Identification and Characterization of MicroRNAs in Normal Equine Tissues by Next Generation Sequencing

The role of microRNAs (miRNAs) as a post-transcriptional gene regulator has been elucidated in a broad range of organisms including domestic animals. Characterization of miRNAs in normal tissues is an important step to investigate the functions of miRNAs in various physiological and pathological conditions. Using Illumina Next Generation Sequencing (NGS) technology, we identified a total of 292 known and 329 novel miRNAs in normal horse tissues including skeletal muscle, colon and liver. Distinct sets of miRNAs were differentially expressed in a tissue-specific manner. The miRNA genes were distributed across all the chromosomes except chromosomes 29 and 31 in the horse reference genome. In some chromosomes, multiple miRNAs were clustered and considered to be polycistronic transcript. A base composition analysis showed that equine miRNAs had a higher frequency of A+U than G+C. Furthermore, U tended to be more frequent at the 59 end of miRNA sequences. This is the first experimental study that identifies and characterizes the global miRNA expression profile in normal horse tissues. The present study enriches the horse miRNA database and provides useful information for further research dissecting biological functions of miRNAs in horse.open2

Myotis rufoniger genome sequence and analyses: M-rufoniger's genomic feature and the decreasing effective population size of Myotis bats

Myotis rufoniger is a vesper bat in the genus Myotis. Here we report the whole genome sequence and analyses of the M. rufoniger. We generated 124 Gb of short-read DNA sequences with an estimated genome size of 1.88 Gb at a sequencing depth of 66x fold. The sequences were aligned to M. brandtii bat reference genome at a mapping rate of 96.50% covering 95.71% coding sequence region at 10x coverage. The divergence time of Myotis bat family is estimated to be 11.5 million years, and the divergence time between M. rufoniger and its closest species M. davidii is estimated to be 10.4 million years. We found 1,239 function-altering M. rufoniger specific amino acid sequences from 929 genes compared to other Myotis bat and mammalian genomes. The functional enrichment test of the 929 genes detected amino acid changes in melanin associated DCT, SLC45A2, TYRP1, and OCA2 genes possibly responsible for the M. rufoniger's red fur color and a general coloration in Myotis. N6AMT1 gene, associated with arsenic resistance, showed a high degree of function alteration in M. rufoniger. We further confirmed that the M. rufoniger also has batspecific sequences within FSHB, GHR, IGF1R, TP53, MDM2, SLC45A2, RGS7BP, RHO, OPN1SW, and CNGB3 genes that have already been published to be related to bat's reproduction, lifespan, flight, low vision, and echolocation. Additionally, our demographic history analysis found that the effective population size of Myotis clade has been consistently decreasing since similar to 30k years ago. M. rufoniger's effective population size was the lowest in Myotis bats, confirming its relatively low genetic diversity

Dysregulated expression of proteins associated with ER stress, autophagy and apoptosis in tissues from nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is categorized into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) and has emerged as a risk factor for more critical clinical conditions. However, the underlying mechanisms of NAFLD pathogenesis are not fully understood. In this study, expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis and autophagy were analyzed in normal, NAFL and NASH human livers by western blotting. Levels of some ER stress-transducing transcription factors, including cleaved activating transcription factor 6, were higher in NASH than in the normal tissues. However, the expression of a majority of the ER chaperones and foldases analyzed, including glucose-regulated protein 78 and ER protein 44, was lower in NASH than in the normal tissues. Levels of apoptosis markers, such as cleaved poly (ADP-ribose) polymerase, were also lower in NASH tissues, in which expression of some B-cell lymphoma-2 family proteins was up-or down-regulated compared to the normal tissues. The level of the autophagy substrate p62 was not different in NASH and normal tissues, although some autophagy regulators were up-or down-regulated in the NASH tissues compared to the normal tissues. Levels of most of the proteins analyzed in NAFL tissues were either similar to those in one of the other two types, NASH and normal, or were somewhere in between. Together, these findings suggest that regulation of certain important tissues processes involved in protein quality control and cell survival were broadly compromised in the NAFLD tissues.Research Institute for Veterinary Science, Seoul National University; National Institutes of Health [ES006694, HD062489]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Piperonyl butoxide and acenaphthylene induce cytochrome P450 1A2 and 1B1 mRNA in aromatic hydrocarbon-responsive receptor knock-out mouse liver

SUMMARY It has been suggested that acenaphthylene (ACN), piperonyl butoxide (PBO) and other methylenedioxyphenyl (benzodioxole) compounds can function as aromatic hydrocarbon-responsive receptor (AHR)-independent inducers of the cytochrome P450 (CYP) 1A2 in mouse liver. Although much indirect evidence has supported this hypothesis, direct proof was lacking until the present study. PBO and ACN were used to examine the expression of CYP1 Al, CYPIA2 and CYP1 Bl in mouse liver. These three CYP isozymes are included in the AHR battery of proteins. In this study, AHR knock-out mice were dosed intraperitoneally with PBO (200 mg/kg) or ACN (100 mg/kg). Induction of hepatic CYP1 Al by PBO or ACN was not detected by northern blots. In contrast, both CYP1A2 and CYP1 Bi mRNA, constitutively expressed at low levels in this tissue, were induced by each compound in the livers of AHR knockout mice. In addition, the use of heterogenous nuclear RNA reverse transcription-polymerase chain reaction procedures revealed that the transcriptional activities of CYP1 A2 were increased by PBO and ACN treatments. These results show that AHR-independent pathway(s) can be involved in induction of CYP1A

Arsenic-induced toxicity and the protective role of ascorbic acid in mouse testis

Oxidative stress has been suggested to be a major cause of male reproductive failure. Here, we investigated whether arsenic, which impairs male reproductive functions in rodent models, acts by inducing oxidative stress. Male 8-week-old ICR mice were given drinking water containing 20 or 40 mg/l sodium arsenite with or without 0.75 or 1.5 g/l of the antioxidant ascorbic acid for 5 weeks. The arsenic-treated mice showed decreased epididymidal sperm counts and testicular weights compared to untreated mice. These effects were reversed in mice that were co-treated with ascorbic acid. Similarly, arsenic treatment lowered the activities of testicular 3β-hydroxysteroid dehydrogenase (HSD) and 17β-HSD, which play important roles in steroidogenesis, and this was reversed by co-treatment with ascorbic acid. The testicles of arsenic-treated mice had decreased glutathione (GSH) levels (which correlate inversely with the degree of cellular oxidative stress) and elevated levels of protein carbonyl (a marker of oxidative damage to tissue proteins). Ascorbic acid co-treatment reversed both of these effects. Thus, ascorbic acid blocks both the adverse effects of arsenic on male reproductive functions and the arsenic-induced testicular oxidative changes. These observations support the notion that arsenic impairs male reproductive function by inducing oxidative stress.This work was supported by the Basic Research Program of the Korea Science and Engineering Foundation (R01-2004-000-10584-0)

Oxidative stress and apoptosis induced by titanium dioxide nanoparticles in cultured BEAS-2B cells

As the applications of industrial nanoparticles are being developed, the concerns on the environmental health are increasing. Cytotoxicities of titanium dioxide nanoparticles of different concentrations (5, 10, 20 and 40 g/ml) were evaluated in this study using a cultured human bronchial epithelial cell line, BEAS-2B. Exposure of the cultured cells to nanoparticles led to cell death, reactive oxygen species (ROS) increase, reduced glutathione (GSH) decrease, and the induction of oxidative stress-related genes such as heme oxygenase-1, thioredoxin reductase, glutathione-S-transferase, catalase, and a hypoxia inducible gene. The ROS increase by titanium dioxide nanoparticles triggered the activation of cytosolic caspase- 3 and chromatin condensation, which means that titanium dioxide nanoparticles exert cytotoxicity by an apoptotic process. Furthermore, the expressions of inflammation-related genes such as interleukin- 1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), TNF-a, and C-X-C motif ligand 2 (CXCL2) were also elevated. The induction of IL-8 by titanium dioxide nanoparticles was inhibited by the pre-treatment with SB203580 and PD98059, which means that the IL-8 was induced through p38 mitogen-acitvated protein kinase (MAPK) pathway and/or extracellular signal (ERK) pathway. Uptake of the nanoparticles into the cultured cells was observed and titanium dioxide nanoparticles seemed to penetrate into the cytoplasm and locate in the peri-region of the nucleus as aggregated particles, which may induce direct interactions between the particles and cellular molecules, to cause adverse biological responses.Thisworkwas supported by the Eco-technopia 21 project of the Korea Ministry of the Environment

Regulation of iron metabolism-related genes in diethylnitrosamine-induced mouse liver tumors

Background: It has been suggested that the altered iron metabolism in liver tumors, characterized by the iron-deficient phenotype, is of importance for tumor growth. Aim: This study was performed to elucidate the mechanisms underlying iron deficiency in liver tumors by examining how the liver tumor development affects the expression of iron metabolism-related genes. Methods: Iron metabolism reference values were analyzed in the sera of diethylnitrosamine-induced hepatocellular adenoma-bearing mice. Expression of iron metabolism-related genes was analyzed in adenomas and surrounding non-tumor tissues, and a subgroup of adenoma-bearing mice loaded with iron 72 h before sacrifice. Results: Iron content of the adenoma tissues was 2.0–2.5-fold lower compared to surrounding and agematched control tissues. There was no significant difference in serum iron levels between the adenomabearing and control mice, while the adenoma-bearing mice exhibited a 2.4-fold lower level of serum transferrin saturation. Expression of iron metabolism-related genes was dysregulated in the adenomas. Iron loading affected protein expression similarly in the adenomas and surrounding tissues suggesting that iron-responsive regulation of the proteins was not impaired. However, the mRNA expression for ceruloplasmin and divalentmetal transporter 1 (DMT1) IRE(+) in the adenomaswas alteredindependently of iron status, and the dysregulation may contribute to diminished iron content. Conclusion: These findings suggest that diethylnitrosamine-induced liver adenoma-bearing mice have abnormal iron metabolism and that dysregulation of iron metabolism-related genes contributes to iron deficiency in the adenomas.This work was supported by research funding from Korea Science and Engineering Foundation (R01-2004-000-10584-0)

Regulation of metal transporters by dietary iron, and the relationship between body iron levels and cadmium uptake

Iron (Fe) plays essential roles in biological processes, whereas cadmium (Cd) is a toxic and non-essential metal. Two metal transporters, divalent metal transporter 1 (DMT1) and metal transporter protein 1 (MTP1), are responsible for Fe transport in mammals. Here, we studied the effect of dietary Fe on the expression of these metal transporters in peripheral tissues, and the uptake by these tissues of Cd. Mice were fed an Fe-sufficient (FeS: 120 mg Fe/kg) or Fe-deficient (FeD: 2–6 mg Fe/kg) diet for 4 weeks. The total Fe levels in the body were evaluated by measuring tissue Fe concentrations. Tissue Cd concentrations were determined 24 h after the mice received a single oral dose of Cd. Animals fed a FeD diet showed depletion of body Fe levels and accumulated 2.8-fold higher levels of Cd than the FeS group. Quantitative real time RT-PCR revealed that whereas DMT1 and MTP1 were both ubiquitously expressed in all FeS peripheral tissues studied, DMT1 was highly expressed in brain, kidney, and testis, whereas MTP1 was highly expressed in liver and spleen. Depletion of the body Fe stores dramatically upregulated DMT1 and MTP1 mRNA expression in the duodenum as well as moderately upregulating their expression in several other peripheral tissues. The iron response element positive isoform of DMT1 was the most prominently upregulated isoform in the duodenum. Thus, DMT1 and MTP1 may play an important role in not only maintaining Fe levels but also facilitating the accumulation of Cd in the body of mammals.This work was supported by grant No. R01-2004-000-10584-0 from the Basic Research Program of the Korea Science and Engineering Foundation