Shuang Shi

Identification of eight genes encoding chemokine-like factor superfamily members 1-8 (CKLFSF1-8) by in silico cloning and experimental validation.

TM4SF11 is only 102 kb from the chemokine gene cluster composed of SCYA22, SCYD1, and SCYA17 on chromosome 16q13. CKLF maps on chromosome 16q22. CKLFs have some characteristics associated with the CCL22/MDC, CX3CL1/fractalkine, CCL17/TARC, and TM4SF proteins. Bioinformatics based on CKLF2 cDNA and protein sequences in combination with experimental validation identified eight novel genes designated chemokine-like factor superfamily members 1-8 (CKLFSF1-8). CKLFSF1-8 form gene clusters; the sequence identities between CKLF2 and CKLFSF1-8 are from 12.5 to 39.7%. Most of the CKLFSFs have alternative RNA splicing forms. CKLFSF1 has a CC motif and higher sequence similarity with chemokines than with any of the other CKLFSFs. CKLFSF8 shares 39.3% amino acid identity with TM4SF11. CKLFSF1 links the CKLFSF family with chemokines, and CKLFSF8 links it with TM4SF. The characteristics of CKLFSF2-7 are intermediate between CKLFSF1 and CKLFSF8. This indicates that CKLFSF represents a novel gene family between the SCY and the TM4SF gene families.

Comparison of four current staging systems for Chinese patients with hepatocellular carcinoma undergoing curative resection: Okuda, CLIP, TNM and CUPI

Background and Aim:  Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. However, there is no general consensus as to which staging system is the most reliable for predicting the survival of patients with HCC. The aims of this study were to compare commonly‐used staging systems in a cohort of Chinese HCC patients undergoing curative resection.

Thermal stability and inactivation of hepatitis C virus grown in cell culture

BackgroundHepatitis C virus (HCV) is a blood-borne flavivirus that infects many millions of people worldwide. Relatively little is known, however, concerning the stability of HCV and reliable procedures for inactivating this virus.MethodsIn the current study, the thermostability of cell culture-derived HCV (HCVcc, JFH-1 strain) under different environmental temperatures (37°C, room temperature, and 4°C) and the ability of heat, UVC light irradiation, and aldehyde and detergent treatments to inactivate HCVcc were evaluated. The infectious titers of treated viral samples were determined by focus-forming unit (FFU) assay using an indirect immunofluorescence assay for HCV NS3 in hepatoma Huh7-25-CD81 cells highly permissive for HCVcc infection. MTT cytotoxicity assay was performed to determine the concentrations of aldehydes or detergents at which they were no longer cytotoxic.ResultsHCVcc in culture medium was found to survive 37°C and room temperature (RT, 25 ± 2°C) for 2 and 16 days, respectively, while the virus was relatively stable at 4°C without drastic loss of infectivity for at least 6 weeks. HCVcc in culture medium was sensitive to heat and could be inactivated in 8 and 4 min when incubated at 60°C and 65°C, respectively. However, at 56°C, 40 min were required to eliminate HCVcc infectivity. Addition of normal human serum to HCVcc did not significantly alter viral stability at RT or its susceptibility to heat. UVC light irradiation (wavelength = 253.7 nm) with an intensity of 450 μW/cm2 efficiently inactivated HCVcc within 2 min. Exposures to formaldehyde, glutaraldehyde, ionic or nonionic detergents all destroyed HCVcc infectivity effectively, regardless of whether the treatments were conducted in the presence of cell culture medium or human serum.ConclusionsThe results provide quantitative evidence for the potential use of a variety of approaches for inactivating HCV. The ability of HCVcc to survive ambient temperatures warrants precautions in handling and disposing of objects and materials that may have been contaminated with HCV.

FAM19A4 is a novel cytokine ligand of formyl peptide receptor 1 (FPR1) and is able to promote the migration and phagocytosis of macrophages.

FAM19A4 is an abbreviation for family with sequence similarity 19 (chemokine (C–C motif)-like) member A4, which is a secretory protein expressed in low levels in normal tissues. The biological functions of FAM19A4 remain to be determined, and its potential receptor(s) is unclarified. In this study, we demonstrated that FAM19A4 was a classical secretory protein and we verified for the first time that its mature protein is composed of 95 amino acids. We found that the expression of this novel cytokine was upregulated in lipopolysaccharide (LPS)-stimulated monocytes and macrophages and was typically in polarized M1. FAM19A4 shows chemotactic activities on macrophages and enhances the macrophage phagocytosis of zymosan both in vitro and in vivo with noticeable increases of the phosphorylation of protein kinase B (Akt). FAM19A4 can also increase the release of reactive oxygen species (ROS) upon zymosan stimulation. Furthermore, based on receptor internalization, radio ligand binding assays and receptor blockage, we demonstrated for the first time that FAM19A4 is a novel ligand of formyl peptide receptor 1 (FPR1). The above data indicate that upon inflammatory stimulation, monocyte/macrophage-derived FAM19A4 may play a crucial role in the migration and activation of macrophages during pathogenic infections.

Simultaneously High Prevalences of Hepatitis B and C Virus Infections in a Population in Putian County, China

ABSTRACT This study investigated a rare area of endemicity with a high prevalence of both hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, in Putian County, China. Among 1,050 subjects, the overall prevalence of HBsAg seropositivity was 15.8%, and that of anti-HCV seropositivity was 28.9%. Intrafamilial viral transmission might be the major cause of the high prevalence of HBV infection in this region. However, HCV infection was shown to be associated with the use of inadequately sterilized medical equipment.

Efficient production of FAM19A4, a novel potential cytokine, in a stable optimized CHO-S cell system.

FAM19A4 is a novel potential cytokine identified by our group, which can chemoattract macrophages, promote phagocytosis against zymosan and increase reactive oxygen species (ROS) release. To further explore the role of FAM19A4 in immune system, abundant recombinant protein with high quality is indispensable. For efficient production of FAM19A4, we used an improved CHO-S cell expression system on the basis of pMH3 vector containing GC-rich regions which were novel ubiquitous chromatin opening elements (UCOEs). We selected CHO-S cells stably expressing FAM19A4 with G418 and screened cell clones with high level of FAM19A4 expression by immune blot and his-ELISA, adapted cell clones to serum-free suspension culture. Afterwards, we obtained the highest FAM19A4 expressing cell clone (2#) through 40 ml batch culture. We optimized the fed-batch culture condition and discovered the final cell viability was critical for FAM19A4 production successfully. Then we scaled 2# clone up to 3 L in fed-batch culture and obtained 22 mg (7.33 mg/L, averagely) endotoxin free FAM19A4 protein with purity over 95% using Ni affinity chromatography and size exclusion chromatography. The final yield was increased 3.6-folds compared to that of our previously reported transient system. Besides, the purified FAM19A4 protein showed chemotactic activity on macrophages. In summary, we developed a stable optimized fed-batch CHO-S cell system to produce FAM19A4, which not only provided sufficient bioactive FAM19A4 protein for further research but also offered an efficient strategy for other recombinant protein production.

A meta-analysis that compares the use of either peginterferon-α2a or peginterferon-α2b plus ribavirin for HCV infection

Background Two kinds of peginterferons, peginterferon-α2a (PEG-IFN-α2a) and peginterferon-α2b (PEG-IFN-α2b), are used in the treatment of chronic hepatitis C virus (HCV) infection. However, it is unclear which is better in terms of virological responses and patient compliance. We conducted a meta-analysis to assess which peginterferon was better when used with ribavirin. Methods Relevant clinical trials were identified through the PubMed and EMBASE databases. Primary outcomes included early virological response (EVR), end of treatment response (ETR) and sustained virological response (SVR). Secondary outcomes included biochemical and histological responses and the discontinuation of treatment after adverse events. Meta-analysis was performed using fixed-effect or random-effect methods, depending on absence or presence of significant heterogeneity. Analyses were performed with Review Manager Version 4.2.2. Results Seven clinical trials were included that involved 3,526 patients in total; six were randomized clinical trials (RCTs) and one was nonrandomized. PEG-IFN-α2a plus ribavirin was better than PEG-IFN-α2b plus ribavirin with regards to ETR (relative risk [RR] = 1.21, 95% confidence interval [CI]: 1.14–1.28). This advantage was less obvious for EVR (RR = 1.12, 95% CI: 1.06–1.19) and SVR (RR = 1.10, 95% CI: 1.02–1.18). Patients who received PEG-IFN-α2a were less likely to discontinue treatment for safety reasons (RR = 0.85, 95% CI: 0.52–1.38). Conclusion We demonstrated that PEG-IFN-α2a was a better choice than PEG-IFN-α2b in terms of virological responses.

A laboratory-adapted HCV JFH-1 strain is sensitive to neutralization and can gradually escape under the selection pressure of neutralizing human plasma

Viral replication and neutralization of hepatitis C viruses (HCV) have been studied using the infectious molecular clone JFH-1. By passaging JFH-1 in hepatoma cells in the absence or presence of HCV neutralizing antibodies (nAbs), we investigated the molecular mechanisms of cell-culture adaptation and sensitivity to nAbs. The cell culture-adapted JFH-1 virus (JFH-1-CA) became more sensitive to nAbs than its parental virus. Sequence analysis revealed that the predominant viruses in the JFH-1-CA population carried two mutations in their envelopes (I414T and V293A). Plasma that could neutralize JFH-1-CA was found in 2 of 7 HCV-infected individuals who have cleared the virus in blood. Plasma 226233 with a higher 50% neutralization titer was used for in vitro selection of neutralization resistant viruses. Under the increasing selection pressure of plasma 226233, the neutralizing sensitivity of JFH-1-CA decreased gradually. Two mutations (T414I and P500S) in envelope were found in all but one sequenced clones in the viral population after eight rounds of selection. Interestingly, the cell-culture adapted mutation I414T reverted back to the wild-type residue (I414) under the selection pressure. By introducing mutations at positions 414 and 500 into the JFH-1 clone, we confirmed that the T414I mutation alone can confer neutralization resistance. The results of this current study suggest that nAbs are present in a subset of HCV-infected individuals who have cleared the virus in blood. Our data also provide the first evidence that, the E2 residue P500, located within a previously identified highly conserved polyclonal epitope, may be a target for neutralizing antibodies present in individual who have spontaneously resolved the HCV infection.