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Zhuling Jisheng decoction is employed for the treatment of bladder urothelial cancer in clinical practice of traditional Chinese medicine. However, there are few studies on its precise mechanism. For the antibladder cancer action of Zhuling Jisheng decoction, a network pharmacological technique was used to design a component/target/pathway molecular regulatory network. The TCMSP dataset was used to identify the chemical makeup of Zhuling Jisheng decoction, which was then analyzed and assessed for oral bioavailability and pharmacological similarity. The chemical composition of Zhuling Jisheng decoction was identified through the TCMSP database, and it was evaluated and screened based on oral bioavailability and drug similarity. The GEO database was searched for genes associated with urothelial bladder carcinoma, and gene targets associated with bladder urothelial cancer resistance were chosen by comparison. The function and linked pathways of the target genes were examined and screened using annotation, visualization, and a comprehensive discovery database. The impact of Zhuling Jisheng decoction on urothelial bladder cancer was studied using Cytoscape software to create a component/target/pathway network. Finally, 69 and 55 target genes were discovered for noninvasive bladder urothelial cancer and invasive bladder urothelial cancer, respectively. In noninvasive urothelial cancer, 118 pathways were highly enriched, including the TNF signaling pathway and the IL-17 signaling route. 103 pathways were highly enriched in invasive urothelial cancer, including the p53 signaling route, bladder cancer route, and calcium signaling route. There were 18 and 15 drug targets associated with noninvasive and invasive bladder urothelial carcinoma prognoses. Many signaling pathways directly act on tumours, and indirect pathways inhibit the development of bladder urothelial carcinoma. This research establishes a scientific foundation for further research into the framework of action of Zhuling Jisheng decoction in the therapy of bladder urothelial cancer., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Chenyu Ma et al.)

Functional studies of organisms and human models have revealed that epigenetic changes can significantly impact the process of aging. Non-coding RNA (ncRNA), one of epigenetic regulators, plays an important role in modifying the expression of mRNAs and their proteins. It can mediate the phenotype of cells. It has been reported that nc886 (=vtRNA2-1 or pre-miR-886), a long ncRNA, can suppress tumor formation and photo-damages of keratinocytes caused by UVB. The aim of this study was to determine the role of nc886 in replicative senescence of fibroblasts and determine whether substances capable of controlling nc886 expression could regulate cellular senescence. In replicative senescence fibroblasts, nc886 expression was decreased while methylated nc886 was increased. There were changes of senescence biomarkers including SA-β-gal activity and expression of p16INK4A and p21Waf1/Cip1 in senescent cells. These findings indicate that the decrease of nc886 associated with aging is related to cellular senescence of fibroblasts and that increasing nc886 expression has potential to suppress cellular senescence. AbsoluTea Concentrate 2.0 (ATC) increased nc886 expression and ameliorated cellular senescence of fibroblasts by inhibiting age-related biomarkers. These results indicate that nc886 has potential as a new target for anti-aging and that ATC can be a potent epigenetic anti-aging ingredient.

Serum supplementation during bovine embryo culture has been demonstrated to promote cell proliferation and preimplantation embryo development. However, these desirable outcomes, have been associated with gene expression alterations of pathways involved in macroautophagy, growth, and development at the blastocyst stage, as well as with developmental anomalies such as fetal overgrowth and placental malformations. In order to start dissecting the molecular pathways by which serum supplementation of the culture medium during the preimplantation stage promotes developmental abnormalities, we examined blastocyst morphometry, inner cell mass and trophectoderm cell allocations, macroautophagy, and endoplasmic reticulum stress. On day 5 post-insemination, > 16 cells embryos were selected and cultured in medium containing 10% serum or left as controls. Embryo diameter, inner cell mass and trophectoderm cell number, and macroautophagy were measured on day 8 blastocysts (BL) and expanded blastocysts (XBL). On day 5 and day 8, we assessed transcript level of the ER stress markers HSPA5, ATF4, MTHFD2, and SHMT2 as well as XBP1 splicing (a marker of the unfolded protein response). Serum increased diameter and proliferation of embryos when compared to the no-serum group. In addition, serum increased macroautophagy of BL when compared to controls, while the opposite was true for XBL. None of the genes analyzed was differentially expressed at any stage, except that serum decreased HSPA5 in day 5 > 16 cells stage embryos. XBP1 splicing was decreased in BL when compared to XBL, but only in the serum group. Our data suggest that serum rescues delayed embryos by alleviating endoplasmic reticulum stress and promotes development of advanced embryos by decreasing macroautophagy., Competing Interests: The authors have declared that no competing interests exist.

Intron retention (IR) is an important regulatory mechanism that affects gene expression and protein functions. Using klotho mice at the pre-symptomatic state, we discovered that retained-introns accumulated in several organs including the liver and that among these retained introns in the liver a subset was recovered to the normal state by a Japanese traditional herbal medicine. This is the first report of IR recovery by a medicine. IR-recovered genes fell into two categories: those involved in liver-specific metabolism and in splicing. Metabolome analysis of the liver showed that the klotho mice were under starvation stress. In addition, our differentially expressed gene analysis showed that liver metabolism was actually recovered by the herbal medicine at the transcriptional level. By analogy with the widespread accumulation of intron-retained pre-mRNAs induced by heat shock stress, we propose a model in which retained-introns in klotho mice were induced by an aging stress and in which this medicine-related IR recovery is indicative of the actual recovery of liver-specific metabolic function to the healthy state. Accumulation of retained-introns was also observed at the pre-symptomatic state of aging in wild-type mice and may be an excellent marker for this state in general., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Coronavirus Disease 2019 (Covid-19) continues to challenge the limits of our knowledge and our healthcare system. Here we sought to define the host immune response, a.k.a, the "cytokine storm" that has been implicated in fatal COVID-19 using an AI-based approach., Method: Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a 'seed' gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. An AI-based approach was used to explore the utility of the signature in navigating the uncharted territory of Covid-19, setting therapeutic goals, and finding therapeutic solutions., Findings: The 166-gene signature was surprisingly conserved across all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determine severity/fatality. Precise therapeutic goals could be formulated; these goals were met in high-dose SARS-CoV-2-challenged hamsters using either neutralizing antibodies that abrogate SARS-CoV-2•ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine prognosticated disease severity., Interpretation: The ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs., Funding: This work was supported by the National Institutes for Health (NIH) [grants CA151673 and GM138385 (to DS) and AI141630 (to P.G), DK107585-05S1 (SD) and AI155696 (to P.G, D.S and S.D), U19-AI142742 (to S., C, Cchi: Cooperative Centers for Human Immunology)]; Research Grants Program Office (RGPO) from the University of California Office of the President (UCOP) (R00RG2628 & R00RG2642 to P.G, D.S and S.D); the UC San Diego Sanford Stem Cell Clinical Center (to P.G, D.S and S.D); LJI Institutional Funds (to S.C); the VA San Diego Healthcare System Institutional funds (to L.C.A). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists., One Sentence Summary: The host immune response in COVID-19., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Acyl-CoA:cholesterol acyltransferase (ACAT) mediates cellular cholesterol esterification. In atherosclerotic plaque macrophages, ACAT promotes cholesteryl ester accumulation, resulting in foam cell formation and atherosclerosis progression. Its complete inactivation in mice, however, showed toxic effects because of an excess of free cholesterol (FC) in macrophages, which can cause endoplasmic reticulum stress, cholesterol crystal formation, and inflammasome activation. Our previous studies showed that long-term partial ACAT inhibition, achieved by dietary supplementation with Fujirebio F1394, delays atherosclerosis progression in apoprotein E-deficient ( Apoe -/- ) mice by reducing plaque foam cell formation without inflammatory or toxic effects. Here, we determined whether short-term partial inhibition of ACAT, in combination with an enhanced systemic FC acceptor capacity, has synergistic benefits. Thus, we crossbred Apoe -/- with human apoprotein A1-transgenic ( APOA1 tg/tg ) mice, which have elevated cholesterol-effluxing high-density lipoprotein particles, and subjected Apoe -/- and APOA1 tg/tg / Apoe -/- mice to an atherogenic diet to develop advanced plaques. Then mice were either euthanized (baseline) or fed purified standard diet with or without F1394 for 4 more weeks. Plaques of APOA1 tg/tg / Apoe -/- mice fed F1394 showed a 60% reduction of macrophages accompanied by multiple other benefits, such as reduced inflammation and favorable changes in extracellular composition, in comparison with Apoe -/- baseline mice. In addition, there was no accumulation of cholesterol crystals or signs of toxicity. Overall, these results show that short-term partial ACAT inhibition, coupled to increased cholesterol efflux capacity, favorably remodels atherosclerosis lesions, supporting the potential of these combined therapies in the treatment of advanced atherosclerosis. SIGNIFICANCE STATEMENT: Short-term pharmacological inhibition of acyl-CoA:cholesterol acyltransferase-mediated cholesterol esterification, in combination with increased free cholesterol efflux acceptors, has positive effects in mice by 1) reducing the inflammatory state of the plaque macrophages and 2) favoring compositional changes associated with plaque stabilization. These effects occur without toxicity, showing the potential of these combined therapies in the treatment of advanced atherosclerosis., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Camellia japonica L. is a flowering tree with several medicinal and cosmetic applications. Here, we investigated the efficacy of C. japonica placenta extract (CJPE) as a potential therapeutic agent for promotion of hair growth and scalp health by using various in vitro and in vivo assays. Moreover, we performed transcriptome analysis to examine the relative expression of human follicle dermal papilla cells (HFDPC) in response to CJPE by RNA-sequencing (RNA-seq). In vitro assays revealed upregulation of the expression of hair growth marker genes in HFDPC after CJPE treatment. Moreover, in vivo clinical tests with 42 adult female participants showed that a solution containing 0.5% CJPE increased the moisture content of the scalp and decreased the scalp's sebum content, dead scalp keratin, and erythema. Furthermore, RNA-seq analysis revealed key genes in HFDPC which are associated with CJPE. Interestingly, genes associated with lipid metabolism and cholesterol efflux were upregulated. Genes upregulated by CJPE are associated with several hormones, including parathyroid, adrenocorticotropic hormone, α-melanocyte-stimulating hormone (alpha-MSH), and norepinephrine, which are involved in hair follicle biology. Furthermore, some upregulated genes are associated with the regulation of axon guidance. In contrast, many genes downregulated by CJPE are associated with structural components of the cytoskeleton. In addition, CJPE suppressed genes associated with muscle structure and development. Taken together, this study provides extensive evidence that CJPE may have potential as a therapeutic agent for scalp treatment and hair growth promotion., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The 3D-printed hybrid biodegradable hydrogels composed of alginate, gelatin, and cellulose nanocrystals (CNCs) were prepared to provide a favorable environment for cell proliferation, adhesion, nutrients exchange, and matrix mineralization for bone tissue engineering (BTE) applications. The hybrid scaffolds exhibited enhanced mechanical strength compared to the pure polymer scaffolds. The biocompatibility, differentiation potential, and bone regeneration potential of the printed scaffolds were evaluated by DAPI staining, live-dead assay, alizarin Red-S (ARS) staining, real-time PCR (qRT-PCR), and μCT analysis, respectively. Enhanced cell proliferation has occurred 1% CNC/Alg/Gel scaffolds compared to the control. The cells were adequately adhered to the scaffold and exhibited the flattened structure. Improved mineralization was observed in the 1% CNC/Alg/Gel scaffolds' presence than the control, showing their mineralization efficiency. A significant enhancement in the expression of osteogenic-specific gene markers (Runx2, ALP, BMP-2, OCN, OPN, BSP, and COL1) has occurred with 1% CNC/Alg/Gel than the control, indicating their osteogenic potential. Furthermore, enhanced bone formation was observed in the scaffolds treated groups than the control in the calvaria critical-sized defects (CCD-1) model, suggesting their improved bone regeneration potential. Therefore, the fabricated scaffolds have the potential to explore as a biomaterial for tissue engineering., Competing Interests: Declaration of competing interest The author(s) declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Patients with inflammatory bowel disease (IBD) show large variability in disease course, and also treatment response. The variability in treatment response has led to many initiatives in search of genetic markers to optimize treatment and avoid severe side effects. This has been very successful for thiopurines, one of the drugs used to induce and maintain remission in IBD. However, for the newer treatment options for IBD, like biologicals, the search for genetic predictors has not yielded any candidate biomarkers with clinical utility. In this review, a summary of recent advances in pharmacogenetics focusing on thiopurines and anti-TNF agents is given.

Predictive indicators of testicular toxicity could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating microRNAs (miRNAs) could serve as testicular toxicity biomarkers in dogs. Herein, we describe the results of an exploratory study conducted to discover biomarkers of drug-induced testicular injury. Following a dose-selection study using the testicular toxicant ethylene glycol monomethyl ether (EGME), we chose a dose of 50 mg/kg/d EGME to avoid systemic toxicity and treated 2 groups of dogs (castrated, non-castrated) for 14 to 28 days. Castrated animals were used as negative controls to identify biomarkers specific for testicular toxicity because EGME can cause toxicity to organ systems in addition to the testis. Blood was collected daily during the dosing period, followed by recovery for 29 to 43 days with less frequent sampling. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Global profiling of serum miRNAs at selected dosing and recovery time points was completed by small RNA sequencing. Bioinformatics data analysis using linear modeling demonstrated several circulating miRNAs that were differentially abundant during the dosing period compared with baseline and/or castrated control samples. Confirmatory reverse transcription quantitative polymerase chain reaction data in these animals was unable to detect sustained alterations of miRNAs in serum, except for 1 potential candidate cfa-miR-146b. Taken together, we report the results of a comprehensive exploratory study and suggest future directions for follow-up research to address the challenge of developing diagnostic biomarkers of testicular toxicity.

Background: Stimulation of ovulation with clomiphene citrate can cause side effects on endometrial receptivity. Formulation with nano-size may be an alternative therapy for women with ovulatory disorders. In this study, we investigated sustained-release clomiphene citrate by using Phosal-based formulation (PBF) and evaluate its decreased side effect on the endometrial receptivity., Methods: In the in-vitro study, CC loaded PBF was analyzed using Zetasizer, Fourier-transform infrared spectroscopy (FTIR), and Transmission electron microscopy (TEM). In the in-vivo study, 24 female mice were randomly divided into three groups: CC (5 mg/kg), CC/PBF (5 mg/kg) and SS (1 ml) daily administered and injected with 5 IU HCG and mated after two days. At day 4.5, pregnant mice were euthanized and endometrial tissue was extracted for quantitative polymerase chain reaction (Q-PCR) analysis., Results: The optimized PBF contained Phosal 50PG/glycerol in a 2:8 ratios (w/w) and the particle size of optimum formulation was 67 ± 0.30551 nm and the release of CC from CC-containing PBF was slightly faster in the first 24 h; wherein, 29% of CC was released, and 76% of CC was released up to 120 h. The mRNA levels of leukemia inhibitory factor (LIF), leukemia inhibitory factor receptor alpha (LIFR), HOXA10, Heparin-binding epidermal growth factor (HB-EGF), and epidermal growth factor (EGF) were significantly upregulated and MUC1 and PGR mRNA levels were significantly downregulated in the CC-containing PBF-treated animals compared with only CC group (P < 0.05)., Conclusion: Sustained release formulation of clomiphene citrate increased its targeting efficiency and improved the impact of the CC on implantation. Graphical abstract A new Phosal Based Formulation (PBF) was designed to decrease the side effects of Clomiphene citrate (CC) on endometrium. This drug formulation could react better during implantation by increasing the expression of genes involved in implantation. The in vivo study demonstrated that the CC-containing PBF in mice has a significantly higher endometrial receptivity, compared with the suspension.

Limited toxicity data on electronic cigarette (ECIG) impede evidence-based policy recommendations. We compared two popular mixed fruit flavored ECIG-liquids with and without nicotine aerosolized at 40 W (E-smoke) with respect to particle number concentrations, chemical composition, and response on physiologically relevant human bronchial and alveolar lung mucosa models cultured at air-liquid interface. E-smoke was characterized by significantly increased particle number concentrations with increased wattage (25, 40, and 55 W) and nicotine presence. The chemical composition of E-smoke differed across the two tested flavors in terms of cytotoxic compounds including p-benzoquinone, nicotyrine, and flavoring agents (for example vanillin, ethyl vanillin). Significant differences in the expression of markers for pro-inflammation, oxidative stress, tissue injury/repair, alarm anti-protease, anti-microbial defense, epithelial barrier function, and epigenetic modification were observed between the flavors, nicotine content, and/ or lung models (bronchial or alveolar). Our findings indicate that ECIG toxicity is influenced by combination of multiple factors including flavor, nicotine content, vaping regime, and the region of respiratory tree (bronchial or alveolar). Toxic chemicals and flavoring agents detected in high concentrations in the E-smoke of each flavor warrant independent evaluation for their specific role in imparting toxicity. Therefore, multi-disciplinary approaches are warranted for comprehensive safety profiling of ECIG.

Glucose-induced oxidative stress is associated with the overproduction of reactive oxygen species (ROS), which may dysregulate the expression of genes controlling insulin secretion leading to β-cell dysfunction, a hallmark of type 2 diabetes mellitus (T2DM). This study investigated the impact of coloured rice phenolic extracts (CRPEs) on the expression of key genes associated with β-cell function in pancreatic β-cells (INS-1E). These genes included glucose transporter 2 ( Glut2 ) , silent mating type information regulation 2 homolog 1 ( Sirt1 ) , mitochondrial transcription factor A ( Tfam ) , pancreatic/duodenal homeobox protein 1 ( Pdx-1 ) and insulin 1 ( Ins1 ). INS-1E cells were cultured in high glucose (25 mM) to induce glucotoxic stress conditions (HGSC) and in normal glucose conditions (NGC-11.1 mM) to represent normal β-cell function. Cells were treated with CRPEs derived from two coloured rice cultivars, Purple and Yunlu29-red varieties at concentrations ranged from 50 to 250 µg/mL. CRPEs upregulated the expression of Glut2, Sirt1 and Pdx-1 significantly at 250 µg/mL under HGSC. CRPEs from both cultivars also upregulated Glut2, Sirt1, Tfam, Pdx-1 and Ins1 markedly at 250 µg/mL under NGC with Yunlu29 having the greatest effect. These data suggest that CRPEs may reduce β-cell dysfunction in T2DM by upregulating the expression of genes involved in insulin secretion pathways.

Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a progressive motor disease with no broadly effective treatment. However, most current therapies are based on symptoms rather than the underlying disease mechanisms. In this review, we describe potential therapeutic strategies based on known pathological biomarkers and related pathogenic processes. The three major conclusions from the current studies are summarized as follows: (i) for the drugs currently being tested in clinical trials; a weak connection was observed between drugs and SCA3/MJD biomarkers. The only two exceptions are the drugs suppressing glutamate-induced calcium influx and chemical chaperon. (ii) For most of the drugs that have been tested in animal studies, there is a direct association with pathological biomarkers. We further found that many drugs are associated with inducing autophagy, which is supported by the evidence of deficient autophagy biomarkers in SCA3/MJD, and that there may be more promising therapeutics. (iii) Some reported biomarkers lack relatively targeted drugs. Low glucose utilization, altered amino acid metabolism, and deficient insulin signaling are all implicated in SCA3/MJD, but there have been few studies on treatment strategies targeting these abnormalities. Therapeutic strategies targeting multiple pathological SCA3/MJD biomarkers may effectively block disease progression and preserve neurological function.

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory neuronal damages and consequent disabilities. Episodic relapses of the disease which lead to brain lesions and irreversible neurological dysfunctions could be decreased by the interferon-beta (IFN-β) therapy in most of the MS patients. However, the efficiency of the drug response is highly variable among patients and the precise mechanism of action of the IFN-β is not clear. To investigate the role of RORA gene as a biomarker of patient's responsiveness, the present study have analyzed the frequency of two polymorphisms (rs4774388 and rs11639084) within this gene between responder (n = 105) and nonresponder (n = 65) groups of MS patients in comparison with 200 healthy controls. The tetra primers-Amplification Refractory Mutation System-PCR method was used for genotyping. The obtained result of the current study showed a significant association between nonresponsiveness and the rs4774388 in dominant model (p = 0.03). However, the allele and genotype frequencies of rs11639084 were not different between controls, nonresponder, and responder patients. In addition, the frequencies of the estimated haplotype blocks were not different between examined groups. The obtained results of the present study suggested the rs4774388 as a possible effective factor in determination of response to IFN-β. However, further studies are needed to confirm the results in a larger sample size.

Since the discovery of the first microRNA (miRNA) decades ago, studies of miRNA biology have expanded in many biomedical research fields, including eye research. The critical roles of miRNAs in normal development and diseases have made miRNAs useful biomarkers or molecular targets for potential therapeutics. In the eye, ocular neovascularization (NV) is a leading cause of blindness in multiple vascular eye diseases. Current anti-angiogenic therapies, such as anti-vascular endothelial growth factor (VEGF) treatment, have their limitations, indicating the need for investigating new targets. Recent studies established the roles of various miRNAs in the regulation of pathological ocular NV, suggesting miRNAs as both biomarkers and therapeutic targets in vascular eye diseases. This review summarizes the biogenesis of miRNAs, and their functions in the normal development and diseases of the eye, with a focus on clinical and experimental retinopathies in both human and animal models. Discovery of novel targets involving miRNAs in vascular eye diseases will provide insights for developing new treatments to counter ocular NV.

Migraine is a disabling and recurrent neurological disorder characterized by headache attacks that are often accompanied by sensory and motor disturbances. The value and importance of reliable biomarkers in migraine have been long recognized and a diverse range of biomarkers from biological samples to electrophysiological patterns and brain imaging has been proposed. There is still no consensus on specific biomarker(s) for migraine. Ideally, not a single but a battery of biomarkers would provide a multidisciplinary way to understand and treat migraine better. Translational research has witnessed an escalating number of studies on microRNAs (miRNAs) during the last decade. Identification of the first miRNA occurred in 1993, and currently more than 2000 human miRNAs have been recognized. miRNAs are a group of endogenous small non-coding molecules that play a key role in post-transcriptional gene processes and hence are involved in health and disease. miRNAs have already been found to be involved in the onset and progression of several human disorders including chronic pain conditions; however, there have been far fewer studies in migraine and other headaches. Current evidence does suggest that miRNAs play a role in migraine and its relief and hence these molecules are proposed as potential migraine biomarkers. This review updates the current evidence for the role of miRNAs in migraine; including their potential as biomarkers, with a role in understanding of its pathogenesis, the population at risk, diagnosis, patient stratification, chronification risk factors, response to treatments, and miRNA-based therapeutic options. Limitations exist and further research is required to completely unwrap the potential of miRNAs in migraine research and practice.

Antiviral susceptibility screening of avian influenza (AI) H9N2 viruses is crucial considering their role at the animal-human interface and potential to cause human infections. The Matrix 2 (M2) inhibitors (amantadine and rimantadine) have been used for prophylaxis and treatment of influenza A virus infections, however, resistance to these drugs has been widely reported. Information about amantadine susceptibility of H9N2 viruses from India is scanty. Matrix genes of 48H9N2 viruses isolated from India during 2009-2017 were sequenced and M2 trans-membrane region sequences were screened for mutations which are known to confer resistance to amantadine namely, L26F, V27A, A30 T/V, S31N and G34E. All the viruses isolated during the year 2009 were sensitive to amantadine. However, resistance started to appear since the year 2010 and all the viruses isolated from the year 2015 onwards showed presence of molecular markers conferring resistance to amantadine. Majority of the resistant viruses exhibited S31 N mutation. Four isolates showed presence of V27A + S31 N dual mutations. Comparison of the M2 sequences from other Asian countries showed different patterns of amantadine resistance wherein phylogenetic analysis of the M genes of the strains from Pakistan formed a separate cluster. In conclusion, the present study reports prevalence and gradual increase of amantadine resistance among AI H9N2 viruses in India, emphasizing the importance of the antiviral surveillance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: Accurate prediction of treatment responses in rheumatoid arthritis (RA) patients can provide valuable information on effective drug selection. Anti-tumor necrosis factor (anti-TNF) drugs are an important second-line treatment after methotrexate, the classic first-line treatment for RA. However, patient heterogeneity hinders identification of predictive biomarkers and accurate modeling of anti-TNF drug responses. This study was undertaken to investigate the usefulness of machine learning to assist in developing predictive models for treatment response., Methods: Using data on patient demographics, baseline disease assessment, treatment, and single-nucleotide polymorphism (SNP) array from the Dialogue on Reverse Engineering Assessment and Methods (DREAM): Rheumatoid Arthritis Responder Challenge, we created a Gaussian process regression model to predict changes in the Disease Activity Score in 28 joints (DAS28) for the patients and to classify them into either the responder or the nonresponder group. This model was developed and cross-validated using data from 1,892 RA patients. It was evaluated using an independent data set from 680 patients. We examined the effectiveness of the similarity modeling and the contribution of individual features., Results: In the cross-validation tests, our method predicted changes in DAS28 (ΔDAS28), with a correlation coefficient of 0.405. It correctly classified responses from 78% of patients. In the independent test, this method achieved a Pearson's correlation coefficient of 0.393 in predicting ΔDAS28. Gaussian process regression effectively remapped the feature space and identified subpopulations that do not respond well to anti-TNF treatments. Genetic SNP biomarkers showed small contributions in the prediction when added to the clinical models. This was the best-performing model in the DREAM Challenge., Conclusion: The model described here shows promise in guiding treatment decisions in clinical practice, based primarily on clinical profiles with additional genetic information., (© 2019, American College of Rheumatology.)

Male factor infertility is increasing and recognized as playing a key role in reproductive health and disease. The current primary diagnostic approach is to assess sperm quality associated with reduced sperm number and motility, which has been historically of limited success in separating fertile from infertile males. The current study was designed to develop a molecular analysis to identify male idiopathic infertility using genome wide alterations in sperm DNA methylation. A signature of differential DNA methylation regions (DMRs) was identified to be associated with male idiopathic infertility patients. A promising therapeutic treatment of male infertility is the use of follicle stimulating hormone (FSH) analogs which improved sperm numbers and motility in a sub-population of infertility patients. The current study also identified genome-wide DMRs that were associated with the patients that were responsive to FSH therapy versus those that were non-responsive. This novel use of epigenetic biomarkers to identify responsive versus non-responsive patient populations is anticipated to dramatically improve clinical trials and facilitate therapeutic treatment of male infertility patients. The use of epigenetic biomarkers for disease and therapeutic responsiveness is anticipated to be applicable for other medical conditions.

To some extent, the use of metformin may improve endometrial receptivity and pregnancy outcomes of women with polycystic ovarian syndrome (PCOS) undergoing in vitro fertilization/intracytoplasmic sperm injection. However, the mechanism is not well-known. The endometrium of metformin-treated group (metformin-treated patients with PCOS) and the control group (non-metformin-treated patients with PCOS) were analyzed for the expression of homeobox A10 (HOXA10) and integrin beta-3 (ITGB3) and differential micro RNA (miRNA) expression profiles. On this basis, miRDB and Target Scan databases were used to predict and screen out that miR-491-3p and miR-1910-3p may target HOXA10 and ITGB3. Furthermore, we verified the effects of metformin on the expression of HOXA10 and ITGB3, and regulatory effects of miR-1910-3p and miR-491-3p on HOXA10 and ITGB3 using Ishikawa cell line. Metformin induced a significant dose-dependent upregulation of HOXA10 and ITGB3. The results from the microarray analyses showed there were 40 differentially expressed miRNAs between the 2 groups. Among them, miR-1910-3p and miR-491-3p were the 2 significantly downregulated miRNAs. Bioinformatics prediction indicated that HOXA10 and ITGB3 are potential target genes for miR-1910-3p and miR-491-3p. In Ishikawa cells transfected with miR-491-3p mimics, the expression of HOXA10 and ITGB3 on both messenger RNA (mRNA) and protein level were lower than those in control group ( P < .001). Also, the expression of HOXA10 mRNA and protein was lower in Ishikawa cells transfected with miR-1910-3p mimics ( P < .001). However, no significant changes in ITGB3 levels were observed in cells transfected with miR-1910-3p mimics ( P > .05). Metformin likely improves endometrial receptivity through downregulating the expression of miR-491-3p and miR-1910-3p, thereby increasing the expression of HOXA10 and ITGB3 in the endometrium of PCOS women.

Compelling preclinical studies indicate that low-dose carbon monoxide (CO) abrogates experimental lung fibrosis. We recently reported the results of a multicenter, double-blinded, clinical trial of inhaled CO in patients with idiopathic pulmonary fibrosis (IPF). Identifying no significantly changes in metalloproteinase-7 (MMP7) serum concentration, or secondary endpoints of physiologic measurements, hospitalization, death, or patient-reported outcomes. In the present study, we evaluated the effect of low dose CO exposure (100-200 ppm) for 12 weeks on genome-wide gene expression in peripheral blood mononuclear cells (PBMC) derived from these IPF study subjects. We conducted transcriptome profiling on 38 IPF subjects with time points available at 0, 12, and 24 weeks. Total RNA isolated from PBMCs was hybridized onto the Affymetrix Human Gene 2.0 ST Array. We identified 621 genes significantly upregulated in the 24-week CO exposed group compared with the 12-week. Pathway analysis demonstrated association with Oxidative Phosphorylation (adjusted P < 0.05). We identified a clear CO signature dominated with 23 oxidative phosphorylation-related genes (FDR <10%). We confirmed the expression of nine selected gene products using Nanostring's nCounter analysis system. These findings suggest this signature may serve as a potential genomic biomarker for CO exposure and for potential titration of dosage to allow precision testing of therapies in future low dose CO therapeutic studies in IPF.

Mouse trophoblast stem cells (TSCs) have been established and maintained using hyperglycemic conditions (11 mM glucose) for no apparent good reason. Because glucose metabolites are used as resources for cellular energy production, biosynthesis, and epigenetic modifications, differences in extracellular glucose levels may widely affect cellular function. Since the hyperglycemic culture conditions used for TSC culture have not been fully validated, the effect of extracellular glucose levels on the properties of TSCs remains unclear. To address this issue, we investigated the gene expression of stemness-related transcription factors in TSCs cultured in the undifferentiated state under various glucose concentrations. We also examined the expression of trophoblast subtype markers during differentiation, after returning the glucose concentration to the conventional culture concentration (11 mM). As a result, it appeared that the extracellular glucose conditions in the stem state not only affected the gene expression of stemness-related transcription factors before differentiation but also affected the expression of marker genes after differentiation, with some line-to-line variation. In the TS4 cell line, which showed the largest glucose concentration-dependent fluctuations in gene expression among all the lines examined, low glucose (1 mM glucose, LG) augmented H3K27me3 levels. An Ezh2 inhibitor prevented these LG-induced changes in gene expression, suggesting the possible involvement of H3K27me3 in the changes in gene expression seen in LG. These results collectively indicate that the response of the TSCs to the change in the extracellular glucose concentration is cell line-dependent and a part of which may be epigenetically memorized.

Purpose: Although association studies of genetic variations with the clinical outcomes of breast cancer patients treated with tamoxifen have been reported, genetic factors which could determine individual response to tamoxifen are not fully clarified. We performed a genome-wide association study (GWAS) to identify novel genetic markers for response to tamoxifen., Experimental Design: We prospectively collected 347 blood samples from patients with hormone receptor-positive and human epidermal growth factor receptor 2-negative, invasive breast cancer receiving preoperative tamoxifen monotherapy for 14 to 28 days. We used Ki-67 response in breast cancer tissues after preoperative short-term tamoxifen therapy as a surrogate marker for response to tamoxifen. We performed GWAS and genotype imputation using 275 patients, and an independent set of 72 patients was used for replication study., Results: The combined result of GWAS and the replication study, and subsequent imputation analysis indicated possible association of three loci with Ki-67 response after tamoxifen therapy (rs17198973 on chromosome 4q34.3, rs4577773 on 6q12, and rs7087428 on 10p13, Pcombined = 5.69 x 10-6, 1.64 x 10-5, and 9.77 x 10-6, respectively). When patients were classified into three groups by the scoring system based on the genotypes of the three SNPs, patients with higher scores showed significantly higher after/before ratio of Ki-67 compared to those with lower scores (P = 1.8 x 10-12), suggesting the cumulative effect of the three SNPs., Conclusion: We identified three novel loci, which could be associated with clinical response to tamoxifen. These findings provide new insights into personalized hormonal therapy for the patients with breast cancer., Competing Interests: The authors have declared that no competing interests exist.

Background: In patients who receive palliative radiation therapy (RT) for painful bone metastases, 40% experience a transient increase in pain known as a pain flare. Prophylactic dexamethasone has been shown to reduce pain flare incidence to 25%. We aimed to identify DNA biomarkers associated with pain flare and dexamethasone response., Methods: Daily pain levels were recorded by 81 patients who received a single 8 Gy RT for painful bone metastases, of which 50 also received prophylactic dexamethasone. To identify single-nucleotide variants (SNVs), patient saliva samples obtained at day of RT were sequenced for 4,813 disease-associated genes, then filtered for genes associated with inflammation, radiation or immune response, and DNA damage. Significant SNVs (P<0.005) identified by the Cochran-Armitage trend test underwent the Penalized LASSO method with minimum Bayesian Information Criterion to select a multi-SNV model that jointly predicted pain flare, and pain flare despite prophylactic dexamethasone (dexamethasone response). The corresponding estimated effects of the multi-SNVs were used to drive the prognostic score of developing pain flare for each patient, who were divided into three risk groups of roughly equal sizes., Results: Risk groups were significantly predictive of pain flare (P<0.0001) and dexamethasone response (P<0.0001). The high-risk patient groups had a 78% chance of developing pain flare, and pain flare despite dexamethasone [OR =24.6, 95% confidence interval (CI): 1.8-342.7, P=0.02]. The multivariable model for pain flare included 15 variants, with effect sizes ranging from -4.97 (NBPF1 rs3872309 C>T) to 5.54 (DNM2 10940838 A>C). The multivariable model for dexamethasone response included 6 variants, with effect sizes ranging from -1.03 (NBPF1 rs3872309 C>T) to 0.85 (TSEN54 rs62088470 C>G)., Conclusions: Significant SNVs associated with pain flare were found in genes with functions in biosynthesis (DHODH, PECR), lipid excretion and metabolism (UGT2A1/2, VLDLR), and intracellular signalling (DNM2, SEC23A). Significant SNVs associated with dexamethasone response were from genes involved in extracellular matrix (HAS1, ADAMTS16) and cytoskeleton regulation (GAS2L2). Identification of SNVs predictive of pain flare and dexamethasone response enables targeted prophylactic therapy according to a patient's predisposed response.

Multiple integrins have been implicated in modulating renal function. Modulation of integrin function can lead to pathophysiological processes associated with diabetic nephropathy such as alterations in the glomerular filtration barrier and kidney fibrosis. The complexity of these pathophysiological changes implies that multiple integrin subtypes might need to be targeted to ameliorate the progression of renal disease. To address this hypothesis, we investigated the effects of MK-0429, a compound that was originally developed as an αvβ3 inhibitor for the treatment of osteoporosis, on renal function and fibrosis. We demonstrated that MK-0429 is an equipotent pan-inhibitor of multiple av integrins. MK-0429 dose-dependently inhibited podocyte motility and also suppressed TGF-β-induced fibrosis marker gene expression in kidney fibroblasts. Moreover, in the obese ZSF1 rat model of diabetic nephropathy, chronic treatment with MK-0429 resulted in significant reduction in proteinuria, kidney fibrosis, and collagen accumulation. In summary, our results suggest that inhibition of multiple integrin subtypes might lead to meaningful impact on proteinuria and renal fibrosis in diabetic nephropathy., (© 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

To understand the process of neurogenesis, generation of functional dopaminergic (DAergic) neurons from human mesenchymal stem cells (hMSCs) is important. BDNF has been reported to be responsible for inducing neuronal maturation and functionality. Previously, we have reported the efficient generation of neurons from human bone marrow derived MSCs using FGF2 alone. We hypothesize that hMSCs from various tissues [(bone marrow (BM), adipose tissue (AD) and dental pulp (DP)], if treated with BDNF on 9 th day of induction, alongwith FGF2 will generate functional DAergic neurons. Hence, cells were characterized at morphometric, transcription and translational levels for various markers like MAP2, TH, NGN2, PITX3, DAT, synaptophysin, Kv4.2 and SCN5A. Functionality of in vitro generated neurons was studied by calcium ion imaging. Result analysis depicted that BDNF has effect on expression of dopaminergic neuronal markers at gene and protein levels and functionality of neurons. Among these hMSCs, DP-MSC showed significantly better neuronal characteristics in terms of morphology, expression of neuronal markers and foremost, functionality of neurons. From the present study, therefore, we concluded that i) BDNF has additive effect on neuronal characteristics and functionality ii) DP-MSC are better MSC candidate to study DAergic neurogenesis and perform future studies.

Therapies that target signalling molecules that are mutated in cancers can often have substantial short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can result from secondary mutations, but in other cases there is no clear genetic cause, raising the possibility of non-genetic rare cell variability. Here we show that human melanoma cells can display profound transcriptional variability at the single-cell level that predicts which cells will ultimately resist drug treatment. This variability involves infrequent, semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins with a loss of SOX10-mediated differentiation followed by activation of new signalling pathways, partially mediated by the activity of the transcription factors JUN and/or AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics in single cells. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general program in which expression is displayed in rare subpopulations of cells.

Studies on adipogenesis may be important for regulating human and/or animal obesity, which causes several complications such as, type II diabetes, hypertension, and cardiovascular disease, thus giving rise to increased economic burden in many countries. Previous reports revealed that various flavonoids have anti-apoptotic, antioxidant, and cell differentiation-regulating activities with a number of physiological benefits, including protection from cardiovascular disease, cancers, and oxidative stress. As we found that the hydroxylation patterns of the flavonoid B ring are known to play a critical role in their function, we screened several flavonoids containing different numbers and positions of OH substitutions in B ring for their modulatory property on adipogenesis. In this study, we revealed the anti-adipogenic activity of the naturally derived flavonoid, 3,4'-dihydroxyflavone (3,4'-DHF) in murine 3T3-L1 pre-adipocytes and equine adipose-derived stromal cells (eADSCs). We found that treatment with 3,4'-dihydroxyflavone (3,4'-DHF) led to decreased expression of adipogenic markers and lipid deposition with differential modulation of ROS and kinase signaling pathways. Regulation of ROS generation through the differential modulation of ROS-regulating gene expression was revealed to have an important role in the suppression of adipogenesis and increase of osteogenesis in eADSCs following 3,4'-DHF treatment. These results suggest that the flavonoid 3,4'-DHF can be used to regulate adipogenesis in ADSCs, which has potential therapeutic application in regenerative medicine or health care for humans and many sport or companion animals. J. Cell. Biochem. 118: 1065-1077, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Both cytogenetic and molecular genetic studies can contribute to the management of patients with cancer. In some cases, genetic markers are specific to particular tumor types and are useful in diagnosis. This can be helpful in distinguishing histologically similar tumors that may respond differently to treatment and can sometimes be of prognostic value. Genetic markers can also be tools for following the response of a tumor to therapy, providing a sensitive means to detect relapse. This introductory unit considers some of the types of genetic changes that occur in association with malignancies, as well as major approaches used in their detection. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

The endothelial-to-mesenchymal transition (EndMT) in endothelial cells contributes to the development of cardiac fibrosis, ultimately leading to cardiac remodeling. In this study, the effects and molecular mechanisms of celastrol (CEL) on transforming growth factor-β1 (TGF-β1)-induced EndMT in human umbilical vein endothelial (HUVEC-12) cells were investigated. The presented data demonstrated that CEL significantly blocked the morphology change of HUVEC-12 cells induced by TGF-β1 without cell cytotoxicity. In accordance with these findings, CEL blocked TGF-β1-induced EndMT as evidenced by the inhibition of the mesenchymal markers, including collagen I, III, α-SMA, fibronectin mRNA expression, and the increase in the mRNA expression of endothelial cell marker CD31. These changes were also confirmed by double immunofluorescence staining of CD31 and vimentin. The in vitro scratch assay showed that CEL inhibited the migration capacity of the transitioned endothelial cells induced by TGF-β1. Further experiments showed that the beneficial effect of CEL on blocking the EndMT in HUVEC-12 cells was associated with the suppression of the TGF-β1/Smads signalling pathway, which was also confirmed by the inhibition of its downstream transcription factor snail1, twist1, twist2, ZEB1 and ZEB2. These results indicate that CEL blocks TGF-β1-induced EndMT through TGF-β1/Smads signalling pathway and suggest that it may be a feasible therapy for cardiac fibrosis diseases.

Our previous studies have shown that maternal high estradiol (E 2 ) environment increased the risk of thyroid dysfunction in offspring. However, the mechanism involved remains unexplored. To evaluate the thyroid function of offspring after high E 2 exposure and to explore the underlying mechanism, we established a high E 2 mouse model of early pregnancy, and detected thyroid hormones of their offspring. In thyroids of offspring, the expressions of Tg, Nis, Tpo, Pax8, and Titf1 and CpG island methylation status of Pax8 and genes involved in methylation were analyzed. We found that thyroxine (T4) and FT4 levels of offspring were obviously increased in the high-E 2 group, especially in females. In both 3- and 8-week-old offspring of the high-E 2 group, Pax8 was significantly up-regulated in thyroid glands, accompanied by the abnormal CpG island methylation status in the promoter region. Furthermore, Dnmt3a and Mbd1 were obviously down-regulated in thyroids of the high E 2 group. Besides, the disturbance of thyroid function in females was more severe than that in males, implying that the effects were related to gender. In summary, our study indicated that maternal high E 2 exposure disturbed the thyroid function of offspring through the dysregulation and abnormal DNA methylation of Pax8.

Previously, we found that Celastrus orbiculatus Thunb. (COT) decreases athero-susceptibility in lipoproteins and the aorta of guinea pigs fed a high-fat diet, and increases high-density lipoprotein (HDL). In the present study, we investigated the effect of COT in reducing lipid accumulation and promoting reverse cholesterol transport (RCT) in vivo and vitro. Healthy male mice were treated with high-fat diet alone, high-fat diet with COT (10.0 g/kg/d), or general fodder for 6 weeks. Serum levels of total cholesterol (TC), triglyceride (TG), HDL-C, non-HDL-C, and (3)H-cholesterol in plasma, liver, bile, and feces were determined. Pathological changes and the levels of TC and TG in liver were examined. The expression of hepatic genes and protein associated with RCT were analyzed. COT administration reduced lipid accumulation in the liver, ameliorated the pathological changes, and lessened liver injury, the levels of TG, TC, and non-HDL-C in plasma were decreased significantly, and COT led to a significant increase in plasma HDL-C and apolipoprotein A (apoA1). (3)H-cholesterol in plasma, liver, bile, and feces was also significantly increased in COT-treated mice compared to controls. Both mRNA and protein expression of SRB1, CYP7A1, LDLR, ATP-binding cassette transporters ABCA1, ABCG5, and LXRα were improved in COT-treated mice. An in vitro isotope tracing experiment showed that COT and its bioactive ingredients, such as celastrol, ursolic acid, oleanolic acid, and quercetin, significantly increased the efflux of (3)H-cholesterol. They also increased the expression of SRB1, ABCA1, and ABCG1 significantly in macrophages. Our findings provided a positive role of COT in reducing lipid accumulation by promoting RCT. These effects may be achieved by activating the SRB1 and ABC transporter pathway and promoting cholesterol metabolism via the CYP7A1 pathway in vivo. The effective ingredients in vitro are celastrol, ursolic acid, oleanolic acid, and quercetin.

A digestibility trial was performed with gilthead sea bream juveniles (IBW = 72 g) fed four diets differing in lipid source (fish oil, FO; or a blend of vegetable oil, VO) and starch content (0 %, CH-; or 20 %, CH+) to evaluate the potential interactive effects between carbohydrates and VO on the processes involved in digestion, absorption and transport of lipids and glucose. In fish fed VO diets a decrease in lipid digestibility and in cholesterol (C), High Density Lipoprotein(HDL)-C and Low Density Lipoprotein (LDL)-C (only in CH+ group) were recorded. Contrarily, dietary starch induced postprandial hyperglycemia and time related alterations on serum triacylglycerol (TAG), phospholipid (PL) and C concentrations. Fish fed a CH+ diet presented lower serum TAG than CH- group at 6 h post-feeding, and the reverse was observed at 12 h post-feeding for TAG and PL. Lower serum C and PL at 6 h post-feeding were recorded only in VOCH+ group. No differences between groups were observed in hepatic and intestinal transcript levels of proteins involved in lipid transport and hydrolysis (FABP, DGAT, GPAT, MTP, LPL, LCAT). Lower transcript levels of proteins related to lipid transport (ApoB, ApoA1, FABP2) were observed in the intestine of fish fed the CH+ diet, but remained unchanged in the liver. Overall, transcriptional mechanisms involved in lipid transport and absorption were not linked to changes in lipid serum and digestibility. Dietary starch affected lipid absorption and transport, probably due to a delay in lipid absorption. This study suggests that a combination of dietary VO and starch may negatively affect cholesterol absorption and transport.

Gene expression changes are linked to air pollutant exposures in in vitro and animal experiments. However, limited data are available on how these outcomes relate to ambient air pollutant exposures in humans. We performed an exploratory analysis testing whether gene expression levels were associated with air pollution exposures in a Los Angeles area cohort of elderly subjects with coronary artery disease. Candidate genes (35) were selected from published studies of gene expression-pollutant associations. Expression levels were measured weekly in 43 subjects (≤ 12 weeks) using quantitative PCR. Exposures included gaseous pollutants O3, nitrogen oxides (NOx), and CO; particulate matter (PM) pollutants elemental and black carbon (EC, BC); and size-fractionated PM mass. We measured organic compounds from PM filter extracts, including polycyclic aromatic hydrocarbons (PAHs), and determined the in vitro oxidative potential of particle extracts. Associations between exposures and gene expression levels were analyzed using mixed-effects regression models. We found positive associations of traffic-related pollutants (EC, BC, primary organic carbon, PM 0.25-2.5 PAH and/or PM 0.25 PAH, and NOx) with NFE2L2, Nrf2-mediated genes (HMOX1, NQO1, and SOD2), CYP1B1, IL1B, and SELP. Findings suggest that NFE2L2 gene expression links associations of traffic-related air pollution with phase I and II enzyme genes at the promoter transcription level.

Scope: The protective effect of fish consumption on the cardiovascular system has primarily been ascribed to n-3 fatty acids, but data investigating the vascular effects of fish protein consumption are scarce. This study aimed to investigate the vascular impact of fish protein in a mouse model of atherosclerosis., Methods and Results: Male apoE null mice were fed a Western diet containing 20% fish (turbot) protein, casein, or soy protein, for 16 wk. Morphometric analysis of the aorta revealed that the atherosclerotic plaque area of fish protein fed mice was twofold larger than that in casein- or soy protein-fed mice. The percentage area of calcification deposits in plaques of fish protein fed mice was higher (7.57%) than that in casein-fed (2.86%) or soy protein-fed (3.46%) mice, and fish protein fed mice exhibited higher plaque expression of CD68, CD36, and IL-6 than the other two groups. Fish protein intake was accompanied by increased serum concentrations of trimethylamine-N-oxide (7.03 ± 2.83 μmol/L), as compared with casein (0.92 ± 0.46 μmol/L) and soy protein (1.32 ± 0.54 μmol/L) intake., Conclusion: The present data indicate adverse effects of fish protein on the vascular system, which could be attributable to the high serum trimethylamine-N-oxide concentrations in these mice., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Interpopulation differences in drug responses are well documented, and in some cases they correspond to differences in the frequency of associated genetic markers. Understanding the diversity of genetic markers associated with drug response across different global populations is essential to infer population rates of drug response or risk for adverse drug reactions, and to guide implementation of pharmacogenomic testing. Sri Lanka is a culturally and linguistically diverse nation, but little is known about the population genetics of the major Sri Lankan ethnic groups. The objective of this study was to investigate the diversity of pharmacogenomic variants in the major Sri Lankan ethnic groups., Methods: We examined the allelic diversity of more than 7000 variants in genes involved in drug biotransformation and response in the three major ethnic populations of Sri Lanka (Sinhalese, Sri Lankan Tamils, and Moors), and compared them with other South Asian, South East Asian, and European populations using Wright's Fixation Index, principal component analysis, and STRUCTURE analysis., Results: We observed overall high levels of similarity within the Sri Lankan populations (median FST=0.0034), and between Sri Lankan and other South Asian populations (median FST=0.0064). Notably, we observed substantial differentiation between Sri Lankan and European populations for important pharmacogenomic variants related to warfarin (VKORC1 rs9923231) and clopidogrel (CYP2C19 rs4986893) response., Conclusion: These data expand our understanding of the population structure of Sri Lanka, provide a resource for pharmacogenomic research, and have implications for the clinical use of genetic testing of pharmacogenomic variants in these populations.

In this study, we evaluated the toxic and genotoxic potential of zinc oxide nanoparticles (ZnO NPs) of 20 nm and the mutagenic potential of these ZnO NPs as well as that of an amorphous ZnO. Toxicity was assessed by XTT colorimetric assay. ZnO NPs were toxic at concentrations equal to or higher than 240.0 μM. Genotoxicity was assessed by in vitro Cytokinesis Block Micronucleus Assay (CBMN) in V79 cells. ZnO NPs were genotoxic at 120.0 μM. The mutagenic potential of amorphous ZnO and the ZnO NPs was assayed using the wing Somatic Mutation and Recombination Test (SMART) of Drosophila melanogaster. In the Standard cross, the amorphous ZnO and ZnO NPs were not mutagenic. Nevertheless, Marker trans-heterozygous individuals from the High bioactivation cross treated with amorphous ZnO (6.25 mM) and ZnO NPs (12.50 mM) displayed a significant increased number of mutant spots when compared with the negative control. In conclusion, the results were not dose related and indicate that only higher concentrations of ZnO NPs were toxic and able to induce genotoxicity in V79 cells. The increase in mutant spots observed in D. melanogaster was generated due to mitotic recombination, rather than mutational events., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Current studies of environmental health suggest a link between air pollution components, such as particulate matter (PM), and various diseases. However, the specific genes and regulatory mechanisms implicated in PM-induced diseases remain largely unknown. Epigenetic systems such as covalent modification of histones in chromatin may mediate environmental factors in gene regulation. Investigating the relationships between PM exposure and histone modification status may help understand the mechanisms underlying environment-associated health conditions., Methods: In this study, we obtained genome-wide profiles of H3K27ac (histone 3 lysine 27 acetylation), known to be an active gene regulatory histone modification marker, in blood samples collected from four Chinese individuals exposed to high or low PM2.5 (particles with diameters up to 2.5 μm)., Results: The genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) data indicated a comprehensive differential H3K27ac landscape across the individual genomes, which was associated with high PM2.5. Moreover, a substantial number of these PM2.5-associated differential H3K27ac markers were in genes involved in immune cell activation, potentially linking these epigenetic changes with air pollution-induced immune and inflammatory responses., Conclusions: Our study provides the first genome-wide characterization of H3K27ac profiles in individuals subjected to different exposure levels of PM2.5. Future systematic investigations of the relationships between air pollutants and histone modifications in large population samples are warranted to elucidate the contributions of histone modifications to environment-associated diseases.

Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity., (Copyright © 2014 John Wiley & Sons, Ltd.)

Di(2-ethylhexyl)phthalate (DEHP) is the most common plasticizer in plastic devices of everyday use. It is a ubiquitous environmental contaminant and primarily known to impair male gonadal development and fertility. Studies concerning the long-term effects of prenatal DEHP exposure on certain diseases [The Developmental Origins of Health and Disease paradigm (DOHaD) hypothesis] are scarce although it is proven that DEHP crosses the placenta. Rising environmental pollution during the last centuries coincides with an increasing prevalence of cardiovascular and metabolic diseases. We have investigated the effects of an early embryonic DEHP exposure at different developmental stages on cardiomyogenesis. We used an in-vitro model, the murine P19 embryonic carcinoma cell line (P19 ECC), mimicking early embryonic stages up to differentiated beating cardiomyocytes. P19 ECC were exposed to DEHP (5, 50, 100 µg ml(-1)) at the undifferentiated stage for 5 days and subsequently differentiated to beating cardiomyocytes. We analyzed the expression of metabolic (Pparg1, Fabp4 and Glut4), cardiac (Myh6, Gja1) and methylation (Dnmt1, Dnmt3a) marker genes by quantitative real-time PCR (qRT-PCR), beating rate and the differentiation velocity of the cells. The methylation status of Pparg1, Ppara and Glut4 was investigated by pyrosequencing. DEHP significantly altered the expression of all investigated genes. The beating rate and differentiation velocity were accelerated. Exposure to DEHP led to small but statistically significant increases in methylation of specific CpGs within Ppara and Pparg1, which otherwise were generally hypomethylated, but methylation of Glut4 was unaltered. Early DEHP exposure of P19 ECC alters the expression of genes associated with cellular metabolism and the functional features of cardiomyocytes., (Copyright © 2014 John Wiley & Sons, Ltd.)

Context: Insecticides are used to control pests. Cypermethrin and fenvalerate are widely used pirethroid insecticides in the world. Rosa canina L. (Rosaceae) is used as a traditional medicinal plant against viral infections and disorders of the kidneys and urinary tract due to its high vitamin C level., Objective: The genotoxic effects of cypermethrin and fenvalerate were examined with the micronucleus (MN) test. Then, we determined the ability of the water (RC(wtr)) and ethanol (RC(eta)) extracts of rosehip (R. canina) to overcome the possible genotoxic effects of the insecticides., Materials and Methods: Preliminary studies determined that the application concentrations were 20, 30, 40, and 50 ppm for cypermethrin, 25, 50, 75, and 100 ppm for fenvalerate, and 100 ppm for rosehip extracts. DMSO (dimethyl sulphoxide) (1%) and 1 mM EMS (ethyl methanesulfonate) were used as negative and positive control groups, respectively. The application groups belonging to insecticides and plant extracts were added to culture tubes including chromosome B medium and peripheral blood for MN test., Results: The MN frequencies were found 0.725 in the negative control group, 2.700 in the positive control groups, 1.275 in the highest application group of cypermethrin, and 1.600 in the highest application group of fenvalerate. The MN frequencies in cypermethrin + RC(wtr), cypermethrin + RC(eta), fenvalerate + RC(wtr), and fenvalerate + RC(eta) application groups were, respectively, determined as 1.000, 1.075, 1.225, and 1.275., Conclusion: According to the results, cypermethrin and fenvalerate have genotoxic effects, the water and ethanol extracts of rosehip reduced the genotoxicity of the both insecticides.

Pharmacogenomic markers in the HLA coding genes have been associated with drug hypersensitivity of multiple drugs, including allopurinol. In this systematic review, we summarize the pharmacogenomic evidence available regarding allopurinol-induced cutaneous adverse drug reactions (cADRs). We found that the HLA-B*5801 allele was significantly associated with the risk of severe cADRs in the Han Chinese, Korean, Thai, Japanese and European populations. The association between less severe cADRs and HLA-B*5801 was less consistent. All SNPs identified in genome-wide association studies of common variants were also located in or nearby HLA-B*5801. Future studies should focus on more common but less severe allopurinol-induced cADRs, as well as the potential role of rare variants as predictors of these cADRs.

Maternal deficiencies in micronutrients affecting one-carbon metabolism before and during pregnancy can influence metabolic status and the degree of insulin resistance and obesity of the progeny in adulthood. Notably, maternal and progeny plasma S-adenosylhomocysteine (SAH) levels are both elevated after vitamin deficiency in pregnancy. Therefore, we investigated whether this key one-carbon cycle intermediate directly affects adipocyte differentiation and function. We found that expansion and differentiation of murine 3T3-L1 preadipocytes in the presence of SAH impaired both basal and induced glucose uptake as well as lipolysis compared with untreated controls. SAH did not alter preadipocyte factor 1 (Dlk1) or peroxisome proliferator-activated receptor-γ 2 (Pparγ2) but significantly reduced expression of CAAT enhancer-binding protein-α (Cebpα), Cebpβ, and retinoid x receptor-α (Rxrα) compared with untreated adipocytes. SAH increased Rxrα methylation on a CpG unit (chr2:27,521,057+, chr2:27,521,049+) and CpG residue (chr2:27,521,080+), but not Cebpβ methylation, relative to untreated adipocytes. Trimethylated histone H3-Lys27 occupancy was significantly increased on Cebpα and Rxrα promoters in SAH-treated adipocytes, consistent with the reduction in gene expression. In conclusion, SAH did not affect adipogenesis per se but altered adipocyte functionality through epigenetic mechanisms, such that they exhibited altered glucose disposal and lipolysis. Our findings implicate micronutrient imbalance in subsequent modulation of adipocyte function., (© 2014 by the American Diabetes Association.)

Busulfan, a cytotoxic alkylating agent used for treatment of chronic myeloid leukemia has effects in mammalian germ cells. In fish species, the use of this compound is of special interest in intra and interspecies germ cell transplants. To determine the effects of busulfan in fish a previous range finding experiment was designed. Survival and growth rate of 150-days after hatching (150DAH) Senegalese sole (Solea senegalensis) juveniles was determined. In a second experiment, the effects of a sublethal busulfan dose in fish germ cell depletion and in somatic tissues were analysed. Sublethal effects of several busulfan treatments (B10-10 days after injection, B20-20 days after injection, B20÷-20 days after injection with double injection) were determined in somatic and gonadal tissues. Alterations were registered through histopathological techniques, TUNEL (cell apoptosis) and quantified at molecular level (Q-PCR analyses) using the vasa mRNAs (Ssvasa1-2 and Ssvasa3-4 mRNAs) as molecular markers for germinal cells in Senegalese sole juveniles. Several sublethal effects were observed with 40 mg kg⁻¹ busulfan, a non-lethal dose, such as: pyknosis in liver, increase of melanomacrophage centres and blood stagnation in spleen and interruption of gonadal development. Females were more affected by busulfan treatments than males in terms of germ cell disruption, since a significant decrease in the expression of both Ssvasa1-2 and Ssvasa3-4 markers was found in the gonad of treated females rather than males. At 10 days post-treatment (B10), females already presented a decrease in germ cell proliferation, as confirmed by Q-PCR. Ssvasa expression proved to be a reliable tool for the direct evaluation of the effects of busulfan on Senegalese sole gonadal development, proving that busulfan can be a suitable treatment for causing transient sterility in recipient gonads for germ cell transplantation.

Background and Objective: The periodontal ligament (PDL) is vital to maintaining the homeostasis of the tooth and periodontal tissue. The influence of iron levels on the cytodifferentiation of PDL cells has not been studied, despite evidence that iron overload or deficiency can have adverse effects on alveolar bone density. The purpose of this study was to examine the effects of altered iron levels on cytodifferentiation in human PDL cells., Material and Methods: Human PDL cells were incubated with culture media supplemented with 10-50 μm ammonium ferric citrate or 5 μm deferoxamine (an iron chelator) during differentiation. Intracellular iron status was assessed by measuring changes in the expression of ferritin RNA and protein. PDL cell differentiation and function were evaluated by measuring osteoblast differentiation gene markers and the capacity of cultures to form mineralized nodules., Results: Iron accumulation resulted in upregulation of light and heavy chain ferritin proteins. Concurrently, osteoblast differentiation gene markers and mineralized nodule formation were suppressed. Iron deficiency resulted in downregulation of light and heavy chain ferritin proteins, suppression of alkaline phosphatase activity and formation of mineralized nodules during PDL cell differentiation., Conclusion: We conclude that iron is critical for normal cell differentiation of human PDL cells., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Serum sclerostin levels have been reported to be inversely associated with serum 25OHD levels, but the effect of vitamin D and calcium supplementation on serum sclerostin levels is unknown. This study was carried out to determine whether vitamin D and calcium supplementation altered serum sclerostin levels in healthy older adults., Design: We measured serum sclerostin levels at baseline and after 2 years in 279 men and women who participated in a placebo-controlled vitamin D (700 IU/day) and calcium (500 mg/day) intervention trial carried out in men and women aged ≥65 years., Method: Serum sclerostin levels were measured using the MesoScale Discovery chemiluminescence assay., Results: In the men, sclerostin levels increased over 2 years by 4.11±1.81 ng/l (13.1%) in the vitamin D plus calcium-supplemented group and decreased by 3.16±1.78 ng/l (10.9%) in the placebo group (P=0.005 for difference in change). Adjustments for the season of measurement, baseline physical activity levels, baseline serum sclerostin levels, and total body bone mineral content did not substantially alter the changes. In the women, there was no significant group difference in change in serum sclerostin levels either before or after the above-mentioned adjustments. In both the sexes, vitamin D and calcium supplementation significantly increased serum ionized calcium levels and decreased parathyroid hormone levels., Conclusion: Men and women appear to have different serum sclerostin responses to vitamin D and calcium supplementation. The reason for this difference remains to be determined.