Biology subjects

Shin, M.

Publications and source records attributed to Shin, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Tiled base editing screens uncover rare second-site mutations that rescue a misfolded disease protein

Therapeutic base editing can directly revert ~30% of disease-causing missense mutations to the wild-type sequence. One strategy to broaden its reach is to install second-site corrector mutations that improve protein stability or function across multiple pathogenic alleles. Here, we use tiled adenine and cytosine base editing screens to identify such corrector mutations in NPC1, a lysosomal cholesterol transporter whose misfolding causes Niemann-Pick type C disease. Tiling mutagenesis of the endogenous NPC1 locus in haploid human cells carrying the prevalent NPC1I1061T disease allele, combined with phenotypic reporters for cholesterol transport and protein abundance, uncovered rare intragenic suppressor mutations distributed across multiple domains. Unexpectedly, we identified a promiscuous second-site variant that can correct many different pathogenic NPC1 mutations. These correctors improve protein stability, promote ER exit and lysosomal trafficking, and restore cholesterol clearance in patient-derived fibroblasts and induced neurons. Our results establish in situ base editing screens for the discovery of second-site corrector mutations that are actionable for therapeutic base editing when the original mutation cannot be corrected.

cell biology↗

bioRxiv2026

Predicting conformational changes induced by biomolecular interactions

Biomolecular interactions trigger structural changes of protein complexes essential for cellular signaling, regulation, and drug action. Modern co-folding models can now generate detailed structures of multi-component complexes directly from sequence. However, recent evaluations reveal that they frequently fail to capture specific conformational states induced by a ligand or partner. Here, we show that explicitly modeling the structural transition from an unbound to a bound state, rather than generating the bound complex alone, better resolves these interaction-induced states. Our generative learning framework, K-Fold, transforms independently predicted unbound component priors into an assembled complex through an ultrafast diffusion bridge, operating entirely without multiple sequence alignments. By eliminating co-evolutionary dependencies, K-Fold accurately recovers non-coevolved interfaces in antibody-antigen and targeted-degrader complexes, as well as ligand-directed regulatory states in GPCRs and kinases. Furthermore, by coupling this diffusion bridge with diverse monomeric equilibrium ensembles, K-Fold provides a computationally tractable foundation for sampling complex multi-state landscapes without retraining. These results demonstrate that formulating molecular assembly as an explicit structural transition moves structure prediction beyond static geometries toward capturing biomolecular dynamics.

molecular biology↗

bioRxiv2026

KRASG12C Inhibition Remodels the Non-Small Cell Lung Cancer Surfaceome to Uncover Targetable Vulnerabilities

Mutant-selective and pan-RAS(ON) multi-selective inhibitors have improved clinical outcomes for patients with RAS-driven cancers; however, adaptive resistance limits the depth and durability of response. Using an integrated genetic, transcriptomic, and mass-spectrometry-based quantitative temporal proteomic platform, we profile the adaptive response to KRASG12C inhibitors (KRASi) in KRASG12C-mutant lung adenocarcinoma models. Our analyses reveal remodeling of signaling pathways and the cell surface proteome, including cell surface proteins selectively upregulated across cell lines, tumor models, and treatment durations. Folate receptor alpha (FOLR1) emerged as a therapeutically actionable target that is significantly upregulated with KRASi and is part of a broader acute cellular state transition characterized by acquisition of an epithelial identity. Combining KRASi with mirvetuximab soravtansine-gynx, a clinically approved FOLR1-targeting antibody drug conjugate, enhanced tumor cell cytotoxicity and deepened response and tumor control in cellular and in vivo models. Additionally, this work represents a comprehensive multi-omic resource characterizing non-genetic temporal adaptation to KRASi for future mechanistic and therapeutic investigations.

cancer biology↗

bioRxiv2026

KRAS inhibition drives adaptive surfaceome remodeling and reveals ALPP/ALPPL2 as a targetable vulnerability in pancreatic cancer

KRAS is mutated in over 90% of pancreatic ductal adenocarcinoma (PDAC) and is required for tumor initiation and maintenance. While clinical trials testing mutant-selective and pan-RAS inhibitors (KRASi) have demonstrated significant clinical benefit, genetic and non-genetic mechanisms of resistance limit the long-term efficacy of monotherapy strategies. Here, we used quantitative temporal analysis of the global, phosphorylation, and surface proteome to comprehensively identify non-genetic adaptive pathways and cell surface biomarkers in response to acute and long-term KRAS inhibition in PDAC. Through integrative machine learning analysis of the KRASi-proteome and publicly available datasets, we discovered top priority cell surface targets for combination therapy with KRASi. We identified Placental Alkaline Phosphatase (ALPP) as upregulated in response to KRASi through compensatory JAK2/STAT3 signaling. We developed a novel clinical grade ALPP/ALPPL2 (ALPG)-targeting antibody drug conjugate (ADC) with site-specific MMAE conjugation and demonstrated single agent efficacy in PDAC models, as well as enhanced internalization and anti-tumor efficacy in combination with KRASi. Together, these studies comprehensively define the KRASi-induced proteome and identify ALPP/ALPPL2 as a promising cell surface target for combination with KRASi to enhance response and forestall KRASi resistance.

cancer biology↗

bioRxiv2026

Evaluation of the Translational Readiness of Pluripotent Stem Cell-Derived Vascular Cell Therapy for Limb Ischemia: A Systematic Review and Meta-analysis

Background: For the first time, pluripotent stem cell (PSC)-derived endothelial cells were administered to a human for peripheral artery disease (PAD), in August 2026. This translation is based on a large preclinical literature claiming that donor cells build blood vessels in animal models of limb ischemia, but whether it supports that claim has not been assessed systematically. Methods: In this systematic review and meta-analysis, we searched MEDLINE, Web of Science, Europe PMC, and Embase to August 2026 for studies of PSC-derived vascular cells in animal models of limb ischemia with an acellular control. Limb perfusion at each experiment's primary timepoint was pooled as Hedges' g in a three-level random-effects model with cluster-robust variance. Donor-cell fate and a seven-level ordinal of vascular contribution were graded for every study. This meta-analysis was registered on PROSPERO (CRD420261465308). Results: Sixty-eight studies in 69 reports met the criteria; 51 contributed 114 comparisons at primary timepoints. Cell therapy increased perfusion (Hedges' g 2.26, 95% CI 1.72-2.79; P=4.1x10-11; I2=87.5%; 95% prediction interval -1.47 to 5.98). No prespecified moderator survived Holm-Bonferroni adjustment, including the donor-cell fate grade (P=0.30). A donor cell was resolved in a vessel wall in 35 studies, and flow through such a vessel in 7. Small-study effects were strong (Egger t=6.25, P=9x10-8) but were not reproduced by a sample-size-based test (P=0.80); trim-and-fill moved the estimate to 1.15. On the natural scale, treated limbs recovered about twice the control perfusion (ratio of means 1.96, 95% CI 1.66-2.33). Limb preservation favored cell therapy in 11 studies with countable events (risk ratio 4.88, 95% CI 2.28-10.45), but 43 of 68 studies reported no functional outcome, and only 22 comparisons from 8 studies were both randomized and blindly assessed. In a post hoc analysis that did not survive multiplicity adjustment, effects were largest in studies whose images could not resolve their incorporation claims (g 3.23) versus those that resolved incorporation (g 2.33). Conclusions: PSC-derived vascular cell therapy improved limb perfusion in animal models, consistently across every sensitivity analysis, although the magnitude is uncertain given heterogeneity and small-study effects. Benefit was not associated with the degree of donor-cell incorporation across studies and was largest where incorporation was least resolvable, a pattern more consistent with reporting and measurement bias than with a true mechanistic effect. The first rigorous, blinded test of that benefit may come not from animals but from the patients now receiving these cells.

cell biology↗

bioRxiv2026

Identification of Lupus Immune Complex-Driven Pathogenic Pro-inflammatory Monocytes and Macrophages in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE or lupus) is an autoimmune disease characterized by anti-nuclear antibody (ANA) production and inflammation, though the mechanisms by which ANAs induce inflammation and tissue injury are incompletely understood. Here, we identified distinct subsets of mononuclear phagocytes (MPs), including monocytes (Mo) and macrophages (M{Phi}), driven by lupus immune complex (IC), comprised of ANAs and their target antigens. scRNA-seq of human Mo incubated with U1-snRNP (snRNP) lupus IC revealed expansion of distinct pro-inflammatory Mo subsets with upregulation of inflammatory genes including those encoding cytokines, NLRP3, and transcription factors. These transcriptomic changes strongly correlated with protein expression, as determined by proteomic analysis. Mo developed similar pro-inflammatory transcriptomic changes in response to other lupus ICs containing anti-dsDNA and Ro60 antibodies. Interrogation of scRNA-seq datasets from the skin, kidney, and peripheral blood of lupus patients revealed the presence and expansion of pro-inflammatory Mo and M{Phi} populations exhibiting transcriptomic signatures similar to those observed in lupus IC-stimulated Mo. Some of these cells expressing the snRNP IC gene signature exhibited low expression of the type I IFN signature, suggesting that lupus IC and type I IFN signaling may independently affect Mo subsets. In lupus nephritis, infiltration of CD68+ M{Phi} expressing NLRP3 was associated with treatment outcomes. Inhibiting activation of the transcription factor ETS2, a master regulator of Mo/M{Phi}-driven inflammation, attenuated lupus IC-induced activation of pro-inflammatory Mo. Collectively, these findings provide novel insights into the role of lupus IC-driven pro-inflammatory MPs in the pathogenesis of SLE and highlight their relevance as therapeutic targets. Significance StatementSystemic lupus erythematosus (SLE or lupus) is a multi-systemic autoimmune inflammatory disease characterized by anti-nuclear antibody (ANA) production. Lupus immune complex (IC), consisting of ANAs and their target antigens, likely play a critical role in the pathogenesis of lupus through activation of mononuclear phagocytes (MPs), including monocytes (Mo) and macrophages (M{Phi}), which can produce an array of inflammatory molecules. Using transcriptomic and proteomic analyses, our study identified distinct pro-inflammatory Mo populations driven by lupus IC, along with expansion of similar pro-inflammatory Mo and M{Phi} subsets in the skin, kidneys, and peripheral blood of lupus patients. These findings provide novel insights into the pathogenic role of lupus IC-driven pro-inflammatory MPs and support a scientific rationale for therapeutically targeting these cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/700902v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1839430org.highwire.dtl.DTLVardef@175dd1dorg.highwire.dtl.DTLVardef@107b54forg.highwire.dtl.DTLVardef@11096b0_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Amenability to Engineering of the Homologation Enzyme, HphA, through Homologous-Based Site-Directed Mutagenesis

Homologation of amino acids, the addition or deletion of a methylene group onto their side chains, has the potential to increase the biostability and bioavailability of peptide natural products. The first enzyme in the homologation pathway, HphA, has been previously characterized and is substrate selective. Bioinformatics studies were used to identify amino acids in the active site of HphA, which may play a role in substrate selection, by comparison to homologous enzymes, homocitrate synthase (HCS) and 2-isopropylmalate synthase (IPMS). Single point mutants to five amino acid residues in the HphAs active site were created to mimic those of HCS and IPMS. Their activities were measured via time-course assays with the natural substrates for HCS and IPMS. Residue A73 was identified as important in the substrate specificity of HphA; therefore, six different additional mutations were generated and tested with nine substrates with various side chains. The HphA A73L mutant exhibited the highest activity compared to the other mutants, showing activity with counterparts of L-Tyr (HphA natural substrate), L-Val (IPMS natural substrate), L-Leu, L-Ser, L-Trp, and L-Asp. Kinetic assays were taken with HphA A73L with the active substrates and compared with kinetic data from HphA WT, HCS, and IPMS. These results demonstrated that the A73L mutation significantly relaxed the substrate specificity of HphA, indicating its amenability to engineering. This research will serve as the foundation for future metabolic engineering studies on the enzymatic homologation pathway of amino acids.

bioengineering↗

bioRxiv2025

Lamin B1 physically regulates neuronal migration by modulating nuclear deformability in the developing cortex

Neuronal migration is a vital process that positions billions of neurons to create a functional brain. To navigate the constrained microenvironments within the cortex, precise control over the nuclear mechanics in migrating neurons is indispensable. Here, we show that Lamin B1 (LB1) regulates neuronal migration by modulating nuclear deformability. Excess LB1 in neurons halted migration without altering laminar identity or overall gene expressions in vivo, while in vitro, it elevated nuclear stiffness and impaired neuronal motility in confined spaces. Moreover, mispositioned neurons resulted in electrophysiological defects in the brain. Computational modeling predicted a temporal relationship between nuclear deformation and enhanced migration velocity, which was validated experimentally through live imaging. Notably, cerebral organoid assays using iPS cells established from patients with LMNB1 duplication exhibited impaired neuronal migration in a human model. Collectively, these findings demonstrate that LB1 is a critical regulator of nuclear mechanics, ensuring the accurate spatiotemporal positioning of neurons.

neuroscience↗

bioRxiv2025

Stem cell-specific NF-kB is required for stem cell survival and epithelial regeneration upon intestinal damage.

Immune signals coordinate the repair of damaged epithelia by intestinal stem cells. However, it is unclear if immune pathways act autonomously within the stem cell to direct the damage response pathway. We consider this an important question, as stem cell dynamics are essential for formation and maintenance of the entire epithelium. We used Drosophila to determine the impact of stem cell-specific loss of NF-{kappa}B on tissue regeneration upon chemical injury. We found that loss of NF-{kappa}B enhanced cell death, impaired enterocyte renewal and increased mortality. Mechanistically, we showed that inhibition of stem cell apoptosis is essential for NF-{kappa}B-dependent maintenance of cell viability and tissue repair. Combined, our data demonstrate that stem cell-intrinsic NF-{kappa}B activity is essential for an orderly repair of damaged intestinal epithelia.

immunology↗

bioRxiv2024

Characterization of newly isolated bacteriophages targeting carbapenem-resistant Klebsiella pneumoniae

Klebsiella pneumoniae, a Gram-negative opportunistic pathogen, is increasingly resistant to carbapenems in clinical settings. This growing problem necessitates the development of alternative antibiotics, with phage therapy being one promising option. In this study, we investigated novel phages targeting carbapenem-resistant Klebsiella pneumoniae (CRKP) and evaluated their lytic capacity against clinical isolates of CRKP. First, 23 CRKP clinical isolates were characterized using Multi-locus Sequence Typing (MLST), carbapenemase test, string test, and capsule typing. MLST classified the 23 K. pneumoniae isolates into 10 sequence types (STs), with the capsule types divided into nine known and one unknown type. From sewage samples collected from a tertiary hospital, 38 phages were isolated. Phenotypic and genotypic characterization of these phages was performed using Random Amplification of Polymorphic DNA-PCR (RAPD-PCR), transmission electron microscopy (TEM), and whole genome sequencing (WGS) analysis. Host spectrum analysis revealed that each phage selectively lysed strains sharing the same STs as their hosts, indicating ST-specific activity. These phages were subtyped based on their host spectrum and RAPD-PCR, identifying nine and five groups, respectively. Fourteen phages were selected for further analysis using TEM and WGS, revealing 13 Myoviruses and one Podovirus. Genomic analysis grouped the phages into three clusters: one closely related to Alcyoneusvirus, one to Autographiviridae, and others to Straboviridae. Our results showed that the host spectrum of K. pneumoniae-specific phages corresponds to the STs of the host strain. These 14 novel phages also hold promise as valuable resources for phage therapy against CRKP.

microbiology↗

bioRxiv2024

Autophagosomes coordinate an AKAP11-dependent regulatory checkpoint that shapes neuronal PKA signaling

Protein Kinase A (PKA) is regulated spatially and temporally via scaffolding of its catalytic (C/{beta}) and regulatory (RI/RII) subunits by the A-kinase-anchoring proteins (AKAP). PKA engages in poorly understood interactions with autophagy, a key degradation pathway for neuronal cell homeostasis, partly via its AKAP11 scaffold. Mutations in AKAP11 drive schizophrenia and bipolar disorders (SZ-BP) through unknown mechanisms. Through proteomic-based analysis of immunopurified lysosomes, we identify the C-RI-AKAP11 holocomplex as a prominent autophagy-associated protein kinase complex. AKAP11 scaffolds C-RI to the autophagic machinery via its LC3-interacting region (LIR), enabling both PKA regulation by upstream signals, and its autophagy-dependent degradation. We identify Ser83 on the RI linker-hinge region as an AKAP11-dependent phospho-residue that modulates RI-C binding and cAMP-induced PKA activation. Decoupling AKAP11-PKA from autophagy alters Ser83 phosphorylation, supporting an autophagy-dependent checkpoint for PKA signaling. Ablating AKAP11 in induced pluripotent stem cell-derived neurons reveals dysregulation of multiple pathways for neuronal homeostasis. Thus, the autophagosome is a novel platform that modulate PKA signaling, providing a possible mechanistic link to SZ/BP pathophysiology.

cell biology↗

bioRxiv2024

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-Treated Adult Zebrafish as a Model for Parkinsons Disease

Dopamine (DA) is a catecholamine neurotransmitter that works to regulate cognitive functions. Patients affected by Parkinsons Disease (PD) experience a loss of dopaminergic neurons and downregulated neural DA production. This leads to cognitive and physical decline that is the hallmark of PD for which no cure currently exists.. Danio rerio, or zebrafish, have become an increasingly popular disease model used in PD pharmaceutical development. This model still requires extensive development to better characterize which PD features are adequately represented. Furthermore, the great majority of PD zebrafish models have been performed in embryos, which may not be relevant towards age-related human PD. As an improvement, mature D. rerio were treated with neurotoxic prodrug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) through intraperitoneal injection to induce parkinsonism. Behavioral analysis confirmed disparities in movement between saline-injected control and the MPTP-injected experimental group, with swim distance and speed significantly lowered seven days after MPTP injection. Simultaneously, cognitive decline was apparent in MPTP-injected zebrafish, demonstrated by decreased alternation in a y-maze. RT-qPCR confirmed trends consistent with downregulation in Parkinsonian genetic markers, specifically DA transporter (DAT), MAO-B, PINK1. In summary, mature zebrafish injected with MPTP present with similar movement and cognitive decline as compared to human disease. Despite its benefits, this model does not appear to recapitulate full pathophysiology of the disease with the full profile of expected gene downregulation. Because of this, it is important that researchers looking for pharmacological interventions for PD only use this zebrafish model when targeting the human-relevant PD symptoms and causes that are represented.

neuroscience↗

bioRxiv2024

Recharacterization of RSL3 reveals that the selenoproteome is a druggable target in colorectal cancer

Ferroptosis is a non-apoptotic form of cell death resulting from the iron-dependent accumulation of lipid peroxides. Colorectal cancer (CRC) cells accumulate high levels of intracellular iron and reactive oxygen species (ROS) and are thus particularly sensitive to ferroptosis. The compound (S)-RSL3 ([1S,3R]-RSL3) is a commonly used ferroptosis inducing compound that is currently characterized as a selective inhibitor of the selenocysteine containing enzyme (selenoprotein) Gluathione Peroxidase 4 (GPx4), an enzyme that utilizes glutathione to directly detoxify lipid peroxides. However, through chemical controls utilizing the (R) stereoisomer of RSL3 ([1R,3R]-RSL3) that does not bind GPx4, combined with inducible genetic knockdowns of GPx4 in CRC cell lines, we revealed that GPx4 dependency does not always align with (S)-RSL3 sensitivity, questioning the current characterization of GPx4 as the central regulator of ferroptosis. Utilizing affinity pull-down mass spectrometry with chemically modified (S)-RSL3 probes we discovered that the effects of (S)-RSL3 extend far beyond GPx4 inhibition, revealing that (S)-RSL3 is a broad and non-selective inhibitor of selenoproteins. To further investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed additional chemical and genetic approaches. We found that the selenoprotein inhibitor auranofin, an FDA approved gold-salt, chemically induced oxidative cell death and ferroptosis in both in-vitro and in-vivo models of CRC. Consistent with these data, we found that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translation of selenoproteins, is essential for the in-vitro growth and xenograft survival of CRC cell lines. In summary, these findings recharacterize the mechanism of action of the most commonly used ferroptosis inducing molecule, (S)-RSL3, and reveal that broad inhibition of selenoproteins is a promising novel therapeutic angle for the treatment of CRC.

cancer biology↗

bioRxiv2024

Oxygen carrying nanoemulsions and respiratory hyperoxia eliminate tumor hypoxia-induced suppression and improve cancer immunotherapy

Hypoxia-HIF-1-driven immunosuppressive transcription and cAMP-elevating signaling through A2A-adenosine receptors (A2AR) represent a major tumor-protecting pathway that enables immune evasion. Recent promising clinical outcomes due to the blockade of the adenosine-generating enzyme CD73 and A2AR in patients refractory to all other therapies have confirmed the importance of targeting hypoxia-adenosinergic signaling. We report a novel and feasible approach to target the upstream stage of hypoxia-adenosinergic immunosuppression using an oxygen-carrying nanoemulsion (perfluorocarbon blood substitute). It is shown that oxygenation agent therapy i) eliminates tumor hypoxia, ii) improves efficacy of endogenously developed and adoptively transferred T cells, and thereby iii) promotes regression of tumors in different anatomical locations. We show that both T cells and NK cells avoid hypoxic tumor areas and that reversal of hypoxia by oxygenation agent therapy increases intratumoral infiltration of activated T cells and NK cells due to re-programming of the tumor microenvironment (TME). Thus, repurposing oxygenation agents in combination with supplemental oxygen may improve current cancer immunotherapies by preventing hypoxia-adenosinergic suppression, promoting immune cell infiltration and enhancing effector responses. These data also suggest that pretreating patients with oxygenation agent therapy may reprogram the TME from immune-suppressive to immune-permissive prior to adoptive cell therapy, or other forms of immunotherapy. SummaryOxygen delivering nanoemulsions and respiratory hyperoxia address limitations of blood vessel-mediated tumor oxygenation and promote anti-tumor immune responses to enhance immunotherapy.

immunology↗

bioRxiv2023

Transcriptomic Profiling Reveals Claudin 18.2 as a Diagnostic Biomarker of Menetrier Disease and the Role of Hedgehog Signaling in Pathogenesis

Both Menetriers disease (MD) and juvenile polyposis syndrome (JPS) are rare premalignant conditions that can lead to gastric cancer development. MD is an acquired disease without known causative mutations. MD patients are characterized by an increased expression of EGF receptor (EGFR) ligand and transforming growth factor alpha (TGF-) in the stomach. JPS is inherited in an autosomal dominant pattern and is caused by BMPR1A or SMAD4 mutations. It is characterized by multiple polyps throughout the gastrointestinal tract along with certain SMAD4 mutations that can result in gastric polyposis. Although there are many distinct clinico- endoscopic and histopathologic features that differ between the two diseases, they also share similar features that often lead to misdiagnosis. This study aimed to identify markers that can help distinguish MD from JPS and to better understand the pathogenesis of MD by comparing differential gene expression patterns. Upon examination of MD and JPS microscopically, we found almost all cases have patchy areas mimicking each other, making it difficult to make a correct diagnosis with histopathologic examination alone. Comparative analysis between MD and JPS using ingenuity pathway analysis (IPA) revealed both common and differential gene signatures. Common gene signatures included estrogen receptor signaling, integrin signaling, mTOR signaling, and others, which may be responsible for histopathologic similarities. Among differential gene signatures, we found that claudin 18 (CLDN18) is upregulated in MD and confirmed that CLDN18.2 (isoform of CLDN18) protein expression is higher in MD than JPS by immunohistochemistry. Comparative analysis between MD and normal control revealed the hedgehog (Hh) signaling pathway is upregulated in MD. Treatment with a hedgehog pathway inhibitor partially rescued the histopathologic phenotypes in a MD mouse model. The current study provides valuable insight into the potential underlying mechanism of why MD and JPS show similar clinico-pathologic features. We also identified a diagnostic marker CLDN18.2 that can help distinguish MD from JPS, genetically. Furthermore, it also shows that Hh signaling plays an important role in the pathogenesis of MD and can function as a potential therapeutic target.

pathology↗

bioRxiv2023

S-nitrosylation-mediated unfolded protein response maintains hematopoietic progenitors

The Drosophila lymph gland houses blood progenitors that give rise to myeloid-like blood cells. Initially, blood progenitors proliferate, but later become quiescent to maintain multipotency before differentiation. Despite the identification of various factors involved in multipotency maintenance, the cellular mechanism regulating blood progenitor quiescence remains elusive. Here, we reveal the expression of nitric oxide synthase in blood progenitors, generating nitric oxide for post-translational S-nitrosylation of protein cysteine residues. S-nitrosylation activates the Ire1-Xbp1-mediated unfolded protein response, leading to G2 cell cycle arrest. Specifically, we identify the epidermal growth factor receptor as a target of S-nitrosylation, resulting in its retention within the endoplasmic reticulum and blockade of its receptor function. Collectively, our findings highlight developmentally programmed S-nitrosylation as a critical mechanism that induces protein quality control in blood progenitors, maintaining their undifferentiated state by inhibiting cell cycle progression and rendering them unresponsive to paracrine factors.

developmental biology↗

bioRxiv2023

The Emergence of Circadian Timekeeping in the Intestine

The circadian clock is a molecular timekeeper, present from cyanobacteria to mammals, that coordinates internal physiology with the external environment. The clock has a 24-hour period however development proceeds with its own timing, raising the question of how these interact. Using the intestine of Drosophila melanogaster as a model for organ development, we track how and when the circadian clock emerges in specific cell types. We find that the circadian clock begins abruptly in the adult intestine and gradually synchronizes to the environment after intestinal development is complete. This delayed start occurs because individual cells at earlier stages lack the complete circadian clock gene network. As the intestine develops, the circadian clock is first consolidated in intestinal stem cells with changes in ecdysone and Bursicon hormone signalling influencing the transcriptional activity of Clk/cyc to drive the expression of tim, Pdp1, and vri. In the mature intestine, stem cell lineage commitment transiently disrupts clock activity in differentiating progeny, mirroring early developmental clock-less transitions. Our data show that clock function and differentiation are incompatible and provide a paradigm for studying circadian clocks in development and stem cell lineages.

developmental biology↗

bioRxiv2023

SINGLE CELL RESOLUTION OF THE ADULT ZEBRAFISH INTESTINE UNDER CONVENTIONAL CONDITIONS, AND IN RESPONSE TO AN ACUTE VIBRIO CHOLERAE INFECTION.

Vibrio cholerae is an aquatic bacterium that primarily infects the gastrointestinal tract, causing the severe and potentially deadly diarrheal disease, cholera. Despite the impact of Vibrio on global health, our understanding of host mucosal responses to the pathogen at the site of infection remains limited, highlighting a critical knowledge gap that must be addressed to develop more effective prevention and treatment strategies. Using a natural infection model, we combined physiological and single-cell transcriptomic studies to characterize adult zebrafish guts raised under conventional conditions and after a challenge with Vibrio. We discovered that Vibrio causes a mild mucosal immune response characterized by T cell activation and enhanced antigen capture in the epithelium. Additionally, we discovered that Vibrio suppresses host interferon signaling, and that ectopic activation of interferon significantly alters the course of infection. Notably, we also found that the adult zebrafish gut shares many similarities with mammalian counterparts, including the presence of previously undescribed Best4+ cells, tuft cells, and a population of basal cycling cells. These discoveries provide important insights into host-pathogen interactions and emphasize the utility of zebrafish as a natural model of Vibrio infection.

immunology↗