Living biointerfaces based on non-pathogenic bacteria to direct cell differentiation

Genetically modified Lactococcus lactis, non-pathogenic bacteria expressing the FNIII7-10 fibronectin fragment as a protein membrane have been used to create a living biointerface between synthetic materials and mammalian cells. This FNIII7-10 fragment comprises the RGD and PHSRN sequences of fibronectin to bind α5β1 integrins and triggers signalling for cell adhesion, spreading and differentiation. We used L. lactis strain to colonize material surfaces and produce stable biofilms presenting the FNIII7-10 fragment readily available to cells. Biofilm density is easily tunable and remains stable for several days. Murine C2C12 myoblasts seeded over mature biofilms undergo bipolar alignment and form differentiated myotubes, a process triggered by the FNIII7-10 fragment. This biointerface based on living bacteria can be further modified to express any desired biochemical signal, establishing a new paradigm in biomaterial surface functionalisation for biomedical applications

Biomarker discovery and redundancy reduction towards classification using a multi-factorial MALDI-TOF MS T2DM mouse model dataset

Diabetes like many diseases and biological processes is not mono-causal. On the one hand multifactorial studies with complex experimental design are required for its comprehensive analysis. On the other hand, the data from these studies often include a substantial amount of redundancy such as proteins that are typically represented by a multitude of peptides. Coping simultaneously with both complexities (experimental and technological) makes data analysis a challenge for Bioinformatics

Chemicals released by male sea cucumber mediate aggregation and spawning behaviours

The importance of chemical communication in reproduction has been demonstrated in many marine broadcast spawners. However, little is known about the use of chemical communication by echinoderms, the nature of the compounds involved and their mechanism(s) of action. Here, the hypothesis that the sea cucumber Holothuria arguinensis uses chemical communication for aggregation and spawning was tested. Water conditioned by males, but not females, attracted both males and females; gonad homogenates and coelomic fluid had no effect on attraction. Male spawning water, but not female spawning water, stimulated males and females to release their gametes; the spermatozoa alone did not induce spawning. H. arguinensis male spawning water also induced spawning in the phylogenetically related H. mammata. This indicates that males release pheromones together with their gametes that induce spawning in conspecifics and possibly sympatric species. Finally, the male pheromone seems to be a mixture with at least one labile compound (biological activity is lost after four hours at ambient temperature) possibly including phosphatidylcholines. The identification of pheromones in sea cucumbers offers a new ecological perspective and may have practical applications for their aquaculture.FCT - Foundation for Science and Technology [UID/Multi/04326/2013, SFRH/BD/90761/2012]info:eu-repo/semantics/publishedVersio

Pharmacogenetic Modulation of Orexin Neurons Alters Sleep/Wakefulness States in Mice

Hypothalamic neurons expressing neuropeptide orexins are critically involved in the control of sleep and wakefulness. Although the activity of orexin neurons is thought to be influenced by various neuronal input as well as humoral factors, the direct consequences of changes in the activity of these neurons in an intact animal are largely unknown. We therefore examined the effects of orexin neuron-specific pharmacogenetic modulation in vivo by a new method called the Designer Receptors Exclusively Activated by Designer Drugs approach (DREADD). Using this system, we successfully activated and suppressed orexin neurons as measured by Fos staining. EEG and EMG recordings suggested that excitation of orexin neurons significantly increased the amount of time spent in wakefulness and decreased both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep times. Inhibition of orexin neurons decreased wakefulness time and increased NREM sleep time. These findings clearly show that changes in the activity of orexin neurons can alter the behavioral state of animals and also validate this novel approach for manipulating neuronal activity in awake, freely-moving animals

CD8+ T-Cell Interleukin-7 Receptor Alpha Expression as a Potential Indicator of Disease Status in HIV-Infected Children

Background: Initiation and modification of antiretroviral therapy in HIV-infected children depend on viral load and CD4+ T-cell count. However, these surrogates have limitations, and complementary immunological markers to assess therapeutic response are needed. Our aim was to evaluate CD8+ T-cell expression of CD127 as a marker of disease status in HIV-infected children, based on adult data suggesting its usefulness. We hypothesized that CD127 expression on CD8+ T-cells is lower in children with more advanced disease. Methods: In a cross-sectional evaluation, we used flow cytometry to measure CD127+ expression on CD8+ T-cells in whole blood from HIV-infected children with varying disease status. This was compared with expression of CD38 on this subset, currently used in clinical practice as a marker of disease status. Results: 51 HIV-infected children were enrolled. There was a strong positive correlation between CD127 expression on CD8+ T-cells and CD4+ T-cell count, and height and weight z-scores, and a strong negative correlation between CD127 expression and viral load. In contrast, we found no association between CD38 expression and these disease status markers. Conclusions: CD8+ T-cell CD127 expression is significantly higher in children with better HIV disease control, and may have a role as an immunologic indicator of disease status. Longitudinal studies are needed to determine the utility of this marker as a potential indicator of HIV disease progression

Increasing the Depth of Current Understanding: Sensitivity Testing of Deep-Sea Larval Dispersal Models for Ecologists

Larval dispersal is an important ecological process of great interest to conservation and the establishment of marine protected areas. Increasing numbers of studies are turning to biophysical models to simulate dispersal patterns, including in the deep-sea, but for many ecologists unassisted by a physical oceanographer, a model can present as a black box. Sensitivity testing offers a means to test the models' abilities and limitations and is a starting point for all modelling efforts. The aim of this study is to illustrate a sensitivity testing process for the unassisted ecologist, through a deep-sea case study example, and demonstrate how sensitivity testing can be used to determine optimal model settings, assess model adequacy, and inform ecological interpretation of model outputs. Five input parameters are tested (timestep of particle simulator (TS), horizontal (HS) and vertical separation (VS) of release points, release frequency (RF), and temporal range (TR) of simulations) using a commonly employed pairing of models. The procedures used are relevant to all marine larval dispersal models. It is shown how the results of these tests can inform the future set up and interpretation of ecological studies in this area. For example, an optimal arrangement of release locations spanning a release area could be deduced; the increased depth range spanned in deep-sea studies may necessitate the stratification of dispersal simulations with different numbers of release locations at different depths; no fewer than 52 releases per year should be used unless biologically informed; three years of simulations chosen based on climatic extremes may provide results with 90% similarity to five years of simulation; and this model setup is not appropriate for simulating rare dispersal events. A step-by-step process, summarising advice on the sensitivity testing procedure, is provided to inform all future unassisted ecologists looking to run a larval dispersal simulation

The Role of Galectin-1 and Galectin-3 in the Mucosal Immune Response to Citrobacter rodentium Infection

Despite their abundance at gastrointestinal sites, little is known about the role of galectins in gut immune responses. We have therefore investigated the Citrobacter rodentium model of colonic infection and inflammation in Galectin-1 or Galectin-3 null mice. Gal-3 null mice showed a slight delay in colonisation after inoculation with C. rodentium and a slight delay in resolution of infection, associated with delayed T cell, macrophage and dendritic cell infiltration into the gut mucosa. However, Gal-1 null mice also demonstrated reduced T cell and macrophage responses to infection. Despite the reduced T cell and macrophage response in Gal-1 null mice, there was no effect on C. rodentium infection kinetics and pathology. Overall, Gal-1 and Gal-3 play only a minor role in immunity to a gut bacterial pathogen

Internalization Dissociates β2-Adrenergic Receptors

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β2-adrenergic receptors (β2ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β2ARs between subcellular compartments. BRET between β2ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β2ARs and endosome markers increases. Energy transfer between β2ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β2ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β2ARs associate transiently with each other in the plasma membrane, or that β2AR dimers or oligomers are actively disrupted during internalization