Effects of fluid-induced shear stress on articular cartilage regeneration under simulated aspects of microgravity

Because a true microgravity environment results in a reduction of mechanical forces on the human body, possibly at each level of physiological organization, we have investigated the effects of applying a stimulus in the form of fluid-induced shear stress to regenerating articular cartilage cultured in an environment hypothesized to simulate aspects of microgravity. Significant alterations to the NASA-designed rotating wall vessel were made to create a unique bioreactor specifically designed for this study. This distinctive reactor not only allowed us to apply fluid-induced shear stress to tissue constructs, it allowed for control of its magnitude, and for independent control of distinct microgravity conditions.;Simulated microgravity conditions were found to decrease the concentration of cells and extracellular matrix components as has been observed in true microgravity. Our results provide support that rotational rate in horizontally rotating cultures affects cellular events and may have a direct relation in the simulation of aspects of microgravity. Negligible to low levels of shear stress had no significant effect on tissue cellularity until a higher level of shear was applied, suggesting a possible threshold value. The application of shear stress was found to slightly decrease glycosaminoglycan levels. We have uncovered evidence that the effects of fluid-induced shear stress and simulated microgravity interact to affect cell numbers and extracellular matrix production in regenerating cartilage tissue. Results of the interaction studies, that are the first of their kind, showed varying response due to shear depending on the condition of microgravity, and varying response due to microgravity conditions depending on the level of shear

Securitization theory and securitization studies

Opposed to the recently fashionable 'moral and ethical' criticism levelled against Ole Wæver's securitization theory this article argues that such criticism fundamentally misconceives the analytical goal of securitization theory, which is namely to offer a tool for practical security analysis. In arguing that being political (critical) on the part of the analyst has no bearing on the type of practical security analysis that can be done using securitization theory, this article proposes that the analytical goal of such criticism and that of securitization theory are incommensurable; in the process rendering obsolete this kind of criticism of securitization theory. By way of reconciling securitization theory with its critics, however, this article takes up Wæver's suggestion of wider securitization studies in which moral and ethical criticism, as well as being political, can play a supplementary role in the analysis of securitization theory

Ground penetrating radar applied to rebar corrosion inspection

In this paper we investigate the use of ground penetrating radar (GPR) to detect corrosion-induced thinning of rebar in concrete bridge structures. We consider a simple pulse/echo amplitude-based inspection, positing that the backscattered response from a thinned rebar will be smaller than the similar response from a fully-intact rebar. Using a commercial 1600-MHz GPR system we demonstrate that, for laboratory specimens, backscattered amplitude measurements can detect a thinning loss of 50% in rebar diameter over a short length. GPR inspections on a highway bridge then identify several rebar with unexpectedly low amplitudes, possibly signaling thinning. To field a practical amplitude-based system for detecting thinned rebar, one must be able to quantify and assess the many factors that can potentially contribute to GPR signal amplitude variations. These include variability arising from the rebar itself (e.g., thinning) and from other factors (concrete properties, antenna orientation and liftoff, etc.). We report on early efforts to model the GPR instrument and the inspection process so as to assess such variability and to optimize inspections. This includes efforts to map the antenna radiation pattern, to predict how backscattered responses will vary with rebar size and location, and to assess detectability improvements via synthetic aperture focusing techniques (SAFT)

Native bradyrhizobial symbionts of Lupinus mariae-josephae, a unique endemism thriving in alkaline soils in Eastern Spain

Lupinus mariae-josephae (Lmj) es una especie de lupino endémica de una pequeña y específica área de Comunidad Valenciana (Este de España), donde prospera en suelos alcalinoscalcáreos, un hábitat singular para los altramuces, que crecen preferentemente en suelos ácidos o neutros. Esto hace de Lmj una especie de lupino única. Cuando se inició este trabajo, la extensión conocida de este endemismo abarcaba unos 700 kilómetros cuadrados, confinados en la provincia de Valencia. En esta área, Lmj prospera en pequeñas poblaciones aisladas que contienen un número reducido de plantas por lo que se la consideró una especie en peligro de extinción. Todos los esfuerzos, utilizando estrategias clásicas dirigidas a ampliar el área de crecimiento de Lmj y garantizar su conservación, han tenido un éxito limitado. El trabajo que se presenta está dirigido a mejorar el conocimiento de la ecología de Lmj, en particular la interacción simbiótica que establece con bacterias del suelo denominadas rizobios y se centra en la caracterización fenotípica, filogenética y genómica de esos rizobios. También se investiga la posible contribución de la simbiosis en mejorar la conservación de Lmj. Para este fin, se han estudiado diferentes aspectos que se describen a continuación. El primero objetivo se centró en aislar y estudiar de la diversidad genética de las bacterias endosimbióticas de Lmj. . Se realizó un análisis filogenético de genes esenciales que mostró que las cepas de Lmj pertenecen al género Bradyrhizobium y que presentan una gran diversidad con características fenotípicas y simbióticas diferentes de cepas de Bradyrhizobium que nodulan otras especies de lupinos nativos de España (cepas ISLU). Las cepas estudiadas se dividieron en dos grupos (Clado I y Clado II). El Clado I, incluye a las cepas Lmj, definiendo un nuevo linaje, filogenéticamente relacionado con otras especies de Bradyrhizobium, como B. jicamae y B. elkanii. El Clado II contiene cepas ISLU relacionadas con cepas de B. canariense y B. japonicum que establecen simbiosis con lupinos de suelos ácidos. Otro análisis filogenético basado en genes simbióticos, distribuyó las cepas de Lmj en sólo dos grupos diferentes. La singularidad y gran diversidad de estas cepas en una pequeña área geográfica, hacen de este, un atractivo sistema para el estudio de la evolución y adaptación de las bacterias simbióticas a su respectiva planta huésped. Adicionalmente, se estudio la presencia de bacterias capaces de nodular Lmj en suelos básicos de Chiapas, México. Sorprendentemente, estos suelos contienen bacterias capaces establecer interacciones simbióticas eficientes con Lmj en ensayos de invernadero. A continuación se investigó la taxonomía de los endosimbiontes de Lmj analizando la secuencia de cuatro genes esenciales (16S rRNA, recA, glnII y atpD) y el promedio de identidad de nucleótidos de genomas completos de algunas cepas representativas de la diversidad (ANIm). Se identificaron nuevas especies de Bradyrhizobium dentro del Clado I y se definió una de ellas: 'Bradyrhizobium valentinum' sp. nov (cepa tipo LmjM3T = CECT 8364T, LMG 2761T). También se abordó cómo conservar Lmj en su hábitat natural mediante inoculación con alguna de las cepas aisladas. Se demostró la ausencia de bacterias capaces de nodular Lmj en suelos rojos alcalinos o ‘‘terra rossa’’ de la Península Ibérica y Baleares. Dos cepas, altamente eficientes en cuanto a la fijación de nitrógeno, LmjC y LmjM3T, fueron seleccionadas para ser empleadas como inoculantes. Dos experimentos de campo llevados a cabo en años consecutivos en áreas con características edafoclimáticas similares a las que presentan las poblaciones de Lmj, lograron la reproducción exitosa de la planta. Se concluyó que un ciclo reproductivo exitoso de Lmj es absolutamente dependiente de la inoculación con sus simbiontes naturales y que la simbiosis debe ser considerada un factor esencial en estrategias de conservación de leguminosas en peligro. La obtención de varias secuencias genómicas de cepas aisladas de Lmj y de otras cepas de Bradyrhizobium reveló una alta similitud entre los genomas de las cepas del Clado I, y permitió la identificación de cinco posibles nuevas especies. Además, se estudiaron tres agrupaciones de genes relacionados con la simbiosis (nod, nif y fix) definiendo un nuevo linaje para las cepas de Lmj, diferente del symbiovar “genistearum” de B. canariense y B. japonicum. La baja diversidad encontrada en el análisis filogenético de los genes simbióticos contrasta con la gran diversidad asociada a genes esenciales. La presencia de plásmidos en cepas del género Bradyrhizobium ha sido descrita en muy pocas ocasiones, sin embargo el análisis de la secuencia genómica de la cepa ISLU101, aislada de Lupinus angustifolius, reveló la presencia de un origen de replicación extracromosómico homólogo al operón repABC, presente en el plásmido de Bradyrhizobium sp BTAi1. Gracias a esta secuencia se identificaron genes homólogos en 19 de 72 cepas ISLU. Filogenéticamente, las secuencias de repABC se agruparon en un grupo monofilético con las de pBTAi1 y separadas de los rizobios de crecimiento rápido. Finalmente, se identificaron sistemas de secreción de proteínas de tipo III (T3SS) en nueve genomas de cepas de Lmj. Los T3SS pueden inyectar proteínas efectoras al interior de células vegetales. Su presencia en rizobios se ha relacionado con la gama de hospedador que pueden nodular y puede tener un efecto beneficioso, neutro o perjudicial en la simbiosis. Los T3SS de las cepas de Lmj codifican para una proteína efectora similar a NopE, un efector dependiente de T3SS descrito en B. diazoefficiens USDA 110T. La proteína NopE de la cepa LmjC se ha caracterizado bioquímicamente. ABSTRACT Lupinus mariae-josephae (Lmj) is a lupine species endemic of a unique small area in Valencia region (Eastern Spain) where the lupine plants thrive in alkaline-limed soils, which preferentially grow in acid or neutral soils. This is the type of soils native lupines of Spain. When this work was initiated, the extension of the endemic area of Lmj was of about 700 squared kilometers confined to the Valencia province. In this area, Lmj thrives in small, isolated patches containing a reduced number of plants, and points to an endemism that can easily became endangered or extinct. Consequently, the Valencia Community authorities gave a ‘‘microreserve” status for conservation of the species. All efforts, using classical strategies directed to extend the area of Lmj growth and ensure its conservation have been so far unsuccessful. The work presented here is directed to improve our knowledge of Lmj ecology and it is centered in the characterization of the rhizobial symbiosis by phenotypic, phylogenetic and genomic analysis as well as in investigate the potential contribution of the symbiosis to improve its conservation. To this end, five different topics have been studied, and results are briefly described here. Extensive details can be followed en the attached, published articles. The first topic deals with the indigenous rhizobial symbionts of the Lmj endemism, and its genetic diversity was investigated. The Lmj root symbionts belong to the Bradyrhizobium genus, and phylogenetic analysis based on core genes identified a large diversity of Bradyrhizobium strains with phenotypic and symbiotic characteristics different from rhizobia nodulating other Lupinus spp. native of Spain. The strains were split in two clades. Clade II contained strains close to classical B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. Clade I included Lmj strains that define a new lineage, close to other Bradyrhizobium species as B. jicamae and B. elkanii. The phylogenetic analysis based on symbiotic genes identified only two distinct clusters. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host. Additionally, the presence of bacteria able to nodulate Lmj in basic soils from Chiapas, Mexico was investigated. Surprisingly, these soils contain bacteria able to effectively nodulate and fix nitrogen with Lmj plants in greenhouse assays. In the second topic, the taxonomic status of the endosymbiotic bacteria of Lmj from Valencia endemism and Chiapas was investigated. Results from phylogenetic analysis of core genes and Average Nucleotide Identity (ANIm) using draft genomic sequences identified new Bradyrhizobium species within strains of Clade I of Lmj endosymbiotic bacteria. Only one of these potentially new species has been defined, meanwhile the others are under process of characterization. The name ‘Bradyrhizobium valentinum’ sp. nov. was proposed for the defined species (type strain LmjM3T= CECT 8364T, LMG 2761T). The third topic was directed to conservation of endangered Lmj in its natural habitat. The relevant conclusion of this experimentation is that the symbiosis should be considered as a relevant factor in the conservation strategies for endangered legumes. First, we showed absence of bacteria able to nodulate Lmj in all the inspected ‘‘terra rossa’’ or alkaline red soils of the Iberian Peninsula and Balearic Islands. Then, two efficient nitrogen fixing strains with Lmj plants, LmjC and LmjM3T, were selected as inoculum for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. The relevant conclusion from these assays was that the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia The forth topic deep into the analysis of the genomic of Lmj representative strains. To this end, draft genomic sequences of selected Lmj strains and type strains of Bradyrhizobium spp. were assembled. The comparison analysis of the draft genomic sequences of Lmj strains and related Bradyrhizobium species grouped in Clade I, revealed a high genomic homology among them, and allowed the definition of five potentially new species of Lmj nodulating bacteria. Also, based on the available draft genomic sequences, only three clusters of nod, fix and nif genes from Lmj strains were identified and showed to define a new symbiotic lineage, distant from that of B. canariense and B. japonicum bv. genistearum. The low diversity exhibited by the phylogenetic analysis of symbiotic genes contrast with the large diversity of strains as regards the housekeeping genes analyzed. Besides, the genomic analysis of a Lupinus angustifolius strain ISLU101, revealed the presence of an extrachromosomal replication origin homologous to repABC cluster from plasmid present in Bradyrhizobium spp BTAi1. This repABC cluster gene sequence allowed the identification of extrachromosomic replication origin in 19 out of 72 Bradyrhizobium strains from Lupinus spp., a highly significant result since the absence of plasmids in the Bradyrhizobium genus was traditionally assumed. The repABC gene sequences of these strains grouped them in a unique monophyletic group, related to B. sp. BTAi1 plasmid, but differentiated from the repABC gene cluster of plasmids in fast growing rhizobium strains. The last topic was focused on characterization of type III secreted effectors present in Lmj endosymbiotic bacteria. Type III secretion systems (T3SS) are specialized protein export machineries which can deliver effector proteins into plant cells. The presence of T3SS in rhizobia has frequently been related to the symbiotic nodulation host-range and may have a beneficial or detrimental effect on the symbiosis with legumes. In this context, the presence of T3SS in genomes of nine Lmj strains was investigated, and it was shown the presence of clusters encoding NopE type III-secreted protein similar to the NopE1 and NopE2 of B. diazoefficiens USDA 110T. The putative NopE protein of LmjC strain is at present being characterized regarding its structure and function

Caracterización de sistemas de secreción tipo VI en la simbiosis Rhizobium-Leguminosa

El análisis de los genomas de cepas de Bradyrhizobium sp. aisladas de Lupinus mariaejosephae, L. angustifolius y Retama sphaerocarpa, así como de cepas de Rhizobium aisladas de Mimosa affinis y Phaseolus vulgaris, ha permitido identificar la presencia de sistemas de secreción tipo VI de proteínas (T6SS). Los T6SS están implicados en la interacción planta-bacteria mediante el transporte de proteínas efectoras al citoplasma de células vegetales a través de una estructura similar a una nanojeringa de bacteriófago. El análisis se centra en las cepas Bradyrhizobium sp. ISLU101, aislada de L. angustifolius y Rhizobium etli Mim1 aislada de M. affinis, capaz de nodular judías. Mim1 e ISLU101 contienen uno y dos T6SS, respectivamente. Una característica distintiva de los T6SS activos es que las proteínas Hcp y VgrG, que forman parte de la nanojeringa, pueden secretarse al medio extracelular; así para determinar cuando son activos los T6SS identificados, se están realizando inmunoensayos

Análisis filogenético de bacterias endosimbióticas de Genista numidica

Los rizobios son bacterias del suelo capaces de formar unas estructuras especializadas en raíces de leguminosas donde reducen dinitrógeno. Algunas de estas leguminosas, como Genista numidica Spach, juegan un importante papel ecológico y económico por la fertilización y la remediación de suelos áridos, lo que ha impulsado el estudio y la caracterización de los rizobios específicos. En la presente investigación se analizan 53 cepas de rizobios aisladas de nódulos de raíces de G. numidica de la costa de Argelia. La diversidad genética de los aislados se llevó a cabo mediante la secuenciación del gen 16S rRNA y del espacio intergénico (ITS), región situada entre los genes 16S y 23S rRNA. Los endosimbiontes de G. numidica muestran una gran diversidad filogenética. Las secuencias de los aislados mostraron proximidad a ?-proteobacterias (Bradyrhizobium sp, Sphingobium sp) y ?-proteobacterias

Lieder, der geselligen Freude geweiht

http://tartu.ester.ee/record=b2409736~S1*es

Kiri Karl Morgensternile, Riga

http://tartu.ester.ee/record=b1777604~S1*es