1,721,076 research outputs found
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
ENGINEERING WATER-SOLUBLE VARIANTS OF THE SINGLE-SUBUNIT OLIGOSACCHARYLTRANSFERASE
No full text49 pagesOligosaccharyltransferase (OST) is a key enzyme in the asparagine-linked (N-linked) protein glycosylation pathway. OSTs exist in all domains of life and are capable of transferring a preassembled glycan from lipid carrier to an acceptor peptide. Bacterial OSTs are an single-subunit enzyme that are amenable to recombinant expression in model organism including Escherichia coli. As a result, bacterial OSTs have been used as models to explore the mechanism of the N-linked glycosylation process in nature. These developments, notwithstanding, recombinant expression and purification of the OST enzymes remain significant challenges. Bacterial OSTs are multi-pass transmembrane protein that requires intricate balance between protein synthesis rate and a pace of membrane insertion. Further, membrane protein purification often necessitates the use of ultracentrifugation and detergent, both of which limit process scalability and compatibility. To address these challenges, we proposed a protein engineering strategy called SIMPLEx or solubilization of integral membrane proteins with high levels of expression to generate water-soluble variants of the bacterial OST. Specifically, we designed several OST chimeras where the N-terminus of the OST is fused with the amphipathic protein including engineered human apolipoprotein A-I. Using E. coli culture as an expression platform, several SIMPLEx-OSTs could be expressed within the cytoplasmic fraction of the E. coli. Importantly, our engineered OSTs retain their biological activity and are able to N-glycosylate several acceptor proteins including therapeutic human growth hormone. Collectively, our OST-engineering strategy is anticipated to generate a new subclass of water-soluble N-OST enzymes with applications in bioproduction of the glycotherapeutics and glycovaccines
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
Co-stimulatory Molecules Improve the Immune Response to a Weak Neoantigen
No full text61 pagesTherapeutic cancer vaccines are an emerging cancer immunotherapy that work by educating the host’s immune system to attack cancer cells within the body. mRNA lipid nanoparticle (LNP) cancer vaccines are one popular method to deliver tumor specific antigens, termed neoantigens. However, they often do not initiate strong enough CD8+ and CD4+ Type 1 helper (Th1) T cell activation to combat the solid tumors. Thus, it is critical for neoantigen-based cancer vaccines to be delivered with adjuvants to effectively activate neoantigen specific T-cells. Co-stimulatory molecule could act as an alternative adjuvant, because co-stimulatory molecule signaling is the second most crucial signal for T cell activation. Here we aim to co-deliver a pancreatic cancer neoantigen with co-stimulatory molecules via mRNA LNPs. Six co-stimulatory molecules known to increase Th1 T-Cell responses were tested as possible adjuvants for a low-immunogenic neoantigen. Results show three of the six co-stimulatory molecules were able to increase the interferon gamma (IFN-g) immune response of the neoantigen: OX40L, GITRL, and CD80. We then tested whether combinations of these three could further increase the IFN-g immune response. Results show that the combination of OX40L and CD80 significantly increased the IFN-g immune response of the neoantigen 3.3-fold. The study showed individual co-stimulatory molecules and combinations of molecules can increase the IFN-g immune response of a weakly stimulating neoantigen. Co-stimulatory molecules could be ideal adjuvants for neoantigen-based mRNA LNP cancer vaccines
Expanding The Utility Of Rna-Based Transcription Activation Mechanisms For Diagnostic Applications
No full textFrom its inception, one of the major aims of the field of synthetic biology was to reliably manipulate microorganisms to create novel solutions in medicine and biotechnology. This is possible due to the remarkable diversity of natural interactions they carry out to regulate gene expression. Bacterial cells for instance contain sophisticated regulatory networks that modulate expression based on environmental cues. This sense and response mechanism could have tremendous utility in resource-limited environments. In such places, lack of access to affordable, rapid diagnostic tools means that viral and bacterial contamination of public water sources present a serious threat to public health. Before applications can be addressed, cellular circuits must be synthetically repurposed. Traditionally, engineers have relied upon protein-based regulatory networks. In recent times however, the mechanistic flexibility of RNA has emerged. It is now recognized as a powerful tool for rewiring biological circuits. In the present paper, combining a toehold-based design with STAR transcriptional activators resulted in the development of a flexible RNA-based sensing device. A colorimetric LacZ assay, in vivo fluorescence assay and a suite of molecular biology techniques demonstrated the sensing capability of the modified RNAs with a fold activation of 1.5. This work provides direction for the development of high-performance flexible RNA sensors. The paper concludes with recommendations for future work in this area including RNA structural approaches and an exploration of in vitro, paper based circuitry
IMPROVING GLYCOSYLATION EFFICIENCY IN ESCHERICHIA COLI BY MAMMALIAN-CELL-INSPIRED PROTEIN-PROTEIN INTERACTIONS
No full text54 pagesAsparagine linked glycosylation (N-glycosylation) is one of the most common post-translational modifications found in proteins. The central enzyme in the N-glycosylation pathway responsible for the transfer of glycans onto asparagine residues is called the oligosaccharyltransferase (OST). In mammalian cells, most proteins are N-glycosylated cotranslationally, mediated by protein-protein interactions between the OST and the general secretion (Sec) pathway. This mechanism is important for efficient glycosylation of many structured proteins. Recombinant N-glycosylation in Escherichia coli also shows a dependence on the structural context of the acceptor protein. However, intentional coordination between protein secretion and glycosylation in this engineered system has yet to be demonstrated. Here we investigated two strategies using artificial protein-protein interactions to couple the protein secretion and glycosylation machinery in E. coli. A novel model acceptor protein based on the human prosaposin was developed. While the proposed designs were ineffective under the conditions tested, we discovered that fusion proteins of the Campylobacter jejuni PglB were able to improve the expression of challenging acceptor proteins without sacrificing glycosylation efficiency. Further investigations reveal significant implications relating OST expression to protein glycosylation efficiency
THE EFFECT OF AMINO ACID CONCENTRATION ON CELL-FREE SYNTHESIS OF MEMBRANE PROTEINS
No full text100 pagesCell-free protein synthesis (CFPS) is often touted as the future of protein production for functional studies. This thesis focuses on how altering the amino acid concentration affects the synthesis of proteins in PUREfrex 2.1, a commercial recombinant CFPS system. These experiments will inform the optimization of a custom recombinant CFPS kit for membrane proteins (MPs), which is currently under development in the Daniel lab. The protein targets analyzed were soluble super folder green fluorescent protein (sfGFP) and MscL-mEGFP, a fusion protein of the membrane protein MscL and a mEGFP folding reporter. The experiments focused on determining how doubling the concentration of select groups of amino acids affects the synthesis of a soluble and MP target in PUREfrex 2.1. The amino acid mixture included with PUREfrex 2.1 has an equimolar concentration of the twenty common amino acids.1,2 Additional amino acids were supplemented into the reaction mixture to determine the effect of increasing the concentrations of several amino acid groups on protein synthesis relative to the equimolar case. The sfGFP and MscL-mEGFP synthesis reactions were run using the equimolar mix in addition to mixes where the concentrations of negative, positive, polar (noncharged), aromatic, and aliphatic amino acids were doubled. sfGFP was the soluble protein target. Increasing the concentration of every amino acid mixture led to increases in average total and fractional sfGFP yield, but only doubling the concentration of negatively charged amino acids led to a statistically significant increase. Doubling the concentration of negatively charged amino acids (aspartate and glutamate) led to a ~23% increase in total yield and a ~9% increase in fractional sfGFP yield relative to the equimolar case. After analyzing the effect of amino acid concentration on soluble sfGFP, the next target was a membrane-bound target, MscL-mEGFP. For this target, I analyzed the effect of using liposomes as a membrane mimetic in addition to altering amino acid concentrations. Adding liposomes to MscL-mEGFP synthesis reactions run using the equimolar amino acid mixture led to a ~200% increase in total yield and ~120% relative increase in fractional yield, both of which were statistically significant increases. Adding DOPC liposomes and doubling the concentration of aromatic compounds led to a ~20% increase in total and fractional MscL-mEGFP yield compared to the equimolar case with DOPC liposomes. Despite statistically significant increases in MscL-mEGFP yield with the addition of liposomes and increases in the concentration of aromatic amino acids, the fractional yield of sfGFP (~37-45%) is still ~17-20 times higher than MscL-mEGFP yield (2.61%) in PUREfrex 2.1 depending on the amino acid mixture used. The gap in fractional yield between soluble and membrane protein targets indicates the need for recombinant CFPS kits optimized for synthesizing MPs. Future work will focus on analyzing whether these synthesis trends are universally applicable to groups of proteins or specific to individual proteins.2026-09-0
Branched peptide-based targeted protein degraders for the modulation of ternary complex geometry
No full text65 pagesProteolysis Targeting Chimeras (PROTACs) are heterobifunctional targeted protein degraders that selectively degrade a protein of interest (POI) by recruiting innate protein degradation machinery. The current PROTAC design landscape is limited by the use of small molecules as ligands which are difficult to design and modify and have a limited range of potential targets. Peptide-based PROTACs (PepTACs) address these limitations through their modularity, standardized synthesis, and the availability of tools—such as phage display, yeast display, and increasingly powerful computational techniques—for designing peptide ligands against nearly any protein target. Here, we aim to expand on the design of PepTACs by proposing a method to modify the linker attachment point utilizing selectively branched, peptide ligands and solid-phase peptide synthesis. As a proof-of-concept, two model branched PepTACs were synthesized and characterized against the oncoprotein known as Cell Cycle-Related and Expression-Elevated Protein in Tumor (CREPT). Lipid nanoparticle delivery of PepTACs was explored, revealing peptide hydrophobicity as a driving force for encapsulation. LNP delivery of the branched constructs into HeLa cells showed dose-dependent cellular uptake. However, a CREPT-luciferase degradation assay yielded inconclusive results regarding their intracellular activity. Circular dichroism indicates branching destabilizes central binding features. Further studies are needed to elucidate the structure-activity relationships of branched PepTACs
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