Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis.

Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.

Hemi-fused structure mediates and controls fusion and fission in live cells.

Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic ß-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

Medication Adherence of Statin Users after Acute Ischemic Stroke.

Although statins are established therapy for the secondary prevention of ischemic stroke, factors associated with adherence to statin treatment following ischemic stroke are not well known. To address this, we assessed the 6-month statin adherence using 8-item Morisky Medication Adherence Scale-8 in patients with acute ischemic stroke. Of 991 patients, 65.6% were adherent to statin at 6-month after discharge. Multiple logistic regression analysis showed that patients' awareness of hyperlipidemia (OR 1.62; 95% CI 1.07-2.43), large artery stroke subtype (versus non-large artery stroke, OR 1.79; 95% CI 1.19-2.68), and alcohol drinking habits (OR 1.64; 95% CI 1.06-2.53) were positively associated, while high statin dose (versus low dose, OR 0.6; 95% CI 0.40-0.90) and higher daily number of medication pills (OR 0.93; 95% CI 0.88-0.97) were found to have a negative association with self-reported good adherence to statin medication after acute ischemic stroke. However, stroke severity and diagnosis of hyperlipidemia were not associated with adherence. These results suggest that educational and motivational interventions may enhance statin adherence because modifiable factors were associated with statin adherence.

Exocytosis and endocytosis: modes, functions, and coupling mechanisms.

Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis.

The SNARE proteins SNAP25 and synaptobrevin are involved in endocytosis at hippocampal synapses.

SNAP25, an essential component of the soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) complex that mediates exocytosis, is not considered to play a role in endocytosis, which couples to exocytosis by retrieving a similar amount of exocytosed vesicles. By knocking down SNAP25 and imaging slow endocytosis at a conventional synapse, the rat cultured hippocampal synapse, we found that SNAP25 is involved in slow, clathrin-dependent endocytosis. With similar techniques, we found that not only SNAP25, but also synaptobrevin is involved in slow endocytosis. These results provide the first evidence showing the dual role of SNAP25 and synaptobrevin in both exocytosis and slow endocytosis at conventional synapses. Such a dual role may contribute to mediate the coupling between exocytosis and clathrin-dependent endocytosis at conventional synapses, a mechanism critical for the maintenance of synaptic transmission and the normal structure of nerve terminals.

Most vesicles in a central nerve terminal participate in recycling.

Studies over the last decade using FM dyes to label vesicles at many terminals, including the calyx-type nerve terminal, led to a well accepted "principle" that only a small fraction of vesicles (∼5-20%) participate in recycling under physiological conditions. This principle imposes a large challenge in maintaining synaptic transmission during repetitive firing, because the small recycling pool may limit the number of available vesicles for release and nerve terminals would have to distinguish the recycling pool from the reserve pool and keep reserve pool vesicles from being used. By recording the presynaptic capacitance changes and the postsynaptic EPSC at rat calyx of Held synapses in the absence or presence of transmitter glutamate in nerve terminals, we developed a new method to count functional recycling vesicles. We found that essentially all vesicles in calyces participated in recycling, challenging the small-recycling-pool principle established by FM dye labeling. Nerve terminals may use all available vesicles to maximize their ability in maintaining synaptic transmission during repetitive firing.

Clathrin mediates membrane fission and budding by constricting membrane pores.

Membrane budding, which underlies fundamental processes like endocytosis, intracellular trafficking, and viral infection, is thought to involve membrane coat-forming proteins, including the most observed clathrin, to form Ω-shape profiles and helix-forming proteins like dynamin to constrict Ω-profiles' pores and thus mediate fission. Challenging this fundamental concept, we report that polymerized clathrin is required for Ω-profiles' pore closure and that clathrin around Ω-profiles' base/pore region mediates pore constriction/closure in neuroendocrine chromaffin cells. Mathematical modeling suggests that clathrin polymerization at Ω-profiles' base/pore region generates forces from its intrinsically curved shape to constrict/close the pore. This new fission function may exert broader impacts than clathrin's well-known coat-forming function during clathrin (coat)-dependent endocytosis, because it underlies not only clathrin (coat)-dependent endocytosis, but also diverse endocytic modes, including ultrafast, fast, slow, bulk, and overshoot endocytosis previously considered clathrin (coat)-independent in chromaffin cells. It mediates kiss-and-run fusion (fusion pore closure) previously considered bona fide clathrin-independent, and limits the vesicular content release rate. Furthermore, analogous to results in chromaffin cells, we found that clathrin is essential for fast and slow endocytosis at hippocampal synapses where clathrin was previously considered dispensable, suggesting clathrin in mediating synaptic vesicle endocytosis and fission. These results suggest that clathrin and likely other intrinsically curved coat proteins are a new class of fission proteins underlying vesicle budding and fusion. The half-a-century concept and studies that attribute vesicle-coat contents' function to Ω-profile formation and classify budding as coat-protein (e.g., clathrin)-dependent or -independent may need to be re-defined and re-examined by considering clathrin's pivotal role in pore constriction/closure.

Mapping the landscape of sleep medicine training across Asia.

STUDY OBJECTIVES: This study assessed the current state of sleep medicine accreditation and training in Asia by conducting a comprehensive survey across 29 Asian countries and regions facilitated by the Asian Society of Sleep Medicine (ASSM) to identify existing gaps and provide recommendations for future enhancements. METHODS: The ASSM Education Task Force Committee designed a survey to gather data on accreditation, education, and training standards in sleep medicine, including information on challenges in enhancing education in the field. RESULTS: With an 86% (25 countries/regions) response rate, the survey showed that sleep medicine is recognized as an independent specialty in just nine countries/regions (36% of the countries/regions surveyed). Ten countries/regions have established sleep medicine training programs, with Japan and Saudi Arabia offering it as a distinct specialty. Significant disparities in training and accreditation standards were identified, with many countries/regions lacking formalized training and practice guidelines. The survey also revealed that most local sleep societies across Asia support the development of an Asian Sleep Medicine Training Curriculum led by the ASSM. However, several barriers significantly impede the establishment and development of sleep medicine training programs, including the scarcity of trained specialists and technologists and the absence of national accreditation for sleep medicine. CONCLUSIONS: The survey highlights the need for standardized sleep medicine training and accreditation across Asia. Developing an Asian Sleep Medicine Training Curriculum and promoting ASSM accreditation guidelines are key recommendations. Implementing these strategies is essential for advancing sleep medicine as a widely recognized discipline throughout Asia.

Effects of levetiracetam monotherapy on the cognitive function of epilepsy patients.

AIMS: To estimate the effect of levetiracetam (LEV) on cognitive function and mood status in newly diagnosed epilepsy patients. METHODS: Fifty-five drug-naïve epilepsy patients (M:F ratio = 31:24; mean age = 30.9 years) were included. All patients underwent two neuropsychological (NP) tests, one before receiving LEV and then another 12.9 ± 5.0 months after starting LEV monotherapy. We evaluated general cognitive function, verbal/visual attention and memory, linguistic and visuospatial functions, frontal lobe function, and mood status. Repeated-measures regression and generalized estimating equation models were applied to assess the effects of all the confounding variables such as seizure control, average LEV dose, duration of epilepsy, inter-test interval, and subtype of epilepsy syndrome. RESULTS: LEV monotherapy over 1 year revealed significant improvements in the following domains of NP tests with the correction of possible confounding variables: verbal and visual attention, psychomotor speed, mental flexibility, executive function, verbal fluency and word generation. No NP domains showed any significant decrease. CONCLUSION: Our study suggested that LEV monotherapy had no harmful effect on cognitive function in drug-naïve patients with epilepsy.

Real-time visualization of exo- and endocytosis membrane dynamics with confocal and super-resolution microscopy.

Real-time confocal and super-resolution imaging reveals membrane dynamics of exo- and endocytosis, including hemi-fusion, fusion pore opening, expansion, constriction, closure (kiss-and-run), fused-vesicle shrinking (shrink fusion), and flat membrane transition to vesicles via intermediate Λ- and Ω-shape structures. Here, we describe a protocol for imaging these membrane dynamics, including primary culture of bovine adrenal chromaffin cells, fluorescent probe application, patch-clamp to deliver depolarization and evoke exo- and endocytosis, electron microscopy (EM), and real-time confocal and stimulated emission depletion (STED) microscopy. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2016), Shin et al. (2018), and Shin et al. (2021).

Respiratory Event Detection During Sleep Using Electrocardiogram and Respiratory Related Signals: Using Polysomnogram and Patch-Type Wearable Device Data.

This paper presents an automatic algorithm for the detection of respiratory events in patients using electrocardiogram (ECG) and respiratory signals. The proposed method was developed using data of polysomnogram (PSG) and those recorded from a patch-type device. In total, data of 1,285 subjects were used for algorithm development and evaluation. The proposed method involved respiratory event detection and apnea-hypopnea index (AHI) estimation. Handcrafted features from the ECG and respiratory signals were applied to machine learning algorithms including linear discriminant analysis, quadratic discriminant analysis, random forest, multi-layer perceptron, and the support vector machine (SVM). High performance was demonstrated when using SVM, where the overall accuracy achieved was 83% and the Cohen's kappa was 0.53 for the minute-by-minute respiratory event detection. The correlation coefficient between the reference AHI obtained using the PSG and estimated AHI as per the proposed method was 0.87. Furthermore, patient classification based on an AHI cutoff of 15 showed an accuracy of 87% and a Cohen's kappa of 0.72. The proposed method increases performance result, as it records the ECG and respiratory signals simultaneously. Overall, it can be used to lower the development cost of commercial software owing to the use of open datasets.

Robust Method for Screening Sleep Apnea With Single-Lead ECG Using Deep Residual Network: Evaluation With Open Database and Patch-Type Wearable Device Data.

This paper proposes a robust method to screen patients with sleep apnea syndrome (SAS) using a single-lead electrocardiogram (ECG). This method consists of minute-by-minute abnormal breathing detection and apnea-hypopnea index (AHI) estimation. Heartbeat interval and ECG-derived respiration (EDR) are calculated using the single-lead ECG and used to train the models, including ResNet18, ResNet34, and ResNet50. The proposed method, using data from 1232 subjects, was developed with two open datasets and experimental data and evaluated using two additional open datasets and data acquired from an abdomen-attached wearable device (in total, data from 189 subjects). ResNet18 showed the best results, having an average Cohen's kappa coefficient of 0.57, in the abnormal breathing detection. Moreover, SAS patient classification, with 15 as the AHI threshold, yielded an average Cohen's kappa coefficient of 0.71. The results of patient classification were biased toward data from the wearable patch-type device, which may be influenced by different ECG waveforms. The proposed method is tuned with a sample of the data from the device, and the performance result of Cohen's kappa increased from 0.54 to 0.91 for SAS patient classification. Our method, proposed in this paper, achieved equivalent performance results with data recorded using an abdomen-attached wearable device and two open datasets used in previous studies, although the method had not used those data during model training. The proposed method could reduce the development costs of commercial software, as it was developed using open datasets, has robust performance throughout all datasets.

Sequential compound fusion and kiss-and-run mediate exo- and endocytosis in excitable cells.

Vesicle fusion at preestablished plasma membrane release sites releases transmitters and hormones to mediate fundamental functions like neuronal network activities and fight-or-flight responses. This half-a-century-old concept-fusion at well-established release sites in excitable cells-needs to be modified to include the sequential compound fusion reported here-vesicle fusion at previously fused Ω-shaped vesicular membrane. With superresolution STED microscopy in excitable neuroendocrine chromaffin cells, we real-time visualized sequential compound fusion pore openings and content releases in generating multivesicular and asynchronous release from single release sites, which enhances exocytosis strength and dynamic ranges in excitable cells. We also visualized subsequent compound fusion pore closure, a new mode of endocytosis termed compound kiss-and-run that enhances vesicle recycling capacity. These results suggest modifying current exo-endocytosis concepts by including rapid release-site assembly at fused vesicle membrane, where sequential compound fusion and kiss-and-run take place to enhance exo-endocytosis capacity and dynamic ranges.

Clathrin-mediated endocytosis cooperates with bulk endocytosis to generate vesicles.

Clathrin-mediated endocytosis, the most prominent endocytic mode, is thought to be generated primarily from relatively flat patches of the plasma membrane. By employing conventional and platinum replica electron microscopy and super-resolution STED microscopy in neuroendocrine chromaffin cells, we found that large Ω-shaped or dome-shaped plasma membrane invaginations, previously thought of as the precursor of bulk endocytosis, are primary sites for clathrin-coated pit generation after depolarization. Clathrin-coated pits are more densely packed at invaginations rather than flat membranes, suggesting that invaginations are preferred sites for clathrin-coated pit formation, likely because their positive curvature facilitates coated-pit formation. Thus, clathrin-mediated endocytosis closely collaborates with bulk endocytosis to enhance endocytic capacity in active secretory cells. This direct collaboration between two classically independent endocytic pathways is of broad importance given the central role of both clathrin-mediated endocytosis and bulk endocytosis in neurons, endocrine cells, immune cells, and many other cell types throughout the body.

Molecular mechanics underlying flat-to-round membrane budding in live secretory cells.

Membrane budding entails forces to transform flat membrane into vesicles essential for cell survival. Accumulated studies have identified coat-proteins (e.g., clathrin) as potential budding factors. However, forces mediating many non-coated membrane buddings remain unclear. By visualizing proteins in mediating endocytic budding in live neuroendocrine cells, performing in vitro protein reconstitution and physical modeling, we discovered how non-coated-membrane budding is mediated: actin filaments and dynamin generate a pulling force transforming flat membrane into Λ-shape; subsequently, dynamin helices surround and constrict Λ-profile's base, transforming Λ- to Ω-profile, and then constrict Ω-profile's pore, converting Ω-profiles to vesicles. These mechanisms control budding speed, vesicle size and number, generating diverse endocytic modes differing in these parameters. Their impact is widespread beyond secretory cells, as the unexpectedly powerful functions of dynamin and actin, previously thought to mediate fission and overcome tension, respectively, may contribute to many dynamin/actin-dependent non-coated-membrane buddings, coated-membrane buddings, and other membrane remodeling processes.

Microwell device for targeting single cells to electrochemical microelectrodes for high-throughput amperometric detection of quantal exocytosis.

Electrochemical microelectrodes are commonly used to detect spikes of amperometric current that correspond to exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. We are developing transparent multielectrochemical electrode arrays on microchips in order to automate measurement of quantal exocytosis. Here, we report development of an improved device to target individual cells to each microelectrode in an array. Efficient targeting (~75%) is achieved using cell-sized microwell traps fabricated in SU-8 photoresist together with patterning of poly(l-lysine) in register with electrodes to promote cell adhesion. The surface between electrodes is made resistant to cell adhesion using poly(ethylene glycol) in order to facilitate movement of cells to electrode "docking sites". We demonstrate the activity of the electrodes using the test analyte ferricyanide and perform recordings of quantal exocytosis from bovine adrenal chromaffin cells on the device. Multiple cell recordings on a single device demonstrate the consistency of spike measurements, and multiple recordings from the same electrodes demonstrate that the device can be cleaned and reused without degradation of performance. The new device will enable high-throughput studies of quantal exocytosis and may also find application in rapidly screening drugs or toxins for effects on exocytosis.

Influence of Residence Area and Basic Livelihood Conditions on the Prevalence and Diagnosis Experience of Osteoporosis in Postmenopausal Women Aged over 50 Years: Evaluation Using Korea National Health and Nutrition Examination Survey Data.

Patients with osteoporosis are asymptomatic and are at risk for fractures. Therefore, early detection and interventions are important. We found that a population with a low socioeconomic status living in rural areas was reported to have a high osteoporosis prevalence but a relatively low diagnosis rate. Research on the disparity of osteoporosis prevalence and treatment from the socioeconomic perspective was conducted. This study aimed to investigate the influence of residence area and basic livelihood conditions on osteoporosis prevalence and diagnosis in postmenopausal women aged over 50 years. The cross-sectional data of 1477 postmenopausal women aged over 50 years obtained from the Korea National Health and Nutrition Examination Survey V-2 were analyzed. Univariate analyses were performed to calculate the prevalence and diagnosis rate according to risk factor categories. A multivariate logistic regression analysis was performed to identify the influence of residence area and basic livelihood conditions after controlling for other factors. The osteoporosis prevalence in basic livelihood beneficiaries (53.7%) and rural area residents (41.9%) was higher than that in non-beneficiaries (33.1%) and urban area residents (31.8%). There was no significant difference in the diagnosis rates in relation to the basic livelihood conditions or residence areas. The adjusted odds ratio for the prevalence among the beneficiaries living in rural areas was 2.08 (95% confidence interval: 1.06-4.10). However, the odds ratio for diagnosis was not significantly different. Earlier screening examination policies for osteoporosis in postmenopausal women with a low socioeconomic status living in rural areas are needed.

Preformed Ω-profile closure and kiss-and-run mediate endocytosis and diverse endocytic modes in neuroendocrine chromaffin cells.

Transformation of flat membrane into round vesicles is generally thought to underlie endocytosis and produce speed-, amount-, and vesicle-size-specific endocytic modes. Visualizing depolarization-induced exocytic and endocytic membrane transformation in live neuroendocrine chromaffin cells, we found that flat membrane is transformed into Λ-shaped, Ω-shaped, and O-shaped vesicles via invagination, Λ-base constriction, and Ω-pore constriction, respectively. Surprisingly, endocytic vesicle formation is predominantly from not flat-membrane-to-round-vesicle transformation but calcium-triggered and dynamin-mediated closure of (1) Ω profiles formed before depolarization and (2) fusion pores (called kiss-and-run). Varying calcium influxes control the speed, number, and vesicle size of these pore closures, resulting in speed-specific slow (more than ∼6 s), fast (less than ∼6 s), or ultrafast (<0.6 s) endocytosis, amount-specific compensatory endocytosis (endocytosis = exocytosis) or overshoot endocytosis (endocytosis > exocytosis), and size-specific bulk endocytosis. These findings reveal major membrane transformation mechanisms underlying endocytosis, diverse endocytic modes, and exocytosis-endocytosis coupling, calling for correction of the half-a-century concept that the flat-to-round transformation predominantly mediates endocytosis after physiological stimulation.

Development and evaluation of a T1 standard brain template for Alzheimer disease.

BACKGROUND: Patients with Alzheimer disease (AD) and mild cognitive impairment (MCI) have high variability in brain tissue loss, making it difficult to use a disease-specific standard brain template. The objective of this study was to develop an AD-specific three-dimensional (3D) T1 brain tissue template and to evaluate the characteristics of the populations used to form the template. METHODS: We obtained 3D T1-weighted images from 294 individuals, including 101 AD, 96 amnestic MCI, and 97 cognitively normal (CN) elderly individuals, and segmented them into different brain tissues to generate AD-specific brain tissue templates. Demographic data and clinical outcome scores were compared between the three groups. Voxel-based analyses and regions-of-interest-based analyses were performed to compare gray matter volume (GMV) and white matter volume (WMV) between the three participant groups and to evaluate the relationship of GMV and WMV loss with age, years of education, and Mini-Mental State Examination (MMSE) scores. RESULTS: We created high-resolution AD-specific tissue probability maps (TPMs). In the AD and MCI groups, losses of both GMV and WMV were found with respect to the CN group in the hippocampus (F >44.60, P<0.001). GMV was lower with increasing age in all individuals in the left (r=-0.621, P<0.001) and right (r=-0.632, P<0.001) hippocampi. In the left hippocampus, GMV was positively correlated with years of education in the CN groups (r=0.345, P<0.001) but not in the MCI (r=0.223, P=0.0293) or AD (r=-0.021, P=0.835) groups. WMV of the corpus callosum was not significantly correlated with years of education in any of the three subject groups (r=0.035 and P=0.549 for left, r=0.013 and P=0.821 for right). In all individuals, GMV of the hippocampus was significantly correlated with MMSE scores (left, r=0.710 and P<0.001; right, r=0.680 and P<0.001), while WMV of the corpus callosum showed a weak correlation (left, r=0.142 and P=0.015; right, r=0.123 and P=0.035). CONCLUSIONS: A 3D, T1 brain tissue template was created using imaging data from CN, MCI, and AD participants considering the participants' age, sex, and years of education. Our disease-specific template can help evaluate brains to promote early diagnosis of MCI individuals and aid treatment of MCI and AD individuals.

Recurrent ischemic stroke in a patient with idiopathic thrombocytopenic purpura.

Recurrent cerebral infarctions developed in a patient with idiopathic thrombocytopenic purpura (ITP). At the time of the first stroke, there were large thrombi in the right proximal internal carotid artery (ICA) and an occlusion of the right terminal ICA. The occlusion was recanalized by intra-arterial infusion of urokinase. After 20 months, he suffered another ischemic attack. The autoantibodies in ITP directed against antigens presented on both platelets and endothelial cells might induce the endothelial damage, thrombus formation, and embolic occlusion of the artery.