Hyperperfusion in the cerebellum lobule VIIb in patients with epileptic seizures.

BACKGROUND: The cerebellum plays an important role in motor control, however, its involvement in epilepsy has not been fully understood. Arterial spin labelling perfusion magnetic resonance image (ASL) is a noninvasive method to evaluate cerebral and cerebellar blood flow. We investigated cerebellar perfusion in patients with epileptic seizures using ASL. METHODS: Adult patients with epileptic seizures who underwent ASL in three post labeling delay (PLD) conditions (1525, 1800, and 2500 msec) and conventional electroencephalography (EEG) on the same day were investigated. Clinical and EEG characteristics of them were retrospectively analyzed. RESULTS: Six patients (6 women, age; 36.2 ± 17.9 years (mean ± SD)) showed hyperperfusion in selective areas in the cerebellar paravermis of lobule VIIb. One patient with generalized epilepsy (tentative diagnosis of juvenile myoclonic epilepsy or epilepsy with myoclonic absences) showed unilateral hypoperfusion in PLD 1525 msec and hyperperfusion in PLD 1800 and 2500 msec at the area while EEG showed generalized spike-wave complexes. After successful treatment, these perfusion abnormalities disappeared. In two patients with focal epilepsy manifesting with asymmetrical motor symptoms, cerebellar hyperperfusion was found on the opposite side to the seizure focus estimated by seizure semiology. Besides hyperperfusion of the VIIb lobule, hypoperfusion at the same area was detected in shorter PLD condition in four patients and in longer PLD condition in one patient. CONCLUSION: The cerebellar paravermis of lobule VIIb can be a component of motor circuit and participate in epileptic network in humans. Cerebellar perfusion abnormalities can be associated with neurovascular coupling via capillary bed.

Complete sequencing of expanded SAMD12 repeats by long-read sequencing and Cas9-mediated enrichment.

A pentanucleotide TTTCA repeat insertion into a polymorphic TTTTA repeat element in SAMD12 causes benign adult familial myoclonic epilepsy. Although the precise determination of the entire SAMD12 repeat sequence is important for molecular diagnosis and research, obtaining this sequence remains challenging when using conventional genomic/genetic methods, and even short-read and long-read next-generation sequencing technologies have been insufficient. Incomplete information regarding expanded repeat sequences may hamper our understanding of the pathogenic roles played by varying numbers of repeat units, genotype-phenotype correlations, and mutational mechanisms. Here, we report a new approach for the precise determination of the entire expanded repeat sequence and present a workflow designed to improve the diagnostic rates in various repeat expansion diseases. We examined 34 clinically diagnosed benign adult familial myoclonic epilepsy patients, from 29 families using repeat-primed PCR, Southern blot, and long-read sequencing with Cas9-mediated enrichment. Two cases with questionable results from repeat-primed PCR and/or Southern blot were confirmed as pathogenic using long-read sequencing with Cas9-mediated enrichment, resulting in the identification of pathogenic SAMD12 repeat expansions in 76% of examined families (22/29). Importantly, long-read sequencing with Cas9-mediated enrichment was able to provide detailed information regarding the sizes, configurations, and compositions of the expanded repeats. The inserted TTTCA repeat size and the proportion of TTTCA sequences among the overall repeat sequences were highly variable, and a novel repeat configuration was identified. A genotype-phenotype correlation study suggested that the insertion of even short (TTTCA)14 repeats contributed to the development of benign adult familial myoclonic epilepsy. However, the sizes of the overall TTTTA and TTTCA repeat units are also likely to be involved in the pathology of benign adult familial myoclonic epilepsy. Seven unsolved SAMD12-negative cases were investigated using whole-genome long-read sequencing, and infrequent, disease-associated, repeat expansions were identified in two cases. The strategic workflow resolved two questionable SAMD12-positive cases and two previously SAMD12-negative cases, increasing the diagnostic yield from 69% (20/29 families) to 83% (24/29 families). This study indicates the significant utility of long-read sequencing technologies to explore the pathogenic contributions made by various repeat units in complex repeat expansions and to improve the overall diagnostic rate.

Cytotoxic lesions of the corpus callosum after COVID-19 vaccination.

A 23-year-old previously healthy man (Patient 1) and a 33-year-old woman with a past history of depression (Patient 2) developed neurological symptoms approximately 1 week after receipt of the first COVID-19 mRNA vaccination and deteriorated over the next week. Patient 1 reported nausea, headache, a high fever, and retrograde amnesia. Patient 2 reported visual disturbance, headache, dysarthria, a left forearm tremor, dysesthesia of the mouth and distal limbs, and visual agnosia. PCR test results for SARS-CoV-2 were negative. Complete blood cell count, biochemistry, and antibody test and cerebrospinal fluid test findings were unremarkable. Diffusion-weighted and fluid-attenuated inversion recovery MRI of the brain showed a high signal intensity lesion at the midline of the splenium of the corpus callosum compatible with cytotoxic lesions of the corpus callosum (CLOCCs). High-dose intravenous methylprednisolone improved their symptoms and imaging findings. CLOCCs should be considered in patients with neurological manifestation after COVID-19 vaccination.

Long-read whole-genome sequencing identified a partial MBD5 deletion in an exome-negative patient with neurodevelopmental disorder.

Whole-exome sequencing (WES) can detect not only single-nucleotide variants in causal genes, but also pathogenic copy-number variations using several methods. However, there may be overlooked pathogenic variations in the out of target genome regions of WES analysis (e.g., promoters), leaving many patients undiagnosed. Whole-genome sequencing (WGS) can potentially analyze such regions. We applied long-read nanopore WGS and our recently developed analysis pipeline "dnarrange" to a patient who was undiagnosed by trio-based WES analysis, and identified a heterozygous 97-kb deletion partially involving 5'-untranslated exons of MBD5, which was outside the WES target regions. The phenotype of the patient, a 32-year-old male, was consistent with haploinsufficiency of MBD5. The transcript level of MBD5 in the patient's lymphoblastoid cells was reduced. We therefore concluded that the partial MBD5 deletion is the culprit for this patient. Furthermore, we found other rare structural variations (SVs) in this patient, i.e., a large inversion and a retrotransposon insertion, which were not seen in 33 controls. Although we considered that they are benign SVs, this finding suggests that our pipeline using long-read WGS is useful for investigating various types of potentially pathogenic SVs. In conclusion, we identified a 97-kb deletion, which causes haploinsufficiency of MBD5 in a patient with neurodevelopmental disorder, demonstrating that long-read WGS is a powerful technique to discover pathogenic SVs.

A Functional Aqp1 Gene Product Localizes on The Contractile Vacuole Complex in Paramecium multimicronucleatum.

In a ciliate Paramecium, the presence of water channels on the membrane of contractile vacuole has long been predicted by both morphological and physiological data, however, to date either the biochemical or the molecular biological data have not been provided. In the present study, to examine the presence of aquaporin in Paramecium, we carried out RT-PCR with degenerated primers designed based on the ParameciumDB, and an aquaporin cDNA (aquaporin 1, aqp1) with a full-length ORF encoding 251 amino acids was obtained from Paramecium multimicronucleatum by using RACE. The deduced amino acid sequence of AQP1 had NPA-NPG motifs, and the prediction of protein secondary structure by CNR5000 and hydropathy plot showed the presence of six putative transmembrane domains and five connecting loops. Phylogenetic analysis results showed that the amino acid sequence of AQP1 was close to that of the Super-aquaporin group. The AQP1-GFP fusion protein clearly demonstrated the subcellular localization of AQP1 on the contractile vacuole complex, except for the decorated spongiome membrane. The functional analyses of aqp1 were done by RNA interference-based gene silencing, using an established feeding method. The aqp1 was found to be crucial for the total fluid output of the cell, the function of contractile vacuole membranes.

Environmental survival of SARS-CoV-2 - A solid waste perspective.

The advent of COVID-19 has kept the whole world on their toes. Countries are maximizing their efforts to combat the virus and to minimize the infection. Since infectious microorganisms may be transmitted by variety of routes, respiratory and facial protection is required for those that are usually transmitted via droplets/aerosols. Therefore this pandemic has caused a sudden increase in the demand for personal protective equipment (PPE) such as gloves, masks, and many other important items since, the evidence of individual-to-individual transmission (through respiratory droplets/coughing) and secondary infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). But the disposal of these personal protective measures remains a huge question mark towards the environmental impact. Huge waste generation demands proper segregation according to waste types, collection, and recycling to minimize the risk of infection spread through aerosols and attempts to implement measures to monitor infections. Hence, this review focuses on the impact of environment due to improper disposal of these personal protective measures and to investigate the safe disposal methods for these protective measures by using the safe, secure and innovative biological methods such as the use of Artificial Intelligence (AI) and Ultraviolet (UV) lights for killing such deadly viruses.

Cough syncope and hyperventilation-induced convulsion in Chiari 1.5 malformation.

Chiari malformation type I (CM1) is defined as cerebellar tonsillar herniation below the level of the foramen magnum. Syncope, especially cough syncope, is a rare but important symptom of CM1 patients. Here, we report a CM1 patient, in combination with brainstem herniation (CM1.5), presenting with repetitive syncope who was successfully treated by decompressive surgery. A 43-year-old right-handed male, with 5-year history of repeated episodes of loss of consciousness in association with cough, was investigated. Neurological examination revealed slight muscle weakness, clumsiness, and sensory disturbance in the left upper limb. There was no sign of orthostatic hypotension or orthostatic intolerance. Cranial and spinal magnetic resonance imaging revealed a herniation of the cerebellar tonsils and a syringomyelia. Forced hyperventilation during electroencephalogram (EEG) induced brief generalized symmetric clonic convulsions with preserved consciousness, but no overt EEG seizure patterns or slow activities were found. Based on the diagnosis of CM1.5 with recurrent episodes of loss of consciousness, he underwent foramen magnum decompression. He has no recurrence of the episode after the surgery on 1 year follow-up. Decompressive surgery was an effective procedure for cough syncope and other symptoms of the current patient with CM1.5. Dissociation of cerebrospinal fluid pressure between the cranial and spinal compartments which leads further herniation of the cerebellar tonsils and subsequent compression on the cerebellum and the brainstem is considered to be the major mechanism of his cough syncope. Analysis of EEG can be useful not only to diagnose epileptic seizures but also to elucidate mechanisms of syncope and concurrent involuntary movements.

Integrative analysis of non-targeted lipidomic data and brain structural imaging identifies phosphatidylethanolamine associated with epileptogenesis.

INTRODUCTION: Epilepsy is a chronic disease, while epileptogenesis is a latent period where brain will be transformed into an epileptic one. Mechanisms of epileptogenesis remain unclear. OBJECTIVES: We aim to provide information of hippocampal lipidomic changes related with epileptogenesis in two kindling models. Combining hippocampal structural imaging indices, our study also attempts to assess biochemical alterations as a function of epileptogenesis in a non-invasive way. METHODS: We constructed two kinds of chemical kindling models, which have long been used as models of epileptogenesis. Two kindling and one control groups were all subjected to structural imaging acquisition after successfully kindled. Voxel-based morphometry, a postprocessing method for brain imaging data, was used to segment and extract hippocampal gray matter volume for subsequent integrative analysis. LC-MS based lipidomic analysis was applied to identify distinct hippocampal lipidomic profiles between kindling and control groups. Further, we regress hippocampal structural indices on lipids to identify those associated with both epileptogenesis and brain structural changes. RESULTS: We report distinct lipidomic profiles between kindling groups and control. A total of 638 lipids were detected in all three groups. Among them were 98 individual lipids, showing significant alterations, in particular lipid class of phosphatidylethanolamine (PE), glucosylceramide and phosphatidylcholine. Hippocampal gray matter volumes were found significant different between groups (P = 0.0223). After combining brain imaging data, we demonstrate several individual PE, namely PE(O-18:1_22:3), PE(O-18:1_22:6) and PE(18:1_18:1), are associated with both epileptogenesis and hippocampal gray matter volume. CONCLUSION: This study suggests metabolic pathway of PE might involve in epileptogenesis. Also, for the first time, we link level of PE with structural brain imaging indices, in an attempt to potentiate the futuristic application of noninvasive brain imaging techniques to identify epileptogenesis in its latent period.

Genetic and pharmacological correction of aberrant dopamine synthesis using patient iPSCs with BH4 metabolism disorders.

Dopamine (DA) is a neurotransmitter in the brain, playing a central role in several disease conditions, including tetrahydrobiopterin (BH4) metabolism disorders and Parkinson's disease (PD). BH4 metabolism disorders present a variety of clinical manifestations including motor disturbance via altered DA metabolism, since BH4 is a cofactor for tyrosine hydroxylase (TH), a rate-limiting enzyme for DA synthesis. Genetically, BH4 metabolism disorders are, in an autosomal recessive pattern, caused by a variant in genes encoding enzymes for BH4 synthesis or recycling, including 6-pyruvoyltetrahydropterin synthase (PTPS) or dihydropteridine reductase (DHPR), respectively. Although BH4 metabolism disorders and its metabolisms have been studied, it is unclear how gene variants cause aberrant DA synthesis in patient neurons. Here, we generated induced pluripotent stem cells (iPSCs) from BH4 metabolism disorder patients with PTPS or DHPR variants, corrected the gene variant in the iPSCs using the CRISPR/Cas9 system, and differentiated the BH4 metabolism disorder patient- and isogenic control iPSCs into midbrain DA neurons. We found that by the gene correction, the BH4 amount, TH protein level and extracellular DA level were restored in DA neuronal culture using PTPS deficiency iPSCs. Furthermore, the pharmacological correction by BH4 precursor sepiapterin treatment also improved the phenotypes of PTPS deficiency. These results suggest that patient iPSCs with BH4 metabolism disorders provide an opportunity for screening substances for treating aberrant DA synthesis-related disorders.

RNF213-related susceptibility of Japanese CADASIL patients to intracranial arterial stenosis.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by NOTCH3, primarily affects small cerebral arteries; however, stenosis of major intracranial arteries has occasionally been reported. Recent studies identified a close association between the c.14576G>A (p.R4859K, rs112735431) variant of the ring finger protein 213 (RNF213) gene and sporadic intracranial arterial stenosis (ICAS). To determine whether RNF213 is associated with ICAS in CADASIL, we genotyped rs112735431 for 124 patients with CADASIL. The c.14576G>A carrier rate in CADASIL patients with ICAS (4/17; 23.5%) was significantly higher compared with those without ICAS (2/107; 1.9%) (P = 0.0032). Among patients with ICAS, frequency of territorial infarction was significantly higher in c.14576G>A carriers (75.0%) than in non-carriers (20.0%) (P = 0.0410). In addition, rate of ≥50% stenosis or occlusion tended to be higher in c.14576G>A carriers (4/4; 100%) than in non-carriers (6/13; 46.2%) (P = 0.1029). We conclude that RNF213 is a gene associated with susceptibility to ICAS in CADASIL patients. MRA follow-up and close observation are necessary for CADASIL patients with the RNF213 variant, as they may be predisposed to ICAS.

Imbalance in multiple sclerosis and neuromyelitis optica: association with deep sensation disturbance.

Abnormality in balance is one of the most important causes of gait disturbance which has a direct impact to disability and medical cost in multiple sclerosis (MS) and neuromyelitis optica (NMO). However, characteristics of imbalance in these two diseases have not been fully elucidated. The aim of this study was to evaluate the degree and features of imbalance using stabilography, the degree of deep sensation disturbance using tibial nerve somatosensory evoked potentials (SEP), and their association with clinical impairment, in patients with MS and NMO. Seven NMO patients and seven MS patients with balance disturbance were examined. The relationship among stabilography measurements representing the degree and features of imbalance, height-adjusted P38 peak latency of SEP, and neurological functional disability, were analyzed. Stabilography evaluation showed a significantly severer degree of imbalance in NMO than in MS. Romberg quotient of the patients with brainstem lesions was significantly larger than those without them. In all patients, length of excursion per second significantly correlated positively with anterio-posterior-axis power spectra at intermediate frequency band. In all patients and in NMO, P38 peak latency adjusted by height significantly correlated positively with anterio-posterior-axis power spectra at intermediate frequency band. These findings suggest that the degree of imbalance of MS and NMO possibly correlate with deep sensation disturbance, which could be evaluated by anterio-posterior-axis power spectra at intermediate frequency band by stabilography. Severer imbalance in NMO than MS may be associated with the severe longitudinally extensive spinal cord lesions.

Increased Signal Intensity in the Dentate Nucleus of Patients with Multiple Sclerosis in Comparison with Neuromyelitis Optica Spectrum Disorder after Multiple Doses of Gadolinium Contrast.

AIMS: This study was conducted to compare the effect of linear gadolinium-based contrast agents in the retention in the cerebellar dentate nucleus of patients with multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOsd). METHODS: The signal intensity ratio of the pons to the cerebellar dentate nucleus was measured on T1-weighted MRI scans of the brain in 21 MS patients and 6 NMOsd patients given at least 10 doses of gadolinium. Linear regression analysis of the number of doses was then conducted for each patient. RESULTS: The mean correlation coefficients of the MS and NMOsd patient groups were 0.0029 and -0.0017, respectively, with positive correlations observed in 17 out of 21 patients (81.0%) in the MS group and 2 out of 6 patients (33.3%) in the NMOsd group. Results suggested that significantly high levels of gadolinium accumulate in MS the more doses to the dentate nucleus are increased (p < 0.05). CONCLUSION: Repeated doses of linear gadolinium cause accumulation of gadolinium in the cerebellar dentate nucleus, but differences observed between MS and NMOsd patients suggested that both the structure of gadolinium and differences in disease pathology affect accumulation.

Effect of CYP2C19 polymorphisms on the clinical outcome of low-dose clobazam therapy in Japanese patients with epilepsy.

PURPOSE: Clobazam (CLB) is metabolized by cytochrome P450 (CYP) 3A4 to yield N-desmethylclobazam (N-CLB), which is further inactivated by CYP2C19. The aim of this study was to retrospectively evaluate the relationship between CYP2C19 polymorphisms and the efficacy of low-dose, add-on CLB therapy in Japanese patients with epilepsy. METHODS: Fifty patients were divided into three groups according to their CYP2C19 polymorphism. CLB and N-CLB serum concentrations and seizure frequency before and after starting CLB were analyzed. RESULTS: Extensive metabolizers (EMs, n=11), intermediate metabolizers (IMs, n=22), and poor metabolizers (PMs, n=17) were included. Although the dose-normalized CLB serum concentrations were not significantly different, the dose-normalized N-CLB serum concentrations were significantly higher in PMs than in EMs or IMs. Seizure frequency was significantly decreased by the CLB therapy in PMs (p<0.01), but not in EMs or IMs. CLB serum concentrations did not correlate with seizure reduction rate, but median N-CLB serum concentrations were significantly higher in patients with excellent seizure control (≧ 90 % seizure reduction) compared to those with ≧50 % seizure reduction or with <50 % seizure reduction (1103, 341, and 570 ng/mL, respectively). CONCLUSIONS: The efficacy of low-dose CLB therapy was significantly influenced by CYP2C19 polymorphisms. Ideally, CLB therapy should be started with a low dose (2.5 mg/day) and dosage increased until N-CLB serum concentration reaches 1100 ng/mL or until the desired effect is acquired, a recommendation that is particularly important for PMs.

Latitude has more significant impact on prevalence of multiple sclerosis than ultraviolet level or sunshine duration in Japanese population.

Higher latitude is known to be associated with higher prevalence of multiple sclerosis (MS). We investigated the degree of impact of latitude, ultraviolet (UV) radiation, and sunshine on the prevalence of MS in Japan, which has 47 prefectures with a variety of climates. MS prevalence in each prefecture was collected from database of the Ministry of Health, Labour, and Welfare of Japan. Latitude of each prefecture was represented by that of the capital city. Data of UV radiation level and annual actual sunshine duration were obtained from databases of Japan Meteorological Agency. We performed linear correlation analyses of MS prevalence against latitude, UV radiation, and annual actual sunshine duration. MS prevalence significantly correlated to latitude (Pearson's correlation, r = 0.69, p < 0.001) and UV radiation level (r = -0.65, p < 0.001) but not to annual actual sunshine duration (r = -0.37, p = 0.011). Stepwise multiple linear regression analyses revealed significant correlation between MS prevalence and only latitude (p < 0.001). While our result shows that both latitude and the UV intensity have significant relationship to MS prevalence, the stronger relevance of the former suggests an existence of risk factors other than UV radiation.

Persistent frequent subclinical seizures and memory impairment after clinical remission in smoldering limbic encephalitis.

AIM: To delineate a possible correlation between clinical course and EEG abnormalities in non-infectious "smoldering" limbic encephalitis. METHODS: Long-term clinical data, including video-EEG monitoring records, were analysed in two patients. RESULTS: The two patients were positive for anti-voltage-gated potassium channel complex antibody and unspecified antineuronal antibody, respectively. The latter patient had small cell lung carcinoma. Both patients had memory impairment and clinical seizures. EEG showed frequent subclinical seizure patterns in the bilateral temporal regions. Subclinical seizure patterns and memory impairment persisted over one to two years after clinical seizure remission. Therapy (prednisolone and chemoradiation in the two patients, respectively) resulted in decreased occurrence of subclinical seizure patterns and memory improvement. CONCLUSIONS: EEG seizure patterns may persist years after clinical seizure remission in "smoldering" limbic encephalitis and lead to memory impairment.

Factors promoting research activities among Japanese pharmacists: a questionnaire survey.

Pharmacists are expected to demonstrate their expertise in clinical practice and conduct research activities to generate new evidence. However, the factors promoting research activities among pharmacists remain unclear. Therefore, we investigated the research activities of Japanese pharmacists through a questionnaire survey and examined the factors contributing to the promotion of research activities. A web-based questionnaire using Google Forms was disseminated across pharmacists working in community pharmacies, drugstores, hospitals, and clinics. The questionnaire included respondents' backgrounds, research activities, and research environments. Logistic regression analysis was used to examine the factors promoting pharmacists' research activities, with experience in research paper acceptance as the objective variable. In total, 401 responses were included in the analysis. Of the respondents, 54.1% were hospital pharmacists, and 77.1% were pharmacists with > 5 years of pharmacist experience. Furthermore, 50.4% of the pharmacists had presented at conferences, and 22.2% had experience in research paper acceptance. The influential factors were "master's degree or higher," "number of affiliated academic societies," "acquisition of specialists/certified pharmacists," and "daily availability of a consultant for writing research papers." This study revealed the factors contributing to the promotion of research activities among pharmacists. We believe that our findings will help promote research among pharmacists.

Elderly Woman With No Autoimmune Disease With Aseptic Meningitis Caused by Celecoxib.

Nonsteroidal anti-inflammatory drug (NSAID)-induced aseptic meningitis (NIAM) is frequently reported in patients with autoimmune disease. Ibuprofen-induced NIAM is the most common case report of NIAM. We report a patient without autoimmune disease who developed NIAM following oral celecoxib administration. A literature review and survey of cases registered in the Japanese Adverse Drug Event Report (JADER) database is also provided. A 73-year-old woman with no autoimmune disease developed a headache the day after taking celecoxib, and NIAM was suspected. The headache resolved quickly following celecoxib discontinuation. Although lumbar puncture was not available in this case, bacterial or viral meningitis was negative, and NIAM could not be ruled out. This case involved an older adult patient without an autoimmune disease, with celecoxib as the causative NSAID. A literature review found numerous cases of autoimmune diseases in younger patients. To date, only one case of celecoxib-induced NIAM has been reported. Analysis of NIAM cases in JADER revealed an onset time of approximately three days. JADER analysis indicated that NIAM tended to occur immediately after administration, although the onset with cyclooxygenase-2 selective agents might be slower.