Immunoassay as an analytical tool in agricultural biotechnology

Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays.     

Small gold nanoparticles presenting linear and looped Cilengitide analogues as radiosensitizers of cells expressing α<Subscript>ν</Subscript>β<Subscript>3</Subscript> integrin

This work uses linear and looped RGDfV sequences attached to the surface of small (1.8 nm in diameter) gold nanoparticles (AuNPs) to enhance the radiosensitizating effects of Cilengitide, a cyclic RGDf (NMe)V pentapeptide that targets α_vβ₃ integrin which is overexpressed in certain cancers. Following synthesis and purification, the AuNPs were evaluated in vitro against HUVEC, H460, and MCF7 cells in clonogenic assays using a ¹³⁷Cs irradiator. Untargeted AuNPs induced no significant dose enhancement factors (DEFs) in any of the cell types when compared to radiation treatment alone, whereas all evaluated AuNPs functionalized with targeting peptides performed at least as well as controls (irradiation after Cilengitide treatment). The observed DEFs also suggest that cyclizing the linear peptides into more spatially constrained, looped structures may facilitate target binding. These greater dose enhancements merit future in vivo studies of drug-AuNP conjugates to assess the ability of the nanostructures to provide an improved therapeutic benefit over treatment with drug candidates and radiation alone.

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X-band Electron Spin Relaxation Times for Four Aromatic Radicals in Fluid Solution and Comparison with Other Organic Radicals

X-band electron spin relaxation times of BDPA (1:1 α,γ-bisdiphenylene-β-phenylallyl), galvinoxyl 2,6-di-tert-butyl-α-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy, DPPH (2,2-diphenyl-1-picrylhydrazyl) and thianthrene radicals in fluid solution were measured by electron spin echo and inversion recovery at ambient temperature. Tumbling correlation times are estimated to be in the range of 20–30 ps. In this fast tumbling regime T ₁ ~ T ₂. Relaxation times are compared with previously reported values for symmetrically substituted triarylmethyl, semiquinone, and nitroxide radicals. The concentration dependence of spin lattice relaxation for neutral BDPA in toluene is about 10³ times greater than for anionic trityl radicals in water. T ₁ decreases in the order carbon-center BDPA > galvinoxyl > DPPH > thianthrene. The dominant relaxation mechanisms are proposed to be a local mode for BDPA, spin rotation, local mode and modulation of anisotropic proton hyperfine coupling for galvinoxyl, modulation of anisotropic nitrogen hyperfine for DPPH, and spin rotation plus modulation of anisotropic proton hyperfine coupling for thianthrene.     

Application of spherical harmonics derived space rotation invariant indices to the analysis of multiple sclerosis lesions' geometry by MRI

In the longitudinal study of multiple sclerosis (MS) lesions, varying position of the patient inside the MRI scanner is one of the major sources of assessment errors. We propose to use analytical indices that are invariant to spatial orientation to describe the lesions, rather than focus on patient repositioning or image realignment. Studies were made on simulated lesions systematically rotated, from in vitro MS lesions scanned on different days, and from in vivo MS lesions from a patient that was scanned five times the same day with short intervals of time between scans. Each of the lesions' 3D surfaces was approximated using spherical harmonics, from which indices that are invariant to space rotation were derived. From these indices, an accurate and highly reproducible volume estimate can be derived, which is superior to the common approach of 2D slice stacking. The results indicate that the suggested approach is useful in reducing part of the errors that affect the analysis of changes of MS lesions during follow-up studies. In conclusion, our proposed method circumvents the need for precise patient repositioning and can be advantageous in MRI longitudinal studies of MS patients.     

Constrained modeling for spectroscopic measurement of bi-exponential spin-lattice relaxation of water in vivo

The (1)H NMR water signal from spectroscopic voxels localized in gray matter contains contributions from tissue and cerebral spinal fluid (CSF). A typically weak CSF signal at short echo times makes separating the tissue and CSF spin-lattice relaxation times (T(1)) difficult, often yielding poor precision in a bi-exponential relaxation model. Simulations show that reducing the variables in the T(1) model by using known signal intensity values significantly improves the precision of the T(1) measurement. The method was validated on studies on eight healthy subjects (four males and four females, mean age 21 +/- 2 years) through a total of twenty-four spectroscopic relaxation studies. Each study included both T(1) and spin-spin relaxation (T(2)) experiments. All volumes were localized along the Sylvian fissure using a stimulated echo localization technique with a mixing time of 10 ms. The T(2) experiment consisted of 16 stimulated echo acquisitions ranging from a minimum echo time (TE) of 20 ms to a maximum of 1000 ms, with a repetition time of 12 s. All T(1) experiments consisted of 16 stimulated echo acquisition, using a homospoil saturation recovery technique with a minimum recovery time of 50 ms and a maximum 12 s. The results of the T(2) measurements provided the signal intensity values used in the bi-exponential T(1) model. The mean T(1) values when the signal intensities were constrained by the T(2) results were 1055.4 ms +/- 7.4% for tissue and 5393.5 ms +/- 59% for CSF. When the signal intensities remained free variables in the model, the mean T(1) values were 1085 ms +/- 19.4% and 5038.8 ms +/- 113.0% for tissue and CSF, respectively. The resulting improvement in precision allows the water tissue T(1) value to be included in the spectroscopic characterization of brain tissue.     

Polyatomic anion assistance in the assembly of [2]pseudorotaxanes

We describe the use of polyatomic anions for the quantitative assembly of ion-paired complexes displaying pseudorotaxane topology. Our approach exploits the unique ion-pair recognition properties exhibited by noncovalent neutral receptors assembled through hydrogen-bonding interactions between a bis-calix[4]pyrrole macrocycle and linear bis-amidepyridyl-N-oxides. The complexation of bidentate polyatomic anions that are complementary in size and shape to the receptor's cavity, in which six NH hydrogen-bond donors converge, induces the exclusive formation of four particle-threaded assemblies.     

Conformational basis for asymmetric seeding barrier in filaments of three- and four-repeat tau

Tau pathology in Alzheimer's disease is intimately linked to the deposition of proteinacious filaments, which akin to infectious prions, have been proposed to spread via seeded conversion. Here we use double electron-electron resonance (DEER) spectroscopy in combination with extensive computational analysis to show that filaments of three- (3R) and four-repeat (4R) tau are conformationally distinct. Distance measurements between spin labels in the third repeat, reveal tau amyloid filaments as ensembles of known β-strand-turn-β-strand U-turn motifs. Whereas filaments seeded with 3R tau are structurally homogeneous, filaments seeded with 4R tau are heterogeneous, composed of at least three distinct conformers. These findings establish a molecular basis for the seeding barrier between different tau isoforms and offer a new powerful approach for investigating the composition and dynamics of amyloid fibril ensembles.     

Click strategies for single-molecule protein fluorescence

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.     

Second-Generation Covalent TMP-Tag for Live Cell Imaging

Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology.