An infecting and propagating parasite relies on its innate defense system to evade the host's immune response and to survive challenges from commensal bacteria. More so for the nematode Anisakis, a marine parasite that during its life cycle encounters both vertebrate and invertebrate hosts and their highly diverse microbiotas. Although much is still unknown about how the nematode mitigates the effects of these microbiota, its antimicrobial peptides likely play an important role in its survival. We identified anisaxins, the first cecropin-like helical antimicrobial peptides originating from a marine parasite, by mining available genomic and transcriptomic data for Anisakis spp. These peptides are potent bactericidal agents in vitro, selectively active against Gram-negative bacteria, including multi-drug resistant strains, at sub-micromolar concentrations. Their interaction with bacterial membranes was confirmed by solid state NMR (ssNMR) and is highly dependent on the peptide concentration as well as peptide to lipid ratio, as evidenced by molecular dynamics (MD) simulations. MD results indicated that an initial step in the membranolytic mode of action involves membrane bulging and lipid extraction; a novel mechanism which may underline the peptides' potency. Subsequent steps include membrane permeabilization leading to leakage of molecules and eventually cell death, but without visible macroscopic damage, as shown by atomic force microscopy and flow cytometry. This membranolytic antibacterial activity does not translate to cytotoxicity towards human peripheral blood mononuclear cells (HPBMCs), which was minimal at well above bactericidal concentrations, making anisaxins promising candidates for further drug development. STATEMENT OF SIGNIFICANCE: Witnessing the rapid spread of antibiotic resistance resulting in millions of infected and dozens of thousands dying worldwide every year, we identified anisaxins, antimicrobial peptides (AMPs) from marine parasites, Anisakis spp., with potent bactericidal activity and selectivity towards multi-drug resistant Gram-negative bacteria. Anisaxins are membrane-active peptides, whose activity, very sensitive to local peptide concentrations, involves membrane bulging and lipid extraction, leading to membrane permeabilization and bacterial cell death. At the same time, their toxicity towards host cells is negligible, which is often not the case for membrane-active AMPs, therefore making them suitable drug candidates. Membrane bulging and lipid extraction are novel concepts that broaden our understanding of peptide interactions with bacterial functional structures, essential for future design of such biomaterials.

• MeSH
• antibakteriální látky farmakologie   MeSH
• antimikrobiální peptidy MeSH
• Bacteria MeSH
• kationické antimikrobiální peptidy chemie   farmakologie   MeSH
• leukocyty mononukleární MeSH
• lidé MeSH
• lipidy farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• paraziti * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations.

• MeSH
• biologické markery krev   MeSH
• kometový test metody   MeSH
• lidé MeSH
• poškození DNA genetika   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between 'desirable' and 'essential' information: 'essential' information refers to the precise details that are necessary to assess the quality of the experimental work, whereas 'desirable' information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.

• MeSH
• dodržování směrnic statistika a číselné údaje   MeSH
• kometový test metody   normy   MeSH
• konsensus MeSH
• laboratoře MeSH
• lidé MeSH
• výzkumný projekt * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

INTRODUCTION: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. AREAS COVERED: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. EXPERT COMMENTARY: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.

• MeSH
• biologické markery analýza   chemie   MeSH
• hmotnostní spektrometrie metody   MeSH
• imunoanalýza metody   MeSH
• lidé MeSH
• proteomika metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH