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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 peritoneal effluent in PD patients with and without peritonitis. Study Design Prospective cohort study Setting & Participants We prospectively characterized cell-free DNA (cfDNA) in 68 peritoneal effluent samples obtained from 33 patients receiving peritoneal dialysis at a single center study from September 2016 to July 2018. Outcomes Peritoneal effluent cfDNA characteristics, microbial and human cfDNA characteristics in culture-confirmed peritonitis and culture-negative peritonitis. Analytical Approach Descriptive statistics, detection of microbial cfDNA 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 not obtained in all peritoneal effluent specimens. Conclusions Metagenomic cfDNA sequencing of peritoneal effluent can identify pathogens in PD patients with peritonitis, including culture-negative peritonitis.

Significance Hematopoietic cell transplantation is the gold standard treatment for many blood disorders, including blood cancers. Nonetheless, frequent post-transplant complications limit the long-term benefit of HCT. Here, we find that circulating cell-free DNA is a highly versatile analyte for monitoring of the most important complications of HCT: Graft-Versus-Host Disease, infection, graft failure and disease relapse. We show that these different therapeutic complications can be informed from a single cell-free DNA sequencing assay followed by disease-specific bioinformatic analyses. This test requires only low coverage DNA sequencing and is compatible with small volumes of blood. Cell-free DNA may improve the care of allogeneic HCT recipients by enabling earlier detection and better prediction of the complex array of complications that occur after HCT.

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 ( 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.

Summary 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 .

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., Graphical Abstract, Context and Significance COVID-19 is a systemic disease with multi-organ involvement. Here, the authors report a blood test to quantify cell-, tissue-, and organ-specific injury due to COVID-19. This is accomplished by profiling methylation marks within circulating cell-free DNA (cfDNA) to trace their tissues of origin and to quantify tissue-specific injury due to COVID-19. The authors assessed the utility of this test to identify subjects with severe disease in two independent, longitudinal cohorts of hospitalized patients and report evidence of injury to the lung and liver and involvement of red blood cell progenitors associated with severe COVID-19. These results support the utility of cfDNA profiling as a prognostic tool for the early detection and monitoring of cell and tissue injury due to COVID-19., COVID-19 affects the entire body. Cheng et al. describe a DNA-based blood test to quantify cell and tissue injury due to COVID-19. They show that severe COVID-19 is associated with increased lung, liver, and erythroblast DNA in blood and that the concentration of cell-free DNA correlates with disease progression.

Allogeneic hematopoietic cell transplantation (HCT) provides effective treatment for hematologic malignancies and immune disorders. Monitoring of post-transplant complications is critical, yet current diagnostic options are limited. Here, we show that cell-free DNA (cfDNA) in blood is a highly 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 different therapeutic complications can be 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 on infections, 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 time points before and after allogeneic HCT. We found that the abundance of solid-organ derived cfDNA in the blood at one-month after HCT is an early predictor of acute graft-versus-host disease (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 cell chimerism, 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.

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.

SUMMARY 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 ~300 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., Graphical Abstract, In brief For transmission, a fusion pore forms when vesicle and target membranes are brought together by SNARE proteins. Delacruz et al. demonstrate that selectivity of the pore accelerates release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin, and acetylcholine, while glutamate release may require a more dilated fusion pore.

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 (

The cytoplasmic face of cell membranes has multiple features, such as cortical actin and tethered vesicles, the nanomechanical properties of which are largely unknown. To study these properties, we prepared plasma membrane sheets from a PC12 cell clone (Lang 2008 Meth.Mol.Biology, 440: 51-9) constitutively expressing ANF-EGFP, GFP-tagged proANF (atrial natriuretic factor) (Han et al 1999 PNAS 96:14577-82) as a secretory vesicle marker. The membrane sheets are firmly attached to the coverslip with their extracellular face while the cytoplasmic face is exposed. We combined a Nikon Ti-E/B total internal reflection fluorescence (TIRF) microscope with an Agilent PicoPlus atomic force microscope (AFM). Soft AFM cantilevers (Olympus Biolevers) were brought into contact with the exposed membrane face, binding spontaneously to the surface and to secretory vesicles. The membrane sheets and the motion of the cantilever tips were imaged by TIRF microscopy while forces of approximately 50pN or more were applied, pulling the tip away from the surface. At 50 ms time resolution, the noise level of the mechanical measurements was ∼1 pN and 0.5 nm (r.m.s.). Dynamic interactions associated with displacements in the 10-40 nm range were recorded. The role of secretory vesicles in this behavior will be investigated.

SNAP25 is a t-SNARE protein mediating exocytosis in neuronal cell types. Previous results with a construct lacking the nine C-terminal residues (SNAP25Δ9) showed changed fusion pore properties, suggesting a model for fusion pore mechanics that couple C-terminal zipping of the SNARE complex to the opening of a proteolipid fusion pore (Fang 2008 PNAS,105:15388). The deleted fragment contains the positively charged residues R198andK201, which are located in layers 7-8 of the SNARE complex at its C-terminal end. To study the role of these two residues in the structure and dynamics of the fusion pore, carbon fiber amperometry and cell-attached capacitance measurement of individual fusion and release events were performed using bovine chromaffin cells overexpressing wild type SNAP-25 or the point mutation constructs R198Q/E, K201Q/E to substitute the positively charged amino acid with a neutral or positively charged one. The R198E/K201E mutants increased fusion pore duration similar to SNAP25Δ9. Furthermore, the R198E/R198Q also showed a smaller fusion pore conductance. These results support the conclusion that fusion pore properties depend on the precise arrangements at the C-terminal end of the SNARE complex. Supported by NIH grant GM085808.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Activity-dependent regulation of protein kinase A (PKA) and protein kinase C (PKC), is essential for modulation of neurosecretion. To study the role of protein phosphorylation in the dynamics of the fusion pore opening and transmitter release, carbon fiber amperometry and cell-attached capacitance measurements were performed in bovine chromaffin cells. In cells treated with the PKC inhibitor, bisindolylmaleimide I (100nM, 10Ki), release frequency was significantly reduced to 5.0±1.2events/min (from control 17.1±2.0events/min). Amperometric spikes also showed increased half width of 22.3±2.0ms (from 11.5±0.9ms control) and smaller amperometric spike amplitude of 21.9±3.7pA (from 56.2±9.4pA control). In the cells treated with PKA inhibitor, KT 5720 (500nM, 10Ki), the duration of foot signals of amperometric events was prolonged to 14.4±1.6ms (from 9.0±1.0 ms control), consistent with prolonged fusion pore duration in cell-attached capacitance measurements of 27.7±6.8ms (from 11.8±1.4ms control). In contrast to PKC inhibitor, KT 5720 did not affect the release frequency.These results indicate that PKC affects the rate of fusion pore formation and release after full fusion but not early fusion pore expansion, while PKA specifically affects the expansion of the early fusion pore as well as release after full fusion.View Large Image | View Hi-Res Image | Download PowerPoint Slide