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Engineering Probiotic E. Coli With a Type III Secretion System for Targeted Delivery of Therapeutic VHH
No full textTo combat severe disease manifestations in the gut from both pathogenic and autoimmune insults, we are engineering probiotic microbes to release therapeutics into the gut as a novel method of treatment delivery. A bacterial protein delivery system was designed based upon the Shigella flexneri type III secretion system (T3SS) to secrete therapeutic proteins both inside and in the vicinity of mammalian cells. The T3SS is a nanomachine that traditionally delivers effector proteins into the host cell to mediate pathogenesis. Fusion of a type III effector secretion sequence to therapeutic proteins results in variants that are recognized as secreted substrates. We recently developed probiotic E. coli strains that express a T3SS. These probiotic T3SS competent E. coli strains can deliver therapeutic proteins modified with type III secretion signals into host cells.
This work describes the development of engineered probiotic T3SS competent E. coli that can secrete VHH proteins. VHH are therapeutic antibody fragments that effectively bind and block activity of target antigens. We demonstrate that DH10B E. coli can use the T3SS to directly secrete VHH, modified with the OspC3 effector secretion sequence, into mammalian cells. These bacteria successfully deliver a variety of VHHs, including those that bind to inflammasome ASC protein and a heterotetrameric VHH-based neutralizing agent (VNA) designed to neutralize Shiga toxin (Stx). A remaining question is whether VHH or VNA secreted via the T3SS maintain their function. If evidence indicates αASC VHH inhibit inflammasome activation, we envision this VHH as a potential anti-inflammatory therapeutic. Whereas, a functional secreted αStx VNA could serve as a therapeutic against enterohemorrhagic E. coli infections. We envision modifying our T3SS platform to secrete the αStx VNA into the intestinal lumen as well as the cytosol. Finally, we engineered probiotic E. coli strains MP, HS and Nissle 1917 for future use in VHH delivery. This research pursues a novel approach of utilizing bacterial type III secretion systems to deliver therapeutic VHH and VNA to the gut.Therapeutic E.coli; VH
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A Genetic analysis of 5' splice site selection in Saccharomyces cerevisiae
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Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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Engineering Symbiotic Microbiota for in Situ Delivery of Therapeutic Proteins
No full textAs symbiotic members of the human microbiome, Lactobacilli are an emerging class of living biotherapeutic agents capable of sustained and targeted drug delivery of protein biologics to the many organs they naturally inhabit. However, progress to this end remains limited by the paucity of genetic tools for manipulating these bacteria. We used synthetic biology techniques to build a tunable, broad host range expression system, and applied in silico techniques for de novo design of robust, strain-optimized secretion signals that direct protein export. Together, these components provide an approach to rapidly engineer niche-adapted Lactobacilli for in situ delivery of therapeutic proteins. We leveraged this platform to engineer model symbionts from the gastrointestinal and female genital tracts to secrete single domain antibody fragments, as candidate vectors for IBD immunotherapy and HIV immunoprophylaxis. This work advances Lactobacillus as a vehicle for drug delivery, and serves as a platform to accelerate development of diverse, microbiome-based medicines capable of addressing myriad disease.Biophysic
The Yeast Saccharomyces cerevisiae: A Versatile Model System for the Identification and Characterization of Bacterial Virulence Proteins
No full textMicrobial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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