RESUMEN

We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N',N'-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.

Asunto(s)

Biotinilación , Medios de Cultivo , Hibridomas , Inmunoglobulina M , Inmunoglobulina M/química , Inmunoglobulina M/aislamiento & purificación , Animales , Medios de Cultivo/química , Ratones , Circonio/química , Biotina/química

RESUMEN

Using the principle of "Magic Bullet", a cisplatin-derived platinum(IV) prodrug heterobimetallic Pt(IV)-Ru(II) complex, cis,cis,trans-[Pt(NH3)2Cl2{Ru(tpy-BODIPY)(tpy-COO)}(biotin)]Cl2 (Pt-Ru-B, 2), having two axial ligands, namely, biotin as water-soluble B-vitamin for enhanced cellular uptake and a BODIPY-ruthenium(II) (Ru-B, 1) photosensitizer having N,N,N-donor tpy (4'-phenyl-2,2':6',2″-terpyridine) bonded to boron-dipyrromethene (BODIPY), is developed as a "Platin Bullet" for targeted photodynamic therapy (PDT). Pt-Ru-B exhibited intense absorption near 500 nm and emission near 513 nm (λex = 488 nm) in a 10% dimethyl sulfoxide-Dulbecco's phosphate-buffered saline medium (pH 7.2). The BODIPY complex on light activation generates singlet oxygen as the reactive oxygen species (ROS) giving a quantum yield (ΦΔ) of ∼0.64 from 1,3-diphenylisobenzofuran experiments. Pt-Ru-B exhibited preferential cellular uptake in cancer cells over noncancerous cells. The dichlorodihydrofluorescein diacetate assay confirmed the generation of cellular ROS. Confocal images revealed its mitochondrial internalization. Pt-Ru-B showed submicromolar photocytotoxicity in visible light (400-700 nm) in A549 and multidrug-resistant MDA-MB-231 cancer cells. It remained nontoxic in the dark and less toxic in nontumorigenic cells. Cellular apoptosis and alteration of the mitochondrial membrane potential were evidenced from the respective Annexin V-FITC/propidium iodide assay and JC-1 dye assay. A wound healing assay using A549 cells and Pt-Ru-B revealed inhibition of cancer cell migration, highlighting its potential as an antimetastatic agent.

Asunto(s)

Antineoplásicos , Biotina , Fotoquimioterapia , Fármacos Fotosensibilizantes , Profármacos , Rutenio , Humanos , Profármacos/farmacología , Profármacos/química , Profármacos/síntesis química , Rutenio/química , Rutenio/farmacología , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Biotina/química , Biotina/farmacología , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/síntesis química , Compuestos de Boro/química , Compuestos de Boro/farmacología , Compuestos de Boro/síntesis química , Porfobilinógeno/análogos & derivados , Porfobilinógeno/química , Porfobilinógeno/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Platino (Metal)/química , Platino (Metal)/farmacología , Estructura Molecular , Supervivencia Celular/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Cisplatino/química

RESUMEN

Cell surface proteins participate in many important biological processes, such as cell-to-cell interaction, signal transduction, cell adhesion, and protein transportation. In-depth study of the cell surface protein group is of great significance. Nevertheless, detection and analysis of the surfaceome remain a significant challenge due to their low abundance and hydrophobicity. Herein, we reported an ultrafast and chemoselective labeling method using our newly developed trifunctional probe, the OPA-S-S-alkyne, which labeled cell surface lysine residues, and then established a novel cell surfaceome profiling approach. According to our experimental results, the OPA-S-S-alkyne probe can react extremely fast with living cells, labeling cells in only 1 min, while traditional NHS (labeling cell surface lysine with N-hydroxysuccinimide ester probe) and CSC (labeling cell surface glycan with hydrazide biotin probe) methods normally take longer time of more than 30 min and 1 h, respectively. Taking advantage of this ultrafast property of the method, we highlight the utility of this method by exploring the temporal dynamic changes of surfaceome upon EGF stimulation in living Hela cells and reported "early" and "late" EGF-regulated cell surface proteins, which are difficult to be distinguished by the current cell surface profiling approaches.

Asunto(s)

Biotinilación , Humanos , Células HeLa , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Membrana Celular/química , Membrana Celular/metabolismo , Biotina/química , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/metabolismo , Factores de Tiempo , Lisina/química

RESUMEN

Stability of conjugated critical reagents supporting ligand binding assays to enable biotherapeutic drug development is a universal concern. Formulation buffer employed for long-term cold storage may be key to mitigate protein aggregation issues. We investigated biophysical and functional attributes of murine mAb and human multispecific drug labeled with biotin, ruthenium, and Alexa fluor 647 frozen at -80 °C in PBS or a protein storage buffer for 3-15 months. Aggregation was observed at 4 months in mAb A-Ru (11.2%) and -Alexa (10%) in PBS followed by precipitation and reduced biological binding at 15 months. Increased aggregation in drug Ru (11.7%, 6 months) and Alexa (6.9%, 15 months) were noted but without impact on performance. There were no observations with biotin labeled reagents.

Asunto(s)

Anticuerpos Monoclonales , Tampones (Química) , Humanos , Animales , Anticuerpos Monoclonales/química , Ratones , Biotina/química , Estabilidad de Medicamentos , Agregado de Proteínas , Rutenio/química , Estabilidad Proteica

RESUMEN

Numerous studies indicate that intrauterine growth restriction (IUGR) can predispose individuals to metabolic syndrome (MetS) in adulthood. Several reports have demonstrated that pharmacological concentrations of biotin have therapeutic effects on MetS. The present study investigated the beneficial effects of prenatal biotin supplementation in a rat model of intrauterine caloric restriction to prevent cardiometabolic risk in adult female offspring fed fructose after weaning. Female rats were exposed to a control (C) diet or global caloric restriction (20%) (GCR), with biotin (GCRB) supplementation (2 mg/kg) during pregnancy. Female offspring were exposed to 20% fructose (F) in drinking water for 16 weeks after weaning (C, C/F, GCR/F, and GCRB/F). The study assessed various metabolic parameters including Lee's index, body weight, feed conversion ratio, caloric intake, glucose tolerance, insulin resistance, lipid profile, hepatic triglycerides, blood pressure, and arterial vasoconstriction. Results showed that GCR and GCRB dams had reduced weights compared to C dams. Offspring of GCRB/F and GCR/F dams had lower body weight and Lee's index than C/F offspring. Maternal biotin supplementation in the GCRB/F group significantly mitigated the adverse effects of fructose intake, including hypertriglyceridemia, hypercholesterolemia, hepatic steatosis, glucose and insulin resistance, hypertension, and arterial hyperresponsiveness. This study concludes that prenatal biotin supplementation can protect against cardiometabolic risk in adult female offspring exposed to postnatal fructose, highlighting its potential therapeutic benefits.

Asunto(s)

Biotina , Restricción Calórica , Suplementos Dietéticos , Retardo del Crecimiento Fetal , Efectos Tardíos de la Exposición Prenatal , Animales , Femenino , Embarazo , Ratas , Restricción Calórica/métodos , Biotina/administración & dosificación , Biotina/farmacología , Efectos Tardíos de la Exposición Prenatal/prevención & control , Retardo del Crecimiento Fetal/prevención & control , Retardo del Crecimiento Fetal/etiología , Resistencia a la Insulina , Modelos Animales de Enfermedad , Síndrome Metabólico/prevención & control , Síndrome Metabólico/etiología , Síndrome Metabólico/metabolismo , Fructosa/efectos adversos , Factores de Riesgo Cardiometabólico , Peso Corporal/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos

RESUMEN

INTRODUCTION: Holocarboxylase synthetase deficiency (HLCS deficiency, OMIM #253270) is an exceedingly rare metabolic disorder resulting in multiple carboxylase deficiencies owing to impaired biotin cycle. Clinical manifestations include severe metabolic acidosis, hyperammonemia, tachypnea, skin rash, alopecia, feeding problems, hypotonia, developmental delay, seizures, and, in severe cases, death. METHODS AND RESULTS: An 8-day-old female neonate presented with severe lactic acidosis, necessitating sedation and mechanical ventilation. Despite receiving supportive care, no evident clinical improvement was observed, accompanied by the onset of generalized ichthyosis. Genetic analysis of actionable metabolic disorders revealed compound heterozygous variants of HLCS (NM_000411.8), specifically c.[710T>C (p.Leu237Pro)]; [1544G>A (p.Ser515Asn)], prompting the initiation of biotin mega-dose therapy (10 mg/day). Remarkably, dramatic clinical improvement in lactic acidosis was observed the day after initiating biotin administration, leading to the discontinuation of mechanical ventilation within 6 days. The patient remained in stable condition during follow-up, exhibiting normal growth and development along with consistently stable laboratory findings up to 18 months of age. CONCLUSION: Our case highlights the significance of early genetic testing in neonates with unexplained metabolic disorders to enable timely diagnosis and therapy initiation. Biotin therapy has demonstrated remarkable efficacy in improving the clinical condition of patients with HLCS deficiency, leading to favorable outcomes.

Asunto(s)

Biotina , Deficiencia de Holocarboxilasa Sintetasa , Humanos , Femenino , Recién Nacido , Biotina/uso terapéutico , Biotina/administración & dosificación , Deficiencia de Holocarboxilasa Sintetasa/genética , Deficiencia de Holocarboxilasa Sintetasa/tratamiento farmacológico , Acidosis Láctica/genética , Acidosis Láctica/tratamiento farmacológico , Complejo Vitamínico B/uso terapéutico , Complejo Vitamínico B/administración & dosificación

RESUMEN

Small-molecule fluorescent probes have emerged as potential tools for cancer cell imaging-based diagnostic and therapeutic applications, but their limited selectivity and poor imaging contrast hinder their broad applications. To address these problems, we present the design and construction of a novel near-infrared (NIR) biotin-conjugated and viscosity-activatable fluorescent probe, named as QL-VB, for selective recognition and imaging of cancer cells. The designed probe exhibited a NIR emission at 680 nm, with a substantial Stokes shift of 100 nm and remarkably sensitive responses toward viscosity changes in solution. Importantly, QL-VB provided an evidently enhanced signal-to-noise ratio (SNR: 6.2) for the discrimination of cancer cells/normal cells, as compared with the control probe without biotin conjugation (SNR: 1.8). Moreover, we validated the capability of QL-VB for dynamic monitoring of stimulated viscosity changes within cancer cells and employed QL-VB for distinguishing breast cancer tissues from normal tissues in live mice with improved accuracy (SNR: 2.5) in comparison with the control probe (SNR: 1.8). All these findings indicated that the cancer-targeting and viscosity-activatable NIR fluorescent probe not only enables the mechanistic investigations of mitochondrial viscosity alterations within cancer cells but also holds the potential as a robust tool for cancer cell imaging-based applications.

Asunto(s)

Colorantes Fluorescentes , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Humanos , Viscosidad , Animales , Ratones , Imagen Óptica , Femenino , Rayos Infrarrojos , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Línea Celular Tumoral , Biotina/química

RESUMEN

Existing strategies use bifunctional chimaeras to mediate extracellular protein degradation. However, these strategies rely on specific lysosome-trafficking receptors to facilitate lysosomal delivery, which may raise resistance concerns due to intrinsic cell-to-cell variation in receptor expression and mutations or downregulation of the receptors. Another challenge is establishing a universal platform applicable in multiple scenarios. Here, we develop MONOTAB (MOdified NanOparticle with TArgeting Binders), a plug-and-play monofunctional degradation platform that can drag extracellular targets into lysosomes for degradation. MONOTAB harnesses the inherent lysosome-targeting ability of certain nanoparticles to obviate specific receptor dependency and the hook effect. To achieve high modularity and programmable target specificity, we utilize the streptavidin-biotin interaction to immobilize antibodies or other targeting molecules on nanoparticles, through an antibody mounting approach or by direct binding. Our study reveals that MONOTAB can induce efficient degradation of diverse therapeutic targets, including membrane proteins, secreted proteins, and even extracellular vesicles.

Asunto(s)

Vesículas Extracelulares , Lisosomas , Nanopartículas , Proteolisis , Vesículas Extracelulares/metabolismo , Humanos , Lisosomas/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Estreptavidina/metabolismo , Estreptavidina/química , Animales , Biotina/metabolismo , Biotina/química , Células HEK293

RESUMEN

In this study, biotin esterified debranched starch (Bio-DBS) nanoparticles with different molecular weights were prepared to improve the stability and antioxidant activity of resveratrol. The molecular weights of branched starch (DBS3, DBS9 and DBSp) determined by high-performance size-exclusion chromatography (HPSEC) were 3306, 3696, and 4688, respectively. Biotin was covalently coupled to DBS through the esterification reaction as a new material to prepare nanoparticles. The morphology, particle size, and loading capacity of Bio-DBS nanoparticles were all related to the molecular weights of DBS. The 1H NMR results indicated that there was a hydrogen bonding interaction between Bio-DBS and resveratrol, which contributed to the photochemical and antioxidant activity of resveratrol in the nanoparticles. The highest encapsulation efficiency (78.9 %) and loading capacity (15.78 %) of resveratrol were observed in Bio-DBS3 nanoparticles. Additionally, the cell viability was over 80 % when the concentration of Bio-DBS3 reached to 200 µg/mL. The Bio-DBS nanoparticles significantly improved the thermal stability, photostability, and antioxidant properties of resveratrol. Therefore, the Bio-DBS nanoparticles prepared in this study can be used as a promising carrier to improve the stability and antioxidant activity of resveratrol and may have potential applications in oral delivery.

Asunto(s)

Antioxidantes , Biotina , Portadores de Fármacos , Nanopartículas , Resveratrol , Almidón , Resveratrol/química , Resveratrol/farmacología , Almidón/química , Antioxidantes/farmacología , Antioxidantes/química , Nanopartículas/química , Portadores de Fármacos/química , Biotina/química , Tamaño de la Partícula , Humanos , Estabilidad de Medicamentos , Esterificación , Administración Oral , Supervivencia Celular/efectos de los fármacos

RESUMEN

Intracellular calcium (Ca2+) is ubiquitous to cell signaling across biology. While existing fluorescent sensors and reporters can detect activated cells with elevated Ca2+ levels, these approaches require implants to deliver light to deep tissue, precluding their noninvasive use in freely behaving animals. Here we engineered an enzyme-catalyzed approach that rapidly and biochemically tags cells with elevated Ca2+ in vivo. Ca2+-activated split-TurboID (CaST) labels activated cells within 10 min with an exogenously delivered biotin molecule. The enzymatic signal increases with Ca2+ concentration and biotin labeling time, demonstrating that CaST is a time-gated integrator of total Ca2+ activity. Furthermore, the CaST readout can be performed immediately after activity labeling, in contrast to transcriptional reporters that require hours to produce signal. These capabilities allowed us to apply CaST to tag prefrontal cortex neurons activated by psilocybin, and to correlate the CaST signal with psilocybin-induced head-twitch responses in untethered mice.

Asunto(s)

Calcio , Animales , Ratones , Calcio/metabolismo , Neuronas/metabolismo , Humanos , Biotina/química , Biotina/metabolismo , Señalización del Calcio , Corteza Prefrontal/metabolismo , Corteza Prefrontal/citología , Ratones Endogámicos C57BL , Masculino

RESUMEN

An ultrasensitive photothermal assay was designed for point-of-care testing (POCT) of tumor markers based on a filter membrane. Firstly, Cu2-xSe was successfully encapsulated in liposome spheres with biotin on the surface and connected to carcinoembryonic antigen (CEA) aptamer with 3'end modified biotin by streptavidin. Secondly, the CEA antibody was successfully modified on the surface of the nitrocellulose membrane through simple incubation. Finally, the assay process was completed using a disposable syringe, and the temperature was recorded using a handheld infrared temperature detector. In the range 0-50 ng mL-1, the temperature change of the nitrocellulose membrane has a strong linear relationship with CEA concentration, and the detection limit is 0.097 ng mL-1. It is worth noting that the entire testing process can be easily performed in 10 min, much shorter than traditional clinical methods. In addition, this method was successfully applied to the quantitative determination of CEA levels in human serum samples with a recovery of 96.2-103.3%. This rapid assay can be performed by "one suction and one push" through a disposable syringe, which is simple to operate, and the excellent sensitivity reveals the great potential of the proposed strategy in the POCT of tumor biomarkers.

Asunto(s)

Aptámeros de Nucleótidos , Biomarcadores de Tumor , Antígeno Carcinoembrionario , Cobre , Límite de Detección , Humanos , Antígeno Carcinoembrionario/sangre , Cobre/química , Aptámeros de Nucleótidos/química , Biomarcadores de Tumor/sangre , Liposomas/química , Técnicas Biosensibles/métodos , Sistemas de Atención de Punto , Temperatura , Biotina/química , Pruebas en el Punto de Atención , Colodión/química

RESUMEN

Photodynamic therapy (PDT) is a clinically-approved cancer treatment that is based on production of cytotoxic reactive oxygen species to induce cell death. However, its efficiency depends on distribution of photosensitizer (PS) and depth of light penetration through the tissues. Tendency of pathological cancer tissues to exhibit lower pH than healthy tissues inspired us to explore dual-targeted pH-activatable photosensitizers based on tunable near-infrared (NIR) boron-dipyrromethene (BODIPY) dyes. Our BODIPY PSs were designed to carry three main attributes: (i) biotin or cRGD peptide as an effective cancer cell targeting unit, (ii) amino moiety that is protonated in acidic (pH <6.5) conditions for pH-activation of the PS based on photoinduced electron transfer (PET) and (iii) hydrophilic groups enhancing the water solubility of very hydrophobic BODIPY dyes. Illumination of such compounds with suitable light (>640nm) allowed for high phototoxicity against HeLa (αvß3 integrin and biotin receptor positive) and A549 (biotin receptor positive) cells compared to healthy MRC-5 (biotin negative) cells. Moreover, no dark toxicity was observed on selected cell lines (>10 µM) providing promising photosensitizers for tumour-targeted photodynamic therapy.

Asunto(s)

Biotina , Compuestos de Boro , Fotoquimioterapia , Fármacos Fotosensibilizantes , Humanos , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Biotina/química , Compuestos de Boro/química , Compuestos de Boro/farmacología , Concentración de Iones de Hidrógeno , Células HeLa , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Rayos Infrarrojos , Células A549 , Supervivencia Celular/efectos de los fármacos

RESUMEN

A series of biotin-functionalized transition metal complexes was prepared by iClick reaction from the corresponding azido complexes with a novel alkyne-functionalized biotin derivative ([Au(triazolatoR,R')(PPh3)], [Pt(dpb)(triazolatoR,R')], [Pt(triazolatoR,R')(terpy)]PF6, and [Ir(ppy)(triazolatoR,R')(terpy)]PF6 with dpb = 1,3-di(2-pyridyl)benzene, ppy = 2-phenylpyridine, and terpy = 2,2':6',2''-terpyridine and R = C6H5, R' = biotin). The complexes were compared to reference compounds lacking the biotin moiety. The binding affinity toward avidin and streptavidin was evaluated with the HABA assay as well as isothermal titration calorimetry (ITC). All compounds exhibit the same binding stoichiometry of complex-to-avidin of 4:1, but the ITC results show that the octahedral Ir(III) compound exhibits a higher binding affinity than the square-planar Pt(II) complex. The antibacterial activity of the compounds was evaluated on a series of Gram-negative and Gram-positive bacterial strains. In particular, the neutral Au(I) and Pt(II) complexes showed significant antibacterial activity against Staphylococcus aureus and Enterococcus faecium at very low micromolar concentrations. The cytotoxicity against a range of eukaryotic cell lines was studied and revealed that the octahedral Ir(III) complex was non-toxic, while the square-planar Pt(II) and linear Au(I) complexes displayed non-selective micromolar activity.

Asunto(s)

Antibacterianos , Biotina , Oro , Iridio , Pruebas de Sensibilidad Microbiana , Platino (Metal) , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Biotina/química , Oro/química , Oro/farmacología , Iridio/química , Iridio/farmacología , Platino (Metal)/química , Platino (Metal)/farmacología , Humanos , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Complejos de Coordinación/síntesis química , Staphylococcus aureus/efectos de los fármacos , Estructura Molecular , Relación Estructura-Actividad

RESUMEN

Cell membrane genetic engineering has been utilized to confer cell membranes with functionalities for diagnostic and therapeutic purposes but concerns over cost and variable modification results. Although nongenetic chemical modification and phospholipid insertion strategies are more convenient, they still face bottlenecks in either biosafety or stability of the modifications. Herein, we show that pyrazolone-bearing molecules can bind to proteins with high stability, which is mainly contributed to by the multiple interactions between pyrazolone and basic amino acids. This new binding model offers a simple and versatile noncovalent approach for cell membrane functionalization. By binding to cell membrane proteins, pyrazolone-bearing dyes enabled precise cell tracking in vitro (>96 h) and in vivo (>21 days) without interfering with the protein function or causing cell death. Furthermore, the convenient anchor of pyrazolone-bearing biotin on cell membranes rendered the biorecognition to avidin, showing the potential for artificially creating cell targetability.

Asunto(s)

Membrana Celular , Pirazolonas , Pirazolonas/química , Pirazolonas/farmacología , Membrana Celular/metabolismo , Membrana Celular/química , Humanos , Biotina/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Unión Proteica

RESUMEN

Biotin, also known as vitamin B7 or vitamin H, is a water-soluble B-complex vitamin and serves as an essential co-enzyme for five specific carboxylases. Holocarboxylase synthase (HCS) activates biotin and facilitates its covalent attachment to these enzymes, while biotinidase releases free biotin in the biotin cycle. The transport of biotin, primarily from the intestine, is mediated by the sodium-dependent multi-vitamin transporter (SMVT). Severe biotin deficiency leads to multiple carboxylase deficiency. Moreover, biotin is crucial to glucose and lipid utilization in cellular energy production because it modulates the expression of metabolic enzymes via various signaling pathways and transcription factors. Biotin also modulates the production of proinflammatory cytokines in the immune system through similar molecular mechanisms. These regulatory roles in metabolic and immune homeostasis connect biotin to conditions such as diabetes, dermatologic manifestations, and multiple sclerosis. Furthermore, deficiencies in biotin and SMVT are implicated in inflammatory bowel disease, affecting intestinal inflammation, permeability, and flora. Notably, HCS and probably biotin directly influence gene expression through histone modification. In this review, we summarize the current knowledge on the molecular aspects of biotin and associated molecules in diseases related to both acute inflammatory responses and chronic inflammation, and discuss the potential therapeutic applications of biotin.

Asunto(s)

Biotina , Inflamación , Humanos , Biotina/metabolismo , Inflamación/metabolismo , Animales , Deficiencia de Biotinidasa/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Citocinas/metabolismo

RESUMEN

Methods that identify protein-protein interactions are essential for understanding molecular mechanisms controlling biological systems. Proximity-dependent labeling has proven to be a valuable method for revealing protein-protein interaction networks in living cells. A mutant form of the biotin protein ligase enzyme from Aquifex aeolicus (BioID2) underpins this methodology by producing biotin that is attached to proteins that enter proximity to it. This labels proteins for capture, extraction, and identification. In this chapter, we present a toolkit for BioID2 specifically adapted for use in E. coli, exemplified by the chemotaxis protein CheA. We have created plasmids containing BioID2 as expression cassettes for proteins (e.g., CheA) fused to BioID2 at either the N or C terminus, optimized with an 8 × GGS linker. We provide a methodology for expression and verification of CheA-BioID2 fusion proteins in E. coli cells, the in vivo biotinylation of interactors by protein-BioID2 fusions, and extraction and analysis of interacting proteins that have been biotinylated.

Asunto(s)

Biotinilación , Escherichia coli , Mapeo de Interacción de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Biotina/metabolismo , Mapas de Interacción de Proteínas , Coloración y Etiquetado/métodos , Plásmidos/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética

RESUMEN

Biotinylation is probably the most frequent and practically useful modification of molecules to facilitate selective and highly affine binding to (strept)avidin for immobilization, enrichment, and purification for further (bio)chemical or (bio)physical investigations. We present a protecting-group-free synthesis of a branched biotin bis-azide that enables dual-payload late-stage functionalization with arbitrary alkynes via click chemistry. Utility of the chassis is briefly showcased on the example of a valuable Pittsburgh B analogue, which binds pathological protein aggregates, commonly found in neurodegenerative diseases.

Asunto(s)

Alquinos , Biotina , Biotinilación , Química Clic , Estructura Molecular , Biotina/química , Alquinos/química , Tiazoles/química , Tiazoles/síntesis química , Azidas/química

RESUMEN

Protein phosphorylation by kinases regulates mammalian cell functions, such as growth, division, and signal transduction. Among human kinases, NME1 and NME2 are associated with metastatic tumor suppression but remain understudied due to the lack of tools to monitor their cellular substrates. In particular, NME1 and NME2 are multispecificity kinases phosphorylating serine, threonine, histidine, and aspartic acid residues of substrate proteins, and the heat and acid sensitivity of phosphohistidine and phosphoaspartate complicates substrate discovery and validation. To provide new substrate monitoring tools, we established the γ-phosphate-modified ATP analog, ATP-biotin, as a cosubstrate for phosphorylbiotinylation of NME1 and NME2 cellular substrates. Building upon this ATP-biotin compatibility, the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates method enabled validation of a known substrate and the discovery of seven NME1 and three NME2 substrates. Given the paucity of methods to study kinase substrates, ATP-biotin and the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates method are valuable tools to characterize the roles of NME1 and NME2 in human cell biology.

Asunto(s)

Biotinilación , Nucleósido Difosfato Quinasas NM23 , Humanos , Nucleósido Difosfato Quinasas NM23/metabolismo , Nucleósido Difosfato Quinasas NM23/química , Nucleósido Difosfato Quinasas NM23/genética , Especificidad por Sustrato , Fosforilación , Biotina/metabolismo , Biotina/química , Biotina/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Células HEK293 , Catálisis

RESUMEN

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.

Asunto(s)

Biotina , Glutatión , Técnicas Fotoacústicas , Fotoquimioterapia , Glutatión/química , Glutatión/metabolismo , Animales , Humanos , Ratones , Biotina/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Imagen Óptica , Femenino , Terapia Fototérmica , Ratones Desnudos , Ratones Endogámicos BALB C , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/uso terapéutico

RESUMEN

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.

Asunto(s)

Biotina , Suplementos Dietéticos , Estro , Vitamina A , Vitamina E , beta Caroteno , Animales , Bovinos , Femenino , Embarazo , Vitamina A/administración & dosificación , Vitamina A/farmacología , beta Caroteno/administración & dosificación , beta Caroteno/farmacología , Vitamina E/administración & dosificación , Vitamina E/farmacología , Estro/efectos de los fármacos , Biotina/administración & dosificación , Biotina/farmacología , Colecalciferol/farmacología , Colecalciferol/administración & dosificación , Folículo Ovárico/efectos de los fármacos , Dieta/veterinaria , Vitaminas/administración & dosificación , Vitaminas/farmacología , Alimentación Animal , Lactancia , Feto/efectos de los fármacos