1,720,953 research outputs found
The characterisation of a recombinant cytosolic shuttle based on ricin toxin
Full text linkCytosolic access is rate limiting for potential therapeutics such as antisense agents. In nature, several entities deliver macromolecules to the cytosol, one example being ricin toxin (RT). In this thesis, a cytosolic drug delivery system based upon attenuated RT was characterised. The toxicological consequences of mutating RT A chain (RTAC) amino acids: Glu177, Ala178, Ala179, Arg180, Phe181, Gln182 to Gly (mut) or deleting (del) them entirely, was evaluated. Epitopes (i.e., 6His, HA) were also incorporated onto either the C- or N-terminus of the RTAC. The resultant plasmid constructs were sequenced, mapped and expressed in E.coli BL21*DE3. A plasmid encoding RT B chain (RTBC) was also characterised, mapped and expressed in E.coli BL21*DE3. Recombinant proteins were affinity purified from bacterial lysate and were soluble. Proteins were detected at approximately the predicted molecular weight using commercial antibodies specific for RTAC, RTBC or the epitopes incorporated into the constructs (i.e. 6His or V5). Optimal culture conditions were obtained by monitoring protein expression in 1.5mL bacterial cultures. Expression levels were established using Western blotting and immuno-detection (using a 6His specific primary antibody). The optimal culture conditions for RTAC with a C-terminal 6His and HA epitope (clone 175) and RTAC with amino acids 177-182 mutated to Gly (and also containing a C-terminal 6His, HA and Lys, Asp, Glu, Leu (KDEL) motif) (clone 189), were found to be 2h pre-induction incubation, induction (with 0.25mM IPTG) and a 4h post-induction incubation, cultured at 37oC. Upon further characterisation, recombinant RTBC (rRTBC) was found not to be lectinic, unlike commercial RTBC (cRTBC). The toxicity of RTAC mutants was characterised in vitro (B16 and Vero cells) using the MTT assay. Using the B16 cell model, at concentrations up to10g/mL, no IC50 values could be established for rRTAC (clone 189). The published wild type RTAC IC50 was 1.4g/mL in B16 cells. The aforementioned rRTAC (clone 189) was re-associated with cRTBC to form a preparation containing enriched heterodimers. Heterodimeric rRTAC (clone 189) and cRTBC did not show any cytotoxicity in B16 cells at RTAC concentrations of up to 1g/mL. The toxicity of heterodimeric rRTAC (clone 189) and cRTBC was measured in Vero cells and the IC50 (0.39g/mL), although statistically different (P<0.001) from cRTBC associated with cRTAC (at concentrations between 0.01 and 100 ng/mL) may be attributed to cRTBC. It was critical to know if the reduced toxicity of the rRTAC (clone 189) - cRTBC, constructs relative to RT holotoxin, was attributable to the RTAC being unable to access the cytosol. Sub-cellular fractionation was performed using Vero cells and the majority of the immuno-reactive rRTAC (clone 189) was detected in a crude cytosolic fraction after 4h. Having ascertained that the mutations introduced into RTAC limit the toxicity of this molecule, the RT based drug delivery system was compared with a previously well-characterised polymer based drug delivery system. In order to make this comparison, a recombinant cargo protein was required. As gelonin (Gel) had been previously used as a cargo for poly(amidoamine) (PAA) mediated cytosolic delivery, three recombinant gelonin analogues were produced. The first was designated mature gelonin (mGel), the second was pre-gelonin with an additional C-terminal cysteine residue (Gel+Cys) and the third contained pre-gelonin sequence with no additional cysteine residue (Gel-Cys). The Gel-Cys construct was necessary to control for any toxicity limiting effects being exerted by additional C-terminal sequence (found on pre-gelonin). The three gelonin constructs were successfully cloned, sequenced and expressed in E.coli BL21*DE3. All of the recombinant gelonin constructs were detected using commercial antibodies (anti-6His and V5). The cytotoxicity of enriched recombinant gelonin proteins was compared with commercial gelonin (cGel). All of the gelonin preparations were non-toxic up to a concentration of 10μg/mL in B16 cells. Gelonin was then used to evaluate two things: 1) the efficiency of PAA mediated delivery relative to cRTBC and 2) the effect of the inclusion of dithiopyridine monomers upon PAA mediated Gel delivery. The PAA FF103 mediated an increase in cytotoxicity when applied to B16 cells in conjunction with a sub-toxic concentration of either with cGel or rGel (i.e. at a Gel concentration of 1.4μg/mL). This indicated that Gel was delivered into the cytosol by PAA FF103. At 10μg/mL of polymer, 93.75% cell viability was recorded and this was reduced to 16% when 1.4μg/mL cGel was added to an equivalent concentration of PAA FF103 (a statistically significant difference (P<0.001)). An investigation into the trafficking of the FITC-conjugated PAAs, ISA1-FITC and ISA23-FITC, supported PAA mediated cytosolic delivery. In this experiment, ISA1-FITC and ISA23-FITC polymers were non-toxic up to a concentration of 3mg/mL in both Vero and B16 cells and the accumulation of ISA1-FITC in the nucleus was observed after 5h. No increase in cGel toxicity was documented after it was mixed with cRTBC relative to cGel delivery mediated by PAAs. Further, despite being able to ablate RTAC toxicity, cRTBC toxicity remains a problem. Lack of lectinic activity of rRTBC (clone 204) suggests that this is unlikely to be resolved, which limits the application of this delivery system
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
R16. Formulation and Evaluation of Doxorubicin HCl Nanoliposomes by Ethanol Injection Method
Full text linkCorresponding author (Pharmaceutics and Drug delivery): Arun Kumar Kotha, [email protected]://egrove.olemiss.edu/pharm_annual_posters/1015/thumbnail.jp
Variations on the Author
Full text link“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
Full text linkWe 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
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe 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
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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