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A recombinant multi-antigen vaccine formulation containing Babesia bovis merozoite surface antigens MSA-2a1, MSA-2b and MSA-2c elicits invasion-inhibitory antibodies and IFN-γ producing cells
No full textAbstract\ud
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Background\ud
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Babesia bovis is a tick-transmitted protozoan hemoparasite and the causative agent of bovine babesiosis, a potential risk to more than 500 million cattle worldwide. The vaccines currently available are based on attenuated parasites, which are difficult to produce, and are only recommended for use in bovines under one year of age. When used in older animals, these vaccines may cause life-threatening clinical symptoms and eventually death. The development of a multi-subunit recombinant vaccine against B. bovis would be attractive from an economic standpoint and, most importantly, could be recommended for animals of any age. In the present study, recombinant ectodomains of MSA-2a1, MSA-2b and MSA-2c antigens were expressed in Pichia pastoris yeast as secreted soluble peptides.\ud
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Results\ud
The antigens were purified to homogeneity, and biochemically and immunologically characterized. A vaccine formulation was obtained by emulsifying a mixture of the three peptides with the adjuvant Montanide ISA 720, which elicited high IgG antibody titers against each of the above antigens. IgG antibodies generated against each MSA-antigen recognized merozoites and significantly inhibited the invasion of bovine erythrocytes. Cellular immune responses were also detected, which were characterized by splenic and lymph node CD4+ T cells producing IFN-γ and TNF-α upon stimulation with the antigens MSA-2a1 or MSA-2c.\ud
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Conclusions\ud
These data strongly suggest the high protective potential of the presented formulation, and we propose that it could be tested in vaccination trials of bovines challenged with B. bovis.This work was supported by grants from CNPq/CONICET (490395/2011-2),\ud
Instituto Nacional de Ciência e Tecnologia de Vacinas (INCTV-CNPq),\ud
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Agencia\ud
Gimenez et al. Parasites & Vectors (2016) 9:577 Page 12 of 13\ud
Nacional de Promocion Cientifica y Tecnologica, Argentina (PICT 2010–0438)\ud
and Instituto Nacional de Tecnologia Agropecuaria, INTA, Argentina (PNBIO\ud
1131034). AMG, ISS and MMR are recipients of fellowships from CNPq
Evaluation of a pharmacogenetic-based warfarin dosing algorithm in patients with low time in therapeutic range – study protocol for a randomized controlled trial
No full textAbstract\ud
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Background\ud
Time in therapeutic range (TTR) is a measurement of quality of warfarin therapy and lower TTR values (<50%) are associated with greater risk of thromboembolic and bleeding events. Recently, we developed a pharmacogenetic-based warfarin dosing algorithm specifically calibrated for a Brazilian patient sample. The aims of this study are: to evaluate the impact of a genetic-based algorithm, compared to traditional anticoagulation, in the time to achieve the therapeutic target and in TTR percentage; and to assess the cost-effectiveness of genotype-guided warfarin dosing in a specific cohort of patients with low TTR (<50%) from a tertiary cardiovascular hospital.\ud
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Methods/design\ud
This study is a randomized controlled trial in patients (n = 300) with atrial fibrillation with TTR < 50%, based on the last three INR values. At the first consultation, patients will be randomized into two groups: TA group (traditional anticoagulation) and PA group (pharmacogenetic anticoagulation). For the first group, the physician will adjust the dose according to current INR value and, for the second group, a pharmacogenetic algorithm will be used. At the second, third, fourth and fifth consultations (with an interval of 7 days each) INR will be measured and, if necessary, the dose will be adjusted based on guidelines. Afterwards, patients who are INR stable will begin measuring their INR in 30 day intervals; if the patient’s INR is not stable, the patient will return in 7 days for a new measurement of the INR. Outcomes measures will include the time to achieve the therapeutic target and the percentage of TTR at 4 and 12 weeks. In addition, as a secondary end-point, pharmacoeconomic analysis will be carried out. Ethical approval was granted by the Ethics Committee for Medical Research on Human Beings of the Clinical Hospital of the University of São Paulo Medical School.\ud
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Discussion\ud
This randomized study will include patients with low TTR and it will evaluate whether a population-specific genetic algorithm might be more effective than traditional anticoagulation for a selected group of poorly anticoagulated patients.\ud
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Trial registration\ud
ClinicalTrials.gov, \ud
NCT02592980\ud
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. Registered on 29 October 2015.This project is funded by the FAPESP, Proc. 2013/09295-3 and CNPQ, Proc.\ud
440324/2014-9
Experimental Lachesis muta rhombeata envenomation and effects of soursop (Annona muricata) as natural antivenom
No full textAbstract\ud
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Background\ud
In the Atlantic forest of the North and Northeast regions of Brazil, local population often uses the fruit juice and the aqueous extract of leaves of soursop (Annona muricata L.) to treat Lachesis muta rhombeata envenomation. Envenomation is a relevant health issue in these areas, especially due to its severity and because the production and distribution of antivenom is limited in these regions. The aim of the present study was to evaluate the relevance of the use of soursop leaf extract and its juice against envenomation by Lachesis muta rhombeata.\ud
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Methods\ud
We evaluated the biochemical, hematological and hemostatic parameters, the blood pressure, the inflammation process and the lethality induced by Lachesis muta rhombeata snake venom. We also assessed the action of the aqueous extract of leaves (AmL) and juice (AmJ) from A. muricata on the animal organism injected with L. m. rhombeata venom (LmrV) in the laboratory environment.\ud
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Results\ud
LmrV induced a decrease of total protein, albumin and glucose; and increase of creatine kinase, aspartate aminotransferase, and urea concentrations. It provoked hemoconcentration followed by reduction of hematocrit, an increase in prothrombin time and partial thromboplastin time and a decrease of the blood pressure. LmrV induced the release of interleukin-6, an increase in neutrophils and changes in the serum protein profile, characteristic of the acute inflammatory process. LD50 values were similar for the groups injected with LmrV and treated or untreated with AmJ and AmL. Both treatments play a role on the maintenance of blood glucose, urea and coagulation parameters and exert a protective action against the myotoxicity. However, they seem to worsen the hypotension caused by LmrV.\ud
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Conclusion\ud
The treatments with AmJ and AmL present some beneficial actions, but they might intensify some effects of the venom. Therefore, additional studies on A. muricata are necessary to enable its use as natural antivenom for bushmaster snakebite.The authors are grateful to Dr. Marcelo Dias Baruffi, Luisa Helena Dias Costa,\ud
Luciana Prado Turin, and Laboratory of Clinical Analysis of School of\ud
Pharmaceutical Sciences of Ribeirão Preto for assistance in clinical analysis. This\ud
study was supported by the following grants: the São Paulo Research\ud
Foundation (FAPESP, grant no. 2005/54855–0 and doctoral scholarship to FAC\ud
2012/13590–8), the National Council for Scientific and Technological\ud
Development (CNPq, masters scholarship to CMC 143472/2011–9) and\ud
Research Support Center in Animal Toxins (NAP-TOXAN-USP, grant no. 12-\ud
125432.1.3). Thanks are also due to the Center for the Study of Venoms and\ud
Venomous Animals (CEVAP) of UNESP for enabling the publication of this paper\ud
(CAPES, grant no\ud
. 23038.006285/2011–21, AUXPE – Toxinologia – 1219/2011)
Hydrogen sulfide modulates chromatin remodeling and inflammatory mediator production in response to endotoxin, but does not play a role in the development of endotoxin tolerance
No full textAbstract\ud
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Background\ud
Pretreatment with low doses of LPS (lipopolysaccharide, bacterial endotoxin) reduces the pro-inflammatory response to a subsequent higher LPS dose, a phenomenon known as endotoxin tolerance. Moreover, hydrogen sulfide (H2S), an endogenous gaseous mediator (gasotransmitter) can exert anti-inflammatory effects. Here we investigated the potential role of H2S in the development of LPS tolerance. THP1 differentiated macrophages were pretreated with the H2S donor NaHS (1 mM) or the H2S biosynthesis inhibitor aminooxyacetic acid (AOAA, 1 mM).\ud
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Methods\ud
To induce tolerance, cells were treated with a low concentration of LPS (0.5 μg/ml) for 4 or 24 h, and then treated with a high concentration of LPS (1 μg/ml) for 4 h or 24 h. In in vivo studies, male wild-type and CSE-/- mice were randomized to the following groups: Control (vehicle); Endotoxemic saline for 3 days before the induction of endotoxemia with 10 mg/kg LPS) mg/kg; Tolerant (LPS at 1 mg/kg for 3 days, followed LPS at 10 mg/kg). Animals were sacrificed after 4 or 12 h; plasma IL-6 and TNF-α levels were measured. Changes in histone H3 and H4 acetylation were analyzed by Western blotting.\ud
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Results\ud
LPS tolerance decreased pro-inflammatory cytokine production. AOAA did not affect the effect of tolerance on reducing cytokine production. Treatment of the cells with the H2S donor reduced cytokine production. Induction of the tolerance increased the acetylation of H3; AOAA reduced histone acetylation. H2S donation increased histone acetylation. Tolerance did not affect the responses to H2S with respect to histone acetylation.\ud
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Conclusions\ud
In conclusion, both LPS tolerance and H2S donation decrease LPS-induced cytokine production in vitro and modulate histone acetylation. However, endogenous, CSE-derived H2S does not appear to play a significant role in the development of LPS tolerance.This work was supported by a grant from the National Institutes of Health\ud
(R01 GM107846) to C.S
Transcriptomic analysis of the stationary phase response regulator SpdR in Caulobacter crescentus
No full textAbstract\ud
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Background\ud
As bacterial cells enter stationary phase, they adjust their growth rate to comply with nutrient restriction and acquire increased resistance to several stresses. These events are regulated by controlling gene expression at this phase, changing the mode of exponential growth into that of growth arrest, and increasing the expression of proteins involved in stress resistance. The two-component system SpdR/SpdS is required for the activation of transcription of the Caulobacter crescentus cspD gene at the onset of stationary phase.\ud
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Results\ud
In this work, we showed that both SpdR and SpdS are also induced upon entry into stationary phase, and this induction is partly mediated by ppGpp and it is not auto-regulated. Global transcriptional analysis at early stationary phase of a spdR null mutant strain compared to the wild type strain was carried out by DNA microarray. Twenty-three genes showed at least twofold decreased expression in the spdR deletion mutant strain relative to its parental strain, including cspD, while five genes showed increased expression in the mutant. The expression of a set of nine genes was evaluated by quantitative real time PCR, validating the microarray data, and indicating an important role for SpdR at stationary phase. Several of the differentially expressed genes can be involved in modulating gene expression, including four transcriptional regulators, and the RNA regulatory protein Hfq. The ribosomal proteins NusE and NusG, which also have additional regulatory functions in transcription and translation, were also downregulated in the spdR mutant, as well as the ParE1 toxin. The purified SpdR protein was shown to bind to the regulatory region of CC0517 by Electrophoretic Mobility Shift Assay, and the SpdR-regulated gene CC0731 was shown to be expressed at a lower level in the null cspD mutant, suggesting that at least part of the effect of SpdR on the expression of this gene is indirect.\ud
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Conclusions\ud
The results indicate that SpdR regulates several genes encoding proteins of regulatory function, which in turn may be required for the expression of other genes important for the transition to stationary phase.We are grateful to Michael T. Laub for making the C. crescentus DNA\ud
microarray slides available, Carla Rosenberg and Andrea Fogaça for\ud
assistance with the microarray experiments, and Julian Munoz for assistance\ud
with antiserum production. This work was supported by São Paulo Research\ud
Foundation (FAPESP, grants 2012/10563-0 and 2014/04046-8). During the\ud
course of this work, CAPTS, RFL and RRM were supported by postdoctoral\ud
fellowship grants 2011/17513-5, 2011/50604-4 and 2011/18847-4 from FAPESP,\ud
respectively. RAR was supported by undergraduate fellowship grant 145213/\ud
2014-5 and MVM was partially supported by a fellowship grant 306558/2013-0,\ud
both from CNPq-Brasil
Microorganisms in the rumen and reticulum of buffalo (Bubalus bubalis) fed two different feeding systems
No full textAbstract\ud
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Background\ud
The community of microorganisms in the rumen and reticulum is influenced by feeding as well as the species and geographical distribution of ruminant animals. Bacteria, methanogenic archaea and ciliate protozoa existing in the rumen and reticulum were evaluated by real-time polymerase chain reaction and light microscopy in buffalo in two feeding systems, grazing and feedlot.\ud
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Results\ud
No significant differences were observed in the total concentrations of bacteria/mL and archaea between rumen and reticulum, and between pasture and feedlots, or interactions between variables. However, the largest density of bacteria and smallest density of archaea was observed in the rumen of grazing animals. The total ciliates protozoa community was higher in grazing buffalo than those in the feedlot on a concentrated diet. There were significant interactions between location in the gastrointestinal tract (rumen vs reticulum) and types of diets (grazing vs feedlot) in the composition of ciliates.\ud
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Conclusions\ud
Our data showed differences in the microbial community of the rumen and reticulum between grazing and feedlot feeding systems demonstrating relevant changes in the microorganism:host relationship existing on rumen–reticulum ecosystem.This work was supported by the Conselho Nacional de Desenvolvimento\ud
Científico e Tecnologico (CNPq) from Federal Government of Brazil
Selection on different genes with equivalent functions: the convergence story told by Hox genes along the evolution of aquatic mammalian lineages
No full textAbstract\ud
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Background\ud
Convergent evolution has been a challenging topic for decades, being cetaceans, pinnipeds and sirenians textbook examples of three independent origins of equivalent phenotypes. These mammalian lineages acquired similar anatomical features correlated to an aquatic life, and remarkably differ from their terrestrial counterparts. Whether their molecular evolutionary history also involved similar genetic mechanisms underlying such morphological convergence nevertheless remained unknown. To test for the existence of convergent molecular signatures, we studied the molecular evolution of Hox genes in these three aquatic mammalian lineages, comparing their patterns to terrestrial mammals. Hox genes are transcription factors that play a pivotal role in specifying embryonic regional identity of nearly any bilateral animal, and are recognized major agents for diversification of body plans.\ud
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Results\ud
We detected few signatures of positive selection on Hox genes across the three aquatic mammalian lineages and verified that purifying selection prevails in these sequences, as expected for pleiotropic genes. Genes found as being positively selected differ across the aquatic mammalian lineages, but we identified a substantial overlap of their developmental functions. Such pattern likely resides on the duplication history of Hox genes, which probably provided different possible evolutionary routes for achieving the same phenotypic solution.\ud
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Conclusions\ud
Our results indicate that convergence occurred at a functional level of Hox genes along three independent origins of aquatic mammals. This conclusion reinforces the idea that different changes in developmental genes may lead to similar phenotypes, probably due to the redundancy provided by the participation of Hox paralogous genes in several developmental functions.MFN holds a grant from Ciencia sem Fronteiras – Atração de Jovens Talentos\ud
CAPES (054/2012). TK was funded by joint grant between FAPESP-Brazil\ud
(2010/52316-3) and CNPq-Brazil (563232/2010-2)
Exploring glycoside hydrolases and accessory proteins from wood decay fungi to enhance sugarcane bagasse saccharification
No full textAbstract\ud
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Background\ud
Glycoside hydrolases (GHs) and accessory proteins are key components for efficient and cost-effective enzymatic hydrolysis of polysaccharides in modern, biochemically based biorefineries. Currently, commercialized GHs and accessory proteins are produced by ascomycetes. However, the role of wood decay basidiomycetes proteins in biomass saccharification has not been extensively pursued. Wood decay fungi degrade polysaccharides in highly lignified tissues in natural environments, and are a promising enzyme source for improving enzymatic cocktails that are designed for in vitro lignocellulose conversion.\ud
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Results\ud
GHs and accessory proteins were produced by representative brown- and white-rot fungi, Laetiporus sulphureus and Pleurotus ostreatus, respectively. Concentrated protein extracts were then used to amend commercial enzymatic cocktails for saccharification of alkaline-sulfite pretreated sugarcane bagasse. The main enzymatic activities found in the wood decay fungal protein extracts were attributed to endoglucanases, xylanases and β-glucosidases. Cellobiohydrolase (CBH) activities in the L. sulphureus and P. ostreatus extracts were low and nonexistent, respectively. The initial glucan conversion rates were boosted when the wood decay fungal proteins were used to replace half of the enzymes from the commercial cocktails. L. sulphureus proteins increased the glucan conversion levels, with values above those observed for the full load of commercial enzymes. Wood decay fungal proteins also enhanced the xylan conversion efficiency due to their high xylanase activities. Proteomic studies revealed 104 and 45 different proteins in the P. ostreatus and L. sulphureus extracts, respectively. The enhancement of the saccharification of alkaline-pretreated substrates by the modified enzymatic cocktails was attributed to the following protein families: GH5- and GH45-endoglucanases, GH3-β-glucosidases, and GH10-xylanases.\ud
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Conclusions\ud
The extracellular proteins produced by wood decay fungi provide useful tools to improve commercial enzyme cocktails that are currently used for the saccharification of alkaline-pretreated lignocellulosic substrates. The relevant proteins encompass multiple glycoside hydrolase families, including the GH5- and GH45-endoglucanases, GH3-β-glucosidases, and GH10-xylanases.The authors thank J.M. Silva and J.C. Tavares for technical assistance. This work\ud
was supported by FAPESP (contract numbers 08/56256-5 and 2014/06923-6),\ud
CNPq (contract numbers 442333/2014-5; 310186/2014-5 and 140796/2013-\ud
4), and CAPES. We gratefully acknowledge the provision of time on the MAS\ud
and NGS facilities (LNBio and CTBE, respectively) at the National Center for\ud
Research in Energy and Materials (CNPEM).\ud
The work was supported by Fundação de Amparo à Pesquisa do Estado de\ud
São Paulo (FAPESP), contract numbers 08/56256-5 and 2014/06923-6, and by\ud
Conselho Nacional de Pesquisa (CNPq), contract numbers 442333/2014-5;\ud
310186/2014-5, 140796/2013-4
Evaluation of AH36 microalloyed steel welded joint by submerged arc welding\ud process with one and two wires
No full textShipbuilding is going through a period of revitalization, growth and technological advancement. One component of these innovations is to improve welding techniques and materials for optimizing processing time, reducing costs and to improve properties and ship performance. The aim of the present work is to evaluate the welding of microalloyed shipbuilding steel, AH36, using the submerged arc welding (SAW) process with one and two wires. The mechanical properties of the welded joint will be presented using microhardness and Charpy V-notch testing. For metallographic characterization, the base metal and welded joint were etched with 2% Nital and Klemm’s I reagent. Scanning electron microscopy, X-ray diffraction and colored etching were used to quantify and verify the presence of martensite and retained austenite, the MA microconstituent. The results obtained from Charpy impact tests and another mechanical test can be correlated with the associated microstructureELAPUSIMINA
E series prostaglandins alter the proliferative, apoptotic and migratory properties of T98G human glioma cells in vitro
No full textAbstract
Background
In many types of cancer, prostaglandin E2 (PGE2) is associated with tumour related processes including proliferation, migration, angiogenesis and apoptosis. However in gliomas the role of this prostanoid is poorly understood. Here, we report on the proliferative, migratory, and apoptotic effects of PGE1, PGE2 and Ibuprofen (IBP) observed in the T98G human glioma cell line in vitro.
Methods
T98G human glioma cells were treated with IBP, PGE1 or PGE2 at varying concentrations for 24–72 hours. Cell proliferation, mitotic index and apoptotic index were determined for each treatment. Caspase-9 and caspase-3 activity was measured using fluorescent probes in live cells (FITC-LEHD-FMK and FITC-DEVD-FMK respectively). The migratory capacity of the cells was quantified using a scratch migration assay and a transwell migration assay.
Results
A significant decrease was seen in cell number (54%) in the presence of 50 μM IBP. Mitotic index and bromodeoxyuridine (BrdU) incorporation were also decreased 57% and 65%, respectively, by IBP. The apoptotic index was increased (167%) and the in situ activity of caspase-9 and caspase-3 was evident in IBP treated cells. The inhibition of COX activity by IBP also caused a significant inhibition of cell migration in the monolayer scratch assay (74%) and the transwell migration assay (36%).
In contrast, the presence of exogenous PGE1 or PGE2 caused significant increases in cell number (37% PGE1 and 45% PGE2). When mitotic index was measured no change was found for either PG treatment. However, the BrdU incorporation rate was significantly increased by PGE1 (62%) and to a greater extent by PGE2 (100%). The apoptotic index was unchanged by exogenous PGs. The addition of exogenous PGs caused an increase in cell migration in the monolayer scratch assay (43% PGE1 and 44% PGE2) and the transwell migration assay (28% PGE1 and 68% PGE2).
Conclusions
The present study demonstrated that treatments which alter PGE1 and PGE2 metabolism influence the proliferative and apoptotic indices of T98G glioma cells. The migratory capacity of the cells was also significantly affected by the change in prostaglandin metabolism. Modifying PG metabolism remains an interesting target for future studies in gliomas