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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.

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 β-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.

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.

Author(s): Kwak, Kyungmin; Park, Jae-Kyung; Shim, Joohyun; Ko, Nayoung; Kim, Hyoung-Joo; Lee, Yongjin; Kim, Jun-Hyeong; Alexander, Michael; Lakey, Jonathan RT; Kim, Hyunil; Choi, Kimyung | Abstract: BackgroundPorcine 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.MethodsAdult porcine islets were isolated from 16 to 17-month-old Yucatan miniature pigs following standard rapid procurement. Pigs weighed on average 48.71n±n2.85nkg, and the produced pancreases were 39.51n±n1.80 grams (meann±nSEM). 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.ResultsIslet yield was highest in the group of adult pigs where Collagenase AF-1 GMP grade was utilized. The mean islet yield was 16n586n±n1391nIEQ/g vs 8302n±n986nIEQ/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.ConclusionsThis 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.

[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.

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.

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.

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.

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 β 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.

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.

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.

Aims S100A8/A9 is an inflammatory mediator secreted by activated neutrophils and monocytes/macrophages. Serum S100A8/A9 levels were previously found to correlate with the severity of coronary artery disease in diabetic patients and to predict cardiovascular (CV) events in healthy postmenopausal women. Our aim was to investigate whether S100A8/A9 correlates with carotid artery disease and whether it can predict CV risk in healthy individuals. Methods We measured baseline levels of S100A8/A9 in 664 individuals with no previous history of CV disease, randomly selected from the Malmö Diet and Cancer cohort. We examined the correlation between S100A8/A9 and traditional CV risk factors, circulating cell populations, plasma cytokines, carotid intima media thickness (IMT) and area (IM area), and the incidence of CV events during a follow-up period of 16.2 years. Results We detected significant positive correlations between baseline S100A8/A9 and the number of circulating neutrophils, IFNγ, TNFα, IL-1β, smoking, BMI, systolic blood pressure and an inverse correlation with HDL. In a multivariate linear regression model adjusted for age and sex, S100A8/A9 and the neutrophil counts were correlated with IMT and IM area in the common carotid artery. The associations with IM area remained significant after further adjustment for CV risk factors. In a Cox regression model corrected for age and sex, S100A8/A9 and circulating neutrophils predicted the incidence of coronary events (fatal or non-fatal myocardial infarction) [HR (95%CI) per SD: 1.28 (1.03-1.59), P =0.026 and 1.26 (1.04-1.53), P =0.020, respectively] and CV death [1.34 (1.06-1.71), P =0.015 and 1.59 (1.33-1.90), P Conclusions S100A8/A9 correlates strongly with circulating neutrophil numbers, but not with monocytes or monocyte sub-populations. Baseline S100A8/A9 concentrations and circulating neutrophils are significantly associated with carotid artery disease and predict coronary events and CV death in apparently healthy individuals. Our study provides further evidence for the involvement of activated neutrophils in atherogenesis and acute coronary events.