Roles of Gβγ in membrane recruitment and activation of p110γ/p101 phosphoinositide 3-kinase γ

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3–binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110γ/p101 PI3Kγ is activated by Gβγ on stimulation of G protein–coupled receptors. It is currently unknown whether in living cells Gβγ acts as a membrane anchor or an allosteric activator of PI3Kγ, and which role its noncatalytic p101 subunit plays in its activation by Gβγ. Using GFP-tagged PI3Kγ subunits expressed in HEK cells, we show that Gβγ recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein–mediated activation of PI3Kγ in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3–binding PH domains. Furthermore, membrane-targeted p110γ displayed basal enzymatic activity, but was further stimulated by Gβγ, even in the absence of p101. Therefore, we conclude that in vivo, Gβγ activates PI3Kγ by a mechanism assigning specific roles for both PI3Kγ subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of Gβγ with p110γ contributes to activation of PI3Kγ

Monovision-based vehicle detection, distance and relative speed measurement in urban traffic

This study presents a monovision-based system for on-road vehicle detection and computation of distance and relative speed in urban traffic. Many works have dealt with monovision vehicle detection, but only a few of them provide the distance to the vehicle which is essential for the control of an intelligent transportation system. The system proposed integrates a single camera reducing the monetary cost of stereovision and RADAR-based technologies. The algorithm is divided in three major stages. For vehicle detection, the authors use a combination of two features: the shadow underneath the vehicle and horizontal edges. They propose a new method for shadow thresholding based on the grey-scale histogram assessment of a region of interest on the road. In the second and third stages, the vehicle hypothesis verification and the distance are obtained by means of its number plate whose dimensions and shape are standardised in each country. The analysis of consecutive frames is employed to calculate the relative speed of the vehicle detected. Experimental results showed excellent performance in both vehicle and number plate detections and in the distance measurement, in terms of accuracy and robustness in complex traffic scenarios and under different lighting conditions

Trace Metals in Soot and PM2.5from Heavy-Fuel-Oil Combustion in a Marine Engine

Heavy fuel oil (HFO) particulate matter (PM) emitted by marine engines is known to contain toxic heavy metals, including vanadium (V) and nickel (Ni). The toxicity of such metals will depend on the their chemical state, size distribution, and mixing state. Using online soot-particle aerosol mass spectrometry (SP-AMS), we quantified the mass of five metals (V, Ni, Fe, Na, and Ba) in HFO-PM soot particles produced by a marine diesel research engine. The in-soot metal concentrations were compared to in-PM2.5measurements by inductively coupled plasma-optical emission spectroscopy (ICP-OES). We found that <3% of total PM2.5metals was associated with soot particles, which may still be sufficient to influence in-cylinder soot burnout rates. Since these metals were most likely present as oxides, whereas studies on lower-temperature boilers report a predominance of sulfates, this result implies that the toxicity of HFO PM depends on its combustion conditions. Finally, we observed a 4-to-25-fold enhancement in the ratio V:Ni in soot particles versus PM2.5, indicating an enrichment of V in soot due to its lower nucleation/condensation temperature. As this enrichment mechanism is not dependent on soot formation, V is expected to be generally enriched within smaller HFO-PM particles from marine engines, enhancing its toxicity

Biased Competition in Visual Processing Hierarchies: A Learning Approach Using Multiple Cues

In this contribution, we present a large-scale hierarchical system for object detection fusing bottom-up (signal-driven) processing results with top-down (model or task-driven) attentional modulation. Specifically, we focus on the question of how the autonomous learning of invariant models can be embedded into a performing system and how such models can be used to define object-specific attentional modulation signals. Our system implements bi-directional data flow in a processing hierarchy. The bottom-up data flow proceeds from a preprocessing level to the hypothesis level where object hypotheses created by exhaustive object detection algorithms are represented in a roughly retinotopic way. A competitive selection mechanism is used to determine the most confident hypotheses, which are used on the system level to train multimodal models that link object identity to invariant hypothesis properties. The top-down data flow originates at the system level, where the trained multimodal models are used to obtain space- and feature-based attentional modulation signals, providing biases for the competitive selection process at the hypothesis level. This results in object-specific hypothesis facilitation/suppression in certain image regions which we show to be applicable to different object detection mechanisms. In order to demonstrate the benefits of this approach, we apply the system to the detection of cars in a variety of challenging traffic videos. Evaluating our approach on a publicly available dataset containing approximately 3,500 annotated video images from more than 1 h of driving, we can show strong increases in performance and generalization when compared to object detection in isolation. Furthermore, we compare our results to a late hypothesis rejection approach, showing that early coupling of top-down and bottom-up information is a favorable approach especially when processing resources are constrained

Influence of wood species on toxicity of log-wood stove combustion aerosols: A parallel animal and air-liquid interface cell exposure study on spruce and pine smoke

Background Wood combustion emissions have been studied previously either by in vitro or in vivo models using collected particles, yet most studies have neglected gaseous compounds. Furthermore, a more accurate and holistic view of the toxicity of aerosols can be gained with parallel in vitro and in vivo studies using direct exposure methods. Moreover, modern exposure techniques such as air-liquid interface (ALI) exposures enable better assessment of the toxicity of the applied aerosols than, for example, the previous state-of-the-art submerged cell exposure techniques. Methods We used three different ALI exposure systems in parallel to study the toxicological effects of spruce and pine combustion emissions in human alveolar epithelial (A549) and murine macrophage (RAW264.7) cell lines. A whole-body mouse inhalation system was also used to expose C57BL/6 J mice to aerosol emissions. Moreover, gaseous and particulate fractions were studied separately in one of the cell exposure systems. After exposure, the cells and animals were measured for various parameters of cytotoxicity, inflammation, genotoxicity, transcriptome and proteome. Results We found that diluted (1:15) exposure pine combustion emissions (PM1 mass 7.7 ± 6.5 mg m− 3, 41 mg MJ$^{Zahl}$) contained, on average, more PM and polycyclic aromatic hydrocarbons (PAHs) than spruce (PM1 mass 4.3 ± 5.1 mg m− 3, 26 mg MJ− 1) emissions, which instead showed a higher concentration of inorganic metals in the emission aerosol. Both A549 cells and mice exposed to these emissions showed low levels of inflammation but significantly increased genotoxicity. Gaseous emission compounds produced similar genotoxicity and a higher inflammatory response than the corresponding complete combustion emission in A549 cells. Systems biology approaches supported the findings, but we detected differing responses between in vivo and in vitro experiments. Conclusions Comprehensive in vitro and in vivo exposure studies with emission characterization and systems biology approaches revealed further information on the effects of combustion aerosol toxicity than could be achieved with either method alone. Interestingly, in vitro and in vivo exposures showed the opposite order of the highest DNA damage. In vitro measurements also indicated that the gaseous fraction of emission aerosols may be more important in causing adverse toxicological effects. Combustion aerosols of different wood species result in mild but aerosol specific in vitro and in vivo effects

Microbial transformations of selenite by methane-oxidizing bacteria

Abstract Methane oxidizing bacteria are well known for their role in the global methane cycle and their potential for microbial transformation of wide range of hydrocarbon and chlorinated hydrocarbon pollution. Recently, it has also emerged that methane-oxidizing bacteria interact with inorganic pollutants in the environment. Here we report what we believe to be the first study of the interaction of pure strains of methane-oxidizing bacteria with selenite. Results indicate that the commonly used laboratory model strains of methane oxidizing bacteria, Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b are both able to reduce the toxic selenite (SeO32-) but not selenate (SeO42-) to red spherical nanoparticulate elemental selenium (Se0), which was characterised via EDX and EXAFS. The cultures also produced volatile selenium-containing species, which suggests that both strains may have an additional activity that can either transform Se0 or selenite into volatile methylated forms of selenium. Transmission electron microscopy (TEM) measurements and experiments with the cell fractions: cytoplasm, cell wall and cell membrane show that the nanoparticles are formed mainly on the cell wall. Collectively these results are promising for the use of methane-oxidizing bacteria for bioremediation or suggest possible uses in the production of selenium nanoparticles for biotechnology

Particulate matter from both heavy fuel oil and diesel fuel shipping emissions show strong biological effects on human lung cells at realistic and comparable in vitro exposure conditions

Background: Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. Objectives: To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. Methods: Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. Results: The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon (“soot”). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. Conclusions: Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices

The gut microbiota: a major player in the toxicity of environmental pollutants?

Exposure to environmental chemicals has been linked to various health disorders, including obesity, type 2 diabetes, cancer and dysregulation of the immune and reproductive systems, whereas the gastrointestinal microbiota critically contributes to a variety of host metabolic and immune functions. We aimed to evaluate the bidirectional relationship between gut bacteria and environmental pollutants and to assess the toxicological relevance of the bacteria–xenobiotic interplay for the host. We examined studies using isolated bacteria, faecal or caecal suspensions—germ-free or antibiotic-treated animals—as well as animals reassociated with a microbiota exposed to environmental chemicals. The literature indicates that gut microbes have an extensive capacity to metabolise environmental chemicals that can be classified in five core enzymatic families (azoreductases, nitroreductases, β-glucuronidases, sulfatases and β-lyases) unequivocally involved in the metabolism of >30 environmental contaminants. There is clear evidence that bacteria-dependent metabolism of pollutants modulates the toxicity for the host. Conversely, environmental contaminants from various chemical families have been shown to alter the composition and/or the metabolic activity of the gastrointestinal bacteria, which may be an important factor contributing to shape an individual’s microbiotype. The physiological consequences of these alterations have not been studied in details but pollutant-induced alterations of the gut bacteria are likely to contribute to their toxicity. In conclusion, there is a body of evidence suggesting that gut microbiota are a major, yet underestimated element that must be considered to fully evaluate the toxicity of environmental contaminants

Expression and purification of recombinant G protein-coupled receptors: A review

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques