C-Terminal Part of Glutamate-Ammonia-Ligase Adenyltransferase Gene Identified by RAPD-HRM with 3H Primer for E. Coli Screening.

A single random oligonucleotide 3H primer has been previously applied in random-amplified- polymorphic-DNA (RAPD)-PCR to distinguish stocked bacteria E. coli within a cocktail mixture also containing Enterococcus faecalis, Bifidobacterium longum and Ruminococcus gnavus. In this study, we demonstrate that a 702 base pair (bp) gene fragment can be amplified as a unique pattern by RAPD-PCR using a 3H primer in human faeces containing E. coli. This unique 702 bp amplicon contained a 687 bp gene fragment identified as the C-terminal region of the glutamate-ammonia-ligase adenyltransferase (glnE) gene of E. coli. By high-resolution melt (HRM) analysis, a mean melt-curve temperature of this 702 bp amplicon was determined to be approximately 88.1 ± 0.22 degrees Celsius (°C). A combination of RAPD with HRM in one single reaction based on this amplicon can achieve semi-quantitative detection of up to 102 CFU/ml of E. coli. To increase the signal intensity of HRM, a primer pair capable of screening E. coli directly from fresh human faeces was re-designed from the 687 bp gene segment, giving a mean peak melt-curve temperature at 88.35 ± 0.11 °C. Finally, single-nucleotide polymorphisms of this 687 bp gene segment were analysed for pathogenic E. coli strains, including UMN026, O83:H1, O104:H4, O157:H7 and O169:H41. We conclude that this 687 bp segment of the glnE gene has a high potential for screening of human faecal E. coli, including pathogenic strains, in contaminated food and water.

Production and characterization of a monoclonal antibody against a late gene encoded by grouper iridovirus 64L.

Viral envelope proteins play important roles in viral infection and assembly. The grouper iridovirus ORF 64L (GIV-64L) was predicted to encode an envelope protein and was conserved in all sequenced Ranaviruses. In this study, the complete nucleotide sequence of the GIV-64L gene (1215 bp) was cloned into the isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction prokaryotic expression vector pET23a. The approximately 50.2 kDa recombinant GIV-64L-His protein was induced, purified and used as an immunogen to immunize BALB/c mice. Three monoclonal antibodies (mAbs), all IgG1 class antibodies against GIV-64L protein, were produced by enzyme-linked immunosorbent assay. Reverse transcription polymerase chain reaction analyses revealed GIV-64L to be a late gene when expressed in grouper kidney cells during GIV infection with cycloheximide (an inhibitor of protein synthesis) or cytosine arabinoside (an inhibitor of DNA synthesis) present. Finally, one of the established mAbs, GIV-64L-mAb-17, was used in Western blotting and an immunofluorescence assay, which showed that GIV-64L protein was expressed at 24 h post-infection and localized only in the cytoplasm in GIV-infected cells, packed into a whole virus particle. The presently characterized GIV-64L mAbs should have widespread applications in GIV immunodiagnostics and other research, and these results should offer important insights into the pathogenesis of GIV.

Development and characterization of two monoclonal antibodies against grouper iridovirus 55L and 97L proteins.

Grouper iridovirus (GIV) is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. The study of GIV pathogenicity has been hampered by the lack of proper immunological reagents to study the expression and function of viral proteins in the infected cells. In this study, two mouse monoclonal antibodies (mAbs) against GIV 55L and 97L proteins were produced. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen these hybridomas, resulting in the identification of two high-affinity mAbs named GIV55L-mAb-2 and GIV97L-mAb-3, respectively. Both mAbs belong to the IgG1 isotype and were effective in detecting their respective target viral protein. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analyses of GIV-infected GK cells revealed that GIV 97L is an immediate early gene, whereas GIV 55L a late one. The localization of 55L and 97L in GIV-infected cells was further characterized by immunofluorescence microscopy with the mAbs. The 55L protein mainly aggregated in the cytoplasm while 97L distributed in both the nucleus and cytoplasm of the infected cells. These studies demonstrate the validity of the two mAbs as immunodiagnostic and research reagents.

Identification and characterization of a late gene encoded by grouper iridovirus 2L (GIV-2L).

Grouper iridovirus (GIV) belongs to the Ranavirus genus and is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. In this study, we identified and characterized the GIV-2L gene, which encodes a protein of unknown function. GIV-2L is 1242 bp in length, with a predicted protein mass of 46.2 kDa. It displayed significant identity only with members of the Ranavirus and Iridovirus genera. We produced mouse monoclonal antibodies against the GIV-2L protein by immunizing mice with GIV-2L-His-tag recombinant protein. By inhibiting de novo protein and DNA synthesis in GIV-infected cells, we showed that GIV-2L was a late gene during the viral replication. Finally, immunofluorescence microscopy revealed that GIV-2L protein accumulated in both the nucleus and cytoplasm of infected cells. These results offer important insights into the pathogenesis of GIV.

Identification and characterization of a late gene of grouper iridovirus 61l and antibody production against the protein encoded by it.

In this study, a late gene encoded by grouper iridovirus, giv-61L, was identified and classified, and mouse monoclonal antibodies (mAbs) were raised against this protein. Giv-61L homologues were found only in the genus Ranavirus. Three mAbs to Giv-61L protein were produced. In drug inhibition assays, giv-61L was identified as a late gene. Finally, GIV-61L-mAb-8 was used in western blotting and immunofluorescence assays to demonstrate that Giv-61L protein was included in the GIV particle, expressed at 18 h, and localized only in the cytoplasm of GIV-infected cells. The results of this study provide insight into GIV pathogenesis and GIV-61L-mAbs will have broad applications in GIV immunodiagnostics.

Prediction of lymph node metastasis in T1/T2 tongue squamous cell carcinoma.

OBJECTIVES: In T1, T2, and clinically NO squamous cell carcinoma of the tongue, there is no reliable predictive variable to determine whether or not neck dissection is needed. Thus, we established a predictive score model based on tumour depth and other pathological variables. METHODS: We retrospectively reviewed 115 patients with T1 and T2 stage squamous cell carcinoma of the tongue. Their pathological variables were used to construct a score model for predicting the risk of cervical lymph node metastasis. RESULTS: A predictive score model was proposed using multivariate logistic regression analysis: Score = (2.694 x tumour depth (cm)) + (1.814 x lymphovascular invasion (yes = 1, no = 0)) + (1.175 x perineural invasion (yes = 1, no = 0)). The cutoff point was set at 2.7427. This predictive score model has a sensitivity of 91.2% and specificity of 65.4%. CONCLUSION: A predictive score model was built and a two-stage surgical approach was suggested for T1 and T2 squamous cell carcinoma of the tongue.

[Assessment of the burden of clonorchiasis and its temporal changes in China].

OBJECTIVE: To assess the burden of clonorchiasis and identify its temporal and spatial changes in China, thus to provide insights into the control and prevention of the diseases. METHODS: The disability-adjusted life years (DALYs) was employed as the primary indicator for the disease burden. The prevalence data of Clonorchis sinensis infection were obtainted from the three national surveys on important human parasitic diseases in China, conducting during the period from 1988 to 1922, from 2001 to 2004 and from 2014 to 2016, respectively, and the demographic data from National Bureau of Statistics of China. DALYs of clonorchiasis were calculated and the temporal changes were analyzed at both national and provincial levels, using the disability weight (DW) obtained from a community study in China. Sensitivity analysis was carried out to compare the resulted DALYs of China calculated under the method adopted in this study and that calculated with other commonly used methods. RESULTS: The national burden of clonorchiasis was 489174.04 [95% confidence interval (CI): (391648.87, 597509.87)] DALYs in China in 2016, indicating 0.36 [95% CI: (0.28, 0.43)] DALYs per 1 000 populations. The regions with a high burden of clonorchiasis were concentrated in southern China and northeastern China, and the provinces with the three highest burdens of clonorchiasis included Guangxi Zhuang Autonomous Region, Guangdong Province and Heilongjiang Province, which accounted for 91.18% of total burdens of clonorchiasis in China. During the periods of the three national surveys on important human parasitic diseases in China, the national burden of clonorchiasis was found to show a tendency of first rise and then decrease in China; however, the burden of clonorchiasis has recently shown a tendency towards a rise in Guangxi Zhuang Autonomous Region, Heilongjiang Province and Jiangxi Province. Sensitivity analysis showed that the calculation of diseases burden with age-stratified prevalence of clonorchiasis was similar to that of our method without age stratification; however, the burden estimates calculated only based on the DW of the severe symptoms were much lower than our estimates. CONCLUSIONS: The burden of clonorchiasis is high in China, with a large regional difference. Recently, the overall burden of clonorchiasis has shown a tendency of decline in China; however, there is a tendency towards a rise in some provinces. Therefore, the control of clonorchiasis requires more adaptations to local circumstances.

[Spatial and temporal characteristics of reported schistosomiasis cases in China based on a Bayesian interrupted time-series model].

OBJECTIVE: To investigate the spatial-temporal characteristics of reported schistosomiasis cases in China from 2004 to 2017, so as to provide insights into the development of different schistosomiasis control strategies at various stages. METHODS: The monthly data of reported schistosomiasis cases at a provincial level of China from 2004 to 2017 were collected from the Public Health Science Data Center, and the spatial-temporal distribution of reported schistosomiasis cases was preliminarily identified using a descriptive statistical method. According to the goals at different stages proposed by the National Mid- and Long-term Program for Schistosomiasis Prevention and Control in China (2004-2015), a Bayesian interrupted time-series model was established to analyze the provincial reported incidence, time trend and seasonal variations of schistosomiasis in China at different stages. RESULTS: The reported schistosomiasis cases were mainly concentrated in 5 provinces of Anhui, Jiangsu, Jiangxi, Hubei and Hunan and 2 provinces of Sichuan and Yunnan in China from 2004 to 2017, and the number of reported cases in endemic areas decreased gradually. The incidence of reported schistosomiasis cases predominantly peaked during the period from May to September in the marshland and lake regions, while no regular seasonality was seen in hilly regions. Bayesian interrupted time-series analysis showed the peak incidence of reported schistosomiasis cases in 4 provinces of Anhui, Hubei, Hunan and Jiangxi between May and September and in Jiangsu Province from July to November; however, no regular seasonal cycle was identified in hilly regions. The number of reported schistosomiasis cases showed a tendency towards an increase in 2 provinces of Hubei and Hunan from 2008 to 2014, with a minor peak during the period between March and April, and since 2015, the seasonality was not remarkable any longer in 3 provinces of Anhui, Jiangsu and Jiangxi with a decline in the incidence of reported schistosomiasis cases, while the seasonality remained in Hubei Province. CONCLUSIONS: The spatial-temporal characteristics of schistosomiasis in China, notably seasonality, vary at different control stages. Bayesian interrupted time-series model is effective to identify the spatial-temporal changes of schistosomiasis, and the schistosomiasis control strategy may be adjusted according to the spatial-temporal changes to improve the schistosomiasis control efficiency.

Establishment, characterization, virus susceptibility and transfection of cell lines from cobia, Rachycentron canadum (L.), brain and fin.

Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate-buffered saline containing 0.25% trypsin at 25 degrees C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 degrees C in Leibovitz-15 medium containing 10% foetal bovine serum. The cells have been sub-cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.

[Trends of female reproductive health status in Bangladesh from 1999 to 2018].

Objective: To analyze the changes of relevant indicators in reproductive health status among Bangladeshi women from 1999 to 2018 and to assess whether the 2030 Sustainable Development Goals (SDGs) can be achieved. Methods: Data were obtained from both the Bangladesh Demographic and Health as well as from the Maternal Mortality and Health Care Surveys. The trends of SDGs indicators related to reproductive health from 1999 to 2018 were analyzed and compared, and the average annual rate of change was calculated. Development index was used to assess the difficulty of achieving the SDGs. Results: The maternal mortality rate increased first and then leveled off from 2001 to 2016. From 1999 to 2018, the coverage of reproductive health care services and the proportion of women who had the right to make the decision on their own health care service, were generally increasing. Proportion of the following areas as: "contraceptive needs, women who consider that partner violence is justified, the rate of early marriage, and the rate of early childbearing etc.", were declining at various degrees. Development index of the antenatal care coverage, rate of delivery in medical facilities, percentage of live births attended by medically trained providers and the rate of postnatal care etc., were less than 1. The development indices of the maternal mortality rates were close to 1, while all the other indicators were greater than 1. Conclusions: The reproductive health-related SDGs indicators in Bangladesh appeared somehow degrees of progress from 1999 to 2018. However, for most indicators, the average annual rate of change was still lower than the expected to achieve the 2030 target which called for acceleration in the next few years.

Caspase-dependent induction of apoptosis in barramundi, Lates calcarifer (Bloch), muscle cells by grouper iridovirus.

We recently reported that grouper iridovirus (GIV) can induce apoptosis in barramundi, Lates calcarifer, muscle (BM) and swim bladder (BSB) cell lines. In this paper, we further characterize the molecular mechanism underlying apoptotic death in BM cells triggered by GIV. DNA-laddering and apoptotic cells were observed in BM cells infected with UV-irradiated or untreated GIV but was absent in cells infected with heat-inactivated GIV, indicating the involvement of viral protein in the apoptosis event. In GIV-infected BM cells, the conversion of procaspase-3 to caspase-3 was evident and the level of caspase-8 and -9 increased as early as 30 min post-infection. When treated with a pancaspase inhibitor, the GIV-induced apoptosis event was abolished. These observations indicate that GIV-induced apoptosis is caspase-dependent, and that both the external and internal routes in the caspase-dependent pathway are likely involved in the apoptosis process.

Lysine demethylase KDM2A inhibits TET2 to promote DNA methylation and silencing of tumor suppressor genes in breast cancer.

The coupling between DNA methylation and histone modification contributes to aberrant expression of oncogenes or tumor suppressor genes that leads to tumor development. Our previous study demonstrated that lysine demethylase 2A (KDM2A) functions as an oncogene in breast cancer by promoting cancer stemness and angiogenesis via activation of the Notch signaling. Here, we demonstrate that knockdown of KDM2A significantly increases the 5'-hydroxymethylcytosine (5'-hmc) level in genomic DNA and expression of tet-eleven translocation 2 (TET2) in various breast cancer cell lines. Conversely, ectopic expression of KDM2A inhibits TET2 expression in KDM2A-depleted cells suggesting TET2 is a transcriptional repression target of KDM2A. Our results show that KDM2A interacts with RelA to co-occupy at the TET2 gene promoter to repress transcription and depletion of RelA or KDM2A restores TET2 expression. Upregulation of TET2 in the KDM2A-depleted cells induces the re-activation of two TET downstream tumor suppressor genes, epithelial cell adhesion molecule (EpCAM) and E-cadherin, and inhibits migration and invasion. On the contrary, knockdown of TET2 in these cells decreases EpCAM and E-cadherin and increases cell invasiveness. More importantly, TET2 expression is negatively associated KDM2A in triple-negative breast tumor tissues, and its expression predicts a better survival. Taken together, we demonstrate for the first time that TET2 is a direct repression target of KDM2A and reveal a novel mechanism by which KDM2A promotes DNA methylation and breast cancer progression via the inhibition of a DNA demethylase.

The SUV39H1 inhibitor chaetocin induces differentiation and shows synergistic cytotoxicity with other epigenetic drugs in acute myeloid leukemia cells.

Epigenetic modifying enzymes have a crucial role in the pathogenesis of acute myeloid leukemia (AML). Methylation of lysine 9 on histone H3 by the methyltransferase G9a and SUV39H1 is associated with inhibition of tumor suppressor genes. We studied the effect of G9a and SUV39H1 inhibitors on viability and differentiation of AML cells and tested the cytotoxicity induced by combination of G9a and SUV39H1 inhibitors and various epigenetic drugs. The SUV39H1 inhibitor (chaetocin) and the G9a inhibitor (UNC0638) caused cell death in AML cells at high concentrations. However, only chaetocin-induced CD11b expression and differentiation of AML cells at non-cytotoxic concentration. HL-60 and KG-1a cells were more sensitive to chaetocin than U937 cells. Long-term incubation of chaetocin led to downregulation of SUV39H1 and reduction of H3K9 tri-methylation in HL-60 and KG-1a cells. Combination of chaetocin with suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor) or JQ (a BET (bromodomain extra terminal) bromodomain inhibitor) showed synergistic cytotoxicity. Conversely, no synergism was found by combining chaetocin and UNC0638. More importantly, chaetocin-induced differentiation and combined cytotoxicity were also found in the primary cells of AML patients. Collectively, the SUV39H1 inhibitor chaetocin alone or in combination with other epigenetic drugs may be effective for the treatment of AML.

Synthesis and protein kinase C inhibitory activities of balanol analogs with replacement of the perhydroazepine moiety.

Balanol is a potent protein kinase C (PKC) inhibitor that is structurally composed of a benzophenone diacid, a 4-hydroxybenzamide, and a perhydroazepine ring. A number of balanol analogs in which the perhydroazepine moiety is replaced have been synthesized and their biological activities evaluated against both PKC and cAMP-dependent kinase (PKA). The results suggested that the activity and the isozyme/kinase selectivity of these compounds are largely related to the conformation about this nonaromatic structural element of the molecules.

Light-induced changes in frog pineal gland N-acetyltransferase activity.

N-Acetyltransferase (NAT) activity was determined in the pineal gland of frogs (Rana tigrina) of different ages using 2-aminofluorene and p-aminobenzoic acid as substrates, and assayed by high-pressure liquid chromatography. Frogs of different ages were either killed during the light phase or exposed to darkness or light for 1 min during the dark phase of the lighting cycle, then returned to their cages in darkness for 30 min before being killed. The pineal gland NAT activity of 1-month-old frogs was inhibited when the animal was nocturnally exposed to 1 min of light. Nocturnal light exposure did not inhibit NAT activity in 1-month-old frogs, even though these animal displayed clear light-dark differences in pineal gland NAT activity. Nocturnal light exposure did not inhibit night-time levels of NAT activity in 1-month-old animals which had been bilaterally enucleated, thus suggesting that this effect is retinally mediated. Pretreatment of 1-month-old and 6-month-old animals with isoproterenol (a beta-adrenoceptor agonist drug) prevented the nocturnal light-induced inhibition of NAT activity. From the different sensitivity of 1-month-old and 6-month-old animals to different intensities or durations of nocturnal light exposure it was found that the duration or intensity of light exposure was not able to inhibit nocturnal NAT activity. The NAT activity was at least 4-5-fold greater in 1-month-old frogs than in 6-month-old frogs. This is the first demonstration of the retino-pineal gland pathway that appears to produce light-induced changes in pineal glands of frogs 1-month-old or older, but this pathway only functions in 1-month-old frogs, and does not appear to function in 6-month-old frogs.

Mucinous biliary cystadenoma with mesenchymal stroma: expressions of CA 19-9 and carcinoembryonic antigen in serum and cystic fluid.

A case of mucinous biliary cystadenoma with mesenchymal stroma (CMS tumor) in a 64-year-old woman is reported. The patient presented with acute abdominal pain and a palpable mass in the upper abdomen. Computed tomography and abdominal sonography showed characteristic multilocular cysts in the left lobe of the liver. Serum CA 19-9 was elevated to 108 U/ml with normal carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) levels. The levels of CA 19-9 and CEA in the cystic fluid were high at 7430 U/ml and 576 ng/ml, respectively. The serum CA 19-9 returned to 35 U/ml 4 weeks after tumor resection. These corresponding findings of both tumor markers in the serum and cystic fluid imply that (1) CA 19-9 and CEA both exist in the epithelial component of CMS tumors as evidenced by immunohistochemical stain, (2) serum CA 19-9 is a valuable marker in the diagnosis and monitoring of CMS, and (3) in cystic fluid, there are more significantly high levels of CA 19-9 in CMS compared with levels in simple cyst and polycystic liver disease. Therefore, measurement of CA 19-9 in cystic fluid and serum may be helpful in the differential diagnosis of hepatic cystic lesions.

Identification of cyprinol and cyprinol sulfate from grass carp bile and their toxic effects in rats.

To elucidate the responsible toxic components of grass carp bile, the bile salt 5 alpha-cyprinol sulfate and its desalted form 5 alpha-cyprinol from grass carp bile were purified and identified by analyses of infrared spectrum, (1)H-, (13)C-nuclear magnetic resonance spectra and mass spectrum. The toxicity of grass carp bile powder, butanol extract of grass carp bile powder, 5 alpha-cyprinol and 5 alpha-cyprinol sulfate in rats were further determined. The kidney and liver functions were significantly affected by grass carp bile powder, butanol extract and 5 alpha-cyprinol sulfate. However, 5 alpha-cyprinol also significantly affected the kidney function, but the toxic effect was less.

Toxic effects of grass carp, snake and chicken bile juices in rats.

To evaluate the toxic effects of different animal bile juices, male Long-Evans rats were used and treated orally with different doses (0.03-0.6 ml) of grass carp, snake and chicken bile juices. After treating with one high dose (0.6 ml) for 6 and 24 h, the levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine in the plasma of rats in the grass carp bile juice group became higher than those of the other two bile treated groups. After 3-days periodic treatment with 0.3 ml of each animal bile juice for 28 days, the levels of GOT, GPT, BUN and creatinine in the plasma of rats were significantly increased, especially the grass carp bile juice-treated rats. It appeared that the rats administered with snake and chicken bile juices for a much longer time were poisoned and had the same symptoms as those treated with grass carp bile juice.

Ulceronodular gastritis in secondary syphilis.

Syphilitic gastric ulcers are rarely diagnosed and are almost always a manifestation of the tertiary stage. We describe the case of a 21-year-old man who developed such an ulcer during the secondary stage.

Expression of cytokeratins in normal and diseased livers and in primary liver carcinomas.

Hepatocytes and bile duct epithelium express several types of cytokeratins, the characteristic intermediate-filament proteins of epithelial cells. The cytokeratin antigen expression was studied in normal and diseased livers, intrahepatic cholangiocarcinomas, and hepatocellular carcinomas by immunohistochemical methods using a panel of polyclonal and monoclonal antibodies to cytokeratins. Ten percent formaldehyde solution-fixed, paraffin-embedded sections obtained from ten patients without liver disease, 18 patients without liver disease, 18 patients with different stages of primary biliary cirrhosis, 14 patients with alcoholic hepatitis, ten patients with fatty liver hepatitis secondary to diabetes mellitus or morbid obesity, five patients with hepatocellular carcinomas, and five patients with cholangiocarcinomas were examined. The results suggested that hepatocytes and bile duct epithelium retain their distinct cytokeratin profiles in liver disease, including malignant transformation. Therefore, demonstration of cytokeratins in the liver is useful in establishing the cellular origin of neoplasms and understanding the pathogenesis of liver diseases.