High affinity immobilization of proteins using the CrAsH/TC tag

Protein microarrays represent important tools for biomedical analysis. We have recently described the use of the biarsenical-tetracysteine (TC) tag for the preparation of protein microarrays. The unique feature of this tag enables the site-specific immobilization of TC-containing proteins on biarsenical-modified surfaces, resulting in a fluorescence enhancement that allows the direct quantification of the immobilized proteins. Moreover, the reversibility of the binding upon incubation with large quantities of thiols permits the detachment of the proteins from the surface, thereby enabling recovery of the substrate to extend the life time of the slide. Herein, we describe our recent results that further extend the applicability of the CrAsH/TC tag to the fabrication of biochips. With this aim, the immobilization of proteins on surfaces has been investigated using two different spacers and two TC tags, the minimal TC sequence (CCPGCC) and an optimized motif (FLNCCPGCCMEP). While the minimal peptide motif enables a rapid recycling of the slide, the optimized TC sequence reveals an increased affinity due to its greater resistance to displacement by thiols. Moreover, the developed methodology was applied to the immobilization of proteins via on-chip ligation of recombinant protein thioesters

Virtual patients design and its effect on clinical reasoning and student experience : a protocol for a randomised factorial multi-centre study

Background Virtual Patients (VPs) are web-based representations of realistic clinical cases. They are proposed as being an optimal method for teaching clinical reasoning skills. International standards exist which define precisely what constitutes a VP. There are multiple design possibilities for VPs, however there is little formal evidence to support individual design features. The purpose of this trial is to explore the effect of two different potentially important design features on clinical reasoning skills and the student experience. These are the branching case pathways (present or absent) and structured clinical reasoning feedback (present or absent). Methods/Design This is a multi-centre randomised 2x2 factorial design study evaluating two independent variables of VP design, branching (present or absent), and structured clinical reasoning feedback (present or absent).The study will be carried out in medical student volunteers in one year group from three university medical schools in the United Kingdom, Warwick, Keele and Birmingham. There are four core musculoskeletal topics. Each case can be designed in four different ways, equating to 16 VPs required for the research. Students will be randomised to four groups, completing the four VP topics in the same order, but with each group exposed to a different VP design sequentially. All students will be exposed to the four designs. Primary outcomes are performance for each case design in a standardized fifteen item clinical reasoning assessment, integrated into each VP, which is identical for each topic. Additionally a 15-item self-reported evaluation is completed for each VP, based on a widely used EViP tool. Student patterns of use of the VPs will be recorded. In one centre, formative clinical and examination performance will be recorded, along with a self reported pre and post-intervention reasoning score, the DTI. Our power calculations indicate a sample size of 112 is required for both primary outcomes

Discovery and Creation: Alternative Theories of Entrepreneurial Action

As oportunidades empreendedoras existem, independentemente, das percepções dos empreendedores, esperando apenas para serem descobertas? Ou, estas oportunidades são criadas pelas ações dos empreendedores? Duas teorias, internamente, consistentes com as oportunidades empreendedoras são: teoria da criação e teoria da descoberta- as quais serão descritas. Enquanto, será sempre possível, descrever a formação de uma oportunidade particular, como exemplo, de um processo da descoberta ou da criação de oportunidade, estas duas teorias têm implicações importantes para a eficácia de uma variedade ampla de ações empreendedoras em contextos diferentes. As implicações destas teorias para sete destas ações serão descritas, acompanhadas de uma discussão sobre algumas das implicações teóricas mais amplas destas duas teorias para os campos do empreendimento e do gerenciamento estratégico

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

The quality of options in strategic decision making: a study about creativity and completeness in business decision making

A qualidade das decisões estratégicas dos empresários está diretamente relacionada à capacidade que eles demonstram para encontrar alternativas criativas quando enfrentam os problemas de suas empresas. Essas alternativas podem ser geradas intuitivamente, utilizando heurísticas. As pesquisas sobre geração de alternativas têm indicado consistentemente que as pessoas não são eficientes nessa atividade. As explicações para esse fato, contidas na literatura sobre decisão, não são conclusivas e permitem especulações a respeito. Para explorar essa questão e relacioná-la ao administrador brasileiro, foi idealizado um experimento com 174 alunos de quatro cursos de MBA para avaliar a originalidade e a completude das alternativas. O experimento e a respectiva análise basearam-se na confluência da pesquisa experimental, oriunda da psicologia cognitiva da decisão, com as visões da ciência da decisão organizacional tradicional e o novo campo de estudo das decisões intuitivas ou naturalísticas. Para mensurar a criatividade das alternativas apresentadas durante o experimento, empregou-se o conceito de árvore hierárquica, que demonstrou ser uma poderosa ferramenta para a tipologia de alternativas. O resultado desse experimento confirmou o baixo desempenho em geração de alternativas dos gerentes e, ao mesmo tempo, indicou que, provavelmente, a etapa de geração de alternativas isolada da etapa de escolha pode melhorar a qualidade das alternativas. A heurística, por sua vez, não demonstrou influenciar o conjunto de alternativas geradas. _________________________________________________________________________________________ ABSTRACT: The quality of strategic decisions of executives is directly related to the ability they have to find creative alternatives when facing business problems. These alternatives could be generated intuitively, using heuristics. On the other hand, the researches on alternatives generation have consistently indicated that people are not efficient on this duty. The argument for that, contained in the decision’s literature, is not conclusive and it allows speculation about it. To explore this issue and relate it to the Brazilian Administration, an experiment was designed for 174 students of four courses of MBA. The experiment and the analysis were resulted from the confluence between the experimental research from decision cognitive psychology with science’s vision of the traditional organizational decision and the new field of study on naturalistic or intuitive decisions. To measure the creativity of the alternatives presented during the experiment, the concept of hierarchical tree was utilized and it has proved a powerful tool to the typology of alternatives. The result of this experiment confirmed the poor performance in alternatives generation by managers and at the same time, indicated that probably, the generation of options isolated of analysis can produce better quality of alternatives. The heuristic, do not demonstrated any influence on options generated