Objective To construct a 293T-CymR cell strain to express cysteine metabolism regulator(CymR) component by gene editing technology, and to regulate the target gene expression with CuO regulatory sequence promoter.Methods Based on clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 technology, the intracellular β factor receptor, C-C chemokine receptor(CCR5) gene, one of the safe harbors in HEK293T cell genome, was chosen as the target site for inserting the desired gene. The selection gene Pac(denoted as PuroR) encoding puromycin N-acetyltransferase was linked to the 3'end of the CymR gene via an internal ribosome entry site(IRES), sharing a cytomegalovirus(CMV) promoter. The 293 TCymR monoclonal cell strain capable of expressing CymR was screened by Puro resistance and limiting dilution method. The CymR gene position and protein function in the 293T-CymR monoclonal cell strain were verified by PCR, Western blot and fluorescence analysis.Results The pY93-CymR plasmid expressing both the target and resistance genes, was successfully constructed. Following co-transfection with the pX330-CCR5 plasmid into HEK293T cells, four 293T-CymR monoclonal cell strains were obtained via Puro resistance selection and the limiting dilution method. The PCR and Western blot analysis indicated that CymR gene was successfully inserted into the intended CCR5 site and CymR protein was efficiently produced in three of these clones. Subsequent transfection tests demonstrated that the produced CymR could successfully suppress the gene expression under the CMV promoter containing CuO element.Conclusion CymRgene was successfully inserted into the CCR5 locus of HEK293T cell genome, and 293T-CymR monoclonal cell strains capable of expressing CymR and regulating gene expression were obtained.

In recent years, the prevalence of myopia has shown a significant upward trend characterized by earlier onset and accelerated progression rates. This epidemic not only imposes an increasing socioeconomic burden but also leads to severe vision impairment through high myopia-related complications that profoundly affect daily life. Current research on the pathogenesis of myopia primarily focuses on four mechanistic theories, including scleral remodeling, choroidal hemodynamic abnormalities, dopamine synthesis and metabolism, and inflammatory responses. The advent of high-throughput sequencing technologies has revolutionized our investigative approaches, enabling deeper exploration into myopia development through multi-omics strategies encompassing genomics, proteomics, and metabolomics. These cutting-edge methodologies have provided novel insights for myopia prevention and control, while simultaneously identifying potential therapeutic targets for precision intervention. This review focuses on summarizing the aforementioned research findings.

S-methyl-L-cysteine sulfoxide (SMCO) is a non-protein sulfur-containing amino acid with a variety of functions. There are few reports on the enzymes catalyzing the biosynthesis of SMCO from S-methyl-L-cysteine (SMC). In this study, the flavin-containing monooxygenase gene derived from Schizosaccharomyces pombe (spfmo) was heterologously expressed in Escherichia coli BL21(DE3) and the enzymatic properties of the expressed protein were analyzed. The optimum catalytic conditions of the recombinant SpFMO were 30 ℃ and pH 8.0, under which the enzyme activity reached 72.77 U/g. An appropriate amount of Mg²⁺ improved the enzyme activity. The enzyme kinetic analysis showed that the K_m and k_cat/K_m of SpFMO on the substrate SMC were 23.89 μmol/L and 61.71 L/(min·mmol), respectively. Under the optimal reaction conditions, the yield of SMCO synthesized from SMC catalyzed by SpFMO was 12.31% within 9 h. This study provides reference for the enzymatic synthesis of SMCO.
Schizosaccharomyces/genetics* ; Escherichia coli/metabolism* ; Recombinant Proteins/metabolism* ; Cysteine/biosynthesis* ; Mixed Function Oxygenases/metabolism* ; Schizosaccharomyces pombe Proteins/metabolism* ; Oxygenases/metabolism* ; Kinetics

Objective To express and identify recombinant adenovirus type 5-Norovirus(NoV)GⅡ.4-VP1 virus-like particles(VLPs)in 293T cells. Methods The recombinant adenovirus plasmids pAd5-eGFP and pAd5-NoV-GⅡ.4-VP1 were transfected into 293T cells respectively,and the recombinant adenovirus rAd5-eGFP and rAd5-NoV-GⅡ.4-VP1 were rescued. The rAd5-eGFP was subcultured in 293T cells to verify the function of the vector. The rAd5-NoV-GⅡ.4-VP1 was subcultured in293T cells,expressed and purified,and then NoV-GⅡ.4-VLP was formed by self-assembly,which was detected by Western blot,ELISA and observed by transmission electron microscope. Results The green fluorescence of the recombinant adenovirus rAd5-eGFP of various generations was observed under microscope,and the brightness increased with the increase of generations. NoV-GⅡ.4-VP1 protein was expressed in the harvested solution of recombinant adenovirus rAd5-NoV-GⅡ.4-VP1 of various generations,with a relative molecular mass of about 58 900. NoV-GⅡ.4-VLP showed specific binding to the rabbit anti-NoV-GⅡ.4-VP1 serum;it had similar conformation to natural NoV virus particles and can effectively identify NoV receptors in volunteer saliva samples;microscopic observation showed that the morphology was complete and spherical,with a diameter of 43. 5 — 58. 3 nm,while there were a few protrusions on the surface of the particles,which might be the P domain exposedon the particle surface during self-assembly of VP1. Conclusion The expressed recombinant adenovirus NoV-GⅡ. 4-VLP has complete VLP structure and good specificity,and is expected to be used in the related research of NoV adenovirus vector vaccine.

Objective To determine the improved effect of methylene blue(MB)on cognitive func-tion in brain-inflammatory-aging rats and investigate the underlying mechanism.Methods A total of 38 healthy 12-month-old SD rats were randomly divided into healthy control group,lipopo-lysaccharide(LPS)group,MB vehicle group and MB group,with 8 rats in the control and 10 rats in the other three groups.LPS was injected into the fourth ventricle with aid of a subcutaneous sustained release pump to establish a rat model of brain chronic inflammatory aging.MB of 0.5 mg/(kg·d)was added into the pump in the rats from the MB group.T-maze test and new object recognition test were employed to evaluate the learning and memory abilities of the rats.The acti-vation of microglia and astrocytes in the hippocampal CA1 region of the rats was detected by im-munofluorescence assay.The release of inflammatory factors IL-1β and IL-6 was measured by ELISA,and neuronal death in the CA1 region was assessed by neuronal nuclei(NeuN)fluores-cence staining.Results There was no significant difference in the exploration time for new and old objects between the LPS group and the MB solvent group(P＞0.05).The MB group spent significantly longer time in exploring the new objects than the old object(22.50±4.32 s vs 11.60± 3.01 s,P=0.000).The alternating selection rate of new arm and expression level of NeuN antigen were significantly decreased,and the expression levels of ionized calcium binding adaptor mole-cule-1(Iba-1)and glial fibrillary acidic protein(GFAP)and the contents of IL-1β and IL-6 were obviously increased(P＜0.05)in the LPS group and the MB vehicle group than the healthy con-trol group.Compared with the MB vehicle group,the MB group had notably increased alternating selection rate of new arms and higher NeuN expression level,and decreased Iba-1 and GFAP ex-pression and IL-1β and IL-6 contents(P＜0.05).Conclusion Subcutaneous administration of MB could significantly inhibit the damages of spatial learning and memory abilities in the LPS-induced brain chronic inflammatory aging rats.The mechanism may be closely associated with MB inhibi-ting inflammatory glial cells and protecting hippocampal pyramidal neurons.

Objective:The human peripheral blood lymphocyte cell line 8E5 is capable of secreting non-infectious HIV-1 viral particles. By targeting the POL region of the HIV-1 proviral gene integrated into the genome of 8E5 cell line and constructing a monoclonal cell line containing a drug resistance mutation site in the POL region using CRISPR/Cas9 point mutation technology, safe and stable HIV-1 genotypic drug resistance test quality control materials can be prepared.Methods:8E5 cells were co-transfected with sgRNA (single guide RNA) and Cas9 coexpression vector and Donor ssODN (donor single-stranded oligonucleotides) carrying the target mutation sites. The positive monoclonal cell lines were obtained through flow microtiter plate sorting, and the editing efficacy of the targeted mutations was validated by Sanger sequencing. Sanger sequencing was performed to verify the editing effect of the targeted mutations on the HIV virus particles secreted into the supernatant of the monoclonal cell lines cultured to the 3rd, 5th and 7th generations.Results:A double sgRNA and Cas9 coexpression vector was successfully constructed and co-transfected with a Donor ssODN carrying the drug-resistant mutation site Q151M to the 8E5 cell line, resulting in the desired outcome. The sequencing result of the target site confirmed the successful mutation at the resistance site and the establishment of a monoclonal homozygous cell line. The Q151M mutation site was detected in non-infectious HIV-1 virus particles secreted by the 8E5 Q151M cell line after transmission. Conclusions:The cell line 8E5 Q151M was successfully constructed using CRISPR/Cas9 point mutation technology to stably carry the Q151M drug resistance mutation site, which provides a new technological platform for the preparation of quality-control materials for testing HIV-1 genotypic drug resistance.

Objective:To explore the clinical characteristics and pathogenic variant in a child with Cantú syndrome (CS).Methods:A male who was admitted to the Children′s Hospital Affiliated to Zhengzhou University on February 23, 2022 was selected as the study subject. Clinical data of the child was collected. Peripheral blood samples of the child and his parents were collected and subjected to whole-exome sequencing (WES). Candidate variant was verified by Sanger sequencing. This study was approved by Medical Ethics Committee of the Children′s Hospital Affiliated to Zhengzhou University (Ethics No. 2023-K-087).Results:The child, a 3-year-and-2-month-old male, was born with hirsutism, with heavy hair all over the body and peculiar facial features. Routine echocardiography 1 month before had discovered atrial septal defect. Sequencing revealed that the child has harbored a heterozygous c. 2438G>C (p.S813T) variant of the ABCC9 gene, which was de novo in origin. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c. 2438G>C variant was classified as likely pathogenic (PS2+ PM2_Supporting+ PP3). Conclusion:The heterozygous c. 2438G>C variant of the ABCC9 gene probably underlay the pathogenesis of CS in this child.

Objective To compare and analyze the differences in CD8+naive,effector,memory,exhausted and regulatory T cells in order to investigate the impact of gender on the differentiation fate of CD8+T cells in the context of type 1 diabetes (T1D)based on female and male non-obese diabetic (NOD)mice and healthy Institute for Cancer Research (ICR)mice.Methods The frequencies and phenotypes of CD8+T cell differentiation subsets including naive T cells (TN),central memory T cells (TCM),effector T cells(TEFF),effector precursor T cells (TEP),exhausted T cells (TEX),precursor exhausted T cells (TPEX)and regulatory T cells (Tregs)in the spleen,pancreatic draining lymph nodes (pLN)and pancreas infiltrating lymphocytes (PIL)of male and female NOD mice were detected by flow cytometry.Results The frequencies of IFN-γ+,CD107a+and CCL5+CD8+TEFF in pLN and PIL of female NOD mice were significantly higher than those of male NOD mice.However,the frequencies of CD8+TN,CD8+TCM,CD8+TEX,CD8+TPEX and CD122+CD8+Tregs subsets in the spleen were significantly decreased.While there were no significant differences in the above CD8+T cell subsets except CD8+Tregs between female and male ICR mice. Conclusion Androgen may inhibit the differentiation of memory T cells into effector T cells and promote the exhaustion of effector T cells,leading to the difference in morbidity between the male and female mice.

Objective To screen the quality evaluation indicators of Bushen Huoxue Prescription(BHP)in the treatment of diabetic retinopathy(DR)by network pharmacology and molecular docking technology,and to establish content determination of active components in BHP by ultra-high-performance liquid chromatography triple-quadrupole mass spectrometry(UHPLC-QqQ-MS/MS).Methods Network pharmacology was used to screen the disease-related targets and key components,followed by molecular docking to further verify the interaction between them and confirm the active ingredients in BHP as the quality evaluation indicators.A UHPLC-QqQ-MS/MS method for the content determination of the active ingredients in BHP was established.A ZORBAX SB-C18 column(2.1 mm×50 mm,1.8 μm)was used,and methanol(A)-0.1％formic acid solution(B)was used as mobile phase.Gradient elution was performed in multiple reaction monitor mode.The flow rate was 0.3 mL·min-1 and the injection volume was 1 μL.Results Network pharmacology and molecular docking revealed five core targets and 12 BHP-related components(verbascoside,echinacoside,isoacteoside,tanshinone ⅡA,cryptotanshinone,dihydrotanshinone I,ginsenoside Re,Rd and Rb1,puerarin,daidzin,biochanin A)for the treatment of DR.There was a strong binding affinity between them(binding energy≤-5.0 kcal·mol-1).The established quantitative method demonstrated each component presented a good linearity within the specified range(r>0.999 5).The average recovery was in the range of 97.57％～101.48％.The contents of 12 components in eight batches of BHP samples were 0.027 9％～0.050 6％,0.006 4％～0.022 0％,0.017 1％～0.041 5％,0.009 2％～0.015 4％,0.012 6％～0.020 5％,0.004 4％～0.007 6％,0.334％～0.643％,0.238％～0.530％,0.353％～0.693％,3.411％～6.048％,1.023％～1.352％,0.000 8％～0.001 8％,respectively.Conclusion Based on network pharmacology,molecular docking and UHPLC-QqQ-MS/MS,a method for rapid screening and determination of 12 active components of BHP in the prevention and treatment of DR was established.This study provided a reference for comprehensive assessment of the quality and effectiveness of BHP.

OBJECTIVE: To analyze the clinical and genetic characteristics of a child with restricted cardiomyopathy (RCM) and phenylketonuria (PKU), and summarize the clinical characteristics and genetic diversity of RCM in children through a literature review. METHODS: A child with RCM in conjunct with PKU who was admitted to the Children's Hospital Affiliated to Zhengzhou University in June 2020 due to edema of eyelids and lower limbs for 1 year and aggravation for over 1 month was selected as the study subject. Relevant clinical data were collected. Peripheral blood samples of the child and his parents were collected for whole exome sequencing (WES). Candidate variants were validated by Sanger sequencing and bioinformatic analysis. Childhood, TNNI3 gene and restricted cardiomyopathy were used as the keywords to search the Wanfang data knowledge service platform, Chinese Journal Full-text database and PubMed database, and the search period was limited to from the time of establishment till August 2022. Clinical manifestations and characteristics of the TNNI3 gene variants were summarized. RESULTS: The child, a 2-year-old-and-4-month-old male, had normal intelligence, facial features and normal hair and skin color, but his motor and physical development was delayed, in addition with edema of bilateral eyelids and lower limbs. The results of WES and Sanger sequencing revealed that he has harbored compound heterozygous variants of the PAH gene, namely c.331C>T (p.R111X) and c.940C>A (p.P341T), which were inherited from his father and mother, respectively. In addition, he has also harbored a de novo heterozygous variant of c.508C>T (p.R170W) of the TNNI3 gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the TNNI3: c.508C>T (p.R170W) was classified as a pathogenic variant (PS2+PS4+PM2_Supporting+PM5), PAH: c.331C>T (p.R111X) as a pathogenic variant (PVS1+PM2_Supporting+PM3+PP4), and c.940C>A (p.P341T) as a likely pathogenic variant (PM2_Supporting+PM3+PM5+PP4). In total 30 children with RCM caused by TNNI3 gene variants were retrieved, with a male-to-female ratio of 1 : 1.55 and manifestations including heart failure, sinus rhythm, bi-atrial enlargement, ST-T wave change, ventricular restricted filling, and decreased ventricular diastolic function. In total 16 variants of the TNNI3 gene were identified, among which c.575G>A was the most common, and all cases had conformed to an autosomal dominant inheritance. CONCLUSION Phenylalanine hydroxylase deficiency and RCM are rare diseases with complex clinical manifestations. The PAH: c.331C>T (p.R111X)/c.940C>A (p.P341T) and TNNI3: c.508C>T (p.R170W) variants probably underlay the RCM and PKU in this child.
Humans ; Male ; Cardiomyopathy, Restrictive ; Computational Biology ; Diastole ; Mutation ; Phenylketonurias ; Child, Preschool