Reversible, High-Affinity Surface Capturing of Proteins Directed by Supramolecular Assembly

The ability to design surfaces with reversible, high-affinity protein binding sites represents a significant step forward in the advancement of analytical methods for diverse biochemical and biomedical applications. Herein, we report a dynamic supramolecular strategy to directly assemble proteins on surfaces based on multivalent host–guest interactions. The host–guest interactions are achieved by one-step nanofabrication of a well-oriented β-cyclodextrin host-derived self-assembled monolayer on gold (β-CD-SAM) that forms specific inclusion complexes with hydrophobic amino acids located on the surface of the protein. Cytochrome c, insulin, α-chymotrypsin, and RNase A are used as model guest proteins. Surface plasmon resonance and static time-of-flight secondary ion mass spectrometry studies demonstrate that all four proteins interact with the β-CD-SAM in a specific manner via the hydrophobic amino acids on the surface of the protein. The β-CD-SAMs bind the proteins with high nanomolar to single-digit micromolar dissociation constants (KD). Importantly, while the proteins can be captured with high affinity, their release from the surface can be achieved under very mild conditions. Our results expose the great advantages of using a supramolecular approach for controlling protein immobilization, in which the strategy described herein provides unprecedented opportunities to create advanced bioanalytic and biosensor technologies

Metabolomic and Proteomic Analysis of ApoE4-Carrying H4 Neuroglioma Cells in Alzheimer’s Disease Using OrbiSIMS and LC-MS/MS

Growing clinical evidence reveals that systematic molecular alterations in the brain occur 20 years before the onset of AD pathological features. Apolipoprotein E4 (ApoE4) is one of the most significant genetic risk factors for Alzheimer’s disease (AD), which is not only associated with the AD pathological features such as amyloid-β deposition, phosphorylation of tau proteins, and neuroinflammation but is also involved in metabolism, neuron growth, and synaptic plasticity. Multiomics, such as metabolomics and proteomics, are applied widely in identifying key disease-related molecular alterations and disease-progression-related changes. Despite recent advances in the development of analytical technologies, screening the entire profile of metabolites remains challenging due to the numerous classes of compounds with diverse chemical properties that require different extraction processes for mass spectrometry. In this study, we utilized Orbitrap Secondary Ion Mass Spectrometry (OrbiSIMS) as a chemical filtering screening tool to examine molecular alterations in ApoE4-carried neuroglioma cells compared to wild-type H4 cells. The findings were compared using liquid chromatography (LC)-MS/MS targeted metabolomics analysis for the confirmation of specific metabolite classes. Detected alterations in peptide fragments by OrbiSIMS provided preliminary indications of protein changes. These were extensively analyzed through proteomics to explore ApoE4’s impact on proteins. Our metabolomics approach, combining OrbiSIMS and LC-MS/MS, revealed disruptions in lipid metabolism, including glycerophospholipids and sphingolipids, as well as amino acid metabolism, encompassing alanine, aspartate, and glutamate metabolism; aminoacyl-tRNA biosynthesis; glutamine metabolism; and taurine and hypotaurine metabolism. Further LC-MS/MS proteomics studies confirmed the dysfunction in amino acid and tRNA aminoacylation metabolic processes, and highlighted RNA splicing alterations influenced by ApoE4

Protein identification by 3D OrbiSIMS to facilitate in situ imaging and depth profiling

Label-free protein characterization at surfaces is commonly achieved using digestion and/or matrix application prior to mass spectrometry. We report the assignment of undigested proteins at surfaces in situ using secondary ion mass spectrometry (SIMS). Ballistic fragmentation of proteins induced by a gas cluster ion beam (GCIB) leads to peptide cleavage producing fragments for subsequent OrbitrapTM analysis. In this work we annotate 16 example proteins (up to 272 kDa) by de novo peptide sequencing and illustrate the advantages of this approach by characterizing a protein monolayer biochip and the depth distribution of proteins in human skin

Disassembly of Self-Assembling Peptide Hydrogels as a Versatile Method for Cell Extraction and Manipulation

Self-assembling peptide hydrogels (SAPHs) are increasingly being used as two-dimensional (2D) cell culture substrates and three-dimensional (3D) matrices due to their tunable properties and biomimicry of native tissues. Despite these advantages, SAPHs often represent an end-point in cell culture, as isolating cells from them leads to low yields and disruption of cells, limiting their use and post-culture analyses. Here, we report on a protocol designed to easily and effectively disassemble peptide amphiphile (PA) SAPHs to retrieve 3D encapsulated cells with high viability and minimal disruption. Due to the pivotal role played by salt ions in SAPH gelation, tetrasodium ethylenediaminetetraacetic acid (Na4EDTA) was used as metal chelator to sequester ions participating in PA self-assembly and induce a rapid, efficient, clean, and gentle gel-to-sol transition. We characterise PA disassembly from the nano- to the macro-scale, provide mechanistic and practical insights into the PA disassembly mechanism, and assess the potential use of the process. As proof-of-concept, we isolated different cell types from cell-laden PA hydrogels and demonstrated the possibility to perform downstream biological analyses including cell re-plating, gene analysis, and flow cytometry with high reproducibility and no material interference. Our work offers new opportunities for the use of SAPHs in cell culture and the potential use of cells cultured on SAPHs, in applications such as cell expansion, analysis of in vitro models, cell therapies, and regenerative medicine

Spatially resolved molecular analysis of host response to medical device implantation using the 3D OrbiSIMS highlights a critical role for lipids

A key goal for implanted medical devices is that they do not elicit a detrimental immune response. Macrophages play critical roles in modulation of the host immune response and are the major cells responsible for persistent inflammatory reactions to implanted biomaterials. We investigate two novel immune-instructive polymers that stimulate pro- or anti-inflammatory responses from macrophages in vitro. These also modulate in vivo foreign body responses (FBR) when implanted subcutaneously in mice as coatings on biomedical grade silicone rubber. The tissue surrounding the implant is mechanically sectioned and imaged to assess the response of the polymers compared to silicone rubber. Immunofluorescent staining reveals responses consistent with pro- or anti-inflammatory responses previously described for these polymers. We apply 3D OrbiSIMS analysis to provide spatial analysis of the metabolite signature in the tissue surrounding the implant for the first time, providing molecular histology insight into the metabolite response in the host tissue. For the pro-inflammatory coating, monoacylglycerols (MG) and diacylglycerols (DG) are observed at increased intensity, while for the anti-inflammatory coating the number of phospholipid species detected decrease and pyridine and pyrimidine levels were elevated. These findings link to observations of small molecule signature from single cell studies of M2 macrophages in vitro where cell and tissue ion intensities were found to correlate suggesting potential for prediction. This illustrates the power of metabolite characterization by the 3D OrbiSIMS to gain insight into the mechanism of bio-instructive materials as medical devices and to inform on the FBR to biomaterials

Ambient and substrate energy influence decomposer diversity differentially across trophic levels

The species-energy hypothesis predicts increasing biodiversity with increasing energy in ecosystems. Proxies for energy availability are often grouped into ambient energy (i.e., solar radiation) and substrate energy (i.e., non-structural carbohydrates or nutritional content). The relative importance of substrate energy is thought to decrease with increasing trophic level from primary consumers to predators, with reciprocal effects of ambient energy. Yet, empirical tests are lacking. We compiled data on 332,557 deadwood-inhabiting beetles of 901 species reared from wood of 49 tree species across Europe. Using host-phylogeny-controlled models, we show that the relative importance of substrate energy versus ambient energy decreases with increasing trophic levels: the diversity of zoophagous and mycetophagous beetles was determined by ambient energy, while non-structural carbohydrate content in woody tissues determined that of xylophagous beetles. Our study thus overall supports the species-energy hypothesis and specifies that the relative importance of ambient temperature increases with increasing trophic level with opposite effects for substrate energy

Ambient and substrate energy influence decomposer diversity differentially across trophic levels.

The species-energy hypothesis predicts increasing biodiversity with increasing energy in ecosystems. Proxies for energy availability are often grouped into ambient energy (i.e., solar radiation) and substrate energy (i.e., non-structural carbohydrates or nutritional content). The relative importance of substrate energy is thought to decrease with increasing trophic level from primary consumers to predators, with reciprocal effects of ambient energy. Yet, empirical tests are lacking. We compiled data on 332,557 deadwood-inhabiting beetles of 901 species reared from wood of 49 tree species across Europe. Using host-phylogeny-controlled models, we show that the relative importance of substrate energy versus ambient energy decreases with increasing trophic levels: the diversity of zoophagous and mycetophagous beetles was determined by ambient energy, while non-structural carbohydrate content in woody tissues determined that of xylophagous beetles. Our study thus overall supports the species-energy hypothesis and specifies that the relative importance of ambient temperature increases with increasing trophic level with opposite effects for substrate energy

Molecular Formula Prediction for Chemical Filtering of 3D OrbiSIMS Datasets

Modern mass spectrometry techniques produce a wealth of spectral data, and although this is an advantage in terms of the richness of the information available, the volume and complexity of data can prevent a thorough interpretation to reach useful conclusions. Application of molecular formula prediction (MFP) to produce annotated lists of ions that have been filtered by their elemental composition and considering structural double bond equivalence are widely used on high resolving power mass spectrometry datasets. However, this has not been applied to secondary ion mass spectrometry data. Here, we apply this data interpretation approach to 3D OrbiSIMS datasets, testing it for a series of increasingly complex samples. In an organic on inorganic sample, we successfully annotated the organic contaminant overlayer separately from the substrate. In a more challenging purely organic human serum sample we filtered out both proteins and lipids based on elemental compositions, 226 different lipids were identified and validated using existing databases, and we assigned amino acid sequences of abundant serum proteins including albumin, fibronectin, and transferrin. Finally, we tested the approach on depth profile data from layered carbonaceous engine deposits and annotated previously unidentified lubricating oil species. Application of an unsupervised machine learning method on filtered ions after performing MFP from this sample uniquely separated depth profiles of species, which were not observed when performing the method on the entire dataset. Overall, the chemical filtering approach using MFP has great potential in enabling full interpretation of complex 3D OrbiSIMS datasets from a plethora of material types

Capric Acid Secreted by S. boulardii Inhibits C. albicans Filamentous Growth, Adhesion and Biofilm Formation

Candidiasis are life-threatening systemic fungal diseases, especially of gastro intestinal track, skin and mucous membranes lining various body cavities like the nostrils, the mouth, the lips, the eyelids, the ears or the genital area. Due to increasing resistance of candidiasis to existing drugs, it is very important to look for new strategies helping the treatment of such fungal diseases. One promising strategy is the use of the probiotic microorganisms, which when administered in adequate amounts confer a health benefit. Such a probiotic microorganism is yeast Saccharomyces boulardii, a close relative of baker yeast. Saccharomyces boulardii cells and their extract affect the virulence factors of the important human fungal pathogen C. albicans, its hyphae formation, adhesion and biofilm development. Extract prepared from S. boulardii culture filtrate was fractionated and GC-MS analysis showed that the active fraction contained, apart from 2-phenylethanol, caproic, caprylic and capric acid whose presence was confirmed by ESI-MS analysis. Biological activity was tested on C. albicans using extract and pure identified compounds. Our study demonstrated that this probiotic yeast secretes into the medium active compounds reducing candidal virulence factors. The chief compound inhibiting filamentous C. albicans growth comparably to S. boulardii extract was capric acid, which is thus responsible for inhibition of hyphae formation. It also reduced candidal adhesion and biofilm formation, though three times less than the extract, which thus contains other factors suppressing C. albicans adherence. The expression profile of selected genes associated with C. albicans virulence by real-time PCR showed a reduced expression of HWP1, INO1 and CSH1 genes in C. albicans cells treated with capric acid and S. boulardii extract. Hence capric acid secreted by S. boulardii is responsible for inhibition of C. albicans filamentation and partially also adhesion and biofilm formation

Population and labour force projections for 27 European countries, 2002-052: impact of international migration on population ageing: Projections de population et de population active pour 27 pays européens 2002-052: impact de la migration internationale sur le vieillissement de la population

Population and labour force projections are made for 27 selected European countries for 2002-052, focussing on the impact of international migration on population and labour force dynamics. Starting from single scenarios for fertility, mortality and economic activity, three sets of assumptions are explored regarding migration flows, taking into account probable policy developments in Europe following the enlargement of the EU. In addition to age structures, various support ratio indicators are analysed. The results indicate that plausible immigration cannot offset the negative effects of population and labour force ageing