The application value of PAX1 and ZNF582 gene methylation in high grade intraepithelial lesion and cervical cancer.

PURPOSE: To investigate the possibility of using the methylation level of PAX1/ZNF582 gene as molecular marker to differentiate the progression of cervical cancer. METHODS: From January 2016 to March 2018, 150 patients, who were admitted to Cervical Disease Diagnosis and Treatment Center of Xuzhu Maternity and Child Care Hospital, were enrolled in this study. Patients were classified into chronic cervicitis (for 19 cases), low-grade squamous intraepithelial lesion (LSIL) (18 cases), high-grade squamous intraepithelial lesion (HSIL) (37 cases) and squamous cell carcinoma (SCC) (31 cases). All patients underwent several tests including Thin-prep cytology test (TCT), HPV DNA detection and detection of methylation level of PAX1/ZNF582 genes. RESULTS: For diagnosis of HSIL, the area under curve (AUC) was 0.878 (95% CI 0.806 ~ 0.950); the threshold for PAX1 was 12.285, the sensitivity and specificity were 91.9% and 72.8%, respectively. The AUC of ZNF582 gene detection was 0.900 (95% CI 0.842 ~ 0.959), the threshold was 11.56, while the sensitivity and specificity were 97.3% and 76.7%, respectively. Among various tests we conducted, PAX gene detection methods showed the highest specificity (97.30%). PAX1/ZNF582 gene detection method demonstrated the highest accuracy. CONCLUSIONS: For patients with high-grade cervical lesion and cervical cancer, the methylation level of PAX1/ZNF582 gene could be applied as a noteworthy biomarker for diagnosis and for cervical cancer classification.

Molecular cloning and functional characterization of cyclin E and CDK2 from Penaeus monodon.

Reduced reproductive performance of the black tiger shrimp (Penaeus monodon) has caused economic losses and hampered the fishing industry. Detailed investigation of the molecular mechanism by which the cell cycle is regulated in this organism is needed to understand the development and maturation of ovaries and oocytes, with a view to improving reproductive capacity. Cell cycle progression is mainly determined by cyclin-dependent kinase (CDK) and cyclin complexes, the cyclin E/CDK2 complex playing a key role in G1/S transition. However, knowledge of the interplay between cyclin E and CDK2 in invertebrates remains limited. In this study, full-length P. monodon cyclin E (Pmcyclin E) and CDK2 (PmCDK2) sequences were cloned. The open reading frame of Pmcyclin E was 1263 bp in length and encoded a 47.9-kDa protein, while that of PmCDK2 was 921 bp, encoding a protein of 34.9 kDa. Recombinant cyclin E and CDK2 proteins were expressed in Escherichia coli and purified by Ni-chelating affinity chromatography. In addition, a pull-down assay was performed to identify any interaction between Pmcyclin E and PmCDK2. This research provides a basis for the study of the functional mechanisms of the cyclin E/CDK2 complex in shrimp, further enriching our knowledge of invertebrate cell cycle regulation.

Polymorphisms in the vascular endothelial growth factor (VEGF) gene associated with asthma.

It has been proven that vascular endothelial growth factor (VEGF) is involved in airway restoration and the development of asthma. We sought to examine the relevance of VEGF gene polymorphisms to asthma in the Chinese Han population. We extracted the whole genomic DNA from the peripheral blood of 471 participants, including 226 patients with asthma and 245 healthy controls. Seven single nucleotide polymorphisms (SNPs) of the VEGF gene were genotyped using the MassARRAY system. The data were then analyzed using HaploView 4.0 and SPSS 20.0 softwares. When comparing the asthma and control groups, significant differences were found in the genotype frequencies of rs3025020 and rs3025039 (P = 0.001 and P = 0.011, respectively). The T alleles in rs3025020 and rs3025039 were significantly more prevalent in the asthma group than in controls (P = 0.0003, P = 0.001, respectively). Furthermore, a strong linkage disequilibrium was observed in three blocks (block1-3). In block3, the asthma group had a significantly lower C-C haplotype frequency of haplotype 1 (P = 0.000015), a higher T-C haplotype frequency of haplotype 2 (P = 0.020), and a significantly higher C-T haplotype frequency of haplotype 3 (P = 0.001). The rs3025020 genotype showed no correlation with the clinical phenotype that may cause asthma. The VEGF SNPs rs3025020 and rs3025039 may be associated with the development of asthma, indicating the role of VEGF in asthma.

Molecular cloning and expression analysis of MAT1 gene in black tiger shrimp (Penaeus monodon).

MAT1 (ménage à trois 1), an assembly factor and targeting subunit of the CDK-dependent kinase (CAK), can regulate the cell cycle, transcription, and DNA repair. This study was intended to investigate the role of MAT1 in the reproductive maturation of black tiger shrimp (Penaeus monodon). In this study, the P. monodon MAT1 (PmMAT1) gene was identified and characterized. The full-length cDNA of PmMAT1 was 1490 bp in length with an open-reading frame of 993 bp corresponding to 330 amino acids. The temporal expression of PmMAT1 in various tissues was measured by quantitative real-time PCR with the highest expression observed in ovaries. In the ovaries, the PmMAT1 gene was continuously but differentially expressed during the maturation stages. Comparative analyses of MAT1, CDK7, and cyclin H in the CAK complex of P. monodon indicated that the expression of CDK7 and cyclin H coincided with that of MAT1 during the ovary maturation stages. Serotonin (5-HT) injection promoted the expression level of PmMAT1 in the ovaries of shrimp at 6-48 h post-injection. These results indicate that PmMat1 plays a prominent role in the process of ovarian maturation.

Characterization and expression analysis of a cyclin B gene from black tiger shrimp (Penaeus monodon).

The open reading frame of black tiger shrimp (Penaeus monodon) cyclin B (Pmcyclin B) was identified, based on cDNA sequence registered in GenBank (accession No. EF015590). The target sequence was 1206 bp, corresponding to 401 amino acids. Two conserved signature sequences of the cyclin B gene family were found in the Pmcyclin B deduced aa sequence. Temporal expression of Pmcyclin B in different tissues, including ovary, lymphoid organ, brain, blood, muscle, heart, gill, hepatopancreas, and intestine, were quantified by quantitative real time PCR. Messenger RNA expression levels of Pmcyclin B were greatest in the ovary, compared to other tissues (P < 0.05). Temporal expression of Pmcyclin B in the ovary at six different developmental stages was investigated by real-time PCR; no significant difference was observed (P < 0.05). Recombinant Pmcyclin B protein and its polyclonal antibody were successfully produced. Western blot analysis revealed differential expression of Pmcyclin B in ovaries in developmental stages II to IV; a positive signal (45 kDa) was observed in all ovarian stages assessed, but was most intense at stage III. Pmcyclin B protein was assessed by immunohistochemistry and was localized to the cytoplasm of prophase oocytes at stage II and enriched in the nuclei of pro-metaphase oocytes at stages III and IV. Results from this study indicate that Pmcyclin B is constitutively expressed and plays an important role in ovarian maturation in P. monodon.

Health and production traits in bovine are associated with single nucleotide polymorphisms in the NOD2 gene.

The NOD2 gene plays a fundamental role in initiating the inflammatory and subsequent immune response. NOD2 was previously identified as a susceptibility locus for inflammatory bowel diseases in humans. In this study, we detected 2 mutations in exon 12 (A→T, G→A) among 5 cattle breeds (N = 315) and analyzed their associations with production traits and genetic resistance against bovine mastitis in Chinese Holstein and Chinese Simmental breeds (N = 218). The transitions (A→T) at position 114 bp were associated with somatic cell score (P < 0.01). The G→A at position 1594 bp plays a critical role in increasing 305-day milk yields. In Chinese Holstein and Chinese Simmental breeds, the BB genotype may contribute to disease susceptibility. Compared to all genotypic combinations, the A, B, and FF genotypes are beneficial not only for reducing somatic cell score but also for increasing production.