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Duchenne muscular dystrophy (DMD) is an intractable X-linked muscular dystrophy caused by mutations in the DMD gene. While many animal models have been used to study the disease, translating findings to humans has been challenging. Microminipigs, with their pronounced physiological similarity to humans and notably compact size amongst pig models, could offer a more representative model for human diseases. Here, we accomplished precise DMD modification in microminipigs by co-injecting embryos with Cas9 protein and a single-guide RNA targeting exon 23 of DMD . The DMD -edited microminipigs exhibited pronounced clinical phenotypes, including perturbed locomotion and body-wide skeletal muscle weakness and atrophy, alongside augmented serum creatine kinase levels. Muscle weakness was observed as of one month of age, respiratory and cardiac dysfunctions emerged by the sixth month, and the maximum lifespan was 29.9 months. Histopathological evaluations confirmed dystrophin deficiency and pronounced dystrophic pathology in the skeletal and myocardial tissues, demonstrating that these animals are an unprecedented model for studying human DMD. The model stands as a distinct and crucial tool in biomedical research, offering deep understanding of disease progression and enhancing therapeutic assessments, with potential to influence forthcoming treatment approaches. The authors present DMD -edited microminipigs showing severe Duchenne muscular dystrophy symptoms; their human-like physiology and small size make them ideal for developing new treatments.

Background/Aim: The reproducibility of athero – sclerotic lesions was evaluated after the production of cloned-microminipigs and their offspring. Materials and Methods: Cloned-microminipig-parents were produced by microminipigsomatic cell nuclei. These parents were crossbred and delivered males (F1-offspring) were divided into two groups: normal chow diet (NcD)-fed and high-fat/high-cholesterol diet (HcD)-fed groups. One of the F1-offsprings was subjected to cloning, and delivered males (F1-clones) were fed with HcD. After 8 weeks, all animals were necropsied for patho – physiological studies compared to non-cloned-microminipigs. Results: HcD-fed F1-offspring and F1-clones, but not NcD-fed F1-offspring, exhibited increased serum lipid levels and systemic atherosclerosis, which were comparable to those of HcD-fed non-cloned-microminipigs. Homogeneity of variance analysis demonstrated that standard deviation values of serum lipoprotein and aortic atherosclerosis area from HcD-fed animals decreased in F1-offspring and F1-clones. Conclusion: HcD-induced atherogenesis was highly reproducible in F1-offsprings and F1-clones, indicating that the atherosclerosis-prone genomic background was preserved in the cloned-microminipigs, which can be used for studies on human atherosclerosis and related diseases.

This study investigated the possibility of the presence of specific receptor for relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) in boar testes. While RLF/INSL3 was produced by Leydig cells in the boar testis, its own receptor RXFP2 was expressed mainly in meiotic and post-meiotic germ cells, but not in Leydig cells, suggesting the existence of RLF/INSL3-RXFP2 signaling in germ cells of boars.

Background and ObjectivesThe effects of seven local anesthetics, mepivacaine, bupivacaine, etidocaine, dibucaine, prilocaine, procaine, and tetracaine, on the central nervous system (CNS) electrical activities were studied in five cats with each agent.MethodsBrain electrodes were implanted chronically in the cortex, amygdala, hippocampus, and midbrain reticular formation. The cortical, amygdala, and hippocampus electroencephalograms (EEGs), reticular multiunit activity (R-MUA), and heart rate were recorded. Drugs were administered intravenously with constant rates of equipotent doses until EEG seizures appeared. Effects of lidocaine, 1 mg·kg−1·min−1, were studied for comparison. The rates of infusion were: mepivacaine, 1 mg·kg−1·min−1; procaine, 4 mg·kg−1·min−1; bupivacaine and tetracaine, 0.25 mg.kg−1·min−1; etidocaine, 0.5 mg·kg−1·min−1; dibucaine, 0.15 mg·kg−1·min−1; and prilocaine, 3 mg·kg−1·min−1. The effects of 10-15 times higher rates of infusion were also studied in two cats with each drug.ResultsDuring the slow rates of infusion, a tetraphasic sequence of changes, common to all agents, was observed: the initial stage represented by diffuse EEG slowing and a suppression of the R-MUA; the second stage by low voltage fast wave EEG and an activation of the R-MUA; the third stage by reappearance of slow wave EEG and a suppression of the R-MUA; and the fourth stage by an epileptiform EEG and an activation of the R-MUA. These changes in EEG were not typical in some cats administered procaine, bupivacaine, prilocaine, and dibucaine. During the high rates of infusion, suppressive stages in the R-MUA were less dominant and activations dominated. High rates of infusion of mepivacaine produced sudden epileptic activities in EEG. Tetracaine, procaine, etidocaine, and bupivacaine in the high rates of infusion produced slow waves for a short period, prior to seizure activities in the EEG. Dibucaine or prilocaine in high rates of infusion could not produce seizure activities in the EEG, but slow waves and isoelectrical EEG, which were accompanied with idioventricular rhythm followed by ventricular fibrillation.ConclusionsAll local anesthetics have similar tetraphasic CNS actions and whether the expression of CNS intoxication with subconvulsive doses is excitation or suppression may possibly be dependent on the brain drug level and its rate of increase.

The mechanistic target of rapamycin complex 1 (mTORC1) is an essential regulator of cell growth and metabolism through the modulation of protein and lipid synthesis, lysosome biogenesis, and autophagy. The activity of mTORC1 is dynamically regulated by several environmental cues, including amino acid availability, growth factors, energy levels, and stresses, to coordinate cellular status with environmental conditions. Dysregulation of mTORC1 activity is closely associated with various diseases, including diabetes, cancer, and neurodegenerative disorders. The discovery of Rag GTPases has greatly expanded our understanding of the regulation of mTORC1 activity by amino acids, especially leucine and arginine. In addition to Rag GTPases, other factors that also contribute to the modulation of mTORC1 activity have been identified. In this review, we discuss the mechanisms of regulation of mTORC1 activity by particular amino acids.

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP- familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP- FD exon 20 has a suboptimal 5′ splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases. Familial dysautonomia is caused by splicing mutation of IKBKAP gene, which induces skipping of exon 20 and subsequent functional loss. Here, the authors report that a synthetic splice modulator RECTAS ameliorates pathogenic exon 20 skipping and shows therapeutic effects in cellular and animal models.

Background Many studies have compared quality of life of post-intensive care syndrome (PICS) patients with age-matched population-based controls. Many studies on PICS used the 36-item Short Form (SF-36) health survey questionnaire version 2, but lack the data for SF-36 values before and after intensive care unit (ICU) admission. Thus, clinically important changes in the parameters of SF-36 are unknown. Therefore, we determined the frequency of co-occurrence of PICS impairments at 6 months after ICU admission. We also evaluated the changes in SF-36 subscales and interpreted the patients’ subjective significance of impairment. Methods A prospective, multicenter, observational cohort study was conducted in 16 ICUs across 14 hospitals in Japan. Adult ICU patients expected to receive mechanical ventilation for > 48 h were enrolled, and their 6-month outcome was assessed using the questionnaires. PICS definition was based on the physical status, indicated by the change in SF-36 physical component score (PCS) ≥ 10 points; mental status, indicated by the change in SF-36 mental component score (MCS) ≥ 10 points; and cognitive function, indicated by the worsening of Short-Memory Questionnaire (SMQ) score and SMQ score at 6 months < 40. Multivariate logistic regression model was used to identify the factors associated with PICS occurrence. The patients’ subjective significance of physical and mental symptoms was assessed using the 7-scale Global Assessment Rating to evaluate minimal clinically important difference (MCID). Results Among 192 patients, 48 (25%) died at 6 months. Among the survivors at 6 months, 96 patients responded to the questionnaire; ≥ 1 PICS impairment occurred in 61 (63.5%) patients, and ≥ 2 occurred in 17 (17.8%) patients. Physical, mental, and cognitive impairments occurred in 32.3%, 14.6% and 37.5% patients, respectively. Population with only mandatory education was associated with PICS occurrence (odds ratio: 4.0, 95% CI 1.1–18.8, P  = 0.029). The MCID of PCS and MCS scores was 6.5 and 8.0, respectively. Conclusions Among the survivors who received mechanical ventilation, 64% had PICS at 6 months; co-occurrence of PICS impairments occurred in 20%. PICS was associated with population with only mandatory education. Future studies elucidating the MCID of SF-36 scores among ICU patients and standardizing the PICS definition are required. Trial registration UMIN000034072.

Transcranial static magnetic stimulation (tSMS) has been focused as a new non-invasive brain stimulation, which can suppress the human cortical excitability just below the magnet. However, the non-regional effects of tSMS via brain network have been rarely studied so far. We investigated whether tSMS over the left primary motor cortex (M1) can facilitate the right M1 in healthy subjects, based on the hypothesis that the functional suppression of M1 can cause the paradoxical functional facilitation of the contralateral M1 via the reduction of interhemispheric inhibition (IHI) between the bilateral M1. This study was double-blind crossover trial. We measured the corticospinal excitability in both M1 and IHI from the left to right M1 by recording motor evoked potentials from first dorsal interosseous muscles using single-pulse and paired-pulse transcranial magnetic stimulation before and after the tSMS intervention for 30 min. We found that the corticospinal excitability of the left M1 decreased, while that of the right M1 increased after tSMS. Moreover, the evaluation of IHI revealed the reduced inhibition from the left to the right M1. Our findings provide new insights on the mechanistic understanding of neuromodulatory effects of tSMS in human.