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In contrast to humans or rabbits, in which maternal IgG is transmitted to offspring prenatally via the placenta or the yolk sac, large domestic animals such as pigs, cows and sheep transmit IgG exclusively through colostrum feeding after delivery. The extremely high IgG content in colostrum is absorbed by newborns via the small intestine. Although it is widely accepted that the neonatal Fc receptor, FcRn, is the receptor mediating IgG transfer across both the placenta and small intestine, it remains unclear whether FcRn also mediates serum IgG transfer across the mammary barrier to colostrum/milk, especially in large domestic animals. In this study, using a FcRn knockout pig model generated with a CRISPR-Cas9-based approach, we clearly demonstrate that FcRn is not responsible for the IgG transfer from serum to colostrum in pigs, although like in other mammals, it is involved in IgG homeostasis and mediates IgG absorption in the small intestine of newborns.

Scope Obesity is a major public health and economic problem of global significance. Here, we investigated the role of diosmetin, a natural flavonoid presents mainly in citrus fruits, in the regulation of obesity and metabolic dysfunctions in mice. Methods and results Eight-week-old male C57BL/6 mice fed a high-fat diet (HFD) or five-week-old male ob/ob mice fed a normal diet were treated with diosmetin (50 mg kg-1 daily) or vehicle for 8 weeks. Diosmetin treatment decreased body weight and fat mass, improved glucose tolerance and insulin resistance in obese mice. These metabolic benefits were mainly attributed to increased energy expenditure via enhancing thermogenesis in brown adipose tissue (BAT) and browning of white adipose tissue (WAT). Mechanistically, diosmetin acted as an agonist for estrogen receptors (ERs), and subsequently elevated adipose expressions of ERs in mice and in cultured adipocytes. When ERs were blocked by their antagonist fulvestrant in mice, diosmetin loses its beneficial effects on activation of BAT, browning of WAT, body weight control and glucose homeostasis, suggesting that ERs are indispensable for the metabolic benefits of diosmetin. Conclusion The results indicate that diosmetin might be a potential anti-obesity nutritional supplement and could be explored for low ERs-related obesity populations. This article is protected by copyright. All rights reserved.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease and the most economically important disease of the swine industry worldwide. Highly pathogenic-PRRS virus (HP-PRRSV) is a variant of PRRSV, which caused high morbidity and mortality. Scavenger receptor CD163, which contains nine scavenger receptor cysteine-rich (SRCR) domains, is a key entry mediator for PRRSV. A previous study demonstrated that SRCR domain 5 (SRCR5), encoded by exon 7, was essential for PRRSV infection in vitro. Here, we substituted exon 7 of porcine CD163 with the corresponding exon of human CD163-like 1 (hCD163L1) using a CRISPR/Cas9 system combined with a donor vector. In CD163Mut/Mut pigs, modifying CD163 gene had no adverse effects on hemoglobin-haptoglobin (Hb-Hp) complex clearance or erythroblast growth. In vitro infection experiments showed that the CD163 mutant strongly inhibited HP-PRRSV replication by inhibiting virus uncoating and genome release. Compared to wild-type (WT) pigs in vivo, HP-PRRSV-infected CD163Mut/Mut pigs showed a substantially decreased viral load in blood and relief from PRRSV-induced fever. While all WT pigs were dead, there of four CD163Mut/Mut pigs survived and recovered at the termination of the experiment. Our data demonstrated that modifying CD163 remarkably inhibited PRRSV replication and protected pigs from HP-PRRSV infection, thus establishing a good foundation for breeding PRRSV-resistant pigs via gene editing technology.

BackgroundThe mutation of insulin-like growth factor 2 (IGF2 mutation) that a single-nucleotide substitution (G→A) in the third intron ofIGF2abrogates the interaction with zinc finger BED-type containing 6 (ZBED6) and leads to increased muscle mass in pigs. IGF2 mutation knock-in (IGF2 KI) and ZBED6 knockout (ZBED6 KO) lead to changes inIGF2expression and increase muscle mass in mice and pigs. Long noncoding RNAs (lncRNAs) may participate in numerous biological processes, including skeletal muscle development. However, the role of the ZBED6-lncRNA axis in skeletal muscle development is poorly characterized.ResultsIn this study, we assembled transcriptomes using RNA-seq data published in previous studies by our group and identified 11,408 known lncRNAs and 2269 potential lncRNAs in seven tissues, heart, longissimus dorsi, gastrocnemius muscle, liver, spleen, lung and kidney, of ZBED6 KO (lean mass model) and WT Bama pigs. ZBED6 affected the expression of 1570 lncRNAs (differentially expressed lncRNAs [DE-lncRNAs]; log2-fold change ≥ 1, nominalp-value ≤ 0.05) in the seven examined tissues. The expressed lncRNAs (FPKM > 0.1) exhibited tissue-specific patterns in WT pigs. Specifically, 3410 lncRNAs were expressed exclusively in only one tissue. Potential functions of lncRNAs were indirectly predicted by searching their target cis- and trans-regulated protein-coding genes. LncRNAs with tissue-specific expression influence numerous genes related to tissue functions. Weighted gene coexpression network analysis (WGCNA) of 1570 DE-lncRNAs between WT and ZBED6 KO pigs was used to define the following six lncRNA modules specific to different tissues: skeletal muscle, heart, lung, spleen, kidney and liver modules. Furthermore, by conjoint analysis of longissimus dorsi data (tissue-specific expression, muscle module and DE-lncRNAs) and ChIP-PCR revealedNONSUSG002145.1(adjustedp-values = 0.044), which is coexpressed with theIGF2gene and binding with ZBED6, may play important roles in ZBED6 KO pig skeletal muscle development.ConclusionsThese findings indicate that the identified lncRNAs may play essential roles in tissue function and regulate the mechanism of ZBED6 action in skeletal muscle development in pigs. To our knowledge, this is the first study describing lncRNAs in ZBED6 KO pigs. These results may open new research directions leading to a better understanding of the global functions of ZBED6 and of lncRNA functions in skeletal muscle development in pigs.

The immunogenicity of heterogeneous pericardium is a major factor causing the failure of commercial biological valves. Both decellularization and glutaraldehyde crosslinking can reduce the immunogenicity of pericardium, but it is difficult to completely remove it. Getting gene knockout pigs is considered a highly efficient method to obtain low-immunogenic biological tissues, including the pericardium. Our results showed that repeated cycles of decellularization could obviously reduce the residual DNA in tissues for pigs without gene knockout, but at the same time they could also break down the components of pericardia, resulting in a decline in mechanical properties. We compared the mechanical and biological properties of the glutaraldehyde crosslinked pericardia of GGTA1 knockout pig, GGTA1/CMAH knockout pig, GGTA1/β4GalNT2 knockout pig and wild type (WT) pig. The mechanical properties of the pericardia of GGTA1 knockout pig and GGTA1/CMAH knockout pig were similar to those of control pig (WT), and the experimental results of subdermal implantation in rats showed significantly reduced immune rejection and calcification. Pulsatile flow and fatigue in vitro tests showed that the fabricated artificial aortic valve met the hydrodynamic requirements after 2×10^8 accelerated fatigue cycles. Gene knockout pigs have great potential in the application of artificial biological valve products.

Genome editing techniques have been implemented in human daily lives, which has created a high demand for the development of new gene-edited product analysis methods. Conventional assays are time-consuming, labor-intensive, and costly. This paper proposes a rapid and low-cost strategy for detecting genome-editing induced deletion which works by integrating rapid-multiplex ligation-dependent probe amplification (MLPA) with a dual-lateral flow nucleic acid biosensor (LFNAB) cascade in a single-copy case. A rapid-MLPA was first introduced to the LFNAB system as a replacement for the conventional PCR for enhanced specificity and accuracy. A dual-LFNAB was applied for the detection of genome-editing induced deletion without any additional instrumentation or complex operation. After optimization, we achieved the specific detection of wildtype alleles and deletion alleles in spiked samples with a detection limit of 0.4 fM, which is comparable to that of electrophoresis-based detection assays and fluorescent biosensors. To confirm the validity and feasibility of our strategy, we assayed two pork samples from two WUZHISHAN pigs successfully. By comparing the detection results from next-generation sequencing analysis, we found that the proposed cascade demonstrates at least 20-fold shorter assay time and at least 100-fold less assay cost. To this effect, the proposed method is a rapid and low-cost solution to sample-to-answer detection of genome-editing induced deletion and shows remarkable potential in regards to international trade, transparency, and freedom of choice.

This study aimed to establish an event-specific multiplex PCR system using microsatellite markers and fluorescently labeled primers to detect six different genetically modified (GM) animal lines, including human lactoferrin GM cattle, human lysozyme GM cattle, human α-lactalbumin GM cattle, myostatin knockout pigs, phytase GM pigs, and ω-3 fatty acid desaturase gene GM pigs. Four different microsatellite loci for species identification, along with six GM animal-specific fragments, were selected as targets for primer design. The capillary gel electrophoresis results of multiplex PCR showed that the target fragments were amplified successfully. This high-throughput multiplex PCR detection system can be applied for the inspection and quarantine of GM animals.

ZBED6 (zinc finger BED domain containing protein 6) is a transcription factor unique to placental mammals and its interaction with the IGF2 (insulin-like growth factor 2) locus plays a prominent role in the regulation of postnatal skeletal muscle growth. Here, we generated lean Bama miniature pigs by generating ZBED6-knockout (ZBED6−/−) and investigated the mechanism underlying ZBED6 in growth of muscle and internal organs of placental mammals. ZBED6−/− pigs show markedly higher lean mass, lean mass rate, larger muscle fiber area and heavier internal organs (heart and liver) than wild-type (WT) pigs. The striking phenotypic changes of ZBED6-/- pigs coincided with remarkable upregulation of IGF2 mRNA and protein expression across three tissues (gastrocnemius muscle, longissimus dorsi, heart). Despite a significant increase in liver weight, ZBED6-/- pigs show comparable levels of IGF2 expression to those of WT controls. A mechanistic study revealed that elevated methylation in the liver abrogates ZBED6 binding at the IGF2 locus, explaining the unaltered hepatic IGF2 expression in ZBED6-/- pigs. These results indicate that a ZBED6-IGF2-independent regulatory pathway exists in the liver. Transcriptome analysis and ChIP-PCR revealed new ZBED6 target genes other than IGF2, including cyclin dependent kinase inhibitor 1A (CDKN1A) and tsukushi, small leucine rich proteoglycan (TSKU), that regulates growth of muscle and liver, respectively., Author summary The lean meat rate is an important economic trait for the swine industry and it is determined by muscle growth and development. A single base change in intron 3 of the insulin-like growth factor 2 (IGF2) gene increases meat production in pigs by disrupting a binding site for zinc finger BED domain containing protein 6 (ZBED6). Chinese indigenous pig breeds carrying the homozygous IGF2 wild-type allele produce low lean meat. We thus generate a lean pig model in Chinese Bama pig by knocking out ZBED6. In this model, we demonstrate that ZBED6 KO increases muscle and internal organ growth through ZBED6-IGF2 axis and other target genes. These results not only open new strategies for lean meat breeding in Chinese indigenous pigs, but also provide new insights to the global function of ZBED6 in organ growth and development.

Molecular characterization of transgenic animals is crucial for the phenotype analysis, unintended effects prediction, commercial and economic requirements. In this study, a comprehensive molecular characterization of a transgenic pig, which expressed hCD46 gene, was verified using next-generation sequencing (NGS). One complete and one incomplete sequence of pBSCD46-neo and multiple backbone fragments were inserted into the host genome. The whole insertion sequence was 28,243bp, and 12bp sequence on the genome was deleted during the transgene. The cost of NGS is higher than other molecular methods, in order to reduce the cost and to make the sequencing strategy more economically effective, we carried out a computer simulation to identify the smallest amount of data that could meet the requirements of the molecular characterization analysis. 15× depth of coverage was shown to be sufficient with good accuracy, while 3× might be effective, 20× was recommended. The obtained molecular characterization information would provide a stronger foundation for the safety evaluation of the GM animals. Meanwhile, the gene integrity of the porcine pathogen PERV was analyzed using the same procedure. No complete sequence of the PERV subtype was observed in the porcine genome, which was the same as the previous research. In consequence, NGS combined bioinformatics analysis is a reliable and accurate approach for the molecular characterization of GMOs, even for the safety evaluation.

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

After hyperacute rejection in pig-to-primate xenotransplantation had been overcome by the introduction of α1,3-galactosyltransferase gene-knockout (GTKO) pigs, acute and chronic antibody-mediated rejection became one of the major barriers to long-term graft survival. This was associated with exposure of non-Gal antigens to the recipient's immune system and indicated that further genetic engineering of the pigs would be necessary. We here report that Gabarapl1, a regulator of tumorigenesis, plays a role in the regulation of immunogenicity of porcine aortic endothelial cells (PAECs). Knockdown of Gabarapl1 in PAECs results in a remarkable reduction in binding of serum antibody from PAEC-immunized monkeys, associated with decreased serum cytotoxicity of pig cells. Expression of swine leukocyte antigens (SLA) II DR was downregulated by Gabarapl1 knockdown. However, suppression of expression of SLA II is associated with less reduction of antibody binding than achieved by Gabarapl1 knockdown, suggesting that other Gabarapl1-regulated xenoantigens may be more important. These findings indicate a hitherto unknown relationship between Gabarapl1 and xenoimmunogenicity, suggesting a potential new strategy to reduce rejection initiated by the presence of non-Gal antigens.

Background In vivo pig liver xenotransplantation preclinical trials appear to have poor efficiency compared to heart or kidney xenotransplantation because of xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia. In contrast, ex vivo pig liver (wild type) perfusion systems have been proven to be effective in "bridging" liver failure patients until subsequent liver allotransplantation, and transgenic (human CD55/CD59) modifications have even prolonged the duration of pig liver perfusion. Despite the fact that hepatocyte cell lines have also been proposed for extracorporeal blood circulation in conditions of acute liver failure, porcine hepatocyte cell lines, and the GalT-KO background in particular, have not been developed and applied in this field. Herein, we established immortalized wild-type and GalT-KO porcine hepatocyte cell lines, which can be used for artificial liver support systems, cell transplantation, and even in vitro studies of xenotransplantation. Methods Primary hepatocytes extracted from GalT-KO and wild-type pigs were transfected with SV40 LT lentivirus to establish immortalized GalT-KO porcine hepatocytes (GalT-KO-hep) and wild-type porcine hepatocytes (WT). Hepatocyte biomarkers and function-related genes were assessed by immunofluorescence, periodic acid-Schiff staining, indocyanine green (ICG) uptake, biochemical analysis, ELISA, and RT-PCR. Furthermore, the tumorigenicity of immortalized cells was detected. In addition, a complement-dependent cytotoxicity (CDC) assay was performed with GalT-KO-hep and WT cells. Cell death and viability rates were assessed by flow cytometry and CCK-8 assay. Results GalT-KO and wild-type porcine hepatocytes were successfully immortalized and maintained the characteristics of primary porcine hepatocytes, including albumin secretion, ICG uptake, urea and glycogen production, and expression of hepatocyte marker proteins and specific metabolic enzymes. GalT-KO-hep and WT cells were confirmed as having no tumorigenicity. In addition, GalT-KO-hep cells showed less apoptosis and more viability than WT cells when exposed to complement and xenogeneic serum. Conclusions Two types of immortalized cell lines of porcine hepatocytes with GalT-KO and wild-type backgrounds were successfully established. GalT-KO-hep cells exhibited higher viability and injury resistance against a xenogeneic immune response.

Pig-to-human organ transplantation provides an alternative for critical shortage of human organs worldwide. Genetically modified pigs are promising donors for xenotransplantation as they show many anatomical and physiological similarities to humans. However, immunological rejection including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), immune cell-mediated rejection, and other barriers associated with xenotransplantation must be overcome with various strategies for the genetic modification of pigs. In this review, we summarize the outcomes of genetically modified and cloned pigs achieved by Chinese scientists to resolve the above-mentioned problems in xenotransplantation. It is now possible to knockout several porcine genes associated with the expression of sugar residues, antigens for (naturally) existing antibodies in humans, including GGTA1, CMAH, and β4GalNT2, and thereby preventing the antigen-antibody response. Moreover, insertion of human complement- and coagulation-regulatory transgenes, such as CD46, CD55, CD59, and hTBM, can further overcome effects of the humoral immune response and coagulation dysfunction, while expression of regulatory factors of immune responses can inhibit the adaptive immune rejection. Furthermore, transgenic strategies have been developed by Chinese scientists to reduce the potential risk of infections by endogenous porcine retroviruses (PERVs). Breeding of multi-gene low-immunogenicity pigs in China is also presented in this review. Lastly, we will briefly mention the preclinical studies on pig-to-non-human primate xenotransplantation conducted in several centers in China.

Significance To investigate the epigenetic mechanism of pregnancy failure in mammals, we exploited the high rate of fetal loss in pig induced pluripotent stem cell (iPSC) nuclear transfer. We generated methylomes of pig iPSCs and associated nuclear transfer embryos from reciprocal crosses between two distinct pig breeds. Our methylome analysis revealed that misregulation of RTL1 as the principal basis of pregnancy failure using pig iPSCs. Remarkably, RTL1 has broad fertility implications across mouse, rat, pig, cattle, and human from nuclear transfer cloning, tetraploid complementation, and artificial insemination, to natural fertilization. In all of these procedures, low RTL1 expression consistently corresponds to pregnancy failures., Substantial rates of fetal loss plague all in vitro procedures involving embryo manipulations, including human-assisted reproduction, and are especially problematic for mammalian cloning where over 90% of reconstructed nuclear transfer embryos are typically lost during pregnancy. However, the epigenetic mechanism of these pregnancy failures has not been well described. Here we performed methylome and transcriptome analyses of pig induced pluripotent stem cells and associated cloned embryos, and revealed that aberrant silencing of imprinted genes, in particular the retrotransposon-derived RTL1 gene, is the principal epigenetic cause of pregnancy failure. Remarkably, restoration of RTL1 expression in pig induced pluripotent stem cells rescued fetal loss. Furthermore, in other mammals, including humans, low RTL1 levels appear to be the main epigenetic cause of pregnancy failure.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease and the most economically important disease of the swine industry worldwide. Highly pathogenic-PRRS virus (HP-PRRSV) is a variant of PRRSV, which caused high morbidity and mortality. Scavenger receptor CD163, which contains nine scavenger receptor cysteine-rich (SRCR) domains, is a key entry mediator for PRRSV. A previous study demonstrated that SRCR domain 5 (SRCR5), encoded by exon 7, was essential for PRRSV infection in vitro. Here, we substituted exon 7 of porcine CD163 with the corresponding exon of human CD163-like 1 (hCD163L1) using a CRISPR/Cas9 system combined with a donor vector. In CD163Mut/Mut pigs, modifying CD163 gene had no adverse effects on hemoglobin-haptoglobin (Hb-Hp) complex clearance or erythroblast growth. In vitro infection experiments showed that the CD163 mutant strongly inhibited HP-PRRSV replication by inhibiting virus uncoating and genome release. Compared to wild-type (WT) pigs in vivo, HP-PRRSV-infected CD163Mut/Mut pigs showed a substantially decreased viral load in blood and relief from PRRSV-induced fever. While all WT pigs were dead, there of four CD163Mut/Mut pigs survived and recovered at the termination of the experiment. Our data demonstrated that modifying CD163 remarkably inhibited PRRSV replication and protected pigs from HP-PRRSV infection, thus establishing a good foundation for breeding PRRSV-resistant pigs via gene editing technology.

Bioprosthetic heart valves (BHVs) originating from pigs are extensively used for heart valve replacement in clinics. However, recipient immune responses associated with chronic calcification lead to structural valve deterioration (SVD) of BHVs. Two well-characterized epitopes on porcine BHVs have been implicated in SVD, including galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) whose synthesis are catalyzed by α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Ac hydroxylase (encoded by the CMAH gene), respectively. It has been reported that BHV from αGal-knockout pigs are associated with a significantly reduced immune response by human serum. Moreover, valves from αGal/Neu5Gc-deficient pigs could further reduce human IgM/IgG binding when compared to BHV from αGal-knockout pigs. Recently, another swine xenoantigen, Sd(a), produced by β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2), has been identified. To explore whether tissue from GGTA1, CMAH, and β4GalNT2 triple gene-knockout (TKO) pigs would further minimize human antibody binding to porcine pericardium, TKO pigs were successfully produced by CRISPR/Cas9 mediated gene targeting. Our results showed that the expression of αGal, Neu5G and Sd(a) on TKO pigs was negative, and that human IgG/IgM binding to pericardium was minimal. Moreover, the analysis of collagen composition and physical characteristics of porcine pericardium from the TKO pigs indicated that elimination of the three xenoantigens had no significant impact on the physical proprieties of porcine pericardium. Our results demonstrated that TKO pigs would be an ideal source of BHVs. Statement of significance Surgical heart valve replacement is an established lifesaving treatment for diseased heart valve. Bioprosthetic heart valves (BHVs) made from glutaraldehyde-fixed porcine or bovine tissues are widely used in clinics but exhibit age-dependent structural valve degeneration (SVD) which is associated with the immune response against BHVs. Three major xenoantigens present on commercial BHVs, Galactosea α1,3 galactose (αGal), N-glycolylneuraminic acid (Neu5Gc) and glycan products of β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2) are eliminated through CRISPR/Cas9 mediated gene targeting in the present study. The genetically modified porcine pericardium showed reduced immunogenicity but comparable collagen composition and physical characteristics of the pericardium from wild-type pigs. Our data suggested that BHVs from TKO pigs is a promising alternative for currently available BHVs from wild-type pigs.

When hyperacute rejection is avoided by deletion of Gal expression in the pig, delayed xenograft rejection (DXR) becomes a major immunologic barrier to successful xenotransplantation. This study was to investigate the potential antigens involved in DXR. We isolated primary renal microvascular endothelial cells (RMEC) and aortic endothelial cells (AEC) from a GGTA1/CMAH double-knockout (DKO) pig (and a GGTA1-KO pig) and immunized cynomolgus monkeys with both of these cells. After sensitization, monkey serum antibody binding and cytotoxicity to RMEC was significantly higher than to AEC(p

Although it is important for the study of graphene, identifying and characterizing the number of graphene layers is challenging. In this paper, we calculate graphene’s transmission. The result shows that the phase change is more sensitive than the intensity change when light passes through graphene in some THz frequencies. Based on this fact, a simple route is presented for identifying the single or few layers of graphene sheets by using terahertz phase contrast microscopy (TPCM). The route is fast, and easy to be carried out.

Cytokines play crucial roles in inflammation, but their role in xenotransplantation remains elusive. We assessed the role of several cytokines using an in vitro model of human antibody-mediated complement-dependent cytotoxicity (CDC). Recombinant human angiopoietin-1 (Ang-1) protected porcine iliac endothelial cells (PIECs) from human antibody-mediated CDC. Interestingly, human angiopoietin-2 (Ang-2) had a similar protective effect on PIECs. By flow cytometry analysis, the extent of human IgM and IgG binding to PIECs did not decrease when PIECs were exposed to Ang-1/Ang-2. The mRNA level of complement regulators (CD46, CD55, CD59) was not upregulated in PIECs treated with Ang-1/Ang-2, both of which activated the PI3K/AKT pathway in PIECs. Wortmannin, which inhibits phosphatidylinositide 3-kinase (PI3K), suppressed Ang-1/Ang-2-induced AKT phosphorylation and consequent Ang-1/Ang-2-mediated protection of PIECs in human antibody-mediated CDC model. Moreover, dominant negative AKT also suppressed Ang-1/Ang-2-mediated protection of PIECs in this model. In conclusion, our data suggest that human Ang-1/Ang-2 induces the protection of PIECs from human antibody-mediated CDC by activating the PI3K/AKT pathway. Ang-1/Ang-2 is likely to protect porcine endothelial cells and may be beneficial in xenotransplantation research.

We present a facile surfactant-free solvothermal method for the fabrication of nearly monodispersed Fe3O4 submicroparticles with tunable particle sizes ranging from 130 to 420 nm by varying the concentration of single iron source FeCl3·6H2O in initial solutions. The morphology and crystal structure of the as-prepared Fe3O4 submicroparticles have been well characterized by using SEM/TEM/HRTEM, XRD, FT-IR, Raman spectroscopy, and XPS methods. It is found that the Fe3O4 particles present single-crystal nature and strong ferromagnetic property with magnetization saturation values ranged in 54.3–88.7 emu·g–1. A complexation–aggregation–phase transformation formation mechanism was first proposed for the nearly monodispersed single-crystal Fe3O4 submicroparticles based upon the quasi-in situ monitoring of the morphology and structure evolution of the samples during the synthesis process. These size-tunable nearly monodispersed Fe3O4 submicroparticles are expected to have promising applications in wide research f...

Mg-Al layered double hydroxide (LDH) nanohybrids intercalated with ibuprofen (IBU) with particle sizes of 150–530 nm have been synthesized through hydrothermal and coprecipitation treatment in aqueous solution without any organic solvent. The in vitro drug release properties of as-prepared IBU-LDH nanohybrids are systematically studied and the kinetic simulation to the release profiles suggests that the release processes of the larger nanohybrids are mainly controlled by intraparticle diffusion. The morphology changes from thick sheet-like into thin margin-curved platelets for the larger nanohybrid particles, induced by the hydrophobic IBU anions aggregations located in the edge region of interlayer via ion-exchange diffusion process, is firstly observed during the release process. Based on the SEM, HRTEM, XRD, FTIR, and UV-vis analyses of the samples recovered at different release time, a release mechanism model of the as-prepared IBU-LDH nanohybrids is tentatively proposed along with their morphology changes during the whole release process. © 2010 American Institute of Chemical Engineers AIChE J, 2011

For fully controlled drug release, it is important to completely understand the microstructure and nature of the layered double hydroxide that ultimately control drug release properties. In this study, a series of novel doxifluridine intercalated Mg–Al-layered double hydroxide (DFUR–LDH) microhybrids were fabricated via the reconstruction method. With increasing aging pH values from 7.2 to 9.5, two different LDH phases with varied basal spacings ( d 003 ) are formed. The DFUR–LDHr1.7p7.2 with d 003 1.91 nm presents continuous worm-like morphology and possible bilayer interlayer arrangement, while DFUR–LDHr2.0p9.5 with d 003 0.82 nm possesses discontinuous plate-like morphology and multi-sites interacted interlayer arrangement. Consequently, the in vitro release shows that the former has much longer release duration and a little faster initial release due to the weaker interaction between DFUR and LDH layers than that of the latter. The in vitro release data can be well described by the Bhaskar equation and modified Freundlich model, revealing that the release mechanism of DFUR from DFUR–LDH microhybrids is heterogeneous particle diffusion. Furthermore, the enteric polymer modified DFUR–LDHr1.7p7.2/L100 microspheres present no obvious release in pH 1.2 HCl solution, but continuous release of 79% in pH 6.8 and 7.4 PBS, suggesting a readily controlled release behavior upon the varied medium pH and potential application in colon specific drug delivery in cancer therapy.

The forward-scattering model is introduced to describe the response of normalized radar cross section (NRCS) of precipitation with synthetic aperture radar (SAR). Since the distribution of near-surface rainfall is related to the rate of near-surface rainfall and horizontal distribution factor, a retrieval algorithm called modified regression empirical and model-oriented statistical (M–M) based on the volterra integration theory is proposed. Compared with the model-oriented statistical and volterra integration (MOSVI) algorithm, the biggest difference is that the M–M algorithm is based on the modified regression empirical algorithm rather than the linear regression formula to retrieve the value of near-surface rainfall rate. Half of the empirical parameters are reduced in the weighted integral work and a smaller average relative error is received while the rainfall rate is less than 100 mm/h. Therefore, the algorithm proposed in this paper can obtain high-precision rainfall information.

In the present study, the DNA methylation patterns of in vitro-derived mouse tetraploid embryos were investigated by immunofluorescence staining with an antibody against 5-methylcytosine (5MeC). Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cytoplasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methylation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribution of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass (ICM) than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts. So the DNA methylation patterns of mouse tetraploid embryos are aberrant, which may lead to subsequent developmental failure and embryo death. This is the first report on the methylation patterns of in vitro-derived mouse tetraploid embryos.

LDH (layered double hydroxides)-based magnetic-sensitive drug−inorganic nanohybrids were assembled by a one-step co-precipitation method. The effect of a magnetic substance on the microstructure and drug release behavior of ibuprofen (IBU)-intercalated Mg−Al−LDH in magnetic nanohybrids was systematically studied via XRD, TEM, XPS, and VSM methods and in vitro release with and without an external magnetic field (MF). The results reveal a well-defined core−shell structure with a gray IBU−LDH shell coated onto the surface of a dark magnetic core and superior magnetic sensitivity of the magnetic nanohybrids. Compared with the clear platelets of the pure IBU−LDH, the IBU−LDH coatings in magnetic nanohybrids exhibit more like a compact stacking film fully covering the magnetic core and their particle sizes and thickness decrease with increasing core contents, explaining an enhanced release rate under no MF. While under a 1500 G MF, the release rate is greatly reduced with increasing core content due to the inst...

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were added into the culture medium in different culturing stages. The effects of EGF or bFGF on the development of porcine parthenogenetic embryos were studied in vitro. The results were as follows: The addition of EGF significantly enhanced the cleavage rate of porcine parthenogenetic embryos (P < 0.05). The addition of EGF or bFGF also significantly enhanced the rate of blastocysts formation of 2–4-cell porcine parthenogenetic embryos (P < 0.05). Additionally, the group of bFGF had more numbers of blastocysts and higher rates of blastocysts formation than the groups of EGF and the control. In conclusion, EGF and bFGF were found propitious to the development of porcine parthenogenetic embryos in vitro, and bFGF increased the quality of blastocysts by increasing the total cell number in porcine parthenogenetic embryos.

In vitro oocyte growth is the essential technology which enables oocytes to achieve maturation and acquire the competence for subsequent manipulation. There is increasing evidence that members of the transforming growth factor-β (TGF-β) superfamily are expressed in a variety of cell types within the ovary in a developmental stage-related manner and function as crucial factors in oocyte growth and follicular development. However, the expression of TGF-β family members has been studied extensively in follicular compartment cells in the ovaries while poorly explored in the cumulus–oocytes complex (COC) within culture systems. Using semi-quantitative RT-PCR, we investigated the temporal and spatial expression patterns of several bone morphogenetic proteins (BMP-4, BMP-6, BMP-15 and GDF-9), as well as BMP receptors (BMPRIA, BMPRIB, BMPRII and ActRII), in porcine COCs throughout in vitro maturation (IVM). In oocytes, the transcription of BMP-6, BMP-15, GDF-9 and BMPRII were down-regulated, while BMP-4, BMPRIA and BMPRIB remained unchanged during IVM. In cumulus cells, BMP-4 mRNA expression increased significantly, while BMP-6 and ActRII was down-regulated during IVM. Nevertheless, mRNAs of BMPRIA, BMPRIB and BMPRII were constantly expressed in cumulus cells in the process. However, BMP-15 was absent in cumulus cells and ActRII was not detected in oocytes. In addition, hardly any transcription of BMP-2, BMP-5, BMP-7, ActRIA was found in porcine COCs throughout IVM. These data demonstrate a complex BMP-signaling system for member gene expression within porcine COCs during IVM and indicate the need for further functional characterization of these factors during oocyte maturation.

An effective isolation and identification method of primordial follicles would greatly benefit the animal production practice, transgenic animal production and endangered species conservation in the future. This study has not only advanced the isolation method but also developed an identification marker of primordial follicles. After enzymatic digestion, Percoll gradient centrifugation and mesh filtrations, the obtained follicular separations were then subjected to a cell sorter in order to collect primordial follicles. The study greatly improved the yield of primordial follicles (from about 1.85 x 10(5) to 7.79 x 10(5) per prepubertal ovary) by means of increasing cell layer number from 1 ml to 2.5 ml after Percoll gradient centrifugation. Based on traditional morphological criteria, the purity of recovered primordial follicles was averagely about 82.43+/-9.41% (n=5) because of their similar size and appearance with somatic cells. To further exactly appreciate the purity of sorted primordial follicles, a germ cell-specific protein, MSY2, was used to recognize the oocytes of primordial follicles. The results of repeat experiments showed that about 98.31+/-0.73% (n=4) of the primordial follicles cluster was MSY2-positive, which indicated the identification method of primordial follicles was more effective and high-yielded than the previous methods because of its higher purity.

To date no authentic embryonic stem cell (ESC) line or germline-competent-induced pluripotent stem cell (iPSC) line has been established for large animals. Despite this fact, there is an impression in the field that large animal ESCs or iPSCs are as good as mouse counterparts. Clarification of this issue is important for a healthy advancement of the stem cell field. Elucidation of the causes of this failure in obtaining high quality iPSCs/ESCs may offer essential clues for eventual establishment of authentic ESCs for large animals including humans. To this end, we first generated porcine iPSCs using nonintegrating replicating episomal plasmids. Although these porcine iPSCs met most pluripotency criteria, they could neither generate cloned piglets through nuclear transfer, nor contribute to later stage chimeras through morula injections or aggregations. We found that the reprogramming genes in iPSCs could not be removed even under negative selection, indicating they are required to maintain self-renewal. The persistent expression of these genes in porcine iPSCs in turn caused differentiation defects in vivo. Therefore, incomplete reprogramming manifested by a reliance on sustained expression of exogenous-reprogramming factors appears to be the main reason for the inability of porcine iPSCs to form iPSC-derived piglets. Stem Cells 2015;33:3228–3238

Background: The activation of tissue factor (TF) is one of the major reasons for coagulation dysregulation after pig-to-primate xenotransplantation. Tissue factor pathway inhibitor (TFPI) is the most important inhibitor of TF. Studies have demonstrated species incompatibility between pig TFPI and human TF. Methods: A pig-to-macaque heterotopic auxiliary liver transplantation model was established to determine the origin of activated TF. Chimeric proteins of human and pig TFPI were constructed to assess the role of Kunitz domains in species incompatibility. Immortalised pig bone marrow mesenchymal stem cells transfected with human TFPI were tested for their ability to inhibit clotting in vitro. Results: TF from recipient was activated early after liver xenotransplantation. Pig TFPI Kunitz domain 2 bound human FXa, but Kunitz domain 1 did not effectively inhibit human TF/FVIIa. Immortalised pig bone marrow mesenchymal cells (BMSCs) transfected with human TFPI showed a prolonged recalcification time in vitro and in a rodent model. Conclusion: Recipient TF is relevant to dysregulated coagulation after xenotransplantation. Kunitz domain 1 plays the most important role in species incompatibility between pig TFPI and human TF, and clotting can be inhibited by human TFPI-transfected pig BMSCs. Our study shows a possible way to resolve the incompatibility of pig TFPI.

SummaryThe present study was designed to evaluate the feasibility of producing pig transgenic blastocysts expressing enhanced green fluorescent protein (GFP) and to examine the effects of shape and preparation methods of donor cells on in vitro developmental ability of pig nuclear transferred embryos (NTEs). In experiment 1, the effect of GFP transfection on development of pig NTEs was evaluated. The cleavage and blastocyst rates showed no significant difference between NTEs derived from transfected and non-transfected donors. In experiment 2, the effect of different nuclear donor preparation methods on in vitro development of NTEs was examined. The cleavage rate showed no statistically significant differences among three preparation methods. The blastocyst rates of donor cells treated once at −4 °C and those of freshly digested cells were similar to each other (26.3% vs 17.9%). The lowest blastocyst rates (5.88%) were observed when cells cryopreserved at −196 °C were used as donors. In experiment 3, the effect of different cell cycle synchronization methods on the in vitro development potential of pig NTEs was evaluated. The cleavage rate of NTEs derived from cycling cells was much better than that of NTEs derived from serum-starved cells (64.4% vs 50.5%, p < 0.05), but no significant difference was observed between the the blastocyst rates of the two groups. In experiment 4, the effect of different shapes of cultured fibroblast cells on the in vitro development of pig NTEs was examined. The fusion rate for couplets derived from rough cells was poorer than that observed in couplets derived from round smooth cells (47.8% vs 76.8%, p < 0.05). However, there were no significant differences observed in the cleavage rate and blastocyst rate. In conclusion, the present study indicated that (i) refrigerated pig GFP-transfected cells could be used as donors in nuclear transfer and these NTEs could be effectively developed to blastocyst stage; (ii) serum starvation of GFP-transfected cells is not required for preimplantation development of pig NTEs; and (iii) a rough surface of GFP-transfected donor cells affects fusion rate negatively but has no influence on the cleavage rate or blastocyst rate of pig NTEs.

Pig cloning has great potential to human xenotransplantation. The present study was designed to establish a more efficient system for producing cloned pigs by somatic cell nuclear transfer (SCNT). Our approach was as follows: SCNT embryos were reconstructed by using fetal fibroblasts of Chinese miniature pig as donors and in vitro matured oocytes of prepubertal gilts as recipients. Reconstructed embryos were induced by electrical fusion/activation and cultured in BSA-containing North Carolina State University 23 medium (NCSU-23) or Porcine Zygote Medium (PZM-3) at the gas condition of 5% CO2, 7% O2, 88% N2. A total of 230 cloned embryos were transferred to three surrogate sows, producing three piglets. One of them is apparently healthy. The clonal provenance of the piglet was indicated by its coat color and confirmed by DNA microsatellite analysis. These results indicate that the use of in vitro matured oocytes from prepubertal gilts as recipient, combined with cloned embryos cultured at low oxygen tension is an effective way to produce cloned pigs.

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (47.5% vs. 71.6%, p0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36 to 42 h (83.6% vs. 96.7%, p0.05). However, no statistical difference was observed between NTEs derived from oocytes of 36 h IVM group and NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p0.05) and blastocyst formation (9.3% vs. 13.2%, p0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O2) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O2) in cleavage rate (70.6% vs. 67.1%, p0.05) and blastocyst rate (11.8% vs. 12.3%, p0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of M II oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.