Probes In Experiments Research Articles

A platform for mapping reactive cysteines within the immunopeptidome

The major histocompatibility complex class I antigen presentation pathways play pivotal roles in orchestrating immune responses. Recent studies have begun to explore the therapeutic potential of cysteines within the immunopeptidome, such as the use of covalent ligands to generate haptenated peptide neoepitopes for immunotherapy. In this work, we report a platform for mapping reactive cysteines on MHC-I-bound peptide antigens. We develop cell-impermeable sulfonated maleimide probes capable of capturing reactive cysteines on these antigens. Using these probes in chemoproteomic experiments, we discover that cysteines on MHC-I-bound antigens exhibit various degrees of reactivity. Moreover, interferon-gamma stimulation enhances the reactivity of cysteines at position 8 of 9-mer MHC-I-bound antigens. Finally, we demonstrate that targeting reactive cysteines on MHC-I-bound antigens with a maleimide-conjugated Fc-binding cyclic peptide contributes to the induction of antibody-dependent cellular phagocytosis.

Signatures of coherent vibrational dynamics in ethylene carbonate.

Despite having practical applications in battery technology and serving as a model system for Fermi resonance coupling, ethylene carbonate (EC) receives little direct attention as a vibrational probe in nonlinear vibrational spectroscopy experiments. EC contains a Fermi resonance that is well-characterized in the linear spectrum, and the environmental sensitivity of its Fermi resonance peaks could make it a good molecular probe for two-dimensional infrared spectroscopy (2DIR) experiments. As a model system, we investigate the linear and 2DIR vibrational spectrum of the carbonyl stretching region of ethylene carbonate in tetrahydrofuran. The 2DIR spectrum reveals peak dynamics that evolve coherently. We characterize these dynamics in the context of Redfield theory and find evidence that EC dynamics proceed through coherent pathways, including singular coherence transfer pathways that have not been widely observed in other studies. We find that coherent contributions play a significant role in the observed dynamics of cross-peaks in the 2DIR spectrum, which must be accounted for to extract accurate measurements of early waiting time dynamics.

Analysis of The Flow-Field Distorsions Induced by Pneumatic Pressure Probes In Test Bench Experiments On Centrifugal Compressors: Are We Testing Right?

Abstract Experimental testing at the test rig still plays a key role in the development of new centrifugal compressor designs for industrial applications. Although a wide set of different probes is used as per common practice, the overall impact of the probes? dimensions on the stage performance and rangeability is often assumed to be negligible. In this study, we aimed at quantifying the intrusiveness effects of pneumatic pressure probes on the flow-field inside a vaned diffuser using a model stage of an industrial centrifugal compressor, for which different experimental setups were tested at the University of Florence test rig. Performance and rangeability were initially assessed using a conventional set of pneumatic probes inserted in the flow path. Then, comparative analyses were realized through dedicated tests by either removing all probes at the diffuser inlet or introducing only a single pressure probe in the upstream section and varying its angular position in the measuring section. Experiments revealed an impact of upstream probes much higher than expected, with a decrease in efficiency and a substantial reduction of stall margin. Moving from this experimental evidence, accurate 3D CFD simulations of the entire stage were carried out including the actual geometry of the probes for two probes configurations and three operating points. Simulations highlighted the effect probes? wakes on downstream vanes and the induced blockage effect, which both appear in line with experimental evidence. As a handout, a recommendation on the optimal probe positioning is suggested.

Evidence for a transfer-to-trap mechanism of fluorophore concentration quenching in lipid bilayers

It is important to understand the behaviors of fluorescent molecules because, firstly, they are often utilized as probes in biophysical experiments and, secondly, they are crucial cofactors in biological processes such as photosynthesis. A phenomenon called “fluorescence quenching” occurs when fluorophores are present at high concentrations, but the mechanisms for quenching are debated. Here, we used a technique called “in-membrane electrophoresis” to generate concentration gradients of fluorophores within a supported lipid bilayer, across which quenching was expected to occur. Fluorescence lifetime imaging microscopy (FLIM) provides images where the fluorescence intensity in each pixel is correlated to fluorescence lifetime: the intensity provides information about the location and concentration of fluorophores and the lifetime reveals the occurrence of energy-dissipative processes. FLIM was used to compare the quenching behavior of three commonly used fluorophores: Texas Red (TR), nitrobenzoaxadiazole (NBD), and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). FLIM images provided evidence of quenching in regions where the fluorophores accumulated, but the degree of quenching varied between the different fluorophores. The relationship between quenching and concentration was quantified and the “critical radius for trap formation,” representing the relative quenching strength, was calculated as 2.70, 2.02, and 1.14 nm, for BODIPY, TR, and NBD, respectively. The experimental data support the theory that quenching takes place via a “transfer-to-trap” mechanism which proposes, firstly, that excitation energy is transferred between fluorophores and may reach a “trap site,” resulting in immediate energy dissipation, and, secondly, that trap sites are formed in a concentration-dependent manner. Some previous work suggested that quenching occurs only when fluorophores aggregate, or form long-lived dimers, but our data and this theory argue that traps may be “statistical pairs” of fluorophores that exist only transiently. Our findings should inspire future work to assess whether these traps can be charge-transfer states, excited-state dimers, or something else.

Effect of photoexcitation on high-harmonic generation in semiconductors

Solid-state high-harmonic generation is intrinsically sensitive to band structure, carrier population, and carrier scattering. As such, solid-state high-harmonic generation is increasingly used as a probe for femtosecond time-resolved pump-probe experiments. So far, most experimental pump-probe studies have reported photoexcitation-induced amplitude suppression of high-harmonic generation in solid-state media, yet the origins of this phenomenon remain elusive. Through simulations based on the semiconductor Bloch equations, we identify the dephasing of the coherent carrier population as the primary mechanism driving this suppression. Furthermore, we find band gap renormalization to be a source for phase shifts of high harmonics. We introduce an analytical model, based on a semi-classical action, that supports our numerical outcomes.

Observation of an acoustic topological Euler insulator with meronic waves

Topological band theory has conventionally been concerned with the topology of bands around a single gap. Only recently non-Abelian topologies that thrive on involving multiple gaps were studied, unveiling a new horizon in topological physics beyond the conventional paradigm. Here, we report on the first experimental realization of a topological Euler insulator phase with unique meronic characterization in an acoustic metamaterial. We demonstrate that this topological phase has several nontrivial features: First, the system cannot be described by conventional topological band theory, but has a nontrivial Euler class that captures the unconventional geometry of the Bloch bands in the Brillouin zone. Second, we uncover in theory and probe in experiments a meronic configuration of the bulk Bloch states for the first time. Third, using a detailed symmetry analysis, we show that the topological Euler insulator evolves from a non-Abelian topological semimetal phase via. the annihilation of Dirac points in pairs in one of the band gaps. With these nontrivial properties, we establish concretely an unconventional bulk-edge correspondence which is confirmed by directly measuring the edge states via. pump-probe techniques. Our work thus unveils a nontrivial topological Euler insulator phase with a unique meronic pattern and paves the way as a platform for non-Abelian topological phenomena.

A highly sensitive biotin-based probe for small RNA northern blot and its application in dissecting miRNA function in pepper.

Small RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope- and biotin- (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope-based probe is limited by strict environmental regulation and availability in many places in the world while biotin-based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase-based method for incorporation of a cluster of 33 biotin-labeled C in small RNA probe (T4BC33 probe). T4BC33 probe reaches similar sensitivity as 32P-labeled probe in dot blot and small RNA northern blot experiments. Addition of locked nucleic acids in T4BC33 probe further enhanced its sensitivity in detecting low-abundance miRNAs. With newly developed northern blot method, expression of miR6027 and miR6149 family members was validated. Northern blot analysis also confirmed the successful application of virus-based miRNA silencing in pepper, knocking down accumulation of Can-miR6027a and Can-miR6149L. Importantly, further analysis showed that knocking-down Can-miR6027a led to upregulation of a nucleotide binding-leucine rich repeat domain protein coding gene (CaRLb1) and increased immunity against Phytophthora capsici in pepper leaves. Our study provided a highly sensitive and convenient method for sRNA research and identified new targets for genetic improvement of pepper immunity against P. capsici.

Probing the reduction of adhesion forces between biofilms and anti-biofouling filtration membrane surfaces using FluidFM technology

Biofouling is a persistent problem in many sectors (healthcare, medicine, marine, and membrane filtration processes). To control the biofouling of surfaces, it is essential to overcome or reduce the adhesion forces between biofilms and surfaces. To access and understand the molecular basis of these interactions, atomic force microscopy (AFM) is a well-suited technology that can measure adhesion forces at the piconewton level. However, AFM-based existing methods only probe interactions between individual cells and surfaces, which is not representative of realistic conditions given that bacteria mainly exist in biofilms. We develop here an original method using FluidFM, a combination of AFM and microfluidics, to probe the adhesion forces between biofilms and filtration membranes modified with an anti-biofouling agent, vanillin. This strategy involves i) growing bacterial biofilms on micrometer-sized polystyrene beads, ii) aspirating these biofilm beads at the aperture of microfluidic cantilevers and iii) using them as probes in force spectroscopy experiments. The results obtained first showed that COOH-functionalized polystyrene beads are more suitable for bacterial growth, and that biofilms obtained after 3 h of incubation could be used with FluidFM. Then, biofilm-scale force spectroscopy experiments showed a significant decrease in adhesion forces, adhesion work, and adhesion events after membrane modification, demonstrating the potential of vanillin-coated membranes to reduce biofouling. In addition, the comparison between results at the individual cell and biofilm scales highlighted the complexity of polymeric matrix unbinding and/or unfolding in the biofilm, showing that individual cells behave differently from biofilms. Overall, this method could have implications in the fields of materials science, chemical engineering, health, and the environment.

Analysis of non-canonical three- and four-way DNA junctions.

The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds-a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively-were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it uses four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (Kd and EC50) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures.

Probing Lattice Oxygen Oxidation in Perovskite Electrocatalysts By Resonant Inelastic X-Ray Scattering

During water electrolysis, the hydrogen evolution reaction that generates hydrogen gas is unavoidably accompanied by the anodic reaction that generates oxygen via the oxygen evolution reaction (OER). However, under OER conditions, many electrocatalysts undergo structural degradation and can become amorphous. Lattice oxygen oxidation was proposed as one of the possible causes for amorphization of perovskite oxides. However, because lattice oxygen oxidation is notoriously challenging to probe in experiments, its unambiguous detection in oxide electrocatalysts has been elusive so far.Here, I will show how oxygen oxidation can be detected in single-crystalline (model) oxide electrocatalysts using high-resolution resonant inelastic X-ray scattering (RIXS). Specifically, I will present our case study of perovskite materials where the emergence of oxidized oxygen depends on the transition metal and the applied potential. An insight into the chemical environment of the oxidized oxygen and how it can be accommodated in the perovskite lattice will be provided based on the results of DFT and molecular dynamics. Finally, I will discuss how lattice oxygen oxidation is related to the degradation of oxide electrocatalysts during OER.

Reversible Redox Probes to Determine the Band Edge Locations for Nano-TiO2

Semiconductor (photo-)electrocatalysts show immense promise in answering the difficulties faced in CO2 electrolysis in relation to the reduction product selectivity. We thus see materials like ZnO[1], Cu2-xSe[2], MoS2 [3], TiO2 [4] and their numerous composites being frequently investigated for their catalytic properties in CO2 (photo-)electroreduction. These catalysts are fabricated via varying techniques and can assume very different thicknesses and morphologies. Although the nano-semiconductors may behave differently, often only the bulk properties are considered when proposing possible reaction pathways. Determining band edge locations of semiconductors in solutions is one such case, where Mott-Schottky (MS) measurements are a widely accepted strategy. The measurement of the potential dependence of the capacitance of the semiconductor space-charge layer under depletion indeed forms an elegant method to determine the flat-band potential and hence the band edge positions for bulk semiconductors in solution. However, the MS method cannot be used for nano-structured semiconductor electrodes in the form of thin films, nanorods or nanoparticles coated on conducting substrates, as their nano-dimensions are typically much smaller than that of a semiconductor depletion layer.In this work, we explored an alternate strategy to determine band edge positions of nano-semiconductors using reversible redox probes. A model thin film system comprised of 30nm anatase TiO2 is used to demonstrate the feasibility of the strategy in aqueous electrolyte solutions. Fe, Ru and Cr based reversible redox species were used as electrochemical probes in cyclic voltammetry experiments with varying solution pH. Based on the presence/absence of the reversible behavior of these probes, we can deduce the TiO2 band edge locations. The procedure also allows us to draw parallels between semiconductor-metal and semiconductor-solution interfaces to gain new insights into the CO2 reduction mechanism on such semiconductor electrodes. Figure 1

Fe-Catalyzed Hydroallylation of Unactivated Alkenes with Vinyl Cyclopropanes.

Catalytic, reductive C-C bond formation between alkenes and vinyl cyclopropane (VCP) through hydrogen atom transfer (MHAT) is developed. Despite VCP's use as probes in radical-clock experiments, translation of this manifold into synthetic methods for accessing elusive C-C bonds remains largely unexplored. This work represents the first foray into this front where the high chemoselectivity of MHAT for alkene over VCP was pivotal for realizing the strategy. This method exhibits a broad scope, high functional group tolerance, and useful applications.

Simultaneous Force and Darkfield Measurements Reveal Solvent-Dependent Axial Control of Optically Trapped Gold Nanoparticles

Single molecule force spectroscopy using optical tweezers (OT) has enabled nanoresolved measurements of dynamic biological processes but not of synthetic molecular mechanisms. Standard OT probes made from silica or polystyrene are incompatible with trapping in organic solvents for solution phase chemistry or with force-detected absorption spectroscopies. Here, we demonstrate optical trapping of gold nanoparticles in both aqueous and organic conditions using a custom OT and darkfield instrument which can uniquely measure force and scattering spectra of single gold nanoparticles (Au NPs) simultaneously. Our work reveals that standard models of trapping developed for aqueous conditions cannot account for the trends observed in different media here. We determine that higher pushing forces mitigate the increase in trapping force in higher index organic solvents and lead to axial displacement of the particle which can be controlled through trap intensity. This work develops a new model framework incorporating axial forces for understanding nanoparticle dynamics in an optical trap. These results establish the combined darkfield OT with Au NPs as an effective OT probe for single molecule and single particle spectroscopy experiments, with three-dimensional nanoscale control over NP location.

Genomic relationships of the polyploid rhizoma peanut (Arachis glabrata Benth.) inferred by genomic in situ hybridization (GISH)

The rhizoma peanut (Arachis glabrata Benth., section Rhizomatosae) is a tetraploid perennial legume. Although several A. glabrata cultivars have been developed as forage and ornamental turf, the origin and genomic constitution of this species are still unknown. In this study, we evaluated the affinity between the genomes of A. glabrata and the probable diploid donors of the sections Rhizomatosae, Arachis, Erectoides and Procumbentes by genomic in situ hybridization (GISH). Single GISH analyses detected that species of the sections Erectoides (E2 subgenome) and Procumbentes (E3 subgenome) were the diploid species with the highest degree of genomic affinity with A. glabrata. Based on single GISH experiments and DNA sequence similarity, three species -A. duranensis, A. paraguariensis subsp. capibarensis, and A. rigonii-, which showed the most uniform and brightest hybridization patterns and lowest genetic distance, were selected as probes for double GISH experiments. Double GISH experiments showed that A. glabrata is constituted by four identical or very similar chromosome complements. In these assays, A. paraguariensis subsp. capibarensis showed the highest brightness onto A. glabrata chromosomes. Thus, our results support the autopolyploid origin of A. glabrata and show that the species with E2 subgenome are the most probable ancestors of this polyploid legume forage.

A monolithic microfluidic probe for ambient mass spectrometry imaging of biological tissues.

Ambient mass spectrometry imaging (MSI) is a powerful technique that allows for the simultaneous mapping of hundreds of molecules in biological samples under atmospheric conditions, requiring minimal sample preparation. We have developed nanospray desorption electrospray ionization (nano-DESI), a liquid extraction-based ambient ionization technique, which has proven to be sensitive and capable of achieving high spatial resolution. We have previously described an integrated microfluidic probe, which simplifies the nano-DESI setup, but is quite difficult to fabricate. Herein, we introduce a facile and scalable strategy for fabricating microfluidic devices for nano-DESI MSI applications. Our approach involves the use of selective laser-assisted etching (SLE) of fused silica to create a monolithic microfluidic probe (SLE-MFP). Unlike the traditional photolithography-based fabrication, SLE eliminates the need for the wafer bonding process and allows for automated, scalable fabrication of the probe. The chamfered design of the sampling port and ESI emitter significantly reduces the amount of polishing required to fine-tune the probe thereby streamlining and simplifying the fabrication process. We have also examined the performance of a V-shaped probe, in which only the sampling port is fabricated using SLE technology. The V-shaped design of the probe is easy to fabricate and provides an opportunity to independently optimize the size and shape of the electrospray emitter. We have evaluated the performance of SLE-MFP by imaging mouse tissue sections. Our results demonstrate that SLE technology enables the fabrication of robust monolithic microfluidic probes for MSI experiments. This development expands the capabilities of nano-DESI MSI and makes the technique more accessible to the broader scientific community.

Biomedical applications of lanthanide complexes

Lanthanide complexes are definitely bioavailable and have a huge number of implementations in medical analysis and in diagnosis and treatment. Due to their chemical and magnetic properties their use in cancer treatment and therapy increases. Luminescent compounds used as light-emitting materials for biosensors, molecular imaging has gotten profound interest attention in biomedical probe. Gadolinium(III) continues to be the dominant starting material for contrast agent design used in MRI response as a function of a specific biomarker. Contrast agents of lanthanides in MRI show a capacity of effective instrument for tumor diagnosis. Lu (3), Eu (3) based complexes operates in chemotherapy act as therapeutic agent. Dysprosium based drug used in treatment of Rheumatoid arthritis. Gd (3), Sm (3), Nd (3) complexes focuses on detailed image of organs, which helps in detecting diseases in body. Lanthanides, which based on physical and spectral, have wide range of uses including fluorescent probes in biological experiments. This review paper goal is to highlight research on lanthanide compounds with biological and biomedical uses.

Design and Evaluation of a Cyclobutane Diazirine Alkyne Tag for Photoaffinity Labeling in Cells.

Alkyl diazirines are frequently used in photoaffinity labeling to map small molecule-protein interactions in target identification studies. However, the alkyl diazirines can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a reactive alkyl diazo intermediate. Here, we explore the use of ring strain to alter these reactivity preferences and report the development of a cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity, termed PALBOX. We show that PALBOX possesses differential reactivity profiles as compared to other diazirine tags in vitro and is readily incorporated into small molecules to profile their binding interactions in cells. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label the known protein targets in cells with reduced labeling of known alkyl diazirine off-targets. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule-protein binding sites. Thus, PALBOX is a versatile diazirine-based photoaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule-protein interactions.

Multicolor single-analyzer high-energy-resolution XES spectrometer for simultaneous examination of different elements.

The present work demonstrates the performance of a von Hámos high-energy-resolution X-ray spectrometer based on a non-conventional conical Si single-crystal analyzer. The analyzer is tested with different primary and secondary X-ray sources as well as a hard X-ray sensitive CCD camera. The spectrometer setup is also characterized with ray-tracing simulations. Both experimental and simulated results affirm that the conical spectrometer can efficiently detect and resolve the two pairs of two elements (Ni and Cu) Kα X-ray emission spectroscopy (XES) peaks simultaneously, requiring a less than 2 cm-wide array on a single position-sensitive detector. The possible applications of this simple yet broad-energy-spectrum crystal spectrometer range from quickly adapting it as another probe for complex experiments at synchrotron beamlines to analyzing X-ray emission from plasma generated by ultrashort laser pulses at modern laser facilities.

Towards a Comprehensive Optical Connectome at Single Synapse Resolution via Expansion Microscopy.

Mapping and determining the molecular identity of individual synapses is a crucial step towards the comprehensive reconstruction of neuronal circuits. Throughout the history of neuroscience, microscopy has been a key technology for mapping brain circuits. However, subdiffraction size and high density of synapses in brain tissue make this process extremely challenging. Electron microscopy (EM), with its nanoscale resolution, offers one approach to this challenge yet comes with many practical limitations, and to date has only been used in very small samples such as C. elegans, tadpole larvae, fruit fly brain, or very small pieces of mammalian brain tissue. Moreover, EM datasets require tedious data tracing. Light microscopy in combination with tissue expansion via physical magnification—known as expansion microscopy (ExM)—offers an alternative approach to this problem. ExM enables nanoscale imaging of large biological samples, which in combination with multicolor neuronal and synaptic labeling offers the unprecedented capability to trace and map entire neuronal circuits in fully automated mode. Recent advances in new methods for synaptic staining as well as new types of optical molecular probes with superior stability, specificity, and brightness provide new modalities for studying brain circuits. Here we review advanced methods and molecular probes for fluorescence staining of the synapses in the brain that are compatible with currently available expansion microscopy techniques. In particular, we will describe genetically encoded probes for synaptic labeling in mice, zebrafish, Drosophila fruit flies, and C. elegans, which enable the visualization of post-synaptic scaffolds and receptors, presynaptic terminals and vesicles, and even a snapshot of the synaptic activity itself. We will address current methods for applying these probes in ExM experiments, as well as appropriate vectors for the delivery of these molecular constructs. In addition, we offer experimental considerations and limitations for using each of these tools as well as our perspective on emerging tools.

Purification of Labeled Oligonucleotides by Precipitation with Ethanol.

If labeled oligonucleotides are to be used only as probes in hybridization experiments, complete removal of unincorporated label is generally not necessary. However, to reduce background to a minimum, the bulk of the unincorporated label should be separated from the labeled oligonucleotide. Most of the residual unincorporated precursors can be removed from the preparation by differential precipitation with ethanol, as described in this protocol, if the oligonucleotide is >18 nt in length.