An easy-to-perform, culture-free Campylobacter point-of-management assay for processing plant applications

Aims Current culture-based methods for detection and determination of Campylobacter levels on processed chickens takes at least two days. Here, we sought to develop a new complete, low-cost and rapid (approximately 2·5 hours) detection system requiring minimal operator input. Methods and Results We observed a strong correlation between culture-based cell counts and our ability to detect either Campylobacter jejuni or Campylobacter coli by Loop mediated isothermal amplification (LAMP) from the same samples. This knowledge was used to develop a rapid and simple five-step assay to quantify Campylobacter, which was subsequently assessed for its specificity, reproducibility and accuracy in quantifying Campylobacter levels from processed chickens. The assay was found to be highly specific for C. jejuni and C. coli and was capable of distinguishing between samples that are either within or exceeding the industry set target of 6,000 Campylobacter colony forming units (cfu) per carcass (equivalent to 12 cfu ml-1 of chicken rinse) with> 90% accuracy relative to culture-based methods. Conclusions Our method can reliably quantify Campylobacter counts of processed chickens with an accuracy comparable to culture-based assays but provides results within hours as opposed to days. Significance and Impact of the Study The research presented here will help improve food safety by providing fast Campylobacter detection that will enable the implementation of real-time risk management strategies in poultry processing plants to rapidly test processed chickens and identify effective intervention strategies. This technology is a powerful tool that can be easily adapted for other organisms and thus could be highly beneficial for a broad range of industries

Perspectives on the Application of Genome-Editing Technologies in Crop Breeding

Most conventional and modern crop-improvement methods exploit natural or artificially induced genetic variations and require laborious characterization of the progeny of multiple generations of time-consuming genetic crosses. Genome-editing systems, in contrast, provide the means to rapidly modify genomes in a precise and predictable way, making it possible to introduce improvements directly into elite varieties. Here, we describe the range of applications available to agricultural researchers using existing genome-editing tools. In addition to providing examples of genome-editing applications in crop breeding, we discuss the technical and social challenges faced by breeders using genome-editing tools for crop improvement

Networking for advanced molecular diagnosis in acute myeloid leukemia patients is possible: the PETHEMA NGS-AML project

Next-generation sequencing (NGS) has recently been introduced to efficiently and simultaneously detect genetic variations in acute myeloid leukemia (AML). However, its implementation in the clinical routine raises new challenges focused on the diversity of assays and variant reporting criteria. In order to overcome this challenge, the PETHEMA group established a nationwide network of reference laboratories aimed to deliver molecular results in the clinics. We report the technical cross-validation results for NGS panel genes during the standardization process and the clinical validation in 823 samples of 751 patients with newly diagnosed or refractory/relapse AML. Two cross-validation rounds were performed in seven nationwide reference laboratories in order to reach a consensus regarding quality metrics criteria and variant reporting. In the pre-standardization cross-validation round, an overall concordance of 60.98% was obtained with a great variability in selected genes and conditions across laboratories. After consensus of relevant genes and optimization of quality parameters the overall concordance rose to 85.57% in the second cross-validation round. We show that a diagnostic network with harmonized NGS analysis and reporting in seven experienced laboratories is feasible in the context of a scientific group. This cooperative nationwide strategy provides advanced molecular diagnostic for AML patients of the PETHEMA group (clinicaltrials gov. Identifier: NCT03311815)

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Can heterotrimeric G proteins help to feed the world?

Recently, two important yield quantitative trait loci (QTLs), GS3 and DEP1, have been cloned in rice (Oryza sativa). Although their relationship has not been established in the 'rice literature', a recent report identified them as heterotrimeric G protein gamma subunits. This identification has profound consequences for our current understanding of both QTLs and the plant G protein signaling network and this opinion article discusses how manipulation of G protein signaling may lead to yield improvements in rice and other crop species. Finally, a mechanistic model to explain the seemingly conflicting phenotypes produced by different GS3 and DEP1 alleles is proposed

Site-directed mutagenesis of the Arabidopsis heterotrimeric G protein beta subunit suggests divergent mechanisms of effector activation between plant and animal G proteins

Heterotrimeric G proteins are integral components of signal transduction in humans and other mammals and have been therefore extensively studied. However, while they are known to mediate many processes, much less is currently known about the effector pathways and molecular mechanisms used by these proteins to regulate effectors in plants. We designed a complementation strategy to study G protein signaling in Arabidopsis thaliana, particularly the mechanism of action of AGB1, the sole identified β subunit. We used biochemical and effector regulation data from human G protein studies to identify four potentially important residues for site-directed mutagenesis (T65, M111, D250 and W361 of AGB1). Each residue was individually mutated and the resulting mutated protein introduced in the agb1-2 mutant background under the control of the native AGB1 promoter. Interestingly, even though these mutations have been shown to have profound effects on effector signaling in humans, all the mutated subunits were able to restore thirteen of the fifteen Gβ-deficient phenotypes characterized in this study. Only one mutated protein, T65A was unable to complement the hypersensitivity to mannitol during germination observed in agb1 mutants; while only D250A failed to restore lateral root numbers in the agb1 mutant to wild-type levels. Our results suggest that the mechanisms used in mammalian G protein signaling are not well conserved in plant G protein signaling, and that either the effectors used by plant G proteins, or the mechanisms used to activate them, are at least partially divergent from the well-studied mammalian G proteins

Conserved Role of Heterotrimeric G Proteins in Plant Defense and Cell Death Progression

Programmed cell death (PCD) is a critical process in plant immunity, enabling the targeted elimination of infected cells to prevent the spread of pathogens. The tight regulation of PCD within plant cells is well-documented; however, specific mechanisms remain elusive or controversial. Heterotrimeric G proteins are multifunctional signaling elements consisting of three distinct subunits, Gα, Gβ, and Gγ. In Arabidopsis, the Gβγ dimer serves as a positive regulator of plant defense. Conversely, in species such as rice, maize, cotton, and tomato, mutants deficient in Gβ exhibit constitutively active defense responses, suggesting a contrasting negative role for Gβ in defense mechanisms within these plants. Using a transient overexpression approach in addition to knockout mutants, we observed that Gβγ enhanced cell death progression and elevated the accumulation of reactive oxygen species in a similar manner across Arabidopsis, tomato, and Nicotiana benthamiana, suggesting a conserved G protein role in PCD regulation among diverse plant species. The enhancement of PCD progression was cooperatively regulated by Gβγ and one Gα, XLG2. We hypothesize that G proteins participate in two distinct mechanisms regulating the initiation and progression of PCD in plants. We speculate that G proteins may act as guardees, the absence of which triggers PCD. However, in Arabidopsis, this G protein guarding mechanism appears to have been lost in the course of evolution