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Abstract Background Various surgical procedures have employed microporous polysaccharide hemosphere (MPH) hemostatic agents. However, data regarding their effectiveness in preventing pocket hematomas (PHs) during the implantation of cardiac implantable electronic devices (CIED) among the Asian population are limited. Therefore, this study aimed to investigate the potential benefits of using MPH hemostatic agents during CIED implantations as a preventive measure against post‐procedural PHs. Methods We conducted a retrospective, single‐center, observational study involving 255 consecutive Japanese patients who underwent CIED implantation between November 2017 and April 2021. We compared PH occurrences within 28 days after CIED implantation between patients who received MPH hemostatic agents (n = 145) and those who did not (n = 110). Results PH development was observed in nine (6.2%) patients who received MPH hemostatic agents and in 13 (11.8%) patients without MPH hemostatic (p = .111). Kaplan–Meier analysis of PH development revealed no significant difference between the two groups (log‐rank p = .102). However, utilizing MPH hemostatic agents among patients taking antithrombotic drugs, including antiplatelet medications, direct oral anticoagulants, and warfarin, significantly reduced PH incidence (log‐rank p = .03). The multivariate Cox proportional hazards model demonstrated that MPH hemostatic agent utilization independently correlated with a decreased PH risk (hazard ratio 0.22, 95% confidence interval 0.08–0.63, p = .004). Conclusions The findings of this study suggest that the incorporation of MPH hemostatic agents into standard practice may benefit to mitigate PH risk during CIED implantations in patients on antithrombotic therapy. This simple and practical measure may be valuable, especially in high‐risk patients, such as those taking antithrombotic medications.

Tauopathy is known to be a major pathognomonic finding in important neurodegenerative diseases such as progressive supranuclear palsy (PSP) and corticobasal degeneration. However, the mechanism by which tauopathy is triggered remains to be elucidated. We previously identified the point mutation c.11596C > G, p.Pro3866Ala in the Bassoon gene (BSN) in a Japanese family with PSP-like syndrome. We showed that mutated BSN may have been involved in its own insolubilization and tau accumulation. Furthermore, BSN mutations have also been related to various neurological diseases. In order to further investigate the pathophysiology of BSN mutation in detail, it is essential to study it in mouse models. We generated a mouse model with the mouse Bassoon p.P3882A mutation, which corresponds to the human BSN p.P3866A mutation, knock-in (KI) and we performed systematic behavioral and histological analyses. Behavioral analyses revealed impaired working memory in a Y-maze test at 3 months of age and decreased locomotor activity in the home cage at 3 and 12 months of age in KI mice compared to those in wild-type mice. Although no obvious structural abnormalities were observed at 3 months of age, immunohistochemical studies showed elevation of Bsn immunoreactivity in the hippocampus and neuronal loss without tau accumulation in the substantia nigra at 12 months of age in KI mice. Although our mice model did not show progressive cognitive dysfunction and locomotor disorder like PSP-like syndrome, dopaminergic neuronal loss was observed in the substantia nigra in 12-month-old KI mice. It is possible that BSN mutation may result in dopaminergic neuronal loss without locomotor symptoms due to the early disease stage. Thus, further clinical course can induce cognitive dysfunction and locomotor symptoms.

BackgroundA rat model of levodopa-induced dyskinesia (LID) showed enlarged axon terminals of striatal direct pathway neurons in the internal segment of the globus pallidus (GPi) with excessive gamma-aminobutyric acid (GABA) storage in them. Massive GABA release to GPi upon levodopa administration determines the emergence of LID.ObjectivesWe examined whether LID and axon terminal hypertrophy gradually develop with repeated levodopa treatment in Parkinsonian rats to examine if the hypertrophy reflects dyskinesia priming.Methods6-hydroxydopamine-lesioned hemiparkinsonian rats were randomly allocated to receive saline injections (placebo group, 14 days; n = 4), injections of 6 mg/kg levodopa methyl ester combined with 12.5 mg/kg benserazide (levodopa-treated groups, 3-day-treatment; n = 4, 7-day-treatment; n = 4, 14-day-treatment; n = 4), or injections of 6 mg/kg levodopa methyl ester with 12.5 mg/kg benserazide and 1 mg/kg 8-hydroxy-2-(di-n-propylamino)tetralin for 14 days (8-OH-DPAT-treated group; n = 4). We evaluated abnormal involuntary movement (AIM) scores and axon terminals in the GPi.ResultsThe AIM score increased with levodopa treatment, as did the hypertrophy of axon terminals in the GPi, showing an increased number of synaptic vesicles in hypertrophied terminals.ConclusionIncreased GABA storage in axon terminals of the direct pathway neurons represents the priming process of LID.

Abstract Background Nonadherence diminishes the efficacy of direct oral anticoagulants (DOACs) in patients with nonvalvular atrial fibrillation (NVAF). This report presents the baseline survey results regarding medication adherence among NVAF patients who were treated with once‐daily edoxaban or twice‐daily apixaban from a randomized control trial of the effect of an educational intervention on DOAC adherence. Methods We prospectively studied 301 NVAF patients who were treated with edoxaban (n = 175) or apixaban (n = 126) during the 12‐week observation period. Adherence was measured with an electronic monitoring system and is expressed as the percentage of days with the correct doses in the measurement period (days). Adherence to DOAC therapy was defined based on the standard threshold (≥80%) or a strict threshold (≥90%). Results Of the 301 patients, 33 had incomplete data or protocol deviations, leaving 268 patients (edoxaban 158 and apixaban 110) for the per‐protocol baseline analysis. There was no difference in adherence (threshold ≥80%) between the groups (edoxaban 95% vs apixaban 91%, P = .2), but there was a lower proportion of patients with strict adherence (threshold ≥90%) among apixaban users than among edoxaban users (edoxaban 87% vs apixaban 76%, P = .02). Multivariate analysis showed a negative relationship between apixaban use and an adherence rate ≥90% (odds ratio 0.49, 95% confidence interval [CI]: 0.25‐0.94). Conclusions Our study showed that the proportion of DOAC users with adherence (≥80%) did not differ between the groups, but the proportion of patients with strict adherence (≥90%) was lower among those using apixaban than among those using edoxaban.

Abstract We aimed to evaluate the effect of baseline low-density lipoprotein cholesterol (LDL-C) on the outcomes of patients with the acute coronary syndrome (ACS) receiving pitavastatin monotherapy or the combination of pitavastatin + ezetimibe. In the HIJ-PROPER study, 1734 ACS patients with dyslipidemia were randomly assigned to receive pitavastatin or pitavastatin + ezetimibe therapy. Statin-naïve participants (n = 1429) were divided into two groups based on the median LDL-C level (131 mg/dL) at enrollment. The primary endpoint was a composite of all-cause death, non-fatal myocardial infarction, non-fatal stroke, unstable angina, and ischemia-driven coronary revascularization. The median follow-up was 3.2 years. In the

Abstract Background Atrial fibrillation and heart failure are common coexisting conditions requiring hospitalisation for heart failure and death. Pulmonary vein isolation is a well-established option for symptomatic atrial fibrillation and for atrial fibrillation concomitant with heart failure with reduced left ventricular ejection fraction. Recently, pulmonary vein isolation using cryoballoon showed non-inferiority to radiofrequency ablation with respect to the treatment of patients with drug-refractory paroxysmal atrial fibrillation. However, the effectiveness of acute-phase rhythm control by semi-urgent pulmonary vein isolation using cryoballoon in patients with haemodynamically unstable atrial fibrillation storm accompanied with low cardiac output syndrome is unclear. Herein, we present a case in which semi-urgent pulmonary vein isolation using cryoballoon was effective for acute-phase rhythm control against drug-resistant and haemodynamically unstable repetitive atrial fibrillation tachycardia accompanied with low cardiac output syndrome. Case presentation A 57-year-old man was hospitalised for New York Heart Association functional class 4 heart failure with atrial fibrillation tachycardia and reduced left ventricular ejection fraction of 20% accompanied with low cardiac output syndrome-induced liver damage. The haemodynamics collapsed during atrial fibrillation tachycardia, which had become resistant to intravenous amiodarone and repeated electrical cardioversions. In addition to atrial fibrillation, atrial tachycardia and common-type atrial flutter appeared on day 3. Multiple organ failure progressed gradually due to haemodynamically unstable atrial fibrillation tachycardia storm accompanied with low cardiac output syndrome. On day 4, to focus on treatment of heart failure and multiple organ failure, semi-urgent rescue pulmonary vein isolation using cryoballoon to atrial fibrillation and cavotricuspid isthmus ablation to common-type atrial flutter were performed for acute-phase rhythm control. Soon after the ablation procedure, atrial fibrillation and common-type atrial flutter were lessened, and sinus rhythm was restored. A stable haemodynamics was successfully achieved with the improvement of hepatorenal function. The patient was discharged on day 77 without complications. Conclusions This case demonstrates that acute-phase rhythm control by semi-urgent pulmonary vein isolation using cryoballoon could be a treatment option in patients with haemodynamically unstable atrial fibrillation tachycardia storm accompanied with low cardiac output syndrome, which is refractory to cardioversion and drug therapy.

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

Abstract Background Amyotrophic lateral sclerosis (ALS) is characterized pathologically by the occurrence of phosphorylated TDP-43 (pTDP-43)-immunoreactive neuronal and glial inclusions in the central nervous system. Recent studies have shown that pTDP-43 aggregates also occur in the skeletal muscles in a certain proportion of ALS patients. Aim The aim of this study was to clarify the distribution and incidence of pTDP-43 aggregates in the skeletal and cardiac muscles of patients with ALS, and also those of patients with neuromuscular diseases (NMDs) and non-NMDs. Material and methods Five regions of muscle (tongue, cervical muscle, diaphragm, iliopsoas muscle and heart) were examined histologically and immunohistochemically in patients with ALS (n = 30), NMDs (n = 13) and non-NMDs (n = 7). Results Two types of pTDP-43 aggregates were distinguishable morphologically: dense filamentous and short linear inclusions. These inclusions were found in at least one of the five muscle regions in all 30 cases of ALS; skeletal muscles in 28 cases and myocardium in 12. pTDP-43 aggregates were also found in 9 of 13 patients with NMDs, including myositis, muscular dystrophy and mitochondrial myopathy, as well as in 3 of 7 patients with non-NMDs. In ALS, pTDP-43 aggregates were most frequent in the diaphragm (19 cases). The mean density of pTDP-43 aggregates in ALS was significantly higher than that in NMDs and non-NMDs. In contiguous sections stained with hematoxylin and eosin and anti-pTDP-43, muscle fibers with dense filamentous inclusions demonstrated single-fiber atrophy with vacuolar degeneration. Conclusion The present findings indicate that pTDP-43 aggregates in skeletal and cardiac muscle are a myogenic pathological marker in multiple diseases including ALS.

Multiple system atrophy (MSA) is an adult-onset neurodegenerative disorder clinically characterized by autonomic failure in addition to various combinations of symptoms of parkinsonism, cerebellar ataxia, and pyramidal dysfunction. Despite extensive research, the mechanisms underlying the progression of MSA remain unknown. Animal models of human diseases that recapitulate their clinical, biochemical and pathological features are indispensable for increasing our understanding of their underlying molecular mechanisms, which allows preclinical studies to be advanced. Because the onset of MSA occurs in middle age, an animal model that first manifests abnormal protein aggregates in adulthood would be most appropriate. We therefore used the Cre-loxP system to express inducible α-synuclein (Syn), a major component of the pathological hallmark of MSA, to generate a mouse model of MSA. Beginning in adulthood, these MSA model mice express excessive levels of Syn in oligodendrocytes, resulting in abnormal Syn accumulation and modifications similar to those observed in human MSA pathology. Additionally, MSA model mice exhibit some clinical features of MSA, including decreased motor activity. These findings suggest that this new mouse model of MSA represents a useful tool for analyzing the pathophysiological alterations that underlie the progression of this disease.

The dynamics of potential oxygen consumption at the sediment surface in a seasonally hypoxic bay were monitored monthly by applying a tetrazolium dye (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride [INT]) reduction assay to intact sediment core samples for two consecutive years (2012–2013). Based on the empirically determined correlation between INT reduction (INT-formazan formation) and actual oxygen consumption of sediment samples, we inferred the relative contribution of biological and non-biological (chemical) processes to the potential whole oxygen consumption in the collected sediment samples. It was demonstrated that both potentials consistently increased and reached a maximum during summer hypoxia in each year. For samples collected in 2012, amplicon sequence variants (ASVs) of the bacterial 16S rRNA genes derived from the sediment surface revealed a sharp increase in the relative abundance of sulfate reducing bacteria toward hypoxia. In addition, a notable shift in other bacterial compositions was observed before and after the INT assay incubation. It was Arcobacter (Arcobacteraceae, Campylobacteria), a putative sulfur-oxidizing bacterial genus, that increased markedly during the assay period in the summer samples. These findings have implications not only for members of Delta- and Gammaproteobacteria that are consistently responsible for the consumption of dissolved oxygen (DO) year-round in the sediment, but also for those that might grow rapidly in response to episodic DO supply on the sediment surface during midst of seasonal hypoxia.

BackgroundIt remains unclear why patients with young-onset Parkinson’s disease more often develop levo-dihydroxyphenylalanine (L-dopa)-induced dyskinesia (LID) and have a more severe form than patients with old-onset Parkinson’s disease. Previous studies using animal models have failed to show young-onset Parkinson’s disease enhances LID.ObjectivesTo evaluate the association of age at dopaminergic denervation (onset age) and initiation of L-dopa treatment (treatment age) with LID development in model rats.MethodsWe established rat models of young- and old-lesioned Parkinson’s disease (6-hydroxydopamine lesions at 10 and 88 weeks of age, respectively). Dopaminergic denervation was confirmed by the rotational behavior test using apomorphine. Rats in the young-lesioned group were allocated to either L-dopa treatment at a young or old age, or saline treatment. Rats in the old-lesioned group were allocated to either L-dopa treatment or saline group. We evaluated L-dopa-induced abnormal involuntary movements during the 14-day treatment period. We also examined preprodynorphin mRNA expression in the striatum (a neurochemical hallmark of LID) and the volume of the medial globus pallidus (a pathological hallmark of LID).ResultsLID-like behavior was enhanced in L-dopa-treated young-lesioned rats compared with L-dopa-treated old-lesioned rats. Preprodynorphin mRNA expression was higher in L-dopa-treated young-lesioned rats than in in L-dopa-treated old-lesioned rats. The volume of the medial globus pallidus was greater in L-dopa-treated young-lesioned rats than in L-dopa-treated old-lesioned rats. Treatment age did not affect LID-like behavior or the degree of medial globus pallidus hypertrophy in the young-lesioned model.ConclusionBoth dopaminergic denervation and L-dopa initiation at a young age contributed to the development of LID; however, the former may be a more important factor.

Levo-dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinson's disease; however, most patients develop uncontrollable abnormal involuntary movements known as L-DOPA-induced dyskinesia. L-DOPA-induced dyskinesia can be reduced by pallidotomy of the medial globus pallidus or pallidal deep brain stimulation, suggesting that the medial globus pallidus plays a significant role in the development of L-DOPA-induced dyskinesia. In the present study, the pathological changes of the medial globus pallidus in L-DOPA-induced dyskinesia were studied in rat models of Parkinson's disease (unilateral 6-hydroxydopamine lesioning) and L-DOPA-induced dyskinesia (L-DOPA injection in Parkinson's disease-model rats twice daily for 2 weeks, confirmed by display of dyskinesia-like abnormal involuntary movements). L-DOPA-induced dyskinesia-model rats displayed medial globus pallidus hypertrophy, enlarged axon terminals surrounding the dendrites of medial globus pallidus neurons, and increased density of synaptic vesicles in enlarged axon terminals on the lesioned side. Synaptic terminal enlargement reversed after discontinuation of L-DOPA. Histological studies revealed the enlarged synaptic terminals were those of GABAergic striatal (direct pathway) neurons. A single injection of L-DOPA enhanced GABA release in the medial globus pallidus on the lesioned side in L-DOPA-induced dyskinesia-model rats compared to Parkinson's disease-model rats. In addition, microinjection of muscimol, a GABAA receptor agonist, into the medial globus pallidus on the lesioned side of Parkinson's disease-model rats induced dyskinesia-like abnormal involuntary movements. Microinjection of bicuculline, a GABAA receptor antagonist, into the medial globus pallidus on the lesioned side alleviated L-DOPA-induced dyskinesia in Parkinson's disease-model rats that had received L-DOPA prior to the microinjection. These results indicate that priming for L-DOPA-induced dyskinesia comprises excessive GABA storage in axon terminals of the direct pathway and that expression of L-DOPA-induced dyskinesia is associated with enhanced GABA release into the medial globus pallidus after L-DOPA dosing and the resultant excessive stimulation of GABAA receptors.

Ubiquitination, a fundamental post-translational modification of intracellular proteins, is enzymatically reversed by deubiquitinase enzymes (deubiquitinases). >90 deubiquitinases have been identified. One of these enzymes, YOD1, possesses deubiquitinase activity and is similar to ovarian tumor domain-containing protein 1, which is associated with regulation of the endoplasmic reticulum (ER)-associated degradation pathway. Indeed, YOD1 is reported to be involved in the ER stress response induced by mislocalization of unfolded proteins in mammalian cells. However, it has remained unclear whether YOD1 is associated with pathophysiological conditions such as mitochondrial damage, impaired proteostasis, and neurodegeneration. We demonstrated that YOD1 possesses deubiquitinating activity and exhibits preference for K48- and K63-linked ubiquitin. Furthermore, YOD1 expression levels increased as a result of various stress conditions. We demonstrated that the neurogenic proteins that cause Huntington disease and Parkinson's disease induced upregulation of YOD1 level. We observed that YOD1 reduced disease cytotoxicity through efficient degradation of mutant proteins, whereas this activity was abolished by catalytically inactive YOD1. Additionally, YOD1 localized to Lewy bodies in Parkinson's disease patients. Collectively, these data suggest that the deubiquitinase YOD1 contributes to pathogenesis of neurodegenerative disease by decreasing ubiquitination of abnormal proteins and their subsequent degradation.

Background This study aimed to examine the impact of baseline eicosapentaenoic acid (EPA) to arachidonic acid (AA) ratio on clinical outcomes of patients with acute coronary syndrome. Methods and Results In the HIJ‐PROPER (Heart Institute of Japan Proper Level of Lipid Lowering With Pitavastatin and Ezetimibe in Acute Coronary Syndrome) study, 1734 patients with acute coronary syndrome and dyslipidemia were randomly assigned to pitavastatin+ezetimibe therapy or pitavastatin monotherapy. We divided the patients into 2 groups based on EPA/AA ratio on admission (cutoff 0.34 μg/mL as median of baseline EPA/AA ratio) and examined their clinical outcomes. The primary end point comprised all‐cause death, nonfatal myocardial infarction, nonfatal stroke, unstable angina pectoris, or ischemia‐driven revascularization. Percentage reduction of low‐density lipoprotein cholesterol and triglyceride from baseline to follow‐up was similar regardless of baseline EPA/AA ratio. Despite the mean low‐density lipoprotein cholesterol level during follow‐up being similar between the low‐ and high‐EPA/AA groups, the mean triglyceride levels during follow‐up were significantly higher in the low‐ than in the high‐EPA/AA group. After 3 years of follow‐up, the cumulative incidence of the primary end point in patients with low EPA/AA was 27.2% in the pitavastatin+ezetimibe group compared with 36.6% in the pitavastatin‐monotherapy group (hazard ratio 0.69; 95% CI, 0.52‐0.93; P=0.015). However, there was no effect of pitavastatin+ezetimibe therapy on the primary end point in patients with high EPA/AA (hazard ratio 0.92; 95% CI, 0.70‐1.20; P=0.52). Conclusions Among acute coronary syndrome patients who have dyslipidemia and low EPA/AA ratio, adding ezetimibe to statin decreases the risk of cardiovascular events compared with statin monotherapy. Clinical Trial Registration URL: http://www.umin.ac.jp/ctr. Unique identifier: UMIN000002742

The sigma-1 receptor (SIGMAR1) is one of the endoplasmic reticulum (ER) chaperones, which participate in the degradation of misfolded proteins via the ER-related degradation machinery linked to the ubiquitin–proteasome pathway. ER dysfunction in the formation of inclusion bodies in various neurodegenerative diseases has also become evident. Recently, we demonstrated that accumulation of SIGMAR1 was common to neuronal nuclear inclusions in polyglutamine diseases including Huntington's disease. Our study also indicated that SIGMAR1 might shuttle between the cytoplasm and the nucleus. In the present study, we investigated the role of SIGMAR1 in nuclear inclusion (NI) formation, using HeLa cells transfected with N-terminal mutant huntingtin. Cell harboring the mutant huntingtin produced SIGMAR1-positive NIs. SIGMAR1 siRNA and a specific inhibitor of the proteasome (epoxomicin) caused significant accumulation of aggregates in the cytoplasm and nucleus. A specific inhibitor of exportin 1 (leptomycin B) also caused NIs. Huntingtin became insolubilized in Western blot analysis after treatments with SIGMAR1 siRNA and epoxomicin. Furthermore, proteasome activity increased chronologically along with the accumulation of mutant huntingtin, but was significantly reduced in cells transfected with SIGMAR1 siRNA. By contrast, overexpression of SIGMAR1 reduced the accumulation of NIs containing mutant huntingtin. Although the LC3-I level was decreased in cells treated with both SIGMAR1 siRNA and control siRNA, the levels of LC3-II and p62 were unchanged. SIGMAR1 agonist and antagonist had no effect on cellular viability and proteasome activity. These findings suggest that the ubiquitin–proteasome pathway is implicated in NI formation, and that SIGMAR1 degrades aberrant proteins in the nucleus via the ER-related degradation machinery. SIGMAR1 might be a promising candidate for therapy of Huntington's disease.

Autophagosomal formation is an initial step for macroautophagy. Similar to the yeast autophagy-related gene 8 (ATG8), mammalian ATG8 is responsible for autophagosomal formation, and categorized into LC3 and GABARAPs/GATE-16. Recent studies have shown that impairment of the autophagy–lysosome system is associated with formation of cytoplasmic inclusions observed in various neurodegenerative disorders including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Although abnormal α-synuclein accumulation is a cardinal neuropathological feature in PD, DLB and multiple system atrophy (MSA), it is unclear whether autophagy is altered in MSA. We here demonstrated that the level of matured GABARAPs was significantly decreased in the cerebellum of MSA relative to controls, and that the higher levels of matured and lipidated LC3 were detected in detergent-insoluble fraction of MSA. Immunohistochemical analysis showed that the vast majority of glial cytoplasmic inclusions, a hallmark of MSA, were positive for LC3, whereas they were unstained or barely stained with anti-GABARAPs or anti-GATE-16 antibodies. Our data suggest that autophagy maturation is impaired through the repressed levels of autophagosomal proteins in MSA.

Macroautophagy is a dynamic process whereby cytoplasmic molecules are sequestered within autophagosomes. Based on amino acid similarity, there exist two groups of mammalian autophagy-related gene (Atg) 8 homologues [microtubule-associated protein 1 light chain 3 (LC3) and γ-aminobutyric-acid type A receptor associated proteins (GABARAPs)], which play essential role in autophagosomal formation. Despite recent progress in studies on LC3, the other Atg8 homologues remain to be poorly understood, especially in pathological condition. In this study, we determined whether Atg8 homologues are affected in Lewy body disease, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Our findings indicated that biochemical and pathological properties of LC3 were altered and that the level of LC3 was increased in an insoluble fraction from the brain of patients with DLB, whereas the level of GABARAPs was decreased in DLB. Furthermore, immunohistochemical staining revealed that both LC3 and GABARAPs were localized in Lewy bodies in PD and DLB. These findings suggest that autophagic function is impaired through alteration of Atg8 homologues in Lewy body disease.

TRIM family proteins are involved in a broad range of biological processes, and their alteration results in many diverse pathological conditions found in genetic diseases, viral infections, and cancers. However, the spatial and temporal expression and function of TRIM9, one of TRIM family proteins, remain obscure. Our results here showed that TRIM9 protein is mainly expressed in the cerebral cortex, and functions as an E3 ubiquitin ligase collaborating with an E2 ubiquitin conjugating enzyme UbcH5b. Immunohistochemical examination revealed that TRIM9 is localized to the neurons in the normal mouse and human brain and that TRIM9 immunoreactivity is severely decreased in the affected brain areas in Parkinson's disease and dementia with Lewy bodies. This repressed level of TRIM9 protein was supported by immunoblotting analysis. Intriguingly, cortical and brainstem-type Lewy bodies were immunopositive for TRIM9. These results suggest that TRIM9 plays an important role in the regulation of neuronal functions and participates in pathological process of Lewy body disease through its ligase activity.

A região dos Campos Gerais do Paraná é importante produtora de leite em sistema confinado e, com isso, também geradora de dejeto líquido bovino. Os objetivos deste trabalho foram avaliar perdas de água, solo, P total, P solúvel e P particulado em chuva simulada, realizada imediatamente após a aplicação das doses de 0, 30, 60 e 90 m³ ha-1 de dejeto líquido bovino, em um Latossolo Vermelho-Amarelo manejado em plantio direto, e inferir os eventuais impactos na qualidade da água. A aplicação de dejeto aumentou a taxa de perda de água por escoamento superficial em mais de 10 vezes, em todos os intervalos de 15 min da chuva durante duas horas. O maior incremento na perda total de água ocorreu com as doses 0 e 30 m³ ha-1. A partir da dose de 60 m³ ha-1, houve tendência de estabilização da perda de água. A taxa de perda de solo nos primeiros 15 min de chuva também aumentou com a aplicação de dejeto, repetindo-se, embora não de forma significativa, até o final da chuva. O maior incremento na perda de solo também ocorreu com a aplicação das doses de 0 e 30 m³ ha-1, seguido por estabilização com as doses maiores. Parte do material sólido perdido veio do próprio dejeto. A aplicação de dejeto, mesmo na dose de 30 m³ ha-1, elevou a concentração de P total, solúvel e particulado no escoamento para valores acima dos níveis críticos relacionados com a eutrofização, especialmente nos primeiros 15 min de chuva. Por aumentar a taxa de escoamento de água e a concentração de P no escoamento, o dejeto também aumentou a perda absoluta das três formas de P, com tendência de estabilização a partir de 60 m³ ha-1. A causa do aumento substancial tanto das taxas como da quantidade absoluta de perda de água, solo e P por escoamento foi possivelmente o selamento superficial do solo promovido pelo próprio dejeto e pelo fato de a chuva ter ocorrido imediatamente após a aplicação deste.Campos Gerais in the State of Paraná (Brazil) is an important region of milk production and consequently of cattle slurry. The purposes of this study were: (a) to evaluate losses of water, soil, total P, soluble P and particulate P under simulated rainfall performed immediately after the application of 0, 30, 60 and 90 m³ ha-1 cattle slurry on a Red-Yellow Latossol (Ferralsol) under no-tillage system, and (b) to discuss the possible impacts on water quality. Slurry application increased the surface runoff rate by more than 10 times in all 15 min intervals of the 2 h rainfall. The increase of total runoff was highest between 0 and 30 m³ ha-1, and tended to stabilize at over 60 m³ ha-1. The soil loss rate in the first 15 min of rainfall also increased with slurry application, and this tendency continued until the end of the 2 h rainfall, although not significantly. The highest increase of total soil loss was also observed between 0 and 30 m³ ha-1, tending to stabilization at the highest slurry dose. Part of the solid runoff was derived from the applied slurry. The application, even at only 30 m³ ha-1 slurry, increased the total, soluble and particulate P concentration in the runoff to over the limit values regarding eutrophication, especially in the first 15 min of rainfall. Besides the increased runoff rate and P concentration, the slurry application also caused higher absolute losses of the three P forms, which tended to stabilize from 60 m³ ha-1 onwards. The increase in runoff and soil and P loss rates were possibly due to soil surface sealing caused by slurry particles and is possibly related to the fact that rainfall was simulated immediately after slurry application.

Objective: To investigate and identify factors associated with successful venoarterial extracorporeal membrane oxygenation (VA-ECMO) while initiating, during, and before VA-ECMO weaning. Design: A single-center retrospective cohort study. Setting: A 510-bed tertiary hospital. Patients and participants: We consecutively enrolled 51 patients treated with VA-ECMO at our center between April 2010 and March 2016. Patients who received venovenous ECMO, backup VA-ECMO, and post-cardiotomy VA-ECMO were excluded. Interventions: VA-ECMO for cardiopulmonary arrest (CPA) or cardiogenic shock. Successful VA-ECMO weaning was defined as survival for more than 24 hours after discontinuation without requiring reintroduction. Measurements and results: Factors associated with VA-ECMO were collected at initiation, during, and before weaning. These were then compared between the successful and unsuccessful VA-ECMO weaning groups. We included 41 patients after 10 exclusions; 17 (41.5%) and 24 (58.5%) were weaned successfully and unsuccessfully, respectively. Among the factors measured at initiation and prior to weaning, higher blood pressure (BP) and pH and lower serum potassium and creatinine were associated with successful weaning. In addition, lower white blood cell counts at initiation, lower lactate levels, and higher pulse pressure before weaning were also associated with successful weaning. Moreover, among the factors analyzed at initiation, successful weaning was associated with CPA during catheterization, percutaneous coronary intervention (PCI), and a shorter CPA-ECMO time. Among the factors studied before weaning, patients successfully weaned from VA-ECMO had lower Kidney Disease: Improving Global Outcomes (KDIGO) urine output stages and intraaortic balloon pump (IABP) use. Conclusion: BP and anaerobic condition-related factors, such as pH or lactate, can be important predictors for successful VA-ECMO weaning. Other clinical factors may also influence VAECMO weaning, such as CPA during catheterization, PCI performance, IABP use, the CPAECMO time, and renal function markers such as the KDIGO urine output stage and serum creatine and potassium levels. [ABSTRACT FROM AUTHOR]

The patient was a 67-year-old female diagnosed with dilated cardiomyopathy. She had chronic atrial fibrillation (AF) with bradycardia and low left ventricular function (left ventricular ejection fraction (LVEF) 40%). She was admitted for congestive heart failure. She remained New York Heart Association (NYHA) functional class III due to AF bradycardia. Pacemaker implantation was necessary for treatment of heart failure and administration of dose intensive β-blockers. As she had normal His-Purkinje activation, we examined the optimal pacing sites. Hemodynamics of His-bundle pacing and biventricular pacing were compared. Pulmonary capillary wedge pressure (PCWP) was significantly lower on Hisbundle pacing than right ventricular (RV) apical pacing and biventricular pacing (13mmHg, 19mmHg, and 19mmHg, respectively) with an almost equal cardiac index. Based on the examination we implanted a permanent pacemaker for Direct His-bundle pacing (DHBP). After the DHBP implantation, the LVEF immediately improved from 40% to 55%, and BNP level decreased from 422 pg/ml to 42 pg/ml. The number of premature ventricular complex (PVC) was decreased, and non sustained ventricular tachycardia (NSVT) disappeared. Pacing threshold for His-bundle pacing has remained at the same level. His-bundle pacing has been maintained during 27 months and her long-term DHBP can improve cardiac function and the NYHA functional class.

Knock-in mouse models have contributed tremendously to our understanding of human disorders. However, generation of knock-in animals requires a significant investment of time and effort. We addressed this problem by developing a novel knock-in system that circumvents several traditional challenges by establishing stem cells with acceptor elements enveloping a particular genomic target. Once established, these acceptor embryonic stem (ES) cells are efficient at directionally incorporating mutated target DNA using modified Cre/lox technology. This is advantageous, because knock-ins are not restricted to one a priori selected variation. Rather, it is possible to generate several mutant animal lines harboring desired alterations in the targeted area. Acceptor ES cell generation is the rate-limiting step, lasting approximately 2 months. Subsequent manipulations toward animal production require an additional 8 weeks, but this delimits the full period from conception of the genetic alteration to its animal incorporation. We call this system a "kick-in" to emphasize its unique characteristics of speed and convenience. To demonstrate the functionality of the kick-in methodology, we generated two mouse lines with separate mutant versions of the voltage-dependent potassium channel Kv7.2 (Kcnq2): p.Tyr284Cys (Y284C) and p.Ala306Thr (A306T); both variations have been associated with benign familial neonatal epilepsy. Adult mice homozygous for Y284C, heretofore unexamined in animals, presented with spontaneous seizures, whereas A306T homozygotes died early. Heterozygous mice of both lines showed increased sensitivity to pentylenetetrazole, possibly due to a reduction in M-current in CA1 hippocampal pyramidal neurons. Our observations for the A306T animals match those obtained with traditional knock-in technology, demonstrating that the kick-in system can readily generate mice bearing various mutations, making it a suitable feeder technology toward streamlined phenotyping.

Many neurodegenerative diseases share a common pathological feature: the deposition of amyloid-like fibrils composed of misfolded proteins. Emerging evidence suggests that these proteins may spread from cell-to-cell and encourage the propagation of neurodegeneration in a prion-like manner. Here, we demonstrated that α-synuclein (αSYN), a principal culprit for Lewy pathology in Parkinson's disease (PD), was present in endosomal compartments and detectably secreted into the extracellular milieu. Unlike prion protein, extracellular αSYN was mainly recovered in the supernatant fraction rather than in exosome-containing pellets from the neuronal culture medium and cerebrospinal fluid. Surprisingly, impaired biogenesis of multivesicular body (MVB), an organelle from which exosomes are derived, by dominant-negative mutant vacuolar protein sorting 4 (VPS4) not only interfered with lysosomal targeting of αSYN but facilitated αSYN secretion. The hypersecretion of αSYN in VPS4-defective cells was efficiently restored by the functional disruption of recycling endosome regulator Rab11a. Furthermore, both brainstem and cortical Lewy bodies in PD were found to be immunoreactive for VPS4. Thus, VPS4, a master regulator of MVB sorting, may serve as a determinant of lysosomal targeting or extracellular secretion of αSYN and thereby contribute to the intercellular propagation of Lewy pathology in PD.