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Goyal, M.

Publications and source records attributed to Goyal, M..

At least 19 recordsLinked to original sources
bioRxiv2026

Identification of the complete pathway for conversion of bilirubin to urobilinogen by human gut bacteria

Bilirubin, the predominant product of heme catabolism in mammals, enters the intestine via the hepatobiliary system and subsequently is metabolized by the gut microbiome. This process consumes bilirubin and generates multiple downstream derivatives, such as urobilinogen and stercobilinogen. Levels of bilirubin and its derivatives are associated with susceptibility to inflammatory and metabolic disorders, but the microbial species and enzymes that metabolize bilirubin have remained largely unknown. Here, demonstrate that metabolism of bilirubin to urobilinogen requires two separate reactions that can occur in either order and identify novel enzymes and pathway intermediates required for conversion. We find that bilirubin reductase (BilR), an enzyme that was recently discovered and proposed to convert bilirubin to urobilinogen, is specific for reducing the methine bridges of bilinoids, converting bilirubin to the novel intermediate divinylurobilinogen and mesobilirubin to urobilinogen. Using transcriptomic profiling, we identify the bilinoid vinyl reductase (BilV) responsible for reducing the vinyl groups of bilirubin and divinylurobilinogen. BilV is a flavin-dependent oxidoreductase of the Old Yellow Enzyme (OYE) superfamily with a broad distribution across human gut bacteria that overlaps with but does not completely mirror the distribution of BilR. These findings establish the complete pathway for bacterial conversion of bilirubin to urobilinogen, enabling defined studies to interrogate how this metabolism contributes to human health and disease.

microbiology↗
bioRxiv2026

The Metaphase Chromatin Unit: A Novel Unit of Higher-Order Chromosome Organization in Human Mitotic Cells

Background and ObjectivesThe linear relation between human metaphase chromosome length and DNA content has never been rigorously reconciled with modern Hi-C models of mitotic chromatin folding. We tested whether the relation implies a quantitative unit of mitotic chromosome organization. MethodsWe pooled metaphase lengths for 24 human chromosomes from five cytogenetic studies of cultured peripheral lymphocytes and regressed length against base-pair content from GRCh38 and T2T-CHM13 v2.0. Pre-specified analyses comprised ordinary least-squares and power-law fits, arm-level decomposition, and reconciliation with the Gibcus 2018 helical loop-array model. Orthogonal validation used Rao 2014 Hi-C boundary counts and Pope 2014 Repli-Seq. ResultsLength scales linearly with DNA content as L (m) = 0.0329 x Mb + 0.043 (R-squared = 0.998, power-law exponent 0.98 +/- 0.01), with a cross-karyotype compaction density of 33.4 +/- 1.0 nm/Mb. T2T-CHM13 reanalysis identifies satellite over-condensation; the arm-level residual correlates with p-arm fraction (r = 0.65, p = 5.6e-4). We propose an operational Metaphase Chromatin Unit (MCU) as a quantitative scaling unit (not a discrete structural quantum, as quantization tests are negative): 1 MCU = 7.6 Mb DNA = 0.25 m axial length, numerically corresponding to one Gibcus late-metaphase helical turn; the human haploid genome scales to 406 MCUs. The pairwise megabase-shift slope of 32.75 nm/Mb (R-squared = 0.996) and the approximately 6 Mb Abbe optical detection threshold correctly classify 9 of 9 microdeletion syndromes as karyotype-visible versus FISH-required. ConclusionsThe MCU provides a unified quantitative unit for human mitotic chromosome organization, integrating cytogenetic, Hi-C, polymer-biophysical, and clinical scales.

genomics↗
bioRxiv2026

Temporal multi-omic profiling of immune, gut, and microbiome responses to ischemic stroke reveals convergence of host and microbial perturbations one week after brain injury

Ischemic stroke poses a significant medical challenge with limited therapy options, therefore detailed understanding of stroke-associated pathophysiological processes across systems and organs is crucial for further research and identification of novel therapeutic targets. In this manuscript, we provide parallel multi-omic host and gut microbiome characterization on several timepoints (day 1, 7 and 14) in the mouse experimental stroke model (middle cerebral artery occlusion, MCAo). Expanding existing host-derived datasets, we profiled transcriptomes from microglia, brain-infiltrating leukocytes and peripheral leukocytes using single cell RNA sequencing. Our data deliver time-resolved characterization of microglial subtypes and highlight heterogeneous dendritic cell populations as main interaction partners of microglia on all timepoints. In peripheral blood, we did not observe large transcriptomic differences when comparing the immune subsets from MCAo and sham-operated control animals. Here, the neutrophils exhibited most transcriptomic changes on day 1 among all blood leukocytes. Parallel proteomic analysis of 5 intestinal segments (duodenum, jejunum, ileum, caecum and colon) and mesenteric lymph nodes highlighted day 7 as the most important timepoint for changes in the gut-related metabolic pathways especially in the jejunum and colon. Specific hypothesis testing revealed compartmentalized regulation of gut-related immune pathways and proteins related to gut permeability. Finally, gut microbiome analyses (longitudinal metatranscriptomics including day -1, 3, 7, 14, and metagenomics from day 14) highlighted temporally matched changes in microbial gene expression (with day 7 emerging again as the most relevant timepoint), larger overall community perturbations when compared to baseline from day -1 in stroke animals and expansion of facultative anaerobes on day 7. Graphical abstractWe performed longitudinal multi-omic host and gut microbiota profiling from experimental stroke and sham control mice. Our data indicate marked differences in brain-infiltrating leukocytes and microglia already on early timepoints after surgery, whereas stroke-specific changes in gut proteomics, bacterial gene expression and community structure emerge on day 7 after middle cerebral artery occlusion/sham surgery. Created in https://BioRender.com O_FIG O_LINKSMALLFIG WIDTH=169 HEIGHT=200 SRC="FIGDIR/small/727504v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@129eff8org.highwire.dtl.DTLVardef@49c401org.highwire.dtl.DTLVardef@e32b22org.highwire.dtl.DTLVardef@24f6df_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗
bioRxiv2026

The nuclear receptor ROR-alpha is a critical myogenic regulator of the cardiomyocyte transcriptome, including the alpha-1A adrenergic receptor

AimsWe recently found that the nuclear receptor retinoic acid-related orphan nuclear receptor alpha (ROR) protects against angiotensin II-induced cardiac hypertrophy and promotes cardiomyocyte mitophagy. The underlying molecular basis for these salutary effects remains unclear. MethodsWe used RNA microarrays to profile the cardiac transcriptomes of "staggerer" (RORsg/sg) mice that carry a naturally occurring mutation in the ligand-binding domain of ROR, resulting in a global loss-of-function genetic model. We then used genetic and pharmacologic loss-and-gain of function studies in cultured cardiomyocytes to ascertain whether ROR regulates transcription of Adra1a, the gene that encodes the alpha-1A-adrenergic receptor (1A-AR). ResultsThe absence of functional ROR results in broad transcriptional changes in the heart providing a likely molecular basis for the RORsg/sg cardiac phenotype. In vivo and in vitro studies confirmed that ROR directly regulates Adra1a transcription. This effect is enhanced by hypoxia. ConclusionsCollectively these findings position ROR as a previously unrecognized central regulator of the cardiac myogenic transcriptome and the first recognized transcriptional regulator of Adra1a in cardiomyocytes. Future studies will probe the contribution of ROR-mediated transcriptional regulation of Adra1a to both the response to cardiomyocyte injury and maintenance of circadian biology.

physiology↗
bioRxiv2025

Clustering of host N-glycans licenses Toxoplasma rhoptry discharge

Apicomplexan parasites must discharge the contents of specialized organelles called rhoptries into host cells to initiate the process of invasion. This process requires the prior recognition and binding of the host cell by proteins released from another set of parasite organelles, the micronemes. However, the host-parasite interactions required for rhoptry discharge are largely unknown. Here we performed a host-cell directed genome-wide screen for host factors required for rhoptry discharge from Toxoplasma gondii, the causative agent of toxoplasmosis. The screen identified host N-glycosylation and cholesterol biosynthesis as pathways required for normal rhoptry discharge. A trimeric microneme complex, MIC1/4/6, interfaces with both pathways by binding host N-glycans to cluster proteins in a process dependent on host plasma membrane cholesterol. The process can be inhibited by depletion of host cholesterol or competition with exogenous glycans. This clustering of host factors by MIC1/4/6 likely prepares the host membrane for rhoptry discharge, delineating a new step in the Toxoplasma invasion process.

microbiology↗
bioRxiv2025

Genetic Determinants of Heart Failure Susceptibility and Response in the Collaborative Cross Mouse Population

Genetic variation and lived experiences shape how our hearts respond to chronic stress. The specific genetic mechanisms which underly cardiac remodeling, however, are still unclear, due in part to the challenge of accounting for environmental effects in human population studies. To overcome this challenge, we used the Collaborative Cross (CC) mouse population to investigate heritable susceptibility to cardiovascular stress by chronic {beta}-adrenergic receptor stimulation. Across 8 founder and 63 CC lines, we measured cardiac structure and function, organ weights, cell and tissue morphology, and left ventricular gene expression. Genome-wide scans detected 49 genome-wide significant loci, collapsing to 20 unique intervals (nine significant for multiple traits and eleven trait-specific), averaging 12.83 Mb in size. To identify high-confidence candidate genes from these loci, we augmented our trait mapping with associations between loci and gene expression, isoproterenol-dependent transcriptional changes, coding variants drawn from sequencing data, tractability in our in vitro rat cardiomyocyte model, and previously reported protein functions and mouse or human phenotypes. This approach recovered both known regulators, such as Hey2, and new candidates. Functional tests in in vitro models highlight three candidate genes that modulate hypertrophic growth: Abcb10, Mrps5 and Lmod3. Abcb10 knockdown increased cell size at baseline and further with isoproterenol, consistent with loss of a mitochondrial stress-buffering role. Mrps5 knockdown blunted stress-induced hypertrophy. Paradoxical upregulation of Lmod3 after siRNA transfection (validated at the protein level) also attenuated hypertrophy, consistent with reinforcement of actin-assembly control under catecholamine stress. Together, these results reveal heritable pathways of {beta}-adrenergic remodeling in mice and provide an interpretable, translational, and stepwise framework to prioritize candidate genes within broad loci for mechanistic studies of heart failure.

genomics↗
bioRxiv2025

The MEK inhibitor trametinib incurs mitochondrial injury and induces innate immune responses in the mouse heart

Trametinib (Trm) is a highly selective MEK inhibitor that potently and persistently abrogates ERK1/2 activation. Trm initially was used to treat BRAF V600E-mutated melanoma but its FDA-approved indications are expanding rapidly. Trm generally is well tolerated but it can cause dose-limiting cardiomyopathy and heart failure. Here we characterize a mouse model of Trm cardiotoxicity using complementary in vitro approaches to show that Trm induces mitochondrial dysfunction in cardiomyocytes and some cancer cell types. In vivo, Trm caused contractile dysfunction within 3 days and heart failure within 2 weeks. High resolution respirometry using isolated cardiac mitochondria revealed that Trm compromises oxidative metabolism, in part through blunted activity of Electron Transport System Complexes. Trm-mediated mitochondrial injury led to the release of mitochondrial Damage-Associated Molecular Patterns including mitochondrial DNA in both mice and humans, triggering activation of canonical innate immune pathways including cGAS-STING. In multiple rodent and human cardiomyocyte platforms, Trm diminished mitochondrial respiratory capacity at nanomolar concentrations but this lesion was reversed by expression of a phosphomimetic STAT3-S727 construct. We also found that Trm induced mitochondrial dysfunction in some but not all cancer cell lines, identifying a previously unrecognized effect that could contribute to Trms anti-cancer efficacy.

physiology↗
bioRxiv2025

Cellulase secretion by engineered Pseudomonas putida enables growth on cellulose oligomers.

Pseudomonas putida is an attractive synthetic biology platform organism for chemical synthesis from low-grade feedstocks due to its high tolerance to chemical solvents and lignin-derived small molecules that are often inhibitory to other biotechnologically relevant microorganisms. However, there are few molecular tools available for engineering P. putida and other gram-negative bacteria to secrete non-native enzymes for extracellular feedstock depolymerisation. In this study P. putida was transformed to secrete cellulase enzymes and evaluated for growth on polymeric or oligomeric cellulose substrates. Active exo- and endocellulase enzymes were secreted into the culture supernatant, and a preferred set of twin-arginine translocase secretion signal peptides were identified. Extracellular cellulase activity was sufficient to support growth of P. putida using cellotriose or cellotetraose as the sole source of carbon and energy. This work supports progress towards consolidated bioprocessing of cellulosic materials using P. putida, and advances the state of engineered protein secretion in gram negative bacteria. Key PointsO_LIEngineered Pseudomonas putida secreted cellulase enzymes into the culture medium C_LIO_LICellulase activity was sufficient to support growth on cellulose oligomers C_LI

synthetic biology↗
bioRxiv2025

Antioxidant role of the GABA shunt in regulating redox balance in blood progenitors during Drosophila hematopoiesis

Redox balance is crucial for normal development of stem and progenitor cells that reside in oxidative environments. This study explores the mechanisms of redox homeostasis in such niches and investigates myeloid-like blood progenitor cells that generate reactive oxygen species (ROS) and moderate it developmentally. Our findings reveal that during lymph gland development, as the blood-progenitor cells oxidize pyruvate via the TCA cycle leading to the generation of ROS, these cell also de novo synthesize GSH to counter excessive ROS and ensure redox balance. GABA metabolism, through GABA-shunt, restricts pyruvate dehydrogenase (PDH) activity and consequently TCA rate. This allows a metabolic route to sustain serine levels in these cells which is the rate limiting precursor controlling de novo GSH production. Disruption of GABA metabolism leads to a metabolic imbalance, characterized by excessive PDH activity, heightened TCA rate leading to impaired serine/GSH production and overall ROS dysregulation. Overall, the study presents a unique metabolic state whereby, in the blood-progenitor cells, by keeping PDH and TCA activity in check and promoting serine/GSH generation, GABA metabolism establishes a metabolic framework that optimises the use of ROS in blood-progenitors, while ensuring redox homeostasis.

developmental biology↗
bioRxiv2024

Novel antibodies detect nucleocytoplasmic O-fucose in protist pathogens, cellular slime molds, and plants

Cellular adaptations to change often involve post-translational modifications of nuclear and cytoplasmic proteins. An example found in protists and plants is the modification of serine and threonine residues of dozens to hundreds of nucleocytoplasmic proteins with a single fucose (O-Fuc). A nucleocytoplasmic O-fucosyltransferase (OFT) occurs in the pathogen Toxoplasma gondii, the social amoeba Dictyostelium, and higher plants, where it is called Spy because mutants have a spindly appearance. O-fucosylation, which is required for optimal proliferation of Toxoplasma and Dictyostelium, is paralogous to the O-GlcNAcylation of nucleocytoplasmic proteins of plants and animals that is involved in stress and nutritional responses. O-Fuc was first discovered in Toxoplasma using Aleuria aurantia lectin, but its broad specificity for terminal fucose residues on N- and O-linked glycans in the secretory pathway limits its use. Here we present affinity purified rabbit antisera that are selective for the detection and enrichment of proteins bearing fucose-O-Ser or fucose-O-Thr. These antibodies detect numerous nucleocytoplasmic proteins in Toxoplasma, Dictyostelium, and Arabidopsis, as well as O-Fuc occurring on secretory proteins of Dictyostelium and mammalian cells, although the latter are frequently blocked by further glycosylation. The antibodies label Toxoplasma, Acanthamoeba, and Dictyostelium in a pattern reminiscent of O-GlcNAc in animal cells including nuclear pores. The O-fucome of Dictyostelium is partially conserved with that of Toxoplasma and is highly induced during starvation-induced development. These antisera demonstrate the unique antigenicity of O-Fuc, document conservation of the O-fucome among unrelated protists, and will enable the study of the O-fucomes of other organisms possessing OFT-like genes. IMPORTANCEO-fucose, a form of mono-glycosylation on serine and threonine residues of nuclear and cytoplasmic proteins of some parasites, other unicellular eukaryotes, and plants, is understudied because it is difficult to detect owing to its neutral charge and lability during mass spectrometry. Yet the O-fucosyltransferase enzyme (OFT) is required for optimal growth of the agent for toxoplasmosis, Toxoplasma gondii, and an unrelated protist, the social amoeba Dictyostelium discoideum. Furthermore, O-fucosylation is closely related to the analogous process of O-GlcNAcylation of thousands of proteins of animal cells, where it plays a central role in stress and nutritional responses. O-Fuc is currently best detected using Aleuria aurantia lectin (AAL), but in most organisms AAL also recognizes a multitude of proteins in the secretory pathway that are modified with fucose in different ways. By establishing the potential to induce highly specific rabbit antisera that discriminate O-Fuc from all other forms of protein fucosylation, this study expands knowledge about the protist O-fucome and opens a gateway to explore the potential occurrence and roles of this intriguing posttranslational modification in bacteria and other protist pathogens such as Acanthamoeba castellanii.

cell biology↗
bioRxiv2024

Identification of new targets for the diagnosis of cysts (four) and trophozoites (one) of the eye pathogen Acanthamoeba

Acanthamoebae, which are free-living amoebae, cause corneal inflammation (keratitis) and blindness, if not diagnosed and effectively treated. While trophozoites adhere to and damage the cornea, Acanthamoeba cysts, the walls of which contain cellulose and have two layers connected by conical ostioles, are the diagnostic form by microscopy of the eye or of corneal scrapings. We recently used structural and experimental methods to characterize cellulose-binding domains of Luke and Leo lectins, which are abundant in the inner layer and ostioles. However, no antibodies have been made to these lectins or to a Jonah lectin and a laccase, which are abundant in the outer layer. Here we used confocal microscopy to show that rabbit antibodies to recombinant Luke, Leo, Jonah, and laccase generally support localizations of GFP-tagged proteins in walls of transfected Acanthamoebae. Rabbit antibodies to all four wall proteins efficiently detected calcofluor white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC, including five T4 genotypes that cause most cases of keratitis. Laccase shed into the medium during encystation was detected by an enzyme-linked immunoassay. We also used structural and experimental methods to characterize the mannose-binding domain of an Acanthamoeba mannose-binding protein and showed that rabbit antibodies to the mannose-binding domain efficiently detected trophozoites of all 11 Acanthamoeba isolates. We conclude that four wall proteins are all excellent targets for diagnosing Acanthamoeba cysts in the eye or corneal scrapings, while the mannose-binding domain is an excellent target for identifying trophozoites in cultures of corneal scrapings. ImportanceFree-living amoeba in the soil or water cause Acanthamoeba keratitis, which is diagnosed by identification of cysts by microscopy of the eye or of corneal scrapings, using calcofluor-white that unfortunately cross-reacts with fungi and plants. Alternatively, Acanthamoeba infections are diagnosed by identification of trophozoites in cultures of scrapings. Here we showed that rabbit antibodies to four abundant cyst wall proteins (Jonah, Luke, Leo, and laccase) each efficiently detect calcofluor-white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC. Further, laccase released into the medium by encysting Acanthamoebae was detected by an enzyme-linked immunoassay. We also showed that rabbit antibodies to the mannose-binding domain of the Acanthamoeba mannose-binding protein, which mediates adherence of trophozoites to keratinocytes, efficiently identifies trophozoites of all 11 ATCC isolates. In summary, four wall proteins and the ManBD appear to be excellent targets for diagnosis of Acanthamoeba cysts and trophozoites, respectively.

molecular biology↗
bioRxiv2024

SUN-domain proteins of the malaria parasite Plasmodium falciparum are essential for proper nuclear division and DNA repair

The protozoan parasite Plasmodium falciparum, which is responsible for the deadliest form of human malaria, accounts for over half a million deaths a year. These parasites proliferate in human red blood cells by consecutive rounds of closed mitoses called schizogony. Their virulence is attributed to their ability to modify the infected red cells to adhere to the vascular endothelium and to evade immunity through antigenic switches. Spatial dynamics at the nuclear periphery were associated with regulation of processes that enable the parasites to establish long-term infection. However, our knowledge of components of the nuclear envelope (NE) in Plasmodium remains limited. One of the major protein complexes at the NE is the LINC complex that forms a connecting bridge between the cytoplasm and the nucleus through the interaction of SUN and KASH domain proteins. Here we have identified two SUN-domain proteins as possible components of the LINC complex of P. falciparum and show that their proper expression is essential for the parasites proliferation in human red blood cells and that their depletion leads to the formation of membranous whorls and morphological changes of the NE. In addition, their differential expression highlight different functions at the nuclear periphery as PfSUN2 is specifically associated with heterochromatin, while PfSUN1 expression is essential for activation of the DNA damage response. Our data provide indications for the involvement of the LINC complex in crucial biological processes in the intraerythrocytic development cycle of malaria parasites.

microbiology↗
bioRxiv2024

Dietary stress induced macrophage metabolic reprogramming, a determinant of animal growth

Nutrient sensing and signaling play pivotal roles in animal growth. However, under dietary stress, this system falters, leading to growth defects. While immune cells are increasingly recognized as key nutrient sensors, their impact on animal growth remains poorly understood. In this study, we investigate how Drosophila larval macrophages respond to excessive dietary sugar and identify a reconfiguration of their metabolic state. They undergo a glycolytic shift, intensify TCA activity, and elevate TAG synthesis. While typical of sugarinduced nutrient stress, these changes interestingly exert contrasting effects on animal growth: glycolysis and increased TCA activity inhibit growth, while the lipogenic shift promotes it. However, the lipogenic response is insufficient to counteract the metabolic events suppressing growth, resulting in an overall reduction in adult fly size under high sugar conditions. Stimulating a pro-lipogenic immune state facilitates growth recovery, suggesting a growth paradigm governed by immune-metabolic transitions. This study unveils the unexpected influence of macrophage metabolic reprogramming on organismal growth homeostasis during Drosophila development, highlighting immune cell states as central determinants of growth, particularly under dietary stress.

developmental biology↗
bioRxiv2024

Basic and translational science of cyst wall proteins of the eye pathogen Acanthamoeba

The cyst wall of the eye pathogen Acanthamoeba castellanii contains cellulose and chitin and has ectocyst and endocyst layers connected by conical ostioles. Previously, we used mass spectrometry of purified walls to identify an abundant laccase and three families of lectins (Jonah, Luke, and Leo). Here we show that frameshifts in the protein prediction in AmoebaDB, which incorrectly add 12 transmembrane helices, cause Jonah to mislocalize to a ring around ostioles rather than to the ectocyst layer. RT-PCR, double labels with GFP and RFP or mCherry, and promoter swaps show that ectocyst localization does not just correlate with but is caused by earlier expression, while localization in the endocyst layer and ostioles is caused by later expression. A chitin-binding domain from an Entamoeba chitinase shows chitin forms thick fibrils in the ectocyst layer and a honeycomb in the endocyst layer. AlphaFold shows Ac wall proteins originate from bacteria by horizontal gene transfer ({beta}-helical folds of Jonah and three cupredoxin-like domains of the laccase), share common ancestry with wall proteins of slime molds ({beta}-jelly-roll folds of Luke), or are unique to Acanthamoeba (four disulfide knots of Leo). Ala mutations show linear arrays of aromatic amino acids in {beta}-jelly-roll folds of Luke and disulfide knots of Leo are necessary for binding cellulose and proper localization of proteins in the cyst wall. Finally, rabbit antibodies to recombinant Jonah, Luke, Leo, and laccase efficiently detect calcoflour white-labeled cysts of 10 of 11 Acanthamoeba isolates tested, suggesting all four proteins are excellent diagnostic targets. IMPORTANCEAcanthamoebae are free-living amoeba in the soil and water that cause Acanthamoeba keratitis in under-resourced countries, where water for washing hands may be scarce. Acanthamoeba is an emerging pathogen in the United States, because of its association with contact lens use. Here we show early expression during encystation causes a Jonah lectin and a laccase to localize to the outer layer of the cyst wall, while later expression cause Luke and Leo lectins to localize to the inner layer and the conical ostioles that connect the layers. We used structural predictions to identify the aromatic amino acids of Luke and Leo necessary for binding cellulose in the wall and to identify domains of Jonah and laccase useful for making recombinant proteins to immunize rabbits. Rabbit antibodies to Jonah, Luke, Leo, and laccase all efficiently detected cysts of ten Acanthamoeba isolates, including five T4 genotypes that cause most keratitis cases.

microbiology↗
bioRxiv2021

Nociceptor subtypes are born continuously over DRG development peaking at E10.5 - E11.5.

Sensory neurogenesis in the dorsal root ganglion (DRG) occurs in two waves of differentiation with larger, myelinated proprioceptive and low-threshold mechanoreceptor (LTMR) neurons differentiating before smaller, unmyelinated (C) nociceptive neurons. This temporal difference was established from early birthdating studies based on DRG soma cell size. However, distinctions in birthdates between molecular subtypes of sensory neurons, particularly nociceptors, is unknown. Here, we assess the birthdate of lumbar DRG neurons in mice using a thymidine analog, EdU, to label developing neurons exiting mitosis combined with co-labeling of known sensory neuron markers. We find that different nociceptor subtypes are born on similar timescales, with continuous births between E9.5 to E13.5, and peak births from E10.5 to E11.5. Notably, we find that thinly myelinated A{delta}-fiber nociceptors and peptidergic C-fibers are born more broadly between E10.5 and E11.5 than previously thought and that non-peptidergic C-fibers and C-LTMRs are born with a peak birth date of E11.5. Moreover, we find that the percentages of nociceptor subtypes born at a particular timepoint are the same for any given nociceptor cell type marker, indicating that intrinsic or extrinsic influences on cell type diversity are occurring similarly across developmental time. Overall, the patterns of birth still fit within the classical "two wave" description, as touch and proprioceptive fibers are born primarily at E10.5, but suggest that nociceptors have a slightly broader wave of birthdates with different nociceptor subtypes continually differentiating throughout sensory neurogenesis irrespective of myelination.

developmental biology↗
bioRxiv2021

Blood progenitor redox homeostasis through GABA control of TCA cycle in Drosophila hematopoiesis

The importance of reactive oxygen species (ROS) in myeloid cell development and function is well-established. However, a comprehensive understanding of metabolic states controlling ROS levels during hematopoiesis remains elusive. Myeloid-like blood progenitor cells of the Drosophila larvae reside in a specialized hematopoietic organ called the lymph gland. We find that these progenitors in homeostasis, utilize TCA to generate ROS. Excessive activation of TCA however raises ROS levels causing them to precociously differentiate and leads to retardation of lymph gland size. Thus, to maintain ROS homeostasis, progenitor cells utilize systemically derived GABA. GABA internalization and catabolism via inhibiting hydroxy prolyl hydroxylase (Hph) activity, promotes pyruvate dehydrogenase kinase enzyme activity (PDK). PDK controls inhibitory phosphorylation of pyruvate dehydrogenase (PDH), the rate-limiting enzyme, connecting pyruvate to TCA cycle and OXPHOS. Thus, by regulating PDK, GABA regulates progenitor TCA activity and ROS levels. In addition to this, GABA-catabolism/Hph axis via Hif/Sima drives a glycolytic state in progenitor cells. The dual control established by GABA on PDK and Sima maintains progenitor cell metabolism and sustains ROS homeostasis necessary for their development. Taken together, our study demonstrates the metabolic underpinnings of GABA in myeloid ROS regulation and their development, the relevance of which may be broadly conserved.

developmental biology↗
bioRxiv2021

An SR protein is essential for the recovery of malaria parasites from DNA damage and exposure to artemisinin

Plasmodium falciparum, the parasite responsible for the deadliest form of human malaria, maintains a complex life cycle with a relatively small number of genes. PfSR1 is an alternative splicing factor that regulates expansion of the P. falciparum protein repertoire. To further investigate PfSR1 functions, we set to unveil its interactome. We found that PfSR1 interacts with proteins, which are linked to various processes of RNA metabolism in a stage-dependent manner. These include: chromatin re-modeling, transcription, splicing and translation. Intriguingly, some of the PfSR1 interacting proteins are orthologues of proteins implicated in the DNA damage response. We demonstrate that PfSR1 expression is important for preventing the accumulation of DNA damage in proliferating parasites. In addition, following parasites exposure to a source of DNA damage, PfSR1 is recruited to damaged foci where it interacts with the phosphorylated core histone PfH2A, which marks damaged chromatin. Furthermore, PfSR1 expression was found to be essential for the ability of the parasite to activate the DNA repair machinery and recover from DNA damage caused by either irradiation or exposure to artemisinin, the first line anti-malarial drug. These findings unveil a novel role of PfSR1 in protecting P. falciparum from DNA damage and artemisinin exposure.

microbiology↗