BIOCHEMICAL CHARACTERIZATION AND CLINICAL SIGNIFICANCE OF COAGULASE-NEGATIVE STAPHYLOCOCCI

Although coagulase-negative staphylococci (C-NS) have been implicated in certain human infections, they are generally regarded as contaminants and their clinical significance is questioned. To assess their role as pathogens, 205 isolates of C-NS from wounds, and body fluids (blood, urine, pleural and peritoneal fluids, etc.) were studied. Patient's charts were reviewed and using strict criteria a determination was made regarding the clinical significance of these isolates. The organisms were then identified using the scheme of Kloos and Schleifer to determine if certain species of C-NS were associated with specific infections. S. epidermidis sensu stricto accounted for 81% of the C-NS isolated; the frequency of other species was S. haemolyticus (6%), S. hominis (5%), S. capitis (4%), S. warneri (3%), and others (1%). Only two isolates were novobiocin resistant; neither was identified as S. saprophyticus. Using these criteria, 22% of C-NS were considered to be clinically significant and the majority of these (93%) were due to S. epidermidis. The most common source of the clinically relevant C-NS isolates was from wounds. These data suggest that identifying C-NS species other than S. epidermidis may be of limited value in predicting clinical significance.^ In addition, selected pathogenic and non-pathogenic strains of C-NS were compared for their ability to adhere to human cells in vitro. Although the results were not conclusive, it appeared that pathogenic C-NS adhered more avidly than non-pathogenic C-NS to buccal cells. Experiments with HeLa cells showed no difference between pathogenic and non-pathogenic C-NS in adherence abilities. ^

INTERACTION OF BACILLUS ANTHRACIS EXOSPORIUM PROTEIN BCLA WITH COMPLEMENT FACTOR H AND SPORE PERSISTENCE IN THE LUNG

Anthrax outbreaks in the United States and Europe and its potential use as a bioweapon have made Bacillus anthracis an interest of study. Anthrax infections are caused by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, cuts or wounds in the skin, and injection. Among these four forms, inhalational anthrax has the highest lethality rate and persistence of spores in the lungs of animals following pulmonary exposure has been noted for decades. However, details or mechanisms of spore persistence were not known. In this study, we investigated spore persistence in a mouse model. The results suggest that B. anthracis spores have special properties that promote persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence. Moreover, recent discoveries from our laboratory suggest that spores evolved a sophisticated mechanism to interact with the host complement system. The complement system is a crucial part of the host defense mechanism against foreign microorganisms. Knowledge of the specific interactions that occur between the complement system and B. anthracis was limited. Studies performed in our laboratory have suggested that spores of B. anthracis can target specific proteins, such as Factor H (fH) of the complement system. Spores of B. anthracis are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called Bacillus collagen-like protein of anthracis (BclA), which comprises a central collagen-like region and a globular C-terminal domain. BclA is the first point of contact with the innate system of an infected host. In this study, we investigated the molecular details of BclA-fH interaction with respect to the specific binding mechanism and the functional significance of this interaction in a murine model of anthrax infection. We hypothesized that the recruitment of fH to the spore surface by BclA limits the extent of complement activation and promotes pathogen survival and persistence in the infected host. Findings from this study are significant to understanding how to treat post-exposure prophylaxis and improve our knowledge of spores with the host immune system.

Delayed Thrombus Resolution and Fibroproliferative Vascular Wound Healing From Deficiency of Type III Collagen: a Paradoxical Mechanism for Tissue Fragility

Vascular Ehlers-Danlos syndrome is a heritable disease of connective tissue caused by mutations in COL3A1, conferring a tissue deficiency of type III collagen. Cutaneous wounds heal poorly in these patients, and they are susceptible to spontaneous and catastrophic rupture of expansible hollow organs like the gut, uterus, and medium-sized to large arteries, which leads to premature death. Although the predisposition for organ rupture is often attributed to inherent tissue fragility, investigation of arteries from a haploinsufficient Col3a1 mouse model (Col3a1+/-) demonstrates that mutant arteries withstand even supraphysiologic pressures comparably to wild-type vessels. We hypothesize that injury that elicits occlusive thrombi instead unmasks defective thrombus resolution resulting from impaired production of type III collagen, which causes deranged remodeling of matrix, persistent inflammation, and dysregulated behavior by resident myofibroblasts, culminating in the development of penetrating neovascular channels that disrupt the mechanical integrity of the arterial wall. Vascular injury and thrombus formation following ligation of the carotid artery reveals an abnormal persistence and elevated burden of occlusive thrombi at 21 post-operative days in vessels from Col3a1+/- mice, as opposed to near complete resolution and formation of a patent and mature neointima in wild-type mice. At only 14 days, both groups harbor comparable burdens of resolving thrombi, but wild-type mice increase production of type III collagen in actively resolving tissues, while mutant mice do not. Rather, thrombi in mutant mice contain higher burdens of macrophages and proliferative myofibroblasts, which persist through 21 days while wild-type thrombi, inflammatory cells, and proliferation all regress. At the same time that increased macrophage burdens were observed at 14 and 21 days post ligation, the medial layer of mutant arterial walls concurrently harbored a significantly higher incidence of penetrating neovessels compared with those in wild-type mice. To assess whether limited type III collagen production alters myofibroblast behavior, fibroblasts from vEDS patients with COL3A1 missense mutations were seeded into three-dimensional fibrin gel constructs and stimulated with transforming growth factor-β1 to initiate myofibroblast differentiation. Although early signaling events occur similarly in all cell lines, late extracellular matrix- and mechanically-regulated events like transcriptional upregulation of type I and type III collagen secretion are delayed in mutant cultures, while transcription of genes encoding intracellular contractile machinery is increased. Sophisticated imaging of collagen synthesized de novo by resident myofibroblasts visualizes complex matrix reorganization by control cells but only meager remodeling by COL3A1 mutant cells, concordant with their compensatory contraction to maintain tension in the matrix. Finally, administration of immunosuppressive rapamycin to mice following carotid ligation sufficiently halts the initial inflammatory phase of thrombus resolution and fully prevents both myofibroblast migration into the thrombus and the differential development of neovessels between mutant and wild-type mice, suggesting that pathological defects in mutant arteries develop secondarily to myofibroblast dysfunction and chronic inflammatory stimulation, rather than as a manifestation of tissue fragility. Together these data establish evidence that pathological defects in the vessel wall architecture develop in mutant arteries as sequelae to abnormal healing and remodeling responses activated by arterial injury. Thus, these data support the hypothesis that events threatening the integrity of type III collagen-deficient vessels develop not as a result of inherent tissue weakness and fragility at baseline but instead as an episodic byproduct of abnormally persistent granulation tissue and fibroproliferative intravascular remodeling.

Método de evaluación a campo de la resistencia al moho azul en clones de ajo tipo colorado

Con el objeto de ajustar un método de evaluación a campo para facilitar la selección de clones de ajo (Allium sativum L.) con resistencia a Penicillium allii, se realizó un ensayo replicado en el cual se midió la respuesta de tres clones experimentales a la infección, en dos épocas de plantación. Los tratamientos incluyeron combinaciones de presencia o ausencia de: a) desinfección de la "semilla", b) heridas artificiales en los bulbillos, c) inoculación artificial. Los bulbillos con la hoja envolvente se desinfectaron con una solución diluida de hipoclorito de sodio. La inoculación se realizó mediante la inmersión en una solución de 106 esporas•ml-1. Las heridas se realizaron con una lanceta (blood lancet). Los bulbillos se incubaron en cámara húmeda durante 24 horas a 20 °C, antes de plantación. Las variables respuestas computadas fueron las siguientes: número de plantas muertas, y vivas con síntomas y sin síntomas, a los 152 días de plantación en la primera época y 118 días en la segunda; número de bulbos por calibres y rendimiento a cosecha. La información obtenida se analizó a través de correspondencia simple, diferencia de proporciones y análisis paramétricos. Se detectaron mayores diferencias entre los tratamientos en la tasa de sobrevivencia y en la proporción de calibres comerciales en la época tardía. No se observaron diferencias (p < 0,05) del rendimiento en la plantación temprana. La herida fue determinante en el ingreso del patógeno. Los tratamientos quedaron limitados a un testigo: desinfectado, no inoculado y sin herida, y a un tratamiento: desinfectado, con inoculación y herida. Con respecto a la época, resultó conveniente la evaluación en plantaciones tardías porque se expresan mejor las diferencias entre los tratamientos.

Trips (Frankliniella brevicaulis Hood) de las erupciones del fruto de banano (Musa acuminata Colla) en la provincia de Formosa, Argentina

El banano en la provincia de Formosa (Argentina) es uno de los principales cultivos de renta y generador de mano de obra para el productor por lo que representa un importante lugar en lo social y económico. La sanidad de los cultivos es fundamental para lograr una mayor rentabilidad. Se han observado en los frutos daños atribuibles a trips. Los objetivos de este trabajo fueron identificar la/s especie/s de trips causal de las erupciones del fruto de banano en la provincia de Formosa, realizar una descripción detallada de su morfología y caracterizar y evaluar los daños producidos. La zona de trabajo correspondió al Área-Laguna Naineck en el Departamento Pilcomayo de la provincia de Formosa considerando un total de 1.103,74 ha pertenecientes a 233 productores. Para la determinación del estado sanitario de las plantaciones con respecto a la presencia del insecto se realizó un diseño de muestreo estadístico. Se recolectaron muestras del insecto y se identificaron con ayuda de claves. La única especie de trips encontrada fue Frankliniella brevicaulis. Los daños producidos por esta especie serían debidos a las heridas causadas por la oviposición, siendo éstas posibles fuentes de entrada para el hongo Colletotrichum musae. Se observaron daños en el 100% de los cachos de banana en todas las plantaciones evaluadas.

Conidial germination of Botryosphaeria dothidea Mough.: Fr (Ces. & De Not.) and histological alterations on stems of pitahaya (Hylocereus undatus H.) (Haworth) Britton & Rose

Conidial germination of Botryosphaeria dothidea (anamorph: Fusicoccum) in sterile distilled water and 1% sterile dextrose solution was evaluated at 4, 6, 12, 24 and 36 h after incubation. Also, it was described the anatomical changes on pitahaya stems induced by this fungus, collected in the field and artificially inoculated in the laboratory. Conidial germination was less than 30% in water and it was improved when 1% dextrose was added to the water. In 1% dextrose solution the germination was 90% after 4h of incubation and 100% at 6 h. Pathogen germ tubes had entered through wounds and sometimes through stomata and hyphae colonized intra and intercellularly in the parenchyma-chlorenchyma tissues. On naturally and artificially diseased stems the main alterations were: destruction of cuticle, hyperplasia of epidermal and collenchymatous hypodermal cells and conform the advance of the pathogen a layer of lignified periderm was formed surrounding the damaged tissues; however, it couldn't stop the advance of the pathogen and the cells that surrounded the lesion suffered necrosis.

La casa vital

RESUMEN. Se trata de una investigación sobre algunas relaciones entre la idea y el ejercicio de la arquitectura y el concepto y la praxis de la herida, tanto física como espiritual: sobre la arquitectura como objeto, destino y origen del trauma. Propongo una definición de estas arquitecturas mediante el estudio de obras que se han visto en algún momento de su vida (concebida como la de un humano) - sometidas a procesos de heridas de varios tipos e intensidades, de forma que estos momentos destructivos las han hecho conocer la agonía y, en ocasiones, las han conducido a la extinción. THIS IS AN INVESTIGATION OF SOME RELATIONSHIPS BETWEEN THE IDEA AND THE PRACTICE OF ARCHITECTURE AND THE CONCEPT AND PRACTICE OF INJURY, PHYSICAL AND SPIRITUAL: ABOUT THE ARCHITECTURE LIKE AN OBJECT, DESTINY AND ORIGIN OF THE TRAUMA. I PROPOSE A DEFINITION OF THESE ARCHITECTURES BY STUDYING WORKS THAT HAVE BEEN AT SOME POINT IN THEIR LIFE (CONCEIVED AS A HUMAN) - UNDER TO PROCESSES OF WOUNDS OF VARIOUS TYPES AND INTENSITIES, SO THAT THESE DESTRUCTIVE TIMES HAVE MADE THEM KNOW THE AGONY AND, ON OCCASION, BEEN DRIVEN TO EXTINCTION.

Análisis de la precisión en la medida del tiempo de reverberación y de los parámetros asociados

El trabajo fin de master “Análisis de la precisión en la medida del tiempo de reverberación y de los parámetros asociados” tiene como objetivo primordial la evaluación de los parámetros y métodos utilizados para la obtención de estos, a través del tiempo de reverberación, tanto de forma global, conjunto de todos los métodos, como cada uno de ellos por separado. Un objetivo secundario es la evaluación de la incertidumbre en función del método de medición usado. Para realizarlo, se van a aprovechar las mediciones realizadas para llevar a cabo el proyecto fin de carrera [1], donde se medía el tiempo de reverberación en dos recintos diferentes usando el método del ruido interrumpido y el método de la respuesta impulsiva integrada con señales distintas. Las señales que han sido utilizadas han sido señales impulsivas de explosión de globos, disparo de pistola, claquetas y, a través de procesado digital, señales periódicas pseudoaleatorias MLS y barridos de tonos puros. La evaluación que se realizará a cada parámetro ha sido extraída de la norma UNE 89002 [2], [3]y [4]. Se determinará si existen valores aberrantes tanto por el método de Grubbs como el de Cochran, e interesará conocer la veracidad, precisión, repetibilidad y reproducibilidad de los resultados obtenidos. Los parámetros que han sido estudiados y evaluados son el tiempo de reverberación con caída de 10 dB, (T10), con caída de 15 dB (T15), con caída de 20 dB (T20), con caída de 30 dB (T30), el tiempo de la caída temprana (EDT), el tiempo final (Ts), claridad (C20, C30, C50 y C80) y definición (D50 y D80). Dependiendo de si el parámetro hace referencia al recinto o si varía en función de la relación entre la posición de fuente y micrófono, su estudio estará sujeto a un procedimiento diferente de evaluación. ABSTRACT. The master thesis called “Analysis of the accuracy in measuring the reverberation time and the associated parameters” has as the main aim the assessment of parameters and methods used to obtain these through reverberation time, both working overall, set of all methods, as each of them separately. A secondary objective is to evaluate the uncertainty depending on the measurement method used. To do this, measurements of [1] will be used, where they were carried on in two different spaces using the interrupted noise method and the method of impulse response integrated with several signals. The signals that have been used are impulsive signals such as balloon burst, gunshot, slates and, through digital processing, periodic pseudorandom signal MLS and swept pure tone. The assessment that will be made to each parameter has been extracted from the UNE 89002 [2], [3] and [4]. It will determine whether there are aberrant values both through Grubbs method and Cochran method, to say so, if a value is inconsistent with the rest of the set. In addition, it is interesting to know the truthfulness, accuracy, repeatability and reproducibility of results obtained from the first part of this rule. The parameters that are going to be evaluated are reverberation time with 10 dB decay, (T10), with 15 dB decay (T15), with 20 dB decay (T20), with 30 dB decay (T30), the Early Decay Time (EDT), the final time (Ts), clarity (C20, C30, C50 y C80) and definition (D50 y D80). Depending on whether the parameter refers to the space or if it varies depending on the relationship between source and microphone positions, the study will be related to a different evaluation procedure.

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal “clip” domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.

Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin–green fluorescent protein

Fibronectin (FN) forms the primitive fibrillar matrix in both embryos and healing wounds. To study the matrix in living cell cultures, we have constructed a cell line that secretes FN molecules chimeric with green fluorescent protein. These FN–green fluorescent protein molecules were assembled into a typical matrix that was easily visualized by fluorescence over periods of several hours. FN fibrils remained mostly straight, and they were seen to extend and contract to accommodate movements of the cells, indicating that they are elastic. When fibrils were broken or detached from cells, they contracted to less than one-fourth of their extended length, demonstrating that they are highly stretched in the living culture. Previous work from other laboratories has suggested that cryptic sites for FN assembly may be exposed by tension on FN. Our results show directly that FN matrix fibrils are not only under tension but are also highly stretched. This stretched state of FN is an obvious candidate for exposing the cryptic assembly sites.

Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells

The α4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the α4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by αv and β3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an αvβ3-integrin–dependent manner. The αvβ3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the αvβ3 integrin-positive focal contacts. We have investigated the function of α4-laminin and αvβ3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

Defect in regulated secretion of P-selectin affects leukocyte recruitment in von Willebrand factor-deficient mice

Stimulation of endothelial cells by various inflammatory mediators leads to release of Weibel–Palade bodies and therefore to exocytosis of both P-selectin (adhesion receptor for leukocytes) and von Willebrand factor (vWf) (platelet ligand). The potential role of vWf in leukocyte recruitment was investigated with the use of vWf-deficient mice. We report a strong reduction of leukocyte rolling in venules of vWf-deficient mice. Similarly, vWf deficiency led to a decrease in neutrophil recruitment in a cytokine-induced meningitis model as well as in early skin wounds. In all instances with an antibody that preferentially recognizes plasma membrane P-selectin, we observed a dramatic reduction in P-selectin expression at the cell surface of vWf-deficient endothelium. With confocal microscopy, we found that the typical rodlike shape of the Weibel–Palade body is missing in vWf −/− endothelial cells and that part of the P-selectin content in the vWf −/− cells colocalized with LAMP-1, a lysosomal marker. However, intracellular P-selectin levels were similar in tumor necrosis factor α- and lipopolysaccharide-activated cells of both genotypes. We conclude that the absence of vWf, as found in severe von Willebrand disease, leads to a defect in Weibel–Palade body formation. This defect results in decreased P-selectin translocation to the cell surface and reduced leukocyte recruitment in early phases of inflammation.

La roca magica: Uses of natural zeolites in agriculture and industry

For nearly 200 years since their discovery in 1756, geologists considered the zeolite minerals to occur as fairly large crystals in the vugs and cavities of basalts and other traprock formations. Here, they were prized by mineral collectors, but their small abundance and polymineralic nature defied commercial exploitation. As the synthetic zeolite (molecular sieve) business began to take hold in the late 1950s, huge beds of zeolite-rich sediments, formed by the alteration of volcanic ash (glass) in lake and marine waters, were discovered in the western United States and elsewhere in the world. These beds were found to contain as much as 95% of a single zeolite; they were generally flat-lying and easily mined by surface methods. The properties of these low-cost natural materials mimicked those of many of their synthetic counterparts, and considerable effort has made since that time to develop applications for them based on their unique adsorption, cation-exchange, dehydration–rehydration, and catalytic properties. Natural zeolites (i.e., those found in volcanogenic sedimentary rocks) have been and are being used as building stone, as lightweight aggregate and pozzolans in cements and concretes, as filler in paper, in the take-up of Cs and Sr from nuclear waste and fallout, as soil amendments in agronomy and horticulture, in the removal of ammonia from municipal, industrial, and agricultural waste and drinking waters, as energy exchangers in solar refrigerators, as dietary supplements in animal diets, as consumer deodorizers, in pet litters, in taking up ammonia from animal manures, and as ammonia filters in kidney-dialysis units. From their use in construction during Roman times, to their role as hydroponic (zeoponic) substrate for growing plants on space missions, to their recent success in the healing of cuts and wounds, natural zeolites are now considered to be full-fledged mineral commodities, the use of which promise to expand even more in the future.

Regulation of Oleoresinosis in Grand Fir (Abies grandis)1 : Differential Transcriptional Control of Monoterpene, Sesquiterpene, and Diterpene Synthase Genes in Response to Wounding

Grand fir (Abies grandis Lindl.) has been developed as a model system for the study of wound-induced oleoresinosis in conifers as a response to insect attack. Oleoresin is a roughly equal mixture of turpentine (85% monoterpenes [C10] and 15% sesquiterpenes [C15]) and rosin (diterpene [C20] resin acids) that acts to seal wounds and is toxic to both invading insects and their pathogenic fungal symbionts. The dynamic regulation of wound-induced oleoresin formation was studied over 29 d at the enzyme level by in vitro assay of the three classes of synthases directly responsible for the formation of monoterpenes, sesquiterpenes, and diterpenes from the corresponding C10, C15, and C20 prenyl diphosphate precursors, and at the gene level by RNA-blot hybridization using terpene synthase class-directed DNA probes. In overall appearance, the shapes of the time-course curves for all classes of synthase activities are similar, suggesting coordinate formation of all of the terpenoid types. However, closer inspection indicates that the monoterpene synthases arise earlier, as shown by an abbreviated time course over 6 to 48 h. RNA-blot analyses indicated that the genes for all three classes of enzymes are transcriptionally activated in response to wounding, with the monoterpene synthases up-regulated first (transcripts detectable 2 h after wounding), in agreement with the results of cell-free assays of monoterpene synthase activity, followed by the coordinately regulated sesquiterpene synthases and diterpene synthases (transcription beginning on d 3–4). The differential timing in the production of oleoresin components of this defense response is consistent with the immediate formation of monoterpenes to act as insect toxins and their later generation at solvent levels for the mobilization of resin acids responsible for wound sealing.