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Chemphyschem : a European Journal of... Jun 2024Scanning probe microscopy (SPM), in particular at low temperature (LT) under ultra-high vacuum (UHV) conditions, offers the possibility of real-space imaging with...
Scanning probe microscopy (SPM), in particular at low temperature (LT) under ultra-high vacuum (UHV) conditions, offers the possibility of real-space imaging with resolution reaching the atomic level. However, its potential for the analysis of complex biological molecules has been hampered by requirements imposed by sample preparation. Transferring molecules onto surfaces in UHV is typically accomplished by thermal sublimation in vacuum. This approach however is limited by the thermal stability of the molecules, i.e. not possible for biological molecules with low vapour pressure. Bypassing this limitation, electrospray ionisation offers an alternative method to transfer molecules from solution to the gas-phase as intact molecular ions. In soft-landing electrospray ion beam deposition (ESIBD), these molecular ions are subsequently mass-selected and gently landed on surfaces which permits large and thermally fragile molecules to be analyzed by LT-UHV SPM. In this concept, we discuss how ESIBD+SPM prepares samples of complex biological molecules at a surface, offering controls of the molecular structural integrity, three-dimensional shape, and purity. These achievements unlock the analytical potential of SPM which is showcased by imaging proteins, peptides, DNA, glycans, and conjugates of these molecules, revealing details of their connectivity, conformation, and interaction that could not be accessed by any other technique.
PubMed: 38945838
DOI: 10.1002/cphc.202400419 -
Angewandte Chemie (International Ed. in... Jun 2024Visualizing lithium (Li) ions and understanding Li plating/stripping processes as well as evolution of solid electrolyte interface (SEI) are critical for optimizing...
Visualizing lithium (Li) ions and understanding Li plating/stripping processes as well as evolution of solid electrolyte interface (SEI) are critical for optimizing all-solid-state Li metal batteries (ASSLMB). However, the buried solid-solid interfaces present a challenge for detection which preclude the employment of multiple analysis techniques. Herein, by employing complementary in situ characterizations, morphological/chemical evolution, Li plating/stripping dynamics and SEI dynamics were efficiently decoupled and Li ion behavior at interface between different solid-state electrolytes (SSE) was successfully detected. The innovative combining experiments of in situ atomic force microscopy and in situ X-ray photoelectron spectroscopy on Li metal anode revealed interfacial morphological/chemical evolution and decoupled Li plating/stripping process from SEI evolution. Though Li plating speed in Li10GeP2S12 (LGPS) was higher than Li3PS4 (LPS), speed of SSE decomposition was similar and ~85% interfacial SSE turned into SEI during plating and remained unchanged in stripping. To leverage strengths of different SSEs, an LPS-LGPS-LPS sandwich electrolyte was developed, demonstrating enhanced ionic conductivity and improved interfacial stability with less SSE decomposition (25%). Using in situ Kelvin Probe Force Microscopy, Li-ion behavior at interface between different SSEs was effectively visualized, uncovering distribution of Li ions at LGPS|LPS interface under different potentials.
PubMed: 38945832
DOI: 10.1002/anie.202409435 -
Experimental Eye Research Jun 2024Ocular complications of diabetes mellitus (DM) are the leading cause of vision loss. Ocular inflammation often occurs in the early stage of DM; however, there are no...
Ocular complications of diabetes mellitus (DM) are the leading cause of vision loss. Ocular inflammation often occurs in the early stage of DM; however, there are no proven quantitative methods to evaluate the inflammatory status of eyes in DM. The 18 kDa translocator protein (TSPO) is an evolutionarily conserved cholesterol binding protein localized in the outer mitochondrial membrane. It is a biomarker of activated microglia/macrophages; however, its role in ocular inflammation is unclear. In this study, fluorine-18-DPA-714 ([F]-DPA-714) was evaluated as a specific TSPO probe by cell uptake, cell binding assays and micro positron emission tomography (microPET) imaging in both in vitro and in vivo models. Primary microglia/macrophages (PMs) extracted from the cornea, retina, choroid or sclera of neonatal rats with or without high glucose (50 mM) treatment were used as the in vitro model. Sprague-Dawley (SD) rats that received an intraperitoneal administration of streptozotocin (STZ, 60 mg/kg once) were used as the in vivo model. Increased cell uptake and high binding affinity of [F]-DPA-714 were observed in primary PMs under hyperglycemic stress. These findings were consistent with cellular morphological changes, cell activation, and TSPO up-regulation. [F]-DPA-714 PET imaging and biodistribution in the eyes of DM rats revealed that inflammation initiates in microglia/macrophages in the early stages (3 weeks and 6 weeks), corresponding with up-regulated TSPO levels. Thus, [F]-DPA-714 microPET imaging may be an effective approach for the early evaluation of ocular inflammation in DM.
PubMed: 38945519
DOI: 10.1016/j.exer.2024.109986 -
Biochemical Pharmacology Jun 2024The peptide hormone relaxin plays a critical role in tissue remodeling in a variety of tissues through activation of its cognate receptor, RXFP1. Relaxin's ability to...
The peptide hormone relaxin plays a critical role in tissue remodeling in a variety of tissues through activation of its cognate receptor, RXFP1. Relaxin's ability to modify extracellular matrices has provided a strong rationale for treating fibrosis in a variety of tissues. Treatment with recombinant relaxin peptides in clinical studies of heart failure has not yet proven useful, likely due to the short half-life of infused peptide. To circumvent this particular pharmacokinetic pitfall we have used a Protein-in-Protein (PiP) antibody technology described previously, to insert a single-chain human relaxin construct into the complementarity-determining region (CDR) of an immunoglobulin G (IgG) backbone, creating a relaxin molecule with a half-life of ∼4-5 days in mice. Relaxin-PiP biologics displaced Europium-labeled human relaxin in RXFP1-expressing cells and demonstrated full agonist activity on both human and mouse RXFP1 receptors. Relaxin-PiPs did not show signal transduction bias, as they activated cAMP in THP-1 cells, and cGMP and pERK signaling in primary human cardiac fibroblasts. In an induced carbon tetrachloride mouse model of liver fibrosis one relaxin-PiP, R2-PiP, caused reduction of liver lesions, ameliorated collagen accumulation in the liver with the corresponding reduction of Collagen1a1 gene expression, and increased cell proliferation in hepatic parenchyma. These relaxin biologics represent a novel approach to the design of a long-acting RXFP1 agonist to probe the clinical utility of relaxin/RXFP1 signaling to treat a variety of human fibrotic diseases.
PubMed: 38945278
DOI: 10.1016/j.bcp.2024.116401 -
Bioorganic & Medicinal Chemistry Jun 2024The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct...
The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5'-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2-8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2-8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4'-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.
PubMed: 38943807
DOI: 10.1016/j.bmc.2024.117815 -
Talanta Jun 2024A novel coumarin-based fluorescent sensor CHE, incorporating 2-hydrazinylbenzothiazole and coumarin aldehyde, has been developed that demonstrated a preferential...
A novel coumarin-based fluorescent sensor CHE, incorporating 2-hydrazinylbenzothiazole and coumarin aldehyde, has been developed that demonstrated a preferential detection of Hg and Ag in presence of interferences. Compared to previously prevalent intensity-based fluorescent probes, CHE exhibited a ratiometric fluorescence response to Hg and Ag, and further accurately differentiated Hg and Ag using the differential extractive ability of EDTA when interacting with ion-CHE complexes. Sensing mechanism was investigated and elucidated. The chemosensor CHE was successfully applied to detect Hg and Ag in six distinct samples with satisfactory results. Additionally, combinatorial logic circuits were constructed utilizing three distinct logic gates (NOT, OR, and INH) based on the sensor's differential output signals in response to Hg/Ag and other cations. Interestingly, utilizing the reversible and reproducible switching behavior of the EDTA interaction with Hg, a conceptual 'Write-Read-Erase-Read' memory function with multi-write capability was proposed, offering a novel perspective for molecular-based memory systems.
PubMed: 38943765
DOI: 10.1016/j.talanta.2024.126478 -
STAR Protocols Jun 2024Super-resolution imaging provides unprecedented visualization of sub-cellular structures, but the two main techniques used, single-molecule localization microscopy...
Super-resolution imaging provides unprecedented visualization of sub-cellular structures, but the two main techniques used, single-molecule localization microscopy (SMLM) and stimulated emission depletion (STED), are not easily reconciled. We present a protocol to super-impose nanoscale protein distribution reconstructed with SMLM to sub-cellular morphology obtained in STED. We describe steps for tracking cells on etched coverslips and registering images from two different microscopes with 30-nm accuracy. In this protocol, synaptic proteins are mapped in the dendritic spines of primary neurons. For complete details on the use and execution of this protocol, please refer to Inavalli et al..
PubMed: 38943646
DOI: 10.1016/j.xpro.2024.103160 -
STAR Protocols Jun 2024Tumor acidosis is one of the hallmarks indicating the initiation and progression of various cancers. Here, we present a protocol for preparing a hyperpolarized (HP)...
Tumor acidosis is one of the hallmarks indicating the initiation and progression of various cancers. Here, we present a protocol for preparing a hyperpolarized (HP) C-bicarbonate tissue pH MRI imaging contrast agent to detect aggressive tumors. We describe the steps for the formulation and polarization of a precursor molecule C-glycerol carbonate (C-GLC), the post-dissolution reaction, and converting HP C-GLC to an injectable HP C-bicarbonate solution. We then detail procedures for MRI data acquisition to generate tumor pH maps for assessing tumor aggressiveness. For complete details on the use and execution of this protocol, please refer to Mu et al..
PubMed: 38943645
DOI: 10.1016/j.xpro.2024.103091 -
Analytical Chemistry Jun 2024Detecting harmful pathogens in food is not only a crucial aspect of food quality management but also an effective way to ensure public health. In this paper, a complete...
Detecting harmful pathogens in food is not only a crucial aspect of food quality management but also an effective way to ensure public health. In this paper, a complete nuclear magnetic resonance biosensor based on a novel gadolinium (Gd)-targeting molecular probe was developed for the detection of Salmonella in milk. First, streptavidin was conjugated to the activated macromolecular polyaspartic acid (PASP) via an amide reaction to generate SA-PASP. Subsequently, the strong chelating and adsorption properties of PASP toward the lanthanide metal gadolinium ions were exploited to generate the magnetic complex (SA-PASP-Gd). Finally, the magnetic complex was linked to biotinylated antibodies to obtain the bioprobe and achieve the capture of Salmonella. Under optimal experimental conditions, the sensor we have constructed can achieve a rapid detection of Salmonella within 1.5 h, with a detection limit of 7.1 × 10 cfu mL.
PubMed: 38943569
DOI: 10.1021/acs.analchem.4c01265 -
Nature Protocols Jun 2024Spatial epigenetic mapping of tissues enables the study of gene regulation programs and cellular functions with the dependency on their local tissue environment. Here we... (Review)
Review
Spatial epigenetic mapping of tissues enables the study of gene regulation programs and cellular functions with the dependency on their local tissue environment. Here we outline a complete procedure for two spatial epigenomic profiling methods: spatially resolved genome-wide profiling of histone modifications using in situ cleavage under targets and tagmentation (CUT&Tag) chemistry (spatial-CUT&Tag) and transposase-accessible chromatin sequencing (spatial-ATAC-sequencing) for chromatin accessibility. Both assays utilize in-tissue Tn5 transposition to recognize genomic DNA loci followed by microfluidic deterministic barcoding to incorporate spatial address codes. Furthermore, these two methods do not necessitate prior knowledge of the transcription or epigenetic markers for a given tissue or cell type but permit genome-wide unbiased profiling pixel-by-pixel at the 10 μm pixel size level and single-base resolution. To support the widespread adaptation of these methods, details are provided in five general steps: (1) sample preparation; (2) Tn5 transposition in spatial-ATAC-sequencing or antibody-controlled pA-Tn5 tagmentation in CUT&Tag; (3) library preparation; (4) next-generation sequencing; and (5) data analysis using our customed pipelines available at: https://github.com/dyxmvp/Spatial_ATAC-seq and https://github.com/dyxmvp/spatial-CUT-Tag . The whole procedure can be completed on four samples in 2-3 days. Familiarity with basic molecular biology and bioinformatics skills with access to a high-performance computing environment are required. A rudimentary understanding of pathology and specimen sectioning, as well as deterministic barcoding in tissue-specific skills (e.g., design of a multiparameter barcode panel and creation of microfluidic devices), are also advantageous. In this protocol, we mainly focus on spatial profiling of tissue region-specific epigenetic landscapes in mouse embryos and mouse brains using spatial-ATAC-sequencing and spatial-CUT&Tag, but these methods can be used for other species with no need for species-specific probe design.
PubMed: 38943021
DOI: 10.1038/s41596-024-01013-y