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Biointerphases Nov 2020The hydrophobically modified glycol chitosan (HGC) nanomicelle has received increasing attention as a promising platform for the delivery of chemotherapeutic drugs. To...
The hydrophobically modified glycol chitosan (HGC) nanomicelle has received increasing attention as a promising platform for the delivery of chemotherapeutic drugs. To improve the tumor selectivity of HGC, here an avidin and biotin functionalization strategy was applied. The hydrodynamic diameter of the biotin-avidin-functionalized HGC (cy5.5-HGC-B4F) was observed to be 104.7 nm, and the surface charge was +3.1 mV. Confocal and structured illumination microscopy showed that at 0.1 mg/ml, cy5.5-HGC-B4F nanomicelles were distributed throughout the cytoplasm of MDA-MB-231 breast cancer cells after 2 h of exposure without significant cytotoxicity. To better understand the intracellular fate of the nanomicelles, entrapment studies were performed and demonstrated that some cy5.5-HGC-B4F nanomicelles were capable of escaping endocytic vesicles, likely via the proton sponge effect. Quantitative analysis of the movements of endosomes in living cells revealed that the addition of HGC greatly enhanced the motility of endosomal compartments, and the nanomicelles were transported by early and late endosomes from cell periphery to the perinuclear region. Our results validate the importance of using live-cell imaging to quantitatively assess the dynamics and mechanisms underlying the complex endocytic pathways of nanosized drug carriers.
Topics: Avidin; Biotin; Carbocyanines; Cell Line, Tumor; Cell Survival; Chitosan; Drug Carriers; Endocytosis; Endosomes; Humans; Microscopy, Fluorescence; Nanoparticles; Particle Size
PubMed: 33187397
DOI: 10.1116/6.0000380 -
Poultry Science Jul 2011The sperm storage tubules (SST) of the turkey hen, which are located in the uterovaginal junction (UVJ) of the oviduct, maintain viable sperm for up to 10 wk after a...
Oviductal expression of avidin, avidin-related protein-2, and progesterone receptor in turkey hens in relation to sperm storage: effects of oviduct tissue type, sperm presence, and turkey line.
The sperm storage tubules (SST) of the turkey hen, which are located in the uterovaginal junction (UVJ) of the oviduct, maintain viable sperm for up to 10 wk after a single insemination. The mechanisms of this in vivo sperm storage are poorly understood. Our objective was to evaluate mRNA and protein expression of avidin and 2 avidin-associated factors, avidin-related protein-2 (AVR2) and progesterone receptor, in the oviducts of 2 different lines to determine the extent to which they were sperm responsive and tissue specific. At 38 wk of age, Hybrid Grade Maker and Converter turkey hens were artificially inseminated with diluted semen (AI) or were sham-inseminated with extender alone (SI). Forty-eight hours after insemination, total RNA was extracted from the UVJ epithelium (containing SST) and vaginal epithelium (VGE) of SI and AI hens. Real time-polymerase chain reaction data showed a clear tissue region-specific effect on gene expression in the turkey hen oviduct, with much greater (P < 0.0001) expression in the UVJ compared with VGE region for avidin and AVR2 mRNA in both lines and for progesterone receptor mRNA in the Converter line. In contrast to real-time PCR data, in situ hybridization of SI and AI tissues showed that the presence of sperm increased avidin mRNA in the SST and UVJ surface epithelium in the Converter hens. Immunohistochemistry confirmed the presence of avidin protein in the epithelium of the UVJ in both lines; however, whereas avidin protein was localized in the SST of SI-Grade Maker hens, this protein was not detected in the SST of Converter hens. The upregulation of avidin and AVR2 mRNA within the sperm storage region indicates the involvement of avidin, and perhaps avidin analogs, in the sustained storage of sperm in the SST, possibly through the binding of biotin to avidin. The absence of avidin protein in the SST and VGE of Converter hens in the presence of increased mRNA may indicate a rapid turnover of protein.
Topics: Animals; Avidin; Female; Gene Expression Regulation; Immunohistochemistry; Least-Squares Analysis; Male; Oviducts; RNA, Messenger; Random Allocation; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; Spermatozoa; Turkeys
PubMed: 21673170
DOI: 10.3382/ps.2010-01159 -
Journal of Proteome Research Oct 2022It is generally believed that vascular endothelial cells (VECs) rely on glycolysis instead of the tricarboxylic acid (TCA) cycle under both normoxic and hypoxic...
It is generally believed that vascular endothelial cells (VECs) rely on glycolysis instead of the tricarboxylic acid (TCA) cycle under both normoxic and hypoxic conditions. However, the metabolic pattern of human umbilical vein endothelial cells (HUVECs) under extreme ischemia (hypoxia and nutrient deprivation) needs to be elucidated. We initiated a lethal ischemic model of HUVECs, performed proteomics and bioinformatics, and verified the metabolic pattern shift of HUVECs. Ischemic HUVECs displayed extensive aerobic respiration, including upregulation of the TCA cycle and mitochondrial respiratory chain in mitochondria and downregulation of glycolysis in cytoplasm. The TCA cycle was enhanced while the cell viability was decreased through the citrate synthase pathway when substrates of the TCA cycle (acetate and/or pyruvate) were added and vice versa when inhibitors of the TCA cycle (palmitoyl-CoA and/or avidin) were applied. The inconsistency of the TCA cycle level and cell viability suggested that the extensive TCA cycle can keep cells alive yet generate toxic substances that reduce cell viability. The data revealed that HUVECs depend on "ischemic TCA cycle" instead of glycolysis to keep cells alive under lethal ischemic conditions, but consideration must be given to relieve cell injury.
Topics: Avidin; Citrate (si)-Synthase; Citric Acid Cycle; Coenzyme A; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia; Ischemia; Pyruvic Acid; Tricarboxylic Acids
PubMed: 36074008
DOI: 10.1021/acs.jproteome.2c00255 -
European Cells & Materials Dec 2017Disease-modifying osteoarthritis drugs (DMOADs) should reach their intra-tissue target sites at optimal doses for clinical efficacy. The dense, negatively charged matrix...
Disease-modifying osteoarthritis drugs (DMOADs) should reach their intra-tissue target sites at optimal doses for clinical efficacy. The dense, negatively charged matrix of cartilage poses a major hindrance to the transport of potential therapeutics. In this work, electrostatic interactions were utilised to overcome this challenge and enable higher uptake, full-thickness penetration and enhanced retention of dexamethasone (Dex) inside rabbit cartilage. This was accomplished by using the positively charged glycoprotein avidin as nanocarrier, conjugated to Dex by releasable linkers. Therapeutic effects of a single intra-articular injection of low dose avidin-Dex (0.5 mg Dex) were evaluated in rabbits 3 weeks after anterior cruciate ligament transection (ACLT). Immunostaining confirmed that avidin penetrated the full cartilage thickness and was retained for at least 3 weeks. Avidin-Dex suppressed injury-induced joint swelling and catabolic gene expression to a greater extent than free Dex. It also significantly improved the histological score of cell infiltration and morphogenesis within the periarticular synovium. Micro-computed tomography confirmed the reduced incidence and volume of osteophytes following avidin-Dex treatment. However, neither treatment restored the loss of cartilage stiffness following ACLT, suggesting the need for a combinational therapy with a pro-anabolic factor for enhancing matrix biosynthesis. The avidin dose used caused significant glycosaminoglycan (GAG) loss, suggesting the use of higher Dex : avidin ratios in future formulations, such that the delivered avidin dose could be much less than that shown to affect GAGs. This charge-based delivery system converted cartilage into a drug depot that could also be employed for delivery to nearby synovium, menisci and ligaments, enabling clinical translation of a variety of DMOADs.
Topics: Animals; Anterior Cruciate Ligament; Anterior Cruciate Ligament Injuries; Anti-Inflammatory Agents; Avidin; Biological Transport; Cartilage, Articular; Dexamethasone; Disease Models, Animal; Drug Carriers; Drug Dosage Calculations; Female; Glycosaminoglycans; Injections, Intra-Articular; Osteoarthritis; Osteophyte; Permeability; Rabbits; Static Electricity
PubMed: 29205258
DOI: 10.22203/eCM.v034a21 -
Analytical Chemistry Sep 2011In this study, a time-of-flight secondary ion mass spectrometer TOF-SIMS, operating in the event-by-event bombardment/detection mode was used to characterize...
In this study, a time-of-flight secondary ion mass spectrometer TOF-SIMS, operating in the event-by-event bombardment/detection mode was used to characterize avidin-biotin assemblies on silane-modified glass substrates. SIMS was used to analyze several variants of the biointerface, including avidin physically adsorbed on a monofunctional acryl silane surface and covalently attached on monofunctional (amine terminated) and bifunctional (amine and acryl terminated) silanes. The goal of these studies was to determine density of avidin and biotin layers chemically or physically adsorbed on silanized glass substrate. An individual impact of a C(60) projectile used in this study creates a hemispherical crater (∼10 nm in diameter) and emits large numbers of secondary ions from the same nanovolume. Thus, a single impact enables one to unfold distinct secondary ions that span the thickness of the assembled film. This method was used to monitor the presence of glass, silane, and protein ions and to estimate the thickness and density of the avidin layer. In addition, we employed the double coincidence mass spectrometry approach to identify ions coemitted from a specific stratum of the biointerface. This approach was used to determine density of biotin and avidin immobilization while eliminating interferences from isobaric ions that originated from other constituents on the surface. Overall, novel TOF-SIMS quantitative approaches employed here were useful for examining complex biointerfaces and determining both lateral and in depth composition of the film.
Topics: Avidin; Biotin; Fullerenes; Glass; Immobilized Proteins; Silanes; Spectrometry, Mass, Secondary Ion
PubMed: 21842883
DOI: 10.1021/ac2016085 -
Biomacromolecules Oct 2021The future success of physiologically relevant three-dimensional (3D) cell/tissue models is dependent on the development of functional biomaterials, which can provide a...
The future success of physiologically relevant three-dimensional (3D) cell/tissue models is dependent on the development of functional biomaterials, which can provide a well-defined 3D environment instructing cellular behavior. To establish a platform to produce tailored hydrogels, we conjugated avidin (Avd) to anionic nanofibrillar cellulose (aNFC) and demonstrated the use of the resulting Avd-NFC hydrogel for 3D cell culture, where Avd-NFC allows easy functionalization biotinylated molecules. Avidin was successfully conjugated to nanocellulose and remained functional, as demonstrated by electrophoresis and titration with fluorescent biotin. Rheological analysis indicated that Avd-NFC retained shear-thinning and gel-forming properties. Topological characterization using AFM revealed the preserved fiber structure and confirmed the binding of biotinylated vitronectin (B-VN) on the fiber surface. The 3D cell culture experiments with mouse embryonic fibroblasts demonstrated the performance of Avd-NFC hydrogels functionalized with biotinylated fibronectin (B-FN) and B-VN. Cells cultured in Avd-NFC hydrogels functionalized with B-FN or B-VN formed matured integrin-mediated adhesions, indicated by phosphorylated focal adhesion kinase. We observed significantly higher cell proliferation rates when biotinylated proteins were bound to the Avd-NFC hydrogel compared to cells cultured in Avd-NFC alone, indicating the importance of the presence of adhesive sites for fibroblasts. The versatile Avd-NFC allows the easy functionalization of hydrogels with virtually any biotinylated molecule and may become widely utilized in 3D cell/tissue culture applications.
Topics: Animals; Avidin; Cellulose; Fibroblasts; Fibronectins; Hydrogels; Mice; Vitronectin
PubMed: 34542997
DOI: 10.1021/acs.biomac.1c00579 -
The Analyst Jul 2020We have developed an immuno-PCR based diagnostic platform which couples detection antibodies to self-assembled, ultra-detectable DNA-avidin nanoparticles stabilized with...
We have developed an immuno-PCR based diagnostic platform which couples detection antibodies to self-assembled, ultra-detectable DNA-avidin nanoparticles stabilized with poly(ethylene glycol) to link DNA amplification to target protein concentration. Electrostatic neutralization and cloaking of the PCR-amplifiable DNA labels by avidin and PEG coating reduces non-specific "stickiness" and enhances assay sensitivity. We further optimized the detectability of the nanoparticles by incorporating four repeats of a unique synthetic DNA PCR target into each nanoparticle. Using human chorionic gonadotropin hormone (hCG) as a model analyte, this platform was able to quantitate the target hCG protein in femtomolar concentrations using only standard laboratory equipment.
Topics: Antibodies; Avidin; DNA; Humans; Nanoparticles; Polymerase Chain Reaction
PubMed: 32500871
DOI: 10.1039/d0an00134a -
Nature Communications Apr 2016Biosensors based on the localized surface plasmon resonance (LSPR) of individual metallic nanoparticles promise to deliver modular, low-cost sensing with high-detection...
Biosensors based on the localized surface plasmon resonance (LSPR) of individual metallic nanoparticles promise to deliver modular, low-cost sensing with high-detection thresholds. However, they continue to suffer from relatively low sensitivity and figures of merit (FOMs). Herein we introduce the idea of sensitivity enhancement of LSPR sensors through engineering of the material dispersion function. Employing dispersion and shape engineering of chiral nanoparticles leads to remarkable refractive index sensitivities (1,091 nm RIU(-1) at λ=921 nm) and FOMs (>2,800 RIU(-1)). A key feature is that the polarization-dependent extinction of the nanoparticles is now characterized by rich spectral features, including bipolar peaks and nulls, suitable for tracking refractive index changes. This sensing modality offers strong optical contrast even in the presence of highly absorbing media, an important consideration for use in complex biological media with limited transmission. The technique is sensitive to surface-specific binding events which we demonstrate through biotin-avidin surface coupling.
Topics: Avidin; Biosensing Techniques; Biotin; Equipment Design; Gold; Metal Nanoparticles; Nanotubes; Refractometry; Surface Plasmon Resonance
PubMed: 27090866
DOI: 10.1038/ncomms11331 -
The FEBS Journal Mar 2009Novel biotin-binding proteins, referred to herein as tamavidin 1 and tamavidin 2, were found in a basidiomycete fungus, Pleurotus cornucopiae, known as the Tamogitake...
Novel biotin-binding proteins, referred to herein as tamavidin 1 and tamavidin 2, were found in a basidiomycete fungus, Pleurotus cornucopiae, known as the Tamogitake mushroom. These are the first avidin-like proteins to be discovered in organisms other than birds and bacteria. Tamavidin 1 and tamavidin 2 have amino acid sequences with 31% and 36% identity, respectively, to avidin, and 47% and 48% identity, respectively, to streptavidin. Unlike any other biotin-binding proteins, tamavidin 1 and tamavidin 2 are expressed as soluble proteins at a high level in Escherichia coli. Recombinant tamavidin 2 was purified as a tetrameric protein in a single step by 2-iminobiotin affinity chromatography, with a yield of 5 mg per 100 mL culture of E. coli. The kinetic parameters measured by a BIAcore biosensor indicated that recombinant tamavidin 2 binds biotin with high affinity, in a similar manner to binding by avidin and streptavidin. The overall crystal structure of recombinant tamavidin 2 is similar to that of avidin and streptavidin. However, recombinant tamavidin 2 is immunologically distinct from avidin and streptavidin. Tamavidin 2 and streptavidin are very similar in terms of the arrangement of the residues interacting with biotin, but different with regard to the number of hydrogen bonds to biotin carboxylate. Recombinant tamavidin 2 is more stable than avidin and streptavidin at high temperature, and nonspecific binding to DNA and human serum by recombinant tamavidin 2 is lower than that for avidin. These findings highlight tamavidin 2 as a probable powerful tool, in addition to avidin and streptavidin, in numerous applications of biotin-binding proteins.
Topics: Amino Acid Sequence; Avidin; Binding Sites; Biotin; Carrier Proteins; Crystallography, X-Ray; Escherichia coli; Fungal Proteins; Kinetics; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Molecular Weight; Pleurotus
PubMed: 19187241
DOI: 10.1111/j.1742-4658.2009.06879.x -
The Journal of Investigative Dermatology Sep 1984Avidin conjugated to the fluorescent dyes rhodamine or fluorescein binds to mast cell granules in rodent and human skin. Sequential staining of tissue mast cells first... (Comparative Study)
Comparative Study
Avidin conjugated to the fluorescent dyes rhodamine or fluorescein binds to mast cell granules in rodent and human skin. Sequential staining of tissue mast cells first with conjugated avidin, and then with a metachromatic stain demonstrated that both techniques identify the same mast cell granules. Specificity for mast cells was confirmed by the absence of avidin-positive cells in the skin of mast cell-deficient (W/Wv) mice. Binding of conjugated avidin to mast cells was inhibited by pretreating tissue specimens with unconjugated avidin but not by pretreating conjugated avidin with biotin, indicating that avidin does not bind to biotin or a biotin-like molecule. Within murine dermis, unique patterns of mast cell distributions were observed, with a prominent perivascular localization in ear skin, and a complete absence of mast cells underlying the scales in tail skin. In tissue sections of guinea pig skin undergoing basophil hypersensitivity reactions and in murine and human skin specimens infiltrated with eosinophils, conjugated avidin selectively stained only dermal mast cells, demonstrating specificity for mast cells in sites of inflammation. Conjugated avidin also readily stained rat peritoneal mast cells, demonstrating its utility for identifying extracutaneous mast cells. Unlike the metachromatic stains, avidin binding to mast cells in tissues is not limited by methods of fixation or special embedding and cutting procedures. Thus, mast cell identification with conjugated avidin is a reliable, specific, and simple method with important clinical and investigative applications.
Topics: Animals; Ascites; Avidin; Fluorescent Dyes; Guinea Pigs; Humans; Mast Cells; Mice; Mice, Inbred BALB C; Ovalbumin; Rats; Rats, Inbred Strains; Skin; Species Specificity
PubMed: 6470526
DOI: 10.1111/1523-1747.ep12263584