Effect of gene transfection timing on TGF-ß1 expression in rabbit mandibular distraction gap.

Transforming growth factor-ß1 (TGF-ß1) is a member of the TGF-ß superfamily, and plays an important role in promoting various stages of intramembranous and endochondral bone formation. It is one of the major growth factors that influence new bone formation in the distraction gap during distraction osteogenesis (DO). The major problem of DO is the time required for the treatment. Reports show that gene therapy accelerates osteogenesis, which can significantly benefit patients with DO. However, the optimal timing of gene transfection has not yet been reported. In this study, we used the New Zealand rabbit mandibular DO model for transfecting recombinant plasmid pIRES-hVEGF165-hBMP2 during the latency, distraction, and consolidation periods of DO. The TGF-ß1 levels in the distraction gap were detected at different time-points by immunohistochemistry and analyzed semi-quantitatively with the CMIAS-2001A computerized image analyzer. The TGF-ß1 levels peaked after 7 days and decreased after 14 days of consolidation in each group. In contrast, the TGF-ß1 levels in the transfected distraction period group were significantly higher than those in the other groups. After 28 days of consolidation, TGF-ß1 levels decreased and there was no significant difference among the groups. These results indicated that the genes transfected in the distraction period up-regulated the expression of TGF-ß1 more than in the latency and consolidation periods, which promoted bone formation in the distraction gap through a series of biological effects. Thus, we obtained a remarkable effect on new bone formation, and showed that the distraction period is optimal for gene therapy.

Expression and clinical significance of glucose transporter 1 mRNA in bronchial brushing liquid-based cytology specimens from patients with and without lung cancer.

OBJECTIVE: The aim of this study was to evaluate the diagnostic utility of glucose transporter 1 (GLUT1) mRNA expression in bronchial brushing specimens from patients with lung cancer. METHODS: GLUT1 mRNA levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) in SurePath(TM) liquid-based cytology bronchial brushing specimens from patients with lung cancer (n=76) and benign lung disease (n=154). RESULTS: Compared with patients with benign disease and compared with cytology, GLUT1 mRNA was found significantly more frequently in patients with all carcinomas, squamous cell carcinomas, adenocarcinomas and small cell carcinomas, as well as central, peripheral and diffuse carcinomas (P<0.01). Minor differences were noted in GLUT1 mRNA and cytology results between histological types and tumour location but were not statistically significant. The diagnostic performance of RT-PCR analysis of GLUT1 mRNA was significantly higher than cytology in terms of sensitivity (97.4 ± 3.6% versus 65.8 ± 10.7) and negative predictive value (98.6 ± 1.9%, versus 85.6 ± 5.1%) but specificity (90.9 ± 4.5%) and positive predictive value (84.1 ± 7.6%) were lower than cytology (100%). CONCLUSIONS: Using liquid-based cytology, RT-PCR can be performed on bronchial brushing specimens to detect GLUT1 mRNA expression, and may be a useful adjunct to cytology diagnosis. It was more sensitive than cytology but its lower specificity should be taken into account.

[Analysis of clinical phenotype and genotype of Chinese children with disorders of sex development].

Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.

Triphalangeal thumb-polysyndactyly syndrome and syndactyly type IV are caused by genomic duplications involving the long range, limb-specific SHH enhancer.

BACKGROUND: The Sonic hedgehog (SHH) protein produced in the zone of polarising activity (ZPA) is a major determinant of the identity and numbers of digits in early limb development. Preaxial polydactyly types II (PPD2) and III (PPD3) have been mapped to a critical region at 7q36, and subsequently shown to be caused by point mutations in the ZPA regulatory sequence (ZRS), a long range cis-regulator for the SHH gene. Triphalangeal thumb-polysyndactyly syndrome (TPTPS) and syndactyly type IV (SD4) were also mapped to the 7q36 region but pathogenic mutations in ZRS have not yet been affirmed. METHODS AND RESULTS: We performed linkage and haplotype analysis in six Han Chinese families with TPTPS and/or SD4, and refined the disease locus to an interval of 646 kb containing ZRS. In all families, the affected individuals heterozygous at rs10254391 (a single nucleotide polymorphism within ZRS) revealed a remarkable allele imbalance on sequence chromatogram. Using real-time quantitative polymerase chain reaction (qPCR), we identified duplication of ZRS and found that this duplication segregated with the limb phenotypes in all families but was not detected in unaffected family members or in unrelated control individuals. The duplication was also confirmed by interphase fluorescence in situ hybridisation (FISH) in an affected individual. We designed 17 additional qPCR assays and defined the minimum duplications in all six families, ranging from 131kb to 398kb. CONCLUSION: Both TPTPS and SD4 are due to duplications involving ZRS, the limb specific SHH enhancer. Point mutations in the ZRS and duplications encompassing the ZRS cause distinctive limb phenotypes.

Immunocytochemical panel for distinguishing carcinoma cells from reactive mesothelial cells in pleural effusions.

OBJECTIVE: The aim of this study was to evaluate the individual and combined diagnostic utility of carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CK19) and HBME-1 in pleural effusions of patients with lung cancer. STUDY DESIGN: CEA, CK19 and HBME-1 were detected by immunocytochemistry in pleural effusions from patients with lung cancer (86 cases) and without lung cancer (40 cases). RESULTS: CEA and CK19 expression were significantly higher in the carcinoma cell group and in three subgrouped as adenocarcinoma (AC), squamous cell carcinoma (SCC) and small cell lung cancer than in the mesothelial cell group, whereas HBME-1 expression was lower in the former group (P < 0.01). In the subgrouped tumours, CEA expression was higher in AC than in SCC (P < 0.05), whereas HBME-1 expression was higher in SCC than in AC (P < 0.01). Used alone, CK19 had the highest sensitivity (95.3%) and accuracy (93.7%), whereas CEA had the highest specificity (97.5%). When combinations of antibodies were evaluated together and membrane staining with HBME-1 taken as a negative outcome, CK19 and HBME-1 gave a high diagnostic performance: sensitivity of 100.0% and accuracy of 95.2% respectively. CONCLUSION: A panel of CEA, CK19 and HBME-1 monoclonal antibodies proved to be suitable for distinguishing carcinoma cells from reactive mesothelial cells in pleural effusions.

[Analysis of allergen spectrum and sensitization factors in children with allergic rhinitis].

Objective:The aim of this study is to analyze the distribution of common allergens in allergic rhinitis patients aged 2-15 in Zhangjiagang area,in order to provide a path to know the epidemic features of children with AR, and supports epidemiological materials to find better prevention, diagnosis and treatment for them.Method:From July.2015 to July.2018,1 320 children aged 2-15 who were suspected with allergic rhinitis by the doctors of ENT clinic in the Zhangjiagang Affiliated Hospital,were enrolled in this project.Using the serum specific IgE detection methods to detect common allergens in those patients,then analyze the relationship among allergens and gender,age and visiting time of every single patient.Result:The total positive rate of 1 320 children was 82.50%,and the total positive rate of 8 common inhaled allergens was 82.50%.The main allergens were dust mites and dogs. The total positive rate of Children aged in 7-12 was the highest,and the positive rate of dust mites of male was higher than female.The common food allergens total positive rate was 9.09%.And the top three food allergens were milk,shrimp,crab.The number of visitors in July and August was the most in a year.The dust mite had the highest positive rate among common inhaled allergen in a year,and the maximum value appeared in July.The rate of patients who were allergic to one inhalant allergen were lower than those who were allergic to two or more inhalant allergens.Conclusion:The dust mite is the major allergen above all.The total positive rate of Children aged in 7-12 was the highest,and the positive rate of dust mites of male was higher than female;the maximum value appeared in July and August.The rate of patients who were allergic to one inhalant allergen were lower than those who were allergic to two or more inhalant allergens.

[Synthesis and antibacterial activity of 1-substituted benzyl quinolone acid derivatives].

The compounds of quinolone class, which were studied extensively and developed very quickly in the last decades, showed high activity, low toxicity and broad antibacterial spectrum. About thirty new compounds of 1,7-disubstituted-6-fluoro-1, 4-dihydro-4-oxoquinoline-3-carboxylic acids were synthesised and evaluated in vitro against S. aureus 25923, E. coli-25922 and P. aeruginosa. Their activities were low. The minimum inhibitory concentrations (MIC microgram/ml) of these compounds against Gram-positive bacteria were higher than those against Gram-negative bacteria. The relative in vitro activity contributed by 1-benzyl was: benzyl greater than p-chlorobenzyl greater than p-nitrobenzyl groups.

[Rejection of physico-chemical indices for pathological matrix in a quantitative drug design].

In a quantitative drug design, correlation of coefficient matrix of physico-chemical indices Xi is often pathological when these indices are highly correlated. The regression equation of biological activity Y about these indices Xi obtained in this case is not stable. In this paper, a method is given to obtain a stable regression equation: giving a critical valne alpha and finding out the indices among which correlation coefficient is not less than alpha. The following are the rules to reject some of them. If Xi and Xj is highly correlated (magnitude of rij greater than or equal to alpha, rij is the correlation coefficient of Xi and Xj), and magnitude of rin greater than magnitude of rjn (rin and rjn are correlation coefficients of Xi and Xj about Y, respectively), than the index Xj is rejected, otherwise, Xi is rejected. Stable equation can be obtained by stepwise regression with the remaining indices.

[Synthesis and antifertility actions of gossypol derivatives and phenol aldehydes].

A series of trihydroxynaphthaldehydes, polyhydroxybiphenol-asdehydes, polyhydroxybinaphthyl aldehydes and some gossypol derivatives were synthesized for antifertility experimental studies.

[Gossypol and its related compounds as contraceptive drugs and drugs for gynecological diseases].

PIP: The article is a summary of research in China and overseas on the effect of gossypol, its compound and its derivatives in contraception and in the treatment of gynecological diseases. Many of these studies demonstrated the antifertility effects of gossypol and its compounds. Nevertheless, some issues related to its use still need to be resolved. The incidence of reduced blood potassium among users and the irreversibility of infertility resulting from oral administration in males have been important concerns. Besides the contraceptive dosage of gossypol has produced harmful effects on the liver. Whether there are longterm toxic effects on the heart and kidney is still unknown. Numerous reforms of the molecular structure of gossypol have eliminated the contraceptive effects. The extracts from the roots of cotton plants may turn out to be a hopeful oral male contraceptive. The implant of gossypol analogue inside the body with slow drug release may be a possible solution against the toxic effects. Gossypol analogues or derivatives may also be made into a vaginal spermicide. Their function in early-stage abortion has also been proved. Gossypol has also been an effective drug for treatment of gynecological diseases, such as menopause functional bleeding, uterine tumor, and endometrial symptoms. Side effects have occurred among some users. Reduced dosage alleviated these problems and resulted in satisfactory long term curative effects.^ieng

Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase.

In these studies we demonstrate that insulin stimulates both tyrosine and serine phosphorylation of the insulin receptor after its partial purification on wheat germ-agarose, and after affinity purification on insulin-agarose. Analysis of the serine phosphate incorporated into partially purified or highly purified insulin receptor suggests that an insulin-sensitive serine kinase (IRSK) copurifies with the insulin receptor. Following trypsin digestion, reversed-phase high pressure liquid chromatography (HPLC) analysis of the phosphorylated, affinity-purified insulin receptor preparation reveals phosphopeptide profiles similar to those of trypsin-digested receptors immunoprecipitated from 32P-labeled fibroblasts overexpressing the human insulin receptor. The major insulin-stimulated HPLC phosphopeptide peak from insulin receptors labeled in intact cells contains a hydrophilic phosphoserine-containing peptide which rapidly elutes from a C18 column. HPLC and two-dimensional separation indicate that the same phosphopeptide is obtained when affinity-purified insulin receptors are phosphorylated by IRSK. The serine containing tryptic peptide within the cytoplasmic domain of the human insulin receptor predicted to elute most rapidly upon HPLC had the sequence SSHCQR corresponding to residues 1293-1298. A synthetic peptide containing this sequence is phosphorylated by the insulin receptor/IRSK preparation. After alkylation and trypsin digestion, the synthetic phosphopeptide comigrates with the alkylated, tryptic phosphopeptide derived from insulin receptor phosphorylated in vitro by IRSK. We propose that serine 1293 or 1294 of the human insulin receptor is a major site(s) phosphorylated on the insulin receptor in intact cells and is phosphorylated by IRSK. Furthermore, insulin added directly to affinity-purified insulin receptor/IRSK preparations stimulates the phosphorylation of synthetic peptides corresponding to this receptor phosphorylation site and another containing threonine 1336. Kemptide phosphorylation is not stimulated by insulin under these conditions. No phosphorylation of peptide substrates for Ca2+/calmodulin-dependent protein kinase, protein kinase C, casein kinase II, or cGMP-dependent protein kinase by IRSK is detected. These data indicate that IRSK exhibits specificity for the insulin receptor and may be activated by the insulin receptor tyrosine kinase in an insulin-dependent manner.

[Antifertility actions of gossypol derivatives and analogues].

This paper reports the results of 32 gossypol derivatives and analogues. Among 12 compounds screened by oral administration to male rats, only dipotassium gossypolate (NC030) exhibited an antifertility activity similar to gossypol. Among 24 compounds screened by injecting into pouches of cauda epididymides of rats, 10 exhibited spermicidal activities.