Biology subjects

Stevens, B.

Publications and source records attributed to Stevens, B..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

IgSeqR: a protocol for the identification, assembly, and characterization of full-length tumor Immunoglobulin transcripts from unselected RNA sequencing data

Immunoglobulin (IG) gene analysis provides fundamental insight into B-cell receptor structure and function. In B-cell tumors, it can inform the cell of origin and clinical outcomes. Its clinical value has been established in the two types of chronic lymphocytic leukemia with unmutated or mutated IGHV genes and is emerging in other B-cell tumors. The traditional PCR-based techniques, which are labor-intensive, rely on the attainment of either a dominant sequence or a small number of subclonal sequences and do not allow automated matching with the clonal phenotypic features. Extraction of the expressed tumor IG transcripts using high-throughput RNA sequencing (RNA-seq) can be faster and allow the collection of multiple sequences matched with the transcriptome profile. Analytical tools are regularly sought to increase the accuracy, depth, and speed of acquisition of the full IGV-(IGD)-IGJ-IGC sequences and combine the IG characteristics with other RNA-seq data. We provide here a user-friendly protocol for the rapid extraction, identification, and accurate determination of the full (leader to constant region) tumor IG templated and non-templated transcript sequence from RNA-seq. The derived amino acid sequences can be interrogated for their physico-chemical characteristics and, in certain lymphomas, predict tumor glycan types occupying acquired N-glycosylation sites. These features will then be available for association studies with the tumor transcriptome. The resulting information can also help refine diagnosis, prognosis, and potential therapeutic targeting in the most common lymphomas.

bioinformatics↗

bioRxiv2024

Synapse protein signatures in cerebrospinal fluid and plasma predict cognitive maintenance versus decline in Alzheimers disease

Rates of cognitive decline in Alzheimers disease (AD) are extremely heterogeneous, with ages of symptom onset ranging from age 40-100 years and conversion from mild cognitive impairment to AD dementia taking 2-20 years. Development of biomarkers for amyloid-beta (A{beta}) and tau protein aggregates, the hallmark pathologies of AD, have improved patient monitoring/stratification and drug development, but they still only explain 20-40% of the variance in cognitive impairment (CI) in AD. To discover additional molecular drivers and biomarkers of AD dementia, we perform cerebrospinal fluid (CSF) proteomics on 3,416 individuals from six deeply phenotyped prospective AD case-control cohorts. We identify synapse proteins as the strongest correlates of CI, independent of A{beta} and tau. Using machine learning we derive the CSF YWHAG:NPTX2 synapse protein ratio, a robust correlate of CI, which explains 27% of the variance in CI beyond CSF PTau181:A{beta}42, 10% beyond tau PET, and 50% beyond CSF NfL in A{beta} positive individuals. We find YWHAG:NPTX2 also increases with normal aging as early as age 20 and increases at a faster rate in APOE4 carriers and autosomal dominant-AD mutation carriers. Most notably, YWHAG:NPTX2+ individuals (top 25th percentile) are 15-times (HR=15.4 [10.6-22.2]) more likely to experience cognitive decline over 15 years compared to YWHAG:NPTX2- individuals (bottom 25th percentile), and this rises to 19-times (HR=18.9 [10.83-32.9]) with additional stratification by A{beta} and phosphorylated tau status. Lastly, we perform plasma proteomics on 4,245 individuals to develop a plasma-based signature of CI which partly recapitulates CSF YWHAG:NPTX2. Overall, our findings underscore CSF YWHAG:NPTX2 and the corresponding plasma signature as robust prognostic biomarkers for AD onset and progression beyond gold-standard biomarkers of A{beta}, tau, and neurodegeneration and implicate synapse dysfunction as a core driver of AD dementia.

neuroscience↗

bioRxiv2024

Developmental maturation of frontal cortical circuits impacts decision-making

In humans, frontal cortical circuits mature in parallel with the development of higher cognitive functions over the course of 15-20 years. In mice, behavior and brain structure change rapidly until reaching sexual maturity ([~]8 weeks of age). In contrast, the degree to which frontal cortices and the behaviors they support continue to develop in mice after this period is unclear. Here, we uncover age-related changes in the acquisition and execution of a probabilistic reward reinforcement task. Both young and old mice complete a similar number of trials per session, but 16-30-week-old mice obtain higher reward rates than younger mice. The older mice adjust their behavior more readily after failing to receive a reward, suggesting that age-dependent changes to circuits relevant to forming action-outcome associations, such as those in frontal associative cortices (FACs). Indeed, we identified cell-type and input-specific refinements of FAC circuits over the first 24 weeks of age, including protracted reductions in excitatory drive and increases in inhibition onto pyramidal cells. Dampening inhibitory activity in FAC alters behavior in a manner that counteracts the age-related differences. Together, these reveal an extended period of synaptic maturation in FAC that directly impacts age-related changes in decision-making.

neuroscience↗

bioRxiv2023

Avian influenza viruses in wild birds in Canada following incursion of the highly pathogenic H5N1 virus from Eurasia in 2021/2022

Following detection of novel highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in Newfoundland, Canada in late 2021, avian influenza surveillance in wild birds was scaled-up across Canada. Herein, we present results of Canadas Interagency Surveillance Program for Avian Influenza in wild birds during the first year (November 2021 - November 2022) following the incursions of HPAIV from Eurasia. Key objectives of the surveillance program were to (i) detect the presence, distribution and spread of HPAIV and other avian influenza viruses (AIVs), (ii) detect wild bird morbidity and mortality associated with HPAIV, (iii) identify the range of wild bird species infected by HPAIV, and (iv) characterize detected AIV. A total of 6,246 sick and dead wild birds were tested, of which 27.4% were HPAIV positive across 12 taxonomic orders and 80 species. Geographically, HPAIV detections occurred in all Canadian provinces and territories, with the highest numbers in the Atlantic and Central flyways. Temporally, peak detections differed across flyways, though the national peak occurred in April 2022. In an additional 11,295 asymptomatic harvested or live captured wild birds, 5.2% were HPAIV positive across 3 taxonomic orders and 19 species. Whole genome sequencing identified HPAIV of Eurasian origin as most prevalent in the Atlantic flyway, along with multiple reassortants of mixed Eurasian and North American origins distributed across Canada, with moderate structuring at the flyway scale. Wild birds were victims and reservoirs of HPAIV H5N1 2.3.4.4b, underscoring the importance of surveillance encompassing samples from sick and dead, as well as live and harvested birds to provide insights into the dynamics and potential impacts of the HPAIV H5N1 outbreak. This dramatic shift in presence and distribution of HPAIV in wild birds in Canada highlights a need for sustained investment in wild bird surveillance and collaboration across One Health partners.

zoology↗

bioRxiv2023

Early Alzheimer's disease pathology in human cortex is associated with a transient phase of distinct cell states

Cellular perturbations underlying Alzheimers disease are primarily studied in human postmortem samples and model organisms. Here we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of Alzheimers disease pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes-which we refer to as the Early Cortical Amyloid Response--were prominent in neurons, wherein we identified a transient state of hyperactivity preceding loss of excitatory neurons, which correlated with the selective loss of layer 1 inhibitory neurons. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathological burden increased. Lastly, both oligodendrocytes and pyramidal neurons upregulated genes associated with amyloid beta production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.

genomics↗

bioRxiv2023

FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity

Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, studies to date have assumed that a few select FFAs are representative of broad structural categories, and there are no scalable approaches to comprehensively assess the biological processes induced by exposure to diverse FFAs circulating in human plasma. Furthermore, assessing how these FFA- mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies) as an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids (MUFAs) with a distinct lipidomic profile associated with decreased membrane fluidity. Furthermore, we developed a new approach to prioritize genes that reflect the combined effects of exposure to harmful FFAs and genetic risk for type 2 diabetes (T2D). Importantly, we found that c-MAF inducing protein (CMIP) protects cells from exposure to FFAs by modulating Akt signaling and we validated the role of CMIP in human pancreatic beta cells. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism. HighlightsO_LIFALCON (Fatty Acid Library for Comprehensive ONtologies) enables multimodal profiling of 61 free fatty acids (FFAs) to reveal 5 FFA clusters with distinct biological effects C_LIO_LIFALCON is applicable to many and diverse cell types C_LIO_LIA subset of monounsaturated FAs (MUFAs) equally or more toxic than canonical lipotoxic saturated FAs (SFAs) leads to decreased membrane fluidity C_LIO_LINew approach prioritizes genes that represent the combined effects of environmental (FFA) exposure and genetic risk for disease C_LIO_LIC-Maf inducing protein (CMIP) is identified as a suppressor of FFA-induced lipotoxicity via Akt-mediated signaling C_LI

cell biology↗

bioRxiv2022

Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of alpha-synuclein inclusions

Intracellular inclusions accompanying neurodegeneration are histopathologically and ultrastructurally heterogeneous but the significance of this heterogeneity is unclear. iPSC models, while promising for disease modeling, do not form inclusions in a reasonable timeframe and suffer from limited tractability. Here, we developed an iPSC toolbox utilizing piggyBac-based or targeted transgenes to rapidly induce CNS cells with concomitant expression of aggregation-prone proteins. This system is amenable to screening and longitudinal tracking at single-cell and single-inclusion resolution. For proof-of-principle, cortical neuron -synuclein "inclusionopathy" models were engineered to form inclusions through exogenous seeding or -synuclein mutation. These models recapitulated known fibril- and lipid-rich inclusion subtypes, uncovering dynamic interactions between them, and refined the classification of inclusions in postmortem brain. Genetic-modifier and protein-interaction screens pinpointed proteins like RhoA whose sequestration into specific inclusion subtypes is likely to be toxic. This iPSC platform should enhance our understanding of proteinaceous pathologies in neurodegeneration and facilitate therapeutics development.

neuroscience↗

bioRxiv2022

A resource for generating and manipulating human microglial states in vitro

Microglia have emerged as key players in the pathogenesis of neurodegenerative conditions such as Alzheimers disease (AD). In response to CNS stimuli, these cells adopt distinct transcriptional and functional subtypes known as states. However, an understanding of the function of these states has been elusive, especially in human microglia, due to lack of tools to model and manipulate this cell-type. Here, we provide a platform for modeling human microglia transcriptional states in vitro. Using single-cell RNA sequencing, we found that exposure of human stem-cell differentiated microglia (iMGLs) to brain-related challenges generated extensive transcriptional diversity which mapped to gene signatures identified in human brain microglia. We identified two in vitro transcriptional clusters that were analogous to human and mouse disease-associated microglia (DAMs), a state enriched in neurodegenerative disease contexts. To facilitate scalable functional analyses, we established a lentiviral approach enabling broad and highly efficient genetic transduction of microglia in vitro. Using this new technology, we demonstrated that MITF (Melanocyte Inducing Transcription Factor), an AD-enriched transcription factor in microglia, drives both a disease-associated transcriptional signature and a highly phagocytic state. Finally, we confirmed these results across iMGLs differentiated from multiple iPSC lines demonstrating the broad utility of this platform. Together, these tools provide a comprehensive resource that enables the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

neuroscience↗

bioRxiv2022

Microglia Detect Externalized Phosphatidylserine on Synapses for Elimination via TREM2 in Alzheimer's Disease Models

Genetic studies implicate phagocytosis pathways in microglia to be a major Alzheimers disease (AD)-associated process. Microglia phagocytose synapses in AD mouse models, suggesting a role for microglia in region-specific synapse loss, a pathological hallmark of AD. However, whether specific synapses are targeted for elimination, and if so, how, remains to be elucidated. Here, we show that synapses externalize phosphatidylserine (PtdSer) upon challenge by {beta}-amyloid oligomers, which are then selectively engulfed by microglia. Mechanistically, we find that Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is critical for microglia to sense and preferentially engulf AD synapses. In brains of mice and humans, TREM2 dysfunction leads to exacerbation of apoptotic synapses. Our work altogether suggests a fundamental role for microglia as brain-resident macrophages to remove damaged synapses in AD. We provide mechanistic insight into how TREM2 variants associated with increased risk of developing AD may contribute to defective microglia-synapse function. One-Sentence summaryMicroglia selectively engulf synapses in Alzheimer-like mouse brains via PtdSer-TREM2 signaling.

neuroscience↗

bioRxiv2021

SARS-CoV-2 wildlife surveillance in Ontario and Quebec, Canada

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, is capable of infecting a variety of wildlife species. Wildlife living in close contact with humans are at an increased risk of SARS-CoV-2 exposure and if infected have the potential to become a reservoir for the pathogen, making control and management more difficult. ObjectiveTo conduct SARS-CoV-2 surveillance in urban wildlife from Ontario and Quebec, Canada, increasing our knowledge of the epidemiology of the virus and our chances of detecting spillover from humans into wildlife. MethodsUsing a One Health approach, we leveraged activities of existing research, surveillance, and rehabilitation programs among multiple agencies to collect samples from 776 animals from 17 different wildlife species between June 2020 and May 2021. Samples from all animals were tested for the presence of SARS-CoV-2 viral RNA, and a subset of samples from 219 animals across 3 species (raccoons, Procyon lotor; striped skunks, Mephitis mephitis; and mink, Neovison vison) were also tested for the presence of neutralizing antibodies. ResultsNo evidence of SARS-CoV-2 viral RNA or neutralizing antibodies was detected in any of the tested samples. ConclusionAlthough we were unable to identify positive SARS-CoV-2 cases in wildlife, continued research and surveillance activities are critical to better understand the rapidly changing landscape of susceptible animal species. Collaboration between academic, public and animal health sectors should include experts from relevant fields to build coordinated surveillance and response capacity.

pathology↗

bioRxiv2021

The evolutionary maintenance of Levy flight foraging: a numerical simulation.

Levy flight is a type of random walk that models the behaviour of many phenomena across a multiplicity of academic disciplines; within biology specifically, the behaviour of fish, birds, insects, mollusks, bacteria, plants, slime molds, t-cells, and human populations. The Levy flight foraging hypothesis states that because Levy flights can maximize an organisms search efficiency, natural selection should result in Levy-like behaviour. Empirical and theoretical research has provided ample evidence of Levy walks in both extinct and extant species, and its efficiency across models with a diversity of resource distributions. However, no model has addressed the maintenance of Levy flight foraging through evolutionary processes, and existing models lack ecological breadth. We use numerical simulations, including lineage-based models of evolution with a distribution of move lengths as a variable and heritable trait, to test the Levy flight foraging hypothesis. We include biological and ecological contexts such as population size, searching costs, lifespan, resource distribution, speed, and consider both energy accumulated at the end of a lifespan and averaged over a lifespan. We demonstrate that selection often results in Levy-like behaviour, although conditional; smaller populations, longer searches, and low searching costs increase the fitness of Levy-like behaviour relative to Brownian behaviour. Interestingly, our results also evidence a bet-hedging strategy; Levy-like behaviour reduces fitness variance, thus maximizing geometric mean fitness over multiple generations. Author summaryIn heterotrophs, incuding animals, survival depends on the net energy gained through foraging. The expectation, then, is that natural selection results in adaptations for efficient foraging that optimize the balance of searching costs and rewards. Levy flight foraging has been proposed as an optimal foraging solution. The hypothesis states, if no information about resource locations are available, and the locations are re-visitable, then selection will result in adaptations for Levy flight foraging, a type of random walk. It has been argued that Levy-like foraging behaviour may simply reflect how resources are distributed, but empirical and theoretical research suggests that this behaviour is intrinsic or innate. However, this research does not address evolutionary mechanisms, and lacks ecological breadth. We extend the current theoretical framework by including evolutionary ecological contexts. We treat an organisms random walk as a heritable trait, and explore ecological contexts such as population size, lifespan, carrying capacity, searching costs, reproductive strategies, and different distributions of food. Our evolutionary simulations overwhelmingly resulted in selection for Levy-like foraging, regardless of the distribution of food, and evidences Levy flight foraging as a bet-hedging strategy. Thus, here we provide some of the first evidence for the evolutionary maintenance of Levy flight foraging.

evolutionary biology↗

bioRxiv2021

Single-nucleus sequencing reveals enriched expression of genetic risk factors sensitises Motor Neurons to degeneration in ALS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterised by a progressive loss of motor function. The eponymous spinal sclerosis observed at autopsy is the result of the degeneration of extratelencephalic neurons, Betz cells (ETNs, Cortico-Spinal Motor Neuron). It remains unclear why this neuronal subtype is selectively affected. To understand the unique molecular properties that sensitise these cells to ALS, we performed RNA sequencing of 79,169 single nuclei from cortices of patients and controls. In unaffected individuals, we found that expression of ALS risk genes was significantly enriched in THY1+-ETNs and not in other cell types. In patients, these genetic risk factors, as well as genes involved in protein homeostasis and stress responses, were significantly induced in a wide collection of ETNs, but not in neurons with more superficial identities. Examination of oligodendroglial and microglial nuclei revealed patient-specific changes that were at least in part a response to alterations in neurons: downregulation of myelinating genes in oligodendrocytes and upregulation of a reactive state connected to endo-lysosomal pathways in microglia. Our findings suggest that the selective vulnerability of extratelencephalic neurons is partly connected to their intrinsic molecular properties sensitising them to genetics and mechanisms of degeneration. Graphical abstract and working model O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/452054v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@11237fdorg.highwire.dtl.DTLVardef@119f3ceorg.highwire.dtl.DTLVardef@d56c69org.highwire.dtl.DTLVardef@1c087e1_HPS_FORMAT_FIGEXP M_FIG Our study highlights cell type specific changes in premotor/motor cortex of sporadic ALS patients. Specifically, we identify upregulation of synaptic molecules in excitatory neurons of upper cortical layers, interestingly correlating to hyperexcitability phenotypes seen in patients. Moreover, excitatory neurons of the deeper layers of the cortex, that project to the spinal cord and are most affected by the disease, show higher levels of cellular stresses than other neuronal types. Correspondently, oligodendrocytes transition from a highly myelinating state to a more neuronally engaged state, probably to counteract stressed phenotypes seen in excitatory neurons. At the same time, microglia show a reactive state with specific upregulation of endo-lysosomal pathways. C_FIG

neuroscience↗

bioRxiv2021

Metamodal Coupling of Vibrotactile and Auditory Speech Processing Systems Through Matched Stimulus Representations

It has been postulated that the brain is organized by "metamodal", sensory-independent cortical modules implementing particular computations, leading to the intriguing hypothesis that brain areas can perform tasks (such as word recognition) not just in "standard" sensory modalities but also in novel sensory modalities. Yet, evidence for this theory, especially in neurotypical subjects, has been variable. We hypothesized that effective metamodal engagement of a brain area requires congruence between the novel and standard sensory modalities not only at the task level (e.g., "word recognition") but critically also a match at the algorithmic level (in Marrs terminology), i.e., at the level of neural representation of the information of interest. To test this hypothesis, we trained participants to recognize vibrotactile versions of auditory words using two encoding schemes. The vocoded approach preserved the dynamics and representational similarities of auditory speech while the token-based approach used an abstract phoneme-based code. Although both groups learned the vibrotactile word recognition task, only in the vocoded group did trained vibrotactile stimuli recruit the auditory speech network and lead to increased coupling between somatosensory and auditory speech areas. In contrast, the token-based encoding appeared to rely on paired-associate learning. Thus, matching neural input representations is a critical factor for assessing and leveraging the metamodal potential of cortical modules.

neuroscience↗

bioRxiv2021

A Developmental Role for Microglial Presenilin 1 in Memory

Microglia, the macrophages of the brain, are increasingly recognized to play a key role in synaptic plasticity and function; however, the underlying mechanisms remain elusive. Presenilin 1 (PS1) is an essential protein involved in learning and memory, through neuronal mechanisms. Loss of Presenilin function in neurons impairs synapse plasticity and causes cognitive deficits in mice. Surprisingly, here we show memory enhancement in mice by deleting PS1 selectively in microglia. We further demonstrate increased synapse transmission and in vivo neuronal activity in mice by depleting PS1 during microglial development, but not after microglial maturation. Remarkably, conditional deletion of PS1 in microglia during development increased memory retention in adulthood and was dependent on the NMDA receptor subunit GluN2B. In vivo calcium imaging of freely behaving mice revealed increased amplitude of neuronal Ca2+ transients in the CA1 hippocampus of PS1 cKO mice compared to control mice, suggesting a greater CA1 engagement during novel object exploration. Finally, loss of PS1 in microglia mitigated synaptic and cognitive deficits in a mouse model of Alzheimers disease. Together our results reveal a novel mechanism and function of PS1 in microglia in which modulation can enhance neuronal activity, learning and memory in mice.

neuroscience↗

bioRxiv2020

Single Cell Sequencing Reveals Glial Specific Responses to Tissue Processing & Enzymatic Dissociation in Mice and Humans

A key aspect of nearly all single cell experiments is the necessity to dissociate intact tissues into single cell suspensions for processing. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression changes during this process. Microglia, the brains resident macrophages, are a highly dynamic population that are extremely sensitive to their microenvironment and have been shown to dramatically alter their transcriptome upon stimulation. We demonstrate that use of enzymatic dissociation methods on mouse central nervous system (CNS) tissue induces an aberrant gene expression signature in microglia that can significantly confound downstream analysis. To minimize this issue, we developed a flexible protocol, that can be used with existing enzymatic protocols for fresh tissue, to eliminate artifactual gene expression while allowing for increased cell type diversity and yield. We demonstrate efficacy of this protocol in analysis of diverse CNS cell types and sorted myeloid populations while using enzymatic dissociation. Generation of new and reanalysis of previously published human brain single nucleus RNAseq (snRNA-seq) datasets reveal that a similar signature is also present in post-mortem tissue. Through novel snRNA-seq analysis of acutely-resected neurosurgical tissue we demonstrate that this signature can be induced in human tissue due to technical differences in sample processing. These results provide key insight into the potential confounds of enzymatic digestion and provide a solution to allow for enzymatic digestion for scRNA-seq while avoiding ex vivo transcriptional artifacts. Analysis of human tissue reveals potential for artifacts in current and future snRNA-seq datasets that will require deeper analysis and careful consideration to separate true biology from artifacts related to post-mortem processes.

neuroscience↗

bioRxiv2020

CUB and Sushi Multiple Domains 1 (CSMD1) opposes the complement cascade in neural tissues

Schizophrenia risk is associated with increased gene copy number and brain expression of complement component 4 (C4). Because the complement system facilitates synaptic pruning, the C4 association has renewed interest in a hypothesis that excessive pruning contributes to schizophrenia pathogenesis. However, little is known about complement regulation in neural tissues or whether such regulation could be relevant to psychiatric illness. Intriguingly, common variation within CSMD1, which encodes a putative complement inhibitor, has consistently associated with schizophrenia at genome-wide significance. We found that Csmd1 is predominantly expressed in the brain by neurons, and is enriched at synapses; that human stem cell-derived neurons lacking CSMD1 are more vulnerable to complement deposition; and that mice lacking Csmd1 have increased brain complement activity, fewer synapses, aberrant complement-dependent development of a neural circuit, and synaptic elements that are preferentially engulfed by cultured microglia. These data suggest that CSMD1 opposes the complement cascade in neural tissues. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/291427v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@172c857org.highwire.dtl.DTLVardef@bee84corg.highwire.dtl.DTLVardef@184da10org.highwire.dtl.DTLVardef@10867c8_HPS_FORMAT_FIGEXP M_FIG Graphic Abstract.Our findings support a model in which CSMD1 opposes actions of the complement cascade in neural tissues (top left). We investigated two models in which Csmd1 was genetically ablated: human cortical neurons derived from embryonic stem cells, and a back-crossed C57bl6-Tac mouse line (top right). Csmd1 is normally expressed by neurons and present at synapses where it can protect them from complement (bottom left); in the absence of Csmd1 (bottom right), we find more deposition of complement (on cultured human cortical neurons and in the mouse visual system), reduced numbers of synapses (in the mouse visual system), and synaptic fractions that are more readily engulfed by microglia (ex vivo). Created with BioRender.com. C_FIG

neuroscience↗

bioRxiv2020

Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens

BackgroundSeveral point-of-care (POC) molecular tests have received emergency use authorization (EUA) from the Food and Drug Administration (FDA) for diagnosis of SARS-CoV-2. The test performance characteristics of the Accula (Mesa Biotech) SARS-CoV-2 POC test need to be evaluated to inform its optimal use. ObjectivesThe aim of this study was to assess test performance of the Accula SARS-CoV-2 test. Study designThe performance of the Accula test was assessed by comparing results of 100 nasopharyngeal swab samples previously characterized by the Stanford Health Care EUA laboratory-developed test (SHC-LDT) targeting the envelope (E) gene. Assay concordance was assessed by overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohens kappa coefficient. ResultsOverall percent agreement between the assays was 84.0% (95% confidence interval [CI] 75.3 to 90.6%), PPA was 68.0% (95% CI 53.3 to 80.5%) and the kappa coefficient was 0.68 (95% CI 0.54 to 0.82). Sixteen specimens detected by the SHC-LDT were not detected by the Accula test, and showed low viral load burden with a median cycle threshold value of 37.7. NPA was 100% (95% CI 94.2 to 100%). ConclusionCompared to the SHC-LDT, the Accula SARS-CoV-2 test showed excellent negative agreement. However, positive agreement was low for samples with low viral load. The false negative rate of the Accula POC test calls for a more thorough evaluation of POC test performance characteristics in clinical settings, and for confirmatory testing in individuals with moderate to high pre-test probability of SARS-CoV-2 who test negative on Accula.

microbiology↗

bioRxiv2020

Local externalization of phosphatidylserine mediates developmental synaptic pruning by microglia

Neuronal circuits assembly requires the fine equilibrium between synapse formation and elimination. Microglia, through the elimination of supernumerary synapses, have an established role in this process. While the microglial receptor TREM2 and the soluble complement proteins C1q and C3 are recognized key players in this process, the neuronal molecular components that tag synapses to be eliminated are still undefined. Here we show that exposed phosphatidylserine (PS) represents a neuronal eat-me signal enabling microglial-mediated synapse pruning. In hippocampal neuron and microglia co-cultures, synapse elimination can be prevented by blocking accessibility of exposed PS using Annexin V or through microglial loss of TREM2. In vivo, exposed PS is detectable at both hippocampal and retinogeniculate synapses, where exposure coincides with the onset of synapse elimination and increased PS engulfment by microglia. Mice deficient in C1q, which fail to properly refine retinogeniculate connections, display elevated exposed PS and reduced PS engulfment by microglia. These data provide mechanistic insight into microglial-mediated synapse pruning and identify a novel role of developmentally regulated PS exposure that is common among developing brain structures.

neuroscience↗