Identifying the molecular basis of Laminin N-terminal domain Ca2+ binding using a hybrid approach.

Ca2+ is a highly abundant ion involved in numerous biological processes, particularly in multicellular eukaryotic organisms where it exerts many of these functions through interactions with Ca2+ binding proteins. The laminin N-terminal (LN) domain is found in members of the laminin and netrin protein families where it plays a critical role in the function of these proteins. The LN domain of laminins and netrins is a Ca2+ binding domain and in many cases requires Ca2+ to perform its biological function. Here, we conduct a detailed examination of the molecular basis of the LN domain Ca2+ interaction combining structural, computational, bioinformatics, and biophysical techniques. By combining computational and bioinformatic techniques with x-ray crystallography we explore the molecular basis of the LN domain Ca2+ interaction and identify a conserved sequence present in Ca2+ binding LN domains. These findings enable a sequence-based prediction of LN domain Ca2+ binding ability. We use thermal shift assays and isothermal titration calorimetry to explore the biophysical properties of the LN domain Ca2+ interaction. We show that the netrin-1 LN domain exhibits a high affinity and specificity for Ca2+, which structurally stabilizes the LN domain. This study elucidates the molecular foundation of the LN domain Ca2+ binding interaction and provides a detailed functional characterization of this essential interaction, advancing our understanding of protein-Ca2+ dynamics within the context of the LN domain.

Clinical outcomes after primary prevention defibrillator implantation are better predicted when the left ventricular ejection fraction is assessed by cardiovascular magnetic resonance.

BACKGROUND: The left ventricular ejection fraction (LVEF) is the key selection criterion for an implanted cardioverter defibrillator (ICD) in primary prevention of sudden cardiac death. LVEF is usually assessed by two-dimensional echocardiography, but cardiovascular magnetic resonance (CMR) imaging is increasingly used. The aim of our study was to evaluate whether LVEF assessment using CMR imaging (CMR-LVEF) or two-dimensional echocardiography (2D echo-LVEF) may predict differently the occurrence of clinical outcomes. METHODS: In this retrospective study, we reviewed patients referred for primary prevention ICD implantation to Caen University Hospital from 2005 to 2014. We included 173 patients with either ischemic (n = 120) or dilated cardiomyopathy (n = 53) and who had undergone pre-ICD CMR imaging. The primary composite end point was the time to death from any cause or first appropriate device therapy. RESULTS: The mean CMR-LVEF was significantly lower than the mean 2D echo-LVEF (24% ± 6 vs 28% ± 6, respectively; p < 0.001). CMR-LVEF was a better independent predictive factor for the occurrence of the primary composite endpoint with a cut-off value of 22% (Hazard Ratio [HR] = 2.22; 95% CI [1.34-3.69]; p = 0.002) than 2D echo-LVEF with a cut-off value of 26% (HR = 1.61; 95% CI [0.99-2.61]; p = 0.056). Combination of the presence of scar with CMR-LVEF< 22% improved the predictive value for the occurrence of the primary outcome (HR = 2.58; 95% CI [1.54-4.30]; p < 0.001). The overall survival was higher among patients with CMR-LVEF≥22% than among patients with CMR-LVEF< 22% (p = 0.026), whereas 2D echo-LVEF was not associated with death. CONCLUSIONS: CMR-LVEF is better associated with clinical outcomes than 2D echo-LVEF in primary prevention using an ICD. Scar identification further improved the outcome prediction. The combination of CMR imaging and echocardiography should be encouraged in addition to other risk markers to better select patients.

One-pot synthesis of a new high vinyl content hybrid silica monolith dedicated to nanoliquid chromatography.

A new vinyltrimethoxysilane-based hybrid silica monolith was developed and used as a reversed-phase capillary column. The synthesis of this rich vinyl hybrid macroporous monolith, by cocondensation of vinyltrimethoxysilane with tetramethoxysilane, was investigated using an unconventional (formamide, nitric acid) porogen/catalyst system. A macroporous hybrid silica monolith with 80% in mass of vinyltrimethoxysilane in the feeding silane solution was obtained and compared to a more conventional low vinyl content hybrid monolith with only of 20% vinyltrimethoxysilane. Monoliths were characterized by scanning electron microscopy, (29) Si nuclear magnetic resonance spectroscopy and N2 adsorption-desorption. About 80% of the vinyl precursor was incorporated in the final materials, leading to 15.9 and 61.5% of Si atoms bonded to vinyl groups for 20% vinyltrimethoxysilane and 80% vinyltrimethoxysilane, respectively. The 80% vinyltrimethoxysilane monolith presents a lower surface area than 20% vinyltrimethoxysilane (159 versus 551 m(2) /g), which is nevertheless compensated by a higher vinyl surface density. Chromatographic properties were evaluated in reversed-phase mode. Plots of ln(k) versus percentage of organic modifier were used to assess the reversed-phase mechanism. Its high content of organic groups leads to high retention properties. Column efficiencies of 170 000 plates/m were measured for this 80% vinyltrimethoxysilane hybrid silica monolith. Long capillary monolithic columns (90 cm) were successfully synthesized (N = 120 000).

The dynamic nature of netrin-1 and the structural basis for glycosaminoglycan fragment-induced filament formation.

Netrin-1 is a bifunctional chemotropic guidance cue that plays key roles in diverse cellular processes including axon pathfinding, cell migration, adhesion, differentiation, and survival. Here, we present a molecular understanding of netrin-1 mediated interactions with glycosaminoglycan chains of diverse heparan sulfate proteoglycans (HSPGs) and short heparin oligosaccharides. Whereas interactions with HSPGs act as platform to co-localise netrin-1 close to the cell surface, heparin oligosaccharides have a significant impact on the highly dynamic behaviour of netrin-1. Remarkably, the monomer-dimer equilibrium of netrin-1 in solution is abolished in the presence of heparin oligosaccharides and replaced with highly hierarchical and distinct super assemblies leading to unique, yet unknown netrin-1 filament formation. In our integrated approach we provide a molecular mechanism for the filament assembly which opens fresh paths towards a molecular understanding of netrin-1 functions.

On Characterizing the Interactions between Proteins and Guanine Quadruplex Structures of Nucleic Acids.

Guanine quadruplexes (G4s) are four-stranded secondary structures of nucleic acids which are stabilized by noncanonical hydrogen bonding systems between the nitrogenous bases as well as extensive base stacking, or pi-pi, interactions. Formation of these structures in either genomic DNA or cellular RNA has the potential to affect cell biology in many facets including telomere maintenance, transcription, alternate splicing, and translation. Consequently, G4s have become therapeutic targets and several small molecule compounds have been developed which can bind such structures, yet little is known about how G4s interact with their native protein binding partners. This review focuses on the recognition of G4s by proteins and small peptides, comparing the modes of recognition that have thus far been observed. Emphasis will be placed on the information that has been gained through high-resolution crystallographic and NMR structures of G4/peptide complexes as well as biochemical investigations of binding specificity. By understanding the molecular features that lead to specificity of G4 binding by native proteins, we will be better equipped to target protein/G4 interactions for therapeutic purposes.