Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving

The nature of certain clinical samples (tissue biopsies, fluids) or the subjects themselves (pediatric subjects, neonates) often constrain the number of cells available to evaluate the breadth of functional T-cell responses to infections or therapeutic interventions. The methods most commonly used to assess this functional diversity ex vivo and to recover specific cells to expand in vitro usually require more than 106 cells. Here we present a process to identify antigen-specific responses efficiently ex vivo from 104–105 single cells from blood or mucosal tissues using dense arrays of subnanoliter wells. The approach combines on-chip imaging cytometry with a technique for capturing secreted proteins—called “microengraving”—to enumerate antigenspecific responses by single T cells in a manner comparable to conventional assays such as ELISpot and intracellular cytokine staining. Unlike those assays, however, the individual cells identified can be recovered readily by micromanipulation for further characterization in vitro. Applying this method to assess HIV-specific T cell responses demonstrates that it is possible to establish clonal CD8+ T-cell lines that represent the most abundant specificities present in circulation using 100- to 1,000-fold fewer cells than traditional approaches require and without extensive genotypic analysis a priori. This rapid (<24 h), efficient, and inexpensive process should improve the comparative study of human T-cell immunology across ages and anatomic compartments

Nonsurgical closure of femoral pseudoaneurysms complicating cardiac catheterization and percutaneous transluminal coronary angioplasty

AbstractObjectives. This study was performed to describe the initial experience and follow-up of ultrasound-guided compression of pseudoaneurysms in patients receiving systemic anticoagulant or antiplatelet therapy, or both, after recent cardiac catheterization or percutaneous transluminal coronary angioplasty.Background. Femoral artery pseudoaneurysm formation after an interventional procedure is becoming more common as larger caliber catheters and prolonged anticoagulant and antiplatelet therapy are being used. Traditional treatment of this complication has been surgical repair. This study describes a new method of closing femoral pseudoaneurysms by using external compression guided by Doppler color flow imaging.Methods. Fifteen patients, 3 undergoing cardiac catheterization and 12 undergoing coronary angioplasty, developed an expansile groin mass at the vascular access site diagnosed as a femoral artery pseudoaneurysm by Doppler ultrasound. Seven of the patients had undergone coronary stenting and were receiving postprocedural anticoagulant therapy. These patients underwent progressive graded mechanical (C-clamp) external compression guided by ultrasound. The mechanical compression was titrated to obliterate the vascular tracts to these aneurysms and maintain adequate flow in the femoral artery.Results. After an average compression time of 30 min (range 10 to 120), these tracts remained closed. Follow-up ultrasound examination at 24 h or later confirmed continued closure in all. Conclusions. This study suggests that nonsurgical closure of femoral pseudoaneurysms is feasible. This technique may be valuable in managing vascular access-related complications after diagnostic and interventional procedures, even in patients requiring prolonged anticoagulant therapy

The 2017 Failed Outburst of GX 339-4: Relativistic X-ray Reflection near the Black Hole Revealed by NuSTAR and Swift Spectroscopy

We report on the spectroscopic analysis of the black hole binary GX 339−4 during its recent 2017–2018 outburst, observed simultaneously by the Swift and NuSTAR observatories. Although during this particular outburst the source failed to make state transitions, and despite Sun constraints during the peak luminosity, we were able to trigger four different observations sampling the evolution of the source in the hard state. We show that even for the lowest-luminosity observations the NuSTAR spectra show clear signatures of X-ray reprocessing (reflection) in an accretion disk. Detailed analysis of the highest signal-to-noise spectra with our family of relativistic reflection models RELXILL indicates the presence of both broad and narrow reflection components. We find that a dual-lamppost model provides a superior fit when compared to the standard single lamppost plus distant neutral reflection. In the dual-lamppost model two sources at different heights are placed on the rotational axis of the black hole, suggesting that the narrow component of the Fe K emission is likely to originate in regions far away in the disk, but still significantly affected by its rotational motions. Regardless of the geometry assumed, we find that the inner edge of the accretion disk reaches a few gravitational radii in all our fits, consistent with previous determinations at similar luminosity levels. This confirms a very low degree of disk truncation for this source at luminosities above ~1% Eddington. Our estimates of R_(in) reinforce the suggested behavior for an inner disk that approaches the innermost regions as the luminosity increases in the hard state

Cardiovascular Risk Factors Are Associated With Future Cancer

BACKGROUND The extent to which co-occurrence of cardiovascular disease (CVD) and cancer is due to shared risk factors or other mechanisms is unknown. OBJECTIVES This study investigated the association of standard CVD risk factors, CVD biomarkers, pre-existing CVD, and ideal cardiovascular (CV) health metrics with the development of future cancer. METHODS This study prospectively followed Framingham Heart Study and PREVEND (Prevention of Renal and Vascular End-Stage Disease) study participants free of cancer at baseline and ascertained histology-proven cancer. This study assessed the association of baseline CV risk factors, 10-year atherosclerotic (ASCVD) risk score, established CVD biomarkers, prevalent CVD, and the American Heart Association (AHA) Life's Simple 7 CV health score with incident cancer using multivariable Cox models. Analyses of interim CVD events with incident cancer used time-dependent covariates. RESULTS Among 20,305 participants (mean age 50 +/- 14 years; 54% women), 2,548 incident cancer cases occurred over a median follow-up of 15.0 years (quartile 1 to 3: 13.3 to 15.0 years). Traditional CVD risk factors, including age, sex, and smoking status, were independently associated with cancer (p < 0.001 for all). Estimated 10-year ASCVD risk was also associated with future cancer (hazard ratio [HR]: 1.16 per 5% increase in risk; 95% confidence interval [CI] 1.14 to 1.17; p < 0.001). The study found that natriuretic peptides (tertile 3 vs. tertite 1; HR: 1.40; 95% 0:1.03 to 1.91; p 0.035) were associated with incident cancer but not high-sensitivity troponin (p 0.47). Prevalent CVD and the development of interim CV events were not associated with higher risk of subsequent cancer. However, ideal CV health was associated with tower future cancer risk (HR: 0.95 per 1-point increase in the AHA health score; 95% CI: 0.92 to 0.99; p = 0.009). CONCLUSIONS CVD risk, as captured by traditional CVD risk factors, 10-year ASCVD risk score, and natriuretic peptide concentrations are associated with increased risk of future cancer. Conversely, a heart healthy lifestyle is associated with a lower risk of future cancer. These data suggest that the association between CVD and future cancer is attributable to shared risk factors. (C) 2021 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation

Anti-Inflammatory Activity of a Potent, Selective Leukotriene A4 Hydrolase Inhibitor in Comparison with the 5-Lipoxygenase Inhibitor Zileuton

ABSTRACT Leukotriene A 4 hydrolase (LTA 4 H) catalyzes production of the proinflammatory lipid mediator, leukotriene (LT) B 4 , which is implicated in a number of inflammatory diseases. We have identified a potent and selective inhibitor of both the epoxide hydrolase and aminopeptidase activities of recombinant human LTA 4 H (IC 50 , approximately 10 nM). In a murine model of arachidonic acid-induced ear inflammation, the LTA 4 H inhibitor, JNJ-26993135 (1-[4-(benzothiazol-2-yloxy)-benzyl]-piperidine-4-carboxylic acid), dose-dependently inhibited ex vivo LTB 4 production in blood, in parallel with dose-dependent inhibition of neutrophil influx (ED 50 , 1-3 mg/kg) and ear edema. In murine whole blood and in zymosan-induced peritonitis, JNJ-26993135 selectively inhibited LTB 4 production, without affecting cysteinyl leukotriene production, while maintaining or increasing production of the anti-inflammatory mediator, lipoxin (LX) A 4 . The 5-lipoxygenase (5-LO) inhibitor zileuton showed inhibition of LTB 4 , LTC 4 , and LXA 4 production. Although zileuton inhibited LTB 4 production in the peritonitis model more effectively than the LTA 4 H inhibitor, the influx of neutrophils into the peritoneum after 1 and 2 h was significantly higher in zileuton-versus JNJ-26993135-treated animals. This difference may have been mediated by the increased LXA 4 levels in the presence of the LTA 4 H inhibitor. The selective inhibition of LTB 4 production by JNJ-26993135, while increasing levels of the anti-inflammatory mediator, LXA 4 , may translate to superior therapeutic efficacy versus 5-LO or 5-LO-activating protein inhibitors in LTB 4 -mediated inflammatory diseases

Mapping Obscured Star Formation in the Host Galaxy of FRB 20201124A

We present high-resolution 1.5--6 GHz Karl G. Jansky Very Large Array (VLA) and $\textit{Hubble Space Telescope}$ ($\textit{HST}$) optical and infrared observations of the extremely active repeating fast radio burst (FRB) FRB$\,$20201124A and its barred spiral host galaxy. We constrain the location and morphology of star formation in the host and search for a persistent radio source (PRS) coincident with FRB$\,$20201124A. We resolve the morphology of the radio emission across all frequency bands and measure a star formation rate SFR $\approx 8.9\,M_{\odot}$ yr$^{-1}$, a factor of $\approx 4-6$ larger than optically-inferred SFRs, demonstrating dust-obscured star formation throughout the host. Compared to a sample of all known FRB hosts with radio emission, the host of FRB$\,$20201124A has the most significant obscured star formation. While ${\it HST}$ observations show the FRB to be offset from the bar or spiral arms, the radio emission extends to the FRB location. We propose that the FRB progenitor could have formed $\textit{in situ}$ (e.g., a magnetar central engine born from the explosion of a massive star). It is still plausible, although less likely, that the progenitor of FRB$\,$20201124A migrated from the central bar of the host, e.g., via a runaway massive star. We further place a limit on the luminosity of a putative PRS at the FRB position of $L_{\rm 6.0 \ GHz}$$\lesssim$2.6$\times$$10^{27}$erg s$^{-1}$Hz$^{-1}$, two orders of magnitude below any PRS known to date. However, this limit is still broadly consistent with both magnetar nebulae and hypernebulae models assuming a constant energy injection rate of the magnetar and an age of$\gtrsim 10^{5}$ yr in each model, respectively.Comment: 21 pages, 6 figures, 3 tables, Submitte

Interactive analysis and quality assessment of single-cell copy-number variations

Single-cell sequencing is emerging as a critical technology for understanding the biology of cancer, neurons, and other complex systems. Here we introduce Ginkgo, a web platform for the interactive analysis and quality assessment of single-cell copy-number alterations. Ginkgo fully automates the process of binning, normalizing, and segmenting mapped reads to infer copy number profiles of individual cells, as well as constructing phylogenetic trees of how those cells are related. We validate Ginkgo by reproducing the results of five major single-cell studies, and discuss how it addresses the wide array of biases that affect single-cell analysis. We also examine the data characteristics of three commonly used single-cell amplification techniques: MDA, MALBAC, and DOP-PCR/WGA4 through comparative analysis of 9 different single-cell datasets. We conclude that DOP-PCR provides the most uniform amplification, while MDA introduces substantial biases into the analysis. Furthermore, given the same level of coverage, our results indicate that data prepared using DOP-PCR can reliably call CNVs at higher resolution than data prepared using either MALBAC or MDA. Ginkgo is freely available at http://qb.cshl.edu/ginkgo.Received November 11, 2014.Accepted November 12, 2014.© 2014, Published by Cold Spring Harbor Laboratory PressThis pre-print is available under a Creative Commons License (Attribution-NonCommercial-NoDerivs 4.0 International), CC BY-NC-ND 4.0, as described at http://creativecommons.org/licenses/by-nc-nd/4.0

Single nucleus genome sequencing reveals high similarity among nuclei of an endomycorrhizal fungus

Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya

Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing

Clear cell renal carcinomas (ccRCCs) can display intratumor heterogeneity (ITH). We applied multiregion exome sequencing (M-seq) to resolve the genetic architecture and evolutionary histories of ten ccRCCs. Ultra-deep sequencing identified ITH in all cases. We found that 73–75% of identified ccRCC driver aberrations were subclonal, confounding estimates of driver mutation prevalence. ITH increased with the number of biopsies analyzed, without evidence of saturation in most tumors. Chromosome 3p loss and VHL aberrations were the only ubiquitous events. The proportion of C>T transitions at CpG sites increased during tumor progression. M-seq permits the temporal resolution of ccRCC evolution and refines mutational signatures occurring during tumor development