Supplementary Material for: Markers CD40, VEGF, AKT, PI3K, and S100 Correlate with Tumor Stage in Gastric Cancer

<b><i>Background: </i></b>To better understand gastric cancer occurrence and prognosis, we explored the expression of molecules in the CD40 pathway and their correlation with gastric cancer prognosis. <b><i>Patients and Methods: </i></b>We measured the expression of CD40, VEGF, AKT, PI3K, and S100 in gastric cancer tissues and adjacent normal tissues from 128 patients by immunohistochemistry. <b><i>Results: </i></b>The expression of CD40, VEGF, AKT, and PI3K were significantly higher in tumor tissue than in normal tissue, while S100 expression in dendritic cells (DC) was lower. Expression of CD40, VEGF, AKT, and PI3K significantly increased with T stage, while S100 expression decreased with T stage. Lymph node metastasis was associated with low or negative S100 expression. PI3K expression increased with clinical stage, while negative S100 expression was associated with higher clinical stages. Multivariate analysis did not indicate significant associations between any of these markers and recurrence or mortality. <b><i>Conclusion: </i></b>The correlation between T stage of gastric cancer and the higher expression of CD40, VEGF, AKT, and PI3K, along with lower S100 expression in DC, may provide insights into future targets for more effective immunotherapy for cancer

Supplementary Material for: Joint Analysis for Integrating Two Related Studies of Different Data Types and Different Study Designs Using Hierarchical Modeling Approaches

<b><i>Background:</i></b> A chronic disease such as asthma is the result of a complex sequence of biological interactions involving multiple genes and pathways in response to a multitude of environmental exposures. However, methods to model jointly all factors are still evolving. Some of the current challenges include how to integrate knowledge from different data types and different disciplines, as well as how to utilize relevant external information such as gene annotation to identify novel disease genes and gene-environment inter-actions. <b><i>Methods:</i></b> Using a Bayesian hierarchical modeling framework, we developed two alternative methods for joint analysis of an epidemiologic study of a disease endpoint and an experimental study of intermediate phenotypes, while incorporating external information. <b><i>Results:</i></b> Our simulation studies demonstrated superior performance of the proposed hierarchical models compared to separate analysis with the standard single-level regression modeling approach. The combined analyses of the Southern California Children's Health Study and challenge study data suggest that these joint analytical methods detected more significant genetic main and gene-environment interaction effects than the conventional analysis. <b><i>Conclusion:</i></b> The proposed prior framework is very flexible and can be generalized for an integrative analysis of diverse sources of relevant biological data

Supplementary Material for: Febuxostat Prevents Renal Interstitial Fibrosis by the Activation of BMP-7 Signaling and Inhibition of USAG-1 Expression in Rats

<b><i>Background:</i></b> Renal interstitial fibrosis (RIF) is a common pathology associated with end-stage renal diseases. The activation of bone morphogenetic protein-7 (BMP-7)-Smad1/5/8 pathway seems to alleviate RIF. Uterine sensitization-associated gene-1 (USAG-1), a kidney-specific BMPs antagonist, is associated with the development and prognosis of several renal diseases. Febuxostat is a xanthine oxidase inhibitor that can attenuate the renal dysfunction of patients. The purpose of this study was to investigate the effects of febuxostat on renal fibrosis and to clarify the mechanisms underlying these effects. <b><i>Methods:</i></b> Rats were randomly divided into 6 groups termed a sham-operated group, a unilateral ureteral obstruction (UUO) group, 3 doses of febuxostat groups (low, intermediate and high doses) and a sham group treated with high-dose febuxostat. After 14 days, renal function, relative kidney weight, accumulation of glycogen and collagens were examined by different methods. Expression of α-SMA, transforming growth factor-β1 (TGF-β1), BMP-7 and USAG-1 was detected by western blotting and RT-PCR, respectively. The phosphorylation level of Smad1/5/8 was also quantified by western blotting. <b><i>Results:</i></b> The renal function was declined, and large amounts of glycogen and collagens were deposited in the kidneys of UUO rats compared with the rats in the sham group. Besides, expression of α-SMA and USAG-1 in these kidneys was elevated, and the TGF-β1 was also activated, while the BMP-7-Smad1/5/8 pathway was inhibited. Febuxostat reversed the changes stated earlier, exhibiting protective effects on RIF induced by UUO. <b><i>Conclusion:</i></b> Febuxostat was able to attenuate RIF caused by UUO, which was associated with the activation of BMP-7-Smad1/5/8 pathway and the inhibition of USAG-1 expression in the kidneys of UUO rats

Supplementary Material for: Serum Human Beta-Defensin-2 Is a Possible Biomarker for Monitoring Response to JAK Inhibitor in Psoriasis Patients

<strong><em>Aims:</em></strong> To analyse the correlation between serum human beta-defensin-2 (hBD-2) levels and response to JAK inhibitor in psoriasis. <b><i>Methods:</i></b> We evaluated the psoriasis area and severity index (PASI) and serum hBD-2 levels of 18 psoriasis patients randomized to receive placebo or tofacitinib 5 or 10 mg b.i.d. at baseline, week 8, and week 16. Serum hBD-2 levels were measured by enzyme-linked immunosorbent assay (ELISA). <b><i>Results:</i></b> The PASI achieved a dramatic reduction after tofacitinib 5 or 10 mg b.i.d. treatment for 16 weeks (<i>p </i>< 0.05). Serum hBD-2 levels significantly decreased in patients treated with tofacitinib 10 mg b.i.d. compared with baseline and the placebo-treated patients (<i>p </i>< 0.05). A significant correlation was found between hBD-2 levels and PASI (<i>r</i> = 0.52, <i>p </i>< 0.01). A serum hBD-2 level of 1,255.45 pg/mL was a cut-off between mild and moderate-to-severe psoriasis in ROC analysis. <b><i>Conclusions:</i></b> Serum hBD-2 level might be a possible biomarker for monitoring psoriasis treatment response and differentiating mild from moderate-to-severe psoriasis

Erratum: Urinary Pigment Epithelium-Derived Factor as a Marker of Diabetic Nephropathy

<i>Background:</i> Pigment epithelium-derived factor (PEDF), a serine protease inhibitor, regulates extracellular matrix production in the kidney. We sought the association between urinary PEDF (uPEDF) and development of nephropathy among patients with type 2 diabetes (T2DM). <i>Methods:</i> Two human studies were performed in which uPEDF was determined by ELISA. These studies included (1) a cross-sectional study of T2DM (n = 228) and healthy controls (n = 49) and (2) a longitudinal study of hypertensive T2DM with microalbuminuria (MA; n = 42) treated with irbesartan for 6 months. An animal study was performed in which PEDF was measured in the kidney and urine samples of control rats, rats rendered diabetic with streptozotocin that were also fed a high-fat diet, and diabetic rats treated with irbesartan for 3 months. <i>Results:</i> Cross-sectional study: compared to controls, uPEDF was significantly higher in patients with diabetic nephropathy. uPEDF independently correlated with MA. In the MA group, uPEDF in patients with diabetic retinopathy was significantly higher than that in patients without diabetic retinopathy. Longitudinal study: irbesartan significantly decreased uPEDF in T2DM with MA. Animal study: in diabetic rats, increased PEDF was observed in both the urine and kidney samples. uPEDF showed a significant correlation with the expression of PEDF in the kidney. Irbesartan could significantly decrease the PEDF expression in the kidneys of diabetic rats as well as uPEDF. <i>Conclusion:</i> uPEDF may serve as a novel marker for screening for nephropathy among patients with T2DM and monitoring the response to therapy

Supplementary Material for: Roles of HDAC2 and HDAC8 in Cardiac Remodeling in Renovascular Hypertensive Rats and the Effects of Valproic Acid Sodium

<p>Recent studies indicate that histone deacetylases (HDACs) activity is associated with the development and progression of cardiac hypertrophy. In this study, we investigated the effects of a HDACs inhibitor, valproic acid sodium (VPA), on cardiac remodeling and the differential expression of HDACs in left ventricles (LVs) of renovascular hypertensive rats. Renovascular hypertension was induced in rats by the two-kidney two-clip (2K2C) method. Cardiac remodeling, heart function and the differential expression of HDACs were examined at different weeks after 2K2C operation. The effects of VPA on cardiac remodeling, the expressions of HDACs, transforming growth factor-beta 1 (TGF-β1) and connective tissue growth factor (CTGF) in LV were investigated. The expressions of atrial natriuretic factor, β-myosin heavy chain, HDAC2 and HDAC8 increased in LV of 2K2C rats at 4, 8, 12 weeks after operation. Cardiac dysfunction, cardiac hypertrophy and fibrosis were markedly attenuated by VPA treatment in 2K2C rats. Further studies revealed that VPA inhibited the expressions of HDAC2, HDAC8, TGF-β1 and CTGF in LV of 2K2C rats. In summary, these data indicate that HDAC2 and HDAC8 play a key role in cardiac remodeling in renovascular hypertensive rats and that VPA attenuates hypertension and cardiac remodeling. The effect of VPA is possibly exerted via decreasing HDAC2, HDAC8, TGF-β1 and CTGF expressions in LV of 2K2C rats.</p

Supplementary Material for: Depression trajectories, genetic risk, and cognitive performance in older adults: multilevel model with a 10-year longitudinal cohort

Background: Cognitive performance in older ages is strongly affected by individuals’ genetic predispositions. We investigated whether depression trajectories were associated with subsequent cognitive performance independent of participants’ genetic predispositions. Methods: Participants from the Health and Retirement Study with European ancestry and aged over 50 were included in the analysis. Depressive symptoms were evaluated using the Center for Epidemiologic Studies Depression Scale, and the 6-year trajectories were fitted using latent class linear mixed models. Linear multilevel regression was applied to model the associations between depression trajectory and subsequent cognitive performance. Stratified analyses were performed to investigate these associations in participants with different genetic predispositions of cognitive performance and APOE ε4 allelic status. Results: A total of 5942 eligible participants were included in the study. Four depression trajectories were identified. Compared with the non-depression trajectory, all other depression trajectories were associated with worse cognitive performance (β [95% CI]: mild-depression trajectory: -0.20 [-0.56, -0.06], p = 0.007; worsening-depression trajectory: -0.29 [-0.47, -0.12], p = 0.001; persistent-depression trajectory: -0.32 [-0.53, -0.13], p = 0.001). Although these associations were independent of participants' inherent genetic risk, the participants with a low polygenetic score for cognitive performance were more likely to have an enhanced association between depression trajectories and cognitive decline. Similar relationships were also found in APOE ε4 noncarriers. Conclusion: Among older participants with European ancestry, even a mild-depression trajectory was associated with worse cognitive performance. Early intervention in participants with any degree of depression might benefit regarding preventing cognitive performance decline

Supplementary Material for: 5’-AMP-Activated Protein Kinase Regulates Goat Sperm Functions via Energy Metabolism <b><i>In Vitro</i></b>

<b><i>Background/Aims:</i></b> ATP is essential for mammalian sperm to survive and maintain fertilizing capacity. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. The aims of the present study were to explore the localization of AMPK in goat sperm and to investigate whether and how AMPK regulates sperm functions <i>in vitro</i>. <b><i>Methods:</i></b> Sperm were treated with AMPK modulators (AICAR, metformin and Compound C) during incubation. Sperm motility was assessed with a computer-assisted spermatozoa analysis system (CASA). Membrane integrity, acrosome reaction and mitochondrial membrane potentials were detected by SYBR-14/PI, FITC-PNA and JC-1 staining, respectively. And the lactate content, ATP content, AMPK activity, activity of pyruvate kinase (PK) and lactate dehydrogenase (LDH) were also measured with the commercial assay kits. Immunofluorescence staining was used to analyze the distribution of PK, LDH, AMPK and phospho-Thr172-AMPK in sperm. The role of AMPK was further studied during induction of capacitation and acrosome reaction. <b><i>Results:</i></b> We found that AMPKα was localized in the entire acrosomal region, the midpiece and the flagellum, while the phospho-Thr172-AMPK was distributed in the head, the midpiece and flagellum. Activation of AMPK by AICAR and metformin significantly improved sperm motility, membrane integrity and acrosome reaction, largely maintained sperm mitochondrial membrane potentials, lactate content and ATP content, and enhanced the activity of AMPK, PK and LDH, whereas inhibition by Compound C triggered the converse effects. Moreover, PK was localized in the acrosomal area and the midpiece, while LDH was distributed in the tail. Induction of capacitation and acrosome reaction led to AMPK phosphorylation. AMPK phosphorylation regulated the activity of energetic enzymes. <b><i>Conclusion:</i></b> This study for the first time provides evidence that AMPK governs goat sperm functions through energy metabolism <i>in vitro</i>. This finding will help to improve assisted reproductive techniques in goats and the other species

Supplementary Material for: Hydroxysafflor Yellow A Promotes Angiogenesis via the Angiopoietin 1/ Tie-2 Signaling Pathway

<i>Background:</i> The flowers of <i>Carthamus tinctorius</i> L. are widely used in traditional Chinese medicine to treat cerebrovascular and cardiovascular diseases. Hydroxysafflor yellow A (HSYA), the main constituent of <i>C. tinctorius</i> L. flowers, is known for its multiple biological activities. The present study investigated the effects of HSYA on angiogenesis in vitro and in a mouse hindlimb ischemia model. <i>Methods:</i>Using human umbilical vein endothelial cells (HUVEC) in vitro and a mouse hindlimb ischemia model in vivo, the angiogenic role of HSYA was evaluated. <i>Results:</i> HSYA significantly increased the capillary-like tube formation and migration of HUVEC. HSYA not only induced a rise in the expression of angiopoietin 1 and Tie-2 but it also increased phosphorylation of Tie-2, Akt, and extracellular signal-regulated kinase 1/2. Furthermore, an anti-Tie-2 neutralizing antibody significantly inhibited HSYA-induced HUVEC tube formation and migration. In vivo, the recovery of perfusion of ischemic hindlimb tissue after femoral artery interruption was significantly increased in HSYA-treated mice compared to vehicle controls. Consistent with these results, the arteriole and capillary densities in ischemic gastrocnemius muscles were significantly increased in HSYA-treated mice. <i>Conclusions:</i> These results indicate the potential utility of HSYA for the treatment of ischemic diseases

Supplementary Material for: Psychotic Symptoms and Attitudes toward Medication Mediate the Effect of Insight on Personal-Social Functions in Patients with Schizophrenia: One-Year Randomized Controlled Trial and Follow-Up

<b><i>Aims:</i></b> This study aimed to investigate the mediating pathway of 3 factors (psychotic symptoms, attitude toward medication, and cognitive processing speed) on the effect of insight on personal-social functioning in patients with schizophrenia. <b><i>Methods:</i></b> Chinese inpatients with schizophrenia (<i>n</i> = 168; mean age 18 ± 50 years) diagnosed according to the DSM-IV were randomly assigned to treatment with antipsychotic medication alone or combined treatment. Positive and Negative Syndrome Scale (PANSS), Drug Attitude Inventory (DAI), Assessment of Insight (SAI), and Social-Personal Performance Scale (PSPS) scores were evaluated at baseline and at 3, 6, and 12 months. Cognitive function was assessed at baseline. Multiple mediation analyses were conducted with baseline data, end point data, and changes-in-scale scores between baseline and the end point, respectively. <b><i>Results:</i></b> At baseline and at 12 months, only psychotic symptoms mediated the effect of insight on personal-social functioning. For changes-in-scale scores over the 12-month follow-up, in patients receiving treatment with medication alone, the effect of improved insight on improved personal-social function was mediated by psychotic symptoms only; in patients receiving a combined treatment, the effect of improved insight on improved personal-social functioning was mediated by both psychotic symptoms and attitudes toward medication, independently. <b><i>Conclusions:</i></b><i></i> The link between insight and personal-social functions is mainly mediated by psychotic symptoms. Psychosocial intervention improves the predicting effect of insight on personal-social function by improving both the attitude toward medication and psychotic symptoms independently