Molecular conformation changes along the malignancy revealed by optical nanosensors

An interdisciplinary approach employing functionalized nanoparticles and ultrasensitive spectroscopic techniques is reported here to track the molecular changes in early stage of malignancy. Melanoma tissue tracking at molecular level using both labelled and unlabelled silver and gold nanoparticles has been achieved using surface enhanced Raman scattering (SERS) technique. We used skin tissue from ex vivo mice with induced melanoma. Raman and SERS molecular characterization of melanoma tissue is proposed here for the first time. Optical nanosensors based on Ag and Au nanoparticles with chemisorbed cresyl violet molecular species as labels revealed sensitive capability to tissues tagging and local molecular characterization. Sensitive information originating from surrounding native biological molecules is provided by the tissue SERS spectra obtained either with visible or NIR laser line. Labelled nanoparticles introduced systematic differences in tissue response compared with unlabelled ones, suggesting that the label functional groups tag specific tissue components revealed by proteins or nucleic acids bands. Vibrational data collected from tissue are presented in conjunction with the immunohistochemical analysis. The results obtained here open perspectives in applied plasmonic nanoparticles and SERS for the early cancer diagnostic based on the appropriate spectral databank.     

The effect of avidin-biotin interactions in detection systems for in situ hybridization.

The effect of avidin-biotin interactions in several detection systems for the non-radioactive in situ hybridization (ISH) technique was studied in a model system using a transitional cell carcinoma line and a biotinylated DNA probe. We performed fluorescence ISH to unravel the individual steps in a sensitive and frequently used amplification method which makes use of the alternating cytochemical detection layers of fluorescein isothiocyanate-conjugated avidin (AvFITC) and biotinylated goat anti-avidin (BioGAA) antibodies to detect the hybridized and biotinylated probe. Our experiments revealed that BioGAA antibodies bind with their antigen binding sites and not with their biotin moieties to avidin molecules that have already interacted with the DNA probe. The probable working mechanism of this amplification method is presented in a model. Furthermore, we used a peroxidase staining technique to compare with each other the sensitivity of several other detection systems in which avidin-biotin interactions play an important role, e.g., the avidin-biotinylated peroxidase complex (ABC) system. The experiments show that avidin molecules can not be efficiently used to interconnect two biotinylated molecular layers, since their introduction leads to firmly closed cytochemical networks. Such a closed network is already formed between the hybridized and biotinylated DNA probe and a first detection layer of avidin molecules, as appears from the finding that biotinylated molecules could hardly be coupled to these avidin molecules in a following detection layer. Therefore, the results presented here provide us with new insight into the molecular basis of cytochemical network formation. This will enable us to choose the proper procedures for increasing the sensitivity of ISH detection systems.     

Study on molecular mechanism and 3D-QSAR of influenza neuraminidase inhibitors

Neuraminidase (NA) is a critical enzyme of the influenza virus and many inhibitors targeting to this enzyme are quite efficient and encouraging as anti-influenza agents. In this paper the binding model of five series of inhibitors to NA was examined using molecular simulation method. The resulted conformation and orientation of the compounds were directly put into CoMSIA study. The most significant amino acid residues at binding sites and the requirement for features of substituents were applied to direct design of new inhibitors. The robust QSAR model and its three-dimensional contour map provided guidelines to building novel compounds with new scaffold and for structural optimization of current molecules.     

Protein scaffold-based molecular probes for cancer molecular imaging

Protein scaffold molecules are powerful reagents for targeting various cell signal receptors, enzymes, cytokines and other cancer-related molecules. They belong to the peptide and small protein platform with distinct properties. For the purpose of development of new generation molecular probes, various protein scaffold molecules have been labeled with imaging moieties and evaluated both in vitro and in vivo. Among the evaluated probes Affibody molecules and analogs, cystine knot peptides, and nanobodies have shown especially good characteristics as protein scaffold platforms for development of in vivo molecular probes. Quantitative data obtained from positron emission tomography, single photon emission computed tomography/CT, and optical imaging together with biodistribution studies have shown high tumor uptakes and high tumor-to-blood ratios for these probes. High tumor contrast imaging has been obtained within 1 h after injection. The success of those molecular probes demonstrates the adequacy of protein scaffold strategy as a general approach in molecular probe development.     

Avidin is a heparin-binding protein. Affinity,specificity and structural analysis

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.     

Use of biotinylated chitosan for substrate-mediated gene delivery

To improve transfection efficiency of nonviral vectors, biotinylated chitosan was applied to complex with DNA in different N/P ratios. The morphologies and the sizes of formed nanoparticles were suitable for cell uptake. The biotinylation decreased the surface charges of nanoparticles and hence reduced the cytotoxicity. The loading capacities of chitosan were slightly decreased with the increase of biotinylation, but most of the DNA molecules were still complexed. Using different avidin-coated surfaces, the interaction between biotinylated nanoparticles to the substrate may be manipulated. The in vitro transfection results demonstrated that biotinylated nanoparticles may be bound to avidin coated surfaces, and the transfection efficiencies were thus increased. Through regulating the N/P ratio, biotinylation levels, and surface avidin, the gene delivery can be optimized. Compared to the nonmodified chitosan, biotinylated nanoparticles on biomaterial surfaces can increase their chances to contact adhered cells. This spatially controlled gene delivery improved the gene transfer efficiency of nonviral vectors and could be broadly applied to different biomaterial scaffolds for tissue engineering applications.     

Converting Sendai virus into a specific fusogen whose cell target can be selected

Covalent intermolecular hybrids of Fab anti-hemagglutinin-neuraminidase (HN) monoclonal antibody and avidin were prepared and characterized. These conjugates were used to block and redirect the fusion activity of Sendai virus (SV). After incubation of SV with Fab anti-HN: avidin conjugate on ice for 1-2 h, the SV fused only those P815 or BW5147 cells which were labeled with biotin-modified anti-cell surface immunoglobulin. The levels of cell-cell fusion obtained were at least as high as those achieved with unmodified SV and unlabeled P815 or BW5147 cells. These results demonstrate that it is possible to block the normal agglutinating activity of the HN molecules of SV and to introduce a new cell recognition feature without negating the fusogenic potential of the virus. Such an approach may be useful in harnessing the fusion activity of SV to a targeted delivery system for microinjection of macromolecules into selected cell populations.     

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.     

DNA condensation by high-affinity interaction with avidin

Avidin, the basic biotin-binding glycoprotein from chicken egg white, is known to interact with DNA, whereas streptavidin, its neutral non-glycosylated bacterial analog, does not. In the present study we investigated the DNA-binding properties of avidin. Its affinity for DNA in the presence and absence of biotin was compared with that of other positively charged molecules, namely the protein lysozyme, the cationic polymers polylysine and polyarginine and an avidin derivative with higher isoelectric point (pI approximately 11) in which most of the lysine residues were converted to homoarginines. Gel-shift assays, transmission electron microscopy and dynamic light scattering experiments demonstrated an unexpectedly strong interaction between avidin and DNA. The most pronounced gel-shift retardation occurred with the avidin-biotin complex, followed by avidin alone and then guanidylated avidin. Furthermore, ultrastructural and light-scattering studies showed that avidin assembles on the DNA molecule in an organized manner. The assembly leads to the formation of nanoparticles that are about 50-100 nm in size (DNA approximately 5 kb) and have a rod-like or toroidal shape. In these particles the DNA is highly condensed and one avidin is bound to each 18 +/- 4 DNA base pairs. The complexes are very stable even at high dilution ([DNA] =10 pM) and are not disrupted in the presence of buffers or salt (up to 200 mM NaCl). The other positively charged molecules also condense DNA and form particles with a globular shape. However, in this case, these particles disassemble by dilution or in the presence of low salt concentration. The results indicate that the interaction of avidin with DNA may also occur under physiological conditions, further enhanced by the presence of biotin. This DNA-binding property of avidin may thus shed light on a potentially new physiological role for the protein in its natural environment.     

Dual-affinity avidin molecules

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.     

Magnetic resonance imaging visualization of targeted cells by the internalization of supramolecular adducts formed between avidin and biotinylated Gd3+ chelates

The high binding affinity between avidin and biotin has been exploited to develop a procedure for magnetic resonance imaging (MRI) visualization of target cells. SHIN3 and PANC1 tumor cell lines have been used as target cells because they possess on their membranes galactosyl receptors able to bind avidin molecules. Avidin–Gd chelate adducts have been built by using two Gd complexes containing one (Gd-I) and two (Gd-II) biotin residues, respectively. The relaxivities of such supramolecular adducts are significantly higher than those shown by free Gd-I and Gd-II. There is evidence of the occurrence of multilayered adducts in which the bis-biotinylated Gd³⁺ complex acts as a bridge between adjacent avidin molecules. MRI differentiation of labeled versus unlabeled cells has been attained when approximately 6×10⁸ Gd units were internalized in each cell. Furthermore, there is a marked decrease in the measured intracellular T₁ relaxivity as the number of internalized Gd complexes increases, probably owing to too short relaxation times of endosomic water protons with respect to their diffusion lifetime.     

Dynamic imaging of glucose flux impedance using FRET sensors in wild-type Arabidopsis plants

Quantitative and dynamic analysis of metabolites and signalling molecules is limited by technical challenges in obtaining temporally resolved information at the cellular and compartmental level. Real-time information on signalling and metabolite levels with subcellular granularity can be obtained with the help of genetically encoded FRET (F?rster resonance energy transfer) nanosensors. FRET nanosensors represent powerful tools for gene discovery, and analysis of regulatory networks, for example by screening mutants. However, RNA silencing has impaired our ability to express FRET nanosensors functionally in Arabidopsis plants. This drawback was overcome here by expressing the nanosensors in RNA silencing mutants. However, the use of silencing mutants requires the generation of homozygous lines deficient in RNA silencing as well as the mutation of interest and co-expression of the nanosensor. Here it is shown that dynamic changes in cytosolic glucose levels can readily be quantified in wild-type Arabidopsis plants at early stages of development (7-15 d) before silencing had a major effect on fluorescence intensity. A detailed protocol for screening 10-20 mutant seedlings per day is provided. The detailed imaging protocol provided here is suitable for analysing sugar flux in young wild-type plants as well as mutants affected in sugar signalling, metabolism, or transport using a wide spectrum of FRET nanosensors.     

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T₁ and T₂-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.     

Dynamic stabilization: Structural plasticity at inhibitory postsynaptic sites

Rearrangement of molecular structures at individual synapses can contribute to network plasticity. At mossy fiber presynaptic terminals, experience regulates both connectivity and structure of individual boutons. Moreover, dendritic spines and postsynaptic densities of glutamatergic synapses rapidly form and remodel in an activity-dependent manner. Recent studies of the postsynaptic scaffold molecule gephyrin have now revealed that also inhibitory shaft synapses undergo rapid remodeling at the postsynaptic scaffold level. Taking into account that also surface membrane receptors are highly mobile, local coincidence of receptors and scaffold elements in adjacent layers at dendritic shafts might depend on regulatory processes underlying synaptic plasticity.     

Two-photon excitation fluorescence resonance energy transfer with small organic molecule as energy donor for bioassay

A fluorescence resonance energy transfer (FRET) model using two-photon excitable small organic molecule DMAHAS as energy donor has been constructed and tried in an assay for avidin. In the FRET model, biotin was conjugated to the FRET donor, and avidin was labeled with a dark quencher DABS-Cl. Binding of DABS-Cl labeled avidin to biotinylated DMAHAS resulted in the quenching of fluorescence emission of the donor, based on which a competitive assay for free avidin was established. With using such donors that are excited in IR region, it is capable of overcoming some primary shortcomings of conventional one-photon FRET methods, especially in bioassays, such as the interference from background fluorescence or scattering light, the coexcitation of the energy acceptor with the donor. And such small molecules also show advantages over inorganic up-converting particles that also give anti-Stokes photoluminescence and have been applied as FRET donor recently. The results of this work suggest that two-photon excitable small molecules could be a promising energy donor for FRET-based bioassays.     

Radiation-force assisted targeting facilitates ultrasonic molecular imaging

Ultrasonic molecular imaging employs contrast agents, such as microbubbles, nanoparticles, or liposomes, coated with ligands specific for receptors expressed on cells at sites of angiogenesis, inflammation, or thrombus. Concentration of these highly echogenic contrast agents at a target site enhances the ultrasound signal received from that site, promoting ultrasonic detection and analysis of disease states. In this article, we show that acoustic radiation force can be used to displace targeted contrast agents to a vessel wall, greatly increasing the number of agents binding to available surface receptors. We provide a theoretical evaluation of the magnitude of acoustic radiation force and show that it is possible to displace micron-sized agents physiologically relevant distances. Following this, we show in a series of experiments that acoustic radiation force can enhance the binding of targeted agents: The number of biotinylated microbubbles adherent to a synthetic vessel coated with avidin increases as much as 20-fold when acoustic radiation force is applied; the adhesion of contrast agents targeted to alpha(v)beta3 expressed on human umbilical vein endothelial cells increases 27-fold within a mimetic vessel when radiation force is applied; and finally, the image signal-to-noise ratio in a phantom vessel increases up to 25 dB using a combination of radiation force and a targeted contrast agent, over use of a targeted contrast agent alone.     

Generic liposome reagent for immunoassays

We have derivatized liposomes with antibodies by using avidin to crosslink biotinylated phospholipid molecules in the liposome membranes with biotinylated antibody molecules. A comparison of the biotin binding activity of avidin in solution and avidin associated with liposomes shows that avidin bound to biotinylated phospholipid in liposome membranes retains full binding activity for additional biotin molecules. Changes in the fluorescence spectrum of avidin have been used to characterize the binding capacity of avidin for biotin in solution, and change in intensity of light scattered due to aggregation of liposomes was used to measure the biotin binding activity of avidin associated with liposomes. Relative amounts of the biotinylated phospholipid, avidin, and biotinylated antibody have been optimized to produce stable liposomes which are derivatized with up to 1.7 nmol of antibody/mumol of lipid. These derivatized liposomes are highly reactive to immunospecific aggregation in the presence of multivalent antigen. A linear increase in light scattering was recorded between 1 and 10 pmol of antigen. This work shows that liposomes containing biotinylated phospholipid can be a successful generic reagent for immunoassays.     

Dual polarization interferometry size and density characterisation of DNA immobilisation and hybridisation

Investigation of nucleic acid interactions was performed using dual polarization interferometry, a novel approach to elucidating molecular interactions. This paper presents a preliminary study of adsorption of single stranded DNA onto functionalised silicon oxynitride, compared with covalent linkage, and avidin-biotin immobilisation. The effect of probe concentration on hybridisation efficiency was also examined. We found that increasing the electrolyte concentration resulted in a decrease of adsorbed DNA and that capture of a biotinylated duplex DNA on an adsorbed avidin layer resulted in four times fewer molecules per cm(2) than for duplex DNA covalently bound via an amine end terminal. The rate of thickness increase of a biotin probe layer on an adsorbed avidin capture layer increased 10-fold when the probe concentration was increased from 0.1 microM to 1 microM. The close grafting density of the higher concentration probe meant that the immobilised probes were unavailable for hybridisation.     

A comprehensive molecular interaction map for Hepatitis B virus and drug designing of a novel inhibitor for Hepatitis B X protein

Hepatitis B virus (HBV) infection is a leading source of liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma. In this study, we use computation methods in order to improve our understanding of the complex interactions that occur between molecules related to Hepatitis B virus (HBV). Due to the complexity of the disease and the numerous molecular players involved, we devised a method to construct a systemic network of interactions of the processes ongoing in patients affected by HBV. The network is based on high-throughput data, refined semi-automatically with carefully curated literature-based information. We find that some nodes in the network that prove to be topologically important, in particular HBx is also known to be important target protein used for the treatment of HBV. Therefore, HBx protein is the preferential choice for inhibition to stop the proteolytic processing. Hence, the 3D structure of HBx protein was downloaded from PDB. Ligands for the active site were designed using LIGBUILDER. The HBx protein's active site was explored to find out the critical interactions pattern for inhibitor binding using molecular docking methodology using AUTODOCK Vina. It should be noted that these predicted data should be validated using suitable assays for further consideration.     

The fibrillin microfibril scaffold: A niche for growth factors and mechanosensation?

The fibrillins, large extracellular matrix molecules, are polymerized to form “microfibrils.” The fibrillin microfibril scaffold is populated by microfibril-associated proteins and by growth factors, which are likely to be latent. The scaffold, associated proteins, and bound growth factors, together with cellular receptors that can sense the microfibril matrix, constitute the fibrillin microenvironment. Activation of TGFβ signaling is associated with the Marfan syndrome, which is caused by mutations in fibrillin-1. Today we know that mutations in fibrillin-1 cause the Marfan syndrome as well as Weill-Marchesani syndrome (and other acromelic dysplasias) and result in opposite clinical phenotypes: tall or short stature; arachnodactyly or brachydactyly; joint hypermobility or stiff joints; hypomuscularity or hypermuscularity. We also know that these different syndromes are associated with different structural abnormalities in the fibrillin microfibril scaffold and perhaps with specific cellular receptors (mechanosensors). How does the microenvironment, framed by the microfibril scaffold and populated by latent growth factors, work? We must await future investigations for the molecular and cellular mechanisms that will answer this question. However, today we can appreciate the importance of the fibrillin microfibril niche as a contextual environment for growth factor signaling and potentially for mechanosensation.