1,720,973 research outputs found
Role of histidine 148 in stability and dynamics of a highly fluorescent GFP variant
No full textThe armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pKa values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein
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
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
Analisi della saliva di neonato sano e infetto: il ruolo di proteine e lipidi come potenziali strumenti diagnostici di infiammazione
No full textWe have carried out a discovery proteomics investigation aimed at identifying disease biomarkers present in saliva, and, more specifically, early biomarkers of inflammation. The proteomic characterization of saliva is possible due to the straightforward and non-invasive sample collection that allows repetitive analyses for pharmacokinetic studies. These advantages are particularly relevant in the case of newborn patients. The study was carried out with samples collected during the first 48 hours of life of the newborns according to an approved Ethic Committee procedure.
In particular, the salivary samples were collected from healthy and infected (n=1) newborns. Proteins were extracted through cycles of sonication, precipitated in ice cold acetone, resuspended and resolved by 2D-electrophoresis. MALDI TOF/TOF mass spectrometry analysis was performed for each spot obtaining the proteins’ identifications. Then we compared healthy newborn salivary proteome and an infected newborn salivary proteome in order to investigate proteins differently expressed in inflammatory condition. In particular the protein alpha-1-antitrypsin (A1AT), correlated with inflammation, was detected differently expressed in the infected newborn saliva. Therefore, in the second part of the project we aimed to develop a robust LC-MS based method that identifies and quantifies this inflammatory protein within saliva that might represent the first relevant step to diagnose a condition of inflammation with a no-invasive assay. The same LC-MS method is also useful to investigate the presence of the F allelic variant of the A1AT in biological samples, which is correlated with the onset of pulmonary diseases. In the last part of the work we analysed newborn saliva samples in order to investigate how phospholipids and mediators of inflammation (eicosanoids) are subject to variations under inflammatory conditions and a trend was observed in lysophosphatidylcholines composition according to the inflammatory conditions.Abbiamo svolto un'indagine proteomica volta ad individuare biomarker di infiammazione presenti nella saliva e, più in particolare, i biomarker dei primi stadi di infiammazione. La caratterizzazione proteica della saliva è possibile grazie alla semplice e non invasiva raccolta del campione, che permette analisi ripetitive per studi farmacocinetici. Questi vantaggi sono particolarmente rilevanti nel caso di pazienti neonati. In particolare, lo studio è stato effettuato con campioni raccolti durante le prime 48 ore di vita dei neonati mediante una procedura approvata da un comitato etico.
I campioni di saliva sono stati raccolti da neonati sani e infetti. Le proteine sono state estratte attraverso cicli di sonicazione, precipitate a freddo con acetone, risospese ed analizzate con la tecnica della 2D-elettroforesi. L’analisi di spettrometria di massa MALDI TOF / TOF è stata effettuata per ogni spot ottenendo l’identificazione delle proteine. Successivamente abbiamo confrontato il proteoma di neonato sano con quello di neonato infetto al fine di indagare le proteine differentemente espresse in condizione infiammatoria. In particolare, la proteina alfa-1-antitripsina (A1AT), correlata con l'infiammazione, è stata rilevata espressa in modo diverso nella saliva di neonato infetto. Pertanto, nella seconda parte del progetto abbiamo sviluppato un robusto metodo basato sull’LC-MS per identificare e quantificare questa proteina infiammatoria all'interno saliva, che potrebbe rappresentare il primo rilevante passo per diagnosticare una condizione di infiammazione nei neonati, con un saggio non invasivo. Lo stesso metodo LC-MS è anche utile per indagare la presenza della variante allelica F della A1AT in campioni biologici, che è correlato con l'insorgenza di malattie polmonari. Nell'ultima parte del lavoro abbiamo analizzato campioni di saliva di neonati al fine di indagare come fosfolipidi e mediatori dell'infiammazione (eicosanoidi) sono soggetti a variazioni in condizioni infiammatorie e una trend è stato osservato per le lisofosfatidilcoline in condizioni infiammatorie
Il ruolo della MicroScale Thermophoresis (MST) nello sviluppo di inibitori di chinasi
No full textMicroScale Thermophoresis (MST) is one of the biophysical techniques commonly used for the characterization of ligand/protein interactions. The phenomenon of "thermophoresis" is the directed movement of molecules along a temperature gradient generated by an IR laser and MST can detect changes in charge, size, and hydration shell or conformation of a biomolecular complex caused by the interaction between a target protein and the cognate ligand. This phenomenon can be quantified by titrating the ligand to obtain a binding curve from which the dissociation constant (KD) can be derived. Even though the main application of MST is to determine binding parameters, the technique can also be employed to gain insights into other aspects of protein interactions such as stoichiometry, conformational states, time dependency, selectivity over mutations, and thermodynamics.
In this project different types of interactions were investigated by using two model protein kinases of pharmaceutical interest, EGFR (Epidermal Growth Factor Receptor) and ROCK (Rho-associated protein kinase) in the presence of their well-known inhibitors. The main purpose was to investigate MST ability to characterize various binding modes with a particular emphasis on potent inhibitors, slow binders, covalent binders, and allosteric binders.
First, EGFR system enabled the characterization of potent inhibitors while facing their intrinsic limitation of resulting in tight binding experimental conditions. Second, a time dependence analysis revealed the MST potential for describing conformational changes in protein kinases, whereas a targeted stoichiometry experiments facilitated the identification of various protein states as well as a difference in the propensity of Type I and Type II inhibitors to bind them. Finally, an alternative method to Jump Dilution for differentiating reversible and irreversible inhibitors was developed using MST.
ROCK system, on the other hand, has been used to differentiate between orthosteric and allosteric binders by combining orthogonal approaches of competition and direct binding assays. Furthermore, an analysis of the best labeling conditions revealed differences between the two examined allosteric compounds, indicating a possible distinct interaction mechanism due to their different sensitivity to the dye position on the protein.
These results enabled the development of a wide range of knowledge in the field of protein kinase interactions, providing a suitable background for future and unknown interaction systems
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
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