Synaptotagmin-1 undergoes phase separation to regulate its calcium-sensitive oligomerization.

Synaptotagmin-1 (Syt1) is a calcium sensor that regulates synaptic vesicle fusion in synchronous neurotransmitter release. Syt1 interacts with negatively charged lipids and the SNARE complex to control the fusion event. However, it remains incompletely understood how Syt1 mediates Ca2+-trigged synaptic vesicle fusion. Here, we discovered that Syt1 undergoes liquid-liquid phase separation (LLPS) to form condensates both in vitro and in living cells. Syt1 condensates play a role in vesicle attachment to the PM and efficiently recruit SNAREs and complexin, which may facilitate the downstream synaptic vesicle fusion. We observed that Syt1 condensates undergo a liquid-to-gel-like phase transition, reflecting the formation of Syt1 oligomers. The phase transition can be blocked or reversed by Ca2+, confirming the essential role of Ca2+ in Syt1 oligomer disassembly. Finally, we showed that the Syt1 mutations causing Syt1-associated neurodevelopmental disorder impair the Ca2+-driven phase transition. These findings reveal that Syt1 undergoes LLPS and a Ca2+-sensitive phase transition, providing new insights into Syt1-mediated vesicle fusion.

Optical coherence tomography analysis of lesion characteristics and thrombus types in non ST-segment elevation myocardial infarction patients.

The precise features of lesions in non-ST-segment elevation myocardial infarction (NSTEMI) patients with total occlusion (TO) of the infarct-related artery (IRA) are still unclear. This study employs optical coherence tomography (OCT) to investigate pathological features in NSTEMI patients with or without IRA TO and explores the relationship between thrombus types and IRA occlusive status. This was a single-center retrospective study. A total of 202 patients diagnosed with NSTEMI were divided into two groups: those with Thrombolysis In Myocardial Infarction (TIMI) flow grade 0 before percutaneous coronary intervention (PCI) (referred to as the TO group, n = 100) and those TIMI flow grade 1-3 (referred to as the Non-TO group, n = 102). Baseline characteristics, coronary angiography findings, and OCT results were collected. Multivariate logistic analysis identified factors influencing TO in NSTEMI. The category of NSTEMI was further subdivided based on the type of electrocardiogram (ECG) into two subgroups: ST segment unoffset myocardial infarction (STUMI) and ST segment depression myocardial infarction (STDMI). This division allows for a more specific classification of NSTEMI cases. The TO group had a younger age, higher male representation, more smokers, lower hypertension and cerebrovascular disease incidence, lower left ventricular ejection fraction (LVEF), and higher creatine kinase myocardial band (CKMB) and creatine kinase (CK) peak levels. In the TO group, LCX served as the main IRA (52.0%), whereas in the Non-TO group, LAD was the predominant IRA (45.1%). Compared to the Non-TO group, OCT findings demonstrated that red thrombus/mixed thrombus was more common in the TO group, along with a lower occurrence of white thrombus (p < 0.001). The TO group exhibited a higher prevalence of STUMI (p = 0.001), whereas STDMI was more commonly observed in the Non-TO group (p = 0.001). NSTEMI presents as STUMI and STDMI distinct entities. Red thrombus/mixed thrombus in IRA often indicates occlusive lesions with STUMI on ECG. White thrombus suggests non-occlusive lesions with STDMI on ECG.

Evaluation of an Optimized Workflow for the Radiofrequency Catheter Ablation of Paroxysmal Atrial Fibrillation.

BACKGROUND A significant number of atrial fibrillation (AF) recurrences occur after initial ablation, often due to pulmonary vein reconnections or triggers from non-pulmonary veins. MATERIAL AND METHODS Patients with paroxysmal AF who underwent radiofrequency catheter ablation for the first time were enrolled. Base on propensity score matching (1: 1 matching), 118 patients were selected for an optimized workflow for the radiofrequency catheter ablation of paroxysmal AF (OWCA) group and a conventional group. Comparative analysis of the acute and 12-month clinical outcomes was conducted. Moreover, an artificial intelligence analytics platform was used to evaluate the quality of pulmonary vein isolation (PVI) circles. RESULTS PVI was successfully achieved in all patients. Incidence of first-pass isolation of bilateral PVI circles was higher (P=0.009) and acute pulmonary vein reconnections was lower (P=0.027) in the OWCA group than conventional group. The OWCA group displayed a significant reduction in the number of fractured points (P<0.001), stacked points (P=0.003), and a greater proportion of cases in which the radiofrequency index achieved the target value (P=0.003). Additionally, the contact force consistently met the force over time criteria (P<0.001) for bilateral PVI circles in the OWCA group, accompanied by a shorter operation time (P=0.017). During the 12-month follow-up period, the OWCA group exhibited a higher atrial arrhythmia-free survival rate following the initial ablation procedure than did the conventional group. CONCLUSIONS The optimized workflow for radiofrequency catheter ablation of paroxysmal AF could play a crucial role in creating higher quality PVI circles. This improvement is reflected in a significantly elevated 12-month atrial arrhythmia-free survival rate.

IL-36 antagonism blunts the proliferation and migration of oral squamous cell carcinoma cells.

IL-36 is known to mediate inflammation and fibrosis. Nevertheless, IL-36 signalling axis has also been implicated in cancer, although understanding of exact contribution of IL-36 to cancer progression is very limited, partly due to existence of multiple IL-36 ligands with agonistic and antagonistic function. Here we explored the role of IL-36 in oral squamous cell carcinoma (OSCC). Firstly, we analyzed expression of IL-36 ligands and receptor and found that the expression of IL-36γ was significantly higher in head and neck cancer (HNSCC) than that of normal tissues, and that the high expression of IL-36γ predicted poor clinical outcomes. Secondly, we investigated the direct effect of IL-36γ on OSCC cells and found that IL-36γ stimulated proliferation of OSCC cells with high expression of IL-36R expression. Interestingly, IL-36γ also promoted migration of OSCC cells with low to high IL-36R expression. Critically, both proliferation and migration of OSCC cells induced by IL-36γ were abrogated by anti-IL-36R mAb. Fittingly, RNA sequence analysis revealed that IL-36γ regulated genes involved in cell cycle and cell division. In summary, our results showed that IL-36γ can be a tumor-promoting factor, and targeting of IL-36R signalling may be a beneficial targeted therapy for patients with abnormal IL-36 signalling.

Munc18c accelerates SNARE-dependent membrane fusion in the presence of regulatory proteins α-SNAP and NSF.

Intracellular vesicle fusion is driven by the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cofactors, including Sec1/Munc18 (SM), α-SNAP, and NSF. α-SNAP and NSF play multiple layers of regulatory roles in the SNARE assembly, disassembling the cis-SNARE complex and the prefusion SNARE complex. How SM proteins coupled with NSF and α-SNAP regulate SNARE-dependent membrane fusion remains incompletely understood. Munc18c, an SM protein involved in the exocytosis of the glucose transporter GLUT4, binds and activates target (t-) SNAREs to accelerate the fusion reaction through a SNARE-like peptide (SLP). Here, using an in vitro reconstituted system, we discovered that α-SNAP blocks the GLUT4 SNAREs-mediated membrane fusion. Munc18c interacts with t-SNAREs to displace α-SNAP, which overcomes the fusion inhibition. Furthermore, Munc18c shields the trans-SNARE complex from NSF/α-SNAP-mediated disassembly and accelerates SNARE-dependent fusion kinetics in the presence of NSF and α-SNAP. The SLP in domain 3a is indispensable in Munc18c-assisted resistance to NSF and α-SNAP. Together, our findings demonstrate that Munc18c protects the prefusion SNARE complex from α-SNAP and NSF, promoting SNARE-dependent membrane fusion through its SLP.

Microvesicles facilitate the differentiation of mesenchymal stem cells into pancreatic beta-like cells via miR-181a-5p/150-5p.

Transplantation of pancreatic islet cells is a promising strategy for the long-term treatment of type 1 diabetes (T1D). The stem cell-derived beta cells showed great potential as substitute sources of transplanted pancreatic islet cells. However, the current efficiency of stem cell differentiation still cannot match the requirements for clinical transplantation. Here, we report that microvesicles (MVs) from insulin-producing INS-1 cells could induce mesenchymal stem cell (MSC) differentiation into pancreatic beta-like cells. The combination of MVs with small molecules, nicotinamide and insulin-transferrin-selenium (ITS), dramatically improved the efficiency of MSC differentiation. Notably, the function of MVs in MSC differentiation requires their entry into MSCs through giant pinocytosis. The MVs-treated or MVs combined with small molecules-treated MSCs show pancreatic beta-like cell morphology and response to glucose stimulation in insulin secretion. Using high throughput small RNA-sequencing, we found that MVs induced MSC differentiation into the beta-like cells through miR-181a-5p/150-5p. Together, our findings reveal the role of MVs or the MV-enriched miR-181a-5p/150-5p as a class of biocompatible reagents to differentiate MSCs into functional beta-like cells and demonstrate that the combined usage of MVs or miR-181a-5p/150-5p with small molecules can potentially be used in making pancreatic islet cells for future clinical purposes.

Relationship between thrombolysis in myocardial infarction blood flow before percutaneous coronary intervention and the morphological characteristics of culprit vessel plaques in patients with STEMI.

The present study aimed to investigate the relationship between morphological characteristics of culprit coronary plaques and thrombolysis in myocardial infarction (TIMI) blood flow grade in patients with ST-segment elevation myocardial infarction (STEMI). According to the TIMI blood flow of the culprit vessel before percutaneous coronary intervention (PCI), 222 patients with STEMI were divided into two groups: TIMI 0/1 group (n=164) and TIMI 2/3 group (n=58). The baseline characteristics, coronary angiographic findings and optical coherence tomography images were collected. Multivariate logistic regression analysis was used to identify factors independently associated with poor initial TIMI blood flow. Compared with TIMI 2/3 group, TIMI 0/1 group had a significantly smaller minimum lumen diameter, greater diameter stenosis and longer lesion length, a higher incidence of lipid plaque, larger lipid length, maximum lipid arc, lipid index and maximum cross-sectional area (CSA) of plaque rupture, as well as a higher prevalence of thin-cap fibroatheroma (TCFA) and healed plaque (P<0.05). Multivariate logistic analysis demonstrated that lipid plaque, lipid length, maximum lipid arc, lipid index, TCFA, maximum CSA of plaque rupture and healed plaque were significantly associated with poor initial TIMI blood flow (P<0.05). In conclusion, the present study revealed that the morphological characteristics of culprit coronary plaques (lipid plaque, lipid length, maximum lipid arc, lipid index, TCFA, maximum CSA of plaque rupture and healed plaque) are significantly associated with poor initial TIMI blood flow before PCI in patients with STEMI.

A novel stepwise catheter ablation method of the mitral isthmus for persistent atrial fibrillation: efficacy and reproducibility.

BACKGROUND: Ethanol infusion of the vein of Marshall (EI-VOM) has been widely used to facilitate mitral isthmus (MI) ablation. According to the literature, the success rate of achieving a bidirectional conduction block across the MI ranges from 51 to 96%, with no standardized strategy or method available for cardiac electrophysiologists. OBJECTIVES: This study aimed to introduce and evaluate a novel ablation method of MI. METHODS: Consecutive patients with persistent atrial fibrillation (PeAF) that underwent catheter ablation were included. The MI ablation procedure followed a stepwise approach. In step 1, ethanol infusion of the vein of Marshall (EI-VOM) was performed. In step 2, a "V-shape" endocardial linear ablation connecting the left inferior pulmonary vein (LIPV) to mitral annulus (MA) was performed. In step 3, earliest activation sites(EASs) near the ablation line were identified using activation mapping followed by reinforced ablation. In step 4, precise epicardial ablation was performed, with the catheter introduced into the coronary sinus(CS) to target key ablation targets (KATs). RESULTS: 135 patients with PeAF underwent catheter ablation with the stepwise ablation method adopted in 119 cases. Bidirectional conduction blocks were achieved in 117 patients (98.3%). The block rates of every step were 0%, 58.0%, 44.0%, and 92.9%, and the cumulative block rates for the four steps were 0%, 58.0%, 76.5%, and 98.3%, respectively. No patient experienced fatal complications. CONCLUSIONS: Our novel stepwise catheter ablation method for MI yielded a high bidirectional block rate with high reproducibility.

Deep Tumor Penetration of CRISPR-Cas System for Photothermal-Sensitized Immunotherapy via Probiotics.

Since central cells are more malignant and aggressive in solid tumors, improving penetration of therapeutic agents and activating immunity in tumor centers exhibit great potential in cancer therapies. Here, polydopamine-coated Escherichia coli Nissle 1917 (EcN) bearing CRISPR-Cas9 plasmid-loaded liposomes (Lipo-P) are applied for enhanced immunotherapy in deep tumors through activation of innate and adaptive immunity simultaneously. After accumulation in the tumor center through hypoxia targeting, Lipo-P could be detached under the reduction of reactive oxygen species (ROS)-responsive linkers, lowering the thermal resistance of cancer cells via Hsp90α depletion. Owing to that, heating induced by polydopamine upon near-infrared irradiation could achieve effective tumor ablation. Furthermore, mild photothermal therapy induces immunogenic cell death, as bacterial infections in tumor tissues trigger innate immunity. This bacteria-assisted approach provides a promising photothermal-sensitized immunotherapy in deep tumors.

The ratio of QRS/RV6-V1: a new electrocardiographic predictor of short- and long-term adverse clinical outcomes in patients with acute myocardial infarction combined with new-onset right bundle branch block.

Background: A few studies have focused on electrocardiography (ECG) parameters correlating with clinical prognosis in patients with acute myocardial infarction (AMI) combined with new-onset right bundle branch block (RBBB). Objective: To assess the prognostic value of a new ECG parameter, namely, the ratio of QRS duration/RV6-V1 interval (QRS/RV6-V1), in patients with AMI combined with new-onset RBBB. Materials and methods: A total of 272 AMI patients combined with new-onset RBBB who received primary percutaneous coronary intervention (P-PCI) were retrospectively enrolled in the study. First, the patients were divided into survival group and non-survival group. Demographic, angiographic, and ECG characteristics were compared between the two groups. Receiver operating characteristic (ROC) curve was used to screen the best ECG parameter for predicting 1-year mortality. Second, the ratio of QRS/RV6-V1, a continuous variable, was converted to the high ratio group and low ratio group according to the optimal cutoff value point determined by the X-tile software. We compared the patient's demographic, angiographic, and ECG characteristics, in-hospital major adverse cardiovascular events (MACE), and 1-year mortality between the two groups. Multivariate logistic and Cox regressions were used to evaluate whether the ratio of QRS/RV6-V1 was an independent prognostic factor of in-hospital MACE and 1-year mortality. Results: The ROC curve showed that the ratio of QRS/RV6-V1 had a higher value for predicting in-hospital MACE and 1-year mortality than the QRS duration, RV6-V1 interval, and RV1 interval. The patients in the high ratio group had significantly higher CK-MB peak and Killip class, lower ejection fraction (EF%), higher ratio of the left anterior (LAD) descending artery as infarct-related artery (IRA), and longer total ischemia time (TIT) than those in the low ratio group. The QRS duration was wider in the high ratio group than that in the low ratio group, whereas RV6-V1 was narrower in the high ratio group compared with that in the low ratio group. The in-hospital MACE rate (93.3% vs. 31.0%, p < 0.001) and 1-year mortality rate (86.7% vs. 13.2%, p < 0.001) in the high ratio group were higher than those in the low ratio group. The higher ratio of QRS/RV6-V1 was an independent predictor of in-hospital MACE (odds ratio, 8.55; 95% CI, 1.40-52.37; p = 0.02) after adjusting other confounders. Cox regression showed that the higher ratio of QRS/RV6-V1 predicted higher 1-year mortality of the patients with AMI combined with new-onset RBBB [hazard ratios (HR), 12.4; 95% CI, 7.26-21.22); p < 0.001] than the lower ratio of QRS/RV6-V1, and the HR still stayed at 2.21 even after a multivariable adjustment (HR, 2.21; 95% CI, 1.05-4.64); p = 0.037). Conclusion: According to the results of our study, the high ratio of QRS/RV6-V1 (>3.0) was a valuable predictor of short- and long-term adverse clinical outcomes in AMI patients combined with new-onset RBBB. The implications of the high ratio of QRS/RV6-V1 were severe ischemia and pseudo synchronization between bi-ventricle.

Distinct Effects of Familial Parkinson's Disease-Associated Mutations on α-Synuclein Phase Separation and Amyloid Aggregation.

The Lewy bodies and Lewy neurites are key pathological hallmarks of Parkinson's disease (PD). Single-point mutations associated with familial PD cause α-synuclein (α-Syn) aggregation, leading to the formation of Lewy bodies and Lewy neurites. Recent studies suggest α-Syn nucleates through liquid-liquid phase separation (LLPS) to form amyloid aggregates in a condensate pathway. How PD-associated mutations affect α-Syn LLPS and its correlation with amyloid aggregation remains incompletely understood. Here, we examined the effects of five mutations identified in PD, A30P, E46K, H50Q, A53T, and A53E, on the phase separation of α-Syn. All other α-Syn mutants behave LLPS similarly to wild-type (WT) α-Syn, except that the E46K mutation substantially promotes the formation of α-Syn condensates. The mutant α-Syn droplets fuse to WT α-Syn droplets and recruit α-Syn monomers into their droplets. Our studies showed that α-Syn A30P, E46K, H50Q, and A53T mutations accelerated the formation of amyloid aggregates in the condensates. In contrast, the α-Syn A53E mutant retarded the aggregation during the liquid-to-solid phase transition. Finally, we observed that WT and mutant α-Syn formed condensates in the cells, whereas the E46K mutation apparently promoted the formation of condensates. These findings reveal that familial PD-associated mutations have divergent effects on α-Syn LLPS and amyloid aggregation in the phase-separated condensates, providing new insights into the pathogenesis of PD-associated α-Syn mutations.

ORP9 and ORP10 form a heterocomplex to transfer phosphatidylinositol 4-phosphate at ER-TGN contact sites.

Oxysterol-binding protein (OSBP) and its related proteins (ORPs) are a family of lipid transfer proteins (LTPs) that mediate non-vesicular lipid transport. ORP9 and ORP10, members of the OSBP/ORPs family, are located at the endoplasmic reticulum (ER)-trans-Golgi network (TGN) membrane contact sites (MCSs). It remained unclear how they mediate lipid transport. In this work, we discovered that ORP9 and ORP10 form a binary complex through intermolecular coiled-coil (CC) domain-CC domain interaction. The PH domains of ORP9 and ORP10 specially interact with phosphatidylinositol 4-phosphate (PI4P), mediating the TGN targeting. The ORP9-ORP10 complex plays a critical role in regulating PI4P levels at the TGN. Using in vitro reconstitution assays, we observed that while full-length ORP9 efficiently transferred PI4P between two apposed membranes, the lipid transfer kinetics was further accelerated by ORP10. Interestingly, our data showed that the PH domains of ORP9 and ORP10 participate in membrane tethering simultaneously, whereas ORDs of both ORP9 and ORP10 are required for lipid transport. Furthermore, our data showed that the depletion of ORP9 and ORP10 led to increased vesicle transport to the plasma membrane (PM). These findings demonstrate that ORP9 and ORP10 form a binary complex through the CC domains, maintaining PI4P homeostasis at ER-TGN MCSs and regulating vesicle trafficking.

Low-dimensional nanomaterials as an emerging platform for cancer diagnosis and therapy.

The burden of cancer is increasing, being widely recognized as one of the main reasons for deaths among humans. Despite the tremendous efforts that have been made worldwide to stem the progression and metastasis of cancer, morbidity and mortality in malignant tumors have been clearly rising and threatening human health. In recent years, nanomedicine has come to occupy an increasingly important position in precision oncotherapy, which improves the diagnosis, treatment, and long-term prognosis of cancer. In particular, LDNs with distinctive physicochemical capabilities have provided great potential for advanced biomedical applications, attributed to their large surface area, abundant surface binding sites, and good cellular permeation properties. In addition, LDNs can integrate CT/MR/US/PAI and PTT/PDT/CDT/NDDS into a multimodal theranostic nanoplatform, enabling targeted therapy and efficacy assessments for cancer. This review attempts to concisely summarize the classification and major properties of LDNs. Simultaneously, we particularly emphasize their applications in the imaging, diagnosis, and treatment of cancerous diseases.

NEDD4 ameliorates myocardial reperfusion injury by preventing macrophages pyroptosis.

OBJECTIVES: The inflammatory cascade and cell death post-myocardial ischemia reperfusion (MI/R) are very complex. Despite the understanding that macrophage inflammation has a pivotal role in the pathophysiology of MI/R, the contribution of macrophage inflammatory signals in tailoring the function of vascular endothelium remains unknown. MATERIALS AND METHODS: In the present study, we analyzed the effects of NEDD4 on the NLRP3 inflammasome activation-mediated pyroptosis in vitro after an acute pro-inflammatory stimulus and in vivo in a MI/R mouse model. TTC and Evan's blue dye, Thioflavin S, immunohistochemistry staining, and ELISA were performed in wild-type and NEDD4 deficiency mice. THP-1 cells were transfected with si-NEDD4 or si-SF3A2. HEK293T cells were transfected with NEDD4 or SF3A2 overexpression plasmid. ELISA analyzed the inflammatory cytokines in the cell supernatant. The levels of NEDD4, SF3A2, and NLRP3/GSDMD pathway were determined by Western blot. Protein interactions were evaluated by immunoprecipitation. The protein colocalization in cells was monitored using a fluorescence microscope. RESULTS: NEDD4 inhibited NLRP3 inflammasome activation and pyroptosis in THP-1 cells treated with lipopolysaccharide (LPS) and nigericin (Nig). Mechanistically, NEDD4 maintained the stability of NLRP3 through direct interaction with the SF3A2, whereas the latter association with NLRP3 indirectly interacted with NEDD4 promoting proteasomal degradation of NLRP3. Deletion of NLRP3 expression further inhibited the caspase cascade to induce pyroptosis. Interestingly, inhibiting NLRP3 inflammasome activation in THP-1 cells could prevent cardiac microvascular endothelial cells (CMECs) injury. In addition, NEDD4 deficiency decreased animal survival and increased myocardial infarct size, no-reflow area, and promoted macrophages infiltration post-MI/R. CONCLUSIONS: NEDD4 could be a potential therapeutic target in microvascular injury following myocardial reperfusion. Video Abstract.

Ablation index-guided high-power ablation for superior vena cava isolation in patients with atrial fibrillation.

Background: The strategy of ablation index (AI)-guided high-power ablation seems to be a novel strategy for performing pulmonary vein isolation (PVI). An AI-guided high-power ablation strategy was used in this study to determine whether superior vena cava isolation (SVCI) after PVI was feasible and safe for patients with AF. Methods: Data from 53 patients with AF were collected. Mapping and ablation of SVC were performed. The applied power was set at 45 W and the procedure was guided by AI. The SVC was divided into six segments in a cranial view. The RF applications and AI values in different segments were compared and analyzed. Using receiver operating characteristic (ROC) analysis, the diagnostic accuracy of AI value for predicting segment block was evaluated. Results: Electrical SVCIs were successfully achieved in all patients. SVCI was performed by segment ablation in most cases, with RF applications in different segments. The mean AI value in non-lateral walls was higher than that of the lateral wall (392 ± 28 vs. 371 ± 37, P < 0.001). Acutely blocked sites had significantly larger AI values compared with no-blocked sites (390 ± 30 vs. 343 ± 23, P < 0.001). The optimal AI cut-off value for non-lateral segments was 379 (sensitivity: 75.9%, specificity: 100%) and for lateral segments was 345 (sensitivity: 82.3%, specificity: 100%). Conclusion: The AI values were predictive of the acute conduction block of SVCI. With AI values of 345 and 379, respectively, conduction block was achieved in the lateral walls at a lower level than in the non-lateral walls.

Reconstitution and biochemical studies of extended synaptotagmin-mediated lipid transport.

Extended synaptotagmins (E-Syts) are a family of lipid transfer proteins (LTPs) located at the endoplasmic reticulum (ER)-plasma membrane (PM) contact sites in eukaryotic cells. They possess a conserved synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain and two to five C2 domains. While the membrane tethering function of E-Syts has been well studied in diverse species, recent studies revealed that the mammalian E-Syt1 and its yeast homolog tricalbin 3 (Tcb3) could transport lipids between the opposed membrane. Mechanical studies suggested SYT1 transfers lipids fundamentally through the SMP domain, but the lipid transport requires the regulation of C2 domain-mediated membrane tethering. In addition, both E-Syt1 and Tcb3 are Ca2+-modulated LTPs, which sense and interact with Ca2+ through the C2 domains. This chapter describes the in vitro reconstitution and biochemical assays for studying the functions and mechanisms of E-Syts, by expressing and purifying recombinant proteins, preparing reconstitution systems, and developing assays for membrane tethering and lipid transport.

The Characterization of the Tobacco-Derived Wild Tomato Mosaic Virus by Employing Its Infectious DNA Clone.

Viral diseases of cultivated crops are often caused by virus spillover from wild plants. Tobacco (N. tabacum) is an important economic crop grown globally. The viral pathogens of tobacco are traditional major subjects in virology studies and key considerations in tobacco breeding practices. A positive-strand RNA virus, wild tomato mosaic virus (WTMV), belonging to the genus potyvirus in the family potyviridae was recently found to infect tobacco in China. In this study, diseased tobacco leaf samples were collected in the Henan Province of China during 2020-2021. Several samples from different locations were identified as WTMV positive. An infectious DNA clone was constructed based on one of the WTMV isolates. By using this clone, we found that WTMV from tobacco could establish infections on natural reservoir hosts, demonstrating a possible route of WTMV spillover and overwintering in the tobacco field. Furthermore, the WTMV infection was found to be accompanied by other tobacco viruses in the field. The co-inoculation experiments indicate the superinfection exclusion (SIE) between WTMV and other potyvirus species that infect tobacco. Overall, our work reveals novel aspects of WTMV evolution and infection in tobacco and provides an important tool for further studies of WTMV.

The Differences in Clinical Characteristic and Outcomes of New Onset Typical versus Atypical Right Branch Bundle Block in Acute Myocardial Infarction.

Objective: The purpose of this study is to explore the clinical characteristics and estimate the new-onset atypical right branch bundle block (ATRBBB) predictive value in short-term and long-term mortality by comparing the typical right branch bundle block (TRBBB) subset in acute myocardial infarction (AMI) patients. Methods: A total of 224 AMI patients combined with new onset RBBB who received primary coronary angiography were included, being admitted to Henan Provincial People's Hospital in China from July 2010 to June 2021. Patients were divided into typical RBBB group (n = 104) and atypical RBBB group (n = 120). The differences in clinical characteristics between the two groups were analyzed. Logistic and Cox regression analysis were performed to identify independent predictors of in-hospital Major Adverse Cardiovascular Events (MACE). Result: The ATRBBB group had a higher proportion of smoking and alcohol consumption, higher body mass index, worse cardiac function (killip ≧ II proportion), higher peak value of CK-MB, lower LVEF%, longer total ischemia time, higher proportion of LAD (left anterior descending coronary artery) occlusion, and multivessel lesions, compared to the TRBBB group. The ATRBBB group had a higher proportion of in-hospital MACE and 1-year all-cause mortality compared to the TRBBB group. ATRBBB was an independent predictor of in-hospital MACE and 1-year mortality in patients with AMI combined with new onset RBBB. Conclusions: ATRBBB group had more serious clinical symptoms and clinical prognosis. New ATRBBB is an independent predictor of in-hospital MACE and 1-year death in patients with AMI combined with RBBB. If the infarct-related vessel was opened immediately, the evolution of TRBBB to ATRBBB may be avoided, leading to a better prognosis.

Epigallocatechin gallate inhibits SNARE-dependent membrane fusion by blocking trans-SNARE assembly.

Insulin secretion is a signal-triggered process that requires membrane fusion between the secretory granules and plasma membrane in pancreatic ß cells. The exocytosis of insulin is mediated by target-soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) on the plasma membrane and vesicle-SNAREs on the vesicles, which assemble into a quaternary trans-SNARE complex to initiate the fusion. Expression of fusion proteins is reduced in the islets of patients with type II diabetes, indicating that SNARE-mediated fusion defect is closely related to insulin-based metabolic diseases. Previous studies have suggested that epigallocatechin gallate (EGCG) has an inhibitory effect on membrane fusion. In the present study, we performed in vitro reconstitution assays to unravel the molecular mechanisms of EGCG in SNARE-mediated insulin secretory vesicle fusion. Our data show that EGCG efficiently inhibits insulin secretory SNARE-mediated membrane fusion. Mechanistic studies indicated that EGCG blocks the formation of the trans-SNARE complex. Furthermore, calcium/synaptotagmin-7-stimulated fusion kinetics were largely reduced by EGCG, confirming that it is a potential regulator of SNARE-dependent insulin secretion. Our findings suggest that the trans-SNARE complex might be a promising target for controlling SNARE-dependent vesicle fusion.