Docosahexaenoic acid supplementation during porcine oocyte in vitro maturation improves oocyte quality and embryonic development by enhancing the homeostasis of energy metabolism.

Although supplementation with docosahexaenoic acid (DHA) during porcine oocyte IVM is well-established, the available data are limited due to the lack of consistency. Moreover, to our knowledge, the anti-oxidant effects of DHA on porcine oocytes have not been reported. Hence, this study aimed to examine the effects of DHA supplementation on the regulation of energy metabolism during porcine oocyte maturation to improve oocyte maturation and embryonic development. By supplementing the IVM medium with various DHA concentrations, 25 µM DHA was identified as the optimal concentration which improved intraoocyte glutathione content and enhanced embryonic development after parthenogenesis. Compared to embryos derived from the control group, those derived from SCNT or IVF showed significantly improved blastocyst formation upon DHA supplementation during IVM. In addition, various transcription factors associated with oocyte development and apoptosis in mature oocytes were beneficially regulated in the DHA-treated oocytes. Moreover, DHA improved the AMP-activated protein kinase (AMPK)-regulatory ability of porcine oocytes and ameliorated nuclear maturation and embryonic development, which were decreased by artificially downregulating AMPK. To our knowledge, this is the first study to examine the effects of DHA as an AMPK regulator on oocyte maturation and embryo development in pigs. Furthermore, DHA addition to the IVM medium upregulated the relative expression of genes associated with mitochondrial potential and lipid metabolism. Therefore, the membrane potential of mitochondria (evaluated based on the JC-1 aggregate/JC-1 monomer ratio) and the levels of fatty acids and lipid droplets in matured oocytes increased, resulting in increased ATP synthesis. In conclusion, the DHA treatment of porcine oocytes with 25 µM DHA during IVM enhances the homeostasis of energy metabolism by improving mitochondrial function and lipid metabolism, leading to improved quality of matured oocytes and enhanced embryonic developmental potential of in vitro produced (IVP) embryos. Thus, 25 µM DHA supplementation could serve as a tool for improving the quality of IVP embryos. The study findings provide a basis for further research on improving the production efficiency of cloned animals by securing high-quality matured oocytes and enhancing energy metabolism in mammalian oocytes, including those of pigs.

Development of novel indole-quinoline hybrid molecules targeting bacterial proton motive force.

AIMS: This study aimed to develop an editable structural scaffold for improving drug development, including pharmacokinetics and pharmacodynamics of antibiotics by using synthetic compounds derived from a (hetero)aryl-quinoline hybrid scaffold. METHODS AND RESULTS: In this study, 18 CF3-substituted (hetero)aryl-quinoline hybrid molecules were examined for their potential antibacterial activity against Staphylococcus aureus by determining minimal inhibitory concentrations. These 18 synthetic compounds represent modifications to key regions of the quinoline N-oxide scaffold, enabling us to conduct a structure-activity relationship analysis for antibacterial potency. Among the compounds, 3 m exhibited potency against with both methicillin resistant S. aureus strains, as well as other Gram-positive bacteria, including Enterococcus faecalis and Bacillus subtilis. We demonstrated that 3 m disrupted the bacterial proton motive force (PMF) through monitoring the PMF and conducting the molecular dynamics simulations. Furthermore, we show that this mechanism of action, disrupting PMF, is challenging for S. aureus to overcome. We also validated this PMF inhibition mechanism of 3 m in an Acinetobacter baumannii strain with weaken lipopolysaccharides. Additionally, in Gram-negative bacteria, we demonstrated that 3 m exhibited a synergistic effect with colistin that disrupts the outer membrane of Gram-negative bacteria. CONCLUSIONS: Our approach to developing editable synthetic novel antibacterials underscores the utility of CF3-substituted (hetero)aryl-quinoline scaffold for designing compounds targeting the bacterial proton motive force, and for further drug development, including pharmacokinetics and pharmacodynamics.

Analysis of production efficiency of cloned transgenic Yucatan miniature pigs according to recipient breeds with embryo transfer conditions.

The purpose of this study was to compare the efficiency of the production of cloned transgenic Yucatan miniature pigs (YMPs) using two recipient breeds, i.e., YMPs and domestic pigs (DPs), under various embryo transfer conditions. We initially assessed the in vitro developmental competence of embryos obtained via somatic cell nuclear transfer (SCNT) from three different transgenic donor cells. No difference was observed among the three groups regarding developmental competence. Furthermore, the cloning efficiency remained consistent among the three groups after the transfer of the SCNT embryos to each surrogate mother. Subsequently, to compare the efficiency of the production of cloned transgenic YMPs between the two recipient breeds using varying parameters, including ovulation status (preovulation and postovulation), duration of in vitro culture (IVC) (incubated within 24 h and 24-48 h), and the number of transferred SCNT embryos (less than and more than 300), we assessed the pregnancy rates, delivery rates, mean offspring counts, and cloning efficiency. Regarding the ovulation status, YMPs exhibited higher pregnancy rates, delivery rates, and cloning efficiency compared with DPs in both statuses. Moreover, the pregnancy rates, delivery rates, and cloning efficiency were affected by the ovulation status in DPs, but not in YMPs. The comparison of IVC duration between groups revealed that YMPs had higher pregnancy rates vs. DPs in both conditions. SCNT embryos cultured for 24-48 h in YMPs yielded higher delivery rates and cloning efficiency compared with those cultured for less than 24 h in DPs. Finally, the analysis based on the number of transferred SCNT embryos showed that both the pregnancy and delivery rates were higher in YMPs vs. DPs. However, the highest average number of offspring was obtained when more than 300 SCNT embryos were transferred into DPs, whereas the cloning efficiency was higher in YMPs vs. DPs. Our results suggest that YMPs are more suitable recipients than are DPs under various conditions for the production of cloned transgenic YMPs.

Catalyst-Controlled C-H Allylation and Annulation of 2-Aryl Quinazolinones with 2-Methylidene Cyclic Carbonate.

The site-selective modification of quinazolinone as a privileged bicyclic N-heterocycle is an attractive topic in medicinal chemistry and material science. We herein report the ruthenium(II)-catalyzed C-H allylation of 2-aryl quinazolinones with 2-methylidene cyclic carbonate. In addition, tandem C-H allylation and annulation are achieved under rhodium(III) catalysis, resulting in the formation of tetracyclic quinazolinones including a tertiary carbon center. Post-transformations of the synthesized products demonstrate the potential of the developed methodology. A series of mechanistic investigations were also performed.

Rhodium(III)-Catalyzed Conjugate Addition of ß-CF3-Enones with Quinoline N-Oxides.

The site-selective incorporation of a trifluoromethyl group into biologically active molecules and pharmaceuticals has emerged as a central topic in medicinal chemistry and drug discovery. Herein, we demonstrate the rhodium(III)-catalyzed conjugate addition of ß-trifluoromethylated enones with quinoline N-oxides, which result in the generation of ß-trifluoromethyl-ß'-quinolinated ketones. The reaction proceeds under mild conditions with complete functional group tolerance. The synthetic applicability was showcased by successful gram-scale experiments and valuable synthetic transformations of coupling products.

Ruthenium(II)-Catalyzed Tandem C-H Allylation and [3 + 2] Dipolar Cycloaddition to Construct Bridged Tetracycles.

The ruthenium(II)-catalyzed tandem C-H allylation and intramolecular dipolar cycloaddition between azomethine imines and 2-methylidenetrimethylene carbonate is described herein. The initially formed ß-substituted allyl fragment could trigger the exotype [3 + 2] cycloaddition with the polar azomethine group, resulting in the formation of bridged tetracycles bearing a hydroxymethylene group at a bridgehead carbon center. A wide substrate scope and broad functional group compatibility were observed. The gram-scale synthesis and synthetic transformations demonstrate the synthetic utility of this process.

A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model.

Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients.

Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid.

Background: Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells. Methods: Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed. Results: The C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs. Conclusion: TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.

Site-selective and metal-free C-H nitration of biologically relevant N-heterocycles.

The site-selective and metal-free C-H nitration reaction of quinoxalinones and pyrazinones as biologically important N-heterocycles with t-butyl nitrite is described. A wide range of quinoxalinones were efficiently applied in this transformation, providing C7-nitrated quinoxalinones without undergoing C3-nitration. From the view of mechanistic point, the radical addition reaction exclusively occurred at the electron-rich aromatic region beyond electron-deficient N-heterocycle ring. This is a first report on the C7-H functionalization of quinoxalinones under metal-free conditions. In contrast, the nitration reaction readily takes place at the C3-position of pyrazinones. This transformation is characterized by the scale-up compatibility, mild reaction conditions, and excellent functional group tolerance. The applicability of the developed method is showcased by the selective reduction of NO2 functionality on the C7-nitrated quinoxalinone product, providing aniline derivatives. Combined mechanistic investigations aided the elucidation of a plausible reaction mechanism.

Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs.

In this study, we investigated the effect of a triple knockout of the genes alpha-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and alpha 1,3-galactosyltransferase 2 (A3GALT2) in Yucatan miniature pigs on human immune reactivity. We used the CRISPR/Cas9 system to create pigs lacking GGTA1 (GTKO) and GGTA1/CMAH/A3GALT2 triple gene knockout (TKO). The expression of all three xenoantigens was absent in TKO pigs, but there was no additional reduction in the level of Galα1,3Gal (αGal) epitopes expression in the A3GALT2 gene KO. Peripheral blood mononuclear cells (PBMCs), aorta endothelial cells (AECs), and cornea endothelial cells (CECs) were isolated from these pigs, and their ability to bind human IgM/IgG and their cytotoxicity in human sera were evaluated. Compared to wild type (WT) pigs, the level of human antibody binding of the PBMCs, AECs, and CECs of the transgenic pigs (GTKO and TKO) was significantly reduced. However, there were significant differences in human antibody binding between GTKO and TKO depending on the cell type. Human antibody binding of TKO pigs was less than that of GTKO on PBMCs but was similar between GTKO and TKO pigs for AECs and CECs. Cytotoxicity of transgenic pig (GTKO and TKO) PBMCs and AECs was significantly reduced compared to that of WT pigs. However, TKO pigs showed a reduction in cytotoxicity compared to GTKO pigs on PBMCs, whereas in AECs from both TKO and GTKO pigs, there was no difference. The cytotoxicity of transgenic pig CECs was significantly decreased from that of WT at 300 min, but there was no significant reduction in TKO pigs from GTKO. Our results indicate that genetic modification of donor pigs for xenotransplantation should be tailored to the target organ and silencing of additional genes such as CMAH or A3GALT2 based on GTKO might not be essential in Yucatan miniature pigs.

Comparison of islet isolation result and clinical applicability according to GMP-grade collagenase enzyme blend in adult porcine islet isolation and culture.

BACKGROUND: Porcine islet xenotransplantation is a promising treatment for type 1 diabetes as an alternative to human pancreatic islet transplantation and long-term insulin therapy. Several research groups have explored porcine islets as an alternative to the inconsistent and chronic shortage of pancreases from human organ donors. Studies have confirmed successful transplant of porcine islets into non-human primate models of diabetes; however, in most cases, they require more than one adult porcine donor to achieve sufficient viable islet mass for sustained function. The importance of GMP-grade reagents includes the following: specific enzymes utilized in the pancreatic isolation process were identified as a key factor in successful human clinical islet transplantation trials using cadaveric islets. As xenotransplantation clinical research progresses, isolation reagents and digestion enzymes play a key role in the consistency of the product and ultimately the outcome of the islet xenotransplant. In this study, we evaluated several commercially available enzyme blends that have been used for islet isolation. We evaluated their impact on islet isolation yield and subsequent islet function as part of our plan to bring xenotransplantation into clinical xenotransplantation trials. METHODS: Adult porcine islets were isolated from 16 to 17-month-old Yucatan miniature pigs following standard rapid procurement. Pigs weighed on average 48.71 ± 2.85 kg, and the produced pancreases were 39.51 ± 1.80 grams (mean ± SEM). After ductal cannulation, we evaluated both GMP-grade enzymes (Collagenase AF-1 GMP grade and Liberase MTF C/T GMP grade) and compared with standard non-GMP enzyme blend (Collagenase P). Islet quality control assessments including islet yield, islet size (IEQ), membrane integrity (acridine orange/propidium iodide), and functional viability (GSIS) were evaluated in triplicate on day 1 post-islet isolation culture. RESULTS: Islet yield was highest in the group of adult pigs where Collagenase AF-1 GMP grade was utilized. The mean islet yield was 16 586 ± 1391 IEQ/g vs 8302 ± 986 IEQ/g from pancreases isolated using unpurified crude Collagenase P. The mean islet size was higher in Collagenase AF-1 GMP grade with neutral protease than in Collagenase P and Liberase MTF C/T GMP grade. We observed no significant difference between the experimental groups, but in vitro islet function after overnight tissue culture was significantly higher in Collagenase AF-1 GMP grade with neutral protease and Liberase MTF C/T GMP grade than the crude control enzyme group. As expected, the GMP-grade enzyme has significantly lower endotoxin levels than the crude control enzyme group when measured. CONCLUSIONS: This study validates the importance of using specifically blended GMP grade for adult pig islet isolation for xenotransplantation trials and the ability to isolate a sufficient number of viable islets from one adult pig to provide a sufficient number for islets for a clinical islet transplantation. GMP-grade enzymes are highly efficient in increasing islet yield, size, viability, and function at a lower and acceptable endotoxin level. Ongoing research transplants these islets into animal models of diabetes to validate in vivo function. Also, these defined and reproducible techniques using GMP-grade enzymes allow for continuance of our plan to advance to xenotransplantation of isolated pig islets for the treatment of type 1 diabetes.

No excessive mutations in transcription activator-like effector nuclease-mediated α-1,3-galactosyltransferase knockout Yucatan miniature pigs.

OBJECTIVE: Specific genomic sites can be recognized and permanently modified by genome editing. The discovery of endonucleases has advanced genome editing in pigs, attenuating xenograft rejection and cross-species disease transmission. However, off-target mutagenesis caused by these nucleases is a major barrier to putative clinical applications. Furthermore, off-target mutagenesis by genome editing has not yet been addressed in pigs. METHODS: Here, we generated genetically inheritable α-1,3-galactosyltransferase (GGTA1) knockout Yucatan miniature pigs by combining transcription activator-like effector nuclease (TALEN) and nuclear transfer. For precise estimation of genomic mutations induced by TALEN in GGTA1 knockout pigs, we obtained the whole-genome sequence of the donor cells for use as an internal control genome. RESULTS: In-depth whole-genome sequencing analysis demonstrated that TALEN-mediated GGTA1 knockout pigs had a comparable mutation rate to homologous recombination-treated pigs and wild-type strain controls. RNA sequencing analysis associated with genomic mutations revealed that TALEN-induced off-target mutations had no discernable effect on RNA transcript abundance. CONCLUSION: Therefore, TALEN appears to be a precise and safe tool for generating genome-edited pigs, and the TALEN-mediated GGTA1 knockout Yucatan miniature pigs produced in this study can serve as a safe and effective organ and tissue resource for clinical applications.

Effect of alanine supplementation during in vitro maturation on oocyte maturation and embryonic development after parthenogenesis and somatic cell nuclear transfer in pigs.

The objective of this study was to examine the effect of alanine treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development in pigs. To this end, we investigated the nuclear maturation, intraoocyte glutathione (GSH) content of IVM oocytes, and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In addition, we analyzed the expression of genes associated with apoptosis and embryonic development in IVM oocytes, 4-cell stage embryos, and blastocysts produced via PA and SCNT. To determine the optimal concentration of alanine to promote the maturation and development of PA and SCNT embryos, various concentrations (0, 0.363, 1, 5, and 10 mM) of alanine were added to IVM medium during oocyte maturation. The proportion of metaphase II (MII) oocytes after IVM did not differ according to the concentration of alanine. However, significantly higher intraoocyte GSH content was observed in oocytes treated with 0.363 mM alanine compared with that in untreated oocytes. However, treatment of recipient oocytes with 5 or 10 mM alanine during IVM decreased the GSH content in mature oocytes compared to that in control oocytes. Oocytes matured in the presence of 0.363 mM alanine showed significantly increased rates of cleavage and blastocyst formation after PA and SCNT compared to untreated oocytes. PA and SCNT embryos from the 0.363 mM alanine-treated group of MII oocytes showed significantly higher transcript levels of POU5F1 and FGFR2, which are associated with oocyte quality and embryonic development, than the untreated group. Our results suggest that treatment of pig oocytes with 0.363 mM alanine during IVM improves embryonic developmental competence after PA and SCNT by increasing intraoocyte GSH content and increasing the mRNA expression of POU5F1 and FGFR2.

The association of locomotive and non-locomotive physical activity measured by an accelerometer with functional fitness in healthy elderly men: a pilot study.

PURPOSE: The purpose of this study was to investigate the relationship of various aspects of daily physical activity, such as the number of steps, time spent in moderate to vigorous intensity physical activity (MVPA), and locomotive and non-locomotive MVPA measured by a triaxial accelerometer, with the functional fitness in healthy elderly men. METHODS: The subjects of this study were 22 healthy elderly men aged over 65 years. The participants wore a triaxial accelerometer for two weeks to estimate their daily physical activities. The level of functional fitness was measured based on "National Fitness Award 100 in Korea" immediately after the measurement of two weeks of daily physical activities. RESULTS: The results showed that active healthy elderly men with more than 6,500 walking steps per day and more than 60 min per day spent in MVPA showed a significantly higher 2-min marching in place and index of cardiorespiratory endurance compared to less physically active participants. Particularly, locomotive MVPA was significantly associated with cardiorespiratory endurance levels (r = 0.50), whereas non-locomotive MVPA was not associated with other measurements of functional fitness. CONCLUSION: Increased MVPA time, especially the locomotive MVPA, can effectively suppress the decrease in cardiorespiratory endurance level in elderly men. However, no association was observed between non-locomotive MVPA, such as household activities, and functional fitness in healthy elderly men.

Production of heterozygous alpha 1,3-galactosyltransferase (GGTA1) knock-out transgenic miniature pigs expressing human CD39.

Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig's cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig's cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.

Analysis of Swine Leukocyte Antigen Haplotypes in Yucatan Miniature Pigs Used as Biomedical Model Animal.

The porcine major histocompatibility complex (MHC) is called swine leukocyte antigen (SLA), which controls immune responses and transplantation reactions. The SLA is mapped on pig chromosome 7 (SSC7) near the centromere. In this study, 3 class I (SLA-1, SLA-3, and SLA-2) and 3 class II (DRB1, DQB1, and DQA) genes were used for investigation of SLA haplotypes in Yucatan miniature pigs in Korea. This pig breed is a well-known model organism for biomedical research worldwide. The current study indicated that Korean Yucatan pig population had 3 Class I haplotypes (Lr-4.0, Lr-6.0, and Lr-25.0) and 3 class II haplotypes (Lr-0.5, Lr-0.7, and Lr-0.25). The combinations of SLA class I and II haplotype together, 2 homozygous (Lr-4.5/4.5 and Lr-6.7/6.7) and 3 heterozygous (Lr-4.5/6.7, Lr-4.5/25.25, and Lr-6.7/25.25) haplotypes were identified, including previously unidentified new heterozygous haplotypes (Lr-4.5/4.7). In addition, a new SLA allele typing method using Agilent 2100 bioanalyzer was developed that permitted more rapid identification of SLA haplotypes. These results will facilitate the breeding of SLA homozygous Yucatan pigs and will expedite the possible use of these pigs for the biomedical research, especially xenotransplantation research.

Plasma S100A8/A9 correlates with blood neutrophil counts, traditional risk factors, and cardiovascular disease in middle-aged healthy individuals.

OBJECTIVE: The S100 alarmins A8, A9, and A8/A9, secreted by activated neutrophils and monocytes/macrophages, are involved in the pathogenesis of various inflammatory diseases. S100A8/A9 has previously been linked to atherogenesis and cardiovascular (CV) disease. We investigated whether S100A8, A9, and A8/A9 correlate with carotid artery disease and CV risk in apparently healthy individuals. APPROACH AND RESULTS: We measured baseline S100A8, A9, and A8/A9 in 664 individuals aged 63 to 68 years, with no previous history of CV disease, randomly selected from the Malmö Diet and Cancer population cohort. We examined the correlations between S100 proteins and circulating cell populations, plasma cytokines, carotid artery disease, and incidence of CV events during a median follow-up period of 16.2 years. We found that plasma S100A8/A9 concentration is positively influenced by circulating neutrophil numbers, smoking, body mass index, glycosylated hemoglobin A1c, and low-density lipoprotein. High-density lipoprotein was negatively associated with S100A8/A9. S100A8/A9 and the neutrophil counts were positively correlated with intima-media area in the common carotid artery, independently of age, sex, and CV risk factors. S100A8/A9 and circulating neutrophils presented similar associations with the incidence of coronary events (hazard ratio [95% confidence interval] per 1 SD: 1.28 [1.03-1.59] and 1.26 [1.04-1.53], respectively) and CV death (1.34 [1.06-1.71] and 1.59 [1.33-1.90], respectively). These relationships were mainly supported by strong associations in women, which were independent of traditional risk factors. There were no independent relationships between S100A8 and S100A9, and CV disease. CONCLUSIONS: Our study supports the value of S100A8/A9 as a potentially important link between neutrophils, traditional CV risk factors, and CV disease.

Nucleofection-mediated α1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation.

Xenotransplantation of pig organs into primates leads to hyperacute rejection (HAR). Functional ablation of the pig α 1,3-galactosyltransferase (GalT) gene, which abrogates expression of the Gal α 1-3Gal ß 1-4GlcNAc-R (Gal) antigen, which inhibits HAR. However, antigens other than Gal may induce immunological rejection by their cognate antibody responses. Ultimately, overexpression of complement regulatory proteins reduces acute humoral rejection by non-Gal antibodies when GalT is ablated. In this study, we developed a vector-based strategy for ablation of GalT function and concurrent expression of membrane cofactor protein (MCP, CD46). We constructed an MCP expression cassette (designated as MCP-IRESneo) and inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Nucleofection of porcine ear skin fibroblasts using the U-023 and V-013 programs resulted in high transfection efficiency and cell survival. We identified 28 clones in which the MCP-IRESneo vector had been successfully targeted to exon 9 of the GalT gene. Two of those clones, with apparent morphologically mitotic fibroblast features were selected through long-term culture. GalT gene expression was downregulated in these 2 clones. Importantly, MCP was shown to be efficiently expressed at the cell surface and to efficiently protect cell lysis against normal human complement serum attack in vitro.

Unique expression of a small IL-32 protein in the Jurkat leukemic T cell line.

Interleukin (IL)-32 was recently identified as a new cytokine which induces various proinflammatory cytokines in human monocytes and macrophages. Therefore, IL-32 has been primarily studied in inflammatory models such as rheumatoid arthritis and inflammatory bowel diseases. The regulation of endogenous IL-32 in other immune cells remains unknown. In the present study, we stimulated Jurkat T cells with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) and examined IL-32 expression at both the mRNA and protein levels. All mRNAs of the four IL-32 isoforms and the 12-15 kDa IL-32 protein were independent of PHA and PMA stimulation, however a 9 kDa molecular weight IL-32 protein in the cell culture supernatant was induced by PHA and PMA after 16 h of stimulation. Compared to other human cell lines, the Jurkat cell line constitutively expressed a 12-15 kDa molecule of IL-32, which is smaller than the known IL-32 isoforms. We used IL-32 shRNA to examine the specificity of the 12-15 kDa molecule. Upon IL-32 shRNA transfection, the 12-15 kDa band was decreased specifically as compared to the control scrambled clone. Thus, the constitutive expression of IL-32 mRNA as well as the predominant production of a smaller sized IL-32 isoform in Jurkat cells may implicate a role for IL-32 in human T cell leukemia.