Biology subjects

Reddy, P.

Publications and source records attributed to Reddy, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Exposure to perfluorooctanoic acid accelerates Drosophila melanogaster juvenile development and disrupts mitochondrial metabolism

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with poorly understood sublethal effects on insects. Perfluorooctanoic acid (PFOA), one of the most widely distributed legacy PFAS is increasingly recognized for altering organismal physiology beyond traditional toxicity endpoints. Here, we use the fruit fly Drosophila melanogaster as a model to examine how PFOA exposure during larval (juvenile) development reshapes insect life-history progression and metabolic homeostasis. Our studies reveal that at environmentally relevant concentrations (nM to low {micro}M), PFOA induces precocious expression of developmentally-regulated genes and leads to metabolic changes that persist into adulthood. At higher concentrations used to probe mechanism, PFOA accelerates larval development, disrupts mitochondrial membrane potential, and increases whole-organism metabolic heat production - results that suggest altered mitochondrial energetic efficiency. Consistent with this tradeoff, PFOA-exposed larvae that develop faster under permissive conditions exhibit heightened sensitivity to environmental stressors, including elevated temperature and reduced food hydration. Together, these findings demonstrate that PFOA disrupts metabolic and developmental processes in a dose- and context-dependent manner, highlighting sublethal effects that may influence insect resilience under environmental stress. SYNOPSIS STATEMENTHere we describe how PFOA alters the growth, development, and metabolism of the fruit fly Drosophila melanogaster. Specifically, we find that PFOA accelerates Drosophila juvenile growth while also rendering exposed larvae sensitive to environmental stress. These observations suggest that widespread PFOA contamination may impair the developmental fitness of insect populations.

pharmacology and toxicology↗

bioRxiv2025

High-resolution quantification of metabolic heat output from individual live Drosophila brains

Quantitative insights into brain metabolism are essential for advancing our understanding of energy dynamics in the brain. However, current approaches for tracking brain metabolism, metabolic profiling and respirometry, provide only static snapshots of metabolite levels or lack the required resolution. Here, we develop a novel nanowatt-resolution biocalorimeter capable of real-time continuous measurements of heat output to quantitatively measure the metabolism of individual live Drosophila melanogaster brains and investigate how sex, genotype, age, and disease affect brain metabolism. We show for the first time that female brains, across multiple wild-type genotypes, exhibit a significantly higher metabolic rate ([~]10%) than male brains at a young age (<10 days old) and follow distinct metabolic trajectories across the lifespan. We also find that parkin mutants, a genetic model for Parkinsons disease, exhibit a [~]15% reduction in brain metabolic output relative to controls, revealing that defective mitophagy due to parkin deficiency affects brain metabolism. Furthermore, we measure the metabolic rate of reproductive tissues of Drosophila, highlighting the broad applicability of our biocalorimeter. Together, these advances open new avenues for investigating how tissue-specific metabolism is impacted by aging, neurodegeneration, and disease states. TeaserDirect measurement of metabolic rate of individual Drosophila brains to investigate how sex, genotype, age, and disease affect brain metabolism.

bioengineering↗

bioRxiv2025

A brainstem-thalamic circuitry for affective-motivational responses to cold pain

The medial thalamus is crucial for the sensory and affective-motivational responses to chronic pain. However, a mechanistic understanding of how the distinct subnuclei of the medial thalamus mediate behavioral responses to pain remains lacking. Taking advantage of intersectional viral genetics, chemogenetics, optogenetics, in-vivo imaging, and ex-vivo physiology, we reveal that the neurons in the parafascicular (PF) nuclei of the medial thalamus receive monosynaptic inputs from the lateral parabrachial nuclei (LPBN) in mice. LPBN is an essential nucleus in the ascending pain pathway, receiving projections from the dorsal horn of the spinal cord. The PF neurons downstream of LPBN (PFpost-LPBN) are nociceptive, sensitized by peripheral neuropathy, acutely aversive, and, when activated, drive both sensory and affective-motivational responses to cold pain. In contrast, the LPBN target neurons in the intralaminar centromedian thalamus (CMpost-LPBN), another nociceptive nucleus of the medial thalamus, are primarily involved in the affective-motivational aspects of pain. Together, we reveal that the LPBN, through two closely related thalamic nuclei, influences behavior in mice with cold hypersensitivity due to peripheral neuropathies.

neuroscience↗

bioRxiv2025

VISTA-induced tumor suppression by a four amino acid intracellular motif

VISTA is a key immune checkpoint receptor under investigation for cancer immunotherapy; however, its signaling mechanisms remain unclear. Here we identify a conserved four amino acid (NPGF) intracellular motif in VISTA that suppresses cell proliferation by constraining cell-intrinsic growth receptor signaling. The NPGF motif binds to the adapter protein NUMB and recruits Rab11 endosomal recycling machinery. We identify and characterize a class of triple-negative breast cancers with high VISTA expression and low proliferative index. In tumor cells with high VISTA levels, the NPGF motif sequesters NUMB at endosomes, which interferes with epidermal growth factor receptor (EGFR) trafficking and signaling to suppress tumor growth. These effects do not require canonical VISTA ligands, nor a functioning immune system. As a consequence of VISTA expression, EGFR receptor remains abnormally phosphorylated and cannot propagate ligand-induced signaling. Mutation of the VISTA NPGF domain reverts VISTA-induced growth suppression in multiple breast cancer mouse models. These results define a mechanism by which VISTA represses NUMB to control malignant epithelial cell growth and signaling. They also define distinct intracellular residues that are critical for VISTA-induced cell-intrinsic signaling that could be exploited to improve immunotherapy.

cancer biology↗

bioRxiv2024

A mitochondria-targeted PPR protein restores cytoplasmic male sterility by post-transcriptional modification of ORF147 in Cajanus cajanifolius

Restoration factors (Rfs) belonging to the pentatricopeptide repeat proteins (PPRs) family play an essential role in plant growth and development including their binding to CMS-associated mitochondrial RNAs leading to fertility restoration. The present study identified 22 mitochondrial-specific PPRs in pigeonpea and explored the underlying mechanisms of restoration of fertility in the A4 CMS system through yeast-three hybrid studies. The identified gene was functionally validated through transgenic expression in Arabidopsis model system and obtained conclusive evidence that the identified Rf-PPR was responsible for fertility restoration. The sub-cellular localization studies implied that the identified Rf-PPR is mitochondrial targeting. The study demonstrated that due to the interaction between mitochondrial CMS mRNA and nuclear Rf-PPR protein, post-transcriptional modification occurred, leading to the inability to translate and accumulate cytotoxic CMS protein resulting in fertility restoration. The study specifically looks into the RNA-protein interaction occurring at the nucleo-cytoplasmic level in the A4 cytoplasm of Cajanus cajanifolius. HighlightsThe study identifies the restoration of fertility genes corresponding to the CMS-causing orf147 gene.

developmental biology↗

bioRxiv2024

Differential control of intestine function by genetically defined enteric neurons

The function of the intestine is regulated by direct innervation from a combination of enteric, sensory, and autonomic neurons. A central question in neurobiology is how these distinct peripheral neuron populations collectively control intestinal function. However, disambiguating the functions of intestine-innervating neuronal populations has been a challenge. Using intersectional genetic approaches in mice, we enable precise manipulations of defined neuronal populations within the intestinal tract. We examined enteric neurons, which represent the majority of intestine-innervating neurons, by genetically isolating neuronal subclasses, identifying their morphological specializations, and defining subclass-specific influences on intestinal functions. We further found that food consumption can be modulated by select enteric neuron populations via the spinal sensory afferent pathway. Taken together, the presented molecular genetic characterization of intestine-innervating neurons establishes a foundation for detailed studies of the enteric nervous system and its interactions with the broader neural networks of the body.

neuroscience↗

bioRxiv2024

Converging inputs compete at the lateral parabrachial nuclei to dictate the affective-motivational responses to cold pain

The neural mechanisms of the affective-motivational symptoms of chronic pain are poorly understood. In chronic pain, our innate coping mechanisms fail to provide relief leading to heightened manifestation of these behaviors. In laboratory animals, such as mice and rats, licking the affected areas is a behavioral coping mechanism and it is sensitized in chronic pain. Hence, we have focused on delineating the brain circuits mediating licking in mice with chemotherapy-induced peripheral neuropathy (CIPN). Mice with CIPN develop intense cold hypersensitivity and lick their paws upon contact with cold stimuli. We studied how the lateral parabrachial nucleus (LPBN) neurons facilitate licking behavior when mice are exposed to noxious thermal stimuli. Taking advantage of transsynaptic viral, optogenetic, and chemogenetic strategies, we observed that the LPBN neurons become hypersensitive to cold in mice with CIPN and facilitate licks. Further, we found that the expression of licks depends on competing excitatory and inhibitory inputs from the spinal cord and lateral hypothalamus (LHA), respectively. We anatomically traced the post-synaptic targets of the spinal cord and LHA in the LPBN and found that they synapse onto overlapping populations. Activation of this LPBN population was sufficient to promote licking due to cold allodynia. In sum, our data indicate that the nociceptive inputs from the spinal cord and information on brain states from the hypothalamus impinge on overlapping LPBN populations to modulate their activity and, in turn, regulate the elevated affective-motivational responses in CIPN.

neuroscience↗

bioRxiv2023

Convergence of aging- and rejuvenation-related epigenetic alterations on PRC2 targets

Rejuvenation of tissues in physiologically aging mice can be accomplished by long-term partial reprogramming via expression of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc). To investigate the epigenetic determinants of partial reprogramming-mediated rejuvenation, we used whole genome bisulfite sequencing to carry out unbiased comprehensive profiling of DNA methylation changes in skin from mice subjected to partial reprogramming, as well as young and untreated old controls. We found a striking convergence of age- and rejuvenation-related epigenetic alterations on targets of the Polycomb repressive complex 2 (PRC2). These results are also supported by a likewise prominent enrichment of PRC2 targets in gene expression data, suggesting that PRC2 activity can modulate aging and mediate tissue rejuvenation.

genomics↗

bioRxiv2023

A DRG genetic toolkit reveals molecular, morphological, and functional diversity of somatosensory neuron subtypes

Mechanical and thermal stimuli acting on the skin are detected by morphologically and physiologically distinct sensory neurons of the dorsal root ganglia (DRG). Achieving a holistic view of how this diverse neuronal population relays sensory information from the skin to the central nervous system (CNS) has been challenging with existing tools. Here, we used transcriptomic datasets of the mouse DRG to guide development and curation of a genetic toolkit to interrogate transcriptionally defined DRG neuron subtypes. Morphological analysis revealed unique cutaneous axon arborization areas and branching patterns of each subtype. Physiological analysis showed that subtypes exhibit distinct thresholds and ranges of responses to mechanical and/or thermal stimuli. The somatosensory neuron toolbox thus enables comprehensive phenotyping of most principal sensory neuron subtypes. Moreover, our findings support a population coding scheme in which the activation thresholds of morphologically and physiologically distinct cutaneous DRG neuron subtypes tile multiple dimensions of stimulus space.

neuroscience↗

bioRxiv2023

A deep-learning driven investigation of circuit basis for reflexive hypersensitivity to pain

Objectively measuring animal behavior is key to understanding the neural circuits underlying pain. Recent progress in machine vision has presented us with unprecedented scope in behavioral analysis. Here, we apply DeeplabCut (DLC) to dissect mouse behavior on the thermal-plate test -- a commonly used paradigm to ascertain supraspinal contributions to noxious thermal sensation and pain hypersensitivity. We determine the signature characteristics of the pattern of mouse movement and posture in 3D in response to a range of temperatures from innocuous to noxious on the thermal-plate test. Next, we test how acute chemical and chronic inflammatory injuries sensitize mouse behaviors. Repeated exposure to noxious temperatures on the thermal-plate can induce learning, and in this study, we design a novel assay and formulate an analytical pipeline that will facilitate the dissection of plasticity mechanisms in pain circuits in the brain. Last, we record and test how activating Tacr1 expressing PBN neurons -- a population responsive to sustained noxious stimuli-affects mouse behavior on the thermal plate test. Taken together, we demonstrate that by tracking a single body part of a mouse, we can reveal the behavioral signatures of mice exposed to noxious surface temperatures, report the alterations of the same when injured, and determine if a molecularly and anatomically defined pain responsive circuit plays a role in the reflexive hypersensitivity to thermal pain.

neuroscience↗

bioRxiv2022

Bursting Translation on Single mRNAs in Live Cells

Stochasticity has emerged as a mechanism to control gene expression. Much of this so-called "noise" has been attributed to bursting transcription. However, the stochasticity of translation has not similarly been investigated due to a lack of enabling imaging technologies. We developed techniques to track single mRNAs and their translation in live cells for hours, allowing measurement of previously uncharacterized translation dynamics. We applied genetic and pharmacological perturbations to control translation kinetics. Like transcription, translation is not a constitutive process but instead cycles between inactive and active states or "bursts". But unlike transcription, which is largely frequency modulated, complex structure in the 5-untranslated region alters burst amplitude. Bursting frequency can be controlled through cap-proximal sequences and trans-acting factors such as eIF4F. We coupled single molecule imaging with stochastic modeling to deduce the fundamental kinetic parameters of translational bursting, a new dimension of translational control. HighlightsO_LILong-term tracking of single mRNAs reveals multi-state, bursting translation C_LIO_LIStructure in the 5-untranslated region modulates translational burst amplitude C_LIO_LI5-cap proximal sequences modulate translational burst frequency C_LIO_LImTOR signaling adjusts translation bursting to respond to environmental cues C_LI

molecular biology↗

bioRxiv2021

A redox cycle with complex II promotes sulfide quinone oxidoreductase dependent H2S oxidation

The dueling roles of H2S as an endogenously synthesized respiratory substrate and as a toxin, raise questions as to how it is cleared when the electron transport chain is inhibited. Sulfide quinone oxidoreductase (SQOR) is a mitochondrial inner membrane flavoprotein that catalyzes the first step in the H2S oxidation pathway and uses coenzyme Q (CoQ) as an electron acceptor. However, complex IV poisoning by H2S inhibits complex III-dependent recycling of CoQH2, which is needed to sustain H2S oxidation. We have discovered that under these conditions, reversal of complex II activity using fumarate as an electron acceptor, establishes a new redox cycle with SQOR. The purine nucleotide cycle and the malate aspartate shuttle are sources of fumarate in H2S treated cells, which accumulate succinate. Complex II knockdown decreases the efficiency of H2S clearance and increases recovery time to the basal respiration rate in H2S treated cells. In contrast, attenuation of complex I, which is a major competitor for the mitochondrial CoQ pool, has the opposite effects. Targeted knockout of complex II in murine intestinal epithelial cells that are routinely exposed to microbiota derived H2S, decreases serum, urine, and fecal thiosulfate, a product of H2S oxidation. Our study identifies a metabolic reprogramming response to H2S that furnishes fumarate as an alternate electron acceptor and supports H2S oxidation independent of complex IV activity. Complex II-linked redox cycling of SQOR has important implications for gut H2S metabolism as colonocytes are routinely exposed to high concentrations of this gas derived from the microbiota. One Sentence SummaryReversal of complex II sustains and prioritizes H2S oxidation when respiration is poisoned.

biochemistry↗

bioRxiv2021

Cohesin Releasing Factor WAPL Regulates Genome Structure and Function of Mature T Cells

The cohesin complex modulates gene expression and cellular functions by shaping three-dimensional (3D) organization of chromatin. WAPL, cohesins DNA release factor, regulates 3D chromatin architecture. The 3D genome structure and its relevance to mature T cell functions in vivo is not well understood. We show that in vivo lymphopenic expansion, and allo-antigen driven proliferation, alters the 3D structure and function of the genome in mature T cells. Conditional deletion of Wapl in T cells reduced long-range genomic interactions, altered chromatin A/B compartments and interactions within topologically associating domains (TADs) of the chromatin in T cells at baseline. Comparison of chromatin structure in normal and WAPL-deficient T cells after lymphopenic and allo-antigen driven stimulation revealed reduced loop extensions with changes in cell cycling genes. WAPL-mediated changes in 3D architecture of chromatin regulated activation, cycling and proliferation of T cells in vitro and in vivo. Finally, WAPL-deficient T cells demonstrated reduced severity of graft-versus-host disease (GVHD) following experimental allogeneic hematopoietic stem cell transplantation. These data collectively characterize 3D genomic architecture of T cells in vivo and demonstrate biological and clinical implications for its disruption by cohesin release factor WAPL.

molecular biology↗

bioRxiv2021

A highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative elicits high titers of neutralizing antibodies

The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of [~]80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 {degrees}C and were stable to long term storage of over 4 weeks at 37 {degrees}C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were [~] three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

microbiology↗

bioRxiv2020

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel, nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100{degrees}C when lyophilized, upto 70{degrees}C in solution and stable for over four weeks at 37{degrees}C. In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

biophysics↗

bioRxiv2020

RNA-seq of Human T-Cells After Hematopoietic Stem Cell Transplantation Identifies Linc00402 as a Novel Regulator of T-Cell Alloimmunity

Mechanisms governing allogeneic T-cell responses after allogeneic hematopoietic stem cell (HSC) and solid organ transplantation are incompletely understood. Long non-coding RNAs (lncRNA) do not code for, but control gene expression with tissue specificity. However, their role in T-cell alloimmunity is unknown. We performed RNA-seq on donor T-cells from HSCT patients and found that increasing strength of allogeneic stimulation caused greater differential expression of lncRNAs. The differential expression was validated in an independent patient cohort, and also following ex vivo allogeneic stimulation of healthy human T-cells. Linc00402, a novel, conserved lncRNA, was identified as the most differentially expressed and was enriched 88 fold in human T-cells. Mechanistically, it was mainly located in the cytoplasm, and its expression was rapidly reduced following T-cell activation. Consistent with this, tacrolimus preserved the expression of Linc00402 following T-cell activation, and lower levels of Linc00402 were found in patients who subsequently went on to develop acute graft versus host disease (GVHD). The dysregulated expression of Linc00402 was also validated in murine T-cells, both in vitro and in vivo. Functional studies using multiple modalities to deplete Linc00402 in both mouse and human T-cells, demonstrated a critical role for Linc00402 in the T-cell proliferative response to an allogeneic stimulus but not a non-specific anti-CD3/CD28 stimulus. Thus, our studies identified Linc00402 as a novel, conserved regulator of allogeneic T-cell function. Because of its T-cell specific expression and its impact on allogeneic T-cell responses, targeting Linc00402 may improve outcomes after allogeneic HSC and solid organ transplantation. One sentence summaryLncRNAs are differentially expressed by allogeneic antigen-stimulated T-cells, and the novel lncRNA, Linc00402, is a specific regulator of mouse and human allogeneic T-cells.

immunology↗

bioRxiv2020

Mitochondrial Complex II In Intestinal Epithelial Cells Regulates T-cell Mediated Immunopathology

Intestinal epithelial cell (IEC) damage by T cells contributes to alloimmune, autoimmune and iatrogenic diseases such as graft-versus-host disease (GVHD), inflammatory bowel disease (IBD) and immune checkpoint blockade (ICB) mediated colitis, respectively. Despite significant advances in understanding the aberrant biology of T cells in these diseases, little is known about how the fundamental biological processes of the target IECs influence the disease severity. Here, through analyses of metabolic pathways of IECs, we identified disruption of oxidative phosphorylation without a concomitant change in glycolysis and an increase in succinate levels in several distinct in vivo models of T cell mediated gastrointestinal damage such as GVHD, IBD and ICB mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses identified a critical role for IEC intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations that contributed to the severity of intestinal damage. The critical mechanistic role of IEC intrinsic SDHA was confirmed by complementary chemical and genetic reduction of SDHA and with IEC specific deletion of SDHA. Further in vitro and in vivo mechanistic studies demonstrated that loss of SDHA in IECs was mediated by the perforin-granzyme from the T cells. The loss of SDHA was also validated in human clinical samples. These data identify a critical role for the alteration of the IEC specific mitochondrial complex II component SDHA in the regulation of the severity of T cell mediated intestinal diseases.

immunology↗

bioRxiv2019

The ER cargo receptor SURF4 facilitates efficient erythropoietin secretion

Erythropoietin (EPO), a glycoprotein produced by specialized peritubular fibroblasts in the kidney, is the master regulator of erythropoiesis. EPO is secreted into the plasma in response to tissue hypoxia and stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels. Using this cell line, we performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent sgRNAs resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, consistent with a role for SURF4 as an ER cargo receptor of EPO, we found that disruption of SURF4 resulted in accumulation of EPO in the ER compartment, and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO, ruling out an artifact of heterologous overexpression. This work suggests that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoeisis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

cell biology↗