Secretome analysis of Trichoderma atroviride T17 biocontrol of Guignardia citricarpa

The fungal species Guignardia citricarpa is an important pathogen in citriculture. Members of the fungal genus Trichoderma are recognized as biocontrol agents but studies on the interactions between both fungi are scarce. This study aimed to identify extracellular proteins secreted by Trichoderma atroviride T17 that are related to the control of G. citricarpa. Two-dimensional gel electrophoresis (2D) was used to study the patterns of proteins secreted by T. atroviride T17 in medium containing glucose (control) and in medium containing G. citricarpa GC3 inactivated mycelium. We identified 59 of the 116 spots differentially expressed (50.86%) by LC–MS/MS. Of these, we highlight the presence of glycoside hydrolases (CAZy families 3, 43, 54, 76 and 93), chitinase, mutanase, a-1,3-glucanase, a-1,2-mannosidase, carboxylic hydrolase ester, carbohydrate-binding module family 13, glucan 1,3-b-glucosidase, a-galactosidase and Neutral protease 2. These proteins are related to mycoparasitism processes, stimuli and therefore to the biological control of pathogens. The results obtained are in agreement with reports describing an increase in the secretion of proteins related to mycoparasitism and biological control and a reduction in the secretion of proteins related to the metabolism of Trichoderma species grown in the presence of the pathogen. Moreover, these results are pioneer in understanding T. atroviride interaction with G. citricarpa. For the first time, we identified potential candidate proteins that may have a role in the antagonism mechanism of G. citricarpa by T. atroviride T17. Thus our results shed a light into the molecular mechanisms that T. atroviride use to control G. citricarpa.

AtDeg2 – a chloroplast protein with dual protease/chaperone activity

Chloroplast protease AtDeg2 (an ATP-independent serine endopeptidase) is cytosolically synthesized as a precursor, which is imported into the chloroplast stroma and deprived of its transit peptide. Then the mature protein undergoes routing to its functional location at the stromal side of thylakoid membrane. In its linear structure AtDeg2 molecule contains the protease domain with catalytic triad (HDS) and two PDZ domains (PDZ1 and PDZ2). In vivo AtDeg2 most probably exists as a supposedly inactive haxamer, which may change its oligomeric stage to form active 12-mer, or 24-mer. AtDeg2 has recently been demonstrated to exhibit dual protease/chaperone function. This review is focused on the current awareness with regard to AtDeg2 structure and functional significance.

Role of Aeromonas hydrophila in bacterial septicemia of cultured carps in Khouzestan Province and investigation on protectivity of bacterin (s) prepared from acute isolate(s) of it

Motile Aeromonas are the most common bacteria of freshwater in the world that cause disease in fish and other cold-blooded and warm-blooded hosts. Among this group of bacteria, Aeromonas hydrophila is important in causing complications such as fin rot, skin ulcers and lethal hemorrhagic septicemia in fish. Several virulence factors involved in the pathogenesis of Aeromonas hydrophila, including extracellular enzymes (protease, lipase, elastase, gelatinase and nuclease) and toxins. From the exotoxins, hemolysin, aerolysin and cytolytic enterotoxin play an important role in pathogenesis. Detection of virulence markers by PCR as a key component of determining the pathogenesis of the bacteria and using indigenous vaccines for better immunization against this disease is important. In this study, a total of 200 fanned carps (126 common carp. 39 silver carp and 35 of grass carp) with symptoms suspected aeromonas septicemia were isolated from Khouzestan province farms. 125 bacteria belong to Aeromonas genus detected by biochemical and PCR methods. 31 of all isolates recognized as Aeromonas hydrophila with biochemical methods, I6srRNA detection and Lipase genes. Results showed that the role of Aeromonas sp. and Aeromonas hydrophila in fish with disease symptoms were 62.5% and 15.5% respectively. By using specific primers, three virulence genes including hemolysin, aerolysine and cytolytic enterotoxin were detected in these confirmed isolates, that 18 isolates (58/06%) hemolysin positive (hlyA +), 16 isolates (51/61%) aerolysine positive (aerA+) and 23 isolates (74/19%) for cytolytic enterotoxin gene (act+) were positive. The result of present study showed that most of the confirmed isolates genotype was hlyA+ act- with frequency equal to 51/61%. For investigating the protection effect of acut strain of bacteria, UV inactivated bacterin was used.

Naturally Occurring Resistance-Associated Variants of Hepatitis C Virus Protease Inhibitors in Poor Responders to Pegylated Interferon-Ribavirin

International audience

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). DLS melting curves were measured for met-HbGp at different concentrations. SAXS temperature studies were performed for oxy-, cyanomet- and met-HbGp forms, at several pH values. At pH 5.0 and 6.0, the scattering curves are identical from 20 to 60 degrees C, and R-g is 108 angstrom, independent of the oxidation form. At pH 7.0, protein denaturation and aggregation occurs above 55 degrees C and 60 degrees C, for oxy and met-HbGp, respectively. Cyanomet-HbGp, at pH 7.0, is stable up to 60 degrees C. At alkaline pH (8.0-9.0) and higher temperature, an irreversible dissociation process is observed, with a decrease of R-g, D-max and I(0). Analysis by p(r), obtained from GNOM, and OLIGOMER, was used to fit the SAXS experimental scattering curves by a combination of theoretical curves obtained for HbLt fragments from the crystal structure. Our results show clearly the increasing contribution of smaller molecular weight fragments, as a function of increasing pH and temperature, as well as, the order of thermal stabilities: cyanomet-> oxy- > met-HbGp. (C) 2012 Elsevier B.V. All rights reserved.

Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth

Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

Estudo do papel desempenhado pelo heterotetrâmero de anexina A2 em células de cancro da mama hipóxicas

Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Associação entre o proteassoma e o inibidor de protease BTCI : caracterização fisico-química e efeitos moleculares em células de câncer de mama (MCF-7)

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2010.

Intra- and extracellular osmotic regulation in the hololimnetic Caridea and Anomura: a phylogenetic perspective on the conquest of fresh water by the decapod Crustacea

We investigate extra- and intracellular osmoregulatory capability in two species of hololimnetic Caridea and Anomura: Macrobrachium brasiliense, a palaemonid shrimp, and Aegla franca, an aeglid anomuran, both restricted to continental waters. We also appraise the sharing of physiological characteristics by the hololimnetic Decapoda, and their origins and role in the conquest of fresh water. Both species survive salinity exposure well. While overall hyperosmoregulatory capability is weak in A. franca and moderate in M. brasiliense, both species strongly hyporegulate hemolymph [Cl-] but not osmolality. Muscle total free amino acids (FAA) increase slowly but markedly in response to the rapid rise in hemolymph osmolality consequent to hyperosmotic challenge: 3.5-fold in A. franca and 1.9-fold in M. brasiliense. Glycine, taurine, arginine, alanine and proline constitute a parts per thousand 85% of muscle FAA pools in fresh water; taurine, arginine, alanine each contribute a parts per thousand 22% in A. franca, while glycine predominates (70%) in M. brasiliense. These FAA also show the greatest increases on salinity challenge. Muscle FAA titers correlate strongly (R = 0.82) with hemolymph osmolalities across the main decapod sub/infraorders, revealing that marine species with high hemolymph osmolalities achieve isosmoticity of the intra- and extracellular fluids partly through elevated intracellular FAA concentrations; freshwater species show low hemolymph osmolalities and exhibit reduced intracellular FAA titers, consistent with isosmoticity at a far lower external osmolality. Given the decapod phylogeny adopted here and their multiple, independent invasions of fresh water, particularly by the Caridea and Anomura, our findings suggest that homoplastic strategies underlie osmotic and ionic homeostasis in the extant freshwater Decapoda.

Unified selective sorting approach to analyse multi-electrode extracellular data

Extracellular data analysis has become a quintessential method for understanding the neurophysiological responses to stimuli. This demands stringent techniques owing to the complicated nature of the recording environment. In this paper, we highlight the challenges in extracellular multi-electrode recording and data analysis as well as the limitations pertaining to some of the currently employed methodologies. To address some of the challenges, we present a unified algorithm in the form of selective sorting. Selective sorting is modelled around hypothesized generative model, which addresses the natural phenomena of spikes triggered by an intricate neuronal population. The algorithm incorporates Cepstrum of Bispectrum, ad hoc clustering algorithms, wavelet transforms, least square and correlation concepts which strategically tailors a sequence to characterize and form distinctive clusters. Additionally, we demonstrate the influence of noise modelled wavelets to sort overlapping spikes. The algorithm is evaluated using both raw and synthesized data sets with different levels of complexity and the performances are tabulated for comparison using widely accepted qualitative and quantitative indicators.

Targeting CD44, ABCG2 and CD133 markers using aptamers: in silico analysis of CD133 extracellular domain 2 and its aptamer

The application of nucleic acid aptamers for the diagnosis and therapy of cancer stem cells (CSCs) is expanding. The current study truncated and probed various existing aptamers against CSC markers CD44, ABCG2 and CD133 in retinoblastoma (RB) primary cells, cell lines, a breast cancer cell line and MCF7-sphere. Truncated CD44 aptamer retained its specific binding to cancer cells, ABCG2+ve MCF7-spheres and CD133+ve RB cells. Similarly, ABCG2 and CD133 aptamers showed higher affinity to ABCG2+ve, CD133+ve cells than the negative population and cell lines. All aptamers appreciably reduced viability of up to 50% and 32% of the primary RB tumor cells and cell lines, respectively. Colony formation of MCF7, RB cell lines and MCF7-sphere growth were inhibited significantly. Structure prediction, simulation of CD133 extracellular domain 2 (ExD2) and A15 followed by docking to comprehend the potential interaction revealed hydrogen bonds and non bonded interactions between them. This information could be used to improve the A15 aptamer to gain more interactions with CD133. Thus approaches undertaken here can be applied universally for cell-specific targeting, and the aptamers studied against CSC markers deserve further in vivo studies.

Determination of Extracellular Molecules Produced by Vibrio Harveyi Using MS/MS

Quorum sensing (QS) is a process that allows bacteria to sense the population density of cells around them by communicating with each other via autoinducer molecules. This cross-communication is crucial in the regulation of bacterial processes such as bioluminescence, virulence, and biofilm formation. Previous research by Milburn and Makemson on Vibrio harveyi suggested that in addition of the known biosynthesis of three well-characterized autoinducers, dozens of unknown molecules are also produced and released to the environment by V. harveyi. This study was performed using electrospray tandem mass spectrometry with the purpose of detection and characterization of the extracellular molecules produced by V. harveyi, and assessment of their relationship to QS. A total of 11 molecules were characterized, from which three could be related to QS. These findings provide a glimpse of the nature of novel secondary metabolites produced by V. harveyi and provide the groundwork for further research.

Candida albicans Modifies the Protein Composition and Size Distribution of THP1 macrophages-derived Extracellular Vesicles.

The effectiveness of macrophages in the response to systemic candidiasis is crucial to an effective clearance of the pathogen. The secretion of proteins, mRNAs, non-coding RNAs and lipids through extracellular vesicles (EVs) is one of the mechanisms of communication between immune cells. EVs change their cargo to mediate different responses, and may play a role in the response against infections. Thus, we have undertaken the first quantitative proteomic analysis on the protein composition of THP1 macrophages-derived EVs during the interaction with Candida albicans. This study revealed changes in EVs sizes and in protein composition, and allowed the identification and quantification of 717 proteins. Of them, 133 proteins changed their abundance due to the interaction. The differentially abundant proteins were involved in functions relating to immune response, signaling, or cytoskeletal reorganization. THP1-derived EVs, both from control and from Candida-infected macrophages, had similar effector functions on other THP1-differenciated macrophages, activating ERK and p38 kinases, and increasing both the secretion of proinflammatory cytokines and the candidacidal activity; while in THP1 non-differenciated monocytes, only EVs from infected macrophages increased significantly the TNF-α secretion. Our findings provide new information on the role of macrophage-derived EVs in response to C. albicans infection and in macrophages communication.