MicroRNA signatures in liver diseases

MicroRNAs (miRNAs) are an emerging class of highly conserved non-coding small RNAs that regulate gene expression at the post-transcriptional level. It is now clear that miRNAs can potentially regulate every aspect of cellular activity, including differentiation and development, metabolism, proliferation, apoptotic cell death, viral infection and tumorigenesis. Recent studies provide clear evidence that miRNAs are abundant in the liver and modulate a diverse spectrum of liver functions. Deregulation of miRNA expression may be a key pathogenetic factor in many liver diseases including viral hepatitis, hepatocellular cancer and polycystic liver diseases. A clearer understanding of the mechanisms involved in miRNA deregulation will offer new diagnostic and therapeutic strategies to treat liver diseases. Moreover, better understanding of miRNA regulation and identification of tissue-specific miRNA targets employing transgenic/knockout models and/or modulating oligonucleotides will improve our knowledge of liver physiology and diseases.

Multiple Strategies for Metabolic Engineering of Escherichia coli for Efficient Production of Coenzyme Q_（10）

Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate （NADPH） availability on CoQ10 production in E.coli were investigated.The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate,increasing CoQ10 production.Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum,together with the overexpression of gapC,could increase NADPH availability and then enhanced CoQ10 production.Three effects,overexpressions of various genes in CoQ biosynthesis and central metabolism,different vectors and culture conditions on CoQ10 production in E.coli,were all investigated.The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ10 production in E.coli.The recombinant E.coli （△ispB：：ddsA,△pykFA and △gapA：：gapC）,harboring the two plasmids encoding pck,dxs,idi and ubiCA genes under the control of PT5 on pQE30,ispA,ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of PBAD on pBAD33,could produce CoQ10 up to 3.24 mg·g-1 dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5.The yield is unprecedented and 1.33 times of the highest production so far in E.coli.

Helicobacter pylori arginase mutant colonizes arginase Ⅱ knockout mice

AIM: To investigate the role of host and bacterial arginases in the colonization of mice by Helicobacter pylori (H.pylori).METHODS: H.pylori produces a very powerful urease that hydrolyzes urea to carbon dioxide and ammonium,which neutralizes acid.Urease is absolutely essential to H.pylori pathogenesis;therefore,the urea substrate must be in ample supply for urease to work efficiently.The urea substrate is most likely provided by arginase activity,which hydrolyzes L-arginine to L-ornithine and urea.Previous work has demonstrated that H.pylori arginase is surprisingly not required for colonization of wild-type mice.Hence,another in vivo source of the critical urea substrate must exist.We hypothesized that the urea source was provided by host arginase Ⅱ,since this enzyme is expressed in the stomach,and H.pylori has previously been shown to induce the expression of murine gastric arginase Ⅱ.To test this hypothesis,wild-type and arginase (rocF) mutant H.pylori strain SS1 were inoculated into arginase Ⅱ knockout mice.RESULTS: Surprisingly,both the wild-type and rocF mutant bacteria still colonized arginase Ⅱ knockout mice.Moreover,feeding arginase Ⅱ knockout mice the host arginase inhibitor S-(2-boronoethyl)L-cysteine (BEC),while inhibiting > 50% of the host arginase Ⅰ?activity in several tissues,did not block the ability of the rocF mutant H.pylori to colonize.In contrast,BEC poorly inhibited H.pylori arginase activity.CONCLUSION: The in vivo source for the essential urea utilized by H.pylori urease is neither bacterial arginase nor host arginase Ⅱ;instead,either residual host arginase Ⅰ?or agmatinase is probably responsible.

Engineering of Corynebacterium glutamicum to Enhance L-ornithine Production by Gene Knockout and Comparative Proteomic Analysis

Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogenase complex(ODHC) on L-ornithine production was also investigated.It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production.The engineered C.glutamicum ATCC13032(ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L-1 from 0.24 g·L-1 of the wild-type strain.In order to understand the mechanism of L-ornithine production in C.glutamicum ATCC13032(ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production,the comparative proteome between the wild-type and the engineered strain was analyzed.L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway.The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further in-crease ornithine production in the engineered C.glutamicum and the ornithine synthesis enzymes(ArgCJBD) may not be the limiting enzymes in the engineered C.glutamicum.

Impact of asialoglycoprotein receptor deficiency on the development of liver injury

The asialoglycoprotein （ASGP） receptor is a wellcharacterized hepatic receptor that is recycled via the common cellular process of receptor-mediated endocytosis （RME）. The RME process plays an integral part in the proper trafficking and routing of receptors and ligands in the healthy cell. Thus, the missorting or altered transport of proteins during RME is thought to play a role in several diseases associated with hepatocyte and liver dysfunction. Previously, we examined in detail alterations that occur in hepatocellular RME and associated receptor functions as a result of one particular liver injury, alcoholic liver disease （ALD）. The studies revealed profound ethanol- mediated impairments to the ASGP receptor and the RME process, indicating the importance of this receptor and the maintenance of proper endocytic events in normal tissue. To further clarify these observations, studies were performed utilizing knockout mice （lacking a functional ASGP receptor） to which were administered several liver toxicants. In addition to alcohol, we examined the effects following administration of anti- Fas （CD95） antibody, carbon tetrachloride （CCh） and lipopolysaccharide （LPS）/galactosamine. The results of these studies demonstrated that the knockout mice sustained enhanced liver injury in response to all of the treatments, as shown by increased indices of liver damage, such as enhancement of serum enzyme levels, histopathological scores, as well as hepatocellular death. Overall, the work completed to date suggests a possible link between hepatic receptors and liver injury. In particular, adequate function and content of the ASGP receptor may provide protection against various toxinmediated liver diseases.

Epigenetic Repression of SATB1 by Polycomb Group Protein EZH2 in Epithelial Cells

Objective To study the regulatory mechanism of SATB1 repression in cells other than T cells or erythroid cells, which have high expression level of SATB1. Methods HeLa epithelial cells were treated with either histone deacetylase inhibitor (HDACi) trichostatin A (TSA) or DNA methylation inhibitor 5-Aza-C before detecting SATB1 expression. Luciferase reporter system was applied to measure effects of EZH2 on SATB1 promoter activity. Over-expression or knockdown of EZH2 and subsequent quantitative reverse transcription-polymerase chain reaction were performed to determine the effect of this Polycomb group protein on SATB1 transcription. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of EZH2 and trimethylated H3K27 (H3K27me3) at SATB1 promoter in HeLa cells. K562 cells and Jurkat cells, both having high-level expression of SATB1, were used in the ChIP experiment as controls. Results Both TSA and 5-Aza-C increased SATB1 expression in HeLa cells. Over-expression of EZH2 reduced promoter activity as well as the mRNA level of SATB1, while knockdown of EZH2 apparently enhanced SATB1 expression in HeLa cells but not in K562 cells and Jurkat cells. ChIP assay results suggested that epigenetic silencing of SATB1 by EZH2 in HeLa cells was mediated by trimethylation modification of H3K27. In contrast, enrichment of EZH2 and H3K27me3 was not detected within proximal promoter region of SATB1 in either K562 or Jurkat cells. Conclusion SATB1 is a bona fide EZH2 target gene in HeLa cells and the repression of SATB1 by EZH2 may be mediated by trimethylation modification on H3K27.

Age-related increase of early afterdepolarization in calsequestrin-2 knock-in mouse cardiomycyte

Objective To characterize early afterdepolarizations （EADs） caused triggered activity （TA） among calsequestrin-2 （CASQ2） knock-in （CASQ2 KI） mice and its relationship with aging. Methods Electrophysiological properties of ventricular myocytes from 3- month （mo, young）, 9-mo （adult-l） and 12-too （adult-2） in wild-type （WT） and CASQ2 KI mice were investigated with patch-clamp technique. Results The incidences of EADs and TA in CASQ2 KI cardiomyocytes increased with increasing age. In contrast, WT mice cardiomyocytes showed no significant change in matched-age groups. Compared with that in 3-mo CASQ2 KI mice, the 50% repolarization of action potential （APD50） showed prolongation in both 9-mo and 12-mo ones （9.2±0.9 ms of 9-mo and 10.3 ± 1.2 ms of 12- mo vs. 5.6± 0.3 ms of 3-mo）, while the 90 % repolarization of action potential （APD90） was similar among 3 age groups. Compared with 3-mo mice, the 9-mo and 12-mo CASQ2 KI mice showed markedly reduced transient outward potassium current （Ito） densities but increased L-type calcium current （ICa-L） densities. Conlcusion This study suggested that events of EADs and TA in CASQ2 KI mice increased with increasing age, It might be associated partly with the augment of cellular calcium concentration and the prolongation of APD50 induced by decrease of Ito and increase of ICa-L in adult CASQ2 KI mice

Loss of CO_2 sensing by the olfactory system of CNGA3 knockout mice

Atmospheric CO2 can signal the presence of food, predators or environmental stress and trigger stereotypical behaviors in both vertebrates and invertebrates. Recent studies have shown that the necklace olfactory system in mice sensitively detects CO2 in the air. Olfactory CO2 neurons are believed to rely on cyclic gnanosine monophosphate （cGMP） as the key second messenger; however, the specific ion channel underlying CO2 responses remains unclear. Here we show that CO2-evoked neuronal and behavioral responses require cyclic nucleotide-gated （CNG） channels consisting of the CNGA3 subunit. Through Ca2＋-imaging, we found that CO2-triggered Ca2＋ influx was abolished in necklace olfactory sensory neurons （OSNs） of CNGA3-knockout mice. Olfactory detection tests using a Go/No-go paradigm showed that these knockout mice failed to detect 0.5% CO2. Thus, sensitive detection of atmospheric CO2 depends on the function of CNG channels consisting of the CNGA3 subunit in necklace OSNs. These data support the important role of the necklace olfactory system in CO2 sensing and extend our understanding of the signal transduction pathway mediating CO2 detection in mammals [Current Zoology 56 （6）： 793-799, 2010].

Constitutively Expressed αB—Crystallin in Heat Schock Transcription Factor 1 Knockout Mice Myocardium

Objective-To investigate the effects of heat shock transcription factor 1) gene on the constitutivety expressed αB-CrystaUin (aBC) in mice myocardium. Methods-The expression levels of constitutive aBC in HSF1 knockout (hsf1 - /- ) and HSFl wild type (As/1 + /+) mice myocardium were evaluated by western blot and immunohistochemistry. Results : The αBC levels in hsfl -/- and hsfl +/+ were 68. 42±4. 16, 100. 00±7. 58, respectively (P<0. 05, cytoso-lic fraction) , and 20. 53±1. 01, 37. 55±1. 91, respectively (P<0. 05, pellet fraction). The aBC signals decreased significantly in hsfl -/- myocardium when compared with those in hsfl +/+ myocardium stained with fluorescence immunohistochemistry. Conclusion-HSF1 is an important, but not the only factor, which mediates the constitutively expressed aBC.

Construction and biological function of Toxoplasma gondii rop41 gene knockout strain

Objective:Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii(T.gondii),which can lead to complications such as encephalitis and ocular toxoplasmosis.The disease becomes more severe when the host’s immune system is compromised.Rhoptry proteins are major virulence factors that enable T.gondii to invade host cells.This study aims to construct a T.gondii rhoptry protein 41(rop41/ROP41)gene knockout strain and preliminarily investigate the biological function of rop41.Methods:Using CRISPR/Cas9 technology,a specific single-guide RNA(sgRNA)for the target gene was designed and linked to a recombinant plasmid.Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes.The recombinant plasmid and the homologous fragments were electroporated into T.gondii,and PCR identification was performed after drug selection and monoclonal screening.Plaque assays were used to comprehensively assess whether rop41 affected the growth and proliferation of T.gondii in host cells.Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity.The STRING database was utilized to construct a protein-protein interaction(PPI)network,and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.Results:The T.gondii rop41 gene knockout strain(RHΔku80Δrop41)was successfully constructed and stably inherited.Plaque assays showed that compared with the parental strain,the number of plaques formed by the rop41 gene knockout strain did not significantly decrease,but the reduction in plaque size was statistically significant(P<0.05).After the rop41 gene was knocked out,the invasion ability of T.gondii was reduced,but there was no statistically significant difference in its proliferation ability(P>0.05).The PPI network revealed that ROP41 was associated with other protein kinases and autophagy related proteins.Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction,biosynthesis,metabolism,and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.Conclusion:The T.gondii RHΔku80Δrop41 strain has been successfully constructed.ROP41 primarily affects the ability of T.gondii to invade host cells and may play a role in signal transduction and autophagy-related pathways between T.gondii and the host.

Deletions are easy detectable in cochlear mitochondrial DNA of Cu/Zn superoxide dismutase gene knockout mice

Abstract Objectives To investigate the tissue specificity of reactive oxygen species (ROS) damage to mitochondrial DNA (mtDNA) and to determine whether cochlear mtDNA is a sensitive target for ROS damage. Methods 10 Cu/ZnSOD gene (Cu/Zn superoxide dismutase gene, Sod1) knockout mice and 16 wild-type mice were analyzed by nested polymerase chain reaction (PCR).Results Three deletions were detected in various tissues of Sod1 knockout mice. MtDNA3867bp and mtDNA3726bp deletions were the most visible, and mtDNA4236bp deletion was barely detected in these tissues. There were obvious differences in the ratio of deleted mtDNA/total mtDNA in different tissue. Deleted mtDNA was most abundant in the liver and kidney and less in cochlea, heart and brain. The lowest was in spleen and skin. The ratio in various tissues was 3-20 times in Sod1 knockout mice over wild-type mice. In cochlea, the ratio was about 15. Conclusions Without the protection of Sod1, ROS can lead to mtDNA deletions in various tissues with significant tissue specificity. Cochlear mtDNA is a sensitive target for ROS damage.

Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer

Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has been shown to be a powerful tool for efficiently inducing mutations in the genome.However,ZFN-mediated targeting in pigs has rarely been achieved.Here,we used ZFNs to knock out the porcineα-1,3-galactosyl-transferase(GGTA1)gene,which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation.Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1.Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4%and 5.2%,respectively.The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer(SCNT).Three GGTA1 null piglets were born,and one knockout primary fibroblast cell line was established from a cloned fetus.Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane.Functionally,GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum.This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs.GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.

Gene editing nuclease and its application in tilapia

Gene editing nucleases including zinc-finger nucleases(ZFNs), transcription activator like effector nucleases(TALENs) and clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated(Cas) system(CRISPR/Cas9) provide powerful tools that improve our ability to understand the physiological processes and their underlying mechanisms. To date, these approaches have already been widely used to generate knockout and knockin models in a large number of species. Fishes comprise nearly half of extant vertebrate species and provide excellent models for studying many aspects of biology. In this review, we present an overview of recent advances in the use of gene editing nucleases for studies of fish species. We focus particularly on the use of TALENs and CRISPR/Cas9 genome editing for studying sex determination in tilapia.

Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum

Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation （ATMT）, but the frequencies are often very low. In the present study, we replaced the Ku80 homolog （a gene of the non-homologous end-joining （NHEJ） pathway） with the hygromycin resistance cassette （hph） by ATMT. No significant change in vegetative growth, conidiation, or pathogenicity was observed in KuSO-deficient strain （△dKuSO） of P. digitatum. However, using △pdKuSO as a targeting strain, the gene-targeting frequencies for both genes PdbrlA and PdmpkA were significantly increased. These results suggest that Ku80 plays an important role in homologous inte- gration and the created △PdKuSO strain would be a good candidate for rapid gene function analysis in P. digitatum.

Zinc finger transcription factor Sp7/Osterix acts on bone formation and regulates col10a1a expression in zebrafish

Sp7/Osterix as a zinc finger transcription factor is expressed specifically in osteoblasts. Embryonic lethality of Sp7 knockout mice, however, has prevented from examining the functions of Sp7 in osteoblast and bone formation in live animals. Here we used TALEN, a versatile genome-editing tool, to generate one zebrafish sp7 mutant line. Homozygous sp7-/- mutant zebrafish are able to survive to adulthood. Alizarin Red staining and Micro-CT analysis showed that sp7-/- larvae and adult fish fail to develop normal opercula, and display curved tail fins and severe craniofacial malformation, while Alcian Blue staining showed no obvious cartilage defects in sp7-/- fish. Quantitative RT-PCR showed that a number of osteoblast markers including sppl, phex, collala, and collalb are significantly down-regulated in sp7-/- fish. Furthermore, col10a1a, whose ortholog is the cartilage marker in mice, was shown to be a novel downstream gene of Sp7 as an osteoblast marker in zebrafish. Together, these results suggest that Sp7 is required for zebrafish bone development and zebrafish sp7 mutants provide animal models for investigating novel aspects of bone development.

Absence of murine CFAP61 causes male infertility due to multiple morphological abnormalities of the flagella

Impaired flagellar development and impaired motility of sperm is a cause of infertility in males. Several genes, including those of the AKAP, CCDC, CFAP, and DNAH families, among others, are involved in the‘‘multiple morphological abnormalities of the flagella"(MMAF) phenotype;these are the most common causes of male infertility. The Cilia-and flagella-associated protein(CFAP) family includes six members reported to cause MMAF phenotypes: CFAP43, CFAP44, CFAP69, CFAP65, CFAP70, and CFAP251. Here, we found that cilia-and flagella-associated protein 61(Cfap61) is highly expressed specifically in murine testes and show that the Cfap61-knockout male mice demonstrate MMAF phenotype, including sperm with short, coiled, and irregular flagella. Deletion of Cfap61 resulted in severe morphological and behavior abnormalities in sperm, reduced total sperm counts, impaired sperm motility, and led to male infertility.Notably, absence of Cfap61 impaired sperm flagella ultrastructural abnormalities on account of numerous distortions in multiple flagellum components. Immunostaining experiments in wild-type mice and healthy adult humans indicated that Cfap61 is initially localized at the neck of sperm, where it potentially functions in flagellum formation, and is later localized to the midpiece of the sperm. Thus, our study provides compelling evidence that dysregulation of Cfap61 affects sperm flagellum development and induces male infertility in mice. Further investigations of the CFAP61 gene in humans alongside clinical evidence showing MMAF phenotype in humans should contribute to our understanding of developmental processes underlying sperm flagellum formation and the pathogenic mechanisms that cause male infertility.

Characterization of a recombinant Akabane mutant virus with knockout of a nonstructural protein NSs in a pregnant goat model

Dear Editor,Akabane virus(AKAV),an orthobunyavirus,is transmitted primarily by biting midges and is widely distributed throughout the world except the Europe.AKAV was first isolated from mosquitoes in Japan(Oya et al.,1961).Although pregnant cows,ewes,and goats infected with AKAV exhibit no clinical signs of disease,in utero infections result in abortion,premature birth,stillbirth。

Biomimetic gold nanocomplexes for gene knockdown： Will gold deliver dividends for small interfering RNA nanomedicines？

RNA interference （RNAi） effectors such as small interfering RNA （siRNA） and micro RNA （miRNA） can selectively downregulate any gene implicated in the pathology of a disease. Therefore, RNAi-based therapies have immense potential for the treatment of a wide range of diseases. However, pharmacokinetic and pharmacodynamic studies have revealed that these therapeutic agents have poor bioactivity due to a number of factors, including insufficient plasma drug levels, short plasma half-lives, renal clearance, and hepatic metabolism. Non-viral delivery may facilitate the clinical application of siRNA-based therapeutics by helping to overcome these barriers. Recently, the potential of gold nanoparticles （AuNPs） as multifunctional carriers for transporting drugs, proteins, and genetic materials has been demonstrated. In this review, some of the key properties of AuNPs relevant to siRNA delivery, such as physical properties and surface chemistry have been described. In addition, the ability of AuNP-based formulation strategies to successfully overcome delivery barriers associated with siRNA, and the potential for this material to translate into safe and effective nanomedicines are critically discussed.