The extent to which proteins in the gas phase retain their condensed-phase structure is a hotly debated issue. Closely related to this is the degree to which the observed charge state reflects ...protein conformation. Evidence from electron capture dissociation, hydrogen/deuterium exchange, ion mobility, and molecular dynamics shows clearly that there is often a strong correlation between the degree of folding and charge state, with the most compact conformations observed for the lowest charge states. In this article, we address recent controversies surrounding the relationship between charge states and folding, focussing also on the manipulation of charge in solution and its effect on conformation. ‘Supercharging’ reagents that have been used to effect change in charge state can promote unfolding in the electrospray droplet. However for several protein complexes, supercharging does not appear to perturb the structure in that unfolding is not detected. Consequently, a higher charge state does not necessarily imply unfolding. Whilst the effect of charge manipulation on conformation remains controversial, there is strong evidence that a folded, compact state of a protein can survive in the gas phase, at least on a millisecond timescale. The exact nature of the side-chain packing and secondary structural elements in these compact states, however, remains elusive and prompts further research.
The early stages of nonalcoholic fatty liver disease (NAFLD) are characterized by the accumulation of fat in the liver (steatosis). This can lead to cell injury and inflammation resulting in ...nonalcoholic steatohepatitis (NASH). To determine whether lipid profiling of liver tissue can identify metabolic signatures associated with disease presence and severity, we explored liquid extraction surface analysis mass spectrometry (LESA–MS) as a novel sampling tool. Using LESA–MS, lipids were extracted directly from the surface of ultrathin slices of liver tissue prior to detection by high-resolution mass spectrometry (MS). An isotopically labeled internal standard mix was incorporated into the extraction solvent to attain semiquantitative data. Data mining and multivariate statistics were employed to evaluate the generated lipid profiles and abundances. With this approach, we were able to differentiate healthy and NAFLD liver in mouse and human tissue samples, finding several triacylglyceride (TAG) and free fatty acid (FFA) species to be significantly increased. Furthermore, LESA–MS was able to successfully differentiate between simple steatosis and more severe NASH, based on a set of short-chain TAGs and FFAs. We compared the data obtained by LESA–MS to that from liquid chromatography (LC)–MS and matrix-assisted laser desorption ionization MS. Advantages of LESA–MS include rapid analysis, minimal sample preparation, and high lipid coverage. Furthermore, since tissue slices are routinely used for diagnostics in clinical settings, LESA–MS is ideally placed to complement traditional histology. Overall LESA–MS is found to be a robust, fast, and discriminating approach for determining NAFLD presence and severity in clinical samples.
Background and Aims
Hepatocytes undergo profound metabolic rewiring when primed to proliferate during compensatory regeneration and in hepatocellular carcinoma (HCC). However, the metabolic control ...of these processes is not fully understood. In order to capture the metabolic signature of proliferating hepatocytes, we applied state‐of‐the‐art systems biology approaches to models of liver regeneration, pharmacologically and genetically activated cell proliferation, and HCC.
Approach and Results
Integrating metabolomics, lipidomics, and transcriptomics, we link changes in the lipidome of proliferating hepatocytes to altered metabolic pathways including lipogenesis, fatty acid desaturation, and generation of phosphatidylcholine (PC). We confirm this altered lipid signature in human HCC and show a positive correlation of monounsaturated PC with hallmarks of cell proliferation and hepatic carcinogenesis.
Conclusions
Overall, we demonstrate that specific lipid metabolic pathways are coherently altered when hepatocytes switch to proliferation. These represent a source of targets for the development of therapeutic strategies and prognostic biomarkers of HCC.
The present paper compares the damage and energy absorption behaviour of composites subjected to low-velocity impact using different frontal geometries for the impactor, with the composites ...possessing a layup of 02/9022s. In this study, the rigid impactors with either round-nosed or flat-ended frontal geometry are employed to perform drop-weight tests at various impact energies ranging from 10 to 30 J. The measured loading response and energy absorption are analysed and compared. Additionally, the types and extent of impact-induced damage in the composite specimens are assessed via ultrasonic C-scan, optical microscopy (OM) and scanning electron microscopy (SEM) studies. It is shown that the impact energy threshold for damage initiation is greater than 20 J when using the flat-ended impactor but is less than 10 J when using the round-nosed impactor. In both cases, delamination initiates between the plies in the composite laminate. However, for the flat-ended impactor, the damage behaviour of the fibres exhibits kinking fracture, which differs from the pull-out fibre-fracture caused by the round-nosed impactor. These differences in behaviour are attributed to impactor/composite contact geometry effects which leads to different extents of indentation damage, which in turn directly affects the degree of delamination and fibre damage in the composite.
Structure determination of macromolecular protein assemblies remains a challenge for well-established methods. Here, we provide an assessment of an emerging structural technique, ion mobility-mass ...spectrometry (IM-MS), and examine the use of collision cross-sections (CCSs), derived from IM-MS, as restraints for structure characterization of heteromeric protein assemblies. Using 15 complexes selected from the Protein Data Bank, we validate the use of low-resolution models by comparing their CCSs with those calculated for all-atom structures. We then select six heteromeric complexes, disrupting them in solution to form subcomplexes. Experimental and calculated CCSs reveal close similarity for 18 of the 21 (sub)complexes. Exploring the use of CCS as a restraint, we incorporate it into a scoring function and show good correlation between the score and similarity to the native structure for heteromers, especially when an additional symmetry restraint was introduced.
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► Robust method for building models of heteromeric protein complexes from IM-MS ► Spatial restraints for low resolution models established ► Subcomplexes generated in solution from heteromers maintain native-like topologies ► IM-MS poses great potential for structural elucidation of protein assemblies
One of the challenges in structural biology is to obtain three-dimensional information of increasingly complex heterogeneous assemblies. Hall et al. critically assess the feasibility of using ion mobility-mass spectrometry (IM-MS) for structure characterization of homomeric and heteromeric protein complexes.
Collapse to compact states in the gas phase, with smaller collision cross sections than calculated for their native-like structure, has been reported previously for some protein complexes although ...not rationalized. Here we combine experimental and theoretical studies to investigate the gas-phase structures of four multimeric protein complexes during collisional activation. Importantly, using ion mobility–mass spectrometry (IM–MS), we find that all four macromolecular complexes retain their native-like topologies at low energy. Upon increasing the collision energy, two of the four complexes adopt a more compact state. This collapse was most noticeable for pentameric serum amyloid P (SAP) which contains a large central cavity. The extent of collapse was found to be highly correlated with charge state, with the surprising observation that the lowest charge states were those which experience the greatest degree of compaction. We compared these experimental results with in vacuo molecular dynamics (MD) simulations of SAP, during which the temperature was increased. Simulations showed that low charge states of SAP exhibited compact states, corresponding to collapse of the ring, while intermediate and high charge states unfolded to more extended structures, maintaining their ring-like topology, as observed experimentally. To simulate the collision-induced dissociation (CID) of different charge states of SAP, we used MS to measure the charge state of the ejected monomer and assigned this charge to one subunit, distributing the residual charges evenly among the remaining four subunits. Under these conditions, MD simulations captured the unfolding and ejection of a single subunit for intermediate charge states of SAP. The highest charge states recapitulated the ejection of compact monomers and dimers, which we observed in CID experiments of high charge states of SAP, accessed by supercharging. This strong correlation between theory and experiment has implications for further studies as well as for understanding the process of CID and for applications to gas-phase structural biology more generally.
We describe a method that integrates data derived from different mass spectrometry (MS)-based techniques with a modeling strategy for structural characterization of protein assemblies. We encoded ...structural data derived from native MS, bottom-up proteomics, ion mobility-MS and chemical cross-linking MS into modeling restraints to compute the most likely structure of a protein assembly. We used the method to generate near-native models for three known structures and characterized an assembly intermediate of the proteasomal base.
Nonalcoholic fatty liver disease (NAFLD) can progress from simple steatosis (i.e., nonalcoholic fatty liver NAFL) to nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. Currently, the driver ...for this progression is not fully understood; in particular, it is not known how NAFLD and its early progression affects the distribution of lipids in the liver, producing lipotoxicity and inflammation. In this study, we used dietary and genetic mouse models of NAFL and NASH and translated the results to humans by correlating the spatial distribution of lipids in liver tissue with disease progression using advanced mass spectrometry imaging technology. We identified several lipids with distinct zonal distributions in control and NAFL samples and observed partial to complete loss of lipid zonation in NASH. In addition, we found increased hepatic expression of genes associated with remodeling the phospholipid membrane, release of arachidonic acid (AA) from the membrane, and production of eicosanoid species that promote inflammation and cell injury. The results of our immunohistochemistry analyses suggest that the zonal location of remodeling enzyme LPCAT2 plays a role in the change in spatial distribution for AA‐containing lipids. This results in a cycle of AA‐enrichment in pericentral hepatocytes, membrane release of AA, and generation of proinflammatory eicosanoids and may account for increased oxidative damage in pericentral regions in NASH. Conclusion: NAFLD is associated not only with lipid enrichment, but also with zonal changes of specific lipids and their associated metabolic pathways. This may play a role in the heterogeneous development of NAFLD. (Hepatology 2017;65:1165‐1180)
The patch-plug configuration has been widely used to repair composite structures and restore the structural integrity of damaged composites. In the present research, single-sided CFRP patch-repaired ...panels, with different patch-plug configurations, are prepared. This is where a circular-shaped damaged area has been removed and a CFRP patch has been adhesively-bonded onto the panel. In some cases, a CFRP plug is inserted into the hole, caused by removal of the damaged area, before the patch is applied. Such patch-repaired panels, and the pristine CFRP panel, are subjected to a low-velocity impact at an energy of 7.5 J. These impacted pristine and repaired panels are then examined using ultrasonic C-scan and optical microscopy to inspect the impact-associated permanent indentation, interlaminar and intralaminar damage. A finite element analysis (FEA) model, which significantly extends a previously validated elastic-plastic (E-P) numerical damage model, has been developed to predict the impact behaviour of the pristine CFRP panel and the various designs of patch-repaired CFRP panels. The comparison between the experimental and numerical results for all the studied cases shows the maximum deviations for the loading response and the damage area are 12% and 15%, respectively. The good agreement between the experimentally-measured impact properties and those predicted using the numerical model demonstrates that the model is a useful design tool.
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•Pristine CFRP panels and Single-sided CFRP patch-repaired panels with and without plugs are prepared and impacted at an energy of 7.5 J;•Effects of the diameter and thickness of the patch as well as the presence of plug on the impacted behaviour of repaired composites are investigated;•Good correlation, between the experiment and simulation demonstrates that the developed numerical model is a useful design tool;•The results revealed that the patch thickness has larger effects on the impact behaviour of repaired composites compared to the patch diameter;•A plug can provide added structural integrity to a composite repair and reduce damage in the adhesive bond.
The mechanisms controlling CD4
T cell switching from an effector to an anti-inflammatory (IL-10
) phenotype play an important role in the persistence of chronic inflammatory diseases. Here, we ...identify the cholesterol biosynthesis pathway as a key regulator of this process. Pathway analysis of cultured cytokine-producing human T cells reveals a significant association between IL-10 and cholesterol metabolism gene expression. Inhibition of the cholesterol biosynthesis pathway with atorvastatin or 25-hydroxycholesterol during switching from IFNγ
to IL-10
shows a specific block in immune resolution, defined as a significant decrease in IL-10 expression. Mechanistically, the master transcriptional regulator of IL10 in T cells, c-Maf, is significantly decreased by physiological levels of 25-hydroxycholesterol. Strikingly, progression to rheumatoid arthritis is associated with altered expression of cholesterol biosynthesis genes in synovial biopsies of predisposed individuals. Our data reveal a link between sterol metabolism and the regulation of the anti-inflammatory response in human CD4
T cells.
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