Temporal association between antibiotic use and resistance in Gram-negative bacteria

Abstract The -lactam/lactamase inhibitors (BLBLIs) combination drugs are considered an effective alternative to carbapenems. However, there is a growing concern that the increased use of BLBLIs may lead to increased resistance. This study determined the temporal association between the consumption of BLBLI and the antimicrobial resistance in Gram-negative bacteria. In this retrospective study, electronic data on the Gram-negative bacterial isolates, including A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae from in-patients and susceptibility testing results were retrieved from the medical records of the clinical laboratory. A linear regression and cross-correlation analysis were performed on the acquired data. Increasing trends (p 0.05) in the consumption of BIBLI and carbapenem with a median use of 27.68 and 34.46 DDD/1000 PD per quarter were observed, respectively. A decreased trend (p=0.023) in the consumption of fluoroquinolones with a median use of 29.13 DDD/1000 PD per quarter was observed. The resistance rate of K. pneumoniae was synchronized with the BIBLI and carbapenem consumptions with a correlation coefficient of 0.893 (p=0.012) and 0.951 (p=0.016), respectively. The cross-correlation analysis against the consumption of BIBLI and meropenem resistant K. pneumoniae was peaked at 0-quarter lag (r=951, p=0.016). There was an increasing trend in the consumption of BLBLI and carbapenems. The increasing trend in the rates of resistance to piperacillin/tazobactam, in line with the increasing consumption of BLBLI, suggests that BLBLI has to be used with caution and cannot be directly considered as a long-term alternative to carbapenems.

Resumo Os medicamentos combinados de -lactâmicos / inibidores da lactamase (BLBLIs) são considerados uma alternativa eficaz aos carbapenêmicos. No entanto, existe uma preocupação crescente de que o aumento do uso de BLBLIs pode levar ao aumento da resistência. Este estudo determinou a associação temporal entre o consumo de BLBLI e a resistência antimicrobiana em bactérias gram-negativas. Neste estudo retrospectivo, os dados eletrônicos sobre as bactérias gram-negativas isoladas, incluindo A. baumannii, P. aeruginosa, E. coli e K. pneumoniae de pacientes internados e os resultados dos testes de suscetibilidade foram recuperados dos registros médicos do laboratório clínico. Uma regressão linear e análise de correlação cruzada foram realizadas nos dados adquiridos. Foram observadas tendências crescentes (p 0,05) no consumo de BIBLI e carbapenem com uma mediana de uso de 27,68 e 34,46 DDD/1000 PD por trimestre, respectivamente. Foi observada uma tendência de diminuição (p = 0,023) no consumo de fluoroquinolonas com uma mediana de uso de 29,13 DDD/1000 PD por trimestre. A taxa de resistência de K. pneumoniae foi sincronizada com os consumos de BIBLI e carbapenem com coeficiente de correlação de 0,893 (p = 0,012) e 0,951 (p = 0,016), respectivamente. A análise de correlação cruzada contra o consumo de BIBLI e K. pneumoniae resistente ao meropenem atingiu o pico no intervalo de 0 quarto (r = 951, p = 0,016). Houve uma tendência de aumento no consumo de BLBLI e carbapenêmicos. A tendência crescente nas taxas de resistência a piperacilina/tazobactam, em linha com o consumo crescente de BLBLI, sugere que BLBLI deve ser usado com cautela e não pode ser considerado diretamente como alternativa de longo prazo aos carbapenêmicos.

Temporal association between antibiotic use and resistance in Gram-negative bacteria / Associação temporal entre o uso de antibióticos e resistência em bactérias gram-negativas

Abstract The β-lactam/lactamase inhibitors (BLBLIs) combination drugs are considered an effective alternative to carbapenems. However, there is a growing concern that the increased use of BLBLIs may lead to increased resistance. This study determined the temporal association between the consumption of BLBLI and the antimicrobial resistance in Gram-negative bacteria. In this retrospective study, electronic data on the Gram-negative bacterial isolates, including A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae from in-patients and susceptibility testing results were retrieved from the medical records of the clinical laboratory. A linear regression and cross-correlation analysis were performed on the acquired data. Increasing trends (p<0.05) in the consumption of BIBLI and carbapenem with a median use of 27.68 and 34.46 DDD/1000 PD per quarter were observed, respectively. A decreased trend (p=0.023) in the consumption of fluoroquinolones with a median use of 29.13 DDD/1000 PD per quarter was observed. The resistance rate of K. pneumoniae was synchronized with the BIBLI and carbapenem consumptions with a correlation coefficient of 0.893 (p=0.012) and 0.951 (p=0.016), respectively. The cross-correlation analysis against the consumption of BIBLI and meropenem resistant K. pneumoniae was peaked at 0-quarter lag (r=951, p=0.016). There was an increasing trend in the consumption of BLBLI and carbapenems. The increasing trend in the rates of resistance to piperacillin/tazobactam, in line with the increasing consumption of BLBLI, suggests that BLBLI has to be used with caution and cannot be directly considered as a long-term alternative to carbapenems.

Resumo Os medicamentos combinados de β-lactâmicos / inibidores da lactamase (BLBLIs) são considerados uma alternativa eficaz aos carbapenêmicos. No entanto, existe uma preocupação crescente de que o aumento do uso de BLBLIs pode levar ao aumento da resistência. Este estudo determinou a associação temporal entre o consumo de BLBLI e a resistência antimicrobiana em bactérias gram-negativas. Neste estudo retrospectivo, os dados eletrônicos sobre as bactérias gram-negativas isoladas, incluindo A. baumannii, P. aeruginosa, E. coli e K. pneumoniae de pacientes internados e os resultados dos testes de suscetibilidade foram recuperados dos registros médicos do laboratório clínico. Uma regressão linear e análise de correlação cruzada foram realizadas nos dados adquiridos. Foram observadas tendências crescentes (p < 0,05) no consumo de BIBLI e carbapenem com uma mediana de uso de 27,68 e 34,46 DDD/1000 PD por trimestre, respectivamente. Foi observada uma tendência de diminuição (p = 0,023) no consumo de fluoroquinolonas com uma mediana de uso de 29,13 DDD/1000 PD por trimestre. A taxa de resistência de K. pneumoniae foi sincronizada com os consumos de BIBLI e carbapenem com coeficiente de correlação de 0,893 (p = 0,012) e 0,951 (p = 0,016), respectivamente. A análise de correlação cruzada contra o consumo de BIBLI e K. pneumoniae resistente ao meropenem atingiu o pico no intervalo de 0 quarto (r = 951, p = 0,016). Houve uma tendência de aumento no consumo de BLBLI e carbapenêmicos. A tendência crescente nas taxas de resistência a piperacilina/tazobactam, em linha com o consumo crescente de BLBLI, sugere que BLBLI deve ser usado com cautela e não pode ser considerado diretamente como alternativa de longo prazo aos carbapenêmicos.

IFN-γ affects pancreatic cancer properties by MACC1-AS1/MACC1 axis via AKT/mTOR signaling pathway.

BACKGROUND: Metastasis-related in colon cancer 1 (MACC1) is highly expressed in a variety of solid tumours, but its role in pancreatic cancer (PC) remains unknown. Interferon gamma (IFN-γ) affecting MACC1 expression was explored as the potential mechanism following its intervention. METHODS: Expressions of MACC1 treated with IFN-γ gradient were confirmed by quantitative real-time PCR (qRT-PCR) and western blot (WB). Proliferation, migration, and invasion abilities of PC cells treated with IFN-γ were analysed by CCK8, EDU, colony formation, Transwell (with or without matrix gel) and wound-healing assays. Expression of antisense long non-coding RNA of MACC1, MACC1-AS1, and proteins of AKT/mTOR pathway, (pho-)AKT, and (pho-)mTOR was also assessed by qRT-PCR and WB. SiRNA kit and lentiviral fluid were conducted for transient expression of MACC1 and stable expression of MACC1-AS1, respectively. Rescue assays of cells overexpressing MACC1-AS1 and of cells silencing MACC1 were performed and cellular properties and proteins were assessed by the above-mentioned assays as well. RESULTS: IFN-γ inhibited MACC1 expression in a time- and dose-dependent manner; 100 ng/mL IFN-γ generally caused downregulation of most significant (p ≤ 0.05). In vitro experiments revealed that IFN-γ decreased cellular proliferation, migration, and invasion abilities and downregulated the expression of pho-AKT and pho-mTOR (p ≤ 0.05). Conversely, overexpression of MACC1-AS1 upregulated pho-AKT and pho-mTOR proteins, and reversed cellular properties (p ≤ 0.05). Rescue assays alleviated the above changes of pho-AKT/ mTOR and cellular properties. CONCLUSION: IFN-γ affected PC properties by MACC1-AS1/MACC1 axis via AKT/mTOR signaling pathway, which provides novel insight for candidate targets for treating PC.

Temporal association between antibiotic use and resistance in Gram-negative bacteria.

The ß-lactam/lactamase inhibitors (BLBLIs) combination drugs are considered an effective alternative to carbapenems. However, there is a growing concern that the increased use of BLBLIs may lead to increased resistance. This study determined the temporal association between the consumption of BLBLI and the antimicrobial resistance in Gram-negative bacteria. In this retrospective study, electronic data on the Gram-negative bacterial isolates, including A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae from in-patients and susceptibility testing results were retrieved from the medical records of the clinical laboratory. A linear regression and cross-correlation analysis were performed on the acquired data. Increasing trends (p<0.05) in the consumption of BIBLI and carbapenem with a median use of 27.68 and 34.46 DDD/1000 PD per quarter were observed, respectively. A decreased trend (p=0.023) in the consumption of fluoroquinolones with a median use of 29.13 DDD/1000 PD per quarter was observed. The resistance rate of K. pneumoniae was synchronized with the BIBLI and carbapenem consumptions with a correlation coefficient of 0.893 (p=0.012) and 0.951 (p=0.016), respectively. The cross-correlation analysis against the consumption of BIBLI and meropenem resistant K. pneumoniae was peaked at 0-quarter lag (r=951, p=0.016). There was an increasing trend in the consumption of BLBLI and carbapenems. The increasing trend in the rates of resistance to piperacillin/tazobactam, in line with the increasing consumption of BLBLI, suggests that BLBLI has to be used with caution and cannot be directly considered as a long-term alternative to carbapenems.

Bioactive metabolites in of Ginkgo biloba leaves: variations by seasonal, meteorological and soil / Metabólitos bioativos em folhas de Ginkgo biloba: variações sazonais, meteorológicas e do solo

Ginkgo biloba is a traditional Chinese herbal medicine containing multiple components that contribute to its notable bioactivities. Variations of seasonal, meteorological and planting soil on the phytochemicals contents in G. biloba leaves due to the effects of growth meteorological and soil parameters were investigated in this study. The leaves of G. biloba were collected from different months and place in Zhejiang province, the contents of flavones (quercetin, kaempferol and isorhamnetin) and terpene lactones (bilobalide, ginkgolides A, B and C) were quantified by high performance liquid chromatography (HPLC) and the evaporative light scattering detector (ELSD) method. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. Comprehensive analysis suggested the proper harvest time for G. biloba was in October of Zhejiang province. The result of correlation analysis with meteorological factors shows that the temperature and precipitation have non-significant effect on the main components of G. biloba. In addition, the type and content (Mn and Zn) of the soil showed significantly effect on the content of flavonoids and terpene lactones. This study enriched the knowledge on the development and utilization value of the G. biloba leaves and was useful for determining the optimal harvest time and growing condition.(AU)

Ginkgo biloba é um fitoterápico tradicional da medicina chinesa que contém vários componentes que contribuem para suas notáveis bioatividades. Variações sazonais, meteorológicos e de plantio do solo sobre os teores fitoquímicos em folhas de G. biloba, devido aos efeitos do crescimento meteorológico e parâmetros do solo, foram investigadas neste estudo. As folhas de G. biloba foram coletadas em diferentes meses e na província de Zhejiang, os teores de flavonas (quercetina, kaempferol e isorhamnetina) e lactonas terpênicas (bilobalida, ginkgolídeos A, B e C) foram quantificados por Cromatografia Líquida de Alta Eficiência (CLAE) e pelo método do detector de dispersão da luz por evaporação. Os métodos estabelecidos foram validados com boa linearidade, precisão, repetibilidade, estabilidade e recuperação. Uma análise abrangente sugeriu que o tempo de colheita adequado para G. biloba foi em outubro na província de Zhejiang. O resultado da análise de correlação com fatores meteorológicos mostra que a temperatura e a precipitação não têm efeito significativo sobre os principais componentes de G. biloba. Além disso, o tipo e composição (Mn e Zn) do solo apresentaram efeito significativo sobre o teor de flavonoides e lactonas terpênicas. Este estudo enriqueceu o conhecimento sobre o valor de desenvolvimento e utilização das folhas de G. biloba e foi útil para determinar o melhor tempo de colheita e condição de crescimento.(AU)

Bioactive metabolites in of Ginkgo biloba leaves: variations by seasonal, meteorological and soil / Metabólitos bioativos em folhas de Ginkgo biloba: variações sazonais, meteorológicas e do solo

Abstract Ginkgo biloba is a traditional Chinese herbal medicine containing multiple components that contribute to its notable bioactivities. Variations of seasonal, meteorological and planting soil on the phytochemicals contents in G. biloba leaves due to the effects of growth meteorological and soil parameters were investigated in this study. The leaves of G. biloba were collected from different months and place in Zhejiang province, the contents of flavones (quercetin, kaempferol and isorhamnetin) and terpene lactones (bilobalide, ginkgolides A, B and C) were quantified by high performance liquid chromatography (HPLC) and the evaporative light scattering detector (ELSD) method. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. Comprehensive analysis suggested the proper harvest time for G. biloba was in October of Zhejiang province. The result of correlation analysis with meteorological factors shows that the temperature and precipitation have non-significant effect on the main components of G. biloba. In addition, the type and content (Mn and Zn) of the soil showed significantly effect on the content of flavonoids and terpene lactones. This study enriched the knowledge on the development and utilization value of the G. biloba leaves and was useful for determining the optimal harvest time and growing condition.

Resumo Ginkgo biloba é um fitoterápico tradicional da medicina chinesa que contém vários componentes que contribuem para suas notáveis bioatividades. Variações sazonais, meteorológicos e de plantio do solo sobre os teores fitoquímicos em folhas de G. biloba, devido aos efeitos do crescimento meteorológico e parâmetros do solo, foram investigadas neste estudo. As folhas de G. biloba foram coletadas em diferentes meses e na província de Zhejiang, os teores de flavonas (quercetina, kaempferol e isorhamnetina) e lactonas terpênicas (bilobalida, ginkgolídeos A, B e C) foram quantificados por Cromatografia Líquida de Alta Eficiência (CLAE) e pelo método do detector de dispersão da luz por evaporação. Os métodos estabelecidos foram validados com boa linearidade, precisão, repetibilidade, estabilidade e recuperação. Uma análise abrangente sugeriu que o tempo de colheita adequado para G. biloba foi em outubro na província de Zhejiang. O resultado da análise de correlação com fatores meteorológicos mostra que a temperatura e a precipitação não têm efeito significativo sobre os principais componentes de G. biloba. Além disso, o tipo e composição (Mn e Zn) do solo apresentaram efeito significativo sobre o teor de flavonoides e lactonas terpênicas. Este estudo enriqueceu o conhecimento sobre o valor de desenvolvimento e utilização das folhas de G. biloba e foi útil para determinar o melhor tempo de colheita e condição de crescimento.

[Polarization of human acute monocytic leukemia THP-1 cells-derived macprophages induced by Nippostrongylus brasiliensis proteins in vitro].

OBJECTIVE: To investigate the polarization of human acute monocytic leukemia THP-1 cells-derived macrophages induced by Nippostrongylus brasiliensis proteins in vitro, so as to provide insights into the elucidation of the mechanisms underlying host immune responses to hookworm infections. METHODS: The in-vitro culture of N. brasiliensis was established and maintained in the laboratory, and the third- (L3) and fifth-stage larvae (L5) were collected under a sterile condition for preparation of L3 and L5 proteins. The in-vitro culture of THP-1 cells was established, stimulated with 500 ng/mL PMA to yield M0 macrophages that were adherent to the plate wall. The LPS + IFN-γ group, IL-4 + IL-13 group, L3 protein group and L5 protein group were given stimulation with 500 ng/mL LPS plus 100 ng/mL IFN-γ, IL-4 and IL-13 (both 100 ng/mL), L3 protein (5 mg/mL) and L5 protein (5 mg/mL), respectively, while the negative control group was given no stimulation. The cell morphology was observed using microscopy, the mRNA expression of M1/M2 macrophages-specific genes was quantified using a quantitative real-time PCR (qPCR) assay, and the surface markers of M1/M2 macrophages were detected using flow cytometry, while the levels of cytokines secreted by M1/M2 macrophages were measured using enzyme-linked immunosorbent assay (ELISA) following stimulations, so as to examine the polarization of THP-1-derived macrophages induced by N. brasiliensis proteins in vitro. RESULTS: Following stimulation with PMA, THP-1 cells appeared wall-adherent M0 macrophages, and polarized to typical M1 macrophages following stimulation with LPS + IFN-γ, and typical M2 macrophages following stimulation with IL-4 + IL-13, IL-3 protein or L5 protein. There was a significant difference in the proportion of M1 macrophages among the negative control group, the LPS + IFN-γ group, the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (χ2 = 3 721.00, P < 0.001), with the highest proportion detected in the LPS + IFN-γ group, and there was also a significant difference in the proportion of M2 macrophages among groups (χ2 = 105.43, P < 0.001). There were significant differences among groups in terms of the mRNA expression of CCL2 (F = 191.95, P < 0.001), TNF-α (F = 129.95, P < 0.001), IL-12b (F = 82.89, P < 0.001), PPARγ (F = 11.30, P < 0.001), IL-10 (F = 9.51, P < 0.001) and Mrc1 genes (F = 12.35, P < 0.001). In addition, there were significant differences in the proportion of positive CD86 and CD206 expression among groups (χ2 = 24 004.33 and 832.50, P < 0.001). Higher IL-1ß and TNF-α levels were measured in the LPS + IFN-γ group than in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (P < 0.001), and greater TGF-ß1 and IL-10 levels were seen in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group than in the negative control group and the LPS + IFN-γ group (P < 0.05). CONCLUSIONS: Both L3 and L5 proteins of N. brasiliensis may induce the polarization of THP-1-derived macrophages to M2 type in vitro.

Bioactive metabolites in of Ginkgo biloba leaves: variations by seasonal, meteorological and soil.

Ginkgo biloba is a traditional Chinese herbal medicine containing multiple components that contribute to its notable bioactivities. Variations of seasonal, meteorological and planting soil on the phytochemicals contents in G. biloba leaves due to the effects of growth meteorological and soil parameters were investigated in this study. The leaves of G. biloba were collected from different months and place in Zhejiang province, the contents of flavones (quercetin, kaempferol and isorhamnetin) and terpene lactones (bilobalide, ginkgolides A, B and C) were quantified by high performance liquid chromatography (HPLC) and the evaporative light scattering detector (ELSD) method. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. Comprehensive analysis suggested the proper harvest time for G. biloba was in October of Zhejiang province. The result of correlation analysis with meteorological factors shows that the temperature and precipitation have non-significant effect on the main components of G. biloba. In addition, the type and content (Mn and Zn) of the soil showed significantly effect on the content of flavonoids and terpene lactones. This study enriched the knowledge on the development and utilization value of the G. biloba leaves and was useful for determining the optimal harvest time and growing condition.

Controlling nutritional status (CONUT) score-based nomogram to predict overall survival of patients with HBV-associated hepatocellular carcinoma after curative hepatectomy.

PURPOSE: As a novel immune-nutritional biomarker, the controlling nutritional status (CONUT) score has been reported to predict outcomes in cancer patients. We aimed to elucidate the prognostic value of preoperative CONUT score and construct a CONUT score-based nomogram to predict individual survival of patients with hepatitis B viral (HBV)-associated hepatocellular carcinoma (HCC) after curative hepatectomy. METHODS: Preoperative CONUT score was retrospectively calculated in 380 HBV-associated HCC patients undergoing radical resection between 2006 and 2012. Patients were assigned to two groups: CONUT-low ( < 2) and CONUT-high ( ≥ 2), according to the optimal cut-off value determined using receiver operating characteristic analysis. Associations of CONUT score with oncological outcomes were evaluated. The Cox proportional hazard model was used to identify predictors of survival and a new nomogram was developed based on the independent prognostic factors for overall survival (OS). RESULTS: The CONUT score exhibited a higher area under the curve value than the other immune-nutritional parameters. The CONUT-high group had significant poorer OS and recurrence-free survival compared with CONUT-low group (P < 0.001 and P = 0.016, respectively). Multivariate analyses identified CONUT score, liver cirrhosis, tumor size and differentiation as independent prognostic factors for OS. And the nomogram based on these four variables had superior discriminative ability to predict survival compared with other conventional staging systems. CONCLUSIONS: Preoperative CONUT score is an effective independent predictor of OS in patients with resected HBV-related HCC. This novel nomogram based on CONUT may provide accurate and individualized survival prediction for HCC patients undergoing surgical resection.

CalliSpheres® drug-eluting beads (DEB) transarterial chemoembolization (TACE) is equally efficient and safe in liver cancer patients with different times of previous conventional TACE treatments: a result from CTILC study.

PURPOSE: To assess the efficacy and safety of drug-eluting beads transarterial chemoembolization (DEB-TACE) in liver cancer patients with different times of previous conventional transarterial chemoembolization (cTACE) treatments. METHODS: 367 liver cancer patients about to receive DEB-TACE treatment were enrolled in this prospective cohort study. All patients were divided into no previous cTACE group (NPC group), 1-2 times previous cTACE group (PC group) and triple or above previous cTACE group (TPC group) according to the times of previous cTACE treatments. RESULTS: There was no difference in complete response (CR) (P = 0.671) and objective response rate (ORR) (P = 0.062) among three groups. Additionally, no difference in overall survival (OS) among groups (P = 0.899) was found. As to liver function, most liver function indexes were deteriorative at 1 week after DEB-TACE operation, but returned to baseline at 1-3 months after DEB-TACE operation in all three groups, while percentage of abnormal total bile acid (TBA) patients was higher in TPC group than NPC and PC groups at 1-3 month post-DEB-TACE (P = 0.018). As for safety profiles, the incidence of pain during DEB-TACE operation was lower in TPC group compared to NPC and PC groups (P = 0.005), while no difference of other adverse events was found during and 1 month post-DEB-TACE treatment among three groups. CONCLUSION: DEB-TACE treatment was equally efficient and tolerated in liver cancer patients with different times of previous cTACE treatments.

Epigallocatechin Gallate (EGCG) Inhibited the Alv-J-Induced Apoptosis in Df-1 Cells by Inactivation of Nuclear Factor b Pathway

Avian leukosis virus subgroup J (ALV-J), a member of the retroviridae family, can infect both broilers and layers and induce a spectrum of different neoplasms, resulting in serious economic losses in poultry production. Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has demonstrated remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays, cell culture systems and epidemiological studies. To assess the antiviral effects of EGCG on ALV-J-induced cell apoptosis in vitro, DF-1 cells were treated with different EGCG concentrations (0, 5, 10, 20 and 40 µg/mL), and their antiviral effects were examined at different time points (0, 24, 48, 72 and 96 h) using a variety of assays. EGCG alleviated the ALV-J-induced apoptosis in a dose-dependent manner. Because high concentrations (20 and 40 µg/mL) inhibited DF-1 cell growth, and low concentration (5 µg/mL) did not suppress the ALV-J virus, 10 µg/mL was the most appropriate concentration. After 96 h of incubation, 10 µg/mL EGCG improved the ALV-J-triggered suppression of the nuclear transcription factor system by enhancing cytoplasmic NF-B p50/p65 expression and inhibiting nuclear NF-B p50/p65 expression, resulting in decreased cell apoptosis. These results demonstrated that EGCG inhibited ALV-J-induced apoptosis in DF-1 cells in a dose-dependent manner via the NF-B signaling pathway, and that 10 µg/mL EGCG is the optimal concentration, which may be useful for therapeutic drug design.(AU)

Bioactive metabolites in of Ginkgo biloba leaves: variations by seasonal, meteorological and soil

Abstract Ginkgo biloba is a traditional Chinese herbal medicine containing multiple components that contribute to its notable bioactivities. Variations of seasonal, meteorological and planting soil on the phytochemicals contents in G. biloba leaves due to the effects of growth meteorological and soil parameters were investigated in this study. The leaves of G. biloba were collected from different months and place in Zhejiang province, the contents of flavones (quercetin, kaempferol and isorhamnetin) and terpene lactones (bilobalide, ginkgolides A, B and C) were quantified by high performance liquid chromatography (HPLC) and the evaporative light scattering detector (ELSD) method. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. Comprehensive analysis suggested the proper harvest time for G. biloba was in October of Zhejiang province. The result of correlation analysis with meteorological factors shows that the temperature and precipitation have non-significant effect on the main components of G. biloba. In addition, the type and content (Mn and Zn) of the soil showed significantly effect on the content of flavonoids and terpene lactones. This study enriched the knowledge on the development and utilization value of the G. biloba leaves and was useful for determining the optimal harvest time and growing condition.

Resumo Ginkgo biloba é um fitoterápico tradicional da medicina chinesa que contém vários componentes que contribuem para suas notáveis bioatividades. Variações sazonais, meteorológicos e de plantio do solo sobre os teores fitoquímicos em folhas de G. biloba, devido aos efeitos do crescimento meteorológico e parâmetros do solo, foram investigadas neste estudo. As folhas de G. biloba foram coletadas em diferentes meses e na província de Zhejiang, os teores de flavonas (quercetina, kaempferol e isorhamnetina) e lactonas terpênicas (bilobalida, ginkgolídeos A, B e C) foram quantificados por Cromatografia Líquida de Alta Eficiência (CLAE) e pelo método do detector de dispersão da luz por evaporação. Os métodos estabelecidos foram validados com boa linearidade, precisão, repetibilidade, estabilidade e recuperação. Uma análise abrangente sugeriu que o tempo de colheita adequado para G. biloba foi em outubro na província de Zhejiang. O resultado da análise de correlação com fatores meteorológicos mostra que a temperatura e a precipitação não têm efeito significativo sobre os principais componentes de G. biloba. Além disso, o tipo e composição (Mn e Zn) do solo apresentaram efeito significativo sobre o teor de flavonoides e lactonas terpênicas. Este estudo enriqueceu o conhecimento sobre o valor de desenvolvimento e utilização das folhas de G. biloba e foi útil para determinar o melhor tempo de colheita e condição de crescimento.

Epigallocatechin Gallate (EGCG) Inhibited the Alv-J-Induced Apoptosis in Df-1 Cells by Inactivation of Nuclear Factor b Pathway

Avian leukosis virus subgroup J (ALV-J), a member of the retroviridae family, can infect both broilers and layers and induce a spectrum of different neoplasms, resulting in serious economic losses in poultry production. Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has demonstrated remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays, cell culture systems and epidemiological studies. To assess the antiviral effects of EGCG on ALV-J-induced cell apoptosis in vitro, DF-1 cells were treated with different EGCG concentrations (0, 5, 10, 20 and 40 µg/mL), and their antiviral effects were examined at different time points (0, 24, 48, 72 and 96 h) using a variety of assays. EGCG alleviated the ALV-J-induced apoptosis in a dose-dependent manner. Because high concentrations (20 and 40 µg/mL) inhibited DF-1 cell growth, and low concentration (5 µg/mL) did not suppress the ALV-J virus, 10 µg/mL was the most appropriate concentration. After 96 h of incubation, 10 µg/mL EGCG improved the ALV-J-triggered suppression of the nuclear transcription factor system by enhancing cytoplasmic NF-B p50/p65 expression and inhibiting nuclear NF-B p50/p65 expression, resulting in decreased cell apoptosis. These results demonstrated that EGCG inhibited ALV-J-induced apoptosis in DF-1 cells in a dose-dependent manner via the NF-B signaling pathway, and that 10 µg/mL EGCG is the optimal concentration, which may be useful for therapeutic drug design.

Cryopreservation of domestic cat (Felis catus) ovarian tissue: Comparison of two vitrification methods.

We aimed to evaluate the effect of two vitrification methods on the morphology and functionality of vitrified feline preantral follicles. Feline ovarian tissue was vitrified with EG + trehalose combined or not with dimethyl sulphoxide (DMSO), using two different techniques (open or closed systems). Morphology, developmental capacity and mRNA expression of markers for follicle survival and quality were assessed before and after in vitro culture (IVC). Both vitrification and culture media were serum-free. Vitrification of feline ovarian tissue from five adult domestic cats was performed with EG + trehalose combined or not with DMSO. Two systems were used: the open system solid-surface vitrification (SSV) and the closed system ovarian tissue cryosystem (OTC). Histological analysis of follicle integrity showed that the percentages of normal follicles in previously vitrified ovarian fragments decreased after 7 days of in vitro culture (IVC), independently of the protocol used. Although follicular activation was observed by Ki-67 labelling, this was accompanied by extensive follicular degeneration as detected by a 3-4-fold decrease in follicular density. Remarkable follicle activation was observed in the ovarian tissue vitrified using OTC and subjected to IVC, probably due to a higher rate of degeneration of developing follicles. Even with such follicular loss, the results are promising for the combination of EG + DMSO + trehalose in a serum-free medium when applying the SSV method, with this approach resulting in the highest rates of normal developing follicles (19%) after 7 days IVC, together with granulosa cells proliferating at the same rate observed in fresh tissue.

Analysis of serum microRNA expression in male workers with occupational noise-induced hearing loss.

Occupational noise-induced hearing loss (ONIHL) is a prevalent occupational disorder that impairs auditory function in workers exposed to prolonged noise. However, serum microRNA expression in ONIHL subjects has not yet been studied. We aimed to compare the serum microRNA expression profiles in male workers of ONIHL subjects and controls. MicroRNA microarray analysis revealed that four serum microRNAs were differentially expressed between controls (n=3) and ONIHL subjects (n=3). Among these microRNAs, three were upregulated (hsa-miR-3162-5p, hsa-miR-4484, hsa-miR-1229-5p) and one was downregulated (hsa-miR-4652-3p) in the ONIHL group (fold change >1.5 and Pbon value <0.05). Real time quantitative PCR was conducted for validation of the microRNA expression. Significantly increased serum levels of miR-1229-5p were found in ONIHL subjects compared to controls (n=10 for each group; P<0.05). A total of 659 (27.0%) genes were predicted as the target genes of miR-1229-5p. These genes were involved in various pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway. Overexpression of miR-1229-5p dramatically inhibited the luciferase activity of 3' UTR segment of MAPK1 (P<0.01). Compared to the negative control, HEK293T cells expressing miR-1229-5p mimics showed a significant decline in mRNA levels of MAPK1 (P<0.05). This preliminary study indicated that serum miR-1229-5p was significantly elevated in ONIHL subjects. Increased miR-1229-5p may participate in the pathogenesis of ONIHL through repressing MAPK1 signaling.

Analysis of serum microRNA expression in male workers with occupational noise-induced hearing loss

Occupational noise-induced hearing loss (ONIHL) is a prevalent occupational disorder that impairs auditory function in workers exposed to prolonged noise. However, serum microRNA expression in ONIHL subjects has not yet been studied. We aimed to compare the serum microRNA expression profiles in male workers of ONIHL subjects and controls. MicroRNA microarray analysis revealed that four serum microRNAs were differentially expressed between controls (n=3) and ONIHL subjects (n=3). Among these microRNAs, three were upregulated (hsa-miR-3162-5p, hsa-miR-4484, hsa-miR-1229-5p) and one was downregulated (hsa-miR-4652-3p) in the ONIHL group (fold change >1.5 and Pbon value <0.05). Real time quantitative PCR was conducted for validation of the microRNA expression. Significantly increased serum levels of miR-1229-5p were found in ONIHL subjects compared to controls (n=10 for each group; P<0.05). A total of 659 (27.0%) genes were predicted as the target genes of miR-1229-5p. These genes were involved in various pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway. Overexpression of miR-1229-5p dramatically inhibited the luciferase activity of 3′ UTR segment of MAPK1 (P<0.01). Compared to the negative control, HEK293T cells expressing miR-1229-5p mimics showed a significant decline in mRNA levels of MAPK1 (P<0.05). This preliminary study indicated that serum miR-1229-5p was significantly elevated in ONIHL subjects. Increased miR-1229-5p may participate in the pathogenesis of ONIHL through repressing MAPK1 signaling.

Renal Dose Dopamine Mediates the Level of Aquaporin-2 Water Channel (Aqp2) in Broiler Chickens

ABSTRACT Aquaporin 2 (AQP2) is a small protein located in the collecting tubules of kidneys; it plays an important role in the concentration and production of urine. The aim of this study was to determine the expression level of the AQP2 gene in the kidney of broiler chickens after the administration of renal­dose dopamine. Broiler chickens (25 days-old) were randomly divided into two groups (n=20 per group): intravenous administration of saline solution (control group) or renal-dose dopamine (dopamine group). The expression and localization of the AQP2 gene were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC), respectively. The protein level of AQP2 was analyzed by western blot analysis. The dopamine group presented no significant difference (p>0.05) in the biochemical criterion or mRNA expression of AQP2 compared with the control group. However, AQP2 protein level was significantly reduced (p 0.05) in the membrane of renal tubular epithelial cells. In contrast, protein level was significantly increased (p 0.05) in the cytoplasm of the dopamine group compared with the control group. Moreover, AQP2 protein was apparently more distributed and localized in the cytoplasmic vacuoles than in the membranes of the kidney in the renal­dose dopamine administered chickens group. In conclusion, present findings suggest that renal­dose dopamine mediates the level of AQP2 protein via shuttle from the cell membrane to the cytoplasm rather than changing the expression of AQP2 gene to adjust the secretion and absorption of water in kidney.(AU)

Renal Dose Dopamine Mediates the Level of Aquaporin-2 Water Channel (Aqp2) in Broiler Chickens

ABSTRACT Aquaporin 2 (AQP2) is a small protein located in the collecting tubules of kidneys; it plays an important role in the concentration and production of urine. The aim of this study was to determine the expression level of the AQP2 gene in the kidney of broiler chickens after the administration of renal­dose dopamine. Broiler chickens (25 days-old) were randomly divided into two groups (n=20 per group): intravenous administration of saline solution (control group) or renal-dose dopamine (dopamine group). The expression and localization of the AQP2 gene were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC), respectively. The protein level of AQP2 was analyzed by western blot analysis. The dopamine group presented no significant difference (p>0.05) in the biochemical criterion or mRNA expression of AQP2 compared with the control group. However, AQP2 protein level was significantly reduced (p 0.05) in the membrane of renal tubular epithelial cells. In contrast, protein level was significantly increased (p 0.05) in the cytoplasm of the dopamine group compared with the control group. Moreover, AQP2 protein was apparently more distributed and localized in the cytoplasmic vacuoles than in the membranes of the kidney in the renal­dose dopamine administered chickens group. In conclusion, present findings suggest that renal­dose dopamine mediates the level of AQP2 protein via shuttle from the cell membrane to the cytoplasm rather than changing the expression of AQP2 gene to adjust the secretion and absorption of water in kidney.

Is endobronchial ultrasound-guided transbronchial needle aspiration with a stylet necessary for lymph node screening in lung cancer patients?

During endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), a needle is commonly used with a stylet, although recently the stylet has been omitted. This prospective study aimed to compare the quality of specimens obtained by EBUS-TBNA performed with and without a stylet. Between November 2013 and November 2014, 131 patients with lung cancer underwent EBUS-TBNA, with a total of 148 mediastinal or hilar lymph nodes sampled both with and without an inner-stylet, yielding 296 cytological specimens. Specimens were scored cytologically using five parameters: background blood or clot, amount of cellular material, degree of cellular degeneration, degree of cellular trauma, and retention of appropriate architecture. The procedure with a stylet required significantly longer operation time than without a stylet (14.5±0.8 vs 12.7±1.1 min, P<0.001). Excellent specimens were obtained in 261/296 and 260/296 samples in the procedures with and without a stylet, respectively (P=0.9), while the remaining 35 and 36 samples, respectively, were adequate. The diagnosing and staging of lung cancer using EBUS-TBNA did not differ significantly between the groups. In conclusion, specimen collection by EBUS-TBNA without a stylet is easier and faster than the procedure using a stylet and absence of a stylet did not alter specimen quality or diagnostic accuracy.

FAMLF is a target of miR-181b in Burkitt lymphoma.

Burkitt lymphoma (BL) is a highly malignant non-Hodgkin's lymphoma that is closely related to the abnormal expression of genes. Familial acute myelogenous leukemia related factor (FAMLF; GenBank accession No. EF413001.1) is a novel gene that was cloned by our research group, and miR-181b is located in the intron of the FAMLF gene. To verify the role of miR-181b and FAMLF in BL, RNAhybrid software was used to predict target site of miR-181b on FAMLF and real-time quantitative PCR (RQ-PCR) was used to detect expression of miR-181b and FAMLF in BL patients, Raji cells and unaffected individuals. miR-181b was then transfected into Raji and CA46 cell lines and FAMLF expression was examined by RQ-PCR and western blotting. Further, Raji cells viability and proliferation were detected by MTT and clone formation, and Raji cell cycle and apoptosis were detected by flow cytometry. The results showed that miR-181b can bind to bases 21-42 of the FAMLF 5' untranslated region (UTR), FAMLF was highly expressed and miR-181b was lowly expressed in BL patients compared with unaffected individuals. FAMLF expression was significantly and inversely correlated to miR-181b expression, and miR-181b negatively regulated FAMLF at posttranscriptional and translational levels. A dual-luciferase reporter gene assay identified that the 5' UTR of FAMLF mRNA contained putative binding sites for miR-181b. Down-regulation of FAMLF by miR-181b arrested cell cycle, inhibited cell viability and proliferation in a BL cell line model. Our findings explain a new mechanism of BL pathogenesis and may also have implications in the therapy of FAMLF-overexpressing BL.