Effects of dietary folic acid supplementation on lactation performance and mammary epithelial cell development of dairy cows and its regulatory mechanism.

The experiment investigated the impacts of FA on the proliferation of bovine mammary gland epithelial cells (BMECs) and to investigate the underlying mechanisms. Supplementation of 10 µM FA elevated the mRNA expression of proliferating cell nuclear antigen (PCNA), cyclin A2 and cyclin D1, and protein expression of PCNA and Cyclin A1. The mRNA and protein expression of B-cell lymphoma-2 (BCL2) and the BCL2 to BCL2 associated X 4 (BAX4) ratio elevated, while that of BAX, Caspase-3 and Caspase-9 reduced by FA. Both Akt and mTOR signaling pathways were activated by FA. Moreover, the stimulation of BMECs proliferation, the alteration of proliferative genes and protein expression, the change of apoptotic genes and protein expression, and the activation of mTOR signaling pathway caused by FA were obstructed by Akt inhibitor. Suppression of mTOR with Rapamycin reversed the FA-modulated promotion of BMECs proliferation and change of proliferous genes and protein expression, with no impact on mRNA or proteins expression related to apoptosis and FA-activated Akt signaling pathway. Supplementation of rumen-protected FA in cow diets evaluated milk yields and serum insulin-like growth factor-1 and estradiol levels. The results implied that the proliferation of BMECs was stimulated by FA through the Akt-mTOR signaling pathway.

Cell death-inducing DFFA-like effector C/CIDEC gene silencing alleviates diabetic cardiomyopathy via upregulating AMPKa phosphorylation.

Cell death-inducing DFFA-like effector C (CIDEC) is responsible for metabolic disturbance and insulin resistance, which are considered to be important triggers in the development of diabetic cardiomyopathy (DCM). To investigate whether CIDEC plays a critical role in DCM, DCM rat model was induced by a high-fat diet and a single injection of low-dose streptozotocin (27.5 mg/kg). DCM rats showed severe metabolic disturbance, insulin resistance, myocardial hypertrophy, interstitial fibrosis, ectopic lipid deposition, inflammation and cardiac dysfunction, accompanied by CIDEC elevation. With CIDEC gene silencing, the above pathophysiological characteristics were significantly ameliorated accompanied by significant improvements in cardiac function in DCM rats. Enhanced AMP-activated protein kinase (AMPK) α activation was involved in the underlying pathophysiological molecular mechanisms. To further explore the underlying mechanisms that CIDEC facilitated collagen syntheses in vitro, insulin-resistant cardiac fibroblast (CF) model was induced by high glucose (15.5 mmol/L) and high insulin (104  µU/mL). We observed that insulin-resistant stimulation dramatically raised CIDEC expression and promoted CIDEC nuclear translocation in CFs. Meanwhile, AMPKα2 was observed to distribute almost completely inside CF nucleus. The results further proved that CIDEC biochemically interacted and co-localized with AMPKα2 rather than AMPKα1 in CF nucleus, which provided a novel mechanism of CIDEC in promoting collagen syntheses. This study suggested that CIDEC gene silencing alleviates DCM via AMPKα signaling both in vivo and in vitro, implicating CIDEC may be a promising target for treatment of human DCM.

Antitumor activity of ginsenoside Rd in gastric cancer via up-regulation of Caspase-3 and Caspase-9.

Ginsenoside Rd (GS-Rd), isolated from the Chinese traditional herbal medicine Panax ginseng, is used for the treatment of cardiovascular diseases, inflammation, different body pains, and trauma. Caspase-3 and Caspase-9 belong to cysteine aspartic acid specific protease (Caspase) family that plays an important role in apoptosis progression of cancers. In the present study, we investigated the anti-tumor effect of GS-Rd by MTT assay, colony formation assessment, flow cytometry, and Western blotting. Our results revealed that ginsenoside Rd significantly inhibits human gastric cancer (GC) growth and cell proliferation. Flow cytometer analysis showed that the GS-Rd could significantly induce apoptosis and arrest the G0/G1 phase in GC cells. Further, GS-Rd was found to increase the ratio of Bax/Bcl-2 and the expression of Caspase-3 and Caspase-9, respectively, and to decrease the expression of Cyclin D1. Taken together, our study suggests that GS-Rd significantly inhibits GC cell proliferation, induces cell apoptosis through increase the expression of Caspase-3, Caspase-9, and the ratio of Bax/Bcl-2. GS-Rd also induces cell cycle arrest at G0/G1 phase by down-regulation Cyclin D1. Thus, GS-Rd could serve as a lead to develop novel therapeutic agents to against human gastric cancer.

L-Carnitine inhibits the senescence-associated secretory phenotype of aging adipose tissue by JNK/p53 pathway.

Senescence-associated secretory phenotype (SASP) plays a role in aging adipose tissue dysfunction by directly promoting chronic inflammation. The JNK/p53 pathway was reported as a potential mechanism that mediates SASP. In this study, we investigated the effects of L-carnitine, an inhibitor of the JNK/p53 pathway in adipose tissue SASP and dysfunction. Young and aging rat were given L-carnitine by gavage. Next, we detected the senescence, cytokines expression, chronic inflammation and insulin resistance of adipose tissue. Additionally, JNK/p53 pathway was estimated. Our results show a significant increase expression of SASP components in the adipose tissue of aging rats compared to young rats. Further, we found that infiltration of immune cells and the expression of pro-inflammatory cytokines were enhanced in aging adipose tissue while insulin signaling activity was reduced in aging adipose tissue. Interestingly, L-carnitine markedly reduced the expression of SASP factors. L-Carnitine could significantly reduce chronic inflammation, improving insulin resistance. Further, L-carnitine inhibited SASP by inhibiting JNK/p53 pathway. L-Carnitine inhibited SASP by JNK/p53 pathway and attenuated adipose tissue dysfunction of aging.

Mongoliibacter ruber gen. nov., sp. nov., a haloalkalitolerant bacterium of the family Cyclobacteriaceae isolated from a haloalkaline lake.

A novel haloalkalitolerant, rod-shaped bacterium, designated strain YIM 4-4T, was isolated from the surface water of the Dugerno lake, a haloalkaline lake in Inner Mongolia. The taxonomy of strain YIM 4-4T was investigated by a polyphasic approach. Strain YIM 4-4T was Gram-stain-negative, strictly aerobic, non-motile and formed red colonies. Optimal growth conditions were 28 °C, pH 8.0-11.0 and 0.5-2 % NaCl. The major respiratory quinone was menaquinone-7 (MK-7). The polar lipid profile was composed predominantly of phosphatidylethanolamine, six unidentified polar lipids, one phospholipid and one aminolipid. The predominant cellular fatty acids (>5 %) were iso-C15 : 0, iso-C17 : 1I/anteiso-C17 : 1B, iso-C16 : 1G, iso-C17 : 0 3-OH, C16 : 1ω7c/C16 : 1ω6c and iso-C16 : 1. The genomic DNA G+C content was 43.0 mol%. 16S rRNA gene sequence analysis indicated that the members of the genera Cecembia, Fontibacter, Aquiflexum and Indibacter of the family Cyclobacteriaceae (phylum Bacteroidetes) were the most closely related, with 16S rRNA gene sequence similarities ranging from 93.6 to 94.2 %. Other members of the family Cyclobacteriaceae showed sequence similarities < 93.0 %. On the basis of phenotypic, chemotaxonomic and phylogenetic properties, strain YIM 4-4T represents a novel species of a new genus, for which the name Mongoliibacter ruber gen. nov., sp. nov. is proposed. The type strain is YIM 4-4T ( = CCTCC AB 2012966T = DSM 27929T).

A Case-control Study of Environmental Risk Factors for Nonsyndromic Cleft of the Lip and/or Palate in Xuzhou, China.

In this study, we sought to determine the association between environmental factors and nonsyndromic cleft of the lip and/or palate (NSCLP) to understand the etiology of the disease. A total of 200 NSCLP cases and 327 controls were recruited at the Maternal and Child Health Hospital of Xuzhou City. We conducted face-to-face interviews with the mothers of both cases and controls. The factors increasing the risk of NSCLP were a positive family history [odds ratio (OR)=56.74], pesticide exposure (OR=8.90), and indoor decoration pollution (OR=4.32). On the other hand, the factors decreasing the risk of NSCLP were a high education level (OR=0.22) and supplementation of folic acid (OR=0.23) and multivitamins (OR=0.16). Positive family history, pesticide exposure, and indoor decoration pollution are associated with the risk of NSCLP. In contrast, high education level and folic acid and multivitamin supplementation are protective factors against NSCLP.

Identification of susceptibility genes in non-syndromic cleft lip with or without cleft palate using whole-exome sequencing.

BACKGROUND: Non-syndromic cleft lip with or without cleft palate (NSCL/P) is among the most common congenital malformations. The etiology of NSCL/P remains poorly characterized owing to its complex genetic heterogeneity. The objective of this study was to identify genetic variants that increase susceptibility to NSCL/P. MATERIAL AND METHODS: Whole-exome sequencing (WES) was performed in 8 fetuses with NSCL/P in China. Bioinformatics analysis was performed using commercially available software. Variants detected by WES were validated by Sanger sequencing. RESULTS: By filtering out synonymous variants in exons, we identified average 8575 nonsynonymous single nucleotide variants (SNVs). We subsequently compared the SNVs against public databases including NCBI dbSNP build 135 and 1000 Genomes Project and obtained an average of 203 SNVs. Total 12 reported candidate genes were verified by Sanger sequencing. Sanger sequencing also confirmed 16 novel SNVs shared by two or more samples. CONCLUSIONS: We have found and confirmed 16 susceptibility genes responsible for NSCL/P, which may play important role in the etiology of NSCL/P. The susceptibility genes identified in this study will not only be useful in revealing the etiology of NSCL/P but also in diagnosis and treatment of the patients with NSCL/P.

[Determination of complexation ability of rhubarb with copper ions by ultra-high performance liquid chromatography].

Gandou decoction (GDD) is a traditional Chinese medicine prescription that has been widely used to treat copper metabolism disorders in China with remarkable clinical effect and lower toxicity. However, evaluation of the complexation ability of copper ions is challenging, which hinders screening and discovery of coordinate active ingredients in GDD. An analytical method is needed to determinate the complexation ability of chemical constituents with copper ions. In this study, a rapid and accurate method based on ultra-high performance liquid chromatography (UHPLC) was developed to determine the complexing ability of rhubarb with copper ions. First, the optimal coordination reaction conditions between active ingredients of rhubarb and copper ions were determined. The samples were separated using an Agilent Eclipse Plus C18 column (50 mm×2.1 mm, 1.8 µm) with 5 µL injection volumes. The mobile phase was gradient eluted with methanol and water containing 0.1% (v/v) phosphoric acid at a flow rate of 0.3 mL/min. The detection wavelength was 254 nm and the column temperature was 30 ℃. Under the optimized chromatographic conditions, the rhubarb constituents were effectively separated. Next, peak areas of rhubarb were calculated before and after the coordination reaction between copper ions. The complexing ability of active ingredients in rhubarb with copper ions was evaluated by calculating the rate of changes of their chromatographic peak areas. Finally, ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used to identify the coordination active ingredients in rhubarb extract. Focusing on the coordination reaction conditions between active ingredients of rhubarb and copper ions revealed that the active ingredients of rhubarb and copper ions reached equilibrium by coordination reaction at pH 9 for 12 h. Methodological evaluation revealed the good stability and repeatability of the method. Under these conditions, 20 major components of rhubarb were identified by UPLC-Q-TOF-MS. According to the coordination rate of each component and copper ions, eight components with strong coordination were screened out (gallic acid 3-O-ß-D-(6'-O-galloyl)-glucopyranoside, aloe emodin-8-O-ß-D-glucoside, sennoside B, l-O-galloyl-2-O-cinnamoyl-glucoside, chysophanol-8-O-ß-D-(6″-O-acetyl)-glucoside, aloe-emodin, rhein and emodin). The respective complexation rates of the components were 62.50%, 29.94%, 70.58%, 32.77%, 34.61%, 26.07%, 28.73% and 31.78%. Compared with other reported methods, the presently developed method can be used to screen the active ingredients of traditional Chinese medicines that have complexing ability with copper ions, especially in complex mixture systems. This study describes an effective detection technology for evaluating and screening the complexing ability of other traditional Chinese medicines with metal ions.

Mongoliicoccus roseus gen. nov., sp. nov., an alkaliphilic bacterium isolated from a haloalkaline lake.

Two pink, non-motile, aerobic, alkaliphilic, halotolerant, Gram-negative cocci, designated MIM28(T) and MIM29, were isolated from the surface water of a haloalkaline lake on the Mongolia Plateau. The isolates grew optimally at 30-33 °C, at pH 8-9 and with 3-4 % (w/v) NaCl. The isolates were chemoheterotrophic and could assimilate carbohydrates, organic acids and amino acids. The major respiratory quinone was menaquinone MK-7. The major polar lipids were phosphatidylcholine and phosphatidylethanolamine. The predominant cellular fatty acids were iso-C(15 : 0) (13.8-17.5 %), anteiso-C(15 : 0) (10.5-11.2 %), iso-C(16 : 0) (9.9-13.0 %), C(16 : 0) (4.3-4.6 %), iso-C(17 : 0) (3.8-5.3 %), anteiso-C(17 : 0) (3.7-7.1 %), C(17 : 1)ω6c (4.6-6.4 %), iso-C(17 : 0) 3-OH (4.6-5.8 %), summed feature 3 (C(16 : 1)ω7c and/or C(16 : 1)ω6c; 4.0-6.4 %) and summed feature 9 (iso-C(17 : 1)ω9c and/or C(16 : 0) 10-methyl; 10.4-12.5 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were most closely related to Litoribacter ruber YIM CH208(T) (93.6 % 16S rRNA gene sequence similarity), the genus Echinicola (90.4-92 %) and other members of the family Cyclobacteriaceae (87.8-90 %). The DNA G+C contents of strains MIM28(T) and MIM29 were 62.8 and 62.2 mol%. On the basis of morphology, physiology, fatty acid composition, phylogeny and 16S rRNA gene sequence analysis, the isolates are assigned to a novel species of a new genus, for which we propose the name Mongoliicoccus roseus gen. nov., sp. nov.; the type strain of the type species is MIM28(T) (= ACCC 05511(T) = KCTC 19808(T)).

Hepcidin gene silencing ameliorated inflammation and insulin resistance in adipose tissue of db/db mice via inhibiting METs formation.

As a major feature of diabetes, inflammation is closely related to macrophage extracellular traps and the expression of hepcidin upregulated by diabetes is reportedly involved in chronic inflammation. Therefore, we aimed to explore whether hepcidin could be implicated in inflammation and macrophage extracellular traps (METs) formation. The diabetic db/db mouse model was established exhibiting insulin resistance (IR), inflammation, macrophages infiltration and higher expression of hepcidin, where samples were obtained from epididymal adipose tissue. We observed that inflammation and IR improved in adipose tissue of mice treated with hepcidin gene silencing. Furthermore, METs formation could be markedly inhibited via hepcidin gene silencing followed by attenuated inflammatory response due to METs, indicating hepcidin gene silencing played a key role in anti-inflammation by inhibiting METs formation. So, we concluded that hepcidin gene silencing has a potential for treatment of diabetes due to its ability to ameliorate inflammation via inhibiting METs formation.

CIDEC silencing attenuates diabetic nephropathy via inhibiting apoptosis and promoting autophagy.

OBJECTIVE: The role of cell death-inducing DFF45-like effector C (CIDEC) in insulin resistance has been established, and it is considered to be an important trigger factor for the progression of diabetic nephropathy (DN). We intend to explore whether CIDEC plays an important role in the regulation of DN and its potential mechanism. METHODS: High-fat diet and low dose streptozotocin were used to establish type 2 diabetic rat model. We investigate the role of CIDEC in the pathogenesis and process of DN through histopathological analysis, western blot and gene silencing. Meanwhile, the effect of CIDEC on renal tubular epithelial cells stimulated by high glucose was also verified. RESULTS: DM group exhibited glucose and lipid metabolic disturbance, with hypertrophy of kidneys, damaged renal function, increased apoptosis, decreased autophagy, glomerulosclerosis and interstitial fibrosis. CIDEC gene silencing improved metabolic disorder and insulin resistance, alleviated renal hypertrophy and renal function damage, decreased glomerular and tubular apoptosis, increased autophagy and inhibited renal fibrosis. At the cellular level, high glucose stimulation increased CIDEC expression in renal tubular epithelial cells, accompanied by increased apoptosis and decreased autophagy. CIDEC gene silencing can improve autophagy and reduce apoptosis. At the molecular level, CIDEC gene silencing also decreased the expression of early growth response factor (EGR)1 and increased the expression of adipose triglyceride lipase (ATGL). CONCLUSION: CIDEC gene silencing may delay the progression of DN by restoring autophagy activity and inhibiting apoptosis with the participation of EGR1and ATGL.

Testosterone ameliorates vascular aging via the Gas6/Axl signaling pathway.

Low serum testosterone level is associated with aging-related vascular stiffness, but the underlying mechanism is unclear. The Growth arrest-specific protein 6 (Gas6) /Axl pathway has been proved to play important roles in cell senescence. In this study, we intend to explore whether Gas6/Axl is involved in the effect of testosterone on vascular aging amelioration. Vascular aging models of wild type and Axl-/- mice were established by natural aging. Mice of these two gene types were randomized into young group, aging group and testosterone undecanoate (TU) treatment group. Mice were treated with TU (37.9 mg/kg) in the TU group, which treated with solvent reagent served as control. The aging mice exhibited decreases in serum testosterone, Gas6 and Axl levels and an increase in cell senescence, manifested age-related vascular remodeling. Testosterone treatment induced testosterone and Gas6 levels in serum, and ameliorated cell senescence and vascular remodeling in aging mice. Furthermore, we uncover the underlying molecular mechanism and show that testosterone treatment restored the phosphorylation of Akt and FoxO1a. Axl knockout accelerated cell senescence and vascular remodeling, and resisted the anti-aging effect of testosterone. Testosterone might exert a protective effect on vascular aging by improving cell senescence and vascular remodeling through the Gas6/Axl pathway.

Sarcopenia is attenuated by TRB3 knockout in aging mice via the alleviation of atrophy and fibrosis of skeletal muscles.

BACKGROUND: Sarcopenia causes several adverse events in elderly people. Muscle fibre atrophy and interstitial fibrosis are the main histopathological changes in sarcopenia and account for decreased muscle function. Tribbles homologue 3 (TRB3) was previously reported to exhibit age-related expression and play a vital role in cell proliferation, differentiation, and fibrosis. We aimed to investigate how TRB3 affects sarcopenia. METHODS: Wild-type and TRB3 knockout C57/BL6J mice were randomly divided into young and old groups. Exercise capacity was evaluated, and single-muscle function was detected by electrophysiological techniques, after which the mice were sacrificed to collect their gastrocnemius muscles for assessment of atrophy and fibrosis by histopathological and molecular biological methods. TRB3 expression, autophagy level, and MAPK signalling pathway activity were evaluated through western blotting. The interaction of TRB3 with P62 and the association between TRB3 and the MAPK signalling pathway were detected by co-immunoprecipitation. RESULTS: In aged mice, exercise capacity and cross-sectional area of skeletal muscle fibres were decreased significantly, whereas TRB3, atrophy-related markers atrogin 1 and MuRF 1, and interstitial fibrosis, including collagen volume fraction, contents of collagens I and III, and ratio of collagens I to III, were increased significantly (P < 0.05 for all). Following TRB3 knockout, the cross-sectional area of muscle fibres, mainly fast fibres, was elevated (P < 0.05 for both), the atrogin 1 expression was decreased (P = 0.0163), and the corresponding tetanic force of fast muscles was increased (P = 0.0398). Conversely, interstitial fibrosis was substantially decreased and exercise capacity was significantly increased in the knockout mice. In terms of the underlying mechanisms, the autophagy receptor p62 was markedly increased and the MAPK signalling pathway was activated in aged skeletal muscles, which might be attributed to the interaction of TRB3 with p62 and MAPKKs, including MEK1/MEK2, MEK3/MEK6, and MEK4/MKK4. Notably, TRB3 knockout reduced the accumulation of p62 and LC3 (P < 0.05 for both), decreased the phosphorylation of JNK (P = 0.0015), and increased p38 phosphorylation (P = 0.0021). CONCLUSIONS: TRB3 knockout in mice attenuated muscle fibre atrophy and reduced skeletal muscle fibrosis by increasing autophagy and inhibiting the MAPK signalling pathway. Correspondingly, in aged knockout mice, exercise capacity was improved. Interfering with TRB3 expression in aged skeletal muscles may serve as a target for the prevention and treatment of age-related sarcopenia.

Anti-androgen effects of the pyrethroid pesticide cypermethrin on interactions of androgen receptor with corepressors.

To clarify whether the mechanism of androgen receptor (AR) antagonism of the pyrethroid pesticide cypermethrin associates with the interactions between the AR and corepressors silencing mediator for thyroid hormone receptors (SMRT) and nuclear receptor corepressor (NCoR), we have developed the mammalian two-hybrid assays. The AR N-terminal domain 1-660 amino acid residues were subcloned into the plasmid pVP16 to construct VP16-ARNTD. The C-terminal receptor interaction domains (RIDs) of SMRT and NCoR were used to construct pM-SMRT and pM-NCoR. The constructed vectors pVP16-ARNTD, pM-SMRT or pM-NCoR, the reporter pG5CAT and the control pCMVß were cotranfected into the CV-1 cells. The cells were treated with cypermethrin at the indicated concentrations. The AR N terminus interacted with RIDs of SMRT and NCoR. The interactions between the AR and corepressors SMRT and NCoR were enhanced by cypermethrin, and the significant enhancement was detected at the concentration of 10(-5)M. The mammalian two-hybrid assays demonstrate the utility to detect the interactions of the AR with SMRT and NCoR. Cypermethrin functions as an anti-androgen by enhancing the associations of the AR with SMRT and NCoR. We provide a novel mechanism in anti-androgen action of cypermethrin associated with the recruitment of SMRT and NCoR to AR.

Effects of cypermethrin on the ligand-independent interaction between androgen receptor and steroid receptor coactivator-1.

The pyrethroid insecticide, cypermethrin has been considered as an environmental anti-androgen by interfering with the androgen receptor (AR) transactivation. In order to clarify the effects of cypermethrin on the ligand-independent interaction between the AR and SRC-1, the mammalian two-hybrid assay has been developed in the study. The AR N-terminal domain 1-660 amino acid residues were subcloned into the plasmid pVP16 to construct the vector pVP16-ARNTD. The SRC-1 C-terminal domain 989-1240 amino acid residues were subcloned into the plasmid pM to construct the vector pM-SRC-1. The fusion vectors pVP16-ARNTD, pM-SRC-1 and the pG5CAT Reporter Vector were cotransfected into the CV-1 cells. The AR AF1 interacted with SRC-1 in the absence of exogenous ligand 5α-dihydrotestosterone (DHT). Furthermore, DHT did not enhance the interaction between AR AF-1 and SRC-1 at the concentrations from 10(-10)M to 10(-8)M. Cypermethrin inhibited the interaction between the AR AF1 and SRC-1, and the significant reduction was detected at the concentration of 10(-5)M. It is suggested that the interaction between the AR AF1 and SRC-1 is ligand-independent. Cypermethrin inhibits AR activity by disrupting the ligand-independent AR-SRC-1 interaction.