Identification of the Porcine Vascular Endothelial Cell-Specific Promoter ESAM1.0 Using Transcriptome Analysis.

The vascular endothelium of xenografted pig organs represents the initial site of rejection after exposure to recipient immune cells. In this study, we aimed to develop a promoter specific to porcine vascular endothelial cells as a step toward overcoming xenograft rejection. Transcriptome analysis was performed on porcine aortic endothelial cells (PAECs), ear skin fibroblasts isolated from GGTA knockout (GTKO) pigs, and the porcine renal epithelial cell line pk-15. RNA sequencing confirmed 243 differentially expressed genes with expression changes of more than 10-fold among the three cell types. Employing the Human Protein Atlas database as a reference, we identified 34 genes exclusive to GTKO PAECs. The endothelial cell-specific adhesion molecule (ESAM) was selected via qPCR validation and showed high endothelial cell specificity and stable expression across tissues. We selected 1.0 kb upstream sequences of the translation start site of the gene as the promoter ESAM1.0. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the porcine intercellular adhesion molecule 2 (ICAM2) promoter, which is frequently used to target endothelial cells in transgenic pigs. Consequently, ESAM1.0 will enable the generation of genetically modified pigs with endothelium-specific target genes to reduce xenograft rejection.

A novel testis-enriched gene, Samd4a, regulates spermatogenesis as a spermatid-specific factor.

Spermatogenesis is the highly orchestrated process involving expression of a series of testicular genes. Testis-enriched genes are critical for cellular processes during spermatogenesis whose disruption leads to impaired spermatogenesis and male infertility. Nevertheless, among poorly investigated testicular genes are the mouse Samd4a and human SAMD4A which were identified in the current study as novel testis-enriched genes through transcriptomic analyses. In particular, as orthologous alternative splicing isoforms, mouse Samd4a E-form and human SAMD4AC-form containing the SAM domain were specific to testes. Western blot analyses revealed that the murine SAMD4AE-form was predominantly found in the testis. Analyses on GEO2R and single-cell RNA-seq datasets revealed that the Samd4a/SAMD4A expression was enriched in spermatids among various types of cells in adult testes. To investigate in vivo functions of Samd4a, Samd4a knockout mice were generated using the CRISPR/Cas9 system. The Samd4a deficiency resulted in lower testis weight, absence of elongated spermatids, and an increased number of apoptotic cells. Profiling of gene expression in human testis samples revealed that the SAMD4A expression was comparable between obstructive azoospermia patients and normal controls, but significantly lowered in nonobstructive azoospermia (NOA) patients. Among three subgroups of NOA, pre-meiotic arrest (NOA-pre), meiotic arrest (NOA-mei), and post-meiotic arrest (NOA-post), expression level of SAMD4A was higher in the NOA-post than the NOA-mei, but there was no difference between the NOA-pre and NOA-mei. The current studies demonstrated spermatid stage-specific expression of Samd4a/SAMD4A, and impairment of the late stages of spermatogenesis by disruption of the mouse Samd4a gene. These data suggest that Samd4a/SAMD4A plays an essential role in normal spermatogenesis, and SAMD4A, as a spermatid specific marker, can be used for subcategorizing NOA patients. Further understanding the molecular role of SAMD4A will advance our knowledge on genetic regulations in male infertility.

cd26 Knockdown Negatively Affects Porcine Parthenogenetic Preimplantation Embryo Development.

cd26 is ubiquitously distributed in the body, particularly in the endothelial and epithelial cells, with the highest expression in the kidney, liver, and small intestine. In humans, cd26 serves as a marker for the embryo implantation phase. However, little is known about the role of cd26 in porcine pre-implantation embryo development. Here, we aimed to examine siRNA-induced cd26 downregulation in the cytoplasm of MII oocytes, to determine whether cd26 is involved in the regulation of porcine pre-implantation embryonic development. The cd26 siRNA was micro-injected into the cytoplasm of MII oocytes, which were then parthenogenetically activated electrically in a medium containing 0.3M Mannitol. Inhibition of the cd26 expression did not affect cleavage but stopped development in the blastocyst stage. Additionally, the cd26 siRNA-treated blastocysts had significantly more apoptotic cells than the untreated blastocysts. Among the 579 transcripts evaluated with transcriptome resequencing, 38 genes were differentially expressed between the treatment and control blastocysts (p < 0.05). Twenty-four genes were upregulated in cd26 siRNA-injected blastocysts, whereas 14 were downregulated. These genes are involved in apoptosis, accumulation of reactive oxygen species, and aberrant expression of ribosomal protein genes. Our results indicate that cd26 is required for proper porcine parthenogenetic activation during embryonic development.

Research Note: Embryonic viability by weight difference between donor and surrogate eggs in a surrogate eggshell incubation system.

A surrogate eggshell incubation system is a well-defined method to apply to avian genetic modification. In this study, we tried to investigate whether the egg weight differences between donor and surrogate eggs have an effect on donor viability. The groups were divided by egg weight differences between the donor and surrogate eggs into 4 in each system. The viability at d 4 was evaluated at the end of System II, the embryos alive were transferred into the second surrogate eggshells, and the viability at d 5, 6 was evaluated at early phase of System III. Then, the viability of System III was evaluated at different incubation period: d 6-12, d 13-18, d 19-21, and hatching rate was evaluated at d 22. Although the effect of egg weight differences between the donor and surrogate eggs was not observed, a specific group in System III showed higher survival and hatching rate than other group (P > 0.05).

Select Porcine Elongation Factor 1α Sequences Mediate Stable High-Level and Upregulated Expression of Heterologous Genes in Porcine Cells in Response to Primate Serum.

Genetically engineered (GE) pigs with various combinations of genetic profiles have been developed using heterologous promoters. This study aimed to identify autologous promoters for high and ubiquitous expression of xenotransplantation relevant genes in GE pigs. A 1.4 kb upstream regulatory sequence of porcine elongation factor 1α (pEF1α) gene was selected and isolated for use as a promoter. Activity of the pEF1α promoter was subsequently compared with that of the cytomegalovirus (CMV) promoter, CMV enhancer/chicken ß-actin (CAG) promoter, and human EF1α (hEF1α) promoter in different types of pig-derived cells. Comparative analysis of luciferase and mutant human leukocyte antigen class E-F2A-ß-2 microglobulin (HLA-E) expression driven by pEF1α, CMV, CAG, and hEF1α promoters revealed the pEF1α promoter mediated comparable expression levels with those of the CAG promoter in porcine ear skin fibroblasts (PEFs) and porcine kidney-15 (PK-15) cells, but lower than those of the CAG promoter in porcine aortic endothelial cells (PAECs). The pEF1α promoter provided long-term stable HLA-E expression in PEFs, but the CAG promoter failed to sustain those levels of expression. For xenogeneic serum-induced cytotoxicity assays, the cells were cultured for several hours in growth medium supplemented with primate serum. Notably, the pEF1α promoter induced significant increases in luciferase and HLA-E expression in response to primate serum in PAECs compared with those driven by the CAG promoter, suggesting the pEF1α promoter could regulate temporal expression of heterologous genes under xenogeneic-cytotoxic conditions. These results suggest the pEF1α promoter may be valuable for development of GE pigs spatiotemporally and stably expressing immunomodulatory genes for xenotransplantation.

N-acetyl-L-cysteine Improves the Developmental Competence of Bovine Oocytes and Embryos Cultured In Vitro by Attenuating Oxidative Damage and Apoptosis.

Oxidative stress has been suggested to negatively affect oocyte and embryo quality and developmental competence, resulting in failure to reach full term. In this study, we investigated the effect of N-acetyl-L-cysteine (NAC), a cell-permeating antioxidant, on developmental competence and the quality of oocytes and embryos upon supplementation (0.1-10 mM) in maturation and culture medium in vitro using slaughterhouse-derived oocytes and embryos. The results show that treating oocytes with 1.0 mM NAC for 8 h during in vitro maturation attenuated the intracellular reactive oxygen species (ROS) (p < 0.05) and upregulated intracellular glutathione levels (p < 0.01) in oocytes. Interestingly, we found that NAC affects early embryonic development, not only in a dose-dependent, but also in a stage-specific, manner. Significantly (p < 0.05) decreased cleavage rates (90.25% vs. 81.46%) were observed during the early stage (days 0-2), while significantly (p < 0.05) increased developmental rates (38.20% vs. 44.46%) were observed during the later stage (from day 3) of embryonic development. In particular, NAC supplementation decreased the proportion of apoptotic blastomeres significantly (p < 0.05), resulting in enhanced hatching capability and developmental rates during the in vitro culture of embryos. Taken together, our results suggest that NAC supplementation has beneficial effects on bovine oocytes and embryos through the prevention of apoptosis and the elimination of oxygen free radicals during maturation and culture in vitro.

Factors Influencing Nurses' Willingness to Speak Up Regarding Patient Safety in East Asia: A Systematic Review.

Speaking up for patient safety among health care professionals is important because it can contribute to the prevention of adverse patient events, such as medication errors, infections, wrong-site surgical procedures, and other sentinel events. This systematic review identified factors that facilitate or inhibit nurses' willingness to speak up regarding patient safety in East Asian hospitals. Following the steps of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, four databases, including PubMed, CINAHL, PsycInfo, and Web of Science, were searched. Nine studies were analyzed in this review, including five qualitative and four quantitative studies published between 2014 and 2019. Quality of included studies were evaluated using the Mixed-Method Appraisal Tool. Data synthesis was based upon qualitative-led synthesis adopting two existing multilevel frameworks on safety voice and employee voice signals. Four studies were conducted in Japan, three in South Korea, one in Hong Kong, and one in Taiwan. We organized factors influencing East Asian nurses' willingness to speak up regarding patient safety according to the following four contexts: individual (motivation toward patient safety, organizational commitment, perceived effectiveness and importance of speaking up, and assertive personality), team (positive relationship and team trust, team culture, and mentoring), organizational (hospital administrative support and organizational culture) and sociocultural (hierarchy and power differential and collectivistic culture). However, due to the limited number of studies conducted in East Asian hospitals, further studies with larger cohort samples of nurses in various East Asian countries should be conducted to deepen our understanding of nurses' willingness to voice their concerns for patient safety.

Isolation and characterization of cultured chicken oviduct epithelial cells and in vitro validation of constructed ovalbumin promoter in these cells.

OBJECTIVE: Transgenic hens hold a great promise to produce various valuable proteins. Through virus transduction into stage X embryo, the transgene expression under the control of constructed chicken ovalbumin promoters has been successfully achieved. However, a validation system that can evaluate differently developed ovalbumin promoters in in vitro, remains to be developed. METHODS: In the present study, chicken oviduct epithelial cells (cOECs) were isolated from oviduct tissue and shortly cultured with keratinocyte complete medium supplemented with chicken serum. The isolated cells were characterized with immunofluorescence, western blot, and flow cytometry using oviduct-specific marker. Chicken mutated ovalbumin promoter (Mut-4.4-kb-pOV) was validated in these cells using luciferase reporter analysis. RESULTS: The isolated cOECs revealed that the oviduct-specific marker, ovalbumin protein, was clearly detected by immunofluorescence, western blot, and flow cytometry analysis revealed that approximately 79.40% of the cells contained this protein. Also, luciferase reporter analysis showed that the constructed Mut-4.4-kb-pOV exhibited 7.1-fold (p<0.001) higher activity in the cOECs. CONCLUSION: Collectively, these results demonstrate the efficient isolation and characterization of cOECs and validate the activity of the constructed ovalbumin promoter in the cultured cOECs. The in vitro validation of the recombinant promoter activity in cOECs can facilitate the production of efficient transgenic chickens for potential use as bioreactors.

Developmental and Degenerative Characterization of Porcine Parthenogenetic Fetuses during Early Pregnancy.

The difference between early pregnancy and delivery rate is quite large in assisted reproduction techniques (ARTs), including animal cloning. However, it is not clear why the implanted fetuses aborted after the early pregnancy stage. In the present study, we tried to evaluate the developmental and morphological characteristics of porcine parthenogenetically activated (PA) embryos or fetuses by electric stimulation during the early pregnancy period. The implanted PA and artificially inseminated (AI) embryos and fetuses were collected at day 26 and 35 after embryo transfer, respectively. The developmental and morphological parameters in the PA embryos at day 26 were similar to the AI embryos. The size, weight, formation of major organs, and apoptotic cells were not statistically different in both embryos at day 26. However, the PA fetuses at day 35 showed ceased fetal development and degenerated with abnormal morphologies in their organs. The day 35 PA fetuses showed significantly higher apoptotic cells and lower methylation status in three differentially methylated regions of the H19 gene compared to their comparators. Therefore, the normal development of PA embryos and fetuses during early gestation could lead to these pregnancies being misinterpreted as normal and become one of the main reasons for the gap between early pregnancy and delivery rate.

Production and Breeding of Transgenic Cloned Pigs Expressing Human CD73.

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.

Syndecan-1 overexpression promotes tumor growth and angiogenesis in an endometrial cancer xenograft model.

OBJECTIVES: Upregulation of syndecan-1, a member of the transmembranous proteoglycans that serves as a coreceptor for a wide pool of extracellular ligands, has been well documented in enabling the promotion of growth and invasion of endometrial cancer. As a step toward understanding a potential role for syndecan-1 in this process, we questioned whether syndecan-1 upregulates tumor-promoting characteristics, particularly, angiogenesis in an in vivo human xenograft tumor model. METHODS: Human syndecan-1 was stably transfected into human endometrial adenocarcinoma 1A cells, and resulting transfectants were subcutaneously grafted into athymic mice; their outcomes were examined with respect to the enhancement of tumor growth and angiogenesis by immunohistochemistry, immunoblotting, and zymography. RESULTS: Overexpression of syndecan-1 promoted tumor growth concomitant with increased angiogenesis in tumor xenografts as evidenced by an increase in immunoreactivity for vascular endothelial growth factor and vascular endothelial cell marker CD34. Furthermore, zymographic studies revealed that syndecan-1 overexpression markedly enhanced activities of matrix metalloproteinases 2 and 9. CONCLUSIONS: This is the first in vivo xenograft analysis providing evidence that supports that syndecan-1 has a critical role in carcinogenic progression, particularly, contributing to the development of angiogenesis and invasive phenotype in association with matrix metalloproteinases 2 and 9 activations in endometrial cancer.