New approach methods for assessing indoor air toxicity

Indoor air is typically a mixture of many chemicals at low concentrations without any adverse health effects alone, but in mixtures they may cause toxicity and risks to human health. The aim of this study was by using new approach methods to assess the potential toxicity of indoor air condensates. In specific, different in vitro test methods including cyto-and immunotoxicity, skin sensitization and endocrine disruption were applied. In addition to biological effects, the indoor air samples were subjected to targeted analysis of 25 volatile organic compounds (VOCs) and Genapol X-80 (a nonionic emulsifier) suspected to be present in the samples, and to a non-targeted "total chemical scan" to find out whether the chemical composition of the samples is associated with the biological effects. The results confirm that assessing health risks of indoor air by analysing individual chemicals is not an adequate approach: We were not able to detect the VOCs and Genapol X-80 in the indoor air samples, yet, several types of toxicity, namely, cytotoxicity, immunotoxicity, skin sensitization and endocrine disruption were detected. In the non-targeted total chemical scan of the indoor air samples, a larger number of compounds were found in the cytotoxic samples than in the non-cytotoxic samples supporting the biological findings. If only one biological method would be selected for the screening of indoor air quality, THP-1 macrophage/WST-1 assay would best fit for the purpose as it is sensitive and serves as a good representative for different sub-toxic end points, including immunotoxicity, (skin) sensitization and endocrine disruption.publishedVersionPeer reviewe

DNA Aptamers against the Lup an 1 Food Allergen

Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs

Dissociation constants obtained in Biacore with relevant statistics of the aptamers obtained.

<p>The values were obtained using duplicates.</p

Schematic steps involved in the ELONA type assay.

<p>In step 1 the target protein is coated on the surface of a microtitre well and incubated with the aptamer pool. In step 2 biotinylated probe complementary to the constant 3′ primer binding end of the 93-mer DNA is added and hybridises to any DNA that has bound to the immobilised target protein. In step 3, streptavidin labelled-horse radish peroxidase (HRP) is added and binds to the biotin label, and finally in step 4, tetramethylbenzidine substrate for the HRP is added and the resulting increase in absorbance is measured.</p

MOWSE scores obtained in the Peptide mass fingerprinting of the protein-conjugated magnetic beads.

<p>MOWSE scores obtained in the Peptide mass fingerprinting of the protein-conjugated magnetic beads.</p

ELONA assay.

<p>Detection of ß-conglutin by ELONA using Sequence 40 in SELEX Binding Buffer at 100 nM final concentration.</p

SPR Sensorgrams of the aptamer sequences obtained.

<p>a) SPR Sensorgram obtained for Sequence 2 at 10 µM passed through the CM5 Biacore chip surface showing the interaction with the immobilized target (Channel 1: ß-conglutin) and negative controls (Channel 2: γ-conglutin, Channel 3: Gliadin Crude Extract); b) SPR Sensorgram obtained for Sequence 40 at 10 µM passed through the CM5 Biacore chip surface showing the interaction with the immobilized target (Channel 1: ß-conglutin) and negative controls (Channel 2: γ-conglutin, Channel 3: Gliadin Crude Extract).</p

SELEX evolution by SPR.

<p>RU reached at the plateau of the association curve for all different channels of the Biacore CM5 chip with DNA isolated from various rounds of SELEX (4, 7, 10, 12 and 14) using channels coated with ß-conglutin, gliadin crude extract and simply blocked with ethanolamine.</p

Hybrid Antibody–Aptamer Assay for Detection of Tetrodotoxin in Pufferfish

The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody–aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated.info:eu-repo/semantics/publishedVersio

H2AX/53BP1 foci as a potential pre-treatment marker of HNSCC tumors radiosensitivity – preliminary methodological study and discussion

In order to improve patients’ post-treatment quality of life, a shift from surgery to non-surgical (chemo)radio-treatment is recognized in head and neck oncology. However, about half of HNSCC tumors are resistant to irradiation and an efficient marker of individual tumor radiosensitivity is still missing. We analyzed whether various parameters of DNA double strand break (DSB) repair determined in vitro can predict, prior to clinical treatment initiation, the radiosensitivity of tumors. We compared formation and decrease of γH2AX/53BP1 foci in 48 h after irradiating tumor cell primocultures with 2 Gy of γ-rays. To better understand complex tumor behavior, three different cell type primocultures – CD90−, CD90+, and a mixed culture of these cells – were isolated from 1 clinically radioresistant, 2 radiosensitive, and 4 undetermined HPV–HNSCC tumors and followed separately. While DSB repair was delayed and the number of persisting DSBs increased in the radiosensitive tumors, the results for the radioresistant tumor were similar to cultured normal human skin fibroblasts. Hence, DSB repair kinetics/efficiency may correlate with clinical response to radiotherapy for a subset of HNSCC tumors but the size (and therefore practical relevance) of this subset remains to be determined. The same is true for contribution of different cell type primocultures to tumor radioresistance