Simultaneous Analysis of Cyanotoxins ß-N-methylamino-L-alanine (BMAA) and Microcystins-RR, -LR, and -YR Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

ß-N-methylamino-L-alanine (BMAA) and its isomers, 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)-glycine (AEG), along with microcystins (MCs)-RR, -LR, and -YR (the major MC congeners), are cyanotoxins that can cause detrimental health and environmental impacts during toxic blooms. Currently, there are no reverse-phase (RP) LC-MS/MS methods for the simultaneous detection and quantification of BMAA, its isomers, and the major MCs in a single analysis; therefore, multiple analyses are required to assess the toxic load of a sample. Here, we present a newly developed and validated method for the detection and quantification of BMAA, 2,4-DAB, AEG, MC-LR, MC-RR, and MC-YR using RP LC-MS/MS. Method validation was performed, assessing linearity (r2 > 0.996), accuracy (>90% recovery for spiked samples), precision (7% relative standard deviation), and limits of detection (LODs) and quantification (LOQs) (ranging from 0.13 to 1.38 ng mL-1). The application of this combined cyanotoxin analysis on a culture of Microcystis aeruginosa resulted in the simultaneous detection of 2,4-DAB (0.249 ng mg-1 dry weight (DW)) and MC-YR (4828 ng mg-1 DW). This study provides a unified method for the quantitative analysis of BMAA, its isomers, and three MC congeners in natural environmental samples.

Inhibition of proinflammatory signaling impairs fibrosis of bone marrow mesenchymal stromal cells in myeloproliferative neoplasms.

Although bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been identified as a major cellular source of fibrosis, the exact molecular mechanism and signaling pathways involved have not been identified thus far. Here, we show that BM-MSCs contribute to fibrosis in myeloproliferative neoplasms (MPNs) by differentiating into αSMA-positive myofibroblasts. These cells display a dysregulated extracellular matrix with increased FN1 production and secretion of profibrotic MMP9 compared to healthy donor cells. Fibrogenic TGFß and inflammatory JAK2/STAT3 and NFκB signaling pathway activity is increased in BM-MSCs of MPN patients. Moreover, coculture with mononuclear cells from MPN patients was sufficient to induce fibrosis in healthy BM-MSCs. Inhibition of JAK1/2, SMAD3 or NFκB significantly reduced the fibrotic phenotype of MPN BM-MSCs and was able to prevent the development of fibrosis induced by coculture of healthy BM-MSCs and MPN mononuclear cells with overly active JAK/STAT signaling, underlining their involvement in fibrosis. Combined treatment with JAK1/2 and SMAD3 inhibitors showed synergistic and the most favorable effects on αSMA and FN1 expression in BM-MSCs. These results support the combined inhibition of TGFß and inflammatory signaling to extenuate fibrosis in MPN.