Timely identification of human papillomavirus (HPV) infection is crucial for the prevention of cervical cancer. Current HPV detection methods mainly rely on polymerase chain reaction (PCR), which often requires bulky equipment and a long assay time. In this work, we report a heating-membrane-assisted multiplexed microfluidics platform that couples recombinase polymerase amplification (RPA) and CRISPR technology (termed M3-CRISPR) for fast and low-cost detection of multiple HPV subtypes. The heating membrane can provide convenient temperature control for the on-chip RPA and CRISPR assays. This stand-alone system allows simultaneous detection of HPV16 and HPV18 with high specificity and detection sensitivity (0.5 nM and 1 × 10–18 M for unamplified and amplified plasmids, respectively) in 30 min with a fluorescence-based readout. Furthermore, we introduced an optimized lateral flow dipstick (LFD) into the portable system to allow visualized detection of HPV DNA. The LFD-based readout also reached a detection sensitivity of 1 × 10–18 M for amplified plasmids and realized successful detection of HPV subtypes in the clinical samples. Finally, we established an automatic microfluidic system that enables the sample-in-answer-out detection of HPV subtypes. We believe that this fast, convenient, and affordable molecular diagnostic platform can serve as a useful tool in point-of-care testing of HPV or other pathogens.
In this study, cell death induced by the oxidant tert-butylhydroperoxide (tBH) was observed in U2OS cells; this phenotype was rescued by Syntaxin 17 (STX17) knockout (KO) but the mechanism is unknown. STX17 plays dual roles in autophagosome–lysosome fusion and mitochondrial fission. However, the contribution of the two functions of STX17 to apoptosis has not been extensively studied. Here, we sought to dissect the dual roles of STX17 in oxidative-stress-induced apoptosis by taking advantage of STX17 knockout cells and an autophagosome–lysosome fusion defective mutant of STX17. We generated STX17 knockout U2OS cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and the STX17 knockout cells were reconstituted with wild-type STX17 and its autophagosome–lysosome fusion defective mutant. Autophagy was assessed by autophagic flux assay, Monomer red fluorescent protein (mRFP)–GFP–LC3 assay and protease protection assay. Golgi, endoplasmic reticulum (ER)/ER–Golgi intermediate compartment (ERGIC) and mitochondrial dynamics were examined by staining the different indicator proteins. Apoptosis was evaluated by caspase cleavage assay. The general reactive oxygen species (ROS) were detected by flow cytometry. In STX17 complete knockout cells, sealed autophagosomes were efficiently formed but their fusion with lysosomes was less defective. The fusion defect was rescued by wild-type STX17 but not the autophagosome–lysosome fusion defective mutant. No obvious defects in Golgi, ERGIC or ER dynamics were observed. Mitochondria were significantly elongated, supporting a role of STX17 in mitochondria fission and the elongation caused by STX17 KO was reversed by the autophagosome–lysosome fusion defective mutant. The clearance of protein aggregation was compromised, correlating with the autophagy defect but not with mitochondrial dynamics. This study revealed a mixed role of STX17 in autophagy, mitochondrial dynamics and oxidative stress response. STX17 knockout cells were highly resistant to oxidative stress, largely due to the function of STX17 in mitochondrial fission rather than autophagy.
ObjectiveHuman papillomavirus (HPV) integration is a crucial genetic step in cervical carcinogenesis. This study aimed to evaluate the performance of an HPV integration test for the triage of HPV‐positive women.DesignAn observational cohort study.SettingA cervical cancer screening programme in China.Population1393 HPV‐positive women aged 25–65 years undergoing routine cervical cancer screening and HPV integration testing with 1‐year follow‐up.MethodsThe sensitivity, specificity, positive predictive value and negative predictive value between HPV integration and cytology were compared.Main outcome measuresCervical intraepithelial neoplasia grade 3 or more severe (CIN3+).ResultsAmong 1393 HPV‐positive patients, 138 (9.9% [8.3–11.5%]) were HPV integration test positive compared with 537 who had abnormal cervical cytology (38.5% [36.0–41.1%]). Compared with cytology, HPV integration exhibited higher specificity (94.5% [93.3–95.8%] versus 63.8% [61.2–66.4%]) and equivalent sensitivity (70.5% [61.4–79.7%] versus 70.5% [61.4–79.7%]) for detection of CIN3+. HPV integration‐negative women accounted for 90.1% (1255/1393) of the total population and had a low immediate CIN3+ risk (2.2%). At 1‐year follow‐up, the progression rate in the HPV integration‐positive women was higher than in the HPV integration‐negative women (12.0% versus 2.1%, odds ratio 5.6, 95% CI, 2.6–11.9). In 10 conservatively managed integration‐negative CIN2 patients, all showed spontaneous regression and seven showed HPV clearance after 1‐year follow‐up.ConclusionThe HPV integration test may be a precise risk stratification tool for HPV‐positive women and could avoid excessive use of invasive biopsies.
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