Depletion of serum-derived exosomes aggravates heat stress-induced damage of bovine mammary epithelial cells.

BACKGROUND: Exosomes are involved in intercellular communication, affecting many physiological and pathological process. The present study evaluated the effects of serum exosomes on the function of bovine mammary epithelial cells (BMECs) and milk synthesis under heat stress. METHODS AND RESULTS: We cultured the BMECs in fetal bovine serum (FBS) or exosome-free FBS medium and examined, their viability using CCK-8 kit. The results showed that culturing the cells in an exosome-free medium decreased viability and increased the levels of reactive oxygen species. The BMECs cultured in the exosome-free medium had reduced mitochondrial membrane potential, decreased manganese superoxide dismutase activity, and disrupted mitochondrial dynamics. They exhibited apoptosis due to upregulated Drp1, Fis1, Bax and HSP70. Lastly, we observed downregulation of milk fat and lactoprotein-related genes: mTOR, PPARγ, p-mTOR and ADD1 and SREBP1, ELF5, and CSN2, respectively, after culturing the cells in an exosome-free medium. These negative effects of the exosome-free medium on the BMECs could be further reinforced under heat stress. CONCLUSION: Our results demonstrated that exosomes from serum are critical for maintaining the normal function of BMECs.

The protective effect of rosmarinic acid on myotube formation during myoblast differentiation under heat stress.

High ambient temperature is one of the most important environmental factors that caused the reduction of livestock productivity and the increase of mortality. It has been shown that heat stress could affect the meat quality characteristics by physiological and metabolic perturbations in live livestock. Rosmarinic acid (RA) is a natural polyphenolic phytochemical compound that has many important biological activities, such as antioxidant, antimutagenic, and antitumor. The purpose of this study was to investigate the possible function and mechanism of RA on myoblast proliferation and differentiation under heat stress condition. The results showed that heat stress reduced the viability of myoblast and increased the percentage of apoptotic cells, and it also disrupted myotube formation by altering the expression of myogenic regulatory factors MyoD, myogenin, and MyHC. However, pretreatment of RA can protect C2C12 cells from heat stress-induced apoptosis, and it also increased the expression level of MyoD, myogenin, and MyHC under heat stress, which indicated that RA have protective effect on heat stress-caused failure of myotube formation during myoblast differentiation. Above all, our finding demonstrated that RA can promote the differentiation of C2C12 myoblast and maintain the formation of myotubes even under heat stress condition.

Tissue Levels of Stefin A and Stefin B in Hepatocellular Carcinoma.

Stefins have been reported to be associated with the progression and metastasis of various malignant tumors. However, the expressions of stefins in hepatocellular carcinoma (HCC) have not been well-defined. In this study, the protein levels of stefin A and stefin B were assessed by immunohistochemical staining, and the mRNA levels were quantified by real-time polymerase chain reaction in 85 primary HCC tissues, 85 surrounding non-cancerous tissues, and 9 normal hepatic tissues. The immunohistochemical staining of cathepsin B and cathepsin D, and the ratio of cathepsins to stefins were assessed. The mRNA expressions of stefin A and stefin B in HCC tissues were significantly higher than surrounding noncancerous tissues and normal hepatic tissues, respectively. A significant positive relationship of stefin A and stefin B was found with node metastasis, tumor size, and Edmondson grade for HCC. Univariate and multivariate analyses revealed that Edmondson grade and stefin B expression were independent factors associated with the risk of lymph node metastasis in HCC. The ratios of cathepsin B to stefin A, cathepsin D to stefin A, cathepsin B to stefin B and cathepsin D to stefin B of the HCC group were significantly higher than that of the surrounding noncancerous group. A significant positive correlation between the ratio of cathepsins to stefins (cathepsin B/stefin A, cathepsin B/stefin B and cathepsin D/stefin B) and node metastasis was demonstrated. We concluded that high expressions of stefin A and stefin B may be an important factor contributing to the development and metastasis of HCC.

Abnormal DNA methylation in the lumbar spinal cord following chronic constriction injury in rats.

Pathogenesis of neuropathic pain is complex and not clearly understood. Glutamate decarboxylase 67 (GAD 67) is a key synthetic enzyme for the main inhibitory transmitter gamma-aminobutyric acid (GABA), and diminishes in the spinal dorsal horn in rats following chronic constriction injury (CCI). GAD 67 is coded by gene GAD 1. DNA methylation can regulate the expression of GAD 67 by regulating the methylation of GAD 1 promoter in the psychotic brain. DNA methylation is primarily mediated by DNA methyltransferases (DNMTs) and methyl-DNA binding domain proteins (MBDs). In this study, in order to discover whether DNA methylation regulates GAD 67 expression in the spinal cord in CCI rats and is involved in neuropathic pain, we examined mRNA levels of DNMTs, MBDs and GAD 67 with real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), and methylation of GAD 1 promoter with Pyromark CpG Assays in the lumbar spinal cord in CCI rats on day 14 after surgery. Our results showed that DNMT3a, DNMT3b and methyl-CpG binding protein 2 (MeCP2) expression increased, MBD2 expression decreased, and DNMT1, MBD1 and MBD3 expression hardly changed in the lumbar spinal cord in CCI rats on day 14 after surgery. GAD 67 expression decreased, and methylation of GAD 1 promoter increased in the lumbar spinal cord in CCI rats on day 14 after surgery. These results indicate that decreased GAD 67 may be associated with increased GAD 1 promoter methylation, which may be mediated by DNMT3a, DNMT3b, MeCP2 and MBD2 in CCI rats. These indicate that abnormal DNA methylation may be highly involved in CCI-induced neuropathic pain.

[Investigation on Clonorchis sinensis infection and its risk factors in Futian District, Shenzhen City].

OBJECTIVE: To understand the status of Clonorchis sinensis infection and its risk factors in Futian District, Shenzhen City, so as to provide the evidence for formulating the strategy of prevention and control. METHODS: Two monitoring points were randomly selected, and the permanent population aged ≥ 3 years were investigated and their stool samples were detected for the eggs of C. sinensis by using Kato-Katz technique. Meanwhile, the risk factors of infection were also investigated. RESULTS: A total of 743 subjects were examined, and 7 persons were infected with C. sinensis and the infection rate was 0.94%. Most of the infected cases were focused on 20-40 and 40-60 age groups. The risk factors were eating raw or semi-cooked fish, and the protective factor was the use of separated cutting board for cooked or uncooked food. CONCLUSION: The infection rate of C. sinensis is low in the population of Futian District. However, the health education, especially for diet health education, still should be strengthened.

The effect of excess Zn on mineral nutrition and antioxidative response in rapeseed seedlings.

Zinc (Zn) is a necessary element for plants, but excess Zn can be detrimental. To investigate Zn toxicity, rapeseed (Brassica napus) seedlings were treated with 0.07-1.12 mM Zn for 7d. Inhibition of plant growth along with root damage, chlorosis and decreased chlorophyll (a and b) content in newly expanded leaves (the second and third leaves formed following cotyledons) were found under Zn stress. The Zn content increased in plants under external Zn stress, while concentrations of phosphorus, copper, iron, manganese and magnesium reduced significantly, especially in roots. Meanwhile, increased lipid peroxidation was detected biochemically and histochemically. Compared with controls, NADH oxidase and peroxidase (POD) activity increased in leaves and roots of plants under high Zn, but superoxide dismutase (SOD), catalase and ascorbate peroxidase activities decreased. The changes in glutathione S-transferase activity and in ascorbic acid, dehydroascorbate, non-protein thiols and glutathione contents were also measured under Zn stress. Isoforms of SOD and POD were separated using non-denaturing polyacrylamide gel electrophoresis and their activities were analyzed. Our results suggested that excess Zn exerts its toxicity partially through disturbing nutrient balance and inducing oxidative stress in plants. These data will be helpful for better understanding of toxicity of Zn and the adaptive mechanism in Zn non-hyperaccumulator plants.

[Relation between distribution of phosphorus form in the sediment of typical urban shallow lakes and eutrophication].

The forms of phosphorus in the surface sediments were extracted and determined sequentially with ethylene dinitrilo tetracetic acid (EDTA) technique in three urban shallow lakes: Lake Xuanwu, Lake Mochou and Lake Daming. The results showed that the iron and calcium-bound phosphate, about accounting for 80%, were the main forms of total phosphorus. The contents of iron bound phosphate in Lake Xuanwu and Lake Mochou were higher than that of Lake Daming, reaching 30%-40%. The organic phosphorus existed mainly in the form of alkali extractable phosphorus, while the contents of acid extractable organic phosphorus were low. However, the proportion of acid extractable organic phosphorus to the total phosphorus can indicate the degree of lake eutrophication.

An efficient and practical synthesis of 2-((1H-indol-3-yl)(aryl)methyl)malononitriles under ultrasound irradiation.

Synthesis of 2-((1H-indol-3-yl)(aryl)methyl)malononitrile via the Michael addition of indole with various arylmethylenemalononitriles was carried out in good yields using anhydrous zinc chloride as the catalyst under ultrasound irradiation.

[Distribution and correlation of P and Fe fractions in sediments of typical urban shallow lakes].

The fractions of phosphorus (P) and Fe in the surface sediments of three typical urban shallow lakes (Xuanwu Lake, Daming Lake and Mouchou Lake) were determined using improved methods of SEDEX and BCR, respectively, and the relationship between P and Fe in sediments was also analyzed. P analyses showed that the relative lower content of Al-P was found in sediments of three urban lakes, while the percentage of Fe-P was in the range of 17.10%-27.88%, 16.68%-27.06% and 0.06%-0.17%, respectively, in Xuanwu Lake, Daming Lake and Mouchou Lake and the contents order was in line with the level of eutrophication. The contents of Ex-P, ACa-P and De-P were highest in Daming Lake while lowest in Mochou Lake. Ca-P was the main fraction, ranging from 32.51% to 75.39% in these lakes. Fe analyses showed that 69.17%-99.88% of Fe in sediments of three urban lakes was present in the residual fraction (F4), suggesting that F4 was the dominant fraction. While the contents of F1 and F2 in sediments of Xuanwu Lake were high, accounting for 6.46%-17.35%. It may be one of the main factors causing the frequent outbreak of cyanobacteria in Xuanwu Lake. There were significant negative correlations (p < 0.01) between TP and F1, F2 and F3, and the correlativity coefficient was - 0.95, - 0.94, - 0.81, respectively. Ex-P, Al-P and Or-P correlated significantly to F1 and F2, suggesting that Ex-P, Al-P and Or-P were sensitive to environment factors due to their active characteristics. ACa-P and De-P were bio-unavailable phosphorus, but they had significantly negative correlativity to F1, F2 and F3. Thus, further study should be performed on the reaction mechanism of ACa-P and De-P.

An improved synthesis of 1,3,5-triaryl-2-pyrazolines in acetic acid aqueous solution under ultrasound irradiation.

BACKGROUND: Pyrazoline derivatives have been found to possess a broad spectrum of biological activities. Among various pyrazoline derivatives, 2-pyrazolines seem to be the most frequently studied. A variety of methods have been reported for the preparation of this class of compound. However, in spite of their potential utility, some of the reported methods suffer from drawbacks such as long reaction times, cumbersome product isolation procedures and environmental concerns. Organic reactions in aqueous media have attracted increasing interest recently because of environmental issues and the understanding of biochemical processes. Ultrasound has increasingly been used in organic synthesis in the last three decades. A large number of organic reactions can be carried out in higher yields, shorter reaction time or milder conditions under ultrasound irradiation. RESULTS: Preparation of a series of 1,3,5-triaryl-2-pyrazolines through the reaction of chalcones and phenylhydrazine hydrochloride was carried out in 83-96% yield within 1.5-2 h in sodium acetate-acetic acid aqueous solution under ultrasound irradiation. CONCLUSION: We have described a practical and convenient procedure for the synthesis of 1,3,5-triaryl-2-pyrazolines in sodium acetate-acetic acid aqueous solution at room temperature under ultrasound irradiation.

Pinacol coupling of aromatic aldehydes and ketones using TiCl3-Mg under ultrasound irradiation.

Titanium trichloride in EtOH can be reduced by Mg to the corresponding low valent titanium complexes, which can reduce some aromatic aldehydes and ketones to the corresponding pinacols in 8-95% yields within 15-40 min under ultrasound irradiation.

[Different effects of illumination with xenon and sulfur lamp on growth and development of cotton plants].

Experiments were carried out with cotton (Gossgpium hirsutum cv. Xuzhou 142) plants to study the effects of illumination with xenon and sulfur lamp on development of cotton plants. The results showed that, compared with xenon lamp, illumination with sulfur lamp inhibited excessive elongation of hypocotyl via promotion of longitudinal elongation of epidermis and cortex cells, increased the numbers of branches, buds and bolls significantly. It suggested that illumination with sulfur lamp rendered cotton photomorphogenesis more favorable to yield formation.

[Determination of the D- and L-configurations of amino deoxy sugars by (S)-TBMB carboxylic acid, a fluorescent chiral derivatization reagent].

D- and L- amino sugars were coupled with (S)-TBMB (S)-TBMB = (S)-2-tert-butyl-2-methyl-1, 3-benzodioxole carbonyl chloride, a fluorescent chiral reagent, followed by per-O-acetylation. The reactions yielded diastereomeric per-O-acetylated N-(S)-TBMB carbonyl amino sugars. Their (1)HNMR signals, especially the strong singlet peaks of tert-Bu and Me groups were diagnostic for the determination of the D-, L- configurations of amino sugar. Furthermore, a simple and highly sensitive method for the determination of the D-, L- configuration of amino deoxy sugars was developed based on the same fluorescent labeling method and reverse phase HPLC. The total time in analysis is less than two hours and the detection limit of the method is 0.2 picomolar.