Immunogenicity of SARS-CoV-2 mRNA intramuscular vaccination in patients with muscular disorders.

Backgrounds: Little clinical data is available on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with muscular disorders (MDs). The immunogenicity of SARS-CoV-2 vaccines against MDs, in particular, remains unknown. Thus, this study aimed to confirm the immunogenicity and safety of the SARS-CoV-2 vaccine against MDs. Methods: All participants were vaccinated with two doses of mRNA vaccines (BNT162b2, Pfizer-BioNTech). The serum samples were collected from each patient on the day of second dose of vaccination, and then, consecutively, after one month, three months, and six months. Anti-SARS-CoV-2 IgG levels were determined using the Abbott SARS-CoV-2 IgG II Quant assay. Results: We evaluated 75 individuals, including 42 patients with MDs and 33 patients with non-muscular disorders (non-MDs). Non-MD patients primarily include those with severe motor and intellectual disabilities. The median age of the patients was 32 years (range 12-64 years). After one and three months following the second immunization, patients with MDs had lower antibody responses. Furthermore, three months following the second immunization, the proportion of high responders among patients with MDs decreased significantly compared to that among patients without MDs (p-value of less than 0.01). No serious adverse events were observed in patients with or without MDs. Conclusion: Intensity and latency of antibody response were suppressed in patients with MDs. Although MDs may be a key contributor in predicting the antibody response to SARS-CoV-2 vaccination, SARS-CoV-2 immunization in MDs needs extensive research.

Efficacy of interferential current transcutaneous electrical sensory stimulation through the neck skin for treating dysphagia in children with disabilities: A case series.

Finding a suitable treatment for dysphagia has been challenging and the efficacy of neuromuscular electrical stimulation has been recognized. Moreover, the beneficial effect of interferential current transcutaneous electrical sensory stimulation has recently been described. However, the efficacy of interferential current transcutaneous electrical sensory stimulation in children with disabilities is unknown. Therefore, the aim of this study was to confirm the efficacy of interferential current transcutaneous electrical sensory stimulation in children with disabilities. Four children with disabilities of various types underwent interferential current transcutaneous electrical sensory stimulation once a week. All patients showed improved symptoms after interferential current transcutaneous electrical sensory stimulation treatment. Videoendoscopic examination showed reduced accumulation of secretion in all patients and decreased residual bolus in two. We also felt an increased forcefulness when swallowing in two. In addition, the questionnaire results regarding dysphagia indicated improvements. No significant side effects were observed. The interferential current transcutaneous electrical sensory stimulation treatment may be effective and safe in children with disabilities. The effect of this treatment on swallowing ability needs to be further investigated by studying more cases.

Assessment of an Automatic Hot Water Sterilizing System-Equipped Dialysis Supply Device's Circuit Degradation, 7 Years Postinstallment.

At most dialysis clinics in Japan, a central dialysis fluid delivery system (CDDS) is used primarily to provide consistent treatment to patients. We incorporated hot water disinfection to a CDDS over a 7 year period, after which we assessed the dialysis purification levels. We observed that adequate levels had been maintained. We rated the internal circuits of the device at the 7 year mark, comparing the insides of both the used and unused new Synflex tubing, silicone blades, Teflon tubing, silicone rubber tubing, and stainless steel parts. No bacteria or contamination (such as endotoxins) was detected, and no degradation or damage were observed. Even after the auto-hot water disinfection system was installed, no internal tubing degradation due to hot water or mechanical issues have occurred, and the system's dialysis fluid purification levels have been maintained.

Difficulty removing dialysis cuff catheter after its adhesion to the right atrium.

We describe the case of an elderly Japanese female who had experienced diabetic nephropathy since the year 20xx and had been undergoing dialysis treatment while receiving vascular access interventional therapy (VAIVT) for arteriovenous fistula (AVF) occlusion. The patient visited the clinic/hospital in 20xx+10 with the AVF occlusion; emergency VAIVT was performed but blood flow could not be resumed. The patient was not admitted and was treated as an outpatient, and thus a cuff catheter (Split stream catheter: SST28 cm, Medcomp) was inserted. An infection developed and was successfully treated with antibiotics. The dialysis treatment continued without issue. One year after the cuff catheter's insertion, the patient was admitted due difficulty breathing. Despite continued dialysis treatment with the catheter, the patient died 15 days post-admission. The removal of the catheter proved to be difficult. An autopsy was approved, and the area around the catheter was examined. The adhesion of the catheter to the right atrium was observed, but no infection was detected in the bloodstream. This case illustrates that dialysis with the use of a cuff catheter can be effective.

Functional maintenance of calcium store by ShcB adaptor protein in cerebellar Purkinje cells.

Intracellular Ca2+ levels are changed by influx from extracellular medium and release from intracellular stores. In the central nervous systems, Ca2+ release is involved in various physiological events, such as neuronal excitability and transmitter release. Although stable Ca2+ release in response to stimulus is critical for proper functions of the nervous systems, regulatory mechanisms relating to Ca2+ release are not fully understood in central neurons. Here, we demonstrate that ShcB, an adaptor protein expressed in central neurons, has an essential role in functional maintenance of Ca2+ store in cerebellar Purkinje cells (PCs). ShcB-knockout (KO) mice showed defects in cerebellar-dependent motor function and long-term depression (LTD) at cerebellar synapse. The reduced LTD was accompanied with an impairment of intracellular Ca2+ release. Although the expression of Ca2+ release channels and morphology of Ca2+ store looked intact, content of intracellular Ca2+ store and activity of sarco/endoplasmic reticular Ca2+-ATPase (SERCA) were largely decreased in the ShcB-deficient cerebellum. Furthermore, when ShcB was ectopically expressed in the ShcB-KO PCs, the Ca2+ release and its SERCA-dependent component were restored. These data indicate that ShcB plays a key role in the functional maintenance of ER Ca2+ store in central neurons through regulation of SERCA activity.

[Usefulness of Nutritional Enforcement by Percutaneous Gastrostomy in Successful Omentoplasty for Empyema with Fistula].

Omentoplasty has been accepted as an effective surgical procedure for fistulated empyema. However, it is difficult for patients with poor nutritional status because their omental volume is often too poor to be applied for omentoplasty. Percutaneous endoscopic gastrostomy(PEG) is useful for long-term nutritional management. There is no report on safety and usefulness of PEG before omentoplasty. We report a case of omentoplasty that was successfully performed after nutritional enforcement by using percutaneous endoscopic gastrostomy in a patient of postoperative empyema with fistula.

Methods We Currently Use for Home Hemodialysis Management.

There are approximately 1,330,000 chronic renal failure patients in Japan, and over 30,000 patients are introduced to dialysis therapy annually. By the end of 2015, there were over 320,000 dialysis patients in Japan. Various groups have been working hard to educate all people involved including the patient, their families, doctors, nurses, and caregivers on three important topics: hemodialysis, peritoneal dialysis, and kidney transplantation. Recently, there has been a growing interest in home hemodialysis. Although peritoneal dialysis has existed in Japan for a long time, the number of peritoneal dialysis patients in Japan has not been increasing. Only 3% of the total number of dialysis treatments done here are peritoneal. Despite these circumstances, home hemodialysis therapy has been gaining attention in Japan, which is a big breakthrough. We are at the frontier of improving dialysis; however, introducing home hemodialysis has been a difficult obstacle to overcome. Here, we would like to present our methods of introducing home hemodialysis and how we have dealt with this challenge.

Evaluation of the functions of the temporary catheter with various tip types.

INTRODUCTION: A temporary catheter (TC) is used short-term and for emergencies. There are some cases when we cannot withdraw blood immediately after inserting the catheter in our patients. The reason is said to be the tips of the TC sticking to the vascular walls. OBJECTIVE: We evaluated examined 3 catheters with different tip shapes in a simulation circuit to assess the effect on the blood flow. METHODS: Water was circulated in the simulation circuit at 1 L/minute. Next, we inserted each TC into the model, and the TCs were connected to the dialysis circuit at 200 mL/minute. We put gold powder into the simulation circuit. We visually observed the movement of the gold power at the head of the catheter and measured the recirculation rate. RESULT: The uplift type TC was able to perform blood removal and reinfusion with the least difficulties. All recirculation rates were less than 1%. The hindrance caused by hitting a vascular wall is believed to have been reduced. CONCLUSION: With the manipulation of the catheter tip shapes, blood was able to circulate smoothly. We expect less blood clots and a decrease in sticking to the vascular wall. We plan to study these 3 catheters at clinical tests in the future.

Severe hyponatremia in a patient with schizophrenia associated with prolonged consciousness disturbance.

Hyponatremia presents with various central nervous system symptoms during its course and treatment. We treated a patient who presented with a prolonged consciousness disorder and was suspected of having complications of neuroleptic malignant syndrome and osmotic demyelination syndrome (ODS) during the treatment for his hyponatremia, which was caused by syndrome of inappropriate secretion of antidiuretic hormone (SIADH). The patient was a 30-year-old Japanese man who had been under treatment for schizophrenia. He presented with profound hyponatremia (96 mEq/L) and a consciousness disorder. Because he was taking a number of antipsychotic drugs and since psychogenic polydipsia was present along with laboratory findings, the patient was diagnosed with SIADH. However, the consciousness disorder reappeared after his serum sodium concentrations were normalized, and it persisted over a long period. Although ODS was suspected from the clinical course and imaging findings, there were several inconsistencies, such as the lack of quadriplegia. The patient also showed muscular rigidity and fever, and we, therefore, diagnosed complications of malignant hyperthermia syndrome caused by the discontinuation of all antipsychotic drugs at the time of onset. There have been no reports of complications of these two conditions, and we report this case for its clinically valuable information.

Involvement of the neuronal phosphotyrosine signal adaptor N-Shc in kainic acid-induced epileptiform activity.

BDNF-TrkB signaling is implicated in experimental seizures and epilepsy. However, the downstream signaling involved in the epileptiform activity caused by TrkB receptor activation is still unknown. The aim of the present study was to determine whether TrkB-mediated N-Shc signal transduction was involved in kainic acid (KA)-induced epileptiform activity. We investigated KA-induced behavioral seizures, epileptiform activities and neuronal cell loss in hippocampus between N-Shc deficient and control mice. There was a significant reduction in seizure severity and the frequency of epileptiform discharges in N-Shc deficient mice, as compared with wild-type and C57BL/6 mice. KA-induced neuronal cell loss in the CA3 of hippocampus was also inhibited in N-Shc deficient mice. This study demonstrates that the activation of N-Shc signaling pathway contributes to an acute KA-induced epileptiform activity and neuronal cell loss in the hippocampus. We propose that the N-Shc-mediated signaling pathway could provide a potential target for the novel therapeutic approaches of epilepsy.

Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression.

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCαS657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCαS657 and FoxO1.

How to monitor vascular access in our hospital.

BACKGROUND: The number of dialysis patients in Japan has amounted to approximately 310,000. Most of the patients undergo hemodialysis. The reason why they can undergo hemodialysis is because maintaining and managing vascular access (VA) has improved. Recently, thanks to the progress of medical equipment, a variety of monitoring systems have been developed. It is important to make good use of these monitoring systems. RESULTS: In our hospital, we have been monitoring with an ultrasonic device and HD02. We measure blood flow of brachial artery with an ultrasonic device during nondialysis treatment. We examine real blood flow and blood recirculation with HD02 and evaluate the function of VA during dialysis. CONCLUSIONS: In order to provide good dialysis care, good use of monitoring devices of VA is significant.

VCP/Cdc48 rescues the growth defect of a GPI10 mutant in yeast.

We identified a yeast mutant with temperature-sensitive growth defects that were rescued by VCP expression. The mutation occurred in GPI10, which encodes a mannosyl transferase for glycosylphosphatidylinositol anchor formation in the endoplasmic reticulum, and caused a Gly469Glu substitution in Gpi10. The mutant exhibited increased unfolded protein response, which was partially rescued by VCP or Cdc48, and showed sensitivity against cell-wall stressors, which were not rescued by VCP. These results suggest a potential link between VCP/Cdc48 and Gpi10 functions in the control of cell growth.

Mdm20 stimulates polyQ aggregation via inhibiting autophagy through Akt-Ser473 phosphorylation.

Mdm20 is an auxiliary subunit of the NatB complex, which includes Nat5, the catalytic subunit for protein N-terminal acetylation. The NatB complex catalyzes N-acetylation during de novo protein synthesis initiation; however, recent evidence from yeast suggests that NatB also affects post-translational modification of tropomyosin, which is involved in intracellular sorting of aggregated proteins. We hypothesized that an acetylation complex such as NatB may contribute to protein clearance and/or proteostasis in mammalian cells. Using a poly glutamine (polyQ) aggregation system, we examined whether the NatB complex or its components affect protein aggregation in rat primary cultured hippocampal neurons and HEK293 cells. The number of polyQ aggregates increased in Mdm20 over-expressing (OE) cells, but not in Nat5-OE cells. Conversely, in Mdm20 knockdown (KD) cells, but not in Nat5-KD cells, polyQ aggregation was significantly reduced. Although Mdm20 directly associates with Nat5, the overall cellular localization of the two proteins was slightly distinct, and Mdm20 apparently co-localized with the polyQ aggregates. Furthermore, in Mdm20-KD cells, a punctate appearance of LC3 was evident, suggesting the induction of autophagy. Consistent with this notion, phosphorylation of Akt, most notably at Ser473, was greatly reduced in Mdm20-KD cells. These results demonstrate that Mdm20, the so-called auxiliary subunit of the translation-coupled protein N-acetylation complex, contributes to protein clearance and/or aggregate formation by affecting the phosphorylation level of Akt indepenently from the function of Nat5.

Spatio-temporal expression pattern of the NatB complex, Nat5/Mdm20 in the developing mouse brain: implications for co-operative versus non-co-operative actions of Mdm20 and Nat5.

The NatB complex, Nat5/Mdm20 acetyltransferase mediates N-acetylation to control cell cycle progression and actin dynamics in yeast. As yet, little is known about the expression pattern of Mdm20 and Nat5 in multi-cellular organisms. Here we show that Mdm20 is highly expressed in mouse embryonic brain. At E11.5, Mdm20 was widely expressed in both neural progenitors and early differentiating neurons, whereas Nat5 was expressed in Sox1/3+/Mdm20+ neural progenitors. By E14.5, both Mdm20 and Nat5 were downregulated in most ventricular zone neural progenitors, whereas both proteins were found in differentiating neurons and co-expression was maintained at E18.5 in derivatives of these cells, such as midbrain dopaminergic (DA) neurons and septal neurons. These data suggest that Nat5/Mdm20 complex-mediated acetylation may play a role in the proliferation and differentiation of neural progenitors. Intriguingly, our data also showed that Mdm20 is not always co-expressed with Nat5 in all differentiated neurons, for example deep cerebellar neurons. Moreover, detailed examination of the subcellular localization of Mdm20 and Nat5 in cultured Nat5+/Mdm20+ midbrain DA neurons revealed that Mdm20 is also not necessarily co-localized with Nat5 within neurons. Given that Nat5 is only a known member of Nat family protein that interacts with Mdm20, our data imply that Mdm20 may function either with an unidentified Nat protein partner(s) or possibly in a Nat-independent manner.

Characterization of stress sensitivity and chaperone activity of Hsp105 in mammalian cells.

Hsp105 is a major mammalian heat shock protein that belongs to the Hsp105/110 family, a diverged subgroup of the Hsp70 family. Hsp105 not only protects the thermal aggregation of proteins, but also regulates the Hsc70 chaperone system in vitro. Recently, it has been shown that Hsp105/110 family members act as nucleotide exchange factors for cytosolic Hsp70s. However, the biological functions of Hsp105/110 family proteins still remain to be clarified. Here, we examined the function of Hsp105 in mammalian cells, and showed that the sensitivity to various stresses was enhanced in the Hsp105-deficient cells compared with that in control cells. In addition, we found that deficiency of Hsp105 impaired the refolding of heat-denatured luciferase in mammalian cells. In contrast, overexpression of Hsp105α enhanced the ability to recover heat-inactivated luciferase in mammalian cells. Thus, Hsp105 may play an important role in the refolding of denatured proteins and protection against stress-induced cell death in mammalian cells.

Maintaining ATP levels via the suppression of PERK-mediated rRNA synthesis at ER stress.

Currently, [(3)H]uridine is most often used to monitor rRNA synthesis in cultured cells. We show here that radiolabeled ribonucleoside triphosphates, such as [alpha-(33)P]UTP, in culture medium were also incorporated efficiently not only into cells but also into de novo RNA, particularly rRNA. Using this method, we first revealed that endoplasmic reticulum (ER) stress inducers such as tunicamycin and thapsigargin suppressed de novo rRNA synthesis, and that PERK, but not IRE1alpha or ATF6, mediated the suppression. PERK is known to mediate the suppression of de novo protein synthesis via phosphorylation of eIF2alpha. Consistently, other translational inhibitors such as PSI, proteasomal inhibitor, and cycloheximide suppressed de novo rRNA synthesis. eIF2alpha knockdown also suppressed both de novo protein and rRNA syntheses. Furthermore, ER stress reduced cellular ATP levels, and the suppression of rRNA synthesis apparently mitigated their reduction. These observations provided a close link between ATP levels and suppression of de novo rRNA synthesis at ER stress, and we proposed a novel feedback mechanism, in which ATP levels were maintained via suppression of de novo rRNA synthesis in ATP-demanding stresses, such as ER stress.

p97/valosin-containing protein (VCP) is highly modulated by phosphorylation and acetylation.

p97/valosin-containing protein (VCP) is a member of the AAA family proteins, which plays various important roles in cells by using its ATPase activity. But mechanism of regulating its ATPase activity is mostly unknown. We report here that VCP is highly modified throughout the protein via acetylation and phosphorylation. In addition to six previously identified phosphorylation sites, we identified at least 14 serines, 14 threonines, 6 tyrosines and 22 lysines as potential modification sites. Interestingly, these sites included Lys251 and Lys524, which are very critical for the ATP binding in Walker A motif of D1 and D2 domains, respectively. It is notable that 16 sites are in the N-terminal region and 16 sites are clustered in D2alpha domain (from Pro646 to Gly765). Indeed, amino acid substitution of Lys696 and Thr761 profoundly affect VCP ATPase activities. From these results, we propose that D2alpha domain acts as a VCP ATPase Regulatory domain or "VAR domain". VCP modifications including those in this VAR domain may endorse adaptive and multiple functions to VCP in different cell conditions such as in the cell cycle and with abnormal protein accumulation.

Targeted disruption of Hsp110/105 gene protects against ischemic stress.

BACKGROUND AND PURPOSE: Hsp110/105 belongs to the HSP110 heat shock protein family, which is a subgroup of the HSP70 family. In mammals, Hsp110/105 is constitutively expressed but exhibits particularly high levels in the brain. It has recently been shown that both Hsp110/105 and Hsp70 are elevated after cerebral ischemia. To study the physiological role of this protein in vivo, we generated hsp110/105 knockout (KO) mice and investigate the effect of reduced Hsp110/105 levels on focal cerebral ischemia. METHODS: hsp110/105 KO and wild-type mice were subjected to 30 minutes of transient middle cerebral artery occlusion followed by reperfusion for 24 hours. The infarct volume and neurological scores were measured and compared. The Hsp70 chaperone activity of thermally denatured firefly luciferase was measured in hsp110/105 KO embryonic fibroblasts. RESULTS: The infarct volume and neurological deficit scores were significantly (P<0.05) reduced in hsp110/105 KO mice compared with wild-type controls. In addition, hsp110/105 KO embryonic fibroblasts exhibited a dose-dependent suppression of Hsp70 chaperone activity by the presence of Hsp110/105. CONCLUSIONS: These results demonstrate that hsp110/105 KO mice are resistant to ischemic injury and that the protective effects of hsp110/105 deficiency in cerebral ischemia may partly be mediated by an increase in the chaperone activity of Hsp70.

Stress protein assay for the evaluation of cytotoxicity of dental amalgam.

To evaluate the cytotoxicity of mercury in dental amalgams, a stress protein assay was performed and the results were compared with the cytotoxicity evaluated by a neutral red uptake assay. The induction of a major stress protein, hsp70, was analyzed at levels of mRNA, synthesis and accumulation in human HeLa cells treated with extracts from amalgam, metal mercury and mercuric chloride. Mercuric chloride induced an increase in the synthesis of hsp70 at concentrations of mercury half those used for the neutral red uptake assay. The extracts from dental amalgam and metal mercury induced an increase in hsp70 mRNA at concentrations of mercury half those causing the inhibition of neutral red uptake into cells. Furthermore, the extracts from dental amalgam or metal mercury increased the synthesis of hsp70 and inhibited the uptake of dye at concentrations of mercury 1/10-1/50 lower than those at which mercuric chloride acted. These results suggest that the stress protein assay is more sensitive than the conventional neutral red assay for the evaluation of the cytotoxicity of mercury in dental amalgams and that the methods used in the preparation of metal solutions seem to be critical to the evaluation of cytotoxicity of dental materials.