Dataset Information

Comparative Assessment of Physical and Chemical Cyanobacteria Cell Lysis Methods for Total Microcystin-LR Analysis.

ABSTRACT: Standardization and validation of alternative cell lysis methods used for quantifying total cyanotoxins is needed to improve laboratory response time goals for total cyanotoxin analysis. In this study, five cell lysis methods (i.e., probe sonication, microwave, freeze-thaw, chemical lysis with Abraxis QuikLyse^TM, and chemical lysis with copper sulfate) were assessed using laboratory-cultured Microcystis aeruginosa (M. aeruginosa) cells. Methods were evaluated for destruction of cells (as determined by optical density of the sample) and recovery of total microcystin-LR (MC-LR) using three M. aeruginosa cell densities (i.e., 1 × 10⁵ cells/mL (low-density), 1 × 10⁶ cells/mL (medium-density), and 1 × 10⁷ cells/mL (high-density)). Of the physical lysis methods, both freeze-thaw (1 to 5 cycles) and pulsed probe sonication (2 to 10 min) resulted in >80% destruction of cells and consistent (>80%) release and recovery of intracellular MC-LR. Microwave (3 to 5 min) did not demonstrate the same decrease in optical density (<50%), although it provided effective release and recovery of >80% intracellular MC-LR. Abraxis QuikLyse^TM was similarly effective for intracellular MC-LR recovery across the different M. aeruginosa cell densities. Copper sulfate (up to 500 mg/L Cu²⁺) did not lyse cells nor release intracellular MC-LR within 20 min. None of the methods appeared to cause degradation of MC-LR. Probe sonication, microwave, and Abraxis QuikLyse^TM served as rapid lysis methods (within minutes) with varying associated costs, while freeze-thaw provided a viable, low-cost alternative if time permits.

SUBMITTER: Greenstein KE

PROVIDER: S-EPMC8473049 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundMany important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly.ResultsHere, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu(1)] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA(2) adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA(2) adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu(1)] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met(1)] microcystin-LR.ConclusionsPhylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu(1)] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.

Project description:BackgroundNanomaterials have been widely used in electrochemistry, sensors, medicine among others applications, causing its inevitable environmental exposure. A raising question is the "carrier" effect due to unique surface properties of nanomaterials, which may collectively impact the bioavailability, toxicokinetic, distribution and biological effects of classic toxicants. Noteworthy, this aspect of information remains largely unexplored.MethodsHere, we deliberately selected two entities to mimic this scenario. One is graphene oxide (GO), which is made in ton quantity with huge surface-area that provides hydrophilicity and π-π interaction to certain chemicals of unique structures. The other is Microcystin-LR (MCLR), a representative double-bond rich liver-toxic endotoxin widely distributed in aquatic-system. Firstly, the adsorption of GO and MCLR after meeting under environmental conditions was explored, and then we focused on the toxicological effect and related mechanism of GO-MCLR complex on human skin cutin forming cells (HaCaT cells) and normal liver cells (L02 cells).ResultsAbiotically, our study demonstrated that GO could effectively adsorb MCLR through hydrogen bonding and π-π interaction, the oxidation degree of GO-MCLR decreased significantly and surface defect level raised. Compared to GO or MCLR, GO-MCLR was found to induce more remarkable apoptosis and ferroptosis in both HaCaT and L02 cells. The underlying mechanism was that GO-MCLR induced stronger intracellular reactive oxygen species (ROS) and mtROS generation, followed by Fe2+ accumulation, mitochondrial dysfunction and cytoskeletal damage.ConclusionsThese results suggest that the GO-MCLR complex formed by GO adsorption of MCLR may exhibit more toxic effects than the single material, which demonstrates the necessity for assessing nano-toxicant complexity. Our discovery may serve as a new toxicological paradigm in which nanomaterial mediated surface adsorption effects could impact the degree of cytotoxicity and toxicological mechanisms of classic toxins.

Project description:Human-driven environmental change has increased the occurrence of harmful cyanobacteria blooms in aquatic ecosystems. Concomitantly, exposure to microcystin (MC), a cyanobacterial toxin that can accumulate in animals, edible plants, and agricultural soils, has become a growing public health concern. For accurate estimation of health risks and timely monitoring, availability of reliable detection methods is imperative. Nonetheless, quantitative analysis of MCs in many types of biological and environmental samples has proven challenging because matrix interferences can hinder sample preparation and extraction procedures, leading to poor MC recovery. Herein, controlled experiments were conducted to enhance the use of ultra-performance liquid-chromatography tandem-mass spectrometry (UPLC-MS/MS) to recover MC-LR and MC-RR at a range of concentrations in seafood (fish), vegetables (lettuce), and environmental (soil) matrices. Although these experiments offer insight into detailed technical aspects of the MC homogenization and extraction process (i.e., sonication duration and centrifugation speed during homogenization; elution solvent to use during the final extraction), they centered on identifying the best (1) solvent system to use during homogenization (2-3 tested per matrix) and (2) single-phase extraction (SPE) column type (3 tested) to use for the final extraction. The best procedure consisted of the following, regardless of sample type: centrifugation speed = 4200 × g; elution volume = 8 mL; elution solvent = 80% methanol; and SPE column type = hydrophilic-lipophilic balance (HLB), with carbon also being satisfactory for fish. For sonication, 2 min, 5 min, and 10 min were optimal for fish, lettuce, and soil matrices, respectively. Using the recommended HLB column, the solvent systems that led to the highest recovery of MCs were methanol:water:butanol for fish, methanol:water for lettuce, and EDTA-Na4P2O7 for soils. Given that the recommended procedures resulted in average MC-LR and MC-RR recoveries that ranged 93 to 98%, their adoption for the preparation of samples with complex matrices before UPLC-MS/MS analysis is encouraged.

Dataset Information

Comparative Assessment of Physical and Chemical Cyanobacteria Cell Lysis Methods for Total Microcystin-LR Analysis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets