Supplementary Material for: Iridoids with Genipin Stem Nucleus Inhibit Lipopolysaccharide-Induced Inflammation and Oxidative Stress by Blocking the NF-κB Pathway in Polycystic Ovary Syndrome

<b><i>Background/Aims:</i></b> Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women, and it is usually characterized by chronic inflammation, oxidative stress, and altered microRNA expression. The aim of this study is to investigate how the effects of iridoids with genipin stem nucleus inhibit PCOS complications. The interactions between iridoids were investigated, as well. <b><i>Methods:</i></b> The chronic inflammation cell model was induced using lipopolysaccharide (LPS) in the RAW 264.7 and KGN cell lines. Levels of mRNA and protein expression were quantified using real time-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. The target of the iridoids was identified using the drug affinity responsive target stability (DARTS) method. The ability to scavenge free radicals was evaluated using the DPPH radical scavenging method and the ultra oxygen anion (O₂^-) radical scavenging method. <b><i>Results:</i></b> The cells recovered from the inflammatory conditions and showed significantly decreased levels of interleukins after treatment with iridoids. The iridoids were demonstrated to target NF-κB, inhibit the phosphorylation and degradation of IκB, inhibit the nuclear entry of NF-κB, and inhibit the expression of inflammatory factors. Though only genipin showed an efficient ability to scavenge O₂^-, the iridoids, IκB inhibitor (BAY 11-7085), and NF-κB inhibitor (PDTC) could inhibit LPS-induced oxidative stress on the cells, indicating that the iridoids exert their anti-oxidant effects via the NF-κB pathway. The expression levels of microRNAs (miRNAs) were also altered by LPS, but the iridoids could scarcely rescue the abnormal condition. <b><i>Conclusion:</i></b> Chronic inflammation may be an important incentive for oxidative stress and abnormal microRNA expression in PCOS, and iridoids can protect patients from inflammatory damage by regulating the NF-κB pathway

Supplementary Material for: Development of a Genetic and Clinical Data-Based (GC) Risk Score for Predicting Survival of Hepatocellular Carcinoma Patients After Tumor Resection

<b><i>Background/Aims:</i></b> Carnitine palmitoyltransferase 1A (CPT1A) is a rate-limiting enzyme in the transport of long-chain fatty acids for β-oxidation. Increasing evidence has indicated that CPT1A plays an important role in carcinogenesis. However, the expression and prognostic value of CPT1A in hepatocellular carcinoma (HCC) have not been extensively studied. <b><i>Methods:</i></b> Here, we collected 66 post-operative liver cancer tissue samples. Gene profile expression was tested by RT-PCR. Receiver operating characteristic (ROC) analysis was performed and multivariate analysis with Cox’s Proportional Hazard Model was used for confirming the selected markers’ predictive efficiency for HCC patients’ survival. A simple risk scoring system was created based on Cox’s regression modeling and bootstrap internal validation. <b><i>Results:</i></b> Cox multivariate regression analysis demonstrated that CPT1A, tumor size, intrahepatic metastasis, TNM stage and histological grade were independent risk factors for the prognosis of HCC patients after surgery. Our genetic and clinical data-based (GC) risk scoring system revealed that HCC patients whose total score≥3 are more likely to relapse and die than patients whose total score < 3. Finally, the good discriminatory power of our risk scoring model was validated by bootstrap internal validation. <b><i>Conclusions:</i></b> The genetic and clinical data-based risk scoring model can be a promising predictive tool for liver cancer patients’ prognosis after operation

Supplementary Material for: Adenosine Triphosphate-Induced Rabbit Corneal Endothelial Cell Proliferation in vitro via the P2Y2-PI3K/Akt Signaling Axis

<b><i>Purpose:</i></b> To investigate the effect of the ATP-P2Y2-PI3K/Akt signaling axis on promoting rabbit corneal endothelial cell (RCEC) proliferation in vitro. <b><i>Methods:</i></b> Five concentrations of adenosine triphosphate (ATP; 1, 10, 25, 50 and 100 μM) were added to RCECs, and the cell proliferation was detected using Cell Counting Kit-8 (CCK8) and Ki67 immunohistochemical staining. Other P2Y2 receptor agonists and antagonists were added to the cells, and the proliferation effect was evaluated using CCK8 to determine the involvement of the P2Y2 receptor. Changes in the expression of phosphorylated Akt in RCECs treated with different concentrations of extracellular ATP and the duration of extracellular ATP on Akt phosphorylation were investigated using Western blotting. The pharmacological profiles with or without the PI3K/Akt pathway inhibitors were also determined using Western blotting. <b><i>Results:</i></b> We found that 10 μM ATP strongly promoted RCEC proliferation in vitro. Additionally, 25 μM ATP had a proliferation effect, whereas other concentrations (1, 50 and 100 μM) had no effect compared with the control group. Selective P2Y2 receptor agonists (UTP, ATPγS and Ap₄A) showed the same promotion effect, while P2Y2 antagonists and PI3K/Akt inhibitors inhibited the effect of ATP. Moreover, phosphorylated Akt could be induced by the addition of extracellular ATP at all five concentrations and lasted for 1 h. This phosphorylation was prevented by PI3K/Akt inhibitors and a P2Y2 antagonist. <b><i>Conclusions:</i></b> These findings showed that 10 μM ATP markedly promoted RCEC proliferation via the P2Y2-PI3K/Akt signaling axis

PowerPoint Slides for: Prognostic Value of the Delivery Dialysis Dose on Twice-Weekly Hemodialysis Patients

<p><b><i>Background:</i></b> Few studies have evaluated the prognostic value of dialysis dose in twice-weekly hemodialysis (HD). A single-pool Kt/V (spKt/V) over 1.70 may benefit patients receiving twice-weekly maintenance HD. <b><i>Methods:</i></b> This is a multicenter randomized controlled trial performed on 163 patients from 17 dialysis centers in Shanghai who were allocated to high- (<i>n</i> = 98) and standard-dose groups (<i>n</i> = 65) and followed through 96 weeks of study period. Therapeutic approaches were given to increase spKt/V to over 1.70 in the high-dose group. Data were collected every 12-24 weeks. The primary outcomes were all-cause mortality and major adverse cardio-cerebrovascular events (MACEs) occurrence, and secondary outcomes included residual kidney function (RKF) and health-related quality of life (HR-QOL). <b><i>Results:</i></b> The spKt/V in high-dose and standard-dose groups were 1.80 ± 0.23 and 1.55 ± 0.19, respectively, after an 8-week intervention (<i>p</i> < 0.001). At the end of the study, SF-36 physical function and total score in high-dose group were 82 (69-90) and 74 (47-84), respectively, both of which were higher than those in the standard-dose group. Decline in urine volume was observed in both groups with no significant difference (<i>p</i> = 0.431). No difference was found in overall survival between the 2 groups (<i>p</i> = 0.580). The 1-year MACE-free survival for high-dose group was 84.49%, better than 76.72% for standard-dose group (<i>p</i> = 0.029). <b><i>Conclusions:</i></b> Higher spKt/V is also associated with MACE-free survival and better HR-QOL, especially in physical function aspect for twice-weekly dialysis patients. Increasing spKt/V over 1.70 in twice-weekly HD population does not cause loss of RKF.</p

Supplementary Material for: Prognostic Value of the Delivery Dialysis Dose on Twice-Weekly Hemodialysis Patients

<p><b><i>Background:</i></b> Few studies have evaluated the prognostic value of dialysis dose in twice-weekly hemodialysis (HD). A single-pool Kt/V (spKt/V) over 1.70 may benefit patients receiving twice-weekly maintenance HD. <b><i>Methods:</i></b> This is a multicenter randomized controlled trial performed on 163 patients from 17 dialysis centers in Shanghai who were allocated to high- (<i>n</i> = 98) and standard-dose groups (<i>n</i> = 65) and followed through 96 weeks of study period. Therapeutic approaches were given to increase spKt/V to over 1.70 in the high-dose group. Data were collected every 12-24 weeks. The primary outcomes were all-cause mortality and major adverse cardio-cerebrovascular events (MACEs) occurrence, and secondary outcomes included residual kidney function (RKF) and health-related quality of life (HR-QOL). <b><i>Results:</i></b> The spKt/V in high-dose and standard-dose groups were 1.80 ± 0.23 and 1.55 ± 0.19, respectively, after an 8-week intervention (<i>p</i> < 0.001). At the end of the study, SF-36 physical function and total score in high-dose group were 82 (69-90) and 74 (47-84), respectively, both of which were higher than those in the standard-dose group. Decline in urine volume was observed in both groups with no significant difference (<i>p</i> = 0.431). No difference was found in overall survival between the 2 groups (<i>p</i> = 0.580). The 1-year MACE-free survival for high-dose group was 84.49%, better than 76.72% for standard-dose group (<i>p</i> = 0.029). <b><i>Conclusions:</i></b> Higher spKt/V is also associated with MACE-free survival and better HR-QOL, especially in physical function aspect for twice-weekly dialysis patients. Increasing spKt/V over 1.70 in twice-weekly HD population does not cause loss of RKF.</p