Temporally distinct roles of ATM and ROS in genotoxic- stress-dependent induction and maintenance of cellular senescence

Cells exposed to genotoxic stress induce cellular senescence through a DNA damage response (DDR) pathway regulated by ATM kinase and reactive oxygen species (ROS). Here, we show that the regulatory roles for ATM kinase and ROS differ during induction and maintenance of cellular senescence. Cells treated with different genotoxic agents were analyzed using specific pathway markers and inhibitors to determine that ATM kinase activation is directly proportional to the dose of the genotoxic stress and that senescence initiation is not dependent on ROS or the p53 status of cells. Cells in which ROS was quenched still activated ATM and initiated the DDR when insulted, and progressed normally to senescence. By contrast, maintenance of a viable senescent state required the presence of ROS as well as activated ATM. Inhibition or removal of either of the components caused cell death in senescent cells, through a deregulated ATM-ROS axis. Overall, our work demonstrates existence of an intricate temporal hierarchy between genotoxic stress, DDR and ROS in cellular senescence. Our model reports the existence of different stages of cellular senescence with distinct regulatory networks.

Remarkable enhancement in photocytotoxicity and hydrolytic stability of curcumin on binding to an oxovanadium(IV) moiety

Oxovanadium(IV) complexes of polypyridyl and curcumin-based ligands, viz. VO(cur)(L)Cl] (1, 2) and VO(scur)(L)Cl] (3, 4), where L is 1,10-phenanthroline (phen in 1 and 3), dipyrido3,2-a:2',3'-c]phenazine (dppz in 2 and 4), Hcur is curcumin and Hscur is diglucosylcurcumin, were synthesized and characterized and their cellular uptake, photocytotoxicity, intracellular localization, DNA binding, and DNA photo-cleavage activity studied. Complex VO(cur)(phen)Cl] (1) has (VN2O3Cl)-N-IV distorted octahedral geometry as evidenced from its crystal structure. The sugar appended complexes show significantly higher uptake into the cancer cells compared to their normal analogues. The complexes are remarkably photocytotoxic in visible light (400-700 nm) giving an IC50 value of <5 mu M in HeLa, HaCaT and MCF-7 cells with no significant dark toxicity. The green emission of the complexes was used for cellular imaging. Predominant cytosolic localization of the complexes 1-4 to a lesser extent into the nucleus was evidenced from confocal imaging. The complexes as strong binders of calf thymus DNA displayed photocleavage of supercoiled pUC19 DNA in red light by generating (OH)-O-center dot radicals as the ROS. The cell death is via an apoptotic pathway involving the ROS. Binding to the VO2+ moiety has resulted in stability against any hydrolytic degradation of curcumin along with an enhancement of its photocytotoxicity.

Unconjugated Bilirubin exerts Pro-Apoptotic Effect on Platelets via p38-MAPK activation

Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro, and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (p < 0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation.

Electrically driven intracellular and extracellular nanomanipulators evoke neurogenic/cardiomyogenic differentiation in human mesenchymal stem cells

Nanomechanical intervention through electroactuation is an effective strategy to guide stem cell differentiation for tissue engineering and regenerative medicine. In the present study, we elucidate that physical forces exerted by electroactuated gold nanoparticles (GNPs) have a strong influence in regulating the lineage commitment of human mesenchymal stem cells (hMSCs). A novel platform that combines intracellular and extracellular GNPs as nano-manipulators was designed to trigger neurogenic/cardiomyogenic differentiation in hMSCs, in electric field stimulated culture condition. In order to mimic the native microenvironment of nerve and cardiac tissues, hMSCs were treated with physiologically relevant direct current electric field (DC EF) or pulsed electric field (PEF) stimuli, respectively. When exposed to regular intermittent cycles of DC EF stimuli, majority of the GNP actuated hMSCs acquired longer filopodial extensions with multiple branch-points possessing neural-like architecture. Such morphological changes were consistent with higher mRNA expression level for neural-specific markers. On the other hand, PEF elicited cardiomyogenic differentiation, which is commensurate with the tubelike morphological alterations along with the upregulation of cardiac specific markers. The observed effect was significantly promoted even by intracellular actuation and was found to be substrate independent. Further, we have substantiated the participation of oxidative signaling, G0/G1 cell cycle arrest and intracellular calcium Ca2+] elevation as the key upstream regulators dictating GNP assisted hMSC differentiation. Thus, by adopting dual stimulation protocols, we could successfully divert the DC EF exposed cells to differentiate predominantly into neural-like cells and PEF treated cells into cardiomyogenic-like cells, via nanoactuation of GNPs. Such a novel multifaceted approach can be exploited to combat tissue loss following brain injury or heart failure. (C) 2015 Elsevier Ltd. All rights reserved.

Stimuli-responsive protamine-based biodegradable nanocapsules for enhanced bioavailability and intracellular delivery of anticancer agents

Enzyme-and pH-responsive polyelectrolyte nanocapsules having diameters in the range of 200 +/- 20 nm were fabricated by means of Layer-by-Layer assembly of biopolymers, protamine, and heparin, and then loaded with anticancer drug doxorubicin. The incorporation of the FDA-approved peptide drug protamine as a wall component rendered the capsules responsive to enzyme stimuli. The stimuli-responsive drug release from these nanocapsules was evaluated, and further modulation of capsule permeability to avoid premature release was demonstrated by crosslinking the wall components. The interaction of the nanocapsules with cancer cells was studied using MCF-7 breast cancer cells. These capsules were readily internalized and disintegrated inside the cells, culminating in the release of the loaded doxorubicin and subsequent cell death as observed by confocal microscopy and MTT Assay. The bioavailability studies performed using BALB/c mice revealed that the encapsulated doxorubicin exhibited enhanced bioavailability compared to free doxorubicin. Our results indicate that this stimuli-responsive system fabricated from clinically used FDA-approved molecules and exhibiting minimal premature release has great potential for drug-delivery applications.

Stimuli-responsive protamine-based biodegradable nanocapsules for enhanced bioavailability and intracellular delivery of anticancer agents

Enzyme-and pH-responsive polyelectrolyte nanocapsules having diameters in the range of 200 +/- 20 nm were fabricated by means of Layer-by-Layer assembly of biopolymers, protamine, and heparin, and then loaded with anticancer drug doxorubicin. The incorporation of the FDA-approved peptide drug protamine as a wall component rendered the capsules responsive to enzyme stimuli. The stimuli-responsive drug release from these nanocapsules was evaluated, and further modulation of capsule permeability to avoid premature release was demonstrated by crosslinking the wall components. The interaction of the nanocapsules with cancer cells was studied using MCF-7 breast cancer cells. These capsules were readily internalized and disintegrated inside the cells, culminating in the release of the loaded doxorubicin and subsequent cell death as observed by confocal microscopy and MTT Assay. The bioavailability studies performed using BALB/c mice revealed that the encapsulated doxorubicin exhibited enhanced bioavailability compared to free doxorubicin. Our results indicate that this stimuli-responsive system fabricated from clinically used FDA-approved molecules and exhibiting minimal premature release has great potential for drug-delivery applications.

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the beta H-beta I loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-a-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

蛋白质吸附和竞争吸附对ROS 17/2.8细胞粘附和生长的影响

细胞在材料表面的粘附是贴壁依赖型细胞生长的前提.本文以胶原和牛血清蛋白(BSA)为模型蛋白,研究了两者的吸附和竞争吸附对细胞粘附和生长的影响.

Intracellular demography and the dynamics of Salmonella enterica infections.

An understanding of within-host dynamics of pathogen interactions with eukaryotic cells can shape the development of effective preventive measures and drug regimes. Such investigations have been hampered by the difficulty of identifying and observing directly, within live tissues, the multiple key variables that underlay infection processes. Fluorescence microscopy data on intracellular distributions of Salmonella enterica serovar Typhimurium (S. Typhimurium) show that, while the number of infected cells increases with time, the distribution of bacteria between cells is stationary (though highly skewed). Here, we report a simple model framework for the intensity of intracellular infection that links the quasi-stationary distribution of bacteria to bacterial and cellular demography. This enables us to reject the hypothesis that the skewed distribution is generated by intrinsic cellular heterogeneities, and to derive specific predictions on the within-cell dynamics of Salmonella division and host-cell lysis. For within-cell pathogens in general, we show that within-cell dynamics have implications across pathogen dynamics, evolution, and control, and we develop novel generic guidelines for the design of antibacterial combination therapies and the management of antibiotic resistance.

A dynamic view of the spread and intracellular distribution of Salmonella enterica.

The events that determine the dynamics of proliferation, spread and distribution of microbial pathogens within their hosts are surprisingly heterogeneous and poorly understood. We contend that understanding these phenomena at a sophisticated level with the help of mathematical models is a prerequisite for the development of truly novel, targeted preventative measures and drug regimes. We describe here recent studies of Salmonella enterica infections in mice which suggest that bacteria resist the antimicrobial environment inside host cells and spread to new sites, where infection foci develop, and thus avoid local escalation of the adaptive immune response. We further describe implications for our understanding of the pathogenic mechanism inside the host.

Bacterial growth rate and host factors as determinants of intracellular bacterial distributions in systemic Salmonella enterica infections.

Bacteria of the species Salmonella enterica cause a range of life-threatening diseases in humans and animals worldwide. The within-host quantitative, spatial, and temporal dynamics of S. enterica interactions are key to understanding how immunity acts on these infections and how bacteria evade immune surveillance. In this study, we test hypotheses generated from mathematical models of in vivo dynamics of Salmonella infections with experimental observation of bacteria at the single-cell level in infected mouse organs to improve our understanding of the dynamic interactions between host and bacterial mechanisms that determine net growth rates of S. enterica within the host. We show that both bacterial and host factors determine the numerical distributions of bacteria within host cells and thus the level of dispersiveness of the infection.

Intracellular demography and the dynamics of Salmonella enterica infections.

An understanding of within-host dynamics of pathogen interactions with eukaryotic cells can shape the development of effective preventive measures and drug regimes. Such investigations have been hampered by the difficulty of identifying and observing directly, within live tissues, the multiple key variables that underlay infection processes. Fluorescence microscopy data on intracellular distributions of Salmonella enterica serovar Typhimurium (S. Typhimurium) show that, while the number of infected cells increases with time, the distribution of bacteria between cells is stationary (though highly skewed). Here, we report a simple model framework for the intensity of intracellular infection that links the quasi-stationary distribution of bacteria to bacterial and cellular demography. This enables us to reject the hypothesis that the skewed distribution is generated by intrinsic cellular heterogeneities, and to derive specific predictions on the within-cell dynamics of Salmonella division and host-cell lysis. For within-cell pathogens in general, we show that within-cell dynamics have implications across pathogen dynamics, evolution, and control, and we develop novel generic guidelines for the design of antibacterial combination therapies and the management of antibiotic resistance.

Autoconcepto y calidad de vida en niños entre 8 y 12 años que concurren a los hogares de tránsito de la ciudad de Concepción del Uruguay, Entre Ríos

Resumen: La presente investigación, de tipo descriptiva-correlacional y transversal, se centró en conocer la relación que existe entre el autoconcepto y la calidad de vida de los niños entre 8 y 12 años que concurren a los hogares de tránsito, en la ciudad de Concepción del Uruguay, Entre Ríos. La muestra (no probabilística, intencional) estuvo conformada por 64 niños, de ambos sexos, 33 varones y 31 mujeres. Los mismos tenían entre 8 y 12 años. Los individuos evaluados eran asistentes a los hogares de tránsito mencionados anteriormente. Las técnicas de recolección de datos utilizadas fueron las siguientes: la Escala Verbal para Niños y Adolescentes del Autoconcepto de Martina Casullo, adaptación de Piers-Harris (1964) y el Cuestionario para Calidad de Vida Kidscreen-27. La Escala Verbal para Niños y Adolescentes del Autoconcepto (Casullo, 1990), cuenta con seis dimensiones: comportamiento, estatus intelectual y escolar, imagen corporal, sentimientos de ansiedad, percepciones acerca del reconocimiento que otros hacen de la propia conducta (popularidad) y bienestar y satisfacción personal. El cuestionario Kidscreen-27 (Quintero, Lugo, García, y Sánchez, 2011) consta de cinco dimensiones: actividad física y salud, estado de ánimo y sentimientos, vida familiar y tiempo libre, apoyo social y amigos, y entorno escolar. Los datos fueron recabados por medio de la concurrencia a ambos hogares, solicitando previamente las autorizaciones correspondientes a los directivos, como así también a los padres y/o tutores de los niños. Respecto a los procedimientos de análisis de los datos, se utilizó el programa estadístico informático SPSS (Statistical Package for the Social Sciences) versión 15.0 para Windows. Se realizó un análisis descriptivo de las dos variables principales de esta investigación (autoconcepto y calidad de vida) con el objetivo de conocer los niveles con que se presentan en niños. Además, para conocer la relación entre ambas variables, se llevó a cabo el análisis de correlación de Pearson. Para estudiar si existe diferencia en el autoconcepto y la calidad de vida en función del sexo de los niños, se realizó análisis multivariados de varianza (MANOVA). Los resultados obtenidos indicaron niveles elevados en la mayoría de las dimensiones de las variables estudiadas (autoconcepto y calidad de vida). En cuanto a las distinciones según el sexo en dichas variables, no se encontraron diferencias significativas, sólo una variación mínima en dos dimensiones del autoconcepto, donde se visualiza que el comportamiento social y la popularidad es más elevada en los niños que en las niñas.Se hallaron, por otro lado, correlaciones estadísticamente significativas en la mayoría de las dimensiones de ambas variables, las mismas son: entre comportamiento y actividad física/salud; entre comportamiento y apoyo social/amigos; entre comportamiento y entorno escolar; entre estatus intelectual/escolar y actividad física/salud; entre estatus intelectual/escolar y estado de ánimo/sentimientos; entre estatus intelectual/escolar y entorno escolar; entre imagen corporal con apoyo social/amigos; entre sentimientos de ansiedad y actividad física/salud; entre sentimientos de ansiedad y estado de ánimo/sentimientos; entre sentimientos de ansiedad y apoyo social/amigos; entre popularidad y estado de ánimo/sentimientos; entre popularidad y apoyo social/amigos; entre bienestar/satisfacción vital y actividad física/salud; entre bienestar/satisfacción vital y estado de ánimo/sentimientos; entre bienestar/satisfacción vital y apoyo social/amigos; entre bienestar/satisfacción vital y entorno escolar; entre el total global del autoconcepto y actividad física/salud; entre el total global del autoconcepto y estado de ánimo/sentimientos; entre el total global del autoconcepto y apoyo social/amigos; entre el total global del autoconcepto y entorno escolar. Sólo una dimensión de la calidad de vida no se relaciona con ninguna de las dimensiones del autoconcepto, a saber, vida familiar/tiempo libre. En lo que respecta a las conclusiones, puede considerarse que los resultados de esta investigación verifican que existe correlación entre el autoconcepto y la calidad de vida de los niños concurrentes a los hogares de tránsito Santa Clara de Asís y la Casa del Menor

Estado actual del bosque de galería en un tramo de la parte media de los ríos Ochomogo y Nandarola, municipio de Nandaime, Granada, Nicaragua, 2014

La Facultad de Recursos Naturales y del Ambiente (FARENA) en conjunto con el proyecto UNA-FAGRO DEPARTIR/ORGANIZACIÓN MUNDIAL PARA LA SALUD Y SEGURIDAD ALIMENTARIA, FAO, consideraron como objetivo principal Diagnosticar el estado actual del bosque de galería en los ríos Ochomogo y Nandarola, del municipio de Nandaime, Granada. En el río Nandarola se inventarió un área de 23.33 ha, encontrándose 321 árboles en 62 especies y 29 familias; la especie más representativa es el Guácimo de ternero(Guazuma ulmifolia Lam) con 30 individuos, y la familia más representativa es Mimosaceae con ocho especies. El área inventariada del río Ochomogo fue de 8.18 ha, se identificaron 154 árboles en 37 especies y 24 familias; la especie más abundante es Tigüilote(Cordia dentata Poir) con 19 individuos, la familiacon mayor representatividad es la Mimosaceae con cinco especies. Las variables silviculturales, iluminación respecto al río Nandarola equivale a un 51% de iluminación vertical plena, un 52% poseen fustes rectos sin ningún daño, y un 65% se encuentra libre de lianas. Por otro lado en el río Ochomogo se encontró un 57% de árboles que recibe iluminación vertical plena, con una calidad de fuste recto sin ningún daño de 45% y el 65% están libres de lianas. En general se puede decir que la población local y circundante ejerce presión sobre el recurso bosque; el caudal del río ha disminuido notoriamente por las actividades de extracción de madera para consumo energético; se evidencia la sustitución de especies nativas por exóticas como Teca (Tectona grandis L.F), Eucalipto (Eucalyptus spp.) y Neem(Azadirachta indica A. Juss) en las áreas de las riveras de los ríos, el aumento de áreas para potreros y el establecimiento de pasto, de plantaciones de cultivos de plátanos y de micro fábricas de ladrillos.

El desarrollo de la cuenca del Plata y la prohibición al uso del "mallón" en la confluencia de los ríos Paraná y Paraguay

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