Leukotriene B4 mediates neutrophil migration induced by heme

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B(4) (LTB(4)). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B(4) receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO(-/-)) mice. Heme administration in vivo increased peritoneal levels of LTB(4) prior to and during neutrophil recruitment. Evidence that LTB(4) was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB(4) was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB(4) production or neutrophil influx; 4) increased levels of LTB(4) were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB(4). Our findings uncover a crucial role of LTB(4) in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.     

Nematode consumption by mite communities varies in different forest microhabitats as indicated by molecular gut content analysis

Soil animals live in complex and heterogeneous habitats including litter of various types but also microhabitats such as mosses, fungal mats and grass patches. Soil food webs have been separated into a slow fungal and a fast bacterial energy channel. Bacterial-feeding nematodes are an important component of the bacterial energy channel by consuming bacteria and forming prey for higher trophic levels such as soil microarthropods. Investigating the role of nematodes as prey for higher trophic level consumers has been hampered by methodological problems related to their small body size and lack in skeletal structures which can be traced in the gut of consumers. Recent studies using molecular gut content analyses suggest that nematodes form major prey of soil microarthropods including those previously assumed to live as detritivores. Using molecular markers we traced nematode prey in fourteen abundant soil microarthropod taxa of Mesostigmata and Oribatida (both Acari) from three different microhabitats (litter, grass and moss). Consumption of nematodes varied between mite species indicating that trophic niche variation contributes to the high diversity of microarthropods in deciduous forests. Further, consumption of nematodes by Mesostigmata (but not Oribatida) differed between microhabitats indicating that trophic niches vary with habitat characteristics. Overall, the results suggest that free-living bacterial-feeding nematodes form important prey for soil microarthropods including those previously assumed to live as detritivores.     

High-pressure SANS and fluorescence unfolding study of calmodulin

Apo-calmodulin, a small soluble mainly α protein, is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80 °C, whereas the calcium-saturated form is stable up to temperatures as high as 100 °C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al., BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000 bar (300 MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form have been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease have been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.     

An old drug and different ways to treat cutaneous leishmaniasis: Intralesional and intramuscular meglumine antimoniate in a reference center,Rio de Janeiro,Brazil

BackgroundTreatment of cutaneous leishmaniasis (CL) remains challenging since the drugs currently used are quite toxic, thus contributing to lethality unrelated to the disease itself but to adverse events (AE). The main objective was to evaluate different treatment regimens with meglumine antimoniate (MA), in a reference center in Rio de Janeiro, Brazil.MethodologyA historical cohort of 592 patients that underwent physical and laboratory examination were enrolled between 2000 and 2017. The outcome measures of effectiveness were epithelialization and complete healing of cutaneous lesions. AE were graded using a standardized scale. Three groups were evaluated: Standard regimen (SR): intramuscular (IM) MA 10–20 mg Sb⁵⁺/kg/day during 20 days (n = 46); Alternative regimen (AR): IM MA 5 mg Sb⁵⁺/kg/day during 30 days (n = 456); Intralesional route (IL): MA infiltration in the lesion(s) through subcutaneous injections (n = 90). Statistical analysis was performed through Fisher exact and Pearson Chi-square tests, Kruskal-Wallis, Kaplan-Meier and log-rank tests.ResultsSR, AR and IL showed efficacy of 95.3%, 84.3% and 75.9%, with abandonment rate of 6.5%, 2.4% and 3.4%, respectively. IL patients had more comorbidities (58.9%; p = 0.001), were mostly over 50 years of age (55.6%), and had an evolution time longer than 2 months (65.6%; p = 0.02). Time for epithelialization and complete healing were similar in IL and IM MA groups (p = 0.9 and p = 0.5; respectively). Total AE and moderate to severe AE that frequently led to treatment interruption were more common in SR group, while AR and IL showed less toxicity.Conclusions/SignificanceAR and IL showed less toxicity and may be good options especially in CL cases with comorbidities, although SR treatment was more effective. IL treatment was an effective and safe strategy, and it may be used as first therapy option as well as a rescue scheme in patients initially treated with other drugs.     

Human Health Risk of Ingested Nanoparticles That Are Added as Multifunctional Agents to Paints: an In Vitro Study

Microorganisms growing on painted surfaces are not only an aesthetic problem, but also actively contribute to the weathering and deterioration of materials. A widely used strategy to combat microbial colonization is the addition of biocides to the paint. However, ecotoxic, non-degradable biocides with a broad protection range are now prohibited in Europe, so the paint industry is considering engineered nanoparticles (ENPs) as an alternative biocide. There is concern that ENPs in paint might be released in run-off water and subsequently consumed by animals and/or humans, potentially coming into contact with cells of the gastrointestinal tract and affecting the immune system. Therefore, in the present study we evaluated the cytotoxic effects of three ENPs (nanosilver, nanotitanium dioxide and nanosilicon dioxide) that have a realistic potential for use in paints in the near future. When exposed to nanotitanium dioxide and nanosilicon dioxide in concentrations up to 243 µg/mL for 48 h, neither the gastrointestinal cells (CaCo-2) nor immune system cells (Jurkat) were significantly affected. However, when exposed to nanosilver, several cell parameters were affected, but far less than by silver ions used as a control. No differences in cytotoxicity were observed when cells were exposed to ENP-containing paint particles, compared with the same paint particles without ENPs. Paint particles containing ENPs did not affect cell morphology, the release of reactive oxygen species or cytokines, cell activity or cell death in a different manner to the same paint particles without ENPs. The results suggest that paints doped with ENPs do not pose an additional acute health hazard for humans.     

Purification of a new cytochrome P-450 from human liver microsomes

Using a classical methodlogy of purification consisting of three chromatographic steps (Octyl-Sepharose, DEAE-cellulose, CM-cellulos) we have purified a new cytochrome P-450 from human liver microsomes. It was called cytochrome P-450₉. It has been proven to be different from all preceedingly purified human liver microsomal cytochrome P-450 isozymes by its immunological and electrophoretical properties. It does not cross-react with any rat liver cytochrome P-450 and anti-cytochrome P-450₉, does not recognize rat liver microsomes; thus this cytochrome P-450₉ is specific to humans. This cytochrome P-450 isozyme exists in low amounts in human liver microsomes and exhibits an important quatitative polymorphism. In reconstituted system, cytochrome P-450₉ is able to hydroxylate all substrates tested but is not specific on any; its exatc role in xenobiotic metabolism in man remains to be elucidated.     

Quantitative phosphoproteomic analysis of the PI3K‐regulated signaling network

The PI3K pathway is commonly activated in cancer. Only a few studies have attempted to explore the spectrum of phosphorylation signaling downstream of the PI3K cascade. Such insight, however, is imperative to understand the mechanisms responsible for oncogenic phenotypes. By applying MS‐based phosphoproteomics, we mapped 2509 phosphorylation sites on 1096 proteins, and quantified their responses to activation or inhibition of PIK3CA using isogenic knock‐in derivatives and a series of targeted inhibitors. We uncovered phosphorylation changes in a wide variety of proteins involved in cell growth and proliferation, many of which have not been previously associated with PI3K signaling. A significant update of the posttranslational modification database PHOSIDA ( http://www.phosida.com ) allows efficient use of the data. All MS data have been deposited in the ProteomeXchange with identifier PXD003899 ( http://proteomecentral.proteomexchange.org/dataset/PXD003899 ).     

Morphological changes in the middle intestine of the rainbow trout,Salmo gairdneri,induced by a hyperosmotic environment

Summary The structural modifications in the middle intestine of the trout, Salmo gairdneri, induced by transfer to seawater have been studied. During the first two days in seawater, significant distensions of the intercellular spaces are observed between the apical tight junctions and the basement membrane. These dilations are more frequent in the apical part of the intestinal folds. At the basal part of the cell, numerous lamellar processes open in the intercellular spaces. They are closely associated with elongated mitochondria, and are often mixed with small clear vesicles. After seven days in seawater, intercellular spaces are less expanded. Numerous mitochondria are observed in the apical part of the cell, and numerous myelinic bodies with dense granules lie near the nucleus. After one month in seawater, the epithelium resembles that of the freshwater controls; mitochondria are more numerous and other organelles are well developed. The most important modifications of the ultrastructure of the intestine mucosa occur during the first two days in seawater, in correlation with important physiological changes following the abrupt increase of environmental salinity.