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Yeast serves as a functional alternative and sustainable protein source in aquaculture. This protocol outlines feeding strategy and intestinal proteome analysis of zebrafish (Danio rerio), using S-trap for digestion, iTRAQ, and mass spectrometry for protein quantification. Additionally, it details the analysis of chemical components in feed and functional assessments via Kyoto Encyclopedia of Genes and Genomes (KEGG), Eukaryotic Orthologous Group (KOG), and Gene Ontology (GO). For complete details on the use and execution of this protocol, please refer to Purushothaman et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: This study investigates the relationship between approaches to learning, self-perceived study burnout, and the level of knowledge among veterinary students. Veterinary educational programs are under regular development and would benefit greatly from detailed feedback on students' knowledge, proficiency, influencing factors, and coping mechanisms., Methods: The VetRepos consortium developed and calibrated an item repository testing knowledge across the entire veterinary curriculum. Two hundred forty-eight students from seven European veterinary institutions took the VetRepos test, comprising a subset of the repository. They also responded to a questionnaire assessing deep and unreflective learning approaches and self-perceived study burnout, represented by exhaustion and cynicism. Structural equation modeling analyzed the relationship between these latent traits and the VetRepos test score., Results: The model failed the exact-fit test but was retained based on global fit indices, inter-item residual correlations, and standardized residual covariances. Root Mean Square Error of Approximation with robust standard errors and scaled test statistic was 0.049 (95% confidence interval 0.033-0.071), scaled and robust Comparative Fit Index 0.95 (0.90-0.98), and scaled Standardized Root Mean Square Residual 0.056 (0.049-0.071). Measurement invariance across study years was not violated (ΔCFI = 0.00, χ 2 = 3.78, Δdf = 4, p = 0.44), but it could not be confirmed between genders or universities. The VetRepos test score regressed on the study year [standardized regression coefficient = 0.68 (0.62-0.73)], showed a negative regression on the unreflective learning approach [-0.25 (-0.47 to -0.03)], and a positive regression on the deep approach [0.16 (0.03-0.28)]. No direct association with perceived burnout was observed; however, a significant, medium-sized association was found between the unreflective approach and self-perceived study burnout. No significant differences in learning approaches or perceived burnout were found between study years., Discussion: The most important source of variance in VetRepos test scores, unrelated to the study year, was the learning approach. The association between the VetRepos test score and self-perceived burnout was indirect. Future research should complement this cross-sectional approach with longitudinal and person-oriented studies, further investigating the relationship between study burnout and learning approaches., Competing Interests: JW was employed by NordicMetrics ApS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Iivanainen, Collares, Wandall, Parpala, Nevgi, Keto-Timonen, Tipold, Schaper, van Haeften, Pihl, Press and Holm.)

Recent pandemics like COVID-19 highlighted the importance of rapidly developing diagnostics to detect evolving pathogens. CRISPR-Cas technology has recently been used to develop diagnostic assays for sequence-specific recognition of DNA or RNA. These assays have similar sensitivity to the gold standard qPCR but can be deployed as easy to use and inexpensive test strips. However, the discovery of diagnostic regions of a genome flanked by conserved regions where primers can be designed requires extensive bioinformatic analyses of genome sequences. We developed the Python package krisp to aid in the discovery of primers and diagnostic sequences that differentiate groups of samples from each other, using either unaligned genome sequences or a variant call format (VCF) file as input. Krisp has been optimized to handle large datasets by using efficient algorithms that run in near linear time, use minimal RAM, and leverage parallel processing when available. The validity of krisp results has been demonstrated in the laboratory with the successful design of a CRISPR diagnostic assay to distinguish the sudden oak death pathogen Phytophthora ramorum from closely related Phytophthora species. Krisp is released open source under a permissive license with all the documentation needed to quickly design CRISPR-Cas diagnostic assays., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Yeasts contain bioactive components that can enhance fish immune robustness and disease resistance. Our study focused on analyzing intestinal immunoregulatory pathways in zebrafish ( Danio rerio ) using iTRAQ and 2D LC-MS/MS to quantify intestinal proteins. Zebrafish were fed either control diet (C) or diet supplemented with autolyzed Cyberlindnera jadinii (ACJ). KEGG analysis revealed that ACJ yeast diet induced increased abundance of proteins related to arginine and proline metabolism, phagosome, C-lectin receptor signaling, ribosome and PPAR signaling pathways, which can modulate and enhance innate immune responses. ACJ yeast diet also showed decreased abundance of proteins associated with inflammatory pathways, including apoptosis, necroptosis and ferroptosis. These findings indicate boosted innate immune response and control of inflammation-related pathways in zebrafish intestine. Our findings in the well annotated proteome of zebrafish enabled a detailed investigation of intestinal responses and provide insight into health-beneficial effects of yeast species C . jadinii , which is relevant for aquaculture species., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

The effect of dietary graded levels of Cyberlindnera jadinii yeast (C. jadinii) on growth performance, nutrient digestibility, and gut health of broilers was evaluated from 1 to 34 d of age. A total of 360 male broiler chicks were randomly allocated to 1 of 4 dietary treatments (6 replicate pens each) consisting of a wheat-soybean meal-based pelleted diet (Control or CJ0), and 3 diets in which 10% (CJ10), 20% (CJ20), and 30% (CJ30) of the crude protein were supplied by C. jadinii, by gradually replacing protein-rich ingredients. Body weight and feed intake were measured at d 1, 11, 22, and 32. Pellet temperature, durability, and hardness increased linearly (P < 0.05) with C. jadinii inclusion, with highest (P < 0.05) values for CJ30. Up until d 22, feed conversion ratio (FCR) was similar between treatments (P = 0.169). Overall, increasing C. jadinii inclusion linearly increased (P = 0.047) feed intake but had no effect on weight gain or mortality. FCR increased (P < 0.05) linearly with increasing C. jadinii inclusion but only birds fed CJ30 had a significantly poorer FCR compared to the Control. Ileal digestibility was not affected by C. jadinii inclusion, however, there was a significant linear decrease in crude protein and phosphorus, and a tendency for a decrease in fat digestibility. Apparent metabolizable energy (AME) decreased (P < 0.001) quadratically with increasing C. jadinii and was significantly lower in CJ30 compared to the Control. Ileal concentrations of volatile fatty acids (VFAs) were not affected by C. jadinii inclusion, but butyric acid and total VFAs were linearly and quadratically increased and were significantly higher in cecal digesta of birds fed CJ20 and CJ30. Increasing C. jadinii inclusion was associated with an increase (P < 0.05) in the relative abundance of lactobacillus in the ileum and cecum. In conclusion, C. jadinii yeast can supply up to 20% of the total dietary protein without negatively affecting performance, digestibility, or gut health of broilers. The potential confounding role of feed processing and C. jadinii cell wall components on broiler performance is discussed., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Progress testing in education is an assessment principle for the measurement of students' progress over time, e.g., from start to graduation. Progress testing offers valid longitudinal formative measurement of the growth in the cognitive skills of the individual students within the subjects of the test as well as a tool for educators to monitor potential educational gaps and mismatches within the curriculum in relation to the basic veterinary learning outcomes., Methods: Six veterinary educational establishments in Denmark, Finland, Germany (Hannover), the Netherlands, Norway, and Sweden established in cooperation with the European Association of Establishments for Veterinary Education (EAEVE) a common veterinary item repository that can be used for progress testing in European Veterinary Education Establishments (VEEs), linear as well as computer adaptive, covering the EAEVE veterinary subjects and theoretical "Day One Competencies." First, a blueprint was created, suitable item formats were identified, and a quality assurance process for reviewing and approving items was established. The items were trialed to create a database of validated and calibrated items, and the responses were subsequently psychometrically analyzed according to Modern Test Theory., Results: In total, 1,836 items were submitted of which 1,342 were approved by the reviewers for trial testing. 1,119 students from all study years and all partners VEEs participated in one or more of six item trials, and 1,948 responses were collected. Responses were analyzed using Rasch Modeling (analysis of item-fit, differential item function, item-response characteristics). A total of 821 calibrated items of various difficulty levels matching the veterinary students' abilities and covering the veterinary knowledge domains have been banked., Discussion: The item bank is now ready to be used for formative progress testing in European veterinary education. This paper presents and discusses possible pitfalls, problems, and solutions when establishing an international veterinary progress test., Competing Interests: JW is part-time employed for the VetRepos project by the University of Copenhagen, as well as employed by NordicMetrics Aps. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schaper, van Haeften, Wandall, Iivanainen, Penell, Press, Lekeux and Holm.)

The interplay between nutrition and the immune system is well recognized, and several studies show that experimental diets elicit local morphological changes and alteration of gene and protein expression in the intestinal mucosa of Atlantic salmon. In this study the pathophysiological effects of experimental diets on mucosal responses in the distal intestine of Atlantic salmon were investigated. Atlantic salmon were fed diets with inclusion of soybean meal (SBM) and Cyberlindnera jadinii (CJ) yeast for 7 days. A standard fish meal (FM) diet was used as a control. Morphological, immunohistochemical and gene expression analyses were used to evaluate the presence of immune cells, proliferating cells, and stem cell populations in mucosal compartments of the simple folds in the distal intestine. Fish fed SBM developed morphological changes consistent with SBM induced enteritis. Immunohistochemistry showed an increased presence of apoptotic cells, CD3ϵ and CD8α labelled cells in the simple fold epithelium of SBM group compared with the CJ group. For the investigated genes, expression levels in all three groups were mostly higher in the epithelial compartment of the simple fold than in the compartment beneath the folds. Most changes within the epithelial compartment were observed in fish fed SBM, where expression of CD3ζ, CD8α, MHC I and MHC II were lower than the FM control group. The CJ group had an increased expression of the stem cell marker Lgr5 in the epithelial compartment compared with SBM group. The division of the simple fold into an apical and basal compartment showed that the increase in Lgr5 was evident along the whole length of the simple folds and not confined to the base of the folds. Similarly, proliferation (PCNA, MCM2) and apoptosis (Caspase-3) gene expression was present in the entire length of the simple folds, suggesting that intestinal epithelial cell turnover is not confined to the basal or apical part of the fold. This study shows that the epithelial compartment is active in the early immunoregulatory response towards dietary stimuli and that the level of an intestinal stem cell marker in salmon was influenced by a diet containing CJ yeast., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Stress related to salmon aquaculture practices (handling, sub-optimal nutrition, diseases, and environmental problems) may compromise fish welfare. This study describes the effects of two hydrolyzed Debaryomyces hansenii yeast-based products (LAN4 and LAN6) on physiological and immune responses of Atlantic salmon ( Salmo salar ) parr exposed to short hypoxia stress. A commercial-like diet (control diet: CD) and two experimental diets (CD supplemented with 0.1% of either component LAN4 or LAN6) were fed to fish for 8 weeks. At the end of the feeding experiment, fish were exposed to 1-min hypoxia and samples were collected at 0, 1, 3, 6, 12, and 24 h post-stress. Results showed that plasma cortisol reached a peak at 1 h post-stress in CD and LAN6 groups, whereas no significant increase in cortisol levels was detected in the LAN4 group. Moreover, the LAN6 group enhanced IL-10 responses to hypoxia, when compared to the control and LAN4 group. This suggests a regulation of immunosuppressive profiles in fish fed LAN4. Hypoxia stress increased TNFα in all groups, which indicates that fish may compensate for the short-term stress response, by modulating innate immune molecules. The apparent suppression of hypoxia responses in the LAN4 group coincided with the detection of differences in goblet cells size and Muc-like proteins production in DI; and upregulation (1 h post-stress) of pathways related to oxygen transport, hemoglobin complex, and glutathione transferase activity and the downregulation of fatty acid metabolism (6 h post-stress) in gills. To conclude, a 1-min hypoxia stress exposure affects the response to stress and immunity; and D. hansenii -based yeast products are promising components in functional aquafeeds for salmon due to their ability to counteract possible consequences of hypoxic stress., Competing Interests: MC was employee in Lallemand SAS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Morales-Lange, Djordjevic, Gaudhaman, Press, Olson, Mydland, Mercado, Imarai, Castex and Øverland.)

The objective of the current study was to examine the effects of yeasts on intestinal health and transcriptomic profiles from the distal intestine and spleen tissue of Atlantic salmon fed SBM-based diets in seawater. Cyberlindnera jadinii (CJ) and Wickerhamomyces anomalus (WA) yeasts were heat-inactivated with spray-drying (ICJ and IWA) or autolyzed at 50 °C for 16 h (ACJ and AWA), followed by spray-drying. Six diets were formulated, one based on fishmeal (FM), a challenging diet with 30% soybean meal (SBM) and four other diets containing 30% SBM and 10% of each of the four yeast fractions (i.e., ICJ, ACJ, IWA and AWA). The inclusion of CJ yeasts reduced the loss of enterocyte supranuclear vacuolization and reduced the population of CD8α labeled cells present in the lamina propria of fish fed the SBM diet. The CJ yeasts controlled the inflammatory responses of fish fed SBM through up-regulation of pathways related to wound healing and taurine metabolism. The WA yeasts dampened the inflammatory profile of fish fed SBM through down-regulation of pathways related to toll-like receptor signaling, C-lectin receptor, cytokine receptor and signal transduction. This study suggests that the yeast species, Cyberlindnera jadinii and Wickerhamomyces anomalus are novel high-quality protein sources with health-beneficial effects in terms of reducing inflammation associated with feeding plant-based diets to Atlantic salmon.

Vaccination against salmon lice ( Lepeophtheirus salmonis ) is a means of control that averts the negative effects of chemical approaches. Here, we studied the immunogenicity and protective effect of a vaccine formulation (based on a salmon lice-gut recombinant protein [P33]) against Lepeophtheirus salmonis infestation in Atlantic salmon in a laboratory-based trial. Our findings revealed that P33 vaccine can provide a measure of protection against immature and adult salmon lice infestation. This protection seemed to be vaccine dose-dependent, where higher doses resulted in lower parasitic infestation rates. We also provide immunological evidence confirming that P33-specific immune response can be triggered in Atlantic salmon after P33 vaccination, and that production of P33-specific antibodies in blood can be detected in vaccinated fish. The negative correlation between P33-specific IgM in salmon plasma and salmon lice numbers on vaccinated fish suggests that protection against lice can be mediated by the specific antibody in salmon plasma. The success of P33 vaccination in protecting salmon against lice confirms the possibility of employing the hematophagous nature of the parasite to deliver salmon-specific antibodies against lice-gut proteins.

Supplying novel feed ingredients for pig production is crucial to enhance food security and decrease the environmental impact of meat production. Several studies have focused on evaluating the beneficial health effects of yeast in pigs. However, its use as a protein source has been partially addressed. Previously, we have shown that yeast at high inclusion levels maintains growth performance and digestibility, while nutrient digestibility, intestinal villi height and fecal consistency were improved. The present study combined microbiome, short-chain fatty acid, and immune parameter analysis to investigate the effect of high inclusion of yeast in diets for post-weaning piglets. Our results showed that yeast did not have a significant impact on the hematological or biochemical parameters in blood. The different immune cell subpopulations isolated from blood and distal jejunal lymph nodes (DJLN) were analyzed by flow cytometry and showed that yeast diet induced an increased number of the subtype of leukocytes CD45+/CD3-/CD8+, a special type of Natural Killer (NK) cells. Also, a very mild to moderate infiltration of neutrophilic granulocytes and lower IgA level were observed in the colon of yeast fed piglets. The microbiome profiling in different compartments of the gastrointestinal tract of piglets was performed using 16S rRNA metabarcoding. The results showed that 40% replacement of dietary protein had a statistically significant effect on the microbial communities in cecum and colon, while the microbial population in ileum and jejunum were not affected. Analysis of predicted microbial metabolic pathways analysis revealed significant upregulation of short-chain fatty acids, ether lipid metabolisms, secondary bile acids, and several other important biosynthesis pathways in cecum and colon of pigs fed yeast. In conclusion, the results showed that diet containing 40% of yeast protein positively shaped microbial community in the large intestine and increased the number of a specific subpopulation of NK cells in the DJLN. These results showed that yeast modulates the microbiome and decreases the secretion of IgA in the colon of post-weaning pigs., (Copyright © 2020 Lagos, Bekkelund, Skugor, Ånestad, Åkesson, Press and Øverland.)

Rapeseed meal is a sustainable feed ingredient that can be used as an alternative to imported soybean meal in European pig production. The gut microbiota plays an important role on pig physiology and health but the impact on microbiota of using rapeseed in diets is still not well known. In this study, 84 purebred Norwegian Landrace pigs with average initial weight of 25 kg were divided into two groups and fed for approximately three months with either a control diet containing soybean meal (CON) or a high-fiber experimental diet where 20% rapeseed meal (RSF) was included as an alternative to soybean meal in CON. The composition and function of microbiome in gut digesta samples were analyzed by performing 16S rRNA gene sequencing and culturing of bacteria. The microbiota diversity and composition were similar between the dietary treatments; however, relative abundance of a variety of bacterial groups and imputed functions of microbiome in the ileum and large intestine were altered when the pigs were fed with a rapeseed-based diet. It was notable that the immune-inducing bacterial group Mucispirillum and anti-inflammatory stimulating bacteria Lachnospira were more abundant in the ileum and large intestine of the RSF group, respectively. Moreover, there was a higher abundance of major amino acid fermenters and amylolytic bacteria in the CON group and a high abundance of putative short chain fatty acid producers in RSF group. In comparison with the CON group, the gut microbiome of RSF group possessed an enhanced potential for carbohydrate and energy metabolism and a reduced potential for bacterial pathogenicity-related pathways.

Background: Dietary yeast inclusions in a pig diet may drive changes both in gut bacterial composition and bacterial functional profile. This study investigated the effect of Cyberlindnera jadinii as a protein to replace 40% of the conventional proteins in a diet for weanling pigs on the microbiota in the small and large intestine, colonic short-chain fatty acid concentration, and colonic histopathology parameters. Seventy-two pigs weaned at 28 days of age were randomly assigned to either a control or a C. jadinii-based diet and followed for 2 weeks., Results: Compared with the controls, higher numbers of cultivable lactic acid-producing bacteria in the small and large intestine were registered in the yeast group. Alpha and beta bacterial diversity were different between the diet groups with lower alpha-diversity and distinct bacterial composition in the large intestine in the yeast group compared with those of the controls. The large intestine microbiota in the yeast group had higher numbers of Prevotella, Mitsuokella and Selenomonas compared with those of the controls. The concentrations of colonic acetate and butyrate were higher in the controls compared with that of the yeast group. The colonic crypt depth was deeper in the control group. The gut histopathology of colonic tissues revealed no differences between the diets. The colonic crypt depth tended to be deeper with higher relative abundance of an unclassified Spirochetes, higher colonic butyrate concentration, and higher bacterial richness. The concentration of colonic butyrate was positively associated with the relative abundance of the Faecalibacterium prausnitzii, Dialister, and an unclassified amplicon of the Spirochaetaceae family in the colon., Conclusions: The replacement of the conventional proteins by proteins from Cyberlindnera jadinii in a weanling pig diet reshaped the large intestine microbiota structure. The novel yeast diet appeared to be selective for Lactobacillus spp., which may represent an added value resulting from using the sustainably produced yeast protein ingredient as an alternative to conventional protein ingredients in animal diets. The large intestine bacterial composition and their metabolites may be involved in an adaptive alteration of the colonic crypts without pathological consequences.

Farmed Atlantic salmon (Salmo salar) are prone to various conditions affecting the quality of the fillet. A well-known but so far poorly understood condition is the focal red changes in muscle, often referred to as haemorrhages. Such changes are characterized by muscle necrosis, haemorrhages and acute inflammation. They can progress into focal melanised changes, a chronic inflammatory condition with melanin-producing leukocytes. The initial cause of intramuscular haemorrhages is unknown. In this study, we aimed to reveal some of their key immunological features. Samples of red focal changes were investigated by immunohistochemistry (IHC), in situ hybridization (ISH) and RT-qPCR for various immune markers. The results were compared with samples of melanised changes and control muscle, subjected to the same analyses. In all red changes, infiltrates with mononuclear cells were detected, consisting mostly of MHC class I/II + cells, but also of CD3 + and CD8 + cells. ISH studies on IgM showed few to moderate amounts of B-cells in red focal changes. Trends in the RT-qPCR showed upregulation of genes related to innate immunity in the red changes, whereas genes related to adaptive immunity were upregulated in the melanised changes. An important result was the significant downregulation of the anti-inflammatory cytokine IL10 in all red changes. Our findings indicate that we can rule out an auto invasive nature of the changes. The downregulation of IL10 at an early phase is a trait for the condition., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Microbial ingredients such as Candida utilis yeast are known to be functional protein sources with immunomodulating effects whereas soybean meal causes soybean meal-induced enteritis in the distal intestine of Atlantic salmon (Salmo salar L.). Inflammatory or immunomodulatory stimuli at the local level in the intestine may alter the plasma proteome profile of Atlantic salmon. These deviations can be helpful indicators for fish health and, therefore potential tools in the diagnosis of fish diseases. The present work aimed to identify local intestinal tissue responses and changes in plasma protein profiles of Atlantic salmon fed inactive dry Candida utilis yeast biomass, soybean meal, or combination of soybean meal based diet with various inclusion levels of Candida utilis. A fishmeal based diet was used as control diet. Inclusion of Candida utilis yeast to a fishmeal based diet did not alter the morphology, immune cell population or gene expression of the distal intestine. Lower levels of Candida utilis combined with soybean meal modulated immune cell populations in the distal intestine and reduced the severity of soybean meal-induced enteritis, while higher inclusion levels of Candida utilis were less effective. Changes in the plasma proteomic profile revealed differences between the diets but did not indicate any specific proteins that could be a marker for health or disease. The results suggest that Candida utilis does not alter intestinal morphology or induce major changes in plasma proteome, and thus could be a high-quality alternative protein source with potential functional properties in diets for Atlantic salmon., Competing Interests: The authors have declared that no competing interests exist.

Rapeseed meal (RSM) is an alternative feed ingredient to soybean meal (SBM) in pig diets. However, knowledge on the effect of RSM on gut health, especially in relation to changes in gut microbiota is still limited. In our study, Norwegian Landrace weaner pigs were fed with either a control diet (CON) based on wheat, barley and SBM, or a high-fiber experimental diet where SBM was replaced by RSM (RSF). We found no large differences in the gut microbiota of pigs fed the two diets, suggesting that RSF does not disturb the gut microbiota and the normal gut function. The relative abundance of SCFA-producing phylotypes and colon-health related phylotypes increased in the large intestine of RSF-fed pigs. Among them, Lachnospira and Coprococcus were negatively associated with the presence of neutrophils in the colon wall. The higher abundance of these bacteria in colon of RSF pigs may suggest an anti-inflammatory stimulus effect of the RSF diet. The gut microbiota of RSF-fed pigs was relatively unaltered following episodes of diarrhea suggesting that the RSF diet may promote robustness in weaner pigs and reduce the risk of dysbiosis., Competing Interests: The authors have declared that no competing interests exist.

Modulation of gene expression through RNA interference is well conserved in eukaryotes and is involved in many cellular processes. In the oomycete Phytophthora , research on the small RNA machinery and function has started to reveal potential roles in the pathogen, but much is still unknown. We examined Argonaute (AGO) homologs within oomycete genome sequences, especially among Phytophthora species, to gain a clearer understanding of the evolution of this well-conserved protein family. We identified AGO homologs across many representative oomycete and stramenopile species, and annotated representative homologs in P. sojae . Furthermore, we demonstrate variable transcript levels of all identified AGO homologs in comparison to previously identified Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) homologs. Our phylogenetic analysis further refines the relationship of the AGO homologs in oomycetes and identifies a conserved tandem duplication of AGO homologs in a subset of Phytophthora species.

This study investigated morphological changes associated with soya bean meal-induced enteritis (SBMIE) in distal intestine (DI) of rainbow trout (Oncorhynchus mykiss) fed a soya bean meal (SBM)-based diet and exposed to normoxia or hypoxia created by optimal and low water flow rates, respectively. A 28-day adaption period was followed by a 42-day challenge period where 600 fish were subjected to dietary challenge and/or hypoxia. Twelve tanks each containing 50 juvenile trout were assigned randomly in triplicate to each treatment. Histopathological and immunohistochemical evaluation revealed pathological features that have not previously been described in association with SBMIE. Vacuolar degeneration of epithelial cells mainly at the base of mucosal folds, epithelial cysts, epithelial dysplasia, necrosis, shedding of necrotic cells, and granulomatous inflammation including infiltration of enlarged, sometimes finely vacuolated or "foamy" macrophages, multinucleated giant cells and increased proliferation of fibroblasts were observed. Acid-fast bacteria were not detected in enlarged macrophages; however, these cells contained AB-PAS- and sometimes cytokeratin-positive material, which was interpreted to be of epithelial/goblet cell origin. Hypoxia did not affect the morphological changes in DI. These results suggest that SBM was associated with a granulomatous form of enteritis in DI of rainbow trout regardless of water oxygen level., (© 2017 John Wiley & Sons Ltd.)

Background: Glenoid component loosening remains a significant issue after anatomic shoulder arthroplasty. Pegged glenoid components have shown better lucency rates than keeled components in the short term; however, midterm to long-term results have not fully been determined. We previously reported early outcomes of the current randomized controlled group of patients, with higher glenoid lucency rates in those with a keeled glenoid. The purpose of this study was to evaluate the radiographic and clinical outcomes of these components at minimum 5-year follow-up., Methods: Fifty-nine total shoulder arthroplasties were performed in patients with primary glenohumeral osteoarthritis. Patients were randomized to receive either a pegged or keeled glenoid component. Three raters graded radiographic glenoid lucencies. Clinical outcome scores and active mobility outcomes were collected preoperatively and at yearly postoperative appointments., Results: Of the 46 shoulders meeting the inclusion criteria, 38 (82.6%) were available for minimum 5-year radiographic follow-up. After an average of 7.9 years, radiographic lucency was present in 100% of pegged and 91% of keeled components (P = .617). Grade 4 or 5 lucency was present in 44% of pegged and 36% of keeled components (P = .743). There were no differences in clinical outcome scores or active mobility outcomes between shoulders with pegged and keeled components at last follow-up. Within the initial cohort, 20% of the keeled shoulders (6 of 30) and 7% of the pegged shoulders (2 of 29) underwent revision surgery (P = .263). Kaplan-Meier analysis showed no significant difference in survival rates between groups (P = .560)., Conclusion: At an average 7.9-year follow-up, non-ingrowth, all-polyethylene pegged glenoid implants are equivalent to keeled implants with respect to radiolucency, clinical outcomes, and need for revision surgery., (Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Phytophthora rubi and P. fragariae are two closely related oomycete plant pathogens that exhibit strong morphological and physiological similarities but are specialized to infect different hosts of economic importance, namely, raspberry and strawberry. Here, we report the draft genome sequences of these two Phytophthora species as a first step toward understanding the genomic processes underlying plant host adaptation in these pathogens.

The interbranchial lymphoid tissue (ILT) of Atlantic salmon originates from an embryological location that in higher vertebrates gives rise to both primary and secondary lymphoid tissues. Still much is unknown about the morphological and functional development of the ILT. In the present work a standardized method of organ volume determination was established to study its development in relation to its containing gill and the thymus. Based on morphological findings and gene transcription data, the ILT shows no signs of primary lymphoid function. In contrast to the thymus, an ILT-complex first became discernible after the yolk-sac period. After its appearance, the ILT-complex constitutes 3-7% of the total volume of the gill (excluding the gill arch) with the newly described distal ILT constituting a major part, and in adult fish it is approximately 13 times larger than the thymus. Confined regions of T-cell proliferation are present within the ILT. Communication with systemic circulation through the distal ILT is also highly plausible thus offering both internal and external recruitment of immune cells in the growing ILT., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Phytophthora infestans, the cause of the devastating late blight disease of potato and tomato, exhibits a clonal reproductive lifestyle in North America. Phenotypes such as fungicide sensitivity and host preference are conserved among individuals within clonal lineages, while substantial phenotypic differences can exist between lineages. Whole P. infestans genomes were aligned and single nucleotide polymorphisms (SNPs) identified as targets for the development of clonal-lineage-specific molecular diagnostic tools. Informative SNPs were used to develop high-resolution melt (HRM) assays and locked nucleic acid (LNA) probes to differentiate lineage US-23, the predominant lineage in the Eastern United States for the past several years, from three other U.S. lineages. Three different primer pairs targeting one to three SNPs were capable of separating lineage US-23 from lineages US-8, US-11, and US-24 using HRM analysis. A fourth HRM primer pair targeted a highly variable genomic region containing nine polymorphisms within 63 bp. These primers separated US-23, US-11, and US-8 plus US-24 into three separate groups following HRM analysis but did not separate US-8 from US-24. Additionally, two LNA probes were designed to target a portion of the P. infestans genome containing two SNPs diagnostic for US-23. A single multiplex quantitative polymerase chain reaction assay containing both differentially labeled LNA probes differentiated individuals belonging to lineage US-23 from those belonging to US-8, US-11, and US-24.

Aims: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA., Methods and Results: Two sets of loop-mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross-reacted with the closely related species Phytophthora nicotianae. Rgn86&#95;2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross-react with P. nicotianae. The specificity of the Rgn86&#95;2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis and P. phaseoli. Cross-reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions., Conclusions: Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86&#95;2 primers had higher specificity., Significance and Impact of the Study: This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories., (© 2016 The Society for Applied Microbiology.)

Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P. sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed.

The amount, distribution and phenotype of ovine NCR1+ cells were investigated during developing GALT from day 70 of gestation. Antibodies against CD3 and CD79 were used to identify the compartments of GALT, and the localization of NCR1+ cells were correlated within these structures. Markers CD34 and c-kit, in addition to Ki67, were used to investigate possible origin and the stage of development of the NCR1+ cells. NCR1+ cells were present as single cells in the subepithelial tissue as early as 70 days of gestation, and were predominantly present in the T cell rich IFAs and domes as these intestinal wall compartments developed. While NCR1+ cells proliferated more intensively at mid-gestation (70-104 days), the number of NCR1+ cells also expressing c-kit, increased at the end of gestation. In conclusion, NCR1+ cells appeared early in T cell areas of the gut and displayed a phenotype consistent with intermediate stages of cNK cells and/or a subpopulation of ILC22., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Inflammatory bowel disease (IBD) in horses is an idiopathic disorder, encompassing different types of chronic intestinal inflammation. The pathogenesis of the disease remains to be established, but it has been suggested that an imbalance between regulatory T cells (Tregs) and T helper 17 (Th17)-associated cytokines and altered toll-like receptor 4 (TLR4) expression is associated with intestinal inflammation in other species. The aim of the present study was to quantify Tregs in rectal biopsies from horses affected with IBD by immunohistochemistry and to evaluate expression of genes encoding interleukin (IL)-12p40, IL-17A, IL-23p19 and TLR4 by real-time quantitative PCR. Rectal biopsies from 11 healthy horses and 11 horses with clinical signs of IBD, showing inflammation classified as chronic simple proctitis (CSP) or chronic active simple proctitis (CASP), were evaluated. Expression of IL-17A mRNA was greater in horses affected with CASP compared with horses with CSP or healthy horses. In contrast, expression of IL-12p40 was lower in horses with CSP compared with horses with CASP or healthy horses. TLR4 expression was greater in horses with CASP compared with healthy horses. A positive correlation was seen between the numbers of Tregs and expression of IL-17A and IL-23p19. An association was demonstrated between the histopathological pattern of inflammation, cytokine profile and number of infiltrating Tregs. The research findings suggest that Th17 cells are involved in active IBD, possibly through recruitment of neutrophils via IL-17A, in combination with inadequate suppression of the inflammatory response by Tregs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The teleost gill forms an extensive, semipermeable barrier that must tolerate intimate contact with the surrounding environment and be able to protect the body from external pathogens. The recent discovery of the interbranchial lymphoid tissue (ILT) has initiated an anatomical and functional investigation of the lymphoid tissue of the salmonid gill. In this article, sectioning of gill arches in all three primary planes revealed an elongation of the ILT outward along the trailing edge of the primary filament to the very distal end, a finding not previously described. This newly found lymphoid tissue was investigated using a range of morphological and transcriptional tools. Avoiding potential salinity-related effects, the study focused on two fresh-water life stages-smoltifying juveniles and mature adults. Aggregates of T-cells continuous with the ILT were found within the thick epithelial lining of the trailing edge of the filament in considerably larger numbers than seen in the epithelium of the leading edge and of the interlamellar area. Only a few of these cells were identified as CD8α(+) -cells, and there was a significantly (P < 0.05) higher relative expression of CD4- than of CD8- related genes in all gill segments investigated. Numerous major histocompatibility complex class II(+) -cells were distributed uniformly throughout the filament epithelial tissue. Few Ig(+) -cells were detected. Overall, the morphological features and comparable immune gene expression of the previously described ILT and the filament trailing edge lymphoid tissue suggest a close functional and anatomical relationship. We propose that the anatomical definition of the ILT must be broadened to include both the previously described ILT (to be renamed proximal ILT) and the trailing edge lymphoid tissue (to be named distal ILT). This extended anatomical localisation identifies the ILT as a widely distributed mucosal lymphoid tissue in the gill of Atlantic salmon., (© 2015 Wiley Periodicals, Inc.)

Root rot of raspberry (Rubus idaeus), thought to be primarily caused by Phytophthora rubi, is an economically important disease in the western United States. The objectives of this study were to determine which Phytophthora species are involved in root rot, examine the efficacy of different isolation methods (cane, root, and root/soil baiting with young raspberry plants), and determine if pathogenicity, fungicide resistance, and/or genetic variation exists among P. rubi isolates collected from raspberry fields in Washington, Oregon, and California. Of 275 samples, direct isolation from cane material resulted in a greater number of P. rubi isolates (39%), whereas root/soil baiting yielded the least (11%). Sequencing of the internal transcribed spacer region of 210 of the total 597 collected Phytophthora isolates showed that all but one isolate (identified as P. bisheria) were P. rubi. Results of the pathogenicity and fungicide resistance to mefenoxam comparing 14 total isolates from Washington, Oregon, and California showed that isolates were similarly virulent against red raspberry and the EC 50 frequency distributions showed no significant difference. These results, combined with amplified fragment length polymorphism results show that P. rubi isolates from Washington, Oregon, and California represent one large mixed population. This work provides novel insights into the isolation and biology of P. rubi in western U.S. raspberry production systems.

Background: On January 1, 2012, the Centers for Medicare & Medicaid Services converted Current Procedural Terminology (CPT) code 29826 (arthroscopic acromioplasty) from a stand-alone code to an add-on code and reduced the relative value units from 19.58 to 5.24. The goal of this study was to quantify the financial impact of this change on a large single-specialty orthopaedic surgery practice., Methods: Custom software was used to query the database that harbors billing data for a large single-specialty orthopaedic group. Data were independently generated based on patient identification data and insurance class, and compared between 2011 and 2012. Codes 29826, 29827 (shoulder arthroscopy, rotator cuff repair), 29822 (shoulder arthroscopy, debridement, limited), 29823 (shoulder arthroscopy, debridement, extensive), and 29824 (shoulder arthroscopy, distal claviculectomy) were all searched independently for each year and cross-referenced with each other and all other shoulder codes. Modifier codes for surgical assistants were analyzed separately and subsequently combined with primary surgeon data for financial analysis. This included assessment of surgeon reimbursement per occurrence of code 29826 and surgeon reimbursement by Medicare compared with non-Medicare payers., Results: Code 29826 was used 1536 times in 2011 and 1410 times in 2012 (-2.59% after correcting for all shoulder arthroscopy cases per year). Code 29822 was used significantly more in 2012 both alone (1.45%, p = 0.001) and in total (2.45%), but the use of 29823 did not change (p = 0.17). A combination of three of the five selected codes was used significantly less in 2012 (p < 0.001), while the use of any combination of four codes was used significantly more in 2012 (p < 0.001). Assistant use did not appreciably change between years. Average reimbursement for code 29826 by all payers in 2011 was $456.84 and $441.64 in 2012. Average payment by Medicare was $268.58 in 2011 and $171.02 in 2012 (-36.3%). Medicare paid 54.3% of other payers per case in 2011 and 33.1% of other payers in 2012., Conclusions: Reimbursement for code 29826 by non-Medicare payers did not decrease dramatically between 2011 and 2012. However, Medicare reimbursement fell substantially., (Copyright © 2014 by The Journal of Bone and Joint Surgery, Incorporated.)

Background: The glenoid vault can be perforated during pegged glenoid preparation in total shoulder arthroplasty. The clinical implications of glenoid vault perforation, however, are unknown. The purpose of this study was to determine the effects of perforation of the glenoid during total shoulder arthroplasty on clinical and radiographic outcomes., Materials and Methods: Eighteen patients with known intraoperative glenoid perforations were prospectively identified and compared with 34 patients matched by age, gender, diagnosis, and arm dominance during the same period. Patients were evaluated with multiple outcome scores. Radiographs were evaluated for glenoid lucency immediately postoperatively and at final follow-up., Results: Average follow-up was 28.1 months for the perforated group and 31.2 months for the matched controls. Both groups had significant improvements in outcome scores postoperatively. American Shoulder and Elbow Surgeons scores increased from 39.8 to 91.0 (P < .001) in the perforated group and from 36.9 to 82.6 (P < .001) in the control group. Constant scores increased from 24.4 to 77.4 (P < .001) in the perforated group and from 36.9 to 75.6 (P < .001) in the control group. Ninety-four percent of the perforated group and 80% of the matched controls were satisfied or very satisfied with their result (P = .896). The presence and number of perforations were not related to the American Shoulder and Elbow Surgeons score (P = .549), Constant score (P = .154), or radiographic lucency grade (P = .584)., Conclusions: Glenoid perforation during pegged glenoid preparation in total shoulder arthroplasty does not seem to have an adverse effect on clinical or radiographic outcomes at an average of 2 years of follow-up., (Copyright © 2014 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.)

When performing revision shoulder surgery, it is important that the surgeon understands why the index procedure failed and has a clear plan to address problems in the revision procedure. The most common cause of failure after anterior instability shoulder surgery is a failure to treat the underlying glenoid bone loss. For most defects, a Latarjet transfer can effectively restore anterior glenoid bone stock and restore shoulder stability. Persistent anterior shoulder pain after rotator cuff surgery may be the result of missed biceps pathology. This can be effectively treated via a biceps tenodesis. The most difficult failures to treat after acromioclavicular joint reconstruction surgery are those involving fractures of either the coracoid or the clavicle. Clavicle hook plates can be used as supplemental fixation during the treatment of these fractures to help offload the fracture site and allow healing while restoring stability to the acromioclavicular articulation. A failed hemiarthroplasty for a proximal humeral fracture frequently results when the tuberosities fail to heal correctly. This complication can be avoided by paying close attention to the implant position and the tuberosity fixation. If hemiarthroplasty is unsuccessful, the patient is best treated with conversion to a reverse shoulder arthroplasty.

Phytophthora plant pathogens contain many hundreds of effectors potentially involved in infection of host plants. Comparative genomic analyses have shown that these effectors evolve rapidly and have been subject to recent expansions. We examined the recent sequence evolution of RXLR-class effector gene families in the sudden oak death pathogen, P. ramorum. We found that P. ramorum RXLR effectors have taken multiple evolutionary paths, including loss or gain of repeated domains, recombination or gene conversion among paralogs, and selection on point mutations. Sequencing of homologs from two subfamilies in P. ramorum's closest known relatives revealed repeated gene duplication and divergence since speciation with P. lateralis. One family showed strong signatures of recombination while the other family has evolved primarily by point mutation. Comparison of a small number of the hundreds of RXLR-class effectors across three clonal lineages of P. ramorum shows striking divergence in alleles among lineages, suggesting the potential for functional differences between lineages. Our results suggest future avenues for examination of rapidly evolving effectors in P. ramorum, including investigation of the functional and coevolutionary significance of the patterns of sequence evolution that we observed.

Background: The purpose of this study was to compare the structural outcomes of a single-row rotator cuff repair and double-row suture bridge fixation after arthroscopic repair of a full-thickness supraspinatus rotator cuff tear., Material and Methods: We evaluated with diagnostic ultrasound a consecutive series of ninety shoulders in ninety patients with full-thickness supraspinatus tears at an average of 10 months (range, 6-12) after operation. A single surgeon at a single hospital performed the repairs. Inclusion criteria were full-thickness supraspinatus tears less than 25 mm in their anterior to posterior dimension. Exclusion criteria were prior operations on the shoulder, partial thickness tears, subscapularis tears, infraspinatus tears, combined supraspinatus and infraspinatus repairs and irreparable supraspinatus tears. Forty-three shoulders were repaired with single-row technique and 47 shoulders with double-row suture bridge technique. Postoperative rehabilitation was identical for both groups. Ultrasound criteria for healed repair included visualization of a tendon with normal thickness and length, and a negative compression test., Results: Eighty-three patients were available for ultrasound examination (40 single-row and 43 suture-bridge). Thirty of 40 patients (75%) with single-row repair demonstrated a healed rotator cuff repair compared to 40/43 (93%) patients with suture-bridge repair (P = .024)., Conclusion: Arthroscopic double-row suture bridge repair (transosseous equivalent) of an isolated supraspinatus rotator cuff tear resulted in a significantly higher tendon healing rate (as determined by ultrasound examination) when compared to arthroscopic single-row repair., (Copyright © 2013 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.)

In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.

When we observe object-directed actions such as grasping, we make predictive eye movements. However, eye movements are reactive when observing similar actions without objects. This reactivity may reflect a lack of attribution of intention to observed actors when they perform actions without 'goals'. Alternatively, it may simply signal that there is no cue present that has been predictive of the subsequent trajectory in the observer's experience. To test this hypothesis, the present study investigated how the time course of eye movements changes as a function of visual experience of predictable, but arbitrary, actions without objects. Participants observed a point-light display of a model performing sequential finger actions in a serial reaction time task. Eye movements became less reactive across blocks. In addition, participants who exhibited more predictive eye movements subsequently demonstrated greater learning when required either to execute, or to recognize, the sequence. No measures were influenced by whether participants had been instructed that the observed movements were human or lever generated. The present data indicate that eye movements when observing actions without objects reflect the extent to which the trajectory can be predicted through experience. The findings are discussed with reference to the implications for the mechanisms supporting perception of actions both with and without objects as well as those mediating inanimate object processing.

The oral route is considered to be the main entry site of several transmissible spongiform encephalopathies or prion diseases of animals and man. Following natural and experimental oral exposure to scrapie, sheep first accumulate disease associated prion protein (PrP (d) ) in Peyer's patch (PP) lymphoid follicles. In this study, recombinant ovine prion protein (rPrP) was inoculated into gut loops of young lambs and the transportation across the intestinal wall studied. In particular, the immunohistochemical phenotypes of cells bearing the inoculated prion protein were investigated. The rPrP was shown to be transported across the villi of the gut, into the lacteals and submucosal lymphatics, mimicking the transport route of PrP (d) from scrapie brain inoculum observed in a previous intestinal loop experiment. The cells bearing the inoculated rPrP were mainly mononuclear cells, and multicolor immunofluorescence procedures were used to show that the rPrP bearing cells were professional antigen presenting cells expressing Major histocompatibility complex II (MHCII). In addition, the rPrP bearing cells labeled with CD205, CD11b and the macrophage marker CD68, and not with the dendritic cell markers CD11c and CD209. Others have reported that cells expressing CD205 and CD11b in the absence of CD11c have been shown to induce T cell tolerance or regulatory T cells. Based on this association, it was speculated that the rPrP and by extension PrP (d) and scrapie infective material may exploit the physiological process of macromolecular uptake across the gut, and that this route of entry may have implications for immune surveillance.

The virulence of an infectious salmon anaemia virus (ISAV) isolate is influenced by the response of the host's immune system to virus infection. Here we report the fate of immune responsive cells in head kidney, spleen and gills of Atlantic salmon during infection with high and low virulent strains of ISAV. A comparison of real-time PCR detection of virus and immunohistochemical detection of immune responsive cells revealed that peak viral load was coincident with both an elevated presence of MHC class I cells and a marked depletion of CD8 alpha cells. There was a larger CD8 alpha population in tissues from salmon infected with the low virulent strain compared with tissues from salmon infected with the high virulent strain at early stages of infection. These findings suggest a protective role for the CD8 alpha cell population in immune defences against ISAV., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Transmissible Spongiform Encephalopathies (TSE) or prion diseases are a threat to food safety and to human and animal health. The molecular mechanisms responsible for prion diseases share similarities with a wider group of neurodegenerative disorders including Alzheimer disease and Parkinson disease and the central pathological event is a disturbance of protein folding of a normal cellular protein that is eventually accompanied by neuronal cell death and the death of the host. Prion protein (PrP) is a constituent of most normal mammalian cells and its presence is essential in the pathogenesis of TSE. However, the function of this normal cellular protein remains unclear. The prevention of PRNP gene expression in mammalian species has been undramatic, implying a functional redundancy. Yet PrP is conserved from mammals to fish. Recent studies of PrP in zebrafish have yielded novel findings showing that PrP has essential roles in early embryonic development. The amenability of zebrafish to global technologies has generated data indicating the existence of "anchorless" splice variants of PrP in the early embryo. This paper will discuss the possibility that the experimentalist's view of PrP functions might be clearer at a greater phylogenetic distance.

Contemporary species identification relies strongly on sequence-based identification, yet resources for identification of many fungal and oomycete pathogens are rare. We developed two web-based, searchable databases for rapid identification of Phytophthora spp. based on sequencing of the internal transcribed spacer (ITS) or the cytochrome oxidase (cox) 1 and 2 spacer region, followed by BLAST searching the databases. Both databases are highly selective. For ITS, only sequences associated with published Phytophthora spp. descriptions or classic Phytophthora phylogenetics references are included. For the cox spacer region, only data obtained by resequencing select isolates reported in published work were included. Novel taxa tentatively named are selectively included in the database and labeled as Phytophthora taxon "X"; as in, for example, P. taxon "asparagi". The database was validated with 700 Phytophthora isolates collected from nursery environments during 2006 to 2009. This resource, found at www.Phytophthora-ID.org , is a robust and validated tool for molecular identification of Phytophthora spp. and is regularly being updated.

In order to gain insight into possible cellular functions of the prion protein (PrP) during normal development, the expression of Prnp (encoding the PrP) and the distribution of the PrP were studied in murine tooth germs. Expression of Prnp in the mouse first molar tooth germ was highly dynamic, increasing several-fold during the secretory phase of odontogenesis, exhibiting a time-course of expression similar to that of genes coding for other extracellular proteins [e.g. enamel matrix proteins (Amelx, Ambn, Enam), Aplp1, Clstn1, and Clu]. Western blot analysis suggested that the amounts of PrP and amyloid beta (A4) precursor-like protein 1 (APLP1) in the tooth germ followed time-courses similar to those of the corresponding mRNAs. Immunohistochemical studies of the distribution of PrP in murine molar and incisor tooth germs at embryonic day (E)18.5 suggested that this protein was located in the cervical loop, outer enamel epithelium, pre-ameloblasts, and dental papilla. Different degrees of immunolabelling of pre-ameloblasts on the mesial and distal aspects of a lower molar cusp may be related to different enamel configurations on the two aspects. It is concluded that the dynamic patterns of expression of Prnp, and of distribution of PrP, suggest that PrP may have functions during secretory odontogenesis, perhaps in relation to amelogenesis., (© 2010 Eur J Oral Sci.)

It is assumed that the mobilisation of a strong cellular immune response is important for the survival of Atlantic salmon infected with infectious salmon anaemia virus (ISAV). In this study, the characterisation of immune cell populations in tissues of non-ISAV infected Atlantic salmon and during the early viraemia of ISAV was undertaken. Immunohistochemical investigations of spleen, head kidney and gills using monoclonal antibodies against recombinant proteins from MHC I, II and CD8 were performed on tissues from Atlantic salmon collected day 17 post-challenge in a cohabitant infection model. The localisations of MHC I and II in control salmon were consistent with previous reports but this study presents novel observations on the distribution of CD8 labelled cell populations in Atlantic salmon including the description of significant mucosal populations in the gills. The distribution of MHC I, MHC II and CD8 positive cell populations differed between control salmon and cohabitant salmon in the early stages of ISAV infection. The changes in MHC I labelled cells differed between organs in ISAV cohabitants but all investigated organs showed a decreased presence of MHC II labelled cells. Together with a clustering of CD8 labelled cells in the head kidney and a reduced presence of CD8 labelled cells in the gills, these observations support the early mobilisation of cellular immunity in the response of Atlantic salmon to ISAV infection. However, differences between the present study and the findings from studies investigating immune gene mRNA expression during ISAV infection suggest that viral strategies to interfere with protein expression and circumvent the host immune response could be operative in the early response to ISAV infection., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Background: In prion disease, the peripheral expression of PrP(C) is necessary for the transfer of infectivity to the central nervous system. The spleen is involved in neuroinvasion and neural dissemination in prion diseases but the nature of this involvement is not known. The present study undertook the investigation of the spatial relationship between sites of PrP(Sc) accumulation, localisation of nerve fibres and PrP(C) expression in the tissue compartments of the spleen of scrapie-inoculated and control sheep., Methodology/principal Findings: Laser microdissection and quantitative PCR were used to determine PrP mRNA levels and results were compared with immunohistochemical protocols to distinguish PrP(C) and PrP(Sc) in tissue compartments of the spleen. In sheep experimentally infected with scrapie, the major sites of accumulation of PrP(Sc) in the spleen, namely the lymphoid nodules and the marginal zone, expressed low levels of PrP mRNA. Double immunohistochemical labelling for PrP(Sc) and the pan-nerve fibre marker, PGP, was used to evaluate the density of innervation of splenic tissue compartments and the intimacy of association between PrP(Sc) and nerves. Some nerve fibres were observed to accompany blood vessels into the PrP(Sc)-laden germinal centres. However, the close association between nerves and PrP(Sc) was most apparent in the marginal zone. Other sites of close association were adjacent to the wall of the central artery of PALS and the outer rim of germinal centres., Conclusions/significance: The findings suggest that the degree of PrP(Sc) accumulation does not depend on the expression level of PrP(C). Though several splenic compartments may contribute to neuroinvasion, the marginal zone may play a central role in being the compartment with most apparent association between nerves and PrP(Sc).

Syndesmotic injuries or high ankle sprains in athletes can occur in elite and recreational athletes. They typically require a longer recovery and a significant delay in return to play compared with routine ankle sprains. Avoiding a misdiagnosis is important and is dependent on being aware of the mechanism of injury, a thorough physical examination, and careful interpretation of imaging studies. Management is guided by the severity of injury and the demands of the athlete. Many mild or stable injuries can be treated nonoperatively; however, acute surgical repair or stabilization in high-grade injuries can provide excellent results and an earlier return to play in selected cases.

Theory and research methodology of self psychology are integrated with the experiences of modern dance choreographers to investigate the importance of creativity, art making, and aesthetics in mental health and our everyday lives. Empathy, as aesthetically based, is explored to understand the capacity of the arts to unite us in our humanity. Connections between aesthetic development, creativity, and infant patterns of learning are drawn. The influence of sensual and exploration/assertion motivational systems upon the contemporary choreographer are highlighted, leading to an understanding of the selfobject function of sensation and movement for the dance artist. Through an examination of the moment to moment ritualized experiences of studio work, the creative process in making dances is discussed. Ultimately understanding creativity and aesthetically based empathy inform our delineation of mental health and the need for aesthetic experience in everyday life.

Unlabelled: Phytophthora ramorum is an oomycete plant pathogen classified in the kingdom Stramenopila. P. ramorum is the causal agent of sudden oak death on coast live oak and tanoak as well as ramorum blight on woody ornamental and forest understorey plants. It causes stem cankers on trees, and leaf blight or stem dieback on ornamentals and understorey forest species. This pathogen is managed in the USA and Europe by eradication where feasible, by containment elsewhere and by quarantine in many parts of the world. Genomic resources provide information on genes of interest to disease management and have improved tremendously since sequencing the genome in 2004. This review provides a current overview of the pathogenicity, population genetics, evolution and genomics of P. ramorum., Taxonomy: Phytophthora ramorum (Werres, De Cock & Man in't Veld): kingdom Stramenopila; phylum Oomycota; class Peronosporomycetidae; order Pythiales; family Pythiaceae; genus Phytophthora., Host Range: The host range is very large and the list of known hosts continues to expand at the time of writing. Coast live oak and tanoak are ecologically, economically and culturally important forest hosts in the USA. Rhododendron, Viburnum, Pieris, Syringa and Camellia are key ornamental hosts on which P. ramorum has been found repeatedly, some of which have been involved in moving the pathogen via nursery shipments. Disease symptoms: P. ramorum causes two different diseases with differing symptoms: sudden oak death (bleeding lesions, stem cankers) on oaks and ramorum blight (twig dieback and/or foliar lesions) on tree and woody ornamental hosts., Useful Websites: http://nature.berkeley.edu/comtf/, http://rapra.csl.gov.uk/, http://www.aphis.usda.gov/plant&#95;health/plant&#95;pest&#95;info/pram/index.shtml, http://genome.jgi-psf.org/Phyra1&#95;1/Phyra1&#95;1.home.html, http://pamgo.vbi.vt.edu/, http://pmgn.vbi.vt.edu/, http://vmd.vbi.vt.edu./, http://web.science.oregonstate.edu/bpp/labs/grunwald/resources.htm, http://www.defra.gov.uk/planth/pramorum.htm, http://www.invasive.org/browse/subject.cfm?sub=4603, http://www.forestry.gov.uk/forestry/WCAS-4Z5JLL.

Phytophthora ramorum S. Werres & A.W.A.M. de Cock is the causal agent of sudden oak death in California and Oregon forests and ramorum blight on a broad range of host species in wildlands and nurseries. It is thought to be an introduced pathogen and only three clonal lineages are known (3). The North American lineage (lineage NA1, mating type A2) is responsible for infections in California and Oregon forests. The European lineage (lineage EU1, predominantly A1) is responsible for infections in Europe, but has also been found in nurseries in Oregon and Washington. A third lineage (NA2) has only been isolated in a few instances from nurseries in Washington and California. In June 2006, P. ramorum was isolated from diseased Viburnum tinus, Osmanthus heterophyllus, and O. fragrans cultivars from a Humboldt County retail nursery in northern California. We genotyped isolates and placed them into clonal lineages using microsatellite markers developed for P. ramorum (3,4). Genomic DNA was extracted from mycelia with the FastDNA SPIN kit (Q-Biogene, Morgan, Irvine, CA). Primers used were PrMS6, Pr9C3, PrMS39, PrMS43a, PrMS43b, and PrMS45 (3) and 18, 64, and 82 (4). We sized fluorescently labeled amplicons using capillary electrophoresis (3100 Avant Genetic Analyzer, Applied Biosystems, Foster City, CA). Isolate genotypes were compared with control isolates of known clonal lineage, including BBA9/95 (EU1), Pr102 (NA1), and WSDA3765 (NA2). Three of four isolates belonged to genotype EU1. The fourth isolate, obtained from O. fragrans, belonged to genotype NA1. We repeated genotyping on independent genomic DNA extractions and obtained identical results. Two EU1 isolates and the single NA1 isolate were tested for mating type (1) and found to be of A1, A1, and A2 mating type, respectively. The coexistence of A1 and A2 mating types in the same retail nursery suggests the potential for sexual reproduction, as is the case in P. infestans where clonal and sexual populations exist (2), although to date, sexual reproduction in nature has not been documented in P. ramorum. The California retail nursery infestation highlights the risks associated with the unintentional transport of host nursery stock infested with P. ramorum. References: (1) C. M. Brasier and S. Kirk. Mycol. Res. 108:823, 2004. (2) N. J. Grünwald and W. G. Flier. Ann. Rev. Phytopathol. 43:171, 2005. (3) K. Ivors et al. Mol. Ecol. 15:1493, 2006. (4) S. Prospero et al. Mol. Ecol. 16:2958, 2007.