Comparative transcriptomics reveals circadian and pluripotency networks as two pillars of longevity regulation.

Mammals differ more than 100-fold in maximum lifespan. Here, we conducted comparative transcriptomics on 26 species with diverse lifespans. We identified thousands of genes with expression levels negatively or positively correlated with a species' maximum lifespan (Neg- or Pos-MLS genes). Neg-MLS genes are primarily involved in energy metabolism and inflammation. Pos-MLS genes show enrichment in DNA repair, microtubule organization, and RNA transport. Expression of Neg- and Pos-MLS genes is modulated by interventions, including mTOR and PI3K inhibition. Regulatory networks analysis showed that Neg-MLS genes are under circadian regulation possibly to avoid persistent high expression, whereas Pos-MLS genes are targets of master pluripotency regulators OCT4 and NANOG and are upregulated during somatic cell reprogramming. Pos-MLS genes are highly expressed during embryogenesis but significantly downregulated after birth. This work provides targets for anti-aging interventions by defining pathways correlating with longevity across mammals and uncovering circadian and pluripotency networks as central regulators of longevity.

The Adhesion G-Protein-Coupled Receptor, GPR56/ADGRG1, Inhibits Cell-Extracellular Matrix Signaling to Prevent Metastatic Melanoma Growth.

Metastatic growth is considered a rate-limiting step in cancer progression, and upregulation of extracellular matrix (ECM) deposition and cell-ECM signaling are major drivers of this process. Mechanisms to reverse ECM upregulation in cancer could potentially facilitate its prevention and treatment but they are poorly understood. We previously reported that the adhesion G-protein-coupled receptor GPR56/ADGRG1 is downregulated in melanoma metastases. Its re-expression inhibited melanoma growth and metastasis and reduced the deposition of fibronectin, a major ECM component. We hypothesize that its effect on fibronectin deposition contributes to its inhibitory role on metastatic growth. To test this, we investigated the function of GPR56 on cell-fibronectin adhesion and its relationship with metastatic growth in melanoma. Our results reveal that GPR56 inhibits melanoma metastatic growth by impeding the expansion of micrometastases to macrometastases. Meanwhile, we present evidence that GPR56 inhibits fibronectin deposition and its downstream signaling, such as phosphorylation of focal adhesion kinase (FAK), during this process. Administration of the FAK inhibitor Y15 perturbed the proliferation of melanoma metastases, supporting a causative link between the cell adhesion defect induced by GPR56 and its inhibition of metastatic growth. Taken together, our results suggest that GPR56 in melanoma metastases inhibits ECM accumulation and adhesion, which contributes to its negative effects on metastatic growth.

Equivalent titanium dioxide nanoparticle deposition by intratracheal instillation and whole body inhalation: the effect of dose rate on acute respiratory tract inflammation.

BACKGROUND: The increased production of nanomaterials has caused a corresponding increase in concern about human exposures in consumer and occupational settings. Studies in rodents have evaluated dose-response relationships following respiratory tract (RT) delivery of nanoparticles (NPs) in order to identify potential hazards. However, these studies often use bolus methods that deliver NPs at high dose rates that do not reflect real world exposures and do not measure the actual deposited dose of NPs. We hypothesize that the delivered dose rate is a key determinant of the inflammatory response in the RT when the deposited dose is constant. METHODS: F-344 rats were exposed to the same deposited doses of titanium dioxide (TiO2) NPs by single or repeated high dose rate intratracheal instillation or low dose rate whole body aerosol inhalation. Controls were exposed to saline or filtered air. Bronchoalveolar lavage fluid (BALF) neutrophils, biochemical parameters and inflammatory mediator release were quantified 4, 8, and 24 hr and 7 days after exposure. RESULTS: Although the initial lung burdens of TiO2 were the same between the two methods, instillation resulted in greater short term retention than inhalation. There was a statistically significant increase in BALF neutrophils at 4, 8 and 24 hr after the single high dose TiO2 instillation compared to saline controls and to TiO2 inhalation, whereas TiO2 inhalation resulted in a modest, yet significant, increase in BALF neutrophils 24 hr after exposure. The acute inflammatory response following instillation was driven primarily by monocyte chemoattractant protein-1 and macrophage inflammatory protein-2, mainly within the lung. Increases in heme oxygenase-1 in the lung were also higher following instillation than inhalation. TiO2 inhalation resulted in few time dependent changes in the inflammatory mediator release. The single low dose and repeated exposure scenarios had similar BALF cellular and mediator response trends, although the responses for single exposures were more robust. CONCLUSIONS: High dose rate NP delivery elicits significantly greater inflammation compared to low dose rate delivery. Although high dose rate methods can be used for quantitative ranking of NP hazards, these data caution against their use for quantitative risk assessment.

GPR56 inhibits melanoma growth by internalizing and degrading its ligand TG2.

Excessive accumulation of extracellular matrix (ECM) is a hallmark of tumor microenvironment and plays active roles during tumor progression. How this process is regulated and whether it is reversible for cancer treatment are outstanding questions. The adhesion G protein-coupled receptor GPR56 inhibits melanoma growth and binds to tissue transglutaminase (TG2), a major crosslinking enzyme in ECM. To understand the function of TG2 in GPR56-mediated melanoma inhibition, we performed xenograft studies in immunodeficient Tg2(-/-) mice. Our results revealed an antagonistic relationship between GPR56 and TG2 in melanoma, although TG2 and its crosslinking activity promote melanoma growth, GPR56 antagonizes this effect by internalizing and degrading it. The negative regulation of TG2 by GPR56 associates with the decreased deposition of a major ECM protein, fibronectin, and impaired accumulation of focal adhesion kinase, indicating that the GPR56-TG2 interaction regulates ECM deposition and cell-ECM adhesion. Taken together, our findings establish the roles of TG2 in GPR56-mediated melanoma inhibition. The uncovered antagonistic relationship between GPR56 and TG2 proposes a mechanism by which ECM accumulation/crosslinking in tumors may be reversed, and thus could have therapeutic potential for cancer control and treatment.

Extraction and quantification of carbon nanotubes in biological matrices with application to rat lung tissue.

Extraction of carbon nanotubes (CNTs) from biological matrices such as rat lung tissue is integral to developing a quantification method for evaluating the environmental and human health exposure and toxicity of CNTs. The ability of various chemical treatment methods, including Solvable (2.5% sodium hydroxide/surfactant mixture), ammonium hydroxide, nitric acid, sulfuric acid, hydrochloric acid, hydrofluoric acid, hydrogen peroxide, and proteinase K, to extract CNTs from rat lung tissue was evaluated. CNTs were quantified using programmed thermal analysis (PTA). Two CNTs were used to represent the lower (500 °C) and upper (800 °C) PTA limit of CNT thermal stability. The recovery efficiency of each of the eight chemical reagents evaluated was found to depend on the ability to (1) minimize oxidation of CNTs, (2) remove interfering background carbon from the rat lung tissue, and (3) separate the solid-phase CNTs from the liquid-phase dissolved tissue via centrifugation. A two-step extraction method using Solvable and proteinase K emerged as the optimal approach, enabling a recovery of 98 ± 15% of a 2.9 ± 0.19 µg CNT loading that was spiked into whole rat lungs. Due to its high yield and applicability to low organ burdens of nanomaterials, this extraction method is particularly well suited for in vivo studies to quantify clearance rates and retained CNTs in lungs and other organs.

Assessing the relevance of in vitro studies in nanotoxicology by examining correlations between in vitro and in vivo data.

There is an urgent need for in vitro screening assays to evaluate nanoparticle (NP) toxicity. However, the relevance of in vitro assays is still disputable. We administered doses of TiO(2) NPs of different sizes to alveolar epithelial cells in vitro and the same NPs by intratracheal instillation in rats in vivo to examine the correlation between in vitro and in vivo responses. The correlations were based on toxicity rankings of NPs after adopting NP surface area as dose metric, and response per unit surface area as response metric. Sizes of the anatase TiO(2) NPs ranged from 3 to 100 nm. A cell-free assay for measuring reactive oxygen species (ROS) was used, and lactate dehydrogenase (LDH) release, and protein oxidation induction were the in vitro cellular assays using a rat lung Type I epithelial cell line (R3/1) following 24 h incubation. The in vivo endpoint was number of PMNs in bronchoalveolar lavage fluid (BALF) after exposure of rats to the NPs via intratracheal instillation. Slope analyses of the dose response curves shows that the in vivo and in vitro responses were well correlated. We conclude that using the approach of steepest slope analysis offers a superior method to correlate in vitro with in vivo results of NP toxicity and for ranking their toxic potency.

Validation of an LDH assay for assessing nanoparticle toxicity.

Studies showed that certain cytotoxicity assays were not suitable for assessing nanoparticle (NP) toxicity. We evaluated a lactate dehydrogenase (LDH) assay for assessing copper (Cu-40, 40nm), silver (Ag-35, 35nm; Ag-40, 40nm), and titanium dioxide (TiO(2)-25, 25nm) NPs by examining their potential to inactivate LDH and interference with ß-nicotinamide adenine dinucleotide (NADH), a substrate for the assay. We also performed a dissolution assay for some of the NPs. We found that the copper NPs, because of their high dissolution rate, could interfere with the LDH assay by inactivating LDH. Ag-35 could also inactivate LDH probably because of the carbon matrix used to cage the particles during synthesis. TiO(2)-25 NPs were found to adsorb LDH molecules. In conclusion, NP interference with the LDH assay depends on the type of NPs and the suitability of the assay for assessing NP toxicity should be examined case by case.

A comparative dose-related response of several key pro- and antiinflammatory mediators in the lungs of rats, mice, and hamsters after subchronic inhalation of carbon black.

OBJECTIVE: The objective of this study was to investigate mechanisms underlying species specificity in particle-induced lung inflammation. METHODS: Rats, mice, and hamsters exposed to air, 1, 7, or 50 mg/m3 of carbon black for 13 weeks were killed at 1 day, 3 months, and 11 months after exposure. Bronchoalveolar lavage was performed and characterized for cell number, cell type, reactive oxygen and nitrogen species, and cytokine levels. Ex vivo mutational analysis of inflammatory cells was evaluated by coincubating with lung epithelial cells. Lung tissue was evaluated for gene expression of various antiinflammatory mediators. RESULTS: There was a dose- and time-related effect with all the parameters. Rats demonstrated greater propensity for generating a proinflammatory response, whereas mice and hamsters demonstrated an increased antiinflammatory response. CONCLUSIONS: These differences in pro- and antiinflammatory responses may contribute to the apparent species differences in inflammation and tumorigenesis.

The rat ear vein model for investigating in vivo thrombogenicity of ultrafine particles (UFP).

Recent studies in rodents indicate that intravenous or intratracheal administration of ultrafine particles (UFP) increases thrombogenesis in a surgically exposed peripheral vein after photodynamic excitation of intravenously injected rose bengal (RB). We sought to adapt the invasive peripheral vein RB model to a noninvasive monitoring of ear veins under an inverted microscope. Animals received one of the following: an intraperitoneal, intravenous bolus, or intravenously infused dose of RB. An ear vein was illuminated by a green laser, and formation of a thrombus was captured with a digital camera. Only continuous intravenous infusion produced a steady-state RB plasma level and reproducible thrombus responses in different ear veins of the same rat. This system was then used to study the thrombogenic effects of iv-administered positively or negatively charged 60-nm ultrafine polystyrene particles (PSP). Significant dose-dependent enhancement of thrombus formation was found, as indicated by decreased laser illumination time to 33% of baseline values at 0.5 mg/kg. Negatively charged PSP of the same size failed to affect thrombus formation. We also studied the thrombogenic effect of PSP without the use of RB. The findings were the same as with RB, although the illumination time had to be increased. When 0.5 mg/kg was instilled intratracheally, the laser illumination time to form a thrombus was decreased to 42% of the baseline value, suggesting translocation of UFP into the bloodstream. These results are consistent with previous findings using the invasive model, and they validate the use of this non-invasive ear vein model to evaluate thrombogenic effects of UFP deposition in the respiratory tract.