The dynamic methylation of human papillomavirus (HPV) 16 DNA is thought to be associated with the progression of cervical lesions. Previous studies that did not consider the physical status of HPV 16 may have incorrectly mapped HPV 16 methylomes. In order to identify reliable biomarkers for squamous cervical cancer (SCC), we comprehensively evaluated the methylation of HPV 16 depending on the integration incidence of each sample. Based on the integration status of 115 HPV 16-infected patients (50 SCC, 30 high-grade squamous intraepithelial lesion [HSIL], and 35 low-grade squamous intraepithelial lesion [LSIL]) and HPV 16-infected Caski cell lines by PCR detection of integrated papillomavirus sequences, we designed a series of primers that would not be influenced by breakpoints for a high-resolution melting (HRM) PCR method to detect the genome methylation. A few regions with recurrent interruptions were identified in E1, E2/E4, L1, and L2 despite scattering of breakpoints throughout all eight genes of HPV 16. Frequent integration sites often occurred concomitantly with methylated CpG sites. The HRM PCR method showed 100% agreement with pyrosequencing when 3% was set as the cutoff value. A panel of CpG sites such as nt5606, nt5609, nt5615, and nt5378 can be combined in reweighing calculations to distinguish SCC from HSIL and LSIL patients which have high sensitivity and specificity (88% and 92.31%, respectively). Our research shows that combination of CpG sites nt5606, nt5609, nt5615, and nt5378 can be used as potential diagnosis biomarkers for SCC, and the HRM PCR method is suitable for clinical methylation analysis.
Background: Ureaplasma parvum and Ureaplasma urealyticum are commonly found in the cervix of women with non-chlamydial and non-gonococcal cervicitis or non-specific cervicitis (NSC). However their contribution to the aetiology of NSC is controversial. Methods: U. parvum and U. urealyticum were identified and quantified in cervical swabs collected from 155 women with NSC and 312 controls without NSC, using real-time PCR. The relative bacterial quantification was then calculated using the Ureaplasma copy number divided by the number of host cells; this is important for the correction of bias linked to the number of cells harvested in different swabs. Results: Ureaplasma was detected in 58.7% (91/155) of NSC patients: U. parvum in 30.3%, U. urealyticum in 16.1%, and mixed infection in 12.3%. It was also detected in 54.5% (170/312) of controls: U. parvum in 33.0%, U. urealyticum in 11.5%, and mixed infection in 9.9%. There were no significant differences for U. parvum, U. urealyticum, or mixed infection between the 2 groups (p > 0.05). However, both biovars were present at higher concentrations in NSC patients than in controls (p < 0.05). Using >10 copies/1000 cells as a reference, the positive rate of U. parvum in NSC patients was 16.1%, significantly higher than that in controls at 5.1% (relative risk 3.145, p < 0.05); positive rates of U. urealyticum in NSC patients and controls were 28.4% and 8.7%, respectively, with a statistically significant difference (relative risk 3.131, p < 0.05). Conclusions: Ureaplasma can adhere to host cells, colonize, internalize, and subsequently produce pathological lesions. A high density of Ureaplasma in the cervix may be associated with the aetiology of NSC.
High-risk human papillomavirus (hr-HPV) infection is a necessary cause of cervical cancer. However, other common lower genital tract microbes may increase hr-HPV infection and their related cervical cytopathy.To confirm this hypothesis, cervical brush and vaginal swab specimens were collected from 826 adult patients who were divided into the hr-HPV-positive group (254) and the negative group (572) by real-time PCR assay. Cervical specimens were tested for Ureaplasma parvum (UP), Ureaplasma urealyticum (UU), and Chlamydia trachomatis (CT) using PCR analysis. Vaginal secretion was detected for Trichomonas vaginalis (TV), Candida spp., and bacterial vaginosis (BV) with conventional assay.Among hr-HPV-positive women, UP was found in 51.6%, UU in 15.4%, CT in 15.7%, Candida spp. in 11.0%, TV in 3.1%, and BV in 20.5%. In the hr-HPV-negative group, UP was positive in 36.2%, UU in 8.6%, CT in 4.0%, Candida spp. in 12.4%, TV in 0.2%, and BV in 7.0%. Multivariate logistic regression analysis with age-adjusted showed that UU (OR, 1.757), UP (OR, 1.804), CT (OR, 3.538), BV (OR, 3.020), and TV (OR, 14.109) were risk factors on hr-HPV infection (P < 0.05).These microbes might induce cervical chronic inflammation that would damage the mucosal barrier and immune protection to promote the infection of hr-HPV.
Abstract Background Ureaplasma spp . are associated with various infectious diseases in females, but there is still limited evidence regarding whether they are related to nonspecific cervicitis. The aim of this study was to develop and evaluate a digital droplet PCR (ddPCR) assay for the detection and quantification of Ureaplasma spp. in cervical swabs. Methods A total of 267 non-specific cervicitis (NSC) patients and 195 asymptomatic females were included in this study. We produced standard curves for Ureaplasma spp . to evaluate the analytical performance of the ddPCR assay. Then, we detected and quantified the bacterial load of Ureaplasma spp. in cervical swabs. Results The prevalences of U. parvum were 37.8% (101/267) and 29.7% (58/195), U. urealyticum were 9.0% (24/267) and 8.7% (17/195) in the NSC group and control group, respectively. In addition, the median copy number of U. parvum was 2.5 × 10 4 copies/ml ( n = 101) in the NSC group and 9.2 × 10 3 copies/ml ( n = 58) in the control group. The U. parvum load in the NSC group was significantly higher than that in the asymptomatic individuals ( P < 0.001). whereas the median load of U. urealyticum was 8.4 × 10 3 copies/ml ( n = 24) and 1.4 × 10 3 ( n = 17) copies/ml in the two groups, respectively, , the difference was not statistically significant ( P = 0.450). Conclusions Our study is the first to develop a droplet digital PCR (ddPCR) method for the detection and quantification of Ureaplasma spp. in clinical samples, and the method has excellent analytical performance and a wide range of clinical application prospects.
The efficacy of artificial neural network (ANN) models employing laboratory variables for predicting fatty liver disease (FLD) remains inadequately established. The study aimed to develop ANN models to precisely predict FLD.
Abstract Background: Ureaplasma spp. are association with a various of infectious diseases in female, but it still limited evidence for the pathogenicity in nonspecific cervicitis. The aim of this study was to develop and evaluate a digital droplet PCR (ddPCR) assay for quantified the load of Ureaplasma spp in cervical swabs. Methods: A total of 293 non-specific cervicitis (NSC) patients and 211 asymptomatic female fulfilled the inclusion criteria. Cervical swabs were identified by qPCR and further absolutely quantified by ddPCR. Results: The prevalence of U.parvum were 51.9% (152/293) and 46.9% (99/211); while U.urealyticum were 8.2% (24/293) and 8.1% (17/211) in the NSC and Control group, respectively. In addition, the average Ct value and median copy number per microliter of U.parvum were 31.33 (n=152) and 599 (n=48) in the NSC group and 33.68 (n=99) and 17.4 (n=33) in control group, respectively, suggest that the load of U.parvum of NSC group were significantly higher than the asymptomatic individual (P<0.001). But, the median load number of U.urealyticum were 1.26 (n=22) and 5.35 (n=14) copies per microliter two groups, the difference was no statistical significance (P>0.05). Conclusions: our study suggests that often carrying U.parvum at a high load but not U.urealyticum may have an important implications on the development and progression of cervicitis among female.
β-defensin is a primary protein immune factor in channel catfish's (Ietalurus punetaus) resistance to pathogenic microorganisms. Its primary structure contains a signal peptide composed of 24 amino acid residues at the N-terminal and a mature peptide composed of 43 amino acid residues at the C-terminal. The mature peptide region is responsible for the biological activity of β-defensin. In the present study, a recombinant strain of Pichia pastoris that produces channel catfish β-defensin, was constructed to realize the biosynthesis of channel catfish β-defensin based on eukaryotic expression. First, the β-defensin gene "IPBD" was isolated from the skin of channel catfish by RT-PCR. After linking it with the expression vector pPICZA, pPICZA-IPBD was transferred into competent P. pastoris X-33 cells to obtain recombinant P. pastoris strains. The yeast transformants with multi-copy gene inserts were obtained by using the culture medium containing 1 000 μg/mL zeocin. Using BMM culture medium (without amino nitrogen culture medium) instead of BMMY culture medium (with amino nitrogen culture medium), the fermentation and culture conditions of the recombinant strain were optimized, and the optimal conditions for producing channel catfish β-defensin were determined as follows: the expression was induced for 96 h with 1.0% methanol at 28 °C , 250 r/min. Purified protein with molecular weight of 5.98 kDa was obtained by nickel affinity chromatography, and MALDI-TOF/TOF mass spectrometry proved that it was the expected recombinant IPBD. The antibacterial test results showed that the inhibitory rates of recombinant IPBD on Gram-positive Staphylococcus aureus and Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa were 69.6%, 71.6% and 65.8%, respectively. This study provides a recombinant DNA technique for the development of small molecule natural antibacterial peptide from fish.β-防御素是斑点叉尾鮰Ietalurus punetaus 抵御病原微生物侵染的首要蛋白质免疫因子，其一级结构包含氨基端24 个氨基酸组成的信号肽和羧基端43 个氨基酸组成的成熟肽，该成熟肽赋予β-防御素的生物学活性。文中首次构建了产斑点叉尾鮰β-防御素的毕赤酵母Pichia pastoris 重组菌株，实现了基于真核表达的斑点叉尾鮰β-防御素的生物合成。首先通过RT-PCR 从斑点叉尾鮰皮肤中分离β-防御素成熟肽基因“IPBD”，将其与表达载体pPICZαA连接并转入毕赤酵母X-33 后，获得重组毕赤酵母菌株；经含1 000 μg/mL 博来霉素的培养基筛选，获得高拷贝重组菌株。以BMM 培养基 (无氨基氮源培养基) 替代BMMY 培养基 (含氨基氮源培养基)，对重组菌株的发酵培养条件进行优化，确定其产斑点叉尾鮰β-防御素的最适条件为：28 ℃、250 r/min、1.0%甲醇诱导表达96 h。重组菌株产物经镍离子亲和层析获得分子量为5.98 kDa 的纯化蛋白，基于MALDI-TOF-TOF 的质谱分析证明该纯化蛋白为重组IPBD。抑菌活性测定结果表明重组IPBD 对革兰氏阳性的金黄色葡萄球菌Staphylococcus aureus、单增李斯特菌Listeria monocytogenes 以及革兰氏阴性的铜绿假单胞菌Pseudomonas aeruginosa 的抑菌率分别为69.6%、71.6%和65.8%。本研究为鱼类来源天然小分子抗菌肽的开发提供了可参考的重组DNA 技术。.
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