Humidified Incubator Research Articles

The Antineuroinflammatory Effect of Simvastatin on Lipopolysaccharide Activated Microglial Cells.

Microglial cells, upon hyperactivation, produce proinflammatory cytokines and other oxidative stress mediators causing neuroinflammation, which is associated with the progress of many neurodegenerative diseases. Suppressing the microglial activation has hence been used as an approach for treating such diseases. In this study, the antineuroinflammatory effect of simvastatin was examined in lipopolysaccharide (LPS)-activated rat C6 glioma cells. The cell proliferation and cytotoxic effect of LPS and simvastatin on C6 glioma cells was evaluated by (MTT) assay. Neuroinflammation was induced in differentiated cell lines by treatment with 3.125 μg/mL of LPS for 12 h. Upon induction, the cell lines were treated with different concentrations (3.125, 6.25, 12.5, 25, 50, 100 μM) of simvastatin and incubated in a humidified CO2 incubator for 24 to 48 h. The optimum concentrations of LPS and simvastatin were found to be 3.125 μg/mL and 25 μM, respectively, with a cell viability of more than 90% at 24 h postincubation. Furthermore, proinflammatory marker expression was analyzed by flow cytometry and showed a decrease in interferon-γ, interleukin 6, nuclear factor-κB p65, and tumor necrosis factor-α in simvastatin-treated and LPS-induced neuroinflammatory cells, and the mean fluorescent values were found to be 21.75 ± 0.76, 20.9 ± 1.90, 19.72 ± 1.29, and 16.82 ± 0.97, respectively, as compared to the untreated cells. Thus, we show that simvastatin has the potential to regulate the anti-inflammatory response in microglial cells upon LPS challenge. Hence, simvastatin can be employed as a potent anti-inflammatory drug against neuroinflammatory diseases and neurodegenerative disorders.

CYTOTOXIC LABDANE DITERPENOIDS FROM ANDROGRAPHIS PANICULATA (BURM.F.) NEES

Objective: The primary objective of this study was to probe the cytotoxic capacity of the labdane diterpenoids andrographolide (1), 14-deoxyandrographolide (2), 14-deoxy-12-hydroxyandrographolide (3), and neoandrographolide (4) on mutant and wild-type immortalized cell lines.Methods: Breast adenocarcinoma (MCF-7), colon carcinomas (HCT-116 and HT-29), small cell lung carcinoma (H69PR), human acute monocytic leukemia (THP-1), and wild-type primary normal human dermal fibroblasts - neonatal cells (HDFn) were incubated with 1-4, and the degree of cytotoxicity was analyzed by employing the in vitro PrestoBlue® cell viability assay. Working solutions of 1-4 were prepared in complete cell culture medium to a final non-toxic dimethyl sulfoxide concentration of 0.2%. The plates were incubated at 37°C with 5% CO2 in a 98% humidified incubator throughout the assay. Nonlinear regression and statistical analyses were done to extrapolate the half maximal inhibitory concentration 50% (IC50). One-way ANOVA (p<0.05) and multiple comparison, Tukey’s post hoc test (p<0.05), were used to compare different pairs of data sets. Results were considered statistically significant at p<0.05.Results: The highest cytotoxicity index was exhibited by the H69PR and 1 trials which displayed the lowest IC50 value of 3.66 μg/mL, followed by HT-29 treated with 2, HCT-116 and 1 trials, and H69PR treated with 4 (IC50=3.81, 3.82, and 4.19 μg/mL, respectively). Only 1 and 4 were detrimental toward MCF-7, while 1, 3, and 4 were degenerative against H69PR. Tukey’s post hoc multiple comparison indicated no significant difference in the cytotoxicity of 1-4 on HCT-116 cells which afforded IC50 values ranging from 3.82 to 5.12 μg/mL. Evaluation of the two colon carcinoma cell lines showed that HCT-116 was categorically more susceptible to cellular damage caused by treatments with 1-4 than was HT-29. Cytotoxicity was not detected in THP-1 and HDFn cells (IC50>100 μg/mL).Conclusion: Diterpenoids 1-4 isolated from the dichloromethane extract of the leaves of A. paniculata exhibited different cytotoxic activities against MCF-7, HCT-116, HT-29, and H69PR. All constituents had comparable action on HCT-116 cells but were not found to be cytotoxic to normal HDFn cells and mutant THP-1 cells.

In vitro progesterone production by luteinized human mural granulosa cells is modulated by activation of AMPK and cause of infertility

BackgroundMural granulosa cells from IVF patients were provided by the West Virginia University Center for Reproductive Medicine in Morgantown, WV. The effect of adenosine monophosphate activated protein kinase (AMPK) activation, primary cause of infertility, age, BMI, and pregnancy outcome on production of progesterone were examined separately.MethodsIsolated mural sheets from IVF patients (n = 26) were centrifuged, supernatant discarded, and the pellet re-suspended in 500 μl of DMEM/F12. Mural granulosa cells were plated at 10,000 cells/well in triplicate per treatment group with 300 μl DMEM/F12 media at 37 °C and 5% CO2 in a humidified incubator to permit luteinization. Four days after initial plating, cells were treated with either an AMPK inhibitor, DM; an AMPK activator, AICAR; or hCG. Cells were cultured for 24 h after treatment when medium was collected and frozen at −20 °C until assayed for P4 by radioimmunoassay.ResultsThe AMPK activator, AICAR, inhibited P4 production (P < 0.001), whereas the AMPK inhibitor, DM, did not affect basal P4 (P < 0.05). Progesterone production increased when cells from patients whose primary cause of infertility was a partner having male infertility were treated with hCG compared to control (P = 0.0045), but not in patients with other primary infertility factors (P > 0.05). Additionally, hCG increased P4 production in patients between the ages 30–35 (P = 0.008) and 36–39 (P = 0.04), but not in patients ages 25–29 (P = 0.73). Patients with normal BMI had increased P4 production when treated with hCG (P < 0.0001), however there was no change in P4 production from cells of patients who were overweight or obese (P > 0.05). Cells from patients who became pregnant to IVF had greater P4 production when stimulated with hCG than those who did not become pregnant when compared to controls (P > 0.05).ConclusionsUnderstanding how AMPK activation is regulated in ovarian cells could lead to alternative or novel infertility treatments. Human mural granulosa cells can serve as a valuable model for understanding how AMPK affects P4 production in steroidogenic cells. Additionally, when stimulated with hCG, P4 production by mural granulosa cells differed among infertility type, age, BMI, and pregnancy outcome.

ID: 1062 The chicken egg tumor model provides an attractive system for evaluating anticancer drugs

Among various tumor models, the chicken egg tumor model provides an attractive system for tumor characterization as well as for characterization of drugs and nanoparticles targeting the tumor. Xenograft tumors can be grown rapidly on a highly vascularized structure known as the chorioallantoic membrane (CAM). In this poster, we describe a method for growing tumors on the CAM membrane. Freshly fertilized chicken eggs are incubated in the rotary humidified incubator. On day 10 of the embryo development, the CAM is detached from the inner eggshell, a window is made in the shell to expose the CAM, a Teflon ring is place above the blood vessel, and cancer cells are grafted and placed inside the ring. Three days post inoculation, solid tumors are ~5mm in diameter and can easily be observed through the shell window. We have used green fluorescent ovarian cancer cells to follow tumor growth. H&amp;E staining of the tumor established by transplanting human ovarian cancer cells showed that the tumor established in the chicken egg closely resembles tumors from ovarian cancer patients. Intravenous injection of doxorubicin led to a dramatic inhibition of tumor growth without affecting egg overall survival, as organs obtained after the doxorubicin treatment exhibited normal appearance. Our study shows that the CAM assay is a promising model to evaluate tumor targeting and bio-distribution of nanoparticles

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Humid versus dry incubator: a prospective, randomized, controlled trial

To evaluate the efficacy of a dry versus humidified incubator on human embryo development exvivo. Prospective, double-blind, randomized, controlled trial. Private fertility centers. A total of 297 women undergoing invitro fertilization randomized into two groups. From days 0 to day 5 or 6 of culture, intervention group embryos exposed to dry culture and control group embryos exposed to humidified culture. Subsequent ongoing pregnancy rate. After transfer of embryos, there were statistically significantly lower rates of clinical and ongoing pregnancy in the dry culture arm than in the humidified culture arm (odds ratio [OR] 0.57; 95% confidence interval [CI], 0.36-0.91; versus OR 0.54; 95% CI, 0.34-0.85). On day 3 of culture, embryo quality and compaction were lower in the dry culture group (OR 0.38; 95% CI, 0.32-0.45) than in the group exposed to humidified culture (OR 0.23; 95% CI, 0.19-0.27). On day 5 of culture, embryos in dry culture had a lower rate of blastocyst formation (OR 0.39; 95% CI, 0.33-0.46), quality (OR 0.34; 95% CI, 0.29-0.40), and cryopreservation (OR 0.41; 95% CI, 0.35-0.48). In this study, human embryos cultivated exvivo in a dry incubator had statistically significantly decreased implantation and clinical and ongoing pregnancy rates. Our findings indicate the need for larger multicenter, randomized, controlled trials. NCT01695096.

The Effect of Histone Hyperacetylation on Viability of Basal-Like Breast Cancer Cells MDA-MB-231

Abstract Background: The Basal-Like breast cancer, is always known for lack of expression of estrogen receptor (ER), progesterone receptor (PR) and as well, absence of epidermal growth factor receptor 2 (HER2) gene amplification. Improper expression pattern of ER, PR, and Her2, makes Basal-Like breast tumors resistant to the current hormonal and anti HER2 treatments. In recent decades, several studies have been conducted to investigate the regulatory role of chemical modifications of core histones in gene expression. Their results have shown that histone acetylation is involved in regulation of cell survival. Acetylation of core histones is regulated by the epigenetic-modifying enzymes named Histone Deacetylases (HDACs). As a new approach to control the viability of breast tumor cells resistant to the hormonal and anti-HER2 treatments, we have targeted the HDACs. Using Trichostatin A (TSA) as a known HDACs inhibitor, we have tried to hyperacetylate the core histones of MDA-MB-231 cells as an in vitro model of Basal-Like breast tumors. Then we have investigated the effect of histone hyperacetylation on viability of MDA-MB-231 cells. Methods: MDA-MB-231 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and were incubated at 37°C, in a humidified incubator with 5% CO2 atmosphere. Then cells were treated with different concentrations of TSA including: 50, 100, 200, 400, 800 and 1000 nM or control (1% DMSO). After 24 and 48 hours, viability of cells was evaluated by MTT assay. Results: After 24 and 48h exposure to different concentrations of TSA, MDA-MB-231 cells showed a maximum tolerable dose. At higher concentrations, TSA decreased the percentage of cell viability through a time-dose dependent manner. IC50 value for 48h treatment was 600 nM. Conclusions: Our results indicate that HDACs inhibition and subsequently hyperacetylation of histones, leads to cytotoxic effects on breast tumor cells resistant to the current treatments. Following this pilot research we are trying to suggest molecular mechanisms underlying the anti-proliferative effects triggered by HDACs inhibition.

Human group 2 innate lymphoid cells do not express the IL-5 receptor

This work was part funded by (for CEB) Airway Disease Predicting Outcomes through Patient Specific Computational Modelling (AirPROM) project (funded through FP7 EU grant), Wellcome Senior Fellowship, (for DJC) Medical Research Council (MRC) and Asthma UK (Centre Grant: G1000758), from the NIHR Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London, an equipment grant from the Midlands Asthma and Allergy Research Association (MAARA) and (for CEB and DJC) the National Institute for Health Research (NIHR) Leicester Respiratory Biomedical Unit. BMJR was funded by an MRC and Asthma UK PhD studentship. This paper presents independent research funded by the NIHR.

The effects of membrane cholesterol on the mechanics of vascular smooth muscle cell

As the major cellular component of blood vessel wall, vascular smooth muscle cell (VSMC) plays important roles at each stage of atherosclerosis by proliferation and migration toward the inner layer of blood vessel wall. It is also known that cholesterol is not only the major factor involved in fatty deposition in the atherosclerotic lesion, but also plays an important role as a regulator of cell mechanics, spreading, and migration. A further complication is the mechanical environment of the vascular wall, which undergoes progressive stiffening with aging and hypertension and thus accelerates development of atherosclerosis. Therefore, it is important to investigate the combined effect of cholesterol and mechanical environment on VSMC and determine how it alters VSMC functional characteristics such as cell stiffness and adhesion. VSMCs used in this study were enzymatically isolated from the descending aorta of male Sprague‐Dawley rats and cultured in DMEM with 10% FBS in a humidified incubator at 37°C and 5% CO2. Cell adhesion was measured for 50–70% confluent cells with an atomic force microscope (AFM) system and the fibronectin (FN) or N‐cadherin coated stylus AFM probes at 100 nm/sec retraction speed. Cell stiffness was tested with spherical AFM probe (5μm diameter) at 1 μm/sec indentation speed. Cells were treated with 5mM Methyl‐β‐cyclodextrin for 1 h for cholesterol depletion prior to the AFM experiment. Our results showed that cholesterol depletion significantly increased N‐cadherin mediated cell adhesion by ~20% and adhesion probability by ~35%, respectively. While α5β1/FN adhesion force significantly decreased upon membrane cholesterol depletion. Moreover, our results suggested that cholesterol depletion significantly reduced VSMCs stiffness by 47% compared to control cells. All together, these results reveal that membrane cholesterol is a key regulator for cellular stiffness and integrin/cadherin‐mediated cell adhesion. The knowledge that we obtained in this project may lead to a novel therapeutic strategy that could potentially control and block VSMC migration and prevent the formation of atherosclerotic plaque by regulating integrin and/or cadherin mediated cell adhesion.Support or Funding Information15SDG25420001 (AHA)

Effect of levamisole on alkaline phosphatase activity and immunoglobulin secretion of lipopolysaccharide-stimulated murine splenic lymphocytes

Background: Alkaline phosphatases (APase) are a group of enzymes whose activity increases above normal levels in conditions of diseases such as cancer. In the present study, the role of APase using mitogen-stimulated murine lymphocytes was investigated. Methods: U266B1 cells and RPMI 8226 cultures were kept at 37°C in a humidified incubator with 5% CO 2. The cultures were pulsed with 0.5 μCi of 3 H-thymidine and were harvested onto glass fiber filter using Skatron automatic cell harvester. The dried filters were transferred into toluene-based scintillation cocktail, and the radioactivity was measured using Beckman scintillation counter. APase activity was determined by p-nitrophenol phosphate hydrolysis. The intracellular immunoglobulin E (IgE) content was quantified by western blot assay. U266 B1 and RPMI 8226 were cultured at 0.25 × 10 6 /ml with and without 1 mM levamisole for 48 h, and 15 ml of the culture supernatant was collected and lyophilized. Electrophoresis was carried out on 30 μl of the dialyzed samples. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and the gels were silver stained. Results: APase may be involved in the constitutive proliferation as well as in Ig secretion of myeloma cells. It was observed that murine splenic lymphocytes showed an increase in proliferative response concomitant with an increase in the APase activity and Ig secretion upon mitogenic stimulation (0.5–2.5 mM, P > 0.05). Conclusion: Levamisole significantly inhibited the APase activity when added to the lipopolysaccharide-stimulated cells at the initiation of the culture. Significance of the present study is discussed in the light of existing literature.

In Vitro Cytotoxicity and Setting Time Assessment of Calcium-Enriched Mixture Cement, Retro Mineral Trioxide Aggregate and Mineral Trioxide Aggregate.

Introduction:The present study sought to evaluate and compare biocompatibility and setting time of Retro mineral trioxide aggregate (MTA), calcium-enriched mixture (CEM) and Angelus MTA. Methods and Materials:CEM cement, Angelus MTA and Retro MTA were assessed in set and fresh states. Extracts transformed to each cavity of three 24-well plates in which 1×104 cell were seeded into each well 24 h earlier. All specimens were incubated in a humidified incubator with 5% CO2 at 37°C. Mosmann’s tetrazolium toxicity (MTT) assay was used to determine in vitro cytotoxicity on L929 mouse fibroblast cell line. Cell viability was determined at 1, 24, and 72 h after exposure. The initial setting time was measured by 113.4 g Gilmore needle testing. Then, final setting times were assessed by the 456.5 g Gilmore needle. Data comparisons were performed using the analysis of variance (ANOVA) and Tukey's post hoc test (α=0.05). Results:All groups in both forms indicated higher cell vitality compared to positive control group (P<0.001). After 24 h, the set Retro MTA showed better biocompatibility compared to set CEM and set Angelus MTA (P<0.001). Retro MTA showed significantly lower initial and final setting time compared to CEM and Angelus MTA (P<0.001). Conclusion:Our results indicated the good cell viability values of Retro MTA and relatively short period of setting time. It seems a promising alternative material in clinical situations where accelerated setting is required. However, more clinical and in vivo investigations are needed for a clear decision making.

Cytotoxicity of Selected Nanoparticles on Human Dental Pulp Stem Cells.

Introduction:Nanoparticles are being increasingly applied in dentistry due to their antimicrobial and mechanical properties. This in vitro study aimed to assess and compare the cytotoxicity of four metal oxide nanoparticles (TiO2, SiO2, ZnO, and Al2O3) on human dental pulp stem cells. Methods and Materials:Four suspension with different concentrations (25, 50, 75, 100 µg/mL) of each nanoparticle were prepared and placed into cavities of three 96-well plates (containing 1×104 cells per well that were seeded 24 earlier). All specimens were incubated in a humidified incubator with 5% CO2 at 37°C. Mosmann’s Tetrazolium Toxicity (MTT) assay was used to determine in vitro cytotoxicity of test materials on pulpal stem cells. Cell viability was determined at 24, 48, and 72 h after exposure. Data comparisons were performed using a general linear model for repeated measures and Tukey's post hoc test. The level of significance was set at 0.05. Results:The tested nanoparticles showed variable levels of cytotoxicity and were dose and time dependant. The minimum cell viability was observed in ZnO followed by TiO2, SiO2 and Al2O3. Conclusion:The results demonstrated that cell viability and morphological modifications occurred at the concentration range of 25 to 100 µg/mL and in all nanoparticles. The higher concentration and longer duration of exposure increased cellular death. Our results highlight the need for a more discrete use of nanoparticles for biomedical applications.

151 EFFECT OF ASCORBIC ACID ON OXIDATIVE STRESS AND ITS THERMOPROTECTANT ROLE ON IN VITRO EMBRYONIC DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) EMBRYOS

The most important factors that lead to stress in farm animals are oxidative and thermal stress, leading to reduced reproductive efficiency. Oxidative stress leads to an increase in proportion of reactive oxygen species, whereas heat stress affects the physiology of animals, which lowers the conception rates of dairy cattle. In vitro culture systems have been enhanced by manipulating media with various supplements such as vitamins, growth factors, and antioxidants that have overcome these problems. Ascorbic acid has been shown to play an antioxidant role in many species such as sheep, goat, and pigs. Keeping this in mind, this study was conducted to investigate the effect of supplementation of in vitro-matured (IVM) and/or in vitro-cultured (IVC) media with ascorbic acid and evaluate its antioxidant role in in vitro development of buffalo embryos. Immature oocytes were collected from visible surface follicles (2 to 8 mm in diameter) in slaughterhouse buffalo ovaries and subjected to IVM, IVF, and IVC in a humidified CO2 incubator at 38.5°C. Ascorbic acid was supplemented to IVM [TCM-199 + 10% featl bovine serum (FBS) + 1 µg mL−1 oestradiol-17β + 5 µg mL−1 pFSH + 0.81 mM sodium pyruvate + 0.68 mM l-glutamine + 50 µg mL−1 gentamicin sulfate] at 50 or 100 µM or IVC (mCR2aa + 0.6% BSA + 10% FBS+ 50 µg mL−1 gentamicin sulfate) at 50 µM or both IVM and IVC media at 50 µM. Oocytes without ascorbic acid were treated as the control group. Cleavage and blastocyst rate was improved when 50 µM (66.67 ± 2.27; 16.67 ± 1.26%) ascorbic acid was supplemented to IVM medium but no significant difference (P &lt; 0.05) was observed at 100 µM (54.04 ± 2.20; 6.16 ± 0.37%) as compared with the control (62.77 ± 2.71; 10.67 ± 0.24%), respectively. When 50 µM ascorbic acid was supplemented in IVM, IVC, or both media, though cleavage rate (66.67 ± 2.27; 69.09 ± 3.22; 66.67 ± 2.23%) was similar in 3 groups, a significant increase was observed in blastocyst rate (16.67 ± 1.26; 20.18 ± 0.86; 28.57 ± 0.37%) when both media were supplemented, respectively. To evaluate the thermoprotectant effect, 4 groups were taken: group 1 without and group 2 with ascorbic acid supplementation, oocytes were given heat treatment at 39.5°C initially for 12 h during IVM; group 3 without and group 4 with ascorbic acid supplementation, oocytes were given heat treatment at 40.5°C initially for 12 h during IVM. No significant difference in developmental rate was observed at elevated temperature of 39.5°C or 40.5°C as compared with the control. Relative mRNA abundance of heat stress-related genes, HSP 70.1 and HSP 70.2, was nonsignificantly higher in oocytes matured at 39.5°C or 40.5°C after supplementation with ascorbic acid as compared to control. Relative mRNA abundance of BAX decreased at 50 µM and increased at 100 µM ascorbic acid compared with control, whereas BID showed similar results between control and treatment. Regarding anti-apoptotic gene expression, significantly higher expression was observed in MCL1 for 50 µM and lower for 100 µM ascorbic acid, and a similar nonsignificant trend was observed for BCL-XL. Developmental genes GDF9 and BMP15 showed a nonsignificant increase in 50 µM, and a nonsignificant decrease in the 100 µM supplemented group as compared with the control. Oxidative stress-related genes SOD and GPX showed a nonsignificant decrease in treated groups as compared to control. From above results, it was concluded that ascorbic acid had an anti-oxidant as well as thermoprotectant role in developmental competence that increased the potential for generation of large domestic animal in vitro embryos for research and applied technologies such as cloning and transgenesis.

EVALUATION OF RENAL EPITHELIAL CELL PROTEIN UNDER STRESS CONDITION

Objective: Proteins are an important component of cells which are involved in various cellular functions. Different kind of stressing conditions has different responses in the components of the protein synthesis system. Super saturation condition in kidney environment leads to crystallization process. Crystals thus formed injure the surrounding cells and result in reactive oxygen species (ROS) formation. There might be some changes in the protein synthesis when the kidney cells enter in oxidative stress. In the present study, kidney cell lines were exposed to oxidative stress and their proteins were analyzed using Bradford analysis and SDS-PAGE.Methods: Vero cells were obtained from NCCS Pune and cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) and maintained in a humidified incubator at 37 °C with 5% CO2. Calcium phosphate (CaP) crystals were prepared by the homogeneous system. After FTIR analysis crystals were used to injure Vero cell line. H2O2 was also used to injure the Vero cells. Intracellular protein was extracted from healthy cells and injured cells (with CaP crystals and H2O2). Ammonium sulfate precipitation method was used for the isolation of extracellular protein from the media of healthy and injured cells. Bradford method was used for the quantitative estimation of protein. Extracted proteins were analyzed by SDS-PAGE.Results: Amount of intracellular and extracellular protein of normal cells was 4.84±0.004µg/ml. Intracellular protein of CaP injured and H2O2 injured cells were 10.59±0.003 µg/ml and 10.78±0.011µg/ml respectively. While extracellular protein of injured cells was nearly 4 µg/ml. Intracellular protein bands ranging from 14.3 to 97.4 kDa was observed in healthy cells. Protein bands of ~40kDa and ~20kDa was absent in H2O2 and CaP injured intracellular protein extract. Two extracellular protein bands of 66kDa and ~60kDa were present in injured cells and healthy cells. Conclusion: When exposed to oxidative stress several proteins are oxidized decreasing the activity of many metabolic pathways. In the present study amount of intracellular protein increases when cells are injured with CaP or H2O2. While extracellular protein remains more or less same in both healthy and injured condition of cells. In SDS-PAGE analysis few bands were missing in the intracellular extract of injured cells. These results indicate that the amount of protein varies when cells are injured with CaP and H2O2.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Mediated Cleft palate by Mouse Embryonic Palate Mesenchymal Cells

ObjectiveTo evaluate the effects of mouse embryonic palatal mesenchymal (MEPM) cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced. Study designAn experimental study at Xinxiang Medical University. MethodsPrimary MEPM cells were derived from palatal tissue on 30 pregnant C57BL/6 female mice (embryonic day13, GD13). The MEPM cells were placed in a humidified incubator at 37°C with 5% CO2 atmosphere with media replaced every other day. The third passage cells were seeded, and one part of cells were treated with 10nM TCDD (TCDD group). And others were treated with DMSO (≤0.05%, as control group). After 72h, MTT assay was analysed cell viability. Scratch wound-healing was analysed cell motility. Flow cytometry was analysed cell apoptosis and cycle. Western blot was analysed the expression of cyclinE, CDK2, P16 and P21. ResultsTCDD inhibited the growth and migration of MEPM cells, while increased cell apoptosis. TCDD exposure inhibited the progression of cells from G1 to S phase and tended to reduce the number of cells entering the G2/M phase. TCDD inhibited expression of cyclinE and CDK2 at the protein level, instead increased the expression of P16 and p21 proteins. ConclusionsTCDD might induce cleft palate by altering MEPM cells.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System.

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

Protective effect of hypoxic preconditioning on SH-SY5Y cells injured by oxygen-glucose deprivation

To investigate the protection effects of hypoxic preconditioning(HPC) on the SH-SY5Y cell injured by oxygen-glu-cose deprivation(OGD),and to discuss the possible mechanism. SH-SY5Y cells were randomly divided into 4 groups. In normal group,the cells were cultured without OGD treatment. In HPC group,the cultured SH-SY5Y cells were treated for 5 days by intermittently ex-posing to hypoxic gas mixture (2% O2,5% CO2) for 30 min in every day. In OGD group,the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 1% O2 and 5% CO2 for 10 h. After that, the cells were fed with glucose-supplemented medium and cultured under normoxic condition for 24 h. In HPC+OGD group,the cultured SH-SY5Y cells were treated for 5 days by intermittently exposing to hypoxic gas mixture for 30 min in each day, then the cells were given the same treatments as those in OGD group. The cell viability was assessed by MTT assay. The degree of the cell damage was evaluated by deter-mining lactate dehydrogenase (LDH) leakage. TUNEL staining were used to detect the variation of cell apoptosis. The expression of Caspase 3 and hypoxia inducible factor-1α(HIF-1α) at protein levels was examined by Western blot. Hypoxic preconditioning relieved the cells apoptosis,decreased the amount of LDH leakage and improved the viability of SH-SY5Y cells injured by OGD (P<0.05). Western blot showed that the expression of Caspase 3 protein in HPC+OGD group was significantly lower than that in OGD group (P<0.05); HIF-1α protein expression was significantly higher than that of OGD group (P<0.05). Hypoxic preconditioning has protective effect on in vitro cultured SH-SY5Y cells injured by OGD. The mechanism may be related to the increase of HIF-1a protein.

Neural Differentiation of Human Umbilical Cord Blood-derived Mesenchymal Stem Cells

Background: Cell therapy is a potential therapeutic approach for neurodegenerative disorders. Human umbilical cord bloodderived mesenchymal stem cells (hUCB-MSCs) are an appropriate source of stem cells for use in various cell-based therapies. Objectives: In this study, we examined a real-time PCR approach for neural differentiation of hUCB-MSCs in vitro. Materials and Methods: MSCs were cultured in DMEM medium supplemented with 10% FBS in a humidified incubator equilibration at 5% CO2 and 37°C. For the neural differentiation of MSCs, the DMEM was removed and replaced with pre-induction media (retinoic acid [RA], basic fibroblast growth factor [bFGF], and epidermal growth factor [EGF]) and basal medium for two days. They were then cultured in nerve growth factor (NGF), 3-isobutyl-1-methylxanthine (IBMX), ascorbic acid (AA), and basal medium for six days. We also monitored the expression of markers for neural differentiation with real-time PCR. Results: The results of real-time PCR showed that the expression of the GFAP, MBP, and MAP-2 genes after two-step induction was significantly increased compared to the common induction protocol. In addition, our study showed that RA should play the main role in the neural differentiation and fate of MSCs compared to other neural inducers. Conclusions: Taken together, the combination of chemical and growth factors in the two-step induction protocol may improve the efficiency of MSC differentiation into neural progenitor cells, and may be a new method for the easy and fast application of MSCs in regenerative medicine in the future.

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

An intriguing relation between periodontal and cardiovascular diseases

Periodontitis is a chronic inflammatory condition promoted by bacterial colonization of the gingiva that causes alveolar bone and tooth loss. Under these conditions, bacteria, as well as bacterial products and inflammatory mediators can move from the gingival pocket to the well vascularised periodontal tissues and into the circulation. In fact, recently a possible connection between oral infection and cardiovascular disease was suggested (1). One of the major pathogens involved in the progression of periodontal disease is the Porphyromonas gingivalis (LPS-G). The aim of the study was to induce endothelial differentiation in human periodontal ligaments stem cells (hPDLSCs) (2) on decellularized pig heart valve as scaffold and evaluate the role of LPS-G on cell cultures in terms of reactive oxygen species (ROS) production and NFKB pathway. Many studies have shown that ROS provoked oxidative stress plays a critical role in the development of cardiovascular disease. Excessive generation of ROS can cause cellular dysfunction and injury by directly oxidizing and damaging proteins, DNA and lipids, which ultimately result in cell death. To induce endothelial differentiation, human periodontal ligament stem cells (hPDLSCs) were cultured with endothelial growth medium (EGM-2MV) supplemented with vascular endothelial growth factor (VEGF) and seeded on decellularized pig heart valve. Valve leaflets were incubated for 30 min with 10 μM DCFH-DA at 37°C in humidified incubator. At the end of loading, the valve leaflets were observed using multiphoton microscope. Results of time lapse experiments revealed that after treatment, with 2,5 mg/ml LPS-G, a rapid sustained increase in ROS generation was observed mainly in differentiated hPDLSCs. Comparing the average response of undifferentiated and differentiated hPDLSCs is clearly evident that the latter showed an six fold increase in ROS production after LPS-G exposure, while on undifferentiated appeared to be negligible. Moreover, in endothelial differentiated cells the NFKB nuclear translocation in presence of LPS-G was evident. A reasonable conclusion could be that the treatment of periodontal disease not only improve dental but presumably also cardiovascular health.