Combination of dasabuvir and PSI-6206 for the treatment of coxsackievirus B3 infection.

Coxsackievirus B3 (CVB3) infections may cause life-threatening diseases and have no approved specific treatment. Some promising approaches to treat viral diseases include drug repurposing and combination therapy. We have selected in this study dasabuvir, an approved antiviral drug, and PSI-6206, an experimental drug and determined their individual and combined antiviral activity against CVB3 replication in vitro. Our results show that the individual drugs inhibited CVB3 infection in a dose-dependent manner, at a selective index >10 with a strong synergetic antiviral effect of the two compounds. Given that dasabuvir has already been approved for the treatment of hepatitis C virus infection, treatment of CVB3-related disease with this drug may represent a promising treatment strategy.

Novel Antiviral Activity of Ethyl 3-Hydroxyhexanoate Against Coxsackievirus B Infection.

Coxsackievirus group B (CVB) is a member of the genus Enterovirus in the family Picornaviridae. CVB infection has been implicated as a major etiologic agent of viral myocarditis, dilated cardiomyopathy, meningitis, and pancreatitis among children and young adults. Until date, no antiviral agent has been licensed for the treatment of Coxsackievirus infection. In an effort to identify antiviral agents against diseases caused by the CVB, we found that ethyl 3-hydroxyhexanoate (EHX), a volatile compound present in fruits and food additives, is a potent antiviral compound. In this study, we demonstrated that EHX treatment significantly inhibits CVB replication both in vivo and in vitro. Furthermore, EHX possesses antiviral activity at 50% effective concentration (EC50) of 1.2 µM and 50% cytotoxicity (CC50) of 25.6 µM, yielding a selective index (SI) value as high as 20.8. Insights into the mechanism of antiviral activity of EHX showed that it acts at the step of viral RNA replication. Since EHX has received approval as food additives, treatment of CVB-related infections with EHX might be a safe therapeutic option and may be a promising strategy for the development of semi-synthetic antiviral drugs for viral diseases.

Functional Annotation of Hypothetical Proteins related to Antibiotic Resistance in Pseudomonas Aeruginosa PA01.

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative bacteria that causes a large range of human infections such as lung infection (cystic fibrosis) and urinary tract infection. Even worse, antibiotic resistant bacteria have become a serious health care problem throughout the last decade, and there is a need for a clear approach to regulate and prevent the spread of pseudomonas aeruginosa resistance. METHODS: A complete analysis of Pseudomonas aeruginosa proteomics data showed that 25% of proteins are hypothetical proteins (HPs) whose function is not precisely defined. HP gene sequence analysis offers a framework for defining sequence-function relationships with a deeper understanding of organisms' molecular mechanisms at the system level. In the current research, we used the power of different bioinformatics tools to assign the potential roles for the HPs based on protein family association, amino acid function, motifs, and pathway analysis. RESULTS: The current findings show that 30 HPs have well-defined functions and are classified as enzymes, DNA binding, periplasmic binding protein, transport, etc. Seven HPs showed virulence characteristics that is to be expected to be essential for Pseudomonas aeruginosa and pathogenesis survival. CONCLUSIONS: This study's findings may encourage a better understanding of virulence mechanisms, drug resistance, pathogenesis, and drug discovery to treat Pseudomonas aeruginosa infections.

RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the common cancers worldwide, especially in developing countries. Although the chronic infections of hepatitis B and C viruses have been established as the etiological factors of HCC, the mechanism for the tumorigenesis and development of HCC is still unclear. The liver-specific microRNA-122 (miR-122), an established tumor-suppressor miRNA, is often down-regulated in HCC, while the underlying mechanism is not well understood. Here we report that the AU-rich element-binding factor AUF1 suppresses the expression of Dicer1, the type III RNase that is required for microRNA maturation, leading to the inhibited biogenesis of miR-122. Overexpression of AUF1 led to the decreased expression of Dicer1 and miR-122, while the level of the miR-122 precursor (pre-miR-122) was increased. On the other hand, siRNA of AUF1 (siAUF1) increased the levels of Dicer1 mRNA and miR-122, but it reduced the abundance of pre-miR-122. Consistent with the reported data, this study demonstrated that AUF1 and Dicer1 showed opposite expression pattern in both human HCC tissues and cell lines. In addition, AUF1 inhibited the expression of Dicer1 by interacting with the 3' untranslated region (3'UTR) and coding region of DICER1 mRNA. Moreover, the knockdown of AUF1 by siRNA altered the expression of other miRNAs and promoted HCC cell death. In conclusion, AUF1 down-regulates the expression miR-122 by interacting with the 3'UTR and coding region of DICER1 mRNA and suppressing Dicer1 expression. The AUF1/Dicer1/miR-122 pathway might play a critical role in the development of HCC.

Fibroblast activation proteins-α suppress tumor immunity by regulating T cells and tumor-associated macrophages.

Fibroblast activation protein-α (FAPα) is a type-II cell-surface-bound integral transmembrane serine protease and selectively overexpressed by tumor-associated stromal fibroblasts (TAFs), which are the main components in the tumor microenvironment, in >90% of malignant epithelial carcinomas. FAPα regulates the immunosuppression of tumor cells in the tumor microenvironment. Regulatory T cells (Tregs) and tumor-associated macrophages (TAMs) are the major immunosuppressive cells in the tumor microenvironment. However, the effect of FAPα on Tregs and TAMs is unknown. The non-enzymatic function of FAPα on Treg and TAM was investigated. In this study, we confirm that FAPα can promote the generation of Tregs and TAMs, which suggests that FAPα plays a immunosuppressive role in the tumor microenvironment and provides evidence for FAP α as a potent immunotherapeutic target for cancer.

Effects of the HIF-1α and NF-κB loop on epithelial-mesenchymal transition and chemoresistance induced by hypoxia in pancreatic cancer cells.

Hypoxia is a microenvironmental factor which plays a critical role in tumor development and chemoresistance. Epithelial-to-mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure and chemoresistance in different types of human cancers. Stabilization of the hypoxia-inducible factor-1α (HIF-1α) transcription complex, caused by intratumoral hypoxia, promotes tumor progression and chemoresistance. Previous evidence suggests that hypoxia can also activate nuclear factor-κB (NF-κB), a known mediator of EMT, which is accompanied by reduced expression of epithelial marker E-cadherin and enhanced expression of the mesenchymal markers Vimentin and N-cadherin as well as overexpression of various transcription factors of EMT, such as Snail and Twist. Based on this evidence, the present study aimed to investigate whether downregulation of the p65 subunit of NF-κB or HIF-1α by small interfering RNA (siRNA) may reverse the EMT phenotype and inhibit the proliferation and induce the apoptosis of pancreatic cancer cell lines (PANC-1, BxPC3) under hypoxic conditions in vitro and enhance the efficacy of gemcitabine in the treatment of pancreatic cancer. These results provide molecular evidence showing that the activation of the HIF-1α and NF-κB loop is mechanistically linked with the chemoresistance phenotype (EMT phenotype) of pancreatic cancer cells under hypoxic conditions, suggesting that the inactivation of HIF-1α and NF-κB signaling by novel strategies may be a potential targeted therapeutic approach for overcoming EMT and chemoresistance induced by hypoxia.

[The suppressive effect of MiR-490 on coxsackievirus B3 replication].

To study the effect of miR-490 on Coxsackievirus B3 (CVB3) replication, HeLa cells were trans- fected with miR-490 in vitro, and infected with a Renilla luciferase (RLuc)-expressing CVB3 variant (RLuc-CVB3). The activities of RLuc in these cells were measured at 8h intervals from 0 to 40 h post-infection (p.i.), and the effects of miR-490 on RLuc-CVB3 replication were observed. In a further study, HeLa cells were transfected with either miR-490 or antisense miR-490 (AMO-miR-490), and were then infected with an enhanced green fluorescence protein (EGFP)-expressing CVB3 variant (EGFP-CVB3). The replication of EGFP-CVB3 was then determined by detecting the expression of EGFP. We observed that miR-490 could significantly inhibit the expression of RLuc in infected cells at 32 h p. i. Furthermore, in HeLa cells infected with EGFP-CVB3 at 32 h p.i., EGFP expression was also significantly inhibited by the presence of mniR-490. The inhibitory effect of miR-490 on EGFP expression in EGFP-CVB3-infected cells could be reversed by tranfection with AMQ-miR-490. These results indicated that miR-490 significantly inhibits the replication and expression of QVB3.

Absence of SOCS3 in the cardiomyocyte increases mortality in a gp130-dependent manner accompanied by contractile dysfunction and ventricular arrhythmias.

BACKGROUND: Suppressor of cytokine signaling-3 (SOCS3) is a key negative-feedback regulator of the gp130 receptor that provides crucial signaling for cardiac hypertrophy and survival; however, an in vivo role of SOCS3 regulation on cardiac gp130 signaling remains obscure. METHODS AND RESULTS: We generated cardiac-specific SOCS3 knockout (SOCS3 cKO) mice. These mice showed increased activation of gp130 downstream signaling targets (STAT3, ERK1/2, AKT, and p38) from 15 weeks of age and developed cardiac dysfunction from approximately 25 weeks of age with signs of heart failure. Surprisingly, SOCS3 cKO failing hearts had minimal histological abnormalities with intact myofibril ultrastructure. In addition, Ca(2+) transients were significantly increased in SOCS3 cKO failing hearts compared with wild-type hearts. We also found that Ser23/24 residues of troponin I were hypophosphorylated in SOCS3 cKO hearts before the manifestation of cardiac dysfunction. These data suggested the presence of abnormalities in myofilament Ca(2+) sensitivity in SOCS3 cKO mice. In addition to the contractile dysfunction, we found various ventricular arrhythmias in SOCS3 cKO nonfailing hearts accompanied by a sarcoplasmic reticulum Ca(2+) overload. To determine the contribution of gp130 signaling to the cardiac phenotype that occurs with SOCS3 deficiency, we generated cardiac-specific gp130 and SOCS3 double KO mice. Double KO mice lived significantly longer and had different histological abnormalities when compared with SOCS3 cKO mice, thus demonstrating the importance of gp130 signaling in the SOCS3 cKO cardiac phenotype. CONCLUSIONS: Our results demonstrate an important role of SOCS3 regulation on cardiac gp130 signaling in the pathogenesis of contractile dysfunction and ventricular arrhythmias.

[Application of nano-sized TiO2 photocatalysis to air purification and sterilization].

OBJECTIVE: To develop and evaluate the efficiency of air purification and sterilization instrument based on nano-sized TiO(2) photocatalytic technique. METHODS: The nano-sized TiO(2) photocatalytic air purification and sterilization instrument was designed and a sample had been prepared. The sterilization efficiencies for E.coli and Klebsiella by the nano-sized TiO(2) photocatalytic instrument and ultraviolet (UV) were measured in closed labs. The on-site efficiency of the instrument was evaluated, too. RESULTS: The nano-sized TiO(2) photocatalytic air purification and sterilization instrument was composed of five units: rough filter, nano-sized TiO(2) photocatalytic unit, activated carbon fiber filter, negative ion generator, and programmed control unit. The E.coli killing rates by the nano-sized TiO(2) photocatalytic instrument were 76.0%, 81.8%, 77.5%, and 80.7% at 30, 60, 90, and 120 minutes, respectively. There was no significant difference between the E.coli killing rates of the instrument and UV (P > 0.05), except the 120 minutes timepoint. The Klebsiella killing rates by the instrument were 78.4%, 79.5%, 67.3%, and 58.5% at 30, 60, 90, and 120 minutes, respectively. The Klebsiella killing efficiencies of the instrument at 30 and 60 minutes were better than that of UV (P < 0.01). There was no significant difference between the Klebsiella killing efficiencies of the instrument and UV (P > 0.05). CONCLUSION: The air sterilization efficiency of the nano-sized TiO(2) photocatalytic instrument should be equivalent or better as compared with the UV. This instrument might be used for the air purification and sterilization of the public locations.

Association of human leukocyte antigen polymorphism with hepatitis B virus infection and genotypes.

Associations were studied between the polymorphism of northern Han Chinese leukocyte antigen (HLA) alleles and the outcomes of hepatitis B virus (HBV) infection and HBV genotypes. HLA-A, B, and DRB1 alleles in peripheral blood mononuclear cells (PBMCs) were detected by polymerase chain reaction (PCR) with sequence-specific primers. The PBMCs were collected from 61 persons who tested positive for hepatitis B surface antigen (HBsAg) for more than 6 months (Persistent group), 32 persons who tested negative for both HBsAg and HBV DNA but positive for both anti-HBc and anti-HBs (Recovered group), and 40 persons who tested negative for all serologic markers of HBV infection (Uninfected group). HBV genotypes in serum specimens from 56 of 61 patients with persistent HBV infection were determined by nested PCR with 6 pairs of HBV genotype-specific primers (A to F). The frequency of HLA-DRB1*12 was significantly higher in the Persistent group than in the Recovered group (P=0.004). HLA-A*02 was significantly higher in the Recovered group than in the Persistent group (P=0.044). HLA-DRB1*15 was significantly higher in the HBV genotype B group than in the C group (P=0.013). These findings suggested that there were associations not only between HLA polymorphisms and outcomes of HBV infection but also between HLA polymorphisms and the infected HBV genotypes.

YMDD mutations and genotypes of hepatitis B virus in northern China.

The objective of this research was to determine the relationship between YMDD mutations and the genotypes of hepatitis B virus (HBV) during lamivudine treatment. HBV genotypes were determined by nested PCR with 6 pairs of HBV genotype-specific primers (A to F) in serum specimens from 142 hepatitis B patients receiving lamivudine antiviral therapy. YMDD mutations were detected by fluorescent hybridization bioprobe PCR and melting curve assay (FH-PCR-MC). Among 142 serum specimens, 13 samples were genotype B (9.2%), 125 samples were genotype C (88%), 4 samples were genotype D (2.8%), and 80 YMDD mutations were found. The YMDD mutation rates were 69.2 and 54.4% in genotype B and genotype C, respectively. There was no significant difference in the YMDD mutation rate between genotypes B and C. Nine genotype B sera with YMDD mutations were found, including 2 YIDD mutations and 7 YVDD (M + V) mutations. Sixty-eight genotype C sera with YMDD mutations were found, including 34 mutations I (M + I) and 17 mutations V (M + V). There was a significant difference in the YMDD mutation types between genotypes B and C. Our results suggested that the YMDD mutation rate was 56.3% in patients treated with lamivudine for 2-4 years. YIDD was the main mutation type. The YMDD mutation rate showed no significant difference between HBV types B and C (P > 0.05), while the YMDD mutation types showed a significant difference between HBV types B and C in Northern China (chi2 test = 4.6, P < 0.05).

Effects of tacrolimus on infection of Friend murine leukemia virus to Fv-4 gene heterozygous mice.

AIM: To investigate the effect of tacrolimus (FK506) on the infection of Friend murine leukemia virus (Friend MuLV) in vivo. METHODS: Three kinds of mice were used including Friend MuLV-sensitive BALB/c mice, Friend MuLV-resistant Fv-4 gene-homozygous mice (Fv-4 mice), and Friend MuLV-resistant Fv-4 gene-heterozygous mice (F1 mice). Tacrolimus was administrated i.p. to those mice in every 2 d. Those treated mice were inoculated i.p. with Friend MuLV once on d 3. The symptoms and viral proliferations in those mice were observed to recognize the Friend MuLV infection. The expression and genotype of Fv-4 gene that resistant against the infection of Friend MuLV were analyzed to confirm the genomic background and related mechanism of the resistance. RESULTS: BALB/c mice and F1 mice, but not Fv-4 mice, appeared obvious early death, spleenomegaly, and viral proliferation after both treatments of viral inoculation and tacrolimus administration, whereas the expression and genotype of Fv-4 gene was not changed in F1 mice and Fv-4 mice with treatment of tacrolimus. Compared to the virus-inoculated control, the Friend MuLV-sensitivity of tacrolimus-treated BALB/c mice and the Friend MuLV-resistance of tacrolimus-treated Fv-4 mice were the same as the controls, but only F1 mice became the symptoms and viral proliferation after both treatments. It suggested the Friend MuLV-resistant F1 mice could be converted to be Friend MuLV-sensitive by treatment of tacrolimus, and this conversion was not depended on the expression and genotype of Fv-4 gene. CONCLUSION: Tacrolimus could not inhibit the infection of Friend MuLV in all mice, furthermore, it could enhance the infection of Friend MuLV in F1 mice. The enhancement may be related to the immunosuppressive effect of tacrolimus.

High prevalence of hepatitis B virus pre-s mutant in countries where it is endemic and its relationship with genotype and chronicity.

It has been reported that hepatitis B virus (HBV) mutants carrying mutations in the pre-S region can be found in infected patients. In this study, we investigated the prevalence of the HBV variant with the pre-S mutant in different geographic regions, including countries with low and high levels of endemic HBV infection, and analyzed the correlation with clinical findings. We examined 387 HBV DNA-positive serum samples from individuals among 12 countries, consisting of Vietnam, Myanmar, Thailand, China, Korea, Nepal, Japan, Russia, Spain, United States, Bolivia, and Ghana. HBV pre-S mutants were detected in 71 (18.3%) of 387 serum samples tested. This mutant was the most prevalent in Vietnam (36%), followed by Nepal (27.3%), Myanmar (23.3%), China (22.4%), Korea (14.3%), Thailand (10.5%), Japan (7.7%), and Ghana (4.3%). In contrast, no case with this mutation was found in Russia, Spain, United States, and Bolivia. Among the HBV deletion mutations, 15.5% (11 of 71) occurred in the pre-S1 and 46.5% (33 of 71) in the pre-S2 regions. Eight (11.3%) cases had a mutation in both the pre-S1 and pre-S2 regions. In addition, a point mutation at the pre-S2 starting codon was observed in 19 (26.7%) cases. The detection rate of the HBV mutant in patients with hepatocellular carcinoma was significantly higher than in other patients (P < 0.05). Furthermore, these mutants were found more frequently in genotype B (25%) and genotype C (24.5%) than in the other genotypes (P < 0.05). Our results indicated that there was a high prevalence of HBV pre-S mutation in regions of endemic HBV infection in Asia. Furthermore, the pre-S mutation appeared to be correlated with hepatocellular carcinoma and HBV genotypes.

Molecular epidemiology of hepatitis viruses and genotypic distribution of hepatitis B and C viruses in Harbin, China.

We carried out a molecular-based epidemiological survey of hepatitis viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis E virus (HEV), in Harbin, China. The study population of 358 subjects consisted of 132 healthy blood donors and 226 liver disease patients residing in Harbin City and surrounding suburbs. The infection rate of each virus among healthy subjects was 14.4% (19/132) for HBV and 2.3% (3/132) for HCV. In contrast, among liver disease patients, the infection rates were 72.6% (164/226) for HBV and 7.5% (17/226) for HCV, respectively (P < 0.01 and P < 0.05, respectively). In particular, nearly 64% of hepatocellular carcinoma patients in Harbin was found to be infected with HBV. The most common viral genotypes were HBV type C (80%) and HCV type 1b (31.3%). Interestingly, a high prevalence of the HBV pre-S1/S2 deletion mutant was found in 13 of 58 (22.4%) subjects. Moreover, testing for HEV among 202 subjects resulted in the detection of anti-HEV IgG in 53 cases (26.2%). The prevalence of anti-HEV IgG has already reached 20% in tested cases aged less than 10 years. These results suggest that HBV infection is widespread in Harbin, China and has led to a high incidence of acute and chronic liver disease in this region.

[Expressions of TNF-alpha and IL-1beta in human ischemic brain tissues].

AIM: To detect the expression of TNF-alpha and IL-1beta in human cerebral ischemic tissues. METHODS: 13 cerebral specimens from patients died of cerebral infarction were divided into three groups, <2 d, 3-5 d, and >5d, according to the lasting time of infarctions. The expression of TNF-alpha and IL-1beta in the cerebral ischemic tissues were examined by immnohistochemical staining. The contralateral tissues were employed as controls. RESULTS: The expression of TNF-alpha and IL-1beta in the cerebral ischemic tissues were significant higher than those in the contralateral tissues. The focal distribution of the TNF-alpha(+) and IL-1beta(+) cells was identical with the ischemic area. The expression of IL-1beta and TNF-alpha peaked at the 3rd to 5th and 2nd day after ichemia, respectively. There were no significant difference between the ischemic and contralateral brains at the 5th day after ischemia for the expression of TNF-alpha and IL-1beta. CONCLUSION: Our results showed the expression of TNF-alpha and IL-1beta in human strok of infarction were similar to those in animal experiments. It is suggested that TNF-alpha and IL-1beta are involved in cerebral ischemic injury, which will be helpful for developing clinically a novel therapy aiming at cerebral ischemic injury.