Melatonin restores the declining maturation quality and early embryonic development of oocytes in aged mice.

With increase in women's age, the reproductive capability of female mammals decreases dramatically caused by age-related oxidative stress, coinciding with the decline in the ovarian reserve, and the quality and quantity of oocytes, which is the main determinant of female fertility. Melatonin, as an effective antioxidant and antiaging substance, is secreted by the pineal gland and been found in the follicular fluid as well, which has been turned out to enable to protect oocytes from oxidative stress during ovulation. However, the beneficial effects of melatonin on meiotic maturation in vitro and early embryo development of aged oocytes are still not fully understood. Thus, the aim of this study is to explore the potential mechanism of melatonin to improve the oocytes maturation and early embryonic development. The results suggested that oocyte quality decreased with age, whereas 10-6 M melatonin supplementation can significantly prompt the maturation quality of oocytes, the rate of fertilization and the formation rate of blastocyst. Mechanistic investigation indicated that melatonin supplementation not only restored the function of mitochondria by reducing reactive oxygen species (ROS) generation and early apoptosis, but also increased the level of ATP and total GSH through enhancing the mRNA expression levels of SIRT1, SIRT3, GPX4, SOD1 and SOD2. In conclusion, melatonin could alleviate the impairment of age-related oxidative stress to meiotic maturation and early embryonic development of oocytes. This study may provide a potential remediation strategy to improve the quality of oocytes from aged women and the efficiency of assisted reproductive technologies.

Identification of miR-192 target genes in porcine endometrial epithelial cells based on miRNA pull-down.

INTRODUCTION: MicroRNAs (miRNAs)-a class of small endogenous non-coding RNAs-are widely involved in post-transcriptional gene regulation of numerous physiological processes. High-throughput sequencing revealed that the miR-192 expression level appeared to be significantly higher in the blood exosomes of sows at early gestation than that in non-pregnant sows. Furthermore, miR-192 was hypothesized to have a regulatory role in embryo implantation; however, the target genes involved in exerting the regulatory function of miR-192 required further elucidation. METHODS: In the present study, potential target genes of miR-192 in porcine endometrial epithelial cells (PEECs) were identified through biotin-labeled miRNA pull-down; functional and pathway enrichment analysis was performed via gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Bioinformatic analyses were concurrently used to predict the potential target genes associated with sow embryo implantation. In addition, double luciferase reporter vectors, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and Western blot were performed to verify the targeting and regulatory roles of the abovementioned target genes. RESULTS: A total of 1688 differentially expressed mRNAs were identified via miRNA pull-down. Through RT-qPCR, the accuracy of the sequencing data was verified. In the bioinformatics analysis, potential target genes of miR-192 appeared to form a dense inter-regulatory network and regulated multiple signaling pathways, such as metabolic pathways and the PI3K-Akt, MAPKs, and mTOR signaling pathways, that are relevant to the mammalian embryo implantation process. In addition, CSK (C-terminal Src kinase) and YY1 (Yin-Yang-1) were predicted to be potential candidates, and we validated that miR-192 directly targets and suppresses the expression of the CSK and YY1 genes. CONCLUSION: We screened 1688 potential target genes of miR-192 were screened, and CSK and YY1 were identified as miR-192 target genes. The outcomes of the present study provide novel insights into the regulatory mechanism of porcine embryo implantation and the identification of miRNA target genes.

Graves' Disease in Children Presenting With Fragility Fracture: A Case Report.

Background: Adults with hyperthyroidism have been found to have decreased bone mineral density (BMD) and higher fracture risk. The most typical cause of hyperthyroidism is Graves' disease. However, there are limited studies on how hyperthyroidism affects bone metabolism and fractures in children. We describe a unique instance of a patient who initially displayed a fragility fracture and was ultimately identified with Graves' disease after biochemical evaluations. Case Summary: A 2-year-8-month-old female presented with fragility fractures three times in only 7 months. A series of examinations were performed to evaluate any possible malformations or abnormalities of bone metabolism. Graves' disease was found, and drug therapies were employed (methimazole, propranolol, calcium carbonate, vitamin D). Since children with Graves' disease and fragility fractures have been uncommonly described in the past, a stringent and thorough long-term follow-up was initiated. Conclusions: Children with undiagnosed Graves' disease had a higher risk of fractures and osteoporosis. This case suggests that BMD measurement may be necessary for the initial evaluation of Graves' disease in children.

Melatonin protects oocytes from cadmium exposure-induced meiosis defects by changing epigenetic modification and enhancing mitochondrial morphology in the mouse.

Cadmium (Cd) is one major environmental pollutant that can cause detrimental impacts on human as well as animal reproductive systems as a result of oxidative stress. It is widely acknowledged that melatonin secreted principally by the pineal gland is not only a natural potent antioxidant but also a free radical scavenger, whereas concerning how to alleviate the toxic effects of Cd on oocyte maturation remains elusive. In this investigation, it was the first time to explore the protective effects and potential mechanism of melatonin on meiotic maturation of mouse oocytes exposed to Cd in vitro medium. We found that Cd exerts adverse effects on meiotic maturation progression by disrupting the normal function of mitochondrion combined with the aberrant mitochondrial distribution and decreased membrane potential and altering epigenetic modification, including H3K9me2 and H3K4me2. Additionally, it was observed that Cd exposure disrupted the morphology of spindle organization and caused chromosome misalignment, which might be through changing the level of acetylated tubulin, whereas melatonin administration alleviated the toxic impacts of Cd on oocytes. Furthermore, the mitochondrial morphology-related genes mRNA expression and protein expression of autophagy-related genes was also investigated. The results suggested that melatonin supplementation significantly altered the mRNA expression of mitochondrial dynamics-related genes, rather than the expression of mitophagy-related proteins. Taken together, our results validated that melatonin administration has a certain protective impact against oocytes meiosis maturation defects induced by cadmium through changing epigenetic modification and enhancing mitochondrial morphology rather than mitophagy.

A glycoprotein from mountain cultivated ginseng: Insights into their chemical characteristics and intracellular antioxidant activity.

A glycoprotein (MGP2) from mountain-cultivated ginseng (MCG) was purified by Tris-HCl extraction followed by DEAE-52 ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The approximate molecular weight (27.0 kDa) and monomeric nature were determined by reduced and non-reduced SDS-PAGE. The structure of MGP2 was characterized by a practical and reliable "protein-polysaccharide analyzed by spectroscopy combined with chemical analysis" strategy. The results showed that MGP2 belonged to Arabinogalactan proteins (AGPs) which contained high amount of Glc (35.1 %). The hemagglutination test concluded that MGP2 was not a lectin. In addition, the MGP2 exhibited antioxidant activity by scavenging radical capacity tests and the ability to protect human erythrocytes and RAW264.7 cells from oxidative damage induced by AAPH. Therefore, these results suggested that glycoprotein MGP2 could be used as a natural antioxidant in drug and food industry.

Identification of SEC14 like lipid binding 2(SEC14L2) sequence and expression profiles in the Chinese tree shrew (Tupaia belangeri chinensis).

BACKGROUND: The product of the SEC14L2 (SEC14 Like Lipid Binding 2) gene belongs to a family of lipid-binding proteins including Sec14p, alpha-tocopherol transfer protein, and cellular retinol-binding protein. SEC14L2 expression enables replication of clinical hepatitis C virus (HCV) isolates in several hepatoma cell lines, and mutations in SEC14L2 may enhance HCV replication in vitro. The Chinese tree shrew (Tupaia belangeri chinensis) is a potential animal model for studying HCV replication, however, the cDNA sequence, protein structure, and expression of the Chinese tree shrew SEC14L2 gene have yet to be characterized. METHODS AND RESULTS: In the present study, we cloned the full-length cDNA sequence of the SEC14L2 in the Chinese tree shrew by using rapid amplification of cDNA ends technology. This led us to determine that, this is 2539 base pairs (bp) in length, the open reading frame sequence is 1212 bp, and encodes 403 amino acids. Following this, we constructed a phylogenetic tree based on SEC14L2 molecules from various species and compared SEC14L2 amino acid sequence with other species. This analysis indicated that the Chinese tree shrew SEC14L2 protein (tsSEC14L2) has 96.28% amino acid similarity to the human protein, and is more closely related to the human protein than either mouse or rat protein. The Chinese tree shrew SEC14L2 mRNA was detected in all tissues, and showed highest expression levels in the pancreas, small intestine and trachea, however the tsSEC14L2 protein abundance was highest in the liver and small intestine. CONCLUSION: The Chinese tree shrew SEC14L2 gene was closer in evolutionary relation to humans and non-human primates and expression of the tsSEC14L2 protein was highest in the liver and small intestine. These results may provide useful information for tsSEC14L2 function in HCV infection.

Coenzyme Q10 improves the quality of sheep sperm stored at room temperature by mitigating oxidative stress.

The purpose of this experiment was to explore whether coenzyme Q10 (CoQ10 ) improves the quality of sheep semen stored at room temperature by attenuating oxidative stress. Semen was diluted without (control group), and with antioxidants (5, 50, 250, and 500 µmol/L CoQ10 ). Sperm kinetic parameters and plasma membrane integrity were determined, and the reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), total antioxidant capacity (TAOC), catalase (CAT), and superoxide dismutase (SOD) activity were evaluated on the fifth day of semen preservation. The results showed that compared with the control group, the progressive motility in the 50 µmol/L group was higher (p < 0.05) within 2-5 days, and the plasma membrane integrity of sperm was higher in the 50 µmol/L group. The ROS content in the 5 and 50 µmol/L groups was reduced. The MDA level was reduced in the CoQ10 supplementation groups (p < 0.05). Additionally, the CAT, SOD, TAOC, ATP and MMP levels in the 50 µmol/L group were higher than those in the control group (p < 0.05). In conclusion, CoQ10 improved the quality of ram semen by alleviating oxidative stress, and 50 µmol/L CoQ10 was the optimum concentration.

Treatment of bilateral granulomatous lobular mastitis during lactation with traditional Chinese medicine: A case report.

BACKGROUND: Granulomatous lobular mastitis (GLM) is a type of benign chronic inflammatory disease that poses therapeutic challenges to healthcare providers. The diagnosis of GLM relies on tissue biopsy, and incorrect treatment may lead to delayed diagnosis, considerable aesthetic damage, and even mastectomy. CASE SUMMARY: We report the case of a 37-year-old Chinese woman who was lactating and had GLM in both breasts. At the time of treatment, the right breast had a mass of approximately 15 cm × 11 cm, which was hard and had poor mobility. Multiple skin ulcerations and pus spills were also observed on the surface of the breast. The left breast had a mass of about 13 cm × 9 cm, which was hard and had poor mobility. CONCLUSION: Herein, we report a case of bilateral GLM in a lactating woman that was successfully treated with traditional Chinese medicine (TCM), without the requirement for surgery or other treatments. Therefore, TCM may have advantages in the nonsurgical treatment of GLM.

Effects of Taurine on Sperm Quality during Room Temperature Storage in Hu Sheep.

The present study aimed to investigate whether the presence of Tau protected Hu sheep sperm from ROS stress during storage at room temperature. The semen was diluted with extender (Tris-based) at room temperature, supplemented with different concentrations of Tau (0, 10, 20, 40, 80, or 100 mM), and stored at 15 °C. Sperm quality parameters (sperm progressive motility, kinetic parameters, plasma membrane integrity rate, acrosome integrity rate, and MMP) and antioxidant parameters (ROS, MDA, SOD, CAT, and T-AOC) were evaluated during the preservation of semen. The addition of Tau, especially at a concentration of 20 mM, exerted positive effects on sperm quality parameters and antioxidant parameters compared to the sperm without Tau treatment (control group). The addition of Tau, especially at a concentration of 100 mM, exerted negative effects on sperm quality parameters and antioxidant parameters compared to the control group. Interestingly, the results indicated that the sperm acrosome integrity rate did not change during storage time. In conclusion, the addition of Tau to sperm preserved at room temperature can enhance the antioxidant ability of sperm, reduce the LPO on the 5th day, and improve the quality of semen preserved at room temperature. These results implied that Tau had potential to enhance Hu sheep sperm reproductive performance.

Plant hormonal changes and differential expression profiling reveal seed dormancy removal process in double dormant plant-herbaceous peony.

Herbaceous peony (Paeonia lactiflora Pall.) is a popular ornamental and medicinal plant. Taking approximately six to seven months, the seeds germination under natural conditions experiences dual dormancies, which seriously affects horticultural cultivation. Few studies have been conducted on exploring both biological and molecular mechanism that regulates dormancy removal process in hypocotyls double dormant plants. Here, we first measured ABA and GA3 content changes at four key dormancy break stages, and then performed transcriptomic analyses to identify the differentially expressed genes (DEGs) using RNA-seq. We subsequently carried out Quantitative real-time PCR (qRT-PCR) to validate RNA-seq data. ABA content decreased during the whole dormancy removal process and GA3 content exhibited decreasing slightly and then increasing trend. RNA sequencing de novo assembly generated a total of 99,577 unigenes. 20,344 unigenes were differentially expressed in the whole dormancy release process. The qPCR results of 54 selected unigenes were consistent with the FPKM values obtained from RNA-seq. Our results summarize a valuable collection of gene expression profiles characterizing the dormancy release process. The DEGs are candidates for functional analyses of genes affecting the dormancy release, which is a precious resource for the on-going physiological and molecular investigation of seeds dormancy removal in other perennial plants.

TKP, a serine protease extracted from Trichosanthes kirilowii, inhibits the migration and invasion of colorectal adenocarcinoma cells by targeting Wnt/ß-catenin and Hedgehog/Gli1 signalings.

Trichosanthes kirilowii, which is a type of Liana from cucurbitaceous family, possesses many bioactive constituents and therefore has multifarious pharmacological functions. TKP, which is a serine protease extracted from the fruit of Trichosanthes kirilowii, has been reported to possess potential anticancer activity. However, the effects of TKP on cancer cell migration and invasion are still unknown. Here, we reported that TKP could inhibit the migration and invasion abilities of colorectal cancer cells. In addition, the mRNA, protein expression levels, and activities of migration and invasion-related proteins MMP2 and MMP9 were decreased in TKP-treated cells. Mechanistically, TKP treatment repressed Wnt/ß-catenin and Hedgehog/Gli1 signaling cascades. However, the addition of lithium chloride or the transfection of plasmid pcDNA3.1-V5-HisA-Gli1 reversed the impacts of TKP on MMP2, MMP9, cell migration, and invasion. These results indicated that TKP suppressed the migration and invasion of colorectal cancer cells through blocking Wnt/ß-catenin and Hedgehog/Gli1 pathways-mediated MMP2 and MMP9.

Characterization of the glycerol-3-phosphate acyltransferase gene and its real-time expression under cold stress in Paeonia lactiflora Pall.

Elucidating the cold tolerance mechanism of Paeonia lactiflora, which is one of the most valuable ornamental and medicinal plants in Asia, fundamentally impacts its breeding and production. The glycerol-3-phosphate acyltransferase (GPAT) gene plays a pivotal role in cold resistance in a variety of plant species. Here, we cloned the P. lactiflora GPAT gene, determined its expression pattern, and tested its role in cold resistance. We obtained the full-length P. lactiflora GPAT gene using tissue-cultured seedlings and real-time polymerase chain reaction and rapid amplification of cDNA ends analyses. We named this gene PlGPAT in P. lactiflora. Phylogenetic analysis indicates that the PlGPAT gene is closely related with the GPAT genes in core eudicots. The phylogenetic tree containing 31 angiosperm species based on GPAT protein sequences is largely consistent with the known phylogeny in flowering plants. We conducted a time-course PlGPAT expression analysis and demonstrated that PlGPAT expression is correlated with low-temperature stress. Our results suggest that the PlGPAT gene plays an important role in regulating cold resistance in P. lactiflora.

Mating patterns and pollen dispersal in a Japanese larch (Larix kaempferi) clonal seed orchard: a case study.

Pollination dynamics highly determines the genetic quality of seed orchard crops. However, there is less research about the effect of mating patterns on seed productivity of orchard crops. So far, clonal seed orchards have been producing genetically improved seedlings used for most Japanese larch (Larix kaempferi (Lamb.) Carr.) plantations in China. In the present study, a total of 17 highly variable simple sequence repeat (SSR) markers were used for genotyping a progeny trial population consisting of 647 open-pollinated progenies germinated from seeds which were collected from 63 maternal clones with 140 potential paternal clones in a Japanese larch clonal seed orchard in China. Paternity analysis was used in the present case study in order to evaluate the level of paternal gametic contribution, estimate pollen contamination and selfing rates, and investigate pollination patterns, pollen dispersal patterns and the impact of mating patterns on seed productivity of orchard crops. We observed 93.7% of the success rate of the parental assignment, unequal paternal gametic contribution (0-12.4%) with 6.3% of the progenies derived from pollen contamination or unsampled pollen donors, and absence of evidence for selfing. We also found that pollination rate highly depended on the distance between pollen donors and maternal parents, the majority of the identified crossing (65.7%) occurred between clones within a 150-m radius, and large variations in growth performance existed among the paternal half-siblings. Progeny growth performance (diameter at breast (DBH) and height (HGT)) was measured at Age-20 in order to investigate the impact of mating patterns on timber production of orchard crops. As either the paternal or maternal, two clones (i. e., clones Z38 and Z62) were identified to have produced progenies with higher average stem volume breeding values than that of all of the progenies. Specifically, the genetic gains for volume were 3.53% for the two clones as paternal parents, and 8.26% as the maternal parents at Age-20. Thus, both elite clones were ideal candidates for the construction of next-generation clonal seed orchards due to their synchronous reproductive phenology with greater crossing rate and higher genetic gain. These results improved the pedigree information to provide solid evidence of mating patterns for future design and effective management of seed orchards and for the development of viable long-term breeding strategies for other coniferous species.

[Development of rapid setting-drying device for herbarium specimens and its application in survey of Chinese materia medica resources in Anhui].

Herbarium specimens are the basis for the plant classification and indispensable media in teaching, scientific research and resources investigation. They have also played an important role in identifying and producing traditional Chinese medicine. High-quality herbarium specimens shall meet high requirements for integrity, smoothness, color and fabricating efficiency. Therefore, we designed a rapid setting and drying device for herbarium specimens, which could make the herbarium specimens smooth, colorful and not easy to mildew. In this paper, we pointed out the deficiency of traditional methods in making herbarium specimens, and introduced the structure and working principle of the device. Besides, we also discussed the effect of the device in setting and drying herbarium specimens and its application in the fourth national survey of the Chinese material medica resources (CMMR) in Anhui province. As a result, the device provides new ideas for producing herbarium specimens, with a reasonable design, good uniformity, high efficiency, safety and portability, and so is worthy of promotion and application in the national survey of CMMR.

[Application of time domain reflectometry for determination of wate content in Xiangsha Yangwei pills].

Xiangsha Yangwei pill was selected as a model drug in this research, and time domain reflectometry (TDR) was used to determine the water content in the pill. The effects of five factors including the number of pill layers, pill packing density, atmospheric moisture, ambient temperature and the ratio of pill formula were investigated on water content. The results showed that the number of pill layers and ambient temperature had significant effects on water content of pills, while the pill packing density, atmospheric moisture and pill formula ratio had little effect on the determination of water content in pills. The reflection value was stable when 6 layers of pills were used. Under the condition of 25 ℃ and 45% relative humidity, the water content of pills ranged from 4.01% to 22.38%, showing good linear relationship between water content and reflection value, and the model equation was as follows: Y=0.279X－21.670 (R²=0.997 0). Verification experiment was used to explain the feasibility of this prediction model. The precision of the method complied with the methodology standard. It is concluded that TDR can be used in determination of water content in Xiangsha Yangwei pills. Additionally, TDR, as a new way to quickly and efficiently determine the water content, has a prospect application in the processing of traditional Chinese medicine pharmacy, especially for concentrated pill.

[Soil microorganisms, nutrients and enzyme activity of Larix kaempferi plantation under different ages in mountainous region of eastern Liaoning Province, China].

We studied the community of soil microorganisms, enzyme activity and soil nutrients under 11-, 20-, 34-and 47-year-old Larix kaempferi plantations in mountainous region of eastern Liaoning Province to discuss the soil biological properties of L. kaempferi plantations of different stand ages and their relationships with soil nutrients. The results showed that the indexes reflecting soil micro-organisms, enzyme activity and soil nutrients of L. kaempferi plantations were the highest under the 11- or 47-year-old stand and the lowest in the 20- or 34-year-old stand. Soil productivity appeared in a decline trend with the increasing stand age, and the changes of soil microbial community structure and enzyme activity were responsive to soil degradation. The difference of fungi community was more noticeable than that of bacteria community among the plantations with different stand ages. The results of CCA showed soil nutrient and pH had no effect on seasonal difference of community structure, but had effects on community, structure among different stand ages. The total N, organic carbon, C/N, available nitrogen, exchangeable Mg2+ and pH had greater effects on bacteria community, while available P, total K and pH had greater effect on fungi community among different age forests. The main T-RFs of bacteria and fungi had higher correlation with N and P, and the fungi community had higher correlation with organic carbon and K than bacteria community. The microor-ganism community of the 11- and 47-year-old stands had greater correlation with soil nutrients and enzyme activity than that of 20- and 34-year-old stands. Consequently, soil organisms, in particular soil fungi, could be used to indicate soil degradation.