Discordant Desire: Morley’s Polyphony in Shakespeare’s Twelfth Night

From Orsino’s opening line, “If music be the food of love, play on,” to Feste’s concluding song of solitude, William Shakespeare’s Twelfth Night, or What You Will is relentlessly full of music, whether explicitly calling for sound or indirectly invoking references to musical ideas (1.1.1). Shakespeare utilizes music not only to entertain the listeners of his plays but also to employ its inane ability to enhance and empower human speech. The musicality of this play, first performed at a feast on February 2, 1602, would have been understood and appreciated by its original audience as Elizabethan England experienced a golden age of music and literature. While music was an essential part of any young, upper-class boy’s schooling, the accessibility to musical education materials was highly limited in the early half of the sixteenth century. Learning was primarily accomplished through listening and remembering tunes such as church hymns, ballads on the streets, and processional ceremony sounds. However, the latter half of the century underwent exponential progress as the music printing industry emerged, allowing musicians’ compositions and theories to become available to the public. As these musical concepts became widespread knowledge, Shakespeare incorporated several theories in accordance with the contemporary philosophical understanding of music into Twelfth Night; the concept of polyphony and its consequent affecting powers upon individuals represents how the play conceptualizes desire as ultimately insatiable

The analisys of possibility laser cutting

Diplomová práce se zabývá použitím laserů ve strojírenství, zejména technologií dělení materiálu laserem. Ta je srovnávána za pomoci kontrolních vzorků s technologií vysekávání a stříhání. Srovnávány jsou parametry struktury povrchu, rozměrová přesnost a vzhled dělící plochy. Na základě výsledků bylo vytvořeno doporučení pro praktickou realizaci.Master’s thesis deals with application of lasers in manufacturing, mainly with the technology of cutting material. This technology is compared with the technology of punching and cutting sheet metal, with the aid of control specimens. The parameters of surface structure, dimension accuracy and appearance of cut-ting surfaces are compared. By consensus of results the recommendation for practical realization was created.

Virion structure and genome delivery mechanism of sacbrood honeybee virus

Infection by sacbrood virus (SBV) from the family Iflaviridae is lethal to honey bee larvae but only rarely causes the collapse of honey bee colonies. Despite the negative effect of SBV on honey bees, the structure of its particles and mechanism of its genome delivery are unknown. Here we present the crystal structure of SBV virion and show that it contains 60 copies of a minor capsid protein (MiCP) attached to the virion surface. No similar MiCPs have been previously reported in any of the related viruses from the order Picornavirales. The location of the MiCP coding sequence within the SBV genome indicates that the MiCP evolved from a C-terminal extension of a major capsid protein by the introduction of a cleavage site for a virus protease. The exposure of SBV to acidic pH, which the virus likely encounters during cell entry, induces the formation of pores at threefold and fivefold axes of the capsid that are 7 angstrom and 12 angstrom in diameter, respectively. This is in contrast to vertebrate picornaviruses, in which the pores along twofold icosahedral symmetry axes are currently considered the most likely sites for genome release. SBV virions lack VP4 subunits that facilitate the genome delivery of many related dicistroviruses and picornaviruses. MiCP subunits induce liposome disruption in vitro, indicating that they are functional analogs of VP4 subunits and enable the virus genome to escape across the endosome membrane into the cell cytoplasm

A molecular clone of Chronic Bee Paralysis Virus (CBPV) causes mortality in honey bee pupae (Apis mellifera)

: Among the many diseases compromising the well-being of the honey bee (Apis mellifera) the chronic paralysis syndrome of adult honey bees is one of the best described. The causative agent, chronic bee paralysis virus (CBPV), is a positive sense, single-stranded RNA virus with a segmented genome. Segment 1 encodes three putative open reading frames (ORFs), including the RNA-dependent RNA polymerase and other non-structural protein coding regions. Segment 2 encodes four putative ORFs, which contain the genes of supposed structural proteins. In this study, we established a reverse genetic system for CBPV by molecular cloning of DNA copies of both genome segments. CBPV rescue was studied in imago and honey bee pupae infection models. Virus replication and progeny virus production was only initiated when capped RNAs of both genome segments were injected in honey bees. As injection of these clonal RNAs caused clinical symptoms similar to wild-type CBPV infection, we conclude that the novel molecular clone fulfilled Koch's postulates. Our virus clone will enable in-depth analysis of CBPV pathogenesis and help to increase knowledge about this important honey bee disease

Virion Structure and In Vitro Genome Release Mechanism of Dicistrovirus Kashmir Bee Virus

Infections with Kashmir bee virus (KBV) are lethal for honeybees and have been associated with colony collapse disorder. KBV and closely related viruses contribute to the ongoing decline in the number of honeybee colonies in North America, Europe, Australia, and other parts of the world. Despite the economic and ecological impact of KBV, its structure and infection process remain unknown. Here, we present the structure of the virion of KBV determined to a resolution of 2.8 angstrom. We show that the exposure of KBV to acidic pH induces a reduction in interpentamer contacts within capsids and the reorganization of its RNA genome from a uniform distribution to regions of high and low density. Capsids of KBV crack into pieces at acidic pH, resulting in the formation of open particles lacking pentamers of capsid proteins. The large openings of capsids enable the rapid release of genomes and thus limit the probability of their degradation by RNases. The opening of capsids may be a shared mechanism for the genome release of viruses from the family Dicistroviridae. IMPORTANCE The western honeybee (Apis mellifera) is indispensable for maintaining agricultural productivity as well as the abundance and diversity of wild flowering plants. However, bees suffer from environmental pollution, parasites, and pathogens, including viruses. Outbreaks of virus infections cause the deaths of individual honeybees as well as collapses of whole colonies. Kashmir bee virus has been associated with colony collapse disorder in the United States, and no cure for the disease is currently available. Here, we report the structure of an infectious particle of Kashmir bee virus and show how its protein capsid opens to release the genome. Our structural characterization of the infection process determined that therapeutic compounds stabilizing contacts between pentamers of capsid proteins could prevent the genome release of the virus.We gratefully acknowledge the Cryoelectron Microscopy and Tomography core facility of CEITEC supported by MEYS CR (LM2018127) . This research was carried out under the project CEITEC 2020 (LQ1601) , with financial support from the MEYS of the Czech Republic under National Sustainability Program II. This work was supported by IT4I project (CZ.1.05/1.1.00/02.0070) , funded by the European Regional Development Fund and the national budget of the Czech Republic via the RDIOP, as well as the MEYS via the grant (LM2011033) . The research of G.A.M. was supported by the grants CONICET (PIP 20150288) , 247 Agencia Nacional de Promocion Cientifica y Tecnica, Argentina (PICT no. 2015-248 0665, PICT No. 20181545) , and Universidad Nacional de La Plata, Argentina. The research of D.M.A.G. was supported by a Grupos Consolidados grant from the University of the Basque Country, Spain (GIU18/172) . The research leading to these results received funding from the Grant Agency of the Czech Republic grant GX19-25982X to P.P

Abstract OR-6: Baseplate Structure of Bacteriophage Phi812 and Mechanism of Cell Wall Binding and Penetration

Background: Antibiotic-resistant strains of Staphylococcus aureus cause human infections that are difficult to treat and can lead to death. Bacteriophage (phage) phi812K1/420 from the family Myoviridae infects 95% of clinical isolates of S. aureus and therefore is a promising candidate for a phage therapy agent. As the native phage particle approaches its host cell, phage receptor-binding proteins make a contact with the host cell wall. This interaction triggers a cascade of structural changes in the baseplate resulting in phage tail contraction and genome ejection. Mechanistic description of the baseplate re-organization, however, remains unknown. Methods: Using cryo-electron microscopy (cryo-EM), we studied the baseplate of the phage phi812K1/420. Also, selected proteins involved in the host cell wall binding and penetration were produced in recombinant form and their structures were solved using X-ray crystallography and cryo-EM single-particle reconstruction. Results: We reconstructed the phage baseplate in native and contracted states. The reconstruction of the native baseplate reaches a resolution of 4 Å, which enables us to discern individual protein structures. Solved protein structures will be fitted into the reconstruction of the contracted baseplate. Conclusion: Our results provide the first structural characterization of contractile phage infecting a Gram-positive bacterium. Comparison of the two distinct baseplate states will allow us to describe the molecular mechanism of the initial stage of phage infection in detail

Structure and dynamics of the RNAPII CTDsome with Rtt103

RNA polymerase II (RNAPII) not only transcribes protein coding genes and many noncoding RNA, but also coordinates transcription and RNA processing. This coordination is mediated by a long C-terminal domain (CTD) of the largest RNAPII subunit, which serves as a binding platform for many RNA/protein-binding factors involved in transcription regulation. In this work, we used a hybrid approach to visualize the architecture of the full-length CTD in complex with the transcription termination factor Rtt103. Specifically, we first solved the structures of the isolated subcomplexes at high resolution and then arranged them into the overall envelopes determined at low resolution by small-angle X-ray scattering. The reconstructed overall architecture of the Rtt103–CTD complex reveals how Rtt103 decorates the CTD platform

Abstract OR-7: Genome Release Mechanism of Picorna-Like Viruses

Protein capsids protect the genomes of viruses from degradation in the extracellular environment. However, virus capsids must release genomes into a host cell to initiate infection. We used cryo-electron microscopy to characterize the genome release of viruses from the order Picornavirales: picornaviruses, dicistroviruses, and iflaviruses. These virus families include numerous human and animal pathogens. The viruses have non-enveloped virions and capsids organized with icosahedral symmetry. Their genome release can be induced in vitro by exposure to acidic pH, mimicking conditions in endosomes. We show that conformational changes of capsids and expansion of viral RNA genomes, which are induced by acidic pH, trigger the opening of picorna-like virus particles. The capsids of the studied viruses crack into pieces or open like flowers to release their genomes. The large openings of capsids enable the virus genomes to exit within microseconds, which limits the probability of their degradation by the RNases. Characterization of the virus genome release is the first step towards developing inhibitors of the process

Virion structure of Iflavirus Slow bee paralysis virus at 2.6-Angstrom resolution

The western honeybee (Apis mellifera) is the most important commercial insect pollinator. However, bees are under pressure from habitat loss, environmental stress, and pathogens, including viruses that can cause lethal epidemics. Slow bee paralysis virus (SBPV) belongs to the Iflaviridae family of nonenveloped single-stranded RNA viruses. Here we present the structure of the SBPV virion determined from two crystal forms to resolutions of 3.4 angstrom and 2.6 angstrom. The overall structure of the virion resembles that of picornaviruses, with the three major capsid proteins VP1 to 3 organized into a pseudo-T3 icosahedral capsid. However, the SBPV capsid protein VP3 contains a C-terminal globular domain that has not been observed in other viruses from the order Picornavirales. The protruding (P) domains form "crowns" on the virion surface around each 5-fold axis in one of the crystal forms. However, the P domains are shifted 36 angstrom toward the 3-fold axis in the other crystal form. Furthermore, the P domain contains the Ser-His-Asp triad within a surface patch of eight conserved residues that constitutes a putative catalytic or receptor-binding site. The movements of the domain might be required for efficient substrate cleavage or receptor binding during virus cell entry. In addition, capsid protein VP2 contains an RGD sequence that is exposed on the virion surface, indicating that integrins might be cellular receptors of SBPV.IMPORTANCEPollination by honeybees is needed to sustain agricultural productivity as well as the biodiversity of wild flora. However, honey-bee populations in Europe and North America have been declining since the 1950s. Honeybee viruses from the Iflaviridae family are among the major causes of honeybee colony mortality. We determined the virion structure of an Iflavirus, slow bee paralysis virus (SBPV). SBPV exhibits unique structural features not observed in other picorna-like viruses. The SBPV capsid protein VP3 has a large C-terminal domain, five of which form highly prominent protruding "crowns" on the virion surface. However, the domains can change their positions depending on the conditions of the environment. The domain includes a putative catalytic or receptor binding site that might be important for SBPV cell entry

Viral discovery and diversity in trypanosomatid protozoa with a focus on relatives of the human parasite <i>Leishmania</i>.

Knowledge of viral diversity is expanding greatly, but many lineages remain underexplored. We surveyed RNA viruses in 52 cultured monoxenous relatives of the human parasite &lt;i&gt;Leishmania&lt;/i&gt; ( &lt;i&gt;Crithidia&lt;/i&gt; and &lt;i&gt;Leptomonas&lt;/i&gt; ), as well as plant-infecting &lt;i&gt;Phytomonas&lt;/i&gt; &lt;i&gt;Leptomonas pyrrhocoris&lt;/i&gt; was a hotbed for viral discovery, carrying a virus (Leptomonas pyrrhocoris ostravirus 1) with a highly divergent RNA-dependent RNA polymerase missed by conventional BLAST searches, an emergent clade of tombus-like viruses, and an example of viral endogenization. A deep-branching clade of trypanosomatid narnaviruses was found, notable as &lt;i&gt;Leptomonas seymouri&lt;/i&gt; bearing Narna-like virus 1 (LepseyNLV1) have been reported in cultures recovered from patients with visceral leishmaniasis. A deep-branching trypanosomatid viral lineage showing strong affinities to bunyaviruses was termed " &lt;i&gt;Leishbunyavirus&lt;/i&gt; " (LBV) and judged sufficiently distinct to warrant assignment within a proposed family termed " &lt;i&gt;Leishbunyaviridae&lt;/i&gt; " Numerous relatives of trypanosomatid viruses were found in insect metatranscriptomic surveys, which likely arise from trypanosomatid microbiota. Despite extensive sampling we found no relatives of the totivirus &lt;i&gt;Leishmaniavirus&lt;/i&gt; (LRV1/2), implying that it was acquired at about the same time the &lt;i&gt;Leishmania&lt;/i&gt; became able to parasitize vertebrates. As viruses were found in over a quarter of isolates tested, many more are likely to be found in the &gt;600 unsurveyed trypanosomatid species. Viral loss was occasionally observed in culture, providing potentially isogenic virus-free lines enabling studies probing the biological role of trypanosomatid viruses. These data shed important insights on the emergence of viruses within an important trypanosomatid clade relevant to human disease