Cytocompatibility, cytotoxicity and genotoxicity analysis of dental implants

Several types of materials are frequently used for dental prostheses in dental medicine. Different treatments with titanium are the most used. The aim of the present study was to analyze by means of cytotoxicity and cytocompatibility techniques the capacity of dental implants to integrate to the bone tissue. Cultures of UMR 106 cell line derived from an osteosarcoma were used for bioassays mainly because they show many of the properties of osteoblasts. Dental implant samples provided by B&W company were compared with others of recognized trademarks. The first ones contain ASTM titanium (8348 GR2) with acid printing. Cytotoxicity was analyzed by means of lysosome activity, using the neutral red technique and alkaline phosphatase enzyme activity. Cell variability was determined by means of the acridine ethidium-orange bromide technique. One-way ANOVA and Bonferroni and Duncan post-ANOVA tests were used for the statistical analysis. The assays did not show significant differences among the dental implants analyzed. Our findings show that the dental prostheses studied present high biocompatibility, quantified by the bioassays performed. The techniques employed revealed that they can be a useful tool for the analysis of other materials for dental medicine use.Instituto Multidisciplinario de Biología Celula

Cytocompatibility, cytotoxicity and genotoxicity analysis of dental implants

Several types of materials are frequently used for dental prostheses in dental medicine. Different treatments with titanium are the most used. The aim of the present study was to analyze by means of cytotoxicity and cytocompatibility techniques the capacity of dental implants to integrate to the bone tissue. Cultures of UMR 106 cell line derived from an osteosarcoma were used for bioassays mainly because they show many of the properties of osteoblasts. Dental implant samples provided by B&W company were compared with others of recognized trademarks. The first ones contain ASTM titanium (8348 GR2) with acid printing. Cytotoxicity was analyzed by means of lysosome activity, using the neutral red technique and alkaline phosphatase enzyme activity. Cell variability was determined by means of the acridine ethidium-orange bromide technique. One-way ANOVA and Bonferroni and Duncan post-ANOVA tests were used for the statistical analysis. The assays did not show significant differences among the dental implants analyzed. Our findings show that the dental prostheses studied present high biocompatibility, quantified by the bioassays performed. The techniques employed revealed that they can be a useful tool for the analysis of other materials for dental medicine use.Instituto Multidisciplinario de Biología Celula

Global environmental consequences of 21st century ice sheet melt

Government policies currently commit us to surface warming of three to four degrees Celsius above pre-industrial levels by 2100, which will lead to enhanced ice-sheet melt. Ice-sheet discharge was not explicitly included in Coupled Model Intercomparison Project phase 5, so effects on climate from this melt are not currently captured in the simulations most commonly used to inform governmental policy. Here we show, using simulations of the Greenland and Antarctic ice sheets constrained by satellite-based measurements of recent changes in ice mass, that increasing meltwater from Greenland will lead to substantial slowing of the Atlantic overturning circulation, and that meltwater from Antarctica will trap warm water below the sea surface, creating a positive feedback that increases Antarctic ice loss. In our simulations, future ice-sheet melt enhances global temperature variability and contributes up to 25 centimetres to sea level by 2100. However, uncertainties in the way in which future changes in ice dynamics are modelled remain, underlining the need for continued observations and comprehensive multi-model assessments

BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the Unfolded Protein Response

Computational modeling and experimentation in the unfolded protein response reveals a role for the ER-resident chaperone protein BiP in fine-tuning the system's response dynamics

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR(+) tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth

Persistent ER Stress Induces the Spliced Leader RNA Silencing Pathway (SLS), Leading to Programmed Cell Death in Trypanosoma brucei

Trypanosomes are parasites that cycle between the insect host (procyclic form) and mammalian host (bloodstream form). These parasites lack conventional transcription regulation, including factors that induce the unfolded protein response (UPR). However, they possess a stress response mechanism, the spliced leader RNA silencing (SLS) pathway. SLS elicits shut-off of spliced leader RNA (SL RNA) transcription by perturbing the binding of the transcription factor tSNAP42 to its cognate promoter, thus eliminating trans-splicing of all mRNAs. Induction of endoplasmic reticulum (ER) stress in procyclic trypanosomes elicits changes in the transcriptome similar to those induced by conventional UPR found in other eukaryotes. The mechanism of up-regulation under ER stress is dependent on differential stabilization of mRNAs. The transcriptome changes are accompanied by ER dilation and elevation in the ER chaperone, BiP. Prolonged ER stress induces SLS pathway. RNAi silencing of SEC63, a factor that participates in protein translocation across the ER membrane, or SEC61, the translocation channel, also induces SLS. Silencing of these genes or prolonged ER stress led to programmed cell death (PCD), evident by exposure of phosphatidyl serine, DNA laddering, increase in reactive oxygen species (ROS) production, increase in cytoplasmic Ca2+, and decrease in mitochondrial membrane potential, as well as typical morphological changes observed by transmission electron microscopy (TEM). ER stress response is also induced in the bloodstream form and if the stress persists it leads to SLS. We propose that prolonged ER stress induces SLS, which serves as a unique death pathway, replacing the conventional caspase-mediated PCD observed in higher eukaryotes

The unfolded protein response and its relevance to connective tissue diseases

The unfolded protein response (UPR) has evolved to counter the stresses that occur in the endoplasmic reticulum (ER) as a result of misfolded proteins. This sophisticated quality control system attempts to restore homeostasis through the action of a number of different pathways that are coordinated in the first instance by the ER stress-senor proteins IRE1, ATF6 and PERK. However, prolonged ER-stress-related UPR can have detrimental effects on cell function and, in the longer term, may induce apoptosis. Connective tissue cells such as fibroblasts, osteoblasts and chondrocytes synthesise and secrete large quantities of proteins and mutations in many of these gene products give rise to heritable disorders of connective tissues. Until recently, these mutant gene products were thought to exert their effect through the assembly of a defective extracellular matrix that ultimately disrupted tissue structure and function. However, it is now becoming clear that ER stress and UPR, because of the expression of a mutant gene product, is not only a feature of, but may be a key mediator in the initiation and progression of a whole range of different connective tissue diseases. This review focuses on ER stress and the UPR that characterises an increasing number of connective tissue diseases and highlights novel therapeutic opportunities that may arise

An intestinal epithelial defect conferring ER stress results in inflammation involving both innate and adaptive immunity

We recently characterized Winnie mice carrying a missense mutation in Muc2, leading to severe endoplasmic reticulum stress in intestinal goblet cells and spontaneous colitis. In this study, we characterized the immune responses due to this intestinal epithelial dysfunction. In Winnie, there was a fourfold increase in activated dendritic cells (DCs; CD11c+ major histocompatibility complex (MHC) class IIhi) in the colonic lamina propria accompanied by decreased colonic secretion of an inhibitor of DC activation, thymic stromal lymphopoietin (TSLP). Winnie also displayed a significant increase in mRNA expression of the mucosal TH17 signature genes Il17a, IL17f, Tgfb, and Ccr6, particularly in the distal colon. Winnie mesenteric lymph node leukocytes secreted multiple TH1, TH2, and TH17 cytokines on activation, with a large increase in interleukin-17A (IL-17A) progressively with age. A major source of mucosal IL-17A in Winnie was CD4+ T lymphocytes. Loss of T and B lymphocytes in Rag1-/- × Winnie (RaW) crosses did not prevent spontaneous inflammation but did prevent progression with age in the colon but not the cecum. Adoptive transfer of naive T cells into RaW mice caused more rapid and severe colitis than in Rag1-/-, indicating that the epithelial defect results in an intestinal microenvironment conducive to T-cell activation. Thus, the Winnie primary epithelial defect results in complex multicytokine-mediated colitis involving both innate and adaptive immune components with a prominent IL-23/TH17 response, similar to that of human ulcerative colitis

Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae

With the increasing demand for biopharmaceutical proteins and industrial enzymes, it is necessary to optimize the production by microbial fermentation or cell cultures. Yeasts are well established for the production of a wide range of recombinant proteins, but there are also some limitations; e.g., metabolic and cellular stresses have a strong impact on recombinant protein production. In this work, we investigated the effect of the specific growth rate on the production of two different recombinant proteins. Our results show that human insulin precursor is produced in a growth-associated manner, whereas alpha-amylase tends to have a higher yield on substrate at low specific growth rates. Based on transcriptional analysis, we found that the difference in the production of the two proteins as function of the specific growth rate is mainly due to differences in endoplasmic reticulum processing, protein turnover, cell cycle, and global stress response. We also found that there is a shift at a specific growth rate of 0.1 h(-1) that influences protein production. Thus, for lower specific growth rates, the alpha-amylase and insulin precursor-producing strains present similar cell responses and phenotypes, whereas for higher specific growth rates, the two strains respond differently to changes in the specific growth rate