Evaluation of oxidative damage and renal distal tubule cell stress response following exposure to lindane

Lindane, or γ-hexachlorocyclohexane, is a chlorinated hydrocarbon pesticide that was banned from U.S. production in 1976, but until recently continued to be imported and applied for occupational and domestic purposes. Lindane is known to cause central nervous system (CNS), immune, cardiovascular, reproductive, liver, and kidney toxicity. The mechanism for which lindane interacts with the CNS has been elucidated, and involves antagonism of the γ-aminobutyric acid/benzodiazepine (GABAA/BZD) receptor. Antagonism of this receptor results in the inhibition of Cl- channel flux, with subsequent convulsions, seizures, and paralysis. This response makes lindane a desirable defense against arthropod pests in agriculture and the home. However, formulation and application of this compound can contribute to human toxicity. In conjunction with this exposure scenario, workers may be subject to both heat and physical stress that may increase their susceptibility to pesticide toxicity by altering their cellular stress response. The kidneys are responsible for maintaining osmotic homeostasis, and are exposed to agents that undergo urinary excretion. The mechanistic action of lindane on the kidneys is not well understood. Lindane, in other organ systems, has been shown to cause cellular damage by generation of free radicals and oxidative stress. Previous research in our laboratory has shown that lindane causes apoptosis in distal tubule cells, and delays renal stress response under hypertonic stress. Characterizing the mechanism of action of lindane under conditions of physiologic stress is necessary to understand the potential hazard cyclodiene pesticides and other organochlorine compounds pose to exposed individuals under baseline conditions, as well as under conditions of physiologic stress. We demonstrated that exposure to lindane results in oxidative damage and dysregulation of glutathione response in renal distal tubule (MDCK) cells. We showed that under conditions of hypertonic stress, lindane-induced oxidative stress resulted in early onset apoptosis and corresponding down-regulated expression of the anti-apoptotic protein, Bcl-xL. Thus, the interaction of lindane with renal peripheral benzodiazepine receptors (PBR) is associated with attenuation of cellular protective proteins, making the cell more susceptible to injury or death. ^

Mechanisms by which nonsense codons downregulate T-cell receptor transcripts

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). Two out of three T-cell receptor β (TCRβ) transcripts carry PTCs as a result of error-prone programmed rearrangements that occur at this locus during lymphocyte maturation. PTCs decrease TCRβ mRNA levels to a much greater extent than mRNAs transcribed from non-rearranging genes. This robust decrease in TCRβ mRNA levels is not a unique characteristic of the T-cell environment or the TCRβ promoter. The simplest explanation for this is that PTC-bearing TCRβ mRNAs elicit a stronger NMD response. An alternative explanation is NMD collaborates with another mechanism to dramatically decrease PTC-bearing TCRβ mRNA levels. ^ In my dissertation, I investigated the molecular mechanism behind the strong decrease in TCRβ mRNA levels triggered by PTCs. To determine the location of this response, I performed mRNA half-life analysis and found that PTCs elicited more rapid TCRβ mRNA decay in the nuclear fraction, not the cytoplasmic fraction. Although decay was restricted to the nuclear fraction, PTC-bearing TCRβ transcript levels were extremely low in the cytoplasm, a phenomenon that I named the nonsense-codon induced partitioning shift (NIPS). I established that NIPS shares several qualities with NMD, including its dependence on translation and NMD factors. Several lines of evidence suggested that NIPS results from PTCs eliciting retention of TCRβ transcripts in the nuclear fraction. This retention, as well as rapid TCRβ mRNA decay, most likely occurs in either the nucleoplasm or the outer nuclear membrane, based on analysis of nuclear and cytoplasmic markers in the highly purified nuclei I used for my studies. To further address the location of decay, I asked whether nuclear or cytoplasmic RNA decay factors mediated the destruction of PTC-bearing mRNAs. My results suggested that a nuclear component of the 3'-to-5' exosome, as well as an endonucleolytic activity, are involved in the destruction of PTC-containing TCRβ mRNAs. Individual endogenous NMD substrates had differential requirements for nuclear and cytoplasmic exonucleases. In summary, my results provide evidence that PTCs trigger multiple mechanisms involving multiple decay factors to remove and regulate mRNAs in mammalian cells. ^

DETERMINING THE GENOTYPE-PHENOTYPE CONNECTION IN SYNTHETIC INDUCIBLE GENE EXPRESSION SYSTEMS

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise. Methods I created probabilistic biochemical reaction models from known behaviors of the tetR and rtTA genes, gene products, and their gene architectures. I then simplified these models to account for essential behaviors of gene expression systems. Finally, I used these models to predict behaviors of modified gene expression systems, which were experimentally verified. Results Cell growth, which is often ignored when formulating chemical kinetics models, was essential for understanding gene expression behavior. Models incorporating growth effects were used to explain unexpected reductions in gene expression noise, design a set of gene expression systems with “linear” dose-responses, and quantify the speed with which cells explored their fitness landscapes due to noisy gene expression. Conclusions Models incorporating noisy gene expression and cell division were necessary to design, understand, and predict the behaviors of synthetic gene expression systems. The methods and models developed here will allow investigators to more efficiently design new gene expression systems, and infer gene expression properties of TetR based systems.

αvβ8 Integrin Interacts with RhoGDI1 to Regulate Rac1 and Cdc42 Activation and Drive Glioblastoma Cell Invasion

As an interface between the circulatory and central nervous systems, the neurovascular unit is vital to the development and survival of tumors. The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are major impediments to surgical resection and targeted therapies. Adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we have utilized human GBM cell lines, primary patient samples, and pre-clinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin associates with Rho GDP Dissociation Inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, suppresses activation of Rho proteins to promote GBM cell invasiveness. Hence, targeting the αvβ8 integrin-RhoGDI1 signaling axis may be an effective strategy for blocking GBM cell invasion.

PIM KINASE INHIBITION: SINGLE AGENT AND COMBINATION APPROACHES IN B-CELL LYMPHOMA

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are Ser/Thr/Tyr kinases. They modulate B-cell development but become oncoproteins and promote cancer development once overexpressed. Containing three isoforms, Pim-1, -2 and -3 are known to phosphorylate various substrates that regulate transcription, translation, cell cycle, and survival pathways in both hematological and solid tumors. Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma. Elevated Pim kinase levels are common in MCL, and it negatively correlates with patient outcome. SGI-1776 is a small molecule inhibitor selective for Pim-1/-3. We hypothesize that SGI-1776 treatment in MCL will inhibit Pim kinase function, and inhibition of downstream substrates phosphorylation will disrupt transcriptional, translational, and cell cycle processes while promoting apoptosis. SGI-1776 treatment induced moderate to high levels of apoptosis in four MCL cell lines (JeKo-1, Mino, SP-53 and Granta-519) and peripheral blood mononuclear cells (PBMCs) from MCL patients. Phosphorylation of transcription and translation regulators, c-Myc and 4E-BP1 declined in both model systems. Additionally, levels of short-lived Mcl-1 mRNA and protein also decreased and correlated with decline of global RNA synthesis. Collectively, our investigations highlight Pim kinases as viable drug targets in MCL and emphasize their roles in transcriptional and translational regulation. We further investigated a combination strategy using SGI-1776 with bendamustine, an FDA-approved DNA-damaging alkylating agent for treating non-Hodgkin’s lymphoma. We hypothesized this combination will enhance SGI-1776-induced transcription and translation inhibition, while promoting bendamustine-triggered DNA damage and inducing additive to synergistic cytotoxicity in B-cell lymphoma. Bendamustine alone resulted in moderate levels of apoptosis induction in MCL cell lines (JeKo-1 and Mino), and in MCL and splenic marginal zone lymphoma (a type of B-cell lymphoma) primary cells. An additive effect in cell killing was observed when combined with SGI-1776. Expectedly, SGI-1776 effectively decreased global RNA and protein synthesis levels, while bendamustine significantly inhibited DNA synthesis and generated DNA damage response. In combination, intensified inhibitory effects in DNA, RNA and protein syntheses were observed. Together, these data suggested feasibility of using Pim kinase inhibitor in combination with chemotherapeutic agents such as bendamustine in B-cell lymphoma, and provided foundation of their mechanism of actions in lymphoma cells.

Investigation of cell fate decision in Schizosaccharomyces pombe by ncRNA annotation and statistical analyses

Studies in the fission yeast Schizosaccharomyces pombe (S. pombe) have done much to inform the view of heterochromatin and its control by the RNA interference (RNAi) machinery. Using cDNA synthesised from poly(A)-enriched RNA samples, numerous novel ncRNA loci were discovered, and the 50 and 30 ends of many other genes were refined in previous studies. Although some of these transcripts may encode novel proteins the function of the majority is yet to be determined. The authors have used strand-specific deep sequencing of RNA, irrespective of poly(A) status, to reveal a highly structured antisense programme that modulates gene expression to dictate cell fate decisions during sexual differentiation. They show that an extensive and elaborate array of ncRNA production accompanies sexual differentiation in the fission yeast S. pombe. Experimental manipulation suggests that these transcripts specifically regulate the function of the target genes.

Percentage of cultured cell-populations that stained positively and/or negatively for apoptotic markers cleaved caspase-3 and cleaved PARP, following DNA damage treatments induced by doxorubicin and TNF-alpha co-treatments

The present dataset contains the source data for Figure 2B of Tentner et al. (2012). The data shows the percentage of cultured cell-populations that stained positively and/or negatively for apoptotic markers cleaved caspase-3 and cleaved PARP, following DNA damage treatments induced by various doses of doxorubicin (0, 2 and 10 µmole/L) in the presence (100 ng/mL) or absence (0 ng/mL) of TNF-alpha co-treatment. For the six treatment conditions investigated, cell counts were made by flow cytometry at times 6, 12, 24, and 48 h following treatment; CULTURE DETAILS: U2OS cells were obtained from ATCC were maintained at 21% oxygen and 5% CO2 in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, 2mM L-glutamine, and used within 15-20 passages. The first thymidine block was released by washing the plates three times with PBS, and incubating them in fresh thymidine-free media for 12 h. A second thymidine block was then performed by re-addition of thymidine to 2.5 mM followed by incubation for an additional 18 h. Media was aspirated, plates were washed 3 with PBS, and replaced with fresh media in the presence or absence of 10 mM aphidicolin; ANALYSIS DETAILS: See supplementary journal publication; RESULT: The authors of the supplementary journal publication conclude that TNF enhances dose-dependent cell death following doxorubicin-induced DNA damage with minimal affect on dose-dependent cell-cycle arrest.

Surface diatom community composition and species-specific contribution to carbon biomass, live and empty cell abundances over the Kerguelen region in the Southern Ocean (KEOPS 2 program)

In the naturally iron-fertilized surface waters of the northern Kerguelen Plateau region, the early spring diatom community composition and contribution to plankton carbon biomass were investigated and compared with the High Nutrient Low Chlorophyll (HNLC) surrounding waters (October-November 2011, KEOPS 2). The large iron-induced blooms were dominated by small diatom species belonging to the genera Chaetoceros (Hyalochaete) and Thalassiosira, which rapidly responded to the onset of favorable light-conditions in the meander of the Polar Front. In comparison, the iron-limited HNLC area was typically characterized by autotrophic nanoeukaryote-dominated communities and by larger and more heavily silicified diatom species (e.g. Fragilariopsis spp.). Our results support the hypothesis that diatoms are valuable vectors of carbon export to depth in naturally iron-fertilized systems of the Southern Ocean. Comparison with the diatom assemblage composition of a sediment trap deployed in the iron-fertilized area suggests that the dominant Chaetoceros (Hyalochaete) cells were less efficiently exported than the less abundant yet heavily silicified cells of Thalassionema nitzschioides and Fragilariopsis kerguelensis. Our observations emphasize the strong influence of species-specific diatom cell properties combined with trophic interactions on matter export efficiency, and illustrate the tight link between the specific composition of phytoplankton communities and the biogeochemical properties characterizing the study area.

Extended Triple-Junction Solar Cell 3D Distributed Model: Application to Chromatic Aberration-Related Losses

An extended 3D distributed model based on distributed circuit units for the simulation of triple‐junction solar cells under realistic conditions for the light distribution has been developed. A special emphasis has been put in the capability of the model to accurately account for current mismatch and chromatic aberration effects. This model has been validated, as shown by the good agreement between experimental and simulation results, for different light spot characteristics including spectral mismatch and irradiance non‐uniformities. This model is then used for the prediction of the performance of a triple‐junction solar cell for a light spot corresponding to a real optical architecture in order to illustrate its suitability in assisting concentrator system analysis and design process.

Computational Schemes for Optimizing Domains of Attraction in Dynamical Systems

In this paper, several computational schemes are presented for the optimal tuning of the global behavior of nonlinear dynamical sys- tems. Specifically, the maximization of the size of domains of attraction associated with invariants in parametrized dynamical sys- tems is addressed. Cell Mapping (CM) tech- niques are used to estimate the size of the domains, and such size is then maximized via different optimization tools. First, a ge- netic algorithm is tested whose performance shows to be good for determining global maxima at the expense of high computa- tional cost. Secondly, an iterative scheme based on a Stochastic Approximation proce- dure (the Kiefer-Wolfowitz algorithm) is eval- uated showing acceptable performance at low cost. Finally, several schemes combining neu- ral network based estimations and optimiza- tion procedures are addressed with promising results. The performance of the methods is illus- trated with two applications: first on the well-known van der Pol equation with stan- dard parametrization, and second the tuning of a controller for saturated systems.

A common mechanism of defective channel trafficking underlying DFNA2 hearing loss result in different cell surface expression levels of KCNQ4 mutants

KCNQ4 mutations underlie DFNA2, a subtype of autosomal dominant hearing loss. We had previously identified the pore-region p.G296S mutation that impaired channel activity in two manners: it greatly reduced surface expression and abolished channel function. Moreover, G296S mutant exerted a strong dominant-negative effect on potassium currents by reducing the channel expression at the cell surface representing the first study to identify a trafficking-dependent dominant mechanism for the loss of KCNQ4 channel function in DFNA2. Here, we have investigated the pathogenic mechanism associated with all the described KCNQ4 mutations (F182L, W242X, E260K, D262V, L274H, W276S, L281S, G285C, G285S and G321S) that are located in different domains of the channel protein. F182L mutant showed a wild type-like cell-surface distribution in transiently transfected NIH3T3 fibroblasts and the recorded currents in Xenopus oocytes resembled those of the wild-type. The remaining KCNQ4 mutants abolished potassium currents, but displayed distinct levels of defective cell-surface expression in NIH3T3 as quantified by flow citometry. Co-localization studies revealed these mutants were retained in the ER, unless W242X, which showed a clear co-localization with Golgi apparatus. Interestingly, this mutation results in a truncated KCNQ4 protein at the S5 transmembrane domain, before the pore region, that escapes the protein quality control in the ER but does not reach the cell surface at normal levels. Currently we are investigating the trafficking behaviour and electrophysiological properties of several KCNQ4 truncated proteins artificially generated in order to identify specific motifs involved in channel retention/exportation. Altogether, our results indicate that a defect in KCNQ4 trafficking is the common mechanism underlying DFNA2

Three-level cell topology for a multilevel power supply to achieve High

This paper presents an envelope amplifier solution for envelope elimination and restoration (EER), that consists of a series combination of a switch-mode power supply (SMPS), based on three-level voltage cells and a linear regulator. This cell topology offers several advantages over a previously presented envelope amplifier based on a different multilevel topology (two-level voltage cells). The topology of the multilevel converter affects to the whole design of the envelope amplifier and a comparison between both design alternatives regarding the size, complexity and the efficiency of the solution is done. Both envelope amplifier solutions have a bandwidth of 2 MHz with an instantaneous maximum power of 50 W. It is also analyzed the linearity of the three-level cell solution, with critical importance in the EER technique implementation. Additionally, considerations to optimize the design of the envelope amplifier and experimental comparison between both cell topologies are included.

Triple-junction solar cell performance under Fresnel-based concentrators taking into account chromatic aberration and off-axis operation

Concentration photovoltaic (CPV) systems might produce quite uneven irradiance distributions (both on their level and on their spectral distribution) on the solar cell. This effect can be even more evident when the CPV system is slightly off-axis, since they are often designed to assure good uniformity only at normal incidence. The non-uniformities both in absolute irradiance and spectral content produced by the CPV systems, can originate electrical losses in multi-junction solar cells (MJSC). This works is focused on the integration of ray-tracing methods for simulating the irradiance and spectrum maps produced by different optic systems throughout the solar cell surface, with a 3D fully distributed circuit model which simulates the electrical behavior of a state-of-the-art triple-junction solar cell under the different light distributions obtained with ray-tracing. In this study four different CPV system (SILO, XTP, RTP, and FK) comprising Fresnel lenses concentrating sunlight onto the same solar cell are modeled when working on-axis and 0.6 degrees off-axis. In this study the impact of non-uniformities on a CPV system behavior is revealed. The FK outperforms other Fresnel-based CPV systems in both on-axis and off-axis conditions.

NGCPV: A new generation of concentrator photovoltaic cells, modules and systems

This work introduces the lines of research that the NGCPV project is pursuing and some of the first results obtained. Sponsored by the European Commission under the 7th Framework Program and NEDO (Japan) within the first collaborative call launched by both Bodies in the field of energy, NGCPV project aims at approaching the cost of the photovoltaic kWh to competitive prices in the framework of high concentration photovoltaics (CPV) by exploring the development and assessment of concentrator photovoltaic solar cells and modules, novel materials and new solar cell structures as well as methods and procedures to standardize measurement technology for concentrator photovoltaic cells and modules. More specific objectives we are facing are: (1) to manufacture a cell prototype with an efficiency of at least 45% and to undertake an experimental activity, (2) to manufacture a 35% module prototype and elaborate the roadmap towards the achievement of 40%, (3) to develop reliable characterization techniques for III-V materials and quantum structures, (4) to achieve and agreement within 5% in the characterization of CPV cells and modules in a round robin scheme, and (5) to evaluate the potential of new materials, devices technologies and quantum nanostructures to improve the efficiency of solar cells for CPV.