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Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein–protein interactions in intact cells. A very attractive reagent for this purpose is formaldehyde, a small molecule which is known to rapidly penetrate into all cellular compartments and to preserve the protein structure. In light of these benefits, it is surprising that identification of formaldehyde cross-links by mass spectrometry has so far been unsuccessful. Here we report mass spectrometry data that reveal formaldehyde cross-links to be the dimerization product of two formaldehyde-induced amino acid modifications. By integrating the revised mechanism into a customized search algorithm, we identify hundreds of cross-links from in situ formaldehyde fixation of human cells. Interestingly, many of the cross-links could not be mapped onto known atomic structures, and thus provide new structural insights. These findings enhance the use of formaldehyde cross-linking and mass spectrometry for structural studies. Formaldehyde (FA) is a popular cross-linking reagent, but applying it for cross-linking mass spectrometry (XLMS) has been largely unsuccessful. Here, the authors show that cross-links in structured proteins are the product of two FA molecules and identify hundreds of FA cross-links by XLMS in vitro and in situ.

Metal chelation can provide structural stability and form reactive centers in metalloproteins. Approximately one third of known protein structures are metalloproteins, and metal binding, or the lack thereof, is often implicated in disease, making it necessary to be able to study these systems in detail. Peptide-metal complexes are both present in nature and can provide a means to focus on the binding region of a protein and control experimental variables to a high degree. Structural studies of peptide complexes with metal ions by nuclear magnetic resonance (NMR) were surveyed for all the essential metal complexes and many non-essential metal complexes. The various methods used to study each metal ion are presented together with examples of recent research. Many of these metal systems have been individually reviewed and this current overview of NMR studies of metallopeptide complexes aims to provide a basis for inspiration from structural studies and methodology applied in the field.

[Display omitted] During pregnancy, the fetal membrane (FM) is subjected to mechanical stretching that may result in preterm labor. The structural integrity of the FM is maintained by its collagenous layer. The disconnection and reconnection of molecular bonds between collagen fibrils are the fundamental processes that govern the irreversible mechanical and supermolecular changes in the FM. Here, we study the activation enthalpy of interfibrillar bonds in ex-vivo human FM. We analyze the strain-rate and temperature dependence of the irreversible deformations in FM subjected to inflation tests, which apply mechanical conditions similar to those experienced by the FM prior to and during the initiation of labor contractions. The obtained activation enthalpy of interfibrillar bonds matches the typical enthalpy values of polyvalent ionic bonds, implying on another important role that ions like Ca and Mg may play in the gestation and labor.

[Display omitted] We study irreversible collagen arrangement processes in ex-vivo human amnions subjected to inflation tests, which simulate the mechanical conditions prior to and during the initiation of labor uterine contractions. The investigation is focused on the center of the membrane where the stresses are maximal and equibiaxial. Second harmonic generation reveals an unexpected collagen rearrangement in the compact layer that is responsible for the structural integrity of the fetal membrane. The observed bundling and alignment of the collagen fibers indicate a deviation from the expected equibiaxial stress state. The statistical analysis of the fiber orientations provides information on two driving forces for collagen alignment: microscale flaws and macroscale deviation from the equibiaxial strain. As the pressure increases, the macroscale effect becomes dominant, and a high density of fibers that are aligned along a specific direction is observed. A model that explains these observations and relates them to the material properties is presented. The results of this study indicate that a temporal increase in intrauterine pressure or uterine cervix dilatation causes irreversible changes in collagen molecular connections that may lead to biological changes, such as the initiation of term and preterm labor.

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated beta-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Abeta oligomerization and fibrillization, as well as the cytotoxic effect of Abeta oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Abeta while immuno-staining of the 3(rd) instar larval brains showed a significant reduction in Abeta accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Abeta. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease.

The intracellular oligomerization of α-synuclein is associated with Parkinson's disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we identified molecular interaction domains within the β-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinson's disease.

Abstract—Monitoring of pore pressure or water level changes in observation wells shows significant variations both during the passage of P- and Rayleigh waves and during the passage of S- and Love waves. Recent borehole measurements have shown an azimuthal dependence of pore pressure variations on the stress orientation and strike direction of the fault zone. In the active fault zone, fracture-induced anisotropy corresponds to the preferred orientation of microcracks and other discontinuities in the medium. This paper is devoted to the development of a modified Skempton equation for a quantitative description of surface wave induced pore pressure variations in a reservoir, related to the orientation and principal values of the stress tensor and rock damage (fracturing). The developed relationships allow the azimuthal dependence of the pore pressure response to be described by a dimensionless parameter defined as the ratio of the amplitudes of the pressure variations caused by the shear component and the bulk strain. According to the proposed theoretical model, the maximum poroelastic response of the reservoir to the passage of a seismic wave is manifested in the case of subparallelism of the directions of predominant rock fracturing and maximum horizontal stress. Pore pressure monitoring data from the Arbuckle wastewater disposal reservoir (Oklahoma, USA) are used to verify the proposed theoretical model. It is shown that the observed diversity of pore pressure response in wells located in the vicinity of a fault zone intersecting the reservoir to the passage of seismic waves from seismic events at different distances is described with high accuracy by the developed model.

Two types of endogenous cannabinoid-receptor agonists have been identified thus far. They are the ethanolamides of polyunsaturated fatty acids—arachidonoyl ethanolamide (anandamide) is the best known compound in the amide series—and 2-arachidonoyl glycerol, the only known endocannabinoid in the ester series. We report now an example of a third, ether-type endocannabinoid, 2-arachidonyl glyceryl ether (noladin ether), isolated from porcine brain. The structure of noladin ether was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by comparison with a synthetic sample. It binds to the CB 1 cannabinoid receptor ( K i = 21.2 ± 0.5 nM) and causes sedation, hypothermia, intestinal immobility, and mild antinociception in mice. It binds weakly to the CB 2 receptor ( K i > 3 μM).

The present study examines the response of groundwater systems to expected changes in the Mediterranean Sea (rise of <1cm/yr) and Dead Sea levels (decline of ∼1 m/yr). A fast response is observed in the Dead Sea coastal aquifer, exhibited both in the drop of the water levels and in the location of the fresh‐saline water interface. No such effect is yet observed in the Mediterranean coastal aquifer, as expected. Numerical simulations, using the FeFlow software, show that the effect of global sea level rise depends on the coastal topography next to the shoreline. A slope of 2.5‰ is expected to yield a shift of the interface by 400 m, after a rise of 1m (∼100 years), whereas a vertical slope will yield no shift. Reduced recharge due to climate change or overexploitation of groundwater also enhances the inland shift of the interface.

Concentrated brine pools are known to exist at the bottom of the Red Sea at depths of 2,000–2,800 m below sea level (bsl). Concentrated continental brines are also known to exist in the nearby continental endorheic base level of the Afar Depression, which is considerably below sea level, attaining 155 m bsl at Lake Asal. According to the modelling that was carried out with the FEFLOW code, the Afar Depression brines have had sufficient time to migrate also northward along the conductive plate boundary of the Red Sea and emerge at the Red Sea submarine pools, since there is evidence of brines in Lake Asal some 6,000 years ago. It is proposed, therefore, that these dense brines descend in the continental brine lakes and subsequently move by density-driven mechanisms along the conductive plate boundary of the Red Sea.