Decision trees and labeling of low noise pavements as support for noise action plans.

Low noise pavements (LNPs) are a market driven trend to mitigate the high road traffic noise exposure levels. Their improvement towards acoustic efficiency and durability over time is a challenge since these factors can conflict with road primary functions, such as safety. LNPs are not always the most cost-effective solution in health effects prevention. Whilst Green Public Procurement (GPP) highlighted the importance of reducing rolling noise emissions by introducing new regulations for new-layed LNPs, the fixed minimum requirements are not exhaustive. Generally, limits are set following the Close ProXimity method, which is only source oriented. This method does not consider real traffic flows and it is not aimed at evaluating citizens' disturbance. This work presents strategy tools that could assist policymakers in choosing LNPs, when truly effective, over other mitigations. The approach includes a variety of indicators that would allow for comparing different facets of noise assessment. The proposed methodology does not require additional efforts from stakeholders because the measurements required for the estimation of the indicators must already be carried out for both verification of legal limits and GPP. The strategy tools are a decisional tree to support the evaluation of the applicability of a LNP before its approval, and an evaluation flowchart applicable after its laying to evaluate its efficiency. Finally, a first LNP labeling approach, based on the same set of indicators, is proposed. As a case study, these tools are applied to measurements performed before and after the laying of twelve LNPs part of the LIFE NEREiDE project.

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system.

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.

Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics.

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular proteins were required to detect biotinylated peptides. In addition, the enriched biotinylated peptide solution contained many contaminant ions. Here, we optimized the workflow for efficient enrichment of biotinylated peptides and removal of contaminant ions. The efficient recovery of biotinylated peptides with fewer contaminant ions was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and using optimal temperature and salt concentration for elution. The optimized workflow enabled identification of nearly 4-fold more biotinylated peptides with higher purity from RAW264.7 macrophages expressing TurboID-fused STING (stimulator of interferon genes). In addition, sequential digestion with Glu-C and trypsin revealed biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon epidermal growth factor (EGF) stimulation. This workflow will be useful for BioID and cell surface proteomics and for various other applications based on protein biotinylation.

Organs in Color: Utilizing Free Software and Emerging Multi Jet Fusion Technology to Color and Surface Label 3D-Printed Anatomical Models.

3D printing (3DP) is a rapidly evolving innovative technology that has already been utilized for the development of educational anatomic models. Until recently, it was difficult and tedious to create multi-colored models and especially labels due to technological constraints. In this technical note, a comprehensive guide for creating labeled and color-coded anatomic models was created using free software, Blender. We have composed a step-by-step process for taking an existing 3D model and adding labeling and color that is compatible with modern high-quality 3D printing technologies (Multi Jet Fusion). We provided colored and labeled 3D renderings of the surface anatomy of the brain, ventricular system of the brain, the segments of the liver, and coronary arteries as examples of the diverse potential of this technology. Additionally, we 3D printed actual models of the surface anatomy of the brain and ventricles of the brain using HP Multi Jet Fusion to demonstrate the potential of this technology in the creation of anatomic models.

Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane.

Localization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. We present a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. Two engineered, monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enables us to quantitatively resolve the stoichiometry and dynamics of the interferon-γ (IFNγ) receptor signaling complex in the plasma membrane of living cells by multicolor single-molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we identify ligand-induced receptor homo- and heterodimerization. Multicolor single-molecule co-tracking and quantitative single-molecule Förster resonance energy transfer moreover reveals transient assembly of IFNγ receptor heterotetramers and confirms its structural architecture.

Site-Specific Protein Modifications by an Engineered Asparaginyl Endopeptidase from Viola canadensis.

Asparaginyl endopeptidases (AEPs) or legumains are Asn/Asp (Asx)-specific proteases that break peptide bonds, but also function as peptide asparaginyl ligases (PALs) that make peptide bonds. This ligase activity can be used for site-specific protein modifications in biochemical and biotechnological applications. Although AEPs are common, PALs are rare. We previously proposed ligase activity determinants (LADs) of these enzymes that could determine whether they catalyze formation or breakage of peptide bonds. LADs are key residues forming the S2 and S1' substrate-binding pockets flanking the S1 active site. Here, we build on the LAD hypothesis with the engineering of ligases from proteases by mutating the S2 and S1' pockets of VcAEP, an AEP from Viola canadensis. Wild type VcAEP yields <5% cyclic product from a linear substrate at pH 6.5, whereas the single mutants VcAEP-V238A (Vc1a) and VcAEP-Y168A (Vc1b) targeting the S2 and S1' substrate-binding pockets yielded 34 and 61% cyclic products, respectively. The double mutant VcAEP-V238A/Y168A (Vc1c) targeting both the S2 and S1' substrate-binding pockets yielded >90% cyclic products. Vc1c had cyclization efficiency of 917,759 M-1s-1, which is one of the fastest rates for ligases yet reported. Vc1c is useful for protein engineering applications, including labeling of DARPins and cell surface MCF-7, as well as producing cyclic protein sfGFP. Together, our work validates the importance of LADs for AEP ligase activity and provides valuable tools for site-specific modification of proteins and biologics.

Extracellular Vesicles Tropism: A Comparative Study between Passive Innate Tropism and the Active Engineered Targeting Capability of Lymphocyte-Derived EVs.

Cellular communications take place thanks to a well-connected network of chemical-physical signals, biomolecules, growth factors, and vesicular messengers that travel inside or between cells. A deep knowledge of the extracellular vesicle (EV) system allows for a better understanding of the whole series of phenomena responsible for cell proliferation and death. To this purpose, here, a thorough immuno-phenotypic characterization of B-cell EV membranes is presented. Furthermore, the cellular membrane of B lymphocytes, Burkitt lymphoma, and human myeloid leukemic cells were characterized through cytofluorimetry assays and fluorescent microscopy analysis. Through cytotoxicity and internalization tests, the tropism of B lymphocyte-derived EVs was investigated toward the parental cell line and two different cancer cell lines. In this study, an innate capability of passive targeting of the native EVs was distinguished from the active targeting capability of monoclonal antibody-engineered EVs, able to selectively drive the vesicles, enhancing their internalization into the target cancer cells. In particular, the specific targeting ability of anti-CD20 engineered EVs towards Daudi cells, highly expressing CD20 marker on their cell membrane, was proved, while almost no internalization events were observed in HL60 cells, since they did not express an appreciable amount of the CD20 marker on their plasma membranes.

Finding Species-Specific Extracellular Surface-Facing Proteomes in Toxic Dinoflagellates.

As a sequel to our previous report of the existence of species-specific protein/peptide expression profiles (PEPs) acquired by mass spectrometry in some dinoflagellates, we established, with the help of a plasma-membrane-impermeable labeling agent, a surface amphiesmal protein extraction method (SAPE) to label and capture species-specific surface proteins (SSSPs) as well as saxitoxins-producing-species-specific surface proteins (Stx-SSPs) that face the extracellular space (i.e., SSSPsEf and Stx-SSPsEf). Five selected toxic dinoflagellates, Alexandrium minutum, A. lusitanicum, A. tamarense, Gymnodinium catenatum, and Karenia mikimotoi, were used in this study. Transcriptomic databases of these five species were also constructed. With the aid of liquid chromatography linked-tandem mass spectrometry (LC-MS/MS) and the transcriptomic databases of these species, extracellularly facing membrane proteomes of the five different species were identified. Within these proteomes, 16 extracellular-facing and functionally significant transport proteins were found. Furthermore, 10 SSSPs and 6 Stx-SSPs were identified as amphiesmal proteins but not facing outward to the extracellular environment. We also found SSSPsEf and Stx-SSPsEf in the proteomes. The potential functional correlation of these proteins towards the production of saxitoxins in dinoflagellates and the degree of species specificity were discussed accordingly.

Vypal2: A Versatile Peptide Ligase for Precision Tailoring of Proteins.

The last two decades have seen an increasing demand for new protein-modification methods from the biotech industry and biomedical research communities. Owing to their mild aqueous reaction conditions, enzymatic methods based on the use of peptide ligases are particularly desirable. In this regard, the recently discovered peptidyl Asx-specific ligases (PALs) have emerged as powerful biotechnological tools in recent years. However, as a new class of peptide ligases, their scope and application remain underexplored. Herein, we report the use of a new PAL, VyPAL2, for a diverse range of protein modifications. We successfully showed that VyPAL2 was an efficient biocatalyst for protein labelling, inter-protein ligation, and protein cyclization. The labelled or cyclized protein ligands remained functionally active in binding to their target receptors. We also demonstrated on-cell labelling of protein ligands pre-bound to cellular receptors and cell-surface engineering via modifying a covalently anchored peptide substrate pre-installed on cell-surface glycans. Together, these examples firmly establish Asx-specific ligases, such as VyPAL2, as the biocatalysts of the future for site-specific protein modification, with a myriad of applications in basic research and drug discovery.

Postsynaptic Targeting and Mobility of Membrane Surface-Localized hASIC1a.

Acid-sensing ion channels (ASICs), the main H+ receptors in the central nervous system, sense extracellular pH fluctuations and mediate cation influx. ASIC1a, the major subunit responsible for acid-activated current, is widely expressed in brain neurons, where it plays pivotal roles in diverse functions including synaptic transmission and plasticity. However, the underlying molecular mechanisms for these functions remain mysterious. Using extracellular epitope tagging and a novel antibody recognizing the hASIC1a ectodomain, we examined the membrane targeting and dynamic trafficking of hASIC1a in cultured cortical neurons. Surface hASIC1a was distributed throughout somata and dendrites, clustered in spine heads, and co-localized with postsynaptic markers. By extracellular pHluorin tagging and fluorescence recovery after photobleaching, we detected movement of hASIC1a in synaptic spine heads. Single-particle tracking along with use of the anti-hASIC1a ectodomain antibody revealed long-distance migration and local movement of surface hASIC1a puncta on dendrites. Importantly, enhancing synaptic activity with brain-derived neurotrophic factor accelerated the trafficking and lateral mobility of hASIC1a. With this newly-developed toolbox, our data demonstrate the synaptic location and high dynamics of functionally-relevant hASIC1a on the surface of excitatory synapses, supporting its involvement in synaptic functions.

Postsynaptic Targeting and Mobility of Membrane Surface-Localized hASIC1a / 神经科学通报·英文版

Acid-sensing ion channels (ASICs), the main H

Cell Surface Labeling by Engineered Extracellular Vesicles.

Extracellular vesicles (EVs) can mediate local and long-range intercellular communication via cell surface signaling. In order to perform in vivo studies of unmanipulated, endogenously released EVs, sensitive but stringent approaches able to detect EV-cell surface interactions are needed. However, isolation and reinfusion of EVs can introduce biases. A rigorous way to study EVs in vivo is by genetically engineering membrane-bound reporters into parental cells. Still, the amount of reporter molecules that EVs can carry is relatively small, and thus, the sensitivity of the approach is suboptimal. This work addresses this issue by engineering EVs to display a membrane-bound form of Sortase A (SrtA), a bacterial transpeptidase that can catalyze the transfer of reporter molecules on the much bigger surface of EV-binding cells. SrtA design and reaction requirements are optimized and validated. Efficient in vitro labeling of EV-binding cells is achieved, even in the presence of only one N-terminal glycine on cell surface proteins. As compared to indirect labeling of EV-binding cells (e.g., using CD63-GFP fusion), the SrtA-based approach shows 1-2 log increase in sensitivity, depending on the EV source. This novel approach will be useful to identify and study the full set of host cells interacting with native EVs in vivo.

Improved sensitivity in cell surface protein detection by combining chemical labeling with mechanical lysis in a colorectal cancer cell model.

OBJECTIVE: To develop a new technique for improved cell surface protein detection and analysis by combining chemical labeling with mild cell lysis using model HCT 116 colorectal cancer cells. RESULTS: We found that Dounce homogenization by hand, rather than the typical sonication or syringe lysis method, recovered surface/membrane proteins more consistently and effectively. This was indicated by marker membrane proteins such as claudin-4 and EGFR (epidermal growth factor receptor) that span the typical 20-200 kD range. As monitored by Western blotting (WB), the Dounce lysis method combined with cell surface biotinylation showed consistent recovery of the marker proteins claudin-4 and EGFR. This lysis method was combined with a cell surface biotinylation strategy to enrich cell surface/membrane proteins using affinity bead-based purification with four-fold less cells compared to prior work. Subsequent LC/MS/MS analysis identified 49 additional surface/membrane proteins for the first time from HCT 116 cells. CONCLUSION: This combination of methodologies may fit into an advanced workflow for identifying new and elusive cell surface proteins. It can increase the protein coverage for biomarker discovery for colorectal cancer or other cancers. This new detection/analysis approach may also promote new applications in surface display systems as well as cell screening, selection, and binding processes.

Sortase A: A Model for Transpeptidation and Its Biological Applications.

Molecular biologists and chemists alike have long sought to modify proteins with substituents that cannot be installed by standard or even advanced genetic approaches. We here describe the use of transpeptidases to achieve these goals. Living systems encode a variety of transpeptidases and peptide ligases that allow for the enzyme-catalyzed formation of peptide bonds, and protein engineers have used directed evolution to enhance these enzymes for biological applications. We focus primarily on the transpeptidase sortase A, which has become popular over the past few years for its ability to perform a remarkably wide variety of protein modifications, both in vitro and in living cells.

Metabolic Profiling of Bacteria by Unnatural C-terminated D-Amino Acids.

Bacterial peptidoglycan is a mesh-like network comprised of sugars and oligopeptides. Transpeptidases cross-link peptidoglycan oligopeptides to provide vital cell wall rigidity and structural support. It was recently discovered that the same transpeptidases catalyze the metabolic incorporation of exogenous D-amino acids onto bacterial cell surfaces with vast promiscuity for the side-chain identity. It is now shown that this enzymatic promiscuity is not exclusive to side chains, but that C-terminus variations can also be accommodated across a diverse range of bacteria. Atomic force microscopy analysis revealed that the incorporation of C-terminus amidated D-amino acids onto bacterial surfaces substantially reduced the cell wall stiffness. We exploited the promiscuity of bacterial transpeptidases to develop a novel assay for profiling different bacterial species.

Studying G protein-coupled receptors: immunoblotting, immunoprecipitation, phosphorylation, surface labeling, and cross-linking protocols.

Primary cilia are signaling organelles that have been shown to coordinate cellular responses to extracellular cues during physiological processes ranging from organ patterning to cell cycle regulation. A variety of receptors, including G protein-coupled receptors (GPCRs), downstream effectors (adenylyl cyclases), and second messengers, such as calcium, accumulate in the ciliary compartment. Isolation of GPCRs is essential for studying posttranslational modifications, intracellular trafficking, and protein-protein interactions that are important in downstream signaling. However, the presence of multiple hydrophobic transmembrane domains, and the inherent conformational flexibility of GPCRs make their extraction from membranes and solubilization particularly challenging. Here, we describe detailed methods for immunoblotting and immunoprecipitation of GPCRs from whole cell extracts. These methods are applicable for studying other multipass transmembrane proteins (such as adenylyl cyclases). We also describe methods for determining GPCR phosphorylation, surface labeling by biotinylation, and cross-linking to detect transient interactions with other proteins. These methods are amenable for studying both ciliary and nonciliary GPCRs in the context of cellular signaling pathways.