Multicenter analysis and a rapid screening model to predict early novel coronavirus pneumonia using a random forest algorithm.

ABSTRACT: Early determination of coronavirus disease 2019 (COVID-19) pneumonia from numerous suspected cases is critical for the early isolation and treatment of patients.The purpose of the study was to develop and validate a rapid screening model to predict early COVID-19 pneumonia from suspected cases using a random forest algorithm in China.A total of 914 initially suspected COVID-19 pneumonia in multiple centers were prospectively included. The computer-assisted embedding method was used to screen the variables. The random forest algorithm was adopted to build a rapid screening model based on the training set. The screening model was evaluated by the confusion matrix and receiver operating characteristic (ROC) analysis in the validation.The rapid screening model was set up based on 4 epidemiological features, 3 clinical manifestations, decreased white blood cell count and lymphocytes, and imaging changes on chest X-ray or computed tomography. The area under the ROC curve was 0.956, and the model had a sensitivity of 83.82% and a specificity of 89.57%. The confusion matrix revealed that the prospective screening model had an accuracy of 87.0% for predicting early COVID-19 pneumonia.Here, we developed and validated a rapid screening model that could predict early COVID-19 pneumonia with high sensitivity and specificity. The use of this model to screen for COVID-19 pneumonia have epidemiological and clinical significance.

Paeonol prevents migration and invasion, and promotes apoptosis of cervical cancer cells by inhibiting 5­lipoxygenase.

Cervical cancer is a common public health issue with high morbidity worldwide. Paeonol (Pae) has been recognized as a traditional Chinese medicine used for the treatment of various cancer types. However, whether Pae could exert a protective effect on cervical cancer remains to be investigated. The aim of the present study was to explore the role of Pae in cervical cancer cells and identify the potential mechanism. Cell Counting Kit­8 and colony­formation assays were conducted to test the proliferation of HeLa cells. Additionally, wound healing and transwell assays were used to detect the migratory and invasive abilities of cells. The plasmid that overexpressed 5­lipoxygenase (5­LO) or control vector was constructed and transfected into the cells. Subsequently, flow cytometry was used to monitor the apoptotic rate of cells. The expression levels of apoptosis­associated proteins and 5­LO were detected using western blot analysis. Reverse transcription­quantitative PCR analysis detected the expression of 5­LO. Pae inhibited the proliferation, invasion and migration of HeLa cells, promoted cell apoptosis and downregulated the expression of 5­LO. Overexpression of 5­LO, however, attenuated these effects. Thus, Pae could inhibit the proliferation, migration and invasion, as well as promote apoptosis of HeLa cells by regulating the expression of 5­LO.

Early and sustained expression of latent and host modulating genes in coordinated transcriptional program of KSHV productive primary infection of human primary endothelial cells.

Coordinated expression of viral genes in primary infection is essential for successful infection of host cells. We examined the expression profiles of Kaposi's sarcoma-associated herpesvirus (KSHV) transcripts in productive primary infection of primary human umbilical vein endothelial cells by whole-genome reverse-transcription real-time quantitative PCR. The latent transcripts were expressed early and sustained at high levels throughout the infection while the lytic transcripts were expressed in the order of immediate early, early, and lytic transcripts, all of which culminated before the production of infectious virions. Significantly, transcripts encoding genes with host modulating functions, including mitogenic and cell cycle-regulatory, immune-modulating, and anti-apoptotic genes, were expressed before those encoding viral structure and replication genes, and sustained at high levels throughout the infection, suggesting KSHV manipulation of host environment to facilitate infection. The KSHV transcriptional program in a primary infection defined in this study should provide a basis for further investigation of virus-cell interactions.

Identification of a novel cellular transcriptional repressor interacting with the latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus.

The latent nuclear antigen (LNA) of Kaposi's sarcoma-associated herpesvirus (KSHV) has an essential role in viral latent infection. LNA maintains the stability of KSHV episomes and modulates the expression of cellular genes. A novel cellular protein KLIP1 was identified to interact with LNA through yeast two-hybrid screening, and confirmed by a glutathione S-transferase pull down assay. Domain mapping showed that KLIP1 interacted with the N-terminal domain of LNA. Northern blot hybridization with a KLIP1 probe identified a major transcript of 1.8 kb and a minor transcript of 2.8 kb. cDNA library screening and 5'-RACE revealed that the major transcript encoded an open-reading-frame of 1,257 bp and had a 5'-untranslated region of 73 nucleotides. The major KLIP1 transcript was ubiquitously present in different cell types examined. A KLIP1 synthetic peptide antibody detected a doublet of 58-kDa and 63-kDa proteins in a Western blot assay. KLIP1 had two putative nuclear localization signals and showed punctate nuclear localization when expressed as a GFP-fusion protein. KLIP1 interacted with LNA in vivo, as demonstrated by coimmunoprecipitation using KSHV-infected cells and colocalization when they were expressed as GFP- and DsRed-fusion proteins, respectively. Consistent with its interaction with LNA, nuclear localization, and possession of two leucine zipper motifs, KLIP1 behaved like a transcriptional factor and repressed herpes simplex virus thymidine kinase (TK) promoter activity in a mammalian one-hybrid assay. In addition, cotransfection with LNA alleviated the transcriptional repression effect of KLIP1 on TK promoter activity. These results suggest that KLIP1 is a new member of cellular transcriptional repressors, and that LNA is involved in deregulating cellular transcription process.

Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis.

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma and several other malignancies. The lack of an efficient infection system has impeded the understanding of KSHV-related pathogenesis. A genetic approach was used to isolate infectious KSHV. Recombinant bacteria artificial chromosome (BAC) KSHV containing hygromycin resistance and green fluorescent protein (GFP) markers was generated by homologous recombination in KSHV-infected BCBL-1 cells. Recombinant KSHV genomes from cell clones that were resistant to hygromycin, expressed GFP, and produced infectious virions after induction with tetradecanoyl phorbol acetate (TPA) were rescued in Escherichia coli and reconstituted in 293 cells. Several 293 cell lines resulting from infection with recombinant virions induced from a full-length recombinant KSHV genome, named BAC36, were obtained. BAC36 virions established stable latent infection in 293 cells, harboring 1 to 2 copies of viral genome per cell and expressing viral latent proteins, with approximately 0.5% of cells undergoing spontaneous lytic replication, which is reminiscent of KSHV infection in Kaposi's sarcoma tumors. TPA treatment induced BAC36-infected 293 cell lines into productive lytic replication, expressing lytic proteins and producing virions that efficiently infected normal 293 cells with a approximately 50% primary infection rate. BAC36 virions were also infectious to HeLa and E6E7-immortalized human endothelial cells. Since BAC36 can be efficiently shuttled between bacteria and mammalian cells, it is useful for KSHV genetic analysis. The feasibility of the system was illustrated through the generation of a KSHV mutant with the vIRF gene deleted. This cellular model is useful for the investigation of KSHV infection and pathogenesis.

Transcriptional regulation of Kaposi's sarcoma-associated herpesvirus-encoded oncogene viral interferon regulatory factor by a novel transcriptional silencer, Tis.

Viral interferon regulatory factor (vIRF) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) has been shown to transform NIH3T3 and Rat-1 cells, inhibit interferon signal transduction, and regulate the expression of KSHV genes. We had previously characterized the vIRF core promoter and defined a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive region in the upstream regulatory sequence of vIRF gene. Here, we have further identified a novel transcriptional silencer, named Tis in this region. Tis represses the promoter activities of vIRF and heterologous herpes simplex virus thymidine kinase genes in both position- and orientation-independent manners. Deletion analysis has identified a cis-element of 23 nucleotides that is essential for the negative regulation. Two Tis-binding protein complexes, named vR1 and vR2, were observed by electrophoretic mobility shift assays using nuclear extracts from both KSHV-negative and -positive cell lines. A sequence fragment GAGTTAATAGGTAGAG in the cis-element was shown to be required for the DNA-protein interactions as well as the repression of vIRF promoter activity. Point-mutation analysis identified TTAAT and GTTAATAG as the core sequence motifs for the binding of vR1 and vR2, respectively. These results define the function of a novel transcriptional silencer in the regulation of vIRF gene expression.