Alpha Transcripts Research Articles

Estrogenic contamination by manure fertilizer in organic farming: a case study with the lizard Podarcis sicula.

In the last years, worldwide organic farming has grown exponentially; as a consequence, the use of animal manure as a soil fertility source has become the principal agricultural choice. However, the use of manure as fertilizer can increase the amount of steroid hormone metabolites in the soil. In southern Italy, lacertidae lizards are the most abundant vertebrate group in agroecosystems and have been identified as potential model species for ecotoxicological studies. The aim of this study was to understand if the manure applied in organic farming has estrogen-like effects in the lizard Podarcis sicula. Adult male lizards were captured in two organic agricultural fields (manure-treated sites) and in an uncultivated field (control site). Lizards from the two organic farms displayed hepatic biosynthetic alterations typical of an estrogenic contamination; hepatocytes contained both vitellogenin and estrogen receptor alpha transcripts and proteins, detected by in situ hybridization and immunocytochemistry. The same cells did not show cadmium, lead and metallothionein accumulation, indicative of the lack of inorganic contamination. These findings suggest that exogenous estrogens, arising from the use of manure, could affect the welfare of wild animals and animal breeding, leading to bioaccumulation of estrogens in food chain, with possible risk for human consumers. For this reason, organic farming should implement the use of sustainable practices such as crop rotation to preserve the soil biological activity, rather than organic manure as fertilizer.

The urodele amphibian Pleurodeles waltl has a diverse repertoire of immunoglobulin heavy chains with polyreactive and species-specific features

Urodele amphibians are an interesting model because although they possess the cardinal elements of the vertebrate immune system, their immune response is apparently subdued. This phenomenon, sometimes regarded as a state of immunodeficiency, has been attributed by some authors to limited antibody diversity. We reinvestigated this issue in Pleurodeles waltl, a metamorphosing urodele, and noted that upsilon transcripts of its IgY repertoire were as diverse as alpha transcripts of the mammalian IgA repertoire. Mu transcripts encoding the IgM repertoire were less diverse, but could confer more plasticity. Both isotypes present potential polyreactive features that may confer urodele antibodies with the ability to bind to a variety of antigens. Finally, we observed additional cysteines in CDR1 and 2 of the IGHV5 and IGHV6 domains, some of which specific to urodeles, that could allow the establishment of a disulfide bond between these CDRs. Together, these data suggest that urodele antibody diversity is not as low as previously thought.

Detection of satellite alpha transcript in sera, as a surrogate marker for the risk of development of multiple cancers in colorectal cancer patients.

e22044 Background: Non-cording RNA, satellite alpha transcript (SAT-a) plays an important role on mitosis, disruption of which leads to genomic damages. We have reported that increased levels of SA...

Genome-Wide Association Study Identifies Novel Loci Associated With Diisocyanate-Induced Occupational Asthma.

Diisocyanates, reactive chemicals used to produce polyurethane products, are the most common causes of occupational asthma. The aim of this study is to identify susceptibility gene variants that could contribute to the pathogenesis of diisocyanate asthma (DA) using a Genome-Wide Association Study (GWAS) approach. Genome-wide single nucleotide polymorphism (SNP) genotyping was performed in 74 diisocyanate-exposed workers with DA and 824 healthy controls using Omni-2.5 and Omni-5 SNP microarrays. We identified 11 SNPs that exceeded genome-wide significance; the strongest association was for the rs12913832 SNP located on chromosome 15, which has been mapped to the HERC2 gene (p = 6.94 × 10(-14)). Strong associations were also found for SNPs near the ODZ3 and CDH17 genes on chromosomes 4 and 8 (rs908084, p = 8.59 × 10(-9) and rs2514805, p = 1.22 × 10(-8), respectively). We also prioritized 38 SNPs with suggestive genome-wide significance (p < 1 × 10(-6)). Among them, 17 SNPs map to the PITPNC1, ACMSD, ZBTB16, ODZ3, and CDH17 gene loci. Functional genomics data indicate that 2 of the suggestive SNPs (rs2446823 and rs2446824) are located within putative binding sites for the CCAAT/Enhancer Binding Protein (CEBP) and Hepatocyte Nuclear Factor 4, Alpha transcription factors (TFs), respectively. This study identified SNPs mapping to the HERC2, CDH17, and ODZ3 genes as potential susceptibility loci for DA. Pathway analysis indicated that these genes are associated with antigen processing and presentation, and other immune pathways. Overlap of 2 suggestive SNPs with likely TF binding sites suggests possible roles in disruption of gene regulation. These results provide new insights into the genetic architecture of DA and serve as a basis for future functional and mechanistic studies.

Adult exposure to bisphenol A (BPA) in Wistar rats reduces sperm quality with disruption of the hypothalamic-pituitary-testicular axis.

Reproductive physiology involves complex biological processes that can be disrupted by exposure to environmental contaminants. The effects of bisphenol A (BPA) on spermatogenesis and sperm quality is still unclear. The objective of this study was to investigate the reproductive toxicity of BPA at dosages considered to be safe (5 or 25mg BPA/kg/day). We assessed multiple sperm parameters, the relative expression of genes involved in the central regulation of the hypothalamic-pituitary-testicular axis, and the serum concentrations of testosterone, estradiol, LH and FSH. BPA exposure reduced sperm production, reserves and transit time. Significant damage to the acrosomes and the plasma membrane with reduced mitochondrial activity and increased levels of defective spermatozoa may have compromised sperm function and caused faster movement through the epididymis. BPA exposure reduced the serum concentrations of testosterone, LH and FSH and increased the concentration of estradiol. The relative gene expression revealed an increase in gonadotropin releasing hormone receptor (Gnrhr), luteinizing hormone beta (Lhb), follicle stimulating hormone beta (Fshb), estrogen receptor beta (Esr2) and androgen receptor (Ar) transcripts in the pituitary and a reduction in estrogen receptor alpha (Esr1) transcripts in the hypothalamus. In this study, we demonstrated for the first time that adult male exposure to BPA caused a reduction in sperm production and specific functional parameters. The corresponding pattern of gene expression is indicative of an attempt by the pituitary to reestablish normal levels of LH, FSH and testosterone serum concentrations. In conclusion, these data suggest that at dosages previously considered nontoxic to reproductive function, BPA compromises the spermatozoa and disrupts the hypothalamic-pituitary-gonadal axis, causing a state of hypogonadotropic hypogonadism.

Early postnatal exposure to a low dose of decabromodiphenyl ether affects expression of androgen and thyroid hormone receptor-alpha and its splicing variants in mouse Sertoli cells.

Decabromodiphenyl ether (decaBDE) adversely affects reproduction and development. Our previous study showed that postnatal exposure to a low dose of decaBDE (0.025 mg/kg body weight/day) by subcutaneous injection on postnatal days (PNDs) 1 through 5 leads to reductions in testicular size and number of Sertoli cells and sperm, while higher dose of decaBDE (2.5 mg/kg body weight/day) had no significant differences about these. In the present study, we examined the molecular mechanism of these effects on mouse testes following postnatal exposure to a low decaBDE dose. We hypothesized that postnatal exposure to decaBDE may alter levels of serum thyroid hormones (THs) and testosterone, or the level of TH receptor alpha (Thra) transcripts and its splicing variants and androgen receptor (Ar) in Sertoli cells, adversely affecting spermatogenesis. To test this hypothesis, we examined serum TH and testosterone levels and the levels of transcripts of the Ar, Thra and its splicing variants, and Thra splicing factors (Hnrnpa1, Srsf1, and Hnrnph1) with qPCR in isolated mouse Sertoli cells exposed postnatally to decaBDE (0.025, 0.25, and 2.5 mg/kg). Levels of serum testosterone and transcripts encoding Ar, Thra, and its variant, Thra1, declined significantly in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg. No significant differences in serum TH level or Thra2, Hnrnph1, or Srsf1 transcript levels were observed between control and decaBDE-exposed mice. However, the Thra1:Thra2 and Hnrnpa1:Srsf1 ratios were altered in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg but not in cells exposed to 0.25 or 2.5 mg decaBDE/kg. These results indicate that postnatal exposure to a low dose of decaBDE on PNDs 1 through 5 lowers the testosterone level and the levels of Ar and Thra transcripts in Sertoli cells, accompanied by an imbalance in the ratios of Thra splicing variants, resulting in smaller testicular size and impaired spermatogenesis.

N-acetylcysteine inhibits the up-regulation of mitochondrial biogenesis genes in livers from rats fed ethanol chronically.

Chronic ethanol (EtOH) administration to experimental animals induces hepatic oxidative stress and up-regulates mitochondrial biogenesis. The mechanisms by which chronic EtOH up-regulates mitochondrial biogenesis have not been fully explored. In this work, we hypothesized that oxidative stress is a factor that triggers mitochondrial biogenesis after chronic EtOH feeding. If our hypothesis is correct, co-administration of antioxidants should prevent up-regulation of mitochondrial biogenesis genes. Rats were fed an EtOH-containing diet intragastrically by total enteral nutrition for 150days, in the absence or presence of the antioxidant N-acetylcysteine (NAC) at 1.7g/kg/d; control rats were administered isocaloric diets where carbohydrates substituted for EtOH calories. EtOH administration significantly increased hepatic oxidative stress, evidenced as decreased liver total glutathione and reduced glutathione/glutathione disulfide ratio. These effects were inhibited by co-administration of EtOH and NAC. Chronic EtOH increased the expression of mitochondrial biogenesis genes including peroxisome proliferator-activated receptor gamma-coactivator-1 alpha and mitochondrial transcription factor A, and mitochondrial DNA; co-administration of EtOH and NAC prevented these effects. Chronic EtOH administration was associated with decreased mitochondrial mass, inactivation and depletion of mitochondrial complex I and complex IV, and increased hepatic mitochondrial oxidative damage, effects that were not prevented by NAC. These results suggest that oxidative stress caused by chronic EtOH triggered the up-regulation of mitochondrial biogenesis genes in rat liver, because an antioxidant such as NAC prevented both effects. Because NAC did not prevent liver mitochondrial oxidative damage, extra-mitochondrial effects of reactive oxygen species may regulate mitochondrial biogenesis. In spite of the induction of hepatic mitochondrial biogenesis genes by chronic EtOH, mitochondrial mass and function decreased probably in association with mitochondrial oxidative damage. These results also predict that the effectiveness of NAC as an antioxidant therapy for chronic alcoholism will be limited by its limited antioxidant effects in mitochondria, and its inhibitory effect on mitochondrial biogenesis.

Evaluation of Cucurbita maxima extract against scopolamine-induced amnesia in rats: implication of tumour necrosis factor alpha.

Cucurbita maxima (CM) seed oil is commonly used in Indian folk medicine to treat various ailments. We have investigated the effect of CM seed oil on memory impairment induced by scopolamine in rats. Male adult Wistar rats were administered scopolamine 1 mg/kg body weight, i.p. or 1.25 mg/kg body weight, s.c. to induce memory impairment. The nootropic agent piracetam 100 mg/kg body weight, i.p. and CM seed oil 100 and 200 mg/kg body weight, p.o. were administered daily for five consecutive days. The memory function was evaluated in the Morris water maze (MWM) test, the social recognition test (SRT), the elevated plus maze (EPM) test, and the pole climbing test (PCT). Acetylcholinesterase (AChE) activity and oxidative stress parameters were estimated in the cortex, hippocampus, and cerebellum of the brains after completion of the behavioural studies. The effects of scopolamine on the levels of the tumour necrosis factor alpha (TNF-α) transcript were also investigated. Scopolamine caused memory impairment in all the behavioural paradigms along with a significant increase in the AChE activity and oxidative stress in the brain. Scopolamine also caused a significant increase in the expression of TNF-α in the hippocampus. CM seed oil exhibited antiamnesic activity as indicated by a significant reduction in the latency time in the MWM test and decreased social interaction during trial 2 in the SRT. Further, treatment with CM seed oil significantly decreased the AChE activity and malondialdehyde levels and increased the glutathione level in brain regions. CM seed oil also significantly decreased the expression of TNF-α in the hippocampus. The effect of CM seed oil on behavioural and biochemical parameters was comparable to that observed in rats treated with piracetam. These results indicate that CM seed oil may exert antiamnesic activity which may be attributed to the inhibition of AChE and inflammation as well as its antioxidant activity in the brain.

Abstract 3387: Upregulation of gene expression in oligodendrogliomas is linked to an increase in GA binding protein alpha transcription

Abstract Lower grade gliomas can be histologically classified into astrocytomas, oligoastrocytomas, or oligodendrogliomas. The latter have come into focus because of their interesting characteristics: they have a more favorable outcome and better response to chemo/radiotherapy. This is correlated with a unique unbalanced translocation der(1;19)(q10;p10) causing a codeletion of the short arm of chromosome 1 and the long arm of chromosome 19. Like the other lower grade gliomas, a large percentage of oligodendrogliomas carry the IDH1 R132 mutation and this is associated with the presence of the codeletion. We identified a high number of mutations in the remaining alleles of the Capicua homolog gene (CIC) and in the far upstream element binding protein (FUBP1) in 1p/19q codeleted oligodendrogliomas (Eisenreich et al. 2013, doi:10.1371/journal.pone.0076623). Both genes are suspected to act as tumor suppressor genes. We then analyzed the expression profiles of 13 oligodendrogliomas and oligoastrocytomas both with the 1p/19q codeletion (nine samples) and without (four samples) using a combination of expression microarrays and transcriptome sequencing (RNA-Seq). Our results showed that all nine samples with the 1p/19q codeletion carried the heterozygous IDH1 R132H mutation while only one tumor without the codeletion carried said mutation. There was a high concordance between the results of RNA-Seq and the microarray experiments and both showed a distinctive expression signature between samples with the 1p/19q codeletion and those without. Using MSigDB, we found that the genes whose expression was altered in in tumors with the 1p/19q codeletion showed significant overlap with those involved in stemness and differentiation and genes that had CpG-rich promoters. Furthermore, there was an overlap with genes with altered expression in chronic myelogenous leukemia (CML). When we compared the expression of genes located on 1p and 19q between the tumors with the 1p/19q codeletion and those without, we found that only 82% of genes on 1p and 19q were downregulated significantly. When we ran the upregulated genes in PathScan, we found enrichment in GA binding protein alpha (GABPA)binding sites. GABPA expression levels were increased in our glioma samples with and without the 1p/19q codeletion. Investigating the expression levels of GABPA in the Cancer Genome Atlas (TCGA) GBM dataset also revealed an upregulation of this transcription factor. An increase in GABPA-mediated transcription was recently reported in CML but has not been yet reported in gliomas. We therefore propose that GABPA is an important transcription factor in gliomas. We are currently evaluating the function of GABPA in glioma what will also be presented. Acknowledgements: This project was partially supported by a MeDDrive grant of the Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden. Citation Format: Barbara Klink, Karol Szafranski, Jaime Campos-Valenzuela, Sophie Eisenreich, Dietmar Krex, Eva-Maria Gerlach, Karl Hackmann, Andreas Rump, Gabriele Schackert, Matthias Platzer, Lars Kaderali, Evelin Schröck, Khalil Abou-El-Ardat. Upregulation of gene expression in oligodendrogliomas is linked to an increase in GA binding protein alpha transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3387. doi:10.1158/1538-7445.AM2014-3387

Adiponectin and Metabolic Syndrome Prevention

Copyright © 2014, Hamadan University of Medical Sciences; Published by Hamadan University of Medical Sciences Corp. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In recent years, adiponectin (APN), a novel adipocytokine, has attracted the scientists attention. Low circulatory concentration of this protein is observed in obese patients and patients with type 2 diabetes and metabolic syndrome (1). Moreover, through its antiatherogenic and anti-inflammatory effects, APN plays a protective role against the development of metabolic disorders (2). Metabolic syndrome (MetS) is a collection of obesityassociated disorders and is very common in different populations (3). Its prevalence in Iran is reported at 34.7% to 41.6% depending on the different diagnostic criteria in rural and urban area (4). Higher prevalence has also been reported in the particular populations; for example, in people older than 60 years old in west of Iran the prevalence was reported at 44.7% (5). An important issue in this field is association of hypoadiponectinemia with MetS (6, 7). Research in this aspect indicated that hypoadiponectinemia is associated with MetS and its association is stronger than any other inflammatory markers (6). We previously reported low concentration of APN in postmenopausal women with type 2 diabetes (8). In addition, we showed that serum APN level is inversely correlated with insulin resistance in obese men with type 2 diabetes (9). APN directly activate AMP-activated protein kinase (AMPK), induces peroxisome proliferator-activated receptor alpha (PPAR-α) transcription (10), and subsequently has an important positive effect on lipid metabolism regulation. On the other hand, antioxidative effect of APN (2) makes it a beneficial agent in oxidative stress–related disorders. The scientists have focused on finding different ways to increase plasma level of APN and therefore, diminishing MetS complications. A recently published review discussed the effect of different intervention on APN and APN receptors (11). In addition to pharmacologic agents, exercise, weight loss, caloric restriction and changing life style, and consumption of some foods are other ways of improving APN level (11). Fish oil, catechin, and green tea extract are compounds can augment APN level in animal models (11). Other than changing the life style and increasing the physical activity, the researchers are interested in using plant products or herbals that increase APN level. An interesting study is a clinical trial conducted by Gomez-Arbelaez et al. on patients with MetS (12). Results of this study indicated that using of aged garlic extract improves APN levels in those with MetS (12). Although garlic is commonly used in different countries, its pungent odor makes it unfavorable for most people. Aged garlic extract that is used in the mentioned study was odorless and rich in antioxidant, and has been shown to contain antioxidant and antithrombotic properties. Nevertheless, there is a report showing no effect of garlic on APN concentration in women with MetS (13). Another natural product that is shown to induce APN secretion is resveratrol (14). It is shown that long-term daily consumption of resveratrol containing grape can increase serum APN (15). We recently examined the effect of resveratrol on the other adipocytokines, namely, apelin, visfatin, and vaspin, resistin gene expression on diabetic rats (unpublished observation). Our preliminary results indicated that resveratrol reduces resistin gene expression. Considering the high prevalence MetS particularly in middleage subjects, implantation of proper policies to prevent this disorder is necessary. Since using herbal medicine is very popular nowadays, it can be a good field of investigation to find the natural products that can increase APN level and subsequently, prevent MetS. The other issue is finding the exact mechanisms of their action.

Chronic cardiovascular disease-associated gene network analysis in human umbilical vein endothelial cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The association of dioxin exposure with increased morbidity or mortality of chronic cardiovascular diseases (CVDs) has been established by many epidemiological studies. However, the precise global gene expression alterations caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the cardiovascular system need to be further elucidated. In this study, we profiled the gene expression of human umbilical vein endothelial cells (HUVECs) exposed to different concentrations of TCDD by high-throughput sequencing. Expression of 1,838 genes was changed significantly after TCDD stimulation. The FunDO analysis suggested that some CVDs were highly associated with TCDD treatment, including atherosclerosis, thromboangiitis obliterans, pulmonary arterial hypertension (PAH), and hypertension. KEGG pathway analysis showed that many genes in the signaling pathways of vascular smooth muscle contraction and apoptosis were altered distinctly. In addition, we revealed evidence regarding the gene network changes of chronic CVDs including atherosclerosis, thrombosis, myocardial infarction (MI), hypertension, and PAH in TCDD-exposed HUVECs. We found that gene expression of β1-adrenoceptors (ADRB1), β2-adrenoceptors (ADRB2), endothelin-converting enzyme 1 (ECE1), and endothelin-1 gene (EDN1) that are involved in the blood pressure regulation pathway decreased apparently under TCDD treatment. Moreover, the transcripts of interleukin 1 beta (IL-1β) and tumor necrosis factor α (TNFα), which are related to atherosclerosis, were up-regulated by TCDD stimulation. In addition, the transcripts of Homo sapiens collagen, type IV, alpha 1 (COL4A1), and isoforms that trigger the MI pathway were up-regulated after TCDD exposure. Finally, we found enhanced platelet-derived growth factor (PDGF) and signal transducer and activator of transcription 5 (Stat5) expression with TCDD treatment in endothelial cells, which are involved in PAH induced by vascular injury.

Effects of water temperature on perchlorate toxicity to the thyroid and reproductive system of Oryzias latipes

Water temperature is expected to increase in many parts of the world due to global climate change. The change in water temperature may affect ecosystems through alterations of the chemical properties or by affecting the susceptibility of organisms. Perchlorate can disrupt thyroid function of an organism by inhibiting iodide uptake. In the present study, the effect of water temperature on perchlorate toxicity was evaluated using Japanese medaka (Oryzias latipes). Pairs of adult medaka fish were exposed to a sublethal concentration of sodium perchlorate (100mg/L) and a control, at a ‘low’ (26°C), ‘medium’ (29°C) or ‘high’ water temperature (33°C) for seven days. The effects of the water temperature on reproduction, thyroid hormones and cortisol concentrations were determined. Transcription of several genes related to thyroid function and stress were also investigated. Significant down-regulation of thyroid hormone receptor alpha (THR-α) and beta (THR-β) transcripts and up-regulation of deiodinase 2 (DIO2) transcripts were observed in the fish exposed to perchlorate. Thyroxine (T4) concentrations were decreased, while triiodothyronine (T3) levels remained constant following exposure to perchlorate, and this effect became more pronounced under the high water temperature conditions (33°C). Up-regulation of the DIO2 gene may explain these observations. The total number of spawned eggs decreased slightly as the water temperature increased, and this reduction became significant when fish were exposed to perchlorate. Our observations indicate that exposure to perchlorate could affect thyroid function and overall reproductive fitness, and these effects could be aggravated under high water temperatures.

PTU-156 Hepatocyte Nuclear Factor 4 Alpha (hnf4a) Is Demonstrated In Barrett’s Metaplasia, But Not In Normal Human Oesophagus

IntroductionBarrett’s metaplasia (BM) is the main risk factor for oesophageal adenocarcinoma, a cancer which carries a mortality of >50% at 12 months. Refluxate containing gastric and bile acids seems to...

Establishment of Three Francisella Infections in Zebrafish Embryos at Different Temperatures

Francisella spp. are facultative intracellular pathogens identified in increasingly diverse hosts, including mammals. F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis infect fish inhabiting warm and cold waters, respectively, while F. tularensis subsp. novicida is highly infectious for mice and has been widely used as a model for the human pathogen F. tularensis. Here, we established zebrafish embryo infection models of fluorescently labeled F. noatunensis subsp. noatunensis, F. noatunensis subsp. orientalis, and F. tularensis subsp. novicida at 22, 28, and 32°C, respectively. All infections led to significant bacterial growth, as shown by reverse transcription-quantitative PCR (RT-qPCR), and to a robust proinflammatory immune response, dominated by increased transcription of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). F. noatunensis subsp. orientalis was the most virulent, F. noatunensis subsp. noatunensis caused chronic infection, and F. tularensis subsp. novicida showed moderate virulence and led to formation of relatively small granuloma-like structures. The use of transgenic zebrafish strains with enhanced green fluorescent protein (EGFP)-labeled immune cells revealed their detailed interactions with Francisella species. All three strains entered preferentially into macrophages, which eventually assembled into granuloma-like structures. Entry into neutrophils was also observed, though the efficiency of this event depended on the route of infection. The results demonstrate the usefulness of the zebrafish embryo model for studying infections caused by different Francisella species at a wide range of temperatures and highlight their interactions with immune cells.

Effects of Switching from Stavudine to Raltegravir on Subcutaneous Adipose Tissue in HIV-Infected Patients with HIV/HAART-Associated Lipodystrophy Syndrome (HALS). A Clinical and Molecular Study

HIV-1/HAART-associated lipodystrophy syndrome (HALS) has been associated with exposure to stavudine (d4T) through mitochondrial dysfunction. We performed a 48-week study to assess the effects of switching from d4T to raltegravir (RAL) on metabolic and fat molecular parameters of patients with HALS. Forty-two patients with HALS and a median exposure to d4T > 7 years were switched to RAL and followed for 48 weeks. Fasting metabolic tests, HIV RNA, CD4 cell count, and fat measured by DEXA were obtained at baseline and week 48. mtDNA and gene transcripts for PPAR gamma, adiponectin, cytochrome b, Cox IV, TNF alpha, MCP-1 and CD68 were assessed in paired subcutaneous fat tissue biopsies. Lipid parameters, fasting glucose, insulin, and HOMA-IR did not change significantly. Whole body fat (P = 0.0027) and limb fat mass (P<0.0001) increased from baseline. Trunk/limb fat ratio (P = 0.0022), fat mass ratio (P = 0.0020), fat mass index (P = 0.0011) and percent leg fat normalized to BMI (P<0.0001) improved after 48 weeks. Relative abundance of mtDNA, expression of PPAR gamma, adiponectin, Cyt b, and MCP-1 genes increased, whereas Cox IV, TNF alpha, and CD68 did not change significantly from baseline. Switching from d4T to RAL in patients with HALS is associated with an increase in limb fat mass and an improvement in markers of adipocyte differentiation and mitochondrial function in SAT.

Dysregulated microRNAs in amyotrophic lateral sclerosis microglia modulate genes linked to neuroinflammation

MicroRNAs (miRNAs) regulate gene expression at post-transcriptional level and are key modulators of immune system, whose dysfunction contributes to the progression of neuroinflammatory diseaseas such as amyotrophic lateral sclerosis (ALS), the most widespread motor neuron disorder. ALS is a non-cell-autonomous disease targeting motor neurons and neighboring glia, with microgliosis directly contributing to neurodegeneration. As limited information exists on miRNAs dysregulations in ALS, we examined this topic in primary microglia from superoxide dismutase 1-G93A mouse model. We compared miRNAs transcriptional profiling of non-transgenic and ALS microglia in resting conditions and after inflammatory activation by P2X7 receptor agonist. We identified upregulation of selected immune-enriched miRNAs, recognizing miR-22, miR-155, miR-125b and miR-146b among the most highly modulated. We proved that miR-365 and miR-125b interfere, respectively, with the interleukin-6 and STAT3 pathway determining increased tumor necrosis factor alpha (TNFα) transcription. As TNFα directly upregulated miR-125b, and inhibitors of miR-365/miR-125b reduced TNFα transcription, we recognized the induction of miR-365 and miR-125b as a vicious gateway culminating in abnormal TNFα release. These results strengthen the impact of miRNAs in modulating inflammatory genes linked to ALS and identify specific miRNAs as pathogenetic mechanisms in the disease.

Effects of triphenyltin on growth and development of the wood frog (Lithobates sylvaticus)

Exposure to contaminants in the environment has been suggested as a contributing cause of ongoing declines in populations of amphibians reported in certain locations around the world. In the current study, responses of the wood frog (Lithobates sylvaticus) to exposure to triphenyltin (TPT), a commonly used fungicide, during the larval period were characterized. Exposure of L. sylvaticus to 0.1, 1.0, or 5.0μgTPT/L significantly affected survival, growth, days to metamorphosis (DTM), and abundances of transcripts of genes of interest. After seven days of exposure there were no significant effects on survival, but masses and snout-ventral length (SVL) of larvae exposed to 5.0μgTPT/L were significantly lesser than controls. Mortality of larvae after exposure to 5.0μgTPT/L was 100% nine days after initiation of the experiment. Larvae exposed to 0.1 or 1.0μgTPT/L were allowed to grow for 100 days or until they reached metamorphic climax, whichever occurred earlier. Mortality of wood frogs exposed to 1.0μgTPT/L was 80%. The LC20 or LC50 after 100 days of exposure was 0.12 or 0.34μgTPT/L, respectively. However, DTM of larvae that survived exposure to 1.0μgTPT/L was significantly less than that of controls. Abundances of transcripts of retinoid-X-receptor (rxr) and perixosomal proliferation receptor gamma (pparγ) were significantly lesser in larvae exposed to either concentration of TPT for seven days. Also, abundances of transcripts of stearoyl-CoA desaturase-1 (scd1), fatty acid synthase (fas), lipoprotein lipase (lpl), and β-hydroxybutyrate dehydrogenase (β-hb-m) were lesser in larvae exposed to 5.0μgTPT/L, which suggested that disruption of lipid metabolism might have affected survival in this exposure group. However, in larvae that survived to metamorphic climax during exposure to TPT for as long as 100 days, abundances of transcripts of perixosomal proliferation receptor alpha (pparα), pparγ, cytochrome p4504B1 (cyp4b1), fas, and lpl were greater than in controls, suggesting that an up-regulation of processes related to metabolism of lipids might have been important for survival and development of these animals. Overall, concentrations of TPT that are found in the environment had a significant effect on the survival and development of L. sylvaticus, and this might have been due, in part, to effects on metabolism of lipids.

The Human RVB Complex Is Required for Efficient Transcription of Type I Interferon-Stimulated Genes

Type I interferons (IFNs) stimulate transcription through a latent heterotrimeric transcription factor composed of tyrosine-phosphorylated STAT1 and STAT2 and the DNA binding partner IRF9, with STAT2 contributing a critical transactivation domain. Human RVB1 and RVB2, which are highly conserved AAA(+) ATP binding proteins contained in chromatin-remodeling complexes such as Ino80, SNF2-related CBP activator protein (SRCAP), and Tip60/NuA4, interacted with the transactivation domain of STAT2 in the nuclei of IFN-stimulated cells. RNA interference (RNAi) experiments demonstrated that RVB proteins were required for robust activation of IFN-α-stimulated genes (ISGs). The requirement for RVB proteins was specific to IFN-α/STAT2 signaling; transcription of tumor necrosis factor alpha (TNF-α)- and IFN-γ-driven genes was not affected by RVB1 depletion. Using RNAi-based depletion, we assessed the involvement of catalytic subunits of the RVB-containing Tip60, BRD8, Ino80, SRCAP, and URI complexes. No component other than RVB1/2 was uniquely required for ISG induction, suggesting that RVB1/2 functions as part of an as yet unidentified complex. Chromatin immunoprecipitation assays indicated that RVB1/2 was required for recruitment of RNA polymerase II (Pol II) to ISG promoters but was dispensable for STAT2 recruitment to chromatin. We hypothesize that an RVB1/2 chromatin-remodeling complex is required for efficient Pol II recruitment and initiation at ISG promoters and is recruited through interaction with the STAT2 transactivation domain.

Keratinocyte-Specific Deletion of the Receptor RAGE Modulates the Kinetics of Skin Inflammation In Vivo

The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor causally related to the pathogenesis of acute and chronic inflammation. In a mouse model of inflammation-driven skin carcinogenesis, RAGE deletion conferred protection from the development of skin tumors due to a severely impaired cutaneous inflammation. Although the impact of RAGE expression in immune cells was shown to be essential for the maintenance of a cutaneous inflammatory reaction, the role of RAGE in keratinocytes remained unsolved. Using mice harboring a keratinocyte-specific deletion of RAGE, we analyzed its role in the regulation of an acute inflammatory response that was induced by topical treatment of the back skin with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We show that RAGE expression in cutaneous keratinocytes modulates the strength and kinetics of acute inflammation and supports the maintenance of epidermal keratinocyte activation. To address the underlying molecular mechanism, we isolated interfollicular epidermis by laser microdissection for gene expression analysis, and identified RAGE as a regulator in the temporal control of TPA-induced epidermal tumor necrosis factor alpha transcript levels. In summary, our data demonstrate that RAGE expression in keratinocytes is critically involved in the perpetuation of acute inflammation and support the central role of RAGE in paracrine communication between keratinocytes and stromal immune cells.

High fat diet induced obesity alters ovarian phosphatidylinositol-3 kinase signaling gene expression

Insulin regulates ovarian phosphatidylinositol-3-kinase (PI3 K) signaling, important for primordial follicle viability and growth activation. This study investigated diet-induced obesity impacts on: (1) insulin receptor (Insr) and insulin receptor substrate 1 (Irs1); (2) PI3K components (Kit ligand (Kitlg), kit (c-Kit), protein kinase B alpha (Akt1) and forkhead transcription factor subfamily 3 (Foxo3a)); (3) xenobiotic biotransformation (microsomal epoxide hydrolase (Ephx1), Cytochrome P450 isoform 2E1 (Cyp2e1), Glutathione S-transferase (Gst) isoforms mu (Gstm) and pi (Gstp)) and (4) microRNA's 184, 205, 103 and 21 gene expression. INSR, GSTM and GSTP protein levels were also measured. Obese mouse ovaries had decreased Irs1, Foxo3a, Cyp2e1, MiR-103, and MiR-21 but increased Kitlg, Akt1, and miR-184 levels relative to lean littermates. These results support that diet-induced obesity potentially impairs ovarian function through aberrant gene expression.