Glutathione-activated biotin-targeted dual-modal imaging probe with improved PDT/PTT synergistic therapy.

BACKGROUND: Glutathione (GSH), a highly abundant thiol compound within cells, plays a critical role in physiological processes and exhibits close correlation with cancer. Among molecular imaging technologies, most probes have relatively short emission wavelengths and lack photoacoustic imaging (PA) capability, resulting in the inability to obtain tissue images with high penetration depth. The presence of GSH in the tumor microenvironment neutralizes ROS, diminishing the therapeutic effect of PDT, thus resulting in often unsatisfactory therapeutic efficacy. Therefore, it is imperative to develop a dual-modal probe for the detection of GSH and the diagnosis and treatment of cancer. RESULTS: In this study, we synthesized a novel dual-modal probe, Cy-Bio-GSH, utilizing near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging techniques for GSH detection. The probe integrates cyanine dye as the fluorophore, nitroazobenzene as the recognition moiety, and biotin as the tumor-targeting moiety. Upon reacting with GSH, the probe emits NIR fluorescence at 820 nm and generates a PA signal. Significantly, this reaction activates the photodynamic and photothermal properties of the probe. By depleting GSH and employing a synergistic photothermal therapy (PTT) treatment, the therapeutic efficacy of photodynamic therapy (PDT) is remarkably enhanced. In-vivo experiments confirm the capability of the probe to detect GSH via NIRF and PA imaging. Notably, the combined tumor-targeting ability and PDT/PTT synergistic therapy enhance therapeutic outcomes for tumors and facilitate their ablation. SIGNIFICANCE: A novel tumor-targeting and dual-modal imaging probe (Cy-Bio-GSH) is synthesized, exhibiting remarkable sensitivity and selectivity to GSH, enabling the visualization of GSH in cells and the differentiation between normal and cancer cells. Cy-Bio-GSH enhances PDT/PTT with effective killing of cancer cells and makes the ablation of tumors in mice. This work represents the first tumor-targeting probe for GSH detection, and provides crucial tool for cancer diagnosis and treatment by dual-modal imaging with improved PDT/PTT synergistic therapy.

Analysis of Lipid GPCR Molecular Interactions by Proximity Labeling.

G-protein-coupled receptors (GPCRs) are hepta-helical transmembrane proteins that mediate various intracellular signaling events in response to their specific ligands including many lipid mediators. Although analyses of GPCR molecular interactions are pivotal to understanding diverse intracellular signaling events, affinity purification of interacting proteins by a conventional co-immunoprecipitation method is challenging due to the hydrophobic nature of GPCRs and their dynamic molecular interactions. Proximity labeling catalyzed by a TurboID system is a powerful technique for defining the molecular interactions of target proteins in living cells. TurboID and miniTurbo (a modified version of TurboID) are engineered biotin ligases that biotinylate neighboring proteins in a promiscuous manner. When fused with a target protein and expressed in living cells, TurboID or miniTurbo mediates the biotin labeling of the proteins with close proximity to the target protein, allowing efficient purification of the biotinylated proteins followed by a shot-gun proteomic analysis. In this chapter, we describe a step-by-step protocol for the labeling of GPCR neighboring proteins by TurboID or miniTurbo, purification of the biotin-labeled proteins, and subsequent sample preparation for proteomic analysis. We utilized S1PR1 as a model GPCR, a receptor for a bioactive lipid molecule sphingosine 1-phosphate (S1P) that plays various roles in physiological and pathological conditions. This analysis pipeline enables the mapping of interacting proteins of lipid GPCRs in living cells.

Effects of supplementation of grazing Nellore cows with ß-carotene and vitamins A + D3 + E + biotin on follicle diameter, oestrus, establishment of pregnancy, and foetal morphometry.

The objectives of this experiment were to evaluate the effects of supplementation of Nellore (Bos indicus) cows with ß-carotene + vitamins A + D3 + E + biotin on body condition score (BCS), oestrus, pregnancy, and foetal morphometry. Lactating cows (n = 497) from two herds were balanced for BCS and calving period [early calving (EC); late calving (LC)] and were assigned randomly to: Control (n = 251)-supplementation with a mineral supplement; and SUP (n = 246)-supplementation with the mineral supplement fed to control + ß-carotene (150 mg/day) + vitamin A (40,000 IU/day) + vitamin D3 (5000 IU/day) + vitamin E (300 mg/day) + biotin (20 mg/day). Cows were supplemented from Days -30 to 30 (Day 0 = timed artificial insemination; TAI). Pregnancy was diagnosed 30 days after TAI and foetal crown-rump distance and thoracic diameter were measured at 30 and 77 days of gestation. Cows in the SUP treatment were more likely to have BCS ≥3.0 on Day 0 (63.0 ± 3.1 vs. 60.2 ± 3.1; p < .01) and were more likely to gain BCS from Days -30 to 30 (57.7 ± 3.3 vs. 44.1 ± 3.3%; p < .01). Fewer LC cows in the SUP treatment were detected in oestrus at the time of the first TAI (Control: LC: 75.4 ± 4.4 vs. SUP: LC: 64.0 ± 5.2 vs. Control: EC: 65.3 ± 4.0 vs. SUP: EC: 71.8 ± 3.7; p = .04). There was a tendency for the SUP treatment to increase pregnancy to the first TAI (64.2 ± 3.0 vs. 56.6 ± 3.1%; p = .08). A greater percentage of SUP cows was detected in oestrus at the time of the second TAI (70.1 ± 5.0 vs. 52.3 ± 4.8%; p = .01). The SUP treatment increased pregnancy to the second TAI among LC cows (SUP: LC: 75.9 ± 8.0% vs. Control: LC: 50.0 ± 8.3% vs. Control: EC: 52.0 ± 5.9% vs. SUP: EC: 41.4 ± 6.5%; p = .02). The SUP treatment increased foetal size (crown-rump; p = .04 and thoracic diameter; p < .01) at 30 days of gestation and, despite decreasing crow-rump length at 77 days after the first TAI among EC cows (p < .01), it increased the thoracic diameter at 77 days after the first TAI independent of calving season. Our results support that pregnancy establishment and foetal growth can be improved when grazing Nellore cows are supplemented with ß-carotene and vitamins A + D3 + E + biotin.

Atypical presentation of biotinidase deficiency: masquerading neuromyelitis optica spectrum disorder.

Biotinidase deficiency (BTD) is a treatable, inherited metabolic disorder commonly characterised by alopecia, dermatitis, seizures and developmental delay. It can also manifest as optic neuritis and myelitis; however, these are infrequently described in the literature. We report three cases who presented with quadriplegia and vision loss, initially managed as neuromyelitis optica spectrum disorder (NMOSD), based on neuroimaging findings. Two of them initially responded to immune therapy but relapsed after a few months, while one case showed no clinical improvement with immune therapy. The clinical presentation and neuroimaging findings in all three cases were consistent with NMOSD, leading to a delayed diagnosis of BTD. Antiaquaporin4 and antimyelin oligodendrocyte glycoprotein antibodies were negative in all patients. Urine organic acids reported raised markers of biotinidase or holocarboxylase synthase deficiency. Two of them had a dramatic response to biotin supplementation, showing significant improvement in motor function and vision.

TurboID-mediated proximity labeling technologies to identify virus co-receptors.

Virus receptors determine the tissue tropism of viruses and have a certain relationship with the clinical outcomes caused by viral infection, which is of great importance for the identification of virus receptors to understand the infection mechanism of viruses and to develop entry inhibitor. Proximity labeling (PL) is a new technique for studying protein-protein interactions, but it has not yet been applied to the identification of virus receptors or co-receptors. Here, we attempt to identify co-receptor of SARS-CoV-2 by employing TurboID-catalyzed PL. The membrane protein angiotensin-converting enzyme 2 (ACE2) was employed as a bait and conjugated to TurboID, and a A549 cell line with stable expression of ACE2-TurboID was constructed. SARS-CoV-2 pseudovirus were incubated with ACE2-TurboID stably expressed cell lines in the presence of biotin and ATP, which could initiate the catalytic activity of TurboID and tag adjacent endogenous proteins with biotin. Subsequently, the biotinylated proteins were harvested and identified by mass spectrometry. We identified a membrane protein, AXL, that has been functionally shown to mediate SARS-CoV-2 entry into host cells. Our data suggest that PL could be used to identify co-receptors for virus entry.

[Progress in research and application of the biotin ligase BirA and its mutants in pathogen-host interaction].

Proteins serve as the primary executors of cellular activities in organisms, and thus investigating the subcellular localization and interactions of proteins is crucial for understanding protein functions and elucidating the molecular mechanisms in organisms. Proximity labeling is a recently developed effective method for detecting protein-protein interactions in live cells. Compared with the conventional methods for studying protein-protein interactions, proximity labeling demonstrates high sensitivity, strong specificity, and low background and is widely employed in the research of protein-protein interactions between pathogens and hosts. This article reviews the recent progress in the development and applications of the biotin ligase BirA and its mutants and elucidates the functioning principles of several classical biotin ligases. This review aims to clarify the role of proximity labeling based on BirA and its mutants in identifying protein-protein interactions between pathogens and hosts.

A tumor-targeting two-photon fluorescent probe with a far-red to NIR emission for imaging basal hypochlorite in cancer cells and tumor.

Cancer cells have a high abundance of hypochlorite compared to normal cells, which can be used as the biomarker for imaging cancer cells and tumor. Developing the tumor-targeting fluorescent probe suitable for imaging hypochlorite in vivo is urgently demanded. In this article, based on xanthene dye with a two-photon excited far-red to NIR emission, a tumor-targeting two-photon fluorescent probe (Biotin-HClO) for imaging basal hypochlorite in cancer cells and tumor was developed. For ClO-, Biotin-HClO (20.0 µM) has a linear response range from 15.0 × 10-8 to 1.1 × 10-5 M with a high selectivity and a high sensitivity, a good detection limit of 50 nM and a 550-fold fluorescence enhancement with high signal-to-noise ratio (20 mM PBS buffer solution with 50 % DMF; pH = 7.4; λex = 605 nm; λem = 635 nm). Morover, Biotin-HClO exhibited excellent performance in monitoring exogenous and endogenous ClO- in cells, and has an outstanding tumor-targeting ability. Subsequently, Biotin-HClO has been applied for imaging ClO- in 4T1 tumor tissue to distinguish from normal tissue. Furthermore, Biotin-HClO was successfully employed for high-contrast imaging 4T1 tumor in mouse based on its tumor-targeting ability. All these results proved that Biotin-HClO is a useful analytical tool to detect ClO- and image tumor in vivo.

Development of sterile platform for quantification of extracellular analytes via single walled carbon nanotubes.

Progress has been made studying cell-cell signaling communication processes. However, due to limitations of current sensors on time and spatial resolution, the role of many extracellular analytes is still unknown. A single walled carbon nanotube (SWNT) platform was previously developed based on the avidin-biotin immobilization of SWNT to a glass substrate. The SWNT platform provides real time feedback about analyte concentration and has a high concentration of evenly distributed sensors, both of which are essential for the study of extracellular analytes. Unfortunately, this initial SWNT platform is synthesized through unsterile conditions and cannot be sterilized post-production due to the delicate nature of the sensors, making it unsuitable for in vitro work. Herein the multiple-step process for SWNT immobilization is modified and the platform's biocompatibility is assessed in terms of sterility, cytotoxicity, cell proliferation, and cell morphology through comparison with non-sensors controls. The results demonstrate the SWNT platform's sterility and lack of toxicity over 72 h. The proliferation rate and morphology profiles for cells growing on the SWNT platform are similar to those grown on tissue culture substrates. This novel nano-sensor platform preserves cell health and cell functionality over time, offering opportunities to study extracellular analytes gradients in cellular communication.

Biotin Homeostasis and Human Disorders: Recent Findings and Perspectives.

Biotin (vitamin B7, or vitamin H) is a water-soluble B-vitamin that functions as a cofactor for carboxylases, i.e., enzymes involved in the cellular metabolism of fatty acids and amino acids and in gluconeogenesis; moreover, as reported, biotin may be involved in gene regulation. Biotin is not synthesized by human cells, but it is found in food and is also produced by intestinal bacteria. Biotin status/homeostasis in human individuals depends on several factors, including efficiency/deficiency of the enzymes involved in biotin recycling within the human organism (biotinidase, holocarboxylase synthetase), and/or effectiveness of intestinal uptake, which is mainly accomplished through the sodium-dependent multivitamin transporter. In the last years, administration of biotin at high/"pharmacological" doses has been proposed to treat specific defects/deficiencies and human disorders, exhibiting mainly neurological and/or dermatological symptoms and including biotinidase deficiency, holocarboxylase synthetase deficiency, and biotin-thiamine-responsive basal ganglia disease. On the other hand, according to warnings of the Food and Drug Administration, USA, high biotin levels can affect clinical biotin-(strept)avidin assays and thus lead to false results during quantification of critical biomarkers. In this review article, recent findings/advancements that may offer new insight in the abovementioned research fields concerning biotin will be presented and briefly discussed.

Biotinylated Theranostic Amphiphilic Polyurethane for Targeted Drug Delivery.

In the area of drug delivery aided by stimuli-responsive polymers, the biodegradability of nanocarriers is one of the major challenges that needs to be addressed with the utmost sincerity. Herein, a hydrogen sulfide (H2S) responsive hydrophobic dansyl-based trigger molecule is custom designed and successfully incorporated into the water-soluble polyurethane backbone, which is made of esterase enzyme susceptible urethane bonds. The amphiphilic polyurethanes, PUx (x = 2 and 3) with a biotin chain end, formed self-assembled nanoaggregates. A hemolysis and cytotoxicity profile of doxorubicin (DOX)-loaded biotinylated PU3 nanocarriers revealed that it is nonhemolytic and has excellent selectivity toward HeLa cells (biotin receptor-positive cell lines) causing ∼60% cell death while maintaining almost 100% cell viability for HEK 293T cells (biotin receptor-negative cell lines). Furthermore, better cellular internalization of DOX-loaded fluorescent nanocarriers in HeLa cells than in HEK 293T cells confirmed receptor-mediated endocytosis. Thus, this work ensures that the synthesized polymers serve as biodegradable nanocarriers for anticancer therapeutics.

Biotin-functionalized nanoparticles: an overview of recent trends in cancer detection.

Electrochemical bio-sensing is a potent and efficient method for converting various biological recognition events into voltage, current, and impedance electrical signals. Biochemical sensors are now a common part of medical applications, such as detecting blood glucose levels, detecting food pathogens, and detecting specific cancers. As an exciting feature, bio-affinity couples, such as proteins with aptamers, ligands, paired nucleotides, and antibodies with antigens, are commonly used as bio-sensitive elements in electrochemical biosensors. Biotin-avidin interactions have been utilized for various purposes in recent years, such as targeting drugs, diagnosing clinically, labeling immunologically, biotechnology, biomedical engineering, and separating or purifying biomolecular compounds. The interaction between biotin and avidin is widely regarded as one of the most robust and reliable noncovalent interactions due to its high bi-affinity and ability to remain selective and accurate under various reaction conditions and bio-molecular attachments. More recently, there have been numerous attempts to develop electrochemical sensors to sense circulating cancer cells and the measurement of intracellular levels of protein thiols, formaldehyde, vitamin-targeted polymers, huwentoxin-I, anti-human antibodies, and a variety of tumor markers (including alpha-fetoprotein, epidermal growth factor receptor, prostate-specific Ag, carcinoembryonic Ag, cancer antigen 125, cancer antigen 15-3, etc.). Still, the non-specific binding of biotin to endogenous biotin-binding proteins present in biological samples can result in false-positive signals and hinder the accurate detection of cancer biomarkers. This review summarizes various categories of biotin-functional nanoparticles designed to detect such biomarkers and highlights some challenges in using them as diagnostic tools.

Contributing to the management of viral infections through simple immunosensing of the arachidonic acid serum level.

A trendsetting direct competitive-based biosensing tool has been developed and implemented for the determination of the polyunsaturated fatty acid arachidonic acid (ARA), a highly significant biological regulator with decisive roles in viral infections. The designed methodology involves a competitive reaction between the target endogenous ARA and a biotin-ARA competitor for the recognition sites of anti-ARA antibodies covalently attached to the surface of carboxylic acid-coated magnetic microbeads (HOOC-MµBs), followed by the enzymatic label of the biotin-ARA residues with streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The resulting bioconjugates were magnetically trapped onto the sensing surface of disposable screen-printed carbon transducers (SPCEs) to monitor the extent of the biorecognition reaction through amperometry. The operational functioning of the exhaustively optimized and characterized immunosensing bioplatform was highly convenient for the quantitative determination of ARA in serum samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-) and respiratory syncytial virus (RSV)-infected individuals in a rapid, affordable, trustful, and sensitive manner.

Laryngeal stridor in children caused by reversible metabolic disease.

We report a case of a boy in his middle childhood who presented with inspiratory stridor and lactic acidosis and was subsequently diagnosed with partial biotinidase deficiency. Fibreoptic laryngoscope showed paradoxical vocal fold mobility.Partial biotidinase deficiency is an inherited disorder in which the body is unable to recycle the vitamin biotin. It may result in clinical consequences and can be easily treated with biotin but need a high index of suspicion to diagnose. The main symptoms include ataxia, seizures, hypotonia, psychomotor retardation, alopecia, skin rash, progressive deafness, optic atrophy and life-threatening episodes of metabolic acidosis. Laryngeal stridor is an uncommon presentation, but it is reversible in case of biotinidase deficiency. Invasive procedure like tracheostomy has not been shown to enhance outcomes.

Learning Protein-Ligand Unbinding Pathways via Single-Parameter Community Detection.

Understanding the dynamics of biomolecular complexes, e.g., of protein-ligand (un)binding, requires the comprehension of paths such systems take between metastable states. In MD simulations, paths are usually not observable per se, but they need to be inferred from simulation trajectories. Here, we present a novel approach to cluster trajectories based on a community detection algorithm that necessitates only the definition of a single parameter. The unbinding of the streptavidin-biotin complex is used as a benchmark system and the A2a adenosine receptor in complex with the inhibitor ZM241385 as an elaborate application. We demonstrate how such clusters of trajectories correspond to pathways and how the approach helps in the identification of reaction coordinates for a considered (un)binding process.

Impact of sulfur substitution on biotin binding affinity to streptavidin.

In this study, we investigated how the replacement of the tetrahydrothiophene ring of biotin with either an oxolane or (methyl)pyrrolidine moiety may affect its molecular interactions, in an effort to identify alternative affinity ligands suitable for in vitro and in vivo applications in synthetic biology. Initial molecular dynamics (MD) simulations suggested the potential formation of a hydrogen bond between either the oxygen or nitrogen atom of the envisaged tetrahydroheteryl analogues and the Thr90 residue of streptavidin, mirroring the sulfur-centered hydrogen bond detected by the crystallographic analysis of the biotin-streptavidin interaction. Therefore, oxy-, aza-, and N-methylazabiotin were readily synthesized starting from chiral five- or six-carbon sugar precursors. Based on fluorescence-based titration experiments using the corresponding fluorescein conjugates, oxybiotin showed a binding behavior similar to biotin with streptavidin, while both amino analogues displayed lower binding capacities. Notably, azabiotin exhibited a pH-dependent interaction profile, demonstrating enhanced binding under acidic conditions but weaker binding under basic pH, which could be exploited for various purposes.

Mycobacterial biotin synthases require an auxiliary protein to convert dethiobiotin into biotin.

Lipid biosynthesis in the pathogen Mycobacterium tuberculosis depends on biotin for posttranslational modification of key enzymes. However, the mycobacterial biotin synthetic pathway is not fully understood. Here, we show that rv1590, a gene of previously unknown function, is required by M. tuberculosis to synthesize biotin. Chemical-generic interaction experiments mapped the function of rv1590 to the conversion of dethiobiotin to biotin, which is catalyzed by biotin synthases (BioB). Biochemical studies confirmed that in contrast to BioB of Escherichia coli, BioB of M. tuberculosis requires Rv1590 (which we named "biotin synthase auxiliary protein" or BsaP), for activity. We found homologs of bsaP associated with bioB in many actinobacterial genomes, and confirmed that BioB of Mycobacterium smegmatis also requires BsaP. Structural comparisons of BsaP-associated biotin synthases with BsaP-independent biotin synthases suggest that the need for BsaP is determined by the [2Fe-2S] cluster that inserts sulfur into dethiobiotin. Our findings open new opportunities to seek BioB inhibitors to treat infections with M. tuberculosis and other pathogens.

Rationalization of a Streptavidin Based Enantioselective Artificial Suzukiase: An Integrative Computational Approach.

An Artificial Metalloenzyme (ArM) built employing the streptavidin-biotin technology has been used for the enantioselective synthesis of binaphthyls by means of asymmetric Suzuki-Miyaura cross-coupling reactions. Despite its success, it remains a challenge to understand how the length of the biotin cofactors or the introduction of mutations to streptavidin leads the preferential synthesis of one atropisomer over the other. In this study, we apply an integrated computational modeling approach, including DFT calculations, protein-ligand dockings and molecular dynamics to rationalize the impact of mutations and length of the biotion cofactor on the enantioselectivities of the biaryl product. The results unravel that the enantiomeric differences found experimentally can be rationalized by the disposition of the first intermediate, coming from the oxidative addition step, and the entrance of the second substrate. The work also showcases the difficulties facing to control the enantioselection when engineering ArM to catalyze enantioselective Suzuki-Miyaura couplings and how the combination of DFT calculations, molecular dockings and MD simulations can be used to rationalize artificial metalloenzymes.

Advances and prospects in microbial production of biotin.

Biotin, serving as a coenzyme in carboxylation reactions, is a vital nutrient crucial for the natural growth, development, and overall well-being of both humans and animals. Consequently, biotin is widely utilized in various industries, including feed, food, and pharmaceuticals. Despite its potential advantages, the chemical synthesis of biotin for commercial production encounters environmental and safety challenges. The burgeoning field of synthetic biology now allows for the creation of microbial cell factories producing bio-based products, offering a cost-effective alternative to chemical synthesis for biotin production. This review outlines the pathway and regulatory mechanism involved in biotin biosynthesis. Then, the strategies to enhance biotin production through both traditional chemical mutagenesis and advanced metabolic engineering are discussed. Finally, the article explores the limitations and future prospects of microbial biotin production. This comprehensive review not only discusses strategies for biotin enhancement but also provides in-depth insights into systematic metabolic engineering approaches aimed at boosting biotin production.

Interface-constrained catalytic hairpin assembly permits highly sensitive SERS signaling of miRNA.

The rapid and precise monitoring of peripheral blood miRNA levels holds paramount importance for disease diagnosis and treatment monitoring. In this study, we propose an innovative research strategy that combines the catalytic hairpin assembly reaction with SERS signal congregation and enhancement. This combination can significantly enhance the stability of SERS detection, enabling stable and efficient detection of miRNA. Specifically, our paper-based SERS detection platform incorporates a streptavidin-modified substrate, biotin-labeled catalytic hairpin assembly reaction probes, 4-ATP, and primer-co-modified gold nanoparticles. In the presence of miRNA, the 4-ATP and primer-co-modified gold nanoparticles can specifically recognize the miRNA and interact with the biotin-labeled CHA probes to initiate an interfacial catalytic hairpin assembly reaction. This enzyme-free high-efficiency catalytic process can accumulate a large amount of biotin on the gold nanoparticles, which then bind to the streptavidin on the substrate with the assistance of the driving liquid, forming red gold nanoparticle stripes. These provide a multitude of hotspots for SERS, enabling enhanced signal detection. This innovative design achieves a low detection limit of 3.47 fM while maintaining excellent stability and repeatability. This conceptually innovative detection platform offers new technological possibilities and solutions for clinical miRNA detection.