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Saunders, A.

Publications and source records attributed to Saunders, A..

At least 19 recordsLinked to original sources

Design and Assembly of Combinatorial DNA Barcodes for Probe-based Genomics Applications

Probe-based genomics technologies are extending molecular analysis into intact tissues and fixed cells, yet strategies to decode complex experimental conditions encoded in cellular RNA remain limited. Here we present a modular framework that integrates custom software tools with purpose-built cloning reagents to design, assemble, validate, and deploy combinatorial DNA barcodes. Combinatorial barcodes comprise spatially adjacent collections of known sequences, enabling millions of unique molecules to be efficiently distinguished using a limited set of probes. Our software tools integrate with optimized assembly plasmids and whole plasmid long-read sequencing for high-fidelity construction and structural validation of diverse combinatorial barcode architectures. Assembled barcode libraries are flexibly transferred into user-modified expression vectors to support diverse downstream experimental applications. We showcase the versatility of this framework by assembling two structurally distinct combinatorial barcode libraries, each containing millions of unique sequences. Following rabies virus-based delivery to the mouse brain, we validate in vivo decoding of a combinatorial barcode architecture capable of distinguishing ~16.3 million expressed RNAs through probe-based in situ sequencing. Our framework for flexible and accurate combinatorial barcode construction fills a technically demanding niche delivering cost-effective molecular reagents for multiplexed experimentation on current and evolving probe-based genomics platforms.

genomics↗

Early postnatal DNA methylation dynamics define neuronal subtypes and are disrupted by MECP2 loss

DNA methylation is the foundational layer of epigenetic regulation underlying mammalian tissue specification. During synaptogenesis, neurons accumulate high levels of a unique type of methylation in which a cytosine precedes a C, A, or T (mCH) instead of the canonical guanine (mCG). Disruption of the mCH reader methyl-CpG binding protein 2 (MECP2) causes the devastating neurodevelopmental disorder Rett syndrome, yet the role of mCH in neurodevelopment remains unclear. In this study, we generated the first large-scale single-cell methylation atlas of the early postnatal mouse brain and resolved subtype-specific trajectories of canonical and noncanonical methylation changes underlying neuronal subtype specification. Each identified population undergoes a rapid maturation event of the noncanonical methylome occurring between the first and second postnatal weeks, with highly subtype-specific changes concentrated at genes involved in synaptic partner establishment. Trajectory analysis resolved that subtype diversification, such as the separation of parvalbumin and somatostatin-positive GABAergic interneurons, is further facilitated by a hierarchical sequence of methylation changes in genes involved in regulation of membrane potential. In parallel, we applied single-cell methylation analysis to a mouse model of Rett syndrome that recapitulates severe human symptoms. We show that while noncanonical methylation accumulation proceeds in each subtype in the absence of MECP2, populations that have been consistently implicated in Rett pathology such as GABAergic interneurons have an increasing number of differentially methylated regions with age and fail to accumulate typical global levels of mCH. Together, our study defines the methylation dynamics that facilitate neuronal subtype specification and resolves subtype-specific, global disruptions of noncanonical methylation in Rett syndrome.

neuroscience↗

Analytic Bounds on GAMLSS Model Variability of Normative White Matter Brain Charts

Brain charts, or normative models of quantitative neuroimaging measures, can identify trajectories of brain development and abnormalities in groups and individuals by leveraging large populations. Recent work has extended these brain charts to model microstructural and macrostructural features of white matter. Assessments of variance for these brain charts are necessary to determine whether the models being used for these data are stable. We implement an analytic approach to characterize variability of the parameters in previously released brain charts created using the generalized additive models for location, scale, and shape (GAMLSS) framework. Additionally, we empirically validate the accuracy of each analytic model through a comparison to a bootstrapping approach from 0.2 to 90 years of age. We find that across all models, the analytic coefficient of variation (COV) remains below 5% for ages greater than 0.25 years, with the maximum empirical observed COV reaching 7% at 0.2 years of age. Further, the empirical assessment shows high agreement with the analytic assessment, with COV estimates averaged across the lifespan for all models having a Pearson correlation coefficient of 0.776 and a mean difference of 4 x 10-4. Both methods exhibit volume and surface area as the features with the largest average COV for the majority of tracts. However, the analytic assessment yields axial diffusivity as the feature most frequently having the smallest COV, whereas the corresponding feature for the empirical assessment is average length. These results suggest that the analytic approach overestimates model stability for WM brain charts when the COV is low and that the validation method is suitable for assessing whether GAMLSS models are unstable.

bioengineering↗

CD200R1 is required for the development of γδ 17 T cells

{gamma}{delta} T cells are enriched at barrier sites such as skin, gut and lung, where they protect against cancer and infections and promote healing. They detect diverse ligands in T cell receptor-dependent or independent manners, producing large quantities of pro-inflammatory cytokines. {gamma}{delta} T cells develop in foetal thymi in temporally controlled waves where, unlike {beta} T cells, many {gamma}{delta} T cells adopt their effector fate, becoming either IFN{gamma} or IL-17A-producers ({gamma}{delta}17 T cells). CD200R1 suppresses myeloid cell activity but has also been shown to promote innate lymphoid cell IL-17A production, enhancing psoriasis-like skin inflammation. {gamma}{delta}17 T cells are potent IL-17A producers in skin therefore, the effect of CD200R1 on IL-17A production by {gamma}{delta}17 T cells was investigated. CD200R1 was found to promote IL-17A production by {gamma}{delta} T cells by supporting the development of {gamma}{delta}17 T cells, enhancing IL-17-producing and ROR{gamma}t+ {gamma}{delta} T cell numbers in foetal thymic organ cultures. To fulfil this role, CD200R1 acts either directly on developing {gamma}{delta} T cells, or indirectly on thymic stromal cells. This identifies CD200R1 as a critical novel regulator of {gamma}{delta}17 T cell development in early life, a key process for ensuring immunity, particularly at barrier sites.

immunology↗

Host and rabies virus gene expression is shaped by human brain cell type and reveals a preexisting pro-viral transcriptional state in astrocytes

How virus-host cell interactions and innate immune antagonism shape neurotropic infection dynamics across diverse brain cell types are largely unknown. To "unmask" and study how innate immune inhibition affects cell type-specific transcriptional regulation of the human and viral genome, we performed single-cell RNA sequencing of human brain cell co-cultures, comparing an isolate of rabies virus (RABV) to its mutant incapable of antagonizing interferon- and NF-{kappa}B-dependent responses. RABV gene expression was shaped by host cell type. RABV induced small-scale, cell-type conserved transcriptional programs that likely support infection by 1) hijacking negative transcriptional feedback of pro-viral factors while 2) reducing anti-viral RNAs. Unexpectedly, disinhibited innate immune signaling increased RABV transcription, most strikingly in an infection-independent "pro-viral" astrocyte subpopulation. Further analysis suggested that "pro-viral"-like astrocytes are a rare subtype in the human brain and are primed to protect the brain during viral infection in concert with interferon-sensitive microglia recalcitrant to infection.

neuroscience↗

Mapping cell-cell fusion at single-cell resolution

Cell-cell fusion is a tightly controlled process in the human body known to be involved in fertilization, placental development, muscle growth, bone remodeling, and viral response. Fusion between cancer cells results first in a whole-genome doubled state, which may be followed by the generation of aneuploidies; these genomic alterations are known drivers of tumor evolution. The role of cell-cell fusion in cancer progression and treatment response has been understudied due to limited experimental systems for tracking and analyzing individual fusion events. To meet this need, we developed a molecular toolkit to map the origins and outcomes of individual cell fusion events within a tumor cell population. This platform, ClonMapper Duo ( CMDuo), identifies cells that have undergone cell-cell fusion through a combination of reporter expression and engineered fluorescence-associated index sequences paired to randomly generated nucleotide barcodes. scRNA-seq of the indexed barcodes enables the mapping of each set of parental cells and fusion progeny throughout the cell population. In triple-negative breast cancer cells CMDuo uncovered subclonal transcriptomic hybridization and unveiled distinct cell-states which arise in direct consequence of homotypic cell-cell fusion. CMDuo is a platform that enables mapping of cell-cell fusion events in high-throughput single cell data and enables the study of cell fusion in disease progression and therapeutic response.

systems biology↗

AnnSQL: A Python SQL-based package for large-scale single-cell genomics analysis on a laptop

As single-cell genomics technologies continue to accelerate biological discovery, software tools that use elegant syntax and minimal computational resources to analyze atlas-scale datasets are increasingly needed. Here we introduce AnnSQL, a Python package that constructs an AnnData-inspired database using the in-process DuckDb engine, enabling orders-of-magnitude performance enhancements for parsing single-cell genomics datasets with the ease of SQL. We highlight AnnSQL functionality and demonstrate transformative runtime improvements by comparing AnnData or AnnSQL operations on a 4.4 million cell single-nucleus RNA-seq dataset: AnnSQL-based operations were executed in minutes on a laptop for which equivalent AnnData operations largely failed (or were [~]700x slower) on a high-performance computing cluster. AnnSQL lowers computational barriers for large-scale single-cell/nucleus RNA-seq analysis on a personal computer, while demonstrating a promising computational infrastructure extendable for complete single-cell workflows across various genome-wide measurements. Availability and ImplementationAnnSQL is a pip installable package that can be found at https://github.com/ArpiarSaundersLab/annsql along with documentation at https://docs.annsql.com.

bioinformatics↗

Transcriptomic diversity of amygdalar subdivisions across humans and nonhuman primates

The amygdaloid complex mediates learning, memory, and emotions. Understanding the cellular and anatomical features that are specialized in the amygdala of primates versus other vertebrates requires a systematic, anatomically-resolved molecular analysis of constituent cell populations. We analyzed five nuclear subdivisions of the primate amygdala with single-nucleus RNA sequencing in macaques, baboons, and humans to examine gene expression profiles for excitatory and inhibitory neurons and confirmed our results with single-molecule FISH analysis. We identified distinct subtypes of FOXP2+ interneurons in the intercalated cell masses and protein-kinase C-{delta} interneurons in the central nucleus. We also establish that glutamatergic, pyramidal-like neurons are transcriptionally specialized within the basal, lateral, or accessory basal nuclei. Understanding the molecular heterogeneity of anatomically-resolved amygdalar neuron types provides a cellular framework for improving existing models of how amygdalar neural circuits contribute to cognition and mental health in humans by using nonhuman primates as a translational bridge.

neuroscience↗

Dietary Bioactive Compounds Trigger Distinct Epigenetic and Metabolic Reprogramming in Lactobacillus acidophilus

Lactobacillus acidophilus ATCC 4356 (LA), a key probiotic in the human gut microbiota, offers several health benefits. While dietary bioactive compounds are known to influence gut microbiota, their specific mechanisms remain unclear. This study investigated how certain dietary bioactive compounds impact LA gene expression and metabolism. Results showed each compound produces unique transcriptional, metabolic, proteomic, and epigenetic profiles in LA. Notably, dietary compounds altered the epigenetic landscape through N4-methylcytosine (4mC) modification, a relatively underexplored form of methyl modification that may play a role in regulating gene transcription. For instance, genistein treatment up-regulated 76 genes and the down-regulated 130 genes in LA. A gene involved in mucus-binding proteins, crucial for bacterial adhesion, was up-regulated 38-fold, likely due to 4mC modifications. Additionally, the gene coding for the melibiose operon regulatory protein increased 78-fold, enhancing melibiose (a prebiotic) production with genistein, but only 1.1-fold with sodium butyrate. This study highlights the potential of dietary compounds for microbial metabolic engineering, offering a non-GMO method for modulating bacterial performance and other biotechnology applications. Graphical AbstractDietary compounds are able to modify the epigenetic landscape of Lactobacillus acidophilus ATCC 4356, resulting in significant transcriptomic, metabolic, and physiological changes. This approach differs from conventional genetic modification techniques for manipulating microbial strains. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/608491v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1d1f547org.highwire.dtl.DTLVardef@13e18a3org.highwire.dtl.DTLVardef@1c37e2dorg.highwire.dtl.DTLVardef@190be49_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

Serial 'deep-sampling PCR of fragmented DNA reveals the wide range of Trypanosoma cruzi burden among chronically infected hosts and allows accurate monitoring of parasite load following treatment

Infection with the protozoan parasite Trypanosoma cruzi is generally well-controlled by host immune responses, but appears to be rarely eliminated. The resulting persistent, low-level infection results in cumulative tissue damage with the greatest impact generally in the heart in the form of chagasic cardiomyopathy. The relative success in immune control of T. cruzi infection usually averts acute phase death but has the negative consequence that the low-level presence of T. cruzi in hosts is challenging to detect unequivocally. Thus, it is difficult to identify those who are actively infected and, as well, problematic to gauge the impact of treatment, particularly in the evaluation of the relative efficacy of new drugs. In this study we employ DNA fragmentation and high numbers of replicate PCR reaction ( deep-sampling) to extend the quantitative range of detecting T. cruzi in blood by at least 3 orders of magnitude relative to current protocols. When combined with sampling blood at multiple time points, deep sampling of fragmented DNA allowed for detection of T. cruzi in all infected hosts in multiple host species. In addition, we provide evidence for a number of characteristics not previously rigorously quantified in the population of hosts with naturally acquired T. cruzi infection, including, a > 6-log variation between chronically infected individuals in the stable parasite levels, a continuing decline in parasite load during the second and third years of infection in some hosts, and the potential for parasite load to change dramatically when health conditions change. Although requiring strict adherence to contamination-prevention protocols and significant resources, deep-sampling PCR provides an important new tool for assessing new therapies and for addressing long-standing questions in T. cruzi infection and Chagas disease. Author SummaryInfection by the protozoan Trypanosoma cruzi normally results in a life-long, but low-level parasitization of muscle tissues, often leading to chagasic heart disease. A major challenge in the Chagas disease field has been the difficulty in detecting and quantifying parasite load in infected hosts. In this study we show that collection of serial blood samples and performance of sometimes high numbers of replicate PCR reactions on fragmented blood DNA, allows detection and quantification of relative parasite load in non-human primates, dogs, and humans with naturally acquired T. cruzi infection. This deep-sampling approach reveals a mostly stable, 100,000-fold or greater difference in parasite load among chronically infected hosts and can detect alterations in parasite levels due to changes in health status or following therapeutic treatment in individual hosts, thus providing a powerful tool for assessing treatment outcomes in T. cruzi infection, including for evaluation of new therapeutics. Additionally, the ability to accurately and sensitively monitor parasite load in hosts provides the means to address highly contentious issues in the Chagas field, including the relative role of parasites and hosts in establishing the persistent parasite burden and the relationship between parasite burden and the presence and severity of clinical disease.

microbiology↗

Dissection of a non-coding risk locus at 1p36.23 identifies ERRFI1 as a novel gene in the pathogenesis of psoriasis and psoriatic arthritis

BackgroundPsoriasis and its associated inflammatory arthritis Psoriatic Arthritis (PsA) are potentially life-ruining conditions associated with numerous comorbidities. A previously-identified genetic risk association for psoriasis and PsA lies in a non-coding region at chromosome 1p36.23, and as such functional validation is required to determine the genetic mechanism contributing to psoriatic disease risk. Resultsrs11121131 - a variant in tight linkage with rs11121129, the lead GWAS variant for the 1p36.23 association - lies in a putative enhancer active in keratinocytes but not in immune cells. Promoter-capture Hi-C and H3K27Ac HiChIP showed keratinocyte-specific interactions between 1p36.23 and the TNFRSF9/PARK7/ERRFI1 gene locus [~]200Kb upstream of the risk locus. Deletion of the enhancer in HaCat keratinocytes led to a reduction in transcript levels of the gene ERRFI1, a negative regulator of Epidermal Growth Factor Receptor (EGFR) signalling. CRISPR activation of the enhancer also affected ERRFI1 levels, but paradoxically showed that steady-state activation led to repression of ERRFI1, accompanied by significant deposition of H3K27Me3 histone marks at both the enhancer and the ERRFI1 gene locus. ERRFI1 levels were shown to be increased in inflamed skin from a mouse model of psoriasis, further suggesting its involvement in disease. ConclusionsThese data indicate rs11121131 lies in an enhancer which modulates ERRFI1 expression in keratinocytes, providing a likely risk mechanism for the 1p36.23 risk association. ERRFI1 represents a novel gene in the pathogenesis of psoriasis and PsA - improving our understanding of these diseases - and the ERRFI1/EGFR signalling axis may therefore be a target for new treatment modalities for psoriatic disease.

genomics↗

Reevaluating the Concept of Aging: Long-Term Stress Adaptation as a Key Factor in Yeast Aging

It has been demonstrated that short-term stress can enhance cellular responses and promote longevity, whereas long-term stress shortens lifespan. Understanding the relationship between short-term and long-term stress could offer new insights into comprehending and modulating age-related diseases. In this study, we investigate this relationship using transcriptomic and metabolomic analyses in the yeast model system (Saccharomyces cerevisiae). We employed three metabolic treatments: firstly, treating yeast cells with threshold levels of benzoic acid for 24 hours (Short-term [ST] Stressed Cells); secondly, treating yeast cells with threshold levels of benzoic acid for 500 hours, with sub-culturing every 24 hours (Long-term [LT] Stressed Cells); and thirdly, allowing the long-term stressed cells to grow for 16 hours without any benzoic acid (Recovered Cells). Here, we propose that aging is an evolutionarily conserved cellular adaptation mechanism in response to long-term stress exposure. Under short-term stressed conditions, prominent lifespan-extending metabolites such as trehalose and metabolites linked to tumor suppression in humans, such as 5-methylthioadenosine, were overexpressed. In contrast, LT Stressed Cells activated genes such as those responsible for epigenetic regulatory enzymes that govern the aging process, and secondary stress response genes, such as heat shock proteins (HSPs) which are associated with adaptation to cell damage but also often associated with aged cells. Chronological lifespan experiments showed that LT stressed cells lived a shorter lifespan compared to ST Stressed Cells. This suggests that the markers of aging (eg. HSPs, certain epigenetic regulators) are expressed in response to long-term stress to enable cell survival but have the long-term effect of reducing lifespan. In support of this hypothesis, we also show that genes exclusively activated in ST Stressed Cells are conserved solely in eukaryotes, while those significantly expressed in LT Stressed Cells (aging related) exhibit high conservation across all domains of life, with a majority having originated from bacteria hinting at the potential evolutionary benefit of aging.

cell biology↗

Tipifarnib potentiates the antitumor effects of PI3Kα inhibition in PIK3CA- and HRAS-dysregulated HNSCC via convergent inhibition of mTOR activity

Outcomes for patients with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) are poor, with median overall survival ranging from 6 to 18 months. For those who progress on standard of care (chemo)immunotherapy, treatment options are limited, necessitating the development of rational therapeutic strategies. Toward this end, we targeted the key HNSCC drivers PI3K-mTOR and HRAS via the combination of tipifarnib, a farnesyltransferase inhibitor, and alpelisib, a PI3K inhibitor, in multiple molecularly defined subsets of HNSCC. We find that tipifarnib synergizes with alpelisib at the level of mTOR in PI3K-or HRAS-dependent HNSCCs, leading to marked cytotoxicity in vitro and tumor regression in vivo. Based on these findings, we have launched the KURRENT-HN trial to evaluate the effectiveness of this combination in PIK3CA-mutant/amplified and/or HRAS-overexpressing R/M HNSCC. Preliminary evidence supports the clinical activity of this molecular biomarker-driven combination therapy. SignificanceBacked by strong mechanistic rationale, the combination of alpelisib and tipifarnib has the potential to benefit >45% of R/M HNSCC patients. By blocking feedback reactivation of mTORC1, tipifarnib may prevent adaptive resistance to additional targeted therapies, thereby enhancing their clinical utility.

cancer biology↗

Collection of triatomines from sylvatic habitats by a Chagas-positive, trained scent-detection canine in Texas, USA

BackgroundTriatomine insects, vectors of the etiologic agent of Chagas disease (Trypanosoma cruzi), are challenging to locate in sylvatic habitats. Collection techniques used in the United States often rely on methods to intercept seasonally dispersing adults or on community scientists encounters. Neither method is suited for detecting nidicolous habitats, which is important for vector control. Furthermore, manual inspection of suspected harborages is unlikely to reveal novel locations and host associations. Similar to a team that used a trained dog to detect sylvatic triatomines in Paraguay, we worked with a trained scent detection dog to detect sylvatic locations of triatomines across Texas. Principle methodology/FindingsZiza, a 3-year-old German Shorthaired Pointer previously naturally infected with T. cruzi, was trained to detect triatomines. Over the course of 6 weeks in the fall of 2017, the dog and her handler visited 18 sites across Texas. The dog detected 60 triatomines at 7 locations; an additional 50 triatomines were contemporaneously collected at these sites without the assistance of the dog. Approximately 0.98 triatomines per hour were found when only humans were conducting searches; when working with the dog, approximately 1.71 triatomines per hour were found. In total, 3 adults and 107 nymphs of four species (Triatoma gerstaeckeri, Triatoma protracta, Triatoma sanguisuga, and Triatoma indictiva) were collected. PCR testing of a subset revealed T. cruzi infection, including DTUs TcI and TcIV, in 28% of nymphs (n=100) and 66% of adults (n=3). Blood meal analysis of a subset of triatomines (n=5) revealed feeding on Virginia opossum (Didelphis virginiana), Southern plains woodrat (Neotoma micropus) and eastern cottontail (Sylvilagus floridanus). Conclusion/SignificanceA trained scent detection dog enhanced triatomine detections in sylvatic habitats. This approach is especially effective at detecting triatomine nymphs and nidicolous locations. With new knowledge of specific sylvatic habitats and key hosts fed upon by triatomines, there are opportunities for continued exploration of novel vector control methods to block the transmission of T. cruzi to humans and domestic animals. Author summaryTriatomine insects, also known as kissing bugs, are vectors of Trypanosoma cruzi, the parasite that causes Chagas disease in humans, dogs, and other mammals. Triatomines are found throughout the Americas, but little is known about where these blood-sucking insects spend their early life stages. Knowing more is important to vector control initiatives aimed at interrupting Chagas disease transmission. Scent detection dogs have been trained to detect many pests, including one study of a triatomine detection dog in South America. In this study, we used a dog to detect triatomines in their natural environments throughout Texas. Over 6 weeks, the dog identified 60 triatomines at 7 different locations; an additional 50 triatomines were collected without the dogs assistance. Nearly twice as many triatomines were collected per hour when the dog was searching as compared to only humans searching. Of all the triatomine nymphs collected, 28% were positive for T. cruzi, and the blood meal analysis revealed kissing bugs had fed on Southern plains woodrat, opossum, and eastern cottontail. This study outlines a strategy that can be replicated in the United States to enhance the detection and control of habitats where triatomines spend their early life stages.

ecology↗

Prophylactic low-dose, bi-weekly benznidazole treatment fails to prevent Trypanosoma cruzi infection in dogs under intense transmission pressure

Trypanosoma cruzi naturally infects a wide variety of wild and domesticated mammals, in addition to humans. Depending on the infection dose and other factors, the acute infection can be life-threatening, and in all cases, the risk of chagasic heart disease is high in persistently infected hosts. Domestic, working, and semi-feral dogs in the Americas are at significant risk of T. cruzi and in certain settings in the southern United States, the risk of new infections can exceed 30% per year, even with the use of vector control protocols. In this study, we explored whether intermittent low-dose treatment with the trypanocidal compound benznidazole (BNZ) during the transmission season, could alter the number of new infections in dogs in an area of known, intense transmission pressure. Preliminary studies in mice suggested that twice-weekly administration of BNZ could prevent or truncate infections when parasites were delivered at the mid-point between BNZ doses. Pre-transmission season screening of 126 dogs identified 53 dogs (42.1%) as T. cruzi infection positive, based upon blood PCR and Luminex-based serology. Serial monitoring of the 67 uninfected dogs during the high transmission season (May to October) revealed 15 (22.4%) new infections, 6 in the untreated control group and 9 in the group receiving BNZ prophylaxis, indicating no impact of this prophylaxis regimen on the incidence of new infections. Although these studies suggest that rigorously timed and more potent dosing regimen may be needed to achieve an immediate benefit of prophylaxis, additional studies would be needed to determine if drug prophylaxis reduced disease severity despite this failure to prevent new infections. Author SummaryTrypanosoma cruzi, the parasite that causes Chagas disease, circulates extensively in the southern U.S. and working dog populations in south-central Texas are at very high risk of infection, and morbidity and early mortality due to this infection. In this study, we used low level administration of an FDA-approved drug during the transmission season to attempt to prevent new infections in these dogs. Although that effort failed, the study revealed new information about the transmission dynamics and generation of antibody responses and immune control of infection in this high-transmission setting.

microbiology↗

CD200R1 promotes IL-17 production by ILC3s, by enhancing STAT3 activation

Psoriasis is a common chronic inflammatory skin disease with no cure. It is driven by the IL-23/IL-17A axis and TH17 cells but, recently group 3 innate lymphoid cells (ILC3s) have also been implicated. However, the development, and factors regulating the activity of ILC3s remain incompletely understood. Immune regulatory pathways are particularly important at barrier sites such as the skin, gut and lung, which are exposed to environmental substances and microbes. CD200R1 is an immune regulatory cell surface receptor which inhibits proinflammatory cytokine production in myeloid cells. CD200R1 is also highly expressed on ILCs, where its function remains largely unexplored. We previously observed reduced CD200R1 signalling in psoriasis skin, suggesting that dysregulation may promote disease. Here we show that contrary to this, psoriasis models are less severe in CD200R1-deficient mice due to reduced IL-17 production. Here we uncover a key cell-intrinsic role for CD200R1 in promoting IL-23-driven IL-17A production by ILC3s, by promoting STAT3 activation. CD200R1 is expressed on ILC precursors and is particularly high on neonatal ILC3s, suggesting CD200R1 may function during ILC development. Therefore, CD200R1 is required on ILC3s, potentially during their development, to promote IL-23-stimulated STAT3 activation triggering optimal IL-17 production.

immunology↗

Reduced cutaneous CD200:CD200R1 signalling in psoriasis enhances neutrophil recruitment to skin

The skin immune system is tightly regulated to prevent inappropriate inflammation in response to harmless environmental substances. This regulation is actively maintained by mechanisms including cytokines and cell surface receptors and its loss results in inflammatory disease. In the case of psoriasis, inappropriate immune activation leads to IL-17-driven chronic inflammation, but molecular mechanisms underlying this loss of regulation are not well understood. We reveal that immunoglobulin superfamily member CD200, and signalling via its receptor, CD200R1 are reduced in non-lesional psoriasis skin. To examine the consequences of this, CD200R1 was blocked in a mouse model of psoriasis demonstrating that the receptor limits psoriasis-like inflammation. Specifically, CD200R1 blockade enhances acanthosis, CCL20 production and neutrophil recruitment but surprisingly, macrophage function and IL-17 production were not affected, and neutrophil reactive oxygen species production was reduced. Collectively, our data show that CD200R1 affects neutrophil function and limits inflammatory responses in healthy skin by restricting neutrophil recruitment. However, the CD200 pathway is reduced in psoriasis, resulting in a loss of immune control, and increased neutrophil recruitment in mouse models. In conclusion, we highlight a pathway that might be targeted to dampen inflammation in patients with psoriasis.

immunology↗

Ascertaining cells' synaptic connections and RNA expression simultaneously with massively barcoded rabies virus libraries

Brain function depends on forming and maintaining connections between neurons of specific types, ensuring neural function while allowing the plasticity necessary for cellular and behavioral dynamics. However, systematic descriptions of how brain cell types organize into synaptic networks and which molecules instruct these relationships are not readily available. Here, we introduce SBARRO (Synaptic Barcode Analysis by Retrograde Rabies ReadOut), a method that uses single-cell RNA sequencing to reveal directional, monosynaptic relationships based on the paths of a barcoded rabies virus from its "starter" postsynaptic cell to that cells presynaptic partners1. Thousands of these partner relationships can be ascertained in a single experiment, alongside genome-wide RNA profiles - and thus cell identities and molecular states - of each host cell. We used SBARRO to describe synaptic networks formed by diverse mouse brain cell types in vitro, leveraging a system similar to those used to identify synaptogenic molecules. We found that the molecular identity (cell type/subtype) of the starter cell predicted the number and types of cells that had synapsed onto it. Rabies transmission tended to occur into cells with RNA-expression signatures related to developmental maturation and synaptic transmission. The estimated size of a cells presynaptic network, relative to that of other cells of the same type, associated with increased expression of Arpp21 and Cdh13. By tracking individual virions and their clonal progeny as they travel among host cells, single-cell, single-virion genomic technologies offer new opportunities to map the synaptic organization of neural circuits in health and disease.

neuroscience↗