Vaginal microbiota molecular profiling and diagnostic performance of artificial intelligence-assisted multiplex PCR testing in women with bacterial vaginosis: a single-center experience.

Background: Bacterial vaginosis (BV) is a most common microbiological syndrome. The use of molecular methods, such as multiplex real-time PCR (mPCR) and next-generation sequencing, has revolutionized our understanding of microbial communities. Here, we aimed to use a novel multiplex PCR test to evaluate the microbial composition and dominant lactobacilli in non-pregnant women with BV, and combined with machine learning algorithms to determine its diagnostic significance. Methods: Residual material of 288 samples of vaginal secretions derived from the vagina from healthy women and BV patients that were sent for routine diagnostics was collected and subjected to the mPCR test. Subsequently, Decision tree (DT), random forest (RF), and support vector machine (SVM) hybrid diagnostic models were constructed and validated in a cohort of 99 women that included 74 BV patients and 25 healthy controls, and a separate cohort of 189 women comprising 75 BV patients, 30 intermediate vaginal microbiota subjects and 84 healthy controls, respectively. Results: The rate or abundance of Lactobacillus crispatus and Lactobacillus jensenii were significantly reduced in BV-affected patients when compared with healthy women, while Lactobacillus iners, Gardnerella vaginalis, Atopobium vaginae, BVAB2, Megasphaera type 2, Prevotella bivia, and Mycoplasma hominis were significantly increased. Then the hybrid diagnostic models were constructed and validated by an independent cohort. The model constructed with support vector machine algorithm achieved excellent prediction performance (Area under curve: 0.969, sensitivity: 90.4%, specificity: 96.1%). Moreover, for subjects with a Nugent score of 4 to 6, the SVM-BV model might be more robust and sensitive than the Nugent scoring method. Conclusion: The application of this mPCR test can be effectively used in key vaginal microbiota evaluation in women with BV, intermediate vaginal microbiota, and healthy women. In addition, this test may be used as an alternative to the clinical examination and Nugent scoring method in diagnosing BV.

Association of co-exposure to metal(loid)s during pregnancy with birth outcomes in the Tibetan plateau.

Maternal metal (loid)s exposure has been related to birth outcomes but the results are still inconclusive. Most previous studies have discussed the single metal (loid)s, neglecting the scene of co-exposure. We examined the associations of both single metal (loid)s and metal mixtures with birth outcomes in a birth cohort from the Tibetan Plateau, including body weight, body length, head circumference, small for gestational age (SGA), and Ponderal index (PI). In our analysis of 1069 women, we measured 29 metal (loid)s in urine samples in the third trimester. The associations of single metal (loid)s with categorical or continuous birth outcomes were evaluated using a generalized linear mixed-effects model or linear mixed-effects model, respectively. The least absolute shrinkage and selection operator, Bayesian kernel machine, and Quantile g-computation regression were used to explore the joint association. We also evaluated the interactive effects of ethnicity and altitude on the effect of metal (loid)s on birth outcomes. Copper (Cu) concentration in maternal urine was positively associated with SGA, birth weight, birth length, and head circumference in the single pollutant models. For instance, Cu was associated with an increased risk of SGA [OR (95% CI) = 1.56 (1.23, 1.97); P < 0.001]. We didn't find significant joint association of metal mixtures with birth outcomes except a positive association between the mixture of Cu, Magnesium (Mg), and Iron (Fe) with the risk of SGA when the exposure level was above its 80th percentile, and Cu dominated the adverse association in a non-linear manner. Living altitude modified the associations of Cu with SGA and the positive association was only found in participants living at high altitude. In conclusion, maternal urinary metal (loid)s, especially Cu, was the dominant harmful metal (loid)s when associated with SGA on the Tibetan Plateau.

Inhibiting TLR9 Signaling Stimulates Apoptosis and Cell Cycle Arrest, and Alleviates Angiogenic Property in Human Cervical Cancer Cells.

AIMS: The aim of the study was to assess the effect of blocking TLR9 signaling on the proliferation of cervical cancer cells and its angiogenic property. BACKGROUND: Toll-Like Receptors (TLRs) have been implicated for their crucial role in not only cervical cancer but also in other malignancies. TLR9 is expressed on an array of cells such as macrophages, dendritic cells, melanocytes, and keratinocytes and is reported to modulate oncogenesis along with tumorigenesis by augmenting NF-κB mediated inflammation within the tumor environment. TLR9 has also been reported to positively regulate oncogenesis within the cervix and as a marker to evaluate malignant remodeling of cervical squamous cells. Therefore, this study was designed to explore the functional relevance of blocking the TLR9signaling pathway in cervical cancer cells. OBJECTIVE: The objective of the current study was to investigate the effect of human TLR9 antagonist, ODN INH-18, on apoptosis and cell cycle regulation, and angiogenic property of human cervical cancer Caski cells. METHODS: MTT assay was performed to measure cell viability and flow cytometry analysis was performed to assess cell cycle arrest. Quantitative Real-Time PCR (qRT-PCR) analysis was performed to measure fold change in the gene expression of various markers of apoptosis, cell cycle regulation, and angiogenesis. RESULTS: The qRT-PCR results showed a higher expression level of TLR9 mRNA in Caski cervical cancer cells as compared to normal cervical keratinocytes. The apoptotic, angiogenic, and cell cycle regulatory factors were also deregulated in Caski cells in comparison to normal keratinocytes. The MTT assay demonstrated that treatment of TLR9 antagonist, ODN INH18, significantly reduced the proliferation of Caski cells in a dose-dependent manner. Treatment of ODN INH18 led to substantial cell cycle arrest in Caski cells at G0/G1 phase. Moreover, the qRT-PCR results demonstrated that ODN INH18 treatment led to suppressed mRNA expression of Bcl-2 and enhanced expression of Bax, signifying the induction of apoptosis in Caski cells. Moreover, the expression of cyclin D1, Cdk4, and Cdc25A was found to be reduced whereas expression of p27 was increased in ODN INH18-treated Caski cells; indicating G0/G1 phase arrest. Interestingly, expression of VEGF and VCAM-1 was found to be significantly inhibited in ODN INH18-treated Caski cells, substantiating alleviation of angiogenic property of cervical cancer cells. CONCLUSION: The results of our study suggest that inhibiting TLR9 signaling might be an interesting therapeutic intervention for the treatment of cervical cancer.

Overexpression of circ_CELSR1 facilitates paclitaxel resistance of ovarian cancer by regulating miR-149-5p/SIK2 axis.

Circular RNAs (circRNAs) have emerged as vital regulators in the chemoresistance of diverse human tumors, including ovarian cancer. In the present study, we attempted to explore the function of circ_CELSR1 in paclitaxel resistance of ovarian cancer. Quantitative real-time PCR (qRT-PCR) was conducted for the expression of circ_CELSR1, miR-149-5p and salt inducible kinase 2 (SIK2). Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the half-maximal inhibitory concentration (IC50) of paclitaxel and cell viability. Colony formation assay was adopted for cell colony formation. Flow cytometry analysis was conducted to analyze cell cycle process and apoptosis. Western blot assay was utilized to determine the protein levels. RNA immunoprecipitation (RIP) and dual-luciferase reporter assays were conducted to verify the association between miR-149-5p and circ_CELSR1 or SIK2. Murine xenograft model assay was carried out to determine the effect of circ_CELSR1 in paclitaxel resistance in vivo. Circ_CELSR1 was upregulated in paclitaxel-resistant ovarian cancer tissues and cells. Circ_CELSR1 knockdown enhanced paclitaxel sensitivity and cell apoptosis and repressed cell viability, colony formation and cell cycle process in paclitaxel-resistant ovarian cancer cells. For mechanism analysis, circ_CELSR1 could positively modulate SIK2 expression via sponging miR-149-5p. MiR-149-5p inhibition effectively restored the impacts of circ_CELSR1 knockdown on paclitaxel resistance and cell progression in paclitaxel-resistant ovarian cancer cells. MiR-149-5p overexpression suppressed paclitaxel resistance and cell progression in paclitaxel-resistant ovarian cancer cells by interacting with SIK2. In addition, circ_CELSR1 silencing impeded paclitaxel resistance of ovarian cancer in vivo. Circ_CELSR1 improved the resistance of ovarian cancer to paclitaxel by regulating miR-149-5p/SIK2 axis.

Four Circulating Long Non-Coding RNAs Act as Biomarkers for Predicting Cervical Cancer.

BACKGROUND/AIMS: Circulation long non-coding RNAs (lncRNAs) have emerged recently as major players in tumor biology and might be applied for cancer diagnosis, prognosis, or potential therapeutic targets. In this study, we aimed to explore whether the circulation lncRNA could predict the tumorigenesis of surgical squamous cervical cancer (CC). METHODS: In this study, we applied the lncRNA microarray to screen the potential biomarker for CC. Real-time quantitate polymerase chain reaction was conducted for further validation in a larger sample size. The multi-stage validation and risk score formula analysis were used to examine the sensitivity and specificity. RESULTS: We discovered 4 lncRNAs including HOTAIR, PVT1, XLOC_000303, and AL592284.1, which were upregulated in CC comparing with the cancer-free controls. Further, the receiver operating characteristic curve (ROC) analysis by risk score formula revealed the combined 4 factors with a high diagnostic ability with the area under ROC curve value of 0.875 and 0.958 in training set and validation set respectively. We finally confirmed the stable detection of the 4 lncRNAs by 5 cycles of freezing and thawing. CONCLUSION: HOTAIR, PVT1, XLOC_000303, and AL592284.1 might be the potential biomarkers for predicting the tumorigenesis of CC in the future.