Extracellular superoxide dismutase (SOD3) regulates oxidative stress at the vitreoretinal interface

Oxidative stress is a pathogenic feature in vitreoretinal disease. However, the ability of the inner retina to manage metabolic waste and oxidative stress is unknown. Proteomic analysis of antioxidants in the human vitreous, the extracellular matrix opposing the inner retina, identified superoxide dismutase-3 (SOD3) that localized to a unique matrix structure in the vitreous base and cortex. To determine the role of SOD3, Sod3-/- mice underwent histological and clinical phenotyping. Although the eyes were structurally normal, at the vitreoretinal interface Sod3-/- mice demonstrated higher levels of 3-nitrotyrosine, a key marker of oxidative stress. Pattern electroretinography also showed physiological signaling abnormalities within the inner retina. Vitreous biopsies and epiretinal membranes collected from patients with diabetic vitreoretinopathy (DVR) and a mouse model of DVR showed significantly higher levels of nitrates and/or 3-nitrotyrosine oxidative stress biomarkers suggestive of SOD3 dysfunction. This study analyzes the molecular pathways that regulate oxidative stress in human vitreous substructures. The absence or dysregulation of the SOD3 antioxidant at the vitreous base and cortex results in increased oxidative stress and tissue damage to the inner retina, which may underlie DVR pathogenesis and other vitreoretinal diseases

Curved Tails in Polymerization-Based Bacterial Motility

The curved actin ``comet-tail'' of the bacterium Listeria monocytogenes is a visually striking signature of actin polymerization-based motility. Similar actin tails are associated with Shigella flexneri, spotted-fever Rickettsiae, the Vaccinia virus, and vesicles and microspheres in related in vitro systems. We show that the torque required to produce the curvature in the tail can arise from randomly placed actin filaments pushing the bacterium or particle. We find that the curvature magnitude determines the number of actively pushing filaments, independent of viscosity and of the molecular details of force generation. The variation of the curvature with time can be used to infer the dynamics of actin filaments at the bacterial surface.Comment: 8 pages, 2 figures, Latex2

Evaluation of natural and tracer fluorescent emission methods for droplet size measurements in a diesel spray

The final publication is available at Springer via http://dx.doi.org/10.1007/s12239-012-0070-zSpray sizing that records fluorescent emission and scattered light has been widely applied to spray diagnostics over the last two decades. Different experimental strategies have been developed, but comparing the different solutions offered has remained of interest to experimentalists. In this work, a comparison of two fluorescence strategies for measuring droplet size in the liquid phase of a last-generation DI diesel spray is conducted. The natural fluorescent emission of a commercial diesel fuel and the fluorescence emitted by a tracer (Rhodamine B) are compared using theoretical and experimental approaches. The LIF/Mie ratio commonly called Planar Droplet Sizing (PDS) technique is applied in two different ways to elucidate the possible advantages of using a fluorescent dopant. The sprays were injected under non-evaporative conditions into a constant pressure vessel that simulates densities present at the moment of injection in currently used passenger car diesel engines. Characterization of the signal properties was performed by measuring the absorption coefficient, fluorescence emission spectrum, quantum yield and lifetime of both configurations. The scattered light and fluorescence intensities were calculated to verify the dependencies of the droplet surface and volume. When applying the two techniques to quantify droplet size in dense diesel sprays, the results show that signal weakness and lack of control over the properties of natural fluorescence produce distortion in the shape of the spray and cause measurements to be unreliable. © 2012 The Korean Society of Automotive Engineers and Springer-Verlag Berlin Heidelberg.This research has been funded in the frame of the project PROFUEL reference TRA2011-26293 from Ministerio de Ciencia e Innovacion. The injectors are part of the ECN international project.Pastor Soriano, JV.; Payri, R.; Salavert Fernandez, J.; Manin, J. (2012). Evaluation of natural and tracer fluorescent emission methods for droplet size measurements in a diesel spray. International Journal of Automotive Technology. 13(5):713-724. https://doi.org/10.1007/s12239-012-0070-zS713724135Albrecht, H. E., Damaschke, N., Borys, M. and Tropea, C. (2003). Laser Doppler and Phase Doppler Measurement Techniques. Springer. Berlin.Barnes, M. D., Whitten, W. B. and Ramsey, J. M. (1994). Enhanced fluorescence yields through cavity quantumelectrodynamic effects in microdroplets. J. Optical Society of America B 11,7, 1297–1304.Benajes, J., Molina, S., Novella, R., Amorim, R., Ben Hadj Hamouda, H. and Hardy, J. (2010). Comparison of two injection systems in an HSDI diesel engine using split injection and different injector nozzles. Int. J. Automotive Technology 11,2, 139–146.Charalampous, G. and Hardalupas, Y. (2011). Method to reduce errors of droplet sizing based on the ratio of fluorescent and scattered light intensities (laser-induced fluorescence/Mie technique). Applied Optics, 50, 3622–3637.Chen, G., Mazumder, M., Chang, R. K., Swindal, J. C. and Acker, W. P. (1996). Laser diagnostics for droplet characterization: Application of morphology dependent resonances. Progress in Energy and Combustion Science 22,2, 163–188.Desantes, J. M., Payri, R., Garcia, J. M. and Salvador, F. J. (2007). A contribution to the understanding of isothermal diesel spray dynamics. Fuel 86,7–8, 1093–1101.Domann, R. and Hardalupas, Y. A. (2000). Study of parameters that influence the accuracy of the planar droplet sizing (PDS) technique. Part. Part. Syst. Charact. 3–11.Domann, R. and Hardalupas, Y. A. (2001). Spatial distribution of fluorescence within large doplets and its dependence on dye concentration. Applied Optics 40,21, 3586–3597.Domann, R. and Hardalupas, Y. A. (2002). Quantitative measurement of planar droplet sauter mean diameter in sprays using planar droplet sizing. 11th Int. Symp. Application of Laser Techniques to Fluid Mechanics, Lisbon, Portugal.Eckbreth, A. C. (1988). Laser Diagnostics for Combustion Species and Temperature. Abacus. Cambridge. Mass.Greenhalgh, D. A. (1999). Planar measurements of fuel vapour, liquid fuel, liquid droplet size and soot. Planar Optical Measurement Methods for Gas Turbine Components, 1–7.Im, K., Lin, K., Lai, M. and Chon, M. (2011). Breakup modeling of a liquid jet in cross flow. Int. J. Automotive Technology 12,4, 489–496.Jermy, M. C. and Greenhalgh, D. A. (2000). Planar dropsizing by elastic and fluorescence scattering in sprays too dense for phase doppler measurement. Appl. Phys. B, 71, 703–710.Kim, Y., Kim, K. and Lee, K. (2011). Effect of a 2-stage injection strategy on the combustion and flame characteristics in a PCCI engine. Int. J. Automotive Technology 12,5, 639–644.Ko, F. H., Weng, L. Y., Ko, C. J. and Chu, T. C. (2006). Characterization of imprinting polymeric temperature variation with fluorescent Rhodamine B molecule. Microelectronic Engineering, 83, 864–868.Lakowicz, J. R. (2006). Principles of Fluorescence Spectroscopy. 3rd Edn. Springer.Lee, S. H., Teong, J., Lee, J. T., Ryou, H. S. and Hong, K. (2005). Investigation on spray characteristics under ultrahigh injection pressure conditions. Int. J. Automotive Technology 6,2, 125–131.Lee, B., Song, J., Chang, Y. and Jeon, C. (2010). Effect of the number of fuel injector holes on characteristics of combustion and emissions in a diesel engine. Int. J. Automotive Technology 11,6, 783–791.LeGal, P., Farrugia, N. and Greenhalgh, D. A. (1999). Laser sheet dropsizing of dense sprays. Optics and Laser Techn., 31, 75–83.Lockett, R. D., Richter, J. and Greenhalgh, D. A. (1998). The characterisation of a diesel spray using combined laser induced fluorescence and laser sheet dropsizing. Conf. Lasers and Electro-Optics Europe.Magde, D., Rojas, G. E. and Seybold, P. (1999). Solvent dependence of the fluorescence lifetimes of xanthene dyes. Photochem. Photobiol., 70, 737.Naber, J. and Siebers, D. (1996). Effects of gas density and vaporization on penetration and dispersion of diesel sprays. SAE Paper No. 960034.Pastor, J. V., López, J. J., Juliá, J. E. and Benajes, J. V. (2002). Planar laser-induced fluorescence fuel concentration measurements in isothermal diesel sprays. Opt. Express 10,7, 309–323.Pastor, J. V., Payri, R., Araneo, L. and Manin, J. (2009). Correction method for droplet sizing by laser-induced fluorescence in a controlled test situation. Optical Engineering 48,1, 013601.Payri, R., Garcia, J. M., Salvador, F. J. and Gimeno, J. (2005a). Using spray momentum flux measurements to understand the influence of diesel nozzle geometry on spray characteristics. Fuel, 84, 551–561.Payri, R., Salvador, F. J., Gimeno, J. and Soare, V. (2005b). Determination of diesel sprays characteristics in real engine in-cylinder air density and pressure conditions. J. Mech. Sci. Technol., 19, 2040–2052.Payri, R., Tormos, B., Salvador, F. J. and Araneo, L. (2008). Spray droplet velocity characterization for convergent nozzles with three different diameters. Fuel 87,15, 3176–3182.Payri, F., Pastor, J., Payri, R. and Manin, J. (2011). Determination of the optical depth of a DI diesel spray. J. Mech. Sci. Technol., 25, 209–219.Potz, D., Chirst, W. and Dittus, B. (2000). Diesel nozzle: The determining interface between injection system and combustion chamber. Conf. Thermo and Fluid-dynamic Processes in Diesel Engines, Valencia, Spain.Ramírez, A. I., Som, S., Aggarwal, S. K., Kastengren, A. L., El-Hannouny, E. M., Longman, D. E. and Powell, C. F. (2009). Quantitative X-ray measurements of highpressure fuel sprays from a production heavy duty diesel injector. Experiments in Fluids 47,1, 119–134.Schulz, C. and Sick, V. (2005). Tracer-LIF diagnostics: quantitative measurement of fuel concentration, temperature and fuel/air ratio in practical combustion systems. Progress in Energy and Combustion Science, 31, 75–121.Sjoback, R. and Nygren, J. and Kubista, M. (1998). Characterization of fluorescein—oligonucleotide conjugates and measurement of local electrostatic potential. Biopolymers, 46, 445–453.Soare, V. (2007). Phase Doppler Measurement in Diesel Dense Sprays: Optimisation of Measurements and Study of the Orifice Geometry Influence Over the Spray at Microscopic Level. Ph.D. Dissertion. E.T.S. Ingenieros Industriales. Universidad Politécnica de Valencia. Spain.Williams, A. T. R., Winfield, S. A. and Miller, J. N. (1983). Relative fluorescence quantum yields using a computer controlled luminescence spectrometer. Analyst., 108, 1067.Yeh, C. N., Kosaka, H. and Kamimoto, T. A. (1993). Fluorescence/scattering imaging technique for instantaneous 2-D measurements of particle size distribution in a transient spray. Proc. 3rd Cong. Opt. Part. Sizing, Yokohama, Japan, 335–361

Mitochondria, Energetics, Epigenetics, and Cellular Responses to Stress

Background: Cells respond to environmental stressors through several key pathways, including response to reactive oxygen species (ROS), nutrient and ATP sensing, DNA damage response (DDR), and epigenetic alterations. Mitochondria play a central role in these pathways not only through energetics and ATP production but also through metabolites generated in the tricarboxylic acid cycle, as well as mitochondria–nuclear signaling related to mitochondria morphology, biogenesis, fission/fusion, mitophagy, apoptosis, and epigenetic regulation. Objectives: We investigated the concept of bidirectional interactions between mitochondria and cellular pathways in response to environmental stress with a focus on epigenetic regulation, and we examined DNA repair and DDR pathways as examples of biological processes that respond to exogenous insults through changes in homeostasis and altered mitochondrial function. Methods: The National Institute of Environmental Health Sciences sponsored the Workshop on Mitochondria, Energetics, Epigenetics, Environment, and DNA Damage Response on 25–26 March 2013. Here, we summarize key points and ideas emerging from this meeting. Discussion: A more comprehensive understanding of signaling mechanisms (cross-talk) between the mitochondria and nucleus is central to elucidating the integration of mitochondrial functions with other cellular response pathways in modulating the effects of environmental agents. Recent studies have highlighted the importance of mitochondrial functions in epigenetic regulation and DDR with environmental stress. Development and application of novel technologies, enhanced experimental models, and a systems-type research approach will help to discern how environmentally induced mitochondrial dysfunction affects key mechanistic pathways. Conclusions: Understanding mitochondria–cell signaling will provide insight into individual responses to environmental hazards, improving prediction of hazard and susceptibility to environmental stressors. Citation: Shaughnessy DT, McAllister K, Worth L, Haugen AC, Meyer JN, Domann FE, Van Houten B, Mostoslavsky R, Bultman SJ, Baccarelli AA, Begley TJ, Sobol RW, Hirschey MD, Ideker T, Santos JH, Copeland WC, Tice RR, Balshaw DM, Tyson FL. 2014. Mitochondria, energetics, epigenetics, and cellular responses to stress. Environ Health Perspect 122:1271–1278; http://dx.doi.org/10.1289/ehp.140841

A DIGE study on the effects of salbutamol on the rat muscle proteome - an exemplar of best practice for data sharing in proteomics

BACKGROUND: Proteomic techniques allow researchers to perform detailed analyses of cellular states and many studies are published each year, which highlight large numbers of proteins quantified in different samples. However, currently few data sets make it into public databases with sufficient metadata to allow other groups to verify findings, perform data mining or integrate different data sets. The Proteomics Standards Initiative has released a series of "Minimum Information About a Proteomics Experiment" guideline documents (MIAPE modules) and accompanying data exchange formats. This article focuses on proteomic studies based on gel electrophoresis and demonstrates how the corresponding MIAPE modules can be fulfilled and data deposited in public databases, using a new experimental data set as an example. FINDINGS: We have performed a study of the effects of an anabolic agent (salbutamol) at two different time points on the protein complement of rat skeletal muscle cells, quantified by difference gel electrophoresis. In the DIGE study, a total of 31 non-redundant proteins were identified as being potentially modulated at 24 h post treatment and 110 non redundant proteins at 96 h post-treatment. Several categories of function have been highlighted as strongly enriched, providing candidate proteins for further study. We also use the study as an example of best practice for data deposition. CONCLUSIONS: We have deposited all data sets from this study in public databases for further analysis by the community. We also describe more generally how gel-based protein identification data sets can now be deposited in the PRoteomics IDEntifications database (PRIDE), using a new software tool, the PRIDESpotMapper, which we developed to work in conjunction with the PRIDE Converter application. We also demonstrate how the ProteoRed MIAPE generator tool can be used to create and share a complete and compliant set of MIAPE reports for this experiment and others

Conjugative IncFI plasmids carrying CTX-M-15 among Escherichia coli ESBL producing isolates at a University hospital in Germany

<p>Abstract</p> <p>Background</p> <p>Multi-drug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, constitute an emerging public-health concern. Little data on the molecular epidemiology of ESBL producing <it>Escherichia coli </it>is available in Germany. Here we describe the prevalence and molecular epidemiology of ESBL producing-<it>Escherichia coli </it>isolates at a German University hospital.</p> <p>Methods</p> <p>We analysed 63 non-duplicate clinical ESBL isolates obtained over an 8-month period using PCR and sequence-based ESBL allele typing, plasmid replicon typing, phylogenetic group typing. Pulsed-field gel electrophoresis (PFGE) based genotyping and plasmid profiling was performed, as well as confirmatory DNA-based hybridization assays.</p> <p>Results</p> <p>Examination of the 63 <it>Escherichia coli </it>isolates revealed an almost equal distribution among the <it>E. coli </it>phylogenetic groups A, B1, B2 and D. High prevalence (36/63) of the CTX-M-15 gene was observed and an analysis of PFGE-based patterns revealed the presence of this CTX-M allele in multiple clones. Resistance to cefotaxime was a transferable trait and a commonly occurring 145.5 kb conjugative IncFI plasmid was detected in 65% of <it>E. coli </it>carrying the CTX-M-15 allele. The rate of transferable antibiotic resistances for GM, SXT, TET, GM-SXT-TET, SXT-TET and GM-TET was 33%, 61%, 61%, 27%, 44% and 11%, respectively. The remaining strains did not have a common IncFI plasmid but harboured transferable IncFI plasmids with sizes that ranged from 97 to 242.5 kb.</p> <p>Conclusion</p> <p>Our data demonstrate the presence of IncFI plasmids within the prevailing <it>E. coli </it>population in a hospital setting and suggest that the dissemination of CTX-M-15 allele is associated to lateral transfer of these well-adapted, conjugative IncFI plasmids among various <it>E. coli </it>genotypes.</p

Epigenetic silencing of DSC3 is a common event in human breast cancer

INTRODUCTION: Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens. METHODS: We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines. RESULTS: DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure. CONCLUSION: These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure

DNA methylation changes in ovarian cancer are cumulative with disease progression and identify tumor stage

<p>Abstract</p> <p>Background</p> <p>Hypermethylation of promoter CpG islands with associated loss of gene expression, and hypomethylation of CpG-rich repetitive elements that may destabilize the genome are common events in most, if not all, epithelial cancers.</p> <p>Methods</p> <p>The methylation of 6,502 CpG-rich sequences spanning the genome was analyzed in 137 ovarian samples (ten normal, 23 low malignant potential, 18 stage I, 16 stage II, 54 stage III, and 16 stage IV) ranging from normal tissue through to stage IV cancer using a sequence-validated human CpG island microarray. The microarray contained 5' promoter-associated CpG islands as well as CpG-rich satellite and Alu repetitive elements.</p> <p>Results</p> <p>Results showed a progressive de-evolution of normal CpG methylation patterns with disease progression; 659 CpG islands showed significant loss or gain of methylation. Satellite and Alu sequences were primarily associated with loss of methylation, while promoter CpG islands composed the majority of sequences with gains in methylation. Since the majority of ovarian tumors are late stage when diagnosed, we tested whether DNA methylation profiles could differentiate between normal and low malignant potential (LMP) compared to stage III ovarian samples. We developed a class predictor consisting of three CpG-rich sequences that was 100% sensitive and 89% specific when used to predict an independent set of normal and LMP samples versus stage III samples. Bisulfite sequencing confirmed the NKX-2-3 promoter CpG island was hypermethylated with disease progression. In addition, 5-aza-2'-deoxycytidine treatment of the ES2 and OVCAR ovarian cancer cell lines re-expressed NKX-2-3. Finally, we merged our CpG methylation results with previously published ovarian expression microarray data and identified correlated expression changes.</p> <p>Conclusion</p> <p>Our results show that changes in CpG methylation are cumulative with ovarian cancer progression in a sequence-type dependent manner, and that CpG island microarrays can rapidly discover novel genes affected by CpG methylation in clinical samples of ovarian cancer.</p