Machado-Joseph disease, or spinocerebellar ataxia type 3 (SCA3), represents the most common autosomal dominant cerebellar ataxia worldwide. Despite its progressive and debilitating nature, disease-modifying therapies remain elusive. Repetitive transcranial magnetic stimulation (rTMS) has emerged as a promising non-invasive intervention; however, its clinical application has been hindered by inconsistent protocols and a lack of mechanistic understanding. A recent landmark study published in Brain Stimulation by Chen et al. addressed these challenges by combining a high-dose intermittent theta-burst stimulation (iTBS) protocol with concurrent transcranial magnetic stimulation-electroencephalography (TMS-EEG). This commentary provides an in-depth analysis of their findings, highlighting the restoration of cerebello-cortical inhibition (CBI) as a key therapeutic mechanism. Furthermore, we discuss the broader implications of this work, proposing that future translational research should integrate accelerated iTBS (aiTBS) paradigms, cortical response measurements (CRM), and individualized neuro-navigation to establish a new era of precision neuromodulation for ataxia.

Machado-Joseph disease, or spinocerebellar ataxia type 3 (SCA3), represents the most common autosomal dominant cerebellar ataxia worldwide. Despite its progressive and debilitating nature, disease-modifying therapies remain elusive. Repetitive transcranial magnetic stimulation (rTMS) has emerged as a promising non-invasive intervention; however, its clinical application has been hindered by inconsistent protocols and a lack of mechanistic understanding. A recent landmark study published in Brain Stimulation by Chen et al. addressed these challenges by combining a high-dose intermittent theta-burst stimulation (iTBS) protocol with concurrent transcranial magnetic stimulation-electroencephalography (TMS-EEG). This commentary provides an in-depth analysis of their findings, highlighting the restoration of cerebello-cortical inhibition (CBI) as a key therapeutic mechanism. Furthermore, we discuss the broader implications of this work, proposing that future translational research should integrate accelerated iTBS (aiTBS) paradigms, cortical response measurements (CRM), and individualized neuro-navigation to establish a new era of precision neuromodulation for ataxia.

This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic acid (5-ALA). Furthermore, this study aimed to explore the underlying mechanism of the protective effects of 5-ALA. First, we collected and stored mouse testicular fragments in different media, including Hank's balanced salt solution (HBSS; n = 5), Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; n = 5), and alpha-minimum essential medium (αMEM; n = 5). Storage of testicular tissue in HBSS preserved the integrity of testicular morphology better than that in the DMEM/F12 group ( P < 0.05) and the αMEM group ( P < 0.01). Testicular fragments were subsequently placed in HBSS with various concentrations of 5-ALA (0 [control], 1 mmol l -1 , 2 mmol l -1 , and 5 mmol l -1 ) to determine the most effective concentration of 5-ALA. The 2 mmol l -1 5-ALA group ( n = 3) presented the highest positive rate of spermatogonial stem cells compared with those in the control, 1 mmol l -1 , and 5 mmol l -1 5-ALA groups. Finally, the tissue fragments were preserved in HBSS with control ( n = 3) and 2 mmol l -1 5-ALA ( n = 3) under low-temperature conditions. A comparative analysis was performed against fresh testes ( n = 3) to elucidate the underlying mechanism of 5-ALA. Gene set enrichment analysis (GSEA) for WikiPathways revealed that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the 2 mmol l -1 5-ALA group compared with that in the control group (normalized enrichment score [NES] = -1.57, false discovery rate [FDR] = 0.229, and P = 0.019). In conclusion, these data suggest that using 2 mmol l -1 5-ALA in HBSS effectively protected the viability of spermatogonial stem cells upon hypothermic transportation.
Male ; Animals ; Testis/cytology* ; Aminolevulinic Acid/pharmacology* ; Mice ; Organ Preservation/methods* ; Organ Preservation Solutions/pharmacology* ; Cryopreservation/methods*

OBJECTIVES: To investigate the molecular epidemiology of children with phenylketonuria (PKU) in Gansu, China, providing foundational data for intervention strategies. METHODS: A retrospective analysis was conducted on 1 159 PKU families who attended Gansu Provincial Maternity and Child Care Hospital from January 2012 to December 2024. Sanger sequencing, multiplex ligation-dependent probe amplification, whole exome sequencing, and deep intronic variant analysis were used to analyze the PAH gene. RESULTS: For the 1 159 children with PKU, 2 295 variants were identified in 2 318 alleles, resulting in a detection rate of 99.01%. The detection rates were 100% (914/914) in 457 classic PKU families, 99.45% (907/912) in 456 mild PKU families, and 96.34% (474/492) in 246 mild hyperphenylalaninemia families. The 2 295 variants detected comprised 208 distinct mutation types, among which c.728G>A (14.95%, 343/2 295) had the highest frequency, followed by c.611A>G (4.88%, 112/2 295) and c.721C>T (4.79%, 110/2 295). The cumulative frequency of the top 23 hotspot variants reached 70.28% (1 613/2 295), and most variant alleles were detected in exon 7 (29.19%, 670/2 295). CONCLUSIONS Deep intronic variant analysis of the PAH gene can improve the genetic diagnostic rate of PKU. The development of targeted detection kits for PAH hotspot variants may enable precision screening programs and enhance preventive strategies for PKU.
Humans ; Phenylketonurias/epidemiology* ; Female ; Male ; Retrospective Studies ; Phenylalanine Hydroxylase/genetics* ; Mutation ; Child, Preschool ; China/epidemiology* ; Child ; Infant

Objective To establish and evaluate a nomogram prediction model for spontaneous peritonitis in HBV-related primary liver cancer.Methods A retrospective study was conducted on 1298 patients with HBV-related primary liver cancer hospitalized in the Kunming Third People's Hospital from January 2012 to December 2022.General data and serological indicators were collected,and patients were divided into infection group(n=262)and control group(n=1036)based on the occurrence of spontaneous peritonitis.Univariate and LASSO regression analyses were used to screen variables,followed by binary logistic regression to analyze the influencing factors of spontaneous peritonitis in HBV-related primary liver cancer patients,leading to the establishment of a nomogram prediction model.Finally,the Hosmer-lemeshow(H-L)goodness of fit test,receiver operating characteristic(ROC)curve,calibration curve,decision curve analysis(DCA)and clinical impact curve(CIC)were utilized to evaluate the fit degree,accuracy,calibration,and clinical practicability of the nomogram prediction model.Results Single factor analysis revealed significant differences between infection group and control group in portal vein cancer thrombus(PVTT),Child-Pugh grade,China Liver Cancer Staging(CNLC)stage,alcohol consumption history,smoking history,white blood cell count(WBC),neutrophil count(NE),hemoglobin(Hb),fibrinogen(FIB),abnormal prothrombin(PIVKA-Ⅱ),aspartate aminotransferase(AST),alanine aminotransferase(ALT),total protein(TP),prealbumin(PA),γ-glutamyltransferase(GGT),alkaline phosphatase(ALP),cholinesterase(CHE),total bile acid(TBA),total cholesterol(TC),low density lipoprotein(LDL),creatinine(Cr),HBV DNA,CD3+T cells count,CD4+T cells count,CD8+T cells count,CD4+T cells/CD8+T cells ratio,procalcitonin(PCT),serum amyloid A(SAA),interleukin-6(IL-6),high-sensitivity C-reactive protein(hs-CRP),alpha-fetoprotein(AFP),and IL-4(P＜0.05).LASSO regression analysis identified 5 variables:Child-Pugh grade,PVTT,WBC,CHE and hs-CRP.Binary logistic regression analysis indicated that Child-Pugh grade(Grade B:OR=5.780,95％CI 3.271-10.213,P＜0.001;Grade C:OR=14.818,95％CI 7.697-28.526,P＜0.001),PVTT(OR=2.893,95％CI 2.037-4.108,P＜0.001),WBC(OR=1.088,95％CI 1.031-1.148,P=0.002),and hs-CRP(OR=1.005,95％CI 1.001-1.010,P=0.026)were the independent risk factors of spontaneous peritonitis in HBV-related primary liver cancer patients.Using these 4 variables,a nomogram prediction model was constructed and evaluated.The P-value of the H-L goodness of fit test was 0.760.Moreover,the area under ROC curve(AUC)was 0.866,with a sensitivity of 0.870 and a specificity of 0.716.The average absolute error of the calibration curve is 0.022.DCA and CIC analyses demonstrated that the nomogram prediction model possessed some clinical utility.Conclusion The nomogram prediction model for spontaneous peritonitis in HBV-related primary liver cancer patients,constructed using Child-Pugh grade,PVTT,WBC and hs-CRP,exhibits a high fitting degree and accuracy,with the prediction probability highly consistent with the actual occurrence probability,and possesses certain clinical practicability.

Objective:This study aims to explore the risk factors of Multi-center Investigator-Initiated Clinical Trials (MIITs), and provide a basis for developing study management strategies.Methods:The original draft of MIIT risk evaluation factors was determined through literature analysis and internal discussions of the research group. Thirty five experts were consulted using the Delphi method, and then the MIIT risk evaluation elements were finally determined. Analytic Hierarchy Process (AHP) was used to calculate the weights of each index.Results:The recovery rates of both rounds of expert consultation were 100%, and the degree of expert authority was 0.856. The study ultimately formed an MIIT risk evaluation framework consisting of three first-class indexes, twelve second-class indexes, and thirty-eight third-class indexes. The weight values of the first-class indexes (start-up period, implementation period, and summary period) were 0.209 8, 0.710 6, and 0.079 6, respectively. Meanwhile, the weight values of the second-class indexes and third-class indexes were determined.Conclusions:Exploring the risk evaluation factors of MIIT provides valuable insights into identifying critical risk points, which, in turn, contributes to enhancing MIIT management efficiency, research progress, and quality.

Aim To study the difference in hepatotox-icity of psoralen on normal rats and Yin-deficiency rats from the perspective of lipid metabolism,so as to help explain the mechanism of psoralen cautiously used in patients with Yin deficiency recorded in ancient books.Methods SD rats were randomly divided into the nor-mal control group(carboxymethyl cellulose-Na,CMC-Na),normal administration group(CMC-Na+psor-alen),Yin-deficiency control group(CMC-Na+thy-roxine)and Yin-deficiency administration group(CMC-Na+thyroxine+psoralen).The model of Yin-deficiency was established by thyroxine(1 mg·kg-1)for ten days,and then psoralen(200 mg·kg-1)was given for three days.The serum indexes related to liver injury were detected by automatic biochemical analy-zer,the morphological changes of liver tissue were ob-served using HE and oil red O staining,and the relative transcription levels of lipid metabolism related enzymes and mRNA of transporter and endoplasmic reticulum stress related factors were detected using Real-time PCR.Results After intragastric administration of psoralen for three days,compared with the normal group,the levels of serum alanine aminotransferase(ALT),aspartate transaminase(AST),total bile acid(TBA)and triglyeride(TG)in Yin deficiency group increased more significantly,while TC,ALB and TP de-creased more significantly,and liver HE and oil red O staining showed more obvious lipid degeneration.TG synthesis factors adrenocortical carcinoma(ACC),fatty acid synthase(FASN)and sterolregulatory element binding protein-1(SREBP-1)were down-regulated more significantly,TG transport factors mili-total pro-tein(MTP)and lipoprotein pipase(LPL)were down-regulated more evidently,fatty acid β-oxidation related factors carnitine palmitoyltransferase 1A(CPT1A),carnitine/organic cation transporter 2(OCTN2)and peroxisome proliferators-activated receptors-alpha(PPARα)were down-regulated more apparently,TC transporter adenosine triphosphate binding cassette transporter G8(ABCG8)and bile acid receptor farne-soid X receptor(FXR)were down-regulated more ob-viously,and endoplasmic reticulum stress factor activa-ting transcription factor 4(ATF4)was up-regulated more significantly.Conclusions Psoralen can cause more severe hepatotoxicity in Yin deficiency rats than that in normal administration group,and its mechanism may be related to the disorder of hepatic lipid metabo-lism,aggravation of hepatic cholestasis and steatosis,and activation of endoplasmic reticulum stress re-sponse.

Aim To investigate the effects of Congrong San(CRS)on learning and memory ability,hippocam-pal neuronal injury,and ferroptosis in rats with Alzhei-mer's disease(AD)and to explore the related mecha-nisms.Methods AD rat models were established and divided into Sham,Model,CRS low-dose,CRS medium-dose,CRS high-dose,and memantine groups.After treatment,Morris water maze,HE and Nissl staining,transmission electron microscopy,immunofluorescence staining,Western blot,and kit assays were performed to assess learning and memory ability,hippocampal neuro-nal injury,ferroptosis-related indicatorsand glucose reg-ulated protein 78 ku(GRP78)-(proteinkinaseR-li-keERkinase)PERK-(activating transcription factor 4)ATF4 pathway protein expression.Results Com-pared with the model group,rats in the CRS medium-and high-dose groups and the memantine group showed significant improvement in learning and memory abili-ty,reduced hippocampal neuronal injury,increased number of Nissl bodies,and ameliorated endoplasmic reticulum swelling and mitochondrial damage.In addi-tion,the expressions of GRP78,p-PERK/PERK,and ATF4 were downregulated,while GPX4 expression was upregulated in the CRS medium-and high-dose groups and the memantine group.Moreover,MDA content de-creased,and SOD and GSH-PX levels increased in these groups.Conclusions CRS can improve the learning and memory ability in AD rats,reduce hipp-ocampal neuronal injury and ferroptosis,and its mecha-nism may be related to the inhibition of the GRP78-PERK-ATF4 pathway,enhancement of GPX4 expres-sion,and reduction of oxidative stress levels,providing a new approach for the clinical treatment of AD.

Aim To study the difference in hepatotox-icity of psoralen on normal rats and Yin-deficiency rats from the perspective of lipid metabolism,so as to help explain the mechanism of psoralen cautiously used in patients with Yin deficiency recorded in ancient books.Methods SD rats were randomly divided into the nor-mal control group(carboxymethyl cellulose-Na,CMC-Na),normal administration group(CMC-Na+psor-alen),Yin-deficiency control group(CMC-Na+thy-roxine)and Yin-deficiency administration group(CMC-Na+thyroxine+psoralen).The model of Yin-deficiency was established by thyroxine(1 mg·kg-1)for ten days,and then psoralen(200 mg·kg-1)was given for three days.The serum indexes related to liver injury were detected by automatic biochemical analy-zer,the morphological changes of liver tissue were ob-served using HE and oil red O staining,and the relative transcription levels of lipid metabolism related enzymes and mRNA of transporter and endoplasmic reticulum stress related factors were detected using Real-time PCR.Results After intragastric administration of psoralen for three days,compared with the normal group,the levels of serum alanine aminotransferase(ALT),aspartate transaminase(AST),total bile acid(TBA)and triglyeride(TG)in Yin deficiency group increased more significantly,while TC,ALB and TP de-creased more significantly,and liver HE and oil red O staining showed more obvious lipid degeneration.TG synthesis factors adrenocortical carcinoma(ACC),fatty acid synthase(FASN)and sterolregulatory element binding protein-1(SREBP-1)were down-regulated more significantly,TG transport factors mili-total pro-tein(MTP)and lipoprotein pipase(LPL)were down-regulated more evidently,fatty acid β-oxidation related factors carnitine palmitoyltransferase 1A(CPT1A),carnitine/organic cation transporter 2(OCTN2)and peroxisome proliferators-activated receptors-alpha(PPARα)were down-regulated more apparently,TC transporter adenosine triphosphate binding cassette transporter G8(ABCG8)and bile acid receptor farne-soid X receptor(FXR)were down-regulated more ob-viously,and endoplasmic reticulum stress factor activa-ting transcription factor 4(ATF4)was up-regulated more significantly.Conclusions Psoralen can cause more severe hepatotoxicity in Yin deficiency rats than that in normal administration group,and its mechanism may be related to the disorder of hepatic lipid metabo-lism,aggravation of hepatic cholestasis and steatosis,and activation of endoplasmic reticulum stress re-sponse.