The Genetics of Primary Familial Brain Calcification: A Literature Review.

Primary familial brain calcification (PFBC), also known as Fahr's disease, is a rare inherited disorder characterized by bilateral calcification in the basal ganglia according to neuroimaging. Other brain regions, such as the thalamus, cerebellum, and subcortical white matter, can also be affected. Among the diverse clinical phenotypes, the most common manifestations are movement disorders, cognitive deficits, and psychiatric disturbances. Although patients with PFBC always exhibit brain calcification, nearly one-third of cases remain clinically asymptomatic. Due to advances in the genetics of PFBC, the diagnostic criteria of PFBC may need to be modified. Hitherto, seven genes have been associated with PFBC, including four dominant inherited genes (SLC20A2, PDGFRB, PDGFB, and XPR1) and three recessive inherited genes (MYORG, JAM2, and CMPK2). Nevertheless, around 50% of patients with PFBC do not have pathogenic variants in these genes, and further PFBC-associated genes are waiting to be identified. The function of currently known genes suggests that PFBC could be caused by the dysfunction of the neurovascular unit, the dysregulation of phosphate homeostasis, or mitochondrial dysfunction. An improved understanding of the underlying pathogenic mechanisms for PFBC may facilitate the development of novel therapies.

Heterogeneous nanoscopic lipid diffusion in the live cell membrane and its dependency on cholesterol.

Cholesterol plays a unique role in the regulation of membrane organization and dynamics by modulating the membrane phase transition at the nanoscale. Unfortunately, due to their small sizes and dynamic nature, the effects of cholesterol-mediated membrane nanodomains on membrane dynamics remain elusive. Here, using ultrahigh-speed single-molecule tracking with advanced optical microscope techniques, we investigate the diffusive motion of single phospholipids in the live cell plasma membrane at the nanoscale and its dependency on the cholesterol concentration. We find that both saturated and unsaturated phospholipids undergo anomalous subdiffusion on the length scale of 10-100 nm. The diffusion characteristics exhibit considerable variations in space and in time, indicating that the nanoscopic lipid diffusion is highly heterogeneous. Importantly, through the statistical analysis, apparent dual-mobility subdiffusion is observed from the mixed diffusion behaviors. The measured subdiffusion agrees well with the hop diffusion model that represents a diffuser moving in a compartmentalized membrane created by the cytoskeleton meshwork. Cholesterol depletion diminishes the lipid mobility with an apparently smaller compartment size and a stronger confinement strength. Similar results are measured with temperature reduction, suggesting that the more heterogeneous and restricted diffusion is connected to the nanoscopic membrane phase transition. Our conclusion supports the model that cholesterol depletion induces the formation of gel-phase, solid-like membrane nanodomains. These nanodomains undergo restricted diffusion and act as diffusion obstacles to the membrane molecules that are excluded from the nanodomains. This work provides the experimental evidence that the nanoscopic lipid diffusion in the cell plasma membrane is heterogeneous and sensitive to the cholesterol concentration and temperature, shedding new light on the regulation mechanisms of nanoscopic membrane dynamics.

Response to Sodium Channel blocking Antiseizure medications and coding polymorphisms of Sodium Channel genes in Taiwanese epilepsy patients.

BACKGROUND: Many antiseizure medications (ASMs) control seizures by blocking voltage-dependent sodium channels. Polymorphisms of sodium channel genes may affect the response to ASMs due to altering the effect of ASMs on blocking sodium channels. METHODS: We conducted a retrospective study of epilepsy patients followed up at the Neurological Department of Kaohsiung Chang Gung Memorial Hospital, Taiwan between January 2010 and December 2018. We categorized the patients into response, partial response, and failure to sodium channel blocking ASM groups. Sodium channel blocking ASMs included phenytoin, carbamazepine, lamotrigine, oxcarbazepine, lacosamide, zonisamide, topiramate, and valproic acid. A subgroup of predominant sodium channel blocking ASMs included phenytoin, carbamazepine, lamotrigine, oxcarbazepine, and lacosamide. Associations between the response of ASMs and single-nucleotide polymorphisms of SCN1A, SCN1B, SCN2A, and SCN9A were analyzed. RESULTS: Two hundred Taiwanese patients and 21 single-nucleotide polymorphisms among SCN1A, SCN1B, SCN2A, and SCN9A were evaluated. We found allele C of rs55742440 in SCN1B was statistically significantly associated with not achieving seizure-free with sodium channel blocking ASMs. For the predominant sodium channel blocking ASMs group, no SNPs were associated with the response of ASMs. CONCLUSION: Single-nucleotide polymorphism in SCN1B was associated with the response to sodium channel blocking ASMs. This highlights the possibility that beta subunits may affect the function of sodium channels and resulted in different responsiveness to ASMs.

Perampanel Treatment for Refractory Status Epilepticus in a Neurological Intensive Care Unit.

BACKGROUND/OBJECTIVE: Perampanel is a novel anti-epileptic drug (AED) which acts as a non-competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist to reduce glutamate-mediated postsynaptic excitation. Previous animal studies and a few case reports/series have suggested that it may be effective to treat refractory status epilepticus (RSE). METHODS: We retrospectively reviewed 67 consecutive patients with RSE, of whom 22 received perampanel. The clinical features, epidemiology-based mortality score in status epilepticus, status epilepticus severity score, seizure control, functional outcome, RSE etiology, and electroencephalogram findings were collected. Responder to perampanel was defined as seizure resolution within 4 days of therapy with perampanel being the last AED used plus no recurrence during hospitalization. RESULTS: Eight of the 22 (36.4%) RSE patients fulfilled the definition of responder to perampanel. An additional 1 patient responded to perampanel after 4 days of treatment. In total, perampanel was the last AED in 9 (40.1%) patients. Among the 8 responders to perampanel, 5 had convulsive SE, 1 had non-convulsive SE, and 2 had focal motor SE. The responders accounted for both of the patients with focal motor SE (100%), 5 (33.3%) of the 15 patients with convulsive SE, and 1 (20%) of the 5 patients with non-convulsive SE. The ictal and inter-ictal activities also decreased after perampanel therapy, and three patients (13.6%) had preferable outcomes at last follow-up. CONCLUSIONS: Perampanel may be an effective add-on treatment for RSE even in patients who failed multiple AEDs. Our study suggests that perampanel may be more effective for focal motor SE and convulsive SE than non-convulsive SE. As most previous studies have focused on non-convulsive SE, further studies are warranted to clarify the effectiveness of perampanel for different subtypes of SE.

Development of a novel engineered antibody targeting Neisseria species.

OBJECTIVES: A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv). RESULTS: Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine. CONCLUSIONS: The recombinant scFv could detect Neisseria strains at 106 CFU/ml.

A pharmacogenetic study of perampanel: association between rare variants of glutamate receptor genes and outcomes.

Introduction: The selection of antiseizure medication usually requires a trial-and-error process. Our goal is to investigate whether genetic markers can predict the outcome of perampanel (PER) use in patients with epilepsy. Method: The studied participants were selected from our previous epilepsy genetics studies where whole exome sequencing was available. We reviewed the medical records of epilepsy patients older than 20 years old treated with PER. The outcome of PER treatment included the response to PER, the occurrence of any adverse drug reaction (ADR), the presence of behavior ADR, and the ability to adhere to PER for more than 1 year. We investigated the association between the rare variants of the glutamate receptor genes and the outcomes of PER use. Result: A total of 83 patients were collected. The gene group burden analysis showed that enriched genetic variants of the glutamate receptor gene group were statistically significantly associated with the occurrence of ADR, while the glutamate ionotropic receptor delta type subunit had a nominal association with the occurrence of ADR. The gene collapse analysis found that GRID1 had a nominal association with the occurrence of ADR and GRIN3A had a nominal association with the occurrence of behavior ADR. However, these nominal associations did not remain statistically significant once adjusted for multiple testing. Discussion: We found that enriched rare genetic variants of the glutamate receptor genes were associated with the occurrence of ADR in patients taking PER. In the future, combining the results of various pharmacogenetic studies may lead to the development of prediction tools for the outcome of antiseizure medications.

Validating Prediction Tools for Autoimmune Encephalitis in Adult Taiwanese Patients: A Retrospective Study.

Autoimmune encephalitis (AE) is a neurological emergency. We aimed to analyze the application and effectiveness of the currently available prediction tools for AE patients in Taiwan. We retrospectively collected 27 AE patients between January 2008 and December 2019. Antibody Prevalence in Epilepsy (APE) score, Response to Immunotherapy in Epilepsy (RITE) score, and anti-NMDAR Encephalitis One Year Functional Status (NEOS) score were applied to validate their usability. Based on the defined cutoff values, the sensitivity and specificity of each score were calculated. A receiver operating characteristic (ROC) curve and the area under the curve (AUC) were generated for each scoring system. The AUC value of APE was 0.571. The AUC value of RITE was 0.550. The AUC values for the NEOS score at discharge and long-term follow-up were 0.645 and 0.796, respectively. The performance of APE and RITE scores was suboptimal in the Taiwanese cohort, probably due to the limitations of the small sample size and single ethnicity. On the other hand, the NEOS score performed better on long-term follow-up than at discharge.

S100A6 participates in initiation of autoimmune encephalitis and is under epigenetic control.

INTRODUCTION: Autoimmune encephalitis (AE) is caused by autoantibodies attacking neuronal cell surface antigens and/or synaptic antigens. We previously demonstrated that S100A6 was hypomethylated in patients with AE and that it promoted B lymphocyte infiltration through the simulated blood-brain barrier (BBB). In this study, we focused on the epigenetic regulation of S100A6, the process by which S100A6 affects B lymphocyte infiltration, and the therapeutic potential of S100A6 antibodies. METHODS: We enrolled and collected serum from 10 patients with AE and 10 healthy control (HC) subjects. Promoter methylation and 5-azacytidine treatment assays were conducted to observe the methylation process of S100A6. The effect of S100A6 on B lymphocytes was analyzed using an adhesion assay and leukocyte transendothelial migration (LTEM) assay. A LTEM assay was also used to compare the effects of the serum of HCs, serum of AE patients, S100A6 recombinant protein, and S100A6 antibodies on B lymphocytes. RESULT: The promoter methylation and 5-azacytidine treatment assays confirmed that S100A6 was regulated by DNA methylation. The adhesion study demonstrated that the addition of S100A6 enhanced adhesion between B lymphocytes and a BBB endothelial cell line in a concentration-dependent manner. The LTEM assay showed that the serum of AE patients, as well as S100A6, promoted B lymphocyte infiltration and that this effect could be attenuated by S100A6 antibodies. CONCLUSION: We clarified that S100A6 was under epigenetic regulation in patients with AE and that it helped B lymphocytes to adhere to and infiltrate the BBB endothelial layer, which could be counteracted by S100A6 antibodies. Therefore, the methylation profile of S100A6 could be a marker of the activity of AE, and countering the effect of S100A6 may be a potential treatment target for AE.

ADCY5-related dyskinesia: a case report.

Adenylyl cyclase 5 (ADCY5) related dyskinesia is a rare disorder characterized by early-onset paroxysmal choreoathetosis, dystonia, myoclonus, or a combination of the above, which primarily involved the limbs, face, and neck. Other common clinical features are axial hypotonia and episodic exacerbation of dyskinesia. Both sporadic and inherited cases have been reported and the predomiant mode of inheritance is autosomal dominant. Herein, we describe the first ADCY5-related dyskinesia patient in Taiwan.

Evaluation of Cardiovascular Concerns of Intravenous Lacosamide Therapy in Epilepsy Patients.

Objective: Voltage-gated sodium channels (VGSCs) play an important role in neuronal excitability and epilepsies. In addition to the brain, VGSCs are also abundant enriched in cardiac tissues and are responsible for normal cardiac rhythm. Theoretically, sodium channel blocking antiseizure medications (SCB-ASMs) may have unwanted cardiac side effects. Lacosamide (LCM) is increasingly used in patients with status epilepticus (SE) due to the availability of intravenous formula. The concerns about the proarrhythmic effect are even higher due to the need for rapid administration of LCM. There were limited data on the cardiac safety of intravenous LCM. Hereby, we performed a study to observe the effect of intravenous loading of LCM in patients with seizures in our Neurological Intensive Care Unit (NICU). Methods: We retrospectively reviewed the patients using parenteral LCM for seizures in NICU. A routine infusion time of 30 min was performed. The electrocardiogram (ECG) and blood pressure were recorded before and after LCM injection. Results: We retrospectively reviewed the clinical data of 38 patients using LCM for treating seizures. Two patients had cardiac side effects after LCM loading, one (3.0%) with new-onset first-degree AV block and the other (3.0%) with atrial premature complex. For the quantitative changes of ECG parameter analysis, there was no change in QRS complex, corrected QT intervals, and heart rate except that the PR interval was mildly increased. A mild decrease in the diastolic blood pressure and mean arterial pressure were also observed. None of the above-mentioned parameter alterations required clinical intervention. Conclusion: We evaluated the cardiac safety concern in real-world epilepsy patients requiring intravenous LCM. Near half of this cohort responded to LCM therapy and there was no life-threatening cardiac adverse effect. Intravenous LCM does have some effects on the ECG parameters and blood pressure but without clinical relevance. Despite the theoretical concern of cardiac adverse effects of LCM, the benefit of seizure control outweighed the risk in patients with status epilepticus or seizure clusters, such as hyperthermia, pulmonary edema, cardiac arrhythmias, or cardiovascular collapse.

The clinical and imaging features of FLNA positive and negative periventricular nodular heterotopia.

BACKGROUND: Periventricular nodular heterotopia (PVNH) is caused by abnormal neuronal migration, resulting in the neurons accumulate as nodules along the surface of the lateral ventricles. PVNH often cause epilepsy, psychomotor development or cognition problem. Mutations in FLNA (Filamin A) is the most common underlying genetic etiology. Our purpose is to delineate the clinical and imaging spectrum that differentiates FLNA-positive and FLNA-negative PVNH patients. METHODS: We included 21 patients with confirmed PVNH. The detailed clinical information, electroencephalography, and other clinical findings were recorded. Detailed brain MR imaging was assessed. Mutation analysis of the FLNA gene was used Sanger sequencing or a next generation sequencing based assay. RESULTS: FLNA mutations were identified in 9 patients (7 females and 2 males), including two nonsense, two splice site, three frameshift, and two missense mutations. In FLNA-positive group, 8 patients had anterior predominant bilateral symmetric presentation and only one had asymmetrical distribution and dilated ventricles. Extra-cerebral features were more often observed in FLNA-positive group than FLNA-negative group. CONCLUSION: Genetics of PVNH is heterogenous, and mutations in FLNA gene account for less than half of the patients in our cohort. Our finding between FLNA-positive and FLNA-negative patients could guide the clinicians to select relevant genetic testing.

Hippocampal Malrotation: A Genetic Developmental Anomaly Related to Epilepsy?

Hippocampal malrotation (HIMAL) is an increasingly recognized neuroimaging feature but the clinical correlation and significance in epilepsies remain under debate. It is characterized by rounded hippocampal shape, deep collateral, or occipitotemporal sulcus, and medial localization of the hippocampus. In this review, we describe the embryonic development of the hippocampus and HIMAL, the qualitative and quantitative diagnosis issues, and the pathological findings of HIMAL. HIMAL can be bilateral or unilateral and more on the left side. Furthermore, the relevance of HIMAL diagnosis in clinical practice, including its role in epileptogenesis and the impact on the pre-surgical decision are reviewed. Finally, the relationship between HIMAL and hippocampal sclerosis (HS) and the possible role of genetics in the etiology of HIMAL are discussed. The evidence so far suggested that HIMAL does not have a significant role in epileptogenesis or surgical decision. HIMAL could be a genetic developmental imaging feature that represents a more diffuse but subtle structural error during brain development. Many questions remain to be explored, such as possible cognitive alteration associated with HIMAL and whether HIMAL predisposes to the development of HS. Further studies using high-quality MRI, unified consensus qualitative and quantitative diagnostic criteria, and comprehensive cognitive assessment are recommended.

Protean neuroimaging presentations in an adult with Klebsiella pneumoniae infection.

PURPOSE: To report on the findings of a series of cranial computed tomography (CT) and magnetic resonance (MR) imaging studies of a 57-years-old woman with Klebsiella (K.) pneumoniae infection. CASE REPORT: The patient had fever, consciousness disturbance and left hemiplegia as the initial presentations of K. pneumoniae infection. Initial blood culture grew K. pneumoniae, and a delayed purulent cerebrospinal fluid profile was found later. The serial neuroimaging studies showed cerebral hemorrhage, cerebritis and subsequent multiple abscess formations. In the meanwhile hepatic and pulmonary abscess were also discovered by imaging studies. With a 77- days intravenous ceftriaxone treatment (4 gm/day), the abscess formations of brain, lung and liver were all resolved. CONCLUSION: This study showed the protean neuroimage features of a woman with K. pneumoniae infection and the confirmation of multiple brain abscesses was made by follow-up neuroimaging studies. These sequential neuroimage findings in one patient are uncommon but deserved special clinical note by the first-line, primary-care physicians including neurologists.

Non-vitamin K Oral Anticoagulants and Anti-seizure Medications: A Retrospective Cohort Study.

Purpose: Concerns of drug-drug interactions (DDIs) between anti-seizure medications (ASMs) and non-vitamin K oral anticoagulants (NOACs) have emerged in recent case reports and guidelines. Theoretically, the induction of hepatic cytochrome P450 3A4 (CYP3A4) enzyme and permeability glycoprotein (P-GP) efflux transporter protein systems may reduce the effect of NOACs. We aimed to investigate whether such DDIs are clinically relevant in a real-world situation. Methods: We retrospectively reviewed 320 ischemic stroke patients with atrial fibrillation (Af) and grouped them according to different potential interactions with CYP3A4 and P-GP. Ischemic stroke events, transient ischemic attack (TIA) events, follow-up duration, baseline characteristics, concomitant ASMs, and stroke risk factors were collected. Statistical analysis included Kaplan-Meier survival curves and the log-rank test. Results: Overall, 320 ischemic stroke with Af patients received NOACs. Among the NOAC users, 75 also took ASMs, including 56 that have potential DDIs: 43 (13.4%) were categorized as potential CYP and P-GP DDIs and 13 (4.1%) as P-GP-only DDIs. The remaining 264 (82.5%) patients were used as controls including 19 exposed to nonsignificant DDI ASMs and 245 patients without ASM exposure. The incidence rates of recurrent stroke/TIA events in both CYP3A4 and P-GP DDIs, P-GP DDIs only, and no DDIs were 7.5, 2.1, and 8.4/100 person-years, respectively. Kaplan-Meier survival curves and the log-rank test did not show significant differences among the groups. Conclusions: The recurrent stroke rate of NOAC users with potential DDIs was not higher than in those without potential DDIs in this single-institute study. Our results suggest that theoretical interactions between ASMs and NOACs may not be as severe as previously thought in a real-world situation.

Idiopathic hypereosinophilia syndrome with loeffler endocarditis, embolic cerebral infarction, and left hydranencephaly: a case report.

PURPOSE: Idiopathic hypereosinophilia syndrome (iHES) is classically defined as prolonged peripheral eosinophilia and multiple organ involvement. The involvement of the heart can lead to intraventricular thrombus because of infiltration of the endomyocardium by eosinophils. Cerebral infarction has been ascribed to thromboembolic events originating from intraventricular thrombus. CASE REPORT: A 67 year-old woman with hypereosinophilia for 6 months presented acute weakness of the right limbs. Left hydranencephaly and absence of the left internal carotid artery were found on brain computed tomography. Brain magnetic resonance imaging (MRI) showed multiple infarctions at bilateral hemispheres. An intraventricular thrombus was detected both in transesophageal echocardiography and in heart MRI. Hypereosinophilia responded well to steroid use and warfarin was used for stroke prevention. CONCLUSIONS: Complete evaluation of systemic involvement in iHES is mandatory and early intervention may prevent deterioration of this disease. Both cardiogenic embolism and endothelial damage related to circulating eosinophils may contribute to the occurrence of stroke in this patient.

Clinical characteristics of spinal involvement in hepatocellular carcinoma.

PURPOSE: To analyze the clinical characteristics of hepatocellular carcinoma (HCC) with spinal metastasis. METHODS: During a period of 14 years, 42 HCC patients with cranial and/or spinal metastasis were identified. Among them, 12 had spinal involvement and thus were included for study. The clinical, laboratory and neuroimaging data of these 12 cases were analyzed. RESULTS: The 12 cases were all male, aged 36-65 years. The time interval from the diagnosis of HCC to the finding of spinal involvement was 0-38 months. Among these 12 cases, four had the features of spinal involvement in the initial presentation of their HCC. Low back pain was the most common symptom followed by weakness and numbness in the lower limbs. A serum biochemical study did not show unique findings. All 12 cases died within nine months after the diagnosis of the HCC spinal involvement. CONCLUSIONS: 28.6% (12/42) of the HCC patients with nervous system metastasis had spinal involvement and the exact incidence rate can be increased by more extensive neuroimaging studies. Viral hepatitis and liver cirrhosis are common preceding events in patients with HCC with spinal involvement. T- and L-spine are the most commonly involved segments and back pain is the most common complaint in patients with HCC with spinal metastasis. The prognosis in this group of patients is grave and most of the patients died soon after the development the HCC's spinal involvement. No specific biomarker can predict the development of spinal involvement in HCC patients and diagnostic consideration can only be emphasized, especially in HCC hyperendemic areas such as Taiwan.

The Different Clinical Features Between Autoimmune and Infectious Status Epilepticus.

Objective: The prognosis of status epilepticus (SE) is highly related to the underlying etiology. Inflammation of the central nervous system (CNS), including infection and autoimmune encephalitis, is one of the treatable conditions causing SE. The initial presentation of infectious and autoimmune CNS disorders can be quite similar, which may be difficult to differentiate at the beginning. However, treatment for these entities can be quite different. In this study, we aim to identify the differences in clinical features among patients with infectious and autoimmune SE, which could help the clinicians to select initial investigation and ensuing therapies that may improve overall outcomes. Methods: This was a retrospective study that included 501 patients with SE within a period of 10.5-years. Patients with inflammatory etiology were collected and separated into infectious and autoimmune SE. The symptoms at onset, SE semiology, status epilepticus severity score, and END-IT score at admission, treatment for SE, and outcome (modified Rankin Scale) on discharge and last follow-up were recorded. Data on the first cerebrospinal fluid, electroencephalography, and magnetic resonance imaging were also collected. Results: Forty-six (9.2%) of the 501 patients had SE with inflammatory etiology. Twenty-five (5%) patients were autoimmune SE and 21 (4.2%) were infectious SE. Patients with autoimmune SE have younger age and female predominance. As for clinical presentations, psychosis, non-convulsive SE, and super refractory SE were more common in patients with autoimmune SE. Nevertheless, the prognosis showed no difference between the two groups. Conclusion: The different initial clinical presentations and patient characteristics may provide some clues about the underlying etiology of SE. When inflammatory etiology is suspected in patients with SE, younger age, female sex, psychosis, non-convulsive SE, and super refractory SE are clinical features that suggest an autoimmune etiology.

Monovalent and Oriented Labeling of Gold Nanoprobes for the High-Resolution Tracking of a Single-Membrane Molecule.

Single-molecule tracking is a powerful method to study molecular dynamics in living systems including biological membranes. High-resolution single-molecule tracking requires a bright and stable signal, which has typically been facilitated by nanoparticles due to their superb optical properties. However, there are concerns about using a nanoparticle to label a single molecule because of its relatively large size and the possibility of cross-linking multiple target molecules, both of which could affect the original molecular dynamics. In this work, using various labeling schemes, we investigate the effects using nanoparticles to measure the diffusion of single-membrane molecules. By conjugating a low density of streptavidin (sAv) to gold nanoparticles (AuNPs) of different sizes (10, 15, 20, 30, and 40 nm), we isolate and quantify the effect of the particle size on the diffusion of biotinylated lipids in supported lipid bilayers (SLBs). We find that single sAv tends to cross-link two biotinylated lipids, leading to a much slower diffusion in SLBs. We further demonstrate a simple and robust strategy for the monovalent and oriented labeling of a single lipid molecule with a AuNP by using naturally dimeric rhizavidin (rAv) as a bridge, thus connecting the biotinylated nanoparticle surface and biotinylated target molecule. The rAv-AuNP conjugate demonstrates fast and free diffusion in SLBs (2-3 µm2/s for rAv-AuNP sizes of 10-40 nm), which is comparable to the diffusion of dye-labeled lipids, indicating that the adverse size and cross-linking effects are successfully avoided. We also note that the diffusion of dye-labeled lipids critically depends on the choice of dye, which could report different diffusion coefficients by about 20% (2.2 µm2/s of ATTO647N and 2.6 µm2/s of ATTO532). By comparing the diffusion of the uniformly and randomly oriented labeling of a single lipid molecule with a AuNP, we conclude that oriented labeling is favorable for measuring the diffusion of single-membrane molecules. Our work shows that the measured diffusion of the membrane molecule is highly sensitive to the molecular design of the cross-linker for labeling. The demonstrated approach of monovalent and oriented AuNP labeling provides the opportunity to study single-molecule membrane dynamics at much higher spatiotemporal resolutions and, most importantly, without labeling artifacts.

Characterization of Single-Protein Dynamics in Polymer-Cushioned Lipid Bilayers Derived from Cell Plasma Membranes.

Native cell-membrane-derived supported lipid bilayers (SLBs) are an emerging platform with broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is the proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact between the bilayer membrane and the supporting substrate, and thus, the mobility of the transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins with distinct structures, i.e., a GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescent protein (GFP), and their dynamics were measured by fluorescent single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length on protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1000 to 5000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were augmented with the polymer length, whereas the diffusion coefficient of the GPI-anchored protein remained almost identical for different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 µm2/s approximately) for the cushioned membrane with the longest polymer length (molecular weight of 5000), indicating that at the microscopic length scale, the SLBs were fully suspended from the substrate by the polymer cushion. Transient confinements were observed for all three proteins, and increasing the polymer length reduced the tendency of transient confinement. The measured dynamics of membrane proteins were found to be nearly unchanged after the depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer-cushioned plasma membrane bilayers that are potentially useful for the future developments of membrane-based biosensors and analytical assays.

Predicting the Functional Outcome of Adult Patients with Status Epilepticus.

Patients that survive status epilepticus (SE) may suffer from neurological and cognitive deficits that cause severe disabilities. An effective scoring system for functional outcome prediction may help the clinician in making treatment decisions for SE patients. Three scoring systems, namely the Status Epilepticus Severity Score (STESS), the Epidemiology-Based Mortality Score in Status Epilepticus (EMSE), and the Encephalitis-Nonconvulsive Status Epilepticus-Diazepam Resistance-Image Abnormalities-Tracheal Intubation (END-IT), have been developed in the past decade to predict the outcomes of patients with SE. Our study aimed at evaluating the effectiveness of these scores in predicting the function outcomes both at and after discharge in SE patients. We retrospectively reviewed the clinical data of 55 patients admitted to our neurological intensive care unit between January 2017 and December 2017. The clinical outcomes at discharge and at last follow-up were graded using the modified Rankin Scale. Our research indicated that STESS was the most sensitive and EMSE was the most specific predictive scoring method for SE outcome prediction. On the other hand, END-IT predicted functional outcomes in SE patients poorly. We concluded that STESS and EMSE can accurately predict the functional outcomes in SE patients both at discharge and the follow-up period.