Selective Labeling Research Articles

Crisis of Trust in International Institutions as a Factor of Russia’s Increasing Influence

The article examines the causes of the crisis of trust in international institutions: the inability to stop crises and wars in the modern world, dependence on the United States and the implementation of U.S. policy as a collective policy of all participating countries of international institutions, the unfair distribution of opportunities for countries to influence collective decisions, working with illegitimate representatives of different countries, selective negative labeling of individual countries and ignoring the violation factor the rights and freedoms of citizens in these countries. The author considers the crisis of trust in international institutions as an opportunity for Russia to strengthen and expand its influence on the formation of the modern world order by creating and increasing the power of existing institutions, primarily BRICS.

Nucleic Acid Covalent Tags.

The selective and site-specific chemical labeling of proteins has emerged as a pivotal research area in chemical biology and cell biology. An effective protein labeling typically meets several criteria, including high specificity, rapid and robust conjugation under physiological conditions, operation at low concentrations with biocompatibility, and minimal perturbation of the protein function and activity. The conjugation of nucleic acids with proteins has garnered significant attention recently due to the rapid advancements in nucleic acid probe technologies, leveraging the programmable nature of nucleic acids alongside the multifaceted functionalities of proteins. It helps to convert protein-specific information into nucleic acid signals, facilitating upstream versatile recognition and downstream signal amplification for the target protein. This review critically evaluates the recent progress in nucleic acid-based protein labeling methodologies, with a specific focus on covalent labeling using aptamer tags, protein fusion tags or the technique of metabolic oligosaccharide engineering. The tags establish covalent linkages with target proteins through various modalities such as small molecules or metabolic glycan engineering. The insights presented in the review highlight promising avenues for the development of highly specific and versatile protein labeling techniques, which is essential for the improvement of protein-targeted detection and imaging across diverse biological contexts.

Sydnthiones are versatile bioorthogonal hydrogen sulfide donors

Hydrogen sulfide (H2S) is an important endogenous gasotransmitter, but the bioorthogonal reaction triggered H2S donors are still rare. Here we show one type of bioorthogonal H2S donors, sydnthiones (1,2,3-oxadiazol-3-ium-5-thiolate derivatives), which was designed with the aid of density functional theory (DFT) calculations. The reactions between sydnthiones and strained alkynes provide a platform for controllable, tunable and mitochondria-targeted release of H2S. We investigate the reactivity of sydnthiones‒dibenzoazacyclooctyne (DIBAC) reactions and their orthogonality with two other bioorthogonal cycloaddition pairs: tetrazine‒norbornene (Nor) and tetrazine‒monohydroxylated cyclooctyne (MOHO). By taking advantage of these mutually orthogonal reactions, we can realize selective labeling or drug release. Furthermore, we explore the role of H2S, which is released from the sydnthione-DIBAC reaction, on doxorubicin-induced cytotoxicity. The results demonstrate that the viability of H9c2 cells can be significantly improved by pretreating with sydnthione 1b and DIBAC for 6 h prior to exposure to Dox.

Modular Assembly of Heterotrifunctional Molecules Enabled by Iodosulfonylation of Allenes and Subsequent Amination.

Beyond the rapid achievements of therapeutic heterobifunctional molecules, some recent efforts have focused on constructing heterotrifunctional molecules, aiming at developing more potent and selective therapeutic agents or emerging additional functions to heterobifunctional molecules. However, the synthesis of these complex molecules requires a specific design and lengthy steps. We have developed a two-step strategy for the modular construction of heterotrifunctional molecules, enabled by the sustainable and convenient iodosulfonylation of allenes followed by SN2'-selective amination. This strategy successfully incorporates a broad range of biologically active molecules, labeling them with a fluorescent group. The applications of the obtained compounds in selective protein labeling, subcellular imaging, and targeted inhibition of tumor cells make this strategy highly appealing.

Changes in Secondary Structure Upon Pr to Pfr Transition in Cyanobacterial Phytochrome Cph1 Detected by DNP NMR.

Phytochromes perceive subtle changes in the light environment and convert them into biological signals by photoconversion between the red-light absorbing (Pr) and the far-red-absorbing (Pfr) states. In the primitive bacteriophytochromes this includes refolding of a tongue-like hairpin loop close to the chromophore, one strand of an antiparallel β-sheet being replaced by an α-helix. However, the strand sequence in the cyanobacterial phytochrome Cph1 is different from that of previously investigated bacteriophytochromes and has a higher β-sheet propensity. We confirm here the transition experimentally and estimate minimum helix length using dynamic nuclear polarisation (DNP) magic angle spinning NMR. Sample conditions were optimized for protein DNP NMR studies at high field, yielding Boltzmann enhancements ϵB of 19 at an NMR field of 18.801 T. Selective labelling of Trp, Ile, Arg, and Val residues with 13C and 15N enabled filtering for pairs of labelled amino acids by the 3D CANCOCA technique to identify signals of the motif 483Ile-Val-Arg485 (IVR) present in both sheet and helix. Those signals were assigned for the Pfr state of the protein. Based on the chemical shift pattern, we confirm for Cph1 the formation of a helix covering the IVR motif.

Magnetic Supraparticles as Identifiers in Single-Layer Lithium-Ion Battery Pouch Cells.

The development of effective recycling technologies is essential for the recovery and reuse of the raw materials required for lithium-ion batteries (LIBs). Future recycling processes depend on accessible information, necessitating the implementation of a digital battery passport. The European battery regulation mandates the use of a machine-readable identifier physically attached to the batteries for accessing digital information. Since externally applied optical labels are vulnerable to mechanical damage, technologies for identification without these restrictions could be beneficial. This study demonstrates that magnetic supraparticles (SPs) can be used for contactless identification of lithium nickel manganese cobalt oxide (NMC) battery pouch cells via magnetic particle spectroscopy (MPS) and that multiple pouch cells can be discriminated based on their specific magnetic code. A comparison of three independent model scenarios revealed that the detection of SPs and the impact on cell performance are dependent on the integration location. The results validate the concept of magnetic identification in metallic environments with MPS as an alternative to optical labeling methods. This study provides a foundation for the development of a new selective labeling and identification technology for batteries, with the potential to facilitate recycling and contribute to a more sustainable future.

Tunable Activated Esters Enable Lysine-Selective Protein Labeling and Profiling.

Lysine residues on protein surfaces are abundant and often found in enzyme active sites, making them critical targets for studying undruggable proteins. However, the varied microenvironment surrounding lysine residues results in a wide range of pKa values, complicating site-specific covalent binding. In this study, we address the challenges posed by the diverse reactivity of amino side chains by modulating the amide reaction activity of heteroaromatic activated esters. By fine-tuning the type, position, and number of heteroatoms, we successfully rationalized the regulation of their amide reaction activity, leading to the design of probes for selective lysine labeling within the proteome for profiling purposes. Systematic optimization of these esters' reactivity and selectivity has yielded a series of effective probes suitable for both in vitro and cellular applications. These findings significantly enhance our understanding of protein functions and mechanisms, facilitated by the precise identification and analysis of protein labeling and profiling.

Selective bioorthogonal probe for N-glycan hybrid structures.

Metabolic incorporation of chemically tagged monosaccharides is a facile means of tagging cellular glycoproteins and glycolipids. However, since the monosaccharide precursors are often shared by several pathways, selectivity has been difficult to attain. For example, N-linked glycosylation is a chemically complex and ubiquitous posttranslational modification, with three distinct classes of GlcNAc-containing N-glycan structures: oligomannose, hybrid and complex. Here we describe the synthesis of 1,3-Pr2-6-OTs GlcNAlk (MM-JH-1) as a next-generation metabolic chemical reporter for the selective labeling of hybrid N-glycan structures. We first developed a general strategy for defining the selectivity of labeling with chemically tagged monosaccharides. We then applied this approach to establish that MM-JH-1 is selectively incorporated into hybrid N-glycans. Using this metabolic chemical reporter as a detection tool, we performed imaging and fractionation to define features of the intracellular localization and trafficking of target proteins bearing hybrid N-glycan structures.

Multi-omic analysis of a mucolipidosis II neuronal cell model uncovers involvement of pathways related to neurodegeneration and drug metabolism

Defining the molecular consequences of lysosomal dysfunction in neuronal cell types remains an area of investigation that is needed to understand many underappreciated phenotypes associated with lysosomal disorders. Here we characterize GNPTAB-knockout DAOY medulloblastoma cells using different genetic and proteomic approaches, with a focus on how altered gene expression and cell surface abundance of glycoproteins may explain emerging neurological issues in individuals with GNPTAB-related disorders, including mucolipidosis II (ML II) and mucolipidosis IIIα/β (ML IIIα/β). The two knockout clones characterized demonstrated all the biochemical hallmarks of this disease, including loss of intracellular glycosidase activity due to impaired mannose 6-phosphate-dependent lysosomal sorting, lysosomal cholesterol accumulation, and increased markers of autophagic dysfunction. RNA sequencing identified altered transcript abundance of several neuronal markers and genes involved in drug metabolism and transport, and neurodegeneration-related pathways. Using selective exo-enzymatic labeling (SEEL) coupled with proteomics to profile cell surface glycoproteins, we demonstrated altered abundance of several glycoproteins in the knockout cells. Most striking was increased abundance of the amyloid precursor protein and apolipoprotein B, indicating that loss of GNPTAB function in these cells corresponds with elevation in proteins associated with neurodegeneration. The implication of these findings on lysosomal disease pathogenesis and the emerging neurological manifestations of GNPTAB-related disorders is discussed.

5-hydroxymethylcytosine sequencing in plasma cell-free DNA identifies unique epigenomic features in prostate cancer patients resistant to androgen deprivation therapies.

Currently, no biomarkers are available to identify resistance to androgen-deprivation therapies (ADT) in men with hormone-naive prostate cancer. Since 5-hydroxymethylcytosines (5hmC) in gene body are associated with gene activation, in this study, we evaluated whether 5hmC signatures in cell-free DNA (cfDNA) predicts early resistance to ADT. We collected a total of 139 serial plasma samples from 55 prostate cancer patients receiving ADT at three time points including baseline (prior to initiating ADT, N=55), 3-month (after initiating ADT, N=55), and disease progression (N=15) within 24 months or 24-month if no progression was detected (N=14). To quantify 5hmC abundance across the genome, we used selective chemical labeling sequencing and mapped sequence reads to individual genes. Differential methylation analysis in baseline samples identified significant 5hmC difference in 1,642 of 23,433 genes between patients with and without progression (false discovery rate, FDR<0.1). Patients with disease progression showed significant 5hmC enrichments in multiple hallmark gene sets with androgen responses as top enriched gene set (FDR=1.19E-13). Interestingly, this enrichment was driven by a subgroup of patients featuring a significant 5hmC hypermethylation in the gene sets involving AR , FOXA1 and GRHL2 . To quantify overall activities of these gene sets, we developed a gene set activity scoring algorithm and observed significant association of high activity scores with poor progression-free survival (P<0.05). Longitudinal analysis showed that the high activity scores were significantly reduced after 3-months of initiating ADT (P<0.0001) but returned to higher levels when the disease was progressed (P<0.05). This study demonstrates that 5hmC-based activity scores from gene sets involved in AR , FOXA1 and GRHL2 may be used as biomarkers to determine early treatment resistance, monitor disease progression, and potentially identify patients who would benefit from upfront treatment intensification.

Oriental covalent immobilization of N-glycan binding protein via N-terminal selective modification

Lectin affinity chromatography is one of powerful tools for the study of protein glycosylation. Different lectin proteins can recognize different structures of monosaccharides or oligosaccharide units, allowing for the selective separation of glycopeptides or glycoproteins containing different polysaccharide structures. However, the N-glycans were only partially captured by most of common lectins, reducing the coverage rate of identifying N-glycoconjugates. Recently, it has been reported that the engineering variant of glycan binding protein Fbs1 has a high affinity for innermost Man3GlcNAc2 structure and is able to bind diverse types of N-glycans, which can be suitable for the analysis of protein N-glycosylation. However, efficient immobilization of protein to separation matrix is particularly challenging as it requires the functionality and integrity of the protein to be preserved. Herein, we describe a simple and robust strategy for oriental covalent immobilization of proteins on magnetic nanoparticles by N-terminal selective labeling techniques. We inserted the enterokinase cleavage site to produce the specific N- terminal glycine of protein. Under physiological conditions, the protein was immobilized on the surface of the MNPs by this glycine tag, and the enrichment process could be completed within 30 min. A whole enrichment and purification of glycan and glycopeptides were completed and analyzed by MALDI TOF-MS. The functional materials achieved stable enrichment of glycan structure in different enzyme digestion systems or complex samples, showing excellent anti-interference and applicability.

Fast and Selective Cysteine Conjugation Using para-Quinone Methides.

An efficient and selective method for cysteine conjugation utilizing para-quinone methides (p-QMs) was developed. p-QM labeling exhibits high specificity toward the cysteine residue, as evidenced by its reactivity with various amino acid derivatives, peptides, and proteins. Notably, the p-QM-cysteine reactions display robust kinetics with rate constants up to 1.67 × 104 M-1·s-1. Furthermore, p-QM conjugation enables us to attach a fluorescent probe to a HER2 nanobody, resulting in selective labeling of HER2-positive SK-BR-3 cells.

Photocatalytic Aldehyde Allylation for Site‐Specific DNA Functionalization

Comprehensive SummaryEnhancing the DNA toolbox with innovative photochemical reactions is pivotal for advancing nucleic acid‐based technologies. Aldehyde groups, versatile bioorthogonal handles for imine formation under acidic conditions, are particularly valuable due to their roles in nucleic acid epigenetics. Here, we present the first photocatalytic on‐DNA aldehyde allylation, enabling precise DNA functionalization under mild, neutral aqueous conditions. Our approach utilizes a photocatalytic polarity‐reversal reaction between DNA‐conjugated benzaldehydes and allyl sulfones. This reaction demonstrates exceptional chemoselectivity while preserving DNA integrity. By varying allyl sulfones, we achieve site‐specific labeling of non‐native DNA with the aldehyde group and cross‐linking with DNA‐bearing allyl sulfones. Furthermore, our method facilitates selective labeling and pull‐down enrichment of 5‐formylpyrimidine nucleotides among complex cellular DNA. This photocatalytic on‐DNA aldehyde transformation expands the limited bioorthogonal photochemical toolboxes, providing novel avenues for functionalizing both non‐native and native aldehyde modifications on DNA.

Developing 1,4-Diethyl-1,2,3,4-tetrahydroquinoxalin-substituted Fluorogens Based on GFP Chromophore for Endoplasmic Reticulum and Lysosome Staining.

In the present study, we demonstrated that the introduction of a 1,4-diethyl-1,2,3,4-tetrahydroquinoxalin moiety into the arylidene part of GFP chromophore-derived compounds results in the formation of environment-sensitive fluorogens. The rationally designed and synthesized compounds exhibit remarkable solvent- and pH-dependence in fluorescence intensity. The solvent-dependent variation in fluorescence quantum yield makes it possible to use some of the proposed compounds as polarity sensors suitable for selective endoplasmic reticulum fluorescent labeling in living cells. Moreover, the pH-dependent emission intensity variation of other fluorogens makes them selective fluorescent labels for the lysosomes in living cells.

Selective Labeling of Small Microplastics with SERS-Tags Based on Gold Nanostars: Method Optimization Using Polystyrene Beads and Application in Environmental Samples.

Microplastics pollution is being unanimously recognized as a global concern in all environments. Routine analysis protocols foresee that samples, which are supposed to contain up to hundreds of microplastics, are eventually collected on nanoporous filters and inspected by microspectroscopy techniques like micro-FTIR or micro-Raman. All particles, whether made of plastic or not, must be inspected one by one to detect and count microplastics. This makes it extremely time-consuming, especially when Raman is adopted, and indeed mandatory for the small microplastic fraction. Inspired by the principles of cell labeling, the present study represents the first report in which gold nanostars (AuNS) are functionalized to act as SERS-tags and used to selectively couple to microplastics. The intrinsic bright signals provided by the SERS-tags are used to run a quick scan over a wide filter area with roughly 2 orders of magnitude shorter analysis time in respect of state of the art in micro- and nanoplastics detection by μ-Raman. The applicability of the present protocol has been validated at the proof-of-concept level on both fabricated and real offshore marine samples. It is indeed worth mentioning that a SERS-based approach is herein successfully applied on filters and protocols routinely adopted in environmental microplastics monitoring, paving the way for future implementations and applications.

A Bis(Acridino)-Crown Ether for Recognizing Oligoamines in Spermine Biosynthesis.

Oligoamines in cellular metabolism carry extremely diverse biological functions (i.e., regulating Ca2+-influx, neuronal nitric oxide synthase, membrane potential, Na+, K+-ATPase activity in synaptosomes, etc.). Furthermore, they also act as longevity agents and have a determinative role in autophagy, cell growth, proliferation, and death, while oligoamines dysregulation is a key in a variety of cancers. However, many of their mechanisms of actions have just begun to be understood. In addition to the numerous biosensing methods, only a very few simple small molecule-based tests are available for their selective but reversible tracking or fluorescent labeling. Motivated by this, we present herein a new fluorescent bis(acridino)-crown ether as a sensor molecule for biogenic oligoamines. The sensor molecule can selectively distinguish oligoamines from aliphatic mono- and diamino-analogues, while showing a reversible 1:2 (host:guest) complexation with a stepwise binding process accompanied by a turn-on fluorescence response. Both computational simulations on molecular docking and regression methods on titration experiments were carried out to reveal the oligoamine-recognition properties of the sensor molecule. The new fluorescent chemosensor molecule has a high potential for molecular-level functional studies on the oligoamine systems in cell processes (cellular uptake, transport, progression in cancers, etc.).

Oxidation-Controlled, Strain-Promoted Tellurophene-Alkyne Cycloaddition (OSTAC): A Bioorthogonal Tellurophene-Dependent Conjugation Reaction.

Tellurophene-bearing small molecules have emerged as valuable tools for localizing cellular activities in vivo using mass cytometry. To broaden the utility of tellurophenes in chemical biology, we have developed a bioorthogonal reaction to facilitate tagging of tellurophene-bearing conjugates for downstream applications. Using TePhe, a tellurophene-based phenylalanine analogue, labeled recombinant proteins were generated for reaction development. Using these proteins, we demonstrate an oxidation-controlled, strain-promoted tellurophene-alkyne cycloaddition (OSTAC) reaction. Mild oxidation of the tellurophene ring with N-chlorosuccinimide produces a reactive Te(IV) species which undergoes rapid (k > 100 M-1 s-1) cycloaddition with bicyclo[6.1.0]nonyne (BCN) yielding a benzo-fused cyclooctane. Selective labeling of TePhe-containing proteins can be achieved in complex protein mixtures and on fixed cells. OSTAC reactions can be combined with strain-promoted azide alkyne cycloaddition (SPAAC) and copper-catalyzed azide alkyne click (CuAAC) reactions. Demonstrating the versatility of this approach, we observe the expected staining patterns for 5-ethynyl-2'-deoxyuridine (DNA synthesis-CuAAC) and immunohistochemistry targets in combination with TePhe (protein synthesis-OSTAC) in fixed cells. The favorable properties of the OSTAC reaction suggest its broad applicability in chemical biology.

Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in gram-negative bacteria.

Lipooligosaccharides (LOS) are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their LOS with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides (LOS) at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their LOS. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the LOS of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering (MOE) and Selective Exo-Enzymatic Labeling (SEEL), and that together they provide tools to modify, label, detect and visualize sialylation of bacterial LOS.

Unsupervised selective labeling for semi-supervised industrial defect detection

In industrial detection scenarios, achieving high accuracy typically relies on extensive labeled datasets, which are costly and time-consuming. This has motivated a shift towards semi-supervised learning (SSL), which leverages labeled and unlabeled data to improve learning efficiency and reduce annotation costs. This work proposes the unsupervised spectral clustering labeling (USCL) method to optimize SSL for industrial challenges like defect variability, rarity, and complex distributions. Integral to USCL, we employ the multi-task fusion self-supervised learning (MTSL) method to extract robust feature representations through multiple self-supervised tasks. Additionally, we introduce the Enhanced Spectral Clustering (ESC) method and a dynamic selecting function (DSF). ESC effectively integrates both local and global similarity matrices, improving clustering accuracy. The DSF maximally selects the most valuable instances for labeling, significantly enhancing the representativeness and diversity of the labeled data. USCL consistently improves various SSL methods compared to traditional instance selection methods. For example, it boosts Efficient Teacher by 5%, 6.6%, and 7.8% in mean Average Precision(mAP) on the Automotive Sealing Rings Defect Dataset, the Metallic Surface Defect Dataset, and the Printed Circuit Boards (PCB) Defect Dataset with 10% labeled data. Our work sets a new benchmark for SSL in industrial settings.

CG-SLENP: A Chemical Genetics Strategy To Selectively Label Existing Proteins and Newly Synthesized Proteins.

Protein synthesis and subsequent delivery to the target locations in cells are essential for their proper functions. Methods to label and distinguish newly synthesized proteins from existing ones are critical to assess their differential properties, but such methods are lacking. We describe the first chemical genetics-based approach for selective labeling of existing and newly synthesized proteins that we termed as CG-SLENP. Using HaloTag in-frame fusion with lamin A (LA), we demonstrate that the two pools of proteins can be selectively labeled using CG-SLENP in living cells. We further employ our recently developed selective small molecule ligand LBL1 for LA to probe the potential differences between newly synthesized and existing LA. Our results show that LBL1 can differentially modulate these two pools of LA. These results indicate that the assembly states of newly synthesized LA are distinct from existing LA in living cells. The CG-SLENP method is potentially generalizable to study any cellular proteins.