RESUMEN

INTRODUCTION: Septic shock is a systemic inflammatory response syndrome associated with organ failures. Earlier clinical diagnosis would be of benefit to a decrease in the mortality rate. However, there is currently a lack of predictive biomarkers. The secretome is the set of proteins secreted by a cell, tissue, or organism at a given time and under certain conditions. The plasma secretome is easily accessible from biological fluids and represents a good opportunity to discover new biomarkers that can be studied with nontargeted "omic" strategies. AIMS: To identify relevant deregulated proteins (DEP) in the secretome of a rat endotoxemic shock model. METHODS: Endotoxemic shock was induced in rats by intravenous injection of lipopolysaccharides (LPS, S. enterica typhi, 0.5 mg/kg) and compared to controls (Ringer Lactate, iv). Under isoflurane anesthesia, carotid cannulation allowed mean arterial blood pressure (MAP) and heart rate (HR) monitoring and blood sampling at different time points (T0 and T50 or T0 and T90, with EDTA and protease inhibitor). Samples were prepared for large-scale tandem mass spectrometry (MS-MS) based on a label-free quantification to allow identification of the proteins deregulated upon endotoxemic conditions. A Gene Ontology (GO) analysis defined several clusters of biological processes (BP) in which the DEP are involved. RESULTS: Ninety minutes after shock induction, the LPS group presents a reduction in MAP (-45%, p < 0.05) and increased lactate levels (+27.5%, p < 0.05) compared to the control group. Proteomic analyses revealed 10 and 33 DEP in the LPS group, respectively, at 50 and 90 minutes after LPS injection. At these time points, GO-BP showed alterations in pathways involved in oxidative stress response and coagulation. CONCLUSION: This study proposes an approach to identify relevant DEP in septic shock and brings new insights into the understanding of the secretome adaptations upon sepsis.

Asunto(s)

RESUMEN

Hospitalized patients often receive oxygen supplementation, which can lead to a supraphysiological oxygen tension (hyperoxia). Hyperoxia can have hemodynamic effects, including an increase in systemic vascular resistance. This increase suggests hyperoxia-induced vasoconstriction, yet reported direct effects of hyperoxia on vessel tone have been inconsistent. Furthermore, hyperoxia-induced changes in vessel diameter have not been studied in mice, currently the most used mammal model of disease. In this study we set out to develop a pressure-myograph model using isolated vessels from mice for investigation of pathways involved in hyperoxic vasoconstriction. Isolated conduit and resistance arteries (femoral artery and gracilis arteriole, respectively) from C57BL/6 mice were exposed to normoxia (PO2 of 80 mmHg) and three levels of hyperoxia (PO2 of 215, 375 and 665 mmHg) in a no-flow pressure myograph setup. Under the different PO2 levels, dose-response agonist induced endothelium-dependent vasodilation (acetylcholine, arachidonic acid), endothelium-independent vasodilation (s-nitroprusside), as well as vasoconstriction (norepinephrine, prostaglandin F2α) were examined. The investigated arteries did not respond to oxygen by a change in vascular tone. In the dose-response studies, maximal responses and EC50 values to any of the aforementioned agonists were not affected by hyperoxia either. We conclude that arteries and arterioles from healthy mice are not intrinsically sensitive to hyperoxic conditions. The present ex-vivo model is therefore not suitable for further research into mechanisms of hyperoxic vasoconstriction.

Asunto(s)

Asunto(s)

RESUMEN

PURPOSE: Palm kernel nut oil(PKNO) from palm fruit of a rare breed of Elaeis guineensis (virescens), and one of the species belonging to the Elaeis genre of arecaceae family was subjected to Gas chromatography - Mass spectroscopic studies as well as anti-seizure and anti-pyretic studies. It is used in South-Eastern part of Nigeria as antipyretic and anti-seizure agents especially in children. The aim of this study was to evaluate the chemical contents of PKNO using GC-MS and to investigate the antiseizure and antipyretic properties of PKNO using animal model.METHOD: 1500 g Palm Kernels (PK) of the rare breed collected from Abia State were cracked open and the nuts (1000 g) poured into steel vessel and heated until oil was produced after charring the nuts. The oil (PKNO) was analyzed using GC-MS to determine its chemical composition. Yeast induced hyperthermia method was used for anti-pyretic studies while strychnine sulphate induced method was used for anti-seizure studies. PKNO and Octanoic acid (0.5 mg/kg ,1 mg/kg, 2 mg/kg) were administered intraperitoneally (IP).Paracetamol (0.15mg/kg) was used as reference standard for anti-pyretic assay while epilim and tegretol (0.14mg/kg, resp.) were used as reference standards for anti-seizure assay.RESULTS: The major component identified in the extract include: Dodecanoic acid,2,3-dihydroxypropyl ester(19.36 %),n-Hexadecanoic acid(15.49 %),Dodecanoic acid( 12.51 %),Myristic acid ( 6.47 %), Dodecanedioic acid (3.93 %),n-Acetylpyrrolidone (3.67 %) , Thiazole (0.98 %) and Octanoic acid, OA( 3.19 %).The anti-seizure results showed that doses of PKNO and OA used had Significant (p<0.02) anti-seizure activities by delaying the on-set of seizure from time of induction to time of first seizure observed. In yeast induced hyperthermia model, PKNO exhibited good level of anti-pyretic activities by direct reduction of pyrexia with the highest dose, 2.0 mg/kg. The PKNO and OA are proven to be safe till 3000 µl/kg as indicated by LD50 results.CONCLUSION: PKNO and its components mainly fatty acids, thiazole, and n- acetylpyrrolidone are potent and promising agents with anti-pyretic and antiseizure activities

Asunto(s)

RESUMEN

BACKGROUND AND OBJECTIVE: Mild therapeutic hypothermia (MTH) is used to limit neurological injury and improve survival after cardiac arrest (CA) and cardiopulmonary resuscitation, but the optimal mode of cooling is controversial. We therefore compared the effectiveness of MTH using invasive intravascular or non-invasive surface cooling with temperature feedback control. METHODS: This retrospective study in post-CA patients studied the effects of intravascular cooling (CoolGard, Zoll, n=97), applied on the intensive care unit (ICU) in one university hospital compared with those of surface cooling (Medi-Therm, Gaymar, n=76) applied in another university hospital. RESULTS: Time to reach target temperature and cooling speeds did not differ between groups. During the maintenance phase, mean core temperature was 33.1°C (range 32.7-33.7°C) versus 32.5°C (range 31.7-33.4°C) at targets of 33.0 and 32.5°C in intravascularly versus surface cooled patients, respectively. The variation coefficient for temperature during maintenance was higher in the surface than the intravascular cooling group (mean 0.85% vs 0.35%, p<0.0001). ICU survival was 60% and 50% in the intravascularly and surface cooled groups, respectively (NS). Lower age (OR 0.95; 95% CI 0.93 to 0.98; p<0.0001), ventricular fibrillation/ventricular tachycardia as presenting rhythm (OR 7.6; 95% CI 1.8 to 8.9; p<0.0001) and lower mean temperature during the maintenance phase (OR 0.52; 95% CI 0.25 to 1.08; p=0.081) might be independent determinants of ICU survival, while cooling technique and temperature variability did not contribute. CONCLUSIONS: In post-CA patients, intravascular cooling systems result in equal cooling speed, but less variation in temperature during the maintenance phase, as surface cooling. This may not affect the outcome.

Asunto(s)

RESUMEN

OBJECTIVES: Mild therapeutic hypothermia (MTH) is being used to improve neurological outcome and survival in patients successfully resuscitated after cardiac arrest. The impact on coagulation may be difficult to assess since most coagulation parameters are measured at 37°C and not at actual body core temperature. Therefore we investigated the effects of MTH both at body core (target) temperature of 32°C and at 37°C. METHODS: Patients admitted at the ICU after cardiac arrest treated with MTH. Baseline blood samples, measured at 37°C were taken directly at arrival. The second and third samples were drawn within 1h and 24h after reaching target temperature and were measured at 32°C and 37°C. A final sample was drawn when the patient returned to normotemperature (measured at 37°C). Clotting time (CT) and maximum clotting formation (MCF) were measured with thromboelastometry. RESULTS: Upon reaching target temperature (32°C) Extem and Intem CT were increased compared to baseline with 57s (49-75) to 65s (59-72) and 165s (144-183) to 193s (167-212) respectively (median with IQR; P<0.05), with a further significant increase after 24h of hypothermia with 68s (57-80) and 221s (196-266). Samples analyzed at 32°C showed a significant longer CT of 12s in Extem and 33s in Intem compared to 37°C. MCF was not affected by MTH or adjustment of temperature. CONCLUSION: The mild effect of MTH on coagulation parameters remains unidentified when measured at 37°C. Although measurements at 32°C differ from those at 37°C, this does not appear to be of clinical relevance as all values were still within the reference range.

Asunto(s)

Asunto(s)

RESUMEN

BACKGROUND: The importance and value of the principles of evidence-based practice (EBP) in the decision-making process is recognized by speech-language therapists (SLTs) worldwide and as a result curricula for speech-language therapy students incorporated EBP principles. However, the willingness actually to use EBP principles in their future profession not only depends on EBP knowledge and skills, but also on self-efficacy and task value students perceive towards EBP. AIMS: To investigate the relation between EBP knowledge and skills, and EBP self-efficacy and task value in different year groups of Dutch SLT students. METHODS & PROCEDURES: Students from three year groups filled in a tool that measured EBP knowledge and skills: the Dutch Modified Fresno (DMF). EBP self-efficacy and task value were assessed by using a 20-item questionnaire. Both tools were validated for this population. Mean scores for the three year groups were calculated and tested for group differences using a one-way analysis of variance (ANOVA) with a post-hoc Games-Howell procedure. With a multiple linear regression technique it was assessed whether EBP self-efficacy and task value predict learning achievement scores on the DMF. Other possible predictors included in the model were: level of prior education, standard of English, having had mathematics in prior education and the SLT study year. OUTCOME & RESULTS: A total of 149 students filled in both measurement tools. Mean scores on EBP knowledge and skills were significantly different for the three year groups, with students who were further along their studies scoring higher on the DMF. Mean scores on the EBP self-efficacy and task value questionnaire were the same for the three year groups: all students valued EBP positive but self-efficacy was low in all groups. Of the possible predictors, only the year in which students study and EBP self-efficacy were significant predictors for learning achievements in EBP. CONCLUSIONS & IMPLICATIONS: Despite a significant increase in EBP knowledge and skills over the years as assessed by the DMF, the integrated EBP curriculum did not raise levels of EBP self-efficacy and task value. This lack of feeling competent might have an impact on students' willingness actually to use EBP. In curricula, therefore, there should be a focus on how to raise EBP self-efficacy in SLT students. This goes even beyond the educational department because a professional culture in which professionals are competent and confident EBP users would have a positive effect on EBP self-efficacy in students.

Asunto(s)

RESUMEN

BACKGROUND: Worldwide speech-language therapy (SLT) students are educated in evidence-based practice (EBP). For students to use EBP in their future day-to-day clinical practice, they must value EBP as positive and must feel confident in using it. For curricula developers it is therefore important to know the impact their teaching has on these aspects of students' motivational beliefs. AIMS: To develop and validate a measurement tool to assess EBP task value and self-efficacy in SLT students. METHODS & PROCEDURES: A 20-item questionnaire was developed based on a review of the literature and an additional group interview with speech-language therapists. Face validity of the questionnaire was established using a Delphi panel consisting of six EBP lecturers. Dutch bachelor SLT students (n = 149) with a different level of EBP knowledge and skills filled in the newly developed questionnaire. Reliability (internal consistency) was assessed using Cronbach's alpha and internal validity using a principal component analysis (PCA). Construct validity was assessed by comparing the bachelor SLT student scores with a group of m students (n = 15) who were highly experienced in EBP. OUTCOMES & RESULTS: The PCA showed that the questionnaire consists of two components, representing EBP task value and self-efficacy, both with good reliability (Cronbach's α = 0.83 and 0.79, respectively). The hypothesis that master's students would score significantly higher on both components than bachelor SLT students was met. CONCLUSIONS & IMPLICATIONS: The study provides evidence on the internal consistency and construct validity of this questionnaire to evaluate EBP task value and self-efficacy in SLT students. As is common with new measures, more research is needed to evaluate further its psychometric properties.

Asunto(s)

RESUMEN

Ochratoxin A (OTA) is a widespread and abundant natural carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium fungi. Due to the ubiquitous presence of these fungi in food and potential risk for human health, a rapid and sensitive in vitro detection assay is required. Analytical methods for OTA detection/identification are generally based on liquid-liquid extraction, clean-up using an immunoaffinity column (IAC), and identification by reversed-phase high pressure liquid chromatography with fluorescence detection (HPLC-FLD). However, IACs are costly and have a short lifespan. Therefore, an interesting approach would appear to be the design and chemical synthesis of a mimotope peptide simulating mycotoxin-specific antibodies. We have developed a promising alternative method that is based on the use of peptides which are able to bind to specific chemical functions and/or molecular structures. Accordingly, a number of peptides (derived from the structures of major redox proteins) were selected and produced by chemical solid phase syntheses. The ability of such peptides to bind to ochratoxin A was evaluated by HPLC. The peptide NF04 (structurally derived from an oxidoreductase enzyme), which was found to be the sole potently reactive compound among tested molecules, was further evaluated in a peptide-based enzyme-linked immunosorbent assay (peptide-based ELISA), thus confirming its specific interaction with ochratoxin A.

Asunto(s)

RESUMEN

BACKGROUND: General practitioner (GP) trainees state that their trainers are not consistent in using evidence-based medicine (EBM) or are even dismissive of it. As trainers are important role models in the Dutch GP training system this could have a large influence on the EBM training of GP trainees. AIM: To establish the motivations and barriers of Dutch GP trainers in using EBM. METHODS: A questionnaire on personal characteristics, knowledge, skills (Berlin, score 0-15) and attitude (McColl, VAS score 0-100), and statements about EBM barriers were presented to 106 GP trainers. Additionally, three focus group sessions with trainers (n = 30) were held. RESULTS: Knowledge and skills were less than half correct (mean 6.1, standard deviation (SD) 2.9); the overall score on attitude was 58.8 (SD 9.4). Factor analysis showed four categories of barriers: EBM competence (mean 3.5 (SD 0.8)), search activities (mean 3.5 (SD 0.8)), motivation (mean 3.8 (SD 0.7)) and time (mean 2.5 (SD 0.9)). After analysis of the focus group sessions, five categories of motivations and barriers predominated: EBM competence, attitude and behaviour, sources, time and logistics. CONCLUSION: GP trainers experience motivations in EBM; however, these motivations can also be barriers, depending on the trainer's level of knowledge and attitude.

Asunto(s)

RESUMEN

BACKGROUND: Both patients and government expect the GP to treat obesity. Previous studies reported a negative attitude of GPs towards this task. Little is known about the attitude of GP trainees. OBJECTIVES: To assess the attitude and other factors that influence the willingness and ability of GP trainees to provide lifestyle interventions for overweight patients. METHODS: A qualitative study was performed using focus groups, consisting of first- and third-year trainees, GP trainers and teachers. Two researchers analysed the data independently. RESULTS: First-year trainees lack knowledge and a positive attitude. Third-year trainees, although trained in motivational interviewing techniques, lack specific knowledge and feel cheated when discussing eating habits. Trainers are despondent as they rarely observe long-lasting results. Teachers warn the trainees not to have high hopes. The trainers and trainees fear ruining the relationship with their patient, and all make a request for evidence-based multidisciplinary treatment programmes, joint responsibility and an image change in society to stop the epidemic. CONCLUSIONS: Trainees do not feel more competent in treating overweight patients successfully over the course of their GP specialty training and GP trainers are not convinced of the success of the treatment of overweight patients. Therefore, it could be equally important to reflect on the GP trainer as a role model as to concentrate on the education of the trainee. Both need a revived attitude and evidence-based treatment programmes, help from policy makers and an attitude change in society are desired.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channel types. It folds according to an alpha/beta scaffold, i.e., a helix connected to a two stranded beta-sheet by two disulphide bridges. In a former study, various parameters that affect the oxidation and folding of the reduced form of synthetic MTX were investigated in vitro. It was found that MTX achieves its final 3-D structure by evolving over time through a series of oxidation intermediates, from the least to the most oxidized species. MTX oxidative intermediates can be studied by iodoacetamide alkylation of free cysteine residues followed by mass spectrometry analysis. Here, we have analysed the effect of Cu2+ (0.1 to 50 mM) on the kinetics of MTX oxidative folding and found that it dramatically speeds up the formation of the four-disulphide bridged, native-like, MTX (maximal production within 30 minutes instead of > 60 hours). This catalysing effect of Cu2+ was found to be concentration-dependent, reaching a plateau at 10 mM copper ions. Cu2+ was also found to prevent the slow transition of a three disulphide-bridged MTX intermediate towards the final four disulphide-bridged product (12% of total MTX). The data are discussed in light of the potential effects of Cu2+ on MTX secondary structure formation, disulphide bridging and peptidyl prolyl cis-trans isomerization.

Asunto(s)

RESUMEN

A Cochrane systematic review of 5 randomised clinical trials compared the safety and efficacy of expectant management versus curettage for early foetal loss. The expectant-care group was more likely to have an incomplete miscarriage, a need of unplanned curettage, and bleeding. In contrast, curettage was associated with a significantly higher risk ofinfection. Given the lack of clear superiority of either approach, the woman's preference should play a dominant role in reaching a decision.

Asunto(s)

RESUMEN

The NHG practice guideline 'Miscarriage' provides guidelines for the diagnosis and management of pregnant women with vaginal bleeding during the period up to and including the 16th week after the first day of the last menstruation. The guideline has been revised on the basis of the developments over the last few years. The most important modifications are: In case of an imminent miscarriage, more consideration than before is given to the patient's preference with regard to ultrasonography, expectant management and curettage. The GP should therefore discuss the advantages and disadvantages of these options with the patient. A midwife was involved in the formulation of the new guideline. Referral from a GP to a midwife for transvaginal ultrasonography is offered as one of the possibilities. The paragraph on 'information' has been expanded on the basis of the results of a patient focus group.

Asunto(s)

RESUMEN

We have studied the role of five ABC transporter genes (MgAtr to MgAtr5) from the wheat pathogen Mycosphaerella graminicola in multidrug resistance (MDR). Complementation of Saccharomyces cerevisiae mutants with the ABC transporter genes from M. graminicola showed that all the genes tested encode proteins that provide protection against chemically unrelated compounds, indicating that their products function as multidrug transporters with distinct but overlapping substrate specificities. Their substrate range in yeast includes fungicides, plant metabolites, antibiotics, and a mycotoxin derived from Fusarium graminearum (diacetoxyscirpenol). Transformants of M. graminicola in which individual ABC transporter genes were deleted or disrupted did not exhibit clear-cut phenotypes, probably due to the functional redundancy of transporters with overlapping substrate specificity. Independently generated MgAtr5 deletion mutants of M. graminicola showed an increase in sensitivity to the putative wheat defence compound resorcinol and to the grape phytoalexin resveratrol, suggesting a role for this transporter in protecting the fungus against plant defence compounds. Bioassays with antagonistic bacteria indicated that MgAtr2 provides protection against metabolites produced by Pseudomonas fluorescens and Burkholderia cepacia. In summary, our results show that ABC transporters from M. graminicola play a role in protection against toxic compounds of natural and artificial origin.

Asunto(s)

RESUMEN

Three single copy ATP-binding cassette (ABC) transporter encoding genes, designated MgAtr3, MgAtr4, and MgAtr5, were cloned and sequenced from the plant pathogenic fungus Mycosphaerella graminicola. The encoded ABC proteins all exhibit the [NBD-TMS(6)](2) configuration and can be classified as novel members of the pleiotropic drug resistance (PDR) class of ABC transporters. The three proteins are highly homologous to other fungal and yeast, ABC proteins involved in multidrug resistance or plant pathogenesis. MgAtr4 and MgAtr5 possess a conserved ABC motif at both the N- and C-terminal domain of the protein. In contrast, the Walker A motif in the N-terminal and the ABC signature in the C-terminal domain of MgAtr3, deviate significantly from the consensus sequence found in other members of the PDR class of ABC transporters. Expression of MgAtr3 could not be detected under any of the conditions tested. However, MgAtr4 and MgAtr5 displayed distinct expression profiles when treated with a range of compounds known to be either substrates or inducers of ABC transporters. These included synthetic fungitoxic compounds, such as imazalil and cyproconazole, natural toxic compounds, such as the plant defence compounds eugenol and psoralen, and the antibiotics cycloheximide and neomycin. The expression pattern of the genes was also dependent on the morphological state of the fungus. The findings suggest a role for MgAtr4 and MgAtr5 during plant pathogenesis and in protection against toxic compounds.

Asunto(s)

RESUMEN

The Xenopus laevis South African frog oocyte is a well suited and widely used system for protein biochemistry and functional studies. So far, two methods are commonly in use for the expression of exogenous proteins in this system. Investigators have the choice between cytoplasmic injections of in vitro synthesized cRNA or nuclear injections of cDNA. Here, we describe a new method for ion channel expression in oocytes, which consists of a coinjection of T7-driven cDNA and T7-RNA polymerase directly into the cytoplasm. This technique uses very limited amounts of purified enzyme and is also applicable to SP6 polymerase. Commercially available polymerases can also conveniently substitute for self-purified enzymes. The technique can be used for electrophysiological and biochemical analysis. In particular, high level expressions have been achieved for potassium (Shaker B, Kv1.2 and Kv1.3) and sodium (P mu 1.2) channels, and we also demonstrate efficient metabolic labeling of the calcium channel auxiliary beta 3 subunit. The properties of the channels expressed by this technique are indistinguishable from those of the channels expressed by classical methods. Expression of multi-subunit proteins was also achieved illustrating that the technique can be used for structure-function analyses. Moreover, this novel expression technique avoids many drawbacks of the two former techniques. It clearly bypasses the costly and time-consuming step of cRNA synthesis in vitro, prevents delicate cRNA manipulation and is easier to perform and more reliable than nuclear injection. Finally, it does not affect cell survival rate. These data indicate that the T7-RNA polymerase expression technique could be widely used in the future for the expression of exogenous proteins in the Xenopus oocyte system.

Asunto(s)