Plasma membrane wounds are repaired by a mechanism involving Ca(2+)-regulated exocytosis. Elevation in intracellular [Ca(2+)] triggers fusion of lysosomes with the plasma membrane, a process regulated by the lysosomal synaptotagmin isoform Syt VII. Here, we show that Ca(2+)-regulated exocytosis of lysosomes is required for the repair of plasma membrane disruptions. Lysosomal exocytosis and membrane resealing are inhibited by the recombinant Syt VII C(2)A domain or anti-Syt VII C(2)A antibodies, or by antibodies against the cytosolic domain of Lamp-1, which specifically aggregate lysosomes. We further demonstrate that lysosomal exocytosis mediates the resealing of primary skin fibroblasts wounded during the contraction of collagen matrices. These findings reveal a fundamental, novel role for lysosomes: as Ca(2+)-regulated exocytic compartments responsible for plasma membrane repair.

Highlights d A comprehensive and quantitative map of the mouse cysteine redox proteome in vivo d Redox networks are highly tissue selective and underlie tissue-specific biology d Cysteine thiol redox sensitivity is encoded by local electrostatic gating d Identification of redox-modified protein disease networks that remodel in aged mice

Summary Naïve T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naïve CD4+ T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture, and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one carbon metabolism that is critical for T cell activation and survival.

Germ-cell tumours (GCTs) are derived from germ cells and occur most frequently in the testes1,2. GCTs are histologically heterogeneous and distinctly curable with chemotherapy3. Gains of chromosome arm 12p and aneuploidy are nearly universal in GCTs4–6, but specific somatic genomic features driving tumour initiation, chemosensitivity and progression are incompletely characterized. Here, using clinical whole-exome and transcriptome sequencing of precursor, primary (testicular and mediastinal) and chemoresistant metastatic human GCTs, we show that the primary somatic feature of GCTs is highly recurrent chromosome arm level amplifications and reciprocal deletions (reciprocal loss of heterozygosity), variations that are significantly enriched in GCTs compared to 19 other cancer types. These tumours also acquire KRAS mutations during the development from precursor to primary disease, and primary testicular GCTs (TGCTs) are uniformly wild type for TP53. In addition, by functional measurement of apoptotic signalling (BH3 profiling) of fresh tumour and adjacent tissue7, we find that primary TGCTs have high mitochondrial priming that facilitates chemotherapy-induced apoptosis. Finally, by phylogenetic analysis of serial TGCTs that emerge with chemotherapy resistance, we show how TGCTs gain additional reciprocal loss of heterozygosity and that this is associated with loss of pluripotency markers (NANOG and POU5F1)8,9 in chemoresistant teratomas or transformed carcinomas. Our results demonstrate the distinct genomic features underlying the origins of this disease and associated with the chemosensitivity phenotype, as well as the rare progression to chemoresistance. These results identify the convergence of cancer genomics, mitochondrial priming and GCT evolution, and may provide insights into chemosensitivity and resistance in other cancers.

Highlights d Mouse and human muscle selectively release succinate during exercise d Muscle cells release succinate by pH-gated secretion via MCT1 d Extracellular succinate regulates paracrine responses to exercise through SUCNR1

Background Protection afforded from prior disease among patients with coronavirus disease 2019 (COVID-19) infection is unknown. If infection provides substantial long-lasting immunity, it may be appropriate to reconsider vaccination distribution plans. Methods This retrospective cohort study of one multi-hospital health system included 150,325 patients tested for COVID-19 infection via PCR from March 12, 2020 to August 30, 2020. Testing performed up to February 24, 2021 in these patients was included for analysis. The main outcome was reinfection, defined as infection ≥ 90 days after initial testing. Secondary outcomes were symptomatic infection and protection of prior infection against reinfection. Results Of 150,325 patients, 8,845 (5.9%) tested positive and 141,480 (94.1%) tested negative prior to August 30. 1,278 (14.4%) of the positive patients were retested after 90 days, and 62 had possible reinfection. Of those, 31 (50%) were symptomatic. Of those with initial negative testing, 5,449 (3.9%) were subsequently positive and 3,191 of those (58.5%) were symptomatic. Protection offered from prior infection was 81.8% (95% confidence interval 76.6 to 85.8), and against symptomatic infection was 84.5% (95% confidence interval 77.9 to 89.1). This protection increased over time. Conclusions Prior infection in patients with COVID-19 was highly protective against reinfection and symptomatic disease. This protection increased over time, suggesting that viral shedding or ongoing immune response may persist beyond 90 days and may not represent true reinfection. As vaccine supply is limited, patients with known history of COVID-19 could delay early vaccination to allow for the most vulnerable to access the vaccine and slow transmission.

All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli β-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.

OBJECTIVES: To determine the association of catecholamine dose, lactate concentration, and timing from shock onset at vasopressin initiation with in-hospital mortality. DESIGN: Retrospective, observational study using segmented and multivariable logistic regression to evaluate the associations of catecholamine dose, lactate concentration, and timing from shock onset at vasopressin initiation with in-hospital mortality. SETTING: Multiple hospitals within the Cleveland Clinic Health System. PATIENTS: Adult patients who met criteria for septic shock based on the U.S. Centers for Disease Control and Prevention Adult Sepsis Event definition. INTERVENTIONS: All patients received continuous infusion vasopressin as an adjunct to catecholamine vasopressors. MEASUREMENTS AND MAIN RESULTS: In total, 1,610 patients were included with a mean Acute Physiology and Chronic Health Evaluation III 109.0 ± 35.1 and Sequential Organ Failure Assessment 14.0 ± 3.5; 41% of patients survived the hospital admission. At the time of vasopressin initiation, patients had median (interquartile range) lactate concentration 3.9 mmol/L (2.3–7.2 mmol/L), norepinephrine-equivalent dose 25 µg/min (18–40 µg/min), and 5.3 hours (2.1–12.2 hr) elapsed since shock onset. The odds of in-hospital mortality increased 20.7% for every 10 µg/min increase in norepinephrine-equivalent dose up to 60 µg/min at the time of vasopressin initiation (adjusted odds ratio, 1.21 [95% CI, 1.09–1.34]), but no association was detected when the norepinephrine-equivalent dose exceeded 60 µg/min (adjusted odds ratio, 0.96 [95% CI, 0.84–1.10]). There was a significant interaction between timing of vasopressin initiation and lactate concentration (p = 0.02) for the association with in-hospital mortality. A linear association between increasing in-hospital mortality was detected for increasing lactate concentration at the time of vasopressin initiation, but no association was detected for time elapsed from shock onset. CONCLUSIONS: Higher norepinephrine-equivalent dose at vasopressin initiation and higher lactate concentration at vasopressin initiation were each associated higher in-hospital mortality in patients with septic shock who received vasopressin.