High-pressure SANS and fluorescence unfolding study of calmodulin

Apo-calmodulin, a small soluble mainly α protein, is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80 °C, whereas the calcium-saturated form is stable up to temperatures as high as 100 °C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al., BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000 bar (300 MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form have been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease have been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.     

Notch3 is necessary for neuronal differentiation and maturation in the adult spinal cord

Notch receptors are key regulators of nervous system development and promoters of neural stem cells renewal and proliferation. Defects in the expression of Notch genes result in severe, often lethal developmental abnormalities. Notch3 is generally thought to have a similar proliferative, anti‐differentiation and gliogenic role to Notch1. However, in some cases, Notch3 has an opposite, pro‐differentiation effect. Here, we show that Notch3 segregates from Notch1 and is transiently expressed in adult rat and mouse spinal cord neuron precursors and immature neurons. This suggests that during the differentiation of adult neural progenitor cells, Notch signalling may follow a modified version of the classical lateral inhibition model, involving the segregation of individual Notch receptors. Notch3 knockout mice, otherwise neurologically normal, are characterized by a reduced number of mature inhibitory interneurons and an increased number of highly excitable immature neurons in spinal cord laminae I–II. As a result, these mice have permanently lower nociceptive thresholds, similar to chronic pain. These results suggest that defective neuronal differentiation, for example as a result of reduced Notch3 expression or activation, may underlie human cases of intractable chronic pain, such as fibromyalgia and neuropathic pain.     

Celiac disease microbiota and its applications

Intestinal microbiota plays an important role in maintaining the overall health of an individual. It can be affected by diet but also inflammation of the intestine due to various causes. In the last decade, particular attention has been paid to the study of the interaction between mucosal cells and intestinal microbiota, and to the host immune response to change in community structure. Here, we review the most significant studies on human microbiota in patients with celiac disease, and also the potential biotechnological use of microorganisms for the production of gluten-free products.     

Prioritizing causal disease genes using unbiased genomic features

Background

Cardiovascular disease (CVD) is the leading cause of death in the developed world. Human genetic studies, including genome-wide sequencing and SNP-array approaches, promise to reveal disease genes and mechanisms representing new therapeutic targets. In practice, however, identification of the actual genes contributing to disease pathogenesis has lagged behind identification of associated loci, thus limiting the clinical benefits.

Results

To aid in localizing causal genes, we develop a machine learning approach, Objective Prioritization for Enhanced Novelty (OPEN), which quantitatively prioritizes gene-disease associations based on a diverse group of genomic features. This approach uses only unbiased predictive features and thus is not hampered by a preference towards previously well-characterized genes. We demonstrate success in identifying genetic determinants for CVD-related traits, including cholesterol levels, blood pressure, and conduction system and cardiomyopathy phenotypes. Using OPEN, we prioritize genes, including FLNC, for association with increased left ventricular diameter, which is a defining feature of a prevalent cardiovascular disorder, dilated cardiomyopathy or DCM. Using a zebrafish model, we experimentally validate FLNC and identify a novel FLNC splice-site mutation in a patient with severe DCM.

Conclusion

Our approach stands to assist interpretation of large-scale genetic studies without compromising their fundamentally unbiased nature.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0534-8) contains supplementary material, which is available to authorized users.     

Productivity, 15N dynamics and water use efficiency in low‐ and high‐input switchgrass systems

Sustainable and environmentally benign switchgrass production systems need to be developed for switchgrass to become a large‐scale dedicated energy crop. An experiment was conducted in California from 2009 to 2011 to determine the sustainability of low‐ and high‐input irrigated switchgrass systems as a function of yield, irrigation requirement, crop N removal, N translocation from aboveground (AG) to belowground (BG) biomass during senescence, and fertilizer ¹⁵N recovery (FNR) in the AG and BG biomass (0–300 cm), and soil (0–300 cm). The low‐input system consisted of a single‐harvest (mid‐fall) irrigated until flowering (early summer), while the high‐input system consisted of a two‐harvest system (early summer and mid‐fall) irrigated throughout the growing season. Three N fertilization rates (0, 100, and 200 kg N ha^?1 yr^?1) were applied as subtreatments in a single application in the spring of each year. A single pulse of ¹⁵N enriched fertilizer was applied in the first year of the study to micro‐plots within the 100 kg N ha^?1 subplots. Average yields across years under optimal N rates (100 and 200 kg ha^?1 yr^?1 for low‐ and high‐input systems, respectively) were 20.7 and 24.8 Mg ha^?1. However, the low input (372 ha mm) required 47% less irrigation than the high‐input system (705 ha mm) and achieved higher irrigation use efficiency. In addition, the low‐input system had 46% lower crop N removal, 53% higher N stored in BG biomass, and a positive N balance, presumably due to 49% of ¹⁵N translocation from AG to BG biomass during senescence. Furthermore, at the end of 3 years, the low‐input system had lower fertilizer ¹⁵N removed by harvest (26%) and higher FNR remaining in the system in BG biomass plus soil (31%) than the high‐input system (45% and 21%, respectively). Based on these findings, low‐input systems are more sustainable than high‐input systems in irrigated Mediterranean climates.     

Backbone resonance assignment of Alt a 1, a unique β-barrel protein and the major allergen of Alternaria alternata

Alt a 1 is the major allergen of the fungus Alternaria alternata and can be found in the cell wall of its spores. It is a cysteine linked homodimeric protein with a unique β-barrel fold as recently revealed by X-ray crystallography. Despite the elucidation of its structure, its biological function remains unknown. For Alternaria-sensitized patients, contact leads to respiratory allergy and in severe cases to asthma-related death. Here we report the sequence-specific Alt a 1 backbone ¹H, ¹⁵N and ¹³C chemical shift assignment.     

Interleukin-21 Is a Critical Regulator of CD4 and CD8 T Cell Survival during Priming under Interleukin-2 Deprivation Conditions

Optimal T cell activation and expansion require binding of the common gamma-chain (γc) cytokine Interleukin-2 (IL-2) to its cognate receptor that in turn engages a γc/Janus tyrosine kinase (Jak)3 signaling pathway. Because of its restricted expression by antigen-activated T cells and its obligatory role in promoting their survival and proliferation, IL-2 has been considered as a selective therapeutic target for preventing T cell mediated diseases. However, in order to further explore IL-2 targeted therapy, it is critical to precisely understand its role during early events of T cell activation. In this study, we delineate the role of IL-2 and other γc cytokines in promoting the survival of CD4 and CD8 T cells during early phases of priming. Under IL-2 inhibitory conditions (by neutralizing anti-IL-2 mAbs), the survival of activated CD8⁺ T cells was reduced, whereas CD4⁺ T cells remained much more resistant. These results correlated with reduced Bcl-2 expression, and mitochondrial membrane potential in CD8⁺ T cells in comparison to CD4⁺ T cells. However, using transwell co-culture assays we have found that CD4⁺ T cells could rescue the survival of CD8⁺ T cells even under IL-2 deprived conditions via secretion of soluble factors. A cytokine screen performed on CD8⁺ T cells cultured alone revealed that IL-21, another γc cytokine, was capable of rescuing their survival under IL-2 deprivation. Indeed, blocking the IL-21 signaling pathway along with IL-2 neutralization resulted in significantly reduced survival of both CD4⁺ and CD8⁺ T cells. Taken together, we have shown that under IL-2 deprivation conditions, IL-21 may act as the major survival factor promoting T cell immune responses. Thus, investigation of IL-2 targeted therapies may need to be revisited to consider blockade of the IL-21 signaling pathways as an adjunct to provide more effective control of T cell immune responses.     

Increased Inflammatory Response in Cytomegalovirus Seropositive Patients with Alzheimer’s Disease

Alzheimer’s disease (AD) has been associated with increased local inflammation in the affected brain regions, and in some studies also with elevated levels of proinflammatory cytokines in peripheral blood. Cytomegalovirus (CMV) is known to promote a more effector-oriented phenotype in the T-cell compartment, increasing with age. The aim of this study was to investigate the inflammatory response of peripheral blood mononuclear cells (PBMCs) from AD patients and non-demented (ND) controls. Using a multiplex Luminex xMAP assay targeting GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10 and TNF-α, cytokine profiles from PBMCs were analysed after stimulation with anti-CD3/CD28 beads, CMV pp65 peptide mix or amyloid β (Aβ) protofibrils, respectively. CMV seropositive AD subjects presented with higher IFN-γ levels after anti-CD3/CD28 and CMV pp65 but not after Aβ stimulation, compared to CMV seropositive ND controls. When analysing IFN-γ response to anti-CD3/CD28 stimulation on a subgroup level, CMV seropositive AD subjects presented with higher levels compared to both CMV seronegative AD and CMV seropositive ND subjects. Taken together, our data from patients with clinically manifest AD suggest a possible role of CMV as an inflammatory promoter in AD immunology. Further studies of AD patients at earlier stages of disease, could provide better insight into the pathophysiology.