Response Surface Methodology Optimization of Resistance Welding Process for Unidirectional Carbon Fiber/PPS Composites.

The use of thermoplastic composites (TPCs) as one of the lightweight solutions will inevitably encounter problems in connection. Resistance welding has the characteristics of high strength, simplicity, and high reliability, and is considered a very potential hot-melt connection technology. The resistance welding technology of unidirectional carbon fiber-reinforced polyphenylene sulfide composites (UCF/PPS) was systematically studied. The experimental results show that the 100-mesh brass mesh has the best resin wetting effect and heating efficiency, and the PPS/oxidized 100-mesh brass mesh composite resistance element (Ox-RE/PPS) has the highest welding strength. The welding failure mode changes from interface failure and RE failure to interlayer structure damage and fiber fracture. The single-factor experimental results show that the maximum welding strength is reached at 310 °C, 1.15 MPa, and 120 kW/m2. According to the conclusion of the single-factor experiment, the Box-Behnken method was further used to design a three-factor, three-level experiment, and a quadratic regression model was established according to the test results. The results of variance analysis, fitting curve analysis, and perturbation plot analysis proved that the model had high fitting and prediction abilities. From the 3D surface diagram analysis, the influence of power density is the largest, and the interaction between welding temperature and power density is the most significant. Combined with the analysis of Design Expert 13 software, the optimal range of process parameters was obtained as follows: welding temperature 313-314 °C, welding pressure 1.04-1.2 MPa, and power density 124-128 kW/m2. The average strength of resistance welding joints prepared in the optimal range of process parameters was 13.58 MPa.

[Comparison of clinical efficacy between percutaneous endoscopic transforaminal discectomy and coblation nucleoplasty in the treatment of inclusive lumbar disc herniation].

OBJECTIVE: To observe clinical efficacy of percutaneous endoscopic transforaminal discectomy (PETD) and target radioffrequency thermal coblation nucleoplasty(CN) on inclusive lumbar disc herniation(LDH) in different age groups, and provide a basis for clinical formulation of precise and individualized treatments. METHODS: A retrospective analysis of 219 patients with lumbar disc herniation treated with PETD and CN between January 2018 and June 2021 was performed, in which 107 patients were treated with PETD and 112 with CN. Patients were stratified by age into young group(≤45 years old), middle-aged group(>45 years old and <60 years old) and older group(≥60 years old). Before treatment, 3 days, 1 month and 6 months after treatment, visual analogue scale (VAS), Japanese Orthopaedic Association (JOA) score, infrared thermal imaging temperature difference (△T) and lumbar range of motion (ROM) were evaluated and clinical efficacy were compared in the different age groups between two treatment methods. RESULTS: ①VAS and JOA score outcomes, in the same age group and the same treatment method, the VAS and JOA scores at different time points postoperatively were obviously improved (P<0.05). For the same age group and the different treatment methods, the older group had lower VAS and higher JOA scores after PETD than after CN (P<0.05), and there was no significant difference between the young group and middle-aged group (P>0.05). There was no significant difference in VAS and JOA scores at the same time between age groups by PETD treatment (P>0.05). The VAS was higher and the JOA score was lower in older group than in young group and middle-aged group at 1, 6 months after CN treatment(P<0.05). ②△T and ROM outcomes, in the same age group and same treatment method, postoperative △T and ROM at different time points were obviously improved(P<0.05). There was no significant difference in △T between two methods of PETD and CN at the same age(P>0.05), there was no significant difference in ROM between young group and middle-aged group(P>0.05), ROM was higher after PETD treatment than after CN treatment(P<0.05). There was no significant difference in △T and ROM at the same time between age groups by PETD treatment(P>0.05). There was no significant difference in △T between age groups by CN treatment, but the ROM was smaller in older group than in young group and middle-aged group after CN treatment(P<0.05). CONCLUSION: Both PETD and CN for inclusive LDH have good efficacy, the curative benefit for older patients receiving PETD within 6 months after surgery more than CN, and CN is more appropriate for young and middle-aged patients.

Research on apoptotic mechanism and related pathways involved in postmortem grass carp (Ctenopharyngodon idellus) muscle.

BACKGROUND: Apoptosis activation is an essential research to reveal the triggering mechanism of flesh quality deterioration. This study was aimed at explaining apoptotic mechanism of postmortem fish in terms of caspases activation, cytochrome c (cyt-c) release, B-cell lymphoma 2 (Bcl-2) and Bcl2-associated X (Bax) protein levels, transcriptional levels of its molecules, and apoptosis-inducing factor (AIF) translocation at 4 °C for 5 days. RESULTS: Activation of caspase-9, caspase-8, caspase-3 and the release of mitochondrial cyt-c were observed during storage. The decreased Bcl-2 protein levels, increased Bax protein expressions and Bax/Bcl-2 ratio were major steps for inducing apoptosis. Collectively, transcriptional regulation of Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), inhibitors of apoptosis proteins (IAPs), myeloid cell leukemia-1 (Mcl-1), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) indicated that extrinsic apoptotic pathways (FasL/caspase-8/caspase-3) and intrinsic pathway [(JNK and p38 MAPK)/(Bcl-2, Bax and Mcl-1)/cyt-c/Apaf-1/caspase-9/caspase-3] were involved in apoptotic process. Mitochondrial AIF translocation to nuclear indicated that AIF mediated caspase-independent pathway. CONCLUSION: Therefore, transcriptional and translational alterations of multiple signaling molecules acted important roles in regulating apoptosis activation in postmortem process. © 2022 Society of Chemical Industry.

Knockdown of CLAUDIN-6 Inhibited Apoptosis and Induced Proliferation of Bovine Cumulus Cells.

This study aims to investigate the effects of CLAUDIN-6 (CLDN6) on cell apoptosis and proliferation of bovine cumulus cells (CCs). Immunofluorescence staining was used to localize CLDN6 protein in CCs. Three pairs of siRNA targeting CLDN6 and one pair of siRNA universal negative sequence as control were transfected into bovine CCs. Then, the effective siRNA was screened by real-time quantitative PCR (RT-qPCR) and Western blotting. The mRNA expression levels of apoptosis related genes (CASPASE-3, BAX and BCL-2) and proliferation related genes (PCNA, CDC42 and CCND2) were evaluated by RT-qPCR in CCs with CLDN6 knockdown. Cell proliferation, apoptosis and cell cycle were detected by flow cytometry with CCK-8 staining, Annexin V-FITC staining and propidium iodide staining, respectively. Results showed that the CLDN6 gene was expressed in bovine CCs and the protein was localized in cell membranes and cytoplasms. After CLDN6 was knocked down in CCs, the cell apoptosis rate significantly decreased and the pro-apoptotic genes BAX and CASPASE-3 were down-regulated significantly, whereas the anti-apoptotic gene BCL-2 was markedly up-regulated (p < 0.05). Additionally, CLDN6 knockdown significantly enhanced cell proliferation of CCs at 72 h after siRNA transfection. The mRNA levels of proliferation-related genes PCNA, CCND2 and CDC42 increased obviously in CCs with CLDN6 knockdown (p < 0.05). After CLDN6 was down-regulated, the percentage of CCs at S phase was significantly increased (p < 0.05). However, there was no remarkable difference in the percentages of cells at the G0/G1 phase and G2/M phase between CCs with or without CLDN6 knockdown (p > 0.05). Therefore, the expression of CLDN6 and its effects on cell proliferation, apoptosis and cell cycle of bovine CCs were first studied. CLDN6 low expression inhibited cell apoptosis, induced cell proliferation and cell cycle arrest of bovine CCs.

Discovery of the thieno[2,3-d]pyrimidine-2,4-dione derivative 21a: A potent and orally bioavailable gonadotropin-releasing hormone receptor antagonist.

The gonadotropin releasing hormone receptor (GnRH-R) is a G protein-coupled receptor (GPCR) belonging to the rhodopsin family. GnRH-R antagonists suppress testosterone to castrate level more rapidly than gonadotropin releasing hormone agonists but lack the flare phenomenon often seen during the early period of GnRH-R agonist treatment. Recently orgovyx (relugolix) was approved as the first oral GnRH-R antagonist for the treatment of advanced prostate cancer. However, orgovyx has demonstrated poor pharmacokinetic profile with low oral bioavailability and high efflux. Here, we rationally designed and synthesized a series of derivatives (13a-m, 21a-i) through the modification and structure-activity relationship study of relugolix, which led to the discovery of 21a as a highly potent GnRH-R antagonist (IC50 = 2.18 nM) with improved membrane permeability (Papp, A-B = 0.98 × 10-6 cm/s) and oral bioavailability (F % = 44.7). Compound 21a showed high binding affinity (IC50 = 0.57 nM) and potent in vitro antagonistic activity (IC50 = 2.18 nM) at GnRH-R. 21a was well tolerated and efficacious in preclinical studies to suppress blood testosterone levels, which merits further investigation as a candidate novel GnRH-R antagonist for clinical studies.

The Microstructure, Antibacterial and Antitumor Activities of Chitosan Oligosaccharides and Derivatives.

Chitosan obtained from abundant marine resources has been proven to have a variety of biological activities. However, due to its poor water solubility, chitosan application is limited, and the degradation products of chitosan oligosaccharides are better than chitosan regarding performance. Chitosan oligosaccharides have two kinds of active groups, amino and hydroxyl groups, which can form a variety of derivatives, and the properties of these derivatives can be further improved. In this review, the key structures of chitosan oligosaccharides and recent studies on chitosan oligosaccharide derivatives, including their synthesis methods, are described. Finally, the antimicrobial and antitumor applications of chitosan oligosaccharides and their derivatives are discussed.

The enhancer rare germline variation rs548071605 contributes to lung cancer development.

Rare germline variations contribute to the missing heritability of human complex diseases including cancers. Given their very low frequency, discovering and testing disease-causing rare germline variations remains challenging. The tag-single nucleotide polymorphism rs17728461 in 22q12.2 is highly associated with lung cancer risk. Here, we identified a functional rare germline variation rs548071605 (A>G) in a p65-responsive enhancer located within 22q12.2. The enhancer significantly promoted lung cancer cell proliferation in vitro and in a xenograft mouse model by upregulating the leukemia inhibitory factor (LIF) gene via the formation of a chromatin loop. Differential expression of LIF and its significant correlation with first progression survival time of patients further supported the lung cancer-driving effects of the 22q-Enh enhancer. Importantly, the rare variation was harbored in the p65 binding sequence and dramatically increased the enhancer activity by increasing responsiveness of the enhancer to p65 and B-cell lymphoma 3 protein, an oncoprotein that assisted the p65 binding. Our study revealed a regulatory rare germline variation with a potential lung cancer-driving role in the 22q12.2 risk region, providing intriguing clues for investigating the "missing heritability" of cancers, and also offered a useful experimental model for identifying causal rare variations.

Vacuum impregnation of chitosan coating combined with water-soluble polyphenol extracts on sensory, physical state, microbiota composition and quality of refrigerated grass carp slices.

Herein, the effects of chitosan (CH) coating with different water-soluble polyphenol extracts (pomegranate peel (PPE), grape seed (GSE) and green tea (GTE)) through vacuum impregnation on the quality retention and microflora of refrigerated grass carp fillets were studied. Generally, the quality degradation of carp fillets was remarkably alleviated using coatings when compared to the control. As suggested by microbial enumeration and high-throughput sequencing, protective coatings were conductive to inhibit bacteria growth, especially spoilage bacteria of Pseudomonas. As a result, the indicator related to bacteria such as total volatile basic nitrogen (TVB-N) and K value had lower levels in coating groups than that in control. In addition, coating also slowed down the deterioration of physical properties of color, texture and water holding capacity in fillets, giving fillets a better edible quality. By contrast, the fillets treated by composite coatings had better quality during storage when compared to chitosan coating alone, and a relatively good synergistic antibacterial effect between chitosan and extracts was also observed, especially for CH-GTE. Overall, the best performance to inhibit quality deterioration was recorded in CH-GTE, with the lowest values of TVB-N, TBARS, K-value and water loss, and highest values of shear force and sensory preference among groups.

Prediction of the Long-Term Effect of Iron on Methane Yield in an Anaerobic Membrane Bioreactor Using Bayesian Network Meta-Analysis.

A method for predicting the long-term effects of ferric on methane production was developed in an anaerobic membrane bioreactor treating food processing wastewater to provide management tools for maximizing methane recovery using ferric based on a batch test. The results demonstrated the accuracy of the predictions for both batch and long-term continuous operations using a Bayesian network meta-analysis based on the Gompertz model. The prediction bias of methane production for batch and continuous operations was minimized, from 11~19% to less than 0.5%. A biochemical methane potential-based Bayesian network meta-analysis suggested a maximum 2.55% ± 0.42% enhancement for Fe2.25. An anaerobic membrane bioreactor improved the methane yield by 2.27% and loading rate by 4.57% for Fe2.25, operating in the sequenced batch mode. The method allowed for a predictable methane yield enhancement based on the biochemical methane potential. Ferric enhanced the biochemical methane potential in batch tests and the methane yield in a continuously operated reactor by a maximum of 8.20% and 7.61% for Fe2.25, respectively. Copper demonstrated a higher methane (18.91%) and sludge yield (17.22%) in batch but faded in the continuous operation (0.32% of methane yield). The enhancement was primarily due to changing the kinetic patterns for the last period, i.e., increasing the second methane production peak (k71), bringing forward the second peak (λ7, λ8), and prolonging the second period (k62). The dual exponential function demonstrated a better fit in the last three stages (after the first peak), which implied that syntrophic methanogenesis with a ferric shuttle played a primary role in the last three methane production periods, in which long-term effects were sustained, as the Bayesian network meta-analysis predicted.

[Effect on Ammonia Inhibition Mitigation in the Anaerobic Digestion Process with Zero-Valent Iron].

Ammonia inhibition is an important factor impacting methane production efficiency during the anaerobic digestion of high-solid organic wastes. This study investigated the effect of micro-sized zero-valent iron (m-ZVI) on the anaerobic digestion of excess sewage sludge and thermal hydrolyzed sludge using batch mode experiments. The effect of m-ZVI on ammonia inhibition mitigation was also studied. Results showed that the kinetic characteristics of the methane production rate, lag phase, and methane production potential of the anaerobic digestion of excess sludge and thermal hydrolyzed sludge were not impacted by the addition of m-ZVI at a dosage of 4 g·L-1 and 10 g·L-1. However, during the inhibited anaerobic digestion process with a high ammonia concentration, the addition of 4 g·L-1 and 10 g·L-1 of m-ZVI was able to shorten the lag phase from 18.61 d (the control) to 17.22 d and 16.18 d, respectively. Moreover, the maximum methane production rate (based on the VS) increased from 6.34 mL·(d·g)-1(the control) to 7.84 mL·(d·g)-1 (4 g·L-1 m-ZVI) and 7.39 mL·(d·g)-1 (10 g·L-1 m-ZVI). The pH buffer system was not influenced by the chemical reaction of m-ZVI in the anaerobic digestion, although the relative abundance of the dominant methanogenic archaea (Methanosarcina) improved greatly from 30.71% (the control) to 53.50% (4 g·L-1 m-ZVI) and 60.30% (10 g·L-1 m-ZVI) at 27 d. This study proved that m-ZVI was incapable of improving the methane production potential of sewage sludge, while the mitigation of ammonia inhibition during anaerobic digestion was enhanced by the stimulating effect on methanogenic archaea.

IL2RG-deficient minipigs generated via CRISPR/Cas9 technology support the growth of human melanoma-derived tumours.

OBJECTIVES: Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti-cancer drug trials for several decades. However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans. Therefore, anti-tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials. Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre-clinical animal model for assessing anti-cancer drugs. However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours. Interleukin (IL)-2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, is required for proper lymphoid development. MATERIALS AND METHODS: IL2RG-/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis. RESULTS: Compared to age-matched wild-type pigs, IL2RG-/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T- and NK-cell deficiency. Human melanoma Mel888 cells generated tumours in IL2RG-/Y pigs but not in wild-type littermates. The human tumours grew faster in IL2RG-/Y pigs than in nude mice. CONCLUSIONS: Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti-cancer drugs.

Relieving ammonia inhibition by zero-valent iron (ZVI) dosing to enhance methanogenesis in the high solid anaerobic digestion of swine manure.

Relieving from ammonia inhibition and enhancing the utilization of thermodynamically unfavorable propionate are crucial for methane harvest in the high solid anaerobic digestion (HSAD) of ammonia-rich swine manure. In this study, the potential of dosing zero-valent iron (ZVI, 150 um) for enhancing the methanogenesis to resist total ammonia (TAN) over 5.0 g-N·L-1 was investigated via batch experiments under mesophilic condition. The cumulative methane production was enhanced by 22.2% at ≥160 mM ZVI dosage and the HSAD duration was further shortened by 50.6% at ≥320 mM ZVI dosage. The enhanced methanogenesis was mainly resulted from the full utilization of propionate and the accelerated collapse of posterior-biodegradable organics which might be driven by ZVI. Results of microbial community and qPCR (mcrA) showed that ZVI might trigger the blooming of Methanosarcina (from 27.9% to 78.3%) and Syntrophomonas (0.5% to 3.7%) and attribute to their possible direct interspecies electron transfer (DIET) to enhance propionate utilization. Besides, the main methanogenesis might remain in the effective aceticlastic pathway even under free ammonia (FAN) almost 1.0 g-N·L-1 because syntrophic acetate oxidizing bacteria (SAOB) decreased to almost none at 320 mM ZVI dosage. Dosing ZVI could relieve HSAD from TAN inhibition and more dosage was required to resist FAN inhibition.

The performance evaluation and kinetics response of advanced anaerobic digestion for sewage sludge under different SRT during semi-continuous operation.

Sludge retention time (SRT) is vital for advanced anaerobic digestion (AD) to realize energy self-sufficient. However, the criteria on reasonable SRT has not been fully understood. This study investigated the performance and kinetics response of AD under different SRT in semi-continuous AD with microwave (MW) pretreatment, according to the long-term operation and methane production during one feeding interval. Results showed that modified Gompertz model better described the kinetics than first-order model. At short SRT (15 d), pretreatment coupled with two-stage AD preserved methane production with the high attainable methane potential (B0) of 257.98 mL/g VS and hydrolysis rate constant (khyd) of 0.075 h-1. But the acceptable decrease of methane production rate seems to be unavoidable, which was possibly derived from the evolution of methanogenesis pathway. This study emphasized the importance of improved methane production rate in semi-continuous AD under short SRT rather than methane production potential obtained from batch experiment.

Inhibition of Mast Cell Degranulation Relieves Visceral Hypersensitivity Induced by Pancreatic Carcinoma in Mice.

Cancer pain induced by pancreatic carcinoma is one of the most common symptoms and is difficult to endure, especially in the advanced stage. Evidence suggests that mast cells are recruited and degranulate in enteric disease-related visceral hypersensitivity. However, whether mast cells promote the visceral pain induced by pancreatic carcinoma remains unclear. Here, using toluidine blue staining and western blotting, we observed that mast cells were dramatically recruited to tissues surrounding pancreatic carcinoma, but not inside the carcinoma in patients with severe visceral pain. The levels of mast cell degranulation products, including tryptase, histamine, and nerve growth factor, were significantly increased in pericarcinoma tissues relative to their levels in normal controls, as evidenced by enzyme-linked immunosorbent assay. We determined that systemic administration of mast cell secretagogue compound 48/80 exacerbated pancreatic carcinoma-induced visceral hypersensitivity in a male BALB/c nude mouse model as assessed by measuring the hunching behavior scores and mechanical withdrawal response frequency evoked by von Frey stimulation. In contrast, the mast cell stabilizer ketotifen dose-dependently alleviated pancreatic cancer pain. In addition, we observed incomplete development of abdominal mechanical hyperalgesia and hunching behavior in mast cell-deficient mice with pancreatic carcinoma. However, ketotifen did not further attenuate visceral hypersensitivity in mast cell-deficient mice with carcinoma. Finally, we confirmed that intraplantar injection of pericarcinoma supernatants from BALB/c nude mice but not mast cell-deficient mice caused acute somatic nociception. In conclusion, our findings suggest that mast cells contribute to pancreatic carcinoma-induced visceral hypersensitivity through enrichment and degranulation in pericarcinoma tissues. The inhibition of mast cell degranulation may be a potential strategy for the therapeutic treatment of pancreatic carcinoma-induced chronic visceral pain.

The inherited variations of a p53-responsive enhancer in 13q12.12 confer lung cancer risk by attenuating TNFRSF19 expression.

BACKGROUND: Inherited factors contribute to lung cancer risk, but the mechanism is not well understood. Defining the biological consequence of GWAS hits in cancers is a promising strategy to elucidate the inherited mechanisms of cancers. The tag-SNP rs753955 (A>G) in 13q12.12 is highly associated with lung cancer risk in the Chinese population. Here, we systematically investigate the biological significance and the underlying mechanism behind 13q12.12 risk locus in vitro and in vivo. RESULTS: We characterize a novel p53-responsive enhancer with lung tissue cell specificity in a 49-kb high linkage disequilibrium block of rs753955. This enhancer harbors 3 highly linked common inherited variations (rs17336602, rs4770489, and rs34354770) and six p53 binding sequences either close to or located between the variations. The enhancer effectively protects normal lung cell lines against pulmonary carcinogen NNK-induced DNA damages and malignant transformation by upregulating TNFRSF19 through chromatin looping. These variations significantly weaken the enhancer activity by affecting its p53 response, especially when cells are exposed to NNK. The effect of the mutant enhancer alleles on TNFRSF19 target gene in vivo is supported by expression quantitative trait loci analysis of 117 Chinese NSCLC samples and GTEx data. Differentiated expression of TNFRSF19 and its statistical significant correlation with tumor TNM staging and patient survival indicate a suppressor role of TNFRSF19 in lung cancer. CONCLUSION: This study provides evidence of how the inherited variations in 13q12.12 contribute to lung cancer risk, highlighting the protective roles of the p53-responsive enhancer-mediated TNFRSF19 activation in lung cells under carcinogen stress.

Generation of Pigs Resistant to Highly Pathogenic-Porcine Reproductive and Respiratory Syndrome Virus through Gene Editing of CD163.

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.

Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos.

Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0-32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.

Differential roles of ice crystal, endogenous proteolytic activities and oxidation in softening of obscure pufferfish (Takifugu obscurus) fillets during frozen storage.

Obscure pufferfish (Takifugu obscurus) softening during frozen storage remains to be solved. This study was therefore aimed to provide explanations by differentiate the roles of three potential factors in fish softening. The influences of ice crystal, endogenous proteolytic activities, and oxidization were distinguished by treatment of fish fillets with liquid nitrogen, iodoacetic acid, and tea polyphenol with ascorbic acid, respectively. This distinguishing method was verified to be effective by investigation in ice crystal microstructure, endogenous proteolytic activities and lipid and protein oxidation. In comparison of three factors, it showed that the shear force of fish fillets with smaller ice crystals was about 15.5% and 13.7% higher than those with the inhibition of endogenous proteolytic activities and oxidation respectively, indicating the dominant role of ice crystal in frozen fish softening. Besides, quality decline of frozen fish was initially fast and then slowed down during the storage.

Silencing of retrotransposon-derived imprinted gene RTL1 is the main cause for postimplantational failures in mammalian cloning.

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.

Inhibition of microbial spoilage of grass carp (Ctenopharyngodon idellus) fillets with a chitosan-based coating during refrigerated storage.

The effects of a chitosan-based coating on the inhibition of microbial spoilage of grass carp (Ctenopharyngodon idellus) fillets were studied during refrigerated storage for 15 days in terms of pH, total volatile base nitrogen (TVB-N), trimethylamine (TMA), adenosine triphosphate (ATP)-related compounds, K value, microbial enumeration and high-throughput sequencing. The results indicated that the fillets treated with chitosan-based coating enriched with 0.3% glycerol monolaurate and 0.5% clove essential oil had lower values of TVB-N, TMA, hypoxanthine riboside (HxR), hypoxanthine (Hx) and K value with the significant reductions (P < 0.05) of nearly 34, 73, 32, 74 and 38%, respectively, when compared to the control at day 15 of storage. Using high-throughput sequencing analysis, the major bacteria phyla of Firmicutes, Cyanobacteria and Bacteroidetes and the bacteria family of Lachnospiraceae and Bacteroidaceae were observed in fresh grass carp. As storage time increased, the coated samples retained bacterial diversity. However, Shewanellaceae, Pseudomonadaceae and Flavobacteriaceae increased and became the predominant microbiota in spoiled control samples. The significant difference between the bacteria species in the control and coated samples showed that the coating had the potential to inhibit microbial growth, especially spoilage microorganisms, and reduced quality deterioration caused by bacteria during refrigerated storage of grass carp fillets.