AIM: To assessed the mRNA transcriptome profiling of human HepG2 cells exposed to Catharanthus roseus G. Don (C. roseus)-AgNPs.

METHODS: The proliferative activity of hepatocellular carcinoma (HepG2) and normal human liver (THLE3) cells treated with C. roseusAgNPs were measured using MTT assay. The RNA samples were extracted and sequenced using BGIseq500 platform. This is followed by data filtering, mapping, gene expression analysis, differentially expression genes analysis, Gene Ontology analysis, and pathway analysis.

RESULTS: The mean IC50 values of C. roseusAgNPs on HepG2 was 4.38 ± 1.59 μg/mL while on THLE3 cells was 800 ± 1.55 μg/mL. Transcriptome profiling revealed an alteration of 296 genes. C. roseusAgNPs induced the expression of stress-associated genes such as MT, HSP and HMOX-1. Cellular signalling pathways were potentially activated through MAPK, TNF and TGF pathways that are responsible for apoptosis and cell cycle arrest. The alteration of ARF6, EHD2, FGFR3, RhoA, EEA1, VPS28, VPS25, and TSG101 indicated the uptake of C. roseus-AgNPs via both clathrin-dependent and clathrin-independent endocytosis.

METHODOLOGY AND PRINCIPAL FINDINGS: A literature search was performed in PubMed, CINAHL Complete, and Scopus databases from the 1st December 2020 until 22nd April 2021. Studies reporting sensitivity and specificity of serological tests against CHIKV that used whole blood, serum, or plasma were included. QUADAS-2 tool was used to assess the risk of bias and applicability, while R software was used for statistical analyses. Thirty-five studies were included in this meta-analysis; 72 index test data were extracted and analysed. Rapid and ELISA-based antigen tests had a pooled sensitivity of 85.8% and 82.2%, respectively, and a pooled specificity of 96.1% and 96.0%, respectively. According to our meta-analysis, antigen detection tests serve as a good diagnostic test for acute-phase samples. The IgM detection tests had more than 90% diagnostic accuracy for ELISA-based tests, immunofluorescence assays, in-house developed tests, and samples collected after seven days of symptom onset. Conversely, low sensitivity was found for the IgM rapid test (42.3%), commercial test (78.6%), and for samples collected less than seven of symptom onset (26.2%). Although IgM antibodies start to develop on day 2 of CHIKV infection, our meta-analysis revealed that the IgM detection test is not recommended for acute-phase samples. The diagnostic performance of the IgG detection tests was more than 93% regardless of the test formats and whether the test was commercially available or developed in-house. The use of samples collected after seven days of symptom onset for the IgG detection test suggests that IgG antibodies can be detected in the convalescent-phase samples. Additionally, we evaluated commercial IgM and IgG tests for CHIKV and found that ELISA-based and IFA commercial tests manufactured by Euroimmun (Lübeck, Germany), Abcam (Cambridge, UK), and Inbios (Seattle, WA) had diagnostic accuracy of above 90%, which was similar to the manufacturers' claim.

Materials and Methods: Pfu polymerase gene sequence, derived from Pyrocuccus furiosus (Pfu) genomic DNA, was cloned and overexpressed in E. coli BL21 (DE3) pLysS. Upon overexpression, bacterial lysate containing the Pfu DNA polymerase was heated at 94°C for 5 minutes. Pfu DNA polymerase having high thermal stability was retained while the other bacterial proteins were denatured. The resulting thermo stable Pfu DNA polymerase was separated from the other debris of the denatured proteins by simple centrifugation.

Results: The enzymatic activity of the resulting Pfu DNA polymerase was estimated by comparing with the commercial Pfu DNA Polymerases. An estimated 50000 units of functional Pfu DNA polymerase was produced from a 400 ml culture.