Rapid and sensitive detection of cholera toxin using gold nanoparticle-based simple colorimetric and dynamic light scattering assay

Herein, a rapid and simple gold nanoparticle based colorimetric and dynamic light scattering (DLS) assay for the sensitive detection of cholera toxin has been developed. The developed assay is based on the distance dependent properties of gold nanoparticles which cause aggregation of antibody-conjugated gold nanoparticles in the presence of cholera toxin resulting discernible color change. This aggregation induced color change caused a red shift in the plasmon band of nanoparticles which was measured by UV–Vis spectroscopy. In addition, we employed DLS assay to monitor the extent of aggregation in the presence of different concentration of cholera toxin. Our assay can visually detect as low as 10 nM of cholera toxin which is lower than the previously reported colorimetric methods. The reported assay is very fast and showed an excellent specificity against other diarrhetic toxins. Moreover, we have demonstrated the feasibility of our method for cholera toxin detection in local lake water.     

Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin B

Ligand-based NMR techniques to study protein–ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi-frequency STD NMR approach, differential epitope mapping (DEEP)-STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so-called DEEP-STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP-STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi-subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.     

The Effect of Botulinum Toxin A on the Vocal Symptoms of Spastic Dysarthria: A Case Study

The vocal symptoms of spastic dysarthria and spasmodic dysphonia have many similar features. Botulinum toxin has been used effectively to treat spasmodic dysphonia. This study was designed to determine what vocal changes occur in an individual with spastic dysarthria following Botulinum toxin A injection into the thyroarytenoid muscles. Measures were obtained preinjection and three times postinjection. Acoustic and aerodynamic results were comparable to those reported for individuals with spasmodic dysphonia. The most marked change was increased DC airflow. Despite persistent breathiness, the participant reported great satisfaction with the result, particularly because of her more appropriate loudness. In addition, everyday listeners perceived significantly less listener burden and more relaxed and pleasant vocal quality postinjection.     

The interaction of the antitoxin DM43 with a snake venom metalloproteinase analyzed by mass spectrometry and surface plasmon resonance

DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non‐covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization‐quadrupole‐time‐of‐flight MS; from native solution conditions, the complex showed a molecular mass of ~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin–antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real‐time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti‐jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k_a = 3.54 ± 0.03 × 10⁴ M^?1 s^?1 and k_d = 1.16 ± 0.07 × 10^?5 s^?1, resulting in an equilibrium dissociation constant (K_D) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification and characterization of new Fusarium masked mycotoxins,T2 and HT2 glycosyl derivatives,in naturally contaminated wheat and oats by liquid chromatography–high‐resolution mass spectrometry

The presence of glucoside derivatives of T‐2 and HT‐2 toxins (type A trichothecene mycotoxins) in naturally contaminated wheat and oats is reported for the first time. The use of advanced high‐resolution mass spectrometry based on Orbitrap technology allowed to obtain molecular structure details by measuring exact masses of main characteristic fragments, with mass accuracy lower than 2.8 ppm (absolute value). A monoglucoside derivative of T‐2 toxin and two monoglucoside derivatives of HT‐2 toxin were identified and characterized. The analysis of their fragmentation patterns provided evidence for glucosylation at C‐3 position for T‐2 toxin and at C‐3 or C‐4 position for HT‐2 toxin. A screening for the presence of these new masked forms of mycotoxins was carried out on a set of naturally contaminated wheat and oats samples. On the basis of peak area ratio between glucoside derivatives and free T‐2 and HT‐2 toxins, the presence of glucoside derivatives was more likely in wheat than in oats samples. The present work confirms the widespread occurrence of trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins, thus suggesting the importance of developing suitable analytical methods for their detection. Besides toxicity studies, tracking down these new masked forms of trichothecenes along the food/feed chain would enable to collect information on their relevance in human/animal exposure to mycotoxin risk. Copyright © 2012 John Wiley & Sons, Ltd.     

2-(Diphenylacetyl)-1,3-Indanedione-1-Hydrazone (Dipain) Derivatives for Detection of Trichothecene Mycotoxins

Abstract

2-(Diphenylacetyl)-1,3-indanedione-1-hydrazone and its derivatives where the NH₂ has been replaced by a substituted NH or an imino group give fluoresence enhancement with trichothecene mycotoxins. Absolute amounts of T-2 toxin as low as 50 ng are detectable. Detection limits for HT-2, Diacetoxyscirpenol, Neosolaniol, and Fusarenon-X range from 0.1 to 4μg.

The advantage in using 2-(diphenylacetyl)-1,3-indanedione-1-hydrazone (DIPAIN) derivatives for the detection of trichothecene mycotoxins is that response time is based on the rate of formation of a molecular association complex between the toxin and the detector reagent rather than on the chemical reactivity of the toxin. Hence, sensitive detection or mycotoxins can be achieved simply and rapidly at 25°C.

Since DlPAlN derivatives have been found to be direct-acting reagents capable of detecting droplets of dissolved trichothecenes, it is speculated that they may be useful as coatings in optical waveguide devices or in other devices that are developed as field detectors for aerosols that contain trichothecene mycotoxins. The DlPAlN reagents may also be used in kits that are designed to detect surface contamination by trichothecene mycotoxins.     

Probing bacterial-toxin inhibition with synthetic glycopolymers prepared by tandem post-polymerization modification: role of linker length and carbohydrate density

Probing the depths: A tandem post-polymerization modification strategy was used to systematically probe the multivalent inhibition of a bacterial toxin as a function of linker length (see scheme), carbohydrate density, and glycopolymer chain length. Guided by structural-biology information, the binding-pocket depth of the toxin was probed and used as a means to specifically improve inhibition of the toxin by the glycopolymer.     

毒素的光纤免疫传感器研究进展

介绍了毒素的光纤免疫传感器及相关固定化技术进展,评述了光纤免疫传感器在毒素检测中的应用。     

Molecular evolution of toxin genes in Elapidae snakes

The venom of the sea krait, Laticauda semifasciata, consists primarily of two toxic proteins, phospholipase A₂ (PLA₂) and a three-finger-structure toxin. We have cloned both toxic protein genes, including the upstream region. PLA₂ genes contain three types of inserted sequences: an AG-rich region, a chicken repeat 1-like long interspersed nucleotide element sequence and an intron II 3′ side repeat sequence. The molecular divergence of L. semifasciata PLA₂ genes was defined on the basis of the inserted sequences and their sequence homology. The length of intron I in the three-finger-structure toxin genes differs from species to species. The alignment analysis of intron I of the three-finger-structure toxin genes revealed that the intron I sequence of the ancestral gene comprised ten genetic regions. A hypothetical evolutionary process for the three-finger-structure toxin genes has also been developed.     

Electrostatic field–induced tip‐electrospray ionization mass spectrometry for direct analysis of raw food materials

Rapid characterization of metabolites and risk compounds such as chemical residues and natural toxins in raw food materials such as vegetables, meats, and edible living plants and animals plays an important part in ensuing food quality and safety. To rapidly characterize the analytes in raw food materials, it is essential to develop in situ method for directly analyzing raw food materials. In this work, raw food materials including biological tissues and living samples were placed between an electrode and mass spectrometric (MS) inlet under a strong electrostatic field; analytes were rapidly induced to generate electrospray ionization (ESI) from the sample tip by adding a drop of solvent onto the sample. Therefore, the electrostatic field–induced tip‐ESI‐MS allows raw samples to avoid contacting high voltage, and thus this method has the advantage for in vivo analysis of food living plants and animals. Metabolite profiling, residues of pesticides and veterinary drugs, and natural toxins from raw food materials have been successfully detected. The analytical performances, including the linear ranges, sensitivity, and reproducibility, were investigated for direct sample analysis. The ionization mechanism of electrostatic field–induced tip‐ESI was also discussed in this work.