Coronavirus disease-2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has lead to a global pandemic with a rising toll in infections and deaths. Better understanding of its pathogenesis will greatly improve the outcomes and treatment of affected patients. Here we compared the inflammatory and cardiovascular disease-related protein cargo of circulating large and small extracellular vesicles (EVs) from 84 hospitalized patients infected with SARS-CoV-2 with different stages of disease severity. Our findings reveal significant enrichment of proinflammatory, procoagulation, immunoregulatory and tissueremodelling protein signatures in EVs, which remarkably distinguished symptomatic COVID-19 patients from uninfected controls with matched comorbidities and delineated those with moderate disease from those who were critically ill. Specifically, EN-RAGE, followed by TF and IL-18R1, showed the strongest correlation with disease severity and length of hospitalization. Importantly, EVs from COVID-19 patients induced apoptosis of pulmonary microvascular endothelial cells in the order of disease severity. In conclusion, our findings support a role for EVs in the pathogenesis of COVID-19 disease and underpin the development of EV-based approaches to predicting disease severity, determining need for patient hospitalization and identifying new therapeutic targets. K E Y WO R D S endothelial injury, inflammation, SARS-CoV-2, thrombosis  INTRODUCTIONThe coronavirus disease-2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (Dashboard WCDC-2020; Wu et al., 2020) is marked by endothelial dysfunction and dysregulated immune responses (Huertas et al., 2020). Like the SARS-CoV pathogen that led to epidemics in 2002 and 2003, SARS-CoV-2 enters cells via binding of its spike protein to angiotensin converting enzyme 2 (ACE2) receptors. ACE2 receptors are abundantly present in pulmonary alveolar type II and endothelial cells, thereby making the lungs and pulmonary vasculature susceptible to SARS-CoV-2-induced inflammation and injury (Zhao et al., 2020). Further, alveolar capillary micro-thrombi and endothelial damage with evidence of intracellular virus have been noted on post-mortem analysis of infected lungs (Ackermann et al., 2020). While there is growing evidence that endothelial injury, vascular remodelling and coagulopathy are key consequences of COVID-19 infection, it remains unclear how the virus induces these changes. Extracellular vesicles (EVs) carry proteins, coding and non-coding RNA, DNA fragments and lipids, which facilitate cross-talk between cells. The transfer of EV cargo plays aThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

COVID-19 infection caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in a global pandemic with the number of deaths growing exponentially. Early evidence points to significant endothelial dysfunction, micro-thromboses, pro-inflammation as well as a dysregulated immune response in the pathogenesis of this disease. In this study, we analyzed the cargo of EVs isolated from the plasma of patients with COVID-19 for the identification of potential biomarkers of disease severity and to explore their role in disease pathogenesis. Plasma-derived EVs were isolated from 53 hospitalized patients with COVID infection and compared according to the severity of the disease. Analysis of inflammatory and cardiovascular protein cargo of large EVs revealed significantly differentially expressed proteins for each disease sub-group. Notably, members of the TNF superfamily and IL-6 family were up-regulated in patients on oxygen support with severe and moderate disease. EVs from the severe group were also enhanced with pro-thrombotic/endothelial injury factors (TF, t-PA, vWF) and proteins associated with cardiovascular pathology (MB, PRSS8, REN, HGF). Significantly higher levels of TF, CD163, and EN-RAGE were observed in EVs from severe patients when compared to patients with a moderate disease requiring supplemental O2. Importantly, we also observed increased caspase 3/7 activity and decreased cell survival in human pulmonary microvascular endothelial cells exposed to EVs from the plasma of patients with severe disease compared to healthy controls. In conclusion, our findings indicate alterations in pro-inflammatory, coagulopathy, and endothelial injury protein cargo in large EVs in response to SARS-CoV-2 infection that may be a causative agent in severe illness.

Imidazobenzodiazepines such as RY-80 have been reported to exhibit both high affinity and selectivity for GABA(A) receptors containing an alpha(5) subunit. A single amino acid residue (alpha(5)Ile215) has been identified that plays a critical role in the high-affinity, subtype-selective effects of RY-80 and structurally related ligands. Thus, substitution of alpha(5)Ile215 with the cognate amino acid contained in the alpha(1) subunit (Val211) reduced the selectivity of RY-80 for alpha(5)beta(3)gamma(2) receptors from approximately 135- to approximately 8-fold compared with alpha(1)beta(3)gamma(2) receptors. This mutation produced a comparable reduction in the selectivity of RY-24 (a structural analog of RY-80) for alpha(5)beta(3)gamma(2) receptors but did not markedly alter the affinities of ligands (e.g., flunitrazepam) that are not subtype-selective. Conversely, substitution of the alpha(1) subunit with the cognate amino acid contained in the alpha(5) subunit (i.e., alpha(1)V211I) increased the affinities of alpha(5)-selective ligands by a approximately 20-fold and reduced by 3-fold the affinity of an alpha(1)-selective agonist (zolpidem). Increasing the lipophilicity (e.g., by substitution of Phe) of alpha(5)215 did not significantly affect the affinities (and selectivities) of RY-80 and RY-24 for alpha(5)-containing GABA(A) receptors. However, the effect of introducing hydrophilic and or charged residues (e.g., Lys, Asp, Thr) at this position was no greater than that produced by the alpha(5)I215V mutation. These data indicate that residue alpha(5)215 may not participate in formation of the lipophilic L(2) pocket that has been proposed to contribute to the unique pharmacological properties of alpha(5)-containing GABA(A) receptors. RY-80 and RY-24 acted as inverse agonists in both wild-type alpha(5)beta(3)gamma(2) and mutant alpha(5)I215Kbeta(3)gamma(2) receptors expressed in Xenopus laevis oocytes. However, both RY-24 and RY-80 acted as antagonists at mutant alpha(5)I215Vbeta(3)gamma(2) and alpha(5)I215Tbeta(3)gamma(2) receptors, whereas the efficacy of flunitrazepam was similar at all three receptor isoforms. The data demonstrate that amino acid residue alpha(5)215 is a determinant of both ligand affinity and efficacy at GABA(A) receptors containing an alpha(5) subunit.

The noninvasive diagnosis of lung cancer remains a formidable challenge. Although tissue diagnosis will remain the gold standard for the foreseeable future, questions pertaining to the risks and costs associated with invasive diagnostic procedures are of prime relevance. This review addresses new modalities for improving the noninvasive evaluation of suspicious lung nodules. Ultimately, the goal is to translate early diagnosis into early treatment. We discuss how biomarkers could assist in distinguishing benign from malignant nodules and aggressive from indolent tumors. The field of biomarkers is rapidly expanding and progressing, and efforts are well underway to apply molecular diagnostics to address the shortcomings of current lung cancer diagnostic tools.

Objectives: Patients who are referred to home health care after an acute care hospitalization may not receive home health care, resulting in incomplete home health referrals. This study examines the prevalence of incomplete referrals to home health, defined as not receiving home health care within 7 days after an initial hospital discharge, and investigates the relationship between home health referral completion and patient outcomes. Design: Retrospective cohort study. Setting and Participants: Medicare beneficiaries who are discharged from short-term acute care hospitals between October 2015 and December 2016 with a discharge status code on the hospital claim indicating home health care. Methods: Patient characteristics and outcomes were compared between Medicare beneficiaries with complete and incomplete home health referrals after hospital discharge. The outcomes included mortality, readmission rate, and total spending over a 1-year episode following hospitalization. These outcomes were risk-adjusted using patient demographic, socioeconomic, clinical characteristic, hospital characteristic, and state fixed effects.Results: Approximately 29% of the 724,700 hospitalizations in the analytic dataset had incomplete home health referrals after discharge. The rate of incomplete home health referrals varied among clinical conditions, ranging from 17% among joint/musculoskeletal patients and 38% among digestive/endocrine patients. Risk-adjusted 1-year mortality and readmission rates were 1.4 and 2.4 percentage points lower and total spending was $1053 higher among patients with complete home health referrals as compared with those with incomplete home health referrals after hospital discharge. Conclusions and Implications: The analysis revealed that almost 1 in 3 patients discharged from a hospital with a discharge status of home health does not receive home health care. In addition, complete home health referrals are associated with lower mortality and readmission rates and higher spending. As home health care utilization increases, policymakers should pay attention to the tradeoff between quality and cost when implementing alternative policies and payment models.