Objective To construct the lentiviral RNA interference vector targeting TPX2 and to obtain the human cervical cancer HeLa cell strain stably infected by TPX2-shRNA for studying the relationship between human cervical carcinoma and TPX2 gene.Methods By targeting TPX2 gene,four double-stranded DNA hairpin structures corresponding to shRNA were designed,synthesized and connected with Pglv2-U6-Puro to construct the recombinant plasmids.Then these recombinant plasmids were transformed into DH5α competent cells.The positive clone was extracted and transfected into 293T cells for virus packages after sequenced correctly.Human cervical carcinoma HeLa cell infected by these recombinant lentiviral was screened by Puromycin,then stable cell strain was obtained.The silencing effect of TPX2 in HeLa cell was detected by RT-fluorescent quantitative PCR and Western blot.Cell cycle and cell apoptosis wer detected by Flow cytometry.Results Sequencing results confirmed that 5 lentiviral was packaged successfully.The steady cell strain transfered TPX2-shRNA was screened with 0.4 μg/mL puromycin.HeLa cells infected by recombinant lentivirus all play the gene silencing effect especially in the group of TPX2-shRNA-1.In the group of TPX2-shRNA-1,TPX2mRNA (0.21 ± 0.07) and protein (0.19 ± 0.28) rela tive expression level is lower than those in the control group (1.08±0.07) (P＜0.01) and(0.64±0.03) (P＜ 0.01)respectively;G2 and S-phase cells are higher than those in the control group (P＜0.05)and the apoptosis rate was significantly more than those in the control group (P＜0.05).Conclusions The effective TPX2 genetic interference sequence was obtained,lentiviral vectors carrying TPX2shRNA was successfully constructed,and the HeLa cell strain with TPX2 silenced was successfully screened,which lay the research foundation for the study of the role of TPX2 in cervical cancer.

Lysine acylation is a ubiquitous protein modification that controls various aspects of protein function. However, it can be challenging to decipher the biological function of site-specific acylation modifications in living cells.The recently developed genetic code expansion (GCE) technology has enabled site-specific incorporation of unnatural amino acids (UAAs) that are structurally consistent with the natural acylation modifications in vivo through orthogonal aminoacyl-tRNA synthetase/tRNA pairs, thus facilitating the study of physicochemical properties and biological behaviors of homogeneously acylated proteins.Besides, GCE technology allows for the targeted introduction of UAAs that mimic acylation modifications but cannot be recognized by deacylases, which improves the stability of lysine acylation modification products.Moreover, the insertion of photo-crosslinked UAAs at specific sites of the target protein has been used to elucidate the reciprocal proteome of acylated modified proteins.Based on the introduction of different structural and functional acylation modifications, we described the novel design of GCE technology combined with three types of UAAs, and their application in studying the functional effects of protein acylation modifications on the enzyme activity, protein stability, cellular localization, protein-DNA interactions and protein-protein interactions of target proteins, with a description of the limitations and prospects of GCE technology in studying protein acylation modification.

Quantitative proteomics is a mass spectrometry-based toolkit used to analyze and quantify entire proteins contained in whole cells, tissues or organisms. It has become an increasingly important element in exploring the mechanism of various biological processes such as discovering novel biomarkers and unknown drug targets. Emerging advances in biological mass spectrometry instrumentation and data acquisition methodologies have provided a state-of-the-art platform for protein quantification, prompting the research of proteomics evolving from the simple qualitative to the accurate quantitative approach. This review aims to introduce the most recent advancements in mass spectrometry instrumentation and methodologies of data acquisition, focusing on their characteristics and applying fields. It also highlights several significant applications of biological mass spectrometry in pharmaceutical research such as quantifitation of drug transporters and metabolizing enzymes, and pharmacokinetic study of therapeutic peptides and proteins.

Objective:To investigate the immunogenicity and safety of a boost dose of measles, mumps, and rubella combined vaccine (MMR) for children 4 to 6 years old.Methods:Children, aged 4 to 6 years old, had vaccinated with 1 dose of measles and rubella combined vaccine(MR) at the age of 8 months and 1 dose of MMR vaccine at 18-months, were recruited in Shanxi, Inner Mongolia, and Beijing, respectively. All children were assigned into 4, 5 and 6-year-old group. The children who met inclusion and exclusion criteria were vaccinated with 1 dose MMR vaccine, and were collected blood samples before vaccination and 35 to 42 d after the vaccination. During the study period, adverse events were collected at 30 min, 1 d, 2 d, 3 d, 4-12 d, and 13 to 42 days after vaccination. Serum was tested for IgG antibodies against measles, mumps and rubella. Geometric mean concentrations (GMC) of measles, mumps, and rubella antibodies were compared among groups by analysis of variance or non-parametric test. Seropositive rates and adverse event rates were compared among groups by Chi-square test or Fisher exact test.Results:A total of 500 children were included in immunogenicity analysis and 535 children were included in safety analysis. The overall adverse event rate was 20.37%, the most of severity for adverse events was mild. The rates of local and systemic adverse events were 0.37% and 20.00%, respectively. Symptoms of local adverse events were redness. The main systemic adverse events were fever, followed by cough, rash and runny nose. Received a dose of MMR vaccine for booster immunization, the seropositive rates of measles antibody, mumps antibody and rubella antibody were above 99% for all 3 age groups, and there was no significant difference between groups. There were significant differences in mumps antibody GMC among groups ( P=0.042), but no significant differences in measles and rubella antibodies GMC. Conclusion:The immunogenicity and safety of a boosted MMR vaccintion in children aged 4, 5 and 6 years were all similar good.

OBJECTIVE: To detect variants of IVD gene among 4 neonates with suspected isovalerate acidemia in order to provide a guidance for clinical treatment. METHODS: 111 986 newborns and 7461 hospitalized children with suspected metabolic disorders were screened for acyl carnitine by tandem mass spectrometry. Those showing a significant increase in serum isovaleryl carnitine (C5) were analyzed for urinary organic acid and variants of the IVD gene. RESULTS: Four cases of isovalerate acidemia were detected, which included 2 asymptomatic newborns (0.018‰, 2/111 986) and 2 children suspected for metabolic genetic diseases (0.268‰, 2/7461). The formers had no obvious clinical symptoms. Analysis of acyl carnitine has suggested a significant increase in C5, and urinary organic acid analysis has shown an increase in isovaleryl glycine and 3-hydroxyisovalerate. Laboratory tests of the two hospitalized children revealed high blood ammonia, hyperglycemia, decreased red blood cells, white blood cells, platelets and metabolic acidosis. The main clinical manifestations have included sweaty foot-like odor, feeding difficulty, confusion, drowsiness, and coma. Eight variants (5 types) were detected, which included c.158G>A (p.Arg53His), c.214G>A (p.Asp72Asn), c.548C>T (p.Ala183Val), c.757A>G (p.Thr253Ala) and 1208A>G (p.Tyr403Cys). Among these, c.548C>T and c.757A>G were unreported previously. None of the variants was detected by next generation sequencing of 2095 healthy newborns, and all variants were predicted to be likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics. CONCLUSION The incidence of isovalerate acidemia in Liuzhou area is quite high. Screening of metabolic genetic diseases is therefore recommended for newborns with abnormal metabolism. The discovery of novel variants has enriched the mutational spectrum of the IVD gene.
Infant, Newborn ; Child ; Humans ; Acidosis ; Carnitine ; Erythrocytes ; High-Throughput Nucleotide Sequencing