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CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20–33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.

The LAMA5 gene encodes laminin α5, an indispensable component of glomerular basement membrane and other types of basement membrane. A homozygous pathological variant in LAMA5 is known to cause a systemic developmental syndrome including glomerulopathy. However, the roles of heterozygous LAMA5 gene variants in human renal and systemic diseases have remained unclear. We performed whole-exome sequencing analyses of a family with slowly progressive nephropathy associated with hereditary focal segmental glomerulosclerosis, and we identified what we believe to be a novel probable pathogenic variant of LAMA5, NP_005551.3:p.Val3687Met. In vitro analyses revealed cell type-dependent changes in secretion of variant laminin α5 laminin globular 4-5 (LG4-5) domain. Heterozygous and homozygous knockin mice with a corresponding variant of human LAMA5, p.Val3687Met, developed focal segmental glomerulosclerosis-like pathology with reduced laminin α5 and increased glomerular vinculin levels, which suggested that impaired cell adhesion may underlie this glomerulopathy. We also identified pulmonary defects such as bronchial deformity and alveolar dilation. Reexaminations of the family revealed phenotypes compatible with reduced laminin α5 and increased vinculin levels in affected tissues. Thus, the heterozygous p.Val3687Met variant may cause a new syndromic nephropathy with focal segmental glomerulosclerosis through possibly defective secretion of laminin α5. Enhanced vinculin may be a useful disease marker.

Circulating lipopolysaccharide (LPS) concentrations are often elevated in patients with sepsis or various endogenous diseases related to bacterial translocation from the gut. Systemic inflammatory responses induced by endotoxemia induce severe involuntary loss of skeletal muscle, termed muscle wasting, which adversely affects the survival and functional outcomes of these patients. Currently, no drugs are available for the treatment of endotoxemia-induced skeletal muscle wasting. Here, we tested the effects of TAK-242, a Toll-like receptor 4 (TLR4)-specific signalling inhibitor, on myotube atrophy in vitro and muscle wasting in vivo induced by endotoxin. LPS treatment of murine C2C12 myotubes induced an inflammatory response (increased nuclear factor-κB activity and interleukin-6 and tumour necrosis factor-α expression) and activated the ubiquitin–proteasome and autophagy proteolytic pathways (increased atrogin-1/MAFbx, MuRF1, and LC-II expression), resulting in myotube atrophy. In mice, LPS injection increased the same inflammatory and proteolytic pathways in skeletal muscle and induced atrophy, resulting in reduced grip strength. Notably, pretreatment of cells or mice with TAK-242 reduced or reversed all the detrimental effects of LPS in vitro and in vivo . Collectively, our results indicate that pharmacological inhibition of TLR4 signalling may be a novel therapeutic intervention for endotoxemia-induced muscle wasting.

Burn injury is the leading cause of death and disability worldwide and places a tremendous economic burden on society. Systemic inflammatory responses induced by thermal burn injury can cause muscle wasting, a severe involuntary loss of skeletal muscle that adversely affects the survival and functional outcomes of these patients. Currently, no pharmacological interventions are available for the treatment of thermal burn-induced skeletal muscle wasting. Elevated levels of inflammatory cytokines, such as interleukin-6 (IL-6), are important hallmarks of severe burn injury. The levels of signal transducer and activator of transcription 3 (STAT3)—a downstream component of IL-6 inflammatory signaling—are elevated with muscle wasting in various pro-catabolic conditions, and STAT3 has been implicated in the regulation of skeletal muscle atrophy. Here, we tested the effects of the STAT3-specific signaling inhibitor C188-9 on thermal burn injury-induced skeletal muscle wasting in vivo and on C2C12 myotube atrophy in vitro after the administration of plasma from burn model mice. In mice, thermal burn injury severity dependently increased IL-6 in the plasma and tibialis anterior muscles and activated the STAT3 (increased ratio of phospho-STAT3/STAT3) and ubiquitin-proteasome proteolytic pathways (increased Atrogin-1/MAFbx and MuRF1). These effects resulted in skeletal muscle atrophy and reduced grip strength. In murine C2C12 myotubes, plasma from burn mice activated the same inflammatory and proteolytic pathways, leading to myotube atrophy. In mice with burn injury, the intraperitoneal injection of C188-9 (50 mg/kg) reduced activation of the STAT3 and ubiquitin-proteasome proteolytic pathways, reversed skeletal muscle atrophy, and increased grip strength. Similarly, pretreatment of murine C2C12 myotubes with C188-9 (10 µM) reduced activation of the same inflammatory and proteolytic pathways, and ameliorated myotube atrophy induced by plasma taken from burn model mice. Collectively, these results indicate that pharmacological inhibition of STAT3 signaling may be a novel therapeutic strategy for thermal burn-induced skeletal muscle wasting.

Road traffic accidents are a global health concern. K-car vehicles, also known as \"mini vehicles,\" are defined as those having an engine displacement <660 cc, vehicle length <3.4 m, width <1.48 m, and height <2.0 m. K-cars have become increasing popular not only in Japan but also in other countries. Compared with standard vehicles, the occupant space of a K-car vehicle is considerably smaller; thus, passengers may be more vulnerable to the external forces generated in a collision, which in turn can lead to deformation of the occupant space and less protection against injuries. However, data are scarce regarding whether K-car vehicles are related to poorer survival outcomes and severe trauma. We conducted a retrospective cohort study involving patients who were injured in four-wheeled vehicle accidents between 2002 and 2023 and admitted to a community teaching hospital in Japan. The vehicle configuration was divided into standard and K-car vehicles. The primary endpoint was in-hospital mortality. Other outcomes included severe trauma, defined as Injury Severity Score (ISS) >15, and anatomically specific severe injury of the head and neck, chest, abdomen, pelvis, and extremities, defined as Abbreviated Injury Scale score ≥3. Of 5331 eligible patients, 2384 (44.7%) were K-car vehicle occupants. In propensity score-matched analysis with 1947 pairs, we observed an increase for in-hospital mortality in the K-car vehicle group (2.6% vs. 4.0%, odds ratio 1.53, 95% confidence interval 1.07–2.19). Compared with standard vehicles, K-car vehicles were associated with a greater risk of severe trauma and serious injuries of the head and neck, chest, abdomen, pelvis, and extremities. These study data should be used to encourage vehicle occupants and automobile manufacturers to consider objective facts regarding the safety of vehicles in a traffic accident.

Muscle mass is an important factor for surviving an illness. Ultrasound has gained increased attention as a muscle mass assessment method because of its noninvasiveness and portability. However, data on the frequency of ultrasound-based muscle mass assessment are limited, and there are some barriers to its implementation. Hence, a web-based cross-sectional survey was conducted on healthcare providers in Japan, which comprised four parts: 1) participant characteristics; 2) general muscle mass assessment; 3) ultrasound-based muscle mass assessment; and 4) the necessity of, interest in, and barriers to its implementation. Necessity and interest were assessed using an 11-point Likert scale, whereas barriers were assessed using a 5-point Likert scale, in which “Strongly agree” and “Agree” were counted for the analysis. Of the 1,058 responders, 1,026 participants, comprising 282 physicians, 489 physical therapists, 84 occupational therapists, 120 nurses, and 51 dieticians, were included in the analysis. In total, 93% of the participants were familiar with general muscle mass assessment, and 64% had conducted it. Ultrasound-based muscle mass assessment was performed by 21% of the participants. Necessity and interest scored 7 (6–8) and 8 (7–10), respectively for ultrasound-based muscle mass assessment. The barriers to its implementation included lack of relevant education (84%), limited staff (61%), and absence of fixed protocol (61%). Regardless of the necessity of and interest in ultrasound-based muscle mass assessment, it was only conducted by one-fifth of the healthcare providers, and the most important barrier to its implementation was lack of education.

Muscle mass is an important factor for surviving an illness. Ultrasound has gained increased attention as a muscle mass assessment method because of its noninvasiveness and portability. However, data on the frequency of ultrasound-based muscle mass assessment are limited, and there are some barriers to its implementation. Hence, a web-based cross-sectional survey was conducted on healthcare providers in Japan, which comprised four parts: 1) participant characteristics; 2) general muscle mass assessment; 3) ultrasound-based muscle mass assessment; and 4) the necessity of, interest in, and barriers to its implementation. Necessity and interest were assessed using an 11-point Likert scale, whereas barriers were assessed using a 5-point Likert scale, in which “Strongly agree” and “Agree” were counted for the analysis. Of the 1,058 responders, 1,026 participants, comprising 282 physicians, 489 physical therapists, 84 occupational therapists, 120 nurses, and 51 dieticians, were included in the analysis. In total, 93% of the participants were familiar with general muscle mass assessment, and 64% had conducted it. Ultrasound-based muscle mass assessment was performed by 21% of the participants. Necessity and interest scored 7 (6–8) and 8 (7–10), respectively for ultrasound-based muscle mass assessment. The barriers to its implementation included lack of relevant education (84%), limited staff (61%), and absence of fixed protocol (61%). Regardless of the necessity of and interest in ultrasound-based muscle mass assessment, it was only conducted by one-fifth of the healthcare providers, and the most important barrier to its implementation was lack of education.

The aim of this study was to clarify whether serum matrix metalloproteinase 3 (MMP-3) levels are associated with an effect of iguratimod as add-on therapy to biological DMARDs (bDMARDs) in patients with rheumatoid arthritis (RA). Forty three patients with RA were treated with iguratimod as add-on therapy to bDMARDs. They were classified into remission and non-remission groups at 24 weeks of iguratimod therapy. Remission was defined as a state with a disease activity score (DAS) <2.6 in 28 joints (termed DAS remission) and total power Doppler ultrasound (US) score <3 (termed US remission). The serum MMP-3 levels at baseline and at 12 weeks were compared between these two groups. There were no significant differences in the serum MMP-3 levels at baseline between the DAS and US remission groups and the non-remission group. The serum MMP-3 levels at 12 weeks in the US remission group were significantly lower than those in the non-remission group. The ratios of the serum MMP-3 levels at baseline to those at 12 weeks in both the DAS and US remission groups were significantly lower than those in the non-remission group. An MMP-3 ratio <0.86 was determined as the cut-off value to predict US remission at 24 weeks. Our findings suggest that the ratios of the serum MMP-3 levels at baseline to those at 12 weeks could be used to predict remission in RA patients who are administered iguratimod as an add-on to bDMARDs.

BackgroundThe clinical severity of very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is difficult to predict using conventional diagnostic methods.MethodsPeripheral blood mononuclear cells obtained from 14 VLCAD deficiency patients and 23 healthy adults were loaded with carbon-13-universally labeled (U-13C-) fatty acids. Differences in acylcarnitine ratios between the patients and healthy groups and correlations between acylcarnitine ratios and a newly established clinical severity score (CSS) in the patient group were statistically examined.ResultsThere was a significant decrease in the 13C-C2/13C-C18 and 13C-C12/13C-C14 ratios in the U-13C-stearic acid loading test and in the 13C-C2/13C-C18:1 and 13C-C12:1/13C-C14:1 ratios in the U-13C-oleic acid loading test in the patient group. The values of each ratio were significantly correlated with the CSS, suggesting that they could predict disease severity. Additionally, patients with a higher 13C-C16/13C-C18 ratio than the 13C-C14/13C-C18 ratio in the U-13C-stearic acid loading test had a significantly higher CSS and were presumed to have more severe disease.ConclusionsOur data indicated that this method could be used to predict the clinical severity of VLCAD deficiency, and identify patients at a risk of severe disease.ImpactWe established a novel method to predict the severity of VLCAD deficiency by performing a loading test with carbon-13-labeled fatty acids on peripheral blood mononuclear cells.The U-13C-oleic acid loading test was useful for comparing the patient group with the control group in terms of disease severity.The U-13C-stearic acid loading test was useful for identifying the more severely affected patients.These methods are relatively less invasive and enable rapid evaluation of the clinical severity.

Sepsis-induced muscle atrophy leads to prolonged physical dysfunction. Although the interaction of muscle atrophy and macrophage has been reported in sepsis, the role of neutrophils in muscle atrophy has not been thoroughly investigated. This study sought to investigate the long-term changes in muscle-localized neutrophils after sepsis induction and their possible role in sepsis. Sepsis was induced in seven-week-old male C57BL/6J mice 8-12 (cecal slurry [CS] model) intraperitoneal injection of 1 mg/g cecal slurry. The percentage change in body weight and grip strength was evaluated. The tibialis anterior muscles were dissected for microscopic examination of the cross-sectional area of myofibers or Fluorescence-activated cell sorting (FACS) analysis of immune cells. These changes were evaluated in the following conditions: (1) Longitudinal change until day 61, (2) CS concentration-dependent change on day 14 at the low (0.3 mg/g), middle (1.0 mg/g), and high (2.0 mg/g) concentrations, and (3) CS mice on day 14 treated with an anti-Ly6G antibody that depletes neutrophils. Body weight and grip strength were significantly lower in the CS model until day 61 (body weight: 123.1% ± 1.8% vs. 130.3% ± 2.5%, p = 0.04; grip strength: 104.5% ± 3.8% vs. 119.3% ± 5.3%, p = 0.04). Likewise, cross-sectional muscle area gradually decreased until day 61 from the CS induction (895.6 [606.0-1304.9] μm vs. 718.8 [536.2-937.0] μm , p < 0.01). The number of muscle-localized neutrophils increased from 2.3 ± 0.6 cell/mg on day 0 to 22.2 ± 13.0 cell/mg on day 14, and decreased thereafter. In terms of CS concentration-dependent change, cross-sectional area was smaller (484.4 ± 221.2 vs. 825.8 ± 436.2 μm [p < 0.001]) and grip strength was lower (71.4% ± 12.8% vs. 116.3% ± 7.4%, p = 0.01) in the CS High group compared with the control, with increased neutrophils (p = 0.03). Ly6G-depleted mice demonstrated significant increase of muscle cross-sectional area and grip strength compared with control mice (p < 0.01). Sepsis causes infiltration of neutrophils in muscles, leading to muscle atrophy and weakness. Depletion of neutrophils in muscle reverses sepsis-induced muscle atrophy and weakness. These results suggest that neutrophils may play a critical role in sepsis-induced muscle atrophy and weakness.