DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA.

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation.

Detection of genomic signatures of recent selection in commercial broiler chickens.

BACKGROUND: Identification of the genomic signatures of recent selection may help uncover causal polymorphisms controlling traits relevant to recent decades of selective breeding in livestock. In this study, we aimed at detecting signatures of recent selection in commercial broiler chickens using genotype information from single nucleotide polymorphisms (SNPs). A total of 565 chickens from five commercial purebred lines, including three broiler sire (male) lines and two broiler dam (female) lines, were genotyped using the 60K SNP Illumina iSelect chicken array. To detect genomic signatures of recent selection, we applied two methods based on population comparison, cross-population extended haplotype homozygosity (XP-EHH) and cross-population composite likelihood ratio (XP-CLR), and further analyzed the results to find genomic regions under recent selection in multiple purebred lines. RESULTS: A total of 321 candidate selection regions spanning approximately 1.45 % of the chicken genome in each line were detected by consensus of results of both XP-EHH and XP-CLR methods. To minimize false discovery due to genetic drift, only 42 of the candidate selection regions that were shared by 2 or more purebred lines were considered as high-confidence selection regions in the study. Of these 42 regions, 20 were 50 kb or less while 4 regions were larger than 0.5 Mb. In total, 91 genes could be found in the 42 regions, among which 19 regions contained only 1 or 2 genes, and 9 regions were located at gene deserts. CONCLUSIONS: Our results provide a genome-wide scan of recent selection signatures in five purebred lines of commercial broiler chickens. We found several candidate genes for recent selection in multiple lines, such as SOX6 (Sex Determining Region Y-Box 6) and cTR (Thyroid hormone receptor beta). These genes may have been under recent selection due to their essential roles in growth, development and reproduction in chickens. Furthermore, our results suggest that in some candidate regions, the same or opposite alleles have been under recent selection in multiple lines. Most of the candidate genes in the selection regions are novel, and as such they should be of great interest for future research into the genetic architecture of traits relevant to modern broiler breeding.

Messenger RNA sequencing and pathway analysis provide novel insights into the biological basis of chickens' feed efficiency.

BACKGROUND: Advanced selection technologies have been developed and continually optimized to improve traits of agricultural importance; however, these methods have been primarily applied without knowledge of underlying biological changes that may be induced by selection. This study aims to characterize the biological basis of differences between chickens with low and high feed efficiency (FE) with a long-term goal of improving the ability to select for FE. RESULTS: High-throughput RNA sequencing was performed on 23 breast muscle samples from commercial broiler chickens with extremely high (n = 10) and low (n = 13) FE. An average of 34 million paired-end reads (75 bp) were produced for each sample, 80% of which were properly mapped to the chicken reference genome (Ensembl Galgal4). Differential expression analysis identified 1,059 genes (FDR < 0.05) that significantly divergently expressed in breast muscle between the high- and low-FE chickens. Gene function analysis revealed that genes involved in muscle remodeling, inflammatory response and free radical scavenging were mostly up-regulated in the high-FE birds. Additionally, growth hormone and IGFs/PI3K/Akt signaling pathways were enriched in differentially expressed genes, which might contribute to the high breast muscle yield in high-FE birds and partly explain the FE advantage of high-FE chickens. CONCLUSIONS: This study provides novel insights into transcriptional differences in breast muscle between high- and low-FE broiler chickens. Our results show that feed efficiency is associated with breast muscle growth in these birds; furthermore, some physiological changes, e.g., inflammatory response and oxidative stress, may occur in the breast muscle of the high-FE chickens, which may be of concern for continued selection for both of these traits together in modern broiler chickens.

Characterization of a novel chicken muscle disorder through differential gene expression and pathway analysis using RNA-sequencing.

BACKGROUND: Improvements in poultry production within the past 50 years have led to increased muscle yield and growth rate, which may be contributing to an increased rate and development of new muscle disorders in chickens. Previously reported muscle disorders and conditions are generally associated with poor meat quality traits and have a significant negative economic impact on the poultry industry. Recently, a novel myopathy phenotype has emerged which is characterized by palpably "hard" or tough breast muscle. The objective of this study is to identify the underlying biological mechanisms that contribute to this emerging muscle disorder colloquially referred to as "Wooden Breast", through the use of RNA-sequencing technology. METHODS: We constructed cDNA libraries from five affected and six unaffected breast muscle samples from a line of commercial broiler chickens. After paired-end sequencing of samples using the Illumina Hiseq platform, we used Tophat to align the resulting sequence reads to the chicken reference genome and then used Cufflinks to find significant changes in gene transcript expression between each group. By comparing our gene list to previously published histology findings on this disorder and using Ingenuity Pathways Analysis (IPA®), we aim to develop a characteristic gene expression profile for this novel disorder through analyzing genes, gene families, and predicted biological pathways. RESULTS: Over 1500 genes were differentially expressed between affected and unaffected birds. There was an average of approximately 98 million reads per sample, across all samples. Results from the IPA analysis suggested "Diseases and Disorders" such as connective tissue disorders, "Molecular and Cellular Functions" such as cellular assembly and organization, cellular function and maintenance, and cellular movement, "Physiological System Development and Function" such as tissue development, and embryonic development, and "Top Canonical Pathways" such as, coagulation system, axonal guidance signaling, and acute phase response signaling, are associated with the Wooden Breast disease. CONCLUSIONS: There is convincing evidence by RNA-seq analysis to support localized hypoxia, oxidative stress, increased intracellular calcium, as well as the possible presence of muscle fiber-type switching, as key features of Wooden Breast Disease, which are supported by reported microscopic lesions of the disease.

Linkage disequilibrium in crossbred and pure line chickens.

BACKGROUND: Both genome-wide association (GWA) studies and genomic selection depend on the level of non-random association of alleles at different loci, i.e. linkage disequilibrium (LD), across the genome. Therefore, characterizing LD is of fundamental importance to implement both approaches. In this study, using a 60K single nucleotide polymorphism (SNP) panel, we estimated LD and haplotype structure in crossbred broiler chickens and their component pure lines (one male and two female lines) and calculated the consistency of LD between these populations. RESULTS: The average level of LD (measured by r(2)) between adjacent SNPs across the chicken autosomes studied here ranged from 0.34 to 0.40 in the pure lines but was only 0.24 in the crossbred populations, with 28.4% of adjacent SNP pairs having an r(2) higher than 0.3. Compared with the pure lines, the crossbred populations consistently showed a lower level of LD, smaller haploblock sizes and lower haplotype homozygosity on macro-, intermediate and micro-chromosomes. Furthermore, correlations of LD between markers at short distances (0 to 10 kb) were high between crossbred and pure lines (0.83 to 0.94). CONCLUSIONS: Our results suggest that using crossbred populations instead of pure lines can be advantageous for high-resolution QTL (quantitative trait loci) mapping in GWA studies and to achieve good persistence of accuracy of genomic breeding values over generations in genomic selection. These results also provide useful information for the design and implementation of GWA studies and genomic selection using crossbred populations.

Knowledge-based IMRT treatment planning for prostate cancer.

PURPOSE: To demonstrate the feasibility of using a knowledge base of prior treatment plans to generate new prostate intensity modulated radiation therapy (IMRT) plans. Each new case would be matched against others in the knowledge base. Once the best match is identified, that clinically approved plan is used to generate the new plan. METHODS: A database of 100 prostate IMRT treatment plans was assembled into an information-theoretic system. An algorithm based on mutual information was implemented to identify similar patient cases by matching 2D beam's eye view projections of contours. Ten randomly selected query cases were each matched with the most similar case from the database of prior clinically approved plans. Treatment parameters from the matched case were used to develop new treatment plans. A comparison of the differences in the dose-volume histograms between the new and the original treatment plans were analyzed. RESULTS: On average, the new knowledge-based plan is capable of achieving very comparable planning target volume coverage as the original plan, to within 2% as evaluated for D98, D95, and D1. Similarly, the dose to the rectum and dose to the bladder are also comparable to the original plan. For the rectum, the mean and standard deviation of the dose percentage differences for D20, D30, and D50 are 1.8% +/- 8.5%, -2.5% +/- 13.9%, and -13.9% +/- 23.6%, respectively. For the bladder, the mean and standard deviation of the dose percentage differences for D20, D30, and D50 are -5.9% +/- 10.8%, -12.2% +/- 14.6%, and -24.9% +/- 21.2%, respectively. A negative percentage difference indicates that the new plan has greater dose sparing as compared to the original plan. CONCLUSIONS: The authors demonstrate a knowledge-based approach of using prior clinically approved treatment plans to generate clinically acceptable treatment plans of high quality. This semiautomated approach has the potential to improve the efficiency of the treatment planning process while ensuring that high quality plans are developed.

Phase II Trial of Enzalutamide and Androgen Deprivation Therapy with Salvage Radiation in Men with High-risk Prostate-specific Antigen Recurrent Prostate Cancer: The STREAM Trial.

BACKGROUND: Salvage external beam radiotherapy (RT) with androgen deprivation therapy (ADT) improves survival over RT in men with prostate cancer (PC) and rising prostate-specific antigen (PSA) levels after radical prostatectomy (RP). OBJECTIVE: To investigate the safety and efficacy of enzalutamide concurrent with salvage RT and ADT. DESIGN, SETTING, AND PARTICIPANTS: This was a three-center prospective phase 2 single-arm trial (NCT02057939) of men with Gleason 7-10 PC and PSA recurrence within 4 yr of RP ranging from 0.2 to 4.0 ng/dl, no prior hormonal therapy, and no radiographic evidence of metastases. We enrolled 38 men; 37 completed therapy and were evaluable with testosterone recovery at 2 yr. INTERVENTION: Six months of ADT with 160 mg/d enzalutamide and 66 Gy RT to the prostate bed. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was improved 2-yr progression-free survival (PFS) over historical controls. Secondary objectives included 3-yr PFS, safety, and patient-reported quality of life (QOL). RESULTS AND LIMITATIONS: The primary endpoint of 2-yr PFS was 65% (95% confidence interval [CI]: 47, 78) versus 51% (95% CI: 33, 67) in a trial of men with similar eligibility treated with salvage RT and adjuvant docetaxel. The 3-yr PFS was 53%. Eleven (29%) men experienced G3 toxicities, and there were no G4-5 or unexpected toxicities. QOL data suggest modest worsening of bowel, bladder, and hormonal symptoms at 3 mo, with recovery by 24 mo in most men. CONCLUSIONS: Salvage RT with enzalutamide and ADT following RP for men with PSA recurrent high-risk PC is safe and demonstrates encouraging efficacy, warranting prospective controlled phase 3 trials of ADT with or without potent androgen receptor inhibition in this curative-intent setting. PATIENT SUMMARY: Addition of 6 mo of oral daily enzalutamide to standard salvage radiation and hormone therapy is safe and may improve prostate cancer remission rates at 2 and 3 yr.

The metabolic characteristics of susceptibility to wooden breast disease in chickens with high feed efficiency.

This study was conducted to characterize metabolic differences between high feed efficiency (HFE) and low feed efficiency (LFE) chickens to investigate why feed efficient chickens are more susceptible to muscle abnormalities such as wooden breast disease. Gene expression profiles were generated by RNA sequencing of pectoralis major muscle samples from 10 HFE and 13 LFE broiler chickens selected from a modern broiler population. Metabolism-associated differentially expressed genes were identified and interpreted by Ingenuity Pathway Analysis and literature mining. Our RNA-seq data indicate decreased glycolytic capacity, increased fatty acid uptake, mitochondrial oxidation of fatty acids, and several other metabolic alterations in the pectoralis major muscle of HFE chickens. We also quantified glycogen content of the pectoralis major muscle and found that the HFE chickens had a significantly (P ≤ 0.05) lower glycogen content. Collectively, this study indicates extensive metabolic differences in the pectoralis major muscle between HFE and LFE chickens and helps identify metabolic features of susceptibility to muscle disorders in modern broiler chickens.

Oxidative Stress and Metabolic Perturbations in Wooden Breast Disorder in Chickens.

This study was conducted to characterize metabolic features of the breast muscle (pectoralis major) in chickens affected with the Wooden Breast myopathy. Live birds from two purebred chicken lines and one crossbred commercial broiler population were clinically examined by manual palpation of the breast muscle (pectoralis major) at 47-48 days of age. Metabolite abundance was determined by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using breast muscle tissue samples from 16 affected and 16 unaffected chickens. Muscle glycogen content was also quantified in breast muscle tissue samples from affected and unaffected chickens. In total, levels of 140 biochemicals were significantly different (FDR<0.1 and fold-change A/U>1.3 or <0.77) between affected and unaffected chickens. Glycogen content measurements were considerably lower (1.7-fold) in samples taken from Wooden Breast affected birds when compared with samples from unaffected birds. Affected tissues exhibited biomarkers related to increased oxidative stress, elevated protein levels, muscle degradation, and altered glucose utilization. Affected muscle also showed elevated levels of hypoxanthine, xanthine, and urate molecules, the generation of which can contribute to altered redox homeostasis. In conclusion, our findings show that Wooden Breast affected tissues possess a unique metabolic signature. This unique profile may identify candidate biomarkers for diagnostic utilization and provide mechanistic insight into altered biochemical processes contributing to tissue hardening associated with the Wooden Breast myopathy in commercial chickens.

RNA-Seq Analysis of Abdominal Fat Reveals Differences between Modern Commercial Broiler Chickens with High and Low Feed Efficiencies.

For economic and environmental reasons, chickens with superior feed efficiency (FE) are preferred in the broiler chicken industry. High FE (HFE) chickens typically have reduced abdominal fat, the major adipose tissue in chickens. In addition to its function of energy storage, adipose tissue is a metabolically active organ that also possesses endocrine and immune regulatory functions. It plays a central role in maintaining energy homeostasis. Comprehensive understanding of the gene expression in the adipose tissue and the biological basis of FE are of significance to optimize selection and breeding strategies. Through gene expression profiling of abdominal fat from high and low FE (LFE) commercial broiler chickens, the present study aimed to characterize the differences of gene expression between HFE and LFE chickens. mRNA-seq analysis was carried out on the total RNA of abdominal fat from 10 HFE and 12 LFE commercial broiler chickens, and 1.48 billion of 75-base sequence reads were generated in total. On average, 11,565 genes were expressed (>5 reads/gene/sample) in the abdominal fat tissue, of which 286 genes were differentially expressed (DE) at q (False Discover Rate) < 0.05 and fold change > 1.3 between HFE and LFE chickens. Expression levels from RNA-seq were confirmed with the NanoString nCounter analysis system. Functional analysis showed that the DE genes were significantly (p < 0.01) enriched in lipid metabolism, coagulation, and immune regulation pathways. Specifically, the LFE chickens had higher expression of lipid synthesis genes and lower expression of triglyceride hydrolysis and cholesterol transport genes. In conclusion, our study reveals the overall differences of gene expression in the abdominal fat from HFE and LFE chickens, and the results suggest that the divergent expression of lipid metabolism genes represents the major differences.

The effects of iohexol administration on technetium thyroid scintigraphy in normal cats.

Administration of iodinated contrast medium interferes with iodide uptake in the human thyroid gland and compromises diagnostic thyroid scintigraphy and radioiodine treatment for 4-6 weeks. However, the degree and duration of inhibition of thyroid uptake of pertechnetate (99mTcO4-) by iodinated contrast medium has not been established in any species. The main objective of this study was to better understand the temporal characteristics and magnitude of inhibition of feline thyroid uptake of 99mTcO4- due to iohexol administration. Routine thyroid scintigraphy was performed in eight cats by intravenous (IV) injection of 185 MBq (5 mCi) of 99mTcO4- both 4 days before and 0, 1, 3, 7, 14, and 28 days after IV administration of 880 mg I/kg iohexol (240 mg I/ml). Thyroid scintigraphy data were used to calculate thyroid:salivary gland ratios (T:S) and the percentage of total injected 99mTcO4- dose uptake within the thyroid (%TU) at 20 min postinjection. After iohexol administration, mean T:S was significantly decreased below baseline only on day 1. At no point during the study did any cat have a T:S that fell below the published normal reference range of 0.71 +/- 0.14. There was a significant decrease in %TU on day 1, 3, and 14; however, at no point during the study, did any cat have a %TU that fell below the published normal reference ranges of 0.64 +/- 0.57, 0.68 +/- 0.9, or 0.75 +/- 1.38.

Clinical evaluation of positioning verification using digital tomosynthesis and bony anatomy and soft tissues for prostate image-guided radiotherapy.

PURPOSE: To evaluate on-board digital tomosynthesis (DTS) for patient positioning vs. two-dimensional (2D) radiography and three-dimensional cone beam (CBCT). METHODS AND MATERIALS: A total of 92 image sessions from 9 prostate cancer patients were analyzed. An on-board image set was registered to a corresponding reference image set. Four pairs of image sets were used: digitally reconstructed radiographs vs. on-board orthogonal paired radiographs for the 2D method, coronal-reference DTS vs. on-board coronal DTS for the coronal-DTS method, sagittal-reference DTS vs. on-board sagittal DTS for the sagittal-DTS method, and planning CT vs. CBCT for the CBCT method. The registration results were compared. RESULTS: The systematic errors in all methods were <1 mm/1 degrees . When registering the bony anatomy, the mean vector difference was 0.21 +/- 0.11 cm between 2D and CBCT, 0.11 +/- 0.08 cm between CBCT and coronal DTS, and 0.14 +/- 0.07 cm between CBCT and sagittal DTS. The correlation between CBCT to DTS was stronger (coefficient = 0.92-0.95) than the correlation between 2D and CBCT or DTS (coefficient = 0.81-0.83). When registering the soft tissue, the mean vector difference was 0.18 +/- 0.11 cm between CBCT and coronal DTS and 0.29 +/- 0.17 cm between CBCT and sagittal DTS. The correlation coefficient of CBCT to sagittal DTS and to coronal DTS was 0.84 and 0.92, respectively. CONCLUSION: DTS could provide equivalent results to CBCT when the bony anatomy is used as landmarks for prostate image-guided radiotherapy. For soft tissue-based positioning verification, coronal DTS produced equivalent results to CBCT, but sagittal DTS alone was insufficient. DTS could allow for comparable soft tissue-based target localization with faster scanning time and a lower imaging dose compared with CBCT.

Prognostic value of cell-cycle markers in ocular adnexal lymphoma: an assessment of 230 cases.

BACKGROUND: To determine the prognostic value of cell-cycle associated markers in ocular adnexal lymphoma (OAL). METHODS: Two hundred sixty-one consecutive cases of ocular adnexal lymphoproliferative lesions were subdivided into reactive lymphoid hyperplasia (RLH), atypical lymphoid hyperplasia (ALH) and OAL. The latter were sub-typed according to the new WHO Lymphoma Classification. All lesions were investigated applying standard immunohistochemical methods with antibodies specific for pRB, p53, p16, p21, BCL-6 and for multiple myeloma oncogene-1-protein (MUM1, also known as IRF4). The main endpoints included the development of a local recurrence, of systemic disease and of lymphoma-related death. The association of prognostic variables with endpoints was assessed by multiple logistic and Cox regression models, respectively. RESULTS: The ocular adnexal lymphoproliferative lesions were categorised as OAL ( n=230; 88%), RLH ( n=29; 11%), and ALH ( n=2; 1%). The major lymphoma subtypes included 136 extranodal marginal zone B-cell lymphoma (EMZL), 31 diffuse large cell B-cell lymphomas, 27 follicular lymphomas, 9 plasmacytomas, 9 lymphoplasmocytic lymphoma/immunocytomas and 8 mantle cell lymphomas. The median follow-up time was 44.5 months. Most OAL patients had Stage IE disease and were treated with radiotherapy. Thirty-seven (25%) Stage IE patients had tumour relapses: these were significantly associated with an increased BCL6 blast percentage. Sixty-two (42%) Stage IE patients developed systemic disease: they had "non-EMZL" with large growth fractions and increased blast percentages for BCL6. Fifty-seven (25%) OAL patients died because of their lymphoma; lymphoma-related death was significantly associated on multivariable analysis with advanced clinical stage, an age >60 years and large tumour growth fractions. CONCLUSION: Subtyping of OAL according to the new WHO Lymphoma Classification, the stage of disease and tumour cell growth fraction aided the prediction of (1) tumour relapse, (2) the development of systemic disease and (3) lymphoma-related death in OAL.

Myxoid leiomyoma of the iris in a dog.

A leiomyoma of the iris is described in an 11-year-old Yorkshire Terrier. This is a rare primary intraocular tumor in dogs and we describe the clinical presentation, gross findings and histopathologic characteristics of this tumor. The diagnosis was made on the basis of light microscopy and immunohistochemical staining using antidesmin antibodies, which is specific for myogenic tissues. An unusual feature of the tumor was the presence of myxoid change. To our knowledge myxoid change has not been previously described in a primary intraocular leiomyoma.

In vitro efficacy of a polyhexamethylene biguanide-impregnated gauze dressing against bacteria found in veterinary patients.

OBJECTIVE: To evaluate the in vitro efficacy of polyhexamethylene biguanide (PHMB)-impregnated gauze dressing in limiting the growth of bacteria both within and underneath the dressing. STUDY DESIGN: In vitro study. METHODS: Squares of PHMB-impregnated and control gauze were placed on agar plates inoculated with 1 of 11 bacterial species, including 8 multi-resistant organisms. Growth under the gauze was assessed qualitatively after 24-hour incubation. Repeated use of sponges was used to evaluate residual inhibitory activity against Micrococcus lutea and Staphylococcus schleiferi ss. schleiferi. In a second procedure, PHMB-impregnated and control gauze squares were placed in sterile plastic wells and inoculated with 1 of 5 bacterial species, including Pseudomonas spp. and Klebsiella spp. Inhibition of bacterial growth within and underneath the dressing after 24-hour incubation was evaluated by quantifying the numbers of bacteria on the well floor and within each square. RESULTS: PHMB-impregnated gauze provided greater inhibition of growth of 4/4 Gram-positive species and 2/6 Gram-negative species on inoculated plates compared with control gauze. Residual inhibitory activity of PHMB-impregnated gauze was significantly greater against M. lutea on all days and against S. schleiferi ss. schleiferi on days 1 and 4 compared with control. No bacteria were recovered from inoculated PHMB-impregnated gauze squares placed in sterile wells or from the well floor underneath. More than 9 x 10(5) colony-forming units (CFU) were recovered from inoculated control samples placed in sterile wells and more than 8.4 x 10(4) CFU were recovered from control well floors. CONCLUSION: PHMB-impregnated gauze dressing, when placed on inoculated agar plates, reduces growth of underlying bacteria, particularly Gram-positive species. Wet-inoculated PHMB-impregnated dressing prevents growth of Gram-positive and Gram-negative bacteria both within and underneath the dressing. CLINICAL RELEVANCE: PHMB-impregnated dressings may be useful for reducing contamination of underlying wounds by bacterial pathogens.