A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images.     

Factors Associated with Primary School Teachers’ Attitudes Towards the Inclusion of Students with Disabilities

Objective

Teachers'' attitudes toward inclusion are often based on the practical implementation of inclusive education rather than a specific ideology and understanding of inclusiveness. This study aimed to identify the factors associated with primary school teachers'' attitudes towards inclusion of students with all disabilities in regular schools.

Method

Seventy four primary school teachers participated in a cross-sectional survey conducted in Western Australia. Teachers'' attitudes and efficacy toward integration of students with disabilities were measured using the Opinions Relative to Integration of Students with Disabilities scale and Bandura''s Teacher Efficacy scale respectively.

Results

Four teacher attributes—age, gender, teaching self-efficacy and training collectively explained 42% of the variability in teachers'' attitude toward including students with disabilities.

Conclusion

The current study further contributes to the accumulation of knowledge that can unpack the complex pattern of factors that should be considered to promote positive attitudes towards inclusive schools.     

A decision-making Fokker–Planck model in computational neuroscience

In computational neuroscience, decision-making may be explained analyzing models based on the evolution of the average firing rates of two interacting neuron populations, e.g., in bistable visual perception problems. These models typically lead to a multi-stable scenario for the concerned dynamical systems. Nevertheless, noise is an important feature of the model taking into account both the finite-size effects and the decision’s robustness. These stochastic dynamical systems can be analyzed by studying carefully their associated Fokker–Planck partial differential equation. In particular, in the Fokker–Planck setting, we analytically discuss the asymptotic behavior for large times towards a unique probability distribution, and we propose a numerical scheme capturing this convergence. These simulations are used to validate deterministic moment methods recently applied to the stochastic differential system. Further, proving the existence, positivity and uniqueness of the probability density solution for the stationary equation, as well as for the time evolving problem, we show that this stabilization does happen. Finally, we discuss the convergence of the solution for large times to the stationary state. Our approach leads to a more detailed analytical and numerical study of decision-making models applied in computational neuroscience.     

CNP budgets of a coral-dominated fringing reef at La Réunion,France: coupling of oceanic phosphate and groundwater nitrate

Productivity, nutrient input, nutrient uptake, and release rates were determined for a coral-dominated reef flat at La Réunion, France, to assess the influence of groundwater nitrogen on carbon and nutrient budgets. Water samples were collected offshore in the ocean, at the reef crest and back reef for nutrients, picoplankton, pH, and total alkalinity. Volume transport of ocean water across the reef flat was measured using both current meters and drogues. Groundwater advected onto the reef flat and mixed with incoming ocean water. Metabolic rates for the reef community were determined to be: gross primary production = 1,000 mmol C m⁻² d⁻¹, community respiration = 960 mmol C m⁻² d⁻¹, and community calcification = 210 mmol C m⁻² d⁻¹. Across the reef flat, silicate behaved conservatively, there was net uptake of phosphate (0.06 mmol P m⁻² d⁻¹) and net release of nitrate, ammonia, dissolved and particulate organic nitrogen (total 7.0 mmol N m⁻² d⁻¹). Groundwater nitrate contributed 37% of the increase in nitrate plus ammonia. The first-order mass transfer coefficient of phosphate was 3.3 m d⁻¹, and for nitrate plus ammonia, 5.9 m d⁻¹. Gross N and P uptake from estimates of mass transfer and uptake of particles were 0.37 mmol P m⁻² d⁻¹ and 7.2 mmol N m⁻² d⁻¹, respectively giving an N:P uptake ratio of 20:1. Thus, the elevation of nitrogen across the reef flat maintains a high N:P flux, enhancing algal growth downstream of the transect. We conclude that net community production (40 mmol C m⁻² d⁻¹) was sustained by net uptake of phosphate from the ocean and net uptake of new nitrogen from groundwater.

     

Comparative proteomic analysis of blood eosinophils reveals redox signaling modifications in patients with FIP1L1-PDGFRA-associated chronic eosinophilic leukemia

The FIP1L1-PDGFRA (F/P) fusion gene, which was identified as a recurrent molecular finding in hypereosinophilic syndrome (HES), lead to a constitutively increased tyrosine kinase activity of the fusion protein. Despite data obtained in animals or cell lines models, the mechanisms underlying the predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. To define more precisely intrinsic molecular events associated with F/P gene, we performed a proteomic analysis comparing F/P+ eosinophils (F/P-Eos) and eosinophils from healthy donors (C-Eos). Using 2D-DIGE and mass spectrometry techniques, we identified 41 proteins significantly overexpressed between F/P-Eos and C-Eos. Among them, 17.8% belonged to the oxidoreductase family. We further observed a down-expression of peroxiredoxin-2 (PRX-2) and an overexpression of src-homology-2 domain containing tyrosine phosphatase (SHP-1), enzymes regulating PDGFR downstream pathways, and especially intracellular reactive oxygen species (ROS) production. This profile, confirmed in immunoblot analysis, appears specific to F/P-Eos compared to controls and patients with idiopathic HES. In this clonal disorder possibly involving a pluripotent hematopoietic stem cell, we postulate that the well documented relationships between PDGFRA downstream signals and intracellular ROS levels might influence the phenotype of this leukemia.     

TRAF2 Must Bind to Cellular Inhibitors of Apoptosis for Tumor Necrosis Factor (TNF) to Efficiently Activate NF-��B and to Prevent TNF-induced Apoptosis

Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-κB-inducing kinase stability and suppress constitutive noncanonical NF-κB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-κB activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-κB signaling and that efficient activation of NF-κB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.     

Maternal environment and the reproductive function of the offspring

Fetal programming of metabolic diseases is now a well established concept. The scope of the Developmental Origins of Health and Disease has, however, widened and led to the identification of new targets of fetal programming, notably effects on reproductive function. Epidemiologic studies about maternal nutrition and effects on offspring's fertility are rare, but a link between impaired fetal growth, possibly caused by maternal malnutrition, and reproductive function, has been established. The methodologic limitations inherent to human epidemiologic studies can be complemented through the use of animal models, which enable experimental studies on maternal environment and its effect on reproductive functions of the offspring. Altogether, an interaction between inappropriate maternal nutrition (excess or reduced nutritional intake, micronutrient unbalance, or alcohol intake) and reproductive maturation of the offspring has been shown in a majority of experiments as summarized in this review. The exact processes through which maternal nutrition or maternal environment affect reproductive function in the offspring remain unclear but epigenetic modifications are a clear link. Further studies are needed to better understand the mechanisms involved, identify the crucial critical periods, and prevent or treat the adverse effects.     

Localized versus systemic effect of arbuscular mycorrhizal fungi on defence responses to Phytophthora infection in tomato plants

Development of biological control for plant diseases is accepted as a durable and environmentally friendly alternative for agrochemicals. Arbuscular mycorrhizal fungi (AMF), which form symbiotic associations with root systems of most agricultural, horticultural and hardwood crop species, have been suggested as widespread potential bioprotective agents. In the present study the ability of two AMF (Glomus mosseae and Glomus intraradices) to induce local or systemic resistance to Phytophthora parasitica in tomato roots have been compared using a split root experimental system. Glomus mosseae was effective in reducing disease symptoms produced by P. parasitica infection, and evidence points to a combination of local and systemic mechanisms being responsible for this bioprotector effect. The biochemical analysis of different plant defence-related enzymes showed a local induction of mycorrhiza-related new isoforms of the hydrolytic enzymes chitinase, chitosanase and beta-1,3-glucanase, as well as superoxide dismutase, an enzyme which is involved in cell protection against oxidative stress. Systemic alterations of the activity of some of the constitutive isoforms were also observed in non-mycorrhizal roots of mycorrhizal plants. Studies on the lytic activity against Phytophthora cell wall of root protein extracts also corroborated a systemic effect of mycorrhizal symbiosis on tomato resistance to Phytophthora.     

Peripheral CD8+CD25+ T lymphocytes from MHC class II-deficient mice exhibit regulatory activity

We characterized CD8(+) T cells constitutively expressing CD25 in mice lacking the expression of MHC class II molecules. We showed that these cells are present not only in the periphery but also in the thymus. Like CD4(+)CD25(+) T cells, CD8(+)CD25(+) T cells appear late in the periphery during ontogeny. Peripheral CD8(+)CD25(+) T cells from MHC class II-deficient mice also share phenotypic and functional features with regulatory CD4(+)CD25(+) T cells: in particular, they strongly express glucocorticoid-induced TNFR family-related gene, CTLA-4 and Foxp3, produce IL-10, and inhibit CD25(-) T cell responses to anti-CD3 stimulation through cell contacts with similar efficiency to CD4(+)CD25(+) T cells. However, unlike CD4(+)CD25(+) T cells CD8(+)CD25(+) T cells from MHC class II-deficient mice strongly proliferate and produce IFN-gamma in vitro in response to stimulation in the absence of exogenous IL-2.