

bioRxiv2020

Arsenic trioxide resistance in acute promyelocytic leukemia: More to it than PML mutations

Acquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain the majority of disease recurrence seen in the clinic. We have generated a stable ATO resistant promyelocytic cell from a ATO sensitive NB4 cell line. We also noted that another ATRA resistant cell line (UF1) was cross resistant to ATO. We have characterized these resistant cell lines and observed that they significantly differed in their immunophenotype, drug transporter expression, drug resistance mutation profile and were also cross-resistant to other conventional chemotherapeutic agents. The NB4 derived resistant cell line had the classical A216V PML-B2 domain mutation while the UF1 cell line did not. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in the resistant cell lines. Glycolytic inhibition by 2-DG was efficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell lines and was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO resistant cell lines. The survival of the resistant cell lines was significantly affected by compounds targeting the mitochondrial respiration irrespective of the existence of ATO resistance-conferring genetic mutations. Our data demonstrate the addition of mitocans can overcome ATO resistance. We further demonstrated that the combination of ATO and mitocans has the potential in the treatment of non-M3 AML and the translation of this approach in the clinic needs to be explored further. Key pointsO_LIMetabolic rewiring promotes ATO resistance, which can be overcome by targeting mitochondrial oxidative phosphorylation. C_LIO_LICombination of ATO and mitocans can be exploited as a potential therapeutic option for relapsed APL and in non-M3 AML patients. C_LI

cancer biology↗