Conjugation Scheme Research Articles

On two numerical schemes of the Monte Carlo method for solving the Boltzmann equation

Two numerical schemes of the Monte Carlo method for solving the Cauchy problem for the Boltzmann equation are constructed and tested. They are based on a well-known relationship between a nonlinear integral equation and a random process. Procedures for modeling special random processes on whose trajectories unbiased estimators for the solution are described. Each scheme has its own domain of applicability, in which its advantages manifest themselves. The conjugate scheme is convenient for calculating the Boltzmann distribution function at high velocities (on “tails”). For the example of the BKW solution, the applicability of the schemes is numerically analyzed.

The e-support function of an e-convex set and conjugacy for e-convex functions

A subset of a locally convex space is called e-convex if it is the intersection of a family of open halfspaces. An extended real-valued function on such a space is called e-convex if its epigraph is e-convex. In this paper we introduce a suitable support function for e-convex sets as well as a conjugation scheme for e-convex functions.

Antigen delivery to dendritic cells by poly(propylene sulfide) nanoparticles with disulfide conjugated peptides: Cross-presentation and T cell activation

Vaccines aiming to activate cytotoxic T cells require cross-presentation of exogenous antigen by antigen-presenting cells (APCs). We recently developed a synthetic nanoparticle vaccine platform that targets lymph node-resident dendritic cells (DCs), capable of mounting an immune response to conjugated antigen. Here, we explore routes of processing and the efficiency of MHC I cross-presentation of OVA peptides conjugated using both reducible and non-reducible linkages, exploring the hypothesis that reduction-sensitive conjugation will lead to better antigen cross-presentation. Both clathrin and macropinocytic pathways were implicated in nanoparticle uptake by colocalization and inhibitor studies. Cross-presentation by DCs was demonstrated by direct antibody staining and in vitro stimulation of CD8+ T cells from OT-I mice and was indeed most efficient with the reduction-sensitive conjugation. Similarly, we observed IFN-γ production by CD4+ T cells from OT-II mice. Finally, immunization with the OVA peptide-bearing nanoparticles resulted in in vivo proliferation and IFN-γ production by adoptively transferred CD8+ OT-I T cells and was also most efficient with reduction-sensitive linking of the peptide antigen. These results demonstrate the relevance of the poly(propylene sulfide) nanoparticle vaccine platform and antigen conjugation scheme for activating both cytotoxic and helper T cell responses.

Surface Ligand Effects on Metal-Affinity Coordination to Quantum Dots: Implications for Nanoprobe Self-Assembly

The conjugation of biomolecules such as proteins and peptides to semiconductor quantum dots (QD) is a critical step in the development of QD-based imaging probes and nanocarriers. Such protein-QD assemblies can have a wide range of biological applications including in vitro protein assays and live-cell fluorescence imaging. One conjugation scheme that has a number of advantages is the self-assembly of biomolecules on a QD surface via polyhistidine coordination. This approach has been demonstrated using QDs that have different coating types, resulting in different interactions between the biomolecule and QD surface. Here, we report the use of a fluorescence resonance energy transfer (FRET) assay to evaluate the self-assembly of fluorescent proteins on the surface of QDs with eight distinct coatings, including several used in commercial preparations. The results of this systematic comparison can provide a basis for rational design of self-assembled biomolecule-QD complexes for biomedical applications.

Effect of Nanoparticle Conjugation on Gene Silencing by RNA Interference

RNA interference (RNAi) is a cellular process whereby the silencing of a particular gene is mediated by short RNAs (siRNAs). Although siRNAs have great therapeutic potential, cellular delivery has been a challenge. Nanoparticle-siRNA conjugates have emerged as potential delivery vehicles; however, reports describing the effects of nanoparticle conjugation on RISC incorporation and subsequent gene silencing have been mixed. In this report, we have systematically evaluated the effect of siRNA coupling strategies using a model nanoparticle system with varying conjugation schemes. We show that the accessibility of the siRNA linked to the nanoparticle and the lability of the cross-linker are critical for efficient gene knockdown.

Synthesis of Pyridyl Disulfide-Functionalized Nanoparticles for Conjugating Thiol-Containing Small Molecules, Peptides, and Proteins

Previously we reported emulsion polymerization of propylene sulfide with Pluronic F127 as an emulsifier, yielding nanoparticles (NPs) in the 25 nm size range. Immunologically functional NPs were prepared by adding an antigen-Pluronic conjugate to the polymerization mixture ( Reddy , S. T. , et al. ( 2007 ) Nat. Biotechnol. 25, 1159 ). We sought a more flexible scheme for conjugation of antigens and other biomolecules to the NP surfaces that would allow for milder reaction conditions than achievable during the polymerization step. Here, we present the synthesis of such functionalizable NPs in the form of NPs that carry thiol-reactive groups, to which thiol-containing antigens (peptide or protein) or other biomolecules can be conjugated under mild conditions to yield immunofunctional NPs. The Pluronic-stabilized poly(propylene sulfide) (PPS) NPs with thiol-reactive pyridyl disulfide groups are prepared in two steps by (1) emulsion polymerization of propylene sulfide in the presence of a carboxylate-Pluronic and (2) reaction of the carboxylic acid groups on the NP surface with cysteamine pyridyl disulfide and a water-soluble carbodiimide reagent. We choose pyridyl disulfide groups to have a reduction-sensitive disulfide bond linking the antigen to the NP surface, allowing efficient release of antigen inside the cell in response to the reductive conditions within the endosome. The functionalizable NPs are characterized by proton NMR, dynamic light scattering (DLS), UV/vis spectroscopy, and transmission electron microscopy (TEM). Conjugation of small molecules and protein to the NP surface is presented.

Ultralong continuously tunable parametric delays via a cascading discrete stage

We report experimental demonstration of an all-optical continuously tunable delay line based on parametric mixing with a total delay range of 7.34 mus. The bit-error rate performance of the delay line was characterized for a 10-Gb/s NRZ data channel. This result is enabled by cascading a discrete delay line that consists of 16 wavelength-dependent delays and a continuously tunable delay stage. Four wavelength conversion stages based on four-wave mixing in silicon waveguides were performed in order to achieve wavelength-preserving operation. The wavelength-optimized optical phase conjugation scheme employed in the delay line is capable of minimizing the residual dispersion for the entire tuning range.

Tunable 503 ns optical delay of 40 Gbit/s RZ-OOK and RZ-DPSK using a wavelength scheme for phase conjugation to reduce residual dispersion and increase delay

We demonstrate a method for a tunable optical delay element that allows for minimal residual dispersion and double the relative delay. A tunable delay of 503 ns for 40 Gbit/s return-to-zero on-off keying and return-to-zero differential phase-shift keying is shown with a reduction in residual dispersion of approximately 95%.

1 μs tunable delay using parametric mixing and optical phase conjugation in Si waveguides

We demonstrate continuously tunable optical delays as large as 1.1 micros range for 10 Gb/s NRZ optical signals based on four-wave mixing (FWM) process in silicon waveguide. The large delay range is made possible by a novel wavelength-optimized optical phase conjugation scheme, which allows for tunable dispersion compensation to minimize the residual group-velocity dispersion (GVD) for the entire tuning range.

Inducing Efficient Cross-priming Using Antigen-coated Yeast Particles

Saccharomyces cerevisiae stimulates dendritic cells (DCs) and represents a promising candidate for cancer vaccine development. Effective cross-presentation of antigen delivered to DCs is necessary for successful induction of cellular immunity. Here, we present a yeast-based vaccine approach that is independent of yeast's ability to express the chosen antigen, which is instead produced separately and conjugated to the yeast cell wall. The conjugation method is site-specific (based on the SNAP-tag) and designed to facilitate antigen release in the DC phagosome and subsequent translocation for cross-presentation. We demonstrate that nonsite-specific chemical conjugation of the same protein hinders cross-presentation. Phagosomal antigen release was further expedited through the insertion of the invariant chain ectodomain as a linker, which is rapidly cleaved by Cathepsin S. The dose of delivered antigen was increased in several ways: by using yeast strains with higher surface amine densities, by using yeast hulls (cell wall fragments) instead of whole cells, and by conjugating multiple layers of antigen. The novel multilayer conjugation scheme takes advantage of Sfp phosphopantetheinyl transferase and remains site-specific; it enables the antigen dose to grow linearly with the number of layers. We show that whole yeast cells coated with 1 layer of the cancer-testis antigen NY-ESO-1 and yeast hulls bearing 3 layers were able to cross-prime naive CD8 T cells in vitro, with the latter resulting in higher frequencies of antigen-specific cells after 10 days. This cross-presentation-efficient antigen conjugation scheme is not limited to yeast and can readily be applied toward the development of other particulate vaccines.

Abstract 4741: Application of nanotechnology for enhanced early detection of prostate cancer in African-American men

Abstract Background: African American men in the US have the highest incidence and mortality from prostate cancer in the world. Five-year survival rates are almost 100% of African American men diagnosed with early stage prostate cancer but only 29% for men diagnosed in the late stages of the disease. Early detection is thus the key to improved mortality and morbidity. We are investigating nanotechnologies to enhance the detection of prostate cancer biomarkers at extremely low levels in blood serum. Our goal is to engineer a multi-biomarker panel that will provide sufficiently high sensitivity and specificity such that it can be applied as a screening test and decrease prostate cancer morbidity and mortality among African-American men. Objectives: 1.To engineer quantum dot - antibody conjugates targeting established prostate cancer biomarkers (antigens): prostate specific antigen (PSA), Kallikrein 2 (KLK2), Kallikrein 14 (KLK14), Osteoprotegerin (OPG), Antip53Ab, Caveolin-1 (Cav-1) and Interleukin-6 (IL-6) 2. To define the photoluminescence signatures of bound versus unbound QDs, reflecting antigen-antibody complex formation. 3. To derive optimal cutpoints for the sensitivity and specificity of the novel QD-conjugated biomarkers individually, and in combination, for the early detection of prostate cancer in African-American men. Results: We have established an African American prostate cancer case-control collection. We have successfully conjugated antibodies to the above biomarkers to quantum dots and these are undergoing characterization. We have observed that the bio-conjugated QD shows a spectral shift of the maximum position on average by 4 nm in comparison to non-conjugated QDs. The shift is towards a shorter wavelengths “blue” shift for conjugated quantum dots. Its existence was confirmed with repetition of the conjugation scheme with identical parameters and the “blue” shift was observed in all repeated experiments. The PL spectral shift observed on bio-conjugated QDs with antigens may be attributed to the existence of an external local electric field that is imposed by the surrounding biomolecules (Stark effect). The shift varied depending on the different antibodies attached and on QDs of different wavelengths. Conclusions: We have observed a spectral shift of the bio-conjugated quantum dots compared to unconjugated quantum dots. The benefit of this observation may lie in the fact that the shift is different for each antibody, thus will show a peak at different wavelengths. This effect may provide a powerful tool for multiplexing proteins for early cancer detection. This study has enormous potential impact on improved detection of this disease in the early and more curable stage which in turn would lead to decreased morbidity and mortality.

Multiplexing encryption technique by combining random amplitude and phase masks

We propose and demonstrate an encryption-selectable undercover multiplexing. We encrypt and multiplex images for storage by means of a random phase mask common to every image, covered with random amplitude masks different for each image. In order to get a correct decryption of the encoded information, we have to use the appropriate random amplitude mask; otherwise fake information is recovered. We employ a phase conjugation scheme to generate the recovering wavefronts. We analyze and compare the different alternatives and degrees of complexity this combination of masks brings to enhance the security of optical encrypting techniques. We also include an analysis on the advantages and disadvantages this undercover multiplexing protocol offers. We present digital simulations to demonstrate the soundness of the proposal.

Jacobi Correction Equation, Line Search, and Conjugate Gradients in Hermitian Eigenvalue Computation II: Computing Several Extreme Eigenvalues

This paper addresses the question of how to efficiently adapt the conjugate gradient (CG) method to the computation of several leftmost or rightmost eigenvalues and corresponding eigenvectors of Hermitian problems. A generic block CG algorithm instantiated by some available block CG algorithms is considered whereby the new approximate eigenpairs are computed by applying the Rayleigh–Ritz procedure in the trial subspace spanning current approximate eigenvectors and the search direction vectors, each of the latter being a linear combination of the respective gradient of the Rayleigh quotient and all search directions from the previous iteration. An approach related to the so-called Jacobi orthogonal complement correction equation is exploited in the local convergence analysis of this CG algorithm. Based on theoretical considerations, a new block conjugation scheme (a way to compute search directions) is suggested that enjoys a certain kind of optimality and has proved to be competitive in practical eigenvalue computation.

Noncovalent, Site-Specific Biotinylation of Histidine-Tagged Proteins

Site-specific conjugation of proteins to surfaces, spectroscopic probes, or other functional units is a key task for implementing biochemical assays. The streptavidin-biotin interaction has proven a highly versatile tool for detection, quantification, and functional analysis of proteins. We have developed an approach for site-specific reversible biotinylation of recombinant proteins through their histidine tag using biotin conjugated to the multivalent chelator trisnitrilotriacetic acid (BTtris-NTA). Stable binding of BTtris-NTA to His-tagged proteins was demonstrated, which is readily reversed by addition of imidazole, enabling versatile conjugation schemes in solution as well as at interfaces. Gel filtration experiments revealed that His-tagged proteins bind to streptavidin doped with BTtris-NTA in a 2:1 stoichiometry. Furthermore, an increased binding affinity toward His-tagged proteins was observed for BTtris-NTA linked to streptavidin compared to tris-NTA in solution and on surfaces. These results indicate an efficient cooperative interaction of two adjacent tris-NTA moieties with a single His-tag, yielding an extremely tight complex with a lifetime of several days. We demonstrate several applications of BTtris-NTA including multiplexed capturing of proteins to biosensor surfaces, cell surface labeling, and Western blot detection. The remarkable selectivity of the His-tag-specific biotinylation, as well as the highly stable, yet reversible complex provides the basis for numerous further applications for functional protein analysis.

AFM Imaging of Immobilized Fibronectin: Does the Surface Conjugation Scheme Affect the Protein Orientation/Conformation?

Covalent grafting of biomolecules could potentially improve the biocompatibility of materials. However, these molecules have to be grafted in an active conformation to play their biological roles. The present work aims at verifying if the surface conjugation scheme of fibronectin (FN) affects the protein orientation/conformation and activity. FN was grafted onto plasma-treated fused silica using two different crosslinkers, glutaric anhydride (GA) or sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Fused silica was chosen as a model surface material because it presents a roughness well below the dimensions of FN, therefore allowing AFM analyses with appropriate depth resolution. Cell adhesion assays were performed to evaluate the bioactivity of grafted FN. Cell adhesion was found to be higher on GA-FN than on SMPB-FN. Since FN-radiolabeling assays allowed us to rule out a surface concentration effect (approximately 80 ng/cm2 of FN on both crosslinkers), it was hypothesized that FN adopted a more active conformation when grafted via GA. In this context, the FN conformation on both crosslinkers was investigated through AFM and contact angle analyses. Before FN grafting, GA- and SMPB-modified surfaces had a similar water contact angle, topography, and roughness. However, water contact angles of GA-FN and SMPB-FN surfaces clearly show differences in surface hydrophilicity, therefore indicating a dependence of protein organization toward the conjugation strategy. Furthermore, AFM results demonstrated that surface topography and roughness of both FN-conjugated surfaces were significantly different. Distribution analysis of FN height and diameter confirmed this observation as the protein dimensions were significantly larger on GA than SMPB. This study confirmed that the covalent immobilization scheme of biomolecules influences their conformation and, hence, their activity. Consequently, selecting the appropriate conjugation strategy is of paramount importance in retaining molecule bioactivity.

Electrochemically Programmed Release of Biomolecules and Nanoparticles

The controlled release of molecules or nanoparticle conjugates is an important tool for a wide range of applications in science and engineering. Here we demonstrate electrochemically programmed release of biomolecules and nanoparticles immobilized on patterned gold electrodes using the thiol-gold linkage. This technique exploits the reductive desorption of self-assembled monolayers and allows both spatially controlled release and regeneration of small molecules (e.g., drugs), biopolymers (e.g., peptides, proteins, DNA), protein assemblies (e.g., viruses), and nanoparticles (e.g., particle-DNA conjugates). Fluorescence microscopy is used to image the release of avidin and nanoparticles in phosphate-buffered saline and to determine the kinetics of desorption. We also demonstrate that the electrodes can be regenerated using the same conjugation scheme.

Control of cell adhesion on poly(methyl methacrylate)

Keratoprostheses have been constructed from a wide variety of transparent materials, including poly(methyl methacrylate) (PMMA). However, the success of keratoprosthesis has been plagued by numerous shortcomings that include the weakening of the implant-host interface due to weak cell adhesion and opaque fibrous membrane formation over the inner surface of the implant due to fibroblast attachment. An effective solution requires a surface modification that would selectively allow enhanced cell attachment at the implant-host interface and reduced cell attachment over the interior surface of the implant. Here, we have developed a novel and simple peptide conjugation scheme to modify PMMA surfaces, which allowed for region-specific control of cell adhesion. This method uses di-amino-PEG, which can be grafted onto PMMA using hydrolysis or aminolysis method. PEG can resist cell adhesion and protein adsorption. The functionalization of grafted di-amino-PEG molecules with RGD peptide not only restored cell adhesion to the surfaces, but also enhanced cell attachment and spreading as compared to untreated PMMA surfaces. Long-term cell migration and micropatterning studies clearly indicated that PEG–PMMA surfaces with and without RGD conjugation can be used to differentiate cell adhesion and control cell attachment spatially on PMMA, which will have potential applications in the modification of keratoprostheses.

All-optical induction of noncentrosymmetry in dyed plastic materials

Two-color holography was performed in a phase conjugation scheme in the 4-dibuthylamino-4′nitrostilbene dye embedded in a polystyrene matrix as well as in an industrial red plastic used for applications in optics. The now classical experiment consists in printing a quasi-permanent second-order nonlinear optical susceptibility χ(2) in a material through the coherent interference of two light beams at ω and 2ω frequencies. The induced nonlinearity in the stilbene-dye doped polymer is attributed to the angular hole burning effect due to the weakly reversible trans–cis photoisomerization. Second harmonic is also induced in the red plastic.

Imparting Mineral Affinity to Fetuin by Bisphosphonate Conjugation: A Comparison of Three Bisphosphonate Conjugation Schemes

Protein conjugation to bisphosphonic acids (BPs), such as 1-amino-1,1-diphosphonate methane (aminoBP) and 3,5-di(ethylamino-2,2-bisphosphono)benzoic acid (diBP), was proposed as a foundation for bone-specific delivery of protein therapeutics. This study was performed to directly compare the mineral affinity of protein-BP conjugates prepared by three different approaches. Fetuin, serving as a model protein, was derivatized with BPs by the following approaches: (i) by attaching the aminoBPs onto protein lysines using succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC); (ii) by attaching the aminoBPs onto protein carbohydrates using 4-(maleimidomethyl)-cyclohexane-1-carboxyl-hydrazide (MMCCH), and (iii) by conjugating diBP to protein lysines using the carbodiimide chemistry. The results indicated that conjugation of aminoBP and diBP to fetuin by all three means unequivocally enhanced the protein's affinity for hydroxyapatite in vitro. Similarly, conjugation of aminoBP and diBP onto fetuin increased the protein's retention in a mineral-containing matrix (Pro-Osteon) when the proteins were implanted in a rat subcutaneous model. Upon parenteral administration, however, no discernible differences were found between the SMCC- or MMCCH-linked conjugates and unmodified fetuin to target to bony tissues. DiBP-fetuin conjugates, however, led to successful bone targeting after intravenous injection in rats. We conclude that all three conjugation schemes were equally effective in imparting an affinity to the proteins toward mineral-containing matrices. Bone targeting, however, was achieved only with diBP conjugation to fetuin, supportive of the superior ability of this BP with a higher density of bisphosphonic acid groups.

Matter-wave amplification and phase conjugation via stimulated dissociation of a molecular Bose-Einstein condensate

We propose a scheme for parametric amplification and phase conjugation of an atomic Bose-Einstein condensate (BEC) via stimulated dissociation of a BEC of molecular dimers consisting of bosonic atoms. This can potentially be realized via coherent Raman transitions or using a magnetic Feshbach resonance. We show that the interaction of a small incoming atomic BEC with a (stationary) molecular BEC can produce two counterpropagating atomic beams -- an amplified atomic BEC and its phase-conjugate or time-reversed replica. The two beams can possess strong quantum correlation in the relative particle number, with squeezed number-difference fluctuations.