Genetic fusion of mussel foot protein to ZZ protein improves target detection in solid-phase immunoassays.

In the process of a solid-phase immunoassay, the stability and binding orientation between the antibody and the solid matrix can substantially influence the results. ZZ protein is a modified peptide of the B domain of Staphylococcus aureus protein A, which can bind to the Fc fragment of an antibody. It is often used for oriented immobilization of antibodies during solid-phase immunoassay. However, the conjugate is often not retained during the process, for example during washing steps. The resulting low stability detracts from reproducibility and sensitivity. Mfp-5 protein comes from mussel, is one of the components of mussel foot silk protein, and has good adhesion and biocompatibility. In this paper, the fusion protein of ZZ and Mfp-5 was constructed and expressed in Escherichia coli. In this method, the ZZ domain was firmly attached to the solid-phase support by Mfp-5, the directional fixation of IgG was realized by binding the ZZ protein to an Fc fragment, and then a Fab fragment was bound to the antigen to realize the solid-phase immunoassay. In addition, a protein adsorption assay confirmed that the adhesion of ZZ-Mfp-5 was significantly higher than that of ZZ protein, and the presence of Mfp-5 improved the ability of ZZ protein to capture antibodies. In conclusion, compared with the passively immobilized ZZ protein, the ZZ-Mfp-5 protein had stronger immobilization and antibody capture, a 10-fold increase in sensitivity and wider linear range, and better stability of detection. This may be an attractive strategy for solid-phase immunoassays or biosensing assays.

TNF-α/NF-κB signaling epigenetically represses PSD4 transcription to promote alcohol-related hepatocellular carcinoma progression.

BACKGROUND: Chronic alcohol consumption is more frequently associated with advanced, aggressive hepatocellular carcinoma (HCC) tumors. Alcohol adversely impacts ER/Golgi membrane trafficking and Golgi protein N-glycosylation in hepatocytes; these effects have been attributed (in part) to dysregulated adenosine diphosphate-ribosylation factor (ARF) GTPase signaling. Here, we investigated the role of the ARF GTPase guanine exchange factor PSD4 in HCC progression. METHODS: R-based bioinformatics analysis was performed on publicly available array data. Modulating gene expression was accomplished via lentiviral vectors. Gene expression was analyzed using quantitative real-time PCR and immunoblotting. PSD4 promoter methylation was assessed using quantitative methylation-specific PCR. Phospho-p65(S276)/DNMT1 binding to the PSD4 promoter was analyzed via chromatin immunoprecipitation. We constructed ethanol/DEN-induced and DEN only-induced transgenic murine models of HCC. RESULTS: We identified PSD4 as a hypermethylated, suppressed gene in alcohol-related HCC tumors; however, PSD4 was not dysregulated in all-cause HCC tumors. Certain HCC cell lines also displayed varying degrees of PSD4 downregulation. PSD4 overexpression or knockdown decreased and increased cell migration and invasiveness, respectively. Mechanistically, PSD4 transcription was repressed by TNF-α-induced phospho-p65(S276)'s recruitment of DNA methyltransferase 1 (DNMT1), resulting in PSD4 promoter methylation. PSD4 inhibited pro-EMT CDC42 activity, resulting in downregulation of E-cadherin and upregulation of N-cadherin and vimentin. Hepatocyte-specific PSD4 overexpression reduced ethanol/DEN-induced HCC tumor progression and EMT marker expression in vivo. CONCLUSIONS: PSD4 is a hypermethylated, suppressed gene in alcohol-related HCC tumors that negatively modulated pro-EMT CDC42 activity. Furthermore, we present a novel phospho-NF-κB p65(S276)/DNMT1-mediated promoter methylation mechanism by which TNF-α/NF-κB signaling represses PSD4 transcription in HCC cells.

High-Fidelity Transfer of Chemical Vapor Deposition Grown 2D Transition Metal Dichalcogenides via Substrate Decoupling and Polymer/Small Molecule Composite.

Current polymeric transfer methods of 2D materials often bring about the presence of wrinkles, cracks, and polymer residue, limiting the quality of the transferred materials and performance of devices. Herein, we report a transfer approach combining pretreatment by liquid nitrogen and lithium ion intercalation with polymer composite of small molecules and polystyrene to achieve high-fidelity transfer of 2D transition metal dichalcogenides (TMDs) grown by chemical vapor deposition. In this method, the as-grown samples were pretreated by liquid nitrogen and lithium ion intercalation to weaken the bonding between the TMD and the substrate. A polymer composite incorporating small molecules, namely camphor or naphthalene, was used to increase the dissolution of the polymer film. These two processes work synergistically to enable nearly 100% transfer of monolayer TMDs virtually free of wrinkles, cracks, or organic residue with retained optical properties. Our technique can be generalized for the efficient and high quality transfer of other 2D materials.

Cancer Stem Cells of Diffuse Large B Cell Lymphoma Are Not Enriched in the CD45+CD19- cells but in the ALDHhigh Cells.

Although the existence of cancer stem cells (CSCs) has been suggested in diffuse large B cell lymphoma (DLBCL), there is still no definitive marker. CD45+CD19- has been regarded as a potential marker of CSCs in mantle cell lymphoma (MCL). So, we explored the role of CD45+CD19- in DLBCL. However, both CD45+CD19- cells and CD45+CD19+ cells did not generate tumors until more than 100,000 cells were inoculated in NOD/SCID mice, even CD45+CD19+ cells generated more and larger tumors, as well as the soft agar colony formation in vitro; The aldehyde dehydrogenase (ALDH) activity was also identified in this study. Only 1,500 ALDHhigh cells were enough to generate tumors in mice while the same number of ALDH- cells were not. Moreover, both groups formed tumors when more cells were inoculated, but ALDHhigh cells formed more and larger tumors. The similar result was obtained in vitro clonogenicity experiments. OCT4, SOX2, Nanog, and ABCG2 genes did not show any difference in CD45+CD19+, CD45+CD19-, ALDHhigh and ALDH- cells. Taken together, CSCs are not enriched in the CD45+CD19- cells but in the ALDHhigh cells in DLBCL cell lines.

Hepatitis B core antigen regulates dendritic cell proliferation and apoptosis through regulation of PKC/NF­κB signaling pathway.

Hepatitis B core antigen (HBcAg) possesses unusual immunologic features. However, the biological roles and mechanisms of HBcAg in dendritic cell proliferation and apoptosis remain to be elucidated. In the present study, DC2.4 cells were treated with different concentrations of HBcAg (10, 20 and 30 µg/ml). MTT assay and flow cytometry (Annexin V/propidium iodide analysis) were performed to investigate changes in cell proliferation and apoptosis. Western blot analysis was conducted to examine the changes in nuclear factor (NF)­κB and protein kinase C (PKC) signaling pathways. NF­κB inhibitor pyrrolidine dithiocarbamate (PDTC) and PKC inhibitor Chelerythrine were used to block these two signaling pathways. It was identified that HBcAg increased proliferation and decreased apoptosis in a dose­dependent manner. Western blotting results demonstrated that HBcAg upregulated p­PKC, p­IκB, p­P65, tumor necrosis factor­α and B­cell lymphoma 2 (Bcl­2) levels, and downregulated cleaved caspase 3, demonstrating that HBcAg activated the PKC and NF­κB signaling pathways. NF­κB inhibitor PDTC reduced the effects of HBcAg on DC2.4 proliferation (0.6 fold vs. 0.25 fold) and apoptosis (0.43 fold vs. 0.17 fold), and on Bcl­2 expression levels. PKC inhibitor Chelerythrine reduced the biological effects of HBcAg; it reduced proliferation (0.67 fold vs. 0.23 fold) and upregulated apoptosis (0.43 fold vs. 0.13 fold). Chelerythrine also blocked NF­κB activity and the HBcAg­induced Bcl­2 increase, suggesting the effect on Bcl­2 from HBcAg was dependent on the PKC/NF­κB signaling pathway. In conclusion, HBcAg promoted proliferation and inhibited apoptosis through the PKC/NF­κB/Bcl­2 signaling pathway in DC2.4 cells.

Hepatitis B Virus Replication in CD34+ Hematopoietic Stem Cells From Umbilical Cord Blood.

BACKGROUND Hepatitis B virus (HBV) is a hepatotropic virus that can infect extrahepatic tissue. Whether hematopoietic stem cells (HSCs) can be infected by HBV and serve as a potential virus reservoir is still unknown. In this study, the susceptibility of CD34+ HSCs to HBV was investigated. MATERIAL AND METHODS Cord blood-derived CD34+ HSCs were exposed to HBV in vitro, and immunocytochemistry, transmission electron microscopy, and RT-PCR were used to identify viral-related proteins and specific viral genomic sequences. Then, CD34+ HSCs were challenged by different titers of HBV, and intracellular and supernatant HBV DNA, and hepatitis B surface antigen (HBsAg) levels, were examined. In addition, CD34+ peripheral blood stem cells (PBSCs) from chronic HBV carriers were isolated and cultured, and HBV DNA levels were measured. RESULTS HBV-infected CD34+ cells showed positive signals for HBsAg by DAB staining and TRITC staining, and HBV particles were identified. RT-PCR results showed that the 403 bp PCR products corresponding to the amplified hepatitis B S gene fragment were observed in CD34+ HSCs infected by HBV. In addition, supernatant and intracellular HBV DNA increased with the proliferation of CD34+ HSCs. Similar results were obtained from intracellular HBsAg quantification tests. In addition, HBV DNA levels both in cells and in supernatants of CD34+ PBSCs increased proportionally, and the increments of HBV DNA in the supernatants paralleled those found in cells. CONCLUSIONS HBV can replicate in CD34+ HSCs in cord blood or peripheral blood of chronic HBV carriers.

Hepatitis B virus replication is upregulated in proliferated peripheral blood lymphocytes.

Increasing evidence indicates that the hepatitis B virus (HBV) replicates in peripheral blood mononuclear cells (PBMCs), but at a low level. The present study aimed to establish a reliable and sensitive method that effectively detects HBV viral products for monitoring antiviral therapy, organ transplantation screening, and diagnosing occult HBV infection. In the present study, PBMCs (obtained from six healthy volunteers) were inoculated with HBV, and cultured with phytohemagglutinin (PHA) and interleukin­2 (IL­2) to stimulate cell proliferation. PBMCs were harvested, and quantitative detection of HBV DNA in cell suspension and intracellular hepatitis B surface antigen (HBsAg) was conducted on days 0, 1, 6 and 12, respectively. In situ hybridization, immunohistochemistry and reverse transcription­polymerase chain reaction (RT­PCR) were performed to analyze the HBV infection. The results demonstrated that HBV DNA increased concurrently with proliferation of PBMCs isolated from three of six healthy volunteers, and the mean number of PBMCs on day 12 was 13.61 times higher than the initially seeded cell number (P<0.01). The mean copies of HBV DNA at day 12 were 2.98 times higher compared with initial levels (P<0.05). Furthermore, intracellular HBsAg levels increased concurrently with proliferation of PBMCs in one group of cultured PBMCs, which was accompanied by increased HBV DNA levels. In addition, HBV nucleic acids were detected in PBMCs using in situ hybridization. Intracellular HBsAg was observed in PBMCs and HBV RNA was also detected by RT­PCR. The present study demonstrated that HBV replicates in proliferating PBMCs, which were induced by PHA and IL­2. This method offers a novel investigative tool to detect HBV infection in PBMCs and to monitor the course of HBV infection.

T-bet expression in CD8+ T cells associated with chronic hepatitis B virus infection.

BACKGROUND: The mechanisms leading to virus-specific CD8+ T cell dysfuction in chronic hepatitis B virus (HBV) infection remain to be elucidated. Our study focused on the role of transcription factor T-bet in HBV infection because it is a crucial regulator of T cell immunity. METHODS: We assessed the expression of T-bet along with PD-1, IFN-γ and perforin, in HBV-specific CD8+ T cells from resolved acute hepatitis B (rAHB) patients, chronic hepatitis B (CHB) patients, as well as asymptomatic HBV carriers (ASCs). We observed dynamic changes of T-bet, PD-1, IFN-γ and perforin in acute stage and recovery stage of acute hepatitis B (AHB). RESULTS: Comparing with other cohorts, HBV-specific CD8+ T cells from rAHB demonstrated a superior ability in T-bet, IFN-γ and perforin expression, but an inferior ability in PD-1 expression. In the CHB group, the level of T-bet has a linear relationship with the level of PD-1, IFN-γ and HBV DNA, respectively. A lower expression of T-bet and PD-1 was observed in ASCs when compared with CHB. A higher expression of T-bet, PD-1, IFN-r and perforin was observed in acute stage when compared with the recovery stage of AHB. CONCLUSIONS: Our results suggest that expression of T-bet may influence the function of HBV-specific CD8+ T cells and thus can be an attractive target for modulation to improve HBV-specific immunity in CHB.