The infection of Pseudomonas syringae pv. actinidiae in kiwifruit is currently assessed by numerous methodologies, each with their own limitations. Most studies are based on either a laborious method of growth quantification of the pathogen or qualitative assessments by visual scoring following stem or cutting inoculation. Additionally, when assessing for resistance against specific pathogen effectors, confounding interactions between multiple genes in the pathogen can make mapping resistance phenotypes nearly impossible. Here we present robust alternative methods to quantify pathogen load based on rapid bacterial DNA quantification by PCR, the use of Pseudomonas fluorescens (Pfo), and a transient reporter eclipse assay, for assessing resistance conferred by isolated bacterial avirulence genes. These assays compare well with bacterial plate counts to assess bacterial colonization as a result of plant resistance activation. The DNA-based quantification, when coupled with the Pfo and reporter eclipse assays to independently identify bacterial avirulence genes, is rapid, highly reproducible, and scalable for high-throughput screens of multiple cultivars or genotypes. Application of these methodologies will allow rapid and high-throughput identification of resistant cultivars and the bacterial avirulence genes they recognize, facilitating resistance gene discovery for plant breeding programs.
Incorporating antibrowning agents into the edible coating is a conventional processing technique for fresh-cut preparation. The main quality attributes of fresh-cuts, i.e., appearance color, taste and texture may be affected by coating formulation and its additives at different and sometime contradictory rates. In this study, the impacts of using alginate coating alone or in combination with citric acid and/or ascorbic acid were studied on browning index, microbial load, textural properties, and some taste and nutritional parameters of apple fresh-cut. Lowering weight loss, decline in browning index, increasing acidic taste and changes in some textural properties (hardness, chewiness, and springiness) in response to adding citric acid to the coating formula can be attributed to the competition of H + ions with calcium cations and therefore physical alteration of forming gel features. On the other side, adding ascorbic acid, as an active antioxidant compound, led to elevated antioxidant capacity, lowered browning index, and least rates of electrolyte leakage. Totally, after 7 days of storage at 4°C, lowest values of weight loss (0.33%), microbial load (2.95 log CFU/g), electrolyte leakage (35.5%) and highest rates of lightness (96.11) and antioxidant capacity (11.03 mmol Fe 2 + /Kg) were detected when the coating formulation was enriched with both citric acid and ascorbic acid compounds.
Glycogen storage diseases (GSDs) are the heterogeneous group of disorders caused by mutations in at least 30 different genes. Different types of GSDs, especially liver GSDs, take overlapping symptoms and can be clinically indistinguishable. This survey evaluated the use of whole-exome sequencing (WES) for the genetic analysis of the liver GSD-suspected patients in three unrelated families. An in-house filtering pipeline was used to assess rare pathogenic variants in GSD-associated genes, autosomal recessive/mendelian disorder genes (carrier status for genetic counseling subjects), and the ACMG’s list of 59 actionable genes. For the interpretation of the causative variants and the incidental/secondary findings, ACMG guidelines were applied. Additionally, we have explored PharmGKB class IA/IB pharmacogenetic variants. The segregation analysis was performed using Sanger sequencing for the novel causative variants. Bioinformatics analysis of the exome data in three individuals revealed three novel homozygous causative variants in the GSD-associated genes. The first variant, c.298_307delATGATCAACC in PYGL gene has related to HERS disease (GSD VI). Both variants of c.1043dupT and c.613-1G > C in SLC2A2 gene have been associated with Fanconi-Bickel syndrome (GSDXI). Eight pathogenic/likely pathogenic medical actionable findings in Mendelian disease genes and 10 pharmacogenetic variants with underlying drug response phenotypes have been identified. No known/expected pathogenic variants were detected in the ACMG’s list of 59 actionable genes. The logical filtering steps can help in finding other medical actionable secondary/incidental findings as well as effectively identifying the causative variants in heterogeneous conditions such as GSDs. Three novel variants related to GSD genes recognized in liver GSD-suspected patients with early infantile and childhood-age onset.
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