Qianqian Ji

Inhibition of HSP70 reduces porcine reproductive and respiratory syndrome virus replication in vitro

BackgroundSuccessful viral infection requires the involvement of host cellular factors in their life cycle. Heat shock protein 70 (HSP70) can be recruited by numerous viruses to promote the folding, maturation, or assembly of viral proteins. We have previously shown that HSP70 is significantly elevated in porcine reproductive and respiratory syndrome virus (PRRSV)-infected lungs, suggesting HSP70 may play a potential role during PRRSV infection. In this study, we tried to investigate the role of HSP70 during PRRSV infection.ResultsIn this study, we observed that PRRSV infection induced the expression of HSP70. The down-regulation of HSP70 using quercetin, a HSPs synthesis inhibitor, or small interfering RNAs (siRNA) reduced the viral protein level and viral production. Notably, these inhibitory effects on PRRSV infection could be attenuated by heat shock treatment. In addition, HSP70 was found to colocalize with the viral double-stranded RNA (dsRNA) and knockdown of HSP70 decreased the dsRNA levels, suggesting HSP70 is involved in the formation of viral replication and transcription complex (RTC) and thus affects the viral replication.ConclusionsOur study revealed that HSP70 is an essential host factor required for the replication of PRRSV. The inhibition of HSP70 significantly reduced PRRSV replication, which may be applied as an effective antiviral strategy.

Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer

Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre-implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P < 0.05), while treating donor cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P < 0.05). However, TSA treatment of both donor cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.

Peripheral administration of TAT-obestatin can influence the expression of liporegulatory genes but fails to affect food intake in mice.

Obestatin is a 23-amino-acid peptide originally regarded as an anorexigenic factor. However, most of the subsequent studies failed to confirm the initially reported anorexigenic properties of obestatin. Obestatin is incapable of crossing the blood brain barrier (BBB), which may affect its biological function. Here, we report the physiological effects of obestatin in mice after intraperitoneal administration of obestatin conjugated to the cell-permeable peptide TAT, which is capable of delivering different types of proteins through the BBB. Acute peripheral administration of 1 μmol/kg of TAT-obestatin did not influence the 24 h cumulative food intake and body weight gain of mice that were fasted for 18 h. Fed mice were injected intraperitoneally with 100 nmol/kg of TAT-obestatin daily for 25 d. Compared with control groups, on day 3, the gain in body weight was significantly altered; on day 7, abdominal fat mass was remarkably reduced; however, on day 25, there was a surprisingly notable increase in abdominal and epididymal fat mass. In comparison with control groups, on day 25, the expression levels of adiponectin, ADD1, C/EBPα, PPARG and GLUT4 were significantly up-regulated in liver tissues; in white adipose tissue, the expression level of C/EBPα was significantly up-regulated, but adiponectin and GLUT4 were significantly down-regulated. In addition, GPR39, the suspected receptor of obestatin, was up-regulated in white adipose tissue on day 25. These findings suggest that TAT-obestatin might play a role in white adipose tissue metabolism, but its physiological effects on food intake and body weight gain regulation remain unclear.

Inhibition of HSP90 attenuates porcine reproductive and respiratory syndrome virus production in vitro.

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) infection leads to substantial economic losses to the swine industry worldwide. However, no effective countermeasures exist to combat this virus infection so far. The most common antiviral strategy relies on directly inhibiting viral proteins. However, this strategy invariably leads to the emergence of drug resistance due to the error-prone nature of viral ploymerase. Targeting cellular proteins required for viral infection for developing new generation of antivirals is gaining concern. Recently, heat shock protein 90 (HSP90) was found to be an important host factor for the replication of multiple viruses and the inhibition of HSP90 showed significant antiviral effects. It is thought that the inhibition of HSP90 could be a promising broad-range antiviral approach. However, the effects of HSP90 inhibition on PRRSV infection have not been evaluated. In the current research, we tried to inhibit HSP90 and test whether the inhibition affect PRRSV infection.MethodsWe inhibit the function of HSP90 with two inhibitors, geldanamycin (GA) and 17- allylamono-demethoxygeldanamycin (17-AAG), and down-regulated the expression of endogenous HSP90 with specific small-interfering RNAs (siRNAs). Cell viability was measured with alamarBlue. The protein level of viral N was determined by western blotting and indirect immunofluorescence (IFA). Besides, IFA was employed to examine the level of viral double-stranded RNA (dsRNA). The viral RNA copy number and the level of IFN-β mRNA were determined by quantitative real-time PCR (qRT-PCR).ResultsOur results indicated that both HSP90 inhibitors showed strong anti-PRRSV activity. They could reduce viral production by preventing the viral RNA synthesis. These inhibitory effects were not due to the activation of innate interferon response. In addition, we observed that individual knockdown targeting HSP90α or HSP90β did not show dramatic inhibitory effect. Combined knockdown of these two isoforms was required to reduce viral infection.ConclusionsOur results shed light on the possibility of developing potential therapeutics targeting HSP90 against PRRSV infection.

Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosis

Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSA-induced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were used to detect apoptosis, and real-time polymerase chain reaction was used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages. Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality.

Exogenous expression of OCT4 facilitates oocyte‐mediated reprogramming in cloned porcine embryos

OCT4 is a well‐established regulator of pluripotency and nuclear reprogramming. To determine if improving OCT4 abundance can facilitate oocyte‐mediated reprogramming in cloned porcine embryos, we artificially increased OCT4 levels by co‐incubating donor cells with 50 ng/µl OCT4 plasmid. We observed higher rates of blastocyst formation (P < 0.05) and lower levels of blastocyst apoptosis in nuclear‐transfer‐derived embryos carrying OCT4‐incubated donor nuclei (OCT4‐SCNT). The beneficial effect caused by exogenous expression of OCT4 involves epigenetic changes, wherein increased histone acetylation (AcH3K9) appeared in OCT4‐SCNT embryos at the one‐cell and blastocyst stages and reduced histone methylation (H3K9me2) was observed at the one‐cell stage (P < 0.05). There was a transient increase in exogenous OCT4 and an up‐regulation of endogenous OCT4 level in OCT4‐SCNT embryos (P < 0.05), while the expression pattern of epigenetic enzymes was changed. These modifications were accompanied by an up‐regulation of CDX2, whose interaction with OCT4 is instrumental for implantation, and a down‐regulation of XIST, a negative indicator of reprogramming (P < 0.05). Taken together, our results support a role for exogenous expression of OCT4 in improving the efficiency of nuclear reprogramming while establishing a convenient and timesaving method to improve nuclear‐transfer outcomes. Mol. Reprod. Dev. 81: 820–832, 2014.

Damage of zona pellucida reduces the developmental potential and quality of porcine circovirus type 2–infected oocytes after parthenogenetic activation

The present aimed to study if porcine circovirus type 2 (PCV2), which adhered to zona pellucida (ZP), was able to enter mature porcine oocytes with intact and damaged ZP. Four groups, including uninfected ZP-intact oocytes (UOZI), uninfected ZP-damaged oocytes (UOZD), PCV2-infected ZP-intact oocytes (POZI), and PCV2-infected ZP-damaged oocytes (POZD) were studied. The oocytes were incubated with 1 mL minimum essential medium, containing 3.1 × 10(8) copies of PCV2 DNA for 1 hour. Mechanical procedure of the insertion by microneedle induced injuries to the ZP of porcine oocytes. At the blastocyst stage, the percentage of PCV2-infected embryos and the ratio of viral antigen-positive cells per embryo were determined by indirect immunofluorescence. To assess the effect of ZP injury on the developmental competence and quality of porcine PCV2-infected oocytes after parthenogenetic activation, blastocyst formation rates and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were analyzed. Moreover, real-time polymerase chain reaction was used to evaluate the expression of genes related to apoptosis and pluripotency at different developmental stages. The results of indirect immunofluorescence showed that only POZD group presented PCV2-infected embryos and viral-positive cells. The blastocyst rate of POZD group dropped down to approximately half of POZI groups (7.1 ± 1.5 vs. 14.5 ± 3.3). At the blastocyst stage, ZP injury increased apoptotic index of PCV2-infected embryos. The relative expression levels of Caspase 3 were higher in POZD group than the ones in POZI group at the two- and four-cell stages (not statistically significant). Compared with the one in POZI group, the ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of the ability to resist apoptosis, was lower in POZD group at the one-cell stage, but higher at the two- and four-cell stages. Expression levels of Oct4 and Nanog associated with pluripotency were lower in POZD group than the ones in POZI group at the morula stage (not statistically significant). Noteworthily, the expression of Nanog was significantly lower in POZD group versus POZI group (P < 0.05), whereas relative expression of Oct4 was significantly higher in the former at the blastocyst stage (P < 0.01). In conclusion, PCV2, which attached to ZP, was able to enter mature porcine oocytes with damaged ZP and subsequently reduced the developmental competence and quality of the oocytes after parthenogenetic activation.

Generation of CD44 gene-deficient mouse derived induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) show good promise for the treatment of defects caused by numerous genetic diseases. Herein, we successfully generated CD44 gene-deficient iPSCs using Oct4, Sox2, Klf4, and vitamin C. The generated iPSCs displayed a characteristic morphology similar to the well-characterized embryonic stem cells. Alkaline phosphatase, cell surface (SSEA1, NANOG, and OCT4), and pluripotency markers were expressed at high levels in these cells. The iPSCs formed teratomas in vivo and supported full-term development of constructed porcine embryos by inter-species nuclear transplantation. Importantly, incubation with trichostatin A increased the efficiency of iPSCs generation by increasing the histone acetylation levels. Moreover, more iPSCs colonies appeared following cell passaging during colony picking, thus increasing the effectiveness of iPSCs selection. Thus, our work provides essential stem cell materials for the treatment of genetic diseases and proposes a novel strategy to enhance the efficiency of induced reprogramming.

Introduction of silent mutations in a codon-optimized xylanase (xynB) results in enhanced protein expression in HEK293A cells

Xylanase is used extensively to improve feed conversion rates to enhance the performance of poultry and pigs. By expressing xylanase in simple-stomached animals, new breeds of genetically modified animals with enhanced feed conversion rates may be obtained. However, expression of heterologous proteins derived from lower organisms in mammalian cells is usually inefficient. When common codons of a ‘‘one amino acid-one codon”-optimized xylanase from Streptomyces olivaceoviridis were replaced with rare codons, xylanase expression in human embryonic kidney 293A cells increased by 1.4- to 2.3-fold as determined by flow cytometry, western blot and enzymatic activity assay. Quantitative RT-PCR assay indicated that the enhanced expression could not be attributed to altered mRNA levels. This study provides an alternative strategy for improving expression levels of heterologous proteins in mammalian cells, which is potentially helpful for generating genetically modified animals with enhanced feed conversion ability.

Correction to: Genetically engineered cell lines for α1-antitrypsin expression

In the original publication of the article, the Acknowledgement section was published incompletely. The complete Acknowledgement is given in this Correction.