Targeted gene disruption coupled with metabolic screen approach to uncover the LEAFY COTYLEDON1-LIKE4 (L1L4) function in tomato fruit metabolism

Key message Functional analysis of tomato L1L4 master transcription factor resulted in important metabolic changes affecting tomato fruit quality. Abstract Tomato fruits from mutant lines bearing targeted disruption of the heterotrimeric nuclear transcription factor Y (NF-Y) transcription factor (TF) gene LEAFY-COTYLEDON1-LIKE4 (L1L4, NF-YB6), a master regulator of biosynthesis for seed storage proteins and fatty acids, were evaluated for metabolites content and morphology. Metabolic screens using LC-MS/MS-based analysis and physico-chemical methods in different L1L4 mutants of the fourth generation allowed a comparative assessment of the effects of the TF disruption. Mutagenesis resulted in fruits phenotypically similar to wild-type with subtle shape differences in the distal end protrusion and symmetry. Conversely, mutant fruits from independent lines had significant variation in moisture content, titratable acidity and overall metabolite profiles including oxalic and citric acid, fructose, beta-carotene, total polyphenols and antioxidants. Lines 6, 7 and 9 were the richest in beta-carotene and antioxidant activity, line 4 in ascorbic acid and lines 4 and 8 in succinic acid. The reduced content of the anti-nutrient oxalic acid in several mutant fruits suggests that L1L4 gene may regulate the accumulation of this compound during fruit development. Detailed LC-MS/MS analysis of mutant seeds showed substantial differences in bioactive compounds compared to wild-type seeds. Taken together, the results suggest that the L1L4 TF is a significant regulator of metabolites both in tomato fruit and seeds providing a molecular target for crop improvement. Elucidation of the candidate genes encoding key enzymes in the affected metabolic pathways aimed to facilitate the L1L4 gene network exploration and eventually lead to systems biology approaches in tomato fruit quality.European funded COST ACTION [FA1106

Polyamine Catabolism in Plants: A Universal Process With Diverse Functions

Polyamine (PA) catabolic processes are performed by copper-containing amine oxidases (CuAOs) and flavin-containing PA oxidases (PAOs). So far, several CuAOs and PAOs have been identified in many plant species. These enzymes exhibit different subcellular localization, substrate specificity, and functional diversity. Since PAs are involved in numerous physiological processes, considerable efforts have been made to explore the functions of plant CuAOs and PAOs during the recent decades. The stress signal transduction pathways usually lead to increase of the intracellular PA levels, which are apoplastically secreted and oxidized by CuAOs and PAOs, with parallel production of hydrogen peroxide (H2O2). Depending on the levels of the generated H2O2, high or low, respectively, either programmed cell death (PCD) occurs or H2O2 is efficiently scavenged by enzymatic/nonenzymatic antioxidant factors that help plants coping with abiotic stress, recruiting different defense mechanisms, as compared to biotic stress. Amine and PA oxidases act further as PA back-converters in peroxisomes, also generating H2O2, possibly by activating Ca2+ permeable channels. Here, the new research data are discussed on the interconnection of PA catabolism with the derived H2O2, together with their signaling roles in developmental processes, such as fruit ripening, senescence, and biotic/abiotic stress reactions, in an effort to elucidate the mechanisms involved in crop adaptation/survival to adverse environmental conditions and to pathogenic infections

Neuronal nicotinic acetylcholine receptor antibodies in autoimmune central nervous system disorders

BackgroundNeuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the central nervous system (CNS), playing critical roles in brain function. Antigenicity of nAChRs has been well demonstrated with antibodies to ganglionic AChR subtypes (i.e., subunit α3 of α3β4-nAChR) and muscle AChR autoantibodies, thus making nAChRs candidate autoantigens in autoimmune CNS disorders. Antibodies to several membrane receptors, like NMDAR, have been identified in autoimmune encephalitis syndromes (AES), but many AES patients have yet to be unidentified for autoantibodies. This study aimed to develop of a cell-based assay (CBA) that selectively detects potentially pathogenic antibodies to subunits of the major nAChR subtypes (α4β2- and α7-nAChRs) and its use for the identification of such antibodies in “orphan” AES cases.MethodsThe study involved screening of sera derived from 1752 patients from Greece, Turkey and Italy, who requested testing for AES-associated antibodies, and from 1203 “control” patients with other neuropsychiatric diseases, from the same countries or from Germany. A sensitive live-CBA with α4β2-or α7-nAChR–transfected cells was developed to detect antibodies against extracellular domains of nAChR major subunits. Flow cytometry (FACS) was performed to confirm the CBA findings and indirect immunohistochemistry (IHC) to investigate serum autoantibodies’ binding to rat brain tissue.ResultsThree patients were found to be positive for serum antibodies against nAChR α4 subunit by CBA and the presence of the specific antibodies was quantitatively confirmed by FACS. We detected specific binding of patient‐derived serum anti‐nAChR α4 subunit antibodies to rat cerebellum and hippocampus tissue. No serum antibodies bound to the α7-nAChR-transfected or control-transfected cells, and no control serum antibodies bound to the transfected cells. All patients positive for serum anti‐nAChRs α4 subunit antibodies were negative for other AES-associated antibodies. All three of the anti‐nAChR α4 subunit serum antibody-positive patients fall into the AES spectrum, with one having Rasmussen encephalitis, another autoimmune meningoencephalomyelitis and another being diagnosed with possible autoimmune encephalitis.ConclusionThis study lends credence to the hypothesis that the major nAChR subunits are autoimmune targets in some cases of AES and establishes a sensitive live-CBA for the identification of such patients

DETECTION OF SMALL UNMANNED AERIAL SYSTEMS USING A 3D LIDAR SENSOR

Small unmanned aerial systems (sUAS) are a rapidly developing technology with countless applications in many areas of human activity, ranging from commercial to military use. In the latter case, counter-UAS operations have become an urgent issue. The problem is that the small size of a sUAS makes its detection quite a challenging task. Many of traditional approaches and technologies may not be applicable at all. This thesis describes a feasibility study for using a stationary 3D 360° Light Detection and Ranging (LiDAR) sensor to detect a fast-moving sUAS. Specifically, a low-end Velodyne Puck Hi-Res LiDAR was used to collect data during a series of flight tests involving different size sUASs at two rural locations. The thesis presents an analysis of the LiDAR output and the developed algorithms to detect a moving sUAS despite several challenges associated with a rich, nonstationary background return. These challenges were overcome by using Principal Components Analysis (PCA) as well as masking. The developed algorithm demonstrated that using a low-end LiDAR with a detection range of about 100 m, it is possible to detect a sUAS of about a 0.3 m cross-section, isolate it from other moving objects, and track it while as it maneuvers within a 25 m range. Obviously, using the same algorithm with a higher resolution LiDAR would allow detection at the higher ranges, thus making LiDAR-based counter-UAS technology a viable candidate for protecting against a UAS threat.Antisyntagmatarhis, Hellenic ArmyApproved for public release. Distribution is unlimited

Metalliska dammtransportsimuleringar för ITER och DEMO

Beryllium and tungsten dust transport is simulated for ITER and DEMO using the dust transport code MIGRAINe. By combining many single-particle simulations a statistical analysis is performed. From that, the evolution of the dust inventories in the reactors is studied. Additionally, predictions are made for the locations at which dust evaporates. Finally, simplified analytical models with a few parameters for fast estimations of the dust inventory evolution are proposed. In the future, these could be integrated into more global models that also take into account dust generation. Dammtransporten för beryllium och volfram simuleras för ITER och DEMO med dammtransportkoden MIGRAINe. En statistisk analys utförs genom att kombinera många enkelpartikelsimuleringar varpå utvecklingen av damminventeringe i reaktorerna studeras. Dessutom genomförs förutsägelser för de platser där damm evaporerar. Slutligen föreslås förenklade analytiska modeller med få parametrar för snabba uppskattningar av utvecklingen av damminventering. I framtiden kan dessa integreras i mer globala modeller som också tar hänsyn till dammgenerering

Metalliska dammtransportsimuleringar för ITER och DEMO

Beryllium and tungsten dust transport is simulated for ITER and DEMO using the dust transport code MIGRAINe. By combining many single-particle simulations a statistical analysis is performed. From that, the evolution of the dust inventories in the reactors is studied. Additionally, predictions are made for the locations at which dust evaporates. Finally, simplified analytical models with a few parameters for fast estimations of the dust inventory evolution are proposed. In the future, these could be integrated into more global models that also take into account dust generation. Dammtransporten för beryllium och volfram simuleras för ITER och DEMO med dammtransportkoden MIGRAINe. En statistisk analys utförs genom att kombinera många enkelpartikelsimuleringar varpå utvecklingen av damminventeringe i reaktorerna studeras. Dessutom genomförs förutsägelser för de platser där damm evaporerar. Slutligen föreslås förenklade analytiska modeller med få parametrar för snabba uppskattningar av utvecklingen av damminventering. I framtiden kan dessa integreras i mer globala modeller som också tar hänsyn till dammgenerering

Pilot Cultivation of the Vulnerable Cretan Endemic Verbascum arcturus L. (Scrophulariaceae): Effect of Fertilization on Growth and Quality Features

The domestication of wild-growing plants, including cultivation and fertilization protocols, is able to alleviate the ecological risks posed by the uncontrolled harvesting of range-restricted local endemic plants. In this field study focused on Verbascum arcturus, a vulnerable local endemic of Crete (Greece), the effect of two kinds of fertilization applied by two methods (foliar/root) was investigated. The foliar application included conventional or integrated nutrient management (INM) fertilization. Root application included the application of conventional fertilizers, biostimulants, or INM with biostimulants. Several properties of plant growth, physiology and nutrition were determined. The results showed that fertilization treatment affected neither leaf color and shape nor plant growth, morphology, dry mass partitioning or nutrient content. However, both kinds of foliar-applied fertilization enhanced Zn and B in leaves and soil-applied biostimulant increased leaf Ca. Considering both chlorophyll and antioxidant compounds’ content, foliar application of the INM fertilizers, as well as soil application of the conventional fertilizers or biostimulants, could be considered as accepted options. This study reports for the first time an assessment of the total phenolic and flavonoids content evidenced in V. arcturus and encourages the use of fertilization in promoting the herbal antioxidant profile without compromising visual quality or yield. The findings of this study could be considered as a documented contribution toward the sustainable exploitation of V. arcturus

Polyamine homeostasis during growth and development of tobacco (Nicotiana tabacum L.) and grapevine (Vitis vinifera L.)

The polyamines (PAs) spermidine (Spd) and spermine (Spm) and their obligatory precursor, putrescine (Put), are small aliphatic amines, ubiquitous in living organisms. Even though their exact role and function is not yet understood, many studies relate them with a wide range of physiological phenomena, from cellular development and differentiation to stress-induced responses. The preliminary and determinant stage of polyamine biosynthesis is the decarboxylation of ornithine and arginine from the homonymous decarboxylases, to putrescine and agmatine, respectively. Their homeostasis in plant cells is regulated by the rate of their biosynthesis, conjugation and catabolism. In the present study, the homeostasis of polyamines was examined, in an easily regenetated species, the tobacco (Nicotiana tabacum L) and in a woody perennial plant species, the grapevine (Vitis vinifera L.), which does not easily respond to in vitro morphogenic manipulations. Detailed spatial and temporal profiles of the endogenous soluble (S-), soluble-conjugated (SH-) and insoluble-conjugated (PH-) fractions of Put, Spd and Spm are shown in nearly all plant organs of both plant species. The profile of expression of enzymes of PA biosynthesis and catabolism at the transcript, protein and specific activity levels along with the endogenous concentrations of the precursor amino acids arginine and ornithine are also presented. The results reveal that PA titers and biosynthesis follow a basipetal decrease along the tobacco plant axis; the transcripts of the genes encoding the arginine decarboxylase (ADC), S-adenosyl-L-methionine decarboxylase (SAMDC) and spermidine synthase (SPDS) enzymes and ADC protein do not coincide with enzyme activities in the youngest tissues; Spd synthesis strongly correlates with intense cell divisions; the S-/tot-PA ratios gradually increase basipetally, whereas the conjugated PAs decrease with Spd to mainly determine these changes; total leaf size negatively correlates with PA titers and synthesis; PA biosynthetic enzymes are down-regulated during development; the titers of Arg and Orn and arginase activity increase with age; the shoot apical meristem is the main site of Spd and Spm biosynthesis, whereas the root synthesizes mostly Put. Analysis of Nicotiana tabacum plants also reveals temporally and spatially discrete patterns of di- and polyamine oxidases (DAO and PAO), peroxidases (POXs) and putrescine N-methyltransferase (PMT) expression in shoot apical cells, leaves (apical, peripheral and central regions), acropetal and basipetal petiole regions, internodes, young and old roots. Enzyme activity assays show that DAO preferentially oxidizes the aliphatic diamine putrescine, whereas PAO oxidizes the triamine spermidine and, preferentially, the tetramine spermine.Οι πολυαμίνες (ΠΑ) σπερμιδίνη (Spd) και σπερμίνη (Spm) και ο υποχρεωτικός πρόδρομος τους, η πουτρεσίνη (Put), είναι μικρές αλειφατικές αμίνες που βρίσκονται σε όλα τα κύτταρα. Αν και ο ακριβής ρόλος τους και ο τρόπος δράσης τους δεν έχει, ακόμα, γίνει κατανοητός, πολυάριθμες μελέτες τις συνδέουν με ποικίλες φυσιολογικές διαδικασίες, που περιλαμβάνουν από την κυτταρική ανάπτυξη και διαφοροποίηση έως και την απόκριση σε συνθήκες καταπόνησης. Το αρχικό και καθοριστικό στάδιο της βιοσύνθεσης των πολυαμινών είναι η αποκαρβοξυλίωση της ορνιθίνης και της αργινίνης από τις ομώνυμες αποκαρβοξυλάσες, προς πουτρεσίνη και αγματίνη, αντίστοιχα. Η ομοιοστασία τους στα κύτταρα των φυτών ρυθμίζεται από το βαθμό βιοσύνθεσης, σύζευξης και καταβολισμού τους. Στην παρούσα εργασία μελετήθηκε η ομοιοστασία των ΠΑ σε ένα εύκολα αναγεννώμενο είδος, τον καπνό (Νicotiana tabacum L.) και σε ένα πολυετές φυτικό είδος, την άμπελο (Vitis vinifera L.), το οποίο δεν αποκρίνεται εύκολα σε in vitro μορφογενετικούς χειρισμούς. Παρουσιάζονται αναλυτικά χωρoταξικά και χρονικά σχεδιαγράμματα των ενδογενών διαλυτών (S-), διαλυτών συζευγμένων (SH-) και αδιάλυτων συζευγμένων (PH-) κλασμάτων της Put, Spd και Spm σχεδόν σε όλα τα όργανα και των δύο φυτικών ειδών. Επίσης, μελετήθηκε η κατατομή της έκφρασης των ενζύμων βιοσύνθεσης και καταβολισμού των ΠΑ σε επίπεδα μεταγράφων, πρωτεϊνών και ειδικών ενεργοτήτων, μαζί με τις ενδογενείς συγκεντρώσεις των προδρόμων αμινοξέων αργινίνης και ορνιθίνης. Τα αποτελέσματα αποκαλύπτουν ότι οι ενδογενείς συγκεντρώσεις και η βιοσύνθεση των ΠΑ ακολουθούν μια βασιπέταλη μείωση κατά μήκος του άξονα των φυτών καπνού. Τα μετάγραφα των γονιδίων που κωδικοποιούν τα ένζυμα ADC, SAMDC και SPDS, καθώς και η πρωτεΐνη της ADC δε συμπίπτουν με τις ενζυμικές ενεργότητες στους νεώτερους ιστούς. Η σύνθεση της Spd συσχετίζεται ισχυρά με τις έντονες κυτταροδιαιρέσεις. Οι σχετικές αναλογίες S/tot-ΠΑς αυξάνονται βαθμιαία βασιπέταλα, ενώ οι συζευγμένες ΠΑς μειώνονται, με τη Spd να καθορίζει κυρίως αυτές τις αλλαγές. Το συνολικό μέγεθος φύλλων συσχετίζεται αρνητικά με τις ενδογενείς συγκεντρώσεις και τη σύνθεση ΠA. Τα βιοσυνθετικά ένζυμα ΠA υποεκφράζονται κατά τη διάρκεια της ανάπτυξης. Οι ποσότητες των Arg και Orn και η ενεργότητα της αργινάσης αυξάνονται με την ηλικία. Το ακραίο μερίστωμα του βλαστού είναι η κύρια περιοχή σύνθεσης των Spd και Spm, ενώ η ρίζα είναι η κύρια περιοχή σύνθεσης της Put

Interaction between concrete linings and surrounding ground in underground excavations: a contribution to the design of temporary and permanent tunnel support systems

The design of underground openings is inherently more difficult and uncertain in comparison to the design of other civil engineering structures, given that part of the rock mass’s self-supporting capacity is utilized, the coupled behavior of the lining and the surrounding rock mass, the inadaptability of equivalent continuum models to discontinuous rock, the poor modelling of attributes of reinforced concrete like cracking and creep by geotechnical software and the 3D nature of the physical problem that is often omitted for computational limitations. An algorithm for the calculation of the stiffness of reinforced concrete sections depending on the concrete class, the applied forces, the cracking extent and creep was formulated, according to the available literature and EC2. Moreover comparative analyses between equivalent continuum and discontinuous models were carried out in 2D and 3D using UDEC, 3DEC and FLAC. The stiffness calculation algorithm was programmed in UDEC using its native language (FISH) and was applied in the modelling of a typical road tunnel section and of a real underground project which had collapsed. The often used method of modelling cracked concrete behavior by globally lowering its stiffness was found to inadequately represent the redistribution of internal forces that takes place and to underestimate design forces by a factor significantly larger than typical safety factors (> 50%). In the 2D comparative study between equivalent continuum and discontinuous models the ground reaction curve was found to be 60% - 80% higher in the case of discontinuous, thus accurate, modelling. In the ensuing 3D analyses, it was observed that the main behavior and stability mechanism was vastly dependent on the properties of the rock joints, particularly when the elastic limit is breached, with a 10fold increase of crown displacements after excavation of the tunnel face. Furthermore, the equivalent continuum model exhibited significant sensitivity to rock strength variation: a 25% drop (from 100 kPa to 75 kPa) of the cohesion value (Mohr-Coulomb) resulted in a 20fold increase in crown displacements. However, the stabilizing effect of the tunnel face was found to extend to no more than one diameter away, after which the ground reaction curves roughly matched the ones from the 2D UDEC analyses.Ο σχεδιασμός των υπόγειων έργων εμφανίζει ιδιαίτερη δυσχέρεια και αβεβαιότητα σε σύγκριση με τα υπόλοιπα έργα του πολιτικού μηχανικού λόγω της αξιοποίησης μέρους της ικανότητας της γεωμάζας να αυτοϋποστηρίζεται, της συζευγμένης συμπεριφοράς του συστήματος γεωμάζας και μέτρων υποστήριξης, των αποκλίσεων που εμφανίζουν τα γεωτεχνικά υπολογιστικά μοντέλα συνεχούς μέσου από την πραγματική εντατική κατάσταση στις περιπτώσεις ασυνεχούς γεωμάζας, της ελλιπούς προσομοίωσης από τα γεωτεχνικά λογισμικά κρίσιμων ιδιοτήτων των επενδύσεων οπλισμένου σκυροδέματος όπως η ρηγμάτωση και ο ερπυσμός και της τρισδιάστατης φύσης του φυσικού προβλήματος, η οποία λόγω υπολογιστικού φόρτου συνήθως αγνοείται. Αναπτύχθηκε αλγόριθμος υπολογισμού της δυσκαμψίας διατομών οπλισμένου σκυροδέματος συναρτήσει των μηχανικών ιδιοτήτων του σκυροδέματος, της επιβαλλόμενης έντασης, του βαθμού ρηγμάτωσης και του ερπυσμού, με βάση τη βιβλιογραφία και τον EC2. Παράλληλα διεξήχθησαν συγκριτικές αριθμητικές αναλύσεις ασυνεχούς και ισοδύναμου συνεχούς μέσου στο επίπεδο και στο χώρο με τα λογισμικά UDEC, 3DEC και FLAC. Ο αλγόριθμος υπολογισμού δυσκαμψίας εισήχθη στο UDEC (ασυνεχές μέσο) μέσω γλώσσας προγραμματισμού (FISH) και εφαρμόστηκε στην ανάλυση τυπικής διατομής υπόγειας σήραγγας και σε πραγματικό υπόγειο έργο όπου εκδηλώθηκε αστοχία. Η συνήθης μέθοδος προσομοίωσης της ρηγμάτωσης μέσω καθολικής μείωσης του μέτρου ελαστικότητας της δυσκαμψίας της διατομής φάνηκε ότι δεν αποτυπώνει ορθά την ανακατανομή των εντατικών μεγεθών ένεκα τοπικής ρηγμάτωσης και δίνει συνήθως ευμενή αποτελέσματα, με σημαντικές υπερβάσεις (> 50%) των συντελεστών ασφαλείας. Στη σύγκριση προσομοίωσης ισοδύναμου συνεχούς και ασυνεχούς μέσου στο επίπεδο, η καμπύλη μετακίνησης - απαιτούμενης υποστήριξης προκύπτει 60% - 80% υψηλότερα στην περίπτωση της μοντελοποίησης ασυνεχούς μέσου. Στη τρισδιάστατη ανάλυση (3DEC) παρατηρήθηκε ότι ο μηχανισμός συμπεριφοράς ένεκα των ασυνεχειών έχει το μεγαλύτερο ποσοστό συνεισφοράς στην τελική εντατική κατάσταση και ότι όταν εκδηλώνεται πλαστικοποίηση, οι τελικές παραμορφώσεις πίσω από το μέτωπο προκύπτουν μία τάξη μεγέθους μεγαλύτερες. Επίσης παρατηρήθηκε σημαντική ευαισθησία του ισοδύναμου συνεχούς μέσου, όπου για μεταβολή της συνοχής κατά 25% (από 100 kPa στα 75 kPa) οι μετακινήσεις της γεωμάζας εικοσαπλασιάστηκαν. Τέλος οι καμπύλες αντίδρασης του εδάφους του 3DEC ταυτίζονται σε απόσταση 1D περίπου από το μέτωπο με αυτές της ανάλυσης στο επίπεδο στο UDEC