ObjectiveThis study aimed to investigate the intervention effect of Renqing Mangjue on the malignant transformation of gastric mucosal epithelial cells induced by N-methyl-N′-nitro-N-nitrosoguanidine （MNNG） and to explore its molecular mechanism in preventing precancerous lesions of gastric cancer based on the cyclic guanosine monophosphate （cGMP）/protein kinase G （PKG）/mitogen-activated protein kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway. MethodsHuman gastric mucosal epithelial cells （GES-1） were initially induced by MNNG to establish a precancerous cell model （MC cells）. The effective concentration of MNNG for inducing malignant transformation in GES-1 cells was screened using the cell proliferation activity decection （CCK-8） assay， and the effective concentration of Renqing Mangjue for inhibiting the proliferation of transformed GES-1 cells was also determined. GES-1 cells were divided into a blank control group， a model group， and treatment groups with Renqing Mangjue at concentrations of 1， 3， 10， and 30 mg·L^-1. Furthermore， the effects of Renqing Mangjue on the migratory ability and epithelial-mesenchymal transition （EMT） characteristics of GES-1 malignant transformed cells were evaluated using Transwell migration assays， wound healing assays， and real-time quantitative reverse transcription polymerase chain reaction （Real-time PCR）. Additionally， candidate chemical components and target sites of Renqing Mangjue were obtained from the TCMIP v2.0 database， and disease targets at various stages of gastric cancer precursors were sourced from the Gene Expression Omnibus （GEO） database. Pathway enrichment analysis was performed using the Metascape database to predict the potential mechanisms of action of Renqing Mangjue. Finally， the protective mechanism of Renqing Mangjue against gastric cancer precursors was validated through Western blot analysis. ResultsAt a concentration of 20 μmol·L^-1， MNNG exhibited an inhibition rate of approximately 50% on GES-1 cells （P<0.01）， and at this concentration， the GES-1 cells displayed biological characteristics indicative of malignant transformation. In contrast， Renqing Mangjue had no significant effect on the proliferation of normal GES-1 cells， but significantly inhibited the proliferation of MC cells （P<0.01） and markedly reduced their migratory capacity （P<0.01）. Moreover， it also increased the mRNA expression level of E-cadherin during the EMT process （P<0.05）， while inhibiting the expression of both N-cadherin and the transcription factor Snail mRNA （P<0.05， P<0.01）. Network predictions suggested that Renqing Mangjue may prevent gastric cancer precursors through modulating the cGMP/PKG and MAPK/ERK signaling pathways. Furthermore， Western blot results indicated that Renqing Mangjue upregulated the expression of PKG and NPRB （B-type natriuretic peptide receptor） proteins in the cGMP/PKG pathway （P<0.01）， while downregulating the expression of the downstream proteins MEK and ERK （P<0.05， P<0.01）. ConclusionIn summary， Renqing Mangjue can prevent gastric cancer precursors by inhibiting the proliferation and migration of malignant transformed GES-1 cells， thereby delaying the EMT process. The underlying mechanisms may be related to the activation of the cGMP/PKG pathway and the inhibition of the MEK/ERK signaling pathway.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent cause of chronic liver disease worldwide, and its intricate pathogenesis presents challenges in the development of new drugs. As a common way of post-translational modification, acetylation regulates protein stability, enzyme activity, and subcellular localization, occurring extensively in MASLD-associated processes such as lipid metabolism, inflammatory response, and oxidative stress. In this paper, we comprehensively review the mechanism of acetylation in MASLD, analyze the expression levels of acetylases in liver tissues of MASLD patients from the gene expression omnibus (GEO), discuss the changes in relevant enzyme expression and mechanisms in animal models, and further explore the feasibility of targeting acetylation for MASLD treatment, in the hope of offering a new perspective for advancing drug discovery in the field of MASLD.

Objective To summarize the diagnosis and treatment of neuronal intranuclear inclusion disease （NIID） with peripheral neuropathy as the initial symptom in a Han Chinese family， and to improve the clinical awareness of this disease through a literature review. Methods A pedigree investigation was performed for a patient with NIID who was admitted to the outpatient service of Department of Neurology， Henan Provincial People’s Hospital， in January 2023， and the clinical and imaging features of the family members were summarized. Various techniques， such as second-generation whole-exome sequencing， third-generation whole-genome sequencing， and PCR capillary electrophoresis， were used for genetic analysis， and a family lineage map was plotted. A literature review was performed to summarize the features of cases with peripheral neuropathy as the initial symptom. Results There were 3 generations and 16 members in this family， among whom 5 had clinical manifestations， 8 underwent blood sampling， and 3 were found to have a GCC repeat expansion mutation in the 5' non-coding region of the NOTCH2NLC gene. The proband was aged 39 years， with the initial symptom of weakness in the extremities， and cranial MRI did not show the characteristic signal of NIID， while electromyography suggested multiple peripheral nerve injuries in the extremities. Genetic testing showed that the proband， his aunt， and his cousin all had a GGC repeat expansion mutation in the NOTCH2NLC gene， with a number of more than 60 repeats （154，144，and 148，respectively）. Conclusion In hereditary peripheral neuropathy， the possibility of NIID should be considered in case of negative results for common pathogenic genes. In addition， third-generation sequencing has a marked diagnostic value in repeat expansion mutation-related disorders.

Objective To identify antibody specificity in an elderly patient with hydronephrosis accompanied by ureter-al stones and shock who had multiple antibodies.Methods Microcolumn gel method was used to screen unexpected anti-bodies of red blood cells and identify antibodies.Enzyme method and antibody absorption method were used to help judge the specificity of antibodies in patients.The ABO blood type,Rh blood type and MNS blood type of patient were determined by saline tube method.Results The patient′s blood types were O,CCDee,NNss,and a combination of anti-E,anti-c,anti-M and anti-S antibodies was detected.Conclusion Repeated blood transfusion may lead to the presence of one or more un-expected antibodies in patients.Patients with multiple or high-frequency antibodies may experience difficulties in identifica-tion and delayed blood use.

Objective:To analyze the functional differences between virus-specific CD4 +T cells and CD8 +T cells in patients infected with Epstein-Barr virus (EBV) who develop liver injury and those who do not. Methods:45 cases of EBV infections were enrolled, including 28 cases developing liver injuries and 17 that did not. Mononuclear cells from peripheral blood were isolated. CD4 +T cells and CD8 +T cells were purified and cultured using recombinant EBV core antigen 2 (EBNA2) for 96 h with stimulation. The CCK-8 method was used to detect cell proliferation. Flow cytometry was used to detect the proportion of CD4 +T cells and CD8 +T cells. An enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of CD4 +T cells secreting cytokines and CD8 +T cells secreting molecular toxicity. Real-time quantitative PCR was used to detect the mRNA levels of transcription factors and molecular toxicity in CD4 +T cell subsets. Flow cytometry was used to detect the immune checkpoints at molecular levels in CD8 +T cells. The inter-group comparison was performed using a t-test or Mann-Whitney test. Results:There was no statistically significant difference ( P > 0.05) in the proliferation proportion of peripheral blood mononuclear cells, CD4 +T cells, and CD8 +T cells after stimulation with recombinant EBNA2 between the EBV-infected non-liver injury group and the infected liver injury group ( P > 0.05). There was no statistically significant difference in the proportion of CD4 +T cells secreting related cytokines and the mRNA levels of transcription factors after stimulation with recombinant EBNA2 between the EBV-infected non-liver injury group and the infected liver injury group ( P > 0.05).The levels of perforin secreted by CD8 +T cells and granzyme B after stimulation with recombinant EBNA2 were higher in the EBV infection-induced liver injury group than those in the non-liver injury group [(75.51±23.33) pg/ml vs. (58.99±18.39) pg/ml, P = 0.017] [(117.8±44.55) pg/ml vs. (90.22±34.21) pg/ml, P = 0.034]. The mRNA levels of Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand in CD8 +T cells in the liver injury group caused by EBV infection were approximately 1.5 and 1.2 times higher than those in the non-liver injury group, respectively, and the difference was statistically significant ( P < 0.001), but there was no statistically significant difference in the proportional expression of programmed cell death-1 and cytotoxic T lymphocyte-associated antigen-4 in CD8 +T cells between the EBV-infected non-liver injury group and infected liver injury group ( P > 0.05) Conclusion:Patients with liver injury caused by EBV infection have strong virus-specific CD8 + T cell toxic effects, which may mediate EBV-induced liver injury.

Objective:To analyze the gene variants of a patient affected with Duchenne/Becker muscular dystrophy in a pedigree and further explore the genotype-phenotype correlation for providing basis for family genetic counseling.Methods:The clinical features and family history of family members were collected.Multiplex ligation-dependent probe amplification(MLPA)was utilized to detect copy number variation of target genes.The pathogenic variations were ana-lyzed by whole exome sequencing(WES).The suspected gene variations were verified by Sanger sequencing.For the splice site mutations,mini-gene was constructed and expressed in vitro to detect the number of transcript and cDNA se-quence.Results:The proband of this family is a male,with no obvious involvement of the lower limbs.Laboratory tests showed an elevated level of creatine kinase(CK)in peripheral blood(700-1600 U/L),and electromyography showed myogenic damage.MLPA did not detect pathogenic exon copy number variation in dystrophin(DMD)gene.Genetic testing showed the proband carried a maternal hemizygotic splicing variation of DMD gene(NM_004006.2):c.2622+2T＞C.An in vitro mini-gene splicing assay confirmed that this splicing mutation could affect RNA splicing.According to clinical features and genetic testing results,the proband was speculated first proof of Duchenne/Becker muscular dys-trophy(DMD/BMD)caused by DMD gene mutation.Conclusion:This study identified the pathogenic variation of a proband with DMD/BMD of DMD gene,which enriched the variation spectrum of DMD/BMD in China.It was con-firmed that the splicing variation of the DMD gene c.2622+2T＞C can produce multiple transcripts leading to different functional impairments,and based on the specificity of temporal and spatial expression,it corresponded to the mild clin-ical manifestations of the patient,providing some reference value for the correlation between genotype and phenotype.

Objective:To explore the clinical phenotype and genetic variant in a Chinese pedigree affected with Hunter syndrome and create immortalized cell lines for the affected pedigree members.Methods:A pedigree of six members who had visited Xi′an Children′s Hospital in July 2022 was selected as the study subject. Clinical data was collected. Whole exome sequencing was carried out for the pedigree members. Candidate variant was verified by Sanger sequencing. In addition, peripheral B lymphocytes were transfected with Epstein-Barr virus to create immortalized cell lines, which were then subjected to enzyme activity analysis.Results:The patient, a five-year-and-seven-month-old boy, had exhibited stiff limbs and enlarged joints. He had developed hernia, scaphocephaly, and barrel chest from 3 months of age. His uncle also had stiff limbs, poor hearing, blindness, and right oblique inguinal hernia. Above features had resembled those of Hunter syndrome. Genetic testing revealed that both the child and his uncle had harbored an IDS (NM_000202.8): c. 823G>A (p.D275N) variant, which was unreported previously. Bioinformatic analysis indicated that the D275 to be a highly conserved site, and the D275N variant may affect the stability of the protein′s spatial conformation, thereby decrease the catalytic activity of the enzyme. The successfully constructed immortalized lymphoblastoid cell lines for the child and his parents showed increased volume, irregular shape, burr structure and cluster growth. And the value of IDS activity of the patient′s immortalized lymphoblastoid cells was below the limit of detection. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic (PS3+ PM2_Supporting+ PM5+ PP1+ PP3). Conclusion:Above finding has enriched the phenotypic and mutational spectra of Hunter syndrome, and provided a basis for the genetic counseling for this pedigree. The creation of immortalized cell lines has offered a model for further investigation of the impact of variant on the function of IDS and development of targeted drugs.

Objective:To analyze the clinical characteristics of a child with Congenital disorder of glycosylation due to compound heterozygous variants of COG6 gene ( COG6-CDG). Methods:A child who was admitted to Xi′an Children′s Hospital on January 10, 2023 was selected as the study subject. Clinical data were collected. Pathogenic variants were analyzed by whole exome sequencing, and candidate variants were verified by Sanger sequencing, in vitro experiments and bioinformatic analysis. This study was approved by the Medical Ethics Committee of Xi′an Children′s Hospital (No. 20230101). Results:The child, a 1-month-8-day-old male, was admitted for diarrhea and weight loss for one month. He had presented with cholestasis, diarrhea, facial dysmorphism, poor response, bilateral Simian crease, and brain atrophy. After discharge, he had continued to have high fever, feeding difficulty, and deceased finally. Whole exome sequencing results showed that he had harbored compound heterozygous variants of the COG6 gene, namely c. 807delT (p.F269Lfs*37) and c. 1746+ 1G>C (p.Gly565_Met582del). Sanger sequencing verified that the variants were inherited from his father and mother, respectively. In vitro experiments verified that the c. 1746+ 1G>C variant could affect the mRNA splicing and produce a truncated protein, whilst the c. 807delT variant could significantly reduce gene expression at both mRNA and protein levels. Based on the guidelines from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP), the variants were classified as pathogenic (PVS1+ PM3+ PM2_Supporting) and likely pathogenic (PVS1+ PM2_Supporting), respectively. Conclusion:The c. 807delT (p.F269Lfs*37) and c. 1746+ 1G>C (p.Gly565_Met582del) compound heterozygous variants of the COG6 gene probably underlay the pathogenesis of this child. Above finding has enriched the mutational spectrum of COG6-CDG and provided a basis for the genetic counseling for this family.

To promote the development of the prescription-based processing of Chinese herbal pieces,the China Association of Chinese Medicine published the social organization standard of the Standard for Prescription-based Processing of Chinese Herbal Pieces(T/CACM 1367-2021)in June 2021.The standard was led by the First Affiliated Hospital of Henan University of Chinese Medicine and Jiangsu Province Hospital of Chinese Medicine.It was jointly drafted by 28 Traditional Chinese medical institutions across the country.This paper introduced the standards in detail to promote the implementation and propel the inheritance and innovation of the processing of Chinese herbal pieces.

Non-alcoholic fatty liver disease (NAFLD) has become a major public health hazard threatening human health worldwide.Yet, due to its complex pathogenesis, new drug development is difficult, with still insufficient clinical medication.Palmitoylation is a universal posttranslational modification of proteins catalyzed by palmitoyltransferase, affecting their stability, membrane localization and function.Recent studies have shown that palmitoylation is closely associated with NAFLD.This review summarizes the mechanisms of palmitoylation in NAFLD and analyzes the expression levels of the palmitoyltransferase family in liver tissues of NAFLD patients from GEO database, aiming to provide important clues to explore new mechanisms for NAFLD.