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Singh, R.

Publications and source records attributed to Singh, R..

At least 73 records · Page 4Linked to original sources

Iterative immunogen optimization to focus elicited responses on a conserved, subdominant influenza hemagglutinin epitope

Designing effective vaccination strategies against genetically diverse viruses, such as HIV or influenza, is hindered by the ability of these pathogens to mutate and readily evade immune responses. While these viruses contain conserved regions that cannot easily mutate without affecting viral fitness, these sites are subdominant, meaning that they are not the primary target of antibodies elicited by natural infection or current vaccines. Here, we integrated recent advances in machine learning methods for protein structure prediction and design to engineer protein immunogens that focus immune responses on a conserved, subdominant HA epitope targeted by broadly cross-reactive influenza antibodies. Iterating between computation-guided optimization and in vivo analyses generated immunogens that faithfully displayed the target epitope and redirected humoral responses toward it upon vaccination. These results provide a blueprint for applying recent protein engineering approaches to immunogen design and may inform the development of broadly protective influenza vaccines.

immunology↗

HypoAD: volumetric and single-cell analysis reveals changes in the human hypothalamus in aging and Alzheimer's disease

Alterations in metabolism, stress response, sleep, circadian rhythms, and neuroendocrine processes are key features of aging and neurodegeneration. These fundamental processes are regulated by the hypothalamus, yet how its functionally distinct subregions and cell types change during human aging and Alzheimers Disease (AD) remains largely unexplored. Here, we present HypoAD, a comprehensive atlas of the human hypothalamus in aging and AD, integrating high-resolution MRI from 202 individuals with single-nucleus RNA-seq (snRNA-seq) of 614,403 nuclei from young, AD, and age-matched non-dementia controls. Our analysis reveals that hypothalamic subregions governing metabolism, stress, and circadian rhythms are particularly vulnerable, exhibiting significant changes in both volumes and gene expression during aging and AD. At the molecular level, machine learning models identified the inflammatory response and regulators of circadian rhythms as key cellular predictors of AD. These signatures were reflected in specific cell types: microglia transitioned to a pro-inflammatory state, while inhibitory neurons within sleep-and circadian-regulating hypothalamic subregions showed the most profound transcriptional alterations, including disruptions in ligand-receptor interactions and G-protein-coupled receptor signaling. Together, HypoAD provides a high-resolution volumetric map and a comprehensive transcriptomic atlas of the human hypothalamus in aging and AD, linking lifestyle and behavioral changes to their underlying volumetric and molecular pathways. Additionally, HypoAD provides a framework to investigate hypothalamic dysfunction and establishes a roadmap for targeted interventions aimed at mitigating physiological disruptions to potentially slow disease progression.

neuroscience↗

PSP-0119: Targeted IRAK4 Degradation as a Novel Therapeutic Strategy for FLT3-Mutant AML

Acute Myeloid Leukemia (AML) is a life-threatening hematologic malignancy. Despite recent therapeutic advances, rising incidence rates emphasize the urgent need for identification of new targets and therapies. Roles of interleukin receptor-associated kinases IRAK1/4 are emerging in hematologic and solid malignancies. In AML, IRAK4 mRNA is overexpressed at diagnosis, relapses, in residual disease, and in FLT3-ITD-mutant cells, MDS, MPN, and MDS/MPN-negative subtypes. Compared with hematopoietic stem cells, IRAK4 is elevated in t(15;17), inv(16)/t(16;16), and t(11q23)/MLL subtypes, correlating with poor survival. Here, we disclose anti-AML activity of PSP-0119, a novel IRAK4 PROTAC degrader. PSP-0119, inhibited IRAK4 kinase activity, NF-{kappa}{beta} activity, and IL-1{beta}-induced IRAK4 phosphorylation. In-silico docking revealed interactions in CRBN/IRAK4/PSP-0119 ternary complex. PSP-0119 degraded IRAK4 in FLT3-mutant AML cell lines sparing FLT3-wild-type AML cells, FLT3-wild-type patient samples, and normal bone-marrow. Bulk-seq of PSP-0119 treated MOLM-13 cells revealed downregulation of eNOS, a poor AML prognosticator. PSP-0119 suppressed colony formation, cell viability, and MOLM-13 xenograft growth, and synergized with IRAK1 covalent inhibitor JH-X-119-01. PSP-0119 is metabolically stable, retaining 71% of parent compound at 60 minutes in human liver microsomes. In summary, IRAK4 degradation via PSP-0119 as a promising therapeutic strategy for treatment of FLT3-mutant AML.

cancer biology↗

Lower Airway Dysbiosis in NTM+ Bronchiectasis is Associated with NET-Predominant Severe Phenotypes

RationaleThe discoveries of neutrophilic inflammation and Pseudomonas-dominant pulmonary dysbiosis have helped pave the way for host-directed therapy in bronchiectasis. Substantial knowledge gaps remain about the interplay between neutrophilic signatures and microbes in non-tuberculous mycobacterial lung disease (NTM-LD), a phenotypically diverse lung infection that is increasingly prevalent in the United States and other parts of the world. ObjectivesEvaluate the lower airway microbiota and neutrophilic traits in NTM- and NTM+ bronchiectasis. Methods16S rRNA gene sequencing, cell counts and neutrophil extracellular trap (NET) immunoassays were performed on bronchoscopic lower airway samples in 200 bronchiectasis subjects (108 NTM-, 92 NTM+). A preclinical model of oral commensal micro-aspiration and NTM infection was used to profile the murine lower airways with flow cytometry and a NET assay. Measurements and Main ResultsLower airways of NTM+ bronchiectasis patients were enriched with Mycobacterium and oral commensals (e.g., Veillonella, Prevotella). NET levels were higher in NTM+ BAL. Mycobacterium and oral commensals co-occurred with NET and neutrophils in network studies. Distinct oral commensal taxa associated with severe disease phenotypes such as cavitary disease and exacerbators. In a murine micro-aspiration model, the combination of oral commensals and Mycobacterium led to a sustained pro-inflammatory immune response marked by an increase in Th17, {gamma}8T cells, PD-1+ T lymphocytes as well as higher NET levels. ConclusionsOur analyses showed that distinct microbiome features beyond the primary pathogen can contribute to neutrophilic inflammation and severe disease phenotypes in bronchiectasis/ NTM-LD.

immunology↗

Mevalonate Biosynthesis is a Metabolic Vulnerability of Gemcitabine-resistant Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a devastating prognosis. Gemcitabine, a pyrimidine anti-metabolite, is a cornerstone in PDAC therapy. However, resistance remains a major hurdle in clinical care. Resistance can arise from microenvironmental metabolites or through direct metabolic reprogramming of pancreatic cancer cells. Here, we generated PDAC models of acquired gemcitabine resistance to determine the relationship between these mechanisms. We observed that physiological levels of exogenous pyrimidines have a diminished ability to impact gemcitabine response in PDAC cells with acquired resistance. This occurs as the metabolic reprogramming of PDAC cells in response to gemcitabine treatment forces a suppression of the pyrimidine salvage pathway. Importantly, this metabolic rewiring renders gemcitabine-resistant PDAC cells highly susceptible to inhibition of the rate limiting enzyme of the mevalonate biosynthesis pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), using statins. Notably, statin treatment inhibits the growth of gemcitabine-resistant tumors in immunocompetent mouse models. Through metabolite rescue experiments, we identified geranylgeranyl pyrophosphate as the critical metabolite lost during statin treatment, resulting in reduced protein geranylation in PDAC cells. Finally, as downregulation of the HMGCR is gradually acquired during gemcitabine resistance, we observed that HMGCR expression predicts patient response to gemcitabine. Collectively, these data demonstrate that the mevalonate biosynthesis pathway represents a promising therapeutic target in gemcitabine resistance and may serve as a biomarker to stratify treatment selection in PDAC patients.

cancer biology↗

Exploiting TGF-β-mediated Stromal Programming in Homologous Recombination-Deficient Pancreatic Cancer

The tumor microenvironment (TME) actively contributes to pancreatic ductal adenocarcinoma (PDAC) pathogenesis through dynamic bidirectional tumor-stroma interactions. Here, we demonstrate that homologous recombination-defective (HRD) tumor epithelium reprograms the TME in a genotype-specific manner to enhance cancer aggressiveness. Using genetically engineered mouse models, pancreatic stellate cell (PSC) and cancer-associated fibroblast (CAF) co-culture systems, single-nucleus multiomics, and human PDAC models, we show that tumoral loss of ATM serine/threonine kinase drives CAFs toward SMA+ myofibroblastic differentiation, independently of P53 status. These myCAFs, in turn, promote cancer aggressiveness and chemoresistance. Mechanistically, ATM deficiency increases reactive oxygen species and contractility signaling, enhancing TGF-{beta}1 secretion. Pharmacological TGF-{beta} inhibition reverses myCAF differentiation, sensitizes tumors to chemotherapy, and impairs tumor progression in both murine and human ATM-null models. Our findings reveal that ATM-deficient tumors shape a cancer-promoting niche via TGF-{beta} signaling and identify dual targeting of intrinsic and extrinsic vulnerabilities as a promising precision oncology strategy. SIGNIFICANCEHRD pancreatic cancers reprogram the tumor microenvironment in a genotype-specific manner through TGF-{beta}-driven myCAF-enrichment. Targeting this stromal axis alongside platinum-based chemotherapy improves therapeutic efficacy in ATM-deficient models. These findings highlight the need to integrate epithelial genotype and stromal context for truly personalized treatment strategies in PDAC.

cancer biology↗

Benzopyrene degrading Bacillus altitudinis AR19 isolated from Digboi oil refinery soil (India)

Benzo[a]pyrene (B[a]P) is a five-ring polycyclic aromatic hydrocarbon (PAH) recognized as a potent carcinogen and persistent environmental pollutant, highly resistant to microbial degradation. In this study, a B[a]P-degrading and biosurfactant producing bacterium, Bacillus altitudinis AR19, was isolated from the Digboi oil refinery site in Assam, India. The isolate was screened using the spray plate technique, and its morphological features and cell viability were examined by scanning electron microscopy (SEM) and flow cytometry. Biosurfactant production was confirmed through drop collapse, oil spreading, and emulsification assays. Taxonomic identity was established using average nucleotide identity (ANI), and comparative pangenome analysis was performed among B. altitudinis (Taxonomy ID: 1178544), Bacillus pumilus (Taxonomy ID: 1408), Mycolicibacterium vanbaalenii (Taxonomy ID: 110539), and B. altitudinis AR19. The results highlight that several mechanisms including osmolyte accumulation, biosurfactant production, and mechanosensitive ion channels play key roles in enabling B. altitudinis AR19 to tolerate and degrade B[a]P. This study demonstrates the potential of B. altitudinis AR19 as a promising candidate for bioremediation of PAH-contaminated environments. Importance of the workThis manuscript describes the morphological and genomic characterization of Bacillus altitudinis AR19, a bacterium capable of degrading benzo[a]pyrene. Benzo[a]pyrene is a five-ring polycyclic aromatic hydrocarbon that is highly recalcitrant to bacterial remediation, making this work important for advancing bioremediation strategies.

microbiology↗

The fecal microbiota transplantation from drug-naive schizophrenia patients distinctively changes gut microbiome and metabolic profiles in male and female mice

BackgroundEmerging evidence suggests a role for the gut microbiome in schizophrenia (SCZ) and antipsychotic-induced metabolic perturbations. Using human fecal microbiota transplantation (FMT) in mice, this study investigated the role of gut microbiome in metabolic changes related to SCZ and antipsychotic (olanzapine) treatment. Methods5-6 weeks old germ-free NIH Swiss mice of both sexes received microbiota from either SCZ patients (SCZ-FMT) or healthy controls (HC-FMT) followed by a diet with or without olanzapine for six-weeks. Food intake and body weight were monitored weekly, and an intraperitoneal glucose tolerance test and open field test were performed. Serum glucose, and insulin were measured. Gut microbiome characterization and short-chain fatty acids (SCFAs) quantification were performed in the cecal samples using 16S rRNA gene sequencing and gas chromatography-mass spectrometry, respectively. ResultsOlanzapine treatment decreased the locomotor activity in the open field test, irrespective of sex or microbiota. Female SCZ-FMT recipient mice exhibited insulin resistance compared to HC-FMT, irrespective of olanzapine treatment. Female SCZ-FMT mice showed significantly lower alpha-diversity compared to HC-FMT, whereas olanzapine treatment increased alpha-diversity. SCZ-FMT and olanzapine treatment differentially altered the microbial abundances, and metabolic pathways in male and female mice. Interestingly, cecal SCFAs, mainly acetate levels, were significantly decreased in female SCZ-FMT mice compared to HC-FMT, while olanzapine treatment increased acetate levels in male mice. Both male and female SCZ-FMT mice showed elevated levels of isovaleric acid compared to HC-FMT. ConclusionThese preliminary findings suggest that gut microbiome could be a predisposing factor contributing to the intrinsic risk of developing type 2 diabetes associated with SCZ in females. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/675957v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@8cfd10org.highwire.dtl.DTLVardef@3a6031org.highwire.dtl.DTLVardef@813428org.highwire.dtl.DTLVardef@87609e_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

neuroscience↗

Comparative Analysis of Primary and Liver Fibroblasts Reveals MET as a Potent Target in Pancreatic Cancer Metastasis

The tumor microenvironment drives many malignant features of pancreatic ductal adenocarcinoma (PDAC). The fibroblasts within pancreatic tumors promote tissue remodeling, immune suppression, and resistance to therapy. However, the interactions between stromal populations and pancreatic cancer cells are less understood in the liver, the most frequent site of PDAC metastasis. To address this, we employ single cell transcriptomics to compare primary pancreatic vs. liver PDAC lesions. Here, we identify the expression of hepatocyte growth factor (HGF) in fibroblasts and its receptor MET in cancer cells are both markedly increased in the PDAC liver niche. Using functional assays, we validate that mitogenic MET signaling is activated in PDAC cells by liver-derived fibroblasts. Importantly, the inhibition of MET signaling leads to reduced tumor growth in immune competent mouse models. Collectively, our data demonstrates that liver stromal-epithelial crosstalk networks engage in signaling pathways distinct from primary pancreatic tumors, highlighting opportunities to develop new treatments for metastatic disease.

cancer biology↗

The dietary emulsifier polysorbate-80 induces lipid accumulation and cell death in intestinal epithelial cells via ferroptosis

Chronic inflammatory and metabolic diseases are major global health issues increasingly linked to dietary factors. Consumption of dietary emulsifiers like polysorbate-80 (p80), common in ultra-processed foods and pharmaceuticals, has raised concerns about gut health. RNA sequencing on intestinal epithelial cells (IECs) exposed to p80 revealed increased expression of ferroptosis-associated genes and disruption of lipid metabolism pathways further demonstrated by mitochondrial dysfunction, including altered membrane potential and architecture, and accumulation of reactive oxygen species, iron, lipid peroxidation, and lipid droplet formation. Lipidomic profiling identified significant alterations in triglyceride species and elevated pro-ferroptotic polyunsaturated fatty acids. These data indicate that p80 disrupts lipid homeostasis in IECs and triggers ferroptotic cell death, mechanisms potentially contributing to the increased incidence of chronic conditions like inflammatory bowel disease and metabolic syndrome. The study highlights critical implications for public health, emphasizing the need for reassessment of emulsifier safety standards while balancing needs with consumer safety.

cell biology↗

Metabolic regulation of copper homeostasis governs the Sec61-dependent protein translocation process in Saccharomyces cerevisiae

Concentration of cellular labile pool of copper must be strictly regulated because disruption in copper homeostasis results in diseases. In Saccharomyces cerevisiae, elevated levels of labile copper impair cell viability by inhibiting Sec61-mediated protein translocation into the endoplasmic reticulum. We investigated how metabolic pathways, specifically mitochondrial respiration and autophagy, contribute to copper homeostasis and the translocation of secretory proteins. We show that copper selectively inhibits protein translocation in yeast cells grown in minimal medium but not in rich medium, highlighting a critical role of nutrients in modulating copper toxicity. Supplementation of specific amino acids suppresses the copper-induced defects in protein translocation and cell death, identifying amino acids suppressors of the copper toxicity. Using a panel of gene deletion mutants affecting mitochondrial functions, autophagy, peroxisomes, and lipid droplets, we demonstrate that metabolic pathways regulate sub-cellular distribution of copper and translocation of secretory proteins. Further, disruption of redox and pH homeostasis, and pharmacological inhibition of respiration, reveals that correct subcellular trafficking of copper is essential to prevent inhibitory effects on protein translocation process. Together, our findings provide mechanistic insights into how metabolic status influences cellular copper homeostasis and the secretory pathway of proteins, with broader implications for understanding diseases of copper metabolism.

cell biology↗

Combined generalist and host-specific transcriptional strategies enable host generalism in the fungal pathogen Botrytis cinerea

How generalist pathogens infect phylogenetically diverse hosts remains a central question in plant-pathogen biology. In particular, the extent to which broad host range is enabled by genetic variation versus transcriptional plasticity is unclear. To investigate how variation and plasticity contribute to generalism, we studied the generalist necrotrophic fungus Botrytis cinerea that infects more than 1,500 plant species. Using a cross-infection matrix of 72 B. cinerea isolates infected on 57 plant genotypes distributed across 15 eudicot species, we identified general and host-dependent fungal components of lesion formation. Transcriptome profiling at 48 hours post-inoculation revealed two distinct pathogen gene modules: (1) a set of general lesion-associated genes enriched in primary metabolism, showing similar expression across hosts but varied among isolates; and (2) a set of high-entropy, host specific-inducible genes, organized into distinct co-regulated modules that respond dynamically to specific host cues. Both gene sets were genomically dispersed, lacking structural clustering, and were under different levels of selective constraints. Our results demonstrate that B. cinerea employs a modular transcriptional strategy that integrates a core metabolic program along with a plastic, host-responsive regulatory network to achieve broad host colonization. This study presents the most comprehensive cross-species co-transcriptomic dataset to date for any fungal phytopathogen, highlighting transcriptional plasticity as a key mechanism underlying generalism in plant-fungal interactions. Moreover, the identification of conserved fungal gene targets across diverse hosts offers a foundation for developing broad-spectrum resistance strategies in multiple crops.

genomics↗

Charged Scanning Mutagenesis as a High Throughput Approach for Epitope Mapping

Identifying neutralizing epitopes is important for developing vaccines and inhibitors against viral pathogens. We describe a rapid method for epitope mapping, employing barcoded charged scanning mutagenesis libraries displayed on the yeast surface, and screening using flow cytometry coupled with deep sequencing. Prior scanning mutagenesis data suggest that mutations to a charged residue, such as Aspartic acid or Arginine, will be well tolerated at exposed positions of an antigen, and minimally affect protein stability and expression. Yet such substitutions at epitope residues strongly perturb binding to a cognate partner. We constructed an Aspartate scanning library of SARS-CoV-2 RBD and linked every mutation in the library to a defined unique barcode. The approach was used to map epitopes targeted in polyclonal sera of mice immunized with different SARS-CoV-2 immunogens. In contrast to complete mutational scans, charged scanning mutagenesis with the introduced barcoding strategy employs libraries with >50-fold lower diversity, facilitating library construction, screening, and downstream analysis, and also allowing for further multiplexing of samples, thus accelerating interaction site identification, as well as vaccine and inhibitor development.

biophysics↗

SAME: Topology-flexible transforms enable robust integration of multimodal spatial omics

Spatial omics technologies provide complementary and layered molecular insights that span proteins, transcripts, and metabolites. However, aligning and integrating these modalities across serial tissue sections remains a computational challenge. Existing alignment methods are primarily unimodal and assume preserved topology, often failing with tissue distortions like tears, folds, or anatomical changes. Here, we present SAME (Spatial Alignment of Multimodal Expression) that introduces space-tearing transforms, a framework for controlling localized topological disruptions during cross-sectional alignment. Using integer linear programming to maximize cell type matches across the modalities, we enhance cell-type alignment accuracy by 20% compared to existing methods while preserving biologically meaningful spatial relationships. Applied to protein-RNA integration in healthy tongue tissue and lung adenocarcinoma, SAME revealed cryptic immune subpopulations that were otherwise missed by RNA-only or protein-only classification. In a separate lung adenocarcinoma study, we assayed and integrated protein and metabolomic profiles, uncovering localized mevalonic acid upregulation specifically within tumor-macrophage spatial niches and identifying targeted metabolic crosstalk invisible to single-modality approaches. SAME enables unprecedented experimental designs that leverage each modality independently while computationally recovering cross-modal spatial structure, unlocking multimodal discoveries in complex tissues.

bioinformatics↗

A 3D iPSC retina model reveals non-cell-autonomous and non-neuronal mechanism of photoreceptor degeneration in a lysosomal storage disorder.

Disruption of photoreceptor-retinal pigment epithelium (RPE) interface with loss of photoreceptor outer segments (POSs) in the retina is a pathological hallmark of several neurodegenerative and retinal diseases including lysosomal storage disorders like CLN3 disease. However, the retina is a functional composite in vivo; and in vitro stem cell models of retina that enable investigation of the photoreceptor-RPE interface in healthy and diseased retina are lacking. Here, we developed a 3D human pluripotent stem cell (hPSC)-derived retina model to investigate the photoreceptor-RPE interface in healthy and disease tissue. Using this 3D hPSC retina model, we demonstrated that the most common disease causing CLN3 mutation (CLN3{Delta}ex7-8) leads to reduced levels of acid ceramidase (AC) and consequently altered sphingolipid metabolism and signaling and POS loss in CLN3 disease. Consistent with the 3D hPSC retina model, altered sphingolipid metabolism and signaling coincided with POS loss in a large animal model of CLN3 disease, CLN3 miniswine. Therapeutically, recombinant human acid ceramidase (rhAC) targeted both altered sphingolipid metabolism and retina degeneration in the CLN3 hPSC retina model and the CLN3 miniswine eye. These findings demonstrate a proof-of-concept that rhAC can rescue disease phenotype in a large animal model of CLN3 disease and suggest that rhAC could be a therapeutic approach for CLN3 disease. One Sentence SummaryAcid ceramidase deficiency and consequently altered sphingolipid signaling promotes disease phenotype(s) in a lysosomal storage disorder, CLN3 disease.

neuroscience↗

Novel algal modular LOV domain proteins expand opto-biotechnological avenues for controlling of eukaryotic riboswitching, translational and proteolytic processes.

Light, Oxygen, or Voltage (LOV) domains mediate blue light-gated signal transduction, regulating diverse opto-biological functions. LOV domain functions either standalone or fused with effector domains, regulating the downstream signalling process. The current repertoire of LOV domain-based tools is limited to a relatively small number of naturally occurring proteins. In this study, we have identified novel algal LOV domain fused with different effector domains as potential light-mediated translational, ribogenetic and proteolytic switches, highlighting their unexplored avenues of opto-biotechnology. LOV-domain fusion with eIF4E suggests its potential as a light-controlled translational switch and as an opto-ribogenetic regulator. LOV-SppA might be used as a light-gated proteolytic switch. Additionally, LOV coupled with UFD1, UbiH, mannosyl-oligosaccharide glucosidase and biosynthetic gene cluster (BCG) molecular chassis pave the way for opto-biomanufacturing strategies of relevant valuable algal bioactive. Here, we report the discovery of 13 novel algal modular LOV domain-containing proteins across the algal system through comprehensive bioinformatics, bio-curation and systems biology approaches. It offers important insights into the structural and functional diversity of LOV photoreceptors in algae. Hence, these newly identified modular LOV domain-containing proteins expand the platform of opto-biotechnology applications. These finding lay the foundation for future research on the mechanistic basis for light-driven signalling cascade of RNA, translational and protein homeostasis in algae, and potentiate development of next-generation opto-biotechnological tools for synthetic biology, optogenetics and opto-biomanufacturing of valuable bioactive via regulation of biosynthetic gene cluster (BGC) in green lineage.

bioinformatics↗

Sex bias determines MERS-CoV infection outcomes in a mouse model of differential pathogenicity

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a spectrum of disease outcomes in infected humans, ranging from asymptomatic or tolerant to lethal. While the virus itself contributes to pathogenesis, disease severity is primarily influenced by the hosts response to infection. One factor observed to impact the host response is sex, as epidemiological data indicates that male persons have a higher case fatality rate than females infected with MERS-CoV. However, the mechanism underlying this sex bias is unknown and disease course remains difficult to predict. This study investigates how male and female transgenic mice expressing humanized dipeptidyl peptidase-4 (hDPP4) respond to MERS-CoV infection following exposure to either a tolerance-inducing low dose or lethal high dose. We observed that female hDPP4 mice display dose-dependent tolerance to infection and males experienced uniformly lethal disease in both dosing groups. Longitudinal transcriptomic analysis revealed that males suppress innate and inflammatory responses early after infection, causing delayed induction of the host antiviral response. In contrast, high dose females mount an immediate and sustained interferon and inflammatory response, activating antiviral effectors and interferon-stimulated genes. Tolerant females displayed the greatest transcriptional control, showing no pathway enrichment and minimal changes in weight throughout infection. Our results suggest that the magnitude of the response is driven by dose while the nature of the response in shaped by sex. Females mount a more robust response to MERS-CoV infection, allowing females to tolerate low-dose infection but causing uncontrolled inflammation after high dose infection. In contrast, males experienced lethal outcomes regardless of dose. By examining the dynamics of sex-biased host transcriptional responses in determining disease severity, this study highlights the importance of sex as a biological variable in coronavirus pathogenesis research.

microbiology↗

Recombinant Expression and Purification of Plasmodium Heme Detoxification Protein in E. coli: Challenges and Discoveries

1.2Heme, a toxic by-product of Plasmodiums proteolytic digestion of host hemoglobin, is detoxified by the malaria parasite through its conversion into hemozoin (Hz)--the malaria pigment. This detoxification pathway is a key target for many antimalarial drugs, which aim to induce heme-mediated toxicity to the parasite. The Heme Detoxification Protein (HDP) plays a central role in heme-to-Hz transformation; however, its precise mechanism remains unclear, largely due to the absence of successful recombinant expression in a native, soluble form. In this study, we aimed to express HDP recombinantly in its native soluble state using an E. coli-based system. A range of strategies were employed, including expression of orthologs, consensus sequence design, fusion to solubility-enhancing partners, co-expression with molecular chaperones, and extensive construct optimization through N-terminal truncations. Despite extensive efforts, most recombinant HDP constructs were either insoluble or formed soluble aggregates. Notably, only a single construct--with a 44-residue N-terminal truncation and a C-terminal 6His tag (HDPpf-C10)--was successfully expressed in a soluble form. Surprisingly, HDPpf-C10, although retaining domains implicated in heme binding and transformation, exhibited no detectable heme-to-Hz transformation activity. This finding highlights the essential role of the flexible-unstructured N-terminal region in mediating both heme binding and its subsequent conversion to Hz, providing new insights into HDP function and guiding future structural and mechanistic studies.

molecular biology↗