Instrumentació i control d'actuadors fluídics per a sistemes analítics: sistema de regulació d'una microbomba de precisió a mode de xeringa

En aquest projecte s’ha implementat un sistema de control per a les bombes microfluídiques LPVX de The Lee Company funcionant a mode de xeringa. El sistema consisteix en un circuit controlador basat en el microxip UDN 296 B de Allegro MicroSystems, que conté dos Ponts en H per a controlar motors pas a pas i dos mòduls de Modulació d’Amplada de Polsos (PWM), governat a partir d’un programa de control com a instrument virtual dissenyat sota l’entorn LabVIEW. El programa de control permet indicar la quantitat de volum a aspirar o dispensar per la bomba i escollir entre una execució simple o una de continuada, podent-ne controlar en aquest segona opció el temps entre execució i execució. El programa també permet visualitzar el procés mitjançant la obtenció de la imatge d’una webcam amb DirectShow. Finalment també permet el control remot de l’Instrument Virtual a través de la xarxa d’Internet.

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study.

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.

Eosinophilic esophagitis: Updated consensus recommendations for children and adults.

Eosinophilic esophagitis (EoE) is a clinicopathologic condition of increasing recognition and prevalence. In 2007, a consensus recommendation provided clinical and histopathologic guidance for the diagnosis and treatment of EoE; however, only a minority of physicians use the 2007 guidelines, which require fulfillment of both histologic and clinical features. Since 2007, the number of EoE publications has doubled, providing new disease insight. Accordingly, a panel of 33 physicians with expertise in pediatric and adult allergy/immunology, gastroenterology, and pathology conducted a systematic review of the EoE literature (since September 2006) using electronic databases. Based on the literature review and expertise of the panel, information and recommendations were provided in each of the following areas of EoE: diagnostics, genetics, allergy testing, therapeutics, and disease complications. Because accumulating animal and human data have provided evidence that EoE appears to be an antigen-driven immunologic process that involves multiple pathogenic pathways, a new conceptual definition is proposed highlighting that EoE represents a chronic, immune/antigen-mediated disease characterized clinically by symptoms related to esophageal dysfunction and histologically by eosinophil-predominant inflammation. The diagnostic guidelines continue to define EoE as an isolated chronic disorder of the esophagus diagnosed by the need of both clinical and pathologic features. Patients commonly have high rates of concurrent allergic diatheses, especially food sensitization, compared with the general population. Proved therapeutic options include chronic dietary elimination, topical corticosteroids, and esophageal dilation. Important additions since 2007 include genetic underpinnings that implicate EoE susceptibility caused by polymorphisms in the thymic stromal lymphopoietin protein gene and the description of a new potential disease phenotype, proton pump inhibitor-responsive esophageal eosinophila. Further advances and controversies regarding diagnostic methods, surrogate disease markers, allergy testing, and treatment approaches are discussed.

Nouveautés en médecine ambutatoire: dépistage, traitement et iatrogénicité [Innovations in ambulatory care: screening, treatment and iatrogenicity]

Cette année, six études utiles pour la pratique ont été retenues. L'indication à la mammographie entre 40 et 49 ans devrait être évaluée individuellement et en tenant compte des risques/bénéfices de cet examen. Au-delà de 65 ans, un dépistage systématique de la fibrillation auriculaire avec prise de pouls puis ECG (si pouls irrégulier) pourrait être réalisé de manière systématique. Les risques de complications postcolonoscopie existent, particulièrement suite à des biopsies/polypectomies, et ce risque devrait être discuté. Les inhibiteurs de la pompe à protons au long court sont un facteur de risque de fracture de hanche. S'il est important de prendre en charge des pressions artérielles élevées au-delà de 80 ans, il faut être prudent (orthostatisme). Une corticothérapie précoce suite à une paralysie faciale périphérique est efficace. This year we have selected six studies useful for the day to day practice. A mammography in women 40 to 49 years of age should be evaluated taking into account the patient's profile and the possible risks and benefits of this exam. In patients over 65 years of age, a systematic atrial fibrillation screening, with pulse rate measuring then ECG (if irregular beat) should be realised on a regular basis. The risks for complications following colonoscopies do exist, especially after biopsies/polypectomies and this risk should be discussed. Long term proton pump inhibitor treatment is a risk factor for hip fracture. It is important to treat high blood pressure problems in the elderly, but the orthostatic risks should be adressed. A corticoid treatment started quickly for Bell's palsy is efficient

Functional effects of Na+,K+-ATPase gene mutations linked to familial hemiplegic migraine.

Familial hemiplegic migraine type 2, an autosomal dominant form of migraine with aura, has been associated with four distinct mutations in the alpha2-subunit of the Na+,K+-ATPase. We have introduced these mutations in the alpha2-subunit of the human Na+,K+-ATPase and the corresponding mutations in the Bufo marinus alpha1-subunit and studied these mutants by expression in Xenopus oocyte. Metabolic labeling studies showed that the mutants were synthesized and associated with the beta-subunit, except for the alpha2HW887R mutant, which was poorly synthesized, and the alpha1BW890R, which was partially retained in the endoplasmic reticulum. [3H]ouabain binding showed the presence of the alpha2HR689Q and alpha2HM731T at the membrane, whereas the alpha2HL764P and alpha2HW887R could not be detected. Functional studies with the mutants of the B. marinus Na+,K+-ATPase showed a reduced or abolished electrogenic activity and a low K+ affinity for the alpha1BW890R mutant. Through different mechanisms, all these mutations result in a strong decrease of the functional expression of the Na+,K+-pump. The decreased activity in alpha2 isoform of the Na+,K+-pump expressed in astrocytes seems an essential component of hemiplegic migraine pathogenesis and may be responsible for the cortical spreading depression, which is one of the first events in migraine attacks.

Influència del context geològic i hidrogeològic en l'eficiència energètica dels sistemes geotèrmics de molt baixa entalpia

L’elevat consum energètic de les societats actuals, així com la impossibilitat de sostenir-lo a llarg termini implica la cerca de noves fonts d’energia. D’aquesta manera, en el camp de la climatització residencial, l’energia geotèrmica de molt baixa entalpia es posiciona com una alternativa als recursos energètics actuals. Així, els primers metres de subsòl presenten una temperatura adequada per al seu aprofitament calorífic, mitjançant els sistemes geotèrmics de bomba de calor. Si bé a nivell energètic, són sistemes, intrínsecament, molt eficients, el seu rendiment pot patir importants variacions davant dels canvis en les condicions del medi geològic i hidrogeològic. Especialment, els col·lectors de calor verticals, treballen, amb freqüència, en el si de les formacions hidrogeològiques. En aquest sentit, els canvis del nivell hidràulic i de la temperatura de l’aigua de l’aqüífer es manifesten amb variacions de la conductivitat tèrmica equivalent i del flux subterrani d’aigua, que alhora, aquestes, es tradueixen en alteracions del flux subterrani de calor. Davant d’aquest fet, l’avaluació quantitativa de l’efecte d’aquestes fluctuacions que es presenta en aquest treball mostra petites variacions del nivell hidràulic i de la temperatura de l’aigua comporten canvis molt notables en l’eficiència dels sistemes verticals de bomba de calor geotèrmica.

Variation between geographical populations of Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho, 1939) sensu lato (Diptera:Psychodidae:Phlebotominae) in Brazil

Phylogenetic analysis of morphometric and biological characters indicated that there are two distinct forms of Lutzomyia whitmani in Brazil: one is present both north and south of the River Amazonas in the State of Pará while the other occurs in northeast Brazil, in the State of Ceará, and further south, including the type locality in State of Bahia. The Amazonian form is reportedly neither strongly anthropophilic nor synanthropic, and it is the vector of Leishmania shawi; whereas the southern form is often collected peridomestically, while biting man, and has been found infected with Le.(V.) braziliensis. The ratio of the length of the genital filaments to that the genital pump was found to be consistently smaller in males of the Amazonian populations. A middle repetitive DNA element was isolated by differentially screening a genomic library made using Amazonian material, and the sequence was diagnostic for this form of Lu. whitmani (being absent or occurring in low copy number in the southern form). The total evidence suggests there are at least two, geographically-isolated forms of Lu. whitmani, which may represent different cryptic species.

Generic drugs in Spain: price competition vs. moral hazard

This paper examines competition between generic and brand-name drugs in the regulated Spanish pharmaceutical market. A nested logit demand model is specified for the three most consumed therapeutic subgroups in Spain: statins (anticholesterol), selective serotonin reuptake inhibitors (antidepressants) and proton pump inhibitors (antiulcers). The model is estimated with instrumental variables from a panel of monthly prescription data from 1999 to 2005. The dataset distinguishes between three different levels of patients’ copayments within the prescriptions and the results show that the greater the level of insurance that the patient has (and therefore the lower the patient’s copayment), the lower the proportion of generic prescriptions made by physicians. It seems that the low level of copayment has delayed the penetration of generics into the Spanish market. Additionally, the estimation of the demand model suggests that the substitution rules and promotional efforts associated with the reference pricing system have increased generic market share, and that being among the first generic entrants has an additional positive effect.

Enteral nutrition in critically ill patients with severe hemodynamic failure after cardiopulmonary bypass.

BACKGROUND & AIMS: The study was designed to investigate and quantify nutritional support, and particularly enteral nutrition (EN), in critically ill patients with severe hemodynamic failure. METHODS: Prospective, descriptive study in a surgical intensive care unit (ICU) in a university teaching hospital: patients aged 67+/-13 yrs (mean+/-SD) admitted after cardiac surgery with extracorporeal circulation, staying 5 days in the ICU with acute cardiovascular failure. Severity of disease was assessed with SAPS II, and SOFA scores. Variables were energy delivery and balance, nutrition route, vasopressor doses, and infectious complications. Artificial feeding delivered according to ICU protocol. EN was considered from day 2-3. Energy target was set 25 kcal/kg/day to be reached stepwise over 5 days. RESULTS: Seventy out of 1114 consecutive patients were studied, aged 67+/-17 years, and staying 10+/-7 days in the ICU. Median SAPS II was 43. Nine patients died (13%). All patients had circulatory failure: 18 patients required intra-aortic balloon-pump support (IABP). Norepinephrine was required in 58 patients (83%). Forty patients required artificial nutrition. Energy delivery was very variable. There was no abdominal complication related to EN. As a mean, 1360+/-620 kcal/kg/day could be delivered enterally during the first 2 weeks, corresponding to 70+/-35% of energy target. Enteral nutrient delivery was negatively influenced by increasing dopamine and norepinephrine doses, but not by the use of IABP. CONCLUSION: EN is possible in the majority of patients with severe hemodynamic failure, but usually results in hypocaloric feeding. EN should be considered in patients with careful abdominal and energy monitoring.

Anatomia e Histologia das Glândulas Salivares nos Triatomíneos

Histological studies upon the salivary glands of ten species of triatomine bugs were performed looking for their number and structural organization in different genera. It was possible to evaluate the celular epithelium type of each gland, as well as the merocrine and apocrine secretions of the glands. Secretion run until the hilo and after to salivary pump and hypofaringe. The glandular components, D1, D2 and D3 are always present in the Triatoma, Panstrongylus and Diptelogaster but in Rhodnius there are only the first two pairs of glands. The salivary channels and the hilo are analyzed by histology. The whole pair D3 has a clear valve that regularizes the exit of the secretions to the hilo. According to the genus the valves appear in different locations. They have low and dense epithelium, and their nucleus are rich in chromatin. The secondary channels leaving these valves, are very different, with clear chitinous ringer, low level of chromatin in the nucleus and homogeneous cytoplasm.

Regulation of horizontal gene transfer of the catabolic island ICEclc

ICEclc is a mobile genetic element found in two copies on the chromosome of the bacterium Pseudomonas knackmussii B13. ICEclc harbors genes encoding metabolic pathways for the degradation of chlorocatechols (CLC) and 2-aminophenol (2AP). At low frequencies, ICEclc excises from the chromosome, closes into a circular DNA molecule which can transfer to another bacterium via conjugation. Once in the recipient cell, ICEclc can reintegrate into the chromosome by site-specific recombination. This thesis aimed at identifying the regulatory network underlying the decisions for ICEclc horizontal transfer (HGT). The first chapter is an introduction on integrative and conjugative elements (ICEs) more in general, of which ICEclc is one example. In particular I emphasized the current knowledge of regulation and conjugation machineries of the different classes of ICE. In the second chapter, I describe a transcriptional analysis using microarrays and other experiments to understand expression of ICEclc in exponential and stationary phase. By overlaying transcriptomic profiles with Northern hybridizations and RT- PCR data, we established a transcription map for the entire core region of ICEclc, a region assumed to encode the ICE conjugation process. We also demonstrated how transcription of the ICEclc core is maximal in stationary phase, which correlates to expression of reporter genes fused to key ICEclc promoters. In the third chapter, I present a transcriptome analysis of ICEclc in a variety of different host species, in order to explore whether there are species-specific differences. In the fourth chapter, I focus on the role of a curious ICEclc-encoded TetR-type transcriptional repressor. We find that this gene, which we name mfsR, not only controls its own expression but that of a set of genes for a putative multi-drug efflux pump (mfsABC) as well. By using a combination of biochemical and molecular biology techniques, I could show that MfsR specifically binds to operator boxes in two ICEclc promoters (PmfsR and PmfsA), inhibiting the transcription of both the mfsR and mfsABC-orf38184 operons. Although we could not detect a clear phenotype of an mfsABC deletion, we discuss the implications of pump gene reorganizations in ICEclc and close relatives. In the fifth chapter, we find that mfsR not only controls its own expression and that of the mfsABC operon, but is also indirectly controlling ICEclc transfer. Using gene deletions, microarrays, transfer assays and microscopy-based reporter fusions, we demonstrate that mfsR actually controls a small operon of three regulatory genes. The last gene of this mfsR operon, orf17162, encodes a LysR-type activator that when deleted strongly impairs ICEclc transfer. Interestingly, deletion of mfsR leads to transfer competence in almost all cells, thereby overruling the bistability process in the wild-type. In the final sixth chapter, I discuss the relevance of the present thesis and the resulting perspectives for future studies.

Extracorporeal membrane oxygenation support in acardia.

In extreme situations, such as hyperacute rejection of heart transplant or major heart trauma, heart preservation may not be possible. Our experimental team works on a project of peripheral extracorporeal membrane oxygenation (ECMO) support in acardia as a bridge to heart transplantation or artificial heart implantation. An ECMO support was established in five calves (58.6 ± 6.9 kg) by the transjugular insertion to the caval axis of a self-expanded cannula, with carotid artery return. After baseline measurements, ventricular fibrillation was induced, great arteries were clamped, heart was excised, and right and left atria remnants, containing pulmonary veins, were sutured together leaving an atrial septal defect over the caval axis cannula. Measurements of pump flow and arterial pressure were taken with the pulmonary artery clamped and anastomosed with the caval axis for a total of 6 hours. Pulmonary artery anastomosis to the caval axis provided an acceptable 6 hour hemodynamic stability, permitting a peripheral access ECMO support in extreme scenarios indicating a heart explantation.

Y-688, a new quinolone active against quinolone-resistant Staphylococcus aureus: lack of in vivo efficacy in experimental endocarditis.

Y-688 is a new fluoroquinolone with increased activity against ciprofloxacin-resistant staphylococci. The MICs of Y-688 and other quinolones were determined for 58 isolates of ciprofloxacin-resistant and methicillin-resistant Staphylococcus aureus (MRSA). The MICs at which 50% and 90% of bacteria were inhibited were >/=128 and >/=128 mg/liter, respectively, for ciprofloxacin, 16 and 32 mg/liter, respectively, for sparfloxacin, and 0.25 and 1 mg/liter, respectively, for Y-688. This new quinolone was further tested in rats with experimental endocarditis due to either of two isolates of ciprofloxacin-resistant MRSA (namely, P8/128 and CR1). Infected animals were treated for 3 days with ciprofloxacin, vancomycin, or Y-688. Antibiotics were administered through a computerized pump to simulate human-like pharmacokinetics in the serum of rats. The anticipated peak and trough levels of Y-688 were 4 and 1 mg/liter at 0.5 and 12 h, respectively. Treatment with ciprofloxacin was ineffective. Vancomycin significantly decreased vegetation bacterial counts for both organisms (P less, similar 0.05). In contrast, Y-688 only marginally decreased vegetation bacterial counts (P greater, similar 0.05). Moreover, several vegetation that failed Y-688 treatment grew staphylococci for which the MICs of the test antibiotic were increased two to eight times. Y-688 also selected for resistance in vitro, and isolates for which the MICs were increased eight times emerged at a frequency of ca. 10(-8). Thus, in spite of its low MIC for ciprofloxacin-resistant MRSA, Y-688 failed in vivo and its use carried the risk of resistance selection. The fact that ciprofloxacin-resistant staphylococci became rapidly resistant to this potent new drug suggests that the treatment of ciprofloxacin-resistant MRSA with new quinolones might be more problematic than expected.

Biometal muscle to restore atrial transport function in a permanent atrial fibrillation animal model: a potential tool in the treatment of end-stage heart failure.

BACKGROUND: Half of the patients with end-stage heart failure suffer from persistent atrial fibrillation (AF). Atrial kick (AK) accounts for 10-15% of the ejection fraction. A device restoring AK should significantly improve cardiac output (CO) and possibly delay ventricular assist device (VAD) implantation. This study has been designed to assess the mechanical effects of a motorless pump on the right chambers of the heart in an animal model. METHODS: Atripump is a dome-shaped biometal actuator electrically driven by a pacemaker-like control unit. In eight sheep, the device was sutured onto the right atrium (RA). AF was simulated with rapid atrial pacing. RA ejection fraction (EF) was assessed with intracardiac ultrasound (ICUS) in baseline, AF and assisted-AF status. In two animals, the pump was left in place for 4 weeks and then explanted. Histology examination was carried out. The mean values for single measurement per animal with +/-SD were analysed. RESULTS: The contraction rate of the device was 60 per min. RA EF was 41% in baseline, 7% in AF and 21% in assisted-AF conditions. CO was 7+/-0.5 l min(-1) in baseline, 6.2+/-0.5 l min(-1) in AF and 6.7+/-0.5 l min(-1) in assisted-AF status (p<0.01). Histology of the atrium in the chronic group showed chronic tissue inflammation and no sign of tissue necrosis. CONCLUSIONS: The artificial muscle restores the AK and improves CO. In patients with end-stage cardiac failure and permanent AF, if implanted on both sides, it would improve CO and possibly delay or even avoid complex surgical treatment such as VAD implantation.

The effect of nitric oxide combined with fluoroquinolones against Salmonella enterica serovar Typhimurium in vitro

Two regulons, soxRS and marRAB, are associated with resistance to quinolones or multiple antibiotic in Salmonella enterica serovar Typhimurium. These regulons are activated by nitric oxide and redox-cycling drugs, such as paraquat and cause on activation of the acrAB-encoded efflux pump. In this study, we investigated the effect of nitric oxide (NO) alone and in combination with ofloxacin, ciprofloxacin, and pefloxacin against S. typhimurium clinical isolates and mutant strains in vitro. We did not observe synergistic effect against clinical isolates and SH5014 (parent strain of acr mutant), while we found synergistic effect against PP120 (soxRS mutant) and SH7616 (an acr mutant) S. typhimurium for all quinolones. Our results suggest that the efficiencies of some antibiotics, including ofloxacin, ciprofloxacin, and pefloxacin are decreased via activation of soxRS and marRAB regulons by NO in S. enterica serovar Typhimurium. Further studies are warranted to establish the interaction of NO with the genes of Salmonella and, with multiple antibiotic resistance.