C-terminal methylation of truncated neuropeptides: An enzyme- assistedextraction artifact involving methanol

Neuropeptides are the largest class of signaling molecules used by nervous systems. Today, neuropeptidediscovery commonly involves chemical extraction from a tissue source followed by mass spectrometriccharacterization. Ideally, the extraction procedure accurately preserves the sequence and any inher-ent modifications of the native peptides. Here, we present data showing that this is not always true.Specifically, we present evidence showing that, in the lobster Homarus americanus, the orcokinin fam-ily members, NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, are non-native peptides generated fromfull-length orcokinin precursors as the result of a highly selective peptide modification (peptide trun-cation with C-terminal methylation) that occurs during extraction. These peptides were observed byMALDI-FTMS and LC-Q-TOFMS analyses when eyestalk ganglia were extracted in a methanolic solvent,but not when tissues were dissected, co-crystallized with matrix, and analyzed directly with methanolexcluded from the sample preparation. The identity of NFDEIDRSGFG-OMe was established using MALDI-FTMS/SORI-CID, LC-Q-TOFMS/MS, and comparison with a peptide standard. Extraction substitutingdeuterated methanol for methanol confirmed that the latter is the source of the C-terminal methyl group,and MS/MS confirmed the C-terminal localization of the added CD3. Surprisingly, NFDEIDRSGFG-OMe isnot produced via a chemical acid-catalyzed esterification. Instead, the methylated peptide appears toresult from proteolytic truncation in the presence of methanol, as evidenced by a reduction in conver-sion with the addition of a protease-inhibitor cocktail; heat effectively eliminated the conversion. Thisunusual and highly specific extraction-derived peptide conversion exemplifies the need to consider bothchemical and biochemical processes that may modify the structure of endogenous neuropeptides. © 2013 The Authors. Published by Elsevier Inc. All rights reserved

The effects of elevated CO2 and eutrophication on surface elevation gain in a European salt marsh.

Salt marshes can play a vital role in mitigating the effects of global environmental change by dissipating incident storm wave energy and, through accretion, tracking increasing water depths consequent upon sea level rise. Atmospheric CO2 concentrations and nutrient availability are two key variables that can affect the biological processes that contribute to marsh surface elevation gain. We measured the effects of CO2 concentrations and nutrient availability on surface elevation change in intact mixed-species blocks of UK salt marsh using six open-top chambers receiving CO2 -enriched (800 ppm) or ambient (400 ppm) air. We found more rapid surface elevation gain in elevated CO2 conditions: an average increase of 3.4 mm over the growing season relative to ambient CO2 . Boosted regression analysis to determine the relative influence of different parameters on elevation change identified that a 10% reduction in microbial activity in elevated CO2 -grown blocks had a positive influence on elevation. The biomass of Puccinellia maritima also had a positive influence on elevation, while other salt marsh species (e.g. Suaeda maritima) had no influence or a negative impact on elevation. Reduced rates of water use by the vegetation in the high CO2 treatment could be contributing to elevation gain, either directly through reduced soil shrinkage or indirectly by decreasing microbial respiration rates due to lower redox levels in the soil. Eutrophication did not influence elevation change in either CO2 treatment despite doubling aboveground biomass. The role of belowground processes (transpiration, root growth and decomposition) in the vertical adjustment of European salt marshes, which are primarily minerogenic in composition, could increase as atmospheric CO2 concentrations rise and should be considered in future wetland models for the region. Elevated CO2 conditions could enhance resilience in vulnerable systems such as those with low mineral sediment supply or where migration upwards within the tidal frame is constrained.Marie Curie Incoming International Fellowship (Grant ID: FP7-PEOPLE-IIF 623720 STORM)This is the author accepted manuscript. The final version is available from Wiley via https://doi.org/10.1111/gcb.1339

Evaluating the viability of coastal wet grassland to a changing management regime through flood hazard modelling

The high-risk nature of low-lying coastal areas means that many are characterised by protective dikes, sea walls, and earthen embankments. Across Europe, such structures are employed to limit landward sea flooding and have been used historically to reclaim wetlands, many of which have since gained international protected habitat status. Sea level rise and storms threaten the future viability of fixed flood defences which will be overtopped with increasing frequency and, in extreme cases, breached. This raises questions surrounding defence maintenance and the fate of the reclaimed wetlands they encircle and support. One approach is to design defences with the expectation that they will be overtopped during extreme events, but that catastrophic failure as a result of breaching will be prevented. Such an approach was recently implemented at Blakeney Freshes, North Norfolk Coast, UK, an embanked area of reedbeds and coastal wet grassland. Lowering and widening of the Freshes embankment was undertaken following extensive breaching during the 5 December 2013 storm surge. In this paper we develop and apply a numerical model chain, comprising storm surge water levels, waves, and overtopping, to explore inundation extent, depth, and duration resulting from storm surge induced flooding under several sea level rise scenarios. Modelling results revealed that 99.5% of the flood volume of the 2013 event resulted from embankment breaching. Simulating the same storm event after embankment reprofiling shows that flooding of the Freshes is reduced by 97%, largely because the lower, wider embankments preclude breaching. However, under future sea level rise scenarios, storm surge induced overtopping results in increased inundation depths and drainage times, raising questions regarding the resilience of vegetation communities within the Freshes. By 2100 under the lowest SLR scenario, and by 2050 under the mid SLR scenario, over half of the Freshes will be inundated for >10 days, a potentially critical threshold for current wet grassland survival. Our findings suggest that while effective defence redesign may increase the viability of reclaimed wetland habitats in the short term, as sea levels rise, lengthened inundation durations may render these habitats increasingly vulnerable to ecosystem change under extreme events

Racial disparities in modifiable risk factors and statin usage in Black patients with familial hypercholesterolemia

Background Black men and women are at higher risk for, and suffer greater morbidity and mortality from, atherosclerotic cardiovascular disease (ASCVD) compared with adults of European Ancestry (EA). Black patients with familial hypercholesterolemia are at particularly high risk for ASCVD complications because of lifelong exposure to elevated levels of low-density-lipoprotein cholesterol. Methods and Results This retrospective study analyzed ASCVD prevalence and risk factors in 808 adults with heterozygous familial hypercholesterolemia from 5 US-based lipid clinics, and compared findings in Black versus EA patients. Multivariate logistic regression models were used to determine the strongest predictors of ASCVD as a function of race. No significant difference was noted in the prevalence of ASCVD in Black versus EA patients with familial hypercholesterolemia (39% versus 32%, respectively

Inhibitors of Foot and Mouth Disease Virus Targeting a Novel Pocket of the RNA-Dependent RNA Polymerase

Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections.Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC(50)s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC(50) = 12 µM and EC(90) = 20 µM).We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD

Midgut epithelial endocrine cells are a rich source of the neuropeptides APSGFLGMRamide (Cancer borealis tachykinin-related peptide Ia) and GYRKPPFNGSIFamide (Gly\u3csup\u3e1\u3c/sup\u3e-SIFamide) in the crabs Cancer borealis, Cancer magister and Cancer productus

Over a quarter of a century ago, Mykles described the presence of putative endocrine cells in the midgut epithelium of the crab Cancer magister (Mykles, 1979). In the years that have followed, these cells have been largely ignored and nothing is known about their hormone content or the functions they play in this species. Here, we used a combination of immunohistochemistry and mass spectrometric techniques to investigate these questions. Using immunohistochemistry, we identified both SIFamide-and tachykinin-related peptide (TRP)-like immunopositive cells in the midgut epithelium of C. magister, as well as in that of Cancer borealis and Cancer productus. In each species, the SIFamide-like labeling was restricted to the anterior portion of the midgut, including the paired anterior midgut caeca, whereas the TRP-like immunoreactivity predominated in the posterior midgut and the posterior midgut caecum. Regardless of location, label or species, the morphology of the immunopositive cells matched that of the putative endocrine cells characterized ultrastructurally by Mykles (Mykles, 1979). Matrix-assisted laser desorption/ ionization-Fourier transform mass spectrometry identified the peptides responsible for the immunoreactivities as GYRKPPFNGSIFamide (Gly 1-SIFamide) and APSGFLGMRamide [Cancer boreatis tachykinin-related peptide Ia (CabTRP Ia)], respectively, both of which are known neuropeptides of Cancer species. Although the function of these midgut-derived peptides remains unknown, we found that both Gly1-SIFamide and CabTRP Ia were released when the midgut was exposed to high-potassium saline. In addition, CabTRP Ia was detectable in the hemolymph of crabs that had been held without food for several days, but not in that of fed animals, paralleling results that were attributed to TRP release from midgut endocrine cells in insects. Thus, one function that midgut-derived CabTRP Ia may play in Cancer species is paracrine/hormonal control of feeding-related behavior, as has been postulated for TRPs released from homologous cells in insects

Bringing the Tiger Back from the Brink—The Six Percent Solution

This article does not have an abstract

Defining myocardial tissue abnormalities in end-stage renal failure with cardiac magnetic resonance imaging using native T1 mapping

Noninvasive quantification of myocardial fibrosis in end-stage renal disease is challenging. Gadolinium contrast agents previously used for cardiac magnetic resonance imaging (MRI) are contraindicated because of an association with nephrogenic systemic fibrosis. In other populations, increased myocardial native T1 times on cardiac MRI have been shown to be a surrogate marker of myocardial fibrosis. We applied this method to 33 incident hemodialysis patients and 28 age- and sex-matched healthy volunteers who underwent MRI at 3.0T. Native T1 relaxation times and feature tracking–derived global longitudinal strain as potential markers of fibrosis were compared and associated with cardiac biomarkers. Left ventricular mass indices were higher in the hemodialysis than the control group. Global, Septal and midseptal T1 times were all significantly higher in the hemodialysis group (global T1 hemodialysis 1171 ± 27 ms vs. 1154 ± 32 ms; septal T1 hemodialysis 1184 ± 29 ms vs. 1163 ± 30 ms; and midseptal T1 hemodialysis 1184 ± 34 ms vs. 1161 ± 29 ms). In the hemodialysis group, T1 times correlated with left ventricular mass indices. Septal T1 times correlated with troponin and electrocardiogram-corrected QT interval. The peak global longitudinal strain was significantly reduced in the hemodialysis group (hemodialysis -17.7±5.3% vs. -21.8±6.2%). For hemodialysis patients, the peak global longitudinal strain significantly correlated with left ventricular mass indices (R = 0.426), and a trend was seen for correlation with galectin-3, a biomarker of cardiac fibrosis. Thus, cardiac tissue properties of hemodialysis patients consistent with myocardial fibrosis can be determined noninvasively and associated with multiple structural and functional abnormalities