Comparison of two photosynthetic biomediators for herbicide detection

Los programas de monitoreo requieren procedimientos de detección rápida, sencilla y a bajo costo para la detección de productos químicos nocivos en el medio ambiente.  Los biosensores fotosintéticos son los que mejor reflejan el impacto de los compuestos tóxicos mediante la medición de la alteración de sus procesos fisiológicos [14,17]. Cuando son expuestos a la iluminación, los materiales fotosintéticos son capaces de transferir electrones a un aceptor de electrones que cambia su estado en reducción, en el cual la corriente eléctrica generada puede ser medida. El presente trabajo se centra en la construcción y la comparación de dos biosensores basados en la fotosíntesis usando la membrana tilacoide o de algas basadas en fotosíntesis.  Esta disposición dio lugar a dos biosensores amperometricos reutilizables para la detección de herbicidas, con límites de detección en el rango nanomolar. El biosensor de algas, como era de esperarse, presentan un tiempo de vitalidad  mayor Abstract Monitoring programs require rapid, simple and low-cost screening procedures for the detection of harmful chemicals in the environment. Photosynthetic biosensors better reflect the impact of toxic compounds by measuring the alteration of a physiological process [14,17]. Under illumination, photosynthetic materials are able to transfer electrons to an electron acceptor changing its redox state; the electric current generated can be measured amperometrically. The present work focuses on the construction and comparison of two photosynthesis-based biosensors using either thylakoid membrane or photosynthetic algae. This setup resulted in two reusable amperometric biosensors for the detection of herbicides both with limits of detection in the nanomolar range. The algal biosensor, as it was expected, presented an increased lifetim

Chondromyxoid Fibroma of the Lateral Malleolus: A Case Report

Chondromyxoid fibromas account for <1% of primary bone neoplasms. We report one such case occurring in the distal fibula of a 27-year-old woman. The patient underwent curettage, followed by phenolisation, insertion of a Steinmann pin, and cementation. This treatment reduced morbidity, restored stability, and enabled rapid functional recovery. There was no recurrence after 2 years

Role of the PSII-H subunit in photoprotection: novel aspects of D1 turnover in Synechocystis 6803.

Photosystem I-less Synechocystis 6803 mutants carrying modified PsbH proteins, derived from different combinations of wild-type cyanobacterial and maize genes, were constructed. The mutants were analyzed in order to determine the relative importance of the intra- and extramembrane domains of the PsbH subunit in the functioning of photosystem (PS) II, by a combination of biochemical, biophysical, and physiological approaches. The results confirmed and extended previously published data showing that, besides D1, the whole PsbH protein is necessary to determine the correct structure of a QB/herbicide-binding site. The different turnover of the D1 protein and chlorophyll photobleaching displayed by mutant cells in response to photoinhibitory treatment revealed for the first time the actual role of the PsbH subunit in photoprotection. A functional PsbH protein is necessary for (i) rapid degradation of photodamaged D1 molecules, which is essential to avoid further oxidative damage to the PSII core, and (ii) insertion of newly synthesized D1 molecules into the thylakoid membrane. PsbH is thus required for both initiation and completion of the repair cycle of the PSII complex in cyanobacteria

The Endolog system for moderate-to-severe hallux valgus

Purpose. To report the midterm outcome of the Endolog system for correction of moderate-to-severe hallux valgus. Methods. 23 women and 2 men (33 feet) aged 35 to 80 (mean, 52) years underwent minimally invasive surgery for moderate (n=25) and severe (n=8) hallux valgus using the Endolog system. The hallux valgus angle (HVA), the intermetatarsal angle (IMA), and the proximal articular set angle (PASA) were measured on radiographs. The feet were also assessed based on the American Orthopaedic Foot and Ankle Society (AOFAS) scale. Results. The mean follow-up duration was 18.2 (range, 12–36) months. The mean HVA, IMA, PASA, and the mean AOFAS score improved significantly after surgery (all p<0.0001). Periosteal reaction was noted by week 4, and callus formation after 3 months. There were no delayed or non-union or other complications. Conclusion. The Endolog system achieved good outcome for moderate-to-severe hallux valgus

Multiparametric system for biosensing technologies applications based on microarray electrodes

La plataforma biosensoristica aquí descrita ha sido diseñada para el uso de nuevos dispositivos eléctricos y versátiles como el MEA (Matriz de Microelectrodos), el sistema ha sido desarrollado aplicando los principios de la  transducción amperometrica en cada dispositivo especifico. Este documento explica las principales técnicas de medición de esta plataforma y se centra  en los dispositivos MEA de 4 pozos, que permiten el uso de hasta 4 tipos diferentes de biomediadores. El diseño y la construcción también son presentados. El documento se ha estructurado con las siguientes secciones: Hardware, sistema mecánico, software y la aplicación bioquímica. La plataforma descrita puede encontrar aplicación en dos campos diferentes: biomédico para la determinación de posibles anomalías en la sangre humana (por ejemplo: A través de la detección de colesterol, las catecolaminas, el glutamato, y de la bilirrubina en muestras de sangre), y en el campo ambiental para la detección de pesticidas o materiales pesados en agua o alimentos (mediante el uso de biomediadores especiales, tales como el fotosistema II (PSII) o en las membranas tilacoidales extraídas de algas verdes o de plantas). Las señales  procedentes del material están asociadas a los niveles de concentración de cada biomedidador acorde a la linea de caibracion y la sensibilidad de cada material  ABSTRACT The biosensor platform here described has been designed for the use of new, versatile, electric devices, such as MEA (Micro Electrodes Array). The system has been developed applying the principles of the amperometric transduction on each specific device. This paper explains the main techniques of measurement of this platform, and is focused on MEA devices with four wells, which allow the use of up to four different types of biomediators. The Design and construction of the platform are also presented. The paper has been structured with the following sections: hardware, mechanical system, software and biochemical application. The described platform can find application in two different fields: biomedical, for the determination of possible anomalies in the human body (e.g. through the detection of cholesterol, catecholamines, glutamate, and bilirubin in blood samples), and environmental, for the detection of pesticides or heavy metals in water or foods (through the use of special biomediators such as photosystem II (PSII), or thylakoid membranes, extracted from plants or green algae). The signals coming from the biological material are correlated to the concentration levels that detect each biomedidadors according to the calibration line and the sensitivity of the material.

CyanoFactory, a European consortium to develop technologies needed to advance cyanobacteria as chassis for production of chemicals and fuels

CyanoFactory, Design, construction and demonstration of solar biofuel production using novel (photo)synthetic cell factories, was an R&D project developed in response to the European Commission FP7-ENERGY-2012-1 call “Future Emerging Technologies” and the need for significant advances in both new science and technologies to convert solar energy into a fuel. CyanoFactory was an example of “purpose driven” research and development with identified scientific goals and creation of new technologies. The present overview highlights significant outcomes of the project, three years after its successful completion. The scientific progress of CyanoFactory involved: (i) development of a ToolBox for cyanobacterial synthetic biology; (ii) construction of DataWarehouse/Bioinformatics web-based capacities and functions; (iii) improvement of chassis growth, functionality and robustness; (iv) introduction of custom designed genetic constructs into cyanobacteria, (v) improvement of photosynthetic efficiency towards hydrogen production; (vi) biosafety mechanisms; (vii) analyses of the designed cyanobacterial cells to identify bottlenecks with suggestions on further improvements; (viii) metabolic modelling of engineered cells; (ix) development of an efficient laboratory scale photobioreactor unit; and (x) the assembly and experimental performance assessment of a larger (1350 L) outdoor flat panel photobioreactor system during two seasons. CyanoFactory - Custom design and purpose construction of microbial cells for the production of desired products using synthetic biology – aimed to go beyond conventional paths to pursue innovative and high impact goals. CyanoFactory brought together ten leading European partners (universities, research organizations and enterprises) with a common goal – to develop the future technologies in Synthetic biology and Advanced photobioreactors

Poly-β-Hydroxybutyrate Production by Rhodopseudomonas sp. Grown in Semi-Continuous Mode in a 4 L Photobioreactor

The synthesis of polyhydroxybutyrate (PHB) by photosynthetic non-sulfur bacteria is a potential approach for producing biodegradable plastics. In this work, acetate was used as a single carbon source to study the effect on PHB formation in Rhodopseudomonas sp. cultured in a cylindrical four-liter photobioreactor under semi-continuous mode. The cultivation process is divided into a symmetrical growth phase and a PHB accumulation phase separated temporally. The symmetrical growth phase (nutrient sufficient conditions) was followed by a sulfur-limited phase to promote PHB accumulation. The main novelty is the progressive lowering of the sulfur concentration into Rhodopseudomonas culture, which was obtained by two concomitant conditions: (1) sulfur consumption during the bacterial growth and (2) semi-continuous growth strategy. This caused a progressive lowering of the sulfur concentration into Rhodopseudomonas culturedue to the sulfur-free medium used to replace 2 L of culture (50% of the total) that was withdrawn from the photobioreactor at each dilution. The PHB content ranged from 9.26% to 15.24% of cell dry weight. At the steady state phase, the average cumulative PHB was &gt;210 mg/L. Sulfur deficiency proved to be one of the most suitable conditions to obtain high cumulative PHB in Rhodopseudomonas culture

Isolation, characterization and crystal structure of charybdin, a new, protein from Charybdis maritima. Production of a biosensor based on photosystem for herbicide detection

Α. In the present work, we describe the purification, characterization and structural determination of charybdin, a novel 29kDa protein from bulbs of Charybdis maritima agg. Charybdin was characterized by biochemical methods and its structure determined by X-ray crystallography. The DNA sequence, and derived amino acid sequence, revealed a significant homology with various ribosome inactivating proteins (RIPs) Although charybdin inhibited the rabbit reticulocyte translation system, the estimated IC50 value of 24nM indicates that it is not such a strong inhibitor of protein synthesis as other RIPs. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 A resolution led to the protein structure with the Molecular Replacement method. The active site of RIPs, which contains four key amino acid residues, is highly conserved. Although the three of the four key residues are present at the active site of charybdin, the fourth residue, which is an invariant Tyr80 (ricin numbering) among more than 360 RIP sequences known to date, is substituted by Val. Β. A screen-printed electrodes based sensor, with immobilised photosynthetic material was successfully developed and tested for detection of herbicides inhibiting photosynthesis. Upon illumination of the photosynthetic material a signal from a reduced mediator was recorded amperometrically. The added herbicide inhibits the photosynthetic process and decreases the reduction of the mediator. The decrease in the measured current is proportional to the herbicide concentration. The spinach thylakoid membranes successfully immobilised onto the sensor surface using cross-linking with glutaraldehyde and bovine serum albumin, gave reproducible and sensitive measurements. The developed system allowed reliable detection of herbicides with a detection limit ranging from 10 to 300nM, depending on the type of herbicide and with a measured lifetime up to 23 hours. In parallel and compared to the thylakoid membranes, Chlamydomonas reinhardtii mutant cells were immobilised and tested to assure a higher biological stability of the biosensor. Preliminary analysis of river water samples using this sensor was also performed indicating good correlation between the data obtain with the biosensor and the ones obtained after GC-MS analysis.Το πρώτο μέρος της παρούσας εργασίας αναφέρεται στο χαρακτηρισμό μια νέας πρωτεΐνης, που απομονώθηκε από βολβούς του οργανισμού Charybdis maritimα, και ονομάστηκε χαρυβδίνη. Το ενδιαφέρον για το συγκεκριμένο οργανισμό οφείλεται στο γεγονός ότι περιέχει ένα αρκετά μεγάλο αριθμό ενώσεων με σημαντικές φαρμακευτικές ιδιότητες. Η χαρυβδίνη απομονώθηκε σε καθαρή μορφή και παρήχθησαν κρύσταλλοι οι οποίοι και έδωσαν δεδομένα περίθλασης σε διακριτικότητα 1,6A. Τα δεδομένα περίθλασης αναλύθηκαν για τον προσδιορισμό των χαρακτηριστικών της δομή της, ενώ προσδιορίστηκε επίσης η αλληλουχία DNA καθώς και η αμινοξική αλληλουχία. Η σύγκριση της αλληλουχίας αμινοξέων της χαρυβδίνης με αλληλουχίες μεγάλου αριθμού πρωτεϊνών έδειξε ότι η χαρυβδίνη είναι μέλος μια σημαντικής οικογένειας πρωτεϊνών οι οποίες αναστέλλουν μη αντιστρεπτά τη δράση των ριβοσωμάτων (Ribosome Inactivating Proteins - RIP). Η χαρυβδίνη αποτελείται από μια ενιαία πολύπεπτιδική αλυσίδα με μοριακό βάρος περίπου 29 kDa, η οποία (μέσω ενός μηχανισμού απογλυκοζιλίωσης) αναστέλλει την πρωτεϊνική μετάφραση σε διαλυμένα δίκτυο-ερυθροκύτταρα με IC50 24nM. Χαρακτηριστικό της χαρυβδίνης είναι η διαφοροποίηση εξαιτίας μετάλλαξης, ενός εκ των τεσσάρων αμινοξέων που σχηματίζουν το ενεργό της κέντρο. Συγκεκριμένα, στη θέση 79 του ενεργού κέντρου μια βαλίνη έχει αντικαταστήσει το κατά τα άλλα συντηρημένο, μεταξύ 360 πρωτεϊνών RIP, κατάλοιπο τυροσίνης. Το δεύτερο μέρος της εργασίας αναφέρεται στην ανάπτυξη ενός αμπερομετρικού αισθητήρα για την ανίχνευση ζιζανιοκτόνων, χρησιμοποιώντας φωτοσυνθετικό υλικό ακινητοποιημένο σε ηλεκτρόδια μεταξοτυπίας. Η ακινητοποίηση πραγματοποιήθηκε με την μέθοδο της μοριακής διασύνδεσης παρουσία BSA και γλουταραλδεΰδης. Η αρχή ανίχνευσης ζιζανιοκτόνου βασίζεται στην παρεμπόδιση της ηλεκτρονιακής ροής στο φωτοσύστημα ΙΙ εξαιτίας της παρουσίας του ζιζανιοκτόνου, το οποίο εμποδίζει τη φωτοσυνθετική διαδικασία μειώνοντας τη αναγωγή του μεταφορέα ηλεκτρονίων με συνέπεια την μείωση του επαγόμενου ρεύματος. Η διαδικασία της φωτοσύνθεσης ελέγχεται μέσω της ποσότητας του ανηγμένου τεχνητού μεταφορέα ηλεκτρονίων, ο οποίος παράγεται κατά τον φωτισμό του φωτοσυνθετικού υλικού. Η οξείδωση του παραπάνω μεταφορέα στο ηλεκτρόδιο παράγει και το καταγραφόμενο σήμα, συσχετίζοντας έτσι την ένταση του ρεύματος με τη συγκέντρωση του ζιζανιοκτόνου. Το συγκεκριμένο σύστημα, χρησιμοποιώντας θυλακοειδείς μεμβράνες, έχει χρόνο ζωής έως και 23 ώρες και επιτρέπει την ανίχνευση των φωτοσυνθετικών ζιζανιοκτόνων με όριο ανίχνευσης της τάξεως των 10-300nM, αναλόγως με τον τύπο ζιζανιοκτόνου. Προκαταρτικές αναλύσεις δειγματοληψιών ύδατος ποταμού έδειξαν καλό συσχετισμό μεταξύ των αποτελεσμάτων που λήφθηκαν με χρωματογραφία-GC και τη χρήση του παραπάνω βιοαισθητήρα. Παράλληλα μελετήθηκε η χρήση ακινητοποιημένων κυττάρων Chlamydomonas, όπου και παρατηρήθηκε αύξηση του χρόνου ζωής σε 42 ώρες και μείωση του ορίου ανίχνευσης των φωτοσυνθετικών ζιζανιοκτόνων στα 20-8nM, ανάλογα με τον τύπο ζιζανιοκτόνου