Search

Abstract Background Human mesenchymal stem cells (MSCs) have been studied and applied extensively because of their ability to self-renew and differentiate into various cell types. Since most human diseases models are murine, mouse MSCs should have been studied in detail. The mdx mouse – a Duchenne muscular dystrophy model – was produced by introducing a point mutation in the dystrophin gene. To understand the role of dystrophin in MSCs, we compared MSCs from mdx and C57BL/10 mice, focusing particularly on the aspects of light and electron microscopic morphology, immunophenotyping, and differentiation potential. Results Our study showed that at passage 10, mdx-MSCs exhibited increased heterochromatin, larger vacuoles, and more lysosomes under electron microscopy compared to C57BL/10-MSCs. C57BL/10-MSCs formed a few myotubes, while mdx-MSCs did not at the same passages. By passage 21, mdx-MSCs but not C57BL/10-MSCs had gradually lost their proliferative ability. In addition, a significant difference in the expression of CD34, not Sca-1 and CD11b, was observed between the MSCs from the 2 mice. Conclusion Our current study reveals that the MSCs from the 2 mice, namely, C57BL/10 and mdx, exhibit differences in proliferative and myogenic abilities. The results suggest that the changes in mouse MSC behavior may be influenced by lack of dystrophin protein in mdx mouse.

Abstract Background Schwann cells (SC) which are myelin-forming cells in peripheral nervous system are very useful for the treatment of diseases of peripheral nervous system and central nervous system. However, it is difficult to obtain sufficient large number of SC for clinical use, so alternative cell systems are desired. Results Using a procedure similar to the one used for propagation of neural stem cells, we could induce rat adipose-derived stem cells (ADSC) into floating neurospheres. In addition to being able to differentiate into neuronal- and glial-like cells, neurospheres could be induced to differentiate into SC-like cells. SC-like cells were bi- or tri-polar in shape and immunopositive for nestin and SC markers p75, GFAP and S-100, identical to genuine SC. We also found that SC-like cells could induce the differentiation of SH-SY5Y neuroblastoma cells efficiently, perhaps through secretion of soluble substances. We showed further that SC-like cells could form myelin structures with PC12 cell neurites in vitro. Conclusion These findings indicated that ADSC could differentiate into SC-like cells in terms of morphology, phenotype and functional capacities. SC-like cells induced from ADSC may be useful for the treatment of neurological diseases.

Abstract Background Duchenne musclar dystrophy (DMD) is an X-linked recessive disease caused by mutations of dystrophin gene, there is no effective treatment for this disorder at present. Plasmid-mediated gene therapy is a promising therapeutical approach for the treatment of DMD. One of the major issues with plasmid-mediated gene therapy for DMD is poor transfection efficiency and distribution. The herpes simplex virus protein VP22 has the capacity to spread from a primary transduced cell to surrounding cells and improve the outcome of gene transfer. To improve the efficiency of plasmid-mediated gene therapy and investigate the utility of the intercellular trafficking properties of VP22-linked protein for the treatment for DMD, expression vectors for C-terminal versions of VP22-microdystrophin fusion protein was constructed and the VP22-mediated shuttle effect was evaluated both in vitro and in vivo. Results Our results clearly demonstrate that the VP22-microdystrophin fusion protein could transport into C2C12 cells from 3T3 cells, moreover, the VP22-microdystrophin fusion protein enhanced greatly the amount of microdystrophin that accumulated following microdystrophin gene transfer in both transfected 3T3 cells and in the muscles of dystrophin-deficient (mdx) mice. Conclusion These results highlight the efficiency of the VP22-mediated intercellular protein delivery for potential therapy of DMD and suggested that protein transduction may be a potential and versatile tool to enhance the effects of gene delivery for somatic gene therapy of DMD.

Objectives: The SOX5 gene has been identified as the pathogenic gene responsible for Lamb-Shaffer syndrome. In this study, we examined the SOX5 variant (c.221C > T, p.Thr74Met) within a Chinese family presenting with intellectual disability and evaluated the functional implications of SOX5 by in vitro experiment. Materials and methods: The family underwent a clinical assessment of intellectual development, which included precise clinical exome sequencing to identify causative genetic variants. The potential deleterious effects and pathogenicity of the variant were predicted using bioinformatics tools such as Mutation Taster, PROVEAN, and SIFT. Additionally, protein stability was evaluated using I-Mutant, and 3D protein structures were modeled with I-TASSER. Western blots and QPCR were employed to assess gene expression and protein stability. Flow cytometry was utilized to compare the cell cycle dynamics between wild-type and mutant cells. Results: A previously identified missense variant (c.221C > T) in the SOX5 gene was determined to be the underlying cause of intellectual disability in a Chinese family. Functional assays demonstrated that mutant cells exhibited increased levels of SOX5 mRNA and protein relative to wild-type cells, accompanied by enhanced protein stability. Additionally, the mutant SOX5 protein was found to alter the cell cycle and downregulate the mRNA expression levels of the ACAN, AXIN2, SOX9, and PDGFRA genes. Conclusions: We confirmed that the SOX5 p.Thr74Met variant is associated with intellectual disability in a second-generation Chinese family. This mutant protein potentially exhibits increased stability, influences the cell cycle, and downregulates genes related to bone and neural functions. [ABSTRACT FROM AUTHOR]

Background & Aims: The effect of transarterial chemoembolization (TACE) plus radiofrequency ablation (RFA) (TACE-RFA) for hepatocellular carcinoma (HCC) in high-risk locations is not satisfactory. The aim of this study was to compare the clinical outcomes of TACE-RFA plus iodine-125 (125I) seed implantation (TACE-RFA-125I) therapy with those of TACE-RFA for unresectable HCC (≤ 5 cm) in high-risk locations. Methods: From January 2010 to June 2023, the clinical data of 126 patients with unresectable HCC (≤ 5 cm) in high-risk locations who received TACE-RFA-125I or TACE-RFA treatment were retrospectively analyzed. The clinical outcomes between the two groups were compared after propensity score matching (PSM) analysis. Results: Forty-six pairs of patients were matched. The local progression-free survival rates at 1-, 2-, 3-, 4-, and 5-years were 100%, 82.4%, 74.8%, 63.5%, and 54% in the TACE-RFA-125I group, which were significantly higher than 91.3%, 69.4%, 50.7%, 29.4%, and 26.7% in the TACE-RFA group, respectively (p = 0.004). The median progression-free survival in the TACE-RFA-125I group was significantly longer than that in the TACE-RFA group (p = 0.002). The overall survival rates at 1-, 2-, 3-, 4-, and 5-years were 100%, 93.4%, 80.7%, 74.9%, and 64.7% in the TACE-RFA-125I group, which were significantly higher than 97.8%, 78%, 68.6%, 51.1%, and 45.3% in the TACE-RFA group, respectively (p = 0.011). There was no occurrence of major complications or procedure-related deaths in the two groups. Conclusion: Compared with the TACE-RFA treatment, TACE-RFA-125I should be a more effective treatment strategy for patients with unresectable HCC (≤ 5 cm) in high-risk locations. [ABSTRACT FROM AUTHOR]

Primary hyperoxaluria type 2 (PH2) is a rare disorder caused by GRHPR mutations. Research on the mutation spectrum and pedigree of PH2 helps in comprehending its pathogenesis and clinical outcomes, guiding clinical diagnosis and treatment. We report a case of PH2 with a three-generational pedigree. The GRHPR genotypes of the family members were confirmed by Sanger sequencing. Urine and blood samples were collected for biochemical analysis. Computational analysis was performed to assess the pathogenicity of the mutations. Cellular experiments based on site-directed mutagenesis were conducted to confirm the effect of mutations on GRHPR expression, activity, and subcellular localization. The proband underwent her first kidney transplantation in 2015, and experienced recurrent urinary tract infections and urolithiasis postoperatively. Graft failure occurred in 2018. Whole exome sequencing identified compound heterozygous GRHPR mutations p.G160E/p.P203Rfs*7. The patient underwent a second kidney transplantation in 2019 and maintained good graft function with urine dilution measures. Notably, her brother and sister carried the same mutations; however, only the proband progressed to renal failure. Computational analysis suggested that p.G160E reduced the affinity of GRHPR for coenzymes. Cellular experiments indicated that p.G160E reduced GRHPR activity (p < 0.001), whereas p.P203Rfs*7 not only suppressed expression (p < 0.001) and reduced activity (p < 0.001), but also facilitated protein aggregation. Based on our results, the variant p.G160E was classified as ‘pathogenic’ according to ACMG guidelines. Our findings suggest that treatment strategies for the long-term prevention of oxalate nephropathy should be developed for patients with PH2 receiving isolated kidney transplantation. Moreover, the pathogenicity of the compound heterozygous GRHPR mutations p.G160E/p.P203Rfs*7 was also validated. [ABSTRACT FROM AUTHOR]

Fabry disease, a lysosomal storage disease, is an uncommon X-linked recessive genetic disorder stemming from abnormalities in the alpha-galactosidase gene (GLA) that codes human alpha-Galactosidase A (α-Gal A). To date, over 800 GLA mutations have been found to cause Fabry disease (FD). Continued enhancement of the GLA mutation spectrum will contribute to a deeper recognition and underlying mechanisms of FD. In this study, a 27-year-old male proband exhibited a typical phenotype of Fabry disease. Subsequently, family screening for Fabry disease was conducted, and high-throughput sequencing was employed to identify the mutated gene. The three-level structure of the mutated protein was analyzed, and its subcellular localization and enzymatic activity were determined. Apoptosis was assessed in GLA mutant cell lines to confirm the functional effects. As a result, a new mutation, c.777_778del (p. Gly261Leufs*3), in the GLA gene was identified. The mutation caused a frameshift during translation and the premature appearance of a termination codon, which led to a partial deletion of the domain in C-terminal region and altered the protein’s tertiary structure. In vitro experiments revealed a significant reduction of the enzymatic activity in mutant cells. The expression was noticeably decreased at the mRNA and protein levels in mutant cell lines. Additionally, the subcellular localization of α-Gal A changed from a homogeneous distribution to punctate aggregation in the cytoplasm. GLA mutant cells exhibited significantly higher levels of apoptosis compared to wild-type cells. [ABSTRACT FROM AUTHOR]

Objective: Thalassemia is a Mendelian‐inherited blood disorder with severe consequences, including disability and mortality, making it a significant public health concern. Therefore, there is an urgent need for precise diagnostic technologies. We introduce two innovative diagnostic techniques for thalassemia, SNPscan and CNVplex, designed to enhance molecular diagnostics of thalassemia. Methods: The SNPscan and CNVplex assays utilize variations in PCR product length and fluorescence to identify multiple mutations. In the SNPscan method, we designed three probes per locus: two 5′ and one 3′, and incorporated allele identification link sequences into one of the 5′ probes to distinguish the alleles. The detection system was designed for 67 previously reported loci in the Chinese population for a specific genetic condition. CNVplex identifies deletion types by analyzing the specific positions of probes within the globin gene. This innovative approach enables the detection of six distinct deletional mutations, enhancing the precision of thalassemia diagnostics. We evaluated and refined the methodologies in a training cohort of 100 individuals with confirmed HBA and HBB genotypes. The validation cohort, consisting of 1647 thalassemia patients and 100 healthy controls, underwent a double‐blind study. Traditional diagnostic techniques served as the control methods. Results: In the training set of 100 samples, 10 mutations (Hb QS, Hb CS, Hb Westmead, CD17, CD26, CD41‐42, IVS‐II‐654, ‐‐SEA, −α3.7 and −α4.2) were identified, consistent with those identified by traditional methods. The validation study showed that SNPscan/CNVplex offered superior molecular diagnostic capabilities for thalassemia, with 100% accuracy compared to 99.43% for traditional methods. Notably, the assay identified three previously undetected mutations in 10 cases, including two deletion mutations (Chinese Gγ(Aγδβ)0 del and SEA‐HPFH), and one non‐deletion mutation (Hb Q‐Thailand). Conclusions: The SNPscan/CNVplex assay is a cost‐effective and user‐friendly tool for diagnosing thalassemia, demonstrating high accuracy and reliability, and showing great potential as a primary diagnostic method in clinical practice. [ABSTRACT FROM AUTHOR]

Background: Contiguous gene deletion in the short arm of chromosome 4 is linked to various neurodevelopmental disorders. Methods: In this study, we conducted peripheral blood chromosome G‐banding karyotyping and whole‐exome sequencing (WES) on a proband presenting with anal atresia, global developmental delay, lymphocytosis, and other multisystem anomalies. Additionally, chromosome G‐banding karyotyping was also carried out on the proband's parents and brother. Results: The 7‐month‐old proband was found to have a 26.738 Mb 4p15.33‐p14 deletion as identified by chromosome G‐banding karyotyping and WES. Conclusion: We identified a patient with proximal 4p deletion syndrome by karyotype and WES analysis, which might explain some of his phenotypes. Our research enhances clinicians' knowledge of this rare condition, and offers valuable genetic counseling to the affected family. Further research is necessary to identify the causative gene or critical region associated with proximal 4p deletion syndrome. [ABSTRACT FROM AUTHOR]

Background: Diarrhea is a leading cause of death in young children worldwide. Vitamin D deficiency impairs the body's ability to clear pathogens, reduces tight junction protein expression in intestinal epithelial cells, and enhances Th1‐mediated intestinal inflammation. This study aimed to investigate the effects of serum vitamin D levels on acute invasive enteritis in children. Methods: This prospective cohort study included 82 children aged 1–3 years with clinically diagnosed acute invasive enteritis at Sichuan Maternal and Child Health Hospital from February 2021 to February 2022, alongside a control group of 80 healthy children. Fecal specimens were collected for routine tests and occult blood analysis, while blood samples were taken for routine tests, C‐reactive protein, and 25‐OHD levels. Comparative analyses were performed between groups, and multifactorial logistic regression was used to identify factors influencing invasive enteritis. Results: The study group showed significantly lower serum 25‐OHD levels (27.95 ± 9.91 ng/mL) compared to controls (32.76 ± 10.23 ng/mL, p <.01). Among the study group, 19.5% (16/82) had levels <20 ng/mL, versus 12.5% (10/80) in controls. Regular vitamin D supplementation was lower in the study group (58.5% vs. 77.5%, p <.05). Outdoor activity duration was also reduced (2.57 ± 0.98 h vs. 3.04 ± 0.88 h, p <.01). Multivariate analysis identified that exclusive breastfeeding, greater outdoor activity time and regular vitamin D supplementation were all associated with reduced risk of invasive enteritis (p <.05). Conclusion: The findings indicate an association between low serum 25‐OHD levels and acute invasive enteritis in children aged 1‐3 years, suggesting that consistent vitamin D supplementation and sufficient outdoor activity may protect against this condition. [ABSTRACT FROM AUTHOR]

This paper presents a study of the multi-objective optimal design of a sliding mode control for an under-actuated nonlinear system with the parallel simple cell mapping method. The multi-objective optimal design of the sliding mode control involves six design parameters and five objective functions. The parallel simple cell mapping method finds the Pareto set and Pareto front efficiently. The parallel computing is done on a graphics processing unit. Numerical simulations and experiments are done on a rotary flexible arm system. The results show that the proposed multi-objective designs are quite effective.