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Human samples from patients with dengue, HCV, HIV and Zika were obtained commercially from Boca Biolistics under their ethical approvals. Influenza samples were obtained from the Centers for Disease Control and Prevention under their ethical approvals. All protocols subsequently performed by the researchers were approved as a Not Human Subjects Research determination no. NHSR-4318 issued by the Broad Institute of MIT and Harvard. General procedures Synthetic targets Conybeare in n. 4, pp. 132-60, ” Homo interior : the inner self”, esp. pp. 147-57 on Ausonius-Paulinus: at 144, on epistolarity and a relational notion of the self; and at 148-9, 153-5, on the interpermeability of god and man as an “attempt to incorporate Ausonius into his new view of the world”. Amplified samples and detection mixes (18 μl) were colour coded using a subset of the 1,050 colour code set. Colour coded solutions were emulsified into droplets, pooled, and loaded onto an mChip (see ‘Colour coding, emulsification and droplet pooling’ and ‘Loading, imaging and merging microwell arrays’ under ‘General procedures’). The chip was imaged with a 1× objective to identify colour codes, droplet pairs were merged, and reporter fluorescence in each well was measured by fluorescence imaging at 1 h and 3 h (see ‘Loading, imaging and merging microwell arrays’ under ‘General procedures’). Data were analysed as described in ‘Data analysis’. A schematic overview of the HV panel sequence design strategy is shown in Extended Data Fig. 5h. In brief, the design pipeline consisted of viral genomes segment alignment and PCR amplicon selection followed by crRNA design that accounts for cross-reactivity. Finally, PCR primers were pooled by genus. All sequences are in Supplementary Data 2. Viral genome segment alignment A 2018 performance at the Teatro Comunale, Florence, changed the ending to take a stand against violence against women. Instead of being killed, Carmen kills Don José with a pistol she grabs from him. [79] Many applauded the change, seeing it as way to break the tradition of representing misogyny in opera while so many women continue to suffer from violence and abuse. [80]
Sets consisting of a small number (1–5) of crRNA sequences were designed to selectively target individual H or N subtypes using ADAPT (Metsky et al., manuscript in preparation). We improved our design approach throughout the process by incorporating new features into each round of design. In the first round of design, we only designed H crRNAs, and required that all crRNAs could hybridize with 90% of all sequences, allowing for up to 1 mismatch. crRNAs in a set could be positioned anywhere in the amplicon. In the second round of design, we designed crRNAs for both H and N and restricted the positions of crRNAs within a set (to within a 91-nt window for H and 35-nt window for N) as some positions within the amplicon were more conserved between subtypes than others. As in round 1, in round 2 we required that all crRNAs could hybridize with 90% of all sequences, allowing for up to 1 mismatch. In addition, we weighted the coverage of our designs towards more recent years by using an exponential decay parameter for sequences from before 2017. In the third round, we used a differential design approach in which all crRNAs were required to have at least 3 mismatches against at least 99% of sequences within any other subtype. In the fourth round, we accounted for G–U pairing in hybridization, and raised the target threshold to 95% of sequences in each subtype, allowing for up to 1 mismatch. Each round of designs was tested experimentally, and high-performing crRNAs between designs were used in combination. H required four rounds of design, while N only required two rounds (rounds two and three). Oligonucleotide sequences are listed in Supplementary Data 2. HIV DRM panel design Primer design Carmen has been the subject of many recordings, beginning with early wax cylinder recordings of excerpts in the 1890s, a nearly complete performance in German from 1908 with Emmy Destinn in the title role, [97] [98] and a complete 1911 Opéra-Comique recording in French. Since then, many of the leading opera houses and artistes have recorded the work, in both studio and live performances. [99] Over the years many versions have been commended and reissued. [100] [101] From the mid-1990s numerous video recordings have become available. These include David McVicar's Glyndebourne production of 2002, and the Royal Opera productions of 2007 and 2010, each designed by Francesca Zambello. [99] Adaptations [ edit ] Vittoria Lepanto [ it] in Carmen (1909)Walsh, Peter. G. (trans.) The Poems of St. Paulinus. New York: Ancient Christian Writers 40, Newman, 1975; Trout in n. 2; R. P. H. Green, The Poetry of Paulinus of Nola, A Study of his Latinity, Brussels: Collection Latomus 120, Latomus, 1971; Catherine Conybeare. Paulinus Noster. Self and Symbols in the Letters of Paulinus of Nola, Oxford: Oxford University Press, 2000.
Capitalizing on the specificity of Cas13 detection, we used CARMEN–Cas13 to discriminate all epidemiologically relevant serotypes of influenza A in parallel. Diversity within a viral species such as influenza A poses a substantial challenge to detection; an assay must correctly identify many distinct sequences within a group of strains, while remaining selective for that group. To discriminate the haemagglutinin (H) and neuraminidase (N) subtypes H1–H16 and N1–N9 of influenza A virus, we designed H and N amplicons that were sufficiently conserved to amplify with two parallel primer sets and used ADAPT to design specific sets of crRNAs to identify subtypes (Fig. 3a, see Methods for details). We tested the optimal crRNA from each set using synthetic consensus sequences from H1–H16 and N1–N9, and successfully identified these subtypes (Fig. 3b, c). We further tested our N-subtyping assay using synthetic sequences that collectively cover more than 90% of the sequence diversity within subtypes N1–N9, and identified 32 out of 35 (91.4%) of these sequences (Extended Data Fig. 8c). Finally, we validated our subtyping assay using 20 throat and nasal swabs from humans infected during the 2018–2019 flu season and were able to successfully subtype all of these infections, showing 100% concordance with results of reverse transcription with quantitative PCR performed by the Centers for Disease Control and Prevention and NGS performed in our laboratory (Fig. 3d, Methods, ‘Cas13-detection reactions’ under ‘General procedures’). On the basis of these results, our assay could potentially identify each of the 144 possible combinations of H1–H16 and N1–N9 subtypes. Each colour code was diluted 1:10 in LB broth (a medium that yields droplets of similar size to droplets made from PCR products and detection reagents) to a final total 3-dye concentration of 20 µM. Each solution was emulsified into droplets as described in ‘Colour coding, emulsification and droplet pooling’ under ‘General procedures’. The 1,050-colour-code set was characterized in 3-colour space and along the 4th colour dimension as described below. Characterization of the 1,050-colour-code set in three-colour space
The popularity of Carmen continued through succeeding generations of American opera-goers; by the beginning of 2011 the Met alone had performed it almost a thousand times. [66] It enjoyed similar success in other American cities and in all parts of the world, in many different languages. [69] Carmen's habanera from act 1, and the toreador's song " Votre toast" from act 2, are among the most popular and best-known of all operatic arias, [70] the latter "a splendid piece of swagger" according to Newman, "against which the voices and the eyebrows of purists have long been raised in vain". [71] Most of the productions outside France followed the example created in Vienna and incorporated lavish ballet interludes and other spectacles, a practice which Mahler abandoned in Vienna when he revived the work there in 1900. [50] In 1919, Bizet's aged contemporary Camille Saint-Saëns was still complaining about the "strange idea" of adding a ballet, which he considered "a hideous blemish in that masterpiece", and he wondered why Bizet's wife had permitted it. [72] It is also telling that Millepied makes de Palma, one of Pedro Almodóvar’s favorite performers, central to the story. It does not have the same kind of melodramatic soap opera storyline; nothing could be simpler than a couple on the run, discovering that they love each other. But as in Almodóvar’s films, the heightened use of color and settings is stunning, and the filmmakers are not afraid to express passionate emotion. That creates movie magic. Cast details are as provided by Curtiss [16] from the original piano and vocal score. The stage designs are credited to Charles Ponchard.
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Ethics statement Carmen: The First Complete Recording". Marston Records. Archived from the original on 15 February 2013 . Retrieved 22 May 2016. The opera is written in the genre of opéra comique with musical numbers separated by dialogue. It is set in southern Spain and tells the story of the downfall of Don José, a naïve soldier who is seduced by the wiles of the fiery gypsy Carmen. José abandons his childhood sweetheart and deserts from his military duties, yet loses Carmen's love to the glamorous torero Escamillo, after which José kills her in a jealous rage. The depictions of proletarian life, immorality, and lawlessness, and the tragic death of the main character on stage, broke new ground in French opera and were highly controversial. March, Ivan (ed.); Greenfield, Edward; Layton, Robert (1993). The Penguin Guide to Opera on Compact Discs. London: Penguin Books. pp. 25–28. ISBN 0-14-046957-5. {{ cite book}}: CS1 maint: multiple names: authors list ( link) N (neuraminidase) primers were based on the majority consensus sequence for each subtype (9 primer pairs) in a single pool. We used ADAPT to design H (hemagglutinin) primers covering at least 95% of the sequences within each subtype. In total, there were 45 primers (15 forward primers and 30 reverse primers) in a single pool. See Supplementary Data 2 for details. crRNA designExtracted viral nucleic acids were prepared for sequencing using library construction methods that have been previously described 30, with a few differences noted below. Following extraction, double-stranded complementary DNA (cDNA) was created using random primers and SuperScript IV (Thermo Fisher Scientific) for first-strand synthesis and Escherichia coli polymerase I (NEB) for second-strand synthesis. Sequencing libraries were generated using the Nextera XT DNA Library Prep Kit (Illumina) with 10–16 cycles of PCR to introduce unique dual-index pairs. Libraries were then quantified using the KAPA Universal Complete Kit (Roche) and 12–18 samples were pooled for sequencing, including a no-input negative control. Samples were sequenced to >0.82 million read-mates using 2 × 75-bp paired-end reads from the Illumina NextSeq Reagent Kit v.2.5. crRNA preparation Of the 169 HV10 species, 164 have designs with 3 or fewer primer pairs (total of 187 primer sequences required to cover these 164 species: 145 have 1 primer pair, 15 have 2 primer pairs, and 4 have 3 primer pairs). There were four species that required more than three primer pairs: lymphocytic choriomeningitis virus (7 primer pairs), norovirus (4 primer pairs), betapapillomavirus 2 (6 primer pairs) and Candiru phlebovirus (6 primer pairs). These four species were combined into a single ‘diverse’ primer pool at 150 nM final concentration. This is pool 2, as shown in Fig. 2c. Degenerate primer pool Then, in line 10, he wrote about a praetor who would molest the people of Bithynia, where he did not care about the people who were under his command. Then, people respond by asking more questions about the area. They wonder if there are bearers for his chair and how the people who would carry his chair are from Bithynia. Catullus said that he convinced the girl that he was more fortunate than other people.
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