Macrophage membrane (MMs) camouflaged near-infrared (NIR) responsive bone defect area targeting nanocarrier delivery system (BTNDS) for rapid repair: promoting osteogenesis via phototherapy and modulating immunity.

Bone defects remain a significant challenge in clinical orthopedics, but no targeted medication can solve these problems. Inspired by inflammatory targeting properties of macrophages, inflammatory microenvironment of bone defects was exploited to develop a multifunctional nanocarrier capable of targeting bone defects and promoting bone regeneration. The avidin-modified black phosphorus nanosheets (BP-Avidin, BPAvi) were combined with biotin-modified Icaritin (ICT-Biotin, ICTBio) to synthesize Icaritin (ICT)-loaded black phosphorus nanosheets (BPICT). BPICT was then coated with macrophage membranes (MMs) to obtain MMs-camouflaged BPICT (M@BPICT). Herein, MMs allowed BPICT to target bone defects area, and BPICT accelerated the release of phosphate ions (PO43-) and ICT when exposed to NIR irradiation. PO43- recruited calcium ions (Ca2+) from the microenvironment to produce Ca3(PO4)2, and ICT increased the expression of osteogenesis-related proteins. Additionally, M@BPICT can decrease M1 polarization of macrophage and expression of pro-inflammatory factors to promote osteogenesis. According to the results, M@BPICT provided bone growth factor and bone repair material, modulated inflammatory microenvironment, and activated osteogenesis-related signaling pathways to promote bone regeneration. PTT could significantly enhance these effects. This strategy not only offers a solution to the challenging problem of drug-targeted delivery in bone defects but also expands the biomedical applications of MMs-camouflaged nanocarriers.

Biotinylated Quinone as a Chemiluminescence Sensor for Biotin-Avidin Interaction and Biotin Detection Application.

Biotin, or vitamin B7, is essential for metabolic reactions. It must be obtained from external sources such as food and biotin/vitamin supplements because it is not biosynthesized by mammals. Therefore, there is a need to monitor its levels in supplements. However, biotin detection methods, which include chromatographic, immune, enzymatic, and microbial assays, are tedious, time-consuming, and expensive. Thus, we synthesized a product called biotin-naphthoquinone, which produces chemiluminescence upon its redox cycle reaction with dithiothreitol and luminol; then it was used as a chemiluminescence sensor for biotin-avidin interaction. When a quinone biotinylated compound binds avidin, the chemiluminescence decreases noticeably due to the proximity between quinone and avidin, and when free biotin is added in a competitive assay, the chemiluminescence returns. The chemiluminescence is regained as the free biotin displaces biotinylated quinone in its complex with avidin, freeing biotin-naphthoquinone. Many experiments, including the use of a biotin-free quinone, proved the competitive nature of the assay. The competitive assay method used in this study was linear in the range of 1.0-100 µM with a detection limit of 0.58 µM. The competitive chemiluminescence assay could detect biotin in vitamin B7 tablets with good recovery of 91.3 to 110% and respectable precision (RSD < 8.7%).

Immune Cells Activating Biotin-Decorated PLGA Protein Carrier.

Nanoparticle formulations have long been proposed as subunit vaccine carriers owing to their ability to entrap proteins and codeliver adjuvants. Poly(lactic-co-glycolic acid) (PLGA) remains one of the most studied polymers for controlled release and nanoparticle drug delivery, and numerous studies exist proposing PLGA particles as subunit vaccine carriers. In this work we report using PLGA nanoparticles modified with biotin (bNPs) to deliver proteins via adsorption and stimulate professional antigen-presenting cells (APCs). We present evidence showing bNPs are capable of retaining proteins through the biotin-avidin interaction. Surface accessible biotin bound both biotinylated catalase (bCAT) through avidin and streptavidin horseradish peroxidase (HRP). Analysis of the HRP found that activity on the bNPs was preserved once captured on the surface of bNP. Further, bNPs were found to have self-adjuvant properties, evidenced by bNP induced IL-1ß, IL-18, and IL-12 production in vitro in APCs, thereby licensing the cells to generate Th1-type helper T cell responses. Cytokine production was reduced in avidin precoated bNPs (but not with other proteins), suggesting that the proinflammatory response is due in part to exposed biotin on the surface of bNPs. bNPs injected subcutaneously were localized to draining lymph nodes detectable after 28 days and were internalized by bronchoalveolar lavage dendritic cells and macrophages in mice in a dose-dependent manner when delivered intranasally. Taken together, these data provide evidence that bNPs should be explored further as potential adjuvanting carriers for subunit vaccines.

DNA building blocks for AFM tip functionalization: An easy, fast and stable strategy.

Biosensing atomic force microscopy (AFM) offers the unique feature to determine the energy landscape of a bimolecular interaction at the real single molecule level. Furthermore, simultaneous and label-free mapping of molecular recognition and the determination of sample topography at the nanoscale gets possible. A prerequisite and one of the major parts in biosensing AFM are the bio-functionalized AFM tips. In the past decades, different approaches for tip functionalization have been developed. Using these functionalization strategies, several biological highly relevant interactions at the single molecule level have been explored. For the most common approach, the use of a heterobifunctional poly(ethylenglycol) crosslinker, a broad range of linkers for different chemical coupling strategies is available. Nonetheless, the time consuming functionalization protocol as well as the broad distribution of rupture length reduces the possibility of automation and may reduce the accuracy of the results. Here we present a stable and fast forward approach based on tetra-functional DNA tetrahedra. A fast functionalization and a sharp defined distribution of rupture length gets possible with low effort and high success rate. We tested the performance on the classical avidin biotin system by using tetrahedra with three disulfide legs for stable and site directed coupling to gold coated tips and a biotinylated end at the fourth vertex. A special advantage appears when working with a DNA aptamer as sensing molecule. In this case, the fourth strand can be extended by a certain DNA sequence complementary to the linkage part of an aptamer. This AFM tip functionalization protocol was applied on thrombin using DNA aptamers directed against the fibrinogen binding side of human thrombin.

The maturation of DNA encoded libraries: opportunities for new users.

DNA-encoded combinatorial libraries (DECLs) represent an exciting new technology for high-throughput screening, significantly increasing its capacity and cost-effectiveness. Historically, DECLs have been the domain of specialized academic groups and industry; however, there has recently been a shift toward more drug discovery academic centers and institutes adopting this technology. Key to this development has been the simplification, characterization and standardization of various DECL subprotocols, such as library design, affinity screening and data analysis of hits. This review examines the feasibility of implementing DECL screening technology as a first-time user, particularly in academia, exploring the some important considerations for this, and outlines some applications of the technology that academia could contribute to the field.

Preparation of Peptide and Recombinant Tissue Plasminogen Activator Conjugated Poly(Lactic-Co-Glycolic Acid) (PLGA) Magnetic Nanoparticles for Dual Targeted Thrombolytic Therapy.

Recombinant tissue plasminogen activator (rtPA) is the only thrombolytic agent that has been approved by the FDA for treatment of ischemic stroke. However, a high dose intravenous infusion is required to maintain effective drug concentration, owing to the short half-life of the thrombolytic drug, whereas a momentous limitation is the risk of bleeding. We envision a dual targeted strategy for rtPA delivery will be feasible to minimize the required dose of rtPA for treatment. For this purpose, rtPA and fibrin-avid peptide were co-immobilized to poly(lactic-co-glycolic acid) (PLGA) magnetic nanoparticles (PMNP) to prepare peptide/rtPA conjugated PMNPs (pPMNP-rtPA). During preparation, PMNP was first surface modified with avidin, which could interact with biotin. This is followed by binding PMNP-avidin with biotin-PEG-rtPA (or biotin-PEG-peptide), which was prepared beforehand by binding rtPA (or peptide) to biotin-PEG-maleimide while using click chemistry between maleimide and the single -SH group in rtPA (or peptide). The physicochemical property characterization indicated the successful preparation of the magnetic nanoparticles with full retention of rtPA fibrinolysis activity, while biological response studies underlined the high biocompatibility of all magnetic nanoparticles from cytotoxicity and hemolysis assays in vitro. The magnetic guidance and fibrin binding effects were also confirmed, which led to a higher thrombolysis rate in vitro using PMNP-rtPA or pPMNP-rtPA when compared to free rtPA after static or dynamic incubation with blood clots. Using pressure-dependent clot lysis model in a flow system, dual targeted pPMNP-rtPA could reduce the clot lysis time for reperfusion by 40% when compared to free rtPA at the same drug dosage. From in vivo targeted thrombolysis in a rat embolic model, pPMNP-rtPA was used at 20% of free rtPA dosage to restore the iliac blood flow in vascular thrombus that was created by injecting a blood clot to the hind limb area.

Versatile nano-platform for tailored immuno-magnetic carriers.

Custom immuno-magnetic devices are desirable tools for biomedical and biotechnological applications. Herein, surface active maghemite nanoparticles (SAMNs) are proposed as a versatile platform for developing tailored immuno-magnetic nano-carriers by simple wet reactions. Two examples for conjugating native and biotinylated antibodies were presented along with their successful applications in the recognition of specific foodborne pathogens. Nanoparticles were functionalized with rhodamine B isothiocyanate (RITC), leading to a fluorescent nano-conjugate, and used for binding anti-Campylobacter fetus antibodies (SAMN@RITC@Anti-Cf). The microorganism was selectively captured in the presence of two other Campylobacter species (C. jejuni and C. coli), as verified by PCR. Alternatively, SAMNs were modified with avidin, forming a biotin-specific magnetic nano-carrier and used for the immobilization of biotinylated anti-Listeria monocytogenes antibodies (SAMN@avidin@Anti-Lm). This immuno-magnetic carrier was integrated in piezoelectric quartz crystal microbalance (QCM) sensor for the detection of L. monocytogenes in milk, showing a detection limit of 3 bacterial cells. The present work presents a new category of customized immuno-magnetic nano-carriers as a competitive option for suiting specific applications. Graphical abstract ᅟ.

Genome-Wide Profiling of DNA Methyltransferases in Mammalian Cells.

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is currently the method of choice to determine binding sites of chromatin-associated factors in a genome-wide manner. Here, we describe a method to investigate the binding preferences of mammalian DNA methyltransferases (DNMT) based on ChIP-seq using biotin-tagging. Stringent ChIP of DNMT proteins based on the strong interaction between biotin and avidin circumvents limitations arising from low antibody specificity and ensures reproducible enrichment. DNMT-bound DNA fragments are ligated to sequencing adaptors, amplified and sequenced on a high-throughput sequencing instrument. Bioinformatic analysis gives valuable information about the binding preferences of DNMTs genome-wide and around promoter regions. This method is unconventional due to the use of genetically engineered cells; however, it allows specific and reliable determination of DNMT binding.

Avidin/Biotin Bioinspired Platform for Dual In Vivo 18F-PET/NIRF Molecular Imaging.

The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of innovative multimodal PET/NIRF probes a very exciting prospect. Herein, the bioinspired design of novel platform exploiting the strength and specificity of interactions between radioactive and fluorescent biotin derivatives and an avidin core is reported. The combination of an original [18F]fluoropyridinylated-biotin derivative and commercially available fluorescent biotin derivatives (Atto-425 and Atto-680) is investigated. The in vivo distribution of such a customized platform is also reported, for the first time, in healthy rodent using PET and ex vivo fluorescence imaging.

Magnetically responsive microbubbles as delivery vehicles for targeted sonodynamic and antimetabolite therapy of pancreatic cancer.

Magnetically responsive microbubbles (MagMBs), consisting of an oxygen gas core and a phospholipid coating functionalised with Rose Bengal (RB) and/or 5-fluorouracil (5-FU), were assessed as a delivery vehicle for the targeted treatment of pancreatic cancer using combined antimetabolite and sonodynamic therapy (SDT). MagMBs delivering the combined 5-FU/SDT treatment produced a reduction in cell viability of over 50% when tested against a panel of four pancreatic cancer cell lines in vitro. Intravenous administration of the MagMBs to mice bearing orthotopic human xenograft BxPC-3 tumours yielded a 48.3% reduction in tumour volume relative to an untreated control group (p<0.05) when the tumour was exposed to both external magnetic and ultrasound fields during administration of the MagMBs. In contrast, application of an external ultrasound field alone resulted in a 27% reduction in tumour volume. In addition, activated caspase and BAX protein levels were both observed to be significantly elevated in tumours harvested from animals treated with the MagMBs in the presence of magnetic and ultrasonic fields when compared to expression of those proteins in tumours from either the control or ultrasound field only groups (p<0.05). These results suggest MagMBs have considerable potential as a platform to enable the targeted delivery of combined sonodynamic/antimetabolite therapy in pancreatic cancer.

Anti-FGFR1 aptamer-tagged superparamagnetic conjugates for anticancer hyperthermia therapy.

Compounds that recognize and strongly bind to molecular targets are one of the cornerstones of modern pharmaceutics. Work has been ongoing for the past 25 years on the therapeutic use of aptamers, nucleic acid molecules, whose three-dimensional structure is the result of interactions between complementary base pairs. The aptamers selection methods allow the oligonucleotides which bind the molecular target in its native environment to be quickly isolated from a large library of random oligonucleotides. The possibilities presented for aptamers in the field of targeted therapy require the application of effective carriers to counter the renal clearance effect and/or functional cargo to exert therapeutic action if the aptamer is only used as a targeting moiety. Lately, a material gaining ground in biomedical research is iron oxide particles, which exhibit a superparamagnetic characteristic at nanoscale levels. This allows the iron oxide nanoparticles to convert external magnetic energy into heat, a mechanism known as hyperthermy, and efficiently supports conventional oncological treatment. In this study, we describe an experimentally confirmed functional model of targeted anticancer hyperthermia therapy. Using the systematic evolution of ligands by exponential enrichment technique, we selected a DNA aptamer that specifically binds to the extracellular domain of recombinant fibroblast growth factor receptor type-1 (FGFR1) with a nanomolar dissociation constant. The chosen target plays an important role in many crucial cellular processes and is also considered a candidate protein that is involved in tumor initiation, survival and progression. Next, we combined the selected aptamer with iron oxide nanoparticles to produce aptamer superparamagnetic conjugates (ASCs). Finally, we found that targeted ASCs selectively destroy FGFR1-overexpressing human osteosarcoma cells U2OS upon magnetic field irradiation.

Synthetic mimics of biotin/(strept)avidin.

Biotin/(strept)avidin self-assembly is a powerful platform for nanoscale fabrication and capture with many different applications in science, medicine, and nanotechnology. However, biotin/(strept)avidin self-assembly has several well-recognized drawbacks that limit performance in certain technical areas and there is a need for synthetic mimics that can either become superior replacements or operational partners with bio-orthogonal recognition properties. The goal of this tutorial review is to describe the recent progress in making high affinity synthetic association partners that operate in water or biological media. The review starts with a background summary of biotin/(strept)avidin self-assembly and the current design rules for creating synthetic mimics. A series of case studies are presented that describe recent success using synthetic derivatives of cyclodextrins, cucurbiturils, and various organic cyclophanes such as calixarenes, deep cavitands, pillararenes, and tetralactams. In some cases, two complementary partners associate to produce a nanoscale complex and in other cases a ditopic host molecule is used to link two partners. The article concludes with a short discussion of future directions and likely challenges.

Highly sensitive avidin-biotin ELISA for detection of nandrolone and testosterone in dietary supplements.

Avidin-biotin technology was used for the implementation of an enzyme-linked immunosorbent assay (AB-ELISA) as a sensitive method for the detection of anabolic androgenic steroids (AAS) present in dietary supplements. Using click chemistry, novel haptens (linker-optimized biotinylated nandrolone (NT) and testosterone (T) at positions C-3 and C-17, respectively) were designed and synthesized to be then applied as four different immobilized competitors in a proposed set of four indirect competitive AB-ELISAs. Four rabbit polyclonal antibodies of various specificities were prepared using four different immunogens synthesized from C-3 and C-17 carboxymethyloxime and hemisuccinate derivatives of NT and T, respectively. Assembled AB-ELISAs were characterized to establish method parameters such as a half-maximum inhibition concentration (0.18-12.99 ng/mL), limit of detection (0.004-0.032 ng/mL) and linear working range (the best with 0.02-1.38 ng/mL). The stability of the set simulating storage in different conditions was demonstrated. Cross reactivity (CR) was tested for 59 steroids including both endogenous and synthetic analogues in four assembled AB-systems. The focus was placed on the practical use of the method in detection of various AAS in 49 samples of counterfeit dietary supplements. The concordance between ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the CR corrected data from AB-ELISA indicated the potential of this method even to quantification of T propionate, NT phenyl propionate, and NT decanoate in such a complex matter. Copyright © 2016 John Wiley & Sons, Ltd.

Coil-like Enzymatic Biohybrid Structures Fabricated by Rational Design: Controlling Size and Enzyme Activity over Sequential Nanoparticle Bioconjugation and Filtration Steps.

Well-defined enzymatic biohybrid structures (BHS) composed of avidin, biotinylated poly(propyleneimine) glycodendrimers, and biotinylated horseradish peroxidase were fabricated by a sequential polyassociation reaction to adopt directed enzyme prodrug therapy to protein-glycopolymer BHS for potential biomedical applications. To tailor and gain fundamental insight into pivotal properties such as size and molar mass of these BHS, the dependence on the fabrication sequence was probed and thoroughly investigated by several complementary methods (e.g., UV/vis, DLS, cryoTEM, AF4-LS). Subsequent purification by hollow fiber filtration allowed us to obtain highly pure and well-defined BHS. Overall, by rational design and control of preparation parameters, e.g., fabrication sequence, ligand-receptor stoichiometry, and degree of biotinylation, well-defined BHS with stable and even strongly enhanced enzymatic activities can be achieved. Open coil-like structures of BHS with few branches are available by the sequential bioconjugation approach between synthetic and biological macromolecules possessing similar size dimensions.

Lentiavidins: Novel avidin-like proteins with low isoelectric points from shiitake mushroom (Lentinula edodes).

A biotin-binding protein with a low isoelectric point (pI), which minimizes electrostatic non-specific binding to substances other than biotin, is potentially valuable. To obtain such a protein, we screened hundreds of mushrooms, and detected strong biotin-binding activity in the fruit bodies of Lentinula edodes, shiitake mushroom. Two cDNAs, each encoding a protein of 152 amino acids, termed lentiavidin 1 and lentiavidin 2 were cloned from L. edodes. The proteins shared sequence identities of 27%-49% with other biotin-binding proteins, and many residues that directly associate with biotin in streptavidin were conserved in lentiavidins. The pI values of lentiavidin 1 and lentiavidin 2 were 3.9 and 4.4, respectively; the former is the lowest pI of the known biotin-binding proteins. Lentiavidin 1 was expressed as a tetrameric protein with a molecular mass of 60 kDa in an insect cell-free expression system and showed biotin-binding activity. Lentiavidin 1, with its pI of 3.9, has a potential for broad applications as a novel biotin-binding protein.

Theranostic Polymeric Micelles for the Diagnosis and Treatment of Hepatocellular Carcinoma.

Theranostics, which combine molecular imaging (diagnostics) and drug delivery (therapeutics) in a single platform, have recently shown great potential in cancer therapy. In this article, a polymeric micelle was designed and prepared for simultaneous magnetic resonance imaging (MRI) and treatment of hepatocellular carcinoma (HCC). Theranostic micelles were assembled using Poly(lactic acid)-poly(ethylene glycol)-poly(L-lysine)-diethylenetriamine pentaacetic acid (PLA-PEG-PLL-DTPA) and PLA-PEG-PLL-Biotin. The HCC therapeutic paclitaxel (PTX) was encapsulated in the cores and Gd ions for imaging were chelated to the DTPA moieties. Biotinylated alpha-fetoprotein (AFP) antibodies were linked to the micelle surface by a biotin-avidin reaction to form targeted Gd/PTX-loaded micelles (TGPM). TGPM were of spherical or ellipsoidal shape with uniform particle size distribution (147.50 ± 4.71 nm), positive zeta potential (24.45 ± 1.04 mV), and high encapsulation efficiency (88.76 ± 1.64%) and drug loading (1.59 ± 0.06%). The cytotoxicity of TGPM in HepG2 cells was superior to that of Taxol or Gd/PTX-loaded micelles (GPM). In MRI tests in vitro, the T1 relaxivity of TGPM was 21.589 mM(-1) s(-1), 4.4 times higher than Magnevist (r1 = 4.8 mM(-1) s(-1)). In H22 tumor-bearing mice, TGPM significantly increased tumor imaging intensity (more than 3 times) and prolonged imaging time (from 1 to 6 h) compared to Magnevist. In vivo, TGPM exhibited higher anti-tumor efficiency than Taxol and GPM. These results indicate that TGPM has great potential in HCC theranostics.

Effect of biotin and pantothenic acid on performance and concentrations of avidin-binding substances in blood and milk of lactating dairy cows.

We hypothesized that pantothenic acid reduces the absorption of biotin in lactating dairy cows. Therefore, the objective of this study was to evaluate the plausible interaction between biotin and pantothenic acid on production performance and concentration of avidin-binding substances (ABS), an indicator of biotin concentration, in blood and milk of lactating dairy cows. Eight primiparous and 16 multiparous Holstein cows were assigned to 1 of 4 diet sequences in a replicated 4×4 Latin square design with 18-d periods. Cows were housed in a freestall barn and fed once daily (0730 h) by means of a Calan gate system (American Calan Inc., Northwood, NH). Treatments consisted of a control diet that contained no B-vitamins, a biotin diet that contained 0.87 mg of biotin per kilogram of dry matter (DM), a pantothenic acid diet that contained 21 mg of pantothenic acid per kilogram of DM, and a biotin plus pantothenic acid diet that contained 0.87 mg of biotin and 21 mg of calcium pantothenic acid per kilogram of DM. Four different concentrates were prepared in a commercial feed mill. These concentrates were mixed with corn silage and grass hay and delivered ad libitum as a total mixed ration. Biotin supplementation did not affect DM intake, milk yield, or milk fat, protein, lactose, and milk-urea-nitrogen concentrations. Fat, protein, and lactose yields were not affected by treatments. The fat-to-protein ratio was <1 and similar among all treatments. Biotin supplementation did not increase the concentration of ABS in plasma. The supplementation of pantothenic acid did not affect the concentration of ABS in plasma when either supplemented alone or in combination with biotin. Biotin supplementation increased the concentration of ABS in milk relative to control. Contrary to our hypothesis, the supplementation of pantothenic acid did not decrease the concentration of ABS in milk relative to the control. When cows were supplemented with both biotin and pantothenic acid, the concentration of ABS in milk was similar to that of cows supplemented with biotin alone. In conclusion, pantothenic acid did not affect the concentrations of ABS in plasma and milk, suggesting that increasing dietary supply of pantothenic acid did not inhibit biotin absorption.

Chemiluminescence-imaging detection of DNA on a solid-phase membrane by using a peroxidase-labeled macromolecular probe.

We have developed a novel method for sensitive chemiluminescence (CL)-imaging detection of DNA by using a macromolecular probe synthesized by attaching multiple molecules of horseradish peroxidase (HRP) and biotin in dextran backbone. The probe formed a macromolecular assembly by binding to streptavidin which specifically recognized biotinylated complementary DNA, which was hybridized to a target DNA on a solid-phase membrane. This methodology was applied to CL-imaging detection of a synthetic telomere DNA (TTAGGG)10 and human telomere DNA by using the CL probe comprising of dextranT2000 (MW=ca. 2000kDa) bonded to approximately 42 molecules of HRP and 210 molecules of biotin. The human telomere DNA in a small number of buccal mucous cells (ca. 70 cell numbers) of cheek tissue was quantitatively determined by the proposed CL detection method that afforded approximately 10 times higher sensitivity than that of the conventional CL method using commercially available HRP-avidin probe.

Oral administration of supplementary biotin differentially influences the fertility rate and oviductal expression of avidin and avidin-related protein-2 in low- and high-fertility broiler line hens.

Probable involvement of avidin and avidin-related protein-2 (AVR2) in sperm viability in the sperm storage tubules of turkeys has been suggested. The high affinity of biotin to avidin and its analogs is also well documented. The present study aimed to determine the effect of oral biotin on reproductive performance and oviductal mRNA expression of avidin and AVR2 in 2 broiler hen lines with different fertility rates. Low-fertility (line B) and high-fertility (line D) hens (n=144) were randomly allotted to receive 0 (T0), 0.30 (T1), or 0.45 (T2) mg/L biotin in drinking water from 30 through 33 wk of age. The reproductive performance of the hens was evaluated using artificial insemination. At the end of the treatment period, 24 hens per line were killed to assay the expression of avidin and AVR2 in the uterovaginal junction. Supplementary biotin increased egg production from 73.5% for T0 to 87.8% for T2. Hens administered with biotin in line B, but not in line D, showed an increase (8.4%) in fertility rate. Hatchability, chick quality, and overall embryonic mortality were not different among the experimental groups. Real-time PCR data showed that both avidin (P=0.0013) and AVR2 (P<0.0001) expressions were influenced by a biotin×line interaction effect, where low-fertility line B hens receiving the high biotin level recorded respectively a 3.9 and 15.3% increase in avidin and AVR2 mRNA expression, although biotin did not affect these traits in line D hens. Control hens in line D had a dramatically higher AVR2 expression record (7.4-fold) compared with the control hens in line B. The correlation coefficients of fertility rate and avidin expression were 0.73 and 0.66 in lines B and D, respectively. However, the correlation of fertility and AVR2 (r=0.65) was significant for line D hens only. Overall, fertility rate and oviductal expression of avidin and AVR2 were dichotomously affected by oral biotin in low- and high-fertility line hens, where only low-fertility birds showed improvements in these attributes.