Investigation of laboratory methods for characterization of aquatic viruses in fish infected experimentally with infectious salmon anemia virus.

Rapid growth in aquaculture has resulted in high-density production systems in ecologically and geographically novel conditions in which the emergence of diseases is inevitable. Well-characterized methods for detection and surveillance of infectious diseases are vital for rapid identification, response, and recovery to protect economic and food security. We implemented a proof-of-concept approach for virus detection using a known high-consequence fish pathogen, infectious salmon anemia virus (ISAV), as the archetypal pathogen. In fish infected with ISAV, we integrated histopathology, virus isolation, whole-genome sequencing (WGS), electron microscopy (EM), in situ hybridization (ISH), and reverse transcription real-time PCR (RT-rtPCR). Fresh-frozen and formalin-fixed tissues were collected from virus-infected, control, and sham-infected Atlantic salmon (Salmo salar). Microscopic differences were not evident between uninfected and infected fish. Viral cytopathic effect was observed in cell cultures inoculated with fresh-frozen tissue homogenates from 3 of 3 ISAV-infected and 0 of 4 uninfected or sham-infected fish. The ISAV genome was detected by shotgun metagenomics in RNA extracted from the medium from 3 of 3 inoculated cell cultures, 3 of 3 infected fish, and 0 of 4 uninfected or sham-infected fish, yielding sufficient coverage for de novo assembly. An ISH probe against ISAV revealed ISAV genome in multiple organs, with abundance in renal hematopoietic tissue. Virus was detected by RT-rtPCR in gill, heart, kidney, liver, and spleen. EM and metagenomic WGS from tissues were challenging and unsuccessful. Our proof-of-concept methodology has promise for detection and characterization of unknown aquatic pathogens and also highlights some associated methodology challenges that require additional investigation.

Distinctive diets of eutherian predators in Australia.

Introduction of the domestic cat and red fox has devastated Australian native fauna. We synthesized Australian diet analyses to identify traits of prey species in cat, fox and dingo diets, which prey were more frequent or distinctive to the diet of each predator, and quantified dietary overlap. Nearly half (45%) of all Australian terrestrial mammal, bird and reptile species occurred in the diets of one or more predators. Cat and dingo diets overlapped least (0.64 ± 0.27, n = 24 location/time points) and cat diet changed little over 55 years of study. Cats were more likely to have eaten birds, reptiles and small mammals than foxes or dingoes. Dingo diet remained constant over 53 years and constituted the largest mammal, bird and reptile prey species, including more macropods/potoroids, wombats, monotremes and bandicoots/bilbies than cats or foxes. Fox diet had greater overlap with both cats (0.79 ± 0.20, n = 37) and dingoes (0.73 ± 0.21, n = 42), fewer distinctive items (plant material, possums/gliders) and significant spatial and temporal heterogeneity over 69 years, suggesting the opportunity for prey switching (especially of mammal prey) to mitigate competition. Our study reinforced concerns about mesopredator impacts upon scarce/threatened species and the need to control foxes and cats for fauna conservation. However, extensive dietary overlap and opportunism, as well as low incidence of mesopredators in dingo diets, precluded resolution of the debate about possible dingo suppression of foxes and cats.

Beyond atrial septal defect closure, it is time to start seriously considering closing ventricular septal defects with devices.

PURPOSE OF REVIEW: The purpose of this review is to discuss the history and current state of transcutaneous device closure of ventricular septal defects. RECENT FINDINGS: Newer devices and techniques have expanded the role of transcutaneous device closure of ventricular septal defects. Device closure of ventricular septal defects is a mainstay of therapy in many parts of the world. Device closure in smaller infants has relatively higher risks than larger infants and older children. The potential for the development of complete heart block remains a concern with this procedure. SUMMARY: Transcutaneous device closure of ventricular septal defects is a safe alternative to surgical repair in many patients.

Draft Genome Sequences of Three Flavobacterium psychrophilum Strains Isolated from Coldwater Disease Outbreaks at Three Production Hatcheries.

We report here the genome sequences of three Flavobacterium psychrophilum strains causing a bacterial coldwater disease (BCWD) outbreak, isolated from infected rainbow trout from hatcheries in Montana and South Dakota. The availability of these virulent outbreak-causing strain genome sequences will help further understand the pathogenesis of BCWD.

The Use of Brainlab Navigation in Le Fort III Osteotomy.

Le Fort III osteotomy is commonly used in the surgical correction of midface hypoplasia, specifically in patients with syndromic craniosynostosis. These osteotomies can be associated with significant complications, which are often the result of incomplete or inaccurate osteotomies. Brainlab, a technology first developed for neurosurgery, has been applied to numerous surgical subspecialties. The aim of this study was to report our initial experience using the Brainlab VectorVision2 and Brainlab Curve (Brainlab, Westchester, IL) as an intraoperative guidance system for osteotomy placement during Le Fort III advancement. Three pediatric patients with syndromic craniosynostosis and midface hypoplasia scheduled to undergo Le Fort III advancement were scanned preoperatively with 0.6-mm computed tomography cuts, which were then uploaded to the Brainlab system. All surgeries commenced with rigid fixation of the Brainlab registration device to the patient's skull. The navigation system was used intraoperatively to accurately determine osteotomy sites and trajectories. External distractors were placed without complication. Mean length of surgery was 331 minutes, and mean estimated blood loss was 500 mL. No transfusion was required with a mean postoperative hemoglobin of 8.3 g/dL. The application of Brainlab technology to Le Fort III advancement proved useful in establishing precise osteotomy lines and trajectories. Looking forward, this technology could be applied to a minimal dissection technique in order to avoid extensive blood loss. Further study would be needed to determine possible benefits such as reduced complications or operative time when using an intraoperative navigation system for image-guided osteotomy placement during Le Fort III advancement.

Health-related quality of life of Canadian Forces veterans after transition to civilian life.

OBJECTIVES: Describe health-related quality of life (HRQoL) of former Canadian Forces (CF) men and women in uniform (Veterans) after transition to civilian life, and compare to age- and sex-adjusted Canadian norms. METHODS: The 2010 Survey on Transition to Civilian Life was a national computer-assisted telephone survey of CF Regular Force personnel who released during 1998-2007. HRQoL was assessed using the SF-12 Physical (PCS) and Mental (MCS) Component Summary scores. Descriptive analysis of HRQoL was conducted for socio-demographic, health, disability and determinants of health characteristics. RESULTS: Mean age was 46 years (range 20-67). Compared to age- and sex-adjusted Canadian averages, PCS (47.3) was low and MCS was similar (52.0). PCS and MCS were variably below average for middle age groups and lowest for non-commissioned ranks, widowed/divorced/separated, 10-19 years of service, physical and mental health conditions, disability, dissatisfaction with finances, seeking work/not working, low social support and difficulty adjusting to civilian life. Among Veterans Affairs Canada clients, 83% had below-average physical PCS, 49% had below-average MCS, and mean PCS (38.2) was significantly lower than mean MCS (48.3). CONCLUSIONS: HRQoL varied across a range of biopsychosocial factors, suggesting possible protective factors and vulnerable subgroups that may benefit from targeted interventions. These findings will be of interest to agencies supporting Veterans in transition to civilian life and to researchers developing hypotheses to better understand well-being in Canadian Veterans.

Work reintegration for veterans with mental disorders: a systematic literature review to inform research.

BACKGROUND: Some veterans, and especially those with mental disorders, have difficulty reintegrating into the civilian workforce. PURPOSE: The objectives of this study were to describe the scope of the existing literature on mental disorders and unemployment and to identify factors potentially associated with reintegration of workers with mental disorders into the workforce. DATA SOURCES: The following databases were searched from their respective inception dates: MEDLINE, EMBASE, Cumulative Index Nursing Allied Health (CINAHL), and PsycINFO. STUDY SELECTION: In-scope studies had quantitative measures of employment and study populations with well-described mental disorders (eg, anxiety, depression, posttraumatic stress disorder, substance-use disorders). DATA EXTRACTION: A systematic and comprehensive search of the relevant published literature up to July 2009 was conducted that identified a total of 5,195 articles. From that list, 81 in-scope studies were identified. An update to July 2012 identified 1,267 new articles, resulting in an additional 16 in-scope articles. DATA SYNTHESIS: Three major categories emerged from the in-scope articles: return to work, supported employment, and reintegration. The literature on return to work and supported employment is well summarized by existing reviews. The reintegration literature included 32 in-scope articles; only 10 of these were conducted in populations of veterans. LIMITATIONS: Studies of reintegration to work were not similar enough to synthesize, and it was inappropriate to pool results for this category of literature. CONCLUSIONS: Comprehensive literature review found limited knowledge about how to integrate people with mental disorders into a new workplace after a prolonged absence (>1 year). Even more limited knowledge was found for veterans. The results informed the next steps for our research team to enhance successful reintegration of veterans with mental disorders into the civilian workplace.

Trainable immunohistochemical HER2/neu image analysis: a multisite performance study using 260 breast tissue specimens.

CONTEXT: Aperio Technologies, Inc (Vista, California) provides a new immunohistochemistry (IHC) HER2 Image Analysis (IA) system that allows tuning of the intensity thresholds of the HER2/ neu scoring scheme to adapt to the staining characteristics of different reagents. OBJECTIVE: To compare the trainable IHC HER2 IA system for different reagents to conventional manual microscopy (MM) in a multisite study. DESIGN: Two hundred sixty formalin-fixed, paraffin-embedded breast cancer specimens from 3 clinical sites were assayed: 180 specimens stained with Dako's HercepTest (Carpinteria, California), and 80 specimens stained with Ventana's PATHWAY HER-2/neu (Tucson, California). At each site, 3 pathologists performed a blinded reading of the glass slides with the use of a light microscope. The glass slides were then scanned and after a wash-out period and randomization, the same pathologists outlined a representative set of tumor regions to be analyzed by IHC HER2 IA. Each of the methods, MM and IA, was evaluated separately and comparatively by using κ statistics of negative HER2/neu scores (0, 1+) versus equivocal HER2/neu scores (2+) versus positive HER2/neu scores (3+) among the different pathologists. RESULTS: κ Values for IA and MM were obtained across all sites. MM: 0.565-0.864; IA: 0.895-0.947; MM versus IA: 0.683-0.892 for site 1; MM: 0.771-0.837; IA: 0.726-0.917; MM versus IA: 0.687-0.877 for site 2; MM: 0.463-0.674; IA: 0.864-0.918; MM versus IA: 0.497-0.626 for site 3. CONCLUSION: Aperio's trainable IHC HER2 IA system shows substantial equivalence to MM for Dako's HercepTest and Ventana's PATHWAY HER-2/neu at 3 clinical sites. Image analysis improved interpathologist agreement in the different clinical sites.

A multisite performance study comparing the reading of immunohistochemical slides on a computer monitor with conventional manual microscopy for estrogen and progesterone receptor analysis.

A multisite study was conducted to assess the performance of the Aperio digital pathology system (Aperio Technologies, Vista, CA) for reading estrogen receptor (ER) and progesterone receptor (PR) slides on a computer monitor. A total of 520 formalin-fixed breast tissue specimens were assayed at 3 clinical sites for ER and PR (260 each). Percentage and average staining intensity of positive nuclei were assessed. At each site, 3 pathologists performed a blinded reading of the glass slides using their microscopes initially and later using digital images on a computer monitor. Comparable percentages of agreements were obtained for manual microscopy (MM) and manual digital slide reading (MDR) (ER, percentage of positive nuclei with cutoffs: MM, 91.3%-99.0%/MDR, 91.3%-100.0%; PR, percentage of positive nuclei with cutoffs: MM, 83.8%-99.0%/MDR, 76.3%-100.0%). Reading ER and PR slides on a computer monitor using the Aperio digital pathology system is equivalent to reading the slides with a conventional light microscope.

The effects of Myxobolus cerebralis on Apache and Gila trout in laboratory exposures.

Whirling disease has been implicated in salmonid population declines in several western states. To determine the risk of a species or strain of salmonid to whirling disease it is critical to establish its relative susceptibility to Myxobolus cerebralis infection. Gila trout Oncorhynchus gilae and Apache trout Oncorhynchus gilae apache were exposed to various doses of M. cerebralis triactinomyxons (TAMs) in laboratory experiments. In trials conducted in consecutive years, fish were exposed to TAMs in doses ranging from 25 to 2,000/fish at ages ranging from 66 to 201 d posthatch (dph). All fish were held for 900 degree-days, and then the infection intensity of each fish was determined by histological examination. In 2002, 98% of the Gila trout died during exposure or within 48 h postexposure. Seventy-four percent of the Apache trout died before the end of the 90-d study period. Those that survived the entire study period had an average histological score of more than 4.0. In subsequent trials, the TAM dosage was decreased to 25-1,000/fish. Also, the age of the fish was increased from 66-72 dph to 89-201 dph. The survival rate increased from 16.0% to 49.1%. Average histological grades ranged from 1.6 to 4.8. Based on this data, it can be concluded that both Apache and Gila trout are highly susceptible to M. cerebralis in laboratory trials.

The development of a novel high-dose pressurized aerosol dry-powder device (PADD) for the delivery of pumactant for inhalation therapy.

The performance of a novel dry powder inhaler designed to deliver exceptionally high doses was investigated using pumactant as a model powder. Pumactant (a synthetic lung surfactant consisting of a phospholipid mixture), with a 90th percentile particle size of 2.92 microm is highly cohesive, has a high moisture affinity (6.2% w/w at 45% RH), and is predominantly amorphous. The device (pressurized aerosol dry-powder delivery [PADD]) utilizes pressurized gas to aerosolize a powder bed from a reservoir and delivers it through a conventional mouthpiece. The influence of loaded dose on dry powder delivery and can pressure on aerosolization efficiency was investigated. Analysis of the delivered dose studies suggested a linear relationship between loaded dose and delivered dose (R(2) = 0.96, for loaded doses of 0-250 mg), with a delivery efficiency of 70%. Analysis of the aerosolization efficiency using a Marple Miller type impactor suggested fine particle fractions (particles with an aerodynamic diameter of <5 microm) of approximately 30% using canister pressures of 8-14 bars. These results indicate that the PADD device may be a useful tool in delivering high-dose medicaments, as a carrier-free formulation, to the deep lung.

Differential expression of thymidylate synthase in colorectal tumors and matched lymph nodes: impact on adjuvant treatment.

Although the expression of thymidylate synthase (TS) in metastatic colorectal cancer (CRC) may be a better predictor of response to 5-fluorouracil chemotherapy than TS expression in primary CRC, this enzyme has not been well studied in tumor-draining regional lymph nodes. We retrospectively examined TS expression in 12 primary CRC lesions (pT3) and matched sentinel lymph nodes. Of the 8 primary tumors that were TS-positive, 50 per cent (4/8) had tumor-positive lymph nodes and 50 per cent (4/8) had tumor-negative nodes. Of the 4 primary tumors that were TS-negative, 75 per cent (3/4) had tumor-positive nodes and 25 per cent (1/4) had tumor-negative nodes [kappa = -0.1386, 95 per cent confidence interval: (-0.4820, 0.2048), P = 0.4284]. Of the 8 TS-positive primaries, 25 per cent (2/8) had TS-positive nodes and 75 per cent (6/8) had TS-negative nodes. Of the 4 TS-negative primaries, 50 per cent (2/4) had TS-positive nodes and 50 per cent (2/4) had TS-negative nodes [kappa = -0.0131, 95 per cent confidence interval: (-0.2958, 0.2696), P = 0.9274]. Two of the three TS-negative primaries that had metastasized to regional lymph nodes were associated with TS-positive lymph nodes. Our findings indicate that expression of TS by a primary CRC does not correlate with nodal metastases or nodal TS expression. Nodal expression of TS may be important in predicting response to 5-fluorouracil when a primary CRC is TS-negative.

Effects of retinoid ligands on RIP140: molecular interaction with retinoid receptors and biological activity.

Receptor interacting protein 140 (RIP140) interacts with retinoic acid receptor (RAR) and retinoid X receptor (RXR) constitutively, but hormone binding enhances this interaction. The ligand-independent interaction is mediated by the amino and central regions of RIP140 which contain a total of nine copies of the LXXLL motif, whereas the agonist-induced interaction is mediated by its carboxyl terminus which contains a novel motif (1063-1076, LTKTNPILYYMLQK). The ligand-independent interaction could be enhanced slightly by agonists, whereas the ligand-dependent interaction was strictly agonist dependent for both RAR and RXR. In the context of heterodimers, ligand occupancy of RXR played a more dominant role for both molecular interaction and biological activity of RIP140. Competition and mutation studies demonstrated an essential role for (1067)Asn and (1073)Met for a ligand-dependent interaction. A model was proposed to address the constitutive and agonist-dependent interaction of RIP140 with RAR/RXR.

Vitrification of human blastocysts using the cryoloop method: successful clinical application and birth of offspring.

PURPOSE: The purpose was to investigate vitrification of human blastocysts using the cryoloop. METHODS: Thirteen couples elected to have blastocysts cryopreserved via vitrification (N = 54 blastocysts). Embryos were exposed to two vitrification solutions, pipetted onto the cryoloop then plunged into liquid nitrogen. On the day of embryo replacement, blastocysts were warmed by passage through three dilution media, and rinsed into culture medium. RESULTS: Four couples returned for cryopreserved embryo replacement. Fifteen blastocysts were warmed; all 15 (15/15; 100%) were recovered. Fifteen (15/15; 100%) blastocysts were deemed viable, and 13 were replaced. Two replacements did not result in pregnancy; one resulted in clinical pregnancy with a blighted ovum; one resulted in clinical pregnancy with the live, term birth of a healthy male infant. Two of 13 embryos implanted yielding an implantation rate of 15.4%. CONCLUSIONS: These preliminary data suggest that blastocysts can be successfully vitrified using the cryoloop method.