Supplementary Material for: Contribution of IVIM to Conventional Dynamic Contrast-Enhanced and Diffusion-Weighted MRI in Differentiating Benign from Malignant Breast Masses

<p><b><i>Background:</i></b> The aim of this study was to determine whether the indicators obtained from intravoxel incoherent motion (IVIM) imaging can improve the characterization of benign and malignant breast masses compared with conventional dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and diffusion-weighted magnetic resonance imaging (DW-MRI). <b><i>Patients and Methods:</i></b> This study included 23 benign and 31 malignant breast masses of 48 patients. Main indicators were initial enhancement ratio (IER), time-signal intensity curve (TIC), apparent diffusion coefficient (ADC), tissue diffusivity (D), pseudodiffusivity (D*), and perfusion fraction (f). The discriminative abilities of the different models were compared by means of receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) analysis. <b><i>Results:</i></b> D had the highest AUC (0.980), sensitivity (93.55%), specificity (100%), and diagnostic accuracy (96.36%). Both D and TIC could provide the independent predicted features for malignant breast masses. The combination of D and TIC had an AUC of up to 0.990. <b><i>Conclusion:</i></b> D of IVIM can effectively complement existing conventional DCE-MRI and DW-MRI in differentiating malignant from benign breast masses. IVIM combined with DCE-MRI is a robust means of evaluating breast masses.</p

Supplementary Material for: Activation of TGR5 increases urine concentration by inducing AQP2 and AQP3 expression in renal medullary collecting ducts

Introduction: G protein-coupled bile acid receptor (TGR5), the first G protein-coupled receptor for bile acids identified, is capable of activating a variety of intracellular signaling pathways after interacting with bile acids. TGR5 plays an important role in multiple physiological processes and is considered to be a potential target for the treatment of various metabolic diseases including type 2 diabetes. Evidence has been emerged that genetically deletion of TGR5 results in an increase in basal urine output, suggesting that it may play a critical role in renal water and salt reabsorption. The present study aims to elucidate the effect and mechanism of TGR5 activation on urine concentration. Methods：Mice were treated with TGR5 agonists (LCA and INT-777) for 3 days. The 24-hour urine of mice was collected and analyzed for urine biochemical parameters. The mRNA expressions were detected by real-time PCR, and the protein expressions were detected by western blot. Immunohistochemistry (IHC) and immunofluorescence (IF) were performed to examine the cellular location of proteins. The primary cultured medullary collecting duct cells were pretreated with H89 (a PKA inhibitor) for 1h, followed by 12-hour treatment of LCA and INT-777. Luciferase reporter assays was used to detect the effect of CREB on gene transcription of AQPs. Gel electrophoretic mobility shift assays were used to analyze DNA–protein interactions. Results: Treatment of mice with the TGR5 agonist LCA and INT-777 markedly reduced urine output and increased urine osmolality, accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation. In cultured primary medullary collecting duct cells, LCA and INT-777 dose-dependently upregulated AQP2 and AQP3 expression in an cAMP/PKA-dependent manner. Mechanistically, both AQP2 and AQP3 gene promoter contains a putative CREB binding site, which can be bound and activated by CREB as assessed by both gene promoter-driven luciferase and gel shift assays. Conclusion: Collectively, our findings demonstrate that activation of TGR5 can promote urine concentration by upregulation of AQP2 and AQP3 expression in renal collecting ducts. TGR5 may represent an attractive target for the treatment of patients with urine concentration defect

Supplementary Material for: A Methyl-Deficient Diet Modifies Early B Cell Development

A functional methyl group donor is essential for the epigenetic regulation of all biological events due to the importance of DNA methylation and histone methylation as an epigenetic marker. However, the epigenetic alterations in the immune system due to methyl donor deficiency are not well known. In this study, we tried to address this question by studying the lymphocyte development and DNA methylation changes caused by a methyl-deficient diet (MDD). We fed one group of C57BL/6J mice with a methyl-sufficient diet (MSD) and the other group with an MDD for 5 months. Flow cytometry analyses of their immune systems showed a decrease in B220+ IgM+ (immature B) cells and an increase in B220+ IgM– (pro/pre-B) cells in the bone marrow of mice fed an MDD. By means of an in vitro OP9 coculture system, we recognized that this B220+ IgM– cell fraction from the MDD has an intrinsic developmental defect. When we quantitatively measured the mRNA expression levels of transcription factors and recombination machinery related to B cell development in the B220+ IgM– cell fraction of their bone marrow, we found that <i>ADA, EBF1, DNTT </i>and <i>Pax5 </i>mRNA expression levels were significantly downregulated in mice fed with an MDD. In addition, there was a drastic decrease in histone methylation profile H3K4me3 in the <i>Pax5 </i>and <i>EBF1 </i>promoters in these B220+ IgM– B cells. However, CpG-DNA methylation profiles had not changed and this revealed that these two promoters are demethylated even under an MSD condition. We also found changed expression levels of the <i>Polycomb </i>group genes <i>(mel18</i>, <i>bmi1</i>, <i>Pc1</i>, <i>Pc2</i>, <i>Ring1A</i>, <i>Ring1B</i>, <i>Ph1)</i> on semi-quantitative RT-PCR. These results indicate that under an MDD condition, early B cell development in bone marrow is easily affected by epigenetic alterations

Supplementary Material for: Corticosteroid for IgA Nephropathy: Are They Really Therapeutic?

<b><i>Background:</i></b> IgA nephropathy (IgAN) is a common chronic glomerular disease that, in most patients, slowly progresses to end-stage kidney disease. The therapy with corticosteroid in IgAN is still a worldwide problem that is confusing the clinicians. <b><i>Methods:</i></b> MEDLINE, EMBASE, the Cochrane Library, and article reference lists were searched for randomized controlled trials (RCTs) that compared corticosteroids with placebo and any other non-immunosuppressive agents in treating IgAN. Twelve RCTs involving 1,057 patients were included. <b><i>Results:</i></b> Overall, we found that steroids had statistically significant effects in preventing the decline in renal function (relative risk 0.42, 95% CI 0.25–0.71, <i>p</i> < 0.001) and reducing proteinuria (SMD: –0.58 g/day, 95% CI –0.80 to –0.36 g/day) in patients with IgAN. The association between glucocorticoid and risk of kidney outcome was not modified by steroids’ type (prednisone or methylprednisone), dose (≤30 or > 30 mg/day), duration (≤8 or > 8 months), or serum creatinine (< 1.10 or ≥1.10 mg/dL). But steroids increased the risk of side effects such as gastrointestinal and endocrinium symptoms. <b><i>Conclusion:</i></b> This study provides the clear beneficial effects of the steroids therapy on the kidney function and proteinuria, although it should be used with caution

Supplementary Material for: MicroRNA-9-1 Attenuates Influenza A Virus Replication via Targeting Tankyrase 1

An unstable influenza genome leads to the virus resistance to antiviral drugs that target viral proteins. Thus, identification of host factors essential for virus replication may pave the way to develop novel antiviral therapies. In this study, we investigated the roles of the poly(ADP-ribose) polymerase enzyme, tankyrase1 (TNKS1) and the endogenous small noncoding RNA, miR-9-1 in influenza A virus (IAV) infection. Increased expression of TNKS1 was observed in IAV-infected human lung epithelial cells and mouse lungs. TNKS1 knock-down by RNA interference repressed influenza viral replication. A screen using TNKS1 3’-untranslation region (3’-UTR) reporter assays and predicted microRNAs identified that miR-9-1 targeted TNKS1. Overexpression of miR-9-1 reduced influenza viral replication in lung epithelial cells as measured by viral mRNA and protein levels as well as virus production. miR-9-1 induced type I interferon production and enhanced the phosphorylation of STAT1 in cell culture. The ectopic expression of miR-9-1 in the lungs of mice by using an adenoviral viral vector enhanced type I interferon response, inhibited viral replication and reduced susceptibility to IAV infection. Our results indicate that miR-9-1 is an antiinfluenza microRNA that targeting TNKS1 and enhanced cellular antiviral state

Supplementary Material for: Baicalein, a Natural Anti-Cancer Compound, Alters MicroRNA Expression Profiles in Bel-7402 Human Hepatocellular Carcinoma Cells

<i>Background/Aims:</i> Baicalein has been shown to possess significant anti-hepatoma activity by inhibiting cell proliferation. Whether the anti-proliferative effect of baicalein is related to its modulation of miRNA expression in hepatocellular carcinoma (HCC) is still unknown. <i>Methods:</i> The anti-proliferative effects of baicalein on HCC cell line Bel-7402 was assessed by detecting the proliferation activity, cell cycle distribution, expression changes of p21/CDKN1A, P27/CDKN1B, total Akt and phosphoryted AKT. Microarray analysis was conducted to determine the miRNA expression profiles in baicalein-treated or untreated Bel-7402 cells and then validated by qRT-PCR in two HCC cell lines (Bel-7402 and Hep3B). The gain-of-function of miR-3127-5p was performed by detecting anti-proliferative effects after transfecting miRNA mimics in cells. Finally, the expression level of miR-3127-5p in different HCC cell lines was determined by qRT-PCR. <i>Results:</i> Baicalein was able to inhibit the proliferation of Bel-7402 cells by inducing cell cycle arrest at the S and G2/M phase via up-regulating the expression of p21/CDKN1A and P27/CDKN1B and suppressing the PI3K/Akt pathway. Baicalein could alter the miRNA expression profiles in Bel-7402 cells. Putative target genes for differentially expressed miRNAs could be enriched in terms of cell proliferation regulation, cell cycle arrest and were mainly involved in MAPK, PI3K-Akt, Wnt, Hippo and mTOR signaling pathways. MiR- 3127-5p, one of up-regulated miRNAs, exhibits low expression level in several HCC cell lines and its overexpression could inhibit cell growth of Bel-7402 and Hep3B cell lines by inducing S phase arrest by up-regulating the expression of p21and P27 and repressing the PI3K/Akt pathway. <i>Conclusions:</i> Modulation of miRNA expression may be an important mechanism underlying the anti-hepatoma effects of baicalein

Supplementary Material for: Identification of 8 Novel Mutations in Nephrogenesis-Related Genes in Chinese Han Patients with Unilateral Renal Agenesis

<p><b><i>Background:</i></b> Few genetic studies have focused on unilateral renal agenesis (URA), which is a disorder with insidious clinical manifestations and a tendency to result in renal failure. We aimed to detect pathogenic mutations in nephrogenesis-related genes, which were identified by a literature review conducted among a large cohort of Chinese Han patients with URA. <b><i>Methods:</i></b> Totally, 86 unrelated URA patients were included. All URA patients were diagnosed by employing radiological methods. Patients with a solitary kidney owing to nephrectomy or renal atrophy due to secondary factors were excluded. Nine (10.5%) patients had a family history of abnormal nephrogenesis. Fifteen (17.4%) had other malformations in the urogenital system. All coding exons and adjacent intron regions of 25 genes were analyzed using next-generation sequencing and validated by Sanger sequencing and 100 ethnically matched healthy controls. <b><i>Results:</i></b> Ten conserved mutations (9 missense mutations and 1 deletion mutation) were identified in <i>SALL1, EYA1, RET, HNF1B, DSTYK, WNT4,</i> and<i> SIX5</i>. All mutations were novel or rare (frequency <0.1%) in the public databases and absent from the 100 healthy controls. Nine patients carried mutations in candidate genes. Most of the patients carried one single heterozygous mutation, except for 2, who respectively carried compound heterozygous mutations and 2 single heterozygous mutations. In addition, 2 patients shared the same mutation in <i>DSTYK</i>. <b><i>Conclusion:</i></b> A total of 10.5% of our URA cases could be explained by mutations in our candidate genes. The mutations in nephrogenesis-related genes in the Chinese Han patients with URA had a decentralized distribution without any hotspot mutations.</p