NSUN2 is critical for mouse oocyte meiosis and early embryonic development.

In brief: The mechanism by which the NSUN2 mutation causes female infertility is still unclear. This study reveals the role and potential mechanism of NSUN2 in mouse oocyte maturation and early embryonic development, and provides a resource for elucidating female infertility with NSUN2 mutations. Abstract: Biallelic variants in the NSUN2 gene cause a rare intellectual disability and female infertility in humans. However, the function and mechanism of NSUN2 during mouse oocyte meiotic maturation and early embryonic development are unknown. Here, we show that NSUN2 is important for mouse oocyte meiotic maturation and early embryonic development. Specifically, NSUN2 is required for ovarian development and oocyte meiosis, and deletion of Nsun2 reduces oocyte maturation and increases the rates of misaligned chromosomes and aberrant spindles. In addition, Nsun2 deficiency results in a low blastocyst rate and impaired blastocyst quality. Strikingly, loss of Nsun2 leads to approximately 35% of embryos being blocked at the 2-cell stage, and Nsun2 knockdown impairs zygotic genome activation at the 2-cell stage. Taken together, these findings suggest that NSUN2 plays a critical role in mouse oocyte meiotic maturation and early embryonic development, and provide key resources for elucidating female infertility with NSUN2 mutations.

P300 regulates histone crotonylation and preimplantation embryo development.

Histone lysine crotonylation, an evolutionarily conserved modification differing from acetylation, exerts pivotal control over diverse biological processes. Among these are gene transcriptional regulation, spermatogenesis, and cell cycle processes. However, the dynamic changes and functions of histone crotonylation in preimplantation embryonic development in mammals remain unclear. Here, we show that the transcription coactivator P300 functions as a writer of histone crotonylation during embryonic development. Depletion of P300 results in significant developmental defects and dysregulation of the transcriptome of embryos. Importantly, we demonstrate that P300 catalyzes the crotonylation of histone, directly stimulating transcription and regulating gene expression, thereby ensuring successful progression of embryo development up to the blastocyst stage. Moreover, the modification of histone H3 lysine 18 crotonylation (H3K18cr) is primarily localized to active promoter regions. This modification serves as a distinctive epigenetic indicator of crucial transcriptional regulators, facilitating the activation of gene transcription. Together, our results propose a model wherein P300-mediated histone crotonylation plays a crucial role in regulating the fate of embryonic development.

Cartilage protective and anti-edema effects of JTF in osteoarthritis via inhibiting NCOA4-HMGB1-driven ferroptosis and aquaporin dysregulation.

BACKGROUND: Preventing joint edema is crucial in halting osteoarthritis (OA) progression. Growing clinical evidence indicate that Jianpi-Tongluo Formula (JTF) may have a promising anti-edema effect. However, the therapeutic properties of JTF and the underlying mechanisms remains unclear. MATERIALS AND METHODS: An OA rat model was established and employed to evaluate pharmacological effects of JTF in vivo based on dynamic histopathologic assessments and micro-CT observations. Then, OA-related genes and potential targets of JTF were identified through clinical transcriptomic data analysis and "disease gene-drug target" network analysis, which were verified by a series of in vivo experiments. RESULTS: JTF administration effectively reduced pain and joint edema, inhibited matrix degradation, chondrocyte apoptosis, and aquaporin expression in OA rats. Notably, JTF dose-dependently reversed damage-associated molecular patterns and inflammatory factor upregulation. Mechanically, our "disease gene-drug target" network analysis indicated that the NCOA4-HMGB1-GSK3B-AQPs axis, implicated in ferroptosis and aquaporin dysregulation, may be potentially served as a target of JTF against OA. Accordingly, JTF mitigated NCOA4, HMGB1, and GSK3B expression, oxidative stress, and iron metabolism aberrations in OA rats. Furthermore, JTF treatment significantly attenuated the aberrant upregulation of AQP1, AQP3, and AQP4 proteins observed in cartilage tissues of OA rats. CONCLUSION: Our data reveal for the first time that JTF may exert cartilage protective and anti-edema effects in osteoarthritis therapy by inhibiting NCOA4-HMGB1-driven ferroptosis and aquaporin dysregulation.

Pien-Tze-Huang prevents hepatocellular carcinoma by inducing ferroptosis via inhibiting SLC7A11-GSH-GPX4 axis.

BACKGROUND: Malignant transformation from hepatic fibrosis to carcinogenesis may be a therapeutic target for hepatocellular carcinoma (HCC). The aim of this study was to evaluate anti-cancer efficacy of Pien-Tze-Huang (PZH), and to investigate the underlying mechanisms by integrating transcriptional regulatory network analysis and experimental validation. METHODS: A diethylnitrosamine (DEN)-induced HCC model in rats was established and used to evaluate the anti-cancer efficacy of PZH. After detecting a transcriptomic profiling, the "disease-related gene-drug effective target" interaction network was constructed, and the candidate targets of PZH against malignant transformation from hepatic fibrosis to HCC were identified and verified in vitro. RESULTS: PZH effectively alleviated the pathological changes of hepatic fibrosis and cirrhosis, and inhibited tumor formation and growth in DEN-induced HCC rats. Additionally, the administration of PZH reduced the levels of various hepatic function-related serological indicators significantly. Mechanically, a ferroptosis-related SLC7A11-GSH-GPX4 axis might be one of potential targets of PZH against malignant transformation from hepatic fibrosis to HCC. Especially, high SLC7A11 expression may be associated with poor prognosis of HCC patients. Experimentally, the administration of PZH markedly increased the trivalent iron and ferrous ion, suppressed the expression levels of SLC7A11 and GPX4 proteins, and reduced the GSH/GSSG ratio in the liver tissues of DEN-induced HCC rats. CONCLUSIONS: Our data offer an evidence that PZH may effectively improve the hepatic fibrosis microenvironment and prevent the occurrence of HCC through promoting ferroptosis in tumor cells via inhibiting the SLC7A11-GSH-GPX4 axis, implying that PZH may be a potential candidate drug for prevention and treatment of HCC at an early stage.

Tongluo Shenggu capsule promotes angiogenesis to ameliorate glucocorticoid-induced femoral head necrosis via upregulating VEGF signaling pathway.

BACKGROUND: Tongluo Shenggu Capsule (TLSGC) is a product of Traditional Chinese patent medicine that has been effective in glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) clinically for many years. It is made from water extracts of a well-used herbal and dietary supplement-pigeon pea leaves. Nevertheless, the material basis and pharmacological mechanisms of TLSGC ameliorating GIONFH needed to be better defined. PURPOSE: To investigate the material basis and pharmacological mechanisms of TLSGC to ameliorate GIONFH. METHODS: The chemical compositions in TLSGC were characterized using the LC-MS system. Based on integrating the relevant targets of TLSGC in MedChem Studio software and GIONFH-related genes in our previous work, a "drug targets-disease genes" interaction network was constructed. The candidate targets of TLSGC ameliorating GIONFH were filtrated by topological characteristic parameters and further experimental validated based on methylprednisolone-induced rat model and dexamethasone-inhibited human umbilical vein endothelial cells (HUVECs). RESULTS: A total of 33 chemical compositions were characterized in TLSGC. Based on these compositions and GIONFH-related genes, 122 hub genes were selected according to topological parameters calculation. Biological functions were mainly enriched in four over-expressed modules of vascular damage, inflammation and apoptosis, bone metabolism and energy metabolism. The hub genes had the maximum enrichment degree in the VEGF-VEGFR2-PKC-Raf1-MEK-ERK signaling axis of the VEGF pathway. Experimentally, the therapeutic effects of TLSGC against GIONFH in rats were proved by micro-CT and pathological examination. Then, the protective effects of TLSGC on vascular damage were determined using angiography, CD31 immunohistochemistry, vascular function indicators in vivo, aortic ring test ex vivo, and the HUVECs activities in vitro including migration, invasion and tube formation. Mechanically, TLSGC effectively suppressed the downregulation of VEGF and VEGFR2 and their downstream targets, including Raf-1, PKC, p-MEK, and p-ERK proteins both in vivo and in vitro. CONCLUSION: TLSGC could promote angiogenesis by upregulating the VEGF-VEGFR2-PKC-Raf-1-MEK-ERK signaling axis, thereby exerting an apparent curative effect on GIONFH.

[Mechanism of Panlongqi Tablets intervening in vertebral artery type of cervical spondylosis in rats through PI3K/AKT signaling pathway based on network pharmacology and experimental verification].

This study aimed to further explore the relevant mechanism of action by network pharmacology integrated with animal experimental verification based on previous proven effective treatment of vertebral artery type of cervical spondylosis(CSA) by Panlongqi Tablets. Bionetwork analysis was performed to establish drug-disease interaction network, and it was found that the key candidate targets of Panlongqi Tablets were enriched in multiple signaling pathways related to CSA pathological links, among which phosphatidylinositol 3-kinase(PI3 K)/serine-threonine kinase(AKT/PKB) signaling pathway was the most significant. Further, mixed modeling method was used to build the CSA rat model, and the rats were divided into normal, model, Panlongqi Tablets low-, medium-and high-dose(0.16, 0.32, 0.64 g·kg~(-1)) and Jingfukang Granules(positive drug, 1.35 g·kg~(-1)) groups. After successful modeling, the rats were administered for 8 consecutive weeks. Pathological changes of rat cervical muscle tissues were detected by hematoxylin-eosin(HE) staining, and the content of interleukin-1ß(IL-1ß), tumor necrosis factor-α(TNF-α), vascular endothelial cell growth factor(VEGF) and chemokine(C-C motif) ligand 2(CCL2) in rat serum and/or cervical tissues was determined by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to detect the protein expression levels of chemokine(C-C motif) receptor 2(CCR2), PI3 K, AKT, phosphorylated AKT(p-AKT), I-kappa-B-kinase beta(IKK-beta/IKKß), nuclear factor kappa B(NF-κB P65) and phosphorylated nuclear factor kappa B(NF-κB p-P65) in rat cervical tissues, and positive expression of p-NF-κB P65 in rat cervical muscle tissues was detected by immunofluorescence. The results showed that Panlongqi Tablets at different doses improved the degree of muscle fibrosis and inflammation in cervical muscle tissues of CSA rats, and reduced the content of inflammatory factors IL-1ß, TNF-α, VEGF, CCL2 and CCR2 in serum and/or cervical tissues. The protein expression levels of PI3 K, p-AKT, IKKß and p-NF-κB P65 as well as the nuclear entry of p-NF-κB P65 in cervical tissues were down-regulated. These findings suggest that Panlongqi Tablets can significantly inhibit the inflammatory response of CSA rats, and the mechanism of action may be related to the down-regulation of the activation of PI3 K/AKT signaling pathway.

Operational Scheduling of Behind-the-Meter Storage Systems Based on Multiple Nonstationary Decomposition and Deep Convolutional Neural Network for Price Forecasting.

In the competitive electricity market, electricity price reflects the relationship between power supply and demand and plays an important role in the strategic behavior of market players. With the development of energy storage systems after watt-hour meter, accurate price prediction becomes more and more crucial in the energy management and control of energy storage systems. Due to the great uncertainty of electricity price, the performance of the general electricity price forecasting models is not satisfactory to be adopted in practice. Therefore, in this paper, we propose a novel electricity price forecasting strategy applied in optimization for the scheduling of battery energy storage systems. At first, multiple nonstationary decompositions are presented to extract the most significant components in price series, which express remarkably discriminative features in price fluctuation for regression prediction. In addition, all extracted components are delivered to a devised deep convolution neural network with multiscale dilated kernels for multistep price forecasting. At last, more advanced price fluctuation detection serves the optimized operation of the battery energy storage system within Ontario grid-connected microgrids. Sufficient ablation studies showed that our proposed price forecasting strategy provides predominant performances compared with the state-of-the-art methods and implies a promising prospect in economic benefits of battery energy storage systems.

[Effect of Jianpi Huogu Formula on function damage of vascular endothelial cells induced by glucocorticoid].

This study aimed to observe the intervention effect of Jianpi Huogu Formula(JPHGF) on the functional damage of vascular endothelial cells caused by glucocorticoid, and explore its action mechanism from the PI3 K/Akt and mitogen activated protein kinase(MAPK) signaling pathways. The extracted thoracic aorta ring of normal SD rats were intervened first with vascularendothelial growth factor(VEGF, 20 µg·L-1) and/or sodium succinate(MPS, 0. 04 g·L-1) in vitro and then with JPHGF(8, 16, and 32 µg·L-1) for five mcontinuous ethylpdays, rednisolofollowed nebythe statistics of the number, length, and area of microvessels budding fromvascular rings. In addition, the human umbilical vein endothelial cells(HUVECs) induced by VEGF(20 µg·L-1) were added with MPS(0. 04 g·L-1) and then with JPHGF(8, 16, and 32 µg·L-1) for observing the migration, invasion, and luminal formation abilities of HUVECs in the migration, invasion and luminal formation experiments. The protein expression levels of PI3 K, p-Akt, p-JN K, and p-ERK in HUVECs were assayed by Western blot. The results showed that JPHGF dose-dependently improved the num-ber,length, and area of microvessels in MPS-induced rat thoracic aortic ring, reversed the migration, invasion and lumen formation abiliti es of HUVECs reduced by MPS, and up-regulated the protein expression levels of PI3 K, p-Akt, and p-JNK in HUVECs. All thesehave suggested that JPHGF exerts the protective effect against hormone-induced damage to the angiogenesis of vascular endothelial cells by activating the PI3 K/Akt and MAPK signaling pathways, which has provided reference for exploring the mechanism of JPHGF in treating s teroid-induced avascular necrosis of femoral head(SANFH) and also the experimental evidence for enriching the scientific connotationof spleen-invigorating and blood-activating therapy.

Dissecting of the AI-2/LuxS Mediated Growth Characteristics and Bacteriostatic Ability of Lactiplantibacillus plantarum SS-128 by Integration of Transcriptomics and Metabolomics.

Lactiplantibacillus plantarum could regulate certain physiological functions through the AI-2/LuxS-mediated quorum sensing (QS) system. To explore the regulation mechanism on the growth characteristics and bacteriostatic ability of L. plantarum SS-128, a luxS mutant was constructed by a two-step homologous recombination. Compared with ΔluxS/SS-128, the metabolites of SS-128 had stronger bacteriostatic ability. The combined analysis of transcriptomics and metabolomics data showed that SS-128 exhibited higher pyruvate metabolic efficiency and energy input, followed by higher LDH level and metabolite overflow compared to ΔluxS/SS-128, resulting in stronger bacteriostatic ability. The absence of luxS induces a regulatory pathway that burdens the cysteine cycle by quantitatively drawing off central metabolic intermediaries. To accommodate this mutations, ΔluxS/SS-128 exhibited lower metabolite overflow and abnormal proliferation. These results demonstrate that the growth characteristic and metabolism of L. plantarum SS-128 are mediated by the AI-2/LuxS QS system, which is a positive regulator involved in food safety. It would be helpful to investigate more bio-preservation control potential of L. plantarum, especially when applied in food industrial biotechnology.

Vitrification of Pronuclear Zygotes Perturbs Porcine Zygotic Genome Activation.

Zygotic genome activation (ZGA) plays an essential role in early embryonic development. Vitrification is a common assisted reproductive technology that frequently reduces the developmental competence of embryos. However, the effect of vitrification on porcine ZGA and gene expression during ZGA remains largely unclear. Here, we found that vitrification of pronuclear zygotes derived from parthenogenetic activation (PA) and in vitro fertilization (IVF) resulted in a significant reduction in the rates of 2-cell, 4-cell, and blastocysts, but did not affect the quality of blastocysts. Functional research revealed that RNA polymerase II Inhibitor (α-amanitin) treatment significantly reduced global transcriptional activity and developmental efficiency of both 4-cell and 8-cell embryos, implying an essential role of ZGA in porcine early embryonic development. Furthermore, vitrification did not affect the synthesis of nascent mRNA of 2-cell embryos, but significantly inhibited global transcriptional activity of both 4-cell and 8-cell embryos, suggesting an impaired effect of vitrification on porcine ZGA. Correspondingly, the single-cell analysis showed that vitrification caused the downregulation or upregulation expression of maternal genes in 4-cell embryos, also significantly decreased the expression of zygotic genes. Taken together, these results indicated that vitrification of pronuclear zygotes impairs porcine zygotic genome activation.

Fengshi Qutong capsule ameliorates bone destruction of experimental rheumatoid arthritis by inhibiting osteoclastogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Bone destruction plays a key role in damaging the joint function of rheumatoid arthritis (RA). Fengshi Qutong capsule (FSQTC) consisting of 19 traditional Chinese medicines has been used for treating RA in China for many years. Preliminary studies show that FSQTC has analgesic activity and inhibits synovial angiogenesis of collagen-induced arthritis (CIA), but its role on bone destruction of RA is still unclear. AIM OF THE STUDY: To explore the effect of FSQTC on bone destruction of RA and the possible mechanism of osteoclastogenesis in vivo and in vitro. MATERIALS AND METHODS: LC-MS system was used to detect the quality control components of FSQTC. The anti-arthritic effect of FSQTC on CIA rats was evaluated by arthritis score, arthritis incidence and histopathology evaluation of inflamed joints. The effect of treatment with FSQTC on bone destruction of joint tissues was determined with X-ray and micro-CT quantification, and on bone resorption marker CTX-I and formation marker osteocalcin in sera were detected by ELISA. Then, osteoclast differentiation and mature were evaluated by TRAP staining, actin ring immunofluorescence and bone resorption assay both in joints and RANKL-induced RAW264.7 cells. In addition, RANKL, OPG, IL-1ß and TNFα in sera were evaluated by ELISA. The molecular mechanisms of the inhibitions were elucidated by analyzing the protein and gene expression of osteoclastic markers CTSK, MMP-9 and ß3-Integrin, transcriptional factors c-Fos and NFATc1, as well as phosphorylation of ERK1/2, JNK and P38 in joints and in RANKL-induced RAW264.7 cells using western blot and/or qPCR. RESULTS: In this study, 12 major quality control components were identified. Our data showed that FSQTC significantly increased bone mineral density, volume fraction, trabecular thickness, and decreased trabecular separation of inflamed joints both at periarticular and extra-articular locations in CIA rats. FSQTC also diminished the level of CTX-I and simultaneously increased osteocalcin in sera of CIA rats. The effects were accompanied by reductions of osteoclast differentiation, bone resorption, and expression of osteoclastic markers (CTSK, MMP-9 and ß3-Integrin) in joints. Interestingly, FSQTC treatment could reduce the protein level of RANKL, increase the expression of OPG, and decrease the ratio of RANKL to OPG in inflamed joints and sera of CIA rats. In addition, FSQTC inhibited the levels of pro-inflammatory cytokines implicated in bone resorption, such as IL-1ß and TNFα in sera. When RAW264.7 cells were treated with RANKL, FSQTC inhibited the formation of TRAP + multinucleated cells, actin ring and the bone-resorbing activity in dose-dependent manners. Furthermore, FSQTC reduced the RANKL-induced expression of osteoclastic genes and proteins and transcriptional factors (c-Fos and NFATc1), as well as phosphorylation of mitogen-activated protein kinases (MAPKs). CONCLUSION: FSQTC may inhibit bone destruction of RA by its anti-osteoclastogenic activity both in vivo and in vitro.

Circular RNA Expression and Regulation Profiling in Testicular Tissues of Immature and Mature Wandong Cattle (Bos taurus).

Wandong cattle are an autochthonous Chinese breed used extensively for beef production. The breed tolerates extreme weather conditions and raw feed and is resistant to tick-borne diseases. However, the genetic basis of testis development and sperm production as well as breeding management is not well established in local cattle. Therefore, improving the reproductive efficiency of bulls via genetic selection is crucial as a single bull can breed thousands of cows through artificial insemination (AI). Testis development and spermatogenesis are regulated by hundreds of genes and transcriptomes. However, circular RNAs (circRNAs) are the key players in many biological developmental processes that have not been methodically described and compared between immature and mature stages in Bovine testes. In this study, we performed total RNA-seq and comprehensively analyzed the circRNA expression profiling of the testis samples of six bulls at 3 years and 3 months of developmental age. In total, 17,013 circRNAs were identified, of which 681 circRNAs (p-adjust < 0.05) were differentially expressed (DE). Among these DE circRNAs, 579 were upregulated and 103 were downregulated in calf and bull testes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the identified target genes were classified into three broad functional categories, including biological process, cellular component, and molecular function, and were enriched in the lysine degradation, cell cycle, and cell adhesion molecule pathways. The binding interactions between DE circRNAs and microRNAs (miRNAs) were subsequently constructed using bioinformatics approaches. The source genes ATM, CCNA1, GSK3B, KMT2C, KMT2E, NSD2, SUCLG2, QKI, HOMER1, and SNAP91 were found to be actively associated with bull sexual maturity and spermatogenesis. In addition, a real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed a strong correlation with the sequencing data. Moreover, the developed model of Bovine testes in the current study provides a suitable framework for understanding the mechanism of circRNAs in the development of testes and spermatogenesis.

High-Temperature Chemical Stability of Cr(III) Oxide Refractories in the Presence of Calcium Aluminate Cement.

Al2O3-CaO-Cr2O3 castables are used in various furnaces due to excellent corrosion resistance and sufficient early strength, but toxic Cr(VI) generation during service remains a concern. Here, we investigated the relative reactivity of analogous Cr(III) phases such as Cr2O3, (Al1-xCrx)2O3 and in situ Cr(III) solid solution with the calcium aluminate cement under an oxidizing atmosphere at various temperatures. The aim is to comprehend the relative Cr(VI) generation in the low-cement castables (Al2O3-CaO-Cr2O3-O2 system) and achieve an environment-friendly application. The solid-state reactions and Cr(VI) formation were investigated using powder XRD, SEM, and leaching tests. Compared to Cr2O3, the stability of (Al1-xCrx)2O3 against CAC was much higher, which improved gradually with the concentration of Al2O3 in (Al1-xCrx)2O3. The substitution of Cr2O3 with (Al1-xCrx)2O3 in the Al2O3-CaO-Cr2O3 castables could completely inhibit the formation of Cr(VI) compound CaCrO4 at 500-1100 °C and could drastically suppress Ca4Al6CrO16 generation at 900 to 1300 °C. The Cr(VI) reduction amounting up to 98.1% could be achieved by replacing Cr2O3 with (Al1-xCrx)2O3 solid solution. However, in situ stabilized Cr(III) phases as a mixture of (Al1-xCrx)2O3 and Ca(Al12-xCrx)O19 solid solution hardly reveal any reoxidation. Moreover, the CA6 was much more stable than CA and CA2, and it did not participate in any chemical reaction with (Al1-xCrx)2O3 solid solution.

Chromatin remodeler INO80 mediates trophectoderm permeability barrier to modulate morula-to-blastocyst transition.

Inositol requiring mutant 80 (INO80) is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells. However, the roles and mechanisms of INO80 in porcine pre-implantation embryonic development remain largely unknown. Here, we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development. The INO80 protein is highly expressed in the nuclei during morula-to-blastocyst transition. Functional studies revealed that RNA interference (RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm. Mechanistically, single-embryo RNA sequencing revealed that INO80 regulates multiple genes, which are important for lineage specification, tight junction assembly, and fluid accumulation. Consistent with the altered expression of key genes required for tight junction assembly, a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts. Importantly, aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium. Taken together, these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification, tight junction assembly, and fluid accumulation.

Dynamic reprogramming and function of RNA N6-methyladenosine modification during porcine early embryonic development.

N6-Methyladenosine (m6A) regulates oocyte-to-embryo transition and the reprogramming of somatic cells into induced pluripotent stem cells. However, the role of m6A methylation in porcine early embryonic development and its reprogramming characteristics in somatic cell nuclear transfer (SCNT) embryos are yet to be known. Here, we showed that m6A methylation was essential for normal early embryonic development and its aberrant reprogramming in SCNT embryos. We identified a persistent occurrence of m6A methylation in embryos between 1-cell to blastocyst stages and m6A levels abruptly increased during the morula-to-blastocyst transition. Cycloleucine (methylation inhibitor, 20 mM) treatment efficiently reduced m6A levels, significantly decreased the rates of 4-cell embryos and blastocysts, and disrupted normal lineage allocation. Moreover, cycloleucine treatment also led to higher levels in both apoptosis and autophagy in blastocysts. Furthermore, m6A levels in SCNT embryos at the 4-cell and 8-cell stages were significantly lower than that in parthenogenetic activation (PA) embryos, suggesting an abnormal reprogramming of m6A methylation in SCNT embryos. Correspondingly, expression levels of m6A writers (METTL3 and METTL14) and eraser (FTO) were apparently higher in SCNT 8-cell embryos compared with their PA counterparts. Taken together, these results indicated that aberrant nuclear transfer-mediated reprogramming of m6A methylation was involved in regulating porcine early embryonic development.

METTL3-mediated m6A methylation negatively modulates autophagy to support porcine blastocyst development‡.

N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Here, we show that METTL3-mediated m6A methylation sustains porcine blastocyst development via negatively modulating autophagy. We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 (a key regulator of autophagy) and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3'UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell qPCR revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Taken together, these results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.

CLAUDIN7 modulates trophectoderm barrier function to maintain blastocyst development in pigs.

Trophectoderm (TE) barrier function is an essential prerequisite for blastocyst development. CLAUDIN7 (CLDN7), a member of CLAUDINS family, is involved in regulating intercellular exchange and cell polarity in epithelium cells. However, the role of CLDN7 in porcine early embryo development is yet to be explored. Here, we found that CLDN7 was highly conserved in different species and was widely expressed in different tissues. Remarkably, CLDN7 expression maintained a low level from GV oocyte to 4-cell stage whereas its expression exhibited a higher level from 8-cell stage onwards. Microinjection of siRNA into cytoplasm effectively knocked down expression of CLDN7 mRNA and protein in porcine embryos. CLDN7 knockdown not only significantly reduced blastocyst rates of embryos derived from parthenogenetic activation and in vitro fertilization, but also reduced number of total cells and TE cells in the resulting blastocysts. Furthermore, CLDN7 knockdown led to a significant reduction in expression of multiple genes associated with tight junction assembly and fluid accumulation. A permeability assay revealed that CLDN7 knockdown disrupted tight junction assembly and paracellular sealing in the TE epithelium. Taken together, these results demonstrate that CLDN7 regulates porcine blastocyst development via modulating trophectoderm barrier function.

Identification and functional annotation of m6A methylation modification in granulosa cells during antral follicle development in pigs.

The N6-methyladenosine (m6A) derivative has the capacity for ubiquitous epigenetic modification of messenger RNA (mRNA) that regulates gene expression through post-transcriptional mRNA modifications. Findings with mapping of m6A methylomes have indicated there are potential functions of this derivative in different cell types of several species. A profile of m6A methylomes and potential functions in granulosa cells of pigs during antral follicle development, however, has not yet occurred. In the present study, there was profiling of an epitranscriptome-wide map of m6A methylation in granulosa cells of pigs derived from small and large follicles using methylated RNA immunoprecipitation techniques, next-generation sequencing and further annotation of the potential functions of m6A utilizing bioinformatic analyses procedures. The m6A modification is abundant in granulosa cells of pigs, and there are dynamic changes in m6A methylomes during the developmental transition from small (< 3 mm) to large (> 5 mm) sized follicles. In particular, there was a prevalence of 7289 and 6882 m6A in granulosa cells from follicles of two different sizes. There was an increased prevalence of m6A in close proximity to the 5' or 3'-untranslated coding regions and a shared conserved consensus motif. Results from further analysis indicated there was significant enrichment of differentially expressed m6A methylated genes in several signaling pathways associated with steroidogenesis, granulosa cell proliferation and follicular development. When considered as a whole, these results indicate there are differential m6A modifications in granulosa cells of pigs during follicle development that are potentially associated with steroidogenesis and folliculogenesis.

Anti-angiogenic effect of Shikonin in rheumatoid arthritis by downregulating PI3K/AKT and MAPKs signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Zicao is the dried root of Lithospermum erythrorhizon Sieb, et Zucc, Arnebia euchroma (Royle) Johnst, or Arnebia guttata Bunge and commonly used to treat viral infection, inflammation, arthritis and cancer in China.Shikonin (SKN) is a major active chemical component isolated from zicao. Previous research showed that SKN has anti-inflammatory, immunomodulatory and analgesic effects, and inhibits the development of arthritis and the condition of collagen arthritis (CIA) mice; nevertheless, its role in the angiogenesis of rheumatoid arthritis (RA) has not been elucidated. AIM OF THE STUDY: The purpose of this study was to investigate the antiangiogenic activity of SKN in CIA rats and various angiogenesis models. MATERIAL AND METHODS: The anti-arthritic effect of SKN on CIA rats was tested by arthritis score, arthritis incidence, radiological observation and histopathology evaluation of inflamed joints. Vessel density evaluated with CD31 immunohistochemistry/immunofluorescence in joint synovial membrane tissues of CIA rats, chick chorioallantoic membrane assay, rat aortic ring assay, and the migration, invasion, adhesion and tube formation of human umbilical vein endothelial (HUVEC) cells induced by tumor necrosis factor (TNF)-α were used to measured the antiangiogenenic activity of SKN. Moreover, the effect of SKN on the expression of angiogenic mediators, such as vascular endothelial growth factor (VEGF), VEGFR2, TNF-α, interleukin (IL)-1ß, platelet derived growth factor (PDGF) and transforming growth factor (TGF)-ß in sera and joint synovia of rats, and in TNF-α-induced MH7A/HUVEC cells were measured by immunohistochemistry, enzyme linked immunosorbent assay, Western blot and/or real-time polymerase chain reaction (PCR). Through the analysis of protein and mRNA levels of phosphoinositide 3-kinase (PI3K), Akt and PTEN, and the autophosphorylation of ERK1/2, JNK and p38 in joint synovia of rats and in TNF-α-induced HUVEC cells, the molecular mechanism of its inhibition was elucidated by using Western blot and/or real-time PCR. RESULTS: SKN significantly reduced the arthritis score and arthritis incidence, and inhibited inflammation, pannus formation, cartilage and bone destruction of inflamed joints in CIA rats. Partially, SKN remarkably decreased the immature blood vessels in synovial membrane tissues of inflamed joints from CIA rats. It also suppressed in vivo angiogenesis in chick embryo and VEGF165-induced microvessel sprout formation ex vivo. Meanwhile, SKN inhibited TNF-α-induced migration, invasion, adhesion and tube formation of HUVEC cells. Moreover, SKN significantly decreased the expression of angiogenic activators including VEGF, VEGFR2, TNF-α, IL-1ß, PDGF and TGF-ß in synovia of CIA rats and/or in MH7A/HUVEC cells. More interestingly, SKN downregulated PI3K and Akt, and simultaneously upregulated PTEN both at protein and mRNA levels in synovia tissues and/or in TNF-α-induced HUVEC cells. It also suppressed the phosphorylation and gene level of TNF-α-induced signaling molecules, as ERK1/2, JNK, and p38 in synovium and/or in TNF-α-induced HUVEC cells. CONCLUSION: These findings indicate for the first time that SKN has the anti-angiogenic effect in RA in vivo, ex vivo and in vitro by interrupting the PI3K/AKT and MAPKs signaling pathways.