Supplementary Material for: Clinical Outcomes of Prolonged Antiplatelet Therapy after Percutaneous Coronary Intervention in Chronic Kidney Disease Patients: A Systematic Review and Meta-Analysis

Background: The optimal duration of dual antiplatelet therapy (DAPT) after percutaneous coronary intervention (PCI) is not established in chronic kidney disease (CKD) patients. Objective: In this meta-analysis, we evaluated the efficacy and safety of long duration DAPT compared with short duration DAPT in CKD patients after PCI. Methods: We searched PubMed, Cochrane, and Embase to identify studies assessing the effect of DAPT duration in CKD patients with PCI. Endpoints included all-cause mortality, major adverse cardiovascular events (MACE), death or myocardial infarction (MI), revascularization, and bleeding. Event rates were compared with a random-effects model expressed by odds ratio (OR) and 95% confidence interval (CI). Results: Six studies were included. CKD patients with extended DAPT duration were at a lower risk of mortality (OR 1.40, 95% CI: 1.11–1.77), MACE (OR 1.33, 95% CI: 1.17–1.51), mortality or MI (OR 1.24, 95% CI: 1.10–1.40), and stroke (OR 1.28, 95% CI: 1.05–1.56). However, there was no significant difference in revascularization and bleeding events between the two groups. Mortality was higher in patients with dialysis or drug-eluting stent comparing short- to long-term DAPT. Conclusions: Prolonged DAPT might decrease the risk of mortality, MACE, and stroke in patients with CKD without any significant difference in bleeding or revascularization. Additional studies are required to determine whether long-term DAPT could be considered for most CKD patients after PCI

Supplementary Material for: Safety of Tigecycline in Patients on Antithrombotic Therapy: A Single Center Retrospective Study

Introduction: To investigate the risk factors of tigecycline-induced hypofibrinogenemia, and to evaluate the safety of tigecycline with concomitant antithrombotic drugs. Methods: We performed a retrospective analysis of patients who received tigecycline for more than 3 days between January 2015 and June 2019. Clinical and laboratory data were collected including fibrinogen concertation, tigecycline dose, duration of treatment, disease severity, complete blood count, indicators of infection, liver and renal function. Risk factors of hypofibrinogenemia were analyzed by univariate and multivariate analysis. To evaluate the safety of tigecycline and concomitant antithrombotic drugs, bleeding events were assessed by comparing the decline in hemoglobin and the amount of red blood cell transfusion in patients with antithrombotic drugs and those without. Results: This study included a total of 68 cases, 20 of which experienced hypofibrinogenemia while receiving tigecycline treatment. Duration of treatment, cefoperazone/sulbactam combination therapy, and fibrinogen levels prior to initiation of tigecycline were risk factors associated with tigecycline-induced hypofibrinogenemia. There were 26 recorded bleeding incidents, 25 of which happened before the start of tigecycline. Antithrombotic and non-antithrombotic patients did not differ in their hemoglobin decline or need for red blood cell transfusions while taking tigecycline. Conclusion: A longer treatment duration, cefoperazone/sulbactam combination therapy, and a lower level of fibrinogen level before tigecycline were associated with an increased risk of tigecycline induced hypofibrinogenemia. A combination of antithrombotic drugs and tigecycline did not aggravate the bleeding events during tigecycline treatment

Supplementary Material for: Molecular Pathways and Functional Analysis of miRNA Expression Associated with Paclitaxel-Induced Apoptosis in Hepatocellular Carcinoma Cells

<b><i>Background:</i></b> We postulated that microRNAs (miRNAs) might be involved in hepatocellular carcinoma (HCC) targeted chemotherapy with paclitaxel. This study sought to generate a list of potential miRNA-based biomarkers and their potential targets to better understand the response to paclitaxel treatment in HCC. <b><i>Methods:</i></b> Cell viability proliferation assays were conducted to test the sensitivity of the HepG2 cells to paclitaxel. The morphological changes of apoptosis were assessed with 4′,6-diamidino-2-phenylindole staining. Differential expression patterns of miRNA in the HepG2 cells either treated or not treated were analyzed using miRNA microarrays. <b><i>Results:</i></b> The array experiments have identified 54 miRNAs whose basal expression levels differed by >2-fold and p < 0.05 between the two phenotypic groups. The data were validated by a quantitative real-time PCR of 8 selected miRNAs (miR-21, miR-1274a, miR-1260, miR-1290, miR-508-5p, miR-877, miR-1246, miR-183*). The PI3K/Akt, mitogen-activated protein kinase (MAPK), TGF-β, ErbB, p53, cell cycle, mammalian target of rapamycin, and Jak-STAT signaling pathways were involved in paclitaxel-induced apoptosis. <b><i>Conclusions:</i></b> The manipulation of one or more of these miRNAs could be an important approach for the improved management of paclitaxel therapy

Supplementary Material for: Secreted Expression of a Hyperthermophilic α-Amylase Gene from <b><i>Thermococcus</i></b> sp. HJ21 in <b><i>Bacillus subtilis</i></b>

The hyperthermophilic α-amylase from <i>Thermococcus</i> sp. HJ21 possesses unique traits (Ca²⁺-independent thermostability and optimal temperature of 95°C) that make it a great potential candidate for use in the food industry. However, this Archaea isolated from a deep-sea thermal vent requires strict control of culture conditions and produces only small amounts of α-amylase. To solve these problems, the α-amylase gene was cloned and expressed in <i>Bacillus subtilis</i>, which is an ideal food-grade host for heterologous protein expression. To express high levels of this α-amylase in <i>B. subtilis</i>, the promoters P_<i>grac</i>, P_<i>xylA</i>, P43, and P_<i>hag</i> were used to construct four different expression vectors for testing. The vector containing the P_<i>xylA</i> promoter was found to have the highest transcriptional activity and produce the highest amylase activity (19.6 U/ml). To test the secretion efficiency of signal peptides in <i>B. subtilis</i>, three signal peptides were cloned and fused to the α-amylase gene (lacking its native signal peptide). The optimal signal peptide was S_<i>amyQ</i>, with a secretion efficiency of approximately 90%. These results indicate that the promoter P_<i>xylA</i> and signal peptide S_<i>amyQ</i> tested in this study may be useful for the expression and secretion of archaeal proteins in <i>B. subtilis</i>

Supplementary Material for: Demineralized Dentin Matrix Induces Odontoblastic Differentiation of Dental Pulp Stem Cells

<br>The aim of this study was to investigate the effect of demineralized dentin matrix (DDM) on dental pulp stem cells (DPSCs) and the potential of complexes with DPSCs and DDM for mineralized tissue formation. Stem cells derived from the dental pulp of healthy pigs aged 18 months were isolated and cultured. DPSCs were incubated with alpha-minimum essential medium treated with DDM extract at 1 mg/ml (DDM1) or 10 mg/ml (DDM10). The concentrations of 3 growth factors in DDM extract was measured by enzyme-linked immunosorbent assay. Adhesion of DPSCs on DDM and hydroxyapatite-tricalcium phosphate (HA-TCP) surfaces was observed using scanning electron microscopy. Cell proliferation was evaluated with cell counting kit-8 and migration by Transwell migration assays. Odontoblastic differentiation was assessed by alkaline phosphatase (ALP) and alizarin red staining, ALP activity and real-time polymerase chain reaction analysis of markers of ALP, runt-related transcription factor 2, type I collagen, dentin matrix acidic phosphoprotein-1, osteonectin and dentin sialophosphoprotein (DSPP). Finally, DPSCs were combined with DDM and placed subcutaneously in nude mice for 12 weeks; DPSCs combined with HA-TCP and DDM alone served as controls. DDM could promote DPSC adhesion, migration and odontoblastic differentiation. Mineralized tissue formation was observed with the DPSC and DDM combination and the DPSC and HA-TCP combination. The mineralized tissue of the DPSC + DDM combination stained positive for DSPP, similar to the dentin tissue. These results indicate that DDM induces DPSC odontoblastic differentiation, suggesting applications for dentin regeneration

Supplementary Material for: Comparison between Fondaparinux and Low-Molecular-Weight Heparin in Patients with Acute Coronary Syndrome: A Meta-Analysis

<b><i>Objective:</i></b> A number of studies have evaluated the efficacy and safety of fondaparinux versus low-molecular-weight heparin (LMWH) in patients with acute coronary syndrome (ACS), but the findings were not consistent across these studies.<b><i> Methods:</i></b> Electronic databases and article references were searched for studies that assessed fondaparinux versus LMWH in ACS patients. <b><i>Results:</i></b> Six studies met the inclusion criteria. There was a lower risk of major adverse cardiac events (MACE) with fondaparinux-based regimens both in randomized controlled trials (RCT; risk ratio, RR: 0.91, p = 0.04) and observational studies (RR: 0.85, p < 0.0001). Mortality decreased in fondaparinux-treated patients in RCT (RR: 0.84, p = 0.02), but not in observational studies (RR: 1.44, p = 0.64). For the analysis of myocardial infarction (MI), recurrent ischemia and stroke, none of the studies showed significant results. In addition, fondaparinux lowered the risk of major bleeding in RCT (RR: 0.62, p < 0.0001) and observational studies (RR: 0.65, p < 0.0001). The net clinical outcome also favored fondaparinux over LMWH in RCT (RR: 0.82, p < 0.0001) and observational studies (RR: 0.84, p < 0.0001). <b><i>Conclusions:</i></b> Among ACS patients, a fondaparinux-based regimen presented advantages regarding MACE and major bleeding, and a net clinical benefit compared with LMWH, although the benefit is minimal regarding MACE. For death, MI, recurrent ischemia and stroke, fondaparinux has not shown significant benefits

Supplementary Material for: Association Study of Three Gene Polymorphisms Recently Identified by a Genome-Wide Association Study with Obesity-Related Phenotypes in Chinese Children

<p><b><i>Objective: </i></b>This study aimed to examine associations of three single-nucleotide polymorphisms (SNPs) with obesity-related phenotypes in Chinese children. These SNPs were identified by a recent genome-wide association (GWA) study among European children. Given that varied genetic backgrounds across different ethnicity may result in different association, it is necessary to study these associations in a different ethnic population. <b><i>Methods: </i></b>A total of 3,922 children, including 2,191 normal-weight, 873 overweight and 858 obese children, from three independent studies were included in the study. Logistic and linear regressions were performed, and meta-analyses were conducted to assess the associations between the SNPs and obesity-related phenotypes. <b><i>Results:</i></b> The pooled odds ratios of the A-allele of rs564343 in <i>PACS1 </i>for obesity and severe obesity were 1.180 (p = 0.03) and 1.312 (p = 0.004), respectively. We also found that rs564343 was nominally associated with BMI, BMI standard deviation score (BMI-SDS), waist circumference, and waist-to-height ratio (p < 0.05). <b><i>Conclusions:</i></b> We showed for the first time that the rs564343 in <i>PACS1</i> was associated with risk of severe obesity in a non-European population. This SNP was also found to be associated with common obesity and various obesity-related phenotypes in Chinese children, which had not been reported in the original study. The results demonstrated the value of conducting genetic researches in populations with different ethnicity.</p

Supplementary Material for: Advanced Glycation End Products-induced Activation of Keratinocytes: a Mechanism Underlying Cutaneous Immune Response in Psoriasis

Psoriasis is a common inflammatory skin disease, in which epidermal keratinocytes play a vital role in its pathogenesis via acting both as the responder and as the accelerator to the cutaneous psoriatic immune response. Advanced glycation end products (AGEs) are a class of proinflammatory metabolites that are commonly accumulating in cardiometabolic disorders. Recent studies have also observed increased level of AGEs in the serum and skin of psoriasis patients, but the role of AGEs in psoriatic inflammation has not been well investigated. In the present study, we initially detected abnormal accumulation of AGEs in epidermal keratinocytes of psoriatic lesion collected from psoriasis patients. Furthermore, AGEs promoted the proliferation of keratinocytes via up-regulated Keratin 17 (K17)-mediated p27KIP1 inhibition followed by accelerated cell cycle progression. More importantly, AGEs facilitated the production of interleukin-36 alpha (IL-36α) in keratinocytes, which could enhance T helper 17 (Th17) immune response. In addition, the induction of both K17 and IL-36α by AGEs in keratinocytes was dependent on the activation of signal transducer and activator of transcription 1/3 (STAT1/3) signaling pathways. At last, the effects of AGEs on keratinocytes were mediated by receptor for AGEs (RAGE). Taken together, these findings support that AGEs potentiate the innate immune function of keratinocytes, which contributes to the formation of psoriatic inflammation. Our study implicates AGEs as a potential pathogenic link between psoriasis and cardiometabolic comorbidities

Supplementary Material for: The consistency between registered acupuncture-moxibustion clinical studies and their published studies and update status of registered information

Background: Few studies have analyzed the consistency between registered acupuncture-moxibustion clinical studies and their published research results as well as their update status of registered information. Methods: We searched for acupuncture-moxibustion clinical studies that were registered at the World Health Organization International Clinical Trials Registry Platform between 2013 and 2015 and collected data regarding their characteristics and update status. Published results of these registered studies were identified and compared with registered information. Results: A total of 425 registered acupuncture-moxibustion clinical studies were included; 379 (89.2%) of them were interventional studies, and the remaining 46 (10.8%) were observational studies. Forty-six studies (10.8%) were found to have published results, and 51 published articles were identified. Overall, 73.2% (311) of registered studies did not update the research status in time; 46.6% (198) stopped updating before recruiting; 21.6% (92) stopped updating after recruiting; and 4.9% (21) stopped updating after completion. Regarding the 46 studies with published results, 29 (63.0%) were considered to be affected by reporting bias. These reporting biases predominantly involved the omission of some predefined outcomes or endpoints (16 studies), contradictions regarding descriptions of sample sizes (9 studies), discrepancies in treatment measurements or group distribution (7 studies), and inconsistent treatment durations (4 studies). When compared with other studies, significant and various reporting biases could also be commonly found in fields other than acupuncture-moxibustion. Conclusions: There were many discrepancies between registered information and published reports on acupuncture-moxibustion, which could also be commonly observed in other fields. Moreover, a large proportion of registered studies did not update their research status in time. Efforts should be made to improve the reporting quality and timely updates

Supplementary Material for: Gene Microarray Integrated with High-Throughput Proteomics for the Discovery of Transthyretin in Rhabdomyolysis-Induced Acute Kidney Injury

<b><i>Background/Aims:</i></b> Rhabdomyolysis, one of the leading causes of acute kidney injury (AKI), develops after trauma, drug toxicity, infections, burns, and physical exertion. The aim of this study was to investigate differences in gene and protein expression to elucidate the pathogenesis of rhabdomyolysis (RM)-induced AKI. <b><i>Methods:</i></b> In this study, we used glycerol induced renal injury as a model of RM-induced AKI. Affymetrix U133 plus 2.0 microarrays were used to perform gene microarray analysis. Isobaric tagging with related and absolute quantitation (iTRAQ) labeling mass spectrometry (MS) was applied to screen and identify differentially expressed proteins between RM-induced AKI and normal murine renal tissue. Verification experiments included immunohistochemistry (IHC), real-time PCR, Western blotting, and the measurement of ATP and ROS production. HK-2 cells were incubated <i>in vitro</i> with ferrous myoglobin and pcDNA-TTR, followed by assays to detect cell proliferation, ROS and apoptosis. <b><i>Results:</i></b> According to gene microarray and iTRAQ-MS analysis, we screened 17 common elements. After multiple analyses, we selected transthyretin (TTR) as our focus and investigated TTR in the kidney. Verification experiments with IHC confirmed differential expression levels of TTR proteins. Furthermore, Western blotting showed a stepwise decrease in TTR in AKI renal tissues. Cell-based experiments showed that overexpression of TTR could improve HK-2 cell viability and inhibit apoptosis. TTR reduced apoptosis by decreasing the accumulation of reactive oxygen species (ROS). <b><i>Conclusion:</i></b> This study reports a possible mechanism for RM-induced AKI and suggests that reductions in TTR could increase the generation of ROS and induce apoptosis. TTR may be a potentially valuable target for RM-induced AKI