Ersonal 4100A microarray scanner. The scan images were processed and data were further analyzed by using GenePix Pro 4.1 software combined with Microsoft Excel software. Spots were analyzed by adaptive quantitation, and the local background was subsequently substracted. Spots with background-corrected signal intensity (median) in both channels of less than twofold of background intensity (median) were rejected from further analysis. Data normalization was performed on the remaining spots by total intensity normalization methods. The normalized log2 ratio of test/reference 10457188 signal for each spot was recorded. Significant changes in gene expression were identified using SAM software. After SAM analysis, only genes with at least 2-fold changes in expression were collected for further analysis. The microarray data 16574785 (GSE46666) had been deposited in Gene Expression Omnibus (GEO).Real-time quantitative PCR Autophagy analysisGene-specific primers (Listed in Table 3) were designed to produce an amplicon of 100?50 bp for each gene tested. The contaminating DNA in RNA samples was removed by using Amibion’s DNA-free Kit (Applied Epigenetic Reader Domain Biosystems, Foster City, CA) cDNAs were generated by random hexamer primers. Using three independent cultures and RNA preparations, real-time RT-PCR was performed in triplicate using the Stepone Plus system together with the SYBR Green master mix. On the basis of the standard curves of 16S rRNA expression, the relative mRNA level was determined by calculating the threshold cycle (DCt) of each gene using the classic DCt method. Negative controls were performed by using cDNA generated without reverse transcriptase asS.suis whole-genome microarray experimentsS.suis strain 05ZYH33 were grown in THB with subinhibitory concentrations (0.25 mg/ml) of LicA to the postexponential growth phase, other aliquots of S.suis strain 05ZYH33 without LicA cultured in the same condition were used as control. Immediately before harvesting, 1 volume of bacterial culture was mixed withInhibition Effect of Licochalcone A on S.suistemplates. Reactions containing primer pairs without template were also included as blank controls. The 16S rRNA gene was used as an internal control to normalize all the other genes.Author ContributionsConceived and designed the experiments: HH YJ. Performed the experiments: WH PL YW X. Zheng X. Zeng. Analyzed the data: HH X. Zheng JL X. Zhou. Contributed reagents/materials/analysis tools: QL HJ YZ. Wrote the paper: HH.
Despite increasing access to HIV treatment in resource-limited settings (RLS), patients commencing anti-retroviral therapy (ART) in these settings have been shown to have an increased risk of mortality in the first months of therapy compared with those in high-income countries [1,2], although this difference in mortality risk reduces with time on ART [1]. A number of factors have been associated with mortality during early ART in RLS. These include WHO clinical stage, CDcount, body weight, anemia, male sex and lack of free access to treatment [3?]. Opportunistic infections have been recognized as important causes of early mortality in those initiating ART in RLS [7,8], but limited data are available regarding the relative impact of specific HIV-associated conditions on mortality. Both prevalent and incident tuberculosis (TB) have been associated with a greater than two fold increased risk of mortality during ART in a South African cohort [9] and cryptococcal meningitis has a high mortality in RLS [10,11].Impact of HIV-.Ersonal 4100A microarray scanner. The scan images were processed and data were further analyzed by using GenePix Pro 4.1 software combined with Microsoft Excel software. Spots were analyzed by adaptive quantitation, and the local background was subsequently substracted. Spots with background-corrected signal intensity (median) in both channels of less than twofold of background intensity (median) were rejected from further analysis. Data normalization was performed on the remaining spots by total intensity normalization methods. The normalized log2 ratio of test/reference 10457188 signal for each spot was recorded. Significant changes in gene expression were identified using SAM software. After SAM analysis, only genes with at least 2-fold changes in expression were collected for further analysis. The microarray data 16574785 (GSE46666) had been deposited in Gene Expression Omnibus (GEO).Real-time quantitative PCR analysisGene-specific primers (Listed in Table 3) were designed to produce an amplicon of 100?50 bp for each gene tested. The contaminating DNA in RNA samples was removed by using Amibion’s DNA-free Kit (Applied Biosystems, Foster City, CA) cDNAs were generated by random hexamer primers. Using three independent cultures and RNA preparations, real-time RT-PCR was performed in triplicate using the Stepone Plus system together with the SYBR Green master mix. On the basis of the standard curves of 16S rRNA expression, the relative mRNA level was determined by calculating the threshold cycle (DCt) of each gene using the classic DCt method. Negative controls were performed by using cDNA generated without reverse transcriptase asS.suis whole-genome microarray experimentsS.suis strain 05ZYH33 were grown in THB with subinhibitory concentrations (0.25 mg/ml) of LicA to the postexponential growth phase, other aliquots of S.suis strain 05ZYH33 without LicA cultured in the same condition were used as control. Immediately before harvesting, 1 volume of bacterial culture was mixed withInhibition Effect of Licochalcone A on S.suistemplates. Reactions containing primer pairs without template were also included as blank controls. The 16S rRNA gene was used as an internal control to normalize all the other genes.Author ContributionsConceived and designed the experiments: HH YJ. Performed the experiments: WH PL YW X. Zheng X. Zeng. Analyzed the data: HH X. Zheng JL X. Zhou. Contributed reagents/materials/analysis tools: QL HJ YZ. Wrote the paper: HH.
Despite increasing access to HIV treatment in resource-limited settings (RLS), patients commencing anti-retroviral therapy (ART) in these settings have been shown to have an increased risk of mortality in the first months of therapy compared with those in high-income countries [1,2], although this difference in mortality risk reduces with time on ART [1]. A number of factors have been associated with mortality during early ART in RLS. These include WHO clinical stage, CDcount, body weight, anemia, male sex and lack of free access to treatment [3?]. Opportunistic infections have been recognized as important causes of early mortality in those initiating ART in RLS [7,8], but limited data are available regarding the relative impact of specific HIV-associated conditions on mortality. Both prevalent and incident tuberculosis (TB) have been associated with a greater than two fold increased risk of mortality during ART in a South African cohort [9] and cryptococcal meningitis has a high mortality in RLS [10,11].Impact of HIV-.
