A Nanoconjugate Apaf-1 Inhibitor Protects Mesothelial Cells from Cytokine-Induced Injury

Background

Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization.

Methodology

Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma.Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice.

Conclusion

Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.     

Covariance between disturbance and soil resources dictates the invasibility of northern fescue prairies

Increasing environmental impacts of exotic organisms have refocused attention on the ability of diverse communities to resist biological invaders. Although resource availability, often related to natural and anthropogenic disturbances, appears central to the invasibility of biological communities, understanding the links between resources, diversity and invasibility is often confounded by the covariance among key variables. To test the hypothesis that community invasibility remains contingent on the type and intensity of disturbance and their impacts on plant community diversity and resource availability, we designed an experiment testing the invasibility of northern fescue prairies by smooth brome (Bromus inermis Leyss.), a Eurasian perennial grass, threatening the structure and function of prairie remnants throughout the Great Plains. Using soil disturbances and herbicide, we imposed treatments manipulating the diversity and resource availability of native prairies. Our observations demonstrate that the vulnerability of native prairies to exotic plant invasions remains contingent on resources. While the establishment of smooth brome seedlings increased with increasing disturbance, its impact depended on the availability of soil nitrogen. As a result, soil burial treatments, simulating disturbance by northern pocket gophers, provided poor recruitment areas for smooth brome, and their low levels of soil moisture and nitrogen, combined with the rapid recovery of the prairie community, compromised seedling establishment. Emphasizing the covariance of diversity and key environmental variables following disturbance, our findings illustrate the importance of disturbance type and intensity on community invasibility. Such a consideration is critical in the conservation and restoration of native prairie remnants throughout the Great Plains.     

Differential expression of pathogenicity- and virulence-related genes of Xanthomonas axonopodis pv. citri under copper stress

In this study, we used real-time quantitative PCR (RT-qPCR) to evaluate the expression of 32 genes of Xanthomonas axonopodis pv. citri related to pathogenicity and virulence that are also involved in copper detoxification. Nearly all of the genes were up-regulated, including copA and copB. Two genes homologous to members of the type II secretion system (xcsH and xcsC) and two involved in the degradation of plant cell wall components (pglA and pel) were the most expressed in response to an elevated copper concentration. The type II secretion system (xcs operon) and a few homologues of proteins putatively secreted by this system showed enhanced expression when the bacteria were exposed to a high concentration of copper sulfate. The enhanced expression of the genes of secretion II system during copper stress suggests that this pathway may have an important role in the adaptative response of X. axonopodis pv. citri to toxic compounds. These findings highlight the potential role of these genes in attenuating the toxicity of certain metals and could represent an important means of bacterial resistance against chemicals used to control diseases.     

Efficient rhizosphere colonization by Pseudomonas fluorescens f113 mutants unable to form biofilms on abiotic surfaces

Motility is a key trait for rhizosphere colonization by Pseudomonas fluorescens. Mutants with reduced motility are poor competitors, and hypermotile, more competitive phenotypic variants are selected in the rhizosphere. Flagellar motility is a feature associated to planktonic, free‐living single cells, and although it is necessary for the initial steps of biofilm formation, bacteria in biofilm lack flagella. To test the correlation between biofilm formation and rhizosphere colonization, we have used P. fluorescens F113 hypermotile derivatives and mutants affected in regulatory genes which in other bacteria modulate biofilm development, namely gacS (G), sadB (S) and wspR (W). Mutants affected in these three genes and a hypermotile variant (V35) isolated from the rhizosphere were impaired in biofilm formation on abiotic surfaces, but colonized the alfalfa root apex as efficiently as the wild‐type strain, indicating that biofilm formation on abiotic surfaces and rhizosphere colonization follow different regulatory pathways in P. fluorescens. Furthermore, a triple mutant gacSsadBwspR (GSW) and V35 were more competitive than the wild‐type strain for root‐tip colonization, suggesting that motility is more relevant in this environment than the ability to form biofilms on abiotic surfaces. Microscopy showed the same root colonization pattern for P. fluorescens F113 and all the derivatives: extensive microcolonies, apparently held to the rhizoplane by a mucigel that seems to be plant produced. Therefore, the ability to form biofilms on abiotic surfaces does not necessarily correlates with efficient rhizosphere colonization or competitive colonization.     

Study of simultaneous experimental colonization of Meriones unguiculatus (Mongolian gerbils) by cagPAI+ and cagPAI− strains of Helicobacter pylori

Helicobacter pylori has a chromosomal pathogenicity island (cagPAI), and the presence or absence of this Island places the microorganism into two types of strains: cagPAI+ which is associated to serious infectious processes, and cagPAI? related to mild to moderate infectious events. Simultaneous colonization by cagPAI+ and cagPAI? strains is frequent and these bacteria can interact among themselves.The aim of this project was to analyze the interaction between cagPAI+ and cagPAI? strains of H. pylori in experimental infection, using the Mongolian gerbil as an experimental animal model.We employed J99 (cagPAI+) and 251F (cagPAI?) strains, and obtained 3 derivate strains in successive isolation from experimentally infected gerbils. By RAPD–PCR we found that cagPAI+ and cagPAI? underwent genetic rearrangement during the gerbil-adaptation process. We identified individual isolates from gerbils, and by in situ hybridization we established that both type of strains were able to colonize the same regions of the host’s stomach, and induce a mild to moderate inflammatory process.We studied the competence between cagPAI+ and cagPAI? strains by simultaneous and sequential infections. The study shows that in both colonization experiments, the cagPAI? strains were more efficient than cagPAI+ strains in colonizing the infected host by displacing cagPAI+.     

Role of IL-13 in a model of Strongyloides venezuelensis infection in rats

IL-13 is a cytokine known to play a role in several pulmonary diseases, including asthma and fibrosis. The role of IL-13 in the context of pulmonary changes induced by helminth infection is unclear. Rats experimentally infected with Strongyloides venezuelensis and treated with anti-IL-13 neutralizing antibody were used to evaluate the role of IL-13 on functional and inflammatory changes of host lungs, and on parasite control. S. venezuelensis-induced airway hyperreactivity was IL-13-independent, but IL-13 played an essential role in driving airway mucus production and eosinophil infiltration. IL-13 was important for the control of egg production but not establishment in the intestine.     

Asymmetric microtubule pushing forces in nuclear centering

Dynamic properties of microtubules contribute to the establishment of spatial order within cells. In the fission yeast Schizosaccharomyces pombe, interphase cytoplasmic microtubules are organized into antiparallel bundles that attach to the nuclear envelope and are needed to position the nucleus at the geometric center of the cell. Here, we show that after the nucleus is displaced by cell centrifugation, these microtubule bundles efficiently push the nucleus back to the center. Asymmetry in microtubule number, length, and dynamics contributes to the generation of force responsible for this unidirectional movement. Notably, microtubules facing the distal cell tip are destabilized when the microtubules in the same bundle are pushing from the proximal cell tip. The CLIP-170-like protein tip1p and the microtubule-bundling protein ase1p are required for this asymmetric regulation of microtubule dynamics, indicating contributions of factors both at microtubule plus ends and within the microtubule bundle. Mutants in these factors are defective in nuclear movement. Thus, cells possess an efficient microtubule-based engine that produces and senses forces for centering the nucleus. These studies may provide insights into mechanisms of asymmetric microtubule behaviors and force sensing in other processes such as chromosome segregation and cell polarization.