Necroptosis is implicated in the pathogenesis of ischemic stroke. However, the mechanism underlying the sequential recruitment of receptor-interacting protein kinase 1 (RIPK1) and N-ethylmaleimide-sensitive fusion ATPase (NSF) in initiating necroptosis remains poorly understood, and the role of NSF in ischemic stroke is a subject of controversy. Here, we utilized a recently emerging RNA-targeting CRISPR system known as CasRx, delivered by AAVs, to knockdown Ripk1 mRNA and Nsf mRNA around the ischemic brain tissue. This approach resulted in a reduction in infarct and edema volume, as well as an improvement in neurological deficits assessed by Bederson score, RotaRod test, and Adhesive removal test, which were achieved by RIPK1/receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein signaling pathway involved in neuronal necroptosis. In conclusion, the downregulation of Ripk1 mRNA and Nsf mRNA mediated by CRISPR-CasRx holds promise for future therapeutic applications aimed at ameliorating cerebral lesions and neurological deficits following the ischemic stroke.

Synaptic vesicle docking and priming are dynamic processes. At the molecular level, SNAREs (soluble NSF attachment protein receptors), synaptotagmins, and other factors are critical for Ca2+-triggered vesicle exocytosis, while disassembly factors, including NSF (N-ethylmaleimide-sensitive factor) and α-SNAP (soluble NSF attachment protein), disassemble and recycle SNAREs and antagonize fusion under some conditions. Here, we introduce a hybrid fusion assay that uses synaptic vesicles isolated from mouse brains and synthetic plasma membrane mimics. We included Munc18, Munc13, complexin, NSF, α-SNAP, and an ATP-regeneration system and maintained them continuously-as in the neuron-to investigate how these opposing processes yield fusogenic synaptic vesicles. In this setting, synaptic vesicle association is reversible, and the ATP-regeneration system produces the most synchronous Ca2+-triggered fusion, suggesting that disassembly factors perform quality control at the early stages of synaptic vesicle association to establish a highly fusogenic state. We uncovered a functional role for Munc13 ancillary to the MUN domain that alleviates an α-SNAP-dependent inhibition of Ca2+-triggered fusion.

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.

The use of conventional gadolinium(Gd)-based contrast agents in magnetic resonance imaging (MRI) poses a significant risk of Nephrogenic Systemic Fibrosis (NSF) syndrome in patients with impaired renal function (grades 4 and 5). To address this issue, a new study has introduced a novel metabolic Gadolinium oxide nanoparticle (Gd2O3 NPs) coated with β-cyclodextrin (βCD). The study aims to investigate NSF syndrome by quantifying tissue Gd deposition biodistribution in renal impairment rats using MR molecular imaging. This is the first study of its kind to use this approach. A group of 20 rats were divided into four groups, each containing five rats that underwent 5/6 nephrectomy. The rats received 12 intravenous injections of a novel homemade synthesized gadolinium oxide polycyclodextrin (Gd2O3@PCD) at a dose of 0.1 mmol/kg, conventional contrast agents (CAs) drugs of Omniscan (Gd-DTPA-BMA) and Dotarem (Gd-DOTA), at a dose of 2.5 mmol/kg, and 250 μl saline for two injections per week during six weeks. T1-weighted MR imaging was performed before the injections and once a week for six weeks to quantify Gd deposition in four different organs (skin, liver, heart, and lung) in rats using inductively coupled plasma mass spectrometry (ICP-MS). The relationship between Signal-to-Noise Ratio (SNR) and biodistribution of Gd deposition due to NSF-induced syndrome was also calculated. The results of the study showed that the Gd concentrations in tissues were significantly higher in the Gd2O3@PCD group compared to the other groups, without any significant histopathological changes (P < 0.05). In the Gd2O3@PCD group, Gd was mainly deposited in the skin, followed by the liver, lung, and heart, without any symptoms of thickening or hardening of the skin. The Gd concentrations in the skin, liver, lung, and heart were significantly lower in the Dotarem group than in the Omniscan group (P < 0.05). In the histopathological examinations, the Omniscan group showed increased cellularity in the dermis. A significant hyperintensity was observed in the Gd2O3@PCD-treated rats compared to the Dotarem and Omniscan groups in the liver, heart, and lung. Compared to conventional Gd-based CAs, the novel metabolically Gd2O3@PCD with increased SNR, biosafety, and a considerably lower probability of developing NSF, has potential applicability for diagnosing patients with renal diseases in clinical MR Molecular Imaging (MRMI).

Neurotransmitters are stored in small membrane-bound vesicles at synapses; a subset of synaptic vesicles is docked at release sites. Fusion of docked vesicles with the plasma membrane releases neurotransmitters. Membrane fusion at synapses, as well as all trafficking steps of the secretory pathway, is mediated by SNARE proteins. The SNAREs are the minimal fusion machinery. They zipper from N-termini to membrane-anchored C-termini to form a 4-helix bundle that forces the apposed membranes to fuse. At synapses, the SNAREs comprise a single helix from syntaxin and synaptobrevin; SNAP-25 contributes the other two helices to complete the bundle. Unc13 mediates synaptic vesicle docking and converts syntaxin into the permissive \"open\" configuration. The SM protein, Unc18, is required to initiate and proofread SNARE assembly. The SNAREs are then held in a half-zippered state by synaptotagmin and complexin. Calcium removes the synaptotagmin and complexin block, and the SNAREs drive vesicle fusion. After fusion, NSF and alpha-SNAP unwind the SNAREs and thereby recharge the system for further rounds of fusion. In this chapter, we will describe the discovery of the SNAREs, their relevant structural features, models for their function, and the central role of Unc18. In addition, we will touch upon the regulation of SNARE complex formation by Unc13, complexin, and synaptotagmin.

Contrast agents is used in magnetic resonance imaging (MRI) to improve the visibility of the details of the organ structures. Gadolinium-based contrast agent (GBCA) has been used since 1988 in MRI for diagnostic and follow-up of patients, the gadolinium good properties make it an effective choice for enhance the signal in MRI by increase its intensity and shortening the relaxation time of the proton. Recently, many studies show a gadolinium deposition in different human organs due to release of free gadolinium various body organs or tissue, which led to increased concern about the use of gadolinium agents, in this study, the potential diseases that may affect the patient and side effects that appear on the patient and related to accumulation of gadolinium were clarified, the study focused on the organs such as brain and bones in which gadolinium deposition was found and the lesions associated with it, and the diseases associated with gadolinium retention includes Nephrogenic Systemic Fibrosis (NSF) and Gadolinium deposition disease (GDD). Some studies tended to improve the contrast agents by developing a new non-gadolinium agents or development of next-generation gadolinium agents. In this review article the latest knowledge about MRI contrast agent.

The AAA+ NSF complex is responsible for SNARE complex disassembly both before and after membrane fusion. Loss of NSF function results in pronounced developmental and degenerative defects. In a genetic screen for sensory deficits in zebrafish, we identified a mutation in nsf, I209N, that impairs hearing and balance in a dosage-dependent manner without accompanying defects in motility, myelination, and innervation. In vitro experiments demonstrate that while the I209N NSF protein recognizes SNARE complexes, the effects on disassembly are dependent upon the type of SNARE complex and I209N concentration. Higher levels of I209N protein produce a modest decrease in binary (syntaxin-SNAP-25) SNARE complex disassembly and residual ternary (syntaxin-1A-SNAP-25-synaptobrevin-2) disassembly, whereas at lower concentrations binary disassembly activity is strongly reduced and ternary disassembly activity is absent. Our study suggests that the differential effect on disassembly of SNARE complexes leads to selective effects on NSF-mediated membrane trafficking and auditory/vestibular function.

Neuropathic pain is a chronic disease that severely afflicts the life and emotional status of patients, but currently available treatments are often ineffective. Novel therapeutic targets for the alleviation of neuropathic pain are urgently needed. Rhodojaponin VI, a grayanotoxin from Rhododendron molle, showed remarkable antinociceptive efficacy in models of neuropathic pain, but its biotargets and mechanisms are unknown. Given the reversible action of rhodojaponin VI and the narrow range over which its structure can be modified, we perforwmed thermal proteome profiling of the rat dorsal root ganglion to determine the protein target of rhodojaponin VI. N-Ethylmaleimide-sensitive fusion (NSF) was confirmed as the key target of rhodojaponin VI through biological and biophysical experiments. Functional validation showed for the first time that NSF facilitated trafficking of the Cav2.2 channel to induce an increase in Ca2+ current intensity, whereas rhodojaponin VI reversed the effects of NSF. In conclusion, rhodojaponin VI represents a unique class of analgesic natural products targeting Cav2.2 channels via NSF.

Major recent advances and previous data have led to a plausible model of how key proteins mediate neurotransmitter release. In this model, the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin form tight complexes that bring the membranes together and are crucial for membrane fusion. NSF and SNAPs disassemble SNARE complexes and ensure that fusion occurs through an exquisitely regulated pathway that starts with Munc18-1 bound to a closed conformation of syntaxin-1. Munc18-1 also binds to synaptobrevin, forming a template to assemble the SNARE complex when Munc13-1 opens syntaxin-1 while bridging the vesicle and plasma membranes. Synaptotagmin-1 and complexin bind to partially assembled SNARE complexes, likely stabilizing them and preventing fusion until Ca2+ binding to synaptotagmin-1 causes dissociation from the SNARE complex and induces interactions with phospholipids that help trigger release. Although fundamental questions remain about the mechanism of membrane fusion, these advances provide a framework to investigate the mechanisms underlying presynaptic plasticity.

After having painstakingly invented a new product, filed patents, published papers in peer-reviewed journals, you reach out to companies to license your product. More often, they reject the product as it is novel and risky. The next option is to sell the product through a startup company setup by yourself. Most inventors think that it is difficult to set up a company, find finance to run the company, and manage it. Fortunately, there are several government entities and private investment firms that can help you with setting up a company. This chapter provides information on resources for setting up and running a company.