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Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection 1 , 2 , but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1β and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1 + pathological fibroblasts 3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand–receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development. Lung samples collected soon after death from COVID-19 are used to provide a single-cell atlas of SARS-CoV-2 infection and the ensuing molecular changes.

AimsCharacterise T-cell receptor gene (TR) repertoires of small intestinal T cells of patients with newly diagnosed (active) coeliac disease (ACD), refractory CD type I (RCD I) and patients with CD on a gluten-free diet (GFD).MethodsNext-generation sequencing of complementarity-determining region 3 (CDR3) of rearranged T cell receptor β (TRB) and γ (TRG) genes was performed using DNA extracted from intraepithelial cell (IEC) and lamina propria cell (LPC) fractions and a small subset of peripheral blood mononuclear cell (PBMC) samples obtained from CD and non-CD (control) patients. Several parameters were assessed, including relative abundance and enrichment.ResultsTRB and TRG repertoires of CD IEC and LPC samples demonstrated lower clonality but higher frequency of rearranged TRs compared with controls. No CD-related differences were detected in the limited number of PBMC samples. Previously published LP gliadin-specific TRB sequences were more frequently detected in LPC samples from patients with CD compared with non-CD controls. TRG repertoires of IECs from both ACD and GFD patients demonstrated increased abundance of certain CDR3 amino acid (AA) motifs compared with controls, which were encoded by multiple nucleotide variants, including one motif that was enriched in duodenal IECs versus the PBMCs of CD patients.ConclusionsSmall intestinal TRB and TRG repertoires of patients with CD are more diverse than individuals without CD, likely due to mucosal recruitment and accumulation of T cells because of protracted inflammation. Enrichment of the unique TRG CDR3 AA sequence in the mucosa of patients with CD may suggest disease-associated changes in the TCRγδ IE lymphocyte (IEL) landscape.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that potently impair immunotherapy responses. The chemokine receptor CXCR4, a central regulator of hematopoiesis, represents an attractive PMN-MDSC target1. Here, we fused a secreted CXCR4 partial agonist TFF2 to mouse serum albumin (MSA) and demonstrated that TFF2-MSA peptide synergized with anti-PD-1 to induce tumor regression or eradication, inhibited distant metastases, and prolonged survival in multiple gastric cancer (GC) models. Using histidine decarboxylase (Hdc)-GFP transgenic mice to track PMN-MDSC , we found TFF2-MSA selectively reduced the immunosuppressive Hdc-GFP CXCR4 tumor PMN-MDSCs while preserving proinflammatory neutrophils, thereby boosting CD8 T cell-mediated anti-tumor response together with anti-PD-1. Furthermore, TFF2-MSA systemically reduced PMN-MDSCs and bone marrow granulopoiesis. In contrast, CXCR4 antagonism plus anti-PD-1 failed to provide a similar therapeutic benefit. In GC patients, expanded PMN-MDSCs containing a prominent CXCR4 LOX-1 subset are inversely correlated with the TFF2 level and CD8 T cells in circulation. Collectively, our studies introduce a strategy of using CXCR4 partial agonism to restore anti-PD-1 sensitivity in GC by targeting PMN-MDSCs and granulopoiesis.

Recent advances in base editing have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) nearby the mutation site further limits the targeting feasibility. Here we modify the NG-targeting adenine base editor (iABE-NGA) to overcome these challenges and demonstrate the high efficiency to precisely edit a Duchenne muscular dystrophy (DMD) mutation in adult mice. Systemic delivery of AAV9-iABE-NGA results in dystrophin restoration and functional improvement. At 10 months after AAV9-iABE-NGA treatment, a near complete rescue of dystrophin is measured in mdx 4cv mouse hearts with up to 15% rescue in skeletal muscle fibers. The off-target activities remains low and no obvious toxicity is detected. This study highlights the promise of permanent base editing using iABE-NGA for the treatment of monogenic diseases. Base editing is one approach used to correct mutations causing cause Duchenne muscular dystrophy (DMD), but limitations are in the requirement for a specific PAM motif and the large size beyond the packaging capacity of adeno-associated virus (AAV). Here, the authors modify the NG-targeting adenine base editor to recognize a broader PAM, devise an intein split strategy to package the otherwise oversized adenine base editor into AAV, and show it efficiently restores dystrophin expression in muscle and heart when systemically injected in a mouse model of DMD

Idiopathic pulmonary fibrosis (IPF) causes considerable global morbidity and mortality, and its mechanisms of disease progression are poorly understood. Recent observational studies have reported associations between lung dysbiosis, mortality, and altered host defense gene expression, supporting a role for lung microbiota in IPF. However, the causal significance of altered lung microbiota in disease progression is undetermined. To examine the effect of microbiota on local alveolar inflammation and disease progression using both animal models and human subjects with IPF. For human studies, we characterized lung microbiota in BAL fluid from 68 patients with IPF. For animal modeling, we used a murine model of pulmonary fibrosis in conventional and germ-free mice. Lung bacteria were characterized using 16S rRNA gene sequencing with novel techniques optimized for low-biomass sample load. Microbiota were correlated with alveolar inflammation, measures of pulmonary fibrosis, and disease progression. Disruption of the lung microbiome predicts disease progression, correlates with local host inflammation, and participates in disease progression. In patients with IPF, lung bacterial burden predicts fibrosis progression, and microbiota diversity and composition correlate with increased alveolar profibrotic cytokines. In murine models of fibrosis, lung dysbiosis precedes peak lung injury and is persistent. In germ-free animals, the absence of a microbiome protects against mortality. Our results demonstrate that lung microbiota contribute to the progression of IPF. We provide biological plausibility for the hypothesis that lung dysbiosis promotes alveolar inflammation and aberrant repair. Manipulation of lung microbiota may represent a novel target for the treatment of IPF.

Background The growing number of cancer survivors combined with a looming shortage of oncology specialists will require greater coordination of post-treatment care responsibilities between oncologists and primary care physicians (PCPs). However, data are limited regarding these physicians’ views of cancer survivors’ care. Objective To compare PCPs and oncologists with regard to their knowledge, attitudes, and practices for follow-up care of breast and colon cancer survivors. Design and Subjects Mailed questionnaires were completed by a nationally representative sample of 1,072 PCPs and 1,130 medical oncologists in 2009 (cooperation rate = 65%). Sampling and non-response weights were used to calculate estimates to reflect practicing US PCPs and oncologists. Main Measures PCPs and oncologists reported their 1) preferred model for delivering cancer survivors’ care; 2) assessment of PCPs’ ability to perform follow-up care tasks; 3) confidence in their knowledge; and 4) cancer surveillance practices. Key Results Compared with PCPs, oncologists were less likely to believe PCPs had the skills to conduct appropriate testing for breast cancer recurrence (59% vs. 23%, P < 0.001) or to care for late effects of breast cancer (75% vs. 38%, P < 0.001). Only 40% of PCPs were very confident of their own knowledge of testing for recurrence. PCPs were more likely than oncologists to endorse routine use of non-recommended blood and imaging tests for detecting cancer recurrence, with both groups departing substantially from guideline recommendations. Conclusion There are significant differences in PCPs’ and oncologists’ knowledge, attitudes, and practices with respect to care of cancer survivors. Improving cancer survivors’ care may require more effective communication between these two groups to increase PCPs’ confidence in their knowledge, and must also address oncologists’ attitudes regarding PCPs’ ability to care for cancer survivors.

The downslope plumes of dense shelf water (DSW) are critical for the formation of Antarctic Bottom Water (AABW), and thus to the exchange of heat and carbon between surface and abyssal ocean. Previous studies have shown that tides and overflow-forced topographic Rossby waves (TRWs) may have strong impact on the downslope transport of DSW, but it remains unclear how the combined action of these two processes influence the descent processes of DSW, and of the resulting AABW properties. Here, with a synthesis of historical in situ observations and a set of numerical model experiments, we show that tides and TRWs play comparable roles in AABW formation: they both act to accelerate DSW descent to the abyss, leading to the formation of colder and denser AABW. Yet, tides have little impact on AABW formation unless the continental slope is steep enough to suppress TRW generation. We further characterize the dynamical regimes of dense overflows around the entire Antarctic continent based on the relative importance of TRWs versus tides. These findings highlight the pervasive role of high-frequency processes, which are not well represented in the present climate models, in the formation of AABW, and thus in the global overturning circulation. This study identifies the key roles of tides and topographic waves in forming Antarctic bottom water in different regions. The Antarctic coastline is divided into four overflow dynamical regimes, providing guidance for future observations.

A bstract The splitting processes of bremsstrahlung and pair production in a medium are coherent over large distances in the very high energy limit, which leads to a suppression known as the Landau-Pomeranchuk-Migdal (LPM) effect. In this paper, we continue analysis of the case when the coherence lengths of two consecutive splitting processes overlap (which is important for understanding corrections to standard treatments of the LPM effect in QCD), avoiding soft-gluon approximations. In particular, this paper analyzes the subtle problem of how to precisely separate overlapping double splitting (e.g. overlapping double bremsstrahlung) from the case of consecutive, independent bremsstrahlung (which is the case that would be implemented in a Monte Carlo simulation based solely on single splitting rates). As an example of the method, we consider the rate of real double gluon bremsstrahlung from an initial gluon with various simplifying assumptions (thick media; q approximation; large N c ; and neglect for the moment of processes involving 4-gluon ver-tices) and explicitly compute the correction Δ d Γ /dx dy due to overlapping formation times.

Multiplexed single cell sequencing allows the identification of functional TCRα-TCRβ pairs Although each T lymphocyte expresses a T-cell receptor (TCR) that recognizes cognate antigen and controls T-cell activation, different T cells bearing the same TCR can be functionally distinct. Each TCR is a heterodimer, and both α- and β-chains contribute to determining TCR antigen specificity. Here we present a methodology enabling integration of information about TCR specificity with information about T cell function. This method involves sequencing of TCRα and TCRβ genes, and amplifying functional genes characteristic of different T cell subsets, in single T cells. Because this approach retains information about individual TCRα-TCRβ pairs, TCRs of interest can be expressed and used in functional studies, for antigen discovery, or in therapeutic applications. We apply this approach to study the clonal ancestry and differentiation of T lymphocytes infiltrating a human colorectal carcinoma.