A self-assembled fusion protein-based surface plasmon resonance biosensor for rapid diagnosis of severe acute respiratory syndrome

A surface plasmon resonance (SPR)-based biosensor was developed for simple diagnosis of severe acute respiratory syndrome (SARS) using a protein created by genetically fusing gold binding polypeptides (GBPs) to a SARS coronaviral surface antigen (SCVme). The GBP domain of the fusion protein serves as an anchoring component onto the gold surface, exploiting the gold binding affinity of the domain, whereas the SCVme domain is a recognition element for anti-SCVme antibody, the target analyte in this study. SPR analysis indicated the fusion protein simply and strongly self-immobilized onto the gold surface, through GBP, without surface chemical modification, offering a stable and specific sensing platform for anti-SCVme detection. AFM and SPR imaging analyses demonstrated that anti-SCVme specifically bound to the fusion protein immobilized onto the gold-micropatterned chip, implying that appropriate orientation of bound fusion protein by GBP resulted in optimal exposure of the SCVme domain to the assay solution, resulting in efficient capture of anti-SCVme antibody. The best packing density of the fusion protein onto the SPR chip was achieved at the concentration of 10 μg mL⁻¹; this density showed the highest detection response (906 RU) for anti-SCVme. The fusion protein-coated SPR chip at the best packing density had a lower limit of detection of 200 ng mL⁻¹ anti-SCVme within 10 min and also allowed selective detection of anti-SCVme with significantly low responses for non-specific mouse IgG at all tested concentrations. The fusion protein provides a simple and effective method for construction of SPR sensing platforms permitting sensitive and selective detection of anti-SCVme antibody.     

Efficient synthesis of phosphonodepsipeptides derived from norleucine

In the present work, we describe in detail an efficient solution synthesis of norleucine-derived phosphonopeptides mimicking the peptide sequences Nle-Gly(Ala) and Nle-Gly(Ala)-Val. The most efficient strategy involved use of the benzyl group. The synthesis was achieved through BOP-catalysed coupling of the monobenzyl ester of the N-Cbz-protected phosphonate derivative of norleucine with the hydroxyl moieties of derivatised l-lactic or glycolic acid. Subsequently, complete deprotection of the products was achieved in good yields by one-step Pd-catalysed hydrogenolysis. We also prepared the Fmoc-Nle-Ψ[PO(OH)O]-CH₂-COOH synthon and demonstrated that this precursor is a suitable building block for the solid-phase synthesis of cysteine-containing phosphonopeptides.     

Determination of major and trace elements in six herbal drugs for relieving heat and toxicity by ICP-AES with microwave digestion

ObjectiveTo establish a method to simultaneously determine major and trace element contents of Na, K, Cu, Fe, Zn, Mn, Ca, Mg, Cr, Ni, Pb, Se, As and Cd in six herbal drugs.MethodsMicrowave digestion procedure was applied under optimized conditions for digesting medicinal herbs. Concentrations of elements were determined by ICP-AES.ResultsThe results indicated that the herbal drugs were abundant in major and trace element contents which are healthy for human body. After optimizing the microwave digestion technology, the recovery of the element was 96.79–103.47% and the RSD < 5.0%.ConclusionICP-AES combined with the microwave digestion technology has a lot of merits in terms of saving time and effort, reducing environmental pollution, fast and accurate determination of results in determining major and trace elements.     

Development of an immunoassay and a sol-gel-based immunoaffinity cleanup method for coplanar polychlorinated biphenyls from soil and sediment samples

Two polychlorinated biphenyls (PCB) enzyme linked immunosorbent assays (ELISAs) were developed using goat PCB purified immunoglobulin (IgG) antibodies (Abs). The IgGs exhibited the highest affinity toward PCB-77 (24 ng mL⁻¹) with sensitivities in the range of 6-11 ng mL⁻¹. The Abs cross-reacted with PCB-126 and the heptachlorodibenzofuran 1,2,3,4,6,7,8-HpCDF but not with PCB-169, PCB-118, Aroclor 1232, 1248, 1260 or the hexachlorodibenzofuran 2,3,4,6,7,8-HxCDF. The IgGs were also used to develop a sol-gel-based immunoaffinity purification (IAP) method for cleanup of PCB-126. Recovery efficiencies depended on the sol-gel formats; a 1:12 format resulted in the highest binding capacity. Net binding capacity ranged from 112 to 257 ng, and 90% of the analyte could be eluted with only 2 mL of ethanol. The method was also very efficient in purifying PCB-126 from spiked soil and sediment samples from contaminated sites; and eliminating matrix interferences to a degree that enabled analysis of the purified samples by ELISA. The approaches developed in the course of the study form a basis for the development of additional IAP methods for other PCBs, and their implementation in high-throughput screening programs for PCB in food, soil, and other environmental and biological samples.     

Fluorescence Properties of Paracetamol During Interactions with Mitochondria

Paracetamol interaction with rat liver mitochondria in respiration media in the presence of succinate was the focus of this experiment. Fluorescence of paracetamol in water was studied by three-dimensional synchronous fluorescence fingerprint (SFF) and by excitation emission matrix (EEM). The direct molecular interactions of paracetamol and mitochondria were studied by fluorescence polarization technique. The paracetamol fluorescence maximum of SFF was Δλ = 110/λ_ex = 320 nm, F_max = 508 nm, and EEM maximum was λ_ex = (320 nm)/λ_em = 425 nm, F_max = 508. The fluorescence polarization results showed nonsignificant elevation of fluorescence polarization after addition of paracetamol into mitochondria in comparison to the control mitochondria group without paracetamol at time point t = 0. Paracetamol probably covalently bound to the mitochondrial surface proteins at time point t = 0, but paracetamol also entered mitochondria, which was observed as nonsignificant decline of fluorescence polarization during 30 min in the paracetamol-treated group. The practical advantages of spectral techniques (EEM, SFF, fluorescence polarization) are high sensitivity, reproducibility, minimal quantity of material, and capability to measure the mitochondrial autofluorescence.     

A Novel RBF Neural Network Training Methodology to Predict Toxicity to Vibrio Fischeri

Summary This work introduces a neural network methodology for developing QSTR predictors of toxicity to Vibrio fischeri. The method adopts the Radial Basis Function (RBF) architecture and the fuzzy means training strategy, which is fast and repetitive, in contrast to most traditional training techniques. The data set that was utilized consisted of 39 organic compounds and their corresponding toxicity values to Vibrio fischeri, while lipophilicity, equalized electronegativity and one topological index were used to provide input information to the models. The performance and predictive ability of the RBF model were illustrated through external validation and various statistical tests. The proposed methodology can be used to successfully model toxicity to Vibrio fischerifor a heterogeneous set of compounds.     

Sanctolide A, a 14-membered PK-NRP hybrid macrolide from the cultured cyanobacterium Oscillatoria sancta (SAG 74.79)

Sanctolide A (1), a 14-membered polyketide-nonribosomal peptide (PK-NRP) hybrid macrolide, was isolated from the cultured cyanobacterium Oscillatoria sancta (SAG 74.79). The planar structure was determined using various spectroscopic techniques including HRESIMS, and 1D and 2D NMR analyses. The relative configuration was assigned by J-based configurational analysis in combination with NOE correlations. The absolute configuration was determined by Mosher ester and enantioselective HPLC analyses. The structure of sanctolide A (1) features a rare N-methyl enamide and a 2-hydroxyisovaleric acid, which are incorporated to form a 14-membered macrolide ring structure, comprising a new type of cyanobacterial macrolides derived from a PKS-NRPS hybrid biosynthetic pathway.