Transcriptome and growth efficiency comparisons of recombinant thermophiles that produce thermolabile and thermostable proteins: implications for burden-based selection of thermostable proteins.

Geobacillus kaustophilus is a thermophilic bacterium that grows at temperatures ranging between 42 and 74 °C. Here, we modified this organism to produce the thermolabile protein (PyrFA) or its thermostable variant (PyrFV) and analyzed the transcriptome and growth efficiency profiles of the resultant strains. In the producer of PyrFA, the transcriptome profile was changed to facilitate ATP synthesis from NADH without pooling reduced quinones. This change implies that PyrFA production at elevated temperatures places an energy burden on cells potentially to maintain protein homeostasis. This was consistent with the observation that the PyrFA producer grew slower than the PyrFV producer at > 45 °C and had a lower cellular fitness. Similar growth profiles were also observed in the PyrFA and PyrFV producers derived from another thermophile (Geobacillus thermodenitrificans) but not in those from Escherichia coli at 30 °C. Thus, we suggest that the production of thermolabile proteins impairs host survival at higher temperatures; therefore, thermophiles are under evolutionary selection for thermostable proteins regardless of whether their functions are associated with survival advantages. This hypothesis provides new insights into evolutionary protein selection in thermophiles and suggests an engineering approach to select thermostable protein variants generated via random gene mutagenesis.

GRP78 antibodies damage the blood-brain barrier and relate to cerebellar degeneration in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the neuromuscular junction caused by autoantibodies binding to P/Q-type voltage-gated calcium channels. Breakdown of the blood-brain barrier and diffusion of cerebellar granule/Purkinje cell-reactive autoantibodies into the CNS are critical for the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome. We recently found evidence that glucose-regulated protein 78 (GRP78) autoantibodies in the plasma of patients with neuromyelitis optica promote the CNS access of AQP4 autoantibodies. In the present study, we investigated whether the GRP78 autoantibodies in PCD-LEMS IgG boost the brain uptake of cerebellar cell-reactive antibodies across the blood-brain barrier and facilitate cerebellar dysfunction. We first evaluated the effects of purified IgG from PCD-LEMS or PCD patients on the blood-brain barrier function in human brain microvascular endothelial cells using a high content imaging system with nuclear factor κB p65 and intracellular adhesion molecule 1 (ICAM1) immunostaining. Next, we identified GRP78 autoantibodies causing blood-brain barrier permeability in PCD-LEMS IgG by co-immunoprecipitation and the living cell-based antibody binding assays. Exposure of brain microvascular endothelial cells to IgG from PCD-LEMS patients induced nuclear factor κB p65 nuclear translocation, ICAM1 upregulation, reduced claudin-5 expression, increased permeability and increased autocrine IL-1ß and IL-8 secretion; the IgG from patients with Lambert-Eaton myasthenic syndrome did not have these effects. We detected GRP78 autoantibodies in the IgG of LEMS-PCD (83.3%, n = 18), but observed fewer in patients with LEMS (6.6%, n = 15) and none were observed in the control subjects (n = 8). The depletion of GRP78 autoantibodies reduced the biological effect of LEMS-PCD IgG on brain microvascular endothelial cells. These findings suggest that GRP78 autoantibodies play a role beyond neuromyelitis optica and that they have direct implications in the phenotypic differences between PCD-LEMS and LEMS.

Biomarkers of Cardiac Dysfunction as Risk Factors in Cryptogenic Stroke.

BACKGROUND: It is unclear whether biomarkers of cardiac dysfunction are associated with cryptogenic stroke (CS). METHODS: We retrospectively evaluated consecutive ischemic stroke patients. Patients underwent transthoracic echocardiography to evaluate left atrial diameter and the peak transmitral filling velocity/mean mitral annular velocity during early diastole (E/e'). Patent foramen ovale (PFO) and left atrial appendage flow velocity were evaluated by transesophageal echocardiography. We compared clinical characteristics and biomarkers of cardiac dysfunction (brain natriuretic peptide [BNP], left atrial diameter, E/e', and left atrial appendage flow velocity) between CS or CS without large PFO and other causative stroke subtypes. RESULTS: Among 1,514 patients with ischemic stroke, 264 patients were classified as having CS. Of these, transesophageal echocardiography revealed 27/158 (17%) large PFOs. In comparison, for the noncardioembolic stroke group, which consisted of large artery and small vessel subtypes, patients with CS without large PFO had higher log10 BNP (adjusted OR 2.70; 95% CI 1.92-3.78; p < 0.001), higher log10 E/e' (3.41; 1.21-13.15; p = 0.019), and lower left atrial appendage flow velocity (0.98; 0.97-1.00; p = 0.031). Left atrial diameter was similar for noncardioembolic stroke and CS without large PFO (p = 0.380). Cutoff values of BNP, E/e', and left atrial appendage flow velocity capable of distinguishing CS without large PFO from noncardioembolic stroke were 65.0 pg/mL (sensitivity 55.3%; specificity 70.9%), 13.0 (45.5%; 68.0%), and 46.0 cm/s (37.1%; 87.5%), respectively. CONCLUSION: Patients with CS without large PFO could have biomarkers of cardiac dysfunction.

[Durable remission attained with plasmapheresis and intravenous immunoglobulin therapy in a patient with acute exacerbation of GVHD-related myasthenia gravis].

A 17-year-old male underwent a second bone marrow transplantation using a 6/8 allele HLA-matched unrelated donor. On day 100 after transplantation, steroid treatment for chronic graft-versus-host disease (GVHD) was started. On day 766, the patient experienced general fatigue, followed by double vision, ptosis, and dysphagia on day 810. Based on the positivity of the acetylcholine receptor antibody and a waning electromyography pattern, he was diagnosed with GVHD-related myasthenia gravis (MG). On day 861, we initiated plasmapheresis (PE), followed by the administration of intravenous immunoglobulin (IVIg) ; this treatment attenuated the bulbar symptoms of MG. Although the steroid treatment was continued, we restarted the administration of tacrolimus. On day 2,739 after transplantation, we stopped the steroid treatment, and the patient remained in remission for MG following the cessation of the steroid treatment on day 2,897. This case suggests that PE followed by IVIg could be an effective therapeutic alternative for MG associated with GVHD.

Abnormal distribution of AQP5 in labial salivary glands is associated with poor saliva secretion in patients with Sjögren's syndrome including neuromyelitis optica complicated patients.

OBJECTIVE: To investigate whether aquaporins (AQPs) are involved in salivary gland dysfunction in patients with neuromyelitis optica (NMO) complicated with Sjögren's syndrome (SS). METHODS: Eight primary SS (pSS) patients, four NMO spectrum disorder (NMOsd) patients complicated with SS (NMOsd-SS), and three control subjects were enrolled. Immunohistochemistry of labial salivary glands (LSGs) was performed to determine the expressions of AQP4, AQP5, and tumor necrosis factor-alpha (TNF-α). In vitro expression of AQP5 was examined by Western blotting in cultured primary salivary gland epithelial cells (SGECs). RESULTS: No expression of AQP4 was shown in all LSGs. AQP5 was clearly expressed in the all acini, but the predominant localization of AQP5 in the apical side was diminished in the patients with pSS or NMOsd-SS compared with the controls and tended to be even lower in NMOsd-SS than pSS. The abnormal localization of AQP5 was associated with poor saliva secretion. No difference was found in TNF-α expression in the LSGs between patients with pSS and NMOsd-SS. AQP5 expression of SGECs in vitro was not changed by TNF-α or interleukin-10. CONCLUSIONS: Our results suggest that AQP5 but not AQP4 contributes to salivary secretion in patients with SS including those with NMO complicated with SS.

Two cases of refractory polymyositis accompanied with steroid myopathy.

Polymyositis (PM) is an inflammatory muscle disease characterized by chronic inflammation in skeletal muscle. Although most patients with PM respond to corticosteroids, some cases show an unsatisfactory response and other therapeutic options must be considered. Furthermore, glucocorticosteroid (GC) toxicity leads to a significant disability known as steroid myopathy, particularly in elderly patients. Here we report two patients with refractory PM. Combined treatment with high-dose GCs, tacrolimus, and intravenous immunoglobulin resulted in beneficial effects against myositis. However, muscle weakness and the disability progressed due to steroid myopathy, and subsequent oral intake became impossible because of swallowing disturbance in these two patients. Nutritional intervention, including branched-chain amino acids (BCAAs) and rehabilitation, was undertaken in addition to treatment against myositis. These treatments finally improved the muscle weakness and activities of daily living, and the two patients were discharged after recovery. The high-dose GC treatment caused elevation of serum levels of amino acids, including BCAAs, but these amino acids subsequently declined during BCAA replacement therapy. These findings suggest that the catabolic effects of the glucocorticoid treatment impair the balance of amino acids, including BCAAs, within the muscle, leading to steroid myopathy.

[Diagnosis and Treatment of Lambert-Eaton Myasthenic Syndrome].

Approximately 90% of patients with Lambert-Eaton myasthenic syndrome (LEMS) show positive P/Q-type voltage-gated calcium channels antibodies, which can be broadly classified clinically as paraneoplastic, particularly with small cell lung carcinoma and non-paraneoplastic without cancer. The first Japanese guideline for LEMS was developed in May 2022 as MG/LEMS Practice Guideline 2022. This article describes the epidemiology, symptoms, diagnosis, examination, treatment, and prognosis of this condition, based on the LEMS guidelines.

[Muscle-Specific Receptor Tyrosine Kinase Antibody Positive Myasthenia Gravis].

Reportedly, patients with muscle-specific kinase (MuSK) antibody-positive myasthenia gravis (MG) account for approximately 3.0% of all patients with MG in Japan. Compared with patients who have acetylcholine receptor antibody-positive MG, those with MuSK antibody-positive MG show young-onset disease with female predominance, a low rate of ocular involvement (5.9%), and greater severity of dysphagia. The aforementioned types of MG are indistinguishable based on clinical symptoms and electrophysiological tests, and measurement of MuSK antibodies is essential for diagnosis. Thymectomy and complement inhibitors are not indicated for treatment, and acetylcholinesterase inhibitors, steroids, immunosuppressants, plasma exchange, intravenous immunoglobulin therapy, and neonatal Fc receptor inhibitors are used.

Right-sided Herpes Zoster Ophthalmicus Complicated by Bilateral Third, Fourth, and Sixth Cranial Nerve Palsies and Syndrome of Inappropriate Antidiuretic Hormone Secretion: A Case Report.

Cases of herpes zoster ophthalmicus (HZO) complicated by bilateral ophthalmoplegia are rare, and no cases of bilateral third, fourth, or sixth cranial nerve palsies have been reported. Syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a rare complication of HZO. We herein report an 80-year-old Japanese woman with right-sided HZO complicated by meningoencephalitis and discuss the pathogenesis of this condition. She developed bilateral third, fourth, and sixth cranial nerve palsies and SIADH almost simultaneously during treatment for HZO. The bilateral cranial palsy spontaneously resolved within a few months.

Streamlined workflow including nurse recognition of conjugate gaze deviation for reduced door-to-puncture time in endovascular thrombectomy: A retrospective study.

BACKGROUND: Endovascular thrombectomy is recognized as a pivotal treatment for acute ischemic stroke due to large vessel occlusion. Prolonged door-to-puncture time correlates with decreased patient independence after acute ischemic stroke. This study aimed to assess whether a streamlined workflow, including nurse recognition of conjugate gaze deviation, could reduce door-to-puncture time in endovascular thrombectomy. METHODS: This study retrospectively reviewed patients with acute ischemic stroke who underwent endovascular thrombectomy between March 2017 and March 2022 and compared a previous workflow with a streamlined workflow implemented in April 2019. In the streamlined workflow, nurses recognized conjugate gaze deviation to identify patients with large vessel occlusions and played a more active role in reducing the door-to-puncture time. We compared time metrics and outcomes, including recanalization status, parenchymal hemorrhage type 2, and favorable outcomes (modified Rankin Scale score 0-2) at three months between the previous and streamlined workflow groups. RESULTS: After the application of the streamlined workflow, the door-to-puncture time was reduced from 76 min to 68 min (p = 0.014), and the number of patients with a door-to-puncture time of less than 60 min increased (15% vs. 36%, p = 0.002). Outcomes including modified thrombolysis in cerebral infarction ≥ 2b (73% vs. 71%, p = 1.000), parenchymal hemorrhage type 2 (7% vs. 2%, p = 0.281), and favorable outcome (33% vs. 34%, p = 1.000) were comparable between the two groups. CONCLUSION: Nurse recognition of conjugate gaze deviation contributed to an 8-minute reduction in the door-to-puncture time, demonstrating the potential benefits of an organized workflow in acute ischemic stroke.

Efficacy of Intrathecal Isoniazid and Steroid Therapy in Refractory Tuberculous Meningitis.

Tuberculous meningitis is an infectious disease with high mortality. Literature describing intrathecal therapy for tuberculous meningitis is scarce. We herein report a case of refractory tuberculous meningitis in a 52-year-old woman with underlying neuropsychiatric systemic lupus erythematosus. Despite systemic treatment with anti-tuberculosis drugs and dexamethasone, her meningeal irritation deteriorated. Intrathecal isoniazid and prednisolone administration was therefore initiated, and the symptoms of severe meningeal irritation improved along with head magnetic resonance imaging and cerebrospinal fluid findings. This case report highlights the efficacy of intrathecal isoniazid and steroid injections for refractory tuberculous meningitis, particularly in patients with severe meningeal irritation.

HTLV-I virological and histopathological analysis in two cases of anti-centromere-antibody-seropositive Sjögren's syndrome.

INTRODUCTION: The aim of this study was to show the clinical and pathological characteristics of anti-centromere-antibody (ACA)-seropositive Sjögren's syndrome (SS) in two anti-human T-cell leukemia virus type I (HTLV-I)-seropositive patients. METHODS: One patient was an HTLV-I carrier whereas the other was diagnosed with HTLV-I-associated myelopathy (HAM). Background data including serum HTLV-I titers, viral loads, and cytokine profiles were recorded. Azocarmine with aniline blue (Azan)-Mallory staining and immunohistochemistry of the labial salivary glands (LSGs) and a muscle biopsy specimen from the HAM patient were performed. RESULTS: Serum transforming growth factor beta (TGF-ß), tumor necrosis factor alpha (TNF-α), and HTLV-I viral load were high in the HAM-SS patient compared with the HTLV-I carrier. Fibrous change in LSG was prominent in the HAM-SS patient. Although TGF-ß expression was similar in the two patients, expression of HTLV-I-related proteins including p12, p28, group-specific antigen (GAG), and nuclear factor kappa-B (NF-κB) in the LSG were dominantly detected in the HAM-SS patient. Frequency of TGF-ß staining in HTLV-I-seropositive SS patients without ACA, HTLV-I-seronegative SS patients with ACA, and HTLV-I-seronegative SS patients without ACA was lower than that of the previous two patients. CONCLUSION: A high HTLV-I viral load in situ is supposed to promote the production of cytokines, especially TGF-ß, resulting in the fibrous change of LSG in ACA-seropositive SS patients.

[Increased rate of serum uric acid levels in Yusho sufferers].

We measured serum uric acid levels in Yusho sufferers annually from 2007 to 2012 in Nagasaki prefecture. We observed an increased rate of serum uric acid levels in 38.2% of the male and 5.5% of the female sufferers. There was no relation among serum uric acid value, Body Mass Index, liver function, blood polychlorinated biphenyls and hypersensitive C reactive protein. We conclude that it is unclear if blood polychlorinated biphenyls may play a role in the increase of serum uric acid levels in Yusho sufferers.

Impact of width of susceptibility vessel sign on recanalization following endovascular therapy.

BACKGROUND AND PURPOSE: We aimed to investigate the relationship between arterial recanalization following endovascular therapy and the susceptibility vessel sign (SVS) length and width on susceptibility-weighted imaging. METHODS: We retrospectively evaluated consecutive patients with anterior circulation ischemic stroke who underwent magnetic resonance imaging preceded endovascular therapy, and measured the SVS length and width. Successful recanalization was defined as expanded thrombolysis in cerebral infarction grade of 2b to 3. Logistic regression analysis was executed to determine the independent predictors of successful recanalization and first-pass reperfusion (FPR) after endovascular therapy. RESULTS: Among 100 patients, successful recanalization and FPR were observed in 77 and 34 patients, respectively. The median SVS length and width were 10.3 mm (interquartile range, 6.8-14.1 mm) and 4.2 mm (interquartile range, 3.1-5.2 mm), respectively. In multivariate logistic regression analysis, SVS width was associated with successful recanalization (odds ratio, 1.88; 95% confidence interval, 1.14-3.07; p = 0.005) and FPR (odds ratio, 1.38; 95% confidence interval, 1.01-1.89; p = 0.039). The optimal cutoff value for the SVS width to predict successful recanalization and FPR were 4.2 mm and 4.0 mm, respectively. CONCLUSIONS: Larger SVS width may predict successful recanalization and FPR following endovascular therapy.

Two Lambert-Eaton Myasthenic Syndrome Patients with Ameliorated Activities of Daily Living Due to Cholinesterase Inhibitors.

We herein report two P/Q-type voltage-gated calcium channel (VGCC) antibody-positive Lambert-Eaton myasthenic syndrome (LEMS) patients who responded dramatically to cholinesterase inhibitors. Patient 1, a 76-year-old man, had small-cell lung cancer and developed LEMS during chemotherapy. When symptomatic treatment was started with pyridostigmine, gait disturbance was ameliorated, and his modified Rankin scale decreased from 4 points to 3 points. Patient 2, a 68-year-old man, had cancer-free LEMS. Distigmine bromide was very effective and ameliorated not only his gait disturbance but also autonomic symptoms, and his modified Rankin scale decreased from 2 points to 1 point. Cholinesterase inhibitors alone may be effective in a small portion of LEMS patients.

Perfusion abnormality in neuronal intranuclear inclusion disease with stroke-like episode: A case report.

Neuronal intranuclear inclusion disease (NIID) is a slowly progressive neurodegenerative disease. Some patients with NIID occasionally present with acute symptoms. However, its mechanism remains unclear. We report a patient with NIID who presented with a stroke-like episode. Arterial spin labeling magnetic resonance imaging revealed hypoperfusion in the focal cerebral region at the onset while no apparent arterial occlusion was observed. The abnormal perfusion area was normalized 6 days after admission. Therefore, the perfusion abnormality was likely the main cause of acute neurologic deficits in NIID. NIID should be considered in the differential diagnosis of stroke mimics.

Motor end-plate analysis to diagnose immune-mediated myasthenia gravis in seronegative patients.

This study aimed to evaluate the diagnostic usefulness of motor end-plate (MEP) analysis along with clustered acetylcholine receptor (AChR) antibody (Ab) assays in patients with myasthenia-like symptoms but negative routine AChR and muscle-specific kinase (MuSK) Ab tests. MEP analysis of muscle biopsies of the biceps brachii was performed in 20 patients to try to differentiate between those with or without immune-mediated myasthenia gravis (MG). Using a quantitative method, complement C3 deposition and AChR densities in MEPs were examined. Independently, cell-based assays were used to detect serum clustered-AChR Abs. Only five of 20 patients had complement deposition at MEPs; four of these patients had reduced AChR densities similar to those in patients with typical AChR Ab positive MG, and distinct from those in the remaining 15 patients. Two of the four serum samples from these patients had clustered-AChR Abs. All complement-positive patients were considered as having immune-mediated MG and improved with appropriate treatments; although one patient presented with MG 3 years later, the remaining patients had other diagnoses during over 10 years of follow-up. These results suggest the usefulness of MEP analysis of muscle biopsies in diagnosing immune-mediated MG in seronegative patients with myasthenia-like symptoms but, due to the invasiveness of the muscle biopsy procedure, clustered AChR Abs should, if possible, be tested first.

Paraneoplastic Cerebellar Degeneration With P/Q-VGCC vs Yo Autoantibodies.

BACKGROUND AND OBJECTIVES: Paraneoplastic cerebellar degeneration (PCD) is characterized by a widespread loss of Purkinje cells (PCs) and may be associated with autoantibodies against intracellular antigens such as Yo or cell surface neuronal antigens such as the P/Q-type voltage-gated calcium channel (P/Q-VGCC). Although the intracellular location of the target antigen in anti-Yo-PCD supports a T cell-mediated pathology, the immune mechanisms in anti-P/Q-VGCC-PCD remain unclear. In this study, we compare neuropathologic characteristics of PCD with anti-P/Q-VGCC and anti-Yo autoantibodies in an archival autopsy cohort. METHODS: We performed neuropathology, immunohistochemistry, and multiplex immunofluorescence on formalin-fixed and paraffin-embedded brain tissue of 1 anti-P/Q-VGCC, 2 anti-Yo-PCD autopsy cases and controls. RESULTS: Anti-Yo-PCD revealed a diffuse and widespread PC loss together with microglial nodules with pSTAT1+ and CD8+granzymeB+ T cells and neuronal upregulation of major histocompatibility complex (MHC) Class I molecules. Some neurons showed a cytoplasmic immunoglobulin G (IgG) staining. In contrast, PC loss in anti-P/Q-VGCC-PCD was focal and predominantly affected the upper vermis, whereas caudal regions and lateral hemispheres were spared. Inflammation was characterized by scattered CD8+ T cells, single CD20+/CD79a+ B/plasma cells, and an IgG staining of the neuropil in the molecular layer of the cerebellar cortex and neuronal cytoplasms. No complement deposition or MHC-I upregulation was detected. Moreover, synaptophysin was reduced, and neuronal P/Q-VGCC was downregulated. In affected areas, axonal spheroids and the accumulation of amyloid precursor protein and glucose-regulated protein 78 in PCs indicate endoplasmatic reticulum stress and impairment of axonal transport. In both PCD types, calbindin expression was reduced or lost in the remaining PCs. DISCUSSION: Anti-Yo-PCD showed characteristic features of a T cell-mediated pathology, whereas this was not observed in 1 case of anti-P/Q-VGCC-PCD. Our findings support a pathogenic role of anti-P/Q-VGCC autoantibodies in causing neuronal dysfunction, probably due to altered synaptic transmission resulting in calcium dysregulation and subsequent PC death. Because disease progression may lead to irreversible PC loss, anti-P/Q-VGCC-PCD patients could benefit from early oncologic and immunologic therapies.

FTY720 Exacerbates Blood-Brain Barrier Dysfunction Induced by IgG Derived from Patients with NMO and MOG Disease.

Neuromyelitis optica (NMO) and myelin oligodendrocyte glycoprotein (MOG) antibody-related disease (MOG disease) are inflammatory demyelinating diseases of the central nervous system (CNS). The disruption of the blood-brain barrier (BBB) is considered a key step in the pathogenesis of NMO and MOG disease. Although a previous report indicated that circulating immunoglobulin G (IgG) from NMO patients disrupts the BBB, the effect of IgG from patients with MOG disease has not been elucidated. In addition, it has been reported that some disease-modifying drugs for multiple sclerosis are harmful to NMO by an unknown mechanism. This study aimed to examine the effects of IgG from patients with NMO or MOG disease on BBB integrity. We also examined the effects of disease-modifying drugs (fingolimod [FTY720] and dimethyl fumarate [DMF]) on IgG-treated brain capillary endothelial cells. We used in vitro BBB models constructed with rat brain capillary endothelial cells (RBECs) to examine the effects on BBB function. The integrity of the RBECs was assessed by measuring transendothelial resistance (TEER) and cell viability. NMO or MOG-IgG treatment decreased TEER and cell viability in the endothelial monolayer model. Although FTY720 and DMF did not affect barrier function or cell viability under normal conditions, disease IgG-induced barrier dysfunctions were worsened by the presence of FTY720. These data indicate that circulating IgG in patients with NMO or MOG disease worsens BBB function. Furthermore, in patients with NMO or MOG disease treated with FTY720, changes in the integrity of the BBB were found to exacerbate the disease.

An adult case of parainfectious optic neuritis associated with genital herpes simplex virus type 2 infection.

Although acute retinal necrosis (ARN) and optic neuritis following herpes encephalitis are known causes of acute visual impairment associated with herpes simplex virus (HSV) infection, there have been no reports of parainfectious optic neuritis associated with genital HSV type 2 (HSV-2) infection. A young Japanese woman developed unilateral optic neuritis 7 days after the onset of genital HSV-2 infection. Ophthalmologic examination revealed no findings suggestive of ARN and both multiple sclerosis and neuromyelitis optica were ruled out by the brain images and serum antibody testing. An oral steroid therapy improved her symptoms. Here, we describe the first case of parainfectious optic neuritis associated with genital HSV-2 infection.