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The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca2+-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex ‘stuttering’ mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores., SNAREs mediate the formation of a fusion pore during exocytosis which connects the lumen of a vesicle with the extracellular space. Here, authors use single molecule approaches to define the role of synaptotagmin 1 and NSF in synaptic pore formation and dissolution.

Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion., The molecular identity of the clamp that arrests the fusion machinery such that synaptic vesicles are docked and primed to release neurotransmitters remains controversial. In this study, the authors use truncation mutants of synaptotagmin (syt) 1 and animal models to demonstrate that the C2B domain of syt1, and not complexin, is solely responsible for the reduction of the spontaneous release at the presynapse

Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an “inside-out” pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] 75%. Reducing nicotine intake by 10-fold decreases activation to ∼20%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

Membrane proteins (MPs) influence all aspects of life, such as tumorigenesis, immune response, and neural transmission. However, characterization of MPs is challenging, as it often needs highly specialized techniques inaccessible to many labs. We herein introduce nanodisc-ID that enables quantitative analysis of membrane proteins using a gel electrophoresis readout. By leveraging the power of nanodiscs and proximity labeling, nanodisc-ID serves both as scaffolds for encasing biochemical reactions and as sensitive reagents for detecting membrane protein-lipid and protein-protein interactions. We demonstrate this label-free and low-cost tool by characterizing a wide range of integral and peripheral membrane proteins from prokaryotes and eukaryotes., Huan Bao describes nanodisc-ID which leverages nanodisc with proximity labeling, for the detection of membrane protein-lipid and protein-protein interactions. Its utility is demonstrated by characterising a wide range of integral and peripheral membrane proteins from prokaryotes and eukaryotes.

Background and purpose The aim of this study was to discover the associations between HMOX-1 and Alzheimer's disease (AD). Methods A total of 500 AD patients and 500 healthy controls were recruited in this study. Polymer chain reaction was used. Results There was a statistically significant difference between AD patients and controls in both the dominant and recessive models of HMOX-1 rs2071746 after adjustment for age, gender and education (dominant model: p = 0.047, odds ratio [OR] 1.34, 95% confidence interval [CI] 1.00-1.78, adjusted; recessive model: p = 0.049, OR 1.34, 95% CI 1.00-1.80, adjusted). There was also a trend for an association between the dominant model and late-onset AD after adjustment for age, gender and education (dominant model: p = 0.084, OR 1.37, 95% CI 0.96-1.95, adjusted). Conclusions We found an association between the dominant and recessive models of HMOX1 rs2071746 and AD.

Thyroid hormones have a profound influence on development, cellular differentiation and metabolism, and are also suspected of playing a role in aggression. We measured territorial aggression, body temperature (Tb) and serum thyroid hormones levels of male striped hamsters (Cricetulus barabensis) acclimated to either cold (5 °C), cool (21 °C) or hot (34 °C) ambient temperatures. The effects of methimazole on territorial aggression, food intake, metabolic rate and serum thyroid hormone levels, were also examined. Territorial aggression was significantly lower in male hamsters acclimated to the hot temperature compared to those acclimated to the cool or cold temperatures. Tb significantly increased during aggressive territorial interactions with intruders but did not significantly differ among the three temperature treatments. Serum T3, T4 and cortisol levels of hamsters acclimated to 34 °C were significantly lower than those acclimated to 21 °C. In addition to significantly reducing territorial aggression, treatment with methimazole also significantly reduced serum T3 and T4 levels, Tb and metabolic rate. These results suggest that exposure to high temperatures reduces the capacity of hamsters to dissipate heat causing them to lower their metabolic rate, which, in turn, causes them to reduce territorial aggression to prevent hyperthermia. The lower metabolic rate mediated by down-regulated thyroid hormones inhibits territorial aggression and could thereby determine the outcome of territorial conflicts.

Compromised protein homeostasis underlies accumulation of plaques and tangles in Alzheimer’s disease (AD); however, little is known about the early mechanisms that contribute to this process. To objectively assess protein turnover at early stages of amyloid beta (Aβ) proteotoxicity, we used dynamic (15)N metabolic labeling followed by proteomic analysis of amyloid precursor protein knock in mouse brains. At initial stages of Aβ accumulation, the turnover of proteins associated with presynaptic terminals is selectively impaired. Presynaptic proteins with impaired turnover, particularly synaptic vesicle (SV) associated proteins, have elevated levels, misfold in both a plaque dependent and independent manner, and interact with APP and Aβ. Concurrent with elevated levels of SV associated proteins, we found an enlargement of the SV pool as well as enhancement of presynaptic potentiation. Together, our findings reveal that the presynaptic terminal is particularly vulnerable and represents a critical site for manifestation of initial AD etiology. A record of this paper’s Transparent Peer Review process is included in the Supplemental Information.

Background: Cerebral ischemic injury is a complicated pathological process. Adipose-derived stromal cells (ADSCs) have been used as a therapeutic strategy, with their therapeutic effects chiefly attributed to paracrine action rather than trans-differentiation. Studies have shown that circAkap7 was found to be downregulated in a mouse model of transient middle cerebral artery occlusion (tMCAO). Methods: To explore whether exosomes derived from circAkap7-modified ADSCs (exo-circAkap7) have therapeutic effects on cerebral ischemic injury, a mouse model of tMCAO, as well as an in vitro model of oxygen and glucose deprivation-reoxygenation (OGD-R) in primary astrocytes, were used. Results: Results showed that treatment with exo-circAkap7 protected against tMCAO in mice, and in vitro experiments confirmed that co-culture with exo-circAkap7 attenuated OGD-R-induced cellular injury by absorbing miR-155-5p, promoting ATG12-mediated autophagy, and inhibiting NRF2-mediated oxidative stress. Conclusion: We demonstrate here that exo-circAkap7 protected against cerebral ischemic injury by promoting autophagy and ameliorating oxidative stress.

The MalFGK $$_2$$ transporter regulates the movement of maltose across the inner membrane of E. coli and serves as a model system for bacterial ATP binding cassette (ABC) importers. Despite the wealth of biochemical and structural data available, a general model describing the various translocation pathways is still lacking. In this study, we formulate a mathematical model with the goal of determining the transporter reaction pathway, specifically looking at the order of binding events and conformation changes by which transport proceeds. Fitting our mathematical model to equilibrium binding data, we estimate the unknown equilibrium parameters of the system, several of which are key determinants of the transport process. Using these estimates along with steady-state ATPase rate data, we determine which of several possible reaction pathways is dominant, as a function of five underdetermined kinetic parameter values. Because neither experimental measurements nor estimates of certain kinetic rate constants are available, the problem of deciding which of the reaction pathways is responsible for transport remains unsolved. However, using the mathematical framework developed here, a firmer conclusion regarding the dominant reaction pathway as a function of MalE and maltose concentration could be drawn once these unknown kinetic parameters are determined.

BACKGROUND Ventilator-associated pneumonia (VAP) is a severe complication that occurs within patients who must use ventilators in the intensive care unit (ICU). Ventilator care bundles (VCB) have been applied across many developed regions and have produced positive results in controlling VAP. In this study, we report on the implementation and effects of using VCBs to manage VAP in a general tertiary hospital in the Inner Mongolia Autonomous Region of China. METHODS A targeted surveillance method was used to survey all the patients (n=4,716) in the ICU from June 1, 2017 to May 31, 2019. Patients from June 1, 2017 to May 31, 2018, and June 1, 2018, to May 31, 2019, were respectively divided into 2 groups: the control group (2,029 patients) and intervention group (2,687 patients). These dates were selected because VCB was implemented from June 1, 2018, in our institution. The variables that were associated with VCB and observed were the head-of-bed elevation, oral care, maintenance of the pressure for the cuff of the endotracheal tube, aspiration of subglottic secretion, daily sedation vacation protocol, daily extubation assessment results, and hand hygiene. After collecting the data, the compliance of VCB, ventilator use ratio, and the incidence rate of VAP in these 2 groups were compared. RESULTS We observed that compliance with all of the intervention measures for VCB improved results in the intervention group compared to the control. Furthermore, the compliance rate of hand hygiene increased from 71.99% to 91.97%, and the head-of-bed elevation of 30°-45° increased from 62.02% to 85.96%. All differences between these two groups were statistically significant, according to the χ 2 -test. The ventilator use ratio was statistically and significantly lower in the intervention group (34.86%) compared to the control group (40.29%) (χ 2 =95.513, P

Background Healthcare-associated infection (HAI) is a crucial factor influencing medical quality. Studies about HAI management situations are rare, especially for the Inner Mongolia region of China. Therefore, this study aimed to investigate management procedures and the overall evaluation of HAI in order to inform HAI management improvement more scientifically. Methods A questionnaire was used to investigate HAI-related prevention and control indicators in tertiary hospitals in the Inner Mongolia region from July 2018 to June 2019. Results The survey showed that the mean incidence rate of HAI was 3.79%. The mean rate of hand hygiene compliance of healthcare workers (HCWs), inpatient's antibiotics-use rate, and the detection of the antibiotic ratio before therapy was 54.34%, 34.33%, and 25.40%, respectively. The mean of the surgical site infection (SSI) rate of the level I incision and the preventive antibiotics-use ratio of the level I incision was 1.31% and 28.89%, respectively. The mean of the multi-drug resistant organism (MDRO) infection rate was 0.40% and the mean of the MDRO detection rate was 18.55%. The mean of the central line-associated bloodstream infection rate was 2.24%, the ventilator-associated pneumonia (VAP) rate was 11.17%, and the catheter-associated urinary tract infection (CAUTI) rate was 1.95‰. As for the overall evaluation, 19 (35.85%) hospitals had a bad grade, 18 (33.96%) hospitals had a medium grade, and 16 (30.19%) hospitals had a good grade. Conclusions The incidence rate of HAI in tertiary hospitals in the Inner Mongolia region is higher than the national level. Also, the overall evaluation of bad-grade hospitals and their deficiencies should be used as an example to improve the HAI management level.

Summary Neurotransmission is a tightly regulated Ca 2+ -dependent process. Upon Ca 2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a ( idh3a ), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.

The function of α-synuclein (α-syn) has been long debated, and two seemingly divergent views have emerged. In one, α-syn binds to VAMP2, acting as a SNARE chaperone—but with no effect on neurotransmission—while another posits that α-syn attenuates neurotransmitter release by restricting synaptic vesicle mobilization and recycling. Here, we show that α-syn–VAMP2 interactions are necessary for α-syn–induced synaptic attenuation. Our data connect divergent views and suggest a unified model of α-syn function.

We sought to investigate electromyographic characteristics of Guillain-Barré syndrome (GBS) patients in the recovery phase by using serial electromyography (EMG). We included seven GBS patients and assessed their neurologic function at admission and 2, 3 and 6 months post onset using Hughes Functional Grading Scale scores. All patients underwent serial electromyographic assessment of compound muscle action potentials (CMAPs), mean conduction velocity (MCV), and distal motor latency (DML) of peripheral nerves. F wave was recorded of the median nerve and ulnar nerve. All seven patients had a Hughes Functional Grading Scale score between 3 and 6 at admission, while three patients at 2 months, one patient at 3 months, and no patient at 6 months post GBS onset had a Hughes Functional Grading Scale score between 3 and 6 ( P

Liu et al. reveal a key mechanism that mediating the transition from cerebrovascular damage to epilepsy. They identify the endothelial cyclin-dependent kinase 5 (CDK5) regulates astrocytic glutamate reuptake and increased glutamate synaptic function through CXCL1/CXCR2-mediated astrogliosis., Blood–brain barrier (BBB) dysfunction has been suggested to play an important role in epilepsy. However, the mechanism mediating the transition from cerebrovascular damage to epilepsy remains unknown. Here, we report that endothelial cyclin-dependent kinase 5 (CDK5) is a central regulator of neuronal excitability. Endothelial-specific Cdk5 knockout led to spontaneous seizures in mice. Knockout mice showed increased endothelial chemokine (C-X-C motif) ligand 1 (Cxcl1) expression, decreased astrocytic glutamate reuptake through the glutamate transporter 1 (GLT1), and increased glutamate synaptic function. Ceftriaxone restored astrocytic GLT1 function and inhibited seizures in endothelial Cdk5-deficient mice, and these effects were also reversed after silencing Cxcl1 in endothelial cells and its receptor chemokine (C-X-C motif) receptor 2 (Cxcr2) in astrocytes, respectively, in the CA1 by AAV transfection. These results reveal a previously unknown link between cerebrovascular factors and epileptogenesis and provide a rationale for targeting endothelial signaling as a potential treatment for epilepsy., Graphical Abstract

International audience; Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

To investigate the risk factors for hemorrhagic transformation (HT) after intravenous thrombolysis using a recombinant tissue plasminogen activator (r-tPA) in acute cerebral infarction.Patients with acute cerebral infarction receiving r-tPA thrombolysis in Shanghai Eastern Hospital were retrospectively studied. Based on the cranial computed tomography or magnetic resonance imaging examination, after the intravenous thrombolysis, the patients were divided into 2 groups: an HT group and a non-HT group. The information was collected before or after thrombolysis.A total of 162 patients were included in the analysis. The age ranged from 25 to 86 years, with an average age of 65.6 ± 10.6 years. The average time from disease onset to thrombolysis was 188 ± 53.1 minutes. Cranial computed tomography or magnetic resonance imaging showed that 20 patients (12.3%) had HT after thrombolysis. Using univariate analysis, history of atrial fibrillation, positive expression of urinary protein, and high National Institutes of Health Stroke Scale (NIHSS) score before thrombolysis, we found that there was a significant difference between the HT and non-HT group (P 0.05) in the level of mean systolic pressure (MSP) 24 hours after thrombolysis. Multivariate logistic regression analysis indicated that age ≥80 years, MSP ≥140 mm Hg, NIHSS score, and fibrinogen concentration before thrombolysis were risk factors for HT after thrombolysis in patients with acute cerebral infarction.Age, MSP, NIHSS score, and fibrinogen concentration before thrombolysis are risk factors for HT after thrombolysis in acute cerebral infarction. These 4 factors should be carefully taken into account before thrombolysis.

We examined the relationship between an early spontaneous type V blood pressure fluctuation and the post-thrombolysis prognosis of patients with acute cerebral infarction. Patients were admitted consecutively. All patients were categorized into the type V blood pressure fluctuation group or non-type V blood pressure group. Their blood pressure was monitored before thrombolysis and until 6 h after thrombolysis. Baseline data and clinical outcomes were compared. Of 170 patients, 43 (25.2%) had an early type V blood pressure fluctuation. The National Institute of Health Stroke Scale (NIHSS) score before thrombolysis and 24 h after thrombolysis and the modified Rankin scale score at 90 days differed significantly between the two groups (P

ATP-binding cassette transporters use an alternating access mechanism to move substrates across cellular membranes. This mode of transport ensures the selective passage of molecules while preserving membrane impermeability. The crystal structures of MalFGK2, inward- and outward-facing, show that the transporter is sealed against ions and small molecules. It has yet to be determined whether membrane impermeability is maintained when MalFGK2 cycles between these two conformations. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that not only is chloride conductance occurring, but also to a degree large enough to compromise cell viability. Introduction of mutations in the periplasmic gate lead to the formation of a channel that is quasi-permanently open. MalFGK2 must therefore stay away from these ion-conducting conformations to preserve the membrane barrier; otherwise, a few mutations that increase access to the ion-conducting states are enough to convert an ATP-binding cassette transporter into a channel.

The purpose of this study is to explore the neuroprotection mechanism of levetiracetam (LEV) with acute focal cerebral ischemia-reperfusion (I/P) mouse. The cerebral artery I/P animal model was prepared with a middle artery cerebral occlusion method. For drug intervention, mice were intraperitoneally injected with LEV with a dose of either 15 or 150 mg/kg. Neuronal injury was evaluated by measuring the infarct area, apoptosis ratio, and observation of blood-brain barrier ultrastructure with transmission electron microscope. CD8(+) antibody and perforin antibody were used to make cross-reference screen through flow cytometry to determine a perforin-positive rate in CD8(+) T lymphocytes (PFN + %). Injection of LEV can reduce infarct area, apoptosis ratio, and blood-brain barrier damage 24 h later after acute I/P in WT mice. In vitro, perforin can lower hippocampal neuron viability. In vivo, removing perforin can relieve neuronal injury. High dose injection of LEV (150 mg/kg) can inhibit perforin secreting from CD8(+)T lymphocytes. In addition, LEV can still protect neurons with perforin knockout mice. Therefore, our results suggested that LEV may contribute to neuron protection after cerebral ischemia reperfusion. The possible mechanism may be related with perforin release. However, we cannot roll out other mechanisms.