Microdroplet PCR in Microfluidic Chip Based on Constant Pressure Regulation.

A device and method for the constant pressure regulation of microdroplet PCR in microfluidic chips are developed to optimize for the microdroplet movement, fragmentation, and bubble generation in microfluidic chips. In the developed device, an air source device is adopted to regulate the pressure in the chip, such that microdroplet generation and PCR amplification without bubbles can be achieved. In 3 min, the sample in 20 µL will be distributed into nearly 50,000 water-in-oil droplets exhibiting a diameter of about 87 µm, and the microdroplet will be subjected to a close arrangement in the chip without air bubbles. The device and chip are adopted to quantitatively detect human genes. As indicated by the experimental results, a good linear relationship exists between the detection signal and DNA concentration ranging from 101 to 105 copies/µL (R2 = 0.999). The microdroplet PCR devices based on constant pressure regulation chips exhibit a wide variety of advantages (e.g., achieving high pollution resistance, microdroplet fragmentation and integration avoidance, reducing human interference, and standardizing results). Thus, microdroplet PCR devices based on constant pressure regulation chips have promising applications for nucleic acid quantification.

Constant Pressure-Regulated Microdroplet Polymerase Chain Reaction in Microfluid Chips: A Methodological Study.

Digital polymerase chain reaction (PCR) technology in microfluidic systems often results in bubble formation post-amplification, leading to microdroplet fragmentation and compromised detection accuracy. To solve this issue, this study introduces a method based on the constant pressure regulation of microdroplets during PCR within microfluidic chips. An ideal pressure reference value for continuous pressure control was produced by examining air solubility in water at various pressures and temperatures as well as modeling air saturation solubility against pressure for various temperature scenarios. Employing a high-efficiency constant pressure device facilitates precise modulation of the microfluidic chip's inlet and outlet pressure. This ensures that air solubility remains unsaturated during PCR amplification, preventing bubble precipitation and maintaining microdroplet integrity. The device and chip were subsequently utilized for quantitative analysis of the human epidermal growth factor receptor (EGFR) exon 18 gene, with results indicating a strong linear relationship between detection signal and DNA concentration within a range of 101-105 copies/µL (R2 = 0.999). By thwarting bubble generation during PCR process, the constant pressure methodology enhances microdroplet stability and PCR efficiency, underscoring its significant potential for nucleic acid quantification and trace detection.

Advanced primary vaginal squamous cell carcinoma: A case report and literature review.

Background: Vaginal carcinoma is a gynecological malignancy with low incidence, and there are few relevant and specific guidelines for vaginal cancer in our country and abroad. Here, we report the case who was diagnosed with advanced, primary vaginal squamous cell carcinoma and underwent integrated treatment successfully. Case introduction: A 64-year-old Chinese woman underwent subtotal hysterectomy for uterine fibroids in 1998 and laparoscopic extensive residual cervical resection, bilateral ovarian salpingectomy, and pelvic lymph node dissection for residual cervical adenocarcinoma (stage IB1) in the First Affiliated Hospital of China Medical University in 2018. There was no postoperative review. The patient experienced vaginal discharge in March 2020, and vaginal bleeding occurred in July 2020. Our patient was diagnosed with stage IVA vaginal squamous cell carcinoma, based on a gynecological examination, colposcopy biopsy with histopathological examination, computed tomography scan, and tumor marker levels by two professors. After three phases of treatment (sequential treatment with chemotherapy plus radiotherapy, chemotherapy combined with immune checkpoint inhibitors, and immune checkpoint inhibitors combined with tyrosine kinase inhibitors therapy), her condition improved. Her current state is generally good, and she has achieved complete remission. Conclusion: We report a rare case of a patient with primary advanced vaginal carcinoma combined with cervical adenocarcinoma. The patient was treated for approximately 2 years, and her personalized treatment showed promising results. We will continue to follow up with the patient and monitor her response to the current treatment process.

PARP1 Is Targeted by miR-519a-3p and Promotes the Migration, Invasion, and Tube Formation of Ovarian Cancer Cells.

Background: Poly ADP-ribose polymerase 1 (PARP1) has been discovered to be implicated in ovarian cancer (OC), but its interaction with microRNA (miR)-519a-3p remained poorly understood. This study aimed to uncover their roles and interactions in OC. Materials and Methods: Clinical tissue from OC patients and adjacent normal tissue were collected, and the survival rates of OC patients with high or low PARP1 expression were analyzed by Kaplan-Meier curve. After transfection, OC cell viability, migration, and tube formation were detected with cell counting kit-8 (CCK-8) assay, scratch assay, and tube formation assay, respectively. The target gene of miR-519a-3p and potential binding sites between them were predicted with TargetScan and confirmed using a dual-luciferase reporter assay. Relative expressions of miR-519a-3p, PARP1, E-cadherin, N-cadherin, SNAIL, vascular endothelial growth factor (VEGF), and p53 were measured by quantitative real-time polymerase chain reaction and Western blot as needed. Results: PARP1 expression was upregulated in OC, which was related to poor prognosis of OC patients. Silencing PARP1 decreased PARP1 expression and suppressed viability, migration, invasion, and tube formation in OC cells, while overexpressed PARP1 did the opposite. PARP1 was the target gene of miR-519a-3p, and it reversed the effects of miR-519a-3p on the migration, invasion, and tube formation of OC cells by upregulating the expressions of PAR, PARP1, N-cadherin, SNAIL, and VEGF and downregulating those of E-cadherin and p53. Conclusion: PARP1, a target gene of miR-519a-3p, promoted the migration, invasion, and tube formation of OC cells, providing a possible therapeutic target for treatment of OC patients.

An efficient strategy for digging protein-protein interactions for rational drug design - A case study with HIF-1α/VHL.

The ubiquitination of the hypoxia-inducible factor-1α (HIF-1α) is mediated by interacting with the von Hippel-Lindau protein (VHL), and is associated with cancer, chronic anemia, and ischemia. VHL, an E3 ligase, has been reported to degrade HIF-1 for decades, however, there are few successful inhibitors currently. Poor understanding of the binding pocket and a lack of in-depth exploration of the interactions between two proteins are the main reasons. Hence, we developed an effective strategy to identify and design new inhibitors for protein-protein interaction targets. The hydroxyproline (Hyp564) of HIF-1α contributed the key interaction between HIF-1α and VHL. In this study, detailed information of the binding pocket were explored by alanine scanning, site-directed mutagenesis and molecular dynamics simulations. Interestingly, we found the interaction(s) between Y565 and H110 played a key role in the binding of VHL/HIF-1α. Based on the interactions, 8 derivates of VH032, 16a-h, were synthesized by introducing various groups bounded to H110. Further assay on protein and cellular level exhibited that 16a-h accessed higher binding affinity to VHL and markable or modest improvement in stabilization of HIF-1α or HIF-1α-OH in HeLa cells. Our work provides a new orientation for the modification or design of VHL/HIF-1α protein-protein interaction inhibitors.

Model constructions of chemosensitivity and prognosis of high grade serous ovarian cancer based on evaluation of immune microenvironment and immune response.

BACKGROUND: The prognosis of high grade serous ovarian cancer (HGSOC) patients is closely related to the immune microenvironment and immune response. Based on this, the purpose of this study was to construct a model to predict chemosensitivity and prognosis, and provide novel biomarkers for immunotherapy and prognosis evaluation of HGSOC. METHODS: GSE40595 (38 samples), GSE18520 (63 samples), GSE26712 (195 samples), TCGA (321 samples) and GTEx (88 samples) were integrated to screen differential expressed genes (DEGs) of HGSOC. The prognosis related DEGs (DEPGs) were screened through overall survival analysis. The DEGs-encoded protein-protein interaction network was constructed and hub genes of DEPGs (DEPHGs) were generated by STRING. Immune characteristics of the samples were judged by ssGSEA, ESTIMATE and CYBERSORT. TIMER was used to analyze the relationship between DEPHGs and tumor-infiltrating immunocytes, as well as the immune checkpoint genes, finally immune-related DEPHGs (IDEPHGs) were determined, and whose expression in 12 pairs of HGSOC tissues and tumor-adjacent tissues were analyzed by histological verification. Furthermore, the chemosensitivity genes in IDEPHGs were screened according to GSE15622 (n = 65). Finally, two prediction models of paclitaxel sensitivity score (PTX score) and carboplatin sensitivity score (CBP score) were constructed by lasso algorithm. The area under curve was calculated to estimate the accuracy of candidate gene models in evaluating chemotherapy sensitivity. RESULTS: 491 DEGs were screened and 37 DEGs were identified as DEPGs, and 11 DEPHGs were further identified. Among them, CXCL13, IDO1, PI3, SPP1 and TRIM22 were screened as IDEPHGs and verified in the human tissues. Further analysis showed that IDO1, PI3 and TRIM22 could independently affect the chemotherapy sensitivity of HGSOC patients. The PTX score was significantly better than TRIM22, PI3, SPP1, IDO1 and CXCL13 in predicting paclitaxel sensitivity, so was CBP score in predicting carboplatin sensitivity. What's more, both of the HGSOC patients with high PTX score or high CBP score had longer survival time. CONCLUSIONS: Five IDEPHGs identified through comprehensive bioinformatics analysis were closely related with the prognosis, immune microenvironment and chemotherapy sensitivity of HGSOC. Two prediction models based on IDEPHGs might have potential application of chemotherapy sensitivity and prognosis for patients with HGSOC.

Numerical Simulation and Experimental Verification of Droplet Generation in Microfluidic Digital PCR Chip.

The generation of droplets is one of the most critical steps in the droplet digital polymerase chain reaction (ddPCR) procedure. In this study, the mechanism of droplet formation in microchannel structure and factors affecting droplet formation were studied. The physical field of laminar two-phase flow level was used to simulate the process of droplet generation through microfluidic technology. The effect of the parameters including flow rate, surface tension, and viscosity on the generated droplet size were evaluated by the simulation. After that, the microfluidic chip that has the same dimension as the simulation was then, fabricated and evaluated. The chip was made by conventional SU-8 photolithography and injection molding. The accuracy of the simulation was validated by comparing the generated droplets in the real scenario with the simulation result. The relative error (RE) between experimentally measured droplet diameter and simulation results under different flow rate, viscosity, surface tension and contact angle was found less than 3.5%, 1.8%, 1.4%, and 1.2%, respectively. Besides, the coefficient of variation (CV) of the droplet diameter was less than 1%, which indicates the experimental droplet generation was of high stability and reliability. This study provides not only fundamental information for the design and experiment of droplet generation by microfluidic technology but also a reliable and efficient investigation method in the ddPCR field.

XT-considered multiple backup paths and resources shared protection scheme based on ring covers.

With the rapid development of space division multiplexing (SDM) and flexible grid technology, the problem of resource allocation in optical network becomes much more complicated. Although there emerge a substantial number of works about link protection or restoration in SDM-EONs mesh networks, the topic of survivability is dug deeper in this work. It is acknowledged that protection schemes based on ring covers bring the advantages of shorter restoration time and lower costs. However, the RSCA problem in SDM-EON for link protection based on ring covers has rarely been investigated. To enhance the survivability of SDM-EON and make the best use of ring covers, we initially select a set of rings for the protection of all the links in a network topology according to constrains, aiming at taking full advantages of network resources. After that, we propose an algorithm to recover the traffic which will break off under link failures, basing on the set of rings. At the end of this protection scheme, we arrange the resources fulfilling the constrains of spectrum contiguity and core continuity constrains by using several different algorithms considering physical impairment. Based on the allocation in both spacial and spectral dimensions, our algorithms achieve better results of survivability. According to the simulations conducted for the evaluation of the proposed algorithms, our algorithms manage to recover at least 72.8% of traffic when the traffic request number is set to be 1000.

MAGI2-AS3 suppresses MYC signaling to inhibit cell proliferation and migration in ovarian cancer through targeting miR-525-5p/MXD1 axis.

Ovarian cancer (OV) is one of the most lethal gynecological malignance in females, and usually diagnosed at advanced stages. Long noncoding RNAs (lncRNAs) exhibit their crucial functions in modulatory mechanisms of cancers. Substantive studies have proven the anti-tumor role of MAGI2-AS3 in multiple cancers, but the physiological functions of MAGI2-AS3 in OV need more detailed explanations. The current study corroborated that overexpression of MAGI2-AS3 executed inhibitory activity in OV via hindering cell proliferation, cell cycle, migration as well as invasion while promoted apoptosis. Moreover MAGI2-AS3 bound with miR-525-5p and negatively regulated the expression of miR-525-5p. Further studies testified that MXD1 was a downstream target of miR-525-5p and the competing relationship between MAGI2-AS3 and MXD1 were confirmed by RNA pull down. Based on the combination between MAX and MYC, we analyzed the effects of MAGI2-AS3 on MXD1 and MYC, unveiling the competing relationship between MXD1 and MYC for binding to MAX. Finally, we constructed rescue assays to certify that MAGI2-AS3 suppressed the course of OV via enhancing MXD1 expression. In summary, MAGI2-AS3 repressed the progression of OV by targeting miR-525-5p/MXD1 axis, offering a novel insight into understanding OV at the molecular level.

NCK1-AS1 promotes NCK1 expression to facilitate tumorigenesis and chemo-resistance in ovarian cancer.

Long non-coding RNAs (lncRNAs) have been unveiled to play crucial parts in tumorigenesis and chemo-resistance of multiple cancers. Herein, we explored the role of NCK1-AS1 in ovarian cancer (OC). As indicated by TCGA, NCK1-AS1 was markedly upregulated in OC tissues. Besides, we found a dramatic upregulation of NCK1-AS1 in OC cell lines relative to the normal IOSE cells. Interestingly, silencing NCK1-AS1 confined cell proliferation, induced apoptosis and suppressed migration and invasion as well as enhanced DDP sensitivity in OC cells. As for mechanistic investigation, starBase (http://starbase.sysu.edu.cn/) suggested that NCK1-AS1 expression in OC tissues was significantly positively correlated with its adjacent gene, NCK adaptor protein 1 (NCK1). Furtherly, we demonstrated that the cytoplasmic NCK1-AS1 competed with NCK1 mRNA for miR-137 binding to boost NCK1 mRNA expression. Importantly, miR-137 inhibition could only offset the suppression of NCK1-AS1 depletion on NCK1 mRNA level but not the protein level. Moreover, NCK1-AS1 stabilized NCK1 protein by hindering c-Cbl-induced degradation via directly interacting with c-Cbl. Furthermore, NCK1 upregulation reversed the influences of NCK1-AS1 inhibition on the biological behaviors and DDP resistance of OC cells. This study disclosed a NCK1-AS1/NCK1 axis in regulating OC progression and chemo-resistance, opening a new path for treatment and chemo-resistance overcoming in OC.

HPV16 E6 upregulates Aurora A expression.

Overexpression of Aurora A kinase occurs in certain types of cancer, and therefore results in chromosome instability and phosphorylation-mediated ubiquitylation and degradation of p53 for tumorigenesis. The high-risk subtype human papillomavirus (HPV)16 early oncoprotein E6 is a major contributor inducing host cell immortalization and transformation through interaction with a number of cellular factors. In the present study, co-immunoprecipitation, glutathione S-transferase pull-down and immunostaining were used to show that HPV16 E6 and Aurora A bind to each other in vivo and in vitro. Western blotting and reverse transcription-polymerase chain reaction were used to reveal that HPV16 E6 inhibited cell apoptosis by stabilizing Aurora A expression. The present study may report a new mechanism for the involvement of HPV16 E6 in carcinogenesis, as HPV16 E6 elevates Aurora A expression and the latter may be a common target for oncogenic viruses that result in cell carcinogenesis.

Unfolded protein response mediated JNK/AP-1 signal transduction, a target for ovarian cancer treatment.

Researches have revealed several stressors, which could activate unfolded protein response (UPR) in cells. However, the survival or death pathway was determined by the duration of UPR exposure. Based on the UPR mediated death pathway, our study was aimed to investigate role of UPR on c-Jun N-terminal kinase (JNK)/activator protein-1 (Ap-1) signal transduction in diindolylmethane (DIM) treated ovarian cancer cell lines. Activation of UPR proteins, UPR mediated apoptotic signaling proteins and expression level of EpCAM, JNK, Ap-1, Caspase-3 and Bcl-2 were measured. Protein and gene expression, transcription factor activity, and protein phosphorylation were measured using standard molecular biology techniques. Our results demonstrated DIM treatment had significantly increased the expression of Endoplasmic reticulum (ER) stress regulators such as Bip, IRE1, CHOP and activation of UPR related apoptotic proteins in ovarian cancer cells. Decreased expression of EpCAM and activity of AP-1 transcription factor were observed in DIM treated cells. The pharmacologic inhibitors of the JNK signal transduction pathway, suggest that the impact of EpCAM expression on AP-1 transcription factor activity is mediated through the JNK pathway. Taken together, these results suggest that UPR mediated JNK/Ap-1 signal transduction has a significant role in the regulation of apoptosis in human ovarian cancer cells, and is a potential molecular target to enhance sensitivity of ovarian cancer to chemotherapy.

Human papillomavirus 16 E6 contributes HIF-1α induced Warburg effect by attenuating the VHL-HIF-1α interaction.

Cervical cancer is still one of the leading causes of cancer deaths in women worldwide, especially in the developing countries. It is a major metabolic character of cancer cells to consume large quantities of glucose and derive more energy by glycolysis even in the presence of adequate oxygen, which is called Warburg effect that can be exaggerated by hypoxia. The high risk subtype HPV16 early oncoprotein E6 contributes host cell immortalization and transformation through interacting with a number of cellular factors. Hypoxia-inducible factor 1α (HIF-1α), a ubiquitously expressed transcriptional regulator involved in induction of numerous genes associated with angiogenesis and tumor growth, is highly increased by HPV E6. HIF-1α is a best-known target of the von Hippel-Lindau tumor suppressor (VHL) as an E3 ligase for degradation. In the present work, we found that HPV16 E6 promotes hypoxia induced Warburg effect through hindering the association of HIF-1α and VHL. This disassociation attenuates VHL-mediated HIF-1α ubiquitination and causes HIF-1α accumulation. These results suggest that oncoprotein E6 plays a major role in the regulation of Warburg effect and can be a valuable therapeutic target for HPV-related cancer.

The ING4 Binding with p53 and Induced p53 Acetylation were Attenuated by Human Papillomavirus 16 E6.

High risk subtype HPV16 early oncoprotein E6 contributes host cell immortalization and transformation through interacting with a number of cellular factors. ING4 is one member of the inhibitor of growth (ING) family of type II tumor suppressors and it has been shown to be involved in regulating p53 function. However, the effect and mechanism of HPV16 E6 on ING4 function remain elusive. In this study, we report HPV16 E6 combines with ING4 in vivo and in vitro. The ING4 induced p53 acetylation and its combining with p53 were attenuated by HPV16 E6 independent of p53 degradation. The enhancing function of ING4 on p53 mediated apoptosis was diminished when HPV16 E6 existed. These findings reveal that ING4 may be a common target of oncogenic viruses for driving host cell carcinogenesis.