Vitamin C enhances the in vitro development of early porcine embryos by improving mitochondrial function.

Mammalian embryos often suffer from oxidative stress in vitro, as the oxygen in the atmosphere is higher than that in the oviductal environment. Vitamin C (Vc) has been proven to enhance early embryonic development in vitro, but the underlying mechanism remains unclear. In this study, we investigated the pathways of action by which Vc promotes the in vitro development of porcine embryos. Comparative analysis of in vitro and in vivo gene expression profiles of morula found that most of the differentially expressed genes were enriched in pathways related to mitochondrial function. The addition of 12.5 µg/mL Vc to the culture medium significantly increased blastocyst production in a dose- and duration-dependent manner. Moreover, ROS levels were significantly higher in embryos cultured in the air (21% oxygen) than cultured in a hypoxic condition (5% oxygen) and were reduced by Vc supplementation. Vc also significantly increased the mitochondrial membrane potential levels and the expression levels of mitochondrial function-related genes (MFN1 and OPA1) and TCA cycle-related genes (PDHA1 and OGDH) in embryos cultured in vitro. These results suggest that the addition of Vc to the in vitro culture medium can increase the developmental potential and improve the mitochondrial function of early porcine embryos.

Establishing a 3D-cultured system based on alginate-hydrogel embedding benefits the in vitro maturation of porcine Oocytes.

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus-oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.

Genome editing: An insight into disease resistance, production efficiency, and biomedical applications in livestock.

One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.

Vitamin C down-regulates the H3K9me3-dependent heterochromatin in buffalo fibroblasts via PI3K/PDK1/SGK1/KDM4A signal axis.

The success of reprogramming is dependent on the reprogramming factors enriched in the cytoplasm of recipient oocytes and the potential of donor nucleus to be reprogrammed. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as a major epigenetic barrier impeding complete reprogramming. Treating donor cell with vitamin C (Vc) can enhance the developmental potential of cloned embryos, but the underlying mechanisms still need to be elucidated. In this study, we found that 20µg/mL Vc could promote proliferation and inhibit apoptosis of BFFs, as well as down-regulate the H3K9me3-dependent heterochromatin and increase chromatin accessibility. Inhibited the expression of KDM4A resulted in increasing apoptosis rate and the H3K9me3-dependent heterochromatin, which can be restored by Vc. Moreover, Vc up-regulated the expression of KDM4A through PI3K/PDK1/SGK1 pathway. Inhibiting any factor in the signal axis of this PI3K pathway not only suppressed the activity of KDM4A but also substantially increased the level of H3K9me3 modification and the expression of the HP1α protein. Finally, Vc can rescue those negative effects induced by the blocking the PI3K/PDK1/SGK1 pathway. Collectively, Vc can down-regulate the H3K9me3-dependent heterochromatin in BFFs via PI3K/PDK1/SGK1/KDM4A signal axis, suggesting that Vc can turn the chromatin status of donor cells to be reprogrammed more easily.

Platelet count and clinical outcomes among ischemic stroke patients with endovascular thrombectomy in DIRECT-MT.

OBJECTIVES: The prognostic role of baseline platelet count (PLT) in acute ischemic stroke patients with large vessel occlusion undergoing endovascular thrombectomy is unclear. Whether PLT modifies alteplase treatment effect on clinical outcome in those patients is also uncertain. METHODS: We derived data from a multicenter randomized clinical trial (DIRECT-MT) comparing intravenous alteplase before endovascular treatment vs. endovascular treatment only. The 654 patients with available PLT data were included. Primary outcome was the ordinal modified Rankin Scale (mRS) score evaluated at 90 days. We also assessed various secondary and safety outcomes. RESULTS: After adjusting for confounding factors, patients in the top tertile of PLT had a significantly lower risk of a worse shift in the distribution of mRS score (Odds Ratio: 0.671, 95% Confidence Interval: 0.473-0.953, p for trend=0.025), major disability and death (Odds Ratio: 0.617, 95% Confidence Interval: 0.393-0.97, p for trend=0.037) as well as death (Odds Ratio: 0.544, 95% Confidence Interval: 0.313-0.947, p for trend=0.031), respectively, compared with the bottom one. Among patients in the bottom tertile of PLT, combination therapy was associated with a better imaging outcome of eTICI score of 2b, 2c or 3 on final angiogram (Odds Ratio: 3.23, 95% Confidence Interval: 1.49-7.002) with a marginally significant interaction effect. CONCLUSIONS: Participants with higher baseline PLT had a decreased risk of poor functional outcomes. Low baseline PLT modified alteplase treatment effect on the eTICI score on final angiogram. Combination therapy was beneficial for patients with low baseline PLT to have a better reperfusion status.

Effect of Platycodon grandiflorus Polysaccharide on M1 Polarization Induced by Autophagy Degradation of SOCS1/2 Proteins in 3D4/21 Cells.

M1-polarized macrophages can improve the body's immune function. This study aimed to explore the mechanism of Platycodon grandiflorus polysaccharide (PGPSt) degrading SOCS1/2 protein through autophagy and promoting M1 polarization in 3D4/21 cells. Immunoprecipitation, confocal laser scanning microscopy, flow cytometry, and intracellular co-localization were used to detect the expression of related phenotypic proteins and cytokines in M1-polarized cells. The results showed that PGPSt significantly promoted the mRNA expression of IL-6, IL-12, and TNF-α and enhanced the protein expression of IL-6, IL-12, TNF-α, IL-1ß, iNOS, CD80, and CD86, indicating that PGPSt promoted M1 polarization in 3D4/21 cells. Next, the effect of the PGPSt autophagy degradation of SOCS1/2 on the M1 polarization of 3D4/21 cells was detected. The results showed that PGPSt significantly downregulated the expression level of SOCS1/2 protein, but had no obvious effect on the mRNA expression level of SOCS1/2, indicating that PGPSt degraded SOCS1/2 protein by activating the lysosome system. Further research found that under the action of 3-MA and BafA1, PGPSt upregulated LC3B II and downregulated SOCS1/2 protein expression, which increased the possibility of LC3B, the key component of autophagy, bridging this connection and degrading SOCS1/2. The interaction between SOCS1/2 and LC3 was identified by indirect immunofluorescence and Co-IP. The results showed that the co-localization percentage of the two proteins increased significantly after PGPSt treatment, and LC3 interacted with SOCS1 and SOCS2. This provides a theoretical basis for the application of PGPSt in the treatment or improvement of diseases related to macrophage polarization by regulating the autophagy level.

The Protective Effect of Lycium Ruthenicum Murr Anthocyanins in Cr (VI)-Induced Mitophagy in DF-1 Cells.

Cr (VI) is an extremely toxic environment and professional pollutant that seriously damages mitochondrial dysfunction when it enters a cell. Anthocyanins possess anti-oxidant, antiaging, and antifatigue properties. The regulatory effect of Lycium ruthenicum Murr anthocyanin (LRMA) on Cr (VI)-induced mitophagy in DF-1 cells was determined. The experimental design was divided into blank group, groups subjected to Cr (VI) and Cr (VI), and LRMA co-treatment groups. Cell viability was determined by the CCK-8 assay. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were assessed by flow cytometry and immunofluorescence. Mitophagy was monitored by ELISA and Western blot. Data showed that Cr (VI) caused the overexpression of autophagy-related proteins (LC3, Beclin-1) and reduced the expressions of autophagy protein p62 and TOMM20. Compared with the Cr (VI) group, the LRMA group showed considerably decreased mitochondrial damage and mitophagy. LRMA decreased the mitochondrial protein expression of PINK1 and Parkin's transfer from the cytoplasm to mitochondria. LRMA may confer protective effects by reducing PINK1/Parkin-mediated mitophagy in Cr (VI)-induced DF-1 cell models.

The surface morphology of Platycodon grandiflorus polysaccharide and its anti-apoptotic effect by targeting autophagy.

BACKGROUND: Fumonisin B1 is categorised as possible carcinogenic to humans which commonly contaminate maize and maize-based products worldwide, FB1, like other environmental pollutants, may activate apoptosis, autophagy, the inflammatory response and oxidative stress. Platycodon grandiflorus polysaccharide (PGPSt) is prepared from a traditional herbal medicine in Asia with tremendous pharmacological activities. However, whether PGPSt could relieve FB1-induced apoptosis has not been elucidated. The study aimed to evaluate the surface morphology of PGPSt and its protective effect on fumonisin B1-induced apoptosis. METHODS: The surface morphology of PGPSt was evaluated by SEM and AFM. Expressions of proteins involved in autophagy and apoptosis were detected by western blot analysis. Western blot, transient transfection, JC-1 and Annexin V-FITC/PI staining, CCK8, Live-cell imaging and autophagy inhibitor were used to observe the effect and explore the mechanism of PGPSt on FB1-induced apoptosis of 3D4/21 cells. RESULTS: PGPSt had triple helix conformation, and had the characteristics of compact, polyporous and agglomerated morphology. PGPSt promoted the expression of LC3-II and Beclin1, reduced the expression of p62, and significantly activated autophagy. PGPSt inhibited the Akt/mTOR signaling pathway at 24 h. Besides, PGPSt increased the expression of Bcl-2 and decreased the expression of Cleaved Caspase-3. PGPSt-mediated autophagy was inhibited by 3-MA, accompanied by the upregulation of Caspase-3 and Cleaved Caspase-3, suggesting that enhanced autophagy inhibited apoptosis. CONCLUSION: PGPSt can activate autophagy, which in turn protects FB1-induced apoptosis. Targeting autophagy may provide a new way to improve the health of humans or animals in FB1 contaminated areas.

[Study on immune status of patients with schistosomiasis japonica in Poyang Lake region III Humoral and cellular immune characteristics between Schistosoma japonicum high and low antibody responders].

OBJECTIVE: To explore the immune mechanism of negative results of immune tests of schistosomiasis japonica patients. METHODS: Totally 142 schistosomiasis patients (positive stool examinations) of Poyang Lake region were tested by ELISA method, and the ROC curve was applied to determine the high and low response of the patients. The levels of cellular immunity and cytokines of high and low responders were compared. RESULTS: Totally eight schistosomiasis patients were found as low responders. Besides SWAP-IgA (t = -1.588, P > 0.1), the levels of isotype antibodies were significantly lower in the low responders compared with those in the high responders (t = -14.517 to -2.866, all P < 0.05). In the low responders, the propor- tion of CD3⁺T was increased; and the proportions of CD4⁺T, CD8⁺T, CD4⁺CD25⁺Treg, and the ratio of CD4⁺/CD8⁺ were all de- creased, but all of them were not significant (t = -1.72 to 0.974, all P > 0.05) compared with those in the high responders. The differences of IFN-γ and IL-10 between the high and low responders were both not significant (t = -2.426 to 0.216, all P > 0.05). CONCLUSIONS: There is a significant difference between the high and low responders only in the levels of isotype antibodies. One of the reasons of low response in the immune tests is the much lower antibody level after the antigen-antibody compound is completely formulated.

The Homocoupling Reaction of Aromatic Terminal Alkynes by a Highly Active Palladium(II)/AgNO3 Cocatalyst in Aqueous Media Under Aerobic Conditions.

A new and efficient Pd(II)/AgNO3-cocatalyzed homocoupling of aromatic terminal alkynes is described. Various symmetrical 1,4-disubstituted-1,3-diynes are obtained in good to excellent yields. This protocol employs a loading with relatively low palladium(II) in aqueous media under aerobic conditions.

A highly efficient catalyst of a nitrogen-based ligand for the Suzuki coupling reaction at room temperature under air in neat water.

Glycine, as a kind of commercially available and inexpensive ligand, is used to prepare an air-stable and water-soluble catalyst for the Suzuki-Miyaura reaction in our study. In the presence of 0.1% [PdCl2(NH2CH2COOH)2] as the catalyst, extremely excellent catalytic activity towards the Suzuki-Miyaura coupling of aryl halides containing the carboxyl group with various aryl boronic acids is observed at room temperature under air in neat water.

A simple hydrophilic palladium(II) complex as a highly efficient catalyst for room temperature aerobic Suzuki coupling reactions in aqueous media.

A study on room temperature Suzuki cross-coupling in an aqueous medium was carried out using a simple hydrophilic palladium (II) complex, trans-PdCl2(NH2CH2COOH)2 as catalyst in the presence of K2CO3 in air. This approach with a comparatively inexpensive and hydrophilic catalyst, mild reaction condition and aqueous media exhibits excellent catalytic activity towards the Suzuki coupling of aryl bromides and arylboronic acids, and good yields were obtained in the Suzuki coupling of activated aryl chlorides.