Generation and Application of Mouse-Rat Allodiploid Embryonic Stem Cells.

Mammalian interspecific hybrids provide unique advantages for mechanistic studies of speciation, gene expression regulation, and X chromosome inactivation (XCI) but are constrained by their limited natural resources. Previous artificially generated mammalian interspecific hybrid cells are usually tetraploids with unstable genomes and limited developmental abilities. Here, we report the generation of mouse-rat allodiploid embryonic stem cells (AdESCs) by fusing haploid ESCs of the two species. The AdESCs have a stable allodiploid genome and are capable of differentiating into all three germ layers and early-stage germ cells. Both the mouse and rat alleles have comparable contributions to the expression of most genes. We have proven AdESCs as a powerful tool to study the mechanisms regulating X chromosome inactivation and to identify X inactivation-escaping genes, as well as to efficiently identify genes regulating phenotypic differences between species. A similar method could be used to create hybrid AdESCs of other distantly related species.

A comprehensive review of matcha: production, food application, potential health benefits, and gastrointestinal fate of main phenolics.

Matcha, a powder processed from tea leaves, has a unique green tea flavor and appealing color, in addition to many other sought after functional properties for a wide range of formulated food applications (e.g., dairy products, bakery products, and beverage). The properties of matcha are influenced by cultivation method and processing post-harvest. The transition from drinking tea infusion to eating whole leaves provides a healthy option for the delivery of functional component and tea phenolics in various food matrix. The aim of this review is to describe the physico-chemical properties of matcha, the specific requirements for tea cultivation and industrial processing. The quality of matcha mainly depends on the quality of fresh tea leaves, which is affected by preharvest factors including tea cultivar, shading treatment, and fertilization. Shading is the key measure to increase greenness, reduce bitterness and astringency, and enhance umami taste of matcha. The potential health benefits of matcha and the gastrointestinal fate of main phenolics in matcha are covered. The chemical compositions and bioactivities of fiber-bound phenolics in matcha and other plant materials are discussed. The fiber-bound phenolics are considered promising components which endow matcha with boosted bioavailability of phenolics and health benefits through modulating gut microbiota.

A comprehensive review of polyphenol oxidase in tea (Camellia sinensis): Physiological characteristics, oxidation manufacturing, and biosynthesis of functional constituents.

Polyphenol oxidase (PPO) is a metalloenzyme with a type III copper core that is abundant in nature. As one of the most essential enzymes in the tea plant (Camellia sinensis), the further regulation of PPO is critical for enhancing defensive responses, cultivating high-quality germplasm resources of tea plants, and producing tea products that are both functional and sensory qualities. Due to their physiological and pharmacological values, the constituents from the oxidative polymerization of PPO in tea manufacturing may serve as functional foods to prevent and treat chronic non-communicable diseases. However, current knowledge of the utilization of PPO in the tea industry is only available from scattered sources, and a more comprehensive study is required to reveal the relationship between PPO and tea obviously. A more comprehensive review of the role of PPO in tea was reported for the first time, as its classification, catalytic mechanism, and utilization in modulating tea flavors, compositions, and nutrition, along with the relationships between PPO-mediated enzymatic reactions and the formation of functional constituents in tea, and the techniques for the modification and application of PPO based on modern enzymology and synthetic biology are summarized and suggested in this article.

Metabolomic differences of seminal plasma between boars with high and low average conception rates after artificial insemination.

Seminal plasma is a complex biological fluid containing many metabolites including amino acids, fructose, carbohydrates and lipids Metabolites play important roles in multiple biological processes, but details and significance of the seminal plasma metabolome related to boar fertility are unknown. The aim of the present study was to compare the comprehensive metabolome of seminal plasma from boars with different conception rate after artificial insemination and to identify the potential biomarkers. Semen samples were collected from boars which divided into two groups according to the conception rates in the offspring. Seminal plasma metabolites were isolated, purified, and then subjected to Ultra-high Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-qTOF-MS) procession. A total of 576 (Positive ion mode) and 377 (Negative ion mode) metabolites were identified in seminal plasma. Metabolites were identified and categorized according to their major chemical classes, including carboxylic acids and derivatives, organooxygen compounds, amino acids, peptides, and alogues, fatty amides, fatty acyls, benzene and substituted derivatives, purine nucleotides, pyrimidine nucleotides, glycosyl compounds, fatty acids and conjugates. The results showed that 4-Aminobenzoate, Pro-Asn, Ile-Tyr, Homoveratric acid and D-Biotin were higher in semen of boar with higher conception rate (HG) versus lower conception rate (LG) (p < .05), whereas L-Serine, Butoxyacetic acid, S-Methyl-5'-thioadenosine, Capsaicin and 1-O-(cis-9-Octadecenyl)-2-O-acetyl-sn-glycero-3-phosphocholine (PAF) were lower in HG than in LG (p < .05). These metabolites may be considered as candidate biomarkers for different fertility in boars.

Tea consumption and colorectal cancer risk: a meta-analysis of prospective cohort studies.

PURPOSE: Data from in vitro and animal studies support the preventive effect of tea (Camellia sinensis) against colorectal cancer. Further, many epidemiologic studies evaluated the association between tea consumption and colorectal cancer risk, but the results were inconsistent. We conducted a meta-analysis of prospective cohort studies to systematically assess the association between tea consumption and colorectal cancer risk. METHODS: A comprehensive literature review was conducted to identify the related articles by searching PubMed and Embase up to June, 2019. Summary relative risks (RRs) and 95% confidence intervals (CIs) were calculated using a fixed effect model. RESULTS: Twenty cohort articles were included in the present meta-analysis involving 2,068,137 participants and 21,437 cases. The combined RR of colorectal cancer for the highest vs. lowest tea consumption was determined to 0.97 (95% CI 0.94-1.01) with marginal heterogeneity (I2 = 24.0%, P = 0.093) among all studies. This indicated that tea consumption had no significant association with colorectal cancer risk. Stratified analysis showed that no significant differences were found in all subgroups. We further conducted the gender-specific meta-analysis for deriving a more precise estimation. No significant association was observed between tea consumption and colorectal cancer risk in male (combined RR = 0.97; 95% CI 0.90-1.04). However, tea consumption had a marginal significant inverse impact on colorectal cancer risk in female (combined RR = 0.93; 95% CI 0.86-1.00). Further, we found a stronger inverse association between tea consumption and risk of colorectal cancer among the female studies with no adjustment of coffee intake (RR: 0.90; 95% CI 0.82-1.00, P < 0.05) compared to the female studies that adjusted for coffee intake (RR = 0.97; 95% CI 0.87-1.09, P > 0.05). CONCLUSIONS: Our finding indicates that tea consumption has no significant impact on the colorectal cancer risk in both genders combined, but gender-specific meta-analysis shows that tea consumption has a marginal significant inverse impact on colorectal cancer risk in female.

µ-TRTX-Ca1a: a novel neurotoxin from Cyriopagopus albostriatus with analgesic effects.

Human genetic and pharmacological studies have demonstrated that voltage-gated sodium channels (VGSCs) are promising therapeutic targets for the treatment of pain. Spider venom contains many toxins that modulate the activity of VGSCs. To date, only 0.01% of such spider toxins has been explored, and thus there is a great potential for discovery of novel VGSC modulators as useful pharmacological tools or potential therapeutics. In the current study, we identified a novel peptide, µ-TRTX-Ca1a (Ca1a), in the venom of the tarantula Cyriopagopus albostriatus. This peptide consisted of 38 residues, including 6 cysteines, i.e. IFECSISCEIEKEGNGKKCKPKKCKGGWKCKFNICVKV. In HEK293T or ND7/23 cells expressing mammalian VGSCs, this peptide exhibited the strongest inhibitory activity on Nav1.7 (IC50 378 nM), followed by Nav1.6 (IC50 547 nM), Nav1.2 (IC50 728 nM), Nav1.3 (IC50 2.2 µM) and Nav1.4 (IC50 3.2 µM), and produced negligible inhibitory effect on Nav1.5, Nav1.8, and Nav1.9, even at high concentrations of up to 10 µM. Furthermore, this peptide did not significantly affect the activation and inactivation of Nav1.7. Using site-directed mutagenesis of Nav1.7 and Nav1.4, we revealed that its binding site was localized to the DIIS3-S4 linker region involving the D816 and E818 residues. In three different mouse models of pain, pretreatment with Cala (100, 200, 500 µg/kg) dose-dependently suppressed the nociceptive responses induced by formalin, acetic acid or heat. These results suggest that Ca1a is a novel neurotoxin against VGSCs and has a potential to be developed as a novel analgesic.

Coadministration of epigallocatechin-3-gallate (EGCG) and caffeine in low dose ameliorates obesity and nonalcoholic fatty liver disease in obese rats.

Epigallocatechin-3-gallate (EGCG) and caffeine in tea exert anti-obesity effects and induces nonalcoholic fatty liver disease (NAFLD) amelioration. However, previous studies usually performed a high-dose EGCG administration, whereas the insecurity was arisen in recent researches. In this study, we treated obese rats with an elaborate dose-40 mg/kg EGCG, 20 mg/kg caffeine, and the coadministration of them as low dose, which were similar to the daily intake; 160 mg/kg EGCG as high dose, which was the maximum safe dose had touched the contentious edge. The results suggested that the coadministration of EGCG and caffeine exerted more remarkable function on suppressing body weight gain, reducing white adipose tissue weight and decreasing the energy intake than single use. This may be due to the variation in serum lipid profile, oxidative stress, and adipose-derived and inflammatory cytokines. The pathological micrographs showed long-term high-fat diets caused severe NAFLD, but it was ameliorated at different levels by all of the administrations. In summary, low dose of EGCG or caffeine only showed a mild effect of anti-obesity and NAFLD amelioration. The coadministration of them could exert a superior curative effect as well as high dose EGCG but no anxiety regarding safety.

[Analysis of SOX2 gene promoter activity in porcine early embryonic development].

SOX2 (sex determining region Y-box2) is one of the critical pluripotent factors that play a crucial role in the first lineage differentiation and maintenance of pluripotency in inner cell mass during early embryonic development. However, there are few researches about the regulation of the SOX2 promoter, especially in Sus scrofa. To analyzed the activity of SOX2 promoter in early porcine embryos, we determined the control system and established the microinjection system for assessing SOX2 promoter activity by analyzing the embryonic development and the expression of enhanced green fluorescence protein (EGFP) after micro-injected different EGFP plasmids at different times after activation of the oocytes. Then, we analyzed the structure of 5000 bp upstream of the SOX2 translation initiation site and found there were four transcription factor binding site clusters. Next, we designed and constructed promoter-containing plasmids to analyze the function of each cluster. To detect the activity of different promoters, we assessed the mCherry expression in protein levels and mRNA levels by analyzing the mCherry fluorescence intensity and qRT-PCR after injecting plasmids into embryos. These results showed that the activity of the shorted promoter, with the region from 2254 bp to 2442 bp upstream of translation initiation site deleted, decreased to 17.8% at 4-cell and 8-cell stages compared with the full-length promoter. This region included two NF-AT transcription factor binding sites, which indicated that the NF-AT binding site is a key region to regulate the activity of the SOX2 promoter. The results provide important data for determination the mechanism of porcine SOX2 regulation.

Glucocorticoid exposure affects female fertility by exerting its effect on the uterus but not on the oocyte: lessons from a hypercortisolism mouse model.

STUDY QUESTION: What is the impact of glucocorticoid (GC) on female reproduction? SUMMARY ANSWER: Corticosterone (CORT) exposure causes little damage to oocyte quality or developmental competence but has an adverse effect on the uterus, which causes decreased implantation, embryo death and subsequent infertility. WHAT IS KNOWN ALREADY: Chronic treatment with high GC doses is effective in controlling most allergic diseases but may lead to metabolic disorders such as obesity that are closely related with reproductive function. STUDY DESIGN, SIZE, DURATION: Hypercortisolism was induced in a female mouse model by supplementing the drinking water with 100 µg/ml of CORT. Controls received vehicle (1% v/v ethanol) only. After 4 weeks treatment mice were either mated or killed in estrus for hormone and organ measurements. In the first experiment, treatment with CORT or control continued during pregnancy but in the second CORT treatment was stopped after mating. To identify the effects of GC exposure on the uterus, blastocysts were generated by IVF of oocytes from CORT and control mice and replaced into recipients receiving the opposite treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects of hypercortisolism on female mice were first characterized by living body fat content, body weight, food intake, hormone and biochemical measurements, a glucose tolerance test and an insulin resistance test. Fertility was determined with or without CORT-treatment during pregnancy. Oocyte quality was assessed by oocyte maturation, mitochondrial distribution, reactive oxygen species production, mitochondrial DNA mutations and morphology of blastocysts produced in vivo or in vitro. Blastocyst cross-transfer was done to evaluate the causes of embryonic development failure. Fetus development and uterus morphology evaluation as well as culture of oocytes in vitro with gradient concentrations of CORT were also carried out. MAIN RESULTS AND THE ROLE OF CHANCE: In the hypercortisolism female mouse model, body weight and food intake were much higher than in the control, and corticosterone, estradiol, cholesterol (CHO) and triglycerides (TG) in the plasma of CORT-treated mice was significantly increased. The hypercortisolism female mice were infertile when CORT-treatment was sustained during pregnancy but fertile if CORT-treatment was stopped after mating. The rate of successful implantation in hypercortisolism mice with sustained CORT-treatment during pregnancy was significantly lower than in the control, and the implanted embryos could not develop beyond 13.5 dpc. Blastocyst cross-transfer showed that blastocysts from CORT-treated mice could develop to term in the uterus of control mice, but blastocysts from control mice failed to develop to term when they were transferred into CORT-treated mice, providing evidence that the infertility was mainly caused by an altered uterine environment. CORT administration did not affect oocyte maturation, mitochondrial distribution, ROS production and blastocyst morphology, but increased mitochondrial DNA mutations. Culture of oocytes in vitro with gradient concentrations of CORT showed that only very high concentrations of CORT caused damage to oocyte developmental competence. LARGE SCALE DATA: NA. LIMITATIONS, REASONS FOR CAUTION: The mouse model has the advantages of a consistent genetic and physiological background and openness to experimental manipulation over clinical studies but may not represent the human situation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings show that special care should be taken when administering CORT during pregnancy, and provide important information concerning female reproduction when treating patients by subjecting them to chronic GC exposure. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Key R&D Program of China (Nos. 2016YFA0100400 and 2017YFC1000600) and the National Natural Science Foundation of China (31472055). The authors have no conflicts of interest.

Recent development in mass spectrometry and its hyphenated techniques for the analysis of medicinal plants.

INTRODUCTION: Medicinal plants are gaining increasing attention worldwide due to their empirical therapeutic efficacy and being a huge natural compound pool for new drug discovery and development. The efficacy, safety and quality of medicinal plants are the main concerns, which are highly dependent on the comprehensive analysis of chemical components in the medicinal plants. With the advances in mass spectrometry (MS) techniques, comprehensive analysis and fast identification of complex phytochemical components have become feasible, and may meet the needs, for the analysis of medicinal plants. OBJECTIVE: Our aim is to provide an overview on the latest developments in MS and its hyphenated technique and their applications for the comprehensive analysis of medicinal plants. METHODOLOGY: Application of various MS and its hyphenated techniques for the analysis of medicinal plants, including but not limited to one-dimensional chromatography, multiple-dimensional chromatography coupled to MS, ambient ionisation MS, and mass spectral database, have been reviewed and compared in this work. RESULTS: Recent advancs in MS and its hyphenated techniques have made MS one of the most powerful tools for the analysis of complex extracts from medicinal plants due to its excellent separation and identification ability, high sensitivity and resolution, and wide detection dynamic range. CONCLUSION: To achieve high-throughput or multi-dimensional analysis of medicinal plants, the state-of-the-art MS and its hyphenated techniques have played, and will continue to play a great role in being the major platform for their further research in order to obtain insight into both their empirical therapeutic efficacy and quality control.