Supplementary Material for: Blood Outgrowth Endothelial Cells Reduce Hypoxia-Mediated Fibroblast to Myofibroblast Conversion by Decreasing Proangiogenic Cytokines

<p><b><i>Purpose:</i></b> Hypoxic conditions cause fibroblasts to differentiate into alpha smooth-muscle cell actin (α -SMA)-positive cells, i.e. myofibroblasts. This process is a hallmark of venous neointimal hyperplasia (VNH) associated with hemodialysis vascular access. The purpose of this study was to determine if blood outgrowth endothelial cells (BOEC) may reduce the conversion of fibroblasts into myofibroblasts under hypoxic conditions, and to determine the potential mechanisms involved. <b><i>Methods:</i></b> An experimental model was used, in which fibroblasts and BOEC were subjected to hypoxia under contact and transwell conditions to determine if BOEC reduce the conversion of fibroblasts into myofibroblasts under hypoxic conditions. Gene expression under different conditions was performed. In addition, functional assays including cell proliferation and migration were determined. <b><i>Results:</i></b> This study demonstrates that contact needs to occur between BOEC and fibroblasts for the reduction of the hypoxia-driven conversion of fibroblasts into α-SMA. This is associated with a decrease in several proangiogenic genes including vascular endothelial growth factor A, platelet-derived growth factor, fibroblast growth factor and matrix metalloproteinase 2 in fibroblasts in contact with BOEC when compared to fibroblasts alone. In addition, migration is significantly reduced while proliferation remains unchanged. <b><i>Conclusion:</i></b> This study helps provide rationale for using BOEC delivered to the adventitia of the outflow vein of hemodialysis vascular access to reduce VNH.</p

PowerPoint Slides for: Fibroblast Growth Factor 23 Predicts All-Cause Mortality in a Dose-Response Fashion in Pre-Dialysis Patients with Chronic Kidney Disease

<p><b><i>Background:</i></b> Quantitative dose-response associations between fibroblast growth factor 23 (FGF23) and risks of mortality, cardiovascular disease (CVD), and renal events in chronic kidney disease (CKD) are not known. This study aimed to summarize and quantify the predictive effects of FGF23 among the pre-dialysis CKD stages 1-5 population. <b><i>Methods:</i></b> Data sources included PubMed, EMBASE, and Web of Science. Prospective cohort studies assessing the associations between FGF23 and all-cause mortality, CVD, and renal events in CKD patients were selected. Summary risk ratios (RRs) and 95% confidence intervals (CIs) were calculated using the random-effects model. The composite higher or the highest level in FGF23 categories of each study was considered the high level. The reference level was regarded as the low level in the overall analysis. The restricted cubic spline model was used to estimate dose-response associations. <b><i>Results:</i></b> Fifteen prospective cohort studies centered around 15,355 subjects were analyzed. A high FGF23 level was associated with increased risks of all-cause mortality (RR 1.46, 95% CI 1.38-1.55, <i>p</i> < 0.001), CVD (RR 1.37, 95% CI 1.15-1.63, <i>p</i> < 0.001), and renal events (RR 1.31, 95% CI 1.07-1.59, <i>p </i>= 0.008), respectively. There was a positive, nonlinear, dose-response relationship between FGF23 and all-cause mortality. The reference level in dose-response analysis was defined as 51 RU/mL of c-terminal FGF23. We then calculated RRs for increments of 20 RU/mL, which was associated with increased risks of mortality (RR 1.04, 95% CI 1.00-1.07, <i>p</i> = 0.038), CVD (RR 1.02, <i>p</i> < 0.001), and renal events (RR 1.01, <i>p</i> < 0.001), respectively. <b><i>Conclusions:</i></b> There may be positive dose-response predictive effects of FGF23 on all-cause mortality, CVD, and renal events in patients with CKD.</p

Supplementary Material for: Tissue inhibitor metalloproteinase-2 (TIMP-2) • IGF-binding protein 7 (IGFBP7) for the prediction of acute kidney injury following cardiac surgery

Introduction: Cardiac surgery-associated acute kidney injury (CSA-AKI) is a common complication associated with increased morbidity and mortality. Tissue inhibitor metalloproteinases-2 • insulin-like growth factor-binding protein 7 (TIMP-2•IGFBP7) determines tubular stress markers, which may occur prior to tubular damage. Previous studies on the use of TIMP-2•IGFBP7 for the prediction of CSA-AKI showed divergent results. Therefore, this study aimed to explore the predictive value of TIMP-2•IGFBP7 measurements for the early detection of acute kidney injury (AKI) and short-term adverse outcomes after cardiac surgery. Methods: In the prospective cohort study, blood and urine samples were collected 6–12 h after cardiac surgery. Blood samples to monitor serum creatinine levels were additionally extracted from days 1 to 7. AKI was defined based on the KDIGO consensus guidelines. AKI within 7 days following surgery was the primary outcome. The initiation of renal replacement therapy, in intensive care unit mortality, and the combination of both were secondary outcomes. Results: A total of 557 patients were enrolled, 134 (24.06%) of them developed AKI and 33 (5.9%) had moderate or severe AKI. AKI developed more frequently in elderly patients with diabetes or with higher baseline serum creatinine levels. Patients with AKI had higher EuroSCORE II, Cleveland clinical score, and simplified renal index than those without AKI. Urinary TIMP-2•IGFBP7 was significantly higher in patients with AKI. The area under the curve was 0.66 in predicting all AKI and 0.70 in predicting stages 2 and 3 AKI. The resulting sensitivity and specificity were 44.0% and 83.9%, respectively, for a calculated threshold TIMP-2•IGFBP7 value of 0.265 (ng/ml)2/1,000. The TIMP-2•IGFBP7 values, simplified renal index (SRI) Score and age were significantly associated with AKI within 7 days postoperatively. A total of 33 patients reached the composite endpoint, the percentage of patients who reached the composite end-point in the TIMP-2•IGFBP7 of >0.265 (ng/ml)2/1,000 group was significantly higher than that of ≤0.265 (ng/ml)2/1,000 group. Conclusions: Postoperative implementation of TIMP-2•IGFBP7 improved prediction of CSA-AKI and may aid in identifying patients at risk of short-term adverse outcomes. We identified an ideal calculated cutoff value of 0.265 (ng/ml))2/1,000 for the prediction of CSA-AKI among all AKI patients

Supplementary Material for: Fibroblast Growth Factor 23 Predicts All-Cause Mortality in a Dose-Response Fashion in Pre-Dialysis Patients with Chronic Kidney Disease

<p><b><i>Background:</i></b> Quantitative dose-response associations between fibroblast growth factor 23 (FGF23) and risks of mortality, cardiovascular disease (CVD), and renal events in chronic kidney disease (CKD) are not known. This study aimed to summarize and quantify the predictive effects of FGF23 among the pre-dialysis CKD stages 1-5 population. <b><i>Methods:</i></b> Data sources included PubMed, EMBASE, and Web of Science. Prospective cohort studies assessing the associations between FGF23 and all-cause mortality, CVD, and renal events in CKD patients were selected. Summary risk ratios (RRs) and 95% confidence intervals (CIs) were calculated using the random-effects model. The composite higher or the highest level in FGF23 categories of each study was considered the high level. The reference level was regarded as the low level in the overall analysis. The restricted cubic spline model was used to estimate dose-response associations. <b><i>Results:</i></b> Fifteen prospective cohort studies centered around 15,355 subjects were analyzed. A high FGF23 level was associated with increased risks of all-cause mortality (RR 1.46, 95% CI 1.38-1.55, <i>p</i> < 0.001), CVD (RR 1.37, 95% CI 1.15-1.63, <i>p</i> < 0.001), and renal events (RR 1.31, 95% CI 1.07-1.59, <i>p </i>= 0.008), respectively. There was a positive, nonlinear, dose-response relationship between FGF23 and all-cause mortality. The reference level in dose-response analysis was defined as 51 RU/mL of c-terminal FGF23. We then calculated RRs for increments of 20 RU/mL, which was associated with increased risks of mortality (RR 1.04, 95% CI 1.00-1.07, <i>p</i> = 0.038), CVD (RR 1.02, <i>p</i> < 0.001), and renal events (RR 1.01, <i>p</i> < 0.001), respectively. <b><i>Conclusions:</i></b> There may be positive dose-response predictive effects of FGF23 on all-cause mortality, CVD, and renal events in patients with CKD.</p

Supplementary Material for: Heterologous Expression of Spinosyn Biosynthetic Gene Cluster in Streptomyces Species Is Dependent on the Expression of Rhamnose Biosynthesis Genes

<p>Spinosyns are a group of macrolide insecticides produced by <i>Saccharopolyspora spinosa</i>. Although <i>S. spinosa</i> can be used for industrial-scale production of spinosyns, this might suffer from several limitations, mainly related to its long growth cycle, low fermentation biomass, and inefficient utilization of starch. It is crucial to generate a robust strain for further spinosyn production and the development of spinosyn derivatives. A BAC vector, containing the whole biosynthetic gene cluster for spinosyn (74 kb) and the elements required for conjugal transfer and site-specific integration, was introduced into different <i>Streptomyces</i> hosts in order to obtain heterologous spinosyn-producing strains. The exconjugants of different <i>Streptomyces</i> strains did not show spinosyn production unless the rhamnose biosynthesis genes from <i>S. spinosa</i> genomic DNA were present and expressed under the control of a strong constitutive <i>ermE</i>*<i>p</i> promoter. Using this heterologous expression system resulted in yields of 1 μg/mL and 1.5 μg/mL spinosyns in <i>Streptomyces coelicolor</i> and <i>Streptomyces lividans</i>, respectively. This report demonstrates spinosyn production in 2 <i>Streptomyces</i> strains and stresses the essential role of rhamnose in this process. This work also provides a potential alternative route for producing spinosyn analogs by means of genetic manipulation in the heterologous hosts.</p

Supplementary Material for: Neohesperidin Exerts Lipid-Regulating Effects in vitro and in vivo via Fibroblast Growth Factor 21 and AMP-Activated Protein Kinase/Sirtuin Type 1/Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1α Signaling Axis

The purpose of this study is to prove the lipid-regulating effects of neohesperidin (NHP) and explore the potential mechanisms related to fibroblast growth factor 21 (FGF21) and AMP-activated protein kinase (AMPK). Free fatty acids (FFAs)-induced lipid-accumulated HepG2 cells, acutely egg yolk-induced dyslipidemia and chronically diet-induced obese (DIO) model mice were treated with NHP. Biochemical analyses were carried out to determine the lipid profiles. Western blotting and real-time PCR were employed to analyze FGF21, AMPK and the related proteins or mRNA expressions. Body weight and food intake were measured in DIO mice. siRNA or inhibitors of FGF21 or AMPK were utilized in further study. NHP showed potent hypolipidemic effect in HepG2 cells loaded with FFAs and reversed the pathological changes of lipid in the acute or chronic dyslipidemia mouse model. It obviously improved the lipid profiles in plasma, liver and gastrocnemius muscles in DIO mice, and led to a significant body weight loss. Simultaneously, FGF21 protein expression or secretion, and AMPK/sirtuin type 1 (SIRT1)/peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) axis or related molecules, was improved by NHP in HepG2 cells and/or DIO mice. Furthermore, the siRNA or inhibitor targeting FGF21 or AMPK rejected the triglyceride-lowering effect of NHP. In conclusion, NHP regulates lipid metabolism in vivo and in vitro via FGF21 and AMPK/SIRT1/PGC-1α signaling axis

Supplementary Material for: Breast Milk Polyamines and Microbiota Interactions: Impact of Mode of Delivery and Geographical Location

<p><b><i>Background/Aims:</i></b> The aim of the present study was to identify and quantify the polyamine levels in human milk obtained from different countries and through different modes of delivery, and to investigate their association with breast milk microbes. <b><i>Methods:</i></b> Mature breast milk samples were obtained from 78 healthy mothers after 1 month of lactation from 4 different geographical locations: Finland, Spain (Europe); South Africa (Africa); and China (Asia). Polyamines were determined using HPLC after dansyl derivatization and milk microbiota was obtained by 16S rRNA gene sequencing. <b><i>Results:</i></b> The mean values of polyamines in breast milk were 70.0, 424.2, and 610.0 nmol/dL for putrescine, spermidine and spermine, respectively, and 1,170.9 nmol/dL of total polyamines. The levels of putrescine were significantly higher in Spain (<i>p < </i>0.05) and spermidine levels were significantly higher in Finland (<i>p < </i>0.05) compared with other countries. Cesarean delivery had an impact on polyamine levels and it was related to an increase in the putrescine concentration being significant in Spanish samples (<i>p < </i>0.01). Furthermore, putrescine levels were correlated positively with Gammaproteobacteria (<i>r</i> = 0.46, <i>p < </i>0.001), especially with <i>Pseudomonas fragi </i>(<i>r</i> = 0.40, <i>p < </i>0.001). <b><i>Conclusions:</i></b> The results demonstrate significant effect of geographical variations in human milk polyamine concentrations, being correlated with human milk microbiota composition. These differences may have an impact on infant development during lactation.</p