Application of Chitosan Microparticles for Reduction of Vibrio Species in Seawater and Live Oysters (Crassostrea virginica)

Human Vibrio infections associated with consumption of raw shellfish greatly impact the seafood industry. Vibrio cholerae-related disease is occasionally attributed to seafood, but V. vulnificus and V. parahaemolyticus are the primary targets of postharvest processing (PHP) efforts in the United States, as they pose the greatest threat to the industry. Most successful PHP treatments for Vibrio reduction also kill the molluscs and are not suitable for the lucrative half-shell market, while nonlethal practices are generally less effective. Therefore, novel intervention strategies for Vibrio reduction are needed for live oyster products. Chitosan is a bioactive derivative of chitin that is generally recognized as safe as a food additive by the FDA, and chitosan microparticles (CMs) were investigated in the present study as a potential PHP treatment for live oyster applications. Treatment of broth cultures with 0.5% (wt/vol) CMs resulted in growth cessation of V. cholerae, V. vulnificus, and V. parahaemolyticus, reducing culturable levels to nondetectable amounts after 3 h in three independent experiments. Furthermore, a similar treatment in artificial seawater at 4, 25, and 37°C reduced V. vulnificus levels by ca. 7 log CFU/ml after 24 h of exposure, but 48 h of exposure and elevated temperature were required to achieve similar results for V. parahaemolyticus and V. cholerae. Live oysters that either were artificially inoculated or contained natural populations of V. vulnificus and V. parahaemolyticus showed significant and consistent reductions following CM treatment (5%) compared to the amounts in the untreated controls. Thus, the results strongly support the promising potential for the application of CMs as a PHP treatment to reduce Vibrio spp. in intact live oysters.     

Local IL-17 Production Exerts a Protective Role in Murine Experimental Glomerulonephritis

IL-17 is a pro-inflammatory cytokine implicated in the pathogenesis of glomerulonephritis and IL-17 deficient mice are protected from nephrotoxic nephritis. However, a regulatory role for IL-17 has recently emerged. We describe a novel protective function for IL-17 in the kidney. Bone marrow chimeras were created using wild-type and IL-17 deficient mice and nephrotoxic nephritis was induced. IL-17 deficient hosts transplanted with wild-type bone marrow had worse disease by all indices compared to wild-type to wild-type bone marrow transplants (serum urea p<0.05; glomerular thrombosis p<0.05; tubular damage p<0.01), suggesting that in wild-type mice, IL-17 production by renal cells resistant to radiation is protective. IL-17 deficient mice transplanted with wild-type bone marrow also had a comparatively altered renal phenotype, with significant differences in renal cytokines (IL-10 p<0.01; IL-1β p<0.001; IL-23 p<0.01), and macrophage phenotype (expression of mannose receptor p<0.05; inducible nitric oxide synthase p<0.001). Finally we show that renal mast cells are resistant to radiation and produce IL-17, suggesting they are potential local mediators of disease protection. This is a novel role for intrinsic cells in the kidney that are radio-resistant and produce IL-17 to mediate protection in nephrotoxic nephritis. This has clinical significance as IL-17 blockade is being trialled as a therapeutic strategy in some autoimmune diseases.     

Stentless Versus Stented Bioprosthetic Aortic Valves: A Consensus Statement of the International Society of Minimally Invasive Cardiothoracic Surgery (ISMICS) 2008

OBJECTIVE:: The purpose of this consensus conference was to determine whether stentless bioprosthetic valves improve clinical and resource outcomes compared with stented valves in patients undergoing aortic valve replacement, and to outline evidence-based recommendations for the use of stentless and stented bioprosthetic valves in adult aortic valve replacement. METHODS:: Before the consensus conference, the best available evidence was reviewed in that systematic reviews, randomized trials, and nonrandomized trials were considered in descending order of validity and importance. At the consensus conference, evidence-based statements were created, and consensus processes were used to determine the ensuing recommendations. The American Heart Association/American College of Cardiology system was used to label the level of evidence and class of recommendation. RESULTS AND RECOMMENDATIONS:: Seventeen randomized studies published in 23 articles involving 1317 patients, and 14 nonrandomized trial published in 18 articles involving 2485 patients were included in the meta-analysis and consensus conference. All randomized trials inserted the stentless bioprosthetic valves in the subcoronary configuration. The consensus panel agreed upon the following statements and recommendations in patients undergoing aortic valve replacement:Because there were no randomized control trial comparing subcoronary stentless prosthetic valve and root replacement, the following recommendations are derived from expert opinion:     

In vitro contractile activity of porcine myometrium during luteolysis and early pregnancy: effect of oxytocin and progesterone

The present study was undertaken to elucidate the role of OT in myometrial contractility in sows. Spontaneous and OT-stimulated contractions of the inner circular (CM) and outer longitudinal (LM) layers isolated from cyclic (Days 14-16) and early pregnant (Days 14-16) sows were examined in six cyclic and six pregnant sows. In addition, the role of P(4) in the modulation of OT-induced uterine contractions was investigated. The contractile activity of the LM and CM layers was recorded in a tissue chamber filled with Krebs-Ringer solution. Myometrial contractility was expressed as area under the contractility curve (AUC) and frequency of contractions. Myometrial longitudinal and circular muscles exhibited spontaneous contractility in sows during both luteolysis and early-pregnancy. The mean AUC was higher (p<0.05) in the LM layer compared to the CM layer in both cyclic and pregnant animals. In addition, pregnant sows were characterized by higher AUC in both LM and CM layers in comparison to cyclic sows. The CM layer was unresponsive to examined treatments. Oxytocin (1-3x10(-8) and 1-3x10(-7)M) increased the AUC and frequency of contractions of the LM layer in both examined animal groups, being more effective during luteolysis (p<0.001) than early pregnancy (p<0.01). Response of the LM layer to OT appeared to be clearly related to the initial spontaneous level of contractility. This response to OT was inhibited (p<0.05) in the presence of OT antagonist (10(-6)M). Moreover, in pregnant sows, OT-stimulated contractile activity of myometrium was inhibited (p<0.05) by P(4) (10(-5)M). In conclusion, OT receptors present in myometrial cells (especially in the LM layer) are involved in the regulation of contractile activity of porcine myometrium during luteolysis and early-pregnancy. In addition, progesterone appears to be involved in this regulation.     

Permeabilization of yeast for in situ determination of α-glucosidase

A quantitative in situ assay of yeast α-glucosidase involving permeabilization of the cells by freezing and thawing is described. The assay was applied to different strains in different physiological states and was shown to give results comparable to those obtained with total cell homogenates. The primary advantage of the in situ assay was the possibility of analyzing a large number of samples from the same culture during a growth curve using a very reduced cell mass.     

Proteogenomic analysis of polymorphisms and gene annotation divergences in prokaryotes using a clustered mass spectrometry-friendly database

Precise annotation of genes or open reading frames is still a difficult task that results in divergence even for data generated from the same genomic sequence. This has an impact in further proteomic studies, and also compromises the characterization of clinical isolates with many specific genetic variations that may not be represented in the selected database. We recently developed software called multistrain mass spectrometry prokaryotic database builder (MSMSpdbb) that can merge protein databases from several sources and be applied on any prokaryotic organism, in a proteomic-friendly approach. We generated a database for the Mycobacterium tuberculosis complex (using three strains of Mycobacterium bovis and five of M. tuberculosis), and analyzed data collected from two laboratory strains and two clinical isolates of M. tuberculosis. We identified 2561 proteins, of which 24 were present in M. tuberculosis H37Rv samples, but not annotated in the M. tuberculosis H37Rv genome. We were also able to identify 280 nonsynonymous single amino acid polymorphisms and confirm 367 translational start sites. As a proof of concept we applied the database to whole-genome DNA sequencing data of one of the clinical isolates, which allowed the validation of 116 predicted single amino acid polymorphisms and the annotation of 131 N-terminal start sites. Moreover we identified regions not present in the original M. tuberculosis H37Rv sequence, indicating strain divergence or errors in the reference sequence. In conclusion, we demonstrated the potential of using a merged database to better characterize laboratory or clinical bacterial strains.     

Structural and ecophysiological adaptations to forest gaps

To survive new microclimatic conditions of a forest gap environment, plant species must physiologically and structurally adjust. A morpho-anatomical, ultrastructural and ecophysiological study was performed at three different times in a forest gap that was created by illegal selective logging. The study followed the early successional Actinostemon verticillatus and the late-successional Metrodorea brevifolia, to elucidate the adaptive strategies of acclimation to gaps. Additionally, Schinus terebinthifolius was included in the study in order to test the plasticity of a pioneer species that grows on forest edges, where this species had higher values of leaf thickness, leaf mass area and succulence. M. brevifolia had succulent leaves, high leaf area and a thin cuticle. A. verticillatus presented the densest leaves and was the only species to show leaf morpho-anatomical plasticity. Ultrastructural and physiological differences were observed only in A. verticillatus and M. brevifolia leaves from the gap: increase in the stroma volume, oil droplets, plastoglobuli, photochemical and non-photochemical quenching. Photosynthetic efficiency showed that the early stages of gap formation are the most critical. Acclimation strategies of A. verticillatus suggest this species invests in the efficiency of photosynthesis by increasing its leaf thickness, leaf mass area and in water content maintenance by increasing the density of its leaves, at the expense of gas exchange, was compensated by a high density of stomata. M. brevifolia compensates for the higher cost of leaves and lower leaf plasticity with ultrastructural changes that are used to adjust the photosynthetic process, which promotes a shorter leaf payback time.     

Expression of connexin 43 mRNA in microisolated murine osteoclasts and regulation of bone resorption in vitro by gap junction inhibitors

Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.     

Fluid pressure gradients, arising from oscillations in intramedullary pressure, is correlated with the formation of bone and inhibition of intracortical porosity

Fluid flow that arises from the functional loading of bone tissue has been proposed to be a critical regulator of skeletal mass and morphology. To test this hypothesis, the bone adaptive response to a physiological fluid stimulus, driven by low magnitude, high frequency oscillations of intramedullary pressure (ImP), were examined, in which fluid pressures were achieved without deforming the bone tissue. The ulnae of adult turkeys were functionally isolated via transverse epiphyseal osteotomies, and the adaptive response to four weeks of disuse (n=5) was compared to disuse plus 10 min per day of a physiological sinusoidal fluid pressure signal (60 mmHg, 20Hz). Disuse alone resulted in significant bone loss (5.7+/-1.9%, p< or =0.05), achieved by thinning the cortex via endosteal resorption and an increase in intracortical porosity. By also subjecting bone to oscillatory fluid flow, a significant increase in bone mass at the mid-diaphysis (18.3+/-7.6%, p<0.05), was achieved by both periosteal and endosteal new bone formation. The spatial distribution of the transcortical fluid pressure gradients (inverted Delta P(r)), a parameter closely related to fluid velocity and fluid shear stress, was quantified in 12 equal sectors across a section at the mid-diaphyses. A strong correlation was found between the inverted Delta P(r) and total new bone formation (r=0.75, p=0.01); and an inverse correlation (r=-0.75, p=0.01) observed between inverted Delta P(r) and the area of increased intracortical porosity, indicating that fluid flow signals were necessary to maintain bone mass and/or inhibit bone loss against the challenge of disuse. By generating this fluid flow in the absence of matrix strain, these data suggest that anabolic fluid movement plays a regulatory role in the modeling and remodeling process. While ImP increases uniformly in the marrow cavity, the distinct parameters of fluid flow vary substantially due to the geometry and ultrastructure of bone, which ultimately defines the spatial non-uniformity of the adaptive process.