A cycle of Vam7p release from and PtdIns 3-P–dependent rebinding to the yeast vacuole is required for homotypic vacuole fusion

Vacuole fusion requires a coordinated cascade of priming, docking, and fusion. SNARE proteins have been implicated in the fusion itself, although their precise role in the cascade remains unclear. We now report that the vacuolar SNAP-23 homologue Vam7p is a mobile element of the SNARE complex, which moves from an initial association with the cis-SNARE complex via a soluble intermediate to the docking site. Soluble Vam7p is specifically recruited to vacuoles and can rescue a fusion reaction poisoned with antibodies to Vam7p. Both the recombinant Vam7p PX domain and a FYVE domain construct of human Hrs block the recruitment of Vam7p and vacuole fusion, demonstrating that phosphatidylinositol 3-phosphate is a primary receptor of Vam7p on vacuoles. We propose that the Vam7p cycle is linked to the availability of a lipid domain on yeast vacuoles, which is essential for coordinating the fusion reaction prior to and beyond docking

Divergent drivers of the microbial methane sink in temperate forest and grassland soils

Aerated topsoils are important sinks for atmospheric methane (CH4) via oxidation by CH4‐oxidizing bacteria (MOB). However, intensified management of grasslands and forests may reduce the CH4 sink capacity of soils. We investigated the influence of grassland land‐use intensity (150 sites) and forest management type (149 sites) on potential atmospheric CH4 oxidation rates (PMORs) and the abundance and diversity of MOB (with qPCR) in topsoils of three temperate regions in Germany. PMORs measurements in microcosms under defined conditions yielded approximately twice as much CH4 oxidation in forest than in grassland soils. High land‐use intensity of grasslands had a negative effect on PMORs (−40%) in almost all regions and fertilization was the predominant factor of grassland land‐use intensity leading to PMOR reduction by 20%. In contrast, forest management did not affect PMORs in forest soils. Upland soil cluster (USC)‐α was the dominant group of MOBs in the forests. In contrast, USC‐γ was absent in more than half of the forest soils but present in almost all grassland soils. USC‐α abundance had a direct positive effect on PMOR in forest, while in grasslands USC‐α and USC‐γ abundance affected PMOR positively with a more pronounced contribution of USC‐γ than USC‐α. Soil bulk density negatively influenced PMOR in both forests and grasslands. We further found that the response of the PMORs to pH, soil texture, soil water holding capacity and organic carbon and nitrogen content differ between temperate forest and grassland soils. pH had no direct effects on PMOR, but indirect ones via the MOB abundances, showing a negative effect on USC‐α, and a positive on USC‐γ abundance. We conclude that reduction in grassland land‐use intensity and afforestation has the potential to increase the CH4 sink function of soils and that different parameters determine the microbial methane sink in forest and grassland soils.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659ESFMinistry of Education, Science and Culture of Mecklenburg‐Western PomeraniaPeer Reviewe

Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation

Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT

Diagnostic Accuracy of a High-Sensitivity Cardiac Troponin Assay with a Single Serum Test in the Emergency Department.

OBJECTIVES: We sought to evaluate diagnostic accuracy of a high-sensitivity cardiac troponin I (hs-cTnI) assay for acute coronary syndromes (ACS) in the emergency department (ED). The assay has high precision at low concentrations and can detect cTnI in 96.8% of healthy individuals. METHODS: In successive prospective multicenter studies ("testing" and "validation"), we included ED patients with suspected ACS. We drew blood for hs-cTnI [Singulex Clarity® cTnI; 99th percentile, 8.67 ng/L; limit of detection (LoD), 0.08 ng/L] on arrival. Patients also underwent hs-cTnT (Roche Elecsys) testing over ≥3 h. The primary outcome was an adjudicated diagnosis of ACS, defined as acute myocardial infarction (AMI; prevalent or incident), death, or revascularization within 30 days. RESULTS: The testing and validation studies included 665 and 2470 patients, respectively, of which 94 (14.1%) and 565 (22.9%) had ACS. At a 1.5-ng/L cutoff, hs-cTnI had good sensitivity for AMI in both studies (98.7% and 98.1%, respectively) and would have "ruled out" 40.1% and 48.9% patients. However, sensitivity was lower for ACS (95.7% and 90.6%, respectively). At a 0.8-ng/L cutoff, sensitivity for ACS was higher (97.5% and 97.9%, ruling out 28.6% patients in each cohort). The hs-cTnT assay had similar performance at the LoD (24.6% ruled out; 97.2% sensitivity for ACS). CONCLUSIONS: The hs-cTnI assay could immediately rule out AMI in 40% of patients and ACS in >25%, with similar accuracy to hs-cTnT at the LoD. Because of its high precision at low concentrations, this hs-cTnI assay has favorable characteristics for this clinical application

Effect of COVID-19 on acute treatment of ST-segment elevation and Non-ST-segment elevation acute coronary syndrome in northwestern Switzerland

To investigate the effect of the corona virus disease 2019 (COVID-19) pandemic on the acute treatment of patients with ST-segment elevation (STEMI) and Non-ST-segment elevation acute coronary syndrome (NSTE-ACS).; We retrospectively identified patients presenting to the emergency department (ED) with suspected ACS. We evaluated the number of percutaneous coronary interventions (PCIs) for STEMI, NSTE-ACS, and elective PCI cases. In STEMI patients, we assessed the time from chest pain onset (cpo) to ED presentation, post-infarction left ventricular ejection fraction (LVEF), and time from ED presentation to PCI. We directly compared cases from two time intervals: January/February 2020 versus March/April 2020 (defined as 2 months before and after the COVID-19 outbreak). In a secondary analysis, we directly compared cases from March/April 2020 with patients from the same time interval in 2019.; From January to April 2020, 765 patients presented with acute chest pain to the ED. A dramatic reduction of ED presentations after compared to before the COVID-19 outbreak (31% relative reduction) was observed. Overall, 398 PCIs were performed, 220/398 PCIs (55.3%) before versus 178/398 PCIs (44.7%) after the outbreak. While numbers for NSTE-ACS and elective interventions declined by 21% and 31%, respectively, the number of STEMI cases remained stable. Time from cpo to ED presentation, post-infarction LVEF, and median door-to-balloon time remained unchanged.; In contrast to previous reports, our findings do not confirm the dramatic drop in STEMI cases and interventions in northwestern Switzerland as observed in other regions and hospitals around the world

The Evolution of Ecological Diversity in Acidobacteria

Acidobacteria occur in a large variety of ecosystems worldwide and are particularly abundant and highly diverse in soils. In spite of their diversity, only few species have been characterized to date which makes Acidobacteria one of the most poorly understood phyla among the domain Bacteria. We used a culture-independent niche modeling approach to elucidate ecological adaptations and their evolution for 4,154 operational taxonomic units (OTUs) of Acidobacteria across 150 different, comprehensively characterized grassland soils in Germany. Using the relative abundances of their 16S rRNA gene transcripts, the responses of active OTUs along gradients of 41 environmental variables were modeled using hierarchical logistic regression (HOF), which allowed to determine values for optimum activity for each variable (niche optima). By linking 16S rRNA transcripts to the phylogeny of full 16S rRNA gene sequences, we could trace the evolution of the different ecological adaptations during the diversification of Acidobacteria. This approach revealed a pronounced ecological diversification even among acidobacterial sister clades. Although the evolution of habitat adaptation was mainly cladogenic, it was disrupted by recurrent events of convergent evolution that resulted in frequent habitat switching within individual clades. Our findings indicate that the high diversity of soil acidobacterial communities is largely sustained by differential habitat adaptation even at the level of closely related species. A comparison of niche optima of individual OTUs with the phenotypic properties of their cultivated representatives showed that our niche modeling approach (1) correctly predicts those physiological properties that have been determined for cultivated species of Acidobacteria but (2) also provides ample information on ecological adaptations that cannot be inferred from standard taxonomic descriptions of bacterial isolates. These novel information on specific adaptations of not-yet-cultivated Acidobacteria can therefore guide future cultivation trials and likely will increase their cultivation success

Veränderungen der mikrobiellen Gemeinschaft in Grünlandböden als Reaktion auf kurz- und langfristiges Flächenmanagement

Im Rahmen des DFG Schwerpunktprogramms Biodiversitäts-Exploratorien (www.biodiversity-exploratories.de) wurden in 150 Grünlandböden die Veränderungen der mikrobiellen Gemeinschaftsstruktur und Enzymaktivität über einen Zeitraum von drei Jahren untersucht. Je 50 der Untersuchungsflächen liegen in der Schwäbischen Alb, dem Hainich-Dün und der Schorfheide-Chorin. Im Mai 2011 und 2014 wurden zeitgleich Oberbodenproben in allen Regionen genommen und die mikrobielle Biomasse (C, N, P), Gemeinschaftsstruktur (Phospholipidfettsäuren) sowie Enzymaktivitäten des C-, N- und P-Kreislaufs bestimmt. Zwischen 2011 und 2014 hat sich die Landnutzungsintensität (LUI) einiger Flächen stark verändert, während die LUI anderer fast identisch blieb. Unsere zentrale Hypothese ist, dass die Veränderung der LUI, durch die direkte Nährstoffzufuhr über Dünger, zu Veränderungen in den mikrobiellen Bodeneigenschaften zwischen den Jahren geführt hat. Tatsächlich konnten Veränderungen der mikrobiellen Bodeneigenschaften im untersuchten Zeitraum detektiert werden. Ob diese direkt durch Veränderungen (V) der LUI, oder durch Variationen in Temperatur, Wasserhaltekapazität, pH-Wert und Pflanzenbestand erklärt werden können oder ob die Änderungen der Mikroorganismen (MO) durch die historischen Bedingungen (H) auf den Flächen beeinflusst wurden, wurde mittels hierarchischer Regressionsanalysen untersucht. Dabei gingen folgende Variablen in fünf Stufen in die Modelle ein: Umwelt: Temperatur (V), Wasserhaltekapazität (V), pH (H); Landmanagement: LUI (V, H); pH-Wertänderung: pH (V); Pflanzenfunktionen: Mykorrhizierungsintensität (V, H), spezifische Blattfläche (V, H), Blatt-P (V, H), Blatt-N (V, H) und Pflanzenbiomasse: Biomasse (V), Cellulose (V), Hemicellulosen (V), Lignin (V), Biomasse P (V), Biomasse N (V), Lignin:N (V). Dabei zeigte sich, dass die funktionellen Pflanzeneigenschaften, insbesondere der Blatt-P-Gehalt, einen erheblichen Einfluss auf die Veränderung der MO im Boden hatten. Am häufigsten signifikant war ihr Einfluss auf die MO der Schwäbischen Alb und des Hainich-Dün, während in der Schorfheide-Chorin die Änderung des pH-Wertes dominierte. Direkt wirkte sich die Änderung der LUI nur auf Pilze aus, nicht auf Bakterien und Enzymaktivitäten. Ob sich die LUI indirekt über die Pflanzen auf Enzyme und Bakterien auswirkte, ist Gegenstand weiterer Analysen

Land‐use intensity and biodiversity effects on infiltration capacity and hydraulic conductivity of grassland soils in southern Germany

Evidence from experimental and established grasslands indicates that plant biodiversity can modify the water cycle. One suspected mechanism behind this is a higher infiltration capacity (ν$_{B}$) and hydraulic conductivity (K) of the soil on species-rich grasslands. However, in established and agriculturally managed grasslands, biodiversity effects cannot be studied independent of land-use effects. Therefore, we investigated in established grassland systems how land-use intensity and associated biodiversity of plants and soil animals affect νB and K at and close to saturation. On 50 grassland plots along a land-use intensity gradient in the Biodiversity Exploratory Schwäbische Alb, Germany, we measured νB with a hood infiltrometer at several matrix potentials and calculated the saturated and unsaturated K. We statistically analysed the relationship between ν$_{B}$ or K and land-use information (e.g., fertilising intensity), abiotic (e.g., soil texture) and biotic data (e.g., plant species richness, earthworm abundance). Land-use intensity decreased and plant species richness increased ν$_{B}$ and K, while the direction of the effects of soil animals was inconsistent. The effect of land-use intensity on ν$_{B}$ and K was mainly attributable to its negative effect on plant species richness. Our results demonstrate that plant species richness was a better predictor of ν$_{B}$ and K at and close to saturation than land-use intensity or soil physical properties in the established grassland systems of the Schwäbische Alb

Capture and release of partially zipped trans-SNARE complexes on intact organelles

Soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptors (SNAREs) are hypothesized to trigger membrane fusion by complexing in trans through their membrane-distal N termini and zippering toward their membrane-embedded C termini, which in turn drives the two membranes together. In this study, we use a set of truncated SNAREs to trap kinetically stable, partially zipped trans-SNARE complexes on intact organelles in the absence of hemifusion and content mixing. We show that the C-terminal zippering of SNARE cytoplasmic domains controls the onset of lipid mixing but not the subsequent transition from hemifusion to full fusion. Moreover, we find that a partially zipped nonfusogenic trans-complex is rescued by Sec17, a universal SNARE cochaperone. Rescue occurs independently of the Sec17-binding partner Sec18, and it exhibits steep cooperativity, indicating that Sec17 engages multiple stalled trans-complexes to drive fusion. These experiments delineate distinct functions within the trans-complex, provide a straightforward method to trap and study prefusion complexes on native membranes, and reveal that Sec17 can rescue a stalled, partially zipped trans-complex

Sequential Analysis of Trans-SNARE Formation in Intracellular Membrane Fusion

SM proteins stabilize cis-SNARE complexes leading to a specific preferred topology for trans-SNARE formation