Improving detection of JC virus by ultrafiltration of cerebrospinal fluid before polymerase chain reaction for the diagnosis of progressive multifocal leukoencephalopathy.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is a demyelinating disorder caused by JC virus (JCV). Although detecting JCV DNA in the cerebrospinal fluid (CSF) by real-time polymerase chain reaction (PCR) is useful, diagnosis is difficult when JCV concentrations are low. We therefore aimed to lower the detection limit of real-time PCR testing by enriching JCV in the CSF via ultrafiltration. METHODS: Virus suspensions and CSF specimens from 20 untreated patients with suspected PML were collected and total DNAs were extracted. The JCV large T gene was detected by quantitative real-time PCR under condition with and without prior centrifugal ultrafiltration. RESULTS: The JCV DNA was reliably detected to a lower limit of 10 copies/mL of virus suspension by real-time PCR with ultrafiltration. When using this method, the quantity of JCV DNA per PCR reaction increased 3.2- to 8.7-fold compared with the standard procedure. Seven patients were positive for JCV when using the standard procedure, and an additional patient was positive when using ultrafiltration. All JCV-positive patients had neurological features and magnetic resonance imaging findings compatible with PML. CONCLUSIONS: The detection limit of JCV DNA by real-time PCR can be lowered by viral enrichment using ultrafiltration. Our simple protocol offers a valuable tool for PML diagnosis when extremely low copy numbers of JCV are released into the CSF or when brain biopsy is not feasible.

Mammalian target of rapamycin (mTOR) complex 2 regulates filamin A-dependent focal adhesion dynamics and cell migration.

The serine/threonine kinase mTOR forms two distinct complexes, mTORC1 and mTORC2, and controls a number of biological processes, including proliferation, survival and autophagy. Although the function of mTORC1 has been extensively studied, the mTORC2 signaling pathway largely remains to be elucidated. Here, we have shown that mTORC2 phosphorylates filamin A, an actin cross-linking protein, at serine 2152 (S2152) both in vivo and in living cells. Treatment of HeLa cells with Torin1 (an mTORC1/mTORC2 inhibitor), but not rapamycin (an mTORC1 inhibitor), suppressed the phosphorylation of filamin A, which decreased the binding of filamin A with ß7-integrin cytoplasmic tail. Torin1 also inhibited focal adhesion formation and cell migration in A7 filamin A-replete melanoma cells but not in M2 filamin A-deficient cells, suggesting a pivotal role for mTORC2 in filamin A function. Finally, reduced focal adhesion formation in M2 cells was significantly rescued by expressing wild type but not S2152A nonphosphorylatable mutant of filamin A. Taken together, our results indicate that mTORC2 regulates filamin A-dependent focal adhesions and cell migration.

Unusual O-alkylation of 2-hydroxy-1,4-naphthoquinone utilizing alkoxymethyl chlorides.

A new type of O-alkylation of 2-hydroxy-1,4-naphthoquinone with alkoxymethyl chlorides is described. The reaction course can be controlled by the choice of base and yields O-alkylated or O-alkoxymethylated products in high yield with high selectivity.

Takotsubo cardiomyopathy in Guillain-Barré syndrome.

BACKGROUND AND PURPOSE: Takotsubo cardiomyopathy (TCM) is a serious autonomic complication of Guillain-Barré syndrome (GBS). However, the association between TCM and GBS has not been investigated in detail. We investigated the characteristics of GBS patients with TCM (GBS-TCM). METHODS: Clinical features and anti-ganglioside antibody between the GBS-TCM patients and 62 classical GBS patients without TCM as control patients were compared. RESULTS: Eight GBS-TCM patients were identified, in whom TCM was diagnosed at a mean of 6.5 [range 3-42] days after the onset of GBS. The age at onset of GBS was elder in the GBS-TCM patients than in the control GBS patients (76.5 [56-87] vs. 52 [20-88] years, p < 0.01). Notably, cranial nerve deficits, particularly in the lower cranial nerves, were observed in all GBS-TCM patients (100% vs. 41.9%, p < 0.01). Additionally, the GBS-TCM patients showed a higher GBS disability score at nadir (5 [4-5] vs. 4 [1-5], p < 0.01), and lower Medical Research Council sum scores at admission and nadir (37 [30-44] vs. 48 [12-60] at admission, p < 0.05, and 20 [12-44] vs. 40 [0-60] at nadir, p < 0.05, respectively). Mechanical ventilation was more frequently required in the GBS-TCM patients (62.5% vs. 11.3%, p < 0.01). Three GBS-TCM patients were positive for anti-ganglioside antibodies. CONCLUSIONS: TCM occurred at a relatively early phase of GBS. The characteristics of GBS-TCM were the elder, lower cranial nerve involvements, severe limb weakness, and respiratory failure.

[Efficacy of FilmArray® ME panel for the rapid diagnosis of meningitis and encephalitis].

Microbial tests are essential for appropriate management for acute meningitis and encephalitis, but it often takes several days to identify the results of culture tests or PCR. BioFire FilmArray® meningitis/encephalitis panel (ME panel) is a rapid multiplex PCR assay that targets 14 bacteria, viruses, and yeast in 1 hour. In this single-center retrospective study, we reviewed adult patients who underwent ME panel test in parallel with conventional microbial tests from January to August 2021. Eighteen of 70 patients (26%) tested positive by ME panel, of which 8 patients (11%) were helpful in altering treatment strategy. Fifty-two patients (74%) could stop empirical treatment such as acyclovir or antibiotics due to negative results on ME panel. All results of ME panel were same as traditional assays. Use of ME panel can contribute to early diagnosis and treatment.

Identification and analysis of function of a novel splicing variant of mouse receptor activator of NF-κB.

Receptor activator of NF-κB (RANK) is a member of the tumor necrosis factor receptor (TNFR) family expressed in osteoclast precursors, and RANK-RANK ligand (RANKL) signaling is a key system for differentiation, activation and survival of osteoclasts. Here, we report the identification of a novel alternative splicing variant of mouse RANK gene (vRANK) that contains a new intervening exon between exon 1 and exon 2 of mouse full-length RANK (fRANK) mRNA. Since this novel exon contains the stop codon, vRANK encodes truncated amino acids that have a portion of the signal peptide of fRANK and an additional 19 amino acids that show no homology to previously reported domains. By transient transfection studies with vRANK-GFP and -Flag expressing constructs, vRANK was found localized mostly in the cytoplasm and partly in the cell membrane, but was not secreted into the culture supernatant. Under the stimulation of various factors, the expression of vRANK mRNA was almost parallel to that of fRANK in RAW264.7 cells not treated with M-CSF. Overexpression of vRANK, on the other hand, decreased TRACP (a marker of osteoclasts) mRNA expression as well as the number of TRACP-positive multinucleated giant cells. While the mRNA expression levels of NFATc1 (a master transcriptional factor of the osteoclast differentiation program) were not affected, apoptotic cells increased significantly in vRAN K-transfected cells treated with sRANKL. Taken together, these results suggest that vRANK is a novel osteoclast suppressor that reduces the number of RANKL-induced mature osteoclasts mainly by negating the anti-apoptotic effect of RANKL.

Electroencephalographic findings in Bickerstaff's brainstem encephalitis: A possible reflection of the dysfunction of the ascending reticular activating system.

OBJECTIVES: Bickerstaff's brainstem encephalitis (BBE) is a rare post-infectious inflammatory disease, which causes impaired consciousness by the dysfunction of the ascending reticular activating system (ARAS). We aimed to clarify EEG changes possibly caused by the dysfunction of the ARAS in BBE. METHODS: We retrospectively investigated 15 EEGs from 5 patients with definite BBE (i.e., the positivity for serum IgG anti-GQ1b antibodies was mandatory for the diagnosis) admitted to our hospital from January 2014 through December 2019, particularly focusing on whether N1 and N2 sleep patterns were maintained. RESULTS: All of the 10 EEGs recorded when patients had consciousness disturbance were abnormal. Stereotypical EEG changes correlating with their level of consciousness were identified: poorly organized posterior dominant rhythms with maintenance of sleep patterns in patients with mild consciousness disturbance (n = 5); predominant N1 and/or N2 sleep patterns even with external stimuli, including spindle coma pattern, in patients with moderate consciousness disturbance ("unarousable sleep-like" EEG) (n = 4); and generalized slow waves without N1 and N2 sleep patterns in patients with severe consciousness disturbance (n = 1). Among 5 patients, 3 (60%) had "unarousable sleep-like" EEG in their clinical course. CONCLUSIONS: Patients with BBE showed stereotypical EEG changes correlating with their level of consciousness, mostly with maintenance of N1 and N2 sleep patterns, and often exhibited characteristic "unarousable sleep-like" EEG. SIGNIFICANCE: This study revealed characteristic EEG changes possibly caused by the dysfunction of the ARAS, which can be a diagnostic clue for BBE.

Neurological Manifestations and Long-term Sequelae in Hospitalized Patients with COVID-19.

Objective Various neurological manifestations have been increasingly reported in coronavirus disease 2019 (COVID-19). We determined the neurological features and long-term sequelae in hospitalized COVID-19 patients. Methods We retrospectively studied 95 consecutive hospitalized patients with COVID-19 between March 1 and May 13, 2020. Acute neurological presentations (within two weeks of the symptom onset of COVID-19) were compared between 60 non-severe and 35 severely infected patients who required high-flow oxygen. In the 12 ventilated patients (the most severe group), we evaluated neurological complications during admission, subacute neurological presentations, and neurological sequelae (51 and 137 days from the onset [median], respectively). Results Of the 95 patients (mean age 53 years old; 40% women), 63% had acute neurological presentations, with an increased prevalence in cases of severe infections (83% vs. 52%, p<0.001). Impaired consciousness and limb weakness were more frequent in severe patients than in non-severe ones (0% vs. 49%; p<0.001, and 0% vs. 54%; p<0.001, respectively). In the most severe group (mean age 72 years old; 42% women), 83% of patients had neurological complications [cerebrovascular disease (17%), encephalopathy (82%), and neuropathy (55%)], and 92% had subacute neurological presentations [impaired consciousness (17%), higher brain dysfunction (82%), limb weakness (75%), and tremor (58%)]. Neurological sequelae were found in 83% of cases, including higher brain dysfunction (73%), limb weakness (50%), and tremor (58%). Conclusions Neurological manifestations are common in COVID-19, with the possibility of long-lasting sequelae.

Structural basis of target recognition by Atg8/LC3 during selective autophagy.

Autophagy is a non-selective bulk degradation process in which isolation membranes enclose a portion of cytoplasm to form double-membrane vesicles, called autophagosomes, and deliver their inner constituents to the lytic compartments. Recent studies have also shed light on another mode of autophagy that selectively degrades various targets. Yeast Atg8 and its mammalian homologue LC3 are ubiquitin-like modifiers that are localized on isolation membranes and play crucial roles in the formation of autophagosomes. These proteins are also involved in selective incorporation of specific cargo molecules into autophagosomes, in which Atg8 and LC3 interact with Atg19 and p62, receptor proteins for vacuolar enzymes and disease-related protein aggregates, respectively. Using X-ray crystallography and NMR, we herein report the structural basis for Atg8-Atg19 and LC3-p62 interactions. Remarkably, Atg8 and LC3 were shown to interact with Atg19 and p62, respectively, in a quite similar manner: they recognized the side-chains of Trp and Leu in a four-amino acid motif, WXXL, in Atg19 and p62 using hydrophobic pockets conserved among Atg8 homologues. Together with mutational analyses, our results show the fundamental mechanism that allows Atg8 homologues, in association with WXXL-containing proteins, to capture specific cargo molecules, thereby endowing isolation membranes and/or their assembly machineries with target selectivity.

Lipopolysaccharide suppresses RANK gene expression in macrophages by down-regulating PU.1 and MITF.

Receptor activator of NF-kappaB (RANK) is a receptor for RANK ligand (RANKL), and signals transduced by RANK-RANKL interaction are prerequisite for the differentiation and activation of osteoclasts. We cloned and characterized a 6-kb fragment containing the 5'-flanking region of the mouse RANK gene. A fragment of 1-kb from the transcription start sites containing four Sp-1 sites and putative binding sites for MITF, CRE/AP-1, and PU.1 was ligated to the pGL3-basic vector, and the promoter activity was confirmed by transfection studies. By electrophoretic gel motility shift assay, both PU.1 and proximal MITF binding site showed specific DNA-protein binding. Co-transfection studies with MITF- and PU.1-expression vectors revealed that MITF and PU.1 increased RANK promoter activity three- and twofold, respectively, and sixfold synergistically. Taken together, these results show that RANK transcription is positively regulated by both PU.1 and MITF. The effect of lipopolysaccharide (LPS) on RANK gene expression, analyzed by in situ hybridization using mouse bone tissue, showed that LPS decreased RANK transcripts of both precursor and mature osteoclasts. Furthermore, LPS treatment of RAW.264.7 cells decreased their RANK mRNA expression by 70%, mirroring the decrease of PU.1 and MITF mRNA. Short-term treatment with LPS decreased the promoter activity of pGL3-WT by 70%. Although LPS has been reported to promote osteoclastogenesis in chronic and local pyogenic inflammation, we speculate that LPS per se may directly suppress RANK expression in the osteoclastic cell lineage by down-regulating the expression of PU.1 and MITF genes in acute and systemic severe endotoxemia, such as in septic shock.

A Punctate Magnetic Resonance Imaging Pattern in a Patient with Systemic Lupus Erythematosus Is an Early Sign of Progressive Multifocal Leukoencephalopathy: A Clinicopathological Study.

A 37-year-old woman with systemic lupus erythematosus presented with gait disturbance and cognitive dysfunction. Brain magnetic resonance imaging (MRI) revealed small, punctate, T2-/fluid-attenuated inversion recovery-hyperintense and T1-hypointense lesions without gadolinium enhancement, which is atypical for progressive multifocal leukoencephalopathy (PML). On a pathological examination of biopsied brain tissues, JC virus-infected cells were hardly detected via immunohistochemistry but were certainly detected via in situ hybridization, conclusively verifying the PML diagnosis. After tapering off the immunosuppressant and mefloquine administration, the MRI findings revealed gradual improvement, and she has been stable for over 18 months. A punctate MRI pattern is not specific to natalizumab-associated PML but may be a ubiquitous early sign useful for the early diagnosis of PML.

Safety and efficacy of eculizumab in Guillain-Barré syndrome: a multicentre, double-blind, randomised phase 2 trial.

BACKGROUND: Despite the introduction of plasmapheresis and immunoglobulin therapy, many patients with Guillain-Barré syndrome still have an incomplete recovery. Evidence from pathogenesis studies suggests the involvement of complement-mediated peripheral nerve damage. We aimed to investigate the safety and efficacy of eculizumab, a humanised monoclonal antibody against the complement protein C5, in patients with severe Guillain-Barré syndrome. METHODS: This study was a 24 week, multicentre, double-blind, placebo-controlled, randomised phase 2 trial done at 13 hospitals in Japan. Eligible patients with Guillain-Barré syndrome were aged 18 years or older and could not walk independently (Guillain-Barré syndrome functional grade 3-5). Patients were randomly assigned (2:1) to receive 4 weeks of intravenous immunoglobulin plus either eculizumab (900 mg) or placebo; randomisation was done via a computer-generated process and web response system with minimisation for functional grade and age. The study had a parallel non-comparative single-arm outcome measure. The primary outcomes were efficacy (the proportion of patients with restored ability to walk independently [functional grade ≤2] at week 4) in the eculizumab group and safety in the full analysis set. For the efficacy endpoint, we predefined a response rate threshold of the lower 90% CI boundary exceeding 50%. This trial is registered with ClinicalTrials.gov, number, NCT02493725. FINDINGS: Between Aug 10, 2015, and April 21, 2016, 34 patients were assigned to receive either eculizumab (n=23) or placebo (n=11). At week 4, the proportion of the patients able to walk independently (functional grade ≤2) was 61% (90% CI 42-78; n=14) in the eculizumab group, and 45% (20-73; n=5) in the placebo group. Adverse events occurred in all 34 patients. Three patients had serious adverse events: two in the eculizumab group (anaphylaxis in one patient and intracranial haemorrhage and abscess in another patient) and one in the placebo group (depression). The possibility that anaphylaxis and intracranial abscess were related to eculizumab could not be excluded. No deaths or meningococcal infections occurred. INTERPRETATION: The primary outcome measure did not reach the predefined response rate. However, because this is a small study without statistical comparison with the placebo group, the efficacy and safety of eculizumab could be investigated in larger, randomised controlled trials. FUNDING: The Japan Agency for Medical Research and Development, Ministry of Health, Labor and Welfare, and Alexion Pharmaceuticals.

Serial EEG findings in anti-NMDA receptor encephalitis: correlation between clinical course and EEG.

Anti-NMDA receptor encephalitis is a paraneoplastic encephalitis characterised by psychiatric features, involuntary movement, and autonomic instability. Various EEG findings in patients with anti-NMDA receptor encephalitis have been reported, however, the correlation between the EEG findings and clinical course of anti-NMDA receptor encephalitis remains unclear. We describe a patient with anti-NMDA receptor encephalitis with a focus on EEG findings, which included: status epilepticus, generalised rhythmic delta activity, excess beta activity, extreme delta brush, and paroxysmal alpha activity upon arousal from sleep, which we term"arousal alpha pattern". Initially, status epilepticus was observed on the EEG when the patient was comatose with conjugate deviation. The EEG then indicated excess beta activity, followed by the emergence of continuous slow activity, including generalised rhythmic delta activity and extreme delta brush, in the most severe phase. Slow activity gradually faded in parallel with clinical amelioration. Excess beta activity persisted, even after the patient became almost independent in daily activities, and finally disappeared with full recovery. In summary, our patient with anti-NMDA receptor encephalitis demonstrated slow activity on the EEG, including extreme delta brush during the most severe phase, which gradually faded in parallel with clinical amelioration, with excess beta activity persisting into the recovery phase.

Recurrent Guillain-Barré syndrome, Miller Fisher syndrome and Bickerstaff brainstem encephalitis.

OBJECTIVE: Guillain-Barré syndrome (GBS), Miller Fisher syndrome (MFS), and Bickerstaff brainstem encephalitis (BBE) are usually monophasic, but some patients experience recurrences after long asymptomatic intervals. We aimed to investigate clinical features of recurrent GBS, MFS, and BBE at a single hospital. METHODS: Records from 97 consecutive patients with GBS, MFS or BBE who were admitted to a tertiary hospital between 2001 and 2013 were reviewed. Clinical and laboratory features of patients with recurrent GBS, MFS, or BBE were investigated. RESULTS: Patients included 55 (32 males) with GBS, 34 (22 males) with MFS, and 8 (6 males) with BBE. Recurrent cases occurred in 2 (4%) of the 55 patients with GBS, 4 (12%) of the 34 patients with MFS, and 2 (25%) of the 8 patients with BBE. Patients with recurrent MFS had a tendency to be younger at the first episode than patients with non-recurrent MFS (median, 22 versus 37years old). Symptoms and signs were less severe during relapses than during the initial episode in recurrent patients. CONCLUSIONS: Recurrences occurred more frequently in patients with MFS or BBE compared with those with GBS. Patients with recurrent MFS might be younger than those with non-recurrent MFS.

Status epilepticus in the elderly: Prognostic implications of rhythmic and periodic patterns in electroencephalography and hyperintensities on diffusion-weighted imaging.

OBJECTIVE: To delineate the clinical characteristics and functional outcome of status epilepticus (SE) in elderly people, and elucidate prognostic implications of SE-associated rhythmic and periodic patterns (RPPs) in electroencephalography and hyperintensities on diffusion-weighted imaging. METHODS: We retrospectively investigated 107 consecutive patients with SE aged≥65years in a comprehensive community hospital. RPPs were classified using the 2012 American Clinical Neurophysiology Society's Standardized Critical Care EEG Terminology. Poor outcome was defined as an increase in modified Rankin Scale (mRS) score at discharge compared with that at baseline, including death. RESULTS: Median age of patients was 80.0years. Median mRS score at baseline was 3. Thirty-four patients (31.8%) had a previous diagnosis of epilepsy. Cerebrovascular disease and dementia were major etiologies. Poor outcome occurred in 41 (38.3%). In electroencephalography, periodic discharges (PDs) were present in 21.0% (22/105), rhythmic delta activity (RDA) in 10.5% (11/105), and conventional seizure patterns in 9.5% (10/105). Diffusion-weighted hyperintensities associated with SE were observed in 28.0% (26/93). With univariate analysis, poor outcome was significantly associated with no previous diagnosis of epilepsy, etiology, refractory SE, specific electroencephalographic patterns (PDs and conventional seizure patterns, but not RDA), and diffusion-weighted hyperintensities. With multivariate logistic regression analysis, diffusion-weighted hyperintensities (OR 6.13 [95% CI 1.72-21.9]) and refractory SE (OR 5.36 [95% CI 1.28-22.4]) were independently associated with poor outcome. CONCLUSIONS: SE often occurred as the first seizure in already disabled elderly people, further worsening their functional disabilities. Diffusion-weighted hyperintensities and refractory SE, but not RPPs in electroencephalography, were independent functional prognostic factors.

[Multiple cerebral infarctions in a patient with hypereosinophilic syndrome with Löffler endocarditis: a case report].

An 82-year-old woman with a history of asthma was admitted to our hospital because of dyspnea. On admission, laboratory testing showed a white blood cell count of 17,700/µl with hypereosinophilia of 9,204/µl (52% of all white blood cells). Various examinations, including a bone marrow biopsy for the cause of eosinophilia, were unremarkable. The patient was diagnosed with hypereosinophilic syndrome (HES). Treatment with intravenous methylprednisolone was initiated. The patient's eosinophil count normalized within 1 day. On the 6th day, she developed left-sided hemiparesis. Magnetic resonance imaging (MRI) of the brain showed acute multiple infarcts in arterial border zones of bilateral cerebral and cerebellar hemispheres, and in bilateral basal ganglia and the thalamus. Magnetic resonance angiography was normal. Coagulation factors were normal, except for an elevated D-dimer level (12.9 µg/ml). A transthoracic echocardiogram showed thickening of the left ventricular endocardium with immobile thrombus, compatible with Löffler endocarditis. Treatment with oral prednisolone was started at 30 mg/day and then tapered to a maintenance dose of 5 mg/day. Anticoagulation was concurrently started for prevention of stroke. Ten months later, an echocardiogram showed that the thrombus had decreased in size, and MRI revealed no new cerebral infarctions. The cause of cerebral infarction in patients with hypereosinophilia is thought to be thromboembolism or cerebrovascular endothelial toxicity of eosinophils. In this patient, the cerebral infarcts may have been the result of embolism from the left ventricular thrombus. Because HES with Löffler endocarditis is frequently associated with a poor prognosis, cardiovascular problems should be evaluated and treatment started as soon as possible.

Atg12-Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site.

Two autophagy-related ubiquitin-like systems have unique features: the E2 enzyme Atg3 conjugates the ubiquitin-like protein Atg8 to the lipid phosphatidylethanolamine, and the other ubiquitin-like protein conjugate Atg12-Atg5 promotes that conjugase activity of Atg3. Here, we elucidate the mode of this action of Atg12-Atg5 as a new E3 enzyme by using Saccharomyces cerevisiae proteins. Biochemical analyses based on structural information suggest that Atg3 requires a threonine residue to catalyze the conjugation reaction instead of the typical asparagine residue used by other E2 enzymes. Moreover, the catalytic cysteine residue of Atg3 is arranged in the catalytic center such that the conjugase activity is suppressed; Atg12-Atg5 induces a reorientation of the cysteine residue toward the threonine residue, which enhances the conjugase activity of Atg3. Thus, this study reveals the mechanism of the key reaction that drives membrane biogenesis during autophagy.