Biology subjects

Mitchell, D.

Publications and source records attributed to Mitchell, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Targeting NAT10 Activates Tumor-Intrinsic Immunity and Suppresses Tumor Progression in Head and Neck Squamos Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) remain a major clinical challenge due to its high heterogeneity and limited therapeutic response, resulting in a 5-year overall survival rate of only [~]50%. Identifying molecular pathways that drive tumor progression while suppressing anti-tumor immunity is therefore critical for developing more effective therapies. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for catalyzing the RNA modification N4-acetylcytidine (ac4C) on rRNA, tRNA, and mRNA, and has been implicated in tumor progression in several cancers. In this study, we identify NAT10 as a key suppressor of tumor-intrinsic immune signaling in HNSCC. NAT10 expression was significantly elevated in tumor tissues and HNSCC cell lines and was associated with poor overall survival. Moreover, high-risk HPV, a major etiological factor in HNSCC, upregulated NAT10 protein expression through the viral oncoproteins E6 and E7. Functional inhibition of NAT10, either by genetic depletion or the small-molecule inhibitor Remodelin, activated tumor-intrinsic innate immune responses, as evidenced by increased IRF3 phosphorylation and induction of type I/II interferons and interferon-stimulated genes. Depletion of NAT10 was able to suppress tumorigenic phenotypes, including cell proliferation, migration, and colony formation in HNSCC cells. Importantly, activation of the STING signaling pathway using agonist cyclic di-GMP further amplified immune activation in NAT10-inhibited cancer cells. Together, our findings establish NAT10 as a previously unrecognized negative regulator of tumor-intrinsic immunity in HNSCC and support NAT10 targeting, particularly in combination with STING agonists, as a promising immunotherapeutic strategy.

Cancer Biology↗

bioRxiv2026

Space-Time Light-Sheet Microscopy

Light-sheet microscopy (LSM) has revolutionized bioimaging by delivering high-contrast volumetric resolution with minimal photodamage. Spatial wavefront shaping, used to gen{-}erate lattice and Airy light-sheets, has been particularly effective in advancing LSM be{-}yond the Rayleigh limit. Despite its broad adoption, most LSM implementations rely on rigid dual-objective geometries that complicate sample handling and impose a trade-off between imaging field of view (FoV) and axial resolution. Here, we introduce space-time light-sheet microscopy (ST-LSM), a single-objective strategy that exploits space-time (ST) correlations for the first time. ST-LSM goes beyond separate spatial or temporal modulation to jointly modulate the spatiotemporal spectral structure of a pulse. This uniquely enabled light-sheets with wavelength-scale thickness over millimeter-scale dis{-}tances. When compared to state-of-the-art approaches, ST-LSM eliminates the dual-objective constraint, expands the sample-accessible volume by 25x, and increases the FoV by 10x without sacrificing sectioning resolution. We demonstrate the versatility of ST-LSM by using a single setup to image specimens across four orders of magnitude in size, from whole roots and developing embryos, down to mammalian cells with sub-cellular axial resolution. These results position ST-LSM as an accessible and high-performance optical microscopy platform at a variety of biological scales, by translating space-time wave-packet physics into a practical imaging modality.

biophysics↗

bioRxiv2026

Kv11.1 (hERG) Protein Interaction Networks Connect Endocytic Trafficking to Polygenic Influences on Cardiac Repolarization

Polygenic scores (PGS) summarize the combined effects of common single-nucleotide polymorphisms and contribute to predictions of disease severity, but biological consequences linked to these common variants remain poorly defined. Here, we focused on polygenic liability for a measurable electrophysiologic trait (the QT interval). Prolonged QT interval, measured on patient electrocardiograms, is associated with an increased risk for cardiac arrhythmia. We investigated human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) from donors with extreme PGS (i.e., high and low) related to QT interval duration. We paired global proteomics with multiplexed affinity purification mass spectrometry (AP-MS) centered on Kv11.1 (hERG), a major determinant of QT-interval repolarization. Global proteomics indicated increased mitochondrial protein abundance in high-PGS cardiomyocytes, but this did not explain the Kv11.1 interactome. In high-PGS cells, Kv11.1 showed increased associations with myosin motor proteins and endosomal recycling machinery, consistent with altered (and potentially increased) recycling/trafficking dynamics rather than trafficking deficiency observed with most pathogenic Kv11.1 variants. This proof-of-concept study underscores a framework for linking polygenic factors to tractable biological consequences by combining patient-specific hiPSCs, proteomics and affinity-purification. Linking polygenic scores to changes in protein networks provides testable mechanisms that can be applied across many diseases. Significance StatementPolygenic scores (PGS) predict disease risk, but how biological pathways are influenced by these common variants remains difficult to define. We generated human induced pluripotent stem cells from individuals with extreme high- and low-PGS for QT interval, a key electrocardiographic measure linked to arrhythmia risk. By combining global proteomics and interactomics for a common ion channel involved in regulating the QT interval (Kv11.1) we found mechanisms that are influenced by common genetic traits in patients. Our work connects polygenic scores to pathway-level molecular mechanisms in human cells and provides a general framework for uncovering how complex genetic architecture drives disease-relevant biology. ClassificationBiological Sciences; Medical Sciences

genomics↗

bioRxiv2025

ShapeUpLMD: An Automated Pipeline for Spatially Optimized Laser Microdissection in Multi-omic Tissue Profiling

Laser microdissection (LMD) enables enrichment of defined cellular populations from heterogeneous tissues, providing histologically-resolved molecular profiles with spatial resolution. However, the absence of standardized methods for generating optimized regions of interest (ROIs) limits reproducibility and scalability for spatial multi-omic workflows. To address this gap, we developed ShapeUpLMD, an open-source software tool and integrated workflow that automates the optimization of spatially defined ROIs for LMD, directly from digital pathology annotations. Fresh-frozen uterine serous carcinoma (USC) tumors (n = 11) were sectioned onto polyethylene naphthalate slides, scanned and subsequently annotated by an expert pathologist. Tumor and non-tumor ROIs were used to train tumor-specific classifiers in HALO (Indica Labs). Classified tumor or unbiased whole tissue ROIs were refined and optimized by ShapeUpLMD prior to automated collection on a Leica LMD7 microscope. Across the cohort, predicted tumor ROIs increased effective tumor purity by 86 {+/-} 6% relative to whole tissue while maintaining high spatial concordance between predicted and collected ROIs (accuracy = 0.9 {+/-} 0.05). Data-independent acquisition mass spectrometry quantified upwards of [~]6,500 proteins across spatially resolved ROIs, revealing regionally coherent clustering of adjacent tumor regions and abundance patterns consistent with tumor and non-tumor cell admixture in an unbiased spatial proteomic application. ShapeUpLMD provides an automated, reproducible, and scalable framework that bridges digital pathology with LMD enabling high-fidelity spatial enrichment for multi-omic analyses. This workflow increases throughput, reduces inter-operator variability, and supports standardized, regionally resolved tissue collection for spatial systems biology applications. The software is available at https://github.com/GYNCOE/ShapeUpLMD.

cancer biology↗

bioRxiv2025

Microglia maintain retinal redox homeostasis following ablation of rod photoreceptors in zebrafish

Microglia rapidly respond to injury, stress, and perturbations to neurons in the brain and retina and perform phagocytosis to clear dying cells and debris. Oxidative stress is a frequent feature of neurodegeneration, and while glia are crucial for managing such stress, microglia may also be dysfunctional in diseased tissue. Here we examine the role of microglia in management of oxidative stress upon death of rod photoreceptors in the larval zebrafish retina. Using rho:nfsb-eGFP transgenic zebrafish and treatment with the pro-drug metronidazole (MTZ), we coupled the generation of reactive oxygen species (ROS) in dying rods to their ablation. Microglia efficiently engulfed and cleared the ROS-laden rods, effectively undertaking the oxidative load. Despite abundant ROS upon MTZ-mediated cell death, oxidative stress overall was minimal in retinal tissue when microglia were present, indicating that they rapidly and efficiently performed redox functions. In irf8-/- mutants, which are deficient in microglia, retinas with MTZ-induced rod ablation showed widespread ROS that localized, at least in part, to Muller glia. Further, there was evidence of increased oxidative stress, and increased numbers of "off-target" inner retinal neurons that stained positive for the cell death marker TUNEL. Supplementation with the antioxidant Glutathione (GSH) reduced the number of off-target TUNEL+ cells detected in microglia-deficient retinas following rod ablation. Our results indicate that microglial redox functions are important in restoring homeostasis following acute retinal damage.

neuroscience↗

bioRxiv2025

Patient-derived Cellular Models of Polygenic Scores: Application to Long QT Syndrome

Polygenic scores (PGS) have emerged as important modifiers of disease risk, drug response, and rare variant penetrance, but the biological mechanisms underlying these associations remain poorly understood. To experimentally investigate these relationships, we derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from individuals at the 1st, 50th, and 99th percentiles of a genome-wide QT interval polygenic score (QT-PGS). We examined baseline repolarization, drug response to the hERG inhibitor E4031, and penetrance of two KCNH2 variants (p.Arg148Trp and p.Arg823Trp) in these cellular models. While baseline field potential durations (FPDs) did not differ across PGS levels, high-PGS iPSC-CMs showed exaggerated prolongation in response to E4031 and increased phenotypic expression of both KCNH2 variants. These findings provide the first experimental demonstration that polygenic background can shape cardiac electrophysiologic phenotypes and modulate the functional impact of both pharmacologic and genetic perturbations. This work establishes a scalable platform for mechanistic studies of polygenic risk.

genetics↗

bioRxiv2025

Endogenous recovery of hippocampal function following global cerebral ischemia in juvenile female mice is influenced by neuroinflammation and circulating sex hormones.

Cardiac arrest-induced global cerebral ischemia (GCI) in childhood often results in learning and memory deficits. We previously demonstrated in a murine cardiac arrest and cardiopulmonary resuscitation (CA/CPR) mouse model that a cellular mechanism of learning and memory, long-term potentiation (LTP), is acutely impaired in the hippocampus of juvenile males, correlating with deficits in memory tasks. However, little is known regarding plasticity impairments in juvenile females. We performed CA/CPR in juvenile (P21-25) female mice and used slice electrophysiology and hippocampal dependent behavior to assess hippocampal function. LTP was and contextual fear were impaired 7-days after GCI and endogenously recovered by 30-days. LTP remained impaired at 30 days in ovariectomized females, suggesting the surge in gonadal sex hormones during puberty mediates endogenous recovery. Unlike juvenile males, recovery of LTP in juvenile females was not associated with BDNF expression. NanoString transcriptional analysis revealed a potential role of neuroinflammatory processes, and specifically Cd68 pathways, in LTP impairment and hormone-dependent recovery. We were able to restore LTP in ovariectomized females with chronic and acute PPT administration, implicating estrogen receptor alpha in recovery mechanisms. This study supports a mechanism of endogenous LTP recovery after GCI in juvenile female mice which differs mechanistically from juvenile males and does not occur in adults of either sex.

neuroscience↗

bioRxiv2025

Multiple lesion inductions intensify central sensitization driven by neuroinflammation in a mouse model of endometriosis.

IntroductionEndometriosis is an inflammatory disease associated with chronic pelvic pain (CPP). Growing evidence indicates that endometriotic lesions are not the sole source of pain. Instead, central nervous system (CNS) dysfunction created by prolonged peripheral and central sensitization plays a role in developing endometriosis-associated CPP. This study investigated how CPP is established using a multiple lesion induction mouse model of endometriosis, as repeated retrograde menstruation is considered underlying endometriosis pathogenesis. MethodsWe generated endometriosis-like lesions by injecting endometrial tissue fragments into the peritoneal cavity in mice. The mice received a single (1x) or multiple inductions (6x) to simulate recurrent retrograde menstruation. Lesion development, hyperalgesia by behavioral testing, signs of peripheral sensitization, chronic inflammation, and neuroinflammation were examined with lesions, peritoneal fluids, dorsal root ganglia (DRG), spinal codes, and brain. ResultsMultiple lesion inductions increased lesion numbers and elevated abdominal and hind paw hypersensitivity compared to single induction mice. Elevated persistent glial cell activation across several brain regions and/or spinal cords was found in the multiple induction mice. Specifically, IBA1+ microglial soma size was increased in the hippocampus and thalamus. IBA1+ cells were abundant in the cortex, hippocampus, thalamus, and hypothalamus of the multiple induction mice. GFAP+ astrocytes were mainly elevated in the hippocampus. Elevated TRPV1, SP, and CGRP expressions in the DRG were persistent in the multiple induction mice. Furthermore, multiple inductions induced the severe disappearance of TIM4hi MHCIIlo residential macrophages and the influx of increased proinflammatory TIM4lo MHCIIhi macrophages in the peritoneal cavity. The single and multiple inductions elevated secreted TNF, IL-1{beta}, and IL-6 levels in the peritoneal cavity at 2 weeks. Elevated cytokine levels returned to the pre-induction levels in the single induction mice at 6 weeks; however, they remained elevated in the multiple induction mice. ConclusionsOur results indicate that the repeatedly occurring lesion inductions (=mimic retrograde menstruation) can be a peripheral stimulus that induces nociceptive pain and creates composite chronic inflammatory stimuli to cause neuroinflammation and sensitize the CNS. The circuits of neuroplasticity and stimulation of peripheral organs via a feedback loop of neuroinflammation may mediate widespread endometriosis-associated CPP.

molecular biology↗

bioRxiv2024

Simultaneous mapping of RNA secondary, tertiary, and quaternary structure in living cells by multi-site DMS probing

Identifying tertiary structures and protein binding sites on RNA molecules remains a key challenge in RNA biology. We report a new chemical probing strategy termed multi-site DMS-MaP (msDMS-MaP) that exploits base tautomerization during mutational profiling reverse-transcription to measure typically invisible DMS-induced modifications at the N7 position of G. These N7-G modifications are now resolved concurrently with conventional N1 and N3 modifications with only minor modifications to established protocols, providing a multi-dimensional, single-molecule readout of RNA structure. We show that N7-G reactivity specifically reports on RNA tertiary and quaternary structure, enabling identification of diverse, functionally significant motifs such as cooperatively folding tertiary domains and protein binding sites in living cells. We apply msDMS-MaP to obtain a map of the quaternary structural ensemble of the 7SK non-coding snRNP, revealing unique protein binding sites across three 7SK structural isoforms. msDMS-MaP represents a broadly applicable strategy for enhanced RNA functional motif discovery and characterization.

biochemistry↗

bioRxiv2024

Systematic Targeting of Protein Complexes with Molecular COUPLrs

Molecular glues that engage protein complexes have transformed the study of cell biology and have had a direct impact on clinical oncology. However, the identification of new glue classes and their corresponding protein complexes has remained largely serendipitous. To overcome this challenge, we report the development of molecular COUPLrs, elaborated small molecules flanked by two cysteine-reactive warheads, as well as CONNECT, an integrated chemical proteomic platform for target deconvolution. By profiling a library of molecular COUPLrs across 13 cancer cell lines, we uncovered hundreds of proteins that can be coupled together, including in some cases in mutant selective fashions. We develop an advanced COUPLr for the oncogene EML4-ALK, which engages the fusion outside of its kinase domain, restricts protein dynamics, and disrupts EML4-ALK signaling. Collectively, molecular COUPLrs substantially expand the scope of proteins that can be chemically connected, providing an unbiased approach to identify small molecules that target protein complexes.

systems biology↗

bioRxiv2024

Meningeal-derived retinoic acid regulates neurogenesis via suppression of Notch and Sox2

The meninges act as a regulator of brain development by secreting ligands that act on neural cells to regulate neurogenesis and neuronal migration. Meningeal-derived retinoic acid (RA) promotes neocortical neural progenitor cell cycle exit; however, the underlying molecular mechanism is unknown. Here, we used spatial transcriptomics and profiling of retinoic-acid receptor- (RAR) DNA binding in Foxc1-mutant embryos that lack meninges-derived ligands to identify the neurogenic transcriptional mechanisms of RA signaling in telencephalic neural progenitors. We determined that meningeal-derived RA controls neurogenesis by suppressing progenitor self-renewal pathways Notch signaling and the transcription factor Sox2. We show that RAR binds in the Sox2ot promoter, a long non-coding RNA that regulates Sox2 expression, and RA promotes Sox2ot expression in neocortical progenitors. Our findings elucidate a previously unknown mechanism of how meningeal RA coordinates neocortical development and insight into how defects in meningeal development may cause neurodevelopmental disorders.

neuroscience↗

bioRxiv2023

Securin Regulates the Spatiotemporal Dynamics of Separase

Separase is a key regulator of the metaphase to anaphase transition with multiple functions. Separase cleaves cohesin to allow chromosome segregation and localizes to vesicles to promote exocytosis in mid-anaphase. The anaphase promoting complex/cyclosome (APC/C) activates separase by ubiquitinating its inhibitory chaperone, securin, triggering its degradation. How this pathway controls the exocytic function of separase has not been investigated. During meiosis I, securin is degraded over several minutes, while separase rapidly relocalizes from kinetochore structures at the spindle and cortex to sites of action on chromosomes and vesicles at anaphase onset. The loss of cohesin coincides with the relocalization of separase to the chromosome midbivalent at anaphase onset. APC/C depletion prevents separase relocalization, while securin depletion causes precocious separase relocalization. Expression of non-degradable securin inhibits chromosome segregation, exocytosis, and separase localization to vesicles but not to the anaphase spindle. We conclude that APC/C mediated securin degradation controls separase localization. This spatiotemporal regulation will impact the effective local concentration of separase for more precise targeting of substrates in anaphase.

cell biology↗

bioRxiv2023

Generation of human induced pluripotent stem cell (hiPSC) lines from patients with extreme high and low polygenic scores for QT interval

Long QT syndrome (LQTS) is an inherited cardiac arrhythmia syndrome with congenital and drug-induced presentations and known monogenic and polygenic contributions. LQTS represents a significant clinical challenge due to its complex genetic underpinning and propensity for fatal arrhythmias. In this study, we generated induced pluripotent stem cells (iPSCs) reprogrammed from peripheral blood mononuclear cells (PBMCs) of six patients with extreme polygenic scores for short and long corrected QT intervals. iPSC lines were rigorously validated for genomic integrity through karyotyping and targeted mutation analysis specific to a lengthened or shortened QT interval. Pluripotency was confirmed by expression of key markers TRA 1-60, TRA 1-81, SOX2, OCT4, NANOG, and REX1 via quantitative PCR and immunofluorescence. Subsequent cardiac induction successfully generated cardiomyocytes that were further characterized. This patient-specific approach will enable us to better understand variable expressivity and penetrance of LQTS. Rigorously validated iPSC lines serve as a vital resource for elucidating the molecular mechanisms underlying LQTS. Our study provides a robust and clinically relevant resource to facilitate our understanding the genetic and cellular complexity of LQTS.

genetics↗

bioRxiv2023

Mutation signature filtering enables high-fidelity RNA structure probing at all four nucleobases with DMS

Chemical probing experiments have transformed RNA structure analysis, enabling high-throughput measurement of base-pairing in living cells. Dimethyl sulfate (DMS) is one of the most widely used structure probing reagents and has played a prominent role in enabling next-generation single-molecule probing analyses. However, DMS has traditionally only been able to probe adenine and cytosine nucleobases. We previously showed that, using appropriate conditions, DMS can also be used to interrogate base-pairing of uracil and guanines in vitro at reduced accuracy. However, DMS remained unable to informatively probe guanines in cells. Here, we develop an improved DMS mutational profiling (MaP) strategy that leverages the unique mutational signature of N1-methylguanine DMS modifications to enable robust, high-fidelity structure probing at all four nucleotides, including in cells. Using information theory, we show that four-base DMS reactivities convey greater structural information than comparable two-base DMS and SHAPE probing strategies. Four-base DMS experiments further enable improved direct base-pair detection by single-molecule PAIR analysis, and ultimately support RNA structure modeling at superior accuracy. Four-base DMS probing experiments are easily performed and will broadly facilitate improved RNA structural analysis in living cells.

biochemistry↗

bioRxiv2023

Genetic modifiers of APOBEC-induced mutagenesis

The cytidine deaminases APOBEC3A and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced mutagenesis are poorly defined. Here, we utilized a screen to identify 61 gene deletions that increase A3B-induced mutations in yeast. Also, we determined whether each deletion was epistatic with UNG1 loss, which indicated whether the encoded factors participate in the error-free bypass of A3B/Ung1-dependent abasic sites or suppress A3B-catalyzed deamination by protecting against aberrant formation of single stranded DNA (ssDNA). Additionally, we determined that the mutation spectra of A3B-induced mutations revealed genotype-specific patterns of strand-specific ssDNA formation and nucleotide incorporation across APOBEC-induced lesions. Combining these three metrics we were able to establish a multifactorial signature of APOBEC-induced mutations specific to (1) failure to remove H3K56 acetylation, which results in extremely high A3B-induced mutagenesis, (2) defective CTF18-RFC complex function, which results in high levels of A3B induced mutations specifically on the leading strand template that synergistically increase with loss of UNG1, and (3) defective HR-mediated bypass of APOBEC-induced lesions, which were epistatic with Ung1 loss and result from increased Rev1-mediated C-to-G substitutions. We extended these results by analyzing mutation data for human tumors and found BRCA1/2-deficient breast cancer tumors display 3- to 4-fold more APOBEC-induced mutations. Mirroring our results in yeast, for BRCA1/2 deficient tumors Rev1-mediated C-to-G substitutions are solely responsible for increased APOBEC-signature mutations and these mutations occur on the lagging strand during DNA replication. Together these results identify important factors that influence the dynamics of DNA replication and likely the abundance of APOBEC-induced mutation during tumor progression as well as a novel mechanistic role for BRCA1/2 during HR-dependent lesion bypass of APOBEC-induced lesions during cancer cell replication.

genomics↗

bioRxiv2023

Three-Dimensional Bioconjugated Liquid-Like Solid (LLS) Enhance Characterization of Solid Tumor - Chimeric Antigen Receptor T cell Interactions

Cancer immunotherapy offers lifesaving treatments for cancers, but the lack of reliable preclinical models that could enable the mechanistic studies of tumor-immune interactions hampers the identification of new therapeutic strategies. We hypothesized 3D confined microchannels, formed by interstitial space between bio-conjugated liquid-like solids (LLS), enable CAR T dynamic locomotion within an immunosuppressive TME to carry out anti-tumor function. Murine CD70-specific CAR T cells cocultured with the CD70-expressing glioblastoma and osteosarcoma demonstrated efficient trafficking, infiltration, and killing of cancer cells. The anti-tumor activity was clearly captured via longterm in situ imaging and supported by upregulation of cytokines and chemokines including IFNg, CXCL9, CXCL10, CCL2, CCL3, and CCL4. Interestingly, target cancer cells, upon an immune attack, initiated an "immune escape" response by frantically invading the surrounding microenvironment. This phenomenon however was not observed for the wild-type tumor samples which remained intact and produced no relevant cytokine response. Single cells collection and transcriptomic profiling of CAR T cells at regions of interest revealed feasibility of identifying differential gene expression amongst the immune subpopulations. Complimentary 3D in vitro platforms are necessary to uncover cancer immune biology mechanisms, as emphasized by the significant roles of the TME and its heterogeneity.

bioengineering↗

bioRxiv2022

Human Surfactant Protein D Facilitates SARS-CoV-2 Pseudotype Binding and Entry in DC-SIGN Expressing Cells, and Downregulates Spike protein Induced Inflammation

Pattern recognition receptors are crucial for innate anti-viral immunity, including C-type lectin receptors. Two such examples are Lung surfactant protein D (SP-D) and Dendritic cell-specific intercellular adhesion molecules-3 grabbing non-integrin (DC-SIGN) which are soluble and membrane-bound C-type lectin receptors, respectively. SP-D has a crucial immune function in detecting and clearing pulmonary pathogens; DC-SIGN is involved in facilitating dendritic cell interaction as an antigen-presenting cell with naive T cells to mount an anti-viral immune response. Both SP-D and DC-SIGN have been shown to interact with various viruses, including HIV-1, Influenza A virus and SARS-CoV-2. SARS-CoV-2 is an enveloped RNA virus that causes COVID-19. A recombinant fragment of human SP-D (rfhSP-D) comprising of -helical neck region, carbohydrate recognition domain, and eight N-terminal Gly-X-Y repeats has been shown to bind SARS-CoV-2 Spike protein and inhibit SARS-CoV-2 replication by preventing viral entry in Vero cells and HEK293T cells expressing ACE2. DC-SIGN has also been shown to act as a cell surface receptor for SARS-CoV-2 independent of ACE2. Since rfhSP-D is known to interact with SARS-CoV-2 Spike protein and DC-SIGN, this study was aimed at investigating the potential of rfhSP-D in modulating SARS-CoV-2 infection. Coincubation of rfhSP-D with Spike protein improved the Spike Protein: DC-SIGN interaction. Molecular dynamic studies revealed that rfhSP-D stabilised the interaction between DC-SIGN and Spike protein. Cell binding analysis with DC-SIGN expressing HEK 293T and THP-1 cells and rfhSP-D treated SARS-CoV-2 Spike pseudotypes confirmed the increased binding. Furthermore, infection assays using the pseudotypes revealed their increased uptake by DC-SIGN expressing cells. The immunomodulatory effect of rfhSP-D on the DC-SIGN: Spike protein interaction on DC-SIGN expressing epithelial and macrophage-like cell lines was also assessed by measuring the mRNA expression of cytokines and chemokines. The RT-qPCR analysis showed that rfhSP-D treatment downregulated the mRNA expression levels of pro-inflammatory cytokines and chemokines such as TNF-, IFN-, IL-1{beta}, IL-6, IL-8, and RANTES (as well as NF-{kappa}B) in DC-SIGN expressing cells challenged by Spike protein. Furthermore, rfhSP-D treatment was found to downregulate the mRNA levels of MHC class II in DC expressing THP-1 when compared to the untreated controls. We conclude that rfhSP-D helps stabilise the interaction of SARS-CoV-2 Spike protein and DC-SIGN and increases viral uptake by macrophages via DC-SIGN, suggesting an additional role for rfhSP-D in SARS-CoV-2 infection.

immunology↗

bioRxiv2022

Jumping out of trouble: Evidence for a cognitive map in guppies (Poecilia reticulata)

Spatial cognitive abilities allow individuals to remember the location of food patches, predator hide-outs, or shelters. Animals typically incorporate learnt spatial information or use external environmental cues to navigate their surroundings. A spectacular example of how some fishes move is through aerial jumping. For instance, fish that are trapped within isolated pools, cut off from the main body of water during dry periods, may jump over obstacles and direct their jumps to return to safe locations. However, what information such re-orientation behaviour during jumping is based on remains enigmatic. Here we combine a lab and field experiment to test if guppies (Poecilia reticulata) incorporate learnt spatial information and external environmental cues (visual and auditory) to determine where to jump. In a spatial memory assay we found that guppies were more likely to jump towards deeper areas, hence incorporating past spatial information to jump to safety. In a matched vs. mismatched spatial cue experiment in the field, we found that animals only showed directed jumping when visual and auditory cues matched. We show that in unfamiliar entrapments guppies direct their jumps by combining visual and auditory cues, while in familiar entrapments they use a cognitive map. We hence conclude that jumping behaviour is a goal-directed behaviour, guided by different sources of information and involving important spatial cognitive skills.

animal behavior and cognition↗