The outbreak of coronavirus disease 2019 (COVID-19) has led to substantial infections and mortality around the world. Fast screening and diagnosis are thus crucial for quick isolation and clinical intervention. In this work, we showed that attenuated total reflection−Fourier transform infrared spectroscopy (ATR−FT-IR) can be a primary diagnostic tool for COVID-19 as a supplement to in-use techniques. It requires only a small volume (∼3 μL) of the serum sample and a shorter detection time (several minutes). The distinct spectral differences and the separability between normal control and COVID-19 were investigated using multivariate and statistical analysis. Results showed that ATR−FT-IR coupled with partial least squares discriminant analysis was effective to differentiate COVID-19 from normal controls and some common respiratory viral infections or inflammation, with the area under the receiver operating characteristic curve (AUROC) of 0.9561 (95% CI: 0.9071−0.9774). Several serum constituents including, but not just, antibodies and serum phospholipids could be reflected on the infrared spectra, serving as "chemical fingerprints" and accounting for good model performances.

Fibroblast activation protein (FAP) is a serine protease that has been reported in fibroblasts and some carcinoma cells, which correlates with poor patient outcomes. FAP can be induced under hypoxia which is also vital in the malignant behaviors of cancer cells. However, the role of FAP and its correlation with hypoxia has not been investigated in HCC cancer cells. In tissues from post-surgical HCC patients in our center, we adopted immunohistochemistry staining (IHC), western blot and quantitative RT-PCR to detect the expression levels of FAP and the hypoxia related marker, hypoxia inducible factor 1α (HIF-1α). X-tile software was used for the determination of high and low expression of FAP and HIF-1α after the IHC analysis. Clinicopathological analysis, Kaplan-Meier analysis and Cox regression model were performed. In-vitro experiments were performed to confirm the relationship between FAP and hypoxia in HCC cancer cell lines (HepG2, Huh7 and MHCC97H). Results revealed that expression levels of FAP and HIF-1α were significantly correlated (Pearson r2 = 0.2753, p < 0.0001) in IHC analysis of the 138-patient cohort. Western blot and quantity RT-PCR indicated parallel changes in 11 post-surgical fresh frozen tissues. The HIF-1α and FAP expression were associated with serum AFP, TNM, tumor size and vascular invasion. Cox regression analysis showed that HIF-1α/ FAP combination were the independent predictor for overall survival (OS) and time-to-recurrence (TTR) in post-surgical HCC patients. Kaplan-Meier analyses revealed that the patient with high levels of HIF-1α, FAP and combined HIF-1α/FAP had the shortest OS and TTR. In-vitro experiments showed that FAP was increased in hypoxic HCC cancer cell lines in parallel with that of HIF-1α and three EMT markers (E-cadherin, Snail and TWIST). In conclusion, the up-regulation of FAP in HCC cancer cells under hypoxia can be indicative of poor prognosis in patients.

Matrix metalloproteinase-1 (MMP1) participates in the metastasis of pancreatic cancer, and its expression can be regulated by endogenous microRNAs (miRs/miRNAs) and exogenous inflammatory factors. Whether miRNAs that potentially modulate MMP1 expression can also attenuate the pro-metastatic effects of its inducer on pancreatic cancer is yet to be completely elucidated. In the present study, a systematic analysis including in silico and bioinformatics analyses, a luciferase reporter assay and an RNA electrophoretic mobility shift assay (EMSA), were used to investigate the interaction between miRNAs and MMP1 mRNA. In addition, wound-healing assays, Transwell assays and xenograft nude mouse models were implemented to investigate the antitumor activities exerted by candidate miRNAs. As a result, hsa-miR-623 was screened as a candidate miRNA that interacts with the MMP1 transcript, and an inverse correlation between the expression of hsa-miR-623 and MMP1 was observed in human pancreatic cancer tissue samples. The EMSA confirmed that hsa-miR-623 was able to directly bind to its cognate target within the 3′-untranslated region of the MMP1 transcript. In addition, transfection of hsa-miR-623 mimics into PANC-1 and BXPC-3 cell lines markedly inhibited the expression of MMP1 at the mRNA and protein levels, and attenuated IL-8-induced MMP1 expression. hsa-miR-623 also decreased IL-8-induced epithelial-mesenchymal transition in PANC-1 and BXPC-3 cells via the underlying mechanism of inhibition of ERK phosphorylation. Consequently, hsa-miR-623 inhibited pancreatic cancer cell migration and invasion in vitro and metastasis in vivo . The results of the present study suggest that hsa-miR-623 represents a novel adjuvant therapeutic target to prevent metastasis in pancreatic cancer.

BackgroundEndothelial progenitor cells (EPCs) contribute to tumor angiogenesis and growth. We previously reported that over-expression of an inhibitor of DNA binding/differentiation 1 (Id1) in EPCs can enhance EPC proliferation, migration, and adhesion. In this study, we investigated the role of Id1 in EPC angiogenesis in patients with ovarian cancer and the underlying signaling pathway.MethodsCirculating EPCs from 22 patients with ovarian cancer and 15 healthy control subjects were cultured. Id1 and matrix metalloproteinase-2 (MMP-2) expression were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot. EPC angiogenesis was detected by tube formation assays. Double-stranded DNA containing the interference sequences was synthesized according to the structure of a pGCSIL-GFP viral vector and then inserted into a linearized vector. Positive clones were identified as lentiviral vectors that expressed human Id1 short hairpin RNA (shRNA).ResultsId1 and MMP-2 expression were increased in EPCs freshly isolated from ovarian cancer patients compared to those obtained from healthy subjects. shRNA-mediated Id1 down-regulation substantially reduced EPC angiogenesis and MMP-2 expression. Importantly, transfection of EPCs with Id1 in vitro induced phosphorylation of Akt (p-Akt) via phosphoinositide 3-kinase and increased the expression of MMP-2 via NF-κB. Blockage of both pathways by specific inhibitors (LY294002 and PDTC, respectively) abrogated Id1-enhanced EPC angiogenesis.ConclusionsId1 can enhance EPC angiogenesis in ovarian cancer, which is mainly mediated by the PI3K/Akt and NF-κB/MMP-2 signaling pathways. Id1 and its downstream effectors are potential targets for treatment of ovarian cancer because of their contribution to angiogenesis.

Pichia pastoris has been widely exploited for the heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value chemicals and pharmaceuticals. Effective synthetic biology tools for genetic engineering are essential for industrial and biotechnological research in this yeast. Here, we describe a novel and efficient genome editing method mediated by the CRISPR-Cpf1 system, which could facilitate the deletion of large DNA fragments and integration of multiplexed gene fragments. The CRISPR-Cpf1 system exhibited a precise and high editing efficiency for single-gene disruption (99 ± 0.8%), duplex genome editing (65 ± 2.5% to 80 ± 3%), and triplex genome editing (30 ± 2.5%). In addition, the deletion of large DNA fragments of 20kb and one-step integration of multiple genes were first achieved using the developed CRISPR-Cpf1 system. Taken together, this study provides an efficient and simple gene editing tool for P. pastoris. The novel multiloci gene integration method mediated by CRISPR-Cpf1 may accelerate the ability to engineer this methylotrophic yeast for metabolic engineering and genome evolution in both biotechnological and biomedical applications.