Declining Trend of Hepatitis A Seroepidemiology in Association with Improved Public Health and Economic Status of Thailand

Hepatitis A virus (HAV) is transmitted via the fecal-oral route from contaminated food or water. As part of the most recent survey of viral hepatitis burden in Thailand, we analyzed the current seroprevalence of HAV in the country and compared with data dating back to 1971. From March to October, 2014, a total of 4,260 individuals between one month and 71 years of age from different geographical regions (North = 961; Central = 1,125; Northeast = 1,109; South = 1,065) were screened for anti-HAV IgG antibody using an automated chemiluminescent microparticle immunoassay. Overall, 34.53% (1,471/4,260) possessed anti-HAV IgG antibody, and the age-standardized seroprevalence was 48.6%. Seroprevalence rates were 27.3% (North), 30.8% (Central), 33.8% (Northeast) and 45.8% (South) and were markedly lower than in the past studies especially among younger age groups. The overall trend showed an increase in the age by which 50% of the population were anti-HAV IgG antibody: 4.48 years (1971–1972), 6 (1976), 12.49 (1990), 36.02 (2004) and 42.03 (2014).This suggests that Thailand is transitioning from low to very low HAV endemicity. Lower prevalence of HAV correlated with improved healthcare system as measured by decreased infant mortality rate and improved national economy based on increased GDP per capita. The aging HAV immuno-naïve population may be rendered susceptible to potential HAV outbreaks similar to those in industrialized countries and may benefit from targeted vaccination of high-risk groups.     

The allelic rice immune receptor Pikh confers extended resistance to strains of the blast fungus through a single polymorphism in the effector binding interface

Arms race co-evolution drives rapid adaptive changes in pathogens and in the immune systems of their hosts. Plant intracellular NLR immune receptors detect effectors delivered by pathogens to promote susceptibility, activating an immune response that halts colonization. As a consequence, pathogen effectors evolve to escape immune recognition and are highly variable. In turn, NLR receptors are one of the most diverse protein families in plants, and this variability underpins differential recognition of effector variants. The molecular mechanisms underlying natural variation in effector recognition by NLRs are starting to be elucidated. The rice NLR pair Pik-1/Pik-2 recognizes AVR-Pik effectors from the blast fungus Magnaporthe oryzae, triggering immune responses that limit rice blast infection. Allelic variation in a heavy metal associated (HMA) domain integrated in the receptor Pik-1 confers differential binding to AVR-Pik variants, determining resistance specificity. Previous mechanistic studies uncovered how a Pik allele, Pikm, has extended recognition to effector variants through a specialized HMA/AVR-Pik binding interface. Here, we reveal the mechanistic basis of extended recognition specificity conferred by another Pik allele, Pikh. A single residue in Pikh-HMA increases binding to AVR-Pik variants, leading to an extended effector response in planta. The crystal structure of Pikh-HMA in complex with an AVR-Pik variant confirmed that Pikh and Pikm use a similar molecular mechanism to extend their pathogen recognition profile. This study shows how different NLR receptor alleles functionally converge to extend recognition specificity to pathogen effectors.     

Scanning electron microscopic examination of a pellet formulation of Bacillus megaterium and B. pumilus,antagonists of Rhizoctonia solani,and survival during storage

Bacterial formulations, produced using both Bacillus megaterium and B. pumilus individually with pharmaceutical technology, were formulated using a wet granular method. Viability testing in the laboratory revealed that bacterial populations rapidly declined during storage at room temperature (26–30 °C) for 6 months. The scanning electron microscope (SEM) was used to observe bacterial formulations. Both endospores and vegetative cells of B. megaterium and B. pumilus were detected on the formulation surfaces. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition analysis of inhibitors derived from lignocellulose pretreatment on the metabolic activity of Zymomonas mobilis biofilm and planktonic cells and the proteomic responses

Lignocellulose pretreatment produces various toxic inhibitors that affect microbial growth, metabolism, and fermentation. Zymomonas mobilis is an ethanologenic microbe that has been demonstrated to have potential to be used in lignocellulose biorefineries for bioethanol production. Z. mobilis biofilm has previously exhibited high potential to enhance ethanol production by presenting a higher viable cell number and higher metabolic activity than planktonic cells or free cells when exposed to lignocellulosic hydrolysate containing toxic inhibitors. However, there has not yet been a systematic study on the tolerance level of Z. mobilis biofilm compared to planktonic cells against model toxic inhibitors derived from lignocellulosic material. We took the first insight into the concentration of toxic compound (formic acid, acetic acid, furfural, and 5‐HMF) required to reduce the metabolic activity of Z. mobilis biofilm and planktonic cells by 25% (IC₂₅), 50% (IC₅₀), 75% (IC₇₅), and 100% (IC₁₀₀). Z. mobilis strains ZM4 and TISTR 551 biofilm were two‐ to three fold more resistant to model toxic inhibitors than planktonic cells. Synergetic effects were found in the presence of formic acid, acetic acid, furfural, and 5‐HMF. The IC₂₅ of Z. mobilis ZM4 biofilm and TISTR 551 biofilm were 57 mm formic acid, 155 mm acetic acid, 37.5 mm furfural and 6.4 mm 5‐HMF, and 225 mm formic acid, 291 mm acetic acid, 51 mm furfural and 41 mm 5‐HMF, respectively. There was no significant difference found between proteomic analysis of the stress response to toxic inhibitors of Z. mobilis biofilm and planktonic cells on ZM4. However, TISTR 551 biofilms exhibited two proteins (molecular chaperone DnaK and 50S ribosomal protein L2) that were up‐regulated in the presence of toxic inhibitors. TISTR 551 planktonic cells possessed two types of protein in the group of 30S ribosomal proteins and motility proteins that were up‐regulated.     

Development and assessment of a real-time pcr assay for rapid and sensitive detection of a novel thermotolerant bacterium, Lactobacillus thermotolerans, in chicken feces

A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345(T) and Lactobacillus fermentum JCM 1173(T), and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 x 10(3) cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 10(4) cells/g feces on day 4 and 10(5) cells/g feces on days 11 to 18. However, cell populations of 10(6) to 10(7) cells/g feces were detected in the second trial. The total bacterial count, measured by 4',6-diamidino-2-phenylindole (DAPI) staining, was approximately 10(11) cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.     

Liver fluke-induced hepatic oxysterols stimulate DNA damage and apoptosis in cultured human cholangiocytes

Oxysterols are cholesterol oxidation products that are generated by enzymatic reactions through cytochrome P450 family enzymes or by non-enzymatic reactions involving reactive oxygen and nitrogen species. Oxysterols have been identified in bile in the setting of chronic inflammation, suggesting that biliary epithelial cells are chronically exposed to these compounds in certain clinical settings. We hypothesized that biliary oxysterols resulting from liver fluke infection participate in cholangiocarcinogenesis. Using gas chromatography/mass spectrometry, we identified oxysterols in livers from hamsters infected with Opisthorchis viverrini that develop cholangiocarcinoma. Five oxysterols were found: 7-keto-cholesta-3,5-diene (7KD), 3-keto-cholest-4-ene (3K4), 3-keto-cholest-7-ene (3K7), 3-keto-cholesta-4,6-diene (3KD), and cholestan-3β,5α,6β-triol (Triol). Triol and 3K4 were found at significantly higher levels in the livers of hamsters with O. viverrini-induced cholangiocarcinoma. We therefore investigated the effects of Triol and 3K4 on induction of cholangiocarcinogenesis using an in vitro human cholangiocyte culture model. Triol- and 3K4-treated cells underwent apoptosis. Western blot analysis showed significantly increased levels of Bax and decreased levels of Bcl-2 in these cells. Increased cytochrome c release from mitochondria was found following treatment with Triol and 3K4. Triol and 3K4 also induced formation of the DNA adducts 1,N(6)-etheno-2'-deoxyadenosine, 3,N(4)-etheno-2'-deoxycytidine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in cholangiocytes. The data suggest that Triol and 3K4 cause DNA damage via oxidative stress. Chronic liver fluke infection increases production of the oxysterols Triol and 3K4 in the setting of chronic inflammation in the biliary system. These oxysterols induce apoptosis and DNA damage in cholangiocytes. Insufficient and impaired DNA repair of such mutated cells may enhance clonal expansion and further drive the change in cellular phenotype from normal to malignant.     

Expression of oxysterol binding protein isoforms in opisthorchiasis-associated cholangiocarcinoma: A potential molecular marker for tumor metastasis

Oxysterols are oxygenated derivatives of cholesterol generated by enzymatic reactions mediated by cytochrome P450 family enzymes or by inflammation-associated non-enzymatic reactions. Oxysterol binding proteins (OSBPs) are cytosolic high affinity receptors for oxysterols. We previously found that OSBPL-8 is upregulated in liver fluke (Opisthorchis viverrini)-induced hamster cholangiocarcinoma (CCA). Our aims were to determine the expression patterns of OSBP isoforms in human CCA tissues and to evaluate whether OSBPs could be used as molecular markers for the identification of blood-borne CCA metastasis. Expression levels of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 in CCA tissues were detected using qRT-PCR and immunohistochemistry. Expression of OSBPs at mRNA level in the blood of CCA patients was also investigated. We confirmed increased expression of OSBPL-8 in O. viverrini -induced hamster CCA tissues. Moreover, increased expression of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 was seen in human CCA tissues. Notably, a significant increased level of OSBPL-7 mRNA was observed in tumor compared to non-tumor liver tissue. Immunohistochemistry supported the mRNA results, in that OSBPL-7 protein was over-expressed in cancer cells and hepatocytes but not in normal biliary cells and surrounding inflammatory cells. Interestingly, in our preliminary results, significantly higher levels of OSBP2 and OSBPL-7 mRNA were seen in blood samples from CCA patients than in healthy controls. These results suggest that OSBP2 and OSBPL-7 might serve as molecular markers for the identification of CCA metastasis in the bloodstream.     

A role for the lymphotoxin/LIGHT axis in the pathogenesis of murine collagen-induced arthritis

A lymphotoxin-beta (LTbeta) receptor-Ig fusion protein (LTbetaR-Ig) was used to evaluate the importance of the lymphotoxin/LIGHT axis in the development and perpetuation of arthritis. Prophylactic treatment with the inhibitor protein LTbetaR-Ig blocked the induction of collagen-induced arthritis in mice and adjuvant arthritis in Lewis rats. Treatment of mice with established collagen-induced arthritis reduced the severity of arthritic symptoms and joint tissue damage. However, in a passive model of anti-collagen Ab-triggered arthritis, joint inflammation was not affected by LTbetaR-Ig treatment precluding LT/LIGHT involvement in the very terminal immune complex/complement/FcR-mediated effector phase. Collagen-II and Mycobacterium-specific T cell responses were not impaired, yet there was evidence that the overall response to the mycobacterium was blunted. Serum titers of anti-collagen-II Abs were reduced especially during the late phase of disease. Treatment with LTbetaR-Ig ablated follicular dendritic cell networks in the draining lymph nodes, suggesting that impaired class switching and affinity maturation may have led to a decreased level of pathological autoantibodies. These data are consistent with a model in which the LT/LIGHT axis controls microenvironments in the draining lymph nodes. These environments are critical in shaping the adjuvant-driven initiating events that impact the subsequent quality of the anti-collagen response in the later phases. Consequently, blockade of the LT/LIGHT axis may represent a novel approach to the treatment of autoimmune diseases such as rheumatoid arthritis that involve both T cell and Ab components.     

Selective cleavage of pepsin by molybdenum metallopeptidase

In this study, the cleavage of protein by molybdenum cluster is reported for the first time. The protein target used is porcine pepsin. The data presented in this study show that pepsin is cleaved to at least three fragments with molecular weights of ～23, ～19 and ～16 kDa when the mixture of the protein and ammonium heptamolybdate tetrahydrate ((NH(4))(6)Mo(7)O(24)·4H(2)O) was incubated at 37°C for 24h. No self cleavage of pepsin occurs at 37 °C, 24h indicating that the reaction is mediated by the metal ions. N-terminal sequencing of the peptide fragments indicated three cleavage sites of pepsin between Leu 112-Tyr 113, Leu 166-Leu 167 and Leu 178-Asn 179. The cleavage reaction occurs after incubation of the mixture of pepsin and (NH(4))(6)Mo(7)O(24)·4H(2)O) only for 2h. However, the specificity of the cleavage decreases when incubation time is longer than 48 h. The mechanism for cleavage of pepsin is expected to be hydrolytic chemistry of the amide bonds in the protein backbone.     

Decreasing Hepatitis C Virus Infection in Thailand in the Past Decade: Evidence from the 2014 National Survey

Hepatitis C virus (HCV) infection affects ≥ 180 million individuals worldwide especially those living in developing countries. Recent advances in direct-acting therapeutics promise effective treatments for chronic HCV carriers, but only if the affected individuals are identified. Good treatment coverage therefore requires accurate epidemiological data on HCV infection. In 2014, we determined the current prevalence of HCV in Thailand to assess whether over the past decade the significant number of chronic carriers had changed. In total, 5964 serum samples from Thai residents between 6 months and 71 years of age were obtained from 7 provinces representing all 4 geographical regions of Thailand and screened for the anti-HCV antibody. Positive samples were further analyzed using RT-PCR, sequencing, and phylogenetic analysis to identify the prevailing HCV genotypes. We found that 56 (0.94%) samples tested positive for anti-HCV antibody (mean age = 36.6±17.6 years), while HCV RNA of the core and NS5B subgenomic regions was detected in 23 (41%) and 19 (34%) of the samples, respectively. The seropositive rates appeared to increase with age and peaked in individuals 41–50 years old. These results suggested that approximately 759,000 individuals are currently anti-HCV-positive and that 357,000 individuals have viremic HCV infection. These numbers represent a significant decline in the prevalence of HCV infection. Interestingly, the frequency of genotype 6 variants increased from 8.9% to 34.8%, while the prevalence of genotype 1b declined from 27% to 13%. These most recent comprehensive estimates of HCV burden in Thailand are valuable towards evidence-based treatment coverage for specific population groups, appropriate allocation of resources, and improvement in the national public health policy.