Gellan gum-coated gold nanorods: an intracellular nanosystem for bone tissue engineering

Gold nanorods (AuNRs) have emerged as an exceptional nanotool for a myriad of applications ranging from cancer therapy to tissue engineering. However, their surface modification with biocompatible and stabilizing biomaterials is crucial to allow their use in a biological environment. Herein, low-acyl gellan gum (GG) was used to coat AuNRs surface, taking advantage of its stabilizing, biocompatible and gelling features. The layer-by-layer based strategy implied the successive deposition of poly(acrylic acid), poly(allylamine hydrochloride) and GG, which allowed the formation of a GG hydrogel-like shell with 7 nm thickness around individual AuNRs. Stability studies in a wide range of pH and salt concentrations showed that the polysaccharide coating can prevent AuNRs aggregation. Moreover, a reversible pH-responsive feature of the nanoparticles was observed. Cytocompatibility and osteogenic ability of GG-coated AuNRs was also addressed. After 14 days of culturing within SaOS-2, an osteoblast-like cell line, in vitro studies revealed that AuNRs-GG exhibit no cytotoxicity, were internalized by the cells and localized inside lysosomes. AuNRs-GG combined with osteogenic media enhanced the mineralization capacity two-fold, as compared to cells exposed to osteogenic media alone. The proposed system has shown interesting features for osteogenesis, and further insights might be relevant for drug delivery, tissue engineering and regenerative medicine.

Aproveitamento de energia em sistemas de abastecimento de água

Dissertação de mestrado integrado em Engenharia Civil

Antidepressant induced excessive yawning and indifference

Introduction Antidepressant induced excessive yawning has been described as a possible side effect of pharmacotherapy. A syndrome of indifference has also been described as another possible side effect. The frequency of those phenomena and their physiopathology are unknown. They are both considered benign and reversible after antidepressant discontinuation but severe cases with complications as temporomandibular lesions, have been described. Methods We report two unprecedented cases in which excessive yawning and indifference occurred simultaneously as side effects of antidepressant therapy, discussing possible physiopathological mechanisms for this co-occurrence. Case 1: A male patient presented excessive yawning (approximately 80/day) and apathy after venlafaxine XR treatment. Symptoms reduced after a switch to escitalopram, with a reduction to 50 yawns/day. Case 2: A female patient presented excessive yawning (approximately 25/day) and inability to react to environmental stressors with desvenlafaxine. Conclusion Induction of indifference and excessive yawning may be modulated by serotonergic and noradrenergic mechanisms. One proposal to unify these side effects would be enhancement of serotonin in midbrain, especially paraventricular and raphe nucleus.

Can the dopaminergic-related effects of general anesthetics be linked to mechanisms involved in drug abuse and addiction?

BACKGROUND: General anesthetics (GA) are well known for the ability to induce a state of reversible loss of consciousness and unresponsiveness to painful stimuli. However, evidence from animal models and clinical studies show that GA exposure may induce behavioral changes beyond acute effects. Most research and concerns are focused on changes in cognition and memory. METHODS: We will look at effects of GA on behavior that is mediated by the dopaminergic system. RESULTS: Pharmacological resemblance of GA with drugs of abuse, and the complexity and importance of dopaminergic systems in both reward seeking and addictive illnesses make us believe that it deserves an overview about what is already known and what matters to us as healthcare workers and specifically as anesthesiologists. CONCLUSION: A review of available evidence strongly suggests that there may be a link between the effects of GA on the brain and substance abuse, partly explained by their influence on the dopaminergic system.

Reduction of QTc interval dispersion. Potential mechanism of cardiac protection of pyridostigmine bromide

OBJECTIVE: Parasympathetic dysfunction is an independent risk factor in individuals with coronary artery disease, and cholinergic stimulation is a potential therapeutical option. We determined the effects of pyridostigmine bromide, a reversible anticholinesterase agent, on electrocardiographic variables of healthy individuals. METHODS: We carried out a cross-sectional, double blind, randomized, placebo-controlled study. We obtained electrocardiographic tracings in 12 simultaneous leads of 10 healthy young individuals at rest before and after oral administration of 45 mg of pyridostigmine or placebo. RESULTS: Pyridostigmine increased RR intervals (before: 886±27 ms vs after: 1054±37 ms) and decreased QTc dispersion (before: 72±9ms vs after: 45±3ms), without changing other electrocardiographic variables (PR segment, QT interval, QTc, and QT dispersion). CONCLUSION: Bradycardia and the reduction in QTc dispersion induced by pyridostigmine may effectively represent a protective mechanism if these results can be reproduced in individuals with cardiovascular diseases.

Exercise stress testing in healthy subjects during cholinergic stimulation after a single dose of pyridostigmine

OBJETIVE: The evaluation, by exercise stress testing, of the cardiorespiratory effects of pyridostigmine (PYR), a reversible acetylcholinesterase inhibitor. METHODS: A double-blind, randomized, cross-over, placebo-controlled comparison of hemodynamic and ventilation variables of 10 healthy subjects who underwent three exercise stress tests (the first for adaptation and determination of tolerance to exercise, the other two after administration of placebo or 45mg of PYR). RESULTS: Heart rate at rest was: 68±3 vs 68±3bpm before and after placebo, respectively (P=0.38); 70±2 vs 59±2bpm, before and after pyridostigmine, respectively (P<0.01). During exercise, relative to placebo: a significantly lower heart rate after PYR at, respectively, 20% (P=0.02), 40% (P=0.03), 80% (P=0.05) and 100% (P=0.02) of peak effort was observed. No significant differences were observed in arterial blood pressure, oxygen consumption at submaximal and maximal effort, exercise duration, respiratory ratio, CO2 production, ventilation threshold, minute ventilation, and oxygen pulse. CONCLUSION: Pyridostigmine, at a dose of 45mg, decreases heart rate at rest and during exercise, with minimal side effects and without interfering with exercise tolerance and ventilation variables.

PARTICIPACIÓN DEL SISTEMA SEROTONINÉRGICO CENTRAL EN LA REGULACIÓN DEL APETITO POR EL SODIO

Estudios previos del laboratorio han demostrado que la depleción de sodio inducida por diálisis peritoneal (DP) produce una rápida caída en la concentración de sodio del suero y del fluido cerebrospinal entre las 1-4hs después de la DP; sin embargo el apetito específico por el sodio (AS) aparece recién 20 hs después cuando se han recuperado los niveles normales de sodio extracelular quizás por medio de fuentes reservorias del cuerpo como hueso e higado. En nuestros trabajos además hemos identificado las distintas áreas y sistemas neuroquímicos involucrados tanto en la fase apetitiva (24hs después de la DP) como en la fase de saciedad del AS (luego del consumo de NaCl hipertónico inducido por DP). En estos estudios la depleción de sodio incrementó la actividad neuronal, evidenciada mediante la inmunoreactividad a Fos (ir-Fos), a lo largo de los órganos circunventriculares (OCVs) de la lamina terminalis (LT): órgano subfornical (SFO) y órgano vasculoso de la lamina terminalis, y diminuyó ir-Fos de las neuronas serotoninérgicas del núcleo del rafe dorsal (DRN), sugiriendo su participación en la génesis del AS. Por otro lado, durante la fase de saciedad la ir-Fos se incrementó dentro de áreas del tronco encefálico como el núcleo del tracto solitario, área postrema, núcleo parabraquial lateral (LPBN), neuronas serotoninérgicas del DRN y a lo largo de las células oxitocinérgicas hipotalámicas indicando su participación en la inhibición del AS. A pesar del conocimiento acumulado acerca de la regulación central del AS, hasta el momento, no se conocen cuales son los mecanismos, áreas y sistemas neuroquímicos involucrados en la disociación temporal existente entre la depleción de sodio y la aparición del AS. Numerosas líneas de evidencia sugieren que el AS es estimulado por la misma señal molecular que la sed hipovolemica, principalmente angiontensina II (ANGII), pero no se manifiesta porque es bloqueada por una señal inhibitoria dominante. Se sugiere que los efectos estimulatorios de ANGII sobre la ingesta de agua y sodio involucran la interacción con el circuito serotoninérgico (5HT) inhibitorio central. El circuito 5HT central que subyace a esta interacción incluye principalmente conexiones bidireccionales entre los OCVs de la LT donde se estimula la sed y AS y los somas 5HT del DRN y los terminales 5HT presentes en el LPBN donde se inhibiría el AS. Consistente con estos datos, la lesión del DRN y la inyección de antagonistas serotoninérgicos en el LPBN provocan un aumento significativo en el consumo de sodio inducido por distintos modelos experimentales.Nuestros resultados recientes indican que ya a las 2hs luego de la DP se incrementa la actividad de renina plasmática y a nivel central se observa una actividad tónica de las neuronas serotoninérgicas del DRN y la activación simultánea de los OCVs de la LT y de los núcleos de tronco encefálicos, estructuras previamente involucradas tanto en la fase apetitiva como de saciedad respectivamente. Con lo cual, es posible hipotetizar que el sistema 5HT a nivel del tronco (DRN, LPBN) estaría impidiendo el consumo de sodio estimulado por ANGII a las 2hs de la depleción de sodio corporal cuando se produce la caída en la natremia y en la volemia pero el AS no se manifiesta. De acuerdo con esto nos proponemos evaluar el efecto de una lesión transitoria y reversible del DRN, y el antagonismo del sistema 5HT a nivel del LPBN a las 2hs de realizada la DP sobre la ingesta de sodio. Además, se utilizará la técnica de registro electrofisiológico in vivo que nos permitirá estudiar la interconexión sináptica funcional entre el DRN y el SFO ante la infusión icv de ANGII. Esperamos observar un incremento en el consumo de sodio 2hs después de la DP, producido por el bloqueo transitorio o antagonismo del sistema serotoninérgico en el DRN o LPBN respectivamente. Además creemos posible que disminuya la actividad eléctrica de las neuronas 5HT del DRN que son moduladas por el SFO ante la inyección icv de AII.

Cell-matrix interactions in Schistosomal portal fibrosis: a dynamic event

In recent years, one of the most significant progress in the understanding of liver diseases was the demonstration that liver fibrosis is a dynamic process resulting from a balance between synthesis and degradation of several matrix components, collagen in particular. Thus, fibrosis has been found to be a very early event during liver diseases, be it of toxic, viral or parasitic origin, and to be spontaneously reversible, either partially or totally. In liver fibrosis cell matrix interactions are dependent on the existence of the many factors (sometimes acting in combination) which produce the same events at the cellular and molecular levels. These events are: (i) the recruitment of fiber-producing cells, (ii) their proliferation, (iii) the secretion of matrix constituents of the extracellular matrix, and (iv) the remodeling and degradation of the newly formed matrix. All these events represent, at least in principle, a target for a therapeutic intervention aimed at influencing the experimentally induced hepatic fibrosis. In this context, hepatosplenic schistosomiasis is of particular interest, being an immune cell-mediated granulomatous disease and a model of liver fibrosis allowing extensive studies in human and animals as well as providing original in vitro models.

Superficial and deep changes of cellular mechanical properties following cytoskeleton disassembly.

The cytoskeleton, composed of actin filaments, intermediate filaments, and microtubules, is a highly dynamic supramolecular network actively involved in many essential biological mechanisms such as cellular structure, transport, movements, differentiation, and signaling. As a first step to characterize the biophysical changes associated with cytoskeleton functions, we have developed finite elements models of the organization of the cell that has allowed us to interpret atomic force microscopy (AFM) data at a higher resolution than that in previous work. Thus, by assuming that living cells behave mechanically as multilayered structures, we have been able to identify superficial and deep effects that could be related to actin and microtubule disassembly, respectively. In Cos-7 cells, actin destabilization with Cytochalasin D induced a decrease of the visco-elasticity close to the membrane surface, while destabilizing microtubules with Nocodazole produced a stiffness decrease only in deeper parts of the cell. In both cases, these effects were reversible. Cell softening was measurable with AFM at concentrations of the destabilizing agents that did not induce detectable effects on the cytoskeleton network when viewing the cells with fluorescent confocal microscopy. All experimental results could be simulated by our models. This technology opens the door to the study of the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.

Vitamin D deficiency and a CYP27B1-1260 promoter polymorphism are associated with chronic hepatitis C and poor response to interferon-alfa based therapy

BACKGROUND: Vitamin D is an important immune modulator and preliminary data indicated an association between vitamin D deficiency and sustained virologic response (SVR) rates in patients with chronic hepatitis C. We therefore performed a comprehensive analysis on the impact of vitamin D serum levels and of genetic polymorphisms within the vitamin D cascade on chronic hepatitis C and its treatment. METHODS: Vitamin D serum levels, genetic polymorphisms within the vitamin D receptor and the 1α- hydroxylase were determined in a cohort of 468 HCV genotype 1, 2 and 3 infected patients who were treated with interferon-alfa based regimens. RESULTS: Chronic hepatitis C was associated with a high incidence of severe vitamin D deficiency compared to controls (25(OH)D3<10 ng/mL in 25% versus 12%, p<0.00001), which was in part reversible after HCV eradication. 25(OH)D3 deficiency correlated with SVR in HCV genotype 2 and 3 patients (63% and 83% SVR for patients with and without severe vitamin D deficiency, respectively, p<0.001). In addition, the CYPB27-1260 promoter polymorphism rs10877012 had substantial impact on 1-25- dihydroxyvitamin D serum levels and SVR rates in HCV genotype 1, 2 and 3 infected patients. CONCLUSIONS: Chronic hepatitis C virus infection is associated with vitamin D deficiency. Reduced 25- hydroxyvitamin D levels and CYPB27-1260 promoter polymorphism are associated with failure to achieve SVR in HCV genotype 1, 2, 3 infected patients.

Cerebral perfusion CT: Current state of the art : P44

Purpose: The aim of this educational poster is to introduce the technical principles of cerebral perfusion CT and to provide examples of its clinical applications and potential limitations in the everyday emergency practice. Methods and materials: Cerebral perfusion CT is a well established investigatory tool for many vascular and parenchymal brain dysfunctions. CT perfusion maps allow a semiquantitative assessment of cerebral perfusion. Results: Currently, cerebral perfusion CT has a pivotal role in differentiating reversible from irreversible ischemic parenchymal insult besides its integral role in grading vasospasm after subarachnoid hemorrhage. Furthermore, cerebral perfusion CT can be coupled to acetazolamide administration in order to assess the cerebrovascular reserve capacity before performing extra-/intra-cranial bypass surgery in patients with cerebral vascular insufficiency. Cerebral perfusion CT can also identify diffuse abnormalities of cerebral perfusion in children with traumatic brain injury showing a low initial GCS in order to predict the final outcome regarding the late occurrence of irreversible parenchymal damage. Cerebral Perfusion CT is also able to detect focal parenchymal perfusion abnormalities in acute epileptic seizures. Conclusion: Cerebral perfusion CT can be integrated in the management of many vascular, traumatic and functional disorders of the brain.

Political Risk, Economic Integration, and the Foreign Direct Investment Decision

In this paper we analyse the impact of policy uncertainty on foreign direct investment strategies. We also consider the impact of economic integration upon FDI decisions. The paper follows the real options approach, which allows investigating the value to a firm of waiting to invest and/or disinvest, when payoffs are stochastic due to political uncertainty and investments are partially reversible. Across the board we find that political uncertainty can be very detrimental to FDI decisions while economic integration leads to an increasing benefit of investing abroad.

In vivo characterization of the circadian clock output gene usp2

Life on earth is subject to the repeated change between day and night periods. All organisms that undergo these alterations have to anticipate consequently the adaptation of their physiology and possess an endogenous periodicity of about 24 hours called circadian rhythm from the Latin circa (about) and diem (day). At the molecular level, virtually all cells of an organism possess a molecular clock which drives rhythmic gene expression and output functions. Besides altered rhythmicity in constant conditions, impaired clock function causes pathophysiological conditions such as diabetes or hypertension. These data unveil a part of the mechanisms underlying the well-described epidemiology of shift work and highlight the function of clock-driven regulatory mechanisms. The post-translational modification of proteins by the ubiquitin polypeptide is a central mechanism to regulate their stability and activity and is capital for clock function. Similarly to the majority of biological processes, it is reversible. Deubiquitylation is carried out by a wide variety of about ninety deubiquitylating enzymes and their function remains poorly understood, especially in vivo. This class of proteolytic enzymes is parted into five families including the Ubiquitin-Specific Proteases (USP), which is the most important with about sixty members. Among them, the Ubiquitin-Specific Protease 2 (Usp2) gene encodes two protein isoforms, USP2-45 and USP2-69. The first is ubiquitously expressed under the control of the circadian clock and displays all features of core clock genes or its closest outputs effectors. Additionally, Usp2-45 was also found to be induced by the mineralocorticoid hormone aldosterone and thought to participate in Na+ reabsorption and blood pressure regulation by Epithelial Na+ Channel ENaC in the kidneys. During my thesis, I aimed to characterize the role of Usp2 in vivo with respect to these two areas, by taking advantage of a total constitutive knockout mouse model. In the first project I aimed to validate the role of USP2-45 in Na+ homeostasis and blood pressure regulation by the kidneys. I found no significant alterations of diurnal Na+ homeostasis and blood pressure in these mice, indicating that Usp2 does not play a substantial role in this process. In urine analyses, we found that our Usp2-KO mice are actually hypercalciuric. In a second project, I aimed to understand the causes of this phenotype. I found that the observed hypercalciuria results essentially from intestinal hyperabsorption. These data reveal a new role for Usp2 as an output effector of the circadian clock in dietary Ca2+ metabolism in the intestine.

Bayesian Model Averaging in the Instrumental Variable Regression Model

This paper considers the instrumental variable regression model when there is uncertainty about the set of instruments, exogeneity restrictions, the validity of identifying restrictions and the set of exogenous regressors. This uncertainty can result in a huge number of models. To avoid statistical problems associated with standard model selection procedures, we develop a reversible jump Markov chain Monte Carlo algorithm that allows us to do Bayesian model averaging. The algorithm is very exible and can be easily adapted to analyze any of the di¤erent priors that have been proposed in the Bayesian instrumental variables literature. We show how to calculate the probability of any relevant restriction (e.g. the posterior probability that over-identifying restrictions hold) and discuss diagnostic checking using the posterior distribution of discrepancy vectors. We illustrate our methods in a returns-to-schooling application.

Chemical genetic analysis of cdc2p : new insights into the regulation of cytokinesis in "Schizosaccharomyces pombe"

RÉSUMÉ La protéine kinase cyciine-cdc2p (Cdk) joue un rôle fondamental dans la progression du cycle cellulaire dans la levure de fission Schizosaccharomyces pombe. Nous avons étudié le rôle de cdc2p dans la régulation de la cascade de septation ou SIN (septation initiation network) en mitose et en méiose. Le SIN contrôle l'initiation de la cytokinèse à la fin de la mitose, et est supposé être négativement régulé par cdc2p. Nous avons mutagénéisé le site actif de cdc2p afin qu'il puisse lier un analogue de l'ATP (PP1) qui agit comme inhibiteur. Cet analogue ne peut pas lier la kinase de type sauvage. Cette approche dite «chemical genetics» permet une meilleure résolution temporelle comparée à l'approche classique utilisant les mutants sensibles à une température élevée. Nous avons montré que ce mutant cdc2-as (analogue sensitive) est fonctionnel et que, in vitro, l'activité kinase est inhibée en présence de l'analogue. Les cellules portant cette mutation, contrairement aux cellules de type sauvage s'arrêtent de manière irréversible soit en G2 soit en G1 et G2, suivant la concentration de l'inhibiteur. L'inactivation de cdc2p-as dans des cellules arrêtées en métaphase conduit au recrutement asymétrique des protéines du SIN sur le pôle du fuseau mitotique et au recrutement des composants du SIN, ainsi que de la ß-(1,3)glucan synthase à l'anneau contractile. De plus, nos résultats montrent que l'orthologue de la phosphatase cdc14p dans S. pombe, fip1p/clp1p, joue un rôle dans la régulation de la localisation des protéines du SIN suite à l'inactivation de cdc2p. Finalement, l'activité de cdc2p est requise pour maintenir la polo-like kinase plo1p sur les pôles du fuseau mitotique dans les premiers stages de la mitose. C'est pourquoi nous concluons que l'inactivation de cdc2p est suffisante pour activer le SIN et promouvoir la cytokinèse. Dans une étude séparée, nous avons caractérisé des potentiellement nouveaux composants ou régulateurs du SIN qui ont été isolés dans deux criblages génétiques visant à isoler des mutants atténuants la signalisation du SIN. Summary : The cyclin dependent protein kinase (Cdk) cdc2p plays a central role in the cell cycle progression of fission yeast Schizosaccharomyces pombe. We have studied the role of cdc2p in regulating the septation initiation network (SIN) in mitosis and meiosis. The SIN regulates the initiation of cytokinesis at the end of mitosis and is thought to be inhibited by cdc2p. We have mutated the active site of cdc2p to permit binding of an inhibitory ATP analogue (PP1), which is unable to bind unmodified kinases. This "chemical genetic" approach provides a much higher temporal resolution than it can be achieved with classical temperature-sensitive mutants. We demonstrate that cdc2-as (analogue sensitive) is functional and that addition of PP1 inhibits cdc2p kinase activity in vitro. Cells carrying the cdc2-as allele, but not cdc2+, undergo reversible cell cycle arrest following addition of PP1 either in G2, or at both major commitment points in the cell cycle (G1 and G2), depending upon the concentration of PP1. Inactivation of cdc2p-as in cells arrested in early mitosis promotes both the asymmetric recruitment of SIN proteins to the spindle pole bodies (SPBs), and the recruitment of the most downstream SIN components and ß-(1,3)-glucan synthase to the contractile ring. Furthermore, our results indicate that the S. pombe orthologue of Cdc14p, flp1p/clp1p, plays a role in regulating the relocalisation of SIN proteins following inactivation of cdc2p, and that cdc2p activity is required to retain the polo like kinase plot p on the SPBs in early mitosis. Thus, we conclude that inactivation of cdc2p is sufficient to activate the SIN and to promote cytokinesis. In a separate study, we have initially characterised potential novel components or regulators of the SIN pathway identified by two genetic screens for mutants attenuating SIN signaling.