Conventional detection of viruses and virus-like diseases of plants is accomplished using a combination of molecular, serological, and biological indexing. These are the primary tools used by plant virologists to monitor and ensure trees are free of known viral pathogens. The biological indexing assay, or bioassay, is considered to be the “gold standard” as it is the only method of the three that can detect new, uncharacterized, or poorly characterized viral disease agents. Unfortunately, this method is also the most labor intensive and can take up to three years to complete. Next generation sequencing (NGS) is a technology with rapidly expanding possibilities including potential applications for the detection of plant viruses. In this study, comparisons are made between tree fruit testing by conventional and NGS methods, to demonstrate the efficacy of NGS. A comparison of 178 infected trees, many infected with several viral pathogens, demonstrated that conventional and NGS were equally capable of detecting known viruses and viroids. Comparable results were obtained for 170 of 178 of the specimens. Of the remaining eight specimens, some discrepancies were observed between viruses detected by the two methods, representing less than 5% of the specimens. NGS was further demonstrated to be equal or superior for the detection of new or poorly characterized viruses when compared with a conventional bioassay. These results validated both the effectiveness of conventional virus testing methods and the use of NGS as an additional or alternative method for plant virus detection.
Transgenic soybean line GTS-40-3-2, marketed under the trade name Roundup Ready (RR) soy, was developed by Monsanto (USA) to allow for the use of glyphosate, the active ingredient of the herbicide Roundup, as a weed control agent. RR soy was first approved in Canada for environmental release and for feed products in 1995 and later for food products in 1996 and is widely grown in Canada. Consumer concern issues have resulted in proposed labeling regulations in Canada for foods derived from genetically engineered crops. One requirement for labeling is the ability to detect and accurately quantify the amount of transgenic material present in foods. Two assays were evaluated. A conventional qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of soy and RR soy and a real-time PCR to quantify the amount of RR soy present in samples that tested positive in the first assay. PCR controls consisted of certified RR soy reference material, single transgenic soybeans, and a processed food sample containing a known amount of RR soy. To test real-world applicability, a number of common grocery store food items that contain soy-based products were tested. For some samples, significant differences in amplification efficiencies during the quantitative PCR assays were observed compared to the controls, resulting in potentially large errors in quantification. A correction factor was used to try to compensate for these differences.
Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of aerolysin. The percentage of lysis of PNH cells after aerolysin exposure paralleled the percentage of CD59+ cells in the samples measured by flow cytometry. The kinetics of red blood cell lysis correlated with the type of PNH erythrocytes. PNH type III cells were completely resistant to aerolysin, whereas PNH type II cells displayed intermediate sensitivity. Importantly, the use of aerolysin allowed us to detect PNH populations that could not be detected by standard flow cytometry. Resistance of PNH cells to aerolysin allows for a simple, inexpensive assay for PNH that is sensitive and specific. Aerolysin should also be useful in studying PNH biology.
SummaryAerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosylphosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.
SummaryAerolysin is a dimeric protein secreted by Aeromonas spp. that binds to glycosylphosphatidylinositol-anchored receptors on target cells and becomes insertion competent by oligomerizing. The protein comprises two lobes joined by a short arm. The large lobe is thought to be responsible for channel formation, whereas the small lobe is believed to stabilize the dimer, and it may also contain the receptor binding site. We cloned and expressed the DNA for both lobes of the toxin separately and together in A. salmonicida. The large lobe produced alone was secreted, although more poorly than native protein. The small lobe with the arm produced by itself was not secreted. When the large lobe without the arm was co-produced with the small lobe with the arm, both were secreted, and they co-purified as a stoichiometric complex. Analytical ultracentrifugation showed that they form a heterotetramer corresponding to the native dimer. The purified product was nearly as active as aerolysin, but lost activity and became trypsin sensitive above 25ЊC. The large lobe with the arm was also purified. It was shown to be monomeric, confirming that the small lobe is responsible for dimer stabilization. The large lobe had very low channel-forming activity, although it was correctly processed by trypsin, and it could form stable oligomers. Surprisingly, the large lobe was found to bind to several glycosylphosphatidylinositol-anchored proteins, indicating that it contains at least part of the receptor-binding domain.
ImportanceIn patients with severe aortic valve stenosis at intermediate surgical risk, transcatheter aortic valve replacement (TAVR) with a self-expanding supra-annular valve was noninferior to surgery for all-cause mortality or disabling stroke at 2 years. Comparisons of longer-term clinical and hemodynamic outcomes in these patients are limited.ObjectiveTo report prespecified secondary 5-year outcomes from the Symptomatic Aortic Stenosis in Intermediate Risk Subjects Who Need Aortic Valve Replacement (SURTAVI) randomized clinical trial.Design, Setting, and ParticipantsSURTAVI is a prospective randomized, unblinded clinical trial. Randomization was stratified by investigational site and need for revascularization determined by the local heart teams. Patients with severe aortic valve stenosis deemed to be at intermediate risk of 30-day surgical mortality were enrolled at 87 centers from June 19, 2012, to June 30, 2016, in Europe and North America. Analysis took place between August and October 2021.InterventionPatients were randomized to TAVR with a self-expanding, supra-annular transcatheter or a surgical bioprosthesis.Main Outcomes and MeasuresThe prespecified secondary end points of death or disabling stroke and other adverse events and hemodynamic findings at 5 years. An independent clinical event committee adjudicated all serious adverse events and an independent echocardiographic core laboratory evaluated all echocardiograms at 5 years.ResultsA total of 1660 individuals underwent an attempted TAVR (n = 864) or surgical (n = 796) procedure. The mean (SD) age was 79.8 (6.2) years, 724 (43.6%) were female, and the mean (SD) Society of Thoracic Surgery Predicted Risk of Mortality score was 4.5% (1.6%). At 5 years, the rates of death or disabling stroke were similar (TAVR, 31.3% vs surgery, 30.8%; hazard ratio, 1.02 [95% CI, 0.85-1.22]; P = .85). Transprosthetic gradients remained lower (mean [SD], 8.6 [5.5] mm Hg vs 11.2 [6.0] mm Hg; P < .001) and aortic valve areas were higher (mean [SD], 2.2 [0.7] cm2 vs 1.8 [0.6] cm2; P < .001) with TAVR vs surgery. More patients had moderate/severe paravalvular leak with TAVR than surgery (11 [3.0%] vs 2 [0.7%]; risk difference, 2.37% [95% CI, 0.17%- 4.85%]; P = .05). New pacemaker implantation rates were higher for TAVR than surgery at 5 years (289 [39.1%] vs 94 [15.1%]; hazard ratio, 3.30 [95% CI, 2.61-4.17]; log-rank P < .001), as were valve reintervention rates (27 [3.5%] vs 11 [1.9%]; hazard ratio, 2.21 [95% CI, 1.10-4.45]; log-rank P = .02), although between 2 and 5 years only 6 patients who underwent TAVR and 7 who underwent surgery required a reintervention.Conclusions and RelevanceAmong intermediate-risk patients with symptomatic severe aortic stenosis, major clinical outcomes at 5 years were similar for TAVR and surgery. TAVR was associated with superior hemodynamic valve performance but also with more paravalvular leak and valve reinterventions.
A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.