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Background: Extensive production and usage of commercially available products containing TiO2 NPs have led to accumulation in the human body. The deposition of TiO2 NPs has even been detected in the human placenta, which raises concerns regarding fetal health. Previous studies regarding developmental toxicity have frequently focused on TiO2 NPs < 50 nm, whereas the potential adverse effects of large-sized TiO2 NPs received less attention. Placental vasculature is essential for maternal–fetal circulatory exchange and ensuring fetal growth. This study explores the impacts of TiO2 NPs (100 nm in size) on the placenta and fetal development and elucidates the underlying mechanism from the perspective of placental vasculature. Pregnant C57BL/6 mice were exposed to TiO2 NPs by gavage at daily dosages of 10, 50, and 250 mg/kg from gestational day 0.5–16.5. Results: TiO2 NPs penetrated the placenta and accumulated in the fetal mice. The fetuses in the TiO2 NP-exposed groups exhibited a dose-dependent decrease in body weight and length, as well as in placental weight and diameter. In vivo imaging showed an impaired placental barrier, and pathological examinations revealed a disrupted vascular network of the labyrinth upon TiO2 NP exposure. We also found an increase in gene expression related to the transforming growth factor-β (TGF-β) -SNAIL pathway and the upregulation of mesenchymal markers, accompanied by a reduction in endothelial markers. In addition, TiO2 NPs enhanced the gene expression responsible for the endothelial-to-mesenchymal transition (EndMT) in cultured human umbilical vein endothelial cells, whereas SNAIL knockdown attenuated the induction of EndMT phenotypes. Conclusion: Our study revealed that maternal exposure to 100 nm TiO2 NPs disrupts placental vascular development and fetal mice growth through aberrant activation of EndMT in the placental labyrinth. These data provide novel insight into the mechanisms of developmental toxicity posed by NPs. [ABSTRACT FROM AUTHOR]

Piglets are susceptible to cold, and piglet death caused by cold stress leads to economic losses in the pig industry in cold areas. Skeletal muscle plays a key role in adaptive thermogenesis in mammals, but the related mechanism in pigs is unclear. In this study, cold-tolerant Tibetan pigs and cold-sensitive Bama pigs were subjected to either a cold environment (4 °C) or a room temperature environment (25 °C) for 3 days. The biceps femoris (BF) and longissimus dorsi muscle (LDM) were collected for phenotypic analysis, and the BF was used for genome-wide transcriptional profiling. Our results showed that Tibetan pigs had a higher body temperature than Bama pigs upon cold stimulation. RNA-seq data indicated a stronger transcriptional response in the skeletal muscle of Tibetan pigs upon cold stimulation, as more differentially expressed genes (DEGs) were identified with the same criteria (p < 0.05 and fold change > 2). In addition, distinct pathway signaling patterns in skeletal muscle upon cold exposure were found between the breeds of pigs. Mitochondrial beta-oxidation-related genes and pathways were significantly upregulated in Tibetan pigs, indicating that Tibetan pigs may use fatty acids as the primary fuel source to protect against cold. However, the significant upregulation of inflammatory response- and glycolysis-related genes and pathways in the skeletal muscle of Bama pigs suggested that these pigs may use glucose as the primary fuel source in cold environments. Together, our study revealed the distinct transcriptional responses of skeletal muscle to cold stimulation in Tibetan pigs and Bama pigs and provided novel insights for future investigation of the cold adaptation mechanism in pigs. [ABSTRACT FROM AUTHOR]

The 3D genome organization and its dynamic modulate genome function, playing a pivotal role in cell differentiation and development. CTCF and cohesin, acting as the core architectural components involved in chromatin looping and genome folding, can also recruit other protein or RNA partners to fine-tune genome structure during development. Moreover, systematic screening for partners of CTCF has been performed through high-throughput approaches. In particular, several novel protein and RNA partners, such as BHLHE40, WIZ, MAZ, Aire, MyoD, YY1, ZNF143, and Jpx, have been identified, and these partners are mostly implicated in transcriptional regulation and chromatin remodeling, offering a unique opportunity for dissecting their roles in higher-order chromatin organization by collaborating with CTCF and cohesin. Here, we review the latest advancements with an emphasis on features of CTCF partners and also discuss the specific functions of CTCF-associated complexes in chromatin structure modulation, which may extend our understanding of the functions of higher-order chromatin architecture in developmental processes. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to develop a predictive tool for live birth in women with adenomyosis undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. Methods: A total of 424 patients with adenomyosis who underwent frozen–thawed embryo transfer (FET) from January 2013 to December 2019 at a public university hospital were included. The patients were randomly divided into training (n = 265) and validation (n = 159) samples for the building and testing of the nomogram, respectively. Multivariate logistic regression (MLR) was developed on the basis of clinical covariates assessed for their association with live birth. Results: In total, 183 (43.16%) patients became pregnant, and 114 (26.88%) had a live birth. The MLR showed that the probability of live birth was significantly correlated with age [odds ratio (OR), 3.465; 95% confidence interval (CI), 1.215–9.885, P = 0.020], uterine volume (OR, 8.141; 95% CI, 2.170–10.542; P = 0.002), blastocyst transfer (OR, 3.231; 95% CI, 1.065–8.819, P = 0.023), twin pregnancy (OR, 0.328; 95% CI, 0.104–0.344, P = 0.005), and protocol in FET (P < 0.001). The statistical nomogram was built based on age, uterine volume, twin pregnancy, stage of the transferred embryo, and protocol of FET, with an area under the curve (AUC) of 0.837 (95% CI: 0.741–0.910) for the training cohort. The AUC for the validation cohort was 0.737 (95% CI: 0.661–0.813), presenting a well-pleasing goodness-of-fit and stability in this model. Conclusions: This visual and easily applied nomogram built on the risk factors of live birth in patients with adenomyosis provides useful and precise information for physicians on individualized decision-making during the IVF/ICSI procedure. [ABSTRACT FROM AUTHOR]

The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors (CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations. Our process resulted in a stop codon in CD163 and myostatin (MSTN) and introduced a beneficial allele in insulin-like growth factor-2 (IGF2). The pigs showed disrupted expression of CD163 and MSTN and increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection, providing a potential new route to produce elite pig breeders. [ABSTRACT FROM AUTHOR]

Background: IgA nephropathy (IgAN) is the most common primary glomerulonephritis globally. Increasing evidence suggests the importance of host immunity in the development of IgAN, but its dynamics during the early stage of IgAN are still largely unclear. Results: Here we successfully resolved the early transcriptomic changes in immune cells of IgAN by conducting single-cell RNA-sequencing (scRNA-seq) with peripheral blood mononuclear cells. The differentially expressed genes (DEGs) between control and IgAN were predominantly enriched in NK cell-mediated cytotoxicity and cell killing pathways. Interestingly, we discovered that the number and cytotoxicity of NK cells are significantly reduced in IgAN patients, where both the number and marker genes of NK cells were negatively associated with the clinical parameters, including the levels of urine protein creatinine ratio (UPCR), serum galactose-deficient IgA1 and IgA. A distinctive B cell subset, which had suppressed NFκB signaling was predominantly in IgAN and positively associated with disease progression. Moreover, the DEGs of B cells were enriched in different viral infection pathways. Classical monocytes also significantly changed in IgAN and a monocyte subset expressing interferon-induced genes was positively associated with the clinical severity of IgAN. Finally, we identified vast dynamics in intercellular communications in IgAN. Conclusions: We dissected the immune landscape of IgAN at the single-cell resolution, which provides new insights in developing novel biomarkers and immunotherapy against glomerulonephritis. [ABSTRACT FROM AUTHOR]

Objectives: RNF20 is recognized as a main E3 ligase for monoubiquitination of histone H2B at lysine 120 (H2Bub). The critical role of RNF20 and H2Bub in various molecular events, such as DNA replication, RNA transcription, and DNA damage response, has been widely investigated and documented. However, its role in porcine adipogenesis remains unknown. In this study, we aimed to clarify the effect of RNF20 on porcine preadipocyte differentiation. Materials and Methods: Backfat tissues from fat‐type pigs (Bama and Meishan) and lean‐type pigs (Yorkshire and Landrace) were collected to detect the expression level of RNF20. Preadipocytes were isolated from Bama piglets and induced to differentiation. Small interfering RNAs were applied to deplete RNF20. Oil Red O staining, quantitative real‐time PCR, RNA‐seq, Western blot analysis, and EdU assays were performed to study the regulatory mechanism of RNF20 during adipogenesis. Results: We found that the expression levels of RNF20 and H2Bub were significantly higher in backfat tissues from fat‐type pigs than in those from lean‐type pigs. Consistently, the significantly induced expression of RNF20 and H2Bub was also observed in porcine differentiated adipocytes. In addition, knockdown of RNF20 greatly inhibited porcine adipogenesis, as evidenced by dramatically decreased lipid droplet formation and lower expression levels of adipogenic transcription masters in RNF20 knockdown cells. Mechanistically, the depletion of RNF20 decreases the cell proliferation and the level of p‐C/EBPβ via the Ras‐Raf‐MEK1/2‐ERK1/2 cascade pathway at the mitotic clonal expansion phase and therefore suppresses cell differentiation. Conclusions: Our results demonstrate that RNF20 is required for porcine preadipocyte differentiation. [ABSTRACT FROM AUTHOR]

Background: Unexplained recurrent spontaneous abortion (URSA) is a common pregnancy complication and the etiology is unknown. URSA-associated lncRNAs are expected to be potential biomarkers for diagnosis, and might be related to the disease pathogenesis. Objective: To investigate differential lncRNAs in peripheral blood of non-pregnant URSA patients and matched healthy control women and to explore the possible mechanism of differential lncRNAs leading to URSA. Methods: We profiled lncRNAs expression in peripheral blood from 5 non-pregnant URSA patients and 5 matched healthy control women by lncRNA microarray analysis. Functions of URSA-associated lncRNAs were further investigated in vitro. Results: RP11-115N4.1 was identified as the most differentially expressed lncRNA which was highly upregulated in peripheral blood of non-pregnant URSA patients (P = 3.63E-07, Fold change = 2.96), and this dysregulation was further validated in approximately 26.67% additional patients (4/15). RP11-115N4.1 expression was detected in both lymphocytes and monocytes of human peripheral blood, and in vitro overexpression of RP11-115N4.1 decreased cell proliferation in K562 cells significantly. Furthermore, heat-shock HSP70 genes (HSPA1A and HSPA1B) were found to be significantly upregulated upon RP11-115N4.1 overexpression by transcriptome analysis (HSPA1A (P = 4.39E-08, Fold change = 4.17), HSPA1B (P = 2.26E-06, Fold change = 2.99)). RNA pull down and RNA immunoprecipitation assay (RIP) analysis demonstrated that RP11-115N4.1 bound to HNRNPH3 protein directly, which in turn activate heat-shock proteins (HSP70) analyzed by protein-protein interaction and HNRNPH3 knockdown assays. Most importantly, the high expression of HSP70 was also verified in the serum of URSA patients and the supernatant of K562 cells with RP11-115N4.1 activation, and HSP70 in supernatant can exacerbate inflammatory responses in monocytes by inducing IL-6, IL-1β, and TNF-α and inhibit the migration of trophoblast cells, which might associate with URSA. Conclusion: Our results demonstrated that the activation of RP11-115N4.1 can significantly increase the protein level of HSP70 via binding to HNRNPH3, which may modulate the immune responses and related to URSA. Moreover, RP11-115N4.1 may be a novel etiological biomarker and a new therapeutic target for URSA. [ABSTRACT FROM AUTHOR]

Chordoma is a rare bone tumor arising from notochordal remnants, but the underlying mechanism remains elusive. By integrated mRNA and microRNA analyses, we found significant downregulation of TGFB3 along with upregulation of its inhibitor, miR-29 family in chordoma comparing with notochord. Somatic copy number gains of miR-29 loci in chordoma highlighted a mechanism of inactivation of TGFB3 signaling in tumor formation. In zebrafish, knockout and knockdown homologous tgfb3 resulted in a chordoma-like neoplasm. On the other hand, Smad7 negative feedback regulation of transforming growth factor-β (TGF-β) signaling is retentive in chordoma cell UM-Chor1 despite its disruption in most cancer cells (e.g. A549). Therefore, contrary to other cancers, exogenous TGF-β activated Smad7 by downregulating miR-182 and inhibited cell migration and invasion in UM-Chor1. Meanwhile, TGF-β decreased chordoma characteristic protein Brachyury. Altogether, downregulation of TGFB3 causes chordomagenesis, showing a feasible target for therapies. The retention of Smad7 negative regulation may maintain the suppressor role of TGF-β in chordoma. [ABSTRACT FROM AUTHOR]

RNF20, an E3 ligase critical for monoubiquitination of histone H2B at lysine 120 (H2Bub), has been implicated in the regulation of various cellar processes; however, its physiological roles in adipocytes remain poorly characterized. Here, we report that the adipocyte-specific knockout of Rnf20 (ASKO) in mice led to progressive fat loss, organomegaly and hyperinsulinemia. Despite signs of hyperinsulinemia, normal insulin sensitivity and improved glucose tolerance were observed in the young and aged CD-fed ASKO mice. In addition, high-fat diet-fed ASKO mice developed severe liver steatosis. Moreover, we observed that the ASKO mice were extremely sensitive to a cold environment due to decreased expression levels of brown adipose tissue (BAT) selective genes, including uncoupling protein 1 (Ucp1), and impaired mitochondrial functions. Significantly decreased levels of peroxisome proliferator-activated receptor gamma (Pparγ) were observed in the gonadal white adipose tissues (gWAT) from the ASKO mice, suggesting that Rnf20 regulates adipogenesis, at least in part, through Pparγ. Rosiglitazone-treated ASKO mice exhibited increased fat mass compared to that of the non-treated ASKO mice. Collectively, our results illustrate the critical role of RNF20 in control of white and brown adipose tissue development and physiological function. [ABSTRACT FROM AUTHOR]

Pig is an important agricultural economic animal, providing large amount of meat products. With the development of functional genomics and bioinformatics, lots of genes and functional single nucleotide polymorphisms (SNPs) related to disease resistance and (or) economic traits in pigs have been identified, which provides the targets for genetic improvement by genome editing. Base editors (BEs), combining Cas9 nickase and cytidine or adenine deaminase, achieve all four possible transition mutations (C-to-T, A-to-G, T-to-C, and G-to-A) efficiently and accurately without double strand breaks (DSBs) under the protospacer adjacent motif (PAM) sequence of NGG. However, the NGG PAM in canonical CRISPR-Cas9 can only cover approximately 8.27% in the whole genome which limits its broad application. In the current study, hA3A-BE3-NG system was constructed with the fusion of SpCas9-NG variant and hA3A-BE3 to create C-to-T conversion at NGN PAM sites efficiently. The editing efficiency and scope of hA3A-BE3-NG were confirmed in HEK293T cells and porcine fetal fibroblast (PFF) cells. Results showed that the efficiency of hA3A-BE3-NG was much higher than that of hA3A-BE3 on NGH (H = A, C, or T) PAM sites (21.27 vs. 2.81% at average). Further, nonsense and missense mutations were introduced efficiently and precisely via hA3A-BE3-NG in multiple pig economic trait-related genes (CD163 , APN , MSTN , and MC4R) in PFF cells by one transfection. The current work indicates the potential applications of hA3A-BE3-NG for pyramid breeding studies in livestock. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: ATPase copper transporting α (ATP7A), also known as Menkes disease protein, is a P-type ATPase that transports copper across cell membranes. The critical role of ATP7A-mediated copper homeostasis has been well recognised in various organs, such as the intestine, macrophages and the nervous system. However, the importance of adipocyte ATP7A-mediated copper homeostasis on fat metabolism is not well understood. Here, we sought to reveal the contribution of adipose ATP7A to whole-body fat metabolism in mice. Methods: We generated adipocyte-specific Atp7a-knockout (ASKO) mice using the Cre/loxP system, with Cre expression driven by the adiponectin promoter. ASKO mice and littermate control mice were aged on a chow diet or fed with a high-fat diet (HFD); body weight, fat mass, and glucose and insulin metabolism were analysed. Histological analysis, transmission electron microscopy and RNA-sequencing (RNA-Seq) analysis of white adipose tissue (WAT) were used to understand the physiological and molecular changes associated with loss of copper homeostasis in adipocytes. Results: Significantly increased copper concentrations were observed in adipose tissues of ASKO mice compared with control mice. Aged or HFD-fed ASKO mice manifested a lipoatrophic phenotype characterised by a progressive generalised loss of WAT. Dysfunction of adipose tissues in these ASKO mice was confirmed by decreased levels of both serum leptin and adiponectin and increased levels of triacylglycerol and insulin. Systemic metabolism was also impaired in these mice, as evidenced by a pronounced glucose intolerance, insulin resistance and hepatic steatosis. Moreover, we demonstrate a significant induction of lipolysis and DNA-damage signalling pathways in gonadal WAT from aged and HFD-fed ASKO mice. In vitro studies suggest that copper overload is responsible for increased lipolysis and DNA damage. Conclusions/interpretation: Our results show a previously unappreciated role of adipocyte Atp7a in the regulation of ageing-related metabolic disease and identify new metallophysiologies in whole-body fat metabolism. Data availability: The datasets generated during the current study are available in the Genome Sequence Archive in BIG Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession number CRA001769 (http://bigd.big.ac.cn/gsa). [ABSTRACT FROM AUTHOR]

Harlequin ichthyosis (HI) is a severe genetic skin disorder and caused by mutation in the ATP-binding cassette A12 (ABCA12) gene. The retinoid administration has dramatically improved long-term survival of HI, but improvements are still needed. However, the ABCA12 null mice failed to respond to retinoid treatment, which impedes the development of novel cure strategies for HI. Here we generated an ethylnitrosourea mutagenic HI pig model (named Z9), which carries a novel deep intronic mutation IVS49-727 A>G in the ABCA12 gene, resulting in abnormal mRNA splicing and truncated protein production. Z9 pigs exhibit significant clinical symptom as human patients with HI. Most importantly, systemic retinoid treatment significantly prolonged the life span of the mutant pigs via improving epidermal maturation, decreasing epidermal apoptosis, and triggering the expression of ABCA6. Taken together, this pig model perfectly resembles the clinical symptom and molecular pathology of patients with HI and will be useful for understanding mechanistic insight and developing therapeutic strategies. [ABSTRACT FROM AUTHOR]

Human Waardenburg syndrome 2A (WS2A) is a dominant hearing loss (HL) syndrome caused by mutations in the microphthalmia-associated transcription factor ( MITF) gene. In mouse models with MITF mutations, WS2A is transmitted in a recessive pattern, which limits the study of hearing loss (HL) pathology. In the current study, we performed ENU (ethylnitrosourea) mutagenesis that resulted in substituting a conserved lysine with a serine (p. L247S) in the DNA-binding domain of the MITF gene to generate a novel miniature pig model of WS2A. The heterozygous mutant pig ( MITF ) exhibits a dominant form of profound HL and hypopigmentation in skin, hair, and iris, accompanied by degeneration of stria vascularis (SV), fused hair cells, and the absence of endocochlear potential, which indicate the pathology of human WS2A. Besides hypopigmentation and bilateral HL, the homozygous mutant pig ( MITF ) and CRISPR/Cas9-mediated MITF bi-allelic knockout pigs both exhibited anophthalmia. Three WS2 patients carrying MITF mutations adjacent to the corresponding region were also identified. The pig models resemble the clinical symptom and molecular pathology of human WS2A patients perfectly, which will provide new clues for better understanding the etiology and development of novel treatment strategies for human HL. [ABSTRACT FROM AUTHOR]

RNF20/Bre1 mediated H2B ubiquitination (H2Bub) has various physiologic functions. Recently, we found that H2Bub participates in meiotic recombination by promoting chromatin relaxation during meiosis. We then analyzed the phylogenetic relationships among the E3 ligase for H2Bub, its E2 Rad6 and their partner WW domain-containing adaptor with a coiled-coil (WAC) or Lge1, and found that the molecular mechanism underlying H2Bub is evolutionarily conserved from yeast to mammals. However, RNF20 has diverse physiologic functions in different organisms, which might be caused by the evolutionary divergency of their domain/motif architectures. In the current extra view, we not only elucidate the evolutionarily conserved molecular mechanism underlying H2Bub, but also discuss the diverse physiologic functions of RNF20 during meiosis. [ABSTRACT FROM PUBLISHER]

Beige adipocytes are a distinct type of fat cells with a thermogenic activity that have gained substantial attention as an alternative cellular anti-obesity target in humans. These cells may provide an alternative strategy for the genetic selection of pigs with reduced fat deposition. Despite the presence of beige adipocytes in piglets, the molecular signatures of porcine beige adipocytes remain unclear. Here, white and beige adipocytes from Tibetan piglets were primarily cultured and differentiated. Compared to the white adipocytes, the beige adipocytes exhibited a stronger thermogenic capacity. RNA-sequencing-based genome-wide comparative analyses revealed distinct gene expression profiles for white and beige adipocytes. In addition, two genes, integrin alpha-2 (ITGA2) and calponin 1 (CNN1), which were specifically differentially expressed in porcine beige adipocytes, were further functionally characterized using a loss-of-function approach. Our data showed that both genes were involved in differentiation and thermogenesis of porcine beige adipocytes. Collectively, these data furthered our understanding of gene expression in porcine white and beige adipocytes. Elucidating the genetic basis of beige adipogenesis in pigs will pave the way for molecular design breeding in both pigs and large animal models of human diseases. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with complex genetic inheritance. Genetic association of signal transducer and activator of transcription 4 (STAT4) with SLE susceptibility has been convincingly established in multiple populations including Asians, whereas studies of genetic relations between STAT4 polymorphisms and subphenotypes of SLE were rarely conducted. In this study, we selected Chinese female population and investigated genetic association between a polymorphism of STAT4 gene (rs7582694) and SLE. Furthermore, genetic association tests based on different subsets classified by 11 clinical manifestations were also performed. A total of 675 SLE female patients and 678 healthy controls were enrolled into this study, and SNP genotyping was performed using Sequenom's MassArray system (Sequenom iPLEX assay). Our study showed strong evidence for genetic predisposition of rs7582694 to SLE ( X = 23.7, OR = 0.68, 95% CI: 0.58-0.79, P = 1.13 × 10), while no association was observed between rs7582694 and any clinical presentations. The results of our study demonstrated that STAT4 rs7582694 SNP was significantly associated with SLE, and these results were in accordance with previous studies. [ABSTRACT FROM AUTHOR]

To "bring carbon emissions to a peak by 2030 and to be carbon-neutral by 2060", the role of renewable energy consumption and carbon emission trading are promoted. As an important energy consumer of regional energy system, it is necessary for integrated energy system to ensure the low-carbon economic operation of the system. Combined with the responsibility of renewable energy consumption, green certificate trading mechanism, carbon emission rights trading, and China Certified Emission Reduction (CCER), a regional integrated energy system operation optimization model was proposed. The model aims to minimize the total cost of the system, which included with electric bus, thermal bus, and cold bus. Setting different scenarios for the given example, the results show that the optimized model could effectively reduce the operating costs of the system. Moreover, the results also provide an effective reference for the system's economic and low-carbon operation. [ABSTRACT FROM AUTHOR]

Beige adipose tissue has been considered to have potential applications in combating obesity and its related metabolic diseases. However, the mechanisms of acute cold-stimulated beige formation still remain largely unknown. Here, transcriptional analysis of acute cold-stimulated (4 °C for 4 h) subcutaneous white adipose tissue (sWAT) was conducted to determine the molecular signatures that might be involved in beige formation. Histological analysis confirmed the appearance of beige adipocytes in acute cold-treated sWAT. The RNA-sequencing data revealed that 714 genes were differentially expressed (p-value < 0.05 and fold change > 2), in which 221 genes were upregulated and 493 genes were downregulated. Gene Ontology (GO) analyses showed that the upregulated genes were enriched in the GO terms related to lipid metabolic process, fatty acid metabolic process, lipid oxidation, fatty acid oxidation, etc. In contrast, downregulated genes were assigned the GO terms of regulation of immune response, regulation of response to stimulus, defense response, etc. The expressions of some browning candidate genes were validated in cold-treated sWAT and 3T3-L1 cell browning differentiation. In summary, our results illustrated the transcriptional response of sWAT to acute cold exposure and identified the genes, including Acad11, Cyp2e1, Plin5, and Pdk2, involved in beige adipocyte formation in mice. [ABSTRACT FROM AUTHOR]

Porcine‐derived xenogeneic sources for transplantation are a promising alternative strategy for providing organs for treatment of end‐stage organ failure in human patients because of the shortage of human donor organs. The recently developed blastocyst or pluripotent stem cell (PSC) complementation strategy opens a new route for regenerating allogenic organs in miniature pigs. Since the eye is a complicated organ with highly specialized constituent tissues derived from different primordial cell lineages, the development of an intact eye from allogenic cells is a challenging task. Here, combining somatic cell nuclear transfer technology (SCNT) and an anophthalmic pig model (MITFL247S/L247S), allogenic retinal pigmented epithelium cells (RPEs) were retrieved from an E60 chimeric fetus using blastocyst complementation. Furthermore, all structures were successfully regenerated in the intact eye from the injected donor blastomeres. These results clearly demonstrate that not only differentiated functional somatic cells but also a disabled organ with highly specialized constituent tissues can be generated from exogenous blastomeres when delivered to pig embryos with an empty organ niche. This system may also provide novel insights into ocular organogenesis. Synopsis: Blastocyst complementation is a new route for regenerating allogeneic organs in pigs for xenotransplantation, but reconstituting complicated organs, like a whole eye, was never done up to now. This strategy offers potentials for generating personalized human patient‐specific organs in large animals. Intact allogenic eyes can be regenerated using exogenous blastocyst complementation in an anophthalmic pig model.Porcine RPEs are isolated and cultured in vitro from 60‐day chimeric foetuses.The chimeric pig with regenerated eyes was derived from the somatic cloned blastocyst complementation and developed to full term. Blastocyst complementation is a new route for regenerating allogeneic organs in pigs for xenotransplantation, but reconstituting complicated organs, like a whole eye, was never done up to now. This strategy offers potentials for generating personalized human patient‐specific organs in large animals. [ABSTRACT FROM AUTHOR]

Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs. [ABSTRACT FROM AUTHOR]