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Retroviral infection involves the reverse transcription of the viral RNA genome into DNA, which is subsequently integrated into the host cell genome. Human immunodeficiency virus type 1 (HIV-1) and other lentiviruses mediate the infection of non-dividing cells through the ability of the capsid protein 1 to engage the cellular nuclear import pathways of the target cell and mediate their nuclear translocation through components of the nuclear pore complex 2 – 4 . Although recent studies have observed the presence of the capsid protein in the nucleus during infection 5 – 8 , reverse transcription and disassembly of the viral core have conventionally been considered to be cytoplasmic events. Here, we use an inducible nuclear pore complex blockade to monitor the kinetics of HIV-1 nuclear import and define the biochemical staging of these steps of infection. Surprisingly, we observe that nuclear import occurs with relatively rapid kinetics (<5 h) and precedes the completion of reverse transcription in target cells, demonstrating that reverse transcription is completed in the nucleus. We also observe that HIV-1 remains susceptible to the capsid-destabilizing compound PF74 following nuclear import, revealing that uncoating is completed in the nucleus. Additionally, we observe that certain capsid mutants are insensitive to a Nup62-mediated nuclear pore complex blockade in cells that potently block infection by wild-type capsid, demonstrating that HIV-1 can use distinct nuclear import pathways during infection. These studies collectively define the spatio-temporal staging of critical steps of HIV-1 infection and provide an experimental system to separate and thereby define the cytoplasmic and nuclear stages of infection by other viruses. HIV-1 reverse transcription is found to be completed in the nucleus of the cell using an HIV-1 nuclear import kinetic assay that takes advantage of a nuclear import blockade method to monitor the kinetics of HIV-1 entry and infection.

In persons living with HIV-1 (PLWH) who start antiretroviral therapy (ART), plasma virus decays in a biphasic fashion to below the detection limit. The first phase reflects the short half-life (<1 d) of cells that produce most of the plasma virus. The second phase represents the slower turnover (t 1/2 = 14 d) of another infected cell population, whose identity is unclear. Using the intact proviral DNA assay (IPDA) to distinguish intact and defective proviruses, we analyzed viral decay in 17 PLWH initiating ART. Circulating CD4⁺ T cells with intact proviruses include few of the rapidly decaying first-phase cells. Instead, this population initially decays more slowly (t 1/2 = 12.9 d) in a process that largely represents death or exit from the circulation rather than transition to latency. This more protracted decay potentially allows for immune selection. After ∼3 mo, the decay slope changes, and CD4⁺ T cells with intact proviruses decay with a half-life of 19 mo, which is still shorter than that of the latently infected cells that persist on long-term ART. Two-long-terminal repeat (2LTR) circles decay with fast and slow phases paralleling intact proviruses, a finding that precludes their use as a simple marker of ongoing viral replication. Proviruses with defects at the 5′ or 3′ end of the genome show equivalent monophasic decay at rates that vary among individuals. Understanding these complex early decay processes is important for correct use of reservoir assays and may provide insights into properties of surviving cells that can constitute the stable latent reservoir.

Antiretroviral therapy (ART) effectively inhibits HIV-1 replication but is not curative due to the persistence of a latent viral reservoir in resting CD4 + T cells. This reservoir is a major barrier to cure. Sequencing studies have revealed that the population of proviruses persisting in ART-treated individuals is dominated by defective proviruses that cannot give rise to viral rebound due to fatal defects including large deletions and APOBEC3-mediated hypermutation. Near full genome sequencing (nFGS) of individual proviruses is used in reservoir assays to provide an estimate of the fraction of proviruses that are intact. nFGS methods rely on a long-distance outer PCR capturing most (~9 kb) of the genome, followed by nested inner PCRs. The outer PCR is carried out at limit dilution, and interpretation of the results is based on the assumption that all proviruses are quantitatively captured. Here, we evaluate nFGS methods using the intact proviral DNA assay (IPDA), a multiplex digital droplet PCR assay that quantitates intact and defective proviruses with single molecule sensitivity using only short, highly efficient amplicons. We analyzed proviral templates of known sequence to avoid the additional complication of sequence polymorphism. With the IPDA, we quantitated molecular yields at each step of nFGS methods. We demonstrate that nFGS methods are inefficient and miss ~70% of full-length proviruses due to amplification failure at the initial outer PCR step. In contrast, proviruses with large internal deletions encompassing 70% of the genome can be quantitatively amplified under the same conditions. Accurate measurement of the latent reservoir of HIV-1 is essential for evaluating the efficacy of cure strategies, and the bias against full length proviruses in nFGS methods must be considered.

Helper (CD4 + ) T cells perform direct therapeutic functions and augment responses of cells such as cytotoxic (CD8 + ) T cells against a wide variety of diseases and pathogens. Nevertheless, inefficient synthetic technologies for expansion of antigen-specific CD4 + T cells hinders consistency and scalability of CD4 +  T cell-based therapies, and complicates mechanistic studies. Here we describe a nanoparticle platform for ex vivo CD4 +  T cell culture that mimics antigen presenting cells (APC) through display of major histocompatibility class II (MHC II) molecules. When combined with soluble co-stimulation signals, MHC II artificial APCs (aAPCs) expand cognate murine CD4 + T cells, including rare endogenous subsets, to induce potent effector functions in vitro and in vivo. Moreover, MHC II aAPCs provide help signals that enhance antitumor function of aAPC-activated CD8 + T cells in a mouse tumor model. Lastly, human leukocyte antigen class II-based aAPCs expand rare subsets of functional, antigen-specific human CD4 + T cells. Overall, MHC II aAPCs provide a promising approach for harnessing targeted CD4 +  T cell responses. Adoptive cell therapies (ACT) hold promise for cancer immunotherapy, but optimization is still an ongoing process. Here the authors report CD4-targeted, nanoparticle-based artificial antigen-presenting cells that expand CD4 + T cells capable of lysing tumor cell lysis in vitro, and CD8 + T cells showing antitumor activity in a mouse melanoma model.

Retroviral infection involves the reverse transcription of the viral RNA genome into DNA, which is subsequently integrated into the host cell genome. HIV-1 and other lentiviruses are able to mediate the infection of non-dividing cells through the ability of the capsid (CA) protein 1 to engage the cellular nuclear import pathways of the target cell and mediate their nuclear translocation through components of the nuclear pore complex (NPC) 2–4. Although recent studies have observed the presence of capsid in the nucleus during infection5–8, reverse transcription and disassembly of the viral core have conventionally been considered to be cytoplasmic events. Here, we use an inducible nuclear pore blockade to monitor the kinetics of HIV-1 nuclear import and define the biochemical staging of these steps of infection. Surprisingly, we observe that nuclear import occurs with relatively rapid kinetics (<5 hours) and precedes the completion of reverse transcription in target cells, demonstrating that reverse transcription completes in the nucleus. We also observe that HIV-1 remains susceptible to a capsid destabilizing compound PF74 following nuclear import, revealing that uncoating completes in the nucleus. We also observe that certain CA mutants are insensitive to a Nup62 mediated nuclear pore blockade in cells which potently block infection by wild type CA, demonstrating that HIV-1 can utilize distinct nuclear import pathways during infection. These studies collectively define the spatiotemporal staging of critical steps of HIV-1 infection and provide an experimental system to separate and thereby define the cytoplasmic and nuclear stages of infection by other viruses.

Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study 1 . Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm 3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer. Quantitative multimodal 3D reconstruction of human pancreatic tissue at single-cell resolution reveals a high burden of multifocal, genetically heterogeneous pancreatic intraepithelial neoplasias in the normal adult pancreas.

This paper analyses the effects of the Federal Reserve’s Quantitative Easing (QE) program on corporate share repurchases in the US between 2008 and 2021. Therefore, it differentiates between the unconventional monetary policy that is QE from standard policies including the Federal Funds Rate (FFR). To isolate the effects, the regression analysis takes into account several corporate finance hypotheses among share repurchases as well as seasonality. The results show that QE is positively correlated with share repurchases while FFR is negatively correlated in all regressions. Thus, this analysis provides evidence that QE positively affects share repurchases. It further finds that the impact QE has on shareholder distribution is similar to the impact a decrease in interest rates has.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.Competing Interest StatementA pending patent application \"COMPUTATIONAL TECHNIQUES FOR THREE-DIMENSIONAL RECONSTRUCTION AND MULTI-LABELING OF SERIALLY SECTIONED TISSUE\" was filed on 6/24/2022 by authors AK, RHH, PHW, DW, and LDW. The other authors report no competing interests.

With an urgent need for bedside imaging of coronavirus disease 2019 (COVID-19), this study’s main goal was to assess inter- and intraobserver agreement in lung ultrasound (LUS) of COVID-19 patients. In this single-center study we prospectively acquired and evaluated 100 recorded ten-second cine-loops in confirmed COVID-19 intensive care unit (ICU) patients. All loops were rated by ten observers with different subspeciality backgrounds for four times by each observer (400 loops overall) in a random sequence using a web-based rating tool. We analyzed inter- and intraobserver variability for specific pathologies and a semiquantitative LUS score. Interobserver agreement for both, identification of specific pathologies and assignment of LUS scores was fair to moderate (e.g., LUS score 1 Fleiss’ κ = 0.27; subpleural consolidations Fleiss’ κ = 0.59). Intraobserver agreement was mostly moderate to substantial with generally higher agreement for more distinct findings (e.g., lowest LUS score 0 vs. highest LUS score 3 (median Fleiss’ κ = 0.71 vs. 0.79) or air bronchograms (median Fleiss’ κ = 0.72)). Intraobserver consistency was relatively low for intermediate LUS scores (e.g. LUS Score 1 median Fleiss’ κ = 0.52). We therefore conclude that more distinct LUS findings (e.g., air bronchograms , subpleural consolidations ) may be more suitable for disease monitoring, especially with more than one investigator and that training material used for LUS in point-of-care ultrasound (POCUS) should pay refined attention to areas such as B-line quantification and differentiation of intermediate LUS scores.

Purpose: The aim of this paper is to study the visual outcomes, changes in higher order aberration (HOA) and corneal densitometry after debridement and excimer laser phototherapeutic keratectomy (PTK) for the treatment of Salzmann nodular degeneration (SND). Methods: This monocentric study includes 69 eyes from 54 patients who underwent debridement and PTK for SND (mean follow-up time of 447.1 ± 597.7 days post-operatively). The following parameters were measured before and after PTK: best corrected visual acuity (BCVA) in logMAR, sphere, cylinder, calculated spherical equivalent (SPHQ), mean and maximum refractive power, astigmatism, HOA, corneal density and thickness. Patients were divided into two cohorts depending on additional visual acuity limitations (VAL). Results: Mean visual acuity improvement was 0.16 ± 0.21 logMAR (p < 0.001), independent of additional VAL, and was associated with normalization of the cornea (hyperopic reduction by 2.13 ± 2.60 dpt, p < 0.001), reductions in cylinder (1.49 ± 2.44 dpt, p < 0.001) and corneal astigmatism (3.01 ± 3.39 dpt, p < 0.001). HOA was reduced by 0.77 ± 1.11 µm (p < 0.001) and corneal density by 6.08 ± 16.45 gray scale units (GSUs) in the center (p = 0.019) and by 9.32 ± 12.08 GSUs in the mid-periphery (p < 0.001). Haze occurred in 26.1% of patients (15.9% mild; 10.1% moderate). Re-PTK was necessary in 5.8%. Conclusions: PTK is a low-complication method for visual improvement in patients with SND, regardless of additional VAL, and is associated with a normalization of corneal parameters. HOA, corneal density and Kmax were reduced significantly and showed a correlation with visual acuity, implying that these objective parameters may have a good predictive value for visual acuity.