mRNA Expression Of Key Genes Research Articles

Cyclin-Dependent Kinase and Antioxidant Gene Expression in Cancers with Poor Therapeutic Response.

Pancreatic cancer, hepatocellular carcinoma (HCC), and mesothelioma are treatment-refractory cancers, and patients afflicted with these cancers generally have a very poor prognosis. The genomics of these tumors were analyzed as part of The Cancer Genome Atlas (TCGA) project. However, these analyses are an overview and may miss pathway interactions that could be exploited for therapeutic targeting. In this study, the TCGA Pan-Cancer datasets were queried via cBioPortal for correlations among mRNA expression of key genes in the cell cycle and mitochondrial (mt) antioxidant defense pathways. Here we describe these correlations. The results support further evaluation to develop combination treatment strategies that target these two critical pathways in pancreatic cancer, hepatocellular carcinoma, and mesothelioma.

Plant Sterol Ester of α-Linolenic Acid Attenuates Nonalcoholic Fatty Liver Disease by Rescuing the Adaption to Endoplasmic Reticulum Stress and Enhancing Mitochondrial Biogenesis.

Nonalcoholic fatty liver disease (NAFLD) is becoming more common in the world and is presenting a great challenge concerning prevention and treatment. Plant sterol ester of α-linolenic acid (PS-ALA) has a potential benefit to NAFLD. To examine the effect of PS-ALA on NAFLD, C57BL/6J mice were given a control diet, high fat and high cholesterol diet (HFD), and HFD plus 2% PS, 1.3% ALA, or 3.3% PS-ALA for 16 weeks. Our results showed that PS-ALA treatment suppressed hepatic steatosis, ameliorated lipid disorder, attenuated inflammatory response, and inhibited oxidative stress. In the molecular level, PS-ALA downregulated high transcriptional and translational levels of endoplasmic reticulum (ER) stress markers (Grp78 and Chop) leading to decreased protein expression of transcription factor and key enzymes involved in de novo lipogenesis (Srebp-1c and Fas) and cholesterol synthesis (Srebp-2 and Hmgcr). In parallel, PS-ALA blocked Nlrp3 activation and reduced release of IL-1β and IL-18 via inhibiting ER stress-induced sensitization of unfolded protein response sensors (Ire1α and Xbp1s). Finally, PS-ALA improved HFD-induced mitochondrial damage and fatty acid accumulation as exhibited by higher protein and mRNA expression of key genes administering mitochondrial biogenesis (Pgc-1α, Nrf1, and Tfam) and fatty acid β-oxidation (Pparα and Cpt1a). In conclusion, our study originally demonstrated that PS-ALA rescued ER stress, enhanced mitochondrial biogenesis, and thus ameliorated NAFLD.

MRNA expression of genes related to fat deposition during in vitro ovine adipogenesis

Fat deposition in animals involves adipogenic differentiation guided by transcriptional factors and other key factors. To understand the molecular mechanism underlying ovine adipogenic differentiation, the dynamic mRNA expression of key genes related to fat deposition, including peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid-binding protein 4 (FABP4), FABP5, and cellular retinoic acid-binding protein 2 (CRABP2), were analyzed during in vitro differentiation of ovine preadipocytes. The stromal vascular cells from underneath the tail fat tissue of 1-wk-old sheep were isolated and cultured, and the preadipocytes were induced using a cocktail of 3-isobutyl-1-methylxanthine, insulin, dexamethasone, and troglitazone. The cultivated cells were collected at different time points after induced differentiation. The expression levels of PPAR-γ, FABP4, FABP5, and CRABP2 were studied by quantitative real-time polymerase chain reaction. The expressions of these genes in sheep were compared with those in human and mouse retrieved from the Gene Expression Omnibus DataSets. We observed that the expression of PPAR-γ, FABP4, and FABP5 was increased upon differentiation of ovine preadipocytes, as in humans and mice. The expression of CRABP2 was sharply increased from days 0 to 2 after induced differentiation and was subsequently decreased. This expression pattern of CRABP2 was different from that observed in humans and mice. Our results provide new insights into the function of these genes in fat deposition.

Somatostatin Receptor Subtype-4 Regulates mRNA Expression of Amyloid-Beta Degrading Enzymes and Microglia Mediators of Phagocytosis in Brains of 3xTg-AD Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder resulting in memory and cognitive impairment. The use of somatostatin receptor subtype-4 (SSTR4) agonists have been proposed for AD treatment. This study investigated the effects of selective SSTR4 agonist NNC 26-9100 on mRNA expression of key genes associated with AD pathology (microglia mediators of Aβ phagocytosis, amyloid-beta (Aβ)-degrading enzymes, anti-oxidant enzymes and pro-inflammatory cytokines) in 3xTg-AD mice. Mice were administered NNC 26-9100 (0.2µg, i.c.v.) or vehicle control, with cortical and subcortical brain tissue collected at 6h and 24h post-treatment. At 6h, NNC 26-9100 treatment decreased cortical expression of cluster of differentiation-33 (Cd33) by 25%, while increasing cortical and subcortical macrophage scavenger receptor-1 (Msr1) by 1.8 and 2.0-fold, respectively. The Cd33 downregulation and Msr1 upregulation support a state of microglia associated Aβ phagocytosis. At 24h, NNC 26-9100 treatment increased the cortical expression of Sstr4 (4.9-fold), Aβ-degrading enzymes neprilysin (9.3-fold) and insulin degrading enzyme (14.8-fold), and the antioxidant catalase (3.6-fold). Similar effects at 24h were found in subcortical tissue with NNC 26-9100 treatment, but did not reach statistical significance. No changes in pro-inflammatory cytokine expression were found. These data demonstrated NNC 26-9100 facilitates transcriptional changes in brain tissue identified with Aβ phagocytosis and clearance, further supporting SSTR4 as a treatment target for AD.

Single-walled carbon nanotubes repress viral-induced defense pathways through oxidative stress

Exposure of lung cells in vitro or mice to single-walled carbon nanotubes (SWCNTs) directly to the respiratory tract leads to a reduced host anti-viral immune response to infection with influenza A virus H1N1 (IAV), resulting in significant increases in viral titers. This suggests that unintended exposure to nanotubes via inhalation may increase susceptibility to notorious respiratory viruses that carry a high social and economic burden globally. However, the molecular mechanisms that contribute to viral susceptibility have not been elucidated. In the present study, we identified the retinoic acid-induced gene I (RIG-I) like receptors (RLRs)/mitochondrial antiviral signaling (MAVS) pathway as a target of SWCNT-induced oxidative stress in small airway epithelial cells (SAEC) that contribute to significantly enhanced influenza viral titers. Exposure of SAEC to SWCNTs increases viral titers while repressing several aspects of the RLR pathway, including mRNA expression of key genes (e.g. IFITs, RIG-I, MDA5, IFNβ1, CCL5). SWCNTs also reduce mitochondrial membrane potential without altering oxygen consumption rates. Our findings also indicate that SWCNTs can impair formation of MAVS prion-like aggregates, which is known to impede downstream activation of the RLR pathway and hence the transcriptional production of interferon-regulated anti-viral genes and cytokines. Furthermore, application of the antioxidant NAC alleviates inhibition of gene expression levels by SWCNTs, as well as MAVS signalosome formation, and increased viral titers. These data provide evidence of targeted impairment of anti-viral signaling networks that are vital to immune defense mechanisms in lung cells, contributing to increased susceptibility to IAV infections by SWCNTs.

Decellularized Human Gut as a Natural 3D Platform for Research in Intestinal Fibrosis.

The current methodologies for the identification of therapeutic targets for inflammatory bowel disease (IBD) are limited to conventional 2-dimensional (2D) cell cultures and animal models. The use of 3D decellularized human intestinal scaffolds obtained from surgically resected intestine and engineered with human intestinal cells may provide a major advancement in the development of innovative intestinal disease models. The aim of the present study was to design and validate a decellularization protocol for the production of acellular 3D extracellular matrix (ECM) scaffolds from the human duodenum. Scaffolds were characterized by verifying the preservation of the ECM protein composition and 3D architecture of the native intestine and were employed for tissue engineering with primary human intestinal myofibroblasts for up to 14 days. Engrafted cells showed the ability to grow and remodel the surrounding ECM. mRNA expression of key genes involved in ECM turnover was significantly different when comparing primary human intestinal myofibroblasts cultured in 3D scaffolds with those cultured in standard 2D cultures on plastic dishes. Moreover, incubation with key profibrogenic growth factors such as TGFβ1 and PDGF-BB resulted in markedly different effects in standard 2D vs 3D cultures, further emphasizing the importance of using 3D cell cultures. These results confirm the feasibility of 3D culture of human intestinal myofibroblasts in intestinal ECM scaffolds as an innovative platform for disease modeling, biomarker discovery, and drug testing in intestinal fibrosis.

The role of nuclear receptors in the differentiation of oligodendrocyte precursor cells derived from fetal and adult neural stem cells

Oligodendrocyte precursor cells (OPCs) differentiation from multipotent neural stem cells (NSCs) into mature oligodendrocytes is driven by thyroid hormone and mediated by thyroid hormone receptors (TRs). We show that several nuclear receptors display strong changes in expression levels between fetal and adult NSCs, with an overexpression of TRβ and a lower expression of RXRγ in adult. Such changes may determine the reduced capacity of adult OPCs to differentiate as supported by reduced yield of maturation and compromised mRNA expression of key genes. RXRγ may be the determinant of these differences, on the evidence of reduced number of mature oligodendrocytes and increased number of proliferating OPCs in RXRγ−/− cultures. Such data also points to RXRγ as an important regulator of the cell cycle exit, as proved by the dysregulation of T3-induced cell cycle exit-related genes.Our data highlight the biological differences between fetal and adult OPCs and demonstrate the essential role of RXRγ in the T3-mediated OPCs maturation process.

Constitutive Ptpn2‐Loss in Mice Decreases Number and Compromises Function of Intestinal Goblet and Paneth cells

Among the ~200 susceptibility loci associated with Inflammatory Bowel Disease (IBD), are loss‐of‐function single nucleotide polymorphisms (SNPs) in the locus (18p11) containing the Protein Tyrosine Phosphatase non‐receptor Type 2 (PTPN2) gene. PTPN2 encodes the T Cell Protein Tyrosine Phosphatase (TCPTP) that functions as a negative regulator of diverse intracellular pathways repressing inflammatory signals. In Ptpn2‐deficient mice, we observed altered intestinal homeostasis in the form of intestinal epithelial barrier defects and altered intestinal microbiome composition. Intestinal epithelial homeostasis is maintained by active‐cycling and slow cycling stem cells confined within the crypt‐based niche and subsequent differentiation into secretory and absorptive epithelial lineages as cells migrate up the crypt‐villus axis. The goal of this study was to determine whether Ptpn2‐loss affects intestinal epithelial cell populations and function that could contribute to altered host‐bacterial interactions.MethodsIntestinal tissues (ileum, cecum, proximal and distal colon) from 21 day old wild type (WT), heterozygous (HET) and constitutive Ptpn2 knockout (KO) Balb/C mice were harvested and processed for imaging to detect proliferation (Ki‐67), apoptosis (TUNEL), Paneth cell (lysozyme), and goblet cell (periodic‐acid Shiff) numbers via staining of specific markers. cDNA was generated by reverse transcriptase, and mRNA expression of key genes associated with Paneth and goblet cells function were measured by PCR.ResultsProximal and distal colon of Ptpn2‐KO mice showed clusters of apoptotic cells at the crypt base in comparison to wildtype. In addition, the number of proliferative cells was higher in the crypts of proximal and distal colon of Ptpn2‐KO mice compared to WT (p=0.0032 and p=0.0005 respectively; n=3). Lysozyme‐positive Paneth cells were significantly reduced in HET mice (p=0.0452 vs. WT; n=2–5) and further reduced in KO mice (p=0.0040; n=4). Moreover, KO mice were almost completely devoid of both lysozyme positive and negative Paneth cells, the latter characterized by large cytosolic granules at the crypt base. mRNA expression of the Paneth cell antimicrobial peptide, Lyz1, was reduced in Ptpn2‐KO mice compared to WT (p=0.017; n=2), whereas expression of antimicrobial peptides Defa5 and Defa6 were not altered. Goblet cell number was slightly reduced in the ileum and distal colon of KO mice (n=2). There was also a preliminary trend towards reduced expression of goblet cell secreted mucin proteins, Muc2, a major mucus layer constituent, and Muc3, a mucin protein homologous to MUC17 in humans shown to be protective against enteroinvasive E. coli, in the cecum of Ptpn2‐KO mice compared to wildtype (n=2).ConclusionPtpn2‐deficiency compromises intestinal epithelial cell differentiation leading to altered numbers and function of Paneth and goblet cells consistent with increased susceptibility to bacterial infection.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM), the Crohn's and Colitis Foundation (DFM), and Science Without Borders Fellowship (VC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Maternal exposure to imazalil disrupts the endocrine system in F1 generation mice

The fungicide imazalil (IMZ), an AR antagonist, has been linked to endocrine disruption in animals. Here, adult female C57BL/6 mice were administered IMZ through their drinking water at levels of 0, 0.025‰ and 0.25‰ during the gestation and lactation periods (the exposed females are marked as F0, and the offspring are marked as F1). Then, we evaluated the physiological, biochemical and gene expression levels in mice after maternal IMZ exposure. The genes involved in sex hormone receptors, cholesterol synthesis and T synthesis were generally inhibited, and the serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were also decreased in the F0 generation female mice. In addition, after F0 IMZ exposure, ovarian androgen receptor (AR) expression was significantly inhibited, and the androgen levels in the serum increased significantly. This may lead to the appearance of progressive virilization during pregnancy. This phenomenon leads to an aromatase deficiency in the F1 generation mice, which results in a decrease in androgen conversion into estrogen and androgen accumulation. In addition, the mRNA expression of key genes and the serum TC, HDL-C, and LDL-C levels increased in the F1 generation after maternal exposure to IMZ. In addition, testicular TC and LDL-C levels also decreased in the F1 generation male mice. Molecular docking analysis revealed that key hydrogen bonds were formed by nitrogen atoms of the imidazole bonds with Trp751 of the ARs. Our data suggests that maternal IMZ exposure could induce endocrine disruption in the next generation of mice.

CA Species in Hepatic Bile Acid Compositions Decreased Largely in the Process of Steatosis When NAFLD Was Not Accompanied by Obesity or Diabetes Mellitus

Introduction: Conclusive findings about hepatic bile acid(BA) composition in NAFLD have not been established. We suppose that findings of hepatic BA composition may be related to both steatosis and accompanied diseases such as obesity or diabetes mellitus, however, it is unknown how hepatic BA composition alter in the process of steatosis. The aim of this study was to investigate alteration of hepatic BA composition in NAFLD model without obesity or diabetes mellitus. Methods: Wistar Kyoto rats were randomly assigned(n=6/group) to be fed with either normal chow diet or choline-deficient(CD) diet for 5 weeks. Two groups were studied: (a)WKY fed with normal diet(WKY-N) and (b)WKY fed with choline-deficient diet(WKY-CD). Hepatic BAs analysis was performed by liquid chromatography-electrospray ionization-tandem mass spectrometry technique. Results: 1. Histopathological findings:Livers of WKY-CD showed steatosis, whereas there was no steatosis in livers of SHR-N. 2. BA composition in WKY—N: In primary BAs of WKY-N, CA species, CDCA species, alpha-MCA species, beta-MCA species, HCA species were 174.9±27.9 nmol/g, 30.3±6.8nmol/g, 22.0±3.4nmol/g, 141.6 ±23.9nmol/g, and 14.0±4.1 nmol/g respectively, whereas in secondary BAs of WKY-N, DCA species, LCA species, UDCA species, HDCA species, omega-species, and MDCA species were 2.6± 0.8nmol/g, 0.0± 0.0nmol/g, 0.5± 0.5nmol/g, 31.0± 9.8nmol/g, 1.1± 0.4nmol/g, and 0± 0nmol/g, respectively. 3. Alteration of BA composition:In BAs of WKY-CD, CA species, CDCA species, alpha-MCA species, beta-MCA species, HCA species, DCA species, LCA species, UDCA species, HDCA species, omega-species, and MDCA were 81.8±16.5 nmol/g, 25.8± 7.9 nmol/g, 19.1±9.2 nmol/g, 125.5±81.3 nmol/g, 9.1±7.2 nmol/g, 4.0±4.7 nmol/g, 0±0 nmol/g, 2.5±1.0 nmol/g, 28.1±31.9 nmol/g, 6.3±3.0 nmol/g, and 0±0 nmol/g, respectively. The alteration of other BAs except for CA species was minor or slight in WKY-CD. 4. Hepatic mRNA expression of key genes involved in BA synthesis:In WKY-CD, mRNA expression of cyp27a1, which is associated with metabolism to CDCA, increased significantly. Conclusion: We demonstrated that CA species in hepatic BA compositions decreased largely in the process of steatosis when NAFLD did not be accompanied with obesity or diabetes mellitus.

Glycine Relieves Intestinal Injury by Maintaining mTOR Signaling and Suppressing AMPK, TLR4, and NOD Signaling in Weaned Piglets after Lipopolysaccharide Challenge

This study was conducted to envaluate whether glycine could alleviate Escherichia coli lipopolysaccharide (LPS)-induced intestinal injury by regulating intestinal epithelial energy status, protein synthesis, and inflammatory response via AMPK, mTOR, TLR4, and NOD signaling pathways. A total of 24 weanling piglets were randomly allotted to 1 of 4 treatments: (1) non-challenged control; (2) LPS-challenged control; (3) LPS + 1% glycine; (4) LPS + 2% glycine. After 28 days feeding, piglets were injected intraperitoneally with saline or LPS. The pigs were slaughtered and intestinal samples were collected at 4 h postinjection. The mRNA expression of key genes in these signaling pathways was measured by real-time PCR. The protein abundance was measured by Western blot analysis. Supplementation with glycine increased jejunal villus height/crypt depth ratio. Glycine also increased the jejunal and ileal protein content, RNA/DNA ratio, and jejunal protein/DNA ratio. The activities of citroyl synthetase in ileum, and α-ketoglutarate dehydrogenase complex in jejunum, were increased in the piglets fed diets supplemented with glycine. In addition, glycine decreased the jejunal and ileal phosphorylation of AMPKα, and increased ileal phosphorylation of mTOR. Furthermore, glycine downregulated the mRNA expression of key genes in inflammatory signaling. Meanwhile, glycine increased the mRNA expression of negative regulators of inflammatory signaling. These results indicate that glycine supplementation could improve energy status and protein synthesis by regulating AMPK and mTOR signaling pathways, and relieve inflammation by inhibiting of TLR4 and NOD signaling pathways to alleviate intestinal injury in LPS-challenged piglets.

D-Serine Metabolism and Its Importance in Development of Dictyostelium discoideum.

In mammals, D-Ser is synthesized by serine racemase (SR) and degraded by D-amino acid oxidase (DAO). D-Ser acts as an endogenous ligand for N-methyl-D-aspartate (NMDA)- and δ2 glutamate receptors, and is involved in brain functions such as learning and memory. Although SR homologs are highly conserved in eukaryotes, little is known about the significance of D-Ser in non-mammals. In contrast to mammals, the slime mold Dictyostelium discoideum genome encodes SR, DAO, and additionally D-Ser specific degradation enzyme D-Ser dehydratase (DSD), but not NMDA- and δ2 glutamate receptors. Here, we studied the significances of D-Ser and DSD in D. discoideum. Enzymatic assays demonstrated that DSD is 460- and 1,700-fold more active than DAO and SR, respectively, in degrading D-Ser. Moreover, in dsd-null cells D-Ser degradation activity is completely abolished. In fact, while in wild-type D. discoideum intracellular D-Ser levels were considerably low, dsd-null cells accumulated D-Ser. These results indicated that DSD but not DAO is the primary enzyme responsible for D-Ser decomposition in D. discoideum. We found that dsd-null cells exhibit delay in development and arrest at the early culmination stage. The efficiency of spore formation was considerably reduced in the mutant cells. These phenotypes were further pronounced by exogenous D-Ser but rescued by plasmid-borne expression of dsd. qRT-PCR analysis demonstrated that mRNA expression of key genes in the cAMP signaling relay is perturbed in the dsd knockout. Our data indicate novel roles for D-Ser and/or DSD in the regulation of cAMP signaling in the development processes of D. discoideum.

Embryonic exposure to valproic acid affects the histaminergic system and the social behaviour of adult zebrafish (Danio rerio).

Histamine modulates several behaviours and physiological functions, and its deficiency is associated with neuropsychiatric disorders. Gestational intake of valproic acid (VPA) is linked to autism spectrum disorder (ASD), characterized by impaired sociability and stereotypies. VPA effects on the neurochemistry and functional morphology of the histaminergic system in ASD are unclear. Zebrafish are highly social, and given the similarities between zebrafish and human neurotransmitter systems, we have studied the effects of VPA on histamine in zebrafish. Histaminergic, dopaminergic and noradrenergic systems of larval and adult zebrafish exposed to VPA from the end of gastrulation until neural tube formation were studied using HPLC, quantitative PCR, immunocytochemistry and in situ hybridization. Sociability, dark-flash response and locomotion were also studied. Zebrafish larvae exposed to VPA showed decreased locomotion and an abnormal dark-flash response. Additionally, a reduced number of histaminergic neurons, low histamine and altered mRNA expression of key genes of the monoaminergic systems were also detected. The reduced mRNA expression of genes of the studied systems persisted until adulthood. Furthermore, adult VPA-exposed animals presented lower brain levels of noradrenaline and 3,4-dihydroxyphenylacetic acid, along with impaired sociability. VPA exposure in early development causes molecular and neurochemical alterations in zebrafish, which persist into adulthood and accompany impaired sociability. These findings will highlight the possible involvement of the histaminergic system in outcomes related to neuropsychiatric disorders. Furthermore, it supports zebrafish as a tool to investigate mechanisms underlying these disorders.

High salt diet decreases reproductive performance in rams and down-regulates gene expression of some components of the renin-angiotensin system in the testis

Although salt-tolerant plants can be used to combat dryland salinity, these plants contain high concentrations of salt (NaCl), which may have deleterious effects if fed to livestock. Twenty-four Merino rams (9 mo of age) with similar body weight were equally allocated to two groups and fed a normal- or high-salt diet (0.5 and 12% NaCl, respectively) for 3 mo. Rams fed the high-salt diet had lower live weight gains, higher water intake, smaller testes (466 ± 48.4 vs 604 ± 51.1 g) and reductions (P <0.05) in spermatogenesis, ejaculate volumes (0.89 ± 0.18 vs 1.24 ± 0.15 mL/ejaculate), sperm concentration (14.3 ± 2.05 vs 22.3 ± 2.33 × 109 sperm/mL) and DNA integrity (DNA fragmentation rate: 5.85 ± 1.09 vs 1.13 ± 0.14%) compared to controls. For in vitro fertilization, although cleavage percentage was not significantly affected, hatching rate was lower (30.8 ± 3.81 vs 52.8 ± 4.08%, P <0.05) for sperm from rams on the high-salt diet. Furthermore, the 12% salt diet decreased plasma concentrations of metabolic (leptin and insulin) and sex (T, FSH and LH) hormones, but did not affect the plasma renin-angiotensin system (RAS) component (REN, Ang II, ACE and ALD). Regarding components of the testicular RAS, the 12% salt diet decreased (P <0.05) expression of REN, Ang II and AT2 mRNA, although ACE and AT1 were unaffected. Furthermore, the 12% salt diet decreased (P <0.05) mRNA expression of key genes for spermatogenesis (Hsp70, c-kit and Cyclin A), and sex hormone receptors (AR, FSHR, LHR, CYP11A1 and CYP17A1), but there were no significant effects on key enzymes (LDH, SDH, AKP and ATPase) in the testis. In conclusion, the high-salt diet reduced ram reproductive performance; we inferred that changes in testis RAS may have had an important role in these reproductive defects.

Effect of amikacin on cell wall glycopeptidolipid synthesis in Mycobacterium abscessus.

Cultivation of the smooth colony Mycobacterium abscessus at the sub-minimum inhibitory concentration (MIC) of amikacin changed its growth pattern including its colony morphology (smooth to rough) and cell arrangement (dispersed to cord formation). In addition, reduced sliding motility and biofilm formation were observed. The amount of glycogpetidolipid (GPL) and mRNA expression of key genes involved in GPL synthesis were decreased in the amikacin-treated M. abscessus strain. An in vitro infection assay revealed that the amikacin-treated smooth M. abscessus strain induced more pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) than that of the smooth strain in murine macrophage cells. These results suggest that long-term exposure to a low concentration of amikacin causes a physical change in the cell wall which may increase its virulence.

Regulation of inflammation, antioxidant production, and methyl-carbon metabolism during methionine supplementation in lipopolysaccharide-challenged neonatal bovine hepatocytes

Supplementation of methionine (Met) may improve immunometabolic status, specifically during a period of inflammatory stress. The aim of the present study was to establish an inflammation model using primary neonatal bovine hepatocytes and to examine the effects of increasing concentrations of dl-Met and a maintained Met to lysine (Lys) ratio on hepatocyte inflammatory responses, antioxidant production, and Met metabolism during lipopolysaccharide (LPS) challenge. Hepatocytes isolated from 4 calves were maintained as monolayer cultures and exposed to 0, 10, or 40 µMdl-Met and 100 µM Lys (0Met100Lys, 10Met100Lys, or 40Met100Lys) or 10 µMdl-Met and 25 µM Lys (10Met25Lys). Cells were exposed to each treatment for 16 h and then challenged with either 0 or 100 ng/mL of LPS for 8 h. In the absence of LPS, glutathione (GSH) was not altered by 10Met100Lys or 10Met25Lys but was increased by 40Met100Lys. With LPS challenge, GSH concentration was decreased with 40Met100Lys and tended to be decreased with 10Met100Lys. Hepatocytes receiving 10Met100Lys treated with 100 ng/mL of LPS showed an inflammatory response with increased mRNA expression of tumor necrosis factor (TNFα), IL-6, IL-1β, and interferon gamma, which was accompanied by increased nuclear factor κB inhibitor and serum amyloid A3 mRNA. The treatment 40Met100Lys was effective for preventing the LPS-induced increase in expression of the above genes except TNFα. Similar preventative effects were observed for 10Met25Lys; however, it did not prevent the LPS-induced increase in TNFα or IL-6 mRNA. Lipopolysaccharide challenge decreased mRNA expression of key genes controlling the transmethylation and Met regeneration pathways, which was not prevented by Met supplementation. The data suggest that bovine hepatocyte cultures can be used as a biological model to study the inflammatory cascade via an LPS challenge. Supplementation of Met prevents the LPS-induced hepatocyte cytokine expression and is associated with elevated intracellular GSH concentration.

Hepatic Glucocorticoid Receptor Plays a Greater Role Than Adipose GR in Metabolic Syndrome Despite Renal Compensation.

Exogenous glucocorticoid administration results in hyperglycemia, insulin resistance, hepatic dyslipidemia, and hypertension, a constellation of findings known as Cushing's syndrome. These effects are mediated by the glucocorticoid receptor (GR). Because GR activation in liver and adipose has been implicated in metabolic syndrome (MS), we wanted to determine the role of GR in these tissues in the development of MS. Because GR knockout (KO) mice (whole-body KO) exhibit perinatal lethality due to respiratory failure, we generated tissue-specific (liver or adipose) GRKO mice using cre-lox technology. Real-time PCR analysis of liver mRNA from dexamethasone-treated wildtype (WT) and liver GRKO mice indicated that hepatic GR regulates the expression of key genes involved in gluconeogenesis and glycogen metabolism. Interestingly, we have observed that liver-specific deletion of GR resulted in a significant increase in mRNA expression of key genes involved in gluconeogenesis and glycogen metabolism in kidney tissue, indicating a compensatory mechanism to maintain glucose homeostasis. We have also observed that GR plays an important role in regulating the mRNA expression of key genes involved in lipid metabolism. Liver GRKO mice demonstrated decreased fat mass and liver glycogen content compared with WT mice administered dexamethasone for 2 weeks. Adipose-specific deletion of GR did not alter glucose tolerance or insulin sensitivity of adipose GRKO mice compared with WT mice administrated dexamethasone. This indicates that liver GR might be more important in development of MS in dexamethasone-treated mice, whereas adipose GR plays a little role in these paradigms.

Brief neonatal nutritional supplementation has sex-specific effects on glucose tolerance and insulin regulating genes in juvenile lambs.

The nutritional plane and composition during fetal life can impact upon growth and epigenetic regulation of genes affecting pancreatic β-cell development and function. However, it is not clear whether β-cell development can be altered by nutritional factors or growth rate after birth. We therefore investigated the effect of neonatal nutritional supplements on growth, glucose tolerance, and pancreatic development in lambs. Newborn lambs were randomized to daily nutritional supplements, calculated to increase macronutrient intake to a similar degree as human breast milk fortifier, or an equivalent volume of water, for 2 wk while continuing to suckle ewe milk. Intravenous glucose tolerance test (IVGTT) was performed at 4 mo of age, and pancreata collected for molecular analysis. Supplemented lambs had slower weight gain than controls. In supplemented lambs, insulin response to IVGTT was increased in males but decreased in females, compared to same sex controls, and was unrelated to growth rate. mRNA expression of key genes in β-cell development showed sexually dimorphic effects. Epigenetic change occurred in the promotor region of PDX1 gene with decreased suppression and increased activation marks in supplemented lambs of both sexes. Nutritional interventions in early life have long-term, sex-specific effects on pancreatic function.

Effects of rosiglitazone on proliferation and differentiation of duck preadipocytes.

Rosiglitazone (RSG), one member of the thiazolidinediones (TZDs), is a type of anti-diabetic drug in diabetic humans and animal models, whose function remains unknown in waterfowl. In this study, effects of RSG on duck preadipocyte differentiation were investigated. We detected cell viability using CCK method and measured the mRNA expression of key genes and protein contents involved in preadipocyte differentiation via qRT-PCR and ELISA kits, respectively. Lipid accumulation was determined via Oil Red O staining extraction, and lipolysis was measured by free fatty acid release in the culture medium. Results showed that high concentrations of RSG (50, 100 μM) significantly decreased cell viability. RSG (0-10 μM) enhanced preadipocyte differentiation in a dose-dependent manner and thus promoted lipid accumulation. With increasing RSG concentrations, cellular lipid content gradually decreased and preadipocyte differentiation was suppressed. mRNA expression of key genes involved in preadipocyte differentiation including FAS, ACC, SCD1, LPL, PLIN, SREBP1c, and ATGL were significantly upregulated by RSG, and the protein content of FAS, ACC, and ATGL were also increased in response to RSG. Meanwhile, RSG exposure increased free fatty acid release in the culture medium. Similar results were obtained in response to RSG plus oleate that was used to induce cell differentiation. These findings suggest that RSG does not promote duck preadipocyte viability, but it does induce duck preadipocyte differentiation, which might influence both lipogenesis and lipolysis pathways.

Cardiac myocardial damage associated with resuscitation

Purpose of study: In the event of sudden cardiac arrest, the 2010 American Heart Association (AHA) guidelines recommend defined cardiopulmonary resuscitation (CPR) compression depths and defibrillation energies for both paediatric (<8 yrs) and adult patients for patient recovery. However there is limited information regarding the damaging effects of resuscitation on the tissues of the heart at a structural and genomic level. This study investigated the extent of gross and microscopic structural cardiac damage associated with implementing the 2010 AHA guidelines in a porcine model of cardiac arrest.Materials and methods: Ventricular fibrillation (VF) was induced in paediatric and adult porcine models. Following defibrillation and CPR as per the recommended guidelines, tissue samples were collected. Structural changes in the myocardial tissue were assessed using standard histological staining and ultrastructural damage to the tissues assessed using electron microscopy (EM). RNA was extracted from tissue samples and quantitative real time-PCR was performed to assess the mRNA expression of key genes associated with early markers of cardiovascular damage (troponin-1, DES, IL6, FABP3, Cx43). Clinical biomarker expression levels of Troponin I were also quantified pre and post treatment via the Vet iStat (Abaxis, USA).Results: All animals sustained gross structural damage including broken ribs, 3 animals had perforated atria and considerable damage to the ventricles was evident in the histological specimens. EM analysis revealed minor modifications in the ultrastructural appearance of the myocardial tissues. An increase in cardiac troponin-1 was observed in both adult and paediatric models post-resuscitation.Conclusions: This study highlights the extent of myocardial damage at a structural and genomic level, associated with implementing current resuscitation guidelines. It is evident that more research is needed to review the specificity and sensitivity of resuscitation to assist in determining the best method of post arrest treatment that increases the chance of resuscitation with minimal consequential damage.