The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells.

Platelets in trauma‐induced coagulopathy (TIC) are impaired, but the mechanism is not known. We performed comprehensive longitudinal platelet function testing in trauma patient samples. Platelets in TIC are widely impaired early after injury, but platelet activatability is intact. This suggests a mechanism of transient platelet cytoskeletal/integrin dysfunction during TIC. Summary BackgroundTrauma‐induced coagulopathy (TIC) is a common and deadly bleeding disorder. Platelet dysfunction is present during TIC, but its mechanisms remain unclear. Platelets are currently thought to become “exhausted,” a state in which they have released their granule contents and can no longer aggregate or contract. MethodsThis prospective observational cohort study tested the hypothesis that platelet exhaustion is present during TIC and characterized the early time course of platelet dysfunction. Blood was collected from 95 adult trauma patients at a Level I trauma center at time of Emergency Department arrival and several time points over 72 h. Platelet activation state and function were characterized using CD62P (P‐selectin) and PAC‐1 surface membrane staining, platelet function analyzer (PFA‐100), aggregometry, viscoelastic platelet mapping, and, to test for exhaustion, their ability to express CD62P after ex vivo adenosine diphosphate (ADP) agonism. Platelet function was compared between patients with and without TIC, defined by prothrombin time ≥18 s. ResultsPlatelets in TIC showed no initial increase in their level of surface activation markers or impairment of their capacity to express CD62P in response to ADP stimulation. However, TIC platelets were impaired in nearly all functional assays, spanning adhesion, aggregation, and contraction. These effects largely remained after controlling for platelet count and fibrinogen concentration and resolved after 8 h. ConclusionThe TIC platelets exhibit early impairment of adhesion, aggregation, and contraction with retained alpha granule secretion ability, suggesting a specific mechanism of cytoskeletal or integrin dysfunction that is not a result of more general platelet exhaustion.

Cystic fibrosis (CF) is characterized by chronic respiratory infections which progressively decrease lung function over time. Affected individuals experience episodes of intensified respiratory symptoms called pulmonary exacerbations (PEx) which accelerate pulmonary function decline and decrease survival. There is no standard classification for PEx, which results in treatments that are heterogeneous. Improving PEx classification and management is a significant priority for people with CF. Previous studies have shown volatile organic compounds (VOCs) in exhaled breath can be used as biomarkers because they are products of metabolic pathways dysregulated by different diseases. To provide insights on PEx classification and other clinical factors, exhaled breath was collected from subjects with CF, with some experiencing PEx and others at baseline. Exhaled breath was collected in Tedlar bags during tidal breathing for VOC analysis by solid phase microextraction coupled to gas chromatography–mass spectrometry. Statistical significance testing between quantitative and categorical clinical variables displayed percent-predicted forced expiratory volume in one second (FEV1pp) was decreased in subjects experiencing PEx. VOCs correlating with other clinical variables (body mass index, age, use of highly effective modulator therapies, and need for antibiotics) were also explored. VOCs correlating to potential confounding variables were removed and analyzed by regression for correlations with FEV1pp measurements. The VOC with the highest correlation with FEV1pp (3,7-dimethyldecane) also gave the lowest p-value when comparing subjects at baseline and during PEx. Receiver operator characteristic curves showed 3,7-dimethyldecane had a higher ability to classify PEx (area under the curve (AUC) = 0.91) relative to FEV1pp values at collection (AUC = 0.83). However, normalized ΔFEV1pp values had the highest capability to distinguish PEx (AUC = 0.93). These results show that exhaled VOCs may be a source of biomarkers for various clinical traits of CF, including PEx, that should be explored in larger sample cohorts and validation studies.

The diagnosis of primary ciliary dyskinesia (PCD) is made through a combination of clinical features supported by a panel of diagnostic tests. Our cases highlight the similarities in the clinical presentation of patients with the specific immunodeficiency activated phosphatidylinositol 3‐kinase delta syndrome 1 (or PIK3CD‐related disorder) and PCD. We highlight the importance of repeating nasal nitric oxide testing when PCD has not been confirmed by genetic or ciliary electron micrograph analysis in the setting of an expanded suppurative lung disease differential that includes considerations for immunodeficiency as well as PCD.