Avidin-biotin Conjugate Research Articles

Decreased Krüppel-like factor 4 in adenomyosis impairs decidualization by repressing autophagy in human endometrial stromal cells

BackgroundPoor decidualization and abnormal autophagy conditions in the endometria of adenomyosis patients have been reported previously. However, the specific regulatory mechanism of decidualization in adenomyosis and its relationship with autophagy levels have not been clarified.MethodsEndometrial tissues from adenomyosis patients and uteri from an adenomyosis mouse model were collected for the detection of different expression patterns of KLF4 and autophagy markers (LC3-B/LC3-A and Beclin-1) compared with control groups. Human endometrial stromal cells (hESCs) isolated from adenomyosis and control endometrial tissues were employed to elucidate the biological functions of KLF4 in autophagy and decidualization. Gene expression regulation was examined by quantitative real-time PCR (qRT-PCR), western blotting and luciferase reporter assays. In addition, DNA promoter-protein interactions were examined by chromatin immunoprecipitation (ChIP)/PCR assay and avidin–biotin conjugate DNA precipitation (ABCD) assay.ResultsKLF4 expression was decreased in endometrial tissues from adenomyosis patients compared with those from fertile controls, especially in stromal compartments. The opposite results were observed for autophagy marker (LC3-B/LC3-A and Beclin-1) expression. At the same time, KLF4 reversed the poor decidualization of hESCs from adenomyosis patients. In addition, KLF4 could induce hESC decidualization by promoting the autophagy level. Mechanistically, KLF4 bound to a conserved site in the autophagy-related 5 (ATG5) promoter region and promoted ATG5 expression. Similar expression patterns of KLF4 and autophagy markers were detected in adenomyotic mice.ConclusionsKLF4 overexpression increases the autophagy level of hESCs by transcriptionally promoting ATG5 expression, and abnormally decreased KLF4 in adenomyosis impairs hESC decidualization by repressing autophagy.

Immunohistochemistry of Immune Cells and Cells Bound to in vivo Administered Antibodies in Liver, Lung, Pancreas, and Colon of B6/lpr Mice.

Employing a novel mouse model of immune related adverse events (irAEs) induced by combination of anti-PD1 and anti-CTLA-4 antibodies, we visualized immune infiltration into the liver, lung, pancreas, and colon. Here, we describe the avidin-biotin conjugate (ABC) method used to stain T cells (CD4 and CD8), B cells (CD19), macrophages (F4/80), and cells bound by the in vivo administered rat anti-mouse antibodies for chromogenic immunohistochemistry (IHC). Using a biotinylated goat anti-rat antibody, we detected the localization of cells bound to the in vivo antibodies for PD-1 and CTLA-4. IHC has advantages over other techniques, namely antibody availability, resistance to photobleaching, and greater sensitivity. Additionally, detection and localization of in vivo antibodies can be used in mice models to infer their therapeutic efficacy, stability, and function. Graphical abstract.

Surface functionalization of dual growth factor on hydroxyapatite-coated nanofibers for bone tissue engineering

To improve bone growth and to imitate the function of natural extracellular matrix for sustained release of multiple growth factors, a scaffold of porous gelatin nanofibers were electrospun, coated with hydroxyapatite using a simulated body fluid solution, and surface-functionalized with avidin to facilitate binding with biotinylated growth factors, namely bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2) at different ratios. Scanning electron microscopy was used for structure characterization and Fourier-transform infrared spectroscopy and calcium assay were performed to evaluate the calcium composition. Immunostaining techniques confirmed the conjugation of multiple growth factors. The effects of the two growth factors and hydroxyapatite on osteogenic cell differentiation were studied using quantitative polymerase chain reaction analysis. Multiple growth factors were successfully conjugated onto the functionalized surface by controlling the FGF-2/BMP-2 ratio. Comparisons of the factor release profiles with those of the physical adsorption showed that avidin-biotin conjugation was more effective for sustained release. Bone regeneration was enhanced via synergism between multiple growth factor delivery and the hydroxyapatite nanofiber coating, as confirmed by the increased expression of osteogenic gene markers. The nanofibers thus provide a promising osteoconductive scaffold with controlled multiple growth factor delivery for bone tissue engineering.

Multivalent Traptavidin-DNA Conjugates for the Programmable Assembly of Nanostructures.

Here, we explore the extended utility of two important functional biomolecules, DNA and protein, by hybridizing them through avidin-biotin conjugation. We report a simple yet scalable technique of successive magnetic separations to synthesize traptavidin-DNA conjugates with four distinct DNA binding sites that can be used as a supramolecular building block for programmable assembly of nanostructures. Using this nanoassembly platform, we fabricate several different plasmonic nanostructures with various metallic as well as semiconductor nanoparticles in predetermined ways. We also use the platform to construct dendrimer nanostructures using valency-controlled traptavidin-DNA conjugates in a programmable manner. These results suggest that our protein-DNA supramolecular building blocks would make a significant contribution to the assembly of multicomponent and complex nanostructures for numerous contemporary and future applications from molecular imaging to drug delivery.

Krüppel-like factor 12 is a novel negative regulator of forkhead box O1 expression: a potential role in impaired decidualization.

BackgroundDecidualization is a prerequisite for successful implantation and the establishment of pregnancy. Krüppel-like factor 12 (KLF12) is a negative regulator of endometrial decidualization in vitro. We investigated whether KLF12 was associated with impaired decidualization under conditions of repeated implantation failure (RIF).MethodsUterine tissues were collected from a mouse model of early pregnancy and artificial decidualization for immunohistochemistry, Western blot and real-time PCR analysis. Reporter gene assays, chromatin immunoprecipitation-PCR and avidin-biotin conjugate DNA precipitation assays were performed to analyze the transcriptional regulation of forkhead box O1 (FOXO1) by KLF12. Furthermore, the protein levels of KLF12 and FOXO1 in patients with RIF were analyzed by Western blot and immunohistochemistry.ResultsKLF12 led to defective implantation and decidualization in the mouse uterine model of early pregnancy and artificial decidualization by directly binding to the FOXO1 promoter region and inhibiting its expression in human endometrial stromal cells. Elevated KLF12 expression was accompanied by decreased FOXO1 expression in the endometria of patients with RIF.ConclusionsAs a novel regulator, KLF12 predominantly controls uterine endometrial differentiation during early pregnancy and leads to implantation failure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-015-0079-z) contains supplementary material, which is available to authorized users.

A magnetic nanobead‐based bioassay provides sensitive detection of single‐ and biplex bacterial DNA using a portable AC susceptometer

Bioassays relying on magnetic read-out using probe-tagged magnetic nanobeads are potential platforms for low-cost biodiagnostic devices for pathogen detection. For optimal assay performance it is crucial to apply an easy, efficient and robust bead-probe conjugation protocol. In this paper, sensitive (1.5 pM) singleplex detection of bacterial DNA sequences is demonstrated in a portable AC susceptometer by a magnetic nanobead-based bioassay principle; the volume-amplified magnetic nanobead detection assay (VAM-NDA). Two bead sizes, 100 and 250 nm, are investigated along with a highly efficient, rapid, robust, and stable conjugation chemistry relying on the avidin–biotin interaction for bead-probe attachment. Avidin-biotin conjugation gives easy control of the number of detection probes per bead; thus allowing for systematic investigation of the impact of varying the detection probe surface coverage upon bead immobilization in rolling circle amplified DNA-coils. The existence of an optimal surface coverage is discussed. Biplex VAM-NDA detection is for the first time demonstrated in the susceptometer: Semi-quantitative results are obtained and it is concluded that the concentration of DNA-coils in the incubation volume is of crucial importance for target quantification. The present findings bring the development of commercial biodiagnostic devices relying on the VAM–NDA further towards implementation in point-of-care and outpatient settings.

Electrochemical DNA biosensor based on avidin–biotin conjugation for influenza virus (type A) detection

An electrochemical DNA biosensor (E-DNA biosensor) was fabricated by avidin–biotin conjugation of a biotinylated probe DNA, 5′-biotin-ATG AGT CTT CTA ACC GAG GTC GAA-3′, and an avidin-modified glassy carbon electrode (GCE) to detect the influenza virus (type A). An avidin-modified GCE was prepared by the reaction of avidin and a carboxylic acid-modified GCE, which was synthesized by the electrochemical reduction of 4-carboxyphenyl diazonium salt. The current value of the E-DNA biosensor was evaluated after hybridization of the probe DNA and target DNA using cyclic voltammetry (CV). The current value decreased after the hybridization of the probe DNA and target DNA. The DNA that was used follows: complementary target DNA, 5′-TTC GAC CTC GGT TAG AAG ACT CAT-3′ and two-base mismatched DNA, 5′-TTC GAC AGC GGT TAT AAG ACT CAT-3′.

Liposome−Hydrogel Bead Complexes Prepared via Biotin−Avidin Conjugation

Liposomes immobilized onto polymeric hydrogel microbeads have potential advantages both in tissue engineering applications and as drug delivery vehicles. Here we demonstrate, quantify, and optimize lipid vesicle binding to polymeric hydrogel microbeads via the avidin-biotin conjugation system and characterize the stability of the resulting microgel-bound liposomes. Microgels consisting of a copolymer of N-isopropylacrylamide (NIPAM) and acrylic acid (AA), cross-linked with bis-acrylamide, that is, p(NIPAM-co-AA), were biotinylated using aqueous carbodiimide chemistry. Extruded liposomes consisting of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) plus a small fraction of a biotin-derivatized phosphatidylethanolamine (B-PE) were saturated with avidin and allowed to bind to biotinylated hydrogel beads. Using a combination of fluorescence spectroscopy, quenching, and microscopy and 31P NMR static and magic angle spinning (MAS) spectroscopies, we demonstrate conditions for near-quantitative liposome binding to p(NIPAM-co-AA) microbeads and show that liposome fusion does not occur under such conditions, that the liposomes remain intact and impermeable when so bound, and that they can function as slow release vehicles for entrapped aqueous species.

Immunolocalization of Androgen Receptor in the Developing Craniofacial Skeleton

Male predominance in metopic and sagittal craniosynostosis and in nonsynostotic plagiocephaly suggests a role for circulating androgens in early craniofacial development. Androgens have been documented to play an important role in postnatal skeletal growth, and the androgen receptor has been recently demonstrated in human and rat osteoblast-like cell lines and in human long bones. The purpose of this study was to describe the expression of androgen receptor in the fetal craniofacial skeleton. The heads of E18 fetal CD-1 male and female mice were fixed in 10% formalin, decalcified, and embedded in paraffin. Four- to 6-mum coronal and sagittal sections were stained with a monoclonal antibody specific to androgen receptor, which was detected by an avidinbiotin conjugate and peroxidase system. The sections were then examined for androgen receptor expression patterns. Strong androgen receptor immunoreactivity was observed in the dura mater of developing fetuses. Androgen receptor expression was also noted in cells lining the osteogenic fronts and in calvarial osteoblasts. Similar androgen receptor expression patterns were found in male and female mice. Androgen receptor is abundantly expressed in fetal dura mater and calvarial bone. This study confirms the presence of androgen receptor in the murine fetal craniofacial skeleton, suggesting a potential role for the anabolic effects of androgens in the developing craniofacial skeleton.

Correlation of Ki-67, p53, and Adnab-9 immunohistochemical staining and ploidy with clinical and histopathologic features of severely dysplastic colorectal adenomas.

Variations of Ki-67, p53, and Adnab-9 monoclonal antibody reactions in colonic adenomas may be associated with colonic cancer risk. We studied the predictive value of these markers for adverse behavior in severely dysplastic colorectal adenomas, such as an associated carcinoma, multiplicity of adenomas, and subsequent development of adenomas. For this purpose we compared theclinical, gross, and histologic characteristics of highly dysplastic index polyps in 42 patients with Ki 67, p53, and Adnab-9 immunostaining and other molecular markers. Polyps were removed endoscopically, and severely dysplastic polyps were stained immunohistochemically with Ki-67, Adnab-9, and p53 protein by the avidin biotin conjugate (ABC) technique. Quantitative DNA (QDNA) was analyzed by computer-assisted image analysis. Ki-67 immunohistochemistry showed reversal of normal distribution of nuclear staining from the normal basal position to the upper third of the colonic crypts. This abnormality of immunostaining in dysplastic adenomas was the earliest detected by the panel we used. A statistically significant correlation was seen between invasiveness of carcinoma in the index polyp and polyp size (P = 0.003), sessile morphology (P = 0.037), and villous or tubulovillous histology (P = 0.019). In the index adenoma, p53 positivity was correlated with multiplicity at initial examination (P = 0.053), villous histology (P = 0.053), invasiveness of carcinoma (P < 0.003), and recurrence of colorectal adenomas (P = 0.025). Although p53 positivity and aneuploidy were correlated with invasiveness of carcinoma in the index polyp (P = 0.025), Adnab-9 positivity was not. However, Adnab-9 positivity in the index polyp was associated with multiplicity of adenomas (P = 0.04) as well as recurrence of adenomas (P < 0.024). In conclusion, in addition to the morphologic and histologic markers already known, Ki-67, Adnab-9 antibody, and p53 protein may be prognostic indicators useful in follow-up of patients with severely dysplastic colorectal adenomas. Adnab-9 antibody may identify a field defect in above-average-risk adenoma-bearing patients.

Glucose transporter 1 gene expression is related to thyroid neoplasms with an unfavorable prognosis: an immunohistochemical study.

An accelerated rate of glucose metabolism mediated by overexpression of key regulatory glycolytic enzymes and glucose transporters is among the most characteristic biochemical marker of malignant transformed cells. In thyroid neoplasms, however, an increased uptake of glucose [measured by 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) and positron emission tomography (PET)] seems to be restricted to more aggressive and high-grade tumors, whereas tumors with favorable prognosis demonstrate no significant tracer uptake. We therefore studied the expression of glucose transporters in thyroid carcinomas with different grades of malignancy. Sections of formalin-fixed and paraffin-embedded tissue obtained from 45 patients with thyroid cancer (5 anaplastic, 20 papillary and 20 follicular tumors) were investigated. Polyclonal rabbit antiglucose transporter antibodies, reactive with glucose transporters 1-5 (GLUT1-5), were used after heat pretreatment of the sections. Staining was performed by the avidin-biotin conjugate immunoperoxidase reaction and evaluated semiquantitatively. Expression of GLUT1 transporter on the cell membrane was closely related to the grade of malignancy in thyroid neoplasms (Fisher exact test p < 0.05). All anaplastic tumors showed a high level of GLUT1 expression in the cytoplasm and on the cell membrane. Positive membranous staining in differentiated tumors was detected predominantly in neoplasms with unfavorable prognosis, e.g., in widely invasive follicular or metastatic tumors, whereas low or no immunoreactivity could be seen in well-differentiated tumors or in normal thyroid epithelium. These data indicate that overexpression of GLUT1 on the cell membrane of thyroid neoplasms is closely related to tumors demonstrating a more aggressive biological behavior. Therefore, determination of GLUT1 expression in thyroid cancer tissue may be a prognostic marker, and FDG-PET may be a helpful technique in identifying patients at a higher risk.

Insights about brain tumors gained through immunohistochemistry and in situ hybridization of nuclear and phenotypic markers.

Immunohistochemistry (IHC) has provided major insights about the classification of brain tumors by identifying cellular markers of phenotype and about tumor growth potential with nuclear markers of proliferation. In situ hybridization (ISH) research shows promise for diagnostic applications in tumor classification. The avidin-biotin conjugate IHC procedure is highlighted for diagnostic use on routinely processed clinical specimens. The immunophenotypes of brain tumors are tabulated in reference to their common IHC markers. Tumors that have been correctly classified by their IHC phenotypes include the giant-cell glioblastoma, primary brain lymphoma, and central neurocytoma. Phenotypes that may be more definitively detected by ISH, such as pituitary hormone, immunoglobulin light chain, and collagen messages are described. IHC of nuclear proliferation markers correlates with grade of malignancy, predicts tumor growth potential, and is prognostic for patient survival. The incorporation of bromodeoxyuridine, the expression of proliferating cell nuclear antigen, and the expression of Ki-67 antigen detected by MIB-1 antibody are compared in regard to their cell cycle activity and labeling index determinations. Fluorescence in situ hybridization (FISH) of brain tumor interphase nuclei and chromosomes is described. Abnormal FISH signals of specific chromosomes are associated with different types of brain tumors, with different grades of malignancy, and with mesenchymal drift of glioma cells in culture.

Avidin targeting of intraperitoneal tumor xenografts.

Lectins (proteins that bind specific sugar molecules on glycoproteins and glycolipids) are expressed at various levels on the surface of tumor cells. Conjugation of cytotoxic agents to glycoproteins recognized by lectins could be useful in the treatment of tumors. Avidin (a highly glycosylated, positively charged protein found in egg white) contains terminal N-acetylglucosamine and mannose residues that bind to some lectins. In this study, we tested the ability of avidin, labeled through conjugation to radioactive biotin (a B vitamin), to target intraperitoneal tumors. Biotin was radioactively labeled with 111In. Four tumor models (one ovarian, one lung, and two colon) were established in nude mice by intraperitoneal injection of cultured cancer cells. The following two approaches were used in the intraperitoneal administration of avidin: 1) radioactive biotin-avidin conjugates were injected and 2) avidin was injected 1-24 hours before the injection of radioactive biotin (avidin pretargeting; avidin-biotin conjugates formed in vivo). The distribution of injected radioactivity in the tissues of treated animals was assessed. Radiolabeled avidin localized highly and rapidly in the tumors. More than 50% of the administered dose of avidin-biotin conjugate accumulated per gram of tumor tissue 2 hours after injection; high tumor uptake of radioactivity was observed up to 24 hours after conjugate injection. In contrast, accumulation of radioactivity in normal tissues was low, yielding high tumor to nontumor ratios. With avidin pretargeting, accumulation of radioactivity in the liver, kidney, and spleen was reduced to a greater extent than that in the tumor, and tumor to nontumor ratios were increased. Avidin may be a promising vehicle for the delivery of radioisotopes, drugs, toxins, or therapeutic genes to intraperitoneal tumors.

IMMUNOHISTOCHEMICAL AND ULTRASTRUCTURAL STUDY OF RHABDOSPHINCTER COMPONENT OF THE PROSTATIC CAPSULE

Purpose There has been no complete agreement on functional anatomy of muscular components of the urethral sphincteric mechanism, particularly in the male patient. The prostatic capsule was studied to define its histological structure and to determine whether its rhabdosphincter component (prostatocapsular rhabdosphincter) consists only of slow twitch or slow and fast twitch striated myofibers. Materials and Methods We studied 11 whole prostates, including 1 obtained at autopsy and 10 by radical prostatectomy. Samples of prostatic capsule from 4 operative specimens were studied by electron microscopy. Whole mount paraffin sections from transverse slices of the remaining 7 prostates were double labeled with avidin biotin conjugate immunostaining using the primary monoclonal antibodies anti-alpha smooth muscle actin plus anti-alpha sarcomeric actin (all striated myofibers) or antiskeletal myosin fast (fast myofibers only). Tissue components of the prostatic capsule, including smooth muscle and slow versus fast twitch striated myofibers, were quantified by computerized image analysis. Results The prostatic capsule consisted of collagen, smooth muscle and striated myofibers. It varied in thickness and proportion of the 3 components among specimens, and in each in relation to transverse circumferential aspect and craniocaudal (horizontal) level of the prostate. Collagen and smooth muscle were equally important components. Striated muscle elements within the capsule consisted of fast twitch and dominant slow twitch myofibers, and were much more abundant in the caudal (distal, lower) than the cranial (proximal, upper) half of the capsule, where they were deficient ventrally (anteriorly) and dorsally (posteriorly). The prostatocapsular rhabdosphincter thus had a butterfly-like appearance, with a thick posteriorly open ring at the apex and 2 thinner, divergent leaflets tapering toward the base at the bladder neck. The fast myofiber population decreased progressively from apex to base of prostate. Conclusions Proof is provided for mixed slow and fast twitch myofiber structure of the prostatocapsular component of human male rhabdosphincter. Sustained (tonic) contraction of slow myofibers probably reinforces the role of urethral smooth muscle in maintaining continence during bladder filling. Swift contraction of fast myofibers that abound caudally in the capsule probably supplements urethral closure by the bulkier membranous urethral part of the rhabdosphincter in preventing leakage of urine under stress when voiding is imminent or willfully withheld.

Insights About Brain Tumors Gained Through Immunohistochemistry and in Situ Hybridization of Nuclear and Phenotypic Markers

Immunohistochemistry (IHC) has provided major insights about the classification of brain tumors by identifying cellular markers of phenotype, and about tumor growth potential with nuclear markers of proliferation. Newer in situ hybridization shows promise in tumor classification and prognostication.Fig. 1 shows the sensitive and reliable avidin-biotin conjugate (ABC) method of localizing glial fibrillary acidic protein (GFAP). GFAP is the most specific marker of gliomas, tumors of brain cells, available today. The ABC method can be used to find any antigen for which a primary antibody is available. A molecular bridge then links this primary antibody bound to tissue with a label that can be seen, a substrate of the horseradish peroxidase (HRP) enzyme like diaminobenzidine (DAB) that produces a brown, insoluble reaction product at the site of the antigen. IHC for GFAP has revealed that brain tumors previously thought to be sarcomas are actually malignant gliomas (Fig. 2).

Development of a site targeted acoustic contrast agent for improved medical ultrasonic diagnosis

A novel site targeted acoustic contrast agent with the potential to define and localize a myriad of pathological tissues for diagnosis or therapy has been developed. The agent is a lipid-coated perfluorocarbon microemulsion modified to accommodate avidin–biotin conjugation. The formulation has low inherent echogenicity in suspension, yet high acoustic reflectivity after binding to a surface or itself. Targeted and control emulsions were suspended in buffered saline and imaged with a 7.5-MHz linear array transducer. Neither suspension manifested significant ultrasonic backscatter until avidin was added. Avidin induced aggregation and produced a marked enhancement in backscatter from the targeted but not from the control emulsion. Targeted and control emulsions were exposed to avidin conjugated nitrocellulose membranes and imaged with a high-resolution 50-MHz acoustic microscope. Bound targeted emulsion produced a fourfold enhancement in the magnitude of ultrasonic backscatter (+6.3 dB) from the membrane relative to the control (p&amp;lt;0.05). Moreover, acoustic contrast targeted to thrombi bothin vitro and in vivo using antifibrin monoclonal antibodies markedly enhanced (p&amp;lt;0.05) their acoustic reflectivity. These data demonstrate the first site targeted acoustic contrast agent and suggest potential improvement in the ultrasonic diagnosis of thrombi and other pathology.