Antioxidant Defenses of Francisella tularensis Modulate Macrophage Function and Production of Proinflammatory Cytokines

Francisella tularensis, the causative agent of a fatal human disease known as tularemia, has been used in the bioweapon programs of several countries in the past, and now it is considered a potential bioterror agent. Extreme infectivity and virulence of F. tularensis is due to its ability to evade immune detection and to suppress the host''s innate immune responses. However, Francisella-encoded factors and mechanisms responsible for causing immune suppression are not completely understood. Macrophages and neutrophils generate reactive oxygen species (ROS)/reactive nitrogen species as a defense mechanism for the clearance of phagocytosed microorganisms. ROS serve a dual role; at high concentrations they act as microbicidal effector molecules that destroy intracellular pathogens, and at low concentrations they serve as secondary signaling messengers that regulate the expression of various inflammatory mediators. We hypothesized that the antioxidant defenses of F. tularensis maintain redox homeostasis in infected macrophages to prevent activation of redox-sensitive signaling components that ultimately result in suppression of pro-inflammatory cytokine production and macrophage microbicidal activity. We demonstrate that antioxidant enzymes of F. tularensis prevent the activation of redox-sensitive MAPK signaling components, NF-κB signaling, and the production of pro-inflammatory cytokines by inhibiting the accumulation of ROS in infected macrophages. We also report that F. tularensis inhibits ROS-dependent autophagy to promote its intramacrophage survival. Collectively, this study reveals novel pathogenic mechanisms adopted by F. tularensis to modulate macrophage innate immune functions to create an environment permissive for its intracellular survival and growth.     

Activation tagging in indica rice identifies ribosomal proteins as potential targets for manipulation of water‐use efficiency and abiotic stress tolerance in plants

We have generated 3900 enhancer‐based activation‐tagged plants, in addition to 1030 stable Dissociator‐enhancer plants in a widely cultivated indica rice variety, BPT‐5204. Of them, 3000 were screened for water‐use efficiency (WUE) by analysing photosynthetic quantum efficiency and yield‐related attributes under water‐limiting conditions that identified 200 activation‐tagged mutants, which were analysed for flanking sequences at the site of enhancer integration in the genome. We have further selected five plants with low Δ¹³C, high quantum efficiency and increased plant yield compared with wild type for a detailed investigation. Expression studies of 18 genes in these mutants revealed that in four plants one of the three to four tagged genes became activated, while two genes were concurrently up‐regulated in the fifth plant. Two genes coding for proteins involved in 60S ribosomal assembly, RPL6 and RPL23A, were among those that became activated by enhancers. Quantitative expression analysis of these two genes also corroborated the results on activating–tagging. The high up‐regulation of RPL6 and RPL23A in various stress treatments and the presence of significant cis‐regulatory elements in their promoter regions along with the high up‐regulation of several of RPL genes in various stress treatments indicate that they are potential targets for manipulating WUE/abiotic stress tolerance.     

Inter‐laboratory validation of a rapid assay for the detection and quantification of Legionella spp. in water samples

Aims: To compare the standard culture method with a new, rapid test (ScanVIT‐Legionella?) using fluorescently labelled gene probes for the detection and enumeration of Legionella spp. The new technique was validated through experiments conducted on both artificially and naturally contaminated water and through an inter‐laboratory comparison. Methods and Results: All samples were processed by the ScanVIT test according to the manufacturer’s instructions and by a culture method (ISO 11731). ScanVIT detected significantly more positive samples, although concentrations were similar and a strong positive correlation between the two methods was observed (r = 0·888, P < 0·001). The new test was more accurate in identifying the co‐presence of Legionella pneumophila and Leg. non‐pneumophila. ScanVIT showed a slightly higher Legionella recovery from water samples artificially contaminated with Leg. pneumophila alone or together with Pseudomonas aeruginosa. Lastly, the inter‐laboratory comparison revealed that the ScanVIT test exhibits a lower variability than the traditional culture test (mean coefficient of variation 8·7 vs 16·1%). Conclusions: The results confirmed that the ScanVIT largely overlaps the reference method and offers advantages in terms of sensitivity, quantitative reliability and reduced assay time. Significance and Impact of the Study: The proposed method may represent a useful validated alternative to traditional culture for the rapid detection and quantification of Legionella spp. in water.     

A blood meal-induced Ixodes scapularis tick saliva serpin inhibits trypsin and thrombin,and interferes with platelet aggregation and blood clotting

Ixodes scapularis is a medically important tick species that transmits causative agents of important human tick-borne diseases including borreliosis, anaplasmosis and babesiosis. An understanding of how this tick feeds is needed prior to the development of novel methods to protect the human population against tick-borne disease infections. This study characterizes a blood meal-induced I. scapularis (Ixsc) tick saliva serine protease inhibitor (serpin (S)), in-house referred to as IxscS-1E1. The hypothesis that ticks use serpins to evade the host’s defense response to tick feeding is based on the assumption that tick serpins inhibit functions of protease mediators of the host’s anti-tick defense response. Thus, it is significant that consistent with hallmark characteristics of inhibitory serpins, Pichia pastoris-expressed recombinant IxscS-1E1 (rIxscS-1E1) can trap thrombin and trypsin in SDS- and heat-stable complexes, and reduce the activity of the two proteases in a dose-responsive manner. Additionally, rIxscS-1E1 also inhibited, but did not apparently form detectable complexes with, cathepsin G and factor Xa. Our data also show that rIxscS-1E1 may not inhibit chymotrypsin, kallikrein, chymase, plasmin, elastase and papain even at a much higher rIxscS-1E1 concentration. Native IxscS-1E1 potentially plays a role(s) in facilitating I. scapularis tick evasion of the host’s hemostatic defense as revealed by the ability of rIxscS-1E1 to inhibit adenosine diphosphate- and thrombin-activated platelet aggregation, and delay activated partial prothrombin time and thrombin time plasma clotting in a dose-responsive manner. We conclude that native IxscS-1E1 is part of the tick saliva protein complex that mediates its anti-hemostatic, and potentially inflammatory, functions by inhibiting the actions of thrombin, trypsin and other yet unknown trypsin-like proteases at the tick–host interface.     

Reusable vs. disposable cups revisited: guidance in life cycle comparisons addressing scenario,model, and parameter uncertainties for the US consumer

Purpose

Despite interest in an environmentally conscious decision between disposable and reusable cups, a comprehensive and current study for US consumers is not yet available. Guidance in favor of single-use cups rely on outdated or non-ISO-compliant results with limited uncertainty information. Such claims are insufficiently generalizable. This article delivers an updated comparative life cycle impact assessment of reusable ceramic cups and single-use expanded polystyrene cups.

Methods

The ReCiPe midpoint model was selected. Scenario uncertainties are addressed by evaluating compliant standard dishwashing appliance models from 2004 to 2013 used in 26 US subregional utility grids. A utility snapshot from 2009 is applied with extension to recent shifts in generation from increased penetration of natural gas and renewable energy. Parameter uncertainty is quantified through statistical methods.

Results

Where there is statistical difference, results almost entirely favor reusable cups in the USA. For climate change, 16 % of users have higher impact for ceramic cups washed in 2013 by minimally compliant dishwashers. Higher climate change impacts for 32 % of reusable cup users is indicated with 2004 average dishwashers, though using a cup twice between washes shifts the impact in favor of the reusable cup.

Conclusions

Disposable cup scenarios do not account for film sleeves, lids, printing, and less conservative shipping weights and distances and therefore reflect a best case scenario. Impact for reusable cups is expected to decrease further as the electricity mix becomes less CO₂-intensive with replacement of coal-fired generators by natural gas, wind, and solar and as less efficient dishwashers are replaced with new units compliant to current laws.