Molecular mechanisms involved in cell response to mechanical forces

New devices were designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured. The stimulation setups were able to apply relatively large strains (30%~50%) at high temporal frequencies (140~207 Hz) in a localized subcellular region. One of the advantages of this technique was to be less invasive to the innate cellular functions because there was no direct contact between the stimulating probe and the cell body. A mechanical vibration induced by the device in the substrate gel where cells were seeded could mainly cause global calcium responses of the cells. This global response was initiated by the influx of calcium across the stretch-activated channels in the plasma membrane. The subsequent production of inositol triphosphate (IP3) via phospholipase C (PLC) activation triggered the calcium release from the endoplasmic reticulum (ER) to cause a global intracellular calcium fluctuation over the whole cell body. This global calcium response was also shown to depend on actomyosin contractility and F-actin integrity, probably controlling the membrane stretch-activated channels. The localized nature of the stimulation is one of the most important features of these new designs as it allowed the observation of the calcium signaling propagation by ER calcium release. The next step was to focus on the calcium influx, more specifically the TRPM7 channels. As TRPM7 expression may modulate cell adhesion, an adhesion assay was developed and tested on HUVECs seeded on gel substrates with different treatments: normal treatment on gels showed highest attachment rate, followed by the partially treated gels (only 5% of usual fibronectin amount) and untreated gels, with the lowest attachment rate. The trend of the attachment rates correlated to the magnitude of the calcium signaling observed after mechanical stimulation. TRPM7 expression inhibition by siRNA caused an increased attachment rate when compared to both control and non-targeting siRNA-treated cells, but resulted in an actual weaker response in terms of calcium signaling. It suggests that TRPM7 channels are indeed important for the calcium signaling in response to mechanical stimulation. A complementary study was also conducted consisting in the mechanical stimulation of a dissected Drosophila embryo. Although ionomycin treatment showed calcium influx in the tissue, the mechanical stimulation delivered as a vertical vibration did not elicited calcium signaling in response. One possible reason is the dissection procedure causing desensitization of the tissue due to the scrapings and manipulations to open the embryo.

Differential mechanisms of angiotensin II and PDGF-BB on migration and proliferation of coronary artery smooth muscle cells

Angiotensin II (Ang II) and platelet-derived growth factor-BB (PDGF-BB) are associated with excessive cell migration, proliferation and many growth-related diseases. However, whether these agents utilise similar mechanisms to trigger vascular pathologies remains to be explored. The effects of Ang II and PDGF-BB on coronary artery smooth muscle cell (CASMC) migration and proliferation were investigated via Dunn chemotaxis assay and the measurement of [3H]thymidine incorporation rates, respectively. Both atherogens produced similar degrees of cell migration which were dramatically inhibited by mevastatin (10 nM). However, the inhibitory effects of losartan (10 nM) and MnTBAP (a free radical scavenger; 50 μM) were found to be unique to Ang II-mediated chemotaxis. In contrast, MnTBAP, apocynin (an antioxidant and phagocytic NADPH oxidase inhibitor; 500 μM), mevastatin and pravastatin (100 nM) equally suppressed both Ang II and PDGF-BB-induced cellular growth. Although atherogens produced similar changes in NADPH oxidase, NOS and superoxide dismutase activities, they differentially regulated antioxidant glutathione peroxidase activity which was diminished by Ang II and unaffected by PDGF-BB. Studies with signal transduction pathway inhibitors revealed the involvement of multiple pathways i.e. protein kinase C, tyrosine kinase and MAPK in Ang II- and/or PDGF-BB-induced aforementioned enzyme activity changes. In conclusion, Ang II and PDGF-BB may induce coronary atherosclerotic disease formation by stimulating CASMC migration and proliferation through agent-specific regulation of oxidative status and utilisation of different signal transduction pathways.

Mechanisms underlying activation of neural stem cells in the adult central nervous system

À la fin du 19e siècle, Dr. Ramón y Cajal, un pionnier scientifique, a découvert les éléments cellulaires individuels, appelés neurones, composant le système nerveux. Il a également remarqué la complexité de ce système et a mentionné l’impossibilité de ces nouveaux neurones à être intégrés dans le système nerveux adulte. Une de ses citations reconnues : “Dans les centres adultes, les chemins nerveux sont fixes, terminés, immuables. Tout doit mourir, rien ne peut être régénérer” est représentative du dogme de l’époque (Ramón y Cajal 1928). D’importantes études effectuées dans les années 1960-1970 suggèrent un point de vue différent. Il a été démontré que les nouveaux neurones peuvent être générés à l’âge adulte, mais cette découverte a créé un scepticisme omniprésent au sein de la communauté scientifique. Il a fallu 30 ans pour que le concept de neurogenèse adulte soit largement accepté. Cette découverte, en plus de nombreuses avancées techniques, a ouvert la porte à de nouvelles cibles thérapeutiques potentielles pour les maladies neurodégénératives. Les cellules souches neurales (CSNs) adultes résident principalement dans deux niches du cerveau : la zone sous-ventriculaire des ventricules latéraux et le gyrus dentelé de l’hippocampe. En condition physiologique, le niveau de neurogenèse est relativement élevé dans la zone sous-ventriculaire contrairement à l’hippocampe où certaines étapes sont limitantes. En revanche, la moelle épinière est plutôt définie comme un environnement en quiescence. Une des principales questions qui a été soulevée suite à ces découvertes est : comment peut-on activer les CSNs adultes afin d’augmenter les niveaux de neurogenèse ? Dans l’hippocampe, la capacité de l’environnement enrichi (incluant la stimulation cognitive, l’exercice et les interactions sociales) à promouvoir la neurogenèse hippocampale a déjà été démontrée. La plasticité de cette région est importante, car elle peut jouer un rôle clé dans la récupération de déficits au niveau de la mémoire et l’apprentissage. Dans la moelle épinière, des études effectuées in vitro ont démontré que les cellules épendymaires situées autour du canal central ont des capacités d’auto-renouvellement et de multipotence (neurones, astrocytes, oligodendrocytes). Il est intéressant de noter qu’in vivo, suite à une lésion de la moelle épinière, les cellules épendymaires sont activées, peuvent s’auto-renouveller, mais peuvent seulement ii donner naissance à des cellules de type gliale (astrocytes et oligodendrocytes). Cette nouvelle fonction post-lésion démontre que la plasticité est encore possible dans un environnement en quiescence et peut être exploité afin de développer des stratégies de réparation endogènes dans la moelle épinière. Les CSNs adultes jouent un rôle important dans le maintien des fonctions physiologiques du cerveau sain et dans la réparation neuronale suite à une lésion. Cependant, il y a peu de données sur les mécanismes qui permettent l'activation des CSNs en quiescence permettant de maintenir ces fonctions. L'objectif général est d'élucider les mécanismes sous-jacents à l'activation des CSNs dans le système nerveux central adulte. Pour répondre à cet objectif, nous avons mis en place deux approches complémentaires chez les souris adultes : 1) L'activation des CSNs hippocampales par l'environnement enrichi (EE) et 2) l'activation des CSNs de la moelle épinière par la neuroinflammation suite à une lésion. De plus, 3) afin d’obtenir plus d’information sur les mécanismes moléculaires de ces modèles, nous utiliserons des approches transcriptomiques afin d’ouvrir de nouvelles perspectives. Le premier projet consiste à établir de nouveaux mécanismes cellulaires et moléculaires à travers lesquels l’environnement enrichi module la plasticité du cerveau adulte. Nous avons tout d’abord évalué la contribution de chacune des composantes de l’environnement enrichi à la neurogenèse hippocampale (Chapitre II). L’exercice volontaire promeut la neurogenèse, tandis que le contexte social augmente l’activation neuronale. Par la suite, nous avons déterminé l’effet de ces composantes sur les performances comportementales et sur le transcriptome à l’aide d’un labyrinthe radial à huit bras afin d’évaluer la mémoire spatiale et un test de reconnaissante d’objets nouveaux ainsi qu’un RNA-Seq, respectivement (Chapitre III). Les coureurs ont démontré une mémoire spatiale de rappel à court-terme plus forte, tandis que les souris exposées aux interactions sociales ont eu une plus grande flexibilité cognitive à abandonner leurs anciens souvenirs. Étonnamment, l’analyse du RNA-Seq a permis d’identifier des différences claires dans l’expression des transcripts entre les coureurs de courte et longue distance, en plus des souris sociales (dans l’environnement complexe). iii Le second projet consiste à découvrir comment les cellules épendymaires acquièrent les propriétés des CSNs in vitro ou la multipotence suite aux lésions in vivo (Chapitre IV). Une analyse du RNA-Seq a révélé que le transforming growth factor-β1 (TGF-β1) agit comme un régulateur, en amont des changements significatifs suite à une lésion de la moelle épinière. Nous avons alors confirmé la présence de cette cytokine suite à la lésion et caractérisé son rôle sur la prolifération, différentiation, et survie des cellules initiatrices de neurosphères de la moelle épinière. Nos résultats suggèrent que TGF-β1 régule l’acquisition et l’expression des propriétés de cellules souches sur les cellules épendymaires provenant de la moelle épinière.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem- resistant Pseudomonas aeruginosa isolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosa isolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXYOprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Fish Reproduction Is Disrupted upon Lifelong Exposure to Environmental PAHs Fractions Revealing Different Modes of Action

Polycyclic aromatic hydrocarbons (PAHs) constitute a large family of organic pollutants emitted in the environment as complex mixtures, the compositions of which depend on origin. Among a wide range of physiological defects, PAHs are suspected to be involved in disruption of reproduction. In an aquatic environment, the trophic route is an important source of chronic exposure to PAHs. Here, we performed trophic exposure of zebrafish to three fractions of different origin, one pyrolytic and two petrogenic. Produced diets contained PAHs at environmental concentrations. Reproductive traits were analyzed at individual, tissue and molecular levels. Reproductive success and cumulative eggs number were disrupted after exposure to all three fractions, albeit to various extents depending on the fraction and concentrations. Histological analyses revealed ovary maturation defects after exposure to all three fractions as well as degeneration after exposure to a pyrolytic fraction. In testis, hypoplasia was observed after exposure to petrogenic fractions. Genes expression analysis in gonads has allowed us to establish common pathways such as endocrine disruption or differentiation/maturation defects. Taken altogether, these results indicate that PAHs can indeed disrupt fish reproduction and that different fractions trigger different pathways resulting in different effects.

Allelopathy: driving mechanisms governing its activity in agriculture.

Allelopathy determines the dynamics of plant species in different environments. Understanding this biological phenomenon could help to develop applications in both natural and agricultural systems. This review summarizes the genetic and environmental characteristics that control the production and release of allelochemicals in agroecosystems. This study highlights the current understanding of the environmental changes caused by allelochemicals and summarizes the knowledge about the mechanisms of action of these compounds. Finally, it reviews novel applications of allelopathy in agricultural production systems, including the role of allelochemicals in consortia and their potential use in no-tillage cropping systems through cover crops or mulches.

A survey on mechanisms and critical parameters on solidification of selective laser melting during fabrication of Ti-6Al-4V prosthetic acetabular cup

Additive Manufacturing (AM) includes a range of approaches that correlate with computer aided design (CAD) and manufacturing by fabrication via precise layers and is a promising method for the production of medical tools. In this study, different aspects and mechanisms of solidification for curved surfaces based on equilibrium at curved interfaces, Monge patch, interfacial and Gibbs energy will be discussed. Also, the effect of capillarity, geometry, substrate temperature, cooling rate and scanning parameters in the solidification of a prosthetic acetabular cup (PAC) using selective laser melting (SLM) is analysed. The contributions of this work are analysing solidification and effective factors in this process to produce parts with a higher quality and mechanical properties such as strength, strain, porosity, relative density and hardness. Results indicate that due to the surface to volume (S/V) ratio, and the increasing effect of the radius on Monge patch, thermal stresses and surface forces are more prevalent on outer surfaces. Moreover, solidification and mechanical properties are related to capillarity, geometry, substrate temperature, cooling rate, scanning power and speed. The results also indicate the interaction of solute diffusion and heat transfer with interatomic forces in large S/V ratio and at small scales tend to improve solidification.

Aqueous peptide-TiO2 interfaces: isoenergetic binding via either entropically or enthalpically driven mechanisms

A major barrier to the systematic improvement of biomimetic peptide-mediated strategies for the controlled growth of inorganic nanomaterials in environmentally benign conditions lies in the lack of clear conceptual connections between the sequence of the peptide and its surface binding affinity, with binding being facilitated by noncovalent interactions. Peptide conformation, both in the adsorbed and in the nonadsorbed state, is the key relationship that connects peptide-materials binding with peptide sequence. Here, we combine experimental peptide-titania binding characterization with state-of-the-art conformational sampling via molecular simulations to elucidate these structure/binding relationships for two very different titania-binding peptide sequences. The two sequences (Ti-1, QPYLFATDSLIK; Ti-2, GHTHYHAVRTQT) differ in their overall hydropathy, yet via quartz-crystal microbalance measurements and predictions from molecular simulations, we show these sequences both support very similar, strong titania-binding affinities. Our molecular simulations reveal that the two sequences exhibit profoundly different modes of surface binding, with Ti-1 acting as an entropically driven binder while Ti-2 behaves as an enthalpically driven binder. The integrated approach presented here provides a rational basis for peptide sequence engineering to achieve the in situ growth and organization of titania nanostructures in aqueous media and for the design of sequences suitable for a range of technological applications that involve the interface between titania and biomolecules.

Anti-inflammatory activity and mechanisms of a lipid extract from hard-shelled mussel (mytilus coruscus) in mice with dextran sulphate sodium-induced colitis

The anti-inflammatory effect of a lipid extract from hard-shelled mussel (HMLE) on dextran sulphate sodium (DSS)-induced colitis in mice was investigated. Salicylazosulphapyridine (SASP) and different doses of HMLE were administered by gastric gavage. HMLE significantly attenuated DSS-induced colitis disease activity index scores, tissue damage, splenic enlargement and colon myeloperoxidase accumulation. In addition, HMLE improved colon oxidative stress and production and expression of anti-inflammatory cytokine, interleukin (IL)-10, while HMLE inhibited the abnormal productions and mRNA expressions of pro-inflammatory cytokines, namely tumour necrosis factor-α, IL-1β, and IL-6, as well as the expression of key molecules in the toll-like receptor (TLR)-4/nuclear factor (NF)-κB signalling pathway. These findings suggest that HMLE has an anti-inflammatory effect on DSS-induced colitis, equivalent to that of SASP, and this effect might be related to the regulation of inflammatory mediators and key molecules in the TLR-4/NF-κB pathway.

Parent-related mechanisms underlying the social gradient of childhood overweight and obesity: a systematic review

BACKGROUND: Low socio-economic status (SES) is a significant risk factor for childhood overweight and obesity (COWOB) in high-income countries. Parents to young children buffer and accentuate social and cultural influences, and are central to the development of this disease. An understanding of the parent-related mechanisms that underlie the SES-COWOB relationship is needed to improve the efficacy of prevention and intervention efforts. OBJECTIVE: A systematic review of relevant literature was conducted to investigate the mechanisms by which levels of SES (low, middle and high) are associated to COWOB, by exploring mediation and interaction effects. METHOD: Six electronic databases were searched yielding 5155 initial records, once duplicates were removed. Studies were included if they investigated COWOB, SES, parent-related factors and the multivariate relationship between these factors. Thirty studies were included. Factors found to be mediating the SES-COWOB relationship or interacting with SES to influence COWOB were categorized according to an ecological systems framework, at child, parent, household and social system level factors. RESULTS: High parent body mass index, ethnicity, child-care attendance, high TV time (mother and child), breastfeeding (early weaning), food intake behaviours and birthweight potentially mediate the relationship between SES and COWOB. Different risk factors for COWOB in different SES groups were found. For low SES families, parental obesity and maternal depressive symptoms were strong risk factors for COWOB, whereas long maternal working hours and a permissive parenting style were risk factors for higher SES families. None of the studies investigated parental psychological attributes such as attitudes, beliefs, self-esteem and so on as potential mechanisms/risk factors. CONCLUSIONS: Families from different SES groups have different risk and protective factors for COWOB. Prevention and intervention efforts may have improved efficacy if they are tailored to address specific risk factors within SES.

Contrast and lustre:a model that accounts for eleven different forms of contrast discrimination in binocular vision

Our goal here is a more complete understanding of how information about luminance contrast is encoded and used by the binocular visual system. In two-interval forced-choice experiments we assessed observers' ability to discriminate changes in contrast that could be an increase or decrease of contrast in one or both eyes, or an increase in one eye coupled with a decrease in the other (termed IncDec). The base or pedestal contrasts were either in-phase or out-of-phase in the two eyes. The opposed changes in the IncDec condition did not cancel each other out, implying that along with binocular summation, information is also available from mechanisms that do not sum the two eyes' inputs. These might be monocular mechanisms. With a binocular pedestal, monocular increments of contrast were much easier to see than monocular decrements. These findings suggest that there are separate binocular (B) and monocular (L,R) channels, but only the largest of the three responses, max(L,B,R), is available to perception and decision. Results from contrast discrimination and contrast matching tasks were described very accurately by this model. Stimuli, data, and model responses can all be visualized in a common binocular contrast space, allowing a more direct comparison between models and data. Some results with out-of-phase pedestals were not accounted for by the max model of contrast coding, but were well explained by an extended model in which gratings of opposite polarity create the sensation of lustre. Observers can discriminate changes in lustre alongside changes in contrast.

Detection of Carbapenemase Production in a Collection of Enterobacteriaceae with Characterized Resistance Mechanisms from Clinical and Environmental Origins by Use of Both Carba NP and Blue-Carba Tests

Rapid-screening methods to confirm the presence of resistance mechanisms in multidrug-resistant bacteria are currently recommended. Carba NP and Blue-Carba tests were evaluated in carbapenemase-producing Enterobacteriaceae from hospital (n = 102) and environmental (n = 57) origins for detecting the different molecular classes among them. Both methods showed to be fast and cost-effective, with high sensitivity (98% to 100%) and specificity (100%), and may be easily introduced in the routine laboratory.

The old and the oldest-old: Do they have different perspectives on adjustment to aging?

Background: Older adults experience varying challenges in old age. This study aims to explore the indicators of adjustment to aging (AtA) and to examine the potential explanatory mechanisms of a correlational model for AtA for the old and oldest-old adults. Methods: This qualitative study comprised demographics and semistructured interviews. Complete information on 152 older adults aged between 75 years and 102 years (mean ¼ 83.76 years; standard deviation ¼ 6.458). Data was subjected to content analysis. The correlational model of indicators of AtA was analyzed using a multiple correspondence analysis. Results: “Occupation and achievement” was the most mentioned indicator of AtA by the old participants (17.7%), whereas “existential meaning and spirituality” was the most verbalized indicator of AtA for the oldest-old participants (16.9%). AtA was explained by a three-factor model for each age group. For the old participants, the largest factor “occupational and social focus” accounted for 33.6% of total variance, whereas for the oldest-old participants, “spirituality and health focus” represented 33.5% of total variance. Conclusion: The outcomes presented in this paper stressed the varied perspectives concerning AtA, contoured in two different models, and the need of considering these when designing and implementing programs in health care for the old and the oldest-old.

Transthyretin Proteins Regulate Angiogenesis by Conferring Different Molecular Identities to Endothelial Cells

Familial amyloidotic polyneuropathy (FAP) has a high prevalence in Portugal, and the most common form of hereditary amyloidosis is caused by an amyloidogenic variant of transthyretin (TTR) with a substitution of methionine for valine at position 30 (V30M). Until now, the available efficient therapy is liver transplantation, when performed in an early phase of the onset of the disease symptoms. However, transplanted FAP patients have a significantly higher incidence of early hepatic artery thrombosis compared with non-FAP transplanted patients. Because FAP was described as an independent risk factor for early hepatic artery thrombosis, more studies to understand the underlying mechanisms involved in this outcome are of the utmost importance. Knowing that the liver is the major site for TTR production, we investigated the biological effects of TTR proteins in the vasculature and on angiogenesis. In this study, we identified genes differentially expressed in endothelial cells exposed to the WT or V30M tetramer. We found that endothelial cells may acquire different molecular identities when exposed to these proteins, and consequently TTR could regulate angiogenesis. Moreover, we show that V30M decreases endothelial survival by inducing apoptosis, and it inhibits migration. These findings provide new knowledge that may have critical implications in the prevention of early hepatic artery thrombosis in FAP patients after liver transplantation.

The initial phase of sodium sulfite pulping of softwood : A comparison of different pulping options

Single stage and two-stage sodium sulfite cooking were carried out on either spruce, pine or pure pine heartwood chips to investigate the influence of several process parameters on the initial phase of such a cook down to about 60 % pulp yield. The cooking experiments were carried out in the laboratory with either a lab-prepared or a mill-prepared cooking acid and the temperature and time were varied. The influences of dissolved organic and inorganic components in the cooking liquor on the final pulp composition and on the extent of side reactions were investigated. Kinetic equations were developed and the activation energies for delignification and carbohydrate dissolution were calculated using the Arrhenius equation. A better understanding of the delignification mechanisms during bisulfite and acid sulfite cooking was obtained by analyzing the lignin carbohydrate complexes (LCC) present in the pulp when different cooking conditions were used. It was found that using a mill-prepared cooking acid beneficial effect with respect to side reactions, extractives removal and higher stability in pH during the cook were observed compared to a lab-prepared cooking acid. However, no significant difference in degrees of delignification or carbohydrate degradation was seen.  The cellulose yield was not affected in the initial phase of the cook however; temperature had an influence on the rates of both delignification and hemicellulose removal. It was also found that the  corresponding activation energies increased in the order:  xylan, glucomannan, lignin and cellulose. The cooking temperature could thus be used to control the cook to a given carbohydrate composition in the final pulp. Lignin condensation reactions were observed during acid sulfite cooking, especially at higher temperatures. The LCC studies indicated the existence of covalent bonds between lignin and hemicellulose components with respect to xylan and glucomannan. LCC in native wood showed the presence of phenyl glycosides, ϒ-esters and α-ethers; whereas the α-ethers  were affected during sulfite pulping. The existence of covalent bonds between lignin and wood polysaccharides might be the rate-limiting factor in sulfite pulping.