Data quality in biofilm high-throughput routine analysis: intralaboratory protocol adaptation and experiment reproducibility

Biofilm research is growing more diverse and dependent on high-throughput technologies and the large-scale production of results aggravates data substantiation. In particular, it is often the case that experimental protocols are adapted to meet the needs of a particular laboratory and no statistical validation of the modified method is provided. This paper discusses the impact of intra-laboratory adaptation and non-rigorous documentation of experimental protocols on biofilm data interchange and validation. The case study is a non-standard, but widely used, workflow for Pseudomonas aeruginosa biofilm development, considering three analysis assays: the crystal violet (CV) assay for biomass quantification, the XTT assay for respiratory activity assessment, and the colony forming units (CFU) assay for determination of cell viability. The ruggedness of the protocol was assessed by introducing small changes in the biofilm growth conditions, which simulate minor protocol adaptations and non-rigorous protocol documentation. Results show that even minor variations in the biofilm growth conditions may affect the results considerably, and that the biofilm analysis assays lack repeatability. Intra-laboratory validation of non-standard protocols is found critical to ensure data quality and enable the comparison of results within and among laboratories.

An efficient chronic unpredictable stress protocol to induce stress-related responses in C57BL/6 mice

Exposure to chronic stress can have broad effects on health ranging from increased predisposition for neuropsychiatric disorders to deregulation of immune responses. The chronic unpredictable stress (CUS) protocol has been widely used to study the impact of stress exposure in several animal models and consists in the random, intermittent, and unpredictable exposure to a variety of stressors during several weeks. CUS has consistently been shown to induce behavioral and immunological alterations typical of the chronic stress-response. Unfortunately C57BL/6 mice, one of the most widely used mouse strains, due to the great variety of genetically modified lines, seem to be resistant to the commonly used 4-week-long CUS protocol. The definition of an alternative CUS protocol allowing the use of C57BL/6 mice in chronic stress experiments is a need. Here, we show that by extending the CUS protocol to 8?weeks is possible to induce a chronic stress-response in C57BL/6 mice, as revealed by abrogated body weight gain, increased adrenals weight, and an overactive hypothalamic-pituitary-adrenal axis with increased levels of serum corticosterone. Moreover, we also observed stress-associated behavioral alterations, including the potentiation of anxious-like and depressive-like behaviors and a reduction of exploratory behavior, as well as subtle stress-related changes in the cell population of the thymus and of the spleen. The present protocol for C57BL/6 mice consistently triggers the spectrum of CUS-induced changes observed in rats and, thus, will be highly useful to researchers that need to use this particular mouse strain as an animal model of neuropsychiatric disorders and/or immune deregulation related to CUS.

Widespread environmental contamination with Mycobacterium tuberculosis complex revealed by a molecular detection protocol

Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70-0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area.

Study of Coronary Flow Reserve with Intravenous Use of Microbubbles (Contrast Echocardiography) and Adenosine: Protocol for Clinical Application in Patients Suspected of Having Coronary Heart Disease

OBJECTIVE: To test the feasibility, safety and accuracy of the adenosine protocol in the study of myocardial perfusion with microbubbles contrast echocardiography. METHODS: 81 pts (64 male, 60+11 years) were submitted to contrast echocardiography with PESDA (sonicated solution of albumin 20%-1ml, dextrose 5%-12ml and deca-fluorobutane gas-8ml) to study the myocardial perfusion at rest and after bolus injection of adenosine (6 to 18mg) and to coronary angiography within 1 month each other. For each patient 3 left ventricle perfusion beds were considered (total of 243 territories). 208 territories were analyzed and 35 territories were excluded. PESDA was continuously infused (1-2ml/min), titrated for best myocardial contrast. Triggered (1:1) second harmonic imaging was used. RESULTS: Coronary angiography showed 70 flow limiting (> 75%) lesions and 138 no flow limiting lesions. At rest an obvious myocardium contrast enhancement was seen in at least 1 segment of a territory in all patients. After adenosine injection an unquestionable further increase in myocardial contrast was observed in 136 territories (99%) related to no flow limiting lesions, lasting < 10 s, and a myocardial perfusion defect was detected in 68 territories (97%) related to flow limiting lesions. It was observed only 4 false results. There were no serious complications. CONCLUSION: Myocardial perfusion study with PESDA and adenosine protocol is a practical, safe and accurate method to analyze the coronary flow reserve.

Relevance to Home Blood Pressure Monitoring Protocol of Blood Pressure Measurements Taken Before First- Morning Micturition and in the Afternoon

Background: The importance of measuring blood pressure before morning micturition and in the afternoon, while working, is yet to be established in relation to the accuracy of home blood pressure monitoring (HBPM). Objective: To compare two HBPM protocols, considering 24-hour ambulatory blood pressure monitoring (wakefulness ABPM) as gold-standard and measurements taken before morning micturition (BM) and in the afternoon (AM), for the best diagnosis of systemic arterial hypertension (SAH), and their association with prognostic markers. Methods: After undergoing 24-hour wakefulness ABPM, 158 participants (84 women) were randomized for 3- or 5-day HBPM. Two variations of the 3-day protocol were considered: with measurements taken before morning micturition and in the afternoon (BM+AM); and with post-morning-micturition and evening measurements (PM+EM). All patients underwent echocardiography (for left ventricular hypertrophy - LVH) and urinary albumin measurement (for microalbuminuria - MAU). Result: Kappa statistic for the diagnosis of SAH between wakefulness-ABPM and standard 3-day HBPM, 3-day HBPM (BM+AM) and (PM+EM), and 5-day HBPM were 0.660, 0.638, 0.348 and 0.387, respectively. The values of sensitivity of (BM+AM) versus (PM+EM) were 82.6% × 71%, respectively, and of specificity, 84.8% × 74%, respectively. The positive and negative predictive values were 69.1% × 40% and 92.2% × 91.2%, respectively. The comparisons of intraclass correlations for the diagnosis of LVH and MAU between (BM+AM) and (PM+EM) were 0.782 × 0.474 and 0.511 × 0.276, respectively. Conclusions: The 3 day-HBPM protocol including measurements taken before morning micturition and during work in the afternoon showed the best agreement with SAH diagnosis and the best association with prognostic markers.

Heart Rate Variability Correlates to Functional Aerobic Impairment in Hemodialysis Patients

Background:Autonomic dysfunction (AD) is highly prevalent in hemodialysis (HD) patients and has been implicated in their increased risk of cardiovascular mortality.Objective:To correlate heart rate variability (HRV) during exercise treadmill test (ETT) with the values obtained when measuring functional aerobic impairment (FAI) in HD patients and controls.Methods:Cross-sectional study involving HD patients and a control group. Clinical examination, blood sampling, transthoracic echocardiogram, 24-hour Holter, and ETT were performed. A symptom-limited ramp treadmill protocol with active recovery was employed. Heart rate variability was evaluated in time domain at exercise and recovery periods.Results:Forty-one HD patients and 41 controls concluded the study. HD patients had higher FAI and lower HRV than controls (p<0.001 for both). A correlation was found between exercise HRV (SDNN) and FAI in both groups. This association was independent of age, sex, smoking, body mass index, diabetes, and clonidine or beta-blocker use, but not of hemoglobin levels.Conclusion:No association was found between FAI and HRV on 24-hour Holter or at the recovery period of ETT. Of note, exercise HRV was inversely correlated with FAI in HD patients and controls. (Arq Bras Cardiol. 2015; [online]. ahead print, PP.0-0)

Analysis Treatment Guideline versus Clinical Practice Protocol in Patients Hospitalized due to Heart Failure

Background: Despite the availability of guidelines for treatment of heart failure (HF), only a few studies have assessed how hospitals adhere to the recommended therapies. Objectives: Compare the rates of adherence to the prescription of angiotensin-converting enzyme inhibitor or angiotensin II receptor blockers (ACEI/ARB) at hospital discharge, which is considered a quality indicator by the Joint Commission International, and to the prescription of beta-blockers at hospital discharge, which is recommended by national and international guidelines, in a hospital with a case management program to supervise the implementation of a clinical practice protocol (HCP) and another hospital that follows treatment guidelines (HCG). Methods: Prospective observational study that evaluated patients consecutively admitted to both hospitals due to decompensated HF between August 1st, 2006, and December 31st, 2008. We used as comparing parameters the prescription rates of beta-blockers and ACEI/ARB at hospital discharge and in-hospital mortality. Results: We analyzed 1,052 patients (30% female, mean age 70.6 ± 14.1 years), 381 (36%) of whom were seen at HCG and 781 (64%) at HCP. The prescription rates of beta-blockers at discharge at HCG and HCP were both 69% (p = 0.458), whereas those of ACEI/ARB were 83% and 86%, respectively (p = 0.162). In-hospital mortality rates were 16.5% at HCP and 27.8% at HCG (p < 0.001). Conclusion: There was no difference in prescription rates of beta-blocker and ACEI/ARB at hospital discharge between the institutions, but HCP had lower in-hospital mortality. This difference in mortality may be attributed to different clinical characteristics of the patients in both hospitals.

Avaluació d’un nou protocol MAC per IEEE 802.11 en un entorn multicel·lular exterior

Estudi realitzat a partir d’una estada al laboratori LSR (Logiciels, Systèmes, Réseaux) – IMAG a Grenoble, França, entre els mesos de setembre del 2005 i febrer del 2006. El grup de recerca Drakkar d’aquest laboratori va desenvolupar un nou mecanisme d’accés al medi per a xarxes d’àrea local sense fils (WLAN) anomenat Idle Sense. S’ha avaluat aquest mecanisme en entorns cel·lulars i s’ha comparat del seu comportament amb l’estàndard IEEE 802.11 DCF mitjançant la implementació en un simulador. En els entorns cel·lulars es troben condicions de transmissió molt allunyades de les ideals, i de les analitzades fins al moment, que consisteixen en escenaris formats per una única cel·la aïllada. Per aquest motiu, primer s’ha estudiat els mecanismes d’accés al medi d’Idle Sense en una cel·la i en condicions de transmissió adverses. Després, s’ha estudiat en entorns multicel·lulars, on les comunicacions entre estacions es veuen influenciades pel problema de solapament de cel·les i per les interferències provocades per les transmissions d’estacions d’altres cel·les. També s’ha comparat els resultats obtinguts amb el comportament d’IEEE 802.11 DCF i d’altres propostes de millora: Asymptotically Optimal Backoff (AOB) i Slow Decrease. Finalment, s’ha realitzat una modificació sobre Idle Sense, que s’ha anomenat Weighted Idle Sense, que resulta més adecuat per a treballar en xarxes d’àrea local sense fils en mode infraestructura.

Disseny i aplicació del protocol d'instal·lació d'energia fotovoltaica en edificis aïllats de la xarxa elèctrica al PNAP

En el perímetre del parc es troben gran nombre d’edificacions amb mancances de subministrament elèctric per la gran dispersió de la població de la comarca. L’objectiu d’aquest estudi és desenvolupar un protocol per determinar la viabilitat de l’energia fotovoltaica en edificis aïllats i aplicar-la de forma pilot a dos edificis gestionats pels responsables del Parc Natural de l’Alt Pirineu: el Centre de Logística i Manteniment (CLM) i el Refugi del Fornet. Aquests han estat escollits per la seva representativitat com a edificis aïllats de la xarxa elèctrica convencional. S’ha elaborat una metodologia pròpia, el Protocol d’instal·lació d’energia fotovoltaica en edificis aïllats de la xarxa elèctrica. S’ha realitzat una anàlisi de sensibilitats del dimensionat fotovoltaic davant la modificació de diversos paràmetres rellevants. Això ha permès determinar les característiques òptimes de viabilitat per als dos edificis en estudi i el cost de la instal·lació fotovoltaica (17.183€ el CLM i 164.815 € el Refugi del Fornet). Els resultats obtinguts en els dos edificis pilot han mostrat el gran cost relatiu entre l’energia fotovoltaica i l’energia elèctrica produïda a partir de combustibles fòssils.

Mobilitat inter-plataforma d'agents software: protocol de migració fragmentada

En aquest projecte s'ha realitzat l'anàlisi, disseny i implementació d'un protocol de migració d'agents software basat en l'enviament del codi dels agents fragmentat en múltiples missatges. Aquest protocol es troba dins d'una arquitectura de migració multi-protocol per a la mobilitat d'agents entre plataformes JADE. Finalment, s'ha realitzat un estudi que compara el rendiment assolit pel protocol i les prestacions que aporta.

Multi-phase post-mortem CT angiography: development of a standardized protocol.

The objective of this work was to develop an easily applicable technique and a standardized protocol for high-quality post-mortem angiography. This protocol should (1) increase the radiological interpretation by decreasing artifacts due to the perfusion and by reaching a complete filling of the vascular system and (2) ease and standardize the execution of the examination. To this aim, 45 human corpses were investigated by post-mortem computed tomography (CT) angiography using different perfusion protocols, a modified heart-lung machine and a new contrast agent mixture, specifically developed for post-mortem investigations. The quality of the CT angiographies was evaluated radiologically by observing the filling of the vascular system and assessing the interpretability of the resulting images and by comparing radiological diagnoses to conventional autopsy conclusions. Post-mortem angiography yielded satisfactory results provided that the volumes of the injected contrast agent mixture were high enough to completely fill the vascular system. In order to avoid artifacts due to the post-mortem perfusion, a minimum of three angiographic phases and one native scan had to be performed. These findings were taken into account to develop a protocol for quality post-mortem CT angiography that minimizes the risk of radiological misinterpretation. The proposed protocol is easy applicable in a standardized way and yields high-quality radiologically interpretable visualization of the vascular system in post-mortem investigations.

A protocol guided by transpulmonary thermodilution and lactate levels for resuscitation of patients with severe burns.

Over-resuscitation is deleterious in many critically ill conditions, including major burns. For more than 15 years, several strategies to reduce fluid administration in burns during the initial resuscitation phase have been proposed, but no single or simple parameter has shown superiority. Fluid administration guided by invasive hemodynamic parameters usually resulted in over-resuscitation. As reported in the previous issue of Critical Care, Sánchez-Sánchez and colleagues analyzed the performance of a 'permissive hypovolemia' protocol guided by invasive hemodynamic parameters (PiCCO, Pulsion Medical Systems, Munich, Germany) and vital signs in a prospective cohort over a 3-year period. The authors' results confirm that resuscitation can be achieved with below-normal levels of preload but at the price of a fluid administration greater than predicted by the Parkland formula (2 to 4 mL/kg per% burn). The classic approach based on an adapted Parkland equation may still be the simplest until further studies identify the optimal bundle of resuscitation goals.

Implementació i simulació funcional en MATLAB del protocol IEEE 802.15.4 de WSN

El principal objectiu que es pretén assolir és dissenyar un simulador de la capa MAC, definida a l’estàndard IEEE 802.15.4. Convé matisar que l’objectiu no és implementar un simulador, sinó utilitzar una plataforma genèrica existent, MATLAB, i definir sobre ella, una metodologia que, permeti utilitzar-la més com a plataforma específica de desenvolupament i simulació de protocols per a xarxes de sensors, que no pas com a simple simulador.

Contribució a la mobilitat inter-plataforma d'agents mòbils: protocol de migració fragmentada

En aquest projecte s’ha dissenyat un protocol de migració d’agents mòbils per a l’arquitectura IPMA basat en l’enviament dels agents fragmentats en diversos missatges FIPA ACL. Aquest s’ha implementat dins el servei de migració JIPMS per a la plataforma JADE. Finalment s’ha dut a terme un conjunt exhaustiu de tests per avaluar-ne el rendiment i comparar-lo amb altres protocols de migració existents.

Establiment d'un protocol de preparació de mostres d'esponja per a l'anàlisi d'isòtops estables de silici

Projecte de recerca elaborat a partir d’una estada a Alfred Wegener Institut für Polar und Meeresforschung, Alemanya, entre juliol i setembre de 2007. Les mostres biològiques usades per a la determinació d’isòtops estables de silici fins a l’actualitat han estat bàsicament provinents de diatomees, i poc se sap de la relació amb el silici que d’altres organismes tenen, com per exemple les esponges (demosponges i hexactinèl.lides). La zona de la Mar de Weddell (Antàrtida) té aigües amb concentracions de silici dissoltes molt elevades, i les seves plataformes continentals presenten una de les faunes bentòniques més ben desenvolupades del planeta, que sovint és dominada per comunitats d’esponges silíciques. L’estudi del cicle del silici en aquestes àrees pot contribuir a respondre preguntes actuals i sobre el passat de les masses d’aigua, així com de l’ecologia dels organismes. El present projecte ha tingut com a objectiu desenvolupar una metodologia per a la preparació de mostres biològiques per a l’anàlisi d’isòtops estables de silici. El desenvolupament del protocol de preparació de les mostres és un procés d’assaig i error lent, però necessari per a la realització de les anàlisis i per a la futura facilització als potencials usuaris d’un protocol estandaritzat. Es presenta el protocol formulat, amb comentaris d’utilitat.