Recognition Events Research Articles

Nucleic Acid Enzyme-Activated CRISPR-Cas12a With Circular CRISPR RNA for Biosensing.

clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non-nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site-specific double-stranded DNA cutting and nonspecific single-stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA-cleaving activity can linearize the circular crRNAs, activating CRISPR-Cas12a functions. Using ligand-responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target-triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as "NAzyme-Activated CRISPR-Cas12a with Circular CRISPR RNA (NA3C)." Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli-responsive RNA-cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated.

Politics, geography and recognition in the emerging multipolar world order

ABSTRACT This article examines international politics of recognition in the increasingly multipolar global environment, with a specific focus on recognition events in Kosovo, Abkhazia and South Ossetia, Crimea, the Occupied Golan Heights, Jerusalem, Western Sahara, Luhansk and Donetsk. It reveals that the recent surge in recognition is propelled by geopolitical and geographical motivations, which undermine the normative significance of territorial integrity, non-recognition and self-determination in recognition politics. The article proposes a thesis that situates recognition within the context of geopolitics, spatial management and power dynamics that expand our understanding of the broader impacts of recognition beyond the traditional emphasis on statehood emergence and prevention. Recognition has become entangled in geopolitical contestations between influential global players seeking to normatively legitimise and normalise conquest, occupation and colonisation. In this context, geopolitical considerations have overridden the normative and legal weight of the well-institutionalised norms of territorial recognition. This underscores the gap between recognition norms and practices and demonstrates the explanatory power of geopolitics.

Species-Specific N-Glycomes and Methylation Patterns of Oysters Crassostrea gigas and Ostrea edulis and Their Possible Consequences for the Norovirus-HBGA Interaction.

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles.

Compassion, collaboration, and community: Strategies for staff well-being.

Compassion, collaboration, and community: Strategies for staff well-being.

Self-Cleaning Recyclable Multiplexed Photoelectrochemical Sensing Strategy Based on Exonuclease III-Assisted Signal Discrimination.

For discriminating the signals of multi-targets, multiplexed photoelectrochemical (PEC) detection is generally accomplished by modulating the light source or voltage, which prospect is usually limited by expensive instrumentation, tedious operational steps, and time-consuming material screening. To realize multiplexed determination on single photoelectric interface using the routine technique, a non-instrument-assisted strategy for signal discrimination needs to be explored. Herein, we propose an exonuclease III-mediated multiple PEC signals resolution strategy and construct a self-cleaning recyclable multiplexed PEC sensor using a porphyrin-bipyridine-based covalent organic framework (Por-Bpy COF) photocathode. Specifically, following the dual-target recognition event, exonuclease III cleaves the DNA strand attached to the magnetic bead so that the two signal labels can be separated. Once the signal label binds to the DNA on the electrode surface (E-DNA), exonuclease III turns to excise the DNA strand of the signal label and consequently the E-DNA can repeatedly bind different signal labels. As a result, distinguishable photocurrent signals of different targets can be generated on a single photoelectric interface. The feasibility of this multiplexed sensor is verified by detecting two coexisting mycotoxins aflatoxin B1 and zearalenone. On account of eliminating the instrumentation constraints and simplifying the experimental procedures, the proposed sensing strategy may provide a brand-new idea for the exploration of portable multiplexed PEC sensing devices.

MOF-derived magnetic trimetallic oxide-carbon nanocomposites for modification-free and dual molecular events-dependent ratiometric electrochemical analysis of intracellular glutathione

The response of ratiometric electrochemical signals toward the targets of interest generally depends on specific molecules-mediated single molecular recognition event, which limits the application in analyzing the targets without specific recognition molecules. In this work, novel dual molecular recognition events were proposed for rapid and reliable electrochemical analysis of glutathione (GSH) using MOF-derived trimetallic oxide-carbon (Fe3O4/CuO/NiO-C) nanocomposites. The binding of GSH to CuO caused a decrease in the oxidation signal of Cu(Ⅱ) due to the formation of nonelectroactive complex, while NiO could electrocatalyze the oxidation of GSH. These GSH-involved dual molecular recognition events guaranteed the readout of ratiometric electrochemical signals with a complementary response range, allowing sensitive and wide-range analysis of GSH. Besides, the difference in spatial conformation gave rise to a different oxidation potential for different biothiols, which guaranteed selective analysis of GSH. The presence of Fe3O4 allowed modification-free detection within 5 min using a magnetic working electrode. Taking advantage of these merits, the developed electrochemical sensor could rapidly and reliably monitor the intracellular GSH.

Structure of the immunoregulatory sialidase NEU1.

Sialic acids linked to glycoproteins and glycolipids are important mediators of cell and protein recognition events. These sugar residues are removed by neuraminidases (sialidases). Neuraminidase-1 (sialidase-1 or NEU1) is a ubiquitously expressed mammalian sialidase located in lysosomes and on the cell membrane. Because of its modulation of multiple signaling processes, it is a potential therapeutic target for cancers and immune disorders. Genetic defects in NEU1 or in its protective protein cathepsin A (PPCA, CTSA) cause the lysosomal storage diseases sialidosis and galactosialidosis. To further our understanding of this enzyme's function at the molecular level, we determined the three-dimensional structure of murine NEU1. The enzyme oligomerizes through two self-association interfaces and displays a wide substrate-binding cavity. A catalytic loop adopts an inactive conformation. We propose a mechanism of activation involving a conformational change in this loop upon binding to its protective protein. These findings may facilitate the development of selective inhibitor and agonist therapies.

Structural Plasticity of NFU1 Upon Interaction with Binding Partners: Insights into the Mitochondrial [4Fe-4S] Cluster Pathway

In humans, the biosynthesis and trafficking of mitochondrial [4Fe-4S]2+ clusters is a highly coordinated process that requires a complex protein machinery. In a mitochondrial pathway among various proposed to biosynthesize nascent [4Fe-4S]2+ clusters, two [2Fe-2S]2+ clusters are converted into a [4Fe-4S]2+ cluster on a ISCA1-ISCA2 complex. Along this pathway, this cluster is then mobilized from this complex to mitochondrial apo recipient proteins with the assistance of accessory proteins. NFU1 is the accessory protein that first receives the [4Fe-4S]2+ cluster from ISCA1-ISCA2 complex. A structural view of the protein–protein recognition events occurring along the [4Fe-4S]2+ cluster trafficking as well as how the globular N-terminal and C-terminal domains of NFU1 act in such process is, however, still elusive. Here, we applied small-angle X-ray scattering coupled with on-line size-exclusion chromatography and paramagnetic NMR to disclose structural snapshots of ISCA1-, ISCA2- and NFU1-containing apo complexes as well as the coordination of [4Fe-4S]2+ cluster bound to the ISCA1-NFU1 complex, which is the terminal stable species of the [4Fe-4S]2+ cluster transfer pathway involving ISCA1-, ISCA2- and NFU1 proteins. The structural modelling of ISCA1-ISCA2, ISCA1-ISCA2-NFU1 and ISCA1-NFU1 apo complexes, here reported, reveals that the structural plasticity of NFU1 domains is crucial to drive protein partner recognition and modulate [4Fe-4S]2+ cluster transfer from the cluster-assembly site in the ISCA1-ISCA2 complex to a cluster-binding site in the ISCA1-NFU1 complex. These structures allowed us to provide a first rational for the molecular function of the N-domain of NFU1, which can act as a modulator in the [4Fe-4S]2+ cluster transfer.

Different electrostatic forces drive the binding kinetics of SARS-CoV, SARS-CoV-2 and MERS-CoV Envelope proteins with the PDZ2 domain of ZO1

The Envelope protein (E) is a structural protein encoded by the genome of SARS-CoV, SARS-CoV-2 and MERS-CoV Coronaviruses. It is poorly present in the virus but highly expressed in the host cell, with prominent role in virus assembly and virulence. The E protein possesses a PDZ-binding motif (PBM) at its C terminus that allows it to interact with host PDZ domain containing proteins. ZO1 is a key protein in assembling the cytoplasmic plaque of epithelial and endothelial Tight Junctions (TJs) as well as in determining cell differentiation, proliferation and polarity. The PDZ2 domain of ZO1 is known to interact with the Coronaviruses Envelope proteins, however the molecular details of such interaction have not been established. In this paper we directly measured, through Fluorescence Resonance Energy Transfer and Stopped-Flow methodology, the binding kinetics of the PDZ2 domain of ZO1 with peptides mimicking the C-terminal portion of the Envelope protein from SARS-CoV, SARS-CoV-2 and MERS-CoV in different ionic strength conditions. Interestingly, the peptide mimicking the E protein from MERS-CoV display much higher microscopic association rate constant with PDZ2 compared to SARS-CoV and SARS-CoV-2 suggesting a stronger contribution of electrostatic forces in the early events of binding. A comparison of thermodynamic and kinetic data obtained at increasing ionic strengths put in evidence different contribution of electrostatics in the recognition and complex formation events for the three peptides. Our data are discussed under the light of available structural data of PDZ2 domain of ZO1 and of previous works about these protein systems.

Coupled protein quality control during nonsense-mediated mRNA decay.

Translation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC containing transcripts. Although the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational and dependent on the ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors but suggested that protein degradation did not depend on the canonical ribosome-quality control (RQC) pathway. A subsequent arrayed screen demonstrated that protein and mRNA branches of NMD rely on a shared recognition event. Our results establish the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a reference for the field to identify and characterize required factors.

Multi-Responsive Eight-State Bis(acridinium-Zn(II) porphyrin) Receptor.

A multi-responsive receptor consisting of two (acridinium-Zn(II) porphyrin) conjugates has been designed. The binding constant between this receptor and a ditopic guest has been modulated (i) upon addition of nucleophiles converting acridinium moieties into the non-aromatic acridane derivatives and (ii) upon oxidation of the porphyrin units. A total of eight states has been probed for this receptor resulting from the cascade of the recognition and responsive events. Moreover, the acridinium/acridane conversion leads to a significant change of the photophysical properties, switching from electron to energy transfer processes. Interestingly, for the bis(acridinium-Zn(II) porphyrin) receptor, charge-transfer luminescence in the near-infrared has been observed.

Exploring Zr-based Metal-Organic Frameworks as Smart Electrochromic Sensors by Coordination-Driven Surface Engineering.

As emerging stimuli-responsive materials, electrochromic metal-organic frameworks (MOFs) are still not utilized in sensing applications due to difficulties in water stability, facile synthesis and functionalization, and efficient translation of specific recognition events. Here, we firstly find that a Zr-based MOF furnished with postsynthetically created viologen-like electron-deficient moiety was electrochromic active. With a coordination-driven surface engineering strategy where phosphate-containing biomolecules are tethered to Zr nodes of the MOF, fine tuning the interface electron transfer was readily achieved, thus benefitting for constructing smart electrochromic sensors through the combination of the sensitivity of electrochemistry with the visuality of colorimetry. Particularly, MOF-coated conductive films enabled label-free detection of phosphoproteins, and aptamer-functionalized ones responded specifically to the target. In two cases distinct color changes allow for visual quantification. This study represents the first example of MOF-based electrochromic sensors developed by an efficient strategy, indicating the generality to electrochromic counterparts for various sensing applications.

A label-free and ratiometric fluorescent immunosensor by integrating CRISPR/Cas 12a and 3D DNA nanomachine

Sensitive, selective, especially label-free detection of disease relevant protein biomarkers is highly valuable for clinical diagnosis and disease process monitoring but remains a challenge. Herein, with prostate specific antigen (PSA) as a model target, we have designed a label-free ratiometric fluorescent immunosensor based on three-dimensional (3D) DNA walker and the powerful clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, with poly(thymine) templated Cu nanoclusters and DAPI as signals. The aptamer-linked activator DNA was designed to convert target recognition event into activate deoxyribonuclease activity of CRISPR/Cas12a system and further cleave oligonucleotides. The cleavage behavior leads to the quenching of copper nanoclusters and stagnation of the cascade amplification progress of 3D DNA nanomachine. The DNA machine is constructed from primer DNA and silica nanospheres loaded by double-stranded DNA (dsDNA) with prominent 3′ end, and movement of the primer DNA is powered by exonuclease (Exo) III via a burnt-bridge mechanism. Without primer DNA, the dyes (DAPI) were embedded in the complementary DNA and emit strong fluorescence. The practical applicability of the biosensor was demonstrated by assay of PSA in human serum with satisfactory result. In addition, this completely label-free method, reduced consumption of antibody, greatly simplify the procedures and saving cost.

Effector-mediated partial and nonhost disease resistance in wheat.

Effector-mediated partial and nonhost disease resistance in wheat.

1,3,5,8-Tetrasubstituted 1,3,4-Benzotriazepin-2-one Scaffolds for β-Turn Mimicry without Stereogenic Carbon Centers: Synthesis and Conformational Analysis.

Topological mimicry of peptide β-turn secondary structures has been investigated using a 1,3,5,8-tetrasubstituted 1,3,4-benzotriazepin-2-one scaffold. Approaches were conceived for the synthesis of tetrasubstituted benzotriazepinones from 4-acetyl-3-aminobenzoate based on aza-amino acid chemistry and different orthogonal protection strategies. Installation of an 8-position carboxylate on the aromatic ring enabled a diverse array of substituents to be introduced for mimicry of the i-position residue. Benzotriazepin-2-one crystallization and X-ray analysis demonstrated that in spite the absence of a stereogenic carbon center, the scaffold could serve as type I and I' β-turn mimics, because pyramidalization of the N3-nitrogen in the benzotriazepin-2-one provides potential for adoptive chirality. 1,3,5,8-Tetrasubstituted 1,3,4-benzotriazepin-2-one scaffolds offer interesting potential for the cost-effective synthesis of nonpeptide β-turn surrogates for peptide mimicry in various recognition events.

Determinants of Superselectivity─Practical Concepts for Application in Biology and Medicine.

Multivalent interactions are common in biological systems and are also widely deployed for targeting applications in biomedicine. A unique feature of multivalent binding is "superselectivity". Superselectivity refers to the sharp discrimination of surfaces (e.g., on cells or cell compartments) by their comparative surface densities of a given receptor. This feature is different from the conventional "type" selectivity, which discriminates surfaces by their distinct receptor types. In a broader definition, a probe is superselective if it converts a gradual change in any one interaction parameter into a sharp on/off dependency in probe binding.This Account describes our systematic experimental and theoretical efforts over the past decade to analyze the determinants of superselective binding. It aims to offer chemical biologists, biophysicists, biologists, and biomedical scientists a set of guidelines for the interpretation of multivalent binding data, and design rules for tuning superselective targeting. We first provide a basic introduction that identifies multiple low-affinity interactions and combinatorial entropy as the minimal set of conditions required for superselective recognition. We then introduce the main experimental and theoretical tools and analyze how salient features of the multivalent probes (i.e., their concentration, size, ligand valency, and scaffold type), of the surface receptors (i.e., their affinity for ligands, surface density, and mobility), and of competitors and cofactors (i.e., their concentration and affinity for the ligands and/or receptors) influence the sharpness and the position of the threshold for superselective recognition.Emerging from this work are a set of relatively simple yet quantitative data analysis guidelines and superselectivity design rules that apply to a broad range of probe types and interaction systems. The key finding is the scaling variable xS which faithfully predicts the influence of the surface receptor density, probe ligand valency, receptor-ligand affinity, and competitor/cofactor concentrations and affinities on superselective recognition. The scaling variable is a simple yet versatile tool to quantitatively tune the on/off threshold of superselective probes. We exemplify its application by reviewing and reinterpreting literature data for selected biological and biomedical interaction systems where superselectivity clearly is important.Our guidelines can be deployed to generate a new mechanistic understanding of multivalent recognition events inside and outside cells and the downstream physiological/pathological implications. Moreover, the design rules can be harnessed to develop novel superselective probes for analytical purposes in the life sciences and for diagnostic/therapeutic intervention in biomedicine.

Make Your Recognition Event Worth Attending

The Volunteer Management ReportVolume 28, Issue 4 p. 7-7 Annual Volunteer Recognition Events Make Your Recognition Event Worth Attending First published: 10 March 2023 https://doi.org/10.1002/vmr.32063Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume28, Issue4April 2023Pages 7-7 RelatedInformation

Computational approaches screening DNA aptamers against conserved outer membrane protein W of Vibrio cholerae O1- an investigation expanding the potential for point-of-care detection with aptasensors

Foodborne outbreaks urge public health domain to upgrade diagnosis by means of simpler, quicker, and more affordable pathogen detection methods. A molecular recognition probe against an analyte of interest makes up a biosensor, along with a method for turning the recognition event into a quantifiable signal. Single-stranded DNA or RNA aptamers are promising bio-recognition molecules for a range of targets, including a wide range of non-nucleic acid targets with which they are highly specific and affine. In the proposed study, 40 DNA aptamers were screened and analyzed interactions using in-silico SELEX procedures, which can selectively interact with active sites at the extracellular region of the Outer membrane Protein W (OmpW) of Vibrio Cholerae. Multiple modeling techniques, like protein structural prediction with I-TASSER, aptamer structural modeling using M-fold, RNA composer, protein-DNA docking using HADDOCK, and large-scale (500 ns) molecular dynamics simulations through GROMACS have been employed. Out of 40, six aptamers having lowest free energy were docked against the predicted active site at the extracellular region of OmpW. VBAPT4-OmpW and VBAPT17-OmpW, the two highest-scoring Aptamer-Protein complexes, were chosen for molecular dynamics simulations. VBAPT4-OmpW is quite unable to attain its structural local minima after 500 ns. But VBAPT17-OmpW is showing great stability and is not destructive even after 500 ns. RMSF, DSSP, PCA, and Essential Dynamics all provided additional confirmation. Current findings, combined with the fabrication of biosensor devices, could pave the way for an innovative pathogen detection platform with high sensitivity, along with an effective and low-impact curative strategy for corresponding diseases.Communicated by Ramaswamy H. Sarma

Activatable G-quadruplex based catalases for signal transduction in biosensing.

Discovery of oxidative catalysis with G-quadruplex•hemin constructs prompted a range of exciting developments in the field of biosensor design. Thus, G-quadruplex based DNAzymes with peroxidase activity found a niche as signal transduction modules in a wide range of analytical applications. The ability of nucleic acid scaffolds to recognise a variety of practically meaningful markers and to translate the recognition events into conformational changes powers numerous sensor design possibilities. In this work, we establish a catalase activity of G-quadruplex•hemin scaffolds. Catalase activated hydrogen peroxide decomposition generates molecular oxygen that forms bubbles. Observation of bubbles is a truly equipment free signal readout platform that is highly desirable in limited resources or do-it-yourself environments. We take a preliminary insight into a G-quadruplex structure-folding topology-catalase activity correlation and establish efficient operating conditions. Further, we demonstrate the platform's potential as a signal transduction modality for reporting on biomolecular recognition using an oligonucleotide as a proof-of-concept target. Ultimately, activatable catalases based on G-quadruplex•hemin scaffolds promise to become valuable contributors towards accessible molecular diagnostics applications.

Long-time-step molecular dynamics can retard simulation of protein-ligand recognition process

Molecular dynamics (MD) simulation of biologically relevant processes at realistic time scale and atomistic precision is generally limited by prohibitively large computational cost, due to its restriction of using an ultrashort integration time step (1–2 fs). A popular numerical recipe to reduce the associated computational burden is adopting schemes that would allow relatively longer-time-step for MD propagation. Here, we explore the perceived potential of one of the most frequently used long-time-step protocols, namely the hydrogen mass repartitioning (HMR) approach, in alleviating the computational overhead associated with simulation of the kinetic process of protein-ligand recognition events. By repartitioning the mass of heavier atoms to their linked hydrogen atoms, HMR leverages around twofold longer time step than regular simulation, holding promise of significant performance boost. However, our probe into direct simulation of the protein-ligand recognition event, one of the computationally most challenging processes, shows that long-time-step HMR MD simulations do not necessarily translate to a computationally affordable solution. Our investigations spanning cumulative 176 μs in three independent proteins (T4 lysozyme, sensor domain of MopR, and galectin-3) show that long-time-step HMR-based MD simulations can catch the ligand in its act of recognizing the native cavity. But, as a major caveat, the ligand is found to require significantly longer time to identify buried native protein cavity in an HMR MD simulation than regular simulation, thereby defeating the purpose of its usage for performance upgrade. A molecular analysis shows that the longer time required by a ligand to recognize the protein in HMR is rooted in faster diffusion of the ligand, which reduces the survival probability of decisive on-pathway metastable intermediates, thereby slowing down the eventual recognition process at the native cavity. Together, the investigation stresses careful assessment of pitfalls of long-time-step algorithms before attempting to utilize them for higher performance for biomolecular recognition simulations.