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Johnson, C.

Publications and source records attributed to Johnson, C..

At least 73 records · Page 4Linked to original sources

Artificial intelligence-driven morphology-based enrichment of malignant cells from body fluid

Cell morphology is a fundamental feature used to evaluate patient specimens in pathological analysis. However, traditional cytopathology analysis of patient effusion samples is limited by low tumor cell abundance coupled with high background of non-malignant cells, restricting the ability for downstream molecular and functional analyses to identify actionable therapeutic targets. We applied the Deepcell platform that combines microfluidic sorting, brightfield imaging, and real-time deep learning interpretations based on multi-dimensional morphology to enrich carcinoma cells from malignant effusions without cell staining or labels. Carcinoma cell enrichment was validated with whole genome sequencing and targeted mutation analysis, which showed higher sensitivity for detection of tumor fractions and critical somatic variant mutations that were initially at low-levels or undetectable in pre-sort patient samples. Combined, our study demonstrates the feasibility and added value of supplementing traditional morphology-based cytology with deep learning, multi-dimensional morphology analysis, and microfluidic sorting.

pathology↗

Comprehensive approach to study branched ubiquitinchains reveals roles for K48-K63 branches inVCP/p97-related processes

Branched ubiquitin (Ub) chains make up a significant proportion of Ub polymers in human cells and are formed when two or more sites on a single Ub molecule are modified with Ub creating bifurcated architectures. Despite their abundance, we have a poor understanding of the cellular functions of branched Ub signals that stems from a lack of facile tools and methods to study them. Here we develop a comprehensive pipeline to define branched Ub function, using K48-K63-branched chains as a case study. We discover branch-specific binders and, by developing a method that monitors cleavage of linkages within complex polyUb, we discover the VCP/p97-associated ATXN3, and MINDY family deubiquitinases to act as debranching enzymes. By engineering and utilizing a branched K48-K63-Ub chain-specific nanobody, we reveal roles for these chains in VCP/p97-related processes. In summary, we provide a blueprint to investigate branched Ub function that can be readily applied to study other branched chain types. HighlightsO_LIAssembly of defined branched ubiquitin chains enables identification of specific binding proteins C_LIO_LIDevelopment of quantitative DUB assay monitoring cleavage of individual Ub linkages within complex ubiquitin chains identifies debranching enzymes C_LIO_LIEngineering specific, high-affinity nanobody against branched K48-K63 ubiquitin reveals roles in VCP/p97 related processes and DNA damage responses C_LIO_LIGeneral blueprint of new methods and tools for in-depth characterization of branched ubiquitin chains and their underlying biology C_LI

biochemistry↗

Mass red blood cell extravasation and tubular uptake results in toxic injury to the tubules during kidney ischemia from venous clamping

Vascular congestion is common in ischemic acute kidney injury (AKI) and represents densely packed red blood cells (RBC) in the kidney circulation. In this study we tested the hypothesis that vascular congestion directly promotes tubular injury. Studies were performed in male and female Wistar-Kyoto rats. Vascular congestion and tubular injury were examined between renal venous clamping, arterial clamping and venous clamping of blood perfused and blood free kidneys. Vessels were occluded for either 15 or 45 minutes without reperfusion. We found that venous clamping resulted in greater vascular congestion than arterial clamping, particularly in the outer-medullary region (P<0.001). Venous clamping resulted in significant tubular injury, including cell swelling, tubular degeneration and luminal cast formation following as little as 15 minutes of occlusion. Tubular injury was significantly less following arterial clamping (P<0.001). Numerous red droplets were observed within tubular cells which were most prominent following venous clamping. Electron microscopy and immunohistochemistry identified these as derived from RBCs and indicated that RBCs from congested renal capillaries were extravasated and phagocytosed by tubular cells. CD235a staining confirmed tubular uptake and secretion of RBCs. Cast formation and tubular swelling were absent from blood free kidneys following venous clamping (P<0.001). Our data demonstrate that congestion of the kidney results in the rapid, mass extravasation and uptake of RBCs by tubular cells causing toxic injury to the tubules. Tubular toxicity from extravasation of RBCs appears to be a major component of tubular injury in ischemic AKI which has not previously been recognized.

physiology↗

TidyMass: An Object-oriented Reproducible Analysis Framework for LC-MS Data

Reproducibility and transparency have been longstanding but significant problems for the metabolomics field. Here, we present the tidyMass project (https://www.tidymass.org/), a comprehensive computational framework that can achieve the shareable and reproducible workflow needs of data processing and analysis for LC-MS-based untargeted metabolomics. TidyMass was designed based on the following strategies to address the limitations of current tools: 1) Cross-platform utility. TidyMass can be installed on all platforms; 2) Uniformity, shareability, traceability, and reproducibility. A uniform data format has been developed, specifically designed to store and manage processed metabolomics data and processing parameters, making it possible to trace the prior analysis steps and parameters; 3) Flexibility and extensibility. The modular architecture makes tidyMass a highly flexible and extensible tool, so other users can improve it and integrate it with their own pipeline easily.

bioinformatics↗

Maternal diet and gut microbiota influence predisposition to cardiovascular disease in the offspring

Cardiovascular disease is one of the most significant causes of death globally, especially in regions where unhealthy diets are prevalent and dietary fibre intake is low.1,2 Fibre, particularly prebiotic types that feed gut microbes, is essential for maintaining healthy gut microbial ecosystems.3 One assumption has been that cardiovascular health relates directly to lifestyle choices in adult life. Here, we show in mice that some of these benefits operate from the prenatal stage and relate to the diet and gut microbiome of the mother. Intake of fibre during pregnancy shaped the mothers gut microbiome, which had a lasting founding effect on the offsprings microbial composition and function. Maternal fibre intake during pregnancy significantly changed the cardiac cellular and molecular landscape in the offspring, protecting them against the development of cardiac hypertrophy, remodelling, and inflammation. These suggest a role for foetal exposure to maternal-derived gut microbial metabolites, which are known to cross the placenta and drive epigenetic changes. Maternal fibre intake led to foetal epigenetic reprogramming of the atrial natriuretic peptide gene (Nppa), protective against heart failure. These results underscore the importance of dietary intake and the gut microbiome of the mother during pregnancy for cardiovascular disease in the offspring.

microbiology↗

Realtime morphological characterization and sorting of unlabeled viable cells using deep learning

Phenotyping of single cells has dramatically lagged advances in molecular characterization and remains a manual, subjective, and destructive process. We introduce COSMOS, a platform for phenotyping and enrichment of cells based on deep learning interpretation of high-content morphology data in realtime. By training models on an atlas of >1.5 billion images, we demonstrate enrichment of unlabeled cells up to 33,000 fold. We apply COSMOS to multicellular tissue biopsy samples demonstrating that it can identify malignant cells with similar accuracy to molecular approaches while enriching viable cells for functional evaluation. We show high-dimensional embedding vectors of morphology generated by COSMOS without any need for complex sample pre-processing, gating, or bioinformatics capabilities, which enables discovery of cellular phenotypes, and integration of morphology into multi-dimensional analyses. One sentence summaryA novel platform capable of high-throughput imaging and gently sorting cells using deep morphological assessment.

cell biology↗

Genetic variation of human myokine signaling is dominated by biologic sex and sex hormones

Abstract/IntroductionProteins secreted from skeletal muscle, termed myokines, allow muscle to impact systemic physiology and disease. Myokines play critical roles in a variety of processes, including metabolic homeostasis, exercise improvements, inflammation, cancer and cognitive functions1-6. Despite the clear relevance of these factors in mediating a multitude of physiological outcomes, the genetic architecture, regulation and functions of myokines, as well as degree of conservation of these communication circuits remains inadequately understood. Given that biologic sex controls critical aspects of nearly every physiologic outcome, it is essential to consider when relating specific mechanisms to complex genetic and metabolic interactions. Specifically, many metabolic traits impacted by myokines show striking sex differences arising from hormonal7-10, genetic7,11 or gene-by-sex interactions12,13. In this study, we performed a genetic survey of myokine gene regulation and cross-tissue signaling in humans where sex as a biological variable was emphasized. While expression levels of a majority of myokines and cell proportions within skeletal muscle showed little differences between males and females, nearly all significant cross-tissue enrichments operated in a sex-specific or hormone-dependent fashion; in particular, with estrogens. These sex- and hormone-specific effects were consistent across key metabolic tissues: liver, pancreas, hypothalamus, intestine, heart, visceral and subcutaneous adipose tissue. Skeletal muscle estrogen receptor enrichments across metabolic tissues appeared stronger than androgen receptor and, surprisingly, ~3-fold higher in males compared to females. To define the causal roles of estrogen signaling on myokine gene expression and functions, we generated male and female mice which lack estrogen receptor (Esr1) specifically in skeletal muscle and integrated global RNA-Sequencing with human data. These analyses highlighted mechanisms of sex-dependent myokine signaling conserved between species, such as myostatin enriched for divergent substrate utilization pathways between sexes. Several other sex-dependent mechanisms of myokine signaling were uncovered, such as muscle-derived TNF exerting stronger inflammatory signaling in females compared to males and GPX3 as a male-specific link between glycolytic fiber abundance and hepatic inflammation. Collectively, we provide the first genetic survey of human myokines and highlight sex and estrogen receptor signaling as critical variables when assaying myokine functions and how changes in cell composition impact other metabolic organs.

physiology↗

Structural Basis for an Unprecedented Enzymatic Alkylation in Cylindrocyclophane Biosynthesis

The cyanobacterial enzyme CylK assembles the cylindrocyclophane natural products by performing two unusual alkylation reactions, forming new carbon-carbon bonds between aromatic rings and secondary alkyl halide substrates. This transformation is unprecedented in biology and the structure and mechanism of CylK are unknown. Here, we report x-ray crystal structures of CylK, revealing a distinctive fusion of a Ca2+ binding domain and a {beta}-propeller fold. We use a mutagenic screening approach to locate CylKs active site at its domain interface, identifying two residues, Arg105 and Tyr473, that are required for catalysis. Anomalous diffraction datasets collected with bound bromide ions, a product analog, suggest these residues interact with the alkyl halide electrophile. Additional mutagenesis and molecular dynamics simulations implicates Asp440 and Glu374 in activating the nucleophilic aromatic ring. Bioinformatic analysis of CylK homologs from other cyanobacteria establishes that they conserve these key catalytic amino acids but they are likely associated with divergent reactivity and altered secondary metabolism. By gaining a molecular understanding of this unusual biosynthetic transformation, this work fills a gap in our understanding of how alkyl halides are activated and used by enzymes as biosynthetic intermediates, informing enzyme engineering, catalyst design, and natural product discovery.

biochemistry↗

Progressive protein aggregation in PRPF31 patient retinal pigment epithelium cells: the mechanism and its reversal through activation of autophagy

Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. Here, we used iPSC technology to generate retinal organoids and RPE models from three patients with severe and very severe PRPF31-adRP, normal individuals and a CRISPR/Cas9-corrected isogenic control. To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells was carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle proteins, which accumulated progressively with time. Mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wildtype levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells with reduced U4/U6 snRNPs and accumulation of U5, smaller nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin resulted in reduction of cytoplasmic aggregates and improved cell survival. Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients. HighlightsO_LIPRPF31 RP mutations lead to formation of insoluble aggregates containing the mutant PRPF31 and misfolded, ubiquitin conjugated proteins including key visual cycle proteins (e.g. RLBP1) in RPE cells, which accumulate progressively with time and affect tight junctions and cell survival. C_LIO_LIMutant PRPF31 is predominantly localised in cytoplasmic aggregates of patient specific RPE and retinal cells and is not able to be incorporated into splicing complexes to cause direct mis-splicing. C_LIO_LIHigh-throughput quantitative proteomics identifies significantly altered RNA splicing, visual perception, retinoid metabolism, waste disposal and unfolded protein response pathways in patient RPE cells, and autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways in photoreceptor cells. C_LIO_LIAccumulation of PRPF31 mutant variant as cytoplasmic aggregates reduces wildtype PRPF31 in the nucleus leading to tri-snRNP assembly defects, characterised by accumulation of U5 and reduction of U4/U6 snRNPs in Cajal bodies, altered morphology of nuclear speckles and consequently downregulation of active spliceosomes (Bact and C complexes) in PRPF31 patient RPE and retinal cells. C_LIO_LIProteomic study of insoluble aggregates identifies other RP-linked splicing factors and multiple key retinal-specific proteins, whose variants are linked to retinitis pigmentosa, within the aggregates of patient RPE cells. C_LIO_LIPRPF31 patient RPE cells have impaired waste disposal and proteasome mediated degradation, which together with the impaired autophagy pathway, further exacerbate aggregate formation. C_LIO_LIPhagocytosis of photoreceptor outer segment fragments (POS) shed daily by RPE cells accelerates aggregation of key proteins indicating enhanced cytoplasmic aggregate formation under physiological conditions in patient RPE cells. C_LIO_LIActivation of autophagy via administration of rapamycin results in reduction of cytoplasmic aggregates in RPE cells, correct localisation of mislocated and misfolded proteins to the nucleus, thereby improving cell survival. C_LI

cell biology↗

A framework for evaluating edited cell libraries created by massively parallel genome engineering

Genome engineering methodologies are transforming biological research and discovery. Approaches based on CRISPR technology have been broadly adopted and there is growing interest in the generation of massively parallel edited cell libraries. Comparing the libraries generated by these varying approaches is challenging and researchers lack a common framework for defining and assessing the characteristics of these libraries. Here we describe a framework for evaluating massively parallel libraries of edited genomes based on established methods for sampling complex populations. We define specific attributes and metrics that are informative for describing a complex cell library and provide examples for estimating these values. We also connect this analysis to generic phenotyping approaches, using either pooled (typically via a selection assay) or isolate (often referred to as screening) phenotyping approaches. We approach this from the context of creating massively parallel, precisely edited libraries with one edit per cell, though the approach holds for other types of modifications, including libraries containing multiple edits per cell (combinatorial editing). This framework is a critical component for evaluating and comparing new technologies as well as understanding how a massively parallel edited cell library will perform in a given phenotyping approach.

genomics↗

Redox-mediated inactivation of the transcriptional repressor C3600 makes uropathogenic Escherichia coli exquisitely resistant to reactive chlorine species

The ability to overcome stressful environments is critical for pathogen survival in the host. One challenge for bacteria is the exposure to reactive chlorine species (RCS), which are generated by innate immune cells as critical part of the oxidative burst. Hypochlorous acid (HOCl) is the most potent antimicrobial RCS and associated with extensive macromolecular damage in the phagocytized pathogen. However, bacteria have evolved defense strategies to alleviate the effects of HOCl-mediated damage. Among these are RCS-sensing transcriptional regulators that control the expression of HOCl-protective genes under non- and HOCl stress. Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils during pathogenesis, however, their responses to and defenses of HOCl are still completely unexplored. Here, we present evidence that UPEC strains tolerate higher levels of HOCl and are better protected from neutrophil-mediated killing compared to other E. coli. Transcriptomic analysis of HOCl-stressed UPEC revealed the upregulation of an operon consisting of three genes, one of which encodes the transcriptional regulator C3600. We identified C3600 as a HOCl-sensing transcriptional repressor, which, under non-stress conditions, is bound to the operator and represses the expression of its target genes. During HOCl exposure, however, the repressor forms reversible intermolecular disulfide bonds and dissociates from the DNA resulting in the de-repression of the operon. Deletion of one of the target genes renders UPEC significantly more susceptible to HOCl indicating that the HOCl-mediated induction of the regulon plays a major role for UPECs HOCl resistance. IMPORTANCEHow do pathogens deal with antimicrobial oxidants produced by the innate immune system during infection? Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils and, therefore, must counter elevated levels of the antimicrobial oxidant HOCl to establish infection. Our study provides fundamentally new insights into a defense mechanism that enables UPEC to fend off the toxic effects of HOCl stress. Intriguingly, the defense system is predominantly found in UPEC and absent in non-invasive enteropathogenic E. coli. Our data suggest that expression of the target gene c3601 is exclusively responsible for UPECs increased HOCl tolerance in culture and therefore potentially contributes to UPECs survival during phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the bodys own capacity to fight UTIs.

microbiology↗

Protein prediction for trait mapping in diverse populations

Genetically regulated gene expression has helped elucidate the biological mechanisms underlying complex traits. Improved high-throughput technology allows similar interrogation of the genetically regulated proteome for understanding complex trait mechanisms. Here, we used the Trans-omics for Precision Medicine (TOPMed) Multi-omics pilot study, which comprises data from Multi-Ethnic Study of Atherosclerosis (MESA), to optimize genetic predictors of the plasma proteome for genetically regulated proteome-wide association studies (PWAS) in diverse populations. We built predictive models for protein abundances using data collected in TOPMed MESA, for which we have measured 1,305 proteins by a SOMAscan assay. We compared predictive models built via elastic net regression to models integrating posterior inclusion probabilities estimated by fine-mapping SNPs prior to elastic net. In order to investigate the transferability of predictive models across ancestries, we built protein prediction models in all four of the TOPMed MESA populations, African American (n=183), Chinese (n=71), European (n=416), and Hispanic/Latino (n=301), as well as in all populations combined. As expected, fine-mapping produced more significant protein prediction models, especially in African ancestries populations, potentially increasing opportunity for discovery. When we tested our TOPMed MESA models in the independent European INTERVAL study, fine-mapping improved cross-ancestries prediction for some proteins. Using GWAS summary statistics from the Population Architecture using Genomics and Epidemiology (PAGE) study, which comprises ~50,000 Hispanic/Latinos, African Americans, Asians, Native Hawaiians, and Native Americans, we applied S-PrediXcan to perform PWAS for 28 complex traits. The most protein-trait associations were discovered, colocalized, and replicated in large independent GWAS using proteome prediction model training populations with similar ancestries to PAGE. At current training population sample sizes, performance between baseline and fine-mapped protein prediction models in PWAS was similar, highlighting the utility of elastic net. Our predictive models in diverse populations are publicly available for use in proteome mapping methods at https://doi.org/10.5281/zenodo.4837328. Author summaryGene regulation is a critical mechanism underlying complex traits. Transcriptome-wide association studies (TWAS) have helped elucidate potential mechanisms because each association connects a gene rather than a variant to the complex trait. Like genome-wide association studies (GWAS), most TWAS are still conducted exclusively in populations of European ancestry, which misses the opportunity to test the full spectrum of human genetic variation for associations with complex traits. Here, move beyond the transcriptome and because protein measurement assays are growing to allow interrogation of the proteome, we use data from TOPMed MESA to develop genetic predictors of protein abundance in diverse ancestry populations. We compare model-building strategies with the goal of providing the best resource for protein association discovery with available data. We demonstrate how these prediction models can be used to perform proteome-wide association studies (PWAS) in diverse populations. We show the most protein-trait associations were discovered, colocalized, and replicated in independent cohorts using proteome prediction model training populations with similar ancestries to individuals in the GWAS. We shared our protein prediction models and performance statistics publicly to facilitate future proteome mapping studies in diverse populations.

genomics↗

Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids

BackgroundAccurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that allows flexibility for high and mid-throughput analysis for three blood-based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and dried blood. MethodsOptimal conditions for sample preparation and DIA-MS analysis were established in plasma then automated and adapted for depleted plasma and whole blood. The MS workflow was modified to facilitate sensitive high-throughput or deep profile analysis with mid-throughput analysis. Analytical performance was evaluated from 5 complete workflows repeated over 3 days as well as a linearity analysis of a 5-6-point dilution curve. ResultUsing our high-throughput workflow, 74%, 93%, 87% of peptides displayed an inter-day CV<30% in plasma, depleted plasma and whole blood. While the mid-throughput workflow had 67%, 90%, 78% of peptides in plasma, depleted plasma and whole blood meeting the CV<30% standard. Lower limits of detection and quantitation were determined for proteins and peptides observed in each biofluid and workflow. Combining the analysis of both high-throughput plasma fractions exceeded the number of reliably identified proteins for individual biofluids in the mid-throughput workflows. ConclusionThe workflow established here allowed for reliable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow on a large scale will facilitate the translation of candidate markers into clinical use.

biochemistry↗

Deficiency of MicroRNA-181a Results in Transcriptome-Wide Cell Specific Changes in the Kidney that Lead to Elevated Blood Pressure and Salt Sensitivity

MicroRNA miR-181a is down-regulated in the kidneys of hypertensive patients and hypertensive mice. In vitro, miR-181a is a posttranslational inhibitor of renin expression, but pleiotropic mechanisms by which miR-181a may influence blood pressure (BP) are unknown. Here we determined whether deletion of miR-181a/b-1 in vivo changes BP and the molecular mechanisms involved at the single-cell level. We developed a knockout mouse model lacking miR-181a/b-1 genes using CRISPR/Cas9 technology. Radio-telemetry probes were implanted in twelve-week-old C57BL/6J wild-type and miR-181a/b-1 knockout mice. Systolic and diastolic BP were 4-5mmHg higher in knockout compared with wild-type mice over 24-hours (P<0.01). Compared with wild-type mice, renal renin was higher in the juxtaglomerular cells of knockout mice. BP was similar in wild-type mice on a high (3.1%) versus low (0.3%) sodium diet (+0.4{+/-}0.8mmHg) but knockout mice showed salt sensitivity (+3.3{+/-}0.8mmHg, P<0.001). Since microRNAs can target several mRNAs simultaneously, we performed single-nuclei RNA-sequencing in 6,699 renal cells. We identified 12 distinct types of renal cells, all of which had genes that were dysregulated. This included genes involved in renal fibrosis and inflammation such as Stat4, Col4a1, Cd81, Flt3l, Cxcl16, Smad4. We observed up-regulation of pathways related to the immune system, inflammatory response, reactive oxygen species and nerve development, consistent with higher tyrosine hydroxylase. In conclusion, downregulation of the miR-181a gene led to increased BP and salt sensitivity in mice. This is likely due to an increase in renin expression in juxtaglomerular cells, as well as microRNA-driven pleiotropic effects impacting renal pathways associated with hypertension.

genomics↗

TRIPLE-NEGATIVE BREAST CANCER CELLS RECRUIT NEUTROPHILS BY SECRETING TGF-β AND CXCR2 LIGANDS

Tumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO (CXCL1/2/3) chemokines and TGF-{beta} cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-{beta}, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-{beta} and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.

immunology↗

A novel SARS-CoV-2 related coronavirus in bats from Cambodia

Knowledge of the origin and reservoir of the coronavirus responsible for the ongoing COVID-19 pandemic is still fragmentary. To date, the closest relatives to SARS-CoV-2 have been detected in Rhinolophus bats sampled in the Yunnan province, China. Here we describe the identification of SARS-CoV-2 related coronaviruses in two Rhinolophus shameli bats sampled in Cambodia in 2010. Metagenomic sequencing identified nearly identical viruses sharing 92.6% nucleotide identity with SARS-CoV-2. Most genomic regions are closely related to SARS-CoV-2, with the exception of a small region corresponding to the spike N terminal domain. The discovery of these viruses in a bat species not found in China indicates that SARS-CoV-2 related viruses have a much wider geographic distribution than previously understood, and suggests that Southeast Asia represents a key area to consider in the ongoing search for the origins of SARS-CoV-2, and in future surveillance for coronaviruses.

microbiology↗

Sahul's megafauna were vulnerable to extinction due to their position in the trophic network

Extinctions stemming from environmental change often trigger trophic cascades and coextinctions. However, it remains unclear whether trophic cascades were a large contributor to the megafauna extinctions that swept across several continents in the Late Pleistocene. The pathways to megafauna extinctions are particularly unclear for Sahul (landmass comprising Australia and New Guinea), where extinctions happened earlier than on other continents. We investigated the role of bottom-up trophic cascades in Late Pleistocene Sahul by constructing pre-extinction (~ 80 ka) trophic network models of the vertebrate community of Naracoorte, south-eastern Australia. These models allowed us to predict vertebrate species vulnerability to cascading extinctions based on their position in the network. We tested whether the observed extinctions could be explained by bottom-up cascades, or if they should be attributed to other external causes. Species that disappeared from the community were more vulnerable, overall, to bottom-up cascades than were species that survived. The position of extinct species in the network - having few or no predators - also suggests they might have been particularly vulnerable to a new predator. These results provide quantitative evidence that trophic cascades and naivety to predators could have contributed to the megafauna extinction event in Sahul.

ecology↗

Distinct VIP interneurons in the cingulate cortex encode anxiogenic and social stimuli

A hallmark of higher-order cortical regions is their functional heterogeneity, but it is not well understood how these areas encode such diverse information. The anterior cingulate cortex (ACC), for example, is important in both emotional regulation and social cognition. Previous work shows activation of the ACC to anxiety-related and social stimuli, but it is unknown how subpopulations or microcircuits within the ACC simultaneously encode these distinct stimuli. One type of inhibitory interneuron, which is positive for vasoactive intestinal peptide (VIP), is known to alter the activity of many cells in local cortical microcircuits, but it is unknown whether the activity of VIP cells in the ACC (VIPACC) encodes anxiety-related or social information. Using in vivo calcium imaging and miniscopes in freely behaving mice to monitor VIPACC activity, we identified distinct, non-overlapping subpopulations of VIPACC that preferentially activated to either anxiogenic, anxiolytic, social, or non-social stimuli. We determined that stimulus-selective cells encode the animals behavioral states and VIP interneuron clusters may co-activate, improving this encoding. Finally, we used trans-synaptic tracing to show that VIPACC receive widespread inputs from regions implicated in emotional regulation and social cognition. These findings demonstrate not only that the ACC is not homogeneous in its function, but also that there is marked functional heterogeneity even within disinhibitory interneuron populations. This work contributes to our understanding of how the cortex encodes information across diverse contexts and provides insight into the complexity of neural processes involved in anxiety and social behavior.

neuroscience↗