DCL-suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology

RNA silencing is a universal mechanism involved in development, epigenetic modifications and responses to biotic and abiotic stresses. The major components of this mechanism are Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins. Understanding the role of each component is of great scientific and agronomic importance. Plants, including Nicotiana benthamiana, an important plant model, usually possess four DCL proteins, each of which has a specific role, namely being responsible for the production of an exclusive small RNA population. Here, we used RNA interference (RNAi) technology to target DCL proteins and produced single and combinatorial mutants for DCL. We analysed the phenotype for each DCL knockdown plant, together with the small RNA profile, by next-generation sequencing (NGS). We also investigated transgene expression, as well as viral infections, and were able to show that DCL suppression results in distinct developmental defects, changes in small RNA populations, increases in transgene expression and, finally, higher susceptibility in certain RNA viruses. Therefore, these plants are excellent tools for the following: (i) to study the role of DCL enzymes; (ii) to overexpress proteins of interest; and (iii) to understand the complex relationship between the plant silencing mechanism and biotic or abiotic stresses.     

Src Mutation Induces Acquired Lapatinib Resistance in ERBB2-Amplified Human Gastroesophageal Adenocarcinoma Models

ERBB2-directed therapy is now a routine component of therapy for ERBB2-amplified metastatic gastroesophageal adenocarcinomas. However, there is little knowledge of the mechanisms by which these tumors develop acquired resistance to ERBB2 inhibition. To investigate this question we sought to characterize cell line models of ERBB2-amplified gastroesophageal adenocarcinoma with acquired resistance to ERBB2 inhibition. We generated lapatinib-resistant (LR) subclones from an initially lapatinib-sensitive ERBB2-amplified esophageal adenocarcinoma cell line, OE19. We subsequently performed genomic characterization and functional analyses of resistant subclones with acquired lapatinib resistance. We identified a novel, acquired Src ^E527K mutation in a subset of LR OE19 subclones. Cells with this mutant allele harbour increased Src phosphorylation. Genetic and pharmacologic inhibition of Src resensitized these subclones to lapatinib. Biochemically, Src mutations could activate both the phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways in the lapatinib-treated LR OE19 cells. Ectopic expression of Src ^E527K mutation also was sufficient to induce lapatinib resistance in drug-naïve cells. These results indicate that pathologic activation of Src is a potential mechanism of acquired resistance to ERBB2 inhibition in ERBB2-amplified gastroesophageal cancer. Although Src mutation has not been described in primary tumor samples, we propose that the Src hyperactivation should be investigated in the settings of acquired resistance to ERBB2 inhibition in esophageal and gastric adenocarcinoma.     

Caprine PrP variants harboring Asp-146, His-154 and Gln-211 alleles display reduced convertibility upon interaction with pathogenic murine prion protein in scrapie infected cells

Scrapie, the prion disease of sheep and goats, is a devastating malady of small ruminants. Due to its infectious nature, epidemic outbreaks may occur in flocks/herds consisting of highly susceptible animals. Field studies identified scrapie-protective caprine PrP variants, harboring specific single amino acid changes (Met-142, Arg-143, Asp-146, Ser-146, His-154, Gln-211 and Lys-222). Their effects are under further evaluation, and aim to determine the most protective allele. We assessed some of these variants (Asp-146, His-154, Gln-211 and Lys-222), after their exogenous expression as murine-caprine chimeras in a scrapie- infected murine cell line. We report that exogenously expressed PrPs undergo conformational conversion upon interaction with the endogenous pathological murine prion protein (PrP^SC), which results in the detection of goat-specific and partially PK-resistant moieties. These moieties display a PK-resistance pattern distinct from the one detected in natural goat scrapie cases. Within this cellular model, distinct conformational conversion potentials were assigned to the tested variants. Molecules carrying the Asp-146, His-154 and Gln-211 alleles showed significantly lower conversion levels compared to wild type, confirming their protective effects against scrapie. Although we utilized a heterologous conversion system, this is to our knowledge, the first study of caprine PrP variants in a cellular context of scrapie, that confirms the protective effects of some of the studied alleles.     

Transcriptome-wide association study reveals two genes that influence mismatch negativity

1. Download : Download high-res image (191KB)
2. Download : Download full-size image

     

Sequential CRISPR gene editing in human iPSCs charts the clonal evolution of myeloid leukemia and identifies early disease targets

1. Download : Download high-res image (194KB)
2. Download : Download full-size image

     

Serum levels of c-myc and c-ras oncogene products in normal subjects and in patients with neoplastic and non neoplastic conditions

A new, simple and sensitive low pH ELISA system has been developed and used to measure serum levels of c-myc and c-ras oncogene products in healthy blood donors and patients with neoplastic and non neoplastic conditions. Blood donors had significantly lower serum levels of oncogene products than patients with cancer or other pathologies (p-value less than 0.01). There was, however, no difference between patients with neoplastic and non-neoplastic conditions. Although c-myc and c-ras oncogene products in the serum appear to discriminate between healthy state and pathological conditions they do not discriminate between neoplastic and non-neoplastic conditions.     

Reducing psychological distress and depression in humanitarian emergencies: An essential role for nonspecialists

John Naslund and Eirini Karyotaki discuss Mark Jordans and colleagues’ accompanying research study on therapy for people with psychological distress in Nepal.

Humanitarian emergencies such as war, natural disasters, or pandemics profoundly disrupt the daily lives of those impacted and result in psychological distress and high risk of mental disorders. With increasing frequency of humanitarian emergencies over the past decade, including the most recent Coronavirus Disease 2019 (COVID-19) pandemic, there is immediate need for brief scalable interventions that can be readily delivered to at-risk population groups [1]. With the dearth of available mental health specialists, especially in low-resource settings susceptible to crises, natural disasters, or displacement, combined with fragmented or poor functioning health systems during emergencies, nonspecialists may be ideally positioned to deliver such programs [2]. Nonspecialists, such as community health workers or lay persons, do not have specialty training in mental healthcare; yet, these frontline providers often play an essential role in delivering primary care services in many low- and middle-income countries [3,4], and they are increasingly being recognized as critical for scaling up access to psychological treatments for mental disorders [5,6]. Further, in a humanitarian crisis, use of nonspecialists from the affected population offers key benefits, such as empowering community members and drawing upon the experience of facilitators [7].In an accompanying study in PLOS Medicine, Mark Jordans and colleagues demonstrate that community members with no prior mental health training could effectively deliver the WHO Group Problem Management Plus (Group PM+) program in a humanitarian setting in Nepal [8]. The research team conducted a cluster randomized controlled trial enrolling 72 wards and found that the 5-session Group PM+ delivered by nonspecialists contributed to reduction in psychological distress and depressive symptoms when compared to usual care. There may be opportunities to expand on these findings and further advance task sharing efforts in humanitarian settings.