Analysis of Escherichia coli nicotinate mononucleotide adenylyltransferase mutants in vivo and in vitro

BACKGROUND: Adenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD(+ )synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD(+), especially for one of the alleles, nadD72. RESULTS: In this study these two mutant proteins have been further characterized together with ten new mutant variants. Of the, in total, twelve mutations four are in a conserved motif in the C-terminus and eight are in the active site. We have tested the activity of the enzymes in vitro and their effect on the growth phenotype in vivo. There is a very good correlation between the two data sets. CONCLUSION: The mutations in the C-terminus did not reveal any function for the conserved motif. On the other hand, our data has lead us to assign amino acid residues His-19, Arg-46 and Asp-109 to the active site. We have also shown that the nadD gene is essential for growth in E. coli

Collaborative software design and modeling in virtual reality

Context: Software engineering is becoming more and more distributed. Developers and other stakeholders are often located in different locations, departments, and countries and operating within different time zones. Most online software design and modeling tools are not adequate for distributed collaboration since they do not support awareness and lack features for effective communication. Objective: The aim of our research is to support distributed software design activities in Virtual Reality (VR). Method: Using design science research methodology, we design and evaluate a tool for collaborative design in VR. We evaluate the collaboration efficiency and recall of design information when using the VR software design environment compared to a non-VR software design environment. Moreover, we collect the perceptions and preferences of users to explore the opportunities and challenges that were incurred by using the VR software design environment. Results: We find that there is no significant difference in the efficiency and recall of design information when using the VR compared to the non-VR environment. Furthermore, we find that developers are more satisfied with collaboration in VR. Conclusion: The results of our research and similar studies show that working in VR is not yet faster or more efficient than working on standard desktops. It is very important to improve the interface in VR (gestures with haptics, keyboard and voice input), as confirmed by the difference in results between the first and second evaluation

Desenvolvimento de uma reação em cadeia pela polimerase e comparação com a imunodifusão em gel de agar na detecção de infecções pelo vírus da leucemia bovina

Polymerase chain reaction (PCR) was used for bovine leukemia virus (BLV) detection in the peripheral leukocytes of the infected bovines. The primers used were designed to amplify a part of env gene of BLV. PCR products were analyzed by agarose gel electrophoresis stained by ethidium bromide. The analytical specificity of PCR was confirmed by enzymatic restriction analysis of the PCR product with Bam HI and also by nucleotide sequence analysis of three PCR samples. Sixty five animals were tested for anti-BLV antibody, by agar gel-immunodiffusion test (AGID) and for direct BLV detection by PCR. There was a 73.80% concordance rate between the two tests. Four animals positive in AGID were PCR negative, while 13 AGID negative animals were found PCR positive. PCR got a 0.87 diagnosis sensitivity and 0.62 specificity. The developed PCR may be complementary tool in the diagnosis of BLV infection, but should have it diagnosis sensitivity improved.A reação em cadeia pela polimerase (PCR) foi utilizada para a detecção do vírus da leucemia bovina (VLB) em leucócitos periféricos de bovinos infectados. Os iniciadores utilizados foram construídos para amplificar uma parte do gene env do VLB. Os produtos da PCR foram analisados por eletroforese em gel de agarose corados por brometo de etídeo. A especificidade analítica da PCR foi confirmada por restrição enzimática dos produtos da reação com Bam HI e também pela análise da seqüência de três amostras. Sessenta e cinco animais foram testados para a presença de anticorpos anti-VLB, pela imunodifusão em gel de agar (IDGA) e pela PCR, para detecção direta do VLB. Houve 73,80% de concordância entre os dois testes. Quatro animais positivos na IDGA foram PCR negativos, enquanto 13 animais negativos na IDGA foram positivos na PCR. A sensibilidade diagnóstica obtida foi de 0,87 e a especificidade diagnóstica 0,62. A PCR desenvolvida pode ser uma ferramenta complementar no diagnóstico de infecções causadas pelo VLB, mas deve ter sua sensibilidade diagnóstica melhorada

Mapping an epitope in EBNA-1 that is recognized by monoclonal antibodies to EBNA-1 that cross-react with dsDNA

Introduction: The Epstein Barr Virus (EBV) has been associated with the autoimmune disease, Systemic Lupus Erythematosus (SLE). EBV nuclear antigen-I (EBNA-1) is the major nuclear protein of EBV. We previously generated an IgG monoclonal antibody (MAb) to EBNA-1, 3D4, and demonstrated that it crossreacts with double stranded DNA (dsDNA) and binds the 148 amino acid viral binding site (VBS) in the carboxyl region of EBNA-1. The aim of the present study was to characterize another antibody to EBNA-1 that cross-reacts with dsDNA, compare its immunoglobulin genes to 3D4, and finely map the epitope in EBNA-1 that is recognized by these cross-reactive antibodies. Methods: We generated an IgM MAb to EBNA-1, 16D2, from EBNA-1 injected mice and demonstrated by ELISA that it cross-reacts with dsDNA and binds the 148 amino acid VBS. We sequenced the variable heavy and light chain genes of 3D4 and 16D2 and compared V gene usage. To more finely map the epitope in EBNA-1 recognized by these MAbs, we examined their binding by ELISA to 15 overlapping peptides spanning the 148 amino acid domain. Results: Sequence analysis revealed that 3D4 and 16D2 utilize different VH and VL genes but identical JH and Jk regions with minimal junctional diversity. This accounts for similarities in their CDR3 regions and may explain their similar dual binding specificity. Epitope mapping revealed 3D4 and 16D2 bind the same peptide in the VBS. Based on the crystal structure of EBNA-1, we observed that this peptide resides at the base of an exposed proline rich loop in EBNA-1. Conclusion: We have demonstrated that two MAbs that bind EBNA-1 and crossreact with dsDNA, recognize the same peptide in the VBS. This peptide may serve as a mimetope for dsDNA and may be of diagnostic and therapeutic value in SLE

Lyric Ear: Romantic Poetics of Listening

My dissertation, Lyric Ear: Romantic Poetics of Listening, turns from a centuries-long critical focus on the “lyric voice” to consider instead what I am calling the lyric ear, or the speaking ear. I offer case studies in four nineteenth-century British and American poets, exploring how each develops poetics of listening that open up authorship, agency, and singularity—into unknown collaboration, receptive action, and multiplicity. By focusing on how lyric constructs the ear, rather than the voice, I argue that poetry expands participation to include bodies, objects, and surroundings that share physical space, rather than simply those who have the agency or the privilege to speak. Although the trope of the speaking ear works differently for each poet, certain characteristics remain constant: 1) the speaking ear involves a description of listening, which goes so far in its intensity or detail that it explicitly or implicitly figures the poem itself as an ear; 2) the construction of the poem-as-ear intensifies into a moment of apparent paradox, in which the actions of the mouth and the ear become interchangeable; 3) the descriptions of listening that extend into imaginations of the poem-as-ear propagate formal repetitions, which in all four chapters include refrain, repetition, and rhyme. The speaking ear makes an organ of receptivity also an instrument of speech, which demonstrates how the body and its parts work in unexpected, unpredictable ways. Unknowable collectivities of listeners construct alternative participations in speaking and in listening, vibrating across registers of music and noise, sharing verbs even as they share the space of a soundscape.My first chapter, on Mary Robinson’s Lyrical Tales, posits gossip as a means of conceiving the speaking ear par excellence. According to gossip’s understudied generic conventions, the speaker is most importantly a listener. Gossip is collaborative, both in its construction and in its reception. Passed from ear to ear, gossip shares authors, just as it spreads in unpredictable ways. Throughout Robinson’s Lyrical Tales, the figure of gossip works to multiply and generalize the speaking voice and its affects, giving the impression that the story at hand is not authored by a single identity, but rather that it approximates and synthesizes the shared impressions of a community. My second chapter, on John Clare’s bird poems, picks up on the collaborative possibilities of the speaking ear to challenge authorship and brings these possibilities into the non-human world. Through poems that variously invite listening attention, and perform poetic impressions of birdsong, Clare asks what the terms of engagement are when there is no common language. In my third chapter, on perversity, occultism, and slow violence in the poetry of Edgar Alan Poe, I trace how the repetitious, inanimate “bells, bells, bells, bells, / Bells, bells, bells” in Poe’s eponymous poem destabilize the environments they simultaneously enliven. I consider poetic effects such as rhyme and repetition as passive or barely-perceptible agents of quiet (but decidedly sonic) violence. My final chapter considers Emily Dickinson’s poem, “The Spirit is the Conscious Ear,” as a prompt to the question: what, then, is the unconscious ear? To say that the Spirit is the Conscious Ear does not isolate the ear’s listening, but expands the ways the ear can work. Through the punning deictic here (hear), Dickinson turns the poem into a speaking ear, an organ of receptivity with diverse capacity for sounding and sensing, and strange powers of speech. The poem, like the ear, is a body for sound, a body of sound. I argue that Dickinson’s vibrational poetics are necessarily permeable, conducting an interpenetration between senses and sounds. By parsing differences between listening and hearing, I argue that the unconscious (in)attention of the dreaming body invites new and slippery ways of understanding how the sonic skips and disjunctions of puns, recurring rhymes, syntactic discontinuities, and fragmentary sound-bites illuminate Dickinson’s poetry and letters more broadly

Lyric Ear: Romantic Poetics of Listening

My dissertation, Lyric Ear: Romantic Poetics of Listening, turns from a centuries-long critical focus on the “lyric voice” to consider instead what I am calling the lyric ear, or the speaking ear. I offer case studies in four nineteenth-century British and American poets, exploring how each develops poetics of listening that open up authorship, agency, and singularity—into unknown collaboration, receptive action, and multiplicity. By focusing on how lyric constructs the ear, rather than the voice, I argue that poetry expands participation to include bodies, objects, and surroundings that share physical space, rather than simply those who have the agency or the privilege to speak. Although the trope of the speaking ear works differently for each poet, certain characteristics remain constant: 1) the speaking ear involves a description of listening, which goes so far in its intensity or detail that it explicitly or implicitly figures the poem itself as an ear; 2) the construction of the poem-as-ear intensifies into a moment of apparent paradox, in which the actions of the mouth and the ear become interchangeable; 3) the descriptions of listening that extend into imaginations of the poem-as-ear propagate formal repetitions, which in all four chapters include refrain, repetition, and rhyme. The speaking ear makes an organ of receptivity also an instrument of speech, which demonstrates how the body and its parts work in unexpected, unpredictable ways. Unknowable collectivities of listeners construct alternative participations in speaking and in listening, vibrating across registers of music and noise, sharing verbs even as they share the space of a soundscape.My first chapter, on Mary Robinson’s Lyrical Tales, posits gossip as a means of conceiving the speaking ear par excellence. According to gossip’s understudied generic conventions, the speaker is most importantly a listener. Gossip is collaborative, both in its construction and in its reception. Passed from ear to ear, gossip shares authors, just as it spreads in unpredictable ways. Throughout Robinson’s Lyrical Tales, the figure of gossip works to multiply and generalize the speaking voice and its affects, giving the impression that the story at hand is not authored by a single identity, but rather that it approximates and synthesizes the shared impressions of a community. My second chapter, on John Clare’s bird poems, picks up on the collaborative possibilities of the speaking ear to challenge authorship and brings these possibilities into the non-human world. Through poems that variously invite listening attention, and perform poetic impressions of birdsong, Clare asks what the terms of engagement are when there is no common language. In my third chapter, on perversity, occultism, and slow violence in the poetry of Edgar Alan Poe, I trace how the repetitious, inanimate “bells, bells, bells, bells, / Bells, bells, bells” in Poe’s eponymous poem destabilize the environments they simultaneously enliven. I consider poetic effects such as rhyme and repetition as passive or barely-perceptible agents of quiet (but decidedly sonic) violence. My final chapter considers Emily Dickinson’s poem, “The Spirit is the Conscious Ear,” as a prompt to the question: what, then, is the unconscious ear? To say that the Spirit is the Conscious Ear does not isolate the ear’s listening, but expands the ways the ear can work. Through the punning deictic here (hear), Dickinson turns the poem into a speaking ear, an organ of receptivity with diverse capacity for sounding and sensing, and strange powers of speech. The poem, like the ear, is a body for sound, a body of sound. I argue that Dickinson’s vibrational poetics are necessarily permeable, conducting an interpenetration between senses and sounds. By parsing differences between listening and hearing, I argue that the unconscious (in)attention of the dreaming body invites new and slippery ways of understanding how the sonic skips and disjunctions of puns, recurring rhymes, syntactic discontinuities, and fragmentary sound-bites illuminate Dickinson’s poetry and letters more broadly