Screening the hub genes and analyzing the mechanisms in discharged COVID-19 patients retesting positive through bioinformatics analysis.

BACKGROUND: After encountering COVID-19 patients who test positive again after discharge, our study analyzed the pathogenesis to further assess the risk and possibility of virus reactivation. METHODS: A separate microarray was acquired from the Gene Expression Omnibus (GEO), and its samples were divided into two groups: a "convalescent-RTP" group consisting of convalescent and "retesting positive" (RTP) patients (group CR) and a "healthy-RTP" group consisting of healthy control and RTP patients (group HR). The enrichment analysis was performed with R software, obtaining the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, the protein-protein interaction (PPI) networks of each group were established, and the hub genes were discovered using the cytoHubba plugin. RESULTS: In this study, 6622 differentially expressed genes were identified in the group CR, among which RAB11B-AS1, DISP1, MICAL3, PSMG1, and DOCK4 were up-regulated genes, and ANAPC1, IGLV1-40, SORT1, PLPPR2, and ATP1A1-AS1 were down-regulated. 7335 genes were screened in the group HR, including the top 5 up-regulated genes ALKBH6, AMBRA1, MIR1249, TRAV18, and LRRC69, and the top 5 down-regulated genes FAM241B, AC018529.3, AL031963.3, AC006946.1, and FAM149B1. The GO and KEGG analysis of the two groups revealed a significant enrichment in immune response and apoptosis. In the PPI network constructed, group CR and group HR identified 10 genes, respectively, and TP53BP1, SNRPD1, and SNRPD2 were selected as hub genes. CONCLUSIONS: Using the messenger ribonucleic acid (mRNA) expression data from GSE166253, we found TP53BP1, SNRPD1, and SNRPD2 as hub genes in RTP patients, which is vital to the management and prognostic prediction of RTP patients.

[Effects of interferon-gamma on Her-2/neu expression and antitumor activity of 131I-Herceptin in breast cancer cell lines].

BACKGROUND & OBJECTIVE: Herceptin plays an important role in treating metastatic breast cancer by targeting Her2/neu, therefore, combining Herceptin with iodine-131 (131I) might enhance its antitumor activity. This study was to up-regulate Her2/neu expression by interferon-gamma (IFN-gamma), and explore its effect on binding and antitumor activity of 131I-Herceptin in breast cancer cell lines MCF-7, SKBR-3 and BT-474. METHODS: MCF-7, SKBR-3 and BT-474 cells were cultured with or without IFN-gamma (500 U/ml) for 48 h. The positive rate and mean fluorescence intensity (MFI) of Her2/neu on the 3 cell lines were tested by flow cytometry. Herceptin was labeled with 131I by Iodogen method, and its radiochemical purity (RCP) was tested by size-exclusion high-pressure liquid chromatography (HPLC). The binding rate of 131I-Herceptin on cells was measured by non-competitive saturation analysis, and its killing effect was estimated by colony-forming assay. The positive rate and MFI of Her2/neu, binding rate of 131I-Herceptin, and colony-forming rate were compared between IFN-gamma-induced group and control group by t test. RESULTS: For MCF-7 cells, the positive rate and MFI of Her2/neu were significantly higher in IFN-gamma-induced cells than in control cells [(15.2+/-4.7)% vs. (8.5+/-1.9)%, t=3.515, P<0.05; 121+/-17 vs. 38+/-7, t=7.823, P<0.002]; for SKBR-3 and BT-474 cells, no obvious difference of Her2/neu positive rate was observed between IFN-gamma-induced cells and control cells [(99.7+/-0.9)% vs. (98.9+/-1.1)%, P>0.05; (99.5+/-1.2)% vs. (98.1+/-0.9)%, P>0.05], but the MFI of Her2/neu was significantly higher in IFN-gamma-induced cells than in control cells (1,608+/-201 vs. 952+/-125, t=4.802, P<0.01; 1,968+/-192 vs. 1,020+/-98, t=7.614, P<0.002). The binding rates of Her2/neu were increased from (5.2+/-1.4)% to (12.3+/-3.4)% by 2.4 folds in MCF-7 cells, from (35.8+/-4.5)% to (48.9+/-7.1)% by 1.4 folds in SKBR-3 cells, and from (37.2+/-3.6)% to (59.5+/-8.7)% by 1.6 folds in BT-474 cells after inducement with IFN-gamma. The colony-forming rates were significantly lower in IFN-gamma-induced MCF-7, SKBR-3 and BT-474 cells than in control cells [(30+/-4)% vs. (49+/-3)%, t=6.574, P<0.05; (23+/-5)% vs. (37+/-6)%, t=3.105, P<0.05; (19+/-6)% vs. (34+/-5)%, t=3.323, P<0.05]. CONCLUSION: IFN-gamma can up-regulate Her-2/neu expression and increase the binding of 131I-Herceptin, hence, improve the inhibitory effect of 131I-Herceptin on proliferation of breast cancer cells.

[Adsorption condition optimization for anti-HBsAg Fab fragment separation and purification from E. coli using Streamline SP].

OBJECTIVE: To optimize the adsorption condition of cation-exchange chromatographic media Streamline SP for separation and purification of anti-HBsAg Fab fragment from E. coli. METHODS: The adsorption of the target protein for separation and purification by the cation-exchange chromatographic media Streamline SP was tested using test tube method in balanced buffer solution with different pH values and ion concentrations. The adsorption effect was then verified by cation-exchange chromatography using 1-ml Streamline SP prepacked column and 28-ml Streamline SP self-assembly column. RESULTS: According to the experiment results of test tube method, the loading buffer with pH of 4.4 and ionic concentration of 100 to 600 mmol/L could achieve optimal target protein adsorption effect by cation-exchange chromatographic media Streamline SP, as verified by cation-exchange chromatography with 1-ml SP prepacked column and 28-ml Streamline SP self-assembly column. CONCLUSION: The optimal condition of cation-exchange chromatography selected by test tube method can be applied for separation and purification of anti-HBsAg Fab fragment from E. coli.

[Preparation, quality control and biodistribution of 131I-herceptin in New Zealand rabbits].

OBJECTIVE: To study the immunoactivity,biodistribution and metabolic pattern of (131)I-Herceptin in rabbits. METHODS: Herceptin was radiolabelled with (131)I and its radiochemicalpurity (RCP) measured by size-exclusion high-pressure liquid chromatography (HPLC). The binding rate to BT-474 cells was measured to evaluate the immunoactivity of (131)I-Herceptin. (131)I-herceptin (2.0 mCi/kg) was injected intravenously into New Zealand rabbits. Scintigraphy on emission computed tomography was performed at 3 h, 1, 3 and 5 days after injection, and the radiocounts of the heart, liver and kidney etc. were compared with that of the muscle to calculate the organ-to-muscle activity ratio (O/M). On the fifth day,the rabbits were killed and the blood, myocardium, lung and other organs were obtained for measuring the radiocounts on gamma-counter to calculate the uptake percentage per gram tissue (ID%/g). RESULTS: The labeling rate of (131)I-herceptin was 93% with RCP of 95% and binding rate to BT-474 cells of 36.9%. After injection of (131)I-herceptin, the heart, lung and liver displayed dense radioactive regions but not the muscles and intestines. Three hours after injection, the O/M ratio of the heart was significantly higher than that of the lung, kidney and intestine (P<0.05), but decreased significantly one day after injection (t=10.817, P<0.001) with further decrement on days 3 and 5 (P<0.05). The O/M ratio of liver on day 1, 3, and 5 reduced significantly in comparison with that at 3 h (P<0.05). The uptake percentage was higher in the blood (11.3 ID/g%) than in the liver (2.8 ID/g%) and the myocardium (1.8 ID/g%). CONCLUSIONS: (131)I-herceptin possesses high immunoactivity which distributes mainly in the blood, liver and kidney, but with low uptake in the myocardium.

[Biological response of B-cell lymphoma cells in vitro to 131I-rituximab].

OBJECTIVE: To study the biological response of B-cell lymphoma cells positive for CD20 expression to (131)I-labeled rituximab. METHODS: Anti-CD20 monoclonal antibody rituximab was labeled with (131)I by means of IODO-GEN method, and its effects on apoptosis of Raji cells were determined by Annexin-V/PI double-labeled cytometry. Its effects on the cell cycles was evaluated by cytometry with PI staining. RESULTS: The cell apoptosis rate measured by Annexin v-FITC/PI was 51.99% in (131)I-rituximab group, significantly higher than that in (131)I group, rituximab group and control group (42.71%, 29.42% and 26.17%, respectively, P<;0.05). The apoptosis rate by flow cytometry with PI staining was 4.32% in (131)I-rituximab group, also significantly higher than that in the other 3 groups (1.47%, 1.39% and 0.37%, respectively, P<0.05). Cell cycle alteration of Raji cells occurred in (131)I-rituximab group, and the majority of cells were arrested at G(1)/G(2) stage. CONCLUSION: (131)I-rituximab can regulate the cell cycle of Raji cells and induce their apoptosis to inhibit their proliferation.

Preventive and therapeutic effects of NF-kappaB inhibitor curcumin in rats colitis induced by trinitrobenzene sulfonic acid.

AIM: To ascertain the molecule mechanism of nuclear factor-kappaB (NF-kappaB) inhibitor curcumin preventive and therapeutic effects in rats' colitis induced by trinitrobenzene sulfonic acid (TNBS). METHODS: Sixty rats with TNBS-induced colitis were treated with 2.0% curcumin in the diet. Thirty positive control rats were treated with 0.5% sulfasalazine (SASP). Thirty negative control rats and thirty model rats were treated with general diet. Changes of body weight together with histological scores were evaluated. Survival rates were also evaluated. Cell nuclear NF-kappaB activity in colonic mucosa was evaluated by using electrophoretic mobility shift assay. Cytoplasmic IkappaB protein in colonic mucosa was detected by using Western Blot analysis. Cytokine messenger expression in colonic tissue was assessed by using semiquantitative reverse-transcription polymerase chain reaction. RESULTS: Treatment with curcumin could prevent and treat both wasting and histopathologic signs of rats with TNBS-induced intestinal inflammation. In accordance with these findings, NF-kappaB activation in colonic mucosa was suppressed in the curcumin-treated groups. Degradations of cytoplasmic IkappaB protein in colonic mucosa were blocked by curcumin treatment. Proinflammatory cytokine messenger RNA expression in colonic mucosa was also suppressed. CONCLUSION: This study shows that NF-kappaB inhibitor curcumin could prevent and improve experimental colitis in murine model with inflammatory bowel disease (IBD). The findings suggest that NF-kappaB inhibitor curcumin could be a potential target for the patients with IBD.