Biology subjects

Smith, S.

Publications and source records attributed to Smith, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Mice regulate their attentional intensity and arousal to exploit increases in task utility

Changes in autonomic arousal, such as mounting sleep pressure, and changes in motivation, such as fluctuating environmental reward statistics, both profoundly influence behavior. Our experience tells us that we have some capacity to control our arousal when doing so is important, such as staying awake while driving a motor vehicle. However, little is known about how decision computations are jointly influenced by arousal and motivation, including whether animals, such as rodents, can adapt their arousal state to their needs. Here, we developed and show results from an auditory feature-based sustained-attention task with intermittently shifting task utility. We use pupil size to estimate arousal across a wide range of states and apply novel signal detection theoretic and accumulation-to-bound modeling approaches in a large behavioral cohort. We find that both pupil-linked arousal and task utility have major impacts on multiple aspects of performance. Although substantial arousal fluctuations persist across utility conditions, mice partially stabilize their arousal near an intermediate, and optimal, level when task utility is high. Behavioral analyses show that multiple elements of behavior improve during high task utility and that arousal influences some, but not all, of them. Specifically, arousal influences the likelihood and timescale of sensory evidence accumulation, but not the quantity of evidence accumulated per time step while attending. In sum, the results establish specific decision-computational signatures of arousal, motivation, and their interaction, in attention. So doing, we provide an experimental and analysis framework for studying arousal self-regulation in neurotypical brains and diseases such as attention-deficit/hyperactivity disorder.

neuroscience↗

bioRxiv2022

Genomic neighbor typing for bacterial outbreaksurveillance

Genomic neighbor typing enables heuristic inference of bacterial lineages and phenotypes from nanopore sequencing data. However, small reference databases may not be sufficiently representative of the diversity of lineages and genotypes present in a collection of isolates. In this study, we explore the use of genomic neighbor typing for surveillance of community-associated Staphylococcus aureus outbreaks in Papua New Guinea (PNG) and Far North Queensland, Australia (FNQ). We developed Sketchy, an implementation of genomic neighbor typing that queries exhaustive whole genome reference databases using MinHash. Evaluations were conducted using nanopore read simulations and six species-wide reference sketches (4832 - 47616 genomes), as well as two S. aureus outbreak data sets sequenced at low depth using a sequential multiplex library protocol on the MinION (n = 160, with matching Illumina data). Heuristic inference of lineages and antimicrobial resistance profiles allowed us to conduct multiplex genotyping in situ at the Papua New Guinea Institute of Medical Research in Goroka, on low-throughput Flongle adapters and using multiple successive libraries on the same MinION flow cell (n = 24 - 48). Comparison to phylogenetically informed genomic neighbor typing with RASE on the dominant outbreak sequence type suggests slightly better performance at predicting lineage-scale genotypes using large sketch sizes, but inferior performance in resolving clade-specific genotypes (methicillin resistance). Sketchy can be used for large-scale bacterial outbreak surveillance and in challenging sequencing scenarios, but improvements to clade-specific genotype inference are needed for diagnostic applications. Sketchy is available open-source at: https://github.com/esteinig/sketchy

bioinformatics↗

bioRxiv2022

USP22 controls type III interferon signaling and SARS-CoV-2 infection through activation of STING

Pattern recognition receptors (PRRs) and interferons (IFNs) serve as essential antiviral defense against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Type III IFN (IFN-{lambda}) exhibit cell-type specific and long-lasting functions in autoinflammation, tumorigenesis and antiviral defense. Here, we identify the deubiquitinating enzyme USP22 as central regulator of basal IFN-{lambda} secretion and SARS-CoV-2 infections in native human intestinal epithelial cells (hIECs). USP22-deficient hIECs strongly upregulate genes involved in IFN signaling and viral defense, including numerous IFN-stimulated genes (ISGs), with increased secretion of IFN-{lambda} and enhanced STAT1 signaling, even in the absence of exogenous IFNs or viral infection. Interestingly, USP22 controls basal and 23-cGAMP-induced STING activation and loss of STING reversed STAT activation and ISG and IFN-{lambda} expression. Intriguingly, USP22-deficient hIECs are protected against SARS-CoV-2 infection, viral replication and the formation of de novo infectious particles, in a STING-dependent manner. These findings reveal USP22 as central host regulator of STING and type III IFN signaling, with important implications for SARS-CoV-2 infection and antiviral defense.

cell biology↗

bioRxiv2022

Decoding the IGF1 Signaling Gene Regulatory Network Behind Alveologenesis from A Mouse Model of Bronchopulmonary Dysplasia

Lung development is precisely controlled by underlying Gene Regulatory Networks (GRN). Disruption of genes in the network can interrupt normal development and cause diseases such as bronchopulmonary dysplasia (BPD)-a chronic lung disease in preterm infants with morbid and sometimes lethal consequences characterized by lung immaturity and reduced alveolarization. Here, we generated a transgenic mouse exhibiting a moderate severity BPD phenotype by blocking IGF1 signaling in secondary crest myofibroblasts (SCMF) at the onset of alveologenesis. Using approaches mirroring the construction of the model GRN in sea urchins development, we constructed the IGF1 signaling network underlying alveologenesis using this mouse model that phenocopies BPD. The constructed GRN, consisting of 43 genes, provides a birds-eye view of how the genes downstream of IGF1 are regulatorily connected. The GRN also reveals a mechanistic interpretation of how the effects of IGF1 signaling are transduced within SCMF from its specification genes to its effector genes and then from SCMF to its neighboring alveolar epithelial cells with WNT5A and FGF10 signaling as the bridge. Consistently, blocking WNT5A signaling in mice phenocopies BPD as inferred by the network. A comparative study on human samples suggests that a GRN of similar components and wiring underlies human BPD. Our network view of alveologenesis is transforming our perspective to understand and treat BPD. This new perspective calls for the construction of the full signaling GRN underlying alveologenesis, upon which targeted therapies for this neonatal chronic lung disease can be viably developed.

developmental biology↗

bioRxiv2022

Genomes of the extinct Sicilian wolf reveal a complex history of isolation and admixture with ancient dogs

The Sicilian wolf represented the only population of wolves living on a Mediterranean island until the first half of the twentieth century (1930s-1960s) 1-7. Previous studies hypothesised that they remained isolated from mainland wolves from the end of the Last Glacial Maximum (LGM) 8,9, until human persecutions led them to extinction 1-7. There are only seven known Sicilian wolf specimens from the 19th and 20th century preserved in museums in Italy and recent morphometric analyses assigned them to the new subspecies Canis lupus cristaldii 10. To better understand the origins of the Sicilian wolf, and its relationship to other wolf populations, we sequenced four whole genomes (3.8x-11.6x) and five mitogenomes. We investigated the relationship between Sicilian wolves and other modern breeds to identify potential admixture. Furthermore, considering that the last land-bridge between Sicily and Italy disappeared after the LGM 11, around 17 kya, we explored the possibility that the Sicilian wolf retained ancestry from ancient wolf and dog lineages. Additionally, we explored whether the long-term isolation might have affected the genomic diversity, inbreeding levels and genetic load of the Sicilian wolf. Our findings show that the Sicilian wolves shared most ancestry with the modern Italian wolf population but are better modelled as admixed with European dog breeds, and shared traces of Eneolithic and Bronze age European dogs. We also find signatures of severe inbreeding and low genomic diversity at population and individual levels due to long-term isolation and drift, suggesting also low effective population size.

genomics↗

bioRxiv2021

Evidence for a Potential Pre-Pandemic SARS-like Coronavirus Among Animals in North America

In late 2019, a novel coronavirus began circulating within humans in central China. It was designated SARS-CoV-2 because of its genetic similarities to the 2003 SARS coronavirus (SARS-CoV). Now that SARS-CoV-2 has spread worldwide, there is a risk of it establishing new animal reservoirs and recombination with native circulating coronaviruses. To screen local animal populations in the United States for exposure to SARS-like coronaviruses, we developed a serological assay using the receptor binding domain (RBD) from SARS-CoV-2. SARS-CoV-2s RBD is antigenically distinct from common human and animal coronaviruses allowing us to identify animals previously infected with SARS-CoV or SARS-CoV-2. Using an indirect ELISA for SARS-CoV-2s RBD, we screened serum from wild and domestic animals for the presence of antibodies against SARS-CoV-2s RBD. Surprisingly pre-pandemic feline serum samples submitted to the University of Tennessee Veterinary Hospital were [~]50% positive for anti-SARS RBD antibodies. Some of these samples were serologically negative for feline coronavirus (FCoV), raising the question of the etiological agent generating anti-SARS-CoV-2 RBD cross-reactivity. We also identified several white-tailed deer from South Carolina with anti-SARS-CoV-2 antibodies. These results are intriguing as cross-reactive antibodies towards SARS-CoV-2 RBD have not been reported to date. The etiological agent responsible for seropositivity was not readily apparent, but finding seropositive cats prior to the current SARS-CoV-2 pandemic highlights our lack of information about circulating coronaviruses in other species. ImportanceWe report cross-reactive antibodies from pre-pandemic cats and post-pandemic South Carolina white-tailed deer that are specific for that SARS-CoV RBD. There are several potential explanations for this cross-reactivity, each with important implications to coronavirus disease surveillance. Perhaps the most intriguing possibility is the existence and transmission of an etiological agent (such as another coronavirus) with similarity to SARS-CoV-2s RBD region. However, we lack conclusive evidence of pre-pandemic transmission of a SARS-like virus. Our findings provide impetus for the adoption of a One Health Initiative focusing on infectious disease surveillance of multiple animal species to predict the next zoonotic transmission to humans and future pandemics.

immunology↗

bioRxiv2021

Structural comparative modeling of multi-domain ΔF508 CFTR

Cystic Fibrosis (CF) is a common genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs - which increases the risk of infection and eventually causes death. CFTR is an ATP binding cassette (ABC) transporter composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 ({Delta}F508), making {Delta}F508 CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain {Delta}F508 CFTR structure has been determined and few studies have modeled {Delta}F508 using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a {Delta}F508 model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and {Delta}F508 CFTR structural models. These models demonstrate the destabilizing effects of {Delta}F508 on NBD1 and the NBD1/TMD interface in both the closed and open conformation of CFTR. Furthermore, we modeled {Delta}F508/R1070W and {Delta}F508 bound to the CFTR corrector VX-809. Our models reveal the stabilizing effects of R1070W and VX-809 on multi-domain models of {Delta}F508 CFTR and pave the way for rational design of additional drugs that target {Delta}F508 CFTR for treatment of CF. Author SummaryProteins three-dimension shape determines their function, so when genetic mutation compromises the shape of vital proteins, it may cause disease. Such is the case in Cystic Fibrosis, a chronic genetic disease caused by mutations in the protein Cystic Fibrosis Transmembrane Conductance Regulator. Here, we work backwards from the shape of the wild-type protein - found in healthy people, to computationally model the shape of the most common Cystic Fibrosis mutant. Our computer models reveal distinct defects in the shape of the mutant Cystic Fibrosis Transmembrane Conductance Regulator protein in the area surrounding the mutation. We also model an important FDA approved Cystic Fibrosis drug, VX-809, into the mutant protein structure and show how VX-809 stabilizes the protein around the location of the mutation. The method we developed will pave the way for computational drug design for Cystic Fibrosis.

bioinformatics↗

bioRxiv2021

Local accessory gene sharing drives lineage-specific acquisition of antimicrobial resistance in Egyptian Campylobacter spp.

Campylobacter is the most common cause of bacterial gastroenteritis worldwide and diarrheal disease is a major cause of child morbidity, growth faltering and mortality in low- and middle-income countries (LMICs). Despite evidence of high incidence and differences in disease epidemiology, there is limited genomic data from studies in developing countries. In this study, we characterised the genetic diversity and accessory genome content of a collection of Campylobacter isolates from Cairo, Egypt. In total, 112 Campylobacter isolates were collected from broiler carcasses (n=31), milk and dairy products (n=24) and patients (n=57) suffering from gastroenteritis. Among the most common sequence types (STs) we identified were the globally disseminated, host generalist ST-21 clonal complex (CC21) and the poultry specialist CC206, CC464 and CC48. Notably, CC45 and the cattle-specialist CC42 were under-represented with a total absence of CC61. Comparative genomics were used to quantify core and accessory genome sharing among isolates from the same country compared to sharing between countries. Lineage-specific accessory genome sharing was significantly higher among isolates from the same country, particularly CC21 which demonstrated greater local geographical clustering. In contrast, no geographic clustering was noted in either the core or accessory genomes of the CC828, suggesting a highly admixed population. A greater proportion of C. coli isolates were multidrug resistant (MDR) compared to C. jejuni. This is a significant public health concern as MDR food chain pathogens are difficult to treat and often pose increased mortality risk demanding enhanced prevention strategies in the Egyptian market to combat such a threat. Impact statementCampylobacter is the leading bacterial cause of gastroenteritis worldwide and despite high incidence in low- and middle-income countries, where infection can be fatal, culture-based isolation is rare and the genotypes responsible for disease are seldom identified. Here, we sequenced the genomes of a collection of isolates from clinical cases and potential infection reservoirs from Cairo in Egypt and characterised their genetic diversity. Among the most common genotypes we identified were globally disseminated lineages implicated in human disease worldwide, including the host generalist ST-21 clonal complex (CC21) and the poultry specialist genotypes CC206, CC464 and CC48. Notably however, some other globally common genotypes were under-represented or entirely absent from our collection, including those from cattle-specialist lineages, CC42 and CC61. By focussing on specific lineages, we demonstrate that there is increased accessory genome sharing in specific clonal complexes. This increased local sharing of genes may have contributed to a greater proportion of C. coli isolates possessing antimicrobial resistance determinants that suggest they could be multidrug resistant (MDR). This is a significant public health concern as MDR food chain pathogens are difficult to treat and often pose increased mortality risk demanding enhanced prevention strategies. Data summaryShort read data are available on the NCBI Sequence Read Archive, associated with BioProject PRJNA576513 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA576513). Assembled genomes, supplementary material and additional analysis files are available from FigShare: https://doi.org/10.6084/m9.figshare.9956597. Phylogenetic trees can be visualised and manipulated on Microreact for C. jejuni (https://next.microreact.org/project/Cjejuni_Egypt) and C. coli (https://next.microreact.org/project/Ccoli_Egypt) separately, or combined Cairo and Oxford data with additional PopPunk network clustering (https://microreact.org/project/Campy-Egypt).

microbiology↗

bioRxiv2021

Accurate predictions of individual differences in task-evoked brain activity from resting-state fMRI using a sparse ensemble learner

Modelling and predicting individual differences in task-evoked FMRI activity can have a wide range of applications from basic to clinical neuroscience. It has been shown that models based on resting-state activity can have high predictive accuracy. Here we propose several improvements to such models. Using a sparse ensemble leaner, we show that (i) features extracted using Stochastic Probabilistic Functional Modes (sPROFUMO) outperform the previously proposed dual-regression approach, (ii) that the shape and overall intensity of individualised task activations can be modelled separately and explicitly, (iii) training the model on predicting residual differences in brain activity further boosts individualised predictions. These results hold for both surface-based analyses of the Human Connectome Project data as well as volumetric analyses of UK-biobank data. Overall, our model achieves state of the art prediction accuracy on par with the test-retest reliability of tfMRI scans, suggesting that it has potential to supplement traditional task localisers.

neuroscience↗

bioRxiv2021

SARS-CoV-2 neutralising antibodies in Dogs and Cats in the United Kingdom

Companion animals are susceptible to SARS-CoV-2 infection and sporadic cases of pet infections have occurred in the United Kingdom. Here we present the first large-scale serological survey of SARS-CoV-2 neutralising antibodies in dogs and cats in the UK. Results are reported for 688 sera (454 canine, 234 feline) collected by a large veterinary diagnostic laboratory for routine haematology during three time periods; pre-COVID-19 (January 2020), during the first wave of UK human infections (April-May 2020) and during the second wave of UK human infections (September 2020-February 2021). Both pre-COVID-19 sera and those from the first wave tested negative. However, in sera collected during the second wave, 1.4% (n=4) of dogs and 2.2% (n=2) cats tested positive for neutralising antibodies. The low numbers of animals testing positive suggests pet animals are unlikely to be a major reservoir for human infection in the UK. However, continued surveillance of in-contact susceptible animals should be performed as part of ongoing population health surveillance initiatives.

microbiology↗

bioRxiv2021

Stat3 co-operates with mutant Ezh2Y641F to regulate gene expression and limit the anti-tumor immune response in melanoma

One of the most frequently genetically altered chromatin modifiers in melanoma is the Enhancer of Zeste Homolog 2 (EZH2), the catalytic component of the Polycomb Repressive Complex 2 (PRC2), which methylates lysine 27 on histone 3 (H3K27me3), a chromatin mark associated with transcriptional repression. Genetic alterations in EZH2 in melanoma include amplifications and activating point mutations at tyrosine 641 (Y641). The oncogenic role of EZH2 in melanoma has previously been identified; however, its downstream mechanisms remain underexplored. We found that in genetically engineered mouse models, Ezh2Y641F expression causes upregulation of a subset of interferon-regulated genes in melanoma cells, suggesting a potential role of the immune system in the pathogenesis of these mutations. Upregulation of these interferon genes was not a result of changes in H3K27me3, but through a direct and non-canonical interaction between Ezh2 and Signal Transducer and Activator of Transcription 3 (Stat3). Ezh2 directly binds Stat3, and, in the presence of Ezh2Y641F, Stat3 protein is hypermethylated. Expression of Stat3 was required to maintain an anti-tumor immune response and its depletion resulted in faster melanoma progression and disease recurrence. Molecularly, Stat3 and Ezh2 bind together at many genomic loci, and can both activate and repress gene expression in association with the rest of the PRC2 complex. These results suggest that Ezh2 can mediate melanomagenesis through evasion of the anti-tumor immune response, and that the immunomodulatory properties of Stat3 are context dependent.

cancer biology↗

bioRxiv2021

Phylodynamic modelling of bacterial outbreaks using nanopore sequencing

Nanopore sequencing and phylodynamic modelling have been used to reconstruct the transmission dynamics of viral epidemics, but their application to bacterial pathogens has remained challenging. Here, we implement Random Forest models for single nucleotide polymorphism (SNP) polishing to estimate divergence and effective reproduction numbers (Re) of two community-associated, methicillin-resistant Staphylococcus aureus (MRSA) outbreaks in remote Far North Queensland and Papua New Guinea (n = 159). Successive bar-coded panels of S. aureus isolates (2 x 12 per MinION) sequenced at low-coverage (> 5x - 10x) provided sufficient data to accurately infer assembly genotypes with high recall when compared with Illumina references. De novo SNP calling with Clair was followed by SNP polishing using intra- and inter-species models trained on Snippy reference calls. Models achieved sufficient resolution on ST93 outbreak sequence types (> 70 - 90% accuracy and precision) for phylodynamic modelling from lineage-wide hybrid alignments and birth-death skyline models in BEAST2. Our method reproduced phylogenetic topology, geographical source of the outbreaks, and indications of sustained transmission (Re > 1). We provide Nextflow pipelines that implement SNP polisher training, evaluation, and outbreak alignments, enabling reconstruction of within-lineage transmission dynamics for infection control of bacterial disease outbreaks using nanopore sequencing.

bioinformatics↗

bioRxiv2021

Phylodynamic signatures in the emergence of community-associated MRSA

Community-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 years. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole genome sequencing data for the Australian sequence type (ST) 93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australia, dissemination of the ST93-MRSA-IV geno-type into population centers on the Australian East Coast, and sub-sequent importation into the highlands of Papua New Guinea and Far North Queensland. Analysis of a ST772-MRSA-V cluster in Pakistan suggests that sustained transmission in the community following importation of resistant genotypes may be more common than previously thought. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia and Europe. Surges in Re were observed at the divergence of antibiotic resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed amongst drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA and MSSA lineages in the late 20th century was driven by a combination of antibiotic resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers.

evolutionary biology↗

bioRxiv2021

Modality-specific tracking of attention and sensory statistics in the human electrophysiological spectral exponent

A hallmark of electrophysiological brain activity is its 1/f-like spectrum - power decreases with increasing frequency. The steepness of this "roll-off" is approximated by the spectral exponent, which in invasively recorded neural populations reflects the balance of excitatory to inhibitory neural activity (E:I balance). Here, we first establish that the spectral exponent of non-invasive electroencephalography (EEG) recordings is highly sensitive to general (i.e., anaesthesia-driven) changes in E:I balance. Building on the EEG spectral exponent as a viable marker of E:I, we then demonstrate its sensitivity to the focus of selective attention in an EEG experiment during which participants detected targets in simultaneous audio-visual noise. In addition to these endogenous changes in E:I balance, EEG spectral exponents over auditory and visual sensory cortices also tracked auditory and visual stimulus spectral exponents, respectively. Individuals degree of this selective stimulus-brain coupling in spectral exponents predicted behavioural performance. Our results highlight the rich information contained in 1/f-like neural activity, providing a window into diverse neural processes previously thought to be inaccessible in non-invasive human recordings.

neuroscience↗

bioRxiv2020

MCOLN1 gene-replacement therapy corrects neurologic dysfunction in the mouse model of mucolipidosis IV.

Mucolipidosis IV (MLIV, OMIM 252650) is an orphan disease leading to debilitating psychomotor deficits and vision loss. It is caused by loss-of-function mutations in the MCOLN1 gene that encodes thethe lysosomal transient receptor potential channel mucolipin 1 (TRPML1). With no existing therapy, the unmet need in this disease is very high. Here we show that AAV-mediated gene transfer of the human MCOLN1 gene rescues motor function and alleviates brain pathology in the Mcoln1-/- MLIV mouse model. Using the AAV-PHP.b vector for initial proof-of-principle experiments in symptomatic mice, we showed long-term reversal of declined motor function and significant delay of paralysis. Next, we designed self-complimentary AAV9 vector for clinical use and showed that its intracerebroventricular administration in post-natal day 1 mice significantly improved motor function and myelination and reduced lysosomal storage load in the MLIV mouse brain. We also showed that CNS targeted gene transfer is necessary to achieve therapeutic efficacy in this disease. Based on our data and general advancements in the gene therapy field, we propose scAAV9-mediated CSF-targeted MCOLN1 gene transfer as a therapeutic strategy in MLIV.

genetics↗

bioRxiv2020

The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice lightsheet microscopy

Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

cell biology↗

bioRxiv2020

Human brain organoids reveal accelerated development of cortical neuron classes as a shared feature of autism risk genes

Genetic risk for autism spectrum disorder (ASD) has been associated with hundreds of genes spanning a wide range of biological functions. The phenotypic alterations in the human brain resulting from mutations in ASD risk genes remain unclear, and the level at which these alterations converge on shared disease pathology is poorly understood. Here, we leveraged reproducible organoid models of the human cerebral cortex to identify cell type-specific developmental abnormalities associated with haploinsufficiency in three ASD risk genes, SUV420H1 (KMT5B), PTEN, and CHD8. We performed comprehensive single-cell RNA-sequencing (scRNA-seq) of over 400,000 cells, and proteomic analysis on individual organoids sampled at different developmental stages to investigate phenotypic convergence among these genes. We find that within a defined period of early cortical development, each of the three mutations demonstrates accelerated development of cortical neurons. Notably, they do so by affecting different neuronal populations: excitatory deep layer (SUV420H1) and callosal (PTEN) neurons, and inhibitory interneurons (CHD8). This work shows that haploinsufficiency in ASD risk genes converge on early developmental defects in the generation of neurons of the cortical microcircuit.

developmental biology↗

bioRxiv2020

Human antigen-specific memory natural killer cell responses develop against HIV-1 and influenza virus and are dependent on MHC-E restriction

For over a decade, multiple studies have disputed the notion of natural killer (NK) cells as purely innate lymphocytes by demonstrating that they are capable of putative antigen-specific immunological memory against multiple infectious agents including two critical global health priorities - HIV and influenza. However, the mechanisms underlying antigen specificity remain unknown. Herein, we demonstrate that antigen-specific human NK cell memory develops upon exposure to both HIV and influenza, unified by a conserved and epitope-specific targetable mechanism firmly dependent on the activating CD94/NKG2C receptor and its ligand HLA-E, and confirm these findings by three rigorous and novel assays. We validated the permanent acquisition of antigen-specificity by individual memory NK cells by single-cell cloning. We identified biomarkers of antigen-specific NK cell memory through RNA-Seq transcriptomic fingerprints and complex immunophenotyping by 30-parameter flow cytometry showing elevated expression of KLRG1, 4{beta}7 and NKG2C. Finally, we show individual HLA-E-restricted peptides that may constitute the dominant response in HIV-1- and influenza-infected persons in vivo. Our findings clarify the mechanisms behind formation of antigen-specific memory NK cells, and suggest they could be targeted for future vaccines, cure strategies, or other therapeutic interventions.

immunology↗