Expression and Purification of a PEDV-Neutralizing Antibody and Its Functional Verification.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious and pathogenic virus causing high morbidity and mortality, especially in newborn piglets. There remain problems with contemporary PEDV vaccines, in part because of the rapid variation of PEDV, poor conferred immunity, and numerous side effects. The ability to produce PEDV-neutralizing antibodies suggests that we may be able to increase the success rate of PEDV prevention in piglets using these antibodies. In this study, we produced an anti-PEDV S protein monoclonal antibody (anti-PEDV mAb-2) that neutralized PEDV-CV777 (a G1 strain), PEDV-SDSX16 and PEDV-Aj1102 (two G2 strains). In vivo challenge experiments demonstrated that anti-PEDV mAb-2 inhibited the PEDV infection in piglets. We also produced three HEK293 cell lines that expressed anti-PEDV mAb-2. Overall, our study showed that anti-PEDV mAb-2 produced from hybridoma supernatants effectively inhibited PEDV infection in piglets, and the recombinant HEK293 cell lines expressed anti-PEDV mAb-2 genes.

Establishment and characterization of immortalized porcine neonatal hepatocytes without the use of viral components.

Porcine hepatocytes are a promising option for xenotransplantation in light of the critical shortage of orthotopic donor livers. Because primary hepatocytes have limited ability to proliferate in vitro, several immortalized hepatocyte lines have been established. However, these cells have typically been generated using a virus-dependent transfection methodology and express viral oncogenes that introduce potential risks in clinical applications. In our study, we established immortalized porcine neonatal hepatocytes by introduction of a plasmid-based hTERT gene expression system by electroporation, without the use of viral components. We detected stable expression of hTERT by RT-PCR and Western blot. The immortalized hepatocytes exhibit a high growth rate, but retain the normal morphology of freshly isolated primary hepatocytes. To date, these immortalized hepatocytes have been expanded for over 80 passages. In addition, no significant differences were detected in glycogen synthesis, secretion of serum albumin, or lipid accumulation between the primary hepatocytes and our immortalized hepatocytes. The cells also exhibit serum-dependent growth and have no capacity for anchorage-independent growth in vitro, demonstrating that they have not been transformed in vitro. Our immortalized porcine hepatocytes will be useful for elucidating the pathogenesis of liver disease and developing efficient treatments. Furthermore, these immortalized hepatocytes may provide a safer source of cells for application in xenotransplantation, compared with immortalized cells generated using viral components.

MC4R deficiency in pigs results in hyperphagia and ultimately hepatic steatosis without high-fat diet.

Melanocortin 4 receptor (MC4R)-deficient mice had been used for several years to study human nonalcoholic steatohepatitis (NASH). However, although liver pathologic and biochemical indicators have been examined, mice models do not always faithfully display the phenotype of the human disease. In this study, we investigated the MC4R knockout phenotype in miniature pigs. We found that pigs lacking MC4R exhibited hyperorexia, insulin resistance, hyperinsulinemia, disordered lipid metabolism and their livers accumulated significant amounts of fat. We have shown that deletion of MC4R results in hyperphagia and increased body fat, ultimately leading to hepatic steatosis without atherogenic diet.

Generation of pig induced pluripotent stem cells using an extended pluripotent stem cell culture system.

BACKGROUND: Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown. METHODS: iPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis. RESULTS: In this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-ß, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%. CONCLUSIONS: Our findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.

Generation of an MC3R knock-out pig by CRSPR/Cas9 combined with somatic cell nuclear transfer (SCNT) technology.

BACKGROUND: Melanocortin 3 receptor (MC3R), a rhodopsin-like G protein-coupled receptor, is an important regulator of metabolism. Although MC3R knock-out (KO) mice and rats were generated in earlier studies, the function of MC3R remains elusive. Since pig models have many advantages over rodents in metabolism research, we generated an MC3R-KO pig using a CRSPR/Cas9-based system combined with somatic cell nuclear transfer (SCNT) technology. METHOD: Four CRSPR/Cas9 target vectors were constructed and then their cleavage efficiency was tested in porcine fetal fibroblasts (PFFs). The pX330-sgRNA1 and pX330-sgRNA4 vectors were used to co-transfect PFFs to obtain positive colonies. PCR screening and sequencing were conducted to identify the genotype of the colonies. The biallelically modified colonies and wild-type control colonies were used simultaneously as donor cells for SCNT. A total of 1203 reconstructed embryos were transferred into 6 surrogates, of which one became pregnant. The genotypes of the resulting piglets were determined by PCR and sequencing, and off-target effects in the MC3R KO piglets were detected by sequencing. Then, offspring were obtained through breeding and six male KO pigs were used for the growth performance analysis. RESULTS: Four vectors were constructed successfully, and their cleavage efficiencies were 27.96, 44.89, 32.72 and 38.86%, respectively. A total of 21 mutant colonies, including 11 MC3R-/- and 10 MC3R+/- clones, were obtained, corresponding to a gene targeting efficiency of 29.17%, with 15.28% biallelic mutations. A total of 6 piglets were born, and only two MC3R KO piglets were generated, one with malformations and a healthy one. No off-target effects were detected by sequencing in the healthy mutant. Six male MC3R KO pigs were obtained in the F2 generation and their body weight and body fat were both increased compared to wild-type full siblings. CONCLUSION: A MC3R KO pig strain was generated using the CRSIPR/Cas9-based system, which makes it possible to study the biological function of MC3R in a non-rodent model.

An FBXO40 knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects.

The regulatory function of Fbxo40 has been well characterized in mice. As a key component of the SCF-E3 ubiquitin ligase complex, Fbxo40 induces IRS1 ubiquitination, thus inactivating the IGF1/Akt pathway. The expression of Fbxo40 is restricted to muscle, and mice with an Fbxo40 null mutation exhibit muscle hypertrophy. However, the function of FBXO40 has not been elucidated in pigs, and it is not known whether FBXO40 mutations affect their health. We therefore generated FBXO40 knockout pigs using somatic cell nuclear transfer (SCNT) technology. CRISPR/Cas9 technology was combined with G418 selection, making it possible to generate donor cells at an efficiency of 75.86%. In muscle from FBXO40 knockout pigs, IRS1 levels were higher, and the IGF1/Akt pathway was stimulated. Mutant animals also had approximately 4% more muscle mass compared to WT controls. The knockout pigs developed normally and no pathological changes were found in major organs. These results demonstrate that FBXO40 is a promising candidate gene for improving production traits in agricultural livestock and for developing therapeutic interventions for muscle diseases.

A data-driven multiplicative fault diagnosis approach for automation processes.

This paper presents a new data-driven method for diagnosing multiplicative key performance degradation in automation processes. Different from the well-established additive fault diagnosis approaches, the proposed method aims at identifying those low-level components which increase the variability of process variables and cause performance degradation. Based on process data, features of multiplicative fault are extracted. To identify the root cause, the impact of fault on each process variable is evaluated in the sense of contribution to performance degradation. Then, a numerical example is used to illustrate the functionalities of the method and Monte-Carlo simulation is performed to demonstrate the effectiveness from the statistical viewpoint. Finally, to show the practical applicability, a case study on the Tennessee Eastman process is presented.