Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus aureus Enterotoxin B

<p>Abstract</p> <p>Background</p> <p>Camelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays.</p> <p>Results</p> <p>Here we describe the isolation of an sdAb against <it>Staphyloccocus aureus </it>enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95°C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL.</p> <p>Conclusion</p> <p>The anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in antibody-based toxin detection technologies.</p

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

The interplay of bottom-up and top-down mechanisms in visual guidance during object naming

An ongoing issue in visual cognition concerns the roles played by low-and high-level information in guiding visual attention, with current research remaining inconclusive about the interaction between the two. In this study, we bring fresh evidence into this long-standing debate by investigating visual saliency and contextual congruency during object naming (Experiment 1), a task in which visual processing interacts with language processing. We then compare the results of this experiment to data of a memorization task using the same stimuli (Experiment 2). In Experiment 1, we find that both saliency and congruency influence visual and naming responses and interact with linguistic factors. In particular, incongruent objects are fixated later and less often than congruent ones. However, saliency is a significant predictor of object naming, with salient objects being named earlier in a trial. Furthermore, the saliency and congruency of a named object interact with the lexical frequency of the associated word and mediate the time-course of fixations at naming. In Experiment 2, we find a similar overall pattern in the eye-movement responses, but only the congruency of the target is a significant predictor, with incongruent targets fixated less often than congruent targets. Crucially, this finding contrasts with claims in the literature that incongruent objects are more informative than congruent objects by deviating from scene context and hence need a longer processing. Overall, this study suggests that different sources of information are interactively used to guide visual attention on the targets to be named and raises new questions for existing theories of visual attention