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Arthropod-borne flaviviruses cause life-threatening diseases associated with endothelial hyperpermeability and vascular leak. We recently found that vascular leak can be triggered by dengue virus (DENV) non-structural protein 1 (NS1) via the disruption of the endothelial glycocalyx-like layer (EGL). However, the molecular determinants of NS1 required to trigger EGL disruption and the cellular pathway(s) involved remain unknown. Here we report that mutation of a single glycosylated residue of NS1 (N207Q) abolishes the ability of NS1 to trigger EGL disruption and induce endothelial hyperpermeability. Intriguingly, while this mutant bound to the surface of endothelial cells comparably to wild-type NS1, it was no longer internalized, suggesting that NS1 binding and internalization are distinct steps. Using endocytic pathway inhibitors and gene-specific siRNAs, we determined that NS1 was endocytosed into endothelial cells in a dynamin- and clathrin-dependent manner, which was required to trigger endothelial dysfunction in vitro and vascular leak in vivo. Finally, we found that the N207 glycosylation site is highly conserved among flaviviruses and is also essential for West Nile and Zika virus NS1 to trigger endothelial hyperpermeability via clathrin-mediated endocytosis. These data provide critical mechanistic insight into flavivirus NS1-induced pathogenesis, presenting novel therapeutic and vaccine targets for flaviviral diseases.

Rickettsia species of the spotted fever group are arthropod-borne obligate intracellular bacteria that can cause mild to severe human disease. These bacteria invade host cells, replicate in the cell cytosol, and spread from cell to cell. To access the host cytosol and avoid immune detection, they escape membrane-bound vacuoles by expressing factors that disrupt host membranes. Here, we show that a patatin-like phospholipase A2 enzyme (Pat1) facilitates Rickettsia parkeri infection by promoting escape from host membranes and cell-cell spread. Pat1 is important for infection in a mouse model and, at the cellular level, is crucial for efficiently escaping from single and double membrane-bound vacuoles into the host cytosol, and for avoiding host galectins that mark damaged membranes. Pat1 is also important for avoiding host polyubiquitin, preventing recruitment of autophagy receptor p62, and promoting actin-based motility and cell-cell spread. Pathogenic Rickettsia species are arthropod-borne, obligate intracellular bacteria that invade host cells, replicate in the cell cytosol, and spread from cell to cell. Here, Borgo et al. identify a Rickettsia phospholipase enzyme that is important for infection by helping the bacteria escape from host cell vacuoles into the host cytosol, preventing targeting by autophagy, and promoting bacterial motility and spread to other cells.

The Flaviviridae are a family of viruses that include the important arthropod-borne human pathogens dengue virus, West Nile virus, Zika virus, Japanese encephalitis virus, and yellow fever virus. Flavivirus nonstructural protein 1 (NS1) is essential for virus replication but is also secreted from virus-infected cells. Extracellular NS1 acts as a virulence factor during flavivirus infection in multiple ways, including triggering endothelial dysfunction and vascular leak via interaction with endothelial cells. While the role of NS1 in inducing vascular leak and exacerbating pathogenesis is well appreciated, if and how NS1-triggered endothelial dysfunction promotes virus infection remains obscure. Flaviviruses have a common need to disseminate from circulation into specific tissues where virus-permissive cells reside. Tissue-specific dissemination is associated with disease manifestations of a given flavivirus, but mechanisms dictating virus dissemination are unclear. Here we show that NS1-mediated endothelial dysfunction promotes virus dissemination in vitro and in vivo . In mouse models of dengue virus infection, we show that anti-NS1 antibodies decrease virus dissemination, while the addition of exogenous NS1 promotes virus dissemination. Using an in vitro system, we show that NS1 promotes virus dissemination in two distinct ways: (1) promoting crossing of barriers and (2) increasing infectivity of target cells in a tissue- and virus-specific manner. The capacity of NS1 to modulate infectivity correlates with a physical association between virions and NS1, suggesting a potential NS1-virion interaction. Taken together, our study indicates that flavivirus NS1 acts as a viral toxin and promotes virus dissemination across endothelial barriers, providing an evolutionary basis for virus-triggered vascular leak.

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of vascular leak are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to induce barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Notably, we show that SARS-CoV-2 infection caused leak in vivo, which was reduced by inhibiting integrins. Our findings offer mechanistic insight into SARS-CoV-2-triggered vascular leak, providing a starting point for development of therapies targeting COVID-19. Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction, however, the molecular pathways resulting in endothelial barrier dysfunction and vascular leakage are only sparsely understood. Here, Biering et al. show that SARS-CoV-2 spike protein is sufficient to induce barrier dysfunction and vascular leak. They show a role for integrins, TGF-beta, ECM remodeling enzymes, and glycosaminoglycans in this S-mediated barrier dysfunction.

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction and vascular leak , independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.

Spotted fever group Rickettsia species are arthropod-borne obligate intracellular bacteria that can cause mild to severe human disease. These bacteria invade host cells, replicate in the cell cytosol, and then spread from cell to cell. To access the host cytosol and avoid detection by immune surveillance mechanisms, these pathogens must have evolved efficient ways to escape membrane-bound vacuoles. Although Rickettsia are predicted to express factors that disrupt host membranes, little is known about how and when these proteins function during infection. Here, we investigated the role of a Rickettsia patatin-like phospholipase A2 enzyme (Pat1) during host cell infection by characterizing a Rickettsia parkeri mutant with a transposon insertion in the pat1 gene. We show that Pat1 is important for infection in a mouse model and in host cells. We further show that Pat1 is critical for efficiently escaping from the single and double membrane-bound vacuoles into the host cytosol, and for avoiding host galectins that mark damaged membranes. In the host cytosol, Pat1 is important for avoiding host polyubiquitin, preventing recruitment of autophagy receptor p62, and promoting actin-based motility and cell-cell spread. Our results show that Pat1 plays critical roles in escaping host membranes and promoting cell-cell spread during R. parkeri infection and suggest diverse roles for patatin-like phospholipases in facilitating microbial infection. Competing Interest Statement The authors have declared no competing interest.

Abstract Study Objectives To investigate the short- and longer-term impact of a 45-min delay in school start time on sleep and well-being of adolescents. Methods The sample consisted of 375 students in grades 7–10 (mean age ± SD: 14.6 ± 1.15 years) from an all-girls’ secondary school in Singapore that delayed its start time from 07:30 to 08:15. Self-reports of sleep timing, sleepiness, and well-being (depressive symptoms and mood) were obtained at baseline prior to the delay, and at approximately 1 and 9 months after the delay. Total sleep time (TST) was evaluated via actigraphy. Results After 1 month, bedtimes on school nights were delayed by 9.0 min, while rise times were delayed by 31.6 min, resulting in an increase in time in bed (TIB) of 23.2 min. After 9 months, the increase in TIB was sustained, and TST increased by 10.0 min relative to baseline. Participants also reported lower levels of subjective sleepiness and improvement in well-being at both follow-ups. Notably, greater increase in sleep duration on school nights was associated with greater improvement in alertness and well-being. Conclusions Delaying school start time can result in sustained benefits on sleep duration, daytime alertness, and mental well-being even within a culture where trading sleep for academic success is widespread.

The role of adjuvant chemotherapy in stage II colon cancer continues to be debated. The presence of circulating tumor DNA (ctDNA) after surgery predicts very poor recurrence-free survival, whereas its absence predicts a low risk of recurrence. The benefit of adjuvant chemotherapy for ctDNA-positive patients is not well understood. We conducted a trial to assess whether a ctDNA-guided approach could reduce the use of adjuvant chemotherapy without compromising recurrence risk. Patients with stage II colon cancer were randomly assigned in a 2:1 ratio to have treatment decisions guided by either ctDNA results or standard clinicopathological features. For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery prompted oxaliplatin-based or fluoropyrimidine chemotherapy. Patients who were ctDNA-negative were not treated. The primary efficacy end point was recurrence-free survival at 2 years. A key secondary end point was adjuvant chemotherapy use. Of the 455 patients who underwent randomization, 302 were assigned to ctDNA-guided management and 153 to standard management. The median follow-up was 37 months. A lower percentage of patients in the ctDNA-guided group than in the standard-management group received adjuvant chemotherapy (15% vs. 28%; relative risk, 1.82; 95% confidence interval [CI], 1.25 to 2.65). In the evaluation of 2-year recurrence-free survival, ctDNA-guided management was noninferior to standard management (93.5% and 92.4%, respectively; absolute difference, 1.1 percentage points; 95% CI, -4.1 to 6.2 [noninferiority margin, -8.5 percentage points]). Three-year recurrence-free survival was 86.4% among ctDNA-positive patients who received adjuvant chemotherapy and 92.5% among ctDNA-negative patients who did not. A ctDNA-guided approach to the treatment of stage II colon cancer reduced adjuvant chemotherapy use without compromising recurrence-free survival. (Supported by the Australian National Health and Medical Research Council and others; DYNAMIC Australian New Zealand Clinical Trials Registry number, ACTRN12615000381583.).

Formic acid is a promising energy carrier for on-demand hydrogen generation. Because the reverse reaction is also feasible, formic acid is a form of stored hydrogen. Here we present a robust, reusable iridium catalyst that enables hydrogen gas release from neat formic acid. This catalysis works under mild conditions in the presence of air, is highly selective and affords millions of turnovers. While many catalysts exist for both formic acid dehydrogenation and carbon dioxide reduction, solutions to date on hydrogen gas release rely on volatile components that reduce the weight content of stored hydrogen and/or introduce fuel cell poisons. These are avoided here. The catalyst utilizes an interesting chemical mechanism, which is described on the basis of kinetic and synthetic experiments. Formic acid is a promising energy carrier for on-demand hydrogen generation. Here, the authors present a robust, reusable iridium catalyst that enables hydrogen release from neat formic acid under mild conditions and in the presence of air.

Study Objectives: To compare the quality and consistency in sleep measurement of a consumer wearable device and a research-grade actigraph with polysomnography (PSG) in adolescents. Methods: Fifty-eight healthy adolescents (aged 15–19 years; 30 males) underwent overnight PSG while wearing both a Fitbit Alta HR and a Philips Respironics Actiwatch 2 (AW2) for 5 nights, with either 5 hours or 6.5 hours time in bed (TIB) and for 4 nights with 9 hours TIB. AW2 data were evaluated using two different wake and immobility thresholds. Discrepancies in estimated total sleep time (TST) and wake after sleep onset (WASO) between devices and PSG, as well as epoch-by-epoch agreements in sleep/wake classification, were assessed. Fitbit-generated sleep staging was compared to PSG. Results: Fitbit and AW2 under default settings similarly underestimated TST and overestimated WASO (TST: medium setting (M10) ≤ 38 minutes, Fitbit ≤ 47 minutes; WASO: M10 ≤ 38 minutes; Fitbit ≤ 42 minutes). AW2 at the high motion threshold setting provided readings closest to PSG (TST: ≤ 12 minutes; WASO: ≤ 18 minutes). Sensitivity for detecting sleep was ≥ 90% for both wearable devices and further improved to 95% by using the high threshold (H5) setting for the AW2 (0.95). Wake detection specificity was highest in Fitbit (≥ 0.88), followed by the AW2 at M10 (≥ 0.80) and H5 thresholds (≤ 0.73). In addition, Fitbit inconsistently estimated stage N1 + N2 sleep depending on TIB, underestimated stage N3 sleep (21–46 min), but was comparable to PSG for rapid eye movement sleep. Fitbit sensitivity values for the detection of N1 + N2, N3 and rapid eye movement sleep were ≥ 0.68, ≥ 0.50, and ≥ 0.72, respectively. Conclusions: A consumer-grade wearable device can measure sleep duration as well as a research actigraph. However, sleep staging would benefit from further refinement before these methods can be reliably used for adolescents. Clinical Trial Registration: Registry: ClinicalTrials.gov ; Title: The Cognitive and Metabolic Effects of Sleep Restriction in Adolescents; Identifier: NCT03333512; URL: https://clinicaltrials.gov/ct2/show/NCT03333512 Citation: Lee XK, Chee NIYN, Ong JL, Teo TB, van Rijn E, Lo JC, Chee MWL. Validation of a consumer sleep wearable device with actigraphy and polysomnography in adolescents across sleep opportunity manipulations. J Clin Sleep Med . 2019;15(9):1337–1346.