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A fluorescent probe for detecting membrane protein binding and remodeling events is presented, which relies on split-GFP technology to generate circularized nanodiscs useful in membrane protein biophysics and structural biology.

Circularised nanodiscs (cNDs) are able to stabilise large lipid bilayer patches and are used for structural and functional studies. Current techniques to build cNDs have numerous steps and low yields; here the authors report a single step construction method using the SpyCatcher-SpyTag system.

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.

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.

Background. There are a large number of stroke patients in China, and there is currently a lack of prehospital acute stroke care training programs. Aim. To develop a prehospital emergency medical service (PEMS) training program to improve the prehospital identification and acute care of acute stroke. Methods. Forty prehospital emergency doctors whose service stations are located within a 10 km radius from Shanghai Pudong New Area Medical Emergency Service Center took this course on November 13, 2014. A questionnaire was designed to evaluate the PEMS personnel’s knowledge in stroke and acute stroke care and was conducted before and after training as an assessment of the effectiveness of training. The patient population in this study included a baseline cohort before training and a prospective cohort after training, each composed of patients who were sent to Shanghai East Hospital South Stoke Center within one year. The transit time, final diagnosis, administration of thrombolysis, and door-to-needle time (DNT) were collected and analyzed. Results. After the training, 100% of the PEMS personnel were competent to identify stroke cases using the Cincinnati prehospital stroke scale (CPSS). All participants realized that intravenous thrombolysis therapy in a time-sensitive manner is the most effective way to treat acute ischemic stroke. Although there was no difference in first-aid transit time before and after training, the stroke diagnosis rate improved by 6.5% after training P=0.03. The thrombolysis rate increased to 29.6% from 24.3% but did not reach statistical significance. Compared to 84.0 minutes (standard deviation: 23.1 minutes) before the training, the average DNT after training was 53 minutes (standard deviation: 15.0 minutes), demonstrating a remarkable reduction P

Purpose: To evaluate the safety and efficacy of mechanical thrombectomy (MT) for acute stroke due to large vessel occlusion (LVO), presenting with mild symptoms.Methods: A prospective cohort study of patients with mild ischemic stroke and LVO was conducted. Patients were divided into two groups: MT group or best medical management (MM) group. Propensity score matching (PSM) was conducted to reduce the confounding bias between the groups. The primary outcome was functional independence at 90 days. The safety outcome was symptomatic intracranial hemorrhage (sICH). Univariate and multivariate logistic regression analyses were used to identify the independent factors associated with outcomes.Results: Among the 105 included patients, 43 were in the MT group and 62 in the MM group. Forty-three pairs of patients were generated after PSM. There were no significant differences in sICH rates between two groups (p = 1.000). The MT group had a higher proportion of independent outcomes (83.7% MT vs. 67.4% MM; OR 2.483; 95% CI 0.886–6.959; p = 0.079) and excellent outcomes (76.7% MT vs. 51.2% MM; OR 3.150; 95% CI 1.247–7.954; p = 0.013) compared to the MM group, especially in patients with stroke of the anterior circulation (p < 0.05). Multivariate logistic regression analysis showed that small infarct core volume (p = 0.015) and MT treatment (p = 0.013) were independently associated with excellent outcomes.Conclusions: Our results suggest that MT in stroke patients, presenting with mild symptoms, due to acute LVO in the anterior circulation may be associated with satisfactory clinical outcomes.Clinical Trial Registration:ClinicalTrials.gov, identifier: NCT04526756.

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

BackgroundCerebral 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).MethodsTo 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.ResultsResults 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.ConclusionWe demonstrate here that exo-circAkap7 protected against cerebral ischemic injury by promoting autophagy and ameliorating oxidative stress.

Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ 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.

This work investigated the impact of three drying methods on structural and functional properties of potato flour (PF), as well as the quality characteristics of fresh noodles made from wheat-potato flours. The results indicated that ethanol drying (ED) and oven drying (OD) had small effects on the properties of starch in potato flour, however, freeze drying (FD) caused some pores and channels on the starch granules and disruption of the long- and short-range ordered structure of starch. The maximum addition of potato flour in fresh noodles was 40% for FD-PF and 50% for both ED-PF and OD-PF. With increasing addition of potato flour in noodles, the L* (lightness) values of noodles decreased gradually, while the a* (redness) and the b* (yellowness) values, as well as the hardness and springiness values of fresh potato noodles increased. This study clearly showed that drying methods have different effects on the properties of potato flour, and in turn the quality of fresh noodles made with potato flour.

Synaptotagmin contains tandem Ca2+-binding C2-domains that interact with target membranes to trigger exocytosis. Here, Bai et al. manipulate the relative orientation of these two domains using a synthetic polyproline linker and show that a parallel orientation is most effective for membrane interaction and exocytosis.

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

The maltose transporter MalFGK(2), together with the substrate-binding protein MalE, is one of the best-characterized ABC transporters. In the conventional model, MalE captures maltose in the periplasm and delivers the sugar to the transporter. Here, using nanodiscs and proteoliposomes, we instead find that MalE is bound with high-affinity to MalFGK2 to facilitate the acquisition of the sugar. When the maltose concentration exceeds the transport capacity, MalE captures maltose and dissociates from the transporter. This mechanism explains why the transport rate is high when MalE has low affinity for maltose, and low when MalE has high affinity for maltose. Transporter-bound MalE facilitates the acquisition of the sugar at low concentrations, but also captures and dissociates from the transporter past a threshold maltose concentration. In vivo, this maltose-forced dissociation limits the rate of transport. Given the conservation of the substrate-binding proteins, this mode of allosteric regulation may be universal to ABC importers.

Abstract Background In mammals, body mass lost during food restriction is often rapidly regained, and fat is accumulated when ad libitum feeding is resumed. Studies in small cold-acclimated mammals have demonstrated significant mobilization of fat deposits during cold exposure to meet the energy requirements of metabolic thermogenesis. However, no studies to our knowledge have examined the effect of cold exposure on fat accumulation during body mass recovery when refed ad libitum. In this study, striped hamsters restricted to 80% of their regular food intake were then refed ad libitum and exposed to one of three conditions: Intermittent cold temperature (5 °C) for 2 h per day (ICE-2 h/d), intermittent cold temperature (5 °C) for 12 h per day (ICE-12 h/d), or persistent cold exposure (PCE) for four weeks. We measured energy intake, fat deposit mass, serum thyroid hormone levels, and uncoupling protein 1 expression in brown adipose tissue. Results There was no significant effect of intermittent or persistent cold exposure on body mass regain, whereas energy intake increased significantly and total fat deposit decreased in the ICE-12 h/d and PCE groups compared to the ICE-2 h/d group and control group maintained at 23 °C (CON). In the ICE-12 h/d and PCE groups, hamsters had 39.6 and 38.3% higher serum 3,3′,5-triiodothyronine levels, respectively, and 81.6 and 71.3% up-regulated expression of uncoupling protein 1, respectively, in brown adipose tissue compared to their counterparts in the CON group. The rate of mitochondrial state III and state IV respiration O2 consumption and the activity of cytochrome c oxidase in BAT and liver were significantly higher in the ICE-12 h/d and PCE groups than in the ICE-2 h/d and CON groups. Conclusions Our findings suggest thyroid hormone-mediated heat production in brown adipose tissue and liver may be involved in preventing fat accumulation during refeeding in animals frequently or persistently exposed to cold conditions.

Disturbance of the cholinergic system plays a crucial role in the pathological progression of neurological diseases that cause dyskinesia-like behaviors. However, the molecular mechanisms underlying this disturbance remain elusive. Here, we showed that cyclin-dependent kinase 5 (Cdk5) was reduced in cholinergic neurons of midbrain according to the single-nucleus RNA sequencing analysis. Serum levels of CDK5 also decreased in patients with Parkinson’s disease accompanied by motor symptoms. Moreover, Cdk5 deficiency in cholinergic neurons triggered paw tremors, abnormal motor coordination, and motor balance deficits in mice. These symptoms occurred along with cholinergic neuron hyperexcitability and increases in the current density of large-conductance Ca2+-activated K+ channels (BK channels). Pharmacological inhibition of BK channels restrained the excessive intrinsic excitability of striatal cholinergic neurons in Cdk5-deficient mice. Furthermore, CDK5 interacted with BK channels and negatively regulated BK channel activity via phosphorylation of threonine-908. Restoration of CDK5 expression in striatal cholinergic neurons reduced dyskinesia-like behaviors in ChAT-Cre;Cdk5f/f mice. Together, these findings indicate that CDK5-induced phosphorylation of BK channels involves in cholinergic-neuron-mediated motor function, providing a potential new therapeutic target for treating dyskinesia-like behaviors arising from neurological diseases.

spi calculus, which was based on the theorems of process algebra, was fit for the proof of concurrent protocol execution. Message origination test was put up and the semantics of message sign operation was successfully expressed base on the test, at last, the non-repudiation protocol ZG was proved so as to prove the validity of the method.