Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells.

Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein.

Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00-13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.

Modification of improved-genome editing via oviductal nucleic acids delivery (i-GONAD)-mediated knock-in in rats.

BACKGROUND: Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency. RESULTS: The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 µM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different. CONCLUSIONS: We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.

Critical role of CREBH-mediated induction of transforming growth factor ß2 by hepatitis C virus infection in fibrogenic responses in hepatic stellate cells.

Mechanisms of hepatic fibrogenesis induced by hepatitis C virus (HCV), one of the leading causes of liver fibrosis, are not fully understood. We studied transcriptional up-regulation of transforming growth factor ß (TGF-ß), especially TGF-ß2, which is mediated by activation of liver-enriched transcription factor cAMP-responsive element-binding protein, hepatocyte specific (CREBH) triggered by HCV infection and its functional significance for induction of profibrogenic phenotypes by interaction of HCV-infected cells with hepatic stellate cells (HSCs). Compared to TGF-ß1, expression of TGF-ß2 mRNA was induced faster and to a higher level upon HCV infection. Serum TGF-ß2 levels in hepatitis C patients were higher compared to those in healthy individuals and were positively correlated with hepatic fibrosis stages F0-F2. TGF-ß2 promoter activity was decreased and increased, respectively, by silencing and overexpression of CREBH. CREBH recognition sites were identified in the TGF-ß2 promoter. CREBH binding to the promoter and its increase in cells expressing HCV Core-NS2 were shown by gel mobility shift and chromatin immunoprecipitation, respectively. The active form of CREBH was detectable in HCV-infected chimeric mice with human livers and cells expressing HCV proteins. Involvement of CREBH in HCV-induced fibrogenic response was further demonstrated in the CREBH null-mutant mouse model. Fibrogenic phenotypes were assessed using co-cultures of HCV-infected cells and HSCs. Expressions of fibrogenic factors and TGF-ß1 increasing in the co-cultures was prevented by TGF-ß2- or CREBH silencing. CONCLUSION: CREBH was identified as a key positive regulator of TGF-ß2 transcription in HCV-infected cells. TGF-ß2 released from infected cells potentially contributes to cross-induction of TGF-ß in an autocrine manner through its own signaling pathway, leading to an increase in fibrogenic responses in adjacent HSCs. (Hepatology 2017;66:1430-1443).

Safety evaluation of glucose oxidase from Penicillium chrysogenum.

Glucose oxidase (ß-d-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) is used in the food and beverage industry as a preservative and stabilizer and is commonly derived from the fungus Aspergillus niger. Although the safety of glucose oxidase preparations from A. niger is well-established, the use of preparations derived from other fungal species is of interest; however, an assessment of their safety is warranted. Here, we report on the safety of a glucose oxidase preparation derived from the fungus Penicillium chrysogenum (designated as PGO) for commercial use in food processing, as well as an ingredient in food. In a repeated dose 90-day oral toxicity study conducted in rats, PGO was without compound-related adverse effects at doses of up to 15,600U/kg body weight/day, equivalent to 193mg total organic solids/kg body weight/day. In addition, PGO was non-genotoxic in a series of genotoxicity tests, including a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and a combined in vivo mammalian erythrocyte micronucleus test and comet assay. The results of these studies support the safe use of PGO in food for human consumption.

Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain.

Improved genome editing via oviductal nucleic acid delivery (i-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. i-GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins via the oviductal wall of pregnant females at 0.7 days post-coitum, followed by in vivo electroporation (EP). Unlike outbred Institute of Cancer Research (ICR) and hybrid mouse strains, genome editing of the most widely used C57BL/6J (B6) strain with i-GONAD has been considered difficult but, recently, setting a constant current of 100 mA upon EP enabled successful i-GONAD in this strain. Unfortunately, the most widely used electroporators employ a constant voltage, and thus we explored conditions allowing the generation of a 100 mA current using two electroporators: NEPA21 (Nepa Gene Co., Ltd.) and GEB15 (BEX Co., Ltd.). When the current and resistance were set to 40 V and 350-400 Ω, respectively, the current was fixed to 100 mA. Another problem in using B6 mice for i-GONAD is the difficulty in obtaining pregnant B6 females consistently because estrous females often fail to be found. A single intraperitoneal injection of low-dose pregnant mare's serum gonadotrophin (PMSG) led to synchronization of the estrous cycle of these mice. Consequently, approximately 51% of B6 females had plugs upon mating with males 2 days after PMSG administration, which contrasts with the case (≈26%) when B6 females were subjected to natural mating. i-GONAD performed on PMSG-treated pregnant B6 females under conditions of average resistance of 367 Ω and average voltage of 116 mA resulted in the production of pregnant females at a rate of 56% (5/9 mice), from which 23 fetuses were successfully delivered. Nine (39%) of these fetuses exhibited successful genome editing at the target locus.

Long Noncoding RNA ELIT-1 Acts as a Smad3 Cofactor to Facilitate TGFß/Smad Signaling and Promote Epithelial-Mesenchymal Transition.

TGFß is involved in various biological processes, including development, differentiation, growth regulation, and epithelial-mesenchymal transition (EMT). In TGFß/Smad signaling, receptor-activated Smad complexes activate or repress their target gene promoters. Smad cofactors are a group of Smad-binding proteins that promote recruitment of Smad complexes to these promoters. Long noncoding RNAs (lncRNA), which behave as Smad cofactors, have thus far not been identified. Here, we characterize a novel lncRNA EMT-associated lncRNA induced by TGFß1 (ELIT-1). ELIT-1 was induced by TGFß stimulation via the TGFß/Smad pathway in TGFß-responsive cell lines. ELIT-1 depletion abrogated TGFß-mediated EMT progression and expression of TGFß target genes including Snail, a transcription factor critical for EMT. A positive correlation between high expression of ELIT-1 and poor prognosis in patients with lung adenocarcinoma and gastric cancer suggests that ELIT-1 may be useful as a prognostic and therapeutic target. RIP assays revealed that ELIT-1 bound to Smad3, but not Smad2. In conjunction with Smad3, ELIT-1 enhanced Smad-responsive promoter activities by recruiting Smad3 to the promoters of its target genes including Snail, other TGFß target genes, and ELIT-1 itself. Collectively, these data show that ELIT-1 is a novel trans-acting lncRNA that forms a positive feedback loop to enhance TGFß/Smad3 signaling and promote EMT progression. SIGNIFICANCE: This study identifies a novel lncRNA ELIT-1 and characterizes its role as a positive regulator of TGFß/Smad3 signaling and EMT.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2821/F1.large.jpg.

i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats.

Zygote-microinjection or in vitro electroporation of isolated zygotes are now widely used methods to produce genome-edited mice. However, these technologies require laborious and time-consuming ex vivo handling of fertilized eggs, including zygote isolation, gene delivery into zygotes and embryo transfer into recipients. We recently developed an alternative method called improved genome-editing via oviductal nucleic acids delivery (i-GONAD), which does not require the above-mentioned ex vivo handing of zygotes, but instead involves intraoviductal instillation of genome-editing components, Cas9 protein and synthetic gRNAs, into the oviducts of pregnant females at the late 1-cell embryo stage under a dissecting microscope and subsequent electroporation. With this method, we succeeded in generating genome-edited mice at relatively high efficiencies (for example, knockout alleles were produced at ~97% efficiency). Here, we extended this improved technology to rats, and found that i-GONAD can create genome-edited rats in various strains, including Sprague Dawley and Lewis, and F1 hybrids (between Sprague Dawley and Brown Norway), with efficiencies of ~62% for indel mutations and ~9% for knock-ins. Thus, i-GONAD will be especially useful for the production of genome-edited rats in small laboratories where expensive micromanipulator systems and highly skilled personnel for embryo manipulation are unavailable.

Gastric mucosal changes induced by polyethylene glycol 400 administered by gavage in rats.

Polyethylene glycol 400 (PEG 400) is widely used with a variety of pharmaceutical formulations, and is often added to dosing formulations in preclinical toxicity studies. The aim of the present study was to characterize the effects of PEG 400 on the rat gastrointestinal tract. Three dosage levels (5, 50 or 100 v/v%) of PEG 400 were administered at a volume of 5 ml/kg/day by gavage for 15 days to the rats (5 males and 5 females in each group). At the end of the treatment, the whole lengths of gastrointestinal tracts were examined pathologically. Although there were no gross abnormalities at necropsy, the histopathological examination revealed several changes localized to the stomach mucosa, but not in the intestine. The changes consisted of infiltration of eosinophils and globule leukocytes, increased in the height of the entire mucosal layer, elongation of the surface mucous epithelial and mucous neck cell layers with increased intracellular mucous in the glandular stomach, and the spongiosis (intercellular edema) of the squamous epithelium in the forestomach. These changes near the limiting ridge tended to increase in severity and extent in a dose-dependent manner. These results suggest that repeated oral administration of concentrated PEG 400 can easily induce the mucosal changes in the stomach of the rats.

Evidence for expression of relaxin hormone-receptor system in the boar testis.

Although the physiological role of relaxin (RLN) in males remains largely unknown, there is limited evidence that the testis might be a candidate source and target of RLN in boars, as RLN transcripts are detected in the boar testis and it contains RLN-binding sites. To determine whether the boar testis acts as a source and target tissue of RLN, we characterised the expression pattern and cellular localisation of both RLN and its own receptor LGR7 (RXFP1) in boar testes during postnatal development by molecular and immunological approaches. Testes were collected from Duroc boars, and partial cDNA sequences of the boar homologue of human RXFP1 were identified. RLN expression increased through puberty onwards, while RXFP1 expression changed little during development. RLN mRNA and protein expression were restricted to the Leydig cells, whereas both Leydig cells and seminiferous epithelial cells expressed RXFP1 mRNA and protein. Interestingly, RLN was expressed in the testis as an 18 kDa form (the expected size of proRLN), but not as the 6 kDa mature form, during development because of a lack of the enzyme required for proRLN processing. In contrast, RXFP1 was detected at all stages as specific bands of 75 and 91-95 kDa (likely non-glycosylated and glycosylated RXFP1 respectively). Thus, we provide evidence for expression of RLN-RXFP1 ligand-receptor system in the boar testis, suggesting that the testis act as a source and possible target tissue of RLN.