Single-cell analysis of MYD88L265P and MYD88WT Waldenström macroglobulinemia patients.

Waldenström macroglobulinemia (WM) is characterized by the expansion of clonal lymphoplasmacytic cells; the MYD88L265P somatic mutation is found in >90% of patients, but malignant B cells may still display intra-clonal heterogeneity. To assess clonal heterogeneity in WM, we generated and performed single-cell RNA sequencing of CD19+ sorted cells from five patients with MYD88 L265P and two patients with MYD88 WT genotype as well as two healthy donors. We identified distinct transcriptional patterns in the clonal subpopulations not only between the two genetically distinct WM subgroups but also among MYD88 L265P patients, which affected the B cell composition in the different subgroups. Comparison of clonal and normal/polyclonal B cells within each patient sample enabled the identification of patient-specific transcriptional changes. We identified gene signatures active in a subset of MYD88L265P patients, while other signatures were active in MYD88 WT patients. Finally, gene expression analysis showed common transcriptional features between patients compared to the healthy control but also differentially expressed genes between MYD88 L265P and MYD88 WT patients involved in distinct pathways, including NFκΒ, BCL2, and BTK. Overall, our data highlight the intra-tumor clonal heterogeneity in WM with potential prognostic and therapeutic implications.

Determination of MYD88L265P mutation fraction in IgM monoclonal gammopathies.

We describe a novel method for the detection of MYD88L265P mutation using a competitive allele-specific polymerase chain reaction (Cast-PCR) assay. This assay has a sensitivity of 1 × 10-3, is applicable in reactions containing very low amounts of DNA (as low as 20 pg), and allowed the detection of MYD88L265P somatic mutation in both tumor-derived DNA (tDNA) and cell-free DNA (cfDNA). In addition, using the Cast-PCR assay, we were able to determine the mutation allele fraction (MAF) in each tested sample. We then analyzed baseline tDNA and cfDNA samples from 163 patients (53 with immunoglobulin M monoclonal gammopathy of undetermined significance and 110 with Waldenström's macroglobulinemia [WM], of whom 54 were asymptomatic and 56 were symptomatic) and also in sequential samples of 37 patients. MAF in both cfDNA and tDNA was higher among patients with symptomatic compared with asymptomatic WM and in those with asymptomatic WM compared with those with immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance. In addition, the evaluation of sequential samples showed that MAF decreased after treatment, whereas it increased in patients who relapsed or progressed to symptomatic WM. Thus, Cast-PCR is a highly sensitive, cost-effective diagnostic tool for MYD88L265P detection, applicable in both tDNA and cfDNA samples, that also provides a quantitative evaluation of the tumor load in patients with IgM monoclonal gammopathies.

Network analysis in aged C. elegans reveals candidate regulatory genes of ageing.

Ageing is a biological process guided by genetic and environmental factors that ultimately lead to adverse outcomes for organismal lifespan and healthspan. Determination of molecular pathways that are affected with age and increase disease susceptibility is crucial. The gene expression profile of the ideal ageing model, namely the nematode Caenorhabditis elegans mapped with the microarray technology initially led to the identification of age-dependent gene expression alterations that characterize the nematode's ageing process. The list of differentially expressed genes was then utilized to construct a network of molecular interactions with their first neighbors/interactors using the interactions listed in the WormBase database. The subsequent network analysis resulted in the unbiased selection of 110 candidate genes, among which well-known ageing regulators appeared. More importantly, our approach revealed candidates that have never been linked to ageing before, thus suggesting promising potential targets/ageing regulators.

Arabidopsis thaliana Plant Natriuretic Peptide Active Domain Forms Amyloid-like Fibrils in a pH-Dependent Manner.

Plant natriuretic peptides (PNPs) are hormones that have been extracted from many different species, with the Arabidopsis thaliana PNP (AtPNP-A) being the most studied among them. AtPNP-A is a signaling molecule that consists of 130 residues and is secreted into the apoplast, under conditions of biotic or abiotic stress. AtPNP-A has distant sequence homology with human ANP, a protein that forms amyloid fibrils in vivo. In this work, we investigated the amyloidogenic properties of a 34-residue-long peptide, located within the AtPNP-A sequence, in three different pH conditions, using transmission electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy, Congo red and Thioflavin T staining assays. We also utilize bioinformatics tools to study its association with known plant amyloidogenic proteins and other A. thaliana proteins. Our results reveal a new case of a pH-dependent amyloid forming peptide in A. thaliana, with a potential functional role.