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BACKGROUND: There is an inflammatory response in the lesion tissue of ischemic cerebral infarction, and the expression of miR-150-5p is significantly decreased. Whether miR-150-5p inhibits the release of inflammatory factors and alleviates the injury of ischemic cerebral infarction tissue through the Toll-like receptor-5/nuclear factor-κB pathway remains unclear. OBJECTIVE: To investigate the role and preliminary mechanism of miR-150-5p in ischemic cerebral infarction in rats. METHODS: (1) The rat models of middle cerebral artery occlusion were constructed and the rat models were divided into five groups: control, miR-150-5p agomir, agomir control, miR-150-5p antagomir and antagomir control groups. (2) The rats in the control group was given the intracerebroventricular injection of normal saline, and the rats in the latter four groups were given the intracerebroventricular injection of miR-150-5p agomir (miR-150-5p agonist), agomir negative control, miR150-5p antagomir (miR150-5p inhibitor) and antagomir negative control, respectively. (3) After 7 days, the brain was graded by modified neurological severity score, the cerebral infarct volume was measured by MRI, and the histopathological changes were observed by hematoxylin-eosin staining. The expression levels of miR-150-5p, interleukin-6, tumor necrosis factor-α, Toll-like receptor-5 and nuclear factor-κB p65 in brain tissues were detected by qRT-PCR, ELISA and western blot assay, respectively. The target relationship between miR150-5p and Toll-like receptor-5 was verified by luciferase assay by retrieving the bioinformatics website Targetscan to predict the binding sites of miR-150-5p and Toll-like receptor-5. RESULTS AND CONCLUSION: (1) Compared with the control group, the modified neurological severity score, and levels of interleukin-6, tumor necrosis factor-α, Toll-like receptor-5 and nuclear factor-κB p65 proteins were significantly decreased in the miR-150-5p agomir group (P < 0.05). The physiological score and biochemical indexes in the miR-150-5p antagomirgroup were significantly increased (P < 0.05). (2) Hematoxylin-eosin staining results showed that the nerve cells in the control group were disordered and ill-defined, and the nerve cells were obviously degenerated and necrotic. The above pathological changes were significantly alleviated in the miR-150-5p agomir group and aggravated in the miR-150-5p antagomir group. There were no significant differences in the indexes between agomir control, the antagomir control and control groups (P > 0.05). (3) TargerScan website prediction results and luciferase reporter gene analysis results showed that miR-150-5p and Toll-like receptor-5 had a targeted binding site. (4) These results imply that miR-150-5p can inhibit the inflammatory signaling pathway of Toll-like receptor-5/nuclear factor-κB p65 in brain injury caused by ischemia and reduce the inflammatory response, thereby alleviating the damage of nerve function and playing a protective role. [ABSTRACT FROM AUTHOR]

Objective: To explore the clinical characteristics and genetic variants in two children with Tuberous sclerosis complex (TSC)., Methods: Two children who had presented at the Children's Hospital Affiliated to Zhengzhou University respectively in June 2020 and July 2021 were selected as the study subjects. Clinical data of the children were collected, and potential pathogenic variants were screened by whole exome sequencing (WES). Candidate variants were verified by Sanger sequencing of their family members., Results: Child 1 was a 7-month-and-29-day-old male, and child 2 was a 2-year-and-6-month-old male. Both children had shown symptoms of epileptic seizures and multiple hypomelanotic macules. Genetic testing revealed that both children had harbored de novo variants of the TSC2 gene, namely c.3239&#95;3240insA and c.3330delC, which were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were rated as pathogenic (PVS1+PS2+PM2&#95;Supporting)., Conclusion: This study has uncovered the genetic etiology for two children with TSC. Above findings have also enriched the phenotypic and mutational spectrum of TSC in the Chinese population.

Objective: To analyze the clinical phenotype and genetic variant in a child with Raynaud-Claes syndrome (RCS)., Methods: A child who was diagnosed with RCS at the Children's Hospital Affiliated to Zhengzhou University for delayed language and motor development in August 2022 was selected as the study subject. Clinical data of the child were collected, and potential genetic variant was detected by next-generation sequencing and Sanger sequencing. The pathogenicity of the candidate variant was analyzed., Results: The child, a 4-year-and-4-month-old male, has manifested global developmental delay, speech disorders, special facial features and behavioral abnormalities. Genetic testing revealed that he has harbored a hemizygous c.1174C>T (p.Gln392Ter) variant of the CLCN4 gene, which was not detected in either of his parents. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was rated as pathogenic (PVS1+PS2+PM2&#95;Supporting)., Conclusion: The c.1174C>T (p.Gln392Ter) variant of the CLCN4 gene probably underlay the PCS in this child. Above finding has expanded the mutational spectrum of the CLCN4 gene and enabled genetic counseling and prenatal diagnosis for his family.

Objective: To carry out Sanger sequencing for MMACHC gene variants among 65 Chinese pedigrees affected with combined methylmalonic aciduria and homocysteinemia, and summarize their genetic and clinical characteristics and prognosis., Methods: Clinical characteristics of the 65 children identified with Methylmalonic acidemia and homocysteinemia at the Children's Hospital Affiliated to Zhengzhou University (Zhengzhou Children's Hospital) from April 2017 to April 2022 were selected as the study subjects. Potential variants of the MMACHC gene were detected by direct sequencing of the PCR products., Results: The median age of the 65 children was 3 months (14 days to 17 years old). These included 28 cases (43.08%) from neonatal screening, 11 cases (16.92%) with a history of jaundice, and 9 cases (13.85%) with various degrees of anemia. The main clinical symptoms included development delay, slow growth, epilepsy, hydrocephalus, lethargy, feeding difficulty, regression or decline in motor ability, recurrent respiratory infections, anemia, jaundice, respiratory and heart failures, hydrocephalus, limb weakness, and hypertension. Blood and urine tandem mass spectrometry screening has revealed increase of methylmalonic acid, propionyl carnitine, propionyl carnitine/acetylcarnitine ratio, and propionyl carnitine/free carnitine ratio to various extents, and blood homocysteine was increased in all patients. The detection rate of genetic variants was 98.46% (128/130), and in total 22 types of MMACHC gene variants were detected. The most common ones have included c.609G>A (W203X) (58/128), c.658-660del (K220del) (19/128), and c.80A>G (Q27A) (16/128). Two novel variants have been identified, namely c.565C>T (p.R189C) and c.624&#95; 625delTG (p.A208Afs), which were respectively predicted as likely pathogenic (PM2&#95;Supporting+PM3+PP2+PP3) and pathogenic (PVS1+PM2&#95;Supporting+PM3+PP2) based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). Exon 4 had the highest frequency for the detection., Conclusion: Identification of MMACHC gene variants has confirmed the diagnosis in the children, among which the c.609G>A variant has the highest frequency. Discovery of the new variants has enriched the mutational spectrum of the MMACHC gene.

Objective: To analyze the clinical characteristics and pathogenic gene in a Chinese pedigree affected with mitochondrial DNA depletion syndrome 8A (MTDPS8A)., Methods: Whole exome sequencing was carried out for the patient. Sanger sequencing was used to verify the results, and PolyPhen-2 and PROVEAN software were used to predict the impact of amino acid changes on the function of the protein., Results: The patient, a two-month-old female, was admitted to the hospital for poor milk intake and poor mental response. Her clinical manifestations included feeding difficulty, shortness of breath and low muscle tone. Auxiliary laboratory test indicated that the infant was underdeveloped with abnormal liver, kidney, and heart functions accompanied by hyperlacticacidemia. She responded poorly to treatment and eventually died. Sequencing revealed that the child has carried compound heterozygous missense variants of the RRM2B gene, namely c.16delA (p.R6Gfs*22) and c.175G>C (p.A59P), which were respectively inherited from her father and mother, and both were newly discovered pathologic variants., Conclusion: The c.16delA and c.175G>C compound heterozygous variants of the RRM2B gene probably underlay the pathogenesis of MTDPS8A. Above finding has strengthened the understanding of the clinical feature and genetic etiology of this disease and expanded the mutation spectrum of the RRM2B gene.

By inserting microRNAs into the intron of EF1α promoter, we constructed a novel lentiviral vector knocking down PD-1 gene via microRNA and applied it to CAR-T cells. Lentiviral transduction efficiency and PD-1-silencing efficiency were detected by flow cytometry. PD-1 expression was detected by Western blotting. Relative expression of microRNA was measured by Q-PCR. Cytotoxicity of CAR-T cells based on this vector was tested by luciferase bioluminescence and flow cytometry. Compared with lentiviral vector with microRNA transcribed by U6 promotor, the transduction efficiency of lentiviral vector with microRNA which was inserted into the intron of EF1α promoter was more significant, and the knockdown rate of PD-1 was more than 90%, which was validated by flow cytometry and Western blotting. And the relative expression level of microRNA in Jurkat cells transduced with this novel lentiviral vector was shown by Q-PCR. Compared with normal CAR-T cells, CAR-T cells based on this vector showed stronger cytotoxicity against PD-L1 positive Raji cells. We successfully constructed a novel lentiviral vector that knocked down PD-1 via microRNA and verified the superiority of its transduction efficiency and knockdown efficiency of PD-1. CAR-T cells based on this vector can exert a more powerful cytotoxicity, thus providing theoretical support for the subsequent treatment of PD-L1 positive tumors.

Based on the color reaction of 2-(2-quinolinylazo)-1,3-dihydroxidebenzene (QADHB) with vanadium (v) and the solid phase extraction of its colored complex with C18 cartridge, a new method for the determination of vanadium was studied. In the presence of CTMAB and citric acid-sodium hydroxide buffer solution (pH 3.5) medium. QADHB reacts with vanadium(II) to form a stable 2:1 complex. The colored complex was extracted by C18 cartridge and eluted the retained chelate from cartridge with ethanol (containing 2% of acetic acid), then can be determined by spectrophotometry at 552 nm. Beer's law is obeyed in range of 0-1.0 mg/l. This method can be applied to the determination of vanadium in environmental samples with good results.