Targeting Extracellular Cyclophilin A via an Albumin-Binding Cyclosporine A Analogue.

An albumin-binding CsA analogue 4MCsA was achieved by attachment of a thiol-reactive maleimide group at the side-chain of P4 position of CsA derivative. 4MCsA was semi-synthesized from CsA, and the cell-impermeability of albumin-4MCsA was detected by mass spectrometry and a competitive flow cytometry. 4MCsA exhibits inhibition of chemotaxis activity and inflammation by targeting extracellular CypA without immunosuppressive effect and cellular toxicity. These combined results suggested that 4MCsA can be restricted extracellularly through covalently binding to Cys34 of albumin with its maleimide group, and regulate the functions of cyclophilin A extracellularly.

Metformin improves boar sperm quality via 5'-AMP-activated protein kinase-mediated energy metabolism in vitro.

Sperm are specialized cells that require adenosine triphosphate (ATP) to support their function. Maintaining sperm energy homeostasis in vitro is vitally important to improve the efficacy of boar sperm preservation. Metformin can activate 5'-AMP-activated protein kinase (AMPK) to improve metabolic flexibility and maintain energy homeostasis. Thus, the aim of the present study was to investigate whether metformin can improve boar sperm quality through AMPK mediation of energy metabolism. Sperm motility parameters, membrane integrity, acrosome integrity, mitochondrial membrane potential (ΔΨ m), ATP content, glucose uptake, and lactate efflux were analyzed. Localization and expression levels of AMPK and phospho-Thr 172-AMPK (p-AMPK) were also detected by immunofluorescence and western blotting. We found that metformin treatment significantly increased sperm motility parameters, ΔΨ m, and ATP content during storage at 17 °C. Moreover, results showed that AMPK was localized at the acrosomal region, connecting piece, and midpiece of sperm and p-AMPK was distributed at the post-acrosomal region, connecting piece, and midpiece. When sperm were incubated with metformin for 4 h at 37 °C, sperm motility parameters, ΔΨ m, ATP content, p-AMPK, glucose uptake, and lactate efflux all significantly increased, whereas the addition of Compound C treatment, an inhibitor of AMPK, counteracted these positive effects. Together, our results suggest that metformin promotes AMPK activation, which contributes to the maintenance of energy hemostasis and mitochondrial activity, thereby maintaining boar sperm functionality and improving the efficacy of semen preservation.

DDAH2 alleviates myocardial fibrosis in diabetic cardiomyopathy through activation of the DDAH/ADMA/NOS/NO pathway in rats.

Diabetic cardiomyopathy (DCM) is a form of idiopathic heart disease, with signs including hypertrophy of myocardial cells, hypertension­independent fibrosis and coronary artery disease. Considering the involvement of dimethylarginine dimethylaminohydrolase 2 (DDAH2) in diabetes, it was hypothesized that DDAH2 may be beneficial to cardiac function and myocardial fibrosis during the progression of DCM with involvement of the DDAH/asymmetric NG, NGdimethyl­L­arginine (ADMA)/nitric oxide synthase (NOS)/nitric oxide (NO) signaling pathway. Following establishment of diabetic rat models, diabetes­related blood biochemical indices and cardiac function were measured in diabetic rats treated with lentivirus expressing DDAH2, short hairpin RNA against DDAH2, or L­NNA (inhibitor of NOS) to identify the roles of DDAH2 in DCM. The functional roles of DDAH2 in DCM were further determined through detection of the levels of collagen I, matrix metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase 2 (TIMP2). The H9C2 myocardial cell line was selected for in vitro experiments. The effects of DDAH2 on the migration of myocardial cells under high glucose conditions were also examined. To further investigate the underlying regulatory mechanism of DDAH2 in DCM, the contents of ADMA and NO, and the activities of DDAH and NOS were observed. The DCM model rats treated with DDAH2 exhibited reduced left ventricular end­diastolic pressure, and decreased blood glucose, total cholesterol, triglyceride, fasting blood glucose, and fasting insulin levels, but exhibited increased left ventricular systolic pressure and maximum rate of left ventricular pressure rise/fall levels in myocardial tissues. Myocardial cells under high glucose conditions treated with DDAH2 showed reductions in collagen I, MMP2 and TIMP2, indicating that DDAH2 reduced cell migration. Decreased levels of ADMA and NO but increased levels of DDAH and NOS were observed following treatment with DDAH2, indicating that the DDAH/ADMA/NOS/NO pathway was activated. These results reveal that the overexpression of DDAH2 attenuates myocardial fibrosis and protects against DCM through activation of the DDAH/ADMA/NOS/NO pathway in DCM rats. These results indicate that DDAH2 is a potential therapeutic candidate for the treatment of DCM.

Effects of microRNA-292-5p on myocardial ischemia-reperfusion injury through the peroxisome proliferator-activated receptor-α/-γ signaling pathway.

Ischemia-reperfusion injury (IRI) is a major cause of cardiac damage following various pathological processes, such as free radical damage and cell apoptosis. This study aims to investigate whether microRNA-292-5p (miR-292-5p) protects against myocardial ischemia-reperfusion injury (IRI) via the peroxisome proliferator-activated receptor (PPAR)-α/-γ signaling pathway in myocardial IRI mice models. Mouse models of myocardial IRI were established. Adult male C57BL/6 mice were divided into different groups. The hemodynamic indexes, levels of related inflammatory factors and serum myocardial enzymes, and malondialdehyde (MDA) content and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected. The 2,3,5-triphenyltetrazolium chloride (TTC) staining was applied to determine infarct size. TUNEL staining was used to detect cardiomyocyte apoptosis. RT-qPCR and western blotting were performed to measure the related gene expressions. Compared with the model group and the T0070907 + miR-292-5p inhibitor, the miR-292-5p inhibitor group exhibited decreased incidence and duration time of ventricular tachycardia and ventricular fibrillation, serum myocardial enzymes, TNF-α, IL-6, IL-1ß, MDA, cardiomyocyte apoptosis, expressions of Bax and p53 in addition to increased SOD and GSH-Px activity, and increased expressions of Bcl-2, PPARα, PPARγ, PLIN5, AQP7, and PCK1. The T0070907 group exhibited opposite results compared to the miR-292-5p inhibitor group. The results indicate that miR-292-5p downregulation protects against myocardial IRI through activation of the PPAR-α/PPAR-γ signaling pathway.

SOCE induced calcium overload regulates autophagy in acute pancreatitis via calcineurin activation.

Acute pancreatitis (AP) is an acute inflammatory process of the pancreas that is characterized by inflammation, edema, vacuolization and necrosis, which has significant morbidity and lethality. The pathogenesis of AP has not been established completely. An early and critical feature of AP is the aberrant signaling of Calcium (Ca2+) within the pancreatic acinar cell, termed Ca2+ overload. Store-operated Ca2+ (SOC) channels are the principal Ca2+ influx channels that contribute to Ca2+ overload in pancreatic acinar cells. Store-operated Ca2+ entry (SOCE) has been proved to be a key pathogenic step in AP development that leads to trypsin activation, inflammation and vacuolization. However, the molecular mechanisms are still poorly understood. By establishing Ca2+ overload model and mouse AP model using caerulein, we found that caerulein triggered SOCE via inducing interaction between STIM1 and Orai1, which activated calcineurin (CaN); CaN activated the nuclear factor of activated T cells (NFAT) and transcription factor EB (TFEB), thus promoting the transcriptional activation of multiple chemokines genes and autophagy-associated genes respectively. To the best of our knowledge, this is the first evidence showing that SOCE activates TFEB via CaN activation, which may have noticeable longer-term effects on autophagy and vacuolization in AP development. Our findings reveal the role for SOCE/CaN in AP development and provide potential targets for AP treatment.

Two genes conferring resistance to Pythium stalk rot in maize inbred line Qi319.

Stalk rots are destructive diseases in maize around the world, and are most often caused by the pathogen Pythium, Fusarium and other fungi. The most efficient management for controlling stalk rots is to breed resistant cultivars. Pythium stalk rot can cause serious yield loss on maize, and to find the resistance genes from the existing germplasm is the basis to develop Pythium-resistance hybrid lines. In this study, we investigated the genetic resistance to Pythium stalk rot in inbred line Qi319 using F2 and F2:3 population, and found that the resistance to Pythium inflatum in Qi319 was conferred by two independently inherited dominant genes, RpiQI319-1 and RpiQI319-2. Linkage analysis uncovered that the RpiQI319-1 co-segregated with markers bnlg1203, and bnlg2057 on chromosome 1, and that the RpiQI319-2 locus co-segregated with markers umc2069 and bnlg1716 on chromosome 10. The RpiQI319-1 locus was further mapped into a ~500-kb interval flanked by markers SSRZ33 and SSRZ47. These results will facilitate marker-assisted selection of Pythium stalk rot-resistant cultivars in maize breeding. To our knowledge, this is the first report on the resistance to P. inflatum in the inbred line Qi319, and is also the first description of two independently inherited dominant genes conferring the resistance of Pythium stalk rot in maize.

Effect of ECRG2 in combination with cisplatin on the proliferation and apoptosis of EC9706 cells.

The aim of the present study was to explore the effect of esophageal cancer-related gene 2 (ECRG2) protein in combination with cisplatin (DDP) on the proliferation and apoptosis of esophageal cancer cells. A 3-(4, 5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the effects of ECRG2 alone and ECRG2 in combination with DDP on the proliferation of EC9706 esophageal cancer cells. Hoechst 33258 staining was performed to analyze the effects of ECRG2 alone and ECRG2 in combination with DDP on apoptosis in the EC9706 cells. The expression levels of Bcl-2-associated X protein (Bax) mRNA and protein were determined by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively. The results from the MTT assay revealed that ECRG2 inhibited the proliferation of EC9706 cells and that ECRG2 in combination with DDP had a greater inhibitory effect on cell proliferation. The antiproliferative effects were time- and concentration-dependent, within a certain range of concentrations. The Hoechst 33258 staining results demonstrated that the number of apoptotic cells following treatment with ECRG2 in combination with DDP for 24 h was higher than that following treatment with ECRG2 alone for the same duration. Western blot analysis and RT-PCR results revealed that the expression levels of Bax mRNA and protein were upregulated in cells treated with ECRG2 in combination with DDP compared with those in cells treated with ECRG2 alone. Thus, ECRG2 in combination with DDP had an enhanced inhibitory effect on EC9706 cell proliferation compared with that of ECRG2 alone, and an increased inductive effect on EC9706 cell apoptosis, possibly due to the upregulation of the expression of Bax.

Rapid development of microsatellite markers for Callosobruchus chinensis using Illumina paired-end sequencing.

BACKGROUND: The adzuki bean weevil, Callosobruchus chinensis L., is one of the most destructive pests of stored legume seeds such as mungbean, cowpea, and adzuki bean, which usually cause considerable loss in the quantity and quality of stored seeds during transportation and storage. However, a lack of genetic information of this pest results in a series of genetic questions remain largely unknown, including population genetic structure, kinship, biotype abundance, and so on. Co-dominant microsatellite markers offer a great resolving power to determine these events. Here, we report rapid microsatellite isolation from C. chinensis via high-throughput sequencing. PRINCIPAL FINDINGS: In this study, 94,560,852 quality-filtered and trimmed reads were obtained for the assembly of genome using Illumina paired-end sequencing technology. In total, the genome with total length of 497,124,785 bp, comprising 403,113 high quality contigs was generated with de novo assembly. More than 6800 SSR loci were detected and a suit of 6303 primer pair sequences were designed and 500 of them were randomly selected for validation. Of these, 196 pair of primers, i.e. 39.2%, produced reproducible amplicons that were polymorphic among 8 C. chinensis genotypes collected from different geographical regions. Twenty out of 196 polymorphic SSR markers were used to analyze the genetic diversity of 18 C. chinensis populations. The results showed the twenty SSR loci were highly polymorphic among these populations. CONCLUSIONS: This study presents a first report of genome sequencing and de novo assembly for C. chinensis and demonstrates the feasibility of generating a large scale of sequence information and SSR loci isolation by Illumina paired-end sequencing. Our results provide a valuable resource for C. chinensis research. These novel markers are valuable for future genetic mapping, trait association, genetic structure and kinship among C. chinensis.

Expression of bcl-2 and p53 in induction of esophageal cancer cell apoptosis by ECRG2 in combination with cisplatin.

AIM: To investigate the mechanisms of induction of apoptosis of esophageal cancer cells by esophageal cancer-related gene 2 (ECRG2) in combination with cisplatin (DDP). METHODS: Hoechest staining was performed to analyze the effects of single ECRG2 and ECRG2 in combination with DDP on apoptosis of EC9706 cells. The expression levels of p53 and bcl-2 mRNA and protein were determined by RT-PCR and Western blotting, respectively. RESULTS: The number of apoptotic cells after the treatment with ECRG2 in combination with DDP for 24 hours was more than that after the treatment with single ECRG2. RT-PCR and Western blotting showed that the expression levels of bcl-2 mRNA and protein were both down-regulated, while p53 mRNA and protein were both up-regulated in the cells treated with ECRG2 in combination with DDP compared with those given ECRG2 alone. CONCLUSION: ECRG2 in combination with DDP can enhance the apoptosis of EC9706 cells, possibly by down-regulating bcl-2 expression and up-regulating p53.

Resistance of Faba Bean and Pea Germplasm to Callosobruchus chinensis (Coleoptera: Bruchidae) and Its Relationship With Quality Components.

In total, 339 faba bean (Vicia faba L.) and 100 pea (Pisum sativum L.) accessions were screened for their ability to resist Callosobruchus chinensis L. in free choice laboratory tests. Four, 15, and 43 faba bean varieties were highly resistant, resistant, and moderately resistant to C. chinensis, respectively. Three immune, three highly resistant, and six resistant accessions were discovered among the pea germplasm. The faba bean and pea varieties presented a hundred-kernel weight reduction varied from 0.18 to 35.36% for faba bean varieties and 0 to 56.53% for pea varieties. Varieties with brown and black seed color had significantly fewer wormholes and higher C. chinensis resistance than varieties with light-color seeds. Resistance to C. chinensis showed a significant, positive correlation with catechin, total polyphenol, and γ-aminobutyric acid contents, but a significant, negative correlation with oligosaccharide content. Correlation coefficients (r) between infestation rate of faba bean and total phenol, catechin, and oligosaccharide contents were -0.9723, -0.8071, and 0.7631, respectively. The values of r for pea resistance and total phenol, catechin, and oligosaccharide content were -0.8846, -0.7666, and 0.8308, respectively. The results suggest that quality components in faba bean and pea have a great role in resistance against C. chinensis.

Zr61Ti2Cu25Al12 metallic glass for potential use in dental implants: biocompatibility assessment by in vitro cellular responses.

In comparison with titanium and its alloys, Zr61Ti2Cu25Al12 (ZT1) bulk metallic glass (BMG) manifests a good combination of high strength, high fracture toughness and lower Young's modulus. To examine its biocompatibility required for potential use in dental implants, this BMG was used as a cell growth subtract for three types of cell lines, L929 fibroblasts, human umbilical vein endothelial cells (HUVEC), and osteoblast-like MG63 cells. For a comparison, these cell lines were in parallel cultured and grown also on commercially pure titanium (CP-Ti) and Ti6-Al4-V alloy (Ti64). Cellular responses on the three metals, including adhesion, morphology and viability, were characterized using the SEM visualization and CCK-8 assay. Furthermore, real-time RT-PCR was used to measure the activity of integrin ß, alkaline phosphatase (ALP) and type I collagen (COL I) in adherent MG63 cells. As indicated, in all cases of three cell lines, no significant differences in the initial attachment and viability/proliferation were found between ZT1, CP-Ti, and Ti64 until 5d of incubation period. It means that the biocompatibility in cellular response for ZT1 BMG is comparable to Ti and its alloys. For gene expression of integrin ß, ALP and COL I, mRNA level from osteoblast cells grown on ZT1 substrates is significantly higher than that on the CP-Ti and Ti64. It suggests that the adhesion and differentiation of osteoblasts grown on ZT1 are even superior to those on the CP-Ti and Ti64 alloy, then promoting bone formation. The good biocompatibility of ZT1 BMG is associated with the formation of zirconium oxide layer on the surface and good corrosion-resistance in physiological environment.

Effect of asymmetric dimethylarginine on MIF expression and TNF-α and IL-8 secretion in THP-1 monocytes-derived macrophages.

OBJECTIVE: To investigate the effect of ADMA on macrophage migration inhibitory factor (MIF) expression and tumor necrosis factor-α (TNF-α) and IL-8 secretion in THP-1 monocyte-derived macrophages. METHIDS: THP-1 monocytes were induced to differentiate into macrophages by a 24-h incubation with 160 nmol/L PMA. The THP-1 monocyte-derived macrophages were exposed to different concentrations of ADMA for 24 h, and the changes in MIF mRNA and protein expressions were analyzed with RT-PCR and Western blotting, respectively. Enzyme-linked immunosorbent assay was used to detect the levels of TNF-α and IL-8 in the supernatant of THP-1-derived macrophages following ADMA treatments. RESULTS: ADMA obviously up-regulated MIF mRNA and protein expressions in THP-1-derived macrophages in a concentration- dependent manner. Exposure of the cells to 15 µmol/L ADMA for 24 h showed the most potent effect in up-regulating MIF mRNA and protein expressions. ADMA treatment also resulted in a dose-dependent increase of the levels of TNF-α and IL-8 in the culture supernatant of the macrophages, and the peak levels occurred following the treatment with 15 µmol/L ADMA. CONCLUSION: ADMA can up-regulate MIF expression and induce TNF-α and IL-8 secretion in THP-1 monocyte-derived macrophages.

Asymmetric dimethylarginine upregulates the expression of ACAT-1 in THP-1 macrophage-derived foam cells.

OBJECTIVE: To investigate the effects of asymmetric dimethylarginine (ADMA) on ACAT-1 expression and cholesterol content in THP-1-derived macrophages and foam cells. METHODS: THP-1 cells were induced to differentiate into macrophages and further into foam cells. The macrophages and foam cells were exposed to different concentrations (0, 3.75, 7.5, 15, and 30 µmol/L) of ADMA for varying time lengths (6, 12, and 24 h), and the changes in ACAT-1 mRNA and protein levels in the cells were measured with RT-PCR and Western blotting. The cellular cholesterol content was measured with enzyme-linked colorimetry assay. RESULTS: In THP-1-derived macrophages and foam cells, the expression levels of ACAT-1 mRNA and protein and cellular cholesterol content increased significantly in response to ADMA treatment in a time- and concentration-dependent manner. CONCLUSION: ADMA may play an important role in inducing foam cell formation from macrophages. ACAT-1 inhibition targeting the macrophages and foam cells may serve as a potential therapeutic target in the treatment of atherosclerosis.

[Primary culture and identification of olfactory ensheathing cells from adult rat].

OBJECTIVE: To explore a simple and inexpensive method or procedure for establishing primary culture and purification of olfactory ensheathing cells (OECs) from the adult rat olfactory bulbs. METHODS: The OECs were dissociated from the first two outer layers of the olfactory bulbs of the adult Sprague-Dawley rat (2.5 months old), cultured in DMEM/F12 nutrient with 20% foetal calf serum, and purified by the method of combining the different rates of cell attachment with the Arabinosylcytosine (AraC) inhibition of cell. The morphological changes of cultured OECs were observed. On 14th culture day, the OECs in culture were identified by the immunocytochemistry technique to glial fibrillary acidic protein (GFAP) and nerve growth factor receptor p75 (NGFRp75), and the purity of the positive cells was calculated. RESULTS: The cultured OECs presented three main morphological types: multipolar, bipolar and flat cells. For GFAP positive cells, the plasma and processes were stained while the nuclei were not, but for NGFRp75 cells the nuclei were stained more deeply than the plasma and processes. More than 9000 cultured cells were identified to be OECs. CONCLUSION: The high purity OECs can be cultured successfully in vitro by the combined purification method. This method is simple and inexpensive.

[Allele frequencies and species specificity of five short tandem repeat loci of Chinese Han population in Chengdu].

OBJECTIVE: To obtain the data in polymorphism distribution of the five short tandem repeat (STR) loci: D18S979, D11S2014, D18S548, D1S1667 and GATA164F07 of Chinese Han population in Chengdu, and to evaluate their usefulness in the field of species specificity in forensic science. METHODS: PCR, polyacrylamide gel electrophoresis (PAGE) and silver staining techniques were used to analyze the DNA samples from 100 unrelated individuals of Chinese Han ethnic group in Chengdu. Twelve different animals: monkey, pig, dog, bull, goat, chicken, duck, eel, mudfish, rabbit, guinea pig and mouse were selected as controls in this study for evaluating the species specificity of the five STR loci. RESULTS: Six alleles and twelve genotypes were observed in D18S979. Five alleles and eleven genotypes were observed in D11S2014. Five alleles and thirteen genotypes were observed in D18S548. Seven alleles and nineteen genotypes were observed in D1S1667. Six alleles and fourteen genotypes were observed in GATA164F07. The genotype distributions of the five loci were analyzed by some related software and no deviation from the Hardy-Weinberg equilibrium was observed. Evaluated by way of using different animals as controls, monkey had amplification products at the extra-typing field of D18S979, D11S2014 and D1S1667. Bull, dog and eel had amplification product at typing field of D18S979, and pig, duck, mouse and rabbit had weak product. Bull had weak product at the typing field of D18S548. Dog, goat and eel had product at the typing field of D1S1667. Dog had weak product at the typing field of GATA164F07. Mudfish, chicken and guinea pig had no amplification product at the five loci. CONCLUSION: These data indicate that D18S979, D18S548, D1S1667 and GATA164F07 are highly polymorphic and D11S2014, D18S548 and GATA164F07 can play a key role in species identification.

[Microsatellite marker development and applications in wheat genetics and breeding].

Microsatellites are simple, tandemly repeated one to six nucleotide sequence motifs. They are very useful as genetic markers because they are co-dominant, detect high levels of allelic diversity, and are easily assayed by the polymerase chain reaction ( PCR ). In wheat, SSR markers have been applied to genetic mapping,detection of genetic diversity, identification of varieties and genotypes,gene tagging, QTL analysis, and marker-assisted selection.