Cyclic mechanical strain causes cAMP-response element binding protein activation by different pathways in cardiac fibroblasts

The transcription factor cAMP-response element binding protein (CREB) mediates the mechanical strain-induced gene expression in the heart. This study investigated which signaling pathways are involved in the straininduced CREB activation using cultured ventricular fibroblasts from adult rat hearts. CREB phosphorylation was analyzed by immunocytochemistry and ELISA. Cyclic mechanical strain (1 Hz and 5% elongation) for 15 min induced CREB phosphorylation in all CREB-positive fibroblasts. Several signaling transduction pathways can contribute to strain-induced CREB activation. The inhibition of PKA, PKC, MEK, p38-MAPK or PI3-kinase partially reduced the strain-induced CREB phosphorylation. Activation of PKA by forskolin or PKC by PMA resulted in a level of CREB phosphorylation comparable to the reduced level of the strain-induced CREB phosphorylation in the presence of PKA or PKC inhibitors. Signaling pathways involving PKC, MEK, p38-MAPK or PI3-kinase seem to converge during strain-induced CREB activation. PKA interacted additively with the investigated signaling pathways. The strain-induced c-Fos expression can be reduced by PKC inhibition but not by PKA inhibition. Our results suggest that the complete strain-induced CREB phosphorylation involves several signaling pathways that have a synergistic effect. The influence on gene expression is dependent on the level and the time of CREB stimulation. These wide-ranging possibilities of CREB activation provide a graduated control system

Investigations on how the enteropathogenic Escherichia coli (EPEC) EspF effector inhibits PI-3 kinase-dependent phagocytosis

PhD ThesisEnteropathogenic E. coli inhibits phosphoinositide 3 (PI-3) kinase dependent phagocytosis via a Type Three Secretion System (T3SS) that delivers up to twenty effector proteins into target cells. The T3SS components are encoded on the Locus of Enterocyte Effacement (LEE) pathogenicity island alongside genes for a surface protein, Intimin, and seven effectors (Tir, Map, EspF, EspG, EspZ, EspH and EspB; latter also needed to deliver effectors). Inhibiting phagocytosis is linked to EspB, EspH, EspG and EspF activities with inhibitory mechanisms described for all except EspF. The aim of this study was to determine if EspF alone could inhibit phagocytosis and define the inhibitory mechanism. Initial anti-phagocytosis studies, with J774A.1 macrophages, not only confirmed EspF and T3SS-dependent inhibition but suggested a T3SS-independent contribution. Moreover, studies with effectors-deficient EPEC and non-pathogenic E. coli carrying LEE on a plasmid argued for LEE sufficiency. Surprisingly, delivering EspF into macrophages without most (EPEC multi-mutant) or all (via T3SS of another pathogen, Yersinia) other EPEC effectors argued against EspF sufficiency. Interestingly, the data also argued against EspG driving the anti-phagocytosis process but suggested that EspF’s contribution required Map, EspH, Tir, and/or the Intimin activities. Surprisingly, screening EspF/Map/EspH/Tir/Intimin single, double, triple, quadruple and quintuple mutants failed to confirm the critical roles for EspF or EspH that are linked to phenotypic instability and/or indirect contributions. Preliminary investigations on EspF’s involvement revealed possible features and domains required for its efficient expression, secretion and/or inhibiting phagocytosis. Crucially, the screening data provided many hypotheses including Tir and Intimin being able to drive T3SS-dependent anti-phagocytosis, an idea supported by complementation studies. Collectively, this study provides important new insights on EPEC’s ability to inhibit its uptake by J774A.1 macrophages and reveals unknown levels of complexity.Higher Committee for Education Development in Iraq (HCED

Die Wertigkeit des sFlt-1/PlGF-Quotienten als Prädiktionsmarker bei Schwangeren mit erhöhtem Präeklampsierisiko: Die Wertigkeit des sFlt-1/PlGF-Quotienten als Prädiktionsmarker beiSchwangeren mit erhöhtem Präeklampsierisiko

Einleitung: Die Dysbalance proangiogener (Placental Growth Factor = PlGF) und antiangiogener Faktoren (soluble fms-like tyrosine kinase 1 = sFlt-1) gilt heute als pathophysiologische Grundlage bei der Entstehung einer Präeklampsie (PE), eines HELLP-Syndroms (Haemolysis, Elevated Liver enzymes, Low Platelets) oder einer intrauterinen Wachstumsretardierung (IUGR). Der sFlt1/PlGF-Quotient, ein sensitiver und robuster diagnostischer Marker, ist bereits Wochen vor der Krankheitsmanifestation erhöht. Ziel dieser Studie war es, die Wertigkeit des sFlt1/PlGFQuotienten als prädiktiven Faktor bei Risikopatientinnen zu untersuchen. Patienten und Methode: In diese prospektive Studie wurden 68 Patientinnen mit einer Einlingsschwangerschaft und mindestens einem Risikofaktor für das Auftreten einer PE, eines HELLP-Syndrom oder einer IUGR im Schwangerschaftsverlauf eingeschlossen. Die Patientinnen wurden je nach Verlauf der Schwangerschaft in eine Gruppe mit Symptomen (Fallgruppe) und eine Gruppe ohne Symptome (Kontrollgruppe) für eine der oben genannten Erkrankungen unterteilt. Der sFlt1/PlGF-Quotient wurde bei der Aufnahme in die Studie und im weiteren Schwangerschaftsverlauf bestimmt. Ergebnisse: Eine PE, ein HELLP-Syndrom oder eine IUGR trat bei 41 % der Risikopatientinnen auf. Der absolute Wert des sFlt-1/PlGF-Quotienten war nur bei der Gruppe mit Symptomen auf ≥ 85 erhöht und zeigte sich in der 25 + 0-31 + 0 SSW (p = 0,005) und ab der 35 + 0 SSW (p = 0,044) als prädiktiver Faktor für eine PE, ein HELLP-Syndrom oder eine IUGR. Ab 7–10 Wochen vor der Entbindung war, in der Fallgruppe stärker als in der Kontrollgruppe, ein Anstieg des sFlt1/PlGFQuotienten zu beobachten. Dieser war 0–2 Wochen vor der Entbindung bei beiden Gruppen (Kontrollgruppe (MW ± SA 66,9 ± 134) vs. Fallgruppe (MW ± SA 393,3 ± 147,4, p = 0,021) am stärksten und zeigte sich ebenfalls als prädiktiver Faktor für eine der genannten Schwangerschaftserkrankungen (p = 0,025). Schlussfolgerung: Bei Risikoschwangeren kann der sFlt1/PlGF-Quotient für die Einschätzung des individuellen Risikos für eine PE, ein HELLP-Syndrom oder eine IUGR im Schwangerschaftsverlauf genutzt werden. Wiederholte Messungen des Quotienten versprechen eine risikoangepasste Betreuung dieser Patientinnen

Dissecting and modeling photic and melanopsin effects to predict sleep disturbances induced by irregular light exposure in mice.

Artificial lighting, day-length changes, shift work, and transmeridian travel all lead to sleep-wake disturbances. The nychthemeral sleep-wake cycle (SWc) is known to be controlled by output from the central circadian clock in the suprachiasmatic nuclei (SCN), which is entrained to the light-dark cycle. Additionally, via intrinsically photosensitive retinal ganglion cells containing the photopigment melanopsin (Opn4), short-term light-dark alternations exert direct and acute influences on sleep and waking. However, the extent to which longer exposures typically experienced across the 24-h day exert such an effect has never been clarified or quantified, as disentangling sustained direct light effects (SDLE) from circadian effects is difficult. Recording sleep in mice lacking a circadian pacemaker, either through transgenesis (Syt10 <sup>cre/cre</sup> Bmal1 <sup>fl/-</sup> ) or SCN lesioning and/or melanopsin-based phototransduction (Opn4 <sup>-/-</sup> ), we uncovered, contrary to prevailing assumptions, that the contribution of SDLE is as important as circadian-driven input in determining SWc amplitude. Specifically, SDLE were primarily mediated (>80%) through melanopsin, of which half were then relayed through the SCN, revealing an ancillary purpose for this structure, independent of its clock function in organizing SWc. Based on these findings, we designed a model to estimate the effect of atypical light-dark cycles on SWc. This model predicted SWc amplitude in mice exposed to simulated transequatorial or transmeridian paradigms. Taken together, we demonstrate this SDLE is a crucial mechanism influencing behavior on par with the circadian system. In a broader context, these findings mandate considering SDLE, in addition to circadian drive, for coping with health consequences of atypical light exposure in our society

Weed suppression greatly increased by plant diversity in intensively managed grasslands:A continental-scale experiment

1. Grassland diversity can support sustainable intensification of grassland production through increased yields, reduced inputs and limited weed invasion. We report the effects of diversity on weed suppression from three years of a 31-site continental-scale field experiment. 2. At each site, fifteen grassland communities comprising four monocultures and eleven 4-species mixtures based on a wide range of species? proportions were sown at two densities and managed by cutting. Forage species were selected according to two crossed functional traits, ?method of nitrogen acquisition? and ?pattern of temporal development?. 3. Averaged across sites, years, and sown densities, annual weed biomass in mixtures and monocultures was 0.5 and 2.0 t DM ha-1 (7% and 33% of total biomass respectively). In over 95% of mixtures (across sites and years) weed biomass was lower than the average of monocultures, and, in about two thirds of cases, lower than in the most suppressive monoculture (transgressive suppression). Suppression was significantly transgressive for 58% of site-years. Transgressive suppression by mixtures was maintained across years and was independent of site productivity. 4. Based on models, predicted average weed biomass in mixture over the whole experiment was 52% less (95% confidence interval 30% to 75%) than in the most suppressive monoculture, and was significantly lower than the most suppressive monoculture across all mixtures and along functional trait axes. Transgressive suppression of weed biomass was significant at each year across all mixtures and for each mixture. 5. Weed biomass was consistently low across all mixtures and years and although, in some years, it increased with increasing sown legume proportion and decreased with increasing sown proportion of persistent species, the level of weed biomass was not largely different from that in the equiproportional mixture. 6. The average variability (standard deviation in t DM ha-1) of annual weed biomass within a site was much lower for mixtures (0.42) than for monocultures (1.77). 7. Synthesis and applications. Weed invasion can be diminished through combining forage species selected for complementarity and persistence traits in systems designed to reduce reliance on fertilizer nitrogen. The effects of mixtures on weed suppression were consistently strong across mixtures varying widely in species proportions and over time. The level of weed biomass did not vary greatly across mixtures varying widely in proportions of sown species. The benefits of plant diversity in intensively managed grasslands are highly relevant for the sustainable intensification of agriculture; importantly, this can be achieved through practical farm-scale actions.publishersversionPeer reviewe

Mechanical stretch stimulates integrin αVβ3-mediated collagen expression in human anterior cruciate ligament cells

Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Here, we focused on the stretch-related responses of different cells derived from the ligament-to-bone interface and midsubstance regions of human anterior cruciate ligaments. Chondroblastic interface cells easily lost their potential to produce collagen genes in non-stretched conditions, rather than fibroblastic midsubstance cells. Uni-axial mechanical stretches increased the type I collagen gene expression of interface and midsubstance cells up to 14- and 6-fold levels of each non-stretched control, respectively. Mechanical stretches also activated the stress fiber formation by shifting the distribution of integrin αVβ3 to the peripheral edges in both interface and midsubstance cells. In addition, integrin αVβ3 colocalized with phosphorylated focal adhesion kinase in stretched cells. Functional blocking analyses using anti-integrin antibodies revealed that the stretch-activated collagen gene expressions on fibronectin were dependent on integrin αVβ3-mediated cellular adhesions in the interface and midsubstance cells. These findings suggest that the integrin αVβ3-mediated stretch signal transduction might have a key role to stimulate collagen gene expression in human anterior cruciate ligament, especially in the ligament-to-bone interface

Innate Attractiveness and Associative Learnability of Odors Can Be Dissociated in Larval Drosophila

We investigate olfactory associative learning in larval Drosophila. A reciprocal training design is used, such that one group of animals receives a reward in the presence of odor X but not in the presence of odor Y (Train: X+ // Y), whereas another group is trained reciprocally (Train: X // Y+). After training, differences in odor preference between these reciprocally trained groups in a choice test (Test: X -- Y) reflect associative learning. The current study, after showing which odor pairs can be used for such learning experiments, 1) introduces a one-odor version of such reciprocal paradigm that allows estimating the learnability of single odors. Regarding this reciprocal one-odor paradigm, we show that 2) paired presentations of an odor with a reward increase odor preference above baseline, whereas unpaired presentations of odor and reward decrease odor preference below baseline; this suggests that odors can become predictive either of reward or of reward absence. Furthermore, we show that 3) innate attractiveness and associative learnability can be dissociated. These data deepen our understanding of odor-reward learning in larval Drosophila on the behavioral level, and thus foster its neurogenetic analysis

Major shifts in species’ relative abundance in grassland mixtures alongside positive effects of species diversity in yield:A continental-scale experiment

Increased species diversity promotes ecosystem function; however, the dynamics of multi-species grassland systems over time and their role in sustaining higher yields generated by increased diversity are still poorly understood. We investigated the development of species’ relative abundances in grassland mixtures over 3 years to identify drivers of diversity change and their links to yield diversity effects.A continental-scale field experiment was conducted at 31 sites using 11 different four-species mixtures each sown at two seed abundances. The four species consisted of two grasses and two legumes, of which one was fast establishing and the other temporally persistent. We modelled the dynamics of the four-species mixtures, and tested associations with diversity effects on yield.We found that species’ dynamics were primarily driven by differences in the relative growth rates (RGRs) of competing species, and secondarily by density dependence and climate. The temporally persistent grass species typically had the highest RGRs and hence became dominant over time. Density dependence sometimes induced stabilising processes on the dominant species and inhibited shifts to monoculture. Legumes persisted at most sites at low or medium abundances and persistence was improved at sites with higher annual minimum temperature.Significant diversity effects were present at the majority of sites in all years and the strength of diversity effects was improved with higher legume abundance in the previous year. Observed diversity effects, when legumes had declined, may be due to (i) important effects of legumes even at low abundance, (ii) interaction between the two grass species or (iii) a store of N because of previous presence of legumes.Synthesis. Alongside major compositional changes driven by RGR differences, diversity effects were observed at most sites, albeit at reduced strength as legumes declined. This evidence strongly supports the sowing of multi-species mixtures that include legumes over the long-standing practice of sowing grass monocultures. Careful and strategic selection of the identity of the species used in mixtures is suggested to facilitate the maintenance of species diversity and especially persistence of legumes over time, and to preserve the strength of yield increases associated with diversity

Human cardiac fibroblasts adaptive responses to controlled combined mechanical strain and oxygen changes in vitro

Upon cardiac pathological conditions such as ischemia, microenvironmental changes instruct a series of cellular responses that trigger cardiac fibroblasts-mediated tissue adaptation and inflammation. A comprehensive model of how early environmental changes may induce cardiac fibroblasts (CF) pathological responses is far from being elucidated, partly due to the lack of approaches involving complex and simultaneous environmental stimulation. Here, we provide a first analysis of human primary CF behavior by means of a multi-stimulus microdevice for combined application of cyclic mechanical strain and controlled oxygen tension. Our findings elucidate differential human CFs responses to different combinations of the above stimuli. Individual stimuli cause proliferative effects (PHH3+ mitotic cells, YAP translocation, PDGF secretion) or increase collagen presence. Interestingly, only the combination of hypoxia and a simulated loss of contractility (2% strain) is able to additionally induce increased CF release of inflammatory and pro-fibrotic cytokines and matrix metalloproteinases

Endothelial cells enhance the in vivo bone-forming ability of osteogenic cell sheets

Addressing the problem of vascularization is of vital importance when engineering three-dimensional (3D) tissues. Endothelial cells are increasingly used in tissue-engineered constructs to obtain prevascularization and to enhance in vivo neovascularization. Rat bone marrow stromal cells were cultured in thermoresponsive dishes under osteogenic conditions with human umbilical vein endothelial cells (HUVECs) to obtain homotypic or heterotypic cell sheets (CSs). Cells were retrieved as sheets from the dishes after incubation at 20 °C. Monoculture osteogenic CSs were stacked on top of homotypic or heterotypic CSs, and subcutaneously implanted in the dorsal flap of nude mice for 7 days. The implants showed mineralized tissue formation under both conditions. Transplanted osteogenic cells were found at the new tissue site, demonstrating CS bone-inductive effect. Perfused vessels, positive for human CD31, confirmed the contribution of HUVECs for the neovascularization of coculture CS constructs. Furthermore, calcium quantification and expression of osteocalcin and osterix genes were higher for the CS constructs, with HUVECs demonstrating the more robust osteogenic potential of these constructs. This work demonstrates the potential of using endothelial cells, combined with osteogenic CSs, to increase the in vivo vascularization of CS-based 3D constructs for bone tissue engineering purposes.We would like to acknowledge Mariana T Cerqueira for the illustration in Figure 1. This study was supported by Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology 'Cell Sheet Tissue Engineering Center (CSTEC)' and the Global CUE program, the Multidisciplinary Education and Research Center for Regenerative Medicine (MERCREM), from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Financial support to RP Pirraco by the Portuguese Foundation for Science and Technology (FCT) through the PhD Grant SFRH/BD/44893/2008 is also acknowledged