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Salman L. Butt Tonya L. Taylor Jeremy D. Volkening Kiril M. Dimitrov Dawn Williams-Coplin Kevin K. Lahmers Patti J. Miller Asif M. Rana David L. Suarez Claudio L. Afonso James B. Stanton 《Virology journal》2018,15(1):179
Background
Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs.Methods
An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow.Results
For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 101 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq.Conclusion
The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.13.
Background
Many high-throughput genomic experiments, such as Synthetic Genetic Array and yeast two-hybrid, use colony growth on solid media as a screen metric. These experiments routinely generate over 100,000 data points, making data analysis a time consuming and painstaking process. Here we describe ScreenMill, a new software suite that automates image analysis and simplifies data review and analysis for high-throughput biological experiments. 相似文献14.
Male stimuli are necessary for female sexual behavior and uterine growth in prairie voles (Microtus ochrogaster) 总被引:3,自引:0,他引:3
In reproductively naive female prairie voles (Microtus ochrogaster) direct contact with male urine or housing in a male-soiled cage, in the absence of physical contact, resulted in increased uterine weights, but did not reliably elicit behavioral estrus (defined by lordosis). Physical contact with an unfamiliar male, for 1 hr or more, followed by 30 or 48 hr of continuous access to a male-soiled cage, induced lordosis in approximately two-thirds of the females tested. When females were physically exposed to a male for 18 hr and tested 6 hr later, 70% showed lordosis. However, when females receiving either 1 or 18 hr of male contact were removed from the presence of the male and placed in a clean cage for 24 hr, only 29-37% of the females subsequently showed lordosis. These results suggest that direct physical contact with the male or chemical stimuli from the male may be necessary to induce and maintain behavioral estrus in female prairie voles. 相似文献
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JB Parentes-Vieira PV Lopes-Costa CG Pires AR dos Santos JD Pereira-Filho BB da Silva 《International Seminars in Surgical Oncology : ISSO》2007,4(1):22
Background
The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.Methods
This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).Results
The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).Conclusion
ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.16.
Teng YK Verburg RJ Verpoort KN Diepenhorst GM Bajema IM van Tol MJ Jol-van der Zijde EC Toes RE Huizinga TW van Laar JM 《Arthritis research & therapy》2007,9(5):R106
In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated
protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy
(HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized
in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against
rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was
measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial
biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with
clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy
were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median
of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to
388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were
differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies
as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative
therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity. 相似文献
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High-throughput approaches are beginning to have an impact on many areas of yeast biology. Two recent studies, using different experimental platforms, provide insight into new pathways involved in the response of yeast to DNA damage. 相似文献
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The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the fungal avirulence gene product avr 9 总被引:12,自引:0,他引:12
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The Cf-9 gene encodes an extracytoplasmic leucine-rich repeat protein that confers resistance in tomato to races of the fungus Cladosporium fulvum that express the corresponding avirulence gene Avr 9. We investigated whether the genomic Cf-9 gene functions in potato and tobacco. Transgenic tobacco and potato plants carrying Cf-9 exhibit a rapid hypersensitive cell death response (HR) to Avr 9 peptide injection. Cf 9 tobacco plants were reciprocally crossed to Avr 9-producing tobacco. A developmentally regulated seedling lethal phenotype occurred in F1 progeny when Cf9 was used as the male parent and Avr 9 as the female parent. However, when Cf9 was inherited in the maternal tissue and a heterozygous Avr 9 plant was used as the pollen donor, a much earlier reaction was caused, leading to no germination of any F1 seed. Detailed analysis of the Avr 9-induced responses in Cf 9 tobacco leaves revealed that (1) most mesophyll cells died within 3 hr (compared with 12 to 16 hr in tomato); (2) the macroscopic HR was visible at an Avr 9 titer five times lower than that which caused visible symptoms in tomato; (3) the HR invariably extended into noninjected panels of the tobacco leaf; (4) no HR occurred in leaves of young tobacco plants; (5) in older plants, the HR was dramatically enhanced by sequential Avr 9 challenges; and (6) coexpression of a salicylate hydroxylase transgene (nahG) from Pseudomonas putida reduced the severity of the macroscopic leaf HR and also restored germination to Cf 9 x 35S:Avr 9 F1 seedlings. Simultaneous introduction of Cf-9 homologs (Hcr 9-9 genes A and B or D) along with the native Cf-9 gene did not alter the responses that were specifically induced by Avr 9. Various ways to use the Cf-9-Avr 9 gene combination to engineer broad-spectrum disease resistance in several solanaceous species are discussed. 相似文献