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Spaceflight induces molecular, cellular and physiological shifts in astronauts and poses myriad biomedical challenges to the human body, which are becoming increasingly relevant as more humans venture into space1-6. Yet current frameworks for aerospace medicine are nascent and lag far behind advancements in precision medicine on Earth, underscoring the need for rapid development of space medicine databases, tools and protocols. Here we present the Space Omics and Medical Atlas (SOMA), an integrated data and sample repository for clinical, cellular and multi-omic research profiles from a diverse range of missions, including the NASA Twins Study7, JAXA CFE study8,9, SpaceX Inspiration4 crew10-12, Axiom and Polaris. The SOMA resource represents a more than tenfold increase in publicly available human space omics data, with matched samples available from the Cornell Aerospace Medicine Biobank. The Atlas includes extensive molecular and physiological profiles encompassing genomics, epigenomics, transcriptomics, proteomics, metabolomics and microbiome datasets, which reveal some consistent features across missions, including cytokine shifts, telomere elongation and gene expression changes, as well as mission-specific molecular responses and links to orthologous, tissue-specific mouse datasets. Leveraging the datasets, tools and resources in SOMA can help to accelerate precision aerospace medicine, bringing needed health monitoring, risk mitigation and countermeasure data for upcoming lunar, Mars and exploration-class missions.
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Tuberculosis (TB) remains a leading cause of death from an infectious disease worldwide, partly due to a lack of effective strategies to screen and triage individuals with potential TB. Whole blood RNA signatures have been tested as biomarkers for TB, but have failed to meet the World Health Organization's (WHO) optimal target product profiles (TPP). Here, we use RNA sequencing and machine-learning to investigate the utility of plasma cell-free RNA (cfRNA) as a host-response biomarker for TB in cohorts from Uganda, Vietnam and Philippines. We report a 6-gene cfRNA signature, which differentiates TB-positive and TB-negative individuals with AUC = 0.95, 0.92, and 0.95 in test, training and validation, respectively. This signature meets WHO TPPs (sensitivity: 97.1% [95% CI: 80.9-100%], specificity: 85.2% [95% CI: 72.4-100%]) regardless of geographic location, sample collection method and HIV status. Overall, our results identify plasma cfRNA as a promising host response biomarker to diagnose TB.
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Biomarcadores , Ácidos Nucleicos Libres de Células , Tuberculosis , Humanos , Ácidos Nucleicos Libres de Células/sangre , Biomarcadores/sangre , Tuberculosis/diagnóstico , Tuberculosis/sangre , Uganda/epidemiología , Masculino , Femenino , Vietnam , Adulto , Aprendizaje Automático , Filipinas , Mycobacterium tuberculosis/genética , Sensibilidad y Especificidad , Persona de Mediana Edad , Análisis de Secuencia de ARN/métodos , Estudios de CohortesRESUMEN
Spaceflight induces an immune response in astronauts. To better characterize this effect, we generated single-cell, multi-ome, cell-free RNA (cfRNA), biochemical, and hematology data for the SpaceX Inspiration4 (I4) mission crew. We found that 18 cytokines/chemokines related to inflammation, aging, and muscle homeostasis changed after spaceflight. In I4 single-cell multi-omics data, we identified a "spaceflight signature" of gene expression characterized by enrichment in oxidative phosphorylation, UV response, immune function, and TCF21 pathways. We confirmed the presence of this signature in independent datasets, including the NASA Twins Study, the I4 skin spatial transcriptomics, and 817 NASA GeneLab mouse transcriptomes. Finally, we observed that (1) T cells showed an up-regulation of FOXP3, (2) MHC class I genes exhibited long-term suppression, and (3) infection-related immune pathways were associated with microbiome shifts. In summary, this study reveals conserved and distinct immune disruptions occurring and details a roadmap for potential countermeasures to preserve astronaut health.
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Análisis de la Célula Individual , Vuelo Espacial , Transcriptoma , Animales , Femenino , Masculino , Humanos , Ratones , Astronautas , Citocinas/metabolismo , Linfocitos T/inmunología , Factores Sexuales , Perfilación de la Expresión Génica , Fosforilación OxidativaRESUMEN
Urine is a rich source of nucleic acid biomarkers including cell-free DNA (cfDNA) and RNA for monitoring the health of kidney allografts. In this study, we aimed to evaluate whether urine filtration can serve as an alternative to the commonly used method of centrifugation to collect urinary fluid and cell pellets for isolating cfDNA and cellular messenger RNA (mRNA). We collected urine specimens from kidney allograft recipients and obtained the urine supernatant and cell pellet from each specimen using both filtration and centrifugation for paired analyses. We performed DNA sequencing to characterize the origin and properties of cfDNA, as well as quantitative PCR of mRNAs extracted from cell fractions. Our results showed that the biophysical properties of cfDNA, the microbial DNA content, and the tissues of origin of cfDNA were comparable between samples processed using filtration and centrifugation method. Similarly, mRNA quality and quantity obtained using both methods met our criteria for downstream application and the Ct values for each mRNA were comparable between the two techniques.The Ct values demonstrated a high degree of correlation. These findings suggest that urine filtration is a viable alternative to urine centrifugation for isolation of nucleic acid biomarkers from urine specimens.
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Biomarcadores , Ácidos Nucleicos Libres de Células , Centrifugación , Filtración , Trasplante de Riñón , Humanos , Centrifugación/métodos , Biomarcadores/orina , Filtración/métodos , Ácidos Nucleicos Libres de Células/orina , Ácidos Nucleicos Libres de Células/aislamiento & purificación , Ácidos Nucleicos Libres de Células/análisis , ARN Mensajero/genética , ARN Mensajero/orina , Masculino , Femenino , Persona de Mediana Edad , Adulto , Orina/químicaRESUMEN
Hematopoietic Stem Cell Transplantation (HSCT) is one of the oldest and most successful immunotherapies. Yet, despite long-standing success in the use of HSCT for the treatment of blood cancers and severe immune disorders, monitoring post-transplant complications remains a challenge due to a lack of informative diagnostic tests. Here, we investigate the utility of cell-free RNA (cfRNA) in plasma as a liquid biopsy to monitor allogeneic HSCT recipients during and after treatment. We assayed longitudinal samples from 92 HSCT recipients by cfRNA sequencing and show that cfRNA provides insight into treatment and recovery trajectories, immune dynamics in response to transplantation, infection, and solid-tissue injury associated with Graft-Versus-Host Disease. Collectively, our results provide support for the use of plasma cfRNA profiling to monitor complications of HSCT.
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Inflammatory syndromes, including those caused by infection, are a major cause of hospital admissions among children and are often misdiagnosed because of a lack of advanced molecular diagnostic tools. In this study, we explored the utility of circulating cell-free RNA (cfRNA) in plasma as an analyte for the differential diagnosis and characterization of pediatric inflammatory syndromes. We profiled cfRNA in 370 plasma samples from pediatric patients with a range of inflammatory conditions, including Kawasaki disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), viral infections and bacterial infections. We developed machine learning models based on these cfRNA profiles, which effectively differentiated KD from MIS-C - two conditions presenting with overlapping symptoms - with high performance (Test Area Under the Curve (AUC) = 0.97). We further extended this methodology into a multiclass machine learning framework that achieved 81% accuracy in distinguishing among KD, MIS-C, viral, and bacterial infections. We further demonstrated that cfRNA profiles can be used to quantify injury to specific tissues and organs, including the liver, heart, endothelium, nervous system, and the upper respiratory tract. Overall, this study identified cfRNA as a versatile analyte for the differential diagnosis and characterization of a wide range of pediatric inflammatory syndromes.
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Differential host responses in coronavirus disease 2019 (COVID-19) and multisystem inflammatory syndrome in children (MIS-C) remain poorly characterized. Here, we use next-generation sequencing to longitudinally analyze blood samples from pediatric patients with COVID-19 or MIS-C across three hospitals. Profiling of plasma cell-free nucleic acids uncovers distinct signatures of cell injury and death between COVID-19 and MIS-C, with increased multiorgan involvement in MIS-C encompassing diverse cell types, including endothelial and neuronal cells, and an enrichment of pyroptosis-related genes. Whole-blood RNA profiling reveals upregulation of similar pro-inflammatory pathways in COVID-19 and MIS-C but also MIS-C-specific downregulation of T cell-associated pathways. Profiling of plasma cell-free RNA and whole-blood RNA in paired samples yields different but complementary signatures for each disease state. Our work provides a systems-level view of immune responses and tissue damage in COVID-19 and MIS-C and informs future development of new disease biomarkers.
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COVID-19 , Ácidos Nucleicos Libres de Células , Ácidos Nucleicos , Humanos , Niño , COVID-19/genética , ARN , BiomarcadoresRESUMEN
Tuberculosis (TB) remains a leading cause of death from an infectious disease worldwide. This is partly due to a lack of tools to effectively screen and triage individuals with potential TB. Whole blood RNA signatures have been extensively studied as potential biomarkers for TB, but they have failed to meet the World Health Organization's (WHOs) target product profiles (TPPs) for a non-sputum triage or diagnostic test. In this study, we investigated the utility of plasma cell-free RNA (cfRNA) as a host response biomarker for TB. We used RNA profiling by sequencing to analyze plasma samples from 182 individuals with a cough lasting at least two weeks, who were seen at outpatient clinics in Uganda, Vietnam, and the Philippines. Of these individuals, 100 were diagnosed with microbiologically-confirmed TB. Our analysis of the plasma cfRNA transcriptome revealed 541 differentially abundant genes, the top 150 of which were used to train 15 machine learning models. The highest performing model led to a 9-gene signature that had a diagnostic accuracy of 89.1% (95% CI: 83.6-93.4%) and an area under the curve of 0.934 (95% CI: 0.8674-1) for microbiologically-confirmed TB. This 9-gene signature exceeds the optimal WHO TPPs for a TB triage test (sensitivity: 96.2% [95% CI: 80.9-100%], specificity: 89.7% [95% CI: 72.4-100%]) and was robust to differences in sample collection, geographic location, and HIV status. Overall, our results demonstrate the utility of plasma cfRNA for the detection of TB and suggest the potential for a point-of-care, gene expression-based assay to aid in early detection of TB.
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Tuberculosis (TB) remains a significant cause of mortality worldwide. Metagenomic next-generation sequencing has the potential to reveal biomarkers of active disease, identify coinfection, and improve detection for sputum-scarce or culture-negative cases. We conducted a large-scale comparative study of 428 plasma, urine, and oral swab samples from 334 individuals from TB endemic and non-endemic regions to evaluate the utility of a shotgun metagenomic DNA sequencing assay for tuberculosis diagnosis. We found that the composition of the control population had a strong impact on the measured performance of the diagnostic test: the use of a control population composed of individuals from a TB non-endemic region led to a test with nearly 100% specificity and sensitivity, whereas a control group composed of individuals from TB endemic regions exhibited a high background of nontuberculous mycobacterial DNA, limiting the diagnostic performance of the test. Using mathematical modeling and quantitative comparisons to matched qPCR data, we found that the burden of Mycobacterium tuberculosis DNA constitutes a very small fraction (0.04 or less) of the total abundance of DNA originating from mycobacteria in samples from TB endemic regions. Our findings suggest that the utility of a minimally invasive metagenomic sequencing assay for pulmonary tuberculosis diagnostics is limited by the low burden of M. tuberculosis and an overwhelming biological background of nontuberculous mycobacterial DNA.
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Mycobacterium tuberculosis , Tuberculosis , Biomarcadores , ADN , Humanos , Mycobacterium tuberculosis/genética , Micobacterias no Tuberculosas/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Esputo/microbiología , Tuberculosis/diagnóstico , Tuberculosis/microbiologíaRESUMEN
Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present Sample-Intrinsic microbial DNA Found by Tagging and sequencing (SIFT-seq) a metagenomic sequencing assay that is robust against environmental DNA contamination introduced during sample preparation. The core idea of SIFT-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. We applied SIFT-seq to screen for infections from microorganisms with low burden in blood and urine, to identify COVID-19 co-infection, to characterize the urinary microbiome, and to identify microbial DNA signatures of sepsis and inflammatory bowel disease in blood.
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COVID-19 , ADN Ambiental , ADN , Contaminación de ADN , ADN Bacteriano/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Metagenómica , Análisis de Secuencia de ADNRESUMEN
Allogeneic hematopoietic cell transplantation (HCT) provides effective treatment for hematologic malignancies and immune disorders. Monitoring of posttransplant complications is critical, yet current diagnostic options are limited. Here, we show that cell-free DNA (cfDNA) in blood is a versatile analyte for monitoring of the most important complications that occur after HCT: graft-versus-host disease (GVHD), a frequent immune complication of HCT, infection, relapse of underlying disease, and graft failure. We demonstrate that these therapeutic complications are informed from a single assay, low-coverage bisulfite sequencing of cfDNA, followed by disease-specific bioinformatic analyses. To inform GVHD, we profile cfDNA methylation marks to trace the cfDNA tissues-of-origin and to quantify tissue-specific injury. To inform infection, we implement metagenomic cfDNA profiling. To inform cancer relapse, we implement analyses of tumor-specific genomic aberrations. Finally, to detect graft failure, we quantify the proportion of donor- and recipient-specific cfDNA. We applied this assay to 170 plasma samples collected from 27 HCT recipients at predetermined timepoints before and after allogeneic HCT. We found that the abundance of solid-organ-derived cfDNA in the blood at 1 mo after HCT is predictive of acute GVHD (area under the curve, 0.88). Metagenomic profiling of cfDNA revealed the frequent occurrence of viral reactivation in this patient population. The fraction of donor-specific cfDNA was indicative of relapse and remission, and the fraction of tumor-specific cfDNA was informative of cancer relapse. This proof-of-principle study shows that cfDNA has the potential to improve the care of allogeneic HCT recipients by enabling earlier detection and better prediction of the complex array of complications that occur after HCT.
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Ácidos Nucleicos Libres de Células , Dermatoglifia del ADN , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Biomarcadores , Metilación de ADN , Progresión de la Enfermedad , Enfermedad Injerto contra Huésped/sangre , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Biopsia Líquida/métodos , Especificidad de Órganos/genética , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/etiología , Recurrencia , Trasplante HomólogoRESUMEN
RATIONALE & OBJECTIVE: Conventional culture can be insensitive for the detection of rare infections and for the detection of common infections in the setting of recent antibiotic usage. Patients receiving peritoneal dialysis (PD) with suspected peritonitis have a significant proportion of negative conventional cultures. This study examines the utility of metagenomic sequencing of peritoneal effluent cell-free DNA (cfDNA) for evaluating the peritoneal effluent in PD patients with and without peritonitis. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: We prospectively characterized cfDNA in 68 peritoneal effluent samples obtained from 33 patients receiving PD at a single center from September 2016 to July 2018. OUTCOMES: Peritoneal effluent, microbial, and human cfDNA characteristics were evaluated in culture-confirmed peritonitis and culture-negative peritonitis. ANALYTICAL APPROACH: Descriptive statistics were analyzed and microbial cfDNA was detected in culture-confirmed peritonitis and culture-negative peritonitis. RESULTS: Metagenomic sequencing of cfDNA was able to detect and identify bacterial, viral, and eukaryotic pathogens in the peritoneal effluent from PD patients with culture-confirmed peritonitis, as well as patients with recent antibiotic usage and in cases of culture-negative peritonitis. LIMITATIONS: Parallel cultures were not obtained in all the peritoneal effluent specimens. CONCLUSIONS: Metagenomic cfDNA sequencing of the peritoneal effluent can identify pathogens in PD patients with peritonitis, including culture-negative peritonitis.
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Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present contamination-free metagenomic DNA sequencing (Coffee-seq), a metagenomic sequencing assay that is robust against environmental contamination. The core idea of Coffee-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. We applied Coffee-seq to screen for infections from microorganisms with low burden in blood and urine, to identify COVID-19 co-infection, to characterize the urinary microbiome, and to identify microbial DNA signatures of inflammatory bowel disease in blood.
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BACKGROUND: Metagenomic sequencing of microbial cell-free DNA (cfDNA) in blood and urine is increasingly used as a tool for unbiased infection screening. The sensitivity of metagenomic cfDNA sequencing assays is determined by the efficiency by which the assay recovers microbial cfDNA vs host-specific cfDNA. We hypothesized that the choice of methods used for DNA isolation, DNA sequencing library preparation, and sequencing would affect the sensitivity of metagenomic cfDNA sequencing. METHODS: We characterized the fragment length biases inherent to select DNA isolation and library preparation procedures and developed a model to correct for these biases. We analyzed 305 cfDNA sequencing data sets, including publicly available data sets and 124 newly generated data sets, to evaluate the dependence of the sensitivity of metagenomic cfDNA sequencing on pre-analytical variables. RESULTS: Length bias correction of fragment length distributions measured from different experimental procedures revealed the ultrashort (<100 bp) nature of microbial-, mitochondrial-, and host-specific urinary cfDNA. The sensitivity of metagenomic sequencing assays to detect the clinically reported microorganism differed by more than 5-fold depending on the combination of DNA isolation and library preparation used. CONCLUSIONS: Substantial gains in the sensitivity of microbial and other short fragment recovery can be achieved by easy-to-implement changes in the sample preparation protocol, which highlights the need for standardization in the liquid biopsy field.
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Ácidos Nucleicos Libres de Células , Fragmentación del ADN , Análisis de Secuencia de ADN , Sesgo , Ácidos Nucleicos Libres de Células/genética , ADN , Humanos , Metagenómica/métodosRESUMEN
Many neurotransmitters are organic ions that carry a net charge, and their release from secretory vesicles is therefore an electrodiffusion process. The selectivity of early exocytotic fusion pores is investigated by combining electrodiffusion theory, measurements of amperometric foot signals from chromaffin cells with anion substitution, and molecular dynamics simulation. The results reveal that very narrow fusion pores are cation selective, but more dilated fusion pores become anion permeable. The transition occurs around a fusion pore conductance of â¼300 pS. The cation selectivity of a narrow fusion pore accelerates the release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin, and acetylcholine, while glutamate release may require a more dilated fusion pore.
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Células Cromafines/metabolismo , Potenciales de la Membrana , Neurotransmisores/metabolismo , Vesículas Secretoras/metabolismo , Animales , BovinosRESUMEN
BACKGROUND: Coronavirus disease 2019 (COVID-19) primarily affects the lungs, but evidence of systemic disease with multi-organ involvement is emerging. Here, we developed a blood test to broadly quantify cell-, tissue-, and organ-specific injury due to COVID-19. METHODS: Our test leverages genome-wide methylation profiling of circulating cell-free DNA in plasma. We assessed the utility of this test to identify subjects with severe disease in two independent, longitudinal cohorts of hospitalized patients. Cell-free DNA profiling was performed on 104 plasma samples from 33 COVID-19 patients and compared to samples from patients with other viral infections and healthy controls. FINDINGS: We found evidence of injury to the lung and liver and involvement of red blood cell progenitors associated with severe COVID-19. The concentration of cell-free DNA correlated with the World Health Organization (WHO) ordinal scale for disease progression and was significantly increased in patients requiring intubation. CONCLUSIONS: This study points to the utility of cell-free DNA as an analyte to monitor and study COVID-19. FUNDING: This work was supported by NIH grants 1DP2AI138242 (to I.D.V.), R01AI146165 (to I.D.V., M.P.C., F.M.M., and J.R.), 1R01AI151059 (to I.D.V.), K08-CA230156 (to W.G.), and R33-AI129455 to C.Y.C., a Synergy award from the Rainin Foundation (to I.D.V.), a SARS-CoV-2 seed grant at Cornell (to I.D.V.), a National Sciences and Engineering Research Council of Canada fellowship PGS-D3 (to A.P.C.), and a Burroughs-Wellcome CAMS Award (to W.G.). D.C.V. is supported by a Fonds de la Recherche en Sante du Quebec Clinical Research Scholar Junior 2 award. C.Y.C. is supported by the California Initiative to Advance Precision Medicine, and the Charles and Helen Schwab Foundation.
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COVID-19 , Ácidos Nucleicos Libres de Células , Virosis , Humanos , Metilación , SARS-CoV-2/genéticaRESUMEN
Mapping the complex biogeography of microbial communities in situ with high taxonomic and spatial resolution poses a major challenge because of the high density1 and rich diversity2 of species in environmental microbiomes and the limitations of optical imaging technology3-6. Here we introduce high-phylogenetic-resolution microbiome mapping by fluorescence in situ hybridization (HiPR-FISH), a versatile technology that uses binary encoding, spectral imaging and decoding based on machine learning to create micrometre-scale maps of the locations and identities of hundreds of microbial species in complex communities. We show that 10-bit HiPR-FISH can distinguish between 1,023 isolates of Escherichia coli, each fluorescently labelled with a unique binary barcode. HiPR-FISH, in conjunction with custom algorithms for automated probe design and analysis of single-cell images, reveals the disruption of spatial networks in the mouse gut microbiome in response to treatment with antibiotics, and the longitudinal stability of spatial architectures in the human oral plaque microbiome. Combined with super-resolution imaging, HiPR-FISH shows the diverse strategies of ribosome organization that are exhibited by taxa in the human oral microbiome. HiPR-FISH provides a framework for analysing the spatial ecology of environmental microbial communities at single-cell resolution.
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Hibridación Fluorescente in Situ/métodos , Microbiota , Algoritmos , Animales , Antibacterianos/farmacología , Biopelículas , Escherichia coli/clasificación , Escherichia coli/citología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Microbioma Gastrointestinal/efectos de los fármacos , Humanos , Ratones , Microbiota/efectos de los fármacos , Boca/efectos de los fármacos , Boca/microbiología , Ribosomas/metabolismo , Análisis de la Célula IndividualRESUMEN
COVID-19 primarily affects the lungs, but evidence of systemic disease with multi-organ involvement is emerging. Here, we developed a blood test to broadly quantify cell, tissue, and organ specific injury due to COVID-19, using genome-wide methylation profiling of circulating cell-free DNA in plasma. We assessed the utility of this test to identify subjects with severe disease in two independent, longitudinal cohorts of hospitalized patients. Cell-free DNA profiling was performed on 104 plasma samples from 33 COVID-19 patients and compared to samples from patients with other viral infections and healthy controls. We found evidence of injury to the lung and liver and involvement of red blood cell progenitors associated with severe COVID-19. The concentration of cfDNA correlated with the WHO ordinal scale for disease progression and was significantly increased in patients requiring intubation. This study points to the utility of cell-free DNA as an analyte to monitor and study COVID-19.
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BACKGROUND: Cell-free DNA (cfDNA) in blood, urine, and other biofluids provides a unique window into human health. A proportion of cfDNA is derived from bacteria and viruses, creating opportunities for the diagnosis of infection via metagenomic sequencing. The total biomass of microbial-derived cfDNA in clinical isolates is low, which makes metagenomic cfDNA sequencing susceptible to contamination and alignment noise. RESULTS: Here, we report low biomass background correction (LBBC), a bioinformatics noise filtering tool informed by the uniformity of the coverage of microbial genomes and the batch variation in the absolute abundance of microbial cfDNA. We demonstrate that LBBC leads to a dramatic reduction in false positive rate while minimally affecting the true positive rate for a cfDNA test to screen for urinary tract infection. We next performed high-throughput sequencing of cfDNA in amniotic fluid collected from term uncomplicated pregnancies or those complicated with clinical chorioamnionitis with and without intra-amniotic infection. CONCLUSIONS: The data provide unique insight into the properties of fetal and maternal cfDNA in amniotic fluid, demonstrate the utility of cfDNA to screen for intra-amniotic infection, support the view that the amniotic fluid is sterile during normal pregnancy, and reveal cases of intra-amniotic inflammation without infection at term. Video abstract.
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Ácidos Nucleicos Libres de Células/análisis , Biología Computacional/métodos , ADN Bacteriano/análisis , Metagenoma , Análisis de Secuencia de ADN/métodos , Líquido Amniótico/microbiología , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/orina , Corioamnionitis/microbiología , Enfermedades Transmisibles/diagnóstico , Enfermedades Transmisibles/microbiología , Enfermedades Transmisibles/orina , Estudios Transversales , Análisis de Datos , Reacciones Falso Positivas , Femenino , Feto/microbiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inflamación , Masculino , Embarazo , Complicaciones del Embarazo/diagnóstico , Complicaciones del Embarazo/microbiología , Programas InformáticosRESUMEN
Although several proteins have been implicated in secretory vesicle tethering, the identity and mechanical properties of the components forming the physical vesicle-plasma membrane link remain unknown. Here we present the first experimental measurements of nanomechanical properties of secretory vesicle-plasma membrane tethers using combined AFM force clamp and TIRF microscopy on membrane sheets from PC12 cells expressing the vesicle marker ANF-eGFP. Application of pulling forces generated tether extensions composed of multiple steps with variable length. The frequency of short (<10 nm) tether extension events was markedly higher when a fluorescent vesicle was present at the cantilever tip and increased in the presence of GTPγS, indicating that these events reflect specifically the properties of vesicle-plasma membrane tethers. The magnitude of the short tether extension events is consistent with extension lengths expected from progressive unfolding of individual helices of the exocyst complex, supporting its direct role in forming the physical vesicle-plasma membrane link.