Steric Hindrance-Mediated Enzymatic Reaction Enable Homogeneous Dual Fluorescence Indicators Aptasensing of Hepatocellular Carcinoma CTCs.

Circulating tumor cells (CTCs) serve as important biomarkers in the liquid biopsy of hepatocellular carcinoma (HCC). Herein, a homogeneous dual fluorescence indicators aptasensing strategy is described for CTCs in HCC, with the core assistance of a steric hindrance-mediated enzymatic reaction. CTCs in the sample could specifically bind to a 5'-biotin-modified glypican-3 (GPC3) aptamer and remove the steric hindrance formed by the biotin-streptavidin system. This influences the efficiency of the terminal deoxynucleotidyl transferase enzymatic reaction. Then, methylene blue (MB) was introduced to react with the main product poly cytosine (polyC) chain, and trivalent cerium ion (Ce3+) was added to react with the byproduct pyrophosphate to form fluorescent pyrophosphate cerium coordination polymeric nanoparticles. Finally, the CTCs were quantified by dual fluorescence indicators analysis. Under optimized conditions, the linear range was 5 to 104 cells/mL, and the limits of detection reached 2 cells/mL. Then, 40 clinical samples (15 healthy and 25 HCC patients) were analyzed. The receiver operating characteristic curve analysis revealed an area under the curve of 0.96, a sensitivity of 92%, and a specificity of 100%. Therefore, this study established a sensitive and accurate CTCs sensing system for clinical HCC patients, promoting early tumor diagnosis.

CCR2+TREM-1+ monocytes promote natural killer T cell dysfunction contributing towards HBV disease progression.

Natural killer T (NKT) cells are amongst the most important innate immune cells against hepatitis B virus (HBV) infection. Moreover, previous studies have shown that HBV infection induced TREM-1+ expression in monocyte and secretion of inflammatory cytokines. Thus, this prompted us to elucidate the role of TREM-1+ monocytes in regulating the function of iNKT cells. Ninety patients and 20 healthy participants were enrolled in the study. The percentage and phenotype of iNKT cells and TREM-1+ monocytes were measured in the peripheral blood of healthy controls (HC), patients with chronic HBV infection (CHB), HBV-related liver cirrhosis (LC), and HBV-related acute-on-chronic liver failure (ACLF) via flow cytometry. Moreover, co-culture experiments with iNKT cells and TREM-1 overexpressing THP-1 cells were performed to determine the role of TREM-1 in the regulation of NKT cell function. We observed that the percentage of iNKT cells and CD4-iNKT cells gradually decreased, whereas the percentage of CCR2+TREM-1+ monocytes increased with the progression of the disease. In addition, activation of the TREM-1 signaling pathway induced the secretion of inflammatory cytokines leading to pyroptosis of iNKT cells and secretion of IL-17 contributing towards disease progression. Therefore, this study suggests that blocking the activation of TREM-1 in monocytes could promote the elimination of HBV by inhibiting pyroptosis of iNKT cells and restoring their function. However, further studies are required to validate these results that would help in developing new treatment strategies for patients with HBV infections.

Lnc-AIFM2-1 promotes HBV immune escape by acting as a ceRNA for miR-330-3p to regulate CD244 expression.

Chronic hepatitis B (CHB) virus infection is a major risk factor for cirrhosis and hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) immune escape is regulated by the exhaustion of virus-specific CD8+ T cells, which is associated with abnormal expression of negative regulatory molecule CD244. However, the underlying mechanisms are unclear. To investigate the important roles of non-coding RNAs play in CD244 regulating HBV immune escape, we performed microarray analysis to determine the differential expression profiles of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs in patients with CHB and patients with spontaneous clearance of HBV. Competing endogenous RNA (ceRNA) was analyzed by bioinformatics methods and confirmed by the dual-luciferase reporter assay. Furthermore, gene silencing and overexpression experiments were used to further identify the roles of lncRNA and miRNA in HBV immune escape through CD244 regulation. The results showed that the expression of CD244 on the surface of CD8+ T cells was significantly increased in CHB patients and in the co-culture system of T cells and HBV-infected HepAD38 cells, which was accompanied by the reduction of miR-330-3p and the elevation of lnc-AIFM2-1. The down-regulated miR-330-3p induced the apoptosis of T cells by lifting the inhibition of CD244, which was reversed by miR-330-3p mimic or CD244-siRNA. Lnc-AIFM2-1 promotes the accumulation of CD244, which is mediated by decreased miR-330-3p, and then reduced the clearance ability of CD8+ T cells to HBV through regulated CD244 expression. And the injury in the ability of CD8+ T cells to clear HBV can be reversed by lnc-AIFM2-1-siRNA, miR-330-3p mimic, or CD244-siRNA. Collectively, our findings indicate that lnc-AIFM2-1 on CD244 by acting as a ceRNA of miR-330-3p contributes to HBV immune escape, which may provide novel insights into the roles of interaction networks among lncRNA, miRNA, and mRNA in HBV immune escape, highlighting potential applications of lnc-AIFM2-1 and CD244 for diagnosis and treatment in CHB.

Surveillance of the Antimicrobial Susceptibility and Molecular Characteristics of Neisseria gonorrhoeae Isolates Collected in Changsha, China from 2016 to 2021.

Antibiotic treatment is critical for individuals infected with gonorrhea and preventing disease transmission. This study aimed to analyze the antimicrobial susceptibility and molecular epidemiological characteristics of Neisseria gonorrhoeae isolates in Changsha, China. A total of 271 N.gonorrhoeae isolates collected from the clinical laboratories of two hospitals between 2016 and 2021 were analyzed for antimicrobial susceptibility using the agar dilution method. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was conducted for genotyping, and phylogenetic analysis was performed using the porB and tbpB sequences. The results showed that antimicrobial resistance against ciprofloxacin, tetracycline, and penicillin was high, and these drugs are no longer recommended for the treatment of gonorrhea. All isolates were susceptible to spectinomycin. However, in 2016-2021, a total of 15 (5.5%) ceftriaxone (CRO)-resistant strains and 31 (11.4%) isolates with decreased susceptibility to CRO were found, and the resistance rate to azithromycin had reached 7.1% in 2016-2017. Epidemiologically, the mosaic penA allele was identified in all CRO-resistant isolates. Based on NG-MAST, ST5061 was the most prevalent ST. Phylogenetic analysis suggested that the resistant isolates did not cluster independently. Despite focus on the local situation, this study raises the need for better gonorrhea medication and highlights that CRO may not be adequate as first-line treatment for gonorrhea in Changsha.