Yuen Fei Wong

In vitro anti-fibrotic activities of herbal compounds and herbs

BACKGROUND We recently developed high-throughput assays of inflammation-independent anti-fibrotic activities based on TGF-beta1-induced total collagen accumulation and nodule formation in normal rat kidney fibroblasts. METHODS These assays were applied to examine the anti-fibrotic activities of 21 compounds isolated from plants used in Chinese medicine and methanol extracts of 12 Chinese herbs. Lactate dehydrogenase release assay and cell detachment index were used to monitor cytotoxicity. Changes in fibrogenic molecular markers were observed by reverse transcriptase quantitative polymerase chain reaction and high-content imaging analysis of immunofluorescence. RESULTS Three flavonoids (quercetin, baicalein and baicalin) and two non-flavonoids (salvianolic acid B and emodin) demonstrated anti-fibrotic activities in both total collagen accumulation and nodule formation assays. The remaining 16 compounds had little anti-fibrotic effect or were cytotoxic. The anti-fibrotic compounds suppressed collagen I expression at both mRNA and protein levels and also variably suppressed alpha-smooth muscle actin expression and bromodeoxyuridine incorporation. Methanol extracts of Scutellaria baicalensis Georgi, Salvia miltiorrhiza Bunge and Rheum palmatum L., which are rich sources of baicalein, baicalin, salvianolic acid B and emodin, respectively, also showed in vitro anti-fibrotic activities. CONCLUSIONS Five herbal compounds and three herbal extracts have in vitro anti-fibrotic activities. These data warrant further studies on these anti-fibrotic entities and suggest it a promising strategy to discover new anti-fibrotic drugs by screening more plant materials.

Maternal Plasma RNA Sequencing for Genome-Wide Transcriptomic Profiling and Identification of Pregnancy-Associated Transcripts

BACKGROUND Analysis of circulating RNA in the plasma of pregnant women has the potential to serve as a powerful tool for noninvasive prenatal testing and research. However, detection of circulating RNA in the plasma in an unbiased and high-throughput manner has been technically challenging. Therefore, only a limited number of circulating RNA species in maternal plasma have been validated as pregnancy- and placenta-specific biomarkers. METHODS We explored the use of massively parallel sequencing for plasma transcriptome profiling in first-, second-, and third-trimester pregnant women. Genotyping was performed for amniotic fluid, placental tissues, and maternal blood cells, with exome-enriched sequencing. RESULTS In the early pregnancy group comprising 1 first- and 1 second-trimester pregnancy cases, the fetal contribution to the RNA pool in maternal plasma was 3.70%. The relative proportion of fetal contribution was increased to 11.28% in the late pregnancy group comprising 2 third-trimester pregnancy cases. The placental biallelic expression pattern of PAPPA (pregnancy-associated plasma protein A, pappalysin 1), a known pregnancy-specific gene, and the monoallelic expression pattern of H19 [H19, imprinted maternally expressed transcript (non-protein coding)], an imprinted maternally expressed gene, were also detected in the maternal plasma. Furthermore, by direct examination of the maternal plasma transcriptomic profiles before and after delivery, we identified a panel of pregnancy-associated genes. CONCLUSIONS Plasma RNA sequencing provides a holistic view of the maternal plasma transcriptomic repertoire. This technology is potentially valuable for using circulating plasma nucleic acids for prenatal testing and research.

Endogenous Retinoic Acid Activity in Principal Cells and Intercalated Cells of Mouse Collecting Duct System

Background Retinoic acid is the bioactive derivative of vitamin A, which plays an indispensible role in kidney development by activating retinoic acid receptors. Although the location, concentration and roles of endogenous retinoic acid in post-natal kidneys are poorly defined, there is accumulating evidence linking post-natal vitamin A deficiency to impaired renal concentrating and acidifying capacity associated with increased susceptibility to urolithiasis, renal inflammation and scarring. The aim of this study is to examine the presence and the detailed localization of endogenous retinoic acid activity in neonatal, young and adult mouse kidneys, to establish a fundamental ground for further research into potential target genes, as well as physiological and pathophysiological roles of endogenous retinoic acid in the post-natal kidneys. Methodology/Principal Findings RARE-hsp68-lacZ transgenic mice were employed as a reporter for endogenous retinoic acid activity that was determined by X-gal assay and immunostaining of the reporter gene product, β-galactosidase. Double immunostaining was performed for β-galactosidase and markers of kidney tubules to localize retinoic acid activity. Distinct pattern of retinoic acid activity was observed in kidneys, which is higher in neonatal and 1- to 3-week-old mice than that in 5- and 8-week-old mice. The activity was present specifically in the principal cells and the intercalated cells of the collecting duct system in all age groups, but was absent from the glomeruli, proximal tubules, thin limbs of Henles loop and distal tubules. Conclusions/Significance Endogenous retinoic acid activity exists in principal cells and intercalated cells of the mouse collecting duct system after birth and persists into adulthood. This observation provides novel insights into potential roles for endogenous retinoic acid beyond nephrogenesis and warrants further studies to investigate target genes and functions of endogenous retinoic acid in the kidney after birth, particularly in the collecting duct system.

Knowledge-Based Discovery of Anti-Fibrotic and Pro-Fibrotic Activities from Chinese Materia Medica

Fibrosis, also known as scarring, sclerosis or cirrhosis, is characterised by excessive accumulation of extracellular matrix (ECM) proteins leading to tissue contraction, disruption of tissue architecture and eventually chronic organ failure (Wynn, 2007; Xu et al., 2007). Research and development of anti-fibrotic drugs are generally based on two distinct but interactive strategies, with one based on mechanism studies and another based on exploring efficacy. In principle, the mechanism-based strategy begins with identification of molecular targets through mechanistic studies, and then development of inhibitors or enhancers targeting the molecules. On the other hand, efficacy-based strategy starts with screening drug candidates in disease models to identify activities and efficacy, with less reliance on analysis of mechanisms of action. There are certain limitations in both the mechanism-based strategy and the efficacy-based strategy, which largely account for the lack of success in development of anti-fibrotic drugs. The former is often associated with identification of multiple molecular targets impeding development of a single drug that tackles multiple targets, while the latter is often hampered by establishment of apt models ideal for efficacy-driven drug screens. Efficacy-based strategy has been employed in development of both traditional and modern medicines. In the context of traditional medicine, the knowledge about efficacy of a given drug is largely derived from a trial-and-error process, namely by assessing patients’ response upon treatment with natural drug candidates. However, in modern medicine, it is impossible to directly test any new drugs in patients. Solid scientific evidence on efficacy and safety of a given drug in experimental models is required prior to clinical trials. Understandably, quality of these models would determine the specificity and efficiency of the tested drug.

Retinoic Acid Receptor-Dependent, Cell-Autonomous, Endogenous Retinoic Acid Signaling and Its Target Genes in Mouse Collecting Duct Cells

Background Vitamin A is necessary for kidney development and has also been linked to regulation of solute and water homeostasis and to protection against kidney stone disease, infection, inflammation, and scarring. Most functions of vitamin A are mediated by its main active form, all-trans retinoic acid (tRA), which binds retinoic acid receptors (RARs) to modulate gene expression. We and others have recently reported that renal tRA/RAR activity is confined to the ureteric bud (UB) and collecting duct (CD) cell lineage, suggesting that endogenous tRA/RARs primarily act through regulating gene expression in these cells in embryonic and adult kidney, respectively. Methodology/Principal Findings To explore target genes of endogenous tRA/RARs, we employed the mIMCD-3 mouse inner medullary CD cell line, which is a model of CD principal cells and exhibits constitutive tRA/RAR activity as CD principal cells do in vivo. Combining antagonism of RARs, inhibition of tRA synthesis, exposure to exogenous tRA, and gene expression profiling techniques, we have identified 125 genes as candidate targets and validated 20 genes that were highly regulated (Dhrs3, Sprr1a, and Ppbp were the top three). Endogenous tRA/RARs were more important in maintaining, rather than suppressing, constitutive gene expression. Although many identified genes were expressed in UBs and/or CDs, their exact functions in this cell lineage are still poorly defined. Nevertheless, gene ontology analysis suggests that these genes are involved in kidney development, renal functioning, and regulation of tRA signaling. Conclusions/Significance A rigorous approach to defining target genes for endogenous tRA/RARs has been established. At the pan-genomic level, genes regulated by endogenous tRA/RARs in a CD cell line have been catalogued for the first time. Such a catalogue will guide further studies on molecular mediators of endogenous tRA/RARs during kidney development and in relation to renal defects associated with vitamin A deficiency.

Kidneys of Alb/TGF-β1 Transgenic Mice Are Deficient in Retinoic Acid and Exogenous Retinoic Acid Shows Dose-Dependent Toxicity

Background: Alb/TGF-β1 transgenic mice overexpress active transforming growth factor-β1 (TGF-β1) in the liver, leading to increased circulating levels of the cytokine and progressive renal fibrosis. This study was designed to explore if exogenous all-trans retinoic acid (tRA) prevents renal fibrosis in this animal model. Methods: The retinoid profile in kidney and liver of wild-type and Alb/TGF-β1 transgenic mice was examined by high-performance liquid chromatography and slow-release pellets containing different amounts of tRA were implanted subcutaneously to treat the Alb/TGF-β1 transgenic mice, starting at 1 week of age; mice were sacrificed 2 weeks later. Results: Kidneys of 3-week-old wild-type mice had abundant tRA, which was completely absent in kidneys of the transgenic mice. Low doses of tRA (6–10.7 mg/kg/day) failed to affect renal fibrosis although it tended to suppress the mRNA expression of some molecular markers of fibrosis and retinal dehydrogenase 2 (RALDH2), a gene encoding a key tRA-synthesising enzyme. These tendencies disappeared, mortality tended to increase and RALDH2 and connective tissue growth factor (CTGF) mRNAs significantly increased in the medium-dose group (12.7–18.8 mg/kg/day). High doses (20.1–27.4 mg/kg/day) showed even higher toxicity with increased renal fibrosis and significant mortality. Conclusions: Alb/TGF-β1 transgenic mice are characterised by depletion of endogenous renal tRA. Exogenous tRA dose-dependently increases mortality and kidney fibrosis, which is associated with dose-dependent regulation of renal RALDH2 and CTGF mRNA expression.

Ablation of klotho and premature aging: is 1,25-dihydroxyvitamin D the key middleman?

The reversal of soft-tissue abnormalities and prolonged lifespan observed in klotho(-/-) mice following genetic inactivation of 1alpha-hydroxylase underscores the pathophysiological role of 1,25-dihydroxyvitamin D in mediating some of the premature aging-like features observed in klotho(-/-) mice.

Development of fine-grained pill identification algorithm using deep convolutional network

OBJECTIVE Oral pills, including tablets and capsules, are one of the most popular pharmaceutical dosage forms available. Compared to other dosage forms, such as liquid and injections, oral pills are very stable and are easy to be administered. However, it is not uncommon for pills to be misidentified, be it within the healthcare institutes or after the pills were dispensed to the patients. Our objective is to develop groundwork for automatic pill identification and verification using Deep Convolutional Network (DCN) that surpasses the existing methods. MATERIALS AND METHODS A DCN model was developed using pill images captured with mobile phones under unconstraint environments. The performance of the DCN model was compared to two baseline methods of hand-crafted features. RESULTS The DCN model outperforms the baseline methods. The mean accuracy rate of DCN at Top-1 return was 95.35%, whereas the mean accuracy rates of the two baseline methods were 89.00% and 70.65%, respectively. The mean accuracy rates of DCN for Top-5 and Top-10 returns, i.e., 98.75% and 99.55%, were also consistently higher than those of the baseline methods. DISCUSSION The images used in this study were captured at various angles and under different level of illumination. DCN model achieved high accuracy despite the suboptimal image quality. CONCLUSION The superior performance of DCN underscores the potential of Deep Learning model in the application of pill identification and verification.