Data base on pelagic ciliates grazing and growth rate as a function of size, prey size and type compiled from the literature

Microzooplankton (the 20 to 200 µm size class of zooplankton) is recognised as an important part of marine pelagic ecosystems. In terms of biomass and abundance pelagic ciliates are one of the important groups of organism in microzooplankton. However, their rates - grazing and growth - , feeding behaviour and prey preferences are poorly known and understood. A set of data was assembled in order to derive a better understanding of pelagic ciliates rates, in response to parameters such as prey concentration, prey type (size and species), temperature and their own size. With these objectives, literature was searched for laboratory experiments with information on one or more of these parameters effect studied. The criteria for selection and inclusion in the database included: (i) controlled laboratory experiment with a known ciliates feeding on a known prey; (ii) presence of ancillary information about experimental conditions, used organisms - cell volume, cell dimensions, and carbon content. Rates and ancillary information were measured in units that meet the experimenter need, creating a need to harmonize the data units after collection. In addition different units can link to different mechanisms (carbon to nutritive quality of the prey, volume to size limits). As a result, grazing rates are thus available as pg C/(ciliate*h), µm**3/(ciliate*h) and prey cell/(ciliate*h); clearance rate was calculated if not given and growth rate is expressed as the growth rate per day.

Análisis de micromovilidad en vástagos femorales mediante ensayo dinámico de oscilaciones libres

La artroplastia de cadera se considera uno de los mayores avances quirúrgicos de la Medicina. La aplicación de esta técnica de Traumatología se ha incrementado notablemente en los últimos anos, a causa principalmente del progresivo incremento de la esperanza de vida. En efecto, con la edad aumentan los problemas de artrosis y osteoporosis, enfermedades típicas de las articulaciones y de los huesos que requieren en muchos casos la sustitución protésica total o parcial de la articulación. El buen comportamiento funcional de una prótesis depende en gran medida de la estabilidad primaria, es decir, el correcto anclaje de la prótesis en el momento de su implantación. Las prótesis no cementadas basan su éxito a largo plazo en la osteointegración que tiene lugar entre el material protésico y el tejido óseo, y para lograrla es imprescindible conseguir unas buenas condiciones de estabilidad primaria. El aflojamiento aséptico es la principal causa de fallo de artroplastia total de cadera. Este es un fenómeno en el que, debido a complejas interacciones de factores mecánicos y biológicos, se producen movimientos relativos que comprometen la funcionalidad del implante. La minimización de los correspondientes danos depende en gran medida de la detección precoz del aflojamiento. Para lograr la detección temprana del aflojamiento aséptico del vástago femoral se han ensayado diferentes técnicas, tanto in vivo como in vitro: análisis numéricos y técnicas experimentales basadas en sensores de movimientos provocados por cargas transmitidas natural o artificialmente, tales como impactos o vibraciones de distintas frecuencias. Los montajes y procedimientos aplicados son heterogéneos y, en muchas ocasiones, complejos y costosos, no existiendo acuerdo sobre una técnica simple y eficaz de aplicación general. Asimismo, en la normativa vigente que regula las condiciones que debe cumplir una prótesis previamente a su comercialización, no hay ningún apartado referido específicamente a la evaluación de la bondad del diseño del vástago femoral con respecto a la estabilidad primaria. El objetivo de esta tesis es desarrollar una metodología para el análisis, in vitro, de la estabilidad de un vástago femoral implantado, a fin de poder evaluar las técnicas de implantación y los diferentes diseños de prótesis previamente a su oferta en el mercado. Además se plantea como requisito fundamental que el método desarrollado sea sencillo, reversible, repetible, no destructivo, con control riguroso de parámetros (condiciones de contorno de cargas y desplazamientos) y con un sistema de registro e interpretación de resultados rápido, fiable y asequible. Como paso previo, se ha realizado un análisis cualitativo del problema de contacto en la interfaz hueso-vástago aplicando una técnica optomecánica del campo continuo (fotoelasticidad). Para ello se han fabricado tres modelos en 2D del conjunto hueso-vástago, simulando tres tipos de contactos en la interfaz: contacto sin adherencia y con holgura, contacto sin adherencia y sin holgura, y contacto con adherencia y homogéneo. Aplicando la misma carga a cada modelo, y empleando la técnica de congelación de tensiones, se han visualizado los correspondientes estados tensionales, siendo estos más severos en el modelo de unión sin adherencia, como cabía esperar. En todo caso, los resultados son ilustrativos de la complejidad del problema de contacto y confirman la conveniencia y necesidad de la vía experimental para el estudio del problema. Seguidamente se ha planteado un ensayo dinámico de oscilaciones libres con instrumentación de sensores resistivos tipo galga extensométrica. Las muestras de ensayo han sido huesos fémur en todas sus posibles variantes: modelos simplificados, hueso sintético normalizado y hueso de cadáver, seco y fresco. Se ha diseñado un sistema de empotramiento del extremo distal de la muestra (fémur) con control riguroso de las condiciones de anclaje. La oscilación libre de la muestra se ha obtenido mediante la liberación instantánea de una carga estética determinada y aplicada previamente, bien con una maquina de ensayo o bien por gravedad. Cada muestra se ha instrumentado con galgas extensométricas convencionales cuya señal se ha registrado con un equipo dinámico comercial. Se ha aplicado un procedimiento de tratamiento de señal para acotar, filtrar y presentar las respuestas de los sensores en el dominio del tiempo y de la frecuencia. La interpretación de resultados es de tipo comparativo: se aplica el ensayo a una muestra de fémur Intacto que se toma de referencia, y a continuación se repite el ensayo sobre la misma muestra con una prótesis implantada; la comparación de resultados permite establecer conclusiones inmediatas sobre los efectos de la implantación de la prótesis. La implantación ha sido realizada por un cirujano traumatólogo utilizando las mismas técnicas e instrumental empleadas en el quirófano durante la práctica clínica real, y se ha trabajado con tres vástagos femorales comerciales. Con los resultados en el dominio del tiempo y de la frecuencia de las distintas aplicaciones se han establecido conclusiones sobre los siguientes aspectos: Viabilidad de los distintos tipos de muestras sintéticas: modelos simplificados y fémur sintético normalizado. Repetibilidad, linealidad y reversibilidad del ensayo. Congruencia de resultados con los valores teóricos deducidos de la teoría de oscilaciones libres de barras. Efectos de la implantación de tallos femorales en la amplitud de las oscilaciones, amortiguamiento y frecuencias de oscilación. Detección de armónicos asociados a la micromovilidad. La metodología se ha demostrado apta para ser incorporada a la normativa de prótesis, es de aplicación universal y abre vías para el análisis de la detección y caracterización de la micromovilidad de una prótesis frente a las cargas de servicio. ABSTRACT Total hip arthroplasty is considered as one of the greatest surgical advances in medicine. The application of this technique on Traumatology has increased significantly in recent years, mainly due to the progressive increase in life expectancy. In fact, advanced age increases osteoarthritis and osteoporosis problems, which are typical diseases of joints and bones, and in many cases require full or partial prosthetic replacement on the joint. Right functional behavior of prosthesis is highly dependent on the primary stability; this means it depends on the correct anchoring of the prosthesis at the time of implantation. Uncemented prosthesis base their long-term success on the quality of osseointegration that takes place between the prosthetic material and bone tissue, and to achieve this good primary stability conditions is mandatory. Aseptic loosening is the main cause of failure in total hip arthroplasty. This is a phenomenon in which relative movements occur, due to complex interactions of mechanical and biological factors, and these micromovements put the implant functionality at risk. To minimize possible damage, it greatly depends on the early detection of loosening. For this purpose, various techniques have been tested both in vivo and in vitro: numerical analysis and experimental techniques based on sensors for movements caused by naturally or artificially transmitted loads, such as impacts or vibrations at different frequencies. The assemblies and methods applied are heterogeneous and, in many cases, they are complex and expensive, with no agreement on the use of a simple and effective technique for general purposes. Likewise, in current regulations for governing the conditions to be fulfilled by the prosthesis before going to market, there is no specific section related to the evaluation of the femoral stem design in relation to primary stability. The aim of this thesis is to develop a in vitro methodology for analyzing the stability of an implanted femoral stem, in order to assess the implantation techniques and the different prosthesis designs prior to its offer in the market. We also propose as a fundamental requirement that the developed testing method should be simple, reversible, repeatable, non-destructive, with close monitoring of parameters (boundary conditions of loads and displacements) and with the availability of a register system to record and interpret results in a fast, reliable and affordable manner. As a preliminary step, we have performed a qualitative analysis of the contact problems in the bone-stem interface, through the application of a continuous field optomechanical technique (photoelasticity). For this proposal three 2D models of bone–stem set, has been built simulating three interface contact types: loosened an unbounded contact, unbounded and fixed contact, and bounded homogeneous contact. By means of applying the same load to each model, and using the stress freezing technique, it has displayed the corresponding stress states, being more severe as expected, in the unbounded union model. In any case, the results clearly show the complexity of the interface contact problem, and they confirm the need for experimental studies about this problem. Afterward a free oscillation dynamic test has been done using resistive strain gauge sensors. Test samples have been femur bones in all possible variants: simplified models, standardized synthetic bone, and dry and cool cadaveric bones. An embedding system at the distal end of the sample with strong control of the anchoring conditions has been designed. The free oscillation of the sample has been obtained by the instantaneous release of a static load, which was previously determined and applied to the sample through a testing machine or using the gravity force. Each sample was equipped with conventional strain gauges whose signal is registered with a marketed dynamic equipment. Then, it has applied a signal processing procedure to delimit, filter and present the time and frequency response signals from the sensors. Results are interpreted by comparing different trials: the test is applied to an intact femur sample which is taken as a reference, and then this test is repeated over the same sample with an implanted prosthesis. From comparison between results, immediate conclusions about the effects of the implantation of the prosthesis can be obtained. It must be said that the implementation has been made by an expert orthopedic surgeon using the same techniques and instruments as those used in clinical surgery. He has worked with three commercial femoral stems. From the results obtained in the time and frequency domains for the different applications the following conclusions have been established: Feasibility of the different types of synthetic samples: simplified models and standardized synthetic femur. Repeatability, linearity and reversibility of the testing method. Consistency of results with theoretical values deduced from the bars free oscillations theory. Effects of introduction of femoral stems in the amplitude, damping and frequencies of oscillations Detection of micromobility associated harmonics. This methodology has been proved suitable to be included in the standardization process of arthroplasty prosthesis, it is universally applicable and it allows establishing new methods for the analysis, detection and characterization of prosthesis micromobility due to functional loads.

NF-κB-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-κB and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-κB was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit IκB that rendered NF-κB inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-κB activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-κB activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell’s apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

Patterns of brain angiogenesis after vascular endothelial growth factor administration in vitro and in vivo

Vascular endothelial growth factor (VEGF) is a secreted endothelial cell mitogen that has been shown to induce vasculogenesis and angiogenesis in many organ systems and tumors. Considering the importance of VEGF to embryonic vascularization and survival, the effects of administered VEGF on developing or adult cerebrovasculature are unknown: can VEGF alter brain angiogenesis or mature cerebrovascular patterns? To examine these questions we exposed fetal, newborn, and adult rat cortical slice explants to graduated doses of recombinant VEGF. The effects of another known angiogenic factor, basic fibroblast growth factor (bFGF), were evaluated in a comparable manner. In addition, we infused VEGF via minipump into the adult cortex. Significant angiogenic effects were found in all VEGF experiments in a dose-responsive manner that were abolished by the addition of VEGF neutralizing antibody. Fetal and newborn explants had a highly complex network of branched vessels that immunoexpressed the flt-1 VEGF receptor, and flk-1 VEGF receptor expression was determined by reverse transcription–PCR. Adult explants had enlarged, dilated vessels that appeared to be an expansion of the existing network. All bFGF-treated explants had substantially fewer vascular profiles. VEGF infusions produced both a remarkable localized neovascularization and, unexpectedly, the expression of flt-1 on reactive astrocytes but not on endothelial cells. The preponderance of neovascularization in vitro and in vivo, however, lacked the blood–brain barrier (BBB) phenotype marker, GLUT-1, suggesting that in brain the angiogenic role of VEGF may differ from a potential BBB functional role, i.e., transport and permeability. VEGF may serve an important capacity in neovascularization or BBB alterations after brain injury.

The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin–serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.

Dissociation between bone resorption and bone formation in osteopenic transgenic mice

Bone mass is maintained constant in vertebrates through bone remodeling (BR). BR is characterized by osteoclastic resorption of preexisting bone followed by de novo bone formation by osteoblasts. This sequence of events and the fact that bone mass remains constant in physiological situation lead to the assumption that resorption and formation are regulated by each other during BR. Recent evidence shows that cells of the osteoblastic lineage are involved in osteoclast differentiation. However, the existence of a functional link between the two activities, formation and resorption, has never been shown in vivo. To define the role of bone formation in the control of bone resorption, we generated an inducible osteoblast ablation mouse model. These mice developed a reversible osteopenia. Functional analyses showed that in the absence of bone formation, bone resorption continued to occur normally, leading to an osteoporosis of controllable severity, whose appearance could be prevented by an antiresorptive agent. This study establishes that bone formation and/or bone mass do not control the extent of bone resorption in vivo.

Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: Structural basis for recognition of B-cell receptors and superantigen activity

Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-Å resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (VH) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human VH3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig VH regions and the T-cell receptor Vβ regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor Vβ backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.

A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K+ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by Gβγ subunits, but instead shows constitutive activation, and (ii) is no longer a K+-selective channel but conducts Na+ as well. The present experiments on weaverGIRK2 (wvGIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na+ concentration. In particular, manipulations that decrease intracellular Na+ produce a component of Na+-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na+ influx through the wvGIRK2 channel, leading to additional Na+ activation. We also show that the wvGIRK2 channel is permeable to Ca2+, providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K+ channels.

Cattle lack vascular receptors for Escherichia coli O157:H7 Shiga toxins

Escherichia coli O157:H7 causes Shiga toxin (Stx)-mediated vascular damage, resulting in hemorrhagic colitis and the hemolytic uremic syndrome in humans. These infections are often foodborne, and healthy carrier cattle are a major reservoir of E. coli O157:H7. We were interested in knowing why cattle are tolerant to infection with E. coli O157:H7. Cattle tissues were examined for the Stx receptor globotriaosylceramide (Gb3), for receptivity to Stx binding in vitro, and for susceptibility to the enterotoxic effects of Stx in vivo. TLC was used to detect Gb3 in tissues from a newborn calf. Gb3 was detected by TLC in kidney and brain, but not in the gastrointestinal tract. Immunohistochemistry was used to define binding of Stx1 and Stx2 overlaid onto sections from cattle tissues. Stx1 and Stx2 bound to selected tubules in the cortex of the kidney of both newborn calves (n = 3) and adult cattle (n = 3). Stx did not bind to blood vessels in any of the six gastrointestinal and five extraintestinal organs examined. The lack of Gb3 and of Stx receptivity in the gastrointestinal tract raised questions about the toxicity of Stx in bovine intestine. We found that neither viable E. coli O157:H7 nor Stx-containing bacterial extracts were enterotoxic (caused fluid accumulation) in ligated ileal loops in newborn calves. The lack of vascular receptors for Stx provides insight into why cattle are tolerant reservoir hosts for E. coli O157:H7.

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.