Density Gradient Centrifugation Method Research Articles

Sperm Dynein AAA1 and AAA2 Expression in Human Sperm : A Regulation in Sperm Preparation

Axoneme structures in sperm tail, is a supramolecular protein complex with motor protein and regulatory which playing a crucial role in determining sperm motility. Dynein, one of the three members of cytoskeletal motor protein, has a ring of six AAA+ which linked together into one large polypeptide that contribute to the formation of sperm flagella bending. The previously research reported that the first two AAA motor domains, AAA1 and AAA2, were a major site in ATP hydrolysis associated with motility in the flagellum. Intrauterine Insemination (IUI) as a management of infertility requires sperm preparation procedures, by Swim-up (SU) and Density Gradient Centrifugation (DGC), in order to enhance the quality regarding concentration and motility of the initial sperm. This study aimed to evaluate the efficiency of the DGC and SU methods in selecting sperm, based the expression of sperm dynein AAA1 and AAA2. Semen samples were obtained from men underwent sperm preparation for IUI and divided into two groups, normozoospermia and asthenozoospermia, according to World Health Organization 2010 guideline. Semen analysis was performed to measure the sperm motility and velocity, before and after sperm preparation. The axoneme was isolated from the obtained samples from SU and DGC methods, while the level of AAA1 and AAA2 was measured by ELISA. This study showed that the percentage of motile sperm and velocity of prepared sperm in both groups in prepared sperm (post-SU and post-DGC) was higher compared to whole semen. The expression of sperm dynein AAA1 of prepared sperm in normozoospermia group showed higher, while in asthenozoospermia group showed lower activities compared to whole semen. The expression of sperm dynein AAA2 of prepared sperm in both groups showed lower activities compared to whole semen. The sperm preparation enhanced the quality of sperm and may increase the expression of sperm dynein AAA1 compared to the whole semen, without the involvement of sperm dynein AAA2.

Interleukin‑10 promotes primary rat hepatic stellate cell senescence by upregulating the expression levels of p53 and p21.

Liver fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) components, and activated hepatic stellate cells (HSCs) are a primary source of ECM. Several studies have revealed that the induction of HSC senescence may reduce liver fibrosis. The effect of interleukin-10 (IL-10) on the senescence of activated HSCs is not fully understood. Therefore, the present study examined its effects and potential mechanisms in activated primary rat HSCs. Collagenase perfusion and density gradient centrifugation methods were used to isolate rat HSCs. HSCs were identified by autofluorescence, Oil Red O staining and immunocytochemical analysis. Activated HSCs were treated with 0, 10, 20 or 40 ng/ml IL-10 for 24 h. Senescence-associated β-galactosidase (SA-β-Gal) staining, flow cytometry analysis and a cell counting kit-8 assay were performed to detect the senescence, apoptosis and viability of rat HSCs, respectively. Reverse transcription-quantitative polymerase chain reaction, western blot analysis and enzyme linked immunosorbent assays were used to detect the expression of senescence-associated proteins and cytokines. Freshly isolated rat HSCs exhibited a striking blue-green autofluorescence and HSC retinoid droplets were stained bright red by Oil Red O. Immunocytochemical analysis demonstrated the cytoplasmic expression of HSC markers desmin and α-smooth muscle actin. The number of SA-β-Gal positive HSCs, the apoptotic rate and the expression levels of p53, p21 and tumor necrosis factor-α were significantly increased following IL-10 treatment. HSC viability and IL-6 and IL-8 expression levels were significantly decreased compared with the control group. In summary, primary rat HSCs were successfully isolated and IL-10 was demonstrated to promote the senescence of activated primary rat HSCs through the upregulation of p53 and p21 expression.

The role of glutathione in steroid induced bone marrow mesenchymal stem cells dysfunction

To investigate the protective effect of the antioxidant glutathione (GSH) on the steroid-induced imbalance between osteogenesis and adipogenesis in human bone marrow mesenchymal stem cells (BMSCs). The BMSCs were isolated from the proximal femur bone marrow from 3 patients of femoral neck fracture and were separated, cultured, and purificated by density gradient centrifugation and adherent wall method in vitro. The third generation BMSCs were divided into 5 groups: group A, BMSCs (1×10 5 cells/mL); group B, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone; group C, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone+5 μmol/L GSH; group D, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone+10 μmol/L GSH; group E, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone+50 μmol/L GSH. After cultured for 7 days, the reactive oxygen species expression was detected by flow cytometry; the superoxide dismutase (SOD) and Catalase mRNA expressions were determined by RT-PCR; the peroxisome proliferator-activated receptors γ (PPAR-γ), CCAAT/enhancer-binding family of proteins (C/EBP), Runx2, and alkaline phosphatase (ALP) mRNA expressions were evaluated by real-time fluorescence quantitative PCR. After cultured for 21 days, Oil red O staining was used to observe the adipogenesis differentiation of cells, and the expressions of related proteins were detected by Western blot. The reactive oxygen species expression in group B was obviously higher than in the other groups, in group C than in groups A, D, and E, and in groups D, E than in group A, all showing significant differences between groups ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). The oil red O staining positive cells in group B were obviously more than the other groups, and groups C, D, E, and A decreased sequentially, the absorbance ( A) values had significant differences between groups ( P<0.05). RT-PCR detection showed that the relative expressions of SOD and Catalase mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E ( P<0.05), but there was no significant difference among groups A, D, and E ( P>0.05). Real-time fluorescence quantitative PCR detection showed that the relative expressions of PPAR-γ and C/EBP mRNA in group B were significantly higher than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). The relative expressions of Runx2 and ALP mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). Western blot detection showed that the relative expression of PPAR-γ and C/EBP protein in group B was significantly higher than those in the other groups, and groups C, D, E, and A decreased sequentially, all showing significant differences between groups ( P<0.05). The relative expression of Runx2 and ALP protein in group B was significantly lower than those in the other groups, and groups C, D, E, and A increased sequentially, all showing significant differences between groups ( P<0.05). GSH can inhibit the adipogenesis differentiation and enhance the osteogenic differentiation of human BMSCs by reducing the intracellular reactive oxygen species level; and in a certain range, the higher the concentration of GSH, the more obvious the effect is.

Effect of Moldey Ion Rhodamine B on viability of Adipose and Bone marrow Derived Mesenchymal Stem cells - an In-vitro Study

The study was performed to access the effect of Moldey ion Rhodamine B on in-vitro labelled Bone marrow and adipose derived derived stem cells in sheep. Perirenal adipose tissue and bone marrow from femur were collected from 28 male sheep. Adipose derived mesenchymal stem cells were isolated from perirenal fat using enzymatic digestion method by using type II collagenase. Bone marrow derived mesenchymal stem cells were separated from femur by density gradient centrifugation method using percoll. These mesenchymal stem cells from bone marrow and adipose tissue were cultured and expanded using DulbeccoÂ’s Modified EagleÂ’s Medium with 10 per-cent of fetal bovine serum, one per-cent of antibiotic-antimycotic solution. Adipose and bone marrow derived mesenchymal stem cells from passage 4 to passage 6 were used for labelling with medium containing 25 µg of MIRB per ml. Cell viability was evaluated by Trypan blue exclusion test in both labelled and control (unlabelled) groups. There was no significant difference in the viability of bone marrow and adipose derived mesenchymal stem cells between the labelled and control groups. It was observed that in-vitro labelling of mesenchymal stem cells with MIRB at a concentration of 25 µg per ml of medium did not affect cell proliferation and viability.

Gelatin Nanoparticle-Coated Silicon Beads for Density-Selective Capture and Release of Heterogeneous Circulating Tumor Cells with High Purity.

Background: Circulating tumor cells (CTCs) are a burgeoning topic in cancer biomarker discovery research with minimal invasive blood draws. CTCs can be used as potential biomarkers for disease prognosis, early cancer diagnosis and pharmacodynamics. However, the extremely low abundance of CTCs limits their clinical utility because of technical challenges such as the isolation and subsequent detailed molecular and functional characterization of rare CTCs from patient blood samples.Methods: In this study, we present a novel density gradient centrifugation method employing biodegradable gelatin nanoparticles coated on silicon beads for the isolation, release, and downstream analysis of CTCs from colorectal and breast cancer patients.Results: Using clinical patient/spiked samples, we demonstrate that this method has significant CTC-capture efficiency (>80%) and purity (>85%), high CTC release efficiency (94%) and viability (92.5%). We also demonstrate the unparalleled robustness of our method in downstream CTC analyses such as the detection of PIK3CA mutations.Conclusion: The efficiency and versatility of the multifunctional density microbeads approach provides new opportunities for personalized cancer diagnostics and treatments.

Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (NETs) Release.

Neutrophils are one of the first cells to arrive at the site of infection, where they apply several strategies to kill pathogens: degranulation, respiratory burst, phagocytosis, and release of neutrophil extracellular traps (NETs). Antibiotics have an immunomodulating effect, and they can influence the properties of numerous immune cells, including neutrophils. The aim of this study was to investigate the effects of azithromycin and chloramphenicol on degranulation, apoptosis, respiratory burst, and the release of NETs by neutrophils. Neutrophils were isolated from healthy donors by density-gradient centrifugation method and incubated for 1 h with the studied antibiotics at different concentrations (0.5, 10 and 50 μg/mL—azithromycin and 10 and 50 μg/mL—chloramphenicol). Next, NET release was induced by a 3 h incubation with 100 nM phorbol 12-myristate 13-acetate (PMA). Amount of extracellular DNA was quantified by fluorometry, and NETs were visualized by immunofluorescent microscopy. Degranulation, apoptosis and respiratory burst were assessed by flow cytometry. We found that pretreatment of neutrophils with azithromycin and chloramphenicol decreases the release of NETs. Moreover, azithromycin showed a concentration-dependent effect on respiratory burst in neutrophils. Chloramphenicol did not affect degranulation, apoptosis nor respiratory burst. It can be concluded that antibiotics modulate the ability of neutrophils to release NETs influencing human innate immunity.

Isolation of membrane vesicles from prokaryotes: a technical and biological comparison reveals heterogeneity

ABSTRACTProkaryotes release membrane vesicles (MVs) with direct roles in disease pathogenesis. MVs are heterogeneous when isolated from bacterial cultures so Density Gradient Centrifugation (DGC) is valuable for separation of MV subgroups from contaminating material. Here we report the technical variability and natural biological heterogeneity seen between DGC preparations of MVs for Mycobacterium smegmatis and Escherichia coli and compare these DGC data with size exclusion chromatography (SEC) columns. Crude preparations of MVs, isolated from cultures by ultrafiltration and ultracentrifugation were separated by DGC with fractions manually collected as guided by visible bands. Yields of protein, RNA and endotoxin, protein banding and particle counts were analysed in these. DGC and SEC methods enabled separation of molecularly distinct MV populations from crude MVs. DGC banding profiles were unique for each of the two species of bacteria tested and further altered by changing culture conditions, for example with iron supplementation. SEC is time efficient, reproducible and cost effective method that may also allow partial LPS removal from Gram-negative bacterial MVs. In summary, both DGC and SEC are suitable for the separation of mixed populations of MVs and we advise trials of both, coupled with complete molecular and single vesicle characterisation prior to downstream experimentation.

Bone mesenchymal stem cells co-expressing VEGF and BMP-6 genes to combat avascular necrosis of the femoral head.

The aim of the present study was to investigate the potential of bone mesenchymal stem cells (BMSCs) treated with a combination of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-6 (BMP-6) genes for the treatment of avascular necrosis of the femoral head (ANFH). Rat BMSCs were isolated and purified using a density gradient centrifugation method. The purity and characteristics of the BMSCs were detected by cell surface antigens identification using flow cytometry. The experimental groups were administered with one of the following adeno-associated virus (AAV) vector constructs: AAV-green fluorescent protein (AAV-GFP), AAV-BMP-6, AAV-VEGF or AAV-VEGF-BMP-6. The expression of VEGF and BMP-6 was detected by reverse transcription-quantitative polymerase chain reaction, western blotting and ELISA assays. The effects of VEGF and BMP-6 on BMSCs were evaluated by angiogenic and osteogenic assays. The transfected BMSCs were combined with a biomimetic synthetic scaffold poly lactide-co-glycolide (PLAGA) and they were then subcutaneously implanted into nude mice. After four weeks, the implants were analyzed with histology and subsequent immunostaining to evaluate the effects of BMSCs on blood vessel and bone formation in vivo. In the AAV-VEGF-BMP-6 group, the expression levels of VEGF and BMP-6 were significantly increased and human umbilical vein endothelial cells tube formation was significantly enhanced compared with other groups. Capillaries and bone formation in the AAV-VEGF-BMP-6 group was significantly higher compared with the other groups. The results of the present study suggest that BMSCs expressing both VEGF and BMP-6 induce an increase in blood vessels and bone formation, which provides theoretical support for ANFH gene therapy.

Hemocytes of the mud crab Scylla paramamosain: Cytometric, morphological characterization and involvement in immune responses

Hemocytes play essential roles in the innate immune system of crustaceans. Characterization of hemocytes from estuary mud crab Scylla paramamosain was performed by flow cytometry and morphological studies such as cytochemical staining and electron microscopy. The hemocyte subsets were further separated using a modified Percoll density gradient centrifugation method. Based on the morphological characteristics of the cells, three distinct categories of hemocytes were identified: granulocytes with abundant large granularity representing 5.27 ± 0.42%, semigranulocytes with small or less granularity representing 76.03 ± 3.34%, and hyalinocytes (18.70 ± 3.92%) which were almost no granularity. The total hemocyte cell count and the percentage of hemocyte subsets varied after pathogen infection, including Vibrio alginolyticus and the viral double-stranded RNA analog Poly (I:C). The phagocytic process is of fundamental importance for crustaceans' cellular immune response as well as development and survival. The results of the in vitro phagocytosis assays analyzed by flow cytometry demonstrated that granulocytes and semigranulocytes had significantly higher phagocytic ability than hyalinocytes. A primary culture system, L-15 medium supplemented with 5–10% fetal bovine serum, was developed to further investigate the immune function of hemocytes. Furthermore, adenovirus can be utilized to effectively transfer GFP gene into hemocytes. Overall, three hemocyte sub-populations of S. paramamosain were successfully discriminated, moreover, their response to pathogen infections, phagocytic activity and adenovirus mediated transfection were also investigated for the first time. This study may contribute to a better understanding of the innate immune system of estuary crabs.

Repair of articular cartilage defects by autologous bone mesenchymal stem cells and allogeneic costal chondrocytes in the knee of Wuzhishan miniature pigs

To investigate the feasibility of construction of tissue engineered cartilage by co-culture of bone marrow mesenchymal stem cells (BMSCs) and costal chondrocytes (CCs), and to provide theoretical basis and experimental basis for clinical repair of articular cartilage defects by Wuzhishan miniature pig knee cartilage defects with co-cultured cells. Methods: Density gradient centrifugation method was used to isolate BMSCs from Wuzhishan miniature pig. The double enzyme digestion method was used to isolate CCs. The passage 3 generation of BMSCs and passage 2 generation of CCs were randomly divided into 3 groups: a co-culture group of BMSCs:CCs for 1:2 (Group A), a simple CCs (Group B), and a simple BMSCs (Group C). The cell growth curve was drawn, and the content of glycosaminoglycan (GAG) of external separation in chondrocytes was determined. The 12 Wuzhishan miniature pigs were randomly divided into a co-culture cells/collagen membrane experimental group, a collagen membrane control group and the blank group. In the co-culture cells/collagen membrane experimental group, the co-cultured cells/collagen membrane were implanted into the cartilage defects of the mandibular condyle; in the collagen membrane control group, only collagen membrane was implanted; while in the blank group, nothing was implanted. Six animals were sacrificed at 8 and 16 weeks after surgery respectively (2 animals in each group). General observation, cartilage histological score and histopathological examination were carried out. Results: The BMSCs and co-culture cells grew well. The biological activity of CCs was good. After 16 weeks of operation, the repair tissues in the co-cultured cells/collagen membrane experimental group showed hyaline cartilage features: smooth, flat, and integrated well with the surrounding cartilage and subchondral bone. The collagen membrane in the collagen membrane control group was fibrously repaired. Repair tissue gross score in the co-culture cells/collagen membrane experimental group was significantly better than that in the collagen membrane control group and the blank group (both P<0.05), but there was no significant difference between the collagen membrane control group and the blank group (P>0.05). Conclusion: BMSCs, CCs and co-cultured cells can function as the seed cells for cartilage tissue engineering, and the co-culture cells (BMSCs:CCs=1:2) possess more advantages; the short-term effect of co-culture cells with collagen membrane on repairing cartilage defects is satisfied.

Ion-exchange chromatography purification of extracellular vesicles.

Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.

Ribosome cycle emerges from DNA replication.

A modification of Meselson and Stahl's density gradient centrifugation method and a rare Texan yeast helped show that eukaryotic ribosomes dissociate and reform during translation.

Regulation of c-Jun N-Terminal Protein Kinase (JNK) Pathway in Apoptosis of Endothelial Outgrowth Cells Induced by Asymmetric Dimethylarginine.

BackgroundEndothelial outgrowth cells (EOCs) are terminal endothelial progenitor cells (EPCs). Asymmetric dimethylarginine (ADMA) has been identified as a novel risk factor for cardiovascular diseases. Our aim in the present study was to investigate the effect of regulation of asymmetric dimethylarginine (ADMA) on EOCs apoptosis and to explore the underlining mechanisms of c-Jun N-terminal protein kinase (JNK) pathway in the process.Material/MethodsEOCs were harvested from umbilical cord blood and obtained by using density gradient centrifugation and adhesive culture methods. Endothelial characteristics were identified by immunohistochemistry and fluorescence staining. EOCs were treated with different concentrations of ADMA and detected by flow cytometry. After JNK specific inhibitor (SP600125) was added, EOCs apoptosis protein expressions were measured by Western blot analysis. Proliferation, migration, and vascularization were detected by CCK-8 assay, wound healing assay, and tube-like formation assay, respectively.ResultsEOCs were successfully extracted from umbilical cord blood and different concentrations of ADMA aggravated EOCs apoptosis. ADMA distinctly activates the phosphorylation activity of JNK. Supplementation of JNK-specific inhibitor (SP600125) decreased expression of Bax and cleaved caspase 3/9, and alleviated ADMA-induced apoptosis. SP600125 also promoted angiogenesis viability.ConclusionThe JNK pathway participates in the apoptosis-promoting process of EOCs, and targeted inhibition of the JNK pathway can alleviate ADMA-induced injury, which Is the potential underlying mechanism of vascular endothelium injury in ischemic stroke.

Effects of PM2.5 exposure on the Notch signaling pathway and immune imbalance in chronic obstructive pulmonary disease

Chronic Obstructive Pulmonary Disease (COPD) is associated with T lymphocytes subset (Th1/Th2, Th17/Treg) imbalance. Notch signaling pathway plays a key role in the development of the adaptive immunity. The immune disorder induced by fine particulate matter (PM2.5) is related to COPD. The aim of this study was to investigate the mechanism by which PM2.5 influences the Notch signaling pathway leading to worsening immune disorder and accelerating COPD development. A COPD mouse model was established by cigarette smoke exposure. PM2.5 exposure was performed by aerosol inhalation. γ-secretase inhibitor (GSI) was given using intraperitoneal injection. Splenic T lymphocytes were purified using a density gradient centrifugation method. CD4+ T lymphocyte subsets (Th1/Th2, Th17/Treg) were detected using flow cytometry. mRNA and proteins of Notch1/2/3/4, Hes1/5, and Hey1 were detected using RT-PCR and Western blot. Serum INF-γ, IL-4, IL-17 and IL-10 concentrations were measured using ELISA. The results showed that in COPD mice Th1% and Th17%, Th1/Th2 and Th17/Treg were increased, and the levels of mRNA and protein in Notch1/2/3/4, Hes1/5, and Hey1 and serum INF-γ and IL-17 concentrations were significantly increased, and Th2%, Treg%, and serum IL-4 and IL-10 concentrations were significantly decreased. COPD Mice have Th1- and Th17-mediated immune disorder, and the Notch signaling pathway is in an overactivated state. PM2.5 promotes the overactivation of the Notch signaling pathway and aggravates the immune disorder of COPD. GSI can partially inhibit the activation of the Notch signaling pathway and alleviate the immune disorder under basal state and the immune disorder of COPD caused by PM2.5. This result suggests that PM2.5 is involved in the immune disorder of mice with COPD by affecting the Notch signaling pathway and that PM2.5 aggravates COPD.

Protective effect and related mechanism of tanshinol borneol ester on homocysteine induced rat bone marrow mesenchymal stem cells damage

Objective: To investigate the protective effect and potential mechanism of tanshinol borneol ester (TBE) on homocysteine(Hcy) induced rat bone marrow mesenchymal stem cells (BMSCs) damage. Methods: BMSCs were isolated and cultured in vitro by density gradient centrifugation and adherent culture method. BMSCs were divided into the control (normal isolation and culture), TBE-1(10 μmol/L TBE-1 solution with 100 μl), TBE-2 (10 μmol/L TBE-2 solution with 100 μl), Hcy (0.5 mmol/L Hcy solution with 100 μl), Hcy + TBE-1(0.5 mmol/L Hcy solution with 100 μl, and 10 μmol/L TBE-1 solution with 100 μl), Hcy + TBE-2 (0.5 mmol/L Hcy solution with 100 μl, and 10 μmol/L TBE-2 solution with 100 μl), Hcy+ TBE-1+ inhibitor group(0.5 mmol/L Hcy solution with 100 μl, 10 μmol/L TBE-1 solution with 100 μl, and 25 μmol/L LY294002(specific blocker of phosphatidylinositol 3 kinase) solution with 100 μl), Hcy+ TBE-2+ inhibitor group(0.5 mmol/L Hcy solution with 100 μl, 10 μmol/L TBE-2 solution with 100 μl, and 25 μmol/L LY294002 solution with 100 μl). Cell proliferation activity was detected by MTT assay. The T-SOD activity and malonaldehyde level of cells were measured by anthineoxidase method and TBA method, respectively, to evaluate cell oxidative and antioxidative activities. The ultrastructure of cells was observed under transmission electron microscope. The expression level of PKB and NF-κB of cells in various groups were detected with the immunocytochemical method. Results: (1)Cell proliferation activity in TBE-1 group and TBE-2 group was significantly increased compared with the control group (both P<0.01), and was similar between TBE-1 group and TBE-2 groups after 1, 12, 24 and 48 hours treatment.(2)The T-SOD activity in TBE-1 group and TBE-2 group was significantly higher than in control group (both P<0.01), while it was significantly lower in Hcy group, Hcy+ TBE-1 group, and Hcy+ TBE-2 group than in control group(all P<0.01), and was similar between control group, Hcy+ TBE-1+ inhibitor group, and Hcy+ TBE-2+ inhibitor group(all P>0.05). The T-SOD activity was higher in Hcy+ TBE-1 group and Hcy+ TBE-2 group than in Hcy group(both P<0.01), and was higher in Hcy+ TBE-1+ inhibitor group than in Hcy+ TBE-1 group(P<0.05) and was higher in Hcy+ TBE-2+ inhibitor group than in Hcy+ TBE-2 group(P<0.05). The malonaldehyde level was lower in TBE-1 group and TBE-2 group than in control group(both P<0.01), was higher in Hcy group, Hcy+ TBE-1 group, Hcy+ TBE-2 group, Hcy+ TBE-1+ inhibitor group, and Hcy+ TBE-2+ inhibitor group than in control group(all P<0.01), was lower in Hcy+ TBE-1 group and Hcy+ TBE-2 group than in Hcy group(both P<0.01), was higher in Hcy+ TBE-1+ inhibitor group than in Hcy+ TBE-1 group(P<0.05), was higher in Hcy+ TBE-2+ inhibitor group than in Hcy+ TBE-2 group(P<0.05). (3)Under electron microscope, BMSCs showed profound swelling, senescence and apoptosis of cells increased significantly in Hcy group, Hcy+ TBE-1+ inhibitor group, and Hcy+ TBE-2+ inhibitor group when compared with control group. BMSCs in the TBE-1 and TBE-2 groups presented with abundant rough endoplasmic reticulum, and very active cell metabolism signs. Compared with Hcy group, BMSCs edema, the number of aging and apoptotic cells, and cell injury severity were significantly less in TBE-1+ Hcy group and TBE-2+ Hcy. (4)The PKB level was higher in TBE-1 group and TBE-2 group than in control group(both P<0.01), was lower in Hcy group, Hcy+ TBE-2 group, Hcy+ TBE-1+ inhibitor group, and Hcy+ TBE-2+ inhibitor group than in control group(all P<0.01), was similar between control group and Hcy+ TBE-1 group(P>0.05), was higher in Hcy+ TBE-1 group and Hcy+ TBE-2 group than in Hcy group(both P<0.05), was lower in Hcy+ TBE-1+ inhibitor group than in Hcy+ TBE-1 group(P<0.05), and was lower in Hcy+ TBE-2+ inhibitor group than in Hcy+ TBE-2 group(P<0.05). The NF-κB level was higher in TBE-1 group and TBE-2 group than in control group(both P<0.01), was lower in Hcy group, Hcy+ TBE-1 group, Hcy+ TBE-2 group, Hcy+ TBE-1+ inhibitor group, and Hcy+ TBE-2+ inhibitor group than in control group(P<0.01 or 0.05), was higher in Hcy+ TBE-1 group and Hcy+ TBE-2 group than in Hcy group(both P<0.05), was lower in Hcy+ TBE-1+ inhibitor group than in Hcy+ TBE-1 group(P<0.05) and was lower in Hcy+ TBE-2+ inhibitor group than in Hcy+ TBE-2 group(P<0.05). Conclusion: Tanshinol borneol ester can promote the proliferation of BMSC, and attenuate the homocysteine induced rat BMSCs damage possibly through activation of phosphatidylinositol 3 kinase/PKB signal transduction and its downstream signal pathway protein NF-κB.

Impact of CD137-CD137L signaling mediated exocytosis of autophagosome within vascular smooth muscle cells on the formation of atherosclerotic calcification

Objective: To explore whether CD137-CD137L signaling mediated exocytosis of autophagosome within vascular smooth muscle cells (VSMCs) could influence the formation of atherosclerotic calcification. Methods: Fifteen 8-week-old male ApoE(-/-)(C57BL/6J-KO) mice fed with high fat diet for 5 weeks were randomly divided into three groups by using stochastic indicator method as follows: control group, n=5; agonist-CD137 group: agonist-CD137 antibody 200 μg/2 weeks for 4 weeks, ip, n=5; anti-CD137 group: 200 μg anti-CD137 antibody+ 200 μg agonist-CD137 antibody/2 weeks for 4 weeks, ip, n=5. Von Kossa staining was applied to observe the calcification of the thoracic aortic atherosclerotic plaque in each group. Immunohistochemistry was used to detect the expression of LC3 and Beclin1 which were the autophage markers of early-to-mid stage; Western blot was adopted to quantify protein level of microtubule-associated proteins 1 light chain 3B(LC3B) and mammalian ortholog of the yeast autophagy-related gene 6 (Beclin1). Transmission electron microscope (TME) was used to observe the formation of autophagosome in plaque. C57BL/6J mouse VSMCs were cultured by using tissue piece inoculation method. Groups of in vitro studies were the same as in vivo study: control group, agonist-CD137 group, anti-CD137 group, the agonist-CD137 groups was treated with agonist-CD137 antibody (10 μg/ml) and anti-CD137 group was treated with anti-CD137 antibody (10 μg/ml) for 30 minutes, followed by agonist-CD137 antibody (10 μg/ml). Von Kossa staining and osteogenesis phenotypic alkaline phosphatase (ALP) activity detection were adopted to observe calcification in VSMCs. Autophagosomes were separated from the supernatant of the agonist-CD137 group with density gradient centrifugation method. VSMCs were divided into two groups: positive group (containing complete medium with above autophagosomes to a final concentration 15 μg/ml) and negative group (only complete medium) after being pretreated with mixed inflammatory cytokines (IL-1β、IFN-γ and TNF-α, final concentration was 25 ng/ml respectively) for 24 hours and calcium deposition and osteogenesis phenotypic marker bone morphogenetic protein 2(BMP2) were then detected. Results: (1) Compared with the control group, activation of the CD137-CD137L signal significantly increased the formation of calcification area in thoracic aortic atherosclerotic plaque of ApoE(-/-) mice((1.82±0.15)×10(4) μm(2) vs. (0.34±0.08)×10(4) μm(2,) P<0.01), this effect was significantly attenuated by inhibiting this signal ((0.83±0.30)×10(4) μm(2) vs. (1.82±0.15)×10(4) μm(2,) P<0.05); positive autophagy makers LC3B and Beclin1 were detected in both agonist-CD137 group and anti-CD137 groups and the expression of LC3B and Beclin1 was substantially higher in anti-CD137 group. Western blot analysis indicated that the expression of LC3B and Beclin1 in agonist-CD137 group was significantly upregulated compared with the control group (0.17±0.01 vs. 0.03±0.08, P<0.05, and 0.12±0.02 vs. 0.06±0.02, P<0.05), which could be significantly downregulated in anti-CD137 group (0.28±0.09 vs. 0.17±0.01, P<0.05 and 0.17±0.02 vs. 0.12±0.02, P<0.05). TME showed that the number (QTY /HP) of autophagosome of agonist-CD137 group and anti-CD137 group in plaque were both increased (14.67±2.52 vs. 3.67±1.53, P<0.01, and 15.33±2.08 vs. 3.67±1.53, P<0.01), while in the agonist-CD137 group, the number of extracellular autophagosome within thoracic aortic atherosclerotic plaque of ApoE(-/-) mice increased more substantially (5.33±1.53 vs. 1.33±0.58, P<0.01). (2) In vitro study showed that activating CD137-CD137L signal could promote calcium deposition in extracellular matrix and the activity of osteogenesis phenotypic ALP((6.73±0.02) μmol/mg protein vs. (1.07±0.03) μmol/mg protein, P<0.05), and ((563.20±0.72) U/mg protein vs. (117.50±0.64) U/mg protein, P<0.05), while these effects were significantly blunted in anti-CD137 group ((1.94±0.05) μmol/mg protein vs. (6.73±0.02) μmol/mg protein, P<0.05, and (236.10±0.14) U/mg protein vs. (563.20±0.72) U/mg protein, P<0.05). TME showed that the number of intracellular autophagosome in agonist-CD137 group and anti-CD137 group was both significantly higher than in control group ((21.65±1.34) μg/ml vs. (8.32±1.58) μg/ml, P<0.01, and (15.42±1.65) μg/ml vs. (8.32±1.58) μg/ml, P<0.05). After the density gradient centrifugation, exocytotic autophagosome in the medium of agonist-CD137 group was markedly higher than in control group ((14.67±1.53) μg/ml vs. (2.33±1.15) μg/ml, P<0.01). (3) Compared with the control group, autophagosomes isolated from culture supernatant (final concentration: 15 μg/ml) could significantly stimulate calcium deposition((2.30±0.10) μmol/mg protein vs. (0.15±0.40) μmol/mg protein, P<0.05) and enhance the expression of bone morphogenetic protein 2 (2.10±0.04 vs. 0.30±0.01, P<0.05). Conclusion: CD137-CD137L signaling could mediate exocytosis of autophagosome within VSMCs, thus influence the formation of atherosclerotic calcification.

Correlative study on the JAK-STAT/PSMβ3 signal transduction pathway in asthenozoospermia.

The aim of the present study was to investigate the possible mechanism of Janus kinase (JAK)-signal transduction and activator of transcription (STAT)/PSMβ3 signaling in the occurrence of asthenozoospermia. We examined seminal fluid samples from 30 cases of asthenozoospermia and 30 healthy controls. Sperm was collected using the Percoll density gradient centrifugation method. The expression of JAK, STAT and PSMβ3 mRNA was assessed by reverse-transcription quantitative PCR and the protein levels of p-JAK, p-STAT and PSMβ3 were measured by western blot analysis. The PSMβ3 mRNA and protein expression levels were also measured after application of a JAK inhibitor, AG-490, to the control group, with a FITC-labeled monoclonal rabbit anti-human PSMβ3 primary antibody. The cells were observed under a laser confocal microscope. The mRNA levels of JAK, STAT and PSMβ3 in asthenozoospermia were decreased significantly (P<0.05). The protein levels of p-JAK, p-STAT and PSMβ3 in asthenozoospermia were also reduced and the differences were statistically significant (P<0.05). The PSMβ3 mRNA and protein expression levels were decreased in the control group after treatment with the JAK inhibitor, and levels were approximately equal to those of the asthenozoospermia group. PSMβ3 was mainly expressed in round-headed sperm, and less in asthenozoospermia. In conclusion, the JAK-STAT/PSMβ3 signaling transduction pathway may be involved in the pathogenic mechanism of asthenozoospermia.

Late Adherent Subpopulation in Umbilical Cord Blood Has the Same Characteristics and Hematopoiesis-Supporting Capacity As Mesenchymal Stromal/Stem Cells

Mesenchymal stromal/stem cells (MSCs) are a major source of cell for cell therapy. MSCs derived from bone marrow (BMMSCs) have been mostly used in clinical applications. BMMSCs can be easily isolated as a cell population that adheres to plastic culture dishes within 1 week of culture. A recent report has demonstrated that cells that remain in suspension and fail to form adherent colonies contain a fraction of late adherent cells that resembles BMMSCs (Biomed Res Int, 2013; 2013: 790842). Umbilical cord blood (UCB) is as accessible as bone marrow for the isolation of MSCs. In this study, we identified a late adherent subpopulation in UCB and determined its hematopoiesis-supporting activity.Forty-five UCB units, which were not matched to the eligibility criterion defined in the Japan UCB donation program, were collected after delivery of placenta. Written informed consent was obtained before delivery from all pregnant women who participated in the study. The study protocol was approved by the ethics committee of the Kyoto University Graduate School of Medicine.Mononuclear cells were isolated from UCB by the density gradient centrifugation method with (n = 19) and without (n = 18) subsequent separation of CD34 negative cells using anti-CD34 immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Nucleated cells were separated by the hydroxyethyl starch sedimentation method from the other eight UCB units. The cells were then seeded into a culture flask and cultured in alpha minimal essential medium supplemented with 15% FBS (Culture 1; C1). After 1 week of culture, non-adherent cells in C1 supernatant were collected and re-seeded into a new flask (C2). The attached cells in C1 were cultured until adherent colonies emerged, after which they were detached using trypsin/EDTA and twice passaged to obtain a sufficient number of cells (C1 cells). In the same way, after 1 week of culture, non-adherent cells in C2 supernatant were collected and re-seeded into a new flask (C3). The attached cells in C2 were cultured to obtain C2 cells. Afterwards, re-seeding and culture (C4, C5c) were repeated until no new colonies were formed. Collected cells were cryopreserved and thawed when required in experiments. BMMSCs were isolated from human bone marrow cells purchased from AllCells (Emeryville, CA).C1 cells, the so-called UCBMSCs, were successfully isolated from 18 units (40 %). Adherent cells isolated from C2 and later were defined as elate adherent cellsf and, were obtained from 9 units: these cells were referred to as C2 cells (from 9 units), C3 cells (from 9 units), C4 cells (from 6 units) and C5 cells (from 2 units). The interval from seeding to the first colony formation in C1 was shorter in these 9 units than that in the other 9 units that contained only C1 cells: 10.8 } 1.4 vs 15.9 } 4.5 days, p < 0.01. The volume of the former 9 units tended to be large compared to the latter 9 units: 49.6 } 10.5 vs 33.7 } 21.0 mL, p = 0.07. These findings indicated that UCB containing late adherent cells was suitable for a cell source of MSCs. Next, we examined whether these late adherent cells (C2 and C3 cells) had properties consistent with those of MSCs. Both C2 and C3 cells showed spindle-shaped fibroblast-like morphology and the same immunophenotype as C1 cells: positive for CD73, CD90 and CD105, and negative for CD34, CD45 and HLA-DR. They had osteogenic, adipogenic and chondrogenic differentiation potentials in vitro. These findings are the minimal criteria for MSCs (Cytotherapy, 2006; 8:315). Finally, we evaluated the hematopoiesis-supporting activity of these cells in vitro and in vivo. CD45-positive hematopoietic cells were expanded when co-cultured of CD34-positive hematopoietic progenitor cells (6 ~ 102 cells) with C2 or C3 cells (2 ~ 104 cells) in vitro as much as when co-cultured with C1 cells (Figure A). In vivo analysis was conducted by using subcutaneous transplantation of MSCs on NOD/SCID mice (Int J Hematol, 2015; 102: 218). C2 cells induced trabecular bone formation and bone marrow hematopoiesis as well as C1 cells, however, C3 cells did not induce hematopoiesis (Figure B).In conclusion, we demonstrated that UCB contains a late adherent cell subpopulation with the same characteristics and hematopoiesis-supporting activity as those of UCBMSCs isolated using the conventional method. The continuance of cell culture without discarding suspension cells could improve the efficiency of isolation of MSCs from UCB. [Display omitted] DisclosuresHirai:Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding. Maekawa:Bristol-Myers K.K.: Research Funding.

Characterization of Outer Membrane Proteome of Akkermansia muciniphila Reveals Sets of Novel Proteins Exposed to the Human Intestine.

Akkermansia muciniphila is a common member of the human gut microbiota and belongs to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum. Decreased levels of A. muciniphila have been associated with many diseases, and thus it is considered to be a beneficial resident of the intestinal mucus layer. Surface-exposed molecules produced by this organism likely play important roles in colonization and communication with other microbes and the host, but the protein composition of the outer membrane (OM) has not been characterized thus far. Herein we set out to identify and characterize A. muciniphila proteins using an integrated approach of proteomics and computational analysis. Sarkosyl extraction and sucrose density-gradient centrifugation methods were used to enrich and fractionate the OM proteome of A. muciniphila. Proteins from these fractions were identified by LC-MS/MS and candidates for OM proteins derived from the experimental approach were subjected to computational screening to verify their location in the cell. In total we identified 79 putative OM and membrane-associated extracellular proteins, and 23 of those were found to differ in abundance between cells of A. muciniphila grown on the natural substrate, mucin, and those grown on the non-mucus sugar, glucose. The identified OM proteins included highly abundant proteins involved in secretion and transport, as well as proteins predicted to take part in formation of the pili-like structures observed in A. muciniphila. The most abundant OM protein was a 95-kD protein, termed PilQ, annotated as a type IV pili secretin and predicted to be involved in the production of pili in A. muciniphila. To verify its location we purified the His-Tag labeled N-terminal domain of PilQ and generated rabbit polyclonal antibodies. Immunoelectron microscopy of thin sections immunolabeled with these antibodies demonstrated the OM localization of PilQ, testifying for its predicted function as a type IV pili secretin in A. muciniphila. As pili structures are known to be involved in the modulation of host immune responses, this provides support for the involvement of OM proteins in the host interaction of A. muciniphila. In conclusion, the characterization of A. muciniphila OM proteome provides valuable information that can be used for further functional and immunological studies.

Zeta Sperm Selection Improves Pregnancy Rate and Alters Sex Ratio in Male Factor Infertility Patients: A Double-Blind, Randomized Clinical Trial.

Selection of sperm for intra-cytoplasmic sperm injection (ICSI) is usually considered as the ultimate technique to alleviate male-factor infertility. In routine ICSI, selection is based on morphology and viability which does not necessarily preclude the chance injection of DNA-damaged or apoptotic sperm into the oocyte. Sperm with high negative surface electrical charge, named "Zeta potential", are mature and more likely to have intact chromatin. In addition, X-bearing spermatozoa carry more negative charge. Therefore, we aimed to compare the clinical outcomes of Zeta procedure with routine sperm selection in infertile men candidate for ICSI. From a total of 203 ICSI cycles studied, 101 cycles were allocated to density gradient centrifugation (DGC)/Zeta group and the remaining 102 were included in the DGC group in this prospective study. Clinical outcomes were com- pared between the two groups. The ratios of Xand Y bearing sperm were assessed by fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) methods in 17 independent semen samples. In the present double-blind randomized clinical trial, a significant increase in top quality embryos and pregnancy rate were observed in DGC/Zeta group compared to DGC group. Moreover, sex ratio (XY/XX) at birth significantly was lower in the DGC/Zeta group compared to DGC group despite similar ratio of X/Y bearings sper- matozoa following Zeta selection. Zeta method not only improves the percentage of top embryo quality and pregnancy outcome but also alters the sex ratio compared to the conventional DGC method, despite no significant change in the ratio of Xand Ybearing sperm population (Registration number: IRCT201108047223N1).