MglA and MglB are required for the intramacrophage growth of Francisella novicida

Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (X-p) and designated GB2. Using an in cis complementation strategy, four strains were isolated that are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for both the intracellular growth defect and the colony morphology on LB (X-p) media. The locus consists of an apparent operon of two genes, designated mglAB , for macrophage growth locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 on LB (X-p) media. Sequencing of mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Cell fractionation studies revealed several differences in the protein profiles of mgl mutants compared with wild-type F. novicida . The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli , SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may affect the expression of genes whose products contribute to survival and growth within macrophages.     

A factorial approach for a sugarcane juice-based low cost culture medium: increasing the astaxanthin production by the red yeast Phaffia rhodozyma

A sugarcane juice-based low cost culture medium was previously explored to produce the carotenoid pigment astaxanthin in liquid culture by the red yeast Phaffia rhodozyma (1300?μg astaxanthin/g of dry yeast and 6500?μg/l whole culture medium). Two peculiar limitations in Phaffia are growth temperature (<26?°C) and lack of sugar osmotolerance. Two advantages are the wide biochemical ability for the assimilation and metabolization of disaccharides and the prompt utilization of simple nitrogen sources. For instance, the sucrolytic/ureolytic enzymatic activities deserves exploration. In order to improve the culture medium composition and the conditions of fermentation for highly oxygenated carotenoids (e.g., astaxanthin) a study was carried out with a factorial design in two steps. As a first step, the production of astaxanthin was studied as a function of the nutrient concentration levels and their interactions. The production increase (μg/l) obtained was 23.0% but at the expense of 16.0% pigment content decrease (μg/g). In the second step, the variables pH and agitation level (OTR, oxygen transfer rate) were optimized and then, both goals were attained: the increase of pigment content (418?μg astaxanthin/g of yeast) as well as the absolute pigment production enhancement (1987?μg/l).     

Monitoring Low Molecular Weight Heparins at Therapeutic Levels: Dose-Responses of,and Correlations and Differences between aPTT,Anti-Factor Xa and Thrombin Generation Assays

Background

Low molecular weight heparins (LMWH’s) are used to prevent and treat thrombosis. Tests for monitoring LMWH’s include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH’s varying affinities for FXa and thrombin.

Aim

To examine the effects of two different LMWH’s on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests’ concordance.

Method

Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents.

Results

Methods’ mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (R_s0.62–0.87), whereas the other aPTT methods had similar correlation coefficients (R_s0.80–0.92).

Conclusions

aPTT displays a linear dose-respone to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa’s present gold standard status. Thrombin generation with tissue factor-rich activator is a promising method for monitoring LMWH’s.     

Antibodies to the Core Proteins of Nairobi Sheep Disease Virus/Ganjam Virus Reveal Details of the Distribution of the Proteins in Infected Cells and Tissues

Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.     

Siaα2-3Galβ1- Receptor Genetic Variants Are Associated with Influenza A(H1N1)pdm09 Severity

Different host genetic variants may be related to the virulence and transmissibility of pandemic Influenza A(H1N1)pdm09, influencing events such as binding of the virus to the entry receptor on the cell of infected individuals and the host immune response. In the present study, two genetic variants of the ST3GAL1 gene, which encodes the Siaα2-3Galβ1- receptor to which influenza A(H1N1)pdm09 virus binds for entry into the host cell, were investigated in an admixed Brazilian population. First, the six exons encoding the ST3GAL1 gene were sequenced in 68 patients infected with strain A(H1N1)pdm09. In a second phase of the study, the rs113350588 and rs1048479 polymorphisms identified in this sample were genotyped in a sample of 356 subjects from the northern and northeastern regions of Brazil with a diagnosis of pandemic influenza. Functional analysis of the polymorphisms was performed in silico and the influence of these variants on the severity of infection was evaluated. The results suggest that rs113350588 and rs1048479 may alter the function of ST3GAL1 either directly through splicing regulation alteration and/or indirectly through LD with SNP with regulatory function. In the study the rs113350588 and rs1048479 polymorphisms were in linkage disequilibrium in the population studied (D’ = 0.65). The GC haplotype was associated with an increased risk of death in subjects with influenza (OR = 4.632, 95% CI = 2.10;1.21). The AT haplotype was associated with an increased risk of severe disease and death (OR = 1.993, 95% CI = 1.09;3.61 and OR 4.476, 95% CI = 2.37;8.44, respectively). This study demonstrated for the first time the association of ST3GAL1 gene haplotypes on the risk of more severe disease and death in patients infected with Influenza A(H1N1)pdm09 virus.     

Ontology Alignment Repair through Modularization and Confidence-Based Heuristics

Ontology Matching aims at identifying a set of semantic correspondences, called an alignment, between related ontologies. In recent years, there has been a growing interest in efficient and effective matching methods for large ontologies. However, alignments produced for large ontologies are often logically incoherent. It was only recently that the use of repair techniques to improve the coherence of ontology alignments began to be explored. This paper presents a novel modularization technique for ontology alignment repair which extracts fragments of the input ontologies that only contain the necessary classes and relations to resolve all detectable incoherences. The paper presents also an alignment repair algorithm that uses a global repair strategy to minimize both the degree of incoherence and the number of mappings removed from the alignment, while overcoming the scalability problem by employing the proposed modularization technique. Our evaluation shows that our modularization technique produces significantly small fragments of the ontologies and that our repair algorithm produces more complete alignments than other current alignment repair systems, while obtaining an equivalent degree of incoherence. Additionally, we also present a variant of our repair algorithm that makes use of the confidence values of the mappings to improve alignment repair. Our repair algorithm was implemented as part of AgreementMakerLight, a free and open-source ontology matching system.     

The NLRP3 inflammasome is activated by nanoparticles through ATP,ADP and adenosine

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO₂) and titanium dioxide (nano-TiO₂) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO₂ or nano-TiO₂ caused a significant increase in P2Y1, P2Y2, A2_A and/or A2_B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2_A or A2_B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.The inflammasome is a major factor of the innate immune system acting as a multiprotein platform to activate caspase-1. We showed recently that nanoparticles of TiO₂ (nano-TiO₂) and SiO₂ (nano-SiO₂) are sensed by the NLRP3 inflammasome to induce the release of mature IL-1β,¹ as observed previously with the environmental irritants asbestos or silica.² Despite the identification and characterisation of numerous sterile or microbial activators, the precise mechanisms mediating NLRP3 inflammasome activation remain to be determined. Here, we investigated whether ATP release and purinergic signalling through ATP, ADP and adenosine may be involved in inflammasome activation by nanoparticles. Intracellular ATP is released after cellular stress and/or activation, and purinergic signalling has been shown to modulate inflammation and immunity.^{3, 4} In the extracellular space, ATP is rapidly hydrolysed in a stepwise manner to ADP, AMP (adenosine monophosphate) and adenosine by ectoenzymes.⁴ Adenosine is then irreversibly hydrolysed to inosine by adenosine deaminase (ADA). Extracellular ATP (eATP) signals through both ATP-gated ion channels P2X and G protein-coupled receptor (GPCR) P2Y membrane receptors, whereas ADP signals through P2Y receptors and adenosine through P1 receptors (or A receptors).⁵ P2Y receptors and A receptors may be coupled to the G_q protein, which activates phospholipase C-beta (PLC-β), to the stimulatory G (G_s) protein, which stimulates adenylate cyclase inducing an increase in cyclic AMP (cAMP) levels, or to the G inhibitory (G_i) protein, which inhibits adenylate cyclase. Extracellular adenosine level is the result of adenosine production from extracellular ATP and ADP, its degradation into inosine and its reuptake by cells. Both ATP and adenosine can be transported outside of the cell via diffusion or active transport, whereas only adenosine can enter the cells through adenosine transporters.⁶ Most cells possess equilibrative and concentrative adenosine transporters (respectively, ENTs and CNTs), which allow adenosine to quickly cross the plasma membrane.⁷ Intracellular adenosine is converted to ATP via phosphorylation steps mediated by adenosine kinase (AK) and AMP kinase (AMPK). The basal physiological level of extracellular adenosine has been estimated to be in the range of 30–200 nM.⁸ ATP-derived adenosine and its subsequent signalling through P1 receptors have beneficial roles in acute disease states.^{4, 9} However, during tissue injury, elevated adenosine levels participate in the progression to chronic diseases by promoting aberrant wound healing leading to fibrosis in different organs including the lungs, liver, skin and kidney. In these conditions the blockade of adenosine signalling is beneficial.^{10, 11, 12, 13, 14, 15, 16} In murine models, ADA-knockout mice present high persistent adenosine levels, which lead to airspace enlargement and fibrosis, cardinal signs of COPD and IPF.^{14, 17, 18}Here we investigate in more detail the critical contribution of purinergic signalling in driving NLRP3 inflammasome activation in response to nanoparticles pointing out the effect of ATP, ADP, as well as adenosine and its receptors. We also identify ATP-derived adenosine as a potential activator of the inflammasome.     

Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins

The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO₂⁻ binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO₂⁻ concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO₂−binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.     

Leaf-cutting ants: an unexpected microenvironment holding human opportunistic black fungi

Fungus-growing ants of the genus Atta are known for their leaf-cutting habit, a lifestyle they have maintained since their 50-million-year-old co-evolution with a mutualistic fungus, cultivated as food. Recent studies have highlighted that, in addition to the mutualistic fungus, nests of ants harbor a great diversity of microbial communities. Such microorganisms include the dematiaceous fungi, which are characterized by their melanized cell walls. In order to contribute to the knowledge of fungal ecology, as well as opportunistic strains that may be dispersed by these social insects, we isolated and identified fungi carried by gynes of Atta capiguara and Atta laevigata, collected from colonies located in Fazenda Santana, Botucatu (São Paulo, Brazil). The isolation was carried out using the oil flotation technique, which is suitable for the growth of black fungi. Inoculated plates were incubated at 25 and 35 °C until black cultures were visible (20–45 days). Isolates were identified based on microscopic and molecular characteristics. Some isolated genera were: Cladophialophora, Cladosporium, Exophiala, Ochroconis, Phaeococcomyces, Phialophora and Penidiella. Hyaline species were also found. The results obtained from this work showed that leaf-cutting gynes may contribute to the dispersal of opportunistic dematiaceous fungi. It is suggested that more attention should be paid to this still unexplored subject.