The R436Q missense mutation in WWP1 disrupts autoinhibition of its E3 ubiquitin ligase activity, leading to self-degradation and loss of function.

Muscular dystrophy in the NH-413 chicken is caused by a missense mutation in the WWP1 gene. WWP1 is a HECT-type E3 ubiquitin ligase containing four tandem WW domains that interact with proline-rich peptide motifs of target proteins, and a short region connecting the second and third WW domains is crucial for the E3 ligase to maintain an autoinhibitory state. A mutation of the arginine in the WW2-WW3 linker to glutamine is thought to affect WWP1 function, but there is little information on this mutation to date. In this study, we generated a transgenic (Tg) mouse model expressing the WWP1 transgene with the R436Q mutation, which corresponds to the missense mutation found in the NH-413 chicken. Tg mice showed marked degradation of mutant WWP1 proteins in various tissues, particularly in striated muscle. Immunoprecipitation analysis using a WWP1-specific antibody demonstrated that the mutant WWP1 proteins lacked the C-terminal catalytic cysteine residue that is required for their binding to the E2-substrate complex during their degradation. In vitro analysis using the R436Q mutant of WWP1 lacking this catalytic cysteine residue showed no autodegradation, indicating that the loss-of-function degradation of this protein is caused by self-ubiquitination. Tg mice expressing R436Q WWP1 did not show stunted growth or premature death. Furthermore, histological analysis did not reveal any obvious changes. These observations suggested that the R436Q mutant WWP1 protein, which is released from autoinhibitory mode by its missense mutation, does not have abnormally activated enzyme function to substrates before its self-degradation and loss of enzyme function.

Evaluation of True Bonding Strength for Adhesive Bonded Carbon Fiber-Reinforced Plastics.

Carbon fiber-reinforced thermoplastics (CFRTPs) have attracted attention in aerospace because of their superior specific strength and stiffness. It can be assembled by adhesive bonding; however, the existing evaluation of bonding strength is inadequate. For example, in a single-lap shear test, the weld zone fails in a combined stress state because of the bending moment. Therefore, the strength obtained experimentally is only the apparent strength. The true bonding strength was obtained via numerical analysis by outputting the local stress state at the initiation point of failure. In this study, the apparent and true bonding strengths were compared with respect to three types of strength evaluation tests to comprehensively evaluate bonding strength. Consequently, the single-lap shear test underestimates the apparent bonding strength by less than 14% of the true bonding strength. This indicates that care should be taken when determining the adhesion properties for use in numerical analyses based on experimental results. We also discussed the thickness dependence of the adhesive on the stress state and found that the developed shear test by compression reduced the discrepancy between apparent and true strength compared with the single-lap shear test and reduced the thickness dependence compared with the flatwise tensile test.

Impact of COVID-19 and Closed Transmission of SARS-CoV-2 during the First Wave in Toyama Prefecture, Japan, March 30 to May 18, 2020.

We studied 226 patients in Toyama Prefecture who were notified of COVID-19 during the first wave between March 30 and May 18, 2020. Of the 226 patients, 22 (9.7%) died, most (95%) of whom were aged ≥65 years. A large cluster comprising 59 patients (41 residents and 18 staff members) was identified in a nursing home on April 17. No deaths occurred among staff members; however, 12 of the 41 residents (29%) died. Although the threshold cycle (Ct) values were significantly lower in the 20-64 and ≥65 years age groups than in the <20 years age group, no correlation was found between the Ct values and severity, fatal outcome, or secondary infection. The haplotype network of 145 SARS-CoV-2 isolates (64%) from 226 patients was analyzed. The viral genomes of the case groups differed by less than five nucleotide bases. These data suggest that the SARS-CoV-2 strains, which were initially introduced into Toyama Prefecture in late March and early April 2020, and their closely related strains, identified as lineage B.1.1, circulated during the first wave. The reduced inter-prefectural mobility of local residents may support the lack of strain diversity in SARS-CoV-2 during the first wave of the state of emergency.

Memories of Professor François Gros.

I joined the laboratory of Professor Francois Gros in 1987 and worked there as a postdoc with Robert Whalen until 1992. I recount the research we carried out and mention that of the other scientists also working on skeletal muscle on the 6th floor of the Molecular Biology Department of the Institut Pasteur at that time. I then present my subsequent research when I returned to Japan. I pay tribute to the influence of Professor Gros and to his support in establishing Japanese/French meetings on muscle biology and muscular dystrophy. I also invoke personal memories of Robert Whalen and Margaret Buckingham and remember the occasions when I returned to Paris to honour François Gros.

J'ai rejoint le laboratoire du professeur François Gros en 1987 et j'y ai travaillé en tant que postdoc avec Robert Whalen jusqu'en 1992. Je raconte les recherches que nous avons menées et je mentionne celles des autres scientifiques qui travaillaient également sur le muscle squelettique au 6 e étage du Département de biologie moléculaire de l'Institut Pasteur à cette époque. Je présente ensuite les recherches ultérieures que j'ai menées de retour au Japon. Je rends hommage à l'influence du professeur Gros et à son soutien lors de la mise en place de réunions franco-japonaises autour de la biologie musculaire et de la dystrophie musculaire. J'évoque également des souvenirs personnels de Robert Whalen et de Margaret Buckingham et je me rappelle les moments où je suis retourné à Paris pour rendre hommage à François Gros.

Antioxidants restore store-operated Ca2+ entry in patient-iPSC-derived myotubes with tubular aggregate myopathy-associated Ile484ArgfsX21 STIM1 mutation via upregulation of binding immunoglobulin protein.

Store-operated Ca2+ entry (SOCE) is indispensable for intracellular Ca2+ homeostasis in skeletal muscle, and constitutive activation of SOCE causes tubular aggregate myopathy (TAM). To understand the pathogenesis of TAM, we induced pluripotent stem cells (iPSCs) from a TAM patient with a rare mutation (c.1450_1451insGA; p. Ile484ArgfsX21) in the STIM1 gene. This frameshift mutation produces a truncated STIM1 with a disrupted C-terminal inhibitory domain (CTID) and was reported to diminish SOCE. Myotubes induced from the patient's-iPSCs (TAM myotubes) showed severely impaired SOCE, but antioxidants greatly restored SOCE partly via upregulation of an endoplasmic reticulum (ER) chaperone, BiP (GRP78), in the TAM myotubes. Our observation suggests that antioxidants are promising tools for treatment of TAM caused by reduced SOCE.

Exon 44 skipping in Duchenne muscular dystrophy: NS-089/NCNP-02, a dual-targeting antisense oligonucleotide.

Exon-skipping therapy mediated by antisense oligonucleotides is expected to provide a therapeutic option for Duchenne muscular dystrophy. Antisense oligonucleotides for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated antisense oligonucleotides for exon 44 skipping (applicable to approximately 6% of all Duchenne muscular dystrophy patients) to improve activity by using a novel antisense oligonucleotide design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to target two splicing regulators in exon 44 simultaneously, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a Duchenne muscular dystrophy patient to whom exon 44 skipping is applicable. We also assessed the in vivo activity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an antisense oligonucleotide with a novel connected-sequence design, showed highly efficient exon skipping both in vitro and in vivo.

Acute hypercapnic respiratory failure in patients with obesity hypoventilation syndrome during the COVID-19 pandemic: Four case reports.

Obesity hypoventilation syndrome (OHS) can cause acute hypercapnic respiratory failure (AHRF). The onset of AHRF in four patients with OHS during the coronavirus disease 2019 (COVID-19) pandemic is reported in this study. Two men (23 and 45 years old) and two women (both 77 years old) presented to our hospital with AHRF. In the two elderly women, a prolonged supine position due to falls seemed to be the cause of AHRF. Treatment was started with bilevel positive airway pressure for all patients. While one patient died, the condition of the other three improved; they were discharged with continuous positive airway pressure. AHRF due to OHS was rarely reported in the rural region of Japan. It is suggested that increased rates of obesity due to lifestyle changes during the COVID-19 pandemic may be responsible for an increase in the prevalence of OHS-associated AHRF.

Large Maf transcription factor family is a major regulator of fast type IIb myofiber determination.

Myofibers are broadly characterized as fatigue-resistant slow-twitch (type I) fibers and rapidly fatiguing fast-twitch (type IIa/IIx/IIb) fibers. However, the molecular regulation of myofiber type is not entirely understood; particularly, information on regulators of fast-twitch muscle is scarce. Here, we demonstrate that the large Maf transcription factor family dictates fast type IIb myofiber specification in mice. Remarkably, the ablation of three large Mafs leads to the drastic loss of type IIb myofibers, resulting in enhanced endurance capacity and the reduction of muscle force. Conversely, the overexpression of each large Maf in the type I soleus muscle induces type IIb myofibers. Mechanistically, a large Maf directly binds to the Maf recognition element on the promoter of myosin heavy chain 4, which encodes the type IIb myosin heavy chain, driving its expression. This work identifies the large Maf transcription factor family as a major regulator for fast type IIb muscle determination.

Full-length human dystrophin on human artificial chromosome compensates for mouse dystrophin deficiency in a Duchenne muscular dystrophy mouse model.

Dystrophin maintains membrane integrity as a sarcolemmal protein. Dystrophin mutations lead to Duchenne muscular dystrophy, an X-linked recessive disorder. Since dystrophin is one of the largest genes consisting of 79 exons in the human genome, delivering a full-length dystrophin using virus vectors is challenging for gene therapy. Human artificial chromosome is a vector that can load megabase-sized genome without any interference from the host chromosome. Chimeric mice carrying a 2.4-Mb human dystrophin gene-loaded human artificial chromosome (DYS-HAC) was previously generated, and dystrophin expression from DYS-HAC was confirmed in skeletal muscles. Here we investigated whether human dystrophin expression from DYS-HAC rescues the muscle phenotypes seen in dystrophin-deficient mice. Human dystrophin was normally expressed in the sarcolemma of skeletal muscle and heart at expected molecular weights, and it ameliorated histological and functional alterations in dystrophin-deficient mice. These results indicate that the 2.4-Mb gene is enough for dystrophin to be correctly transcribed and translated, improving muscular dystrophy. Therefore, this technique using HAC gives insight into developing new treatments and novel humanized Duchenne muscular dystrophy mouse models with human dystrophin gene mutations.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

[A Case of Pulmonary Pleomorphic Carcinoma in a Hemodialysis Patient Treated with Pembrolizumab].

The patient was a 67-year-old male undergoing maintenance hemodialysis due to chronic renal failure caused by diabetic nephropathy. A left upper lobe resection was carried out for non-small cell lung cancer of the left upper lobe. It was histologically confirmed as pleomorphic carcinoma pT3N0M0, Stage ⅡB. He suffered a relapse with multiple metastases occurring in both lungs 3 months following surgery. The PD-L1 tumor proportion score(TPS)was 90%, indicating a high level of expression; 200mg of pembrolizumab was administered every 3 weeks on non-dialysis days. Two courses of administration achieved a partial response. A total of 17 courses were administered until discontinuation due to drug-induced lung injury.

Dataset for surface and internal damage after impact on CFRP laminates.

Various foreign objects can collide with CFRP structures, such as CFRP aircraft. Once something impacts with CFRP laminates, both surface damage and internal damage can occur. Even if the external damage is such invisible as called barely visible impact damage, there are matrix cracks or delamination that are the main cause of compressive strength reduction, so it is difficult to find the relationship between external and internal damage on CFRP laminates. This dataset is prepared for predicting impact information only from surface damage profiles using Machine Learning (Hasebe et al., 2022). It includes three data, surface damage image (png), surface depth contour image(png), and internal damage image after ultrasound C-scanning (jpg) after low-velocity impact testing under various impact conditions. The data are helpful for researchers and engineers who deal with the impact behavior of CFRP or data science.

Hansen Solubility Parameter Analysis on Dispersion of Oleylamine-Capped Silver Nanoinks and their Sintered Film Morphology.

Optimizing stabilizers and solvents is crucial for obtaining highly dispersed nanoparticle inks. Generally, nonpolar (hydrophobic) ligand-stabilized nanoparticles show superior dispersibility in nonpolar solvents, whereas polar ligand (hydrophilic)-stabilized nanoparticles exhibit high dispersibility in polar solvents. However, these properties are too qualitative to select optimum stabilizers and solvents for stable nanoparticle inks, and researchers often rely on their experiences. This study presents a Hansen solubility parameter (HSP)-based analysis of the dispersibility of oleylamine-capped silver nanoparticle (OAm-Ag NP) inks for optimizing ink preparation. We determined the HSP sphere of the OAm-Ag NPs, defined as the center coordinate, and the interaction radius in 3D HSP space. The solvent's HSP inside the HSP sphere causes high dispersibility of the OAm-Ag NPs in the solvent. In contrast, the HSPs outside the sphere resulted in low dispersibility in the solvent. Thus, we can quantitatively predict the dispersibility of the OAm-Ag NPs in a given solvent using the HSP approach. Moreover, the HSP sphere method can establish a correlation between the dispersibility of the particles in inks and the sintered film morphology, facilitating electronic application of the nanoparticle inks. The HSP method is also helpful for optimizing stabilizers and solvents for stable nanoparticle inks in printed electronics.

Brain Dp140 alters glutamatergic transmission and social behaviour in the mdx52 mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a muscle disorder caused by DMD mutations and is characterized by neurobehavioural comorbidities due to dystrophin deficiency in the brain. The lack of Dp140, a dystrophin short isoform, is clinically associated with intellectual disability and autism spectrum disorders (ASDs), but its postnatal functional role is not well understood. To investigate synaptic function in the presence or absence of brain Dp140, we utilized two DMD mouse models, mdx23 and mdx52 mice, in which Dp140 is preserved or lacking, respectively. ASD-like behaviours were observed in pups and 8-week-old mdx52 mice lacking Dp140. Paired-pulse ratio of excitatory postsynaptic currents, glutamatergic vesicle number in basolateral amygdala neurons, and glutamatergic transmission in medial prefrontal cortex-basolateral amygdala projections were significantly reduced in mdx52 mice compared to those in wild-type and mdx23 mice. ASD-like behaviour and electrophysiological findings in mdx52 mice were ameliorated by restoration of Dp140 following intra-cerebroventricular injection of antisense oligonucleotide drug-induced exon 53 skipping or intra-basolateral amygdala administration of Dp140 mRNA-based drug. Our results implicate Dp140 in ASD-like behaviour via altered glutamatergic transmission in the basolateral amygdala of mdx52 mice.

Viral isolation analysis of SARS-CoV-2 from clinical specimens of COVID-19 patients.

Genetic testing using reverse transcriptase real-time polymerase chain reaction (rRT-PCR) is the mainstay of diagnosis of COVID-19. However, it has not been fully investigated whether infectious viruses are contained in SARS-CoV-2 genome-positive specimens examined using the rRT-PCR test. In this study, we examined the correlation between the threshold Cycle (Ct) value obtained from the rRT-PCR test and virus isolation in cultured cells, using 533 consecutive clinical specimens of COVID-19 patients. The virus was isolated from specimens with a Ct value of less than 30 cycles, and the lower the Ct value, the more efficient the isolation rate. A cytopathic effect due to herpes simplex virus type 1 contamination was observed in one sample with a Ct value of 35 cycles. In a comparison of VeroE6/TMPRSS2 cells and VeroE6 cells used for virus isolation, VeroE6/TMPRSS2 cells isolated the virus 1.7 times more efficiently than VeroE6 cells. There was no significant difference between the two cells in the mean Ct value of the detectable sample. In conclusion, Lower Ct values in the PCR test were associated with higher virus isolation rates, and VeroE6/TMPRSS2 cells were able to isolate viruses more efficiently than VeroE6 cells.

A new mouse model of Ehlers-Danlos syndrome generated using CRISPR/Cas9-mediated genomic editing.

Musculocontractural Ehlers-Danlos syndrome (mcEDS) is caused by generalized depletion of dermatan sulfate (DS) due to biallelic pathogenic variants in CHST14 encoding dermatan 4-O-sulfotransferase 1 (D4ST1) (mcEDS-CHST14). Here, we generated mouse models for mcEDS-CHST14 carrying homozygous mutations (1 bp deletion or 6 bp insertion/10 bp deletion) in Chst14 through CRISPR/Cas9 genome engineering to overcome perinatal lethality in conventional Chst14-deleted knockout mice. DS depletion was detected in the skeletal muscle of these genome-edited mutant mice, consistent with loss of D4ST1 activity. The mutant mice showed common pathophysiological features, regardless of the variant, including growth impairment and skin fragility. Notably, we identified myopathy-related phenotypes. Muscle histopathology showed variation in fiber size and spread of the muscle interstitium. Decorin localized diffusely in the spread endomysium and perimysium of skeletal muscle, unlike in wild-type mice. The mutant mice showed lower grip strength and decreased exercise capacity compared to wild type, and morphometric evaluation demonstrated thoracic kyphosis in mutant mice. The established CRISPR/Cas9-engineered Chst14 mutant mice could be a useful model to further our understanding of mcEDS pathophysiology and aid in the development of novel treatment strategies.

Mesenchymal stem cells derived from human induced pluripotent stem cells improve the engraftment of myogenic cells by secreting urokinase-type plasminogen activator receptor (uPAR).

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by mutations in the dystrophin gene. Transplantation of myogenic stem cells holds great promise for treating muscular dystrophies. However, poor engraftment of myogenic stem cells limits the therapeutic effects of cell therapy. Mesenchymal stem cells (MSCs) have been reported to secrete soluble factors necessary for skeletal muscle growth and regeneration. METHODS: We induced MSC-like cells (iMSCs) from induced pluripotent stem cells (iPSCs) and examined the effects of iMSCs on the proliferation and differentiation of human myogenic cells and on the engraftment of human myogenic cells in the tibialis anterior (TA) muscle of NSG-mdx4Cv mice, an immunodeficient dystrophin-deficient DMD model. We also examined the cytokines secreted by iMSCs and tested their effects on the engraftment of human myogenic cells. RESULTS: iMSCs promoted the proliferation and differentiation of human myogenic cells to the same extent as bone marrow-derived (BM)-MSCs in coculture experiments. In cell transplantation experiments, iMSCs significantly improved the engraftment of human myogenic cells injected into the TA muscle of NSG-mdx4Cv mice. Cytokine array analysis revealed that iMSCs produced insulin-like growth factor-binding protein 2 (IGFBP2), urokinase-type plasminogen activator receptor (uPAR), and brain-derived neurotrophic factor (BDNF) at higher levels than did BM-MSCs. We further found that uPAR stimulates the migration of human myogenic cells in vitro and promotes their engraftment into the TA muscles of immunodeficient NOD/Scid mice. CONCLUSIONS: Our results indicate that iMSCs are a new tool to improve the engraftment of myogenic progenitors in dystrophic muscle.

Myopathy Associated With Dermatan Sulfate-Deficient Decorin and Myostatin in Musculocontractural Ehlers-Danlos Syndrome: A Mouse Model Investigation.

Carbohydrate sulfotransferase 14 (CHST14) encodes dermatan 4-O-sulfotransferase 1, a critical enzyme for dermatan sulfate (DS) biosynthesis. Musculocontractural Ehlers-Danlos syndrome (mcEDS) is associated with biallelic pathogenic variants of CHST14 and is characterized by malformations and manifestations related to progressive connective tissue fragility. We identified myopathy phenotypes in Chst14-deficient mice using an mcEDS model. Decorin is a proteoglycan harboring a single glycosaminoglycan chain containing mainly DS, which are replaced with chondroitin sulfate (CS) in mcEDS patients with CHST14 deficiency. We studied the function of decorin in the skeletal muscle of Chst14-deficient mice because decorin is important for collagen-fibril assembly and has a myokine role in promoting muscle growth. Although decorin was present in the muscle perimysium of wild-type (Chst14+/+ ) mice, decorin was distributed in the muscle perimysium as well as in the endomysium of Chst14-/- mice. Chst14-/- mice had small muscle fibers within the spread interstitium; however, histopathological findings indicated milder myopathy in Chst14-/- mice. Myostatin, a negative regulator of protein synthesis in the muscle, was upregulated in Chst14-/- mice. In the muscle of Chst14-/- mice, decorin was downregulated compared to that in Chst14+/+ mice. Chst14-/- mice showed altered cytokine/chemokine balance and increased fibrosis, suggesting low myogenic activity in DS-deficient muscle. Therefore, DS deficiency in mcEDS causes pathological localization and functional abnormalities of decorin, which causes disturbances in skeletal muscle myogenesis.

Increasing numbers of CD19 + CD24highCD38high regulatory B cells and pre-germinal center B cells reflect activated autoimmunity and predict future treatment response in patients with untreated immune thrombocytopenia.

The pathophysiology of immune thrombocytopenia (ITP) is poorly understood, particularly aspects regarding abnormal homeostasis and dysregulation of B cells. In this study, we analyzed peripheral lymphocyte subsets in patients with untreated ITP and healthy controls, and examined correlations between cell percentages/counts and titers of serum cytokines and antibodies. We also compared ITP patients who later required second-line therapies and those who did not. The percentages of CD19 + CD24highCD38high regulatory B cells, pre-germinal center (GC) B cells, and plasmablast-like B cells were significantly higher in ITP patients than in healthy controls. Absolute counts of regulatory B cells and pre-GC B cells were significantly higher in those who needed second-line therapies. In addition, serum B cell-activating factor belonging to the tumor necrosis factor family (BAFF) levels and platelet-associated immune globulin G antibody titers correlated positively with regulatory B cell, pre-GC B cell, and auto-reactive B cell counts. Serum interferon-α (IFN-α) levels were elevated in four ITP patients with high auto-reactive B cell counts. These results indicate that increases in regulatory B cells and pre-GC B cells may reflect activated autoimmunity induced by BAFF and/or IFN-α. Consequently, evaluation of B cell subsets in untreated ITP patients may predict treatment response.