An Efficient Triplex TaqMan Quantitative PCR to Detect a Blackleg-Causing Lineage of Pectobacterium brasiliense in Potato Based on a Pangenome Analysis.

P. brasiliense is an important bacterial pathogen causing blackleg (BL) in potatoes. Nevertheless, P. brasiliense is often detected in seed lots that do not develop any of the typical blackleg symptoms in the potato crop when planted. Field bioassays identified that P. brasiliense strains can be categorized into two distinct classes, some able to cause blackleg symptoms and some unable to do it. A comparative pangenomic approach was performed on 116 P. brasiliense strains, of which 15 were characterized as BL-causing strains and 25 as non-causative. In a genetically homogeneous clade comprising all BL-causing P. brasiliense strains, two genes only present in the BL-causing strains were identified, one encoding a predicted lysozyme inhibitor Lprl (LZI) and one encoding a putative Toll/interleukin-1 receptor (TIR) domain-containing protein. TaqMan assays for the specific detection of BL-causing P. brasiliense were developed and integrated with the previously developed generic P. brasiliense assay into a triplex TaqMan assay. This simultaneous detection makes the scoring more efficient as only a single tube is needed, and it is more robust as BL-causing strains of P. brasiliense should be positive for all three assays. Individual P. brasiliense strains were found to be either positive for all three assays or only for the P. brasiliense assay. In potato samples, the mixed presence of BL-causing and not BL-causing P. brasiliense strains was observed as shown by the difference in Ct value of the TaqMan assays. However, upon extension of the number of strains, it became clear that in recent years additional BL-causing lineages of P. brasiliense were detected for which additional assays must be developed.

Comparative Study of Variations in Quantum Approximate Optimization Algorithms for the Traveling Salesman Problem.

The traveling salesman problem (TSP) is one of the most often-used NP-hard problems in computer science to study the effectiveness of computing models and hardware platforms. In this regard, it is also heavily used as a vehicle to study the feasibility of the quantum computing paradigm for this class of problems. In this paper, we tackle the TSP using the quantum approximate optimization algorithm (QAOA) approach by formulating it as an optimization problem. By adopting an improved qubit encoding strategy and a layer-wise learning optimization protocol, we present numerical results obtained from the gate-based digital quantum simulator, specifically targeting TSP instances with 3, 4, and 5 cities. We focus on the evaluations of three distinctive QAOA mixer designs, considering their performances in terms of numerical accuracy and optimization cost. Notably, we find that a well-balanced QAOA mixer design exhibits more promising potential for gate-based simulators and realistic quantum devices in the long run, an observation further supported by our noise model simulations. Furthermore, we investigate the sensitivity of the simulations to the TSP graph. Overall, our simulation results show that the digital quantum simulation of problem-inspired ansatz is a successful candidate for finding optimal TSP solutions.

The development of a decision aid for shared decision making in the Dutch implantable cardioverter defibrillator patient population: A novel approach to patient education.

Background: Counseling of Implantable Cardioverter-defibrillator (ICD) patients with regard to individual risks and benefits is challenging. An evidence-based decision aid tailored to the needs of Dutch ICD patients is not yet available. The objective of this pilot project was to structurally evaluate the current clinical practice in The Netherlands and the ICD patient experience, in order to develop an online decision aid to facilitate shared decision making in ICD procedures. Methods: Between June 2016 and December 2017, a Dutch web-based decision aid was developed according to the Patient Decision Aid Standards (IPDAS) using the RAND-UCLA/multi-stepped Delphi model. Development process consisted of 5 stages in which the Dutch clinical practice was reviewed (stage 1), patients' needs and their history of decision making was structurally assessed (stages 2A and B) and a modified Delphi consensus process was performed with an expert panel consisting of representatives from different medical fields (stage 3). Results from stages 1-3 were used to design and structure the content of an online-based decision aid (stage 4) which was finally evaluated in a usability testing by patients in stage 5. Results and conclusion: This study describes the evidence-based approach to the development of the Dutch ICD decision aid. In our population, levels of shared decision-making experience were low. The ICD decision aid was structurally developed for the Dutch ICD patient population. Our upcoming multicenter stepped wedge clustered randomized trial will further evaluate the ICD decision aid in clinical practice.

Specific recommendations (or lack thereof) for older patients with cardiovascular disease in the current European Society of Cardiology guidelines : From the Dutch Working Group of Geriatric Cardiology of the Netherlands Society of Cardiology (NVVC) and Special Interest Group Geriatric Cardiology of the Netherlands Society for Clinical Geriatrics (NVKG).

Due to population ageing, the number of older and frail patients with cardiovascular disease is increasing. In the current guidelines of the European Society of Cardiology specific recommendations for this older population are missing or scarce, probably due to limited evidence concerning diagnosis and treatment of cardiovascular disease in older patients. Moreover, recommendations on shared decision making, palliative care and advanced care planning are also essential in these guidelines. In this article we evaluate the current European of Society of Cardiology guidelines (2013-2020) to determine whether specific recommendations for older patients have been included.

Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls.

BACKGROUND AND AIMS: The programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by ß-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested. METHODS: We developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action. KEY RESULTS: In model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide ('ΔUA-GalA'), taken as diagnostic of PL action. ΔUA-GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA-GalA from higher homologues. The ΔUA-GalA was confirmed as 4-deoxy-ß-l-threo-hex-4-enopyranuronosyl-(1→4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA-GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA-GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA-GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol-1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action. CONCLUSIONS: The results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.

The Pectobacterium pangenome, with a focus on Pectobacterium brasiliense, shows a robust core and extensive exchange of genes from a shared gene pool.

BACKGROUND: Bacterial plant pathogens of the Pectobacterium genus are responsible for a wide spectrum of diseases in plants, including important crops such as potato, tomato, lettuce, and banana. Investigation of the genetic diversity underlying virulence and host specificity can be performed at genome level by using a comprehensive comparative approach called pangenomics. A pangenomic approach, using newly developed functionalities in PanTools, was applied to analyze the complex phylogeny of the Pectobacterium genus. We specifically used the pangenome to investigate genetic differences between virulent and avirulent strains of P. brasiliense, a potato blackleg causing species dominantly present in Western Europe. RESULTS: Here we generated a multilevel pangenome for Pectobacterium, comprising 197 strains across 19 species, including type strains, with a focus on P. brasiliense. The extensive phylogenetic analysis of the Pectobacterium genus showed robust distinct clades, with most detail provided by 452,388 parsimony-informative single-nucleotide polymorphisms identified in single-copy orthologs. The average Pectobacterium genome consists of 47% core genes, 1% unique genes, and 52% accessory genes. Using the pangenome, we zoomed in on differences between virulent and avirulent P. brasiliense strains and identified 86 genes associated to virulent strains. We found that the organization of genes is highly structured and linked with gene conservation, function, and transcriptional orientation. CONCLUSION: The pangenome analysis demonstrates that evolution in Pectobacteria is a highly dynamic process, including gene acquisitions partly in clusters, genome rearrangements, and loss of genes. Pectobacterium species are typically not characterized by a set of species-specific genes, but instead present themselves using new gene combinations from the shared gene pool. A multilevel pangenomic approach, fusing DNA, protein, biological function, taxonomic group, and phenotypes, facilitates studies in a flexible taxonomic context.

The learning curve of video-assisted mediastinoscopic lymphadenectomy for staging of non-small-cell lung carcinoma.

OBJECTIVES: The objective of this study was to define the learning process of video-assisted mediastinoscopic lymphadenectomy (VAMLA) by the assessment of consecutive procedural metrics. METHODS: We conducted a single-centre retrospective observational study of all consecutive VAMLAs performed between 2011 and 2018 for the staging of non-small-cell lung carcinoma. Learning curves were assessed using non-risk adjusted cumulative observed minus expected (CUSUM) failure charts of complications. Boundary lines were defined by the acceptable and unacceptable complication rates of 4.5% and 15.0%. The Kruskal-Wallis test with post hoc analysis was used to assess trends in operation time and blood loss. RESULTS: Two-hundred-thirty-six unique VAMLAs by 4 surgeons performing their first procedures were evaluated. CUSUM charts of surgeons A and B showed a typical learning curve with an initial incline, followed by a turning point towards lower complications rates after 16-17 cases, whereas surgeons C and D showed an average performance. The median time between consecutive VAMLAs was shorter for surgeons A and B (13.0 vs 28.5-38.0 days for surgeons C and D). Overcoming the learning curve, complication rates of surgeons A and B decreased from 19% to 3% and from 18% to 5%, respectively. Operation time and blood loss showed a significant improvement after, respectively, 81-100 and 61-80 procedures compared to the first 20 procedures. CONCLUSIONS: VAMLA is a safe procedure to adopt and perform with acceptable complication rates from the first operation onward, regardless of the caseload. To overcome its learning curve, 16-17 cases are required, preferably at least 1 per 2 weeks.

Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method.

BACKGROUND AND AIMS: Many fruits soften during ripening, which is important commercially and in rendering the fruit attractive to seed-dispersing animals. Cell-wall polysaccharide hydrolases may contribute to softening, but sometimes appear to be absent. An alternative hypothesis is that hydroxyl radicals ((•)OH) non-enzymically cleave wall polysaccharides. We evaluated this hypothesis by using a new fluorescent labelling procedure to 'fingerprint' (•)OH-attacked polysaccharides. METHODS: We tagged fruit polysaccharides with 2-(isopropylamino)-acridone (pAMAC) groups to detect (a) any mid-chain glycosulose residues formed in vivo during (•)OH action and (b) the conventional reducing termini. The pAMAC-labelled pectins were digested with Driselase, and the products resolved by high-voltage electrophoresis and high-pressure liquid chromatography. KEY RESULTS: Strawberry, pear, mango, banana, apple, avocado, Arbutus unedo, plum and nectarine pectins all yielded several pAMAC-labelled products. GalA-pAMAC (monomeric galacturonate, labelled with pAMAC at carbon-1) was produced in all species, usually increasing during fruit softening. The six true fruits also gave pAMAC·UA-GalA disaccharides (where pAMAC·UA is an unspecified uronate, labelled at a position other than carbon-1), with yields increasing during softening. Among false fruits, apple and strawberry gave little pAMAC·UA-GalA; pear produced it transiently. CONCLUSIONS: GalA-pAMAC arises from pectic reducing termini, formed by any of three proposed chain-cleaving agents ((•)OH, endopolygalacturonase and pectate lyase), any of which could cause its ripening-related increase. In contrast, pAMAC·UA-GalA conjugates are diagnostic of mid-chain oxidation of pectins by (•)OH. The evidence shows that (•)OH radicals do indeed attack fruit cell wall polysaccharides non-enzymically during softening in vivo. This applies much more prominently to drupes and berries (true fruits) than to false fruits (swollen receptacles). (•)OH radical attack on polysaccharides is thus predominantly a feature of ovary-wall tissue.

A genetic anomaly of oriented collagen biosynthesis and cross-linking: Keratoconus.

Oriented collagen biosynthesis is one of the major mechanisms involved in tissue and organ formation during development. Corneal biogenesis is one example. Defects in this process lead to anomalies in tissue structure and function. The transparency of cornea and its achievement are a good example as well as its pathological modifications. Keratoconus is one example of this type of pathologies, involving also inappropriate cross-linking of collagen fibers. Among the tentatives to correct this anomaly, the riboflavin-potentiated UV-cross-linking (CXL) of keratoconus corneas appears clinically satisfactory, although none of the experiments and clinical results published prove effective cross-linking. The published results are reviewed in this article.

Fingerprinting of hydroxyl radical-attacked polysaccharides by N-isopropyl-2-aminoacridone labelling.

Hydroxyl radicals (•OH) cause non-enzymic scission of polysaccharides in diverse biological systems. Such reactions can be detrimental (e.g. causing rheumatic and arthritic diseases in mammals) or beneficial (e.g. promoting the softening of ripening fruit, and biomass saccharification). Here we present a method for documenting •OH action, based on fluorescent labelling of the oxo groups that are introduced as glycosulose residues when •OH attacks polysaccharides. The method was tested on several polysaccharides, especially pectin, after treatment with Fenton reagents. 2-Aminoacridone plus cyanoborohydride reductively aminated the oxo groups in treated polysaccharides; the product was then reacted with acetone plus cyanoborohydride, forming a stable tertiary amine with the carbohydrate linked to N-isopropyl-2-aminoacridone (pAMAC). Digestion of labelled pectin with 'Driselase' yielded several fluorescent products which on electrophoresis and HPLC provided a useful 'fingerprint' indicating •OH attack. The most diagnostic product was a disaccharide conjugate of the type pAMAC·UA-GalA (UA=unspecified uronic acid), whose UA-GalA bond was Driselase-resistant (product 2A). 2A was clearly distinguishable from GalA-GalA-pAMAC (disaccharide labelled at its reducing end), which was digestible to GalA-pAMAC. The methodology is applicable, with appropriate enzymes in place of Driselase, for detecting natural and artificial •OH attack in diverse plant, animal and microbial polysaccharides.

Xanthine oxido-reductase, free radicals and cardiovascular disease. A critical review.

Free radical mediated pathologies occupy a special place in medical semiology and in mechanistic interpretation of diseases. Free radicals, or better reactive oxygen species (ROS) or reactive nitrogen species (RNS) play also an important role in cell signaling. This is the basis of the ambivalent (Jekyll-Hyde) situation of ROS in biology and pathology. Aging itself is attributed by a popular theory to free radicals. A number of ROS-scavenging substances and procedures were described without however reaching credibility for their therapeutic value. An interesting exception is the xanthine oxido-reductase produced ROS and their role in cardiovascular disease. Allopurinol inhibition of xanthine oxido-reductase was shown to be efficient in some cases of cardiovascular diseases. Another important aspect of xanthine oxido-reductase produced ROS is their antibacterial capacity considered to be of importance with newborns fed on milk rich in this enzyme as well as at the gastrointestinal barrier. This ambivalent role of xanthine oxido-reductase justifies this review on the basic enzymatic mechanisms involved, derived ROS production, their role in the above mentioned biological processes and especially the interest of the inhibition of this enzyme as a preventive or curative measure in some cardiovascular pathologies.

Symmetry-breaking, grouped images and multistability with transient unconsciousness.

Multistability in consciousness is characterised by transient switches in which the attributes of space and time are locally absent. An extensive number of studies has attempted to describe and predict the causes and duration of such switches, and many are obviously incomplete models or wrong, but some show promise. Models have, for example, drawn on neural network theory, psychophysics, signal detection theory, Markov matrices, and Shilnikov dynamics. Levels of macro-, meso- and micro-dynamics are employed by writers and contrasted. We compare some of those models and find problems in attempting to identify their properties and causality. Discontinuities in the observed local evolution of dynamical time series may be modelled in various ways; they are observed in multistability switches, in saddle-node bifurcations, and in cusp catastrophes. Three models, involving psychophysics, rapid recurrence, and neural networks, are considered as complementing rather than competing for representation.

Decision making with complex nonlinear systems: inference and identification in the context of DS22q11.2.

A rare gene deletion syndrome, that has in its associated phenome some possible cognitive and psychotic features, has been examined with DNA and fMRI for its causal basis within families and its statistical distribution in populations. Identification of its presence without DNA evidence is problematic as the condition is not stationary nor linear in its properties as the carrier grows older. Within a family its distribution is Mendelian, but there are also complications due to its complexity. A combined approach using both signal detection and an extension of Bayes theorem is a possible approach to discriminating between symptoms that have potentially a multi-causal basis, of which 22q11.2 deletion is only one possibility. Two later issues have arisen, one involving possibly at least two genetically different syndromes that result in similar autism in infancy, the other in statistical problems of prediction. Diagnosis of probable early DS 22q11.2 independent deaths as opposed to survival into adulthood can be wrongly thought to be a case of infanticide, and legal disputes have consequently arisen in the U.K., the USA, and Australia.

[The irido-corneo-endothelial syndrome. The loss of the control of corneal endothelial cell cycle. A review]. / Le syndrome irido-cornéo-endothélial ou la perte du contrôle du cycle cellulaire de l'endothélium cornéen. Une revue.

The three major symptoms of the irido-corneo-endothelial syndrome are the alterations of the corneal endothelium and of the iris with a loss of the regulation of the cell cycle, and the progressive obstruction of the irido-corneal angle. This rare pathology attacks mainly young adult women. Most of the symptoms and complications originate from the excessive proliferation of the corneal endothelial cells accompanied by the evolution of their phenotype towards that of the epithelial cells. In normal conditions the corneal endothelial cells do not divide, they are blocked in the G1 stage of the cell cycle, mainly because of the action of the inhibitors of cyclin-dependent kinases. Still these cells retain a good capacity for proliferation, which can be induced by the down-regulation of the expression of the inhibitors of the cyclin-dependent kinases. This proliferative capacity declines with age and is also different according to the localization of the cells: it is more intense with those originating from the central area then in those from the peripheral area of the cornea. The age-related decline of the proliferative capacity is not due to the shortening of the telomers, but to the stress-induced accelerated senescence of the cells.

Substrate-protecting antiproteolytic agents for the prevention of pathological degradation of connective tissues. A review.

Connective tissues play an important role in the physiological functions of the organism. The integrity of the macromolecular components of these tissues, also called extracellular matrix, is necessary for their functional efficiency. A number of proteinases present in the organism, and the activity of which increases with age and with several pathologies, specifically degrade the components of the extracellular matrix. For a long time, tentatives for the protection of the matrix-components against degradation were made with low molecular weight inhibitors, not very efficient in vivo and not devoid of inconveniencies. We initiated a different approach for the preservation of the macromolecules of the extracellular matrix against proteolytic degradation with substances which exert an intense antiproteolytic activity not only in vitro, but also in vivo. The particularity of these substances is the fact that they do not act on the enzymes, but combine with the macromolecules. This is the type of combination of substances with the macromolecules of the matrix that prevents their degradation by the proteinases. Because of this affinity of such antiproteolytic agents not for the enzymes but for the substrates, we called them "substrate protectors" (Robert et al., 1979). The aim of the present review is to summarise the essential of our experiments which led to the description of substrate protectors.

Celebration of the 50th anniversary of the foundation of the French society for connective tissue research. Its short history in the frame of the origin and development of this discipline.

The science of connective tissues has (at least) a double origin. Collagen, their major constituent was first studied in conjunction with the leather industry. Acid mucopolysaccharides (now glycosaminoglycans) were characterised by (bio)-chemists interested in glycoconjugates. They joined mainly hospital-based rheumatology departments. Later started the study of elastin with the discovery of elastases and of connective tissue-born (structural) glycoproteins. Besides rhumatologists and leather-chemists mainly pathologists became involved in this type of research, followed closely by ophthalmology research. The first important meetings of these diverse specialists were organised under the auspices of NATO, first in Saint-Andrew's in GB in 1964 and a few years later (1969) in Santa Margareta, Italy. With the discovery of fibronectin, a "structural glycoprotein", started the study of cell-matrix interactions, reinforced by the identification of cell-receptors mediating them and the "cross-talk" between cells and matrix constituents. The first initiative to organise societies for this rapidly growing discipline was that of Ward Pigman in New York in 1961, restricted however to glycol-conjugates. Next year, in 1962 was founded the first European Connective Tissue Society in Paris: the "Club français du tissu conjonctif", which played a crucial role in the establishment of schools, laboratories, national and international meetings in the major cities of France: Paris, Lyon, Reims, Caen,Toulouse. A second European society was born in Great Britain, and at a joint meeting with the French society at the Paris Pasteur Institute, was founded in 1967 by these societies the Federation of European Connective Tissue Societies (FECTS). Their meetings, organised every second year, drained a wide attendance from all over the world. An increasing number of young scientists joined since then this branch of biomedical discipline with several international journals devoted to connective tissue research, to matrix biology. The increasing number and quality of the young generation of scientists engaged in research related to the extracellular matrix or better Biomatrix and cell-matrix interactions is a further guarantee for the continued interest in this crucial field of science at the interface of basic and medically oriented research.

Apple extract induces increased epithelial resistance and claudin 4 expression in Caco-2 cells.

BACKGROUND: The small intestinal epithelium functions both to absorb nutrients, and to provide a barrier between the outside, luminal, world and the human body. One of the passageways across the intestinal epithelium is paracellular diffusion, which is controlled by the properties of tight junction complexes. We used a differentiated Caco-2 monolayer as a model for small intestinal epithelium to study the effect of crude apple extracts on paracellular permeability. RESULTS: Exposure of crude apple homogenate to the differentiated Caco-2 cells increased the paracellular resistance, determined as trans-epithelial electrical resistance (TEER). This increase was linearly related to the concentration of apple present. The TEER-enhancing effect of apple extract was due to factors mainly present in the cortex, and the induction was not inhibited by protein kinase inhibitors. Apple-induced resistance was accompanied by increased expression of several tight junction related genes, including claudin 4 (CLDN4). CONCLUSION: Crude apple extract induces a higher paracellular resistance in differentiated Caco-2 cells. Future research will determine whether these results can be extrapolated to human small intestinal epithelia.

Physiology of skin aging.

Skin is the most voluminous organ of the body. It assumes several important physiological functions and represents also a "social interface" between an individual and other members of society. This is the main reason its age-dependent modifications are in the forefront of dermatological research and of the "anti-aging" cosmetic industry. Here we concentrate on some aspects only of skin aging, as far as the cellular and extracellular matrix components of skin are concerned. Most well studied mechanisms of skin aging can be situated at the postgenetic level, both epigenetic and post-translational mechanisms being involved. Some of these mechanisms will be reviewed as well as the capacity of fucose- and rhamnose-rich oligo- and polysaccharides (FROP and RROP) to counteract several of the mechanisms involved in skin aging.

Multistable switching series in chaotic nets.

Examples of conscious and interpretable responses that have two or more forms alternating to the same stimuli have been known for centuries, and methods of describing how such situations arise have evolved in biological science. When switches between transient, perceptual or cognitive responses can occur and are mixed serially within time series exhibiting local terminal stability, then patterns arise where psychological data series are too brief to analyse empirically, and neurophysiological data and mathematical simulation are necessary. Modelling such conditions can be approached by using one modified Markov matrix, which we illustrate if we allow some singularities to exist in the dynamics. As soon as networks cease to be homogeneous and have a number of attractors present and operate with different local structures, then one or more response patterns may potentially exist at the same time. The patterns may be addressed within the behavioural dynamics by incorporating in turn very short transients that can be voluntary or involuntary, in sensory and cognitive data. Related software work for modelling, employing hierarchical Dirichlet structures projected into hidden Markov matrices is noted.