Essai du triméthylamine N-oxyde et du sodium 4-phénylbutyrate, chaperonnes à potentiel thérapeutique, dans l'épidermolyse bulleuse simplex causée par une mutation dans la kératine 14

L'épidermolyse bulleuse simplex (EBS) est une maladie orpheline dermatologique qui a une prévalence mondiale de 1/50 000 à 1/30 000. Elle est caractérisée par des mutations transmises de manière autosomale dominante dans les gènes de la kératine 5 (KRT5) et 14 (KRT14), menant à une agrégation des filaments intermédiaires. Phénotypiquement, il y a présence d'ampoules causées par un décollement de l'épiderme. Jusqu'à présent, aucune thérapie efficace n'est disponible. Dans cette étude, des essais cellulaires évaluant le potentiel thérapeutique de deux chaperonnes chimiques, le triméthylamine N-oxyde (TMAO) et le sodium 4-phénylbutyrate (4-PBA), ont été réalisés. Pour ce faire, trois lignées immortalisées de kératinocytes de patients EBS ayant une mutation dans KRT14 et une lignée contrôle ont été utilisées pour déterminer la capacité des molécules à réduire le nombre de cellules ayant des agrégats. Les résultats ont montré un effet bénéfique significatif du TMAO seulement, et ce dans deux lignées uniquement. Ainsi, le TMAO se qualifie comme une approche thérapeutique potentielle

Transcriptomics-Based Approach Identifies Spinosad-Associated Targets in the Colorado Potato Beetle, Leptinotarsa decemlineata

The Colorado potato beetle Leptinotarsa decemlineata is an insect pest that threatens potato crops globally. The primary method to control its damage on potato plants is the use of insecticides, including imidacloprid, chlorantraniliprole and spinosad. However, insecticide resistance has been frequently observed in Colorado potato beetles. The molecular targets and the basis of resistance to imidacloprid and chlorantraniliprole have both been previously quantified. This work was undertaken with the overarching goal of better characterizing the molecular changes associated with spinosad exposure in this insect pest. Next-generation sequencing was conducted to identify transcripts that were differentially expressed between Colorado potato beetles exposed to spinosad versus control insects. Results showed several transcripts that exhibit different expression levels between the two conditions, including ones coding for venom carboxylesterase-6, chitinase 10, juvenile hormone esterase and multidrug resistance-associated protein 4. In addition, several microRNAs, such as miR-12-3p and miR-750-3p, were also modulated in the investigated conditions. Overall, this work reveals a molecular footprint underlying spinosad response in Colorado potato beetles and provides novel leads that could be targeted as part of RNAi-based approaches to control this insect pest

Reduction in keratin aggregates in epidermolysis bullosa simplex keratinocytes after pretreatment with trimethylamine N-oxide

Epidermolysis bullosa simplex (EBS) is a dominantly inherited skin disease caused by mutations in the keratin 5 (KRT5) or KRT14 genes 1. Some reports suggested that fever and/or hot weather may exacerbate EBS phenotype 2. Effective EBS therapies are still lacking. Molecular chaperones are proteins whose main function is to promote the correct folding of polypeptides (s1). Molecules such as trimethylamine N-oxide (TMAO) and sodium 4-phenylbutyrate (4-PBA) act as chemical chaperones (s2) with protein folding and stabilization activities (s3, s4, s5). Treatment of affected epidermal cells by chemical chaperones to correct the misfolded and aggregated keratins that characterize EBS seems a viable therapeutic option 3. Furthermore, the type I keratins K16 and K17 polymerize with K5, and upregulation of these proteins could replace the mutant K14 in the heterodimer and improve disease pathology (s6). Hence, chemical chaperones which can reduce keratin aggregates formation and upregulate K16 and K17 in EBS-affected cells would be ideal therapeutic candidates for EBS

Gene expression analysis of epidermolysis bullosa simplex with mottled pigmentation

Epidermolysis bullosa simplex with mottled pigmentation (EBS-MP) is a subtype of epidermolysis bullosa simplex first reported in 1979. The disease has its onset in early childhood and manifests with either much localized skin blistering, resembling the Weber-Cockayne subtype of EBS, or with more extensive bulla formation as seen in the Koebner subtype of EBS. Associated features include palmoplantar keratoderma and reticular hyperpigmentation unrelated to the blistering

Small RNA sequencing analysis of peptide-affinity isolated plasma extracellular vesicles distinguishes pancreatic cancer patients from non-affected individuals

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a high fatality rate, mainly due to its asymptomatic nature until late-stage disease and therefore delayed diagnosis that leads to a lack of timely treatment intervention. Consequently, there is a significant need for better methods to screen populations that are at high risk of developing PDAC. Such advances would result in earlier diagnosis, more treatment options, and ultimately better outcomes for patients. Several recent studies have applied the concept of liquid biopsy, which is the sampling of a biofluid (such as blood plasma) for the presence of disease biomarkers, to develop screening approaches for PDAC; several of these studies have focused on analysis of extracellular vesicles (EVs) and their cargoes. While these studies have identified many potential biomarkers for PDAC that are present within EVs, their application to clinical practice is hindered by the lack of a robust, reproducible method for EV isolation and analysis that is amenable to a clinical setting. Our previous research has shown that the Vn96 synthetic peptide is indeed a robust and reproducible method for EV isolation that has the potential to be used in a clinical setting. We have therefore chosen to investigate the utility of the Vn96 synthetic peptide for this isolation of EVs from human plasma and the subsequent detection of small RNA biomarkers of PDAC by Next-generation sequencing (NGS) analysis. We find that analysis of small RNA from Vn96-isolated EVs permits the discrimination of PDAC patients from non-affected individuals. Moreover, analyses of all small RNA species, miRNAs, and lncRNA fragments are most effective at segregating PDAC patients from non-affected individuals. Several of the identified small RNA biomarkers have been previously associated with and/or characterized in PDAC, indicating the validity of our findings, whereas other identified small RNA biomarkers may have novel roles in PDAC or cancer in general. Overall, our results provide a basis for a clinically-amendable detection and/or screening strategy for PDAC using a liquid biopsy approach that relies on Vn96-mediated isolation of EVs from plasma

Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS