sj-pdf-1-imr-10.1177_03000605221149870 - Supplemental material for Efficacy and safety of cyclosporine-based regimens for primary immune thrombocytopenia: a systematic review and meta-analysis

Supplemental material, sj-pdf-1-imr-10.1177_03000605221149870 for Efficacy and safety of cyclosporine-based regimens for primary immune thrombocytopenia: a systematic review and meta-analysis by Xiaojing Li, Wenwei Zhu, Jizhang Bao, Jiekai Li, Yongming Zhou in Journal of International Medical Research</p

Connected network of the enriched differentially expressed genes following exposure to 0.2 mM H₂O₂.

<p>(A) Connected network of enriched differentially expressed genes involved in fatty acid metabolism (RM018 and RM020). (B) Partial fatty acid metabolism in <i>M</i>. <i>smegmatis</i>. Genes expressed differentially after 0.2 mM H₂O₂ treatment assigned to RM018 and RM020 are marked in red.</p

Quantitative RT-PCR validation of RNA-sequencing results.

<p>(A) Quantitative RT-PCR analysis of the mRNA expression of genes differentially expressed after treatment with different levels of H₂O₂. <i>M</i>. <i>smegmatis</i> cultures were treated with 2 mM or 7 mM H₂O₂ for 30 min before extraction of RNA for qRT-PCR. The data represent 3 independent experiments. (B) Fold changes of selected genes differentially expressed genes after treatment with 0.2 mM and 7 mM H₂O₂ obtained by the RNA-sequencing.</p

Distinct Responses of <i>Mycobacterium smegmatis</i> to Exposure to Low and High Levels of Hydrogen Peroxide

<div><p>Hydrogen peroxide (H₂O₂) is a natural oxidant produced by aerobic organisms and gives rise to oxidative damage, including DNA mutations, protein inactivation and lipid damage. The genus <i>Mycobacterium</i> utilizes redox sensors and H₂O₂ scavenging enzymes for the detoxification of H₂O₂. To date, the precise response to oxidative stress has not been fully elucidated. Here, we compared the effects of different levels of H₂O₂ on transcription in <i>M</i>. <i>smegmatis</i> using RNA-sequencing. A 0.2 mM H₂O₂ treatment had little effect on the growth and viability of <i>M</i>. <i>smegmatis</i> whereas 7 mM H₂O₂ was lethal. Analysis of global transcription showed that 0.2 mM H₂O₂ induced relatively few changes in gene expression, whereas a large proportion of the mycobacterial genome was found to be differentially expressed after treatment with 7 mM H₂O₂. Genes differentially expressed following treatment with 0.2 mM H₂O₂ included those coding for proteins involved in glycolysis-gluconeogenesis and fatty acid metabolism pathways, and expression of most genes encoding ribosomal proteins was lower following treatment with 7 mM H₂O₂. Our analysis shows that <i>M</i>. <i>smegmatis</i> utilizes the sigma factor MSMEG_5214 in response to 0.2 mM H₂O₂, and the RpoE1 sigma factors MSMEG_0573 and MSMEG_0574 in response to 7 mM H₂O₂. In addition, different transcriptional regulators responded to different levels of H₂O₂: MSMEG_1919 was induced by 0.2 mM H₂O₂, while high-level induction of DevR occurred in response to 7 mM H₂O₂. We detected the induction of different detoxifying enzymes, including genes encoding KatG, AhpD, TrxB and Trx, at different levels of H₂O₂ and the detoxifying enzymes were expressed at different levels of H₂O₂. In conclusion, our study reveals the changes in transcription that are induced in response to different levels of H₂O₂ in <i>M</i>. <i>smegmatis</i>.</p></div

RNA-sequencing mapping statistics.

<p>RNA-sequencing mapping statistics.</p

Overview of the differential expression profiles in response to 0.2 mM H₂O₂ in <i>M</i>. <i>smegmatis</i>.

<p>(A) Enrichment analysis. The differently colored bars indicate the gene number for the enrichment of the annotations. (B) Interaction network of the differentially expressed genes of <i>M</i>. <i>smegmatis</i> induced by 0.2 mM H₂O₂ using STRING (9.1) at confidence scores ≥ 0.4. The network is enriched among the 634 differentially expressed genes and 111 interactions were observed (p value = 0).</p

Fold changes of genes differentially expressed after treatment with 0.2 mM and 7 mM H₂O₂ (treated vs untreated).

<p>Fold changes of genes differentially expressed after treatment with 0.2 mM and 7 mM H₂O₂ (treated vs untreated).</p

Connected network of the enriched differentially expressed genes following exposure to 0.2 mM H₂O₂.

<p>(A) Connected network of enriched differentially expressed genes involved in fatty acid metabolism (RM018 and RM020). (B) Partial fatty acid metabolism in <i>M</i>. <i>smegmatis</i>. Genes expressed differentially after 0.2 mM H₂O₂ treatment assigned to RM018 and RM020 are marked in red.</p

Functional Characterization of Sirtuin-like Protein in <i>Mycobacterium smegmatis</i>

Nicotinamide adenine dinucleotide (NAD)-dependent deacetylases (sirtuins) are well conserved from prokaryotes to eukaryotes. Functions and regulations of mammalian sirtuins have been extensively studied and indicate that sirtuins play an important role in regulation of biological processes, whereas functions of mycobacterial sirtuins were less explored. To examine functions of the sirtuin-like protein in mycobacteria, a <i>Mycobacterium smegmatis</i> sirtuin, MSMEG_5175, was overexpressed in a <i>M. smegmatis</i> strain mc²155 to generate an MSMEG_5175-overexpression strain (mc²155-MS5175) in the present study. The physiological aspects of mc²155-MS5175 strain were characterized showing that they had a lower intracellular NAD level and a higher resistance to isoniazid (INH) as compared to mc²155 containing empty pMV261 plasmid (mc²155-pMV261). Quantitative proteomic analysis was carried out to determine differentially expressed proteins between mc²155-pMV261 and mc²155-MS5175. Among 3032 identified proteins, overexpression of MSMEG_5175 results in up-regulation of 34 proteins and down-regulation of 72 proteins, which involve in diverse cellular processes including metabolic activation, transcription and translation, antioxidant, and DNA repair. Down-regulation of catalase peroxidase (KatG) expression in both mRNA and protein levels were observed in mc²155-MS5175 strain, suggesting that a decrease in cellular NAD content and down-regulation of KatG expression contribute to the higher resistance to INH in mc²155-MS5175. Using a combination of immunoprecipitation and proteomic analysis, we found that acetylation in 27 proteins was decreased in mc²155-MS5175 as compared to those in mc²155-pMV261, suggesting that these proteins including the beta prime subunit of RNA polymerase (rpoC), ribosomal proteins, and metabolic enzymes were substrates of MSMEG_5175. Acetylation changes in rpoC may affect its function and cause changes in global gene transcription. Taken together, these results suggest that MSMEG_5175 regulates diverse cellular processes resulting in an increase in INH resistance in mycobacteria, and provide a useful resource to further biological exploration into functions of protein acetylation in mycobacteria

Protocol.

BackgroundToday, patients with coronary heart disease (CHD) are becoming younger and younger, and after percutaneous coronary intervention (PCI), most patients want to resume their occupations. The return to work of patients with CHD post PCI in China, however, has received little research attention. So, the goal of this study was to investigate the variables impacting the return to work following PCI in young and middle-aged patients with CHD in Wuxi and to offer a reference basis for the development of targeted interventions.MethodsThis study was executed at the Affiliated Hospital of Jiangnan University. We selected 280 young and middle-aged patients who underwent PCI for CHD as the study subjects and gathered general data about them while they were hospitalized. At 3 months after PCI, we surveyed the subjects with the return to work self-efficacy questionnaire, the Chinese version of the brief fatigue inventory, and the social support rating scale, and obtained information about their return to work. The factors affecting patients’ returning to work were analyzed using binary logistic regression.ResultsThe final 255 cases were included in the study, of which 155 (60.8%) were successfully returned to work. Binary logistic regression showed that women (OR = 0.379, 95%CI:0.169,0.851), ejection fraction ≥50% (OR = 2.053, 95%CI:1.085,3.885), the brain-based job types (OR = 2.902, 95%CI:1.361,6.190), the kind of employment requiring both mental and physical capacity (OR = 2.867, 95%CI:1.224,6.715), moderate fatigue (OR = 6.023, 95%:1.596,22.7251), mild fatigue (OR = 4.035, 95%:1.104,14.751), return to work efficacy (OR = 1.839, 95%:1.140,3.144), and social support (OR = 1.060, 95%:1.003,1.121) were independent influences on patients’ return to work at 3 months after PCI (All PConclusionIn order to help patient return to work as soon as possible, healthcare professionals should focus on those who are female, have worked mainly in physical activities, have low return-to-work self-efficacy, have severe fatigue, have low social support, and have poor ejection fraction.</div