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Find video protocols related to scientific articles indexed in Pubmed.
In Vivo Monitoring of Staphylococcus aureus Biofilm Infections and Antimicrobial Therapy by [18F]Fluoro-Deoxyglucose-MicroPET in a Mouse Model.
Antimicrob. Agents Chemother.
PUBLISHED: 08-25-2014
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A mouse model was developed for in vivo monitoring of infection and the effect of antimicrobial treatment against Staphylococcus aureus biofilms, using the [(18)F]fluoro-deoxyglucose-MicroPET ([(18)F]FDG-MicroPET) image technique. In the model, sealed Vialon catheters were briefly precolonized with S. aureus strains ATCC 15981 or V329, which differ in cytotoxic properties and biofilm matrix composition. After subcutaneous implantation of catheters in mice, the S. aureus strain differences found in bacterial counts and the inflammatory reaction triggered were detected by the regular bacteriological and histological procedures and also by [(18)F]FDG-MicroPET image signal intensity determinations in the infection area and regional lymph node. Moreover, [(18)F]FDG-MicroPET imaging allowed the monitoring of the rifampin treatment effect, identifying the periods of controlled infection and those of reactivated infection due to the appearance of bacteria naturally resistant to rifampin. Overall, the mouse model developed may be useful for noninvasive in vivo determinations in studies on S. aureus biofilm infections and assessment of new therapeutic approaches.
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Brucella abortus depends on pyruvate phosphate dikinase and malic enzyme but not on Fbp and GlpX fructose-1,6-bisphosphatases for full virulence in laboratory models.
J. Bacteriol.
PUBLISHED: 06-16-2014
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The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolpyruvate carboxykinase (pckA), pyruvate phosphate dikinase (ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ?ppdK and ?mae mutants was severely impaired and growth of the double ?fbp-?glpX mutant was reduced. In macrophages, only the ?ppdK and ?mae mutants showed reduced multiplication, and studies with the ?ppdK mutant confirmed that it reached the replicative niche. Similarly, only the ?ppdK and ?mae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt.
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The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus.
Microb. Pathog.
PUBLISHED: 05-03-2014
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Brucellosis is a worldwide extended zoonosis caused by Brucella spp. These gram-negative bacteria are not readily detected by innate immunity, a virulence-related property largely linked to their surface lipopolysaccharide (LPS). The role of the LPS lipid A and O-polysaccharide in virulence is well known. Moreover, mutation of the glycosyltransferase gene wadC of Brucella abortus, although not affecting O-polysaccharide assembly onto the lipid-A core section causes a core oligosaccharide defect that increases recognition by innate immunity. Here, we report on a second gene (wadB) encoding a LPS core glycosyltransferase not involved in the assembly of the O-polysaccharide-linked core section. As compared to wild-type B. abortus, a wadB mutant was sensitive to bactericidal peptides and non-immune serum, and was attenuated in mice and dendritic cells. These observations show that as WadC, WadB is also involved in the assembly of a branch of Brucella LPS core and support the concept that this LPS section is a virulence-related structure.
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Acupuncture in the management of acute dental pain.
J Acupunct Meridian Stud
PUBLISHED: 04-22-2014
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Acute dental pain is the main reason for seeking dental services to provide urgent dental care; there is consensus about the use of alternative therapies, such as acupuncture, to control dental pain in pre-dental care. This study aimed to evaluate the use of acupuncture in reducing the intensity of acute dental pain in pre-dental care in patients waiting for emergency dental care, and was conducted at the After-Hours Emergency Dental Clinic of Piracicaba Dental School, and at the Emergency Center Dental Specialties I in Piracicaba, São Paulo, Brazil. The sample consisted of 120 patients. The Visual Analog Scale (VAS) was used to measure pain intensity. All patients underwent one session of acupuncture; the points LI4, ST44 and CV23 were selected and were used alone or in combinations. Reduction in pain was observed in 120 patients (mean initial VAS=6.558±1.886, p<0; mean final VAS=0.962±2.163, p<0.00001). The results of this study indicate that acupuncture analgesia could be a technical adjunct to pain control in patients with acute dental pain, contributing to the restoration of health with social benefit.
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Simultaneous infections by different Salmonella strains in mesenteric lymph nodes of finishing pigs.
BMC Vet. Res.
PUBLISHED: 03-03-2014
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Salmonellosis is a major worldwide zoonosis, and Salmonella-infected finishing pigs are considered one of the major sources of human infections in developed countries. Baseline studies on salmonellosis prevalence in fattening pigs in Europe are based on direct pathogen isolation from mesenteric lymph nodes (MLN). This procedure is considered the most reliable for diagnosing salmonellosis in apparently healthy pigs. The presence of simultaneous infections by different Salmonella strains in the same animal has never been reported and could have important epidemiological implications.
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Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice.
Vet. Res.
PUBLISHED: 01-23-2014
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Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.
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Relative contributions of lipooligosaccharide inner and outer core modifications to nontypeable Haemophilus influenzae pathogenesis.
Infect. Immun.
PUBLISHED: 08-26-2013
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Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.
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Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine.
Vet. Res.
PUBLISHED: 08-17-2013
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Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production.
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Hypertension management algorithm for type 2 diabetic patients applied in primary care.
Diabetol Metab Syndr
PUBLISHED: 06-26-2013
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Hypertension frequently coexists with type 2 diabetes (DM), and increases the risk of cardiovascular outcomes. The aim of the study was to obtain/maintain blood pressure (BP) goals (ADA/JNC 7) according to a stepwise algorithm using the medication supplied by the Brazilian government.
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Expansion of undergraduate courses in nursing: dilemmas and contradictions facing the labor market.
Rev Esc Enferm USP
PUBLISHED: 06-24-2013
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We sought to analyze, from the perspective of professors and students, the reasons and consequences of the expansion of undergraduate courses in nursing, discussing the dilemmas and the contradictions confronting the labor market. It was a qualitative study with data obtained from focus groups, conducted in 18 undergraduate nursing courses in the state of Minas Gerais, during the period of February to October of 2011. The narratives were submitted to critical discourse analysis. The results indicated that the education of the nurse was permeated by insecurity as to the future integration into the labor market. The insecurity translates into dilemmas that referred to employability and the precariousness of the working conditions. In this context, employment in the family health strategy emerges as a mirage. One glimpses the need for a political agenda with the purpose of discussion about education, the labor market and the determinants of these processes.
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Effect of different types of self-management education in patients with diabetes.
Rev Assoc Med Bras
PUBLISHED: 02-11-2013
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Education plays an important role in diabetes mellitus (DM) treatment, as it enables patients to manage their disease. There is a wide range of tested educational interventions, and, to date, no universal model that can be standardized and recognized as effective for all individuals with the disease has been defined. This article aims to review the effect of different types of educational interventions for self-management of glycemic control in patients with DM type 2, in addition to define general recommendations for this treatment strategy.
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Predictive value of electrocardiogram in diagnosing acute coronary artery lesions among patients with out-of-hospital-cardiac-arrest.
Resuscitation
PUBLISHED: 01-14-2013
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Acute coronary lesions are known to be the most common trigger of out of hospital cardiac arrest (OHCA). Aim of the present study was to assess the predictive value of ST-segment changes in diagnosing the presence of acute coronary lesions among OHCA patients
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Effect of peroxides on spermine transport in rat brain and liver mitochondria.
Amino Acids
PUBLISHED: 03-16-2011
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The polyamine spermine is transported into the matrix of various types of mitochondria by a specific uniporter system identified as a protein channel. This mechanism is regulated by the membrane potential; other regulatory effectors are unknown. This study analyzes the transport of spermine in the presence of peroxides in both isolated rat liver and brain mitochondria, in order to evaluate the involvement of the redox state in this mechanism, and to compare its effect in both types of mitochondria. In liver mitochondria peroxides are able to inhibit spermine transport. This effect is indicative of redox regulation by the transporter, probably due to the presence of critical thiol groups along the transport pathway, or in close association with it, with different accessibility for the peroxides and performing different functions. In brain mitochondria, peroxides have several effects, supporting the hypothesis of a different regulation of spermine transport. The fact that peroxovanadate can inhibit tyrosine phosphatases in brain mitochondria suggests that mitochondrial spermine transport is regulated by tyrosine phosphorylation in this organ. In this regard, the evaluation of spermine transport in the presence of Src inhibitors suggests the involvement of Src family kinases in this process. It is possible that phosphorylation sites for Src kinases are present in the channel pathway and have an inhibitory effect on spermine transport under regulation by Src kinases. The results of this study suggest that the activity of the spermine transporter probably depends on the redox and/or tyrosine phosphorylation state of mitochondria, and that its regulation may be different in distinct organs.
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Further characterization of agmatine binding to mitochondrial membranes: involvement of imidazoline I2 receptor.
Amino Acids
PUBLISHED: 03-14-2011
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Agmatine, a divalent diamine with two positive charges at physiological pH, is transported into the matrix of liver mitochondria by an energy-dependent mechanism, the driving force of which is the electrical membrane potential. Its binding to mitochondrial membranes is studied by applying a thermodynamic treatment of ligand-receptor interactions on the analyses of Scatchard and Hill. The presence of two mono-coordinated binding sites S(1) and S(2), with a negative influence of S(2) on S(1), has been demonstrated. The calculated binding energy is characteristic for weak interactions. S(1) exhibits a lower binding capacity and higher binding affinity both of about two orders of magnitude than S(2). Experiments with idazoxan, a ligand of the mitochondrial imidazoline receptor I(2), demonstrate that S(1) site is localized on this receptor while S(2) is localized on the transport system. S(1) would act as a sensor of exogenous agmatine concentration, thus modulating the transport of the amine by its binding to S(2).
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Brucella abortus ornithine lipids are dispensable outer membrane components devoid of a marked pathogen-associated molecular pattern.
PLoS ONE
PUBLISHED: 01-07-2011
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The brucellae are ?-Proteobacteria facultative intracellular parasites that cause an important zoonosis. These bacteria escape early detection by innate immunity, an ability associated to the absence of marked pathogen-associated molecular patterns in the cell envelope lipopolysaccharide, lipoproteins and flagellin. We show here that, in contrast to the outer membrane ornithine lipids (OL) of other Gram negative bacteria, Brucella abortus OL lack a marked pathogen-associated molecular pattern activity. We identified two OL genes (olsB and olsA) and by generating the corresponding mutants found that olsB deficient B. abortus did not synthesize OL or their lyso-OL precursors. Liposomes constructed with B. abortus OL did not trigger IL-6 or TNF-? release by macrophages whereas those constructed with Bordetella pertussis OL and the olsB mutant lipids as carriers were highly active. The OL deficiency in the olsB mutant did not promote proinflammatory responses or generated attenuation in mice. In addition, OL deficiency did not increase sensitivity to polymyxins, normal serum or complement consumption, or alter the permeability to antibiotics and dyes. Taken together, these observations indicate that OL have become dispensable in the extant brucellae and are consistent within the trend observed in ?-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity.
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The use of green fluorescent protein as a marker for Brucella vaccines.
Vaccine
PUBLISHED: 04-24-2010
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Brucellosis is an important malady of productive and wildlife animals and a worldwide zoonosis. The use of effective vaccines and the corresponding diagnostic tests that allow differentiating infected from vaccinated animals are essential tools to control the disease. For this, a prototype of Brucella abortus S19 vaccine expressing green fluorescent protein (S19-GFP) was constructed. The S19-GFP was readily identified under ultraviolet light by macroscopic and microscopic examination and maintained all the biochemical characteristics of the parental S19 vaccine. S19-GFP replicated ex vivo and in vivo, and protected mice against challenge with virulent B. abortus to the same extent as the isogenic S19. An immunoenzymatic assay designed to measure anti-GFP antibodies allowed the discrimination between mice vaccinated with S19-GFP and those immunized with S19. Both vaccines raised antibodies against lipopolysaccharide molecule to similar levels. This experimental model constitutes a "proof of concept" for the use of Brucella-GFP vaccines and associated diagnostic tests to distinguish vaccinated from naturally Brucella infected animals.
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Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures.
Int. J. Biochem. Cell Biol.
PUBLISHED: 12-16-2009
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It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process. Cobalt, in the ionized species Co(2+), is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co(2+) induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca(2+) transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co(2+) also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent DeltaPsi drop, suggesting that Co(2+) also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co(2+) of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co(2+) also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co(2+) results in apoptosis induction with caspase activation and increased level of expression of HIF-1alpha. All these observations allow us to state that, in the presence of calcium, Co(2+) is an inducer of apoptosis triggered by mitochondrial oxidative stress.
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Right ventricle in pulmonary arterial hypertension: haemodynamics, structural changes, imaging, and proposal of a study protocol aimed to assess remodelling and treatment effects.
Eur J Echocardiogr
PUBLISHED: 10-07-2009
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Although right ventricular (RV) failure is the main cause of death in patients with pulmonary arterial hypertension (PAH), there is insufficient data about the effects of PAH treatment on RV geometry and function mainly because the RV assessment has been hampered by its complex crescentic shape, large infundibulum, and its trabecular nature. Echocardiography is a widely available imaging technique particularly suitable for follow-up studies, because of its non-invasive nature, low cost, and lack of ionizing radiation or radioactive agent. Real-time three-dimensional echocardiography (RT3DE) has been shown to be accurate in assessing RV and left ventricular (LV) volumes, stroke volumes, and ejection fractions in comparison with cardiac magnetic resonance imaging. In this review, we describe RV structural and functional changes which occur in patients with PAH and strengths and weaknesses of current non-invasive imaging techniques to assess them. Finally, we describe an ongoing multicentre, prospective observational study involving seven centres expert in treating patients with PAH from four different countries. Investigators will use conventional and advanced echo parameters from RT3DE and speckle-tracking echocardiography to assess the extent of LV and RV remodelling before symptom onset and during pharmacological treatment in patients with PAH. Seventy patients who will survive for at least 1 year will be recruited. All the participating institutions will perform comprehensive standard 2D and Doppler as well as RT3DE examinations with a pre-defined imaging protocol. Measurements will be performed at the core echocardiography laboratory by experienced observers who will be unaware of each patients treatment assignment and whether the examination was a baseline or a follow-up study. Enrolment duration is expected to be 1 year.
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First-principles study of low Miller index Ni3S2 surfaces in hydrotreating conditions.
J Phys Chem B
PUBLISHED: 08-27-2009
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Density functional theory (DFT) calculations combined with surface thermodynamic arguments and the Gibbs-Curie-Wulff equilibrium morphology formalism have been employed to explore the effect of the reaction conditions, temperature (T), and gas-phase partial pressures (PH2 and PH2S) on the stability of nickel sulfide (Ni3S2) surfaces. Furthermore, the strength and nature of chemical bonds for selected Ni3S2 surface cuts were investigated with the quantum theory of atoms in molecules methodology. A particular analysis of the electrostatic potential within this theoretical framework is performed to study the potential activity of nickel sulfide nanoparticles as hydrodesulfurization (HDS) catalysts. The calculated thermodynamic surface stabilities and the resulting equilibrium morphology model suggest that unsupported Ni3S2 nanoparticles mainly expose (111) and (111) type surface faces in HDS conditions. Analysis of the electrostatic potential mapped onto a selected electron density isocontour (0.001 au) on those expose surface reveals a poor potential reactivity toward electron-donating reagents (i.e., low Lewis acidity). Consequently, a very low attraction between coordinatively unsaturated active sites (Lewis sites) exposed at the catalytic particles and the S atoms coming from reagent polluting molecules does inactive these kinds of particles for HDS.
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Long-term results of early condylar fracture correction: case report.
Dent Traumatol
PUBLISHED: 02-22-2009
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Condylar fractures in childhood occur frequently, often with minimal pain and discomfort and therefore the diagnosis is not made at the time of injury. Management may be surgical or non-surgical. Non-surgical therapy in children is the method of choice if the condyle can translate normally. In fact, there is an excellent chance of regeneration and continued normal development after fracture in growing patients. The purpose of this article is to describe the long-term clinical and radiological evaluation of a conservatively treated unilateral condylar fracture, a result of trauma, in a 6-year-old patient. In addition, she presented a congenitally missing lower incisor ipsilateral to the fracture and a class II malocclusion. She was treated with functional jaw orthopedics using a splint and an activator and subsequent orthodontic treatment with fixed appliances [J Orofac Orthop5 (2002) 429]. The remodeling process of the condylar head and neck is clearly observed in the panoramic radiographs of the 12-year follow-up records presented.
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[Nursing education and the challenges for health promotion].
Rev Bras Enferm
PUBLISHED: 02-17-2009
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This is a descriptive-exploratory study with qualitative approach whose objective was analyze the health promotion on nursing education. The subjects were teachers, students & professionals whom receive the students on the scenarios of learningship of two courses of graduation in Minas Gerais/Brazil. The results indicate the incorporation of health promotion as a referencing for nursing formation favored by schoolservice articulation as well as an important strategy. In the mean time, it needs major valorization as a condition to maintain the changes occurred in nursing formation. It concludes that is necessary strategic actions for change the sanitary and education practices involving the good practices of health promotion on overcoming the challenges to change the nursing education.
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Rough mutants defective in core and O-polysaccharide synthesis and export induce antibodies reacting in an indirect ELISA with smooth lipopolysaccharide and are less effective than Rev 1 vaccine against Brucella melitensis infection of sheep.
Vaccine
PUBLISHED: 01-11-2009
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Classical brucellosis vaccines induce antibodies to the O-polysaccharide section of the lipopolysaccharide that interfere in serodiagnosis. Brucella rough (R) mutants lack the O-polysaccharide but their usefulness as vaccines is controversial. Here, Brucella melitensis R mutants in all main lipopolysaccharide biosynthetic pathways were evaluated in sheep in comparison with the reference B. melitensis Rev 1 vaccine. In a first experiment, these mutants were tested for ability to induce anti-O-polysaccharide antibodies, persistence and spread through target organs, and innocuousness. Using the data obtained and those of genetic studies, three candidates were selected and tested for efficacy as vaccines against a challenge infecting 100% of unvaccinated ewes. Protection by R vaccines was 54% or less whereas Rev 1 afforded 100% protection. One-third of R mutant vaccinated ewes became positive in an enzyme-linked immunosorbent assay with smooth lipopolysaccharide due to the core epitopes remaining in the mutated lipopolysaccharide. We conclude that R vaccines interfere in lipopolysaccharide immunosorbent assays and are less effective than Rev 1 against B. melitensis infection of sheep.
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Lipopolysaccharide as a target for brucellosis vaccine design.
Microb. Pathog.
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The gram-negative bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a world wide-distributed zoonotic disease that represents a serious problem for animal and human health. There is no human-to-human contagion and, since there is no human vaccine, animal vaccination is essential to control brucellosis. However, current vaccines (all developed empirically) do not provide 100% protection and are infectious in humans. Attempts to generate new vaccines by obtaining mutants lacking the lipopolysaccharide O-polysaccharide, in purine metabolism or in Brucella type IV secretion system have not been successful. Here we propose a new approach to develop brucellosis vaccines based on the concept that Brucella surface molecules evade efficient detection by innate immunity, thus delaying protective Th1 responses and opening a time window to reach sheltered intracellular compartments. We showed recently that a branch of the core oligosaccharide section of Brucella lipopolysaccharide hampers recognition by TLR4-MD2. Mutation of glycosyltransferase WadC, involved in the synthesis of this branch, results in a lipopolysaccharide that, while keeping the O-polysaccharide essential for optimal protection, shows a truncated core, is more efficiently recognized by MD2 and triggers an increased cytokine response. In keeping with this, the wadC mutant is attenuated in dendritic cells and mice. In the mouse model of brucellosis vaccines, the Brucella abortus wadC mutant conferred protection similar to that provided by S19, the best cattle vaccine available. The properties of the wadC mutant provide the proof of concept for this new approach and open the way for more effective brucellosis vaccines.
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The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition.
PLoS Pathog.
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Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.
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The extradomain a of fibronectin enhances the efficacy of lipopolysaccharide defective Salmonella bacterins as vaccines in mice.
Vet. Res.
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The Extradomain A from fibronectin (EDA) has an immunomodulatory role as fusion protein with viral and tumor antigens, but its effect when administered with bacteria has not been assessed. Here, we investigated the adjuvant effect of EDA in mice immunizations against Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis). Since lipopolysaccharide (LPS) is a major virulence factor and the LPS O-polysaccharide (O-PS) is the immunodominant antigen in serological diagnostic tests, Salmonella mutants lacking O-PS (rough mutants) represent an interesting approach for developing new vaccines and diagnostic tests to differentiate infected and vaccinated animals (DIVA tests). Here, antigenic preparations (hot-saline extracts and formalin-inactivated bacterins) from two Salmonella Enteritidis rough mutants, carrying either intact (SE?waaL) or deep-defective (SE?gal) LPS-Core, were used in combination with EDA. Biotinylated bacterins, in particular SE?waaL bacterin, decorated with EDAvidin (EDA and streptavidin fusion protein) improved the protection conferred by hot-saline or bacterins alone and prevented significantly the virulent infection at least to the levels of live attenuated rough mutants. These findings demonstrate the adjuvant effect of EDAvidin when administered with biotinylated bacterins from Salmonella Enteritidis lacking O-PS and the usefulness of BEDA-SE?waaL as non-live vaccine in the mouse model.
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What have we learned from brucellosis in the mouse model?
Vet. Res.
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Brucellosis is a zoonosis caused by Brucella species. Brucellosis research in natural hosts is often precluded by practical, economical and ethical reasons and mice are widely used. However, mice are not natural Brucella hosts and the course of murine brucellosis depends on bacterial strain virulence, dose and inoculation route as well as breed, genetic background, age, sex and physiological statu of mice. Therefore, meaningful experiments require a definition of these variables. Brucella spleen replication profiles are highly reproducible and course in four phases: i), onset or spleen colonization (first 48?h); ii), acute phase, from the third day to the time when bacteria reach maximal numbers; iii), chronic steady phase, where bacterial numbers plateaus; and iv), chronic declining phase, during which brucellae are eliminated. This pattern displays clear physiopathological signs and is sensitive to small virulence variations, making possible to assess attenuation when fully virulent bacteria are used as controls. Similarly, immunity studies using mice with known defects are possible. Mutations affecting INF-?, TLR9, Myd88, T?? and TNF-? favor Brucella replication; whereas IL-1?, IL-18, TLR4, TLR5, TLR2, NOD1, NOD2, GM-CSF, IL/17r, Rip2, TRIF, NK or Nramp1 deficiencies have no noticeable effects. Splenomegaly development is also useful: it correlates with IFN-? and IL-12 levels and with Brucella strain virulence. The genetic background is also important: Brucella-resistant mice (C57BL) yield lower splenic bacterial replication and less splenomegaly than susceptible breeds. When inoculum is increased, a saturating dose above which bacterial numbers per organ do not augment, is reached. Unlike many gram-negative bacteria, lethal doses are large (? 108 bacteria/mouse) and normally higher than the saturating dose. Persistence is a useful virulence/attenuation index and is used in vaccine (Residual Virulence) quality control. Vaccine candidates are also often tested in mice by determining splenic Brucella numbers after challenging with appropriate virulent brucellae doses at precise post-vaccination times. Since most live or killed Brucella vaccines provide some protection in mice, controls immunized with reference vaccines (S19 or Rev1) are critical. Finally, mice have been successfully used to evaluate brucellosis therapies. It is concluded that, when used properly, the mouse is a valuable brucellosis model.
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The extradomain A of fibronectin (EDA) combined with poly(I:C) enhances the immune response to HIV-1 p24 protein and the protection against recombinant Listeria monocytogenes-Gag infection in the mouse model.
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The development of effective vaccines against HIV-1 infection constitutes one of the major challenges in viral immunology. One of the protein candidates in vaccination against this virus is p24, since it is a conserved HIV antigen that has cytotoxic and helper T cell epitopes as well as B cell epitopes that may jointly confer enhanced protection against infection when used in immunization-challenge approaches. In this context, the adjuvant effect of EDA (used as EDAp24 fusion protein) and poly(I:C), as agonists of TLR4 and TLR3, respectively, was assessed in p24 immunizations using a recombinant Listeria monocytogenes HIV-1 Gag proteins (Lm-Gag, where p24 is the major antigen) for challenge in mice. Immunization with EDAp24 fusion protein together with poly(I:C) adjuvant induced a specific p24 IFN-? production (Th1 profile) as well as protection against a Lm-Gag challenge, suggesting an additive or synergistic effect between both adjuvants. The combination of EDA (as a fusion protein with the antigen, which may favor antigen targeting to dendritic cells through TLR4) and poly(I:C) could thus be a good adjuvant candidate to enhance the immune response against HIV-1 proteins and its use may open new ways in vaccine investigations on this virus.
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