Development of a multi-recombinase polymerase amplification assay for rapid identification of COVID-19, influenza A and B.

The coronavirus disease 2019 (COVID-19) pandemic caused extensive loss of life worldwide. Further, the COVID-19 and influenza mix-infection had caused great distress to the diagnosis of the disease. To control illness progression and limit viral spread within the population, a real-time reverse-transcription PCR (RT-PCR) assay for early diagnosis of COVID-19 was developed, but detection was time-consuming (4-6 h). To improve the diagnosis of COVID-19 and influenza, we herein developed a recombinase polymerase amplification (RPA) method for simple and rapid amplification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 and Influenza A (H1N1, H3N2) and B (influenza B). Genes encoding the matrix protein (M) for H1N1, and the hemagglutinin (HA) for H3N2, and the polymerase A (PA) for Influenza B, and the nucleocapsid protein (N), the RNA-dependent-RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) for SARS-CoV-2 were selected, and specific primers were designed. We validated our method using SARS-CoV-2, H1N1, H3N2 and influenza B plasmid standards and RNA samples extracted from COVID-19 and Influenza A/B (RT-PCR-verified) positive patients. The method could detect SARS-CoV-2 plasmid standard DNA quantitatively between 102 and 105 copies/ml with a log linearity of 0.99 in 22 min. And this method also be very effective in simultaneous detection of H1N1, H3N2 and influenza B. Clinical validation of 100 cases revealed a sensitivity of 100% for differentiating COVID-19 patients from healthy controls when the specificity was set at 90%. These results demonstrate that this nucleic acid testing method is advantageous compared with traditional PCR and other isothermal nucleic acid amplification methods in terms of time and portability. This method could potentially be used for detection of SARS-CoV-2, H1N1, H3N2 and influenza B, and adapted for point-of-care (POC) detection of a broad range of infectious pathogens in resource-limited settings.

RON in hepatobiliary and pancreatic cancers: Pathogenesis and potential therapeutic targets.

The receptor protein tyrosine kinase RON belongs to the c-MET proto-oncogene family. Research has shown that RON has a role in cancer pathogenesis, which places RON on the frontline of the development of novel cancer therapeutic strategies. Hepatobiliary and pancreatic (HBP) cancers have a poor prognosis, being reported as having higher rates of cancer-related death. Therefore, to combat these malignant diseases, the mechanism underlying the aberrant expression and signaling of RON in HBP cancer pathogenesis, and the development of RON as a drug target for therapeutic intervention should be investigated. Abnormal RON expression and signaling have been identified in HBP cancers, and also act as tumorigenic determinants for HBP cancer malignant behaviors. In addition, RON is emerging as an important mediator of the clinical prognosis of HBP cancers. Thus, not only is RON significant in HBP cancers, but also RON-targeted therapeutics could be developed to treat these cancers, for example, therapeutic monoclonal antibodies and small-molecule inhibitors. Among them, antibody-drug conjugates have become increasingly popular in current research and their potential as novel anti-cancer biotherapeutics will be determined in future clinical trials.

Pathological significance of abnormal recepteur d'origine nantais and programmed death ligand 1 expression in colorectal cancer.

BACKGROUND: Programmed death ligand 1 (PD-L1) immunotherapy remains poorly efficacious in colorectal cancer (CRC). The recepteur d'origine nantais (RON) receptor tyrosine kinase plays an important role in regulating tumor immunity. AIM: To identify the patterns of RON and PD-L1 expression and explore their clinical significance in CRC. METHODS: Gene expression data from the Gene Expression Omnibus database (GEO; n = 290) and patients at the First Affiliated Hospital, Zhejiang University School of Medicine (FAHZUSM; n = 381) were analyzed to determine the prognostic value of RON and PD-L1 expression within the tumor microenvironment of CRC. HT29 cell line was treated with BMS-777607 to explore the relationship between RON activity and PD-L1 expression. Signaling pathways and protein expression perturbed by RON inhibition were evaluated by cellular immunofluorescence and Western blot. RESULTS: In the GEO patient cohort, cut-off values for RON and PD-L1 expression were determined to be 7.70 and 4.3, respectively. Stratification of patients based on these cutoffs demonstrated that high expression of RON and PD-L1 was associated with a poor prognosis. In the FAHZUSM cohort, rates of high expression of RON in tumor cells, high PD-L1 expression in tumor cells and tumor infiltrating monocytes, and both high RON and high PD-L1 expression in the tumor microenvironment were 121 (32%), 43 (11%), 91 (24%), and 51 (13.4%), respectively. High expression of RON was significantly correlated with high expression of PD-L1 in the tumor cell compartment (P < 0.001). High expression of RON and that of PD-L1 were independent prognostic factors for poorer overall survival. Concurrent high expression of both RON and PD-L1 in the tumor microenvironment was significantly associated with a poor prognosis. In vitro, BMS-777607 inhibited the phosphorylation of RON, inhibited PD-L1 expression, and attenuated activation of the ERK1/2 and AKT signaling pathways in CRC cells. CONCLUSION: RON, PD-L1, and their crosstalk are significant in predicting the prognostic value of CRC. Moreover, phosphorylation of RON upregulates PD-L1 expression, which provides a novel approach to immunotherapy in CRC.