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.

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.

Assembly-promoting protein Munc18c stimulates SNARE-dependent membrane fusion through its SNARE-like peptide.

Intracellular vesicle fusion requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cognate Sec1/Munc18 (SM) proteins. How SM proteins act in concert with trans-SNARE complexes to promote membrane fusion remains incompletely understood. Munc18c, a broadly distributed SM protein, selectively regulates multiple exocytotic pathways, including GLUT4 exocytosis. Here, using an in vitro reconstituted system, we discovered a SNARE-like peptide (SLP), conserved in Munc18-1 of synaptic exocytosis, is crucial to the stimulatory activity of Munc18c in vesicle fusion. The direct stimulation of the SNARE-mediated fusion reaction by SLP further supported the essential role of this fragment. Interestingly, we found SLP strongly accelerates the membrane fusion rate when anchored to the target membrane but not the vesicle membrane, suggesting it primarily interacts with t-SNAREs in cis to drive fusion. Furthermore, we determined the SLP fragment is competitive with the full-length Munc18c protein and specific to the cognate v-SNARE isoforms, supporting how it could resemble Munc18c's activity in membrane fusion. Together, our findings demonstrate that Munc18c facilitates SNARE-dependent membrane fusion through SLP, revealing that the t-SNARE-SLP binding mode might be a conserved mechanism for the stimulatory function of SM proteins in vesicle fusion.

A Bayesian phase I-II clinical trial design to find the biological optimal dose on drug combination.

In recent years, combined therapy shows expected treatment effect as they increase dose intensity, work on multiple targets and benefit more patients for antitumor treatment. However, dose -finding designs for combined therapy face a number of challenges. Therefore, under the framework of phase I-II, we propose a two-stage dose -finding design to identify the biologically optimal dose combination (BODC), defined as the one with the maximum posterior mean utility under acceptable safety. We model the probabilities of toxicity and efficacy by using linear logistic regression models and conduct Bayesian model selection (BMS) procedure to define the most likely pattern of dose-response surface. The BMS can adaptively select the most suitable model during the trial, making the results robust. We investigated the operating characteristics of the proposed design through simulation studies under various practical scenarios and showed that the proposed design is robust and performed well.

Calcium-dependent and -independent lipid transfer mediated by tricalbins in yeast.

Membrane contact sites (MCSs) formed between the endoplasmic reticulum (ER) and the plasma membrane (PM) provide a platform for nonvesicular lipid exchange. The ER-anchored tricalbins (Tcb1, Tcb2, and Tcb3) are critical tethering factors at ER-PM MCSs in yeast. Tricalbins possess a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain and multiple Ca2+-binding C2 domains. Although tricalbins have been suggested to be involved in lipid exchange at the ER-PM MCSs, it remains unclear whether they directly mediate lipid transport. Here, using in vitro lipid transfer assays, we discovered that tricalbins are capable of transferring phospholipids between membranes. Unexpectedly, while its lipid transfer activity was markedly elevated by Ca2+, Tcb3 constitutively transferred lipids even in the absence of Ca2+. The stimulatory activity of Ca2+ on Tcb3 required intact Ca2+-binding sites on both the C2C and C2D domains of Tcb3, while Ca2+-independent lipid transport was mediated by the SMP domain that transferred lipids via direct interactions with phosphatidylserine and other negatively charged lipid molecules. These findings establish tricalbins as lipid transfer proteins, and reveal Ca2+-dependent and -independent lipid transfer activities mediated by these tricalbins, providing new insights into their mechanism in maintaining PM integrity at ER-PM MCSs.

Acidiluteibacter ferrifornacis gen. nov., sp. nov., a new member of the Flavobacteriales from Tielu Harbour, Hainan, PR China.

A novel bacterial strain of the family 'Vicingaceae' was isolated from mangrove of Tielu Harbour, Hainan, PR China. Strain S-15T was a Gram-stain-negative, short-rod-shaped, yellow-pigmented that could grow at 10-42 °C (optimum, 26-35 °C), at pH 5.0-9.0 (optimum, pH 5.5) and in 0.5-10.0 % w/v sea salt (optimum, 3.5-4.0 %). Cells of strain S-15T were 0.9-1.4 µm long, 0.8-0.9 µm wide, catalase-positive and oxidase-positive. Colonies on modified marine agar 2216 were 0.5-2.0 mm in diameter after incubation for 72 h at 28 °C. Analysis of 16S rRNA gene sequences revealed that strain S-15T was most closely related to Vicingus serpentipes ANORD5T (89.8 %). The major respiratory quinone of strain S-15T was menaquinone MK-7, and the dominant fatty acids were C15:0 iso, C15:1 iso G and C17:0 iso 3-OH. The major polar lipids were two unidentified aminolipids, phosphatidylethanolamine and six unidentified lipids. Analyses showed that the genome size was 3.52 Mb and the DNA G+C content was 35.6 mol%, which were higher than V. serpentipes ANORD5T with 2.92 Mb genome size and 31.0 mol% G+C content, respectively. Based on morphological, physiological and phylogenetic data, strain S-15T is considered a type strain of a new species and a new genus of the family 'Vicingaceae' for which the name Acidiluteibacter ferrifornacis gen. nov., sp. nov. is proposed. The type strain of Acidiluteibacter ferrifornacis is S-15T (=MCCC 1K03817T=JCM 33804T).

Perspective of Signal Processing-Based on Brain-Computer Interfaces Using Machine Learning Methods.

The application of artificial intelligence (AI) algorithms is an indispensable portion of developing brain-computer interfaces (BCI). With the continuous development of AI concepts and related technologies. AI algorithms such as neural networks play an increasingly powerful and extensive role in brain-computer interfaces. However, brain-computer interfaces are still facing many technical challenges. Due to the limitations of AI algorithms, brain-computer interfaces not only work with limited accuracy, but also can only be applied to certain simple scenarios. In order to explore the future directions and improvements of AI algorithms in the area of brain-computer interfaces, this paper will review and analyse the advanced applications of AI algorithms in the field of brain-computer interfaces in recent years and give possible future enhancements and development directions for the controversial parts of them. This review first presents the effects of different AI algorithms in BCI applications. A multi-objective classification method is compared with evolutionary algorithms in feature extraction of data. Then, a kind of supervised learning algorithm based on Event Related Potential (ERP) tags is presented to achieve a high accuracy in the process of pattern recognition. Finally, as an important experimental paradigm for BCI, a combined TFD-PSR-CSP feature extraction method, is explained for the problem of motor imagery. The "Discussion" part comprehensively analyses the advantages and disadvantages of the above algorithms and proposes a deep learning-based artificial intelligence algorithm in order to solve the problems arising from the above algorithms.

Optimizing dose-schedule regimens with bayesian adaptive designs: opportunities and challenges.

Due to the small sample sizes in early-phase clinical trials, the toxicity and efficacy profiles of the dose-schedule regimens determined for subsequent trials may not be well established. The recent development of novel anti-tumor treatments and combination therapies further complicates the problem. Therefore, there is an increasing recognition of the essential place of optimizing dose-schedule regimens, and new strategies are now urgently needed. Bayesian adaptive designs provide a potentially effective way to evaluate several doses and schedules simultaneously in a single clinical trial with higher efficiency, but real-world implementation examples of such adaptive designs are still few. In this paper, we cover the critical factors associated with dose-schedule optimization and review the related innovative Bayesian adaptive designs. The assumptions, characteristics, limitations, and application scenarios of those designs are introduced. The review also summarizes some unresolved issues and future research opportunities for dose-schedule optimization.

Enhancing phase I dose-finding trials design through dynamic borrowing information and handling late-onset toxicity.

Introduction: In recent years, there has been a growing trend among regulatory agencies to consider the use of historical controls in clinical trials as a means of improving the efficiency of trial design. In this paper, to enhance the statistical operating characteristic of Phase I dose-finding trials, we propose a novel model-assisted design method named "MEM-Keyboard". Methods: The proposed design is based on the multisource exchangeability models (MEMs) that allows for dynamic borrowing of information from multiple supplemental data sources, including historical trial data, to inform the dose-escalation process. Furthermore, with the frequent occurrence of delayed toxicity in novel anti-cancer drugs, we extended our proposed method to handle late-onset toxicity by incorporating historical data. This extended method is referred to as "MEM-TITE-Keyboard" and aims to improve the efficiency of early clinical trials. Results: Simulation studies have indicated that the proposed methods can improve the probability of correctly selecting the maximum tolerated dose (MTD) with an acceptable level of risk, compared to designs that do not account for information borrowing and late-onset toxicity. Discussion: The MEM-Keyboard and MEM-TITE-Keyboard, easy to implement in practice, provide a useful tool for identifying MTD and accelerating drug development.

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.

In Vitro Reconstitution Studies of SNAREs and Their Regulators Mediating GLUT4 Vesicle Fusion.

The GLUT4 vesicle fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and a variety of regulatory proteins. For example, synip and tomosyn negatively regulate GLUT4 SNARE-mediated membrane fusion. Here we describe in vitro reconstituted assays to determine the molecular mechanisms of SNAREs, synip, and tomosyn. These methods can also be extended to the studies of other types of membrane fusion events.

Endoplasmic Reticulum-Plasma Membrane Contact Sites: Regulators, Mechanisms, and Physiological Functions.

The endoplasmic reticulum (ER) forms direct membrane contact sites with the plasma membrane (PM) in eukaryotic cells. These ER-PM contact sites play essential roles in lipid homeostasis, ion dynamics, and cell signaling, which are carried out by protein-protein or protein-lipid interactions. Distinct tethering factors dynamically control the architecture of ER-PM junctions in response to intracellular signals or external stimuli. The physiological roles of ER-PM contact sites are dependent on a variety of regulators that individually or cooperatively perform functions in diverse cellular processes. This review focuses on proteins functioning at ER-PM contact sites and highlights the recent progress in their mechanisms and physiological roles.