DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K_d = 68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.     

Mathematical Models for Cell Migration with Real-Time Cell Cycle Dynamics

The fluorescent ubiquitination-based cell cycle indicator, also known as FUCCI, allows the visualization of the G1 and S/G2/M cell cycle phases of individual cells. FUCCI consists of two fluorescent probes, so that cells in the G1 phase fluoresce red and cells in the S/G2/M phase fluoresce green. FUCCI reveals real-time information about cell cycle dynamics of individual cells, and can be used to explore how the cell cycle relates to the location of individual cells, local cell density, and different cellular microenvironments. In particular, FUCCI is used in experimental studies examining cell migration, such as malignant invasion and wound healing. Here we present, to our knowledge, new mathematical models that can describe cell migration and cell cycle dynamics as indicated by FUCCI. The fundamental model describes the two cell cycle phases, G1 and S/G2/M, which FUCCI directly labels. The extended model includes a third phase, early S, which FUCCI indirectly labels. We present experimental data from scratch assays using FUCCI-transduced melanoma cells, and show that the predictions of spatial and temporal patterns of cell density in the experiments can be described by the fundamental model. We obtain numerical solutions of both the fundamental and extended models, which can take the form of traveling waves. These solutions are mathematically interesting because they are a combination of moving wavefronts and moving pulses. We derive and confirm a simple analytical expression for the minimum wave speed, as well as exploring how the wave speed depends on the spatial decay rate of the initial condition.     

Evolutionary Genomics of Immunoglobulin-Encoding Loci in Vertebrates

Immunoglobulins (or antibodies) are an essential element of the jawed vertebrate adaptive immune response system. These molecules have evolved over the past 500 million years and generated highly specialized proteins that recognize an extraordinarily large number of diverse substances, collectively known as antigens. During vertebrate evolution the diversification of the immunoglobulin-encoding loci resulted in differences in the genomic organization, gene content, and ratio of functional genes and pseudogenes. The tinkering process in the immunoglobulin-encoding loci often gave rise to lineage-specific characteristics that were formed by selection to increase species adaptation and fitness. Immunoglobulin loci and their encoded antibodies have been shaped repeatedly by contrasting evolutionary forces, either to conserve the prototypic structure and mechanism of action or to generate alternative and diversified structures and modes of function. Moreover, evolution favored the development of multiple mechanisms of primary and secondary antibody diversification, which are used by different species to effectively generate an almost infinite collection of diverse antibody types. This review summarizes our current knowledge on the genomics and evolution of the immunoglobulin-encoding loci and their protein products in jawed vertebrates.     

Human embryonal epithelial cells of the developing small intestinal crypts can express the Hodgkin-cell associated antigen Ki-1 (CD30) in spontaneous abortions during the first trimester of gestation

Background  

Ki-1 (CD30) antigen expression is not found on peripheral blood cells but its expression can be induced in vitro on T and B lymphocytes by viruses and lectins. Expression of CD30 in normal tissues is very limited, being restricted mainly to a subpopulation of large lymphoid cells; in particular, cells of the recently described anaplastic large cell lymphoma (ALCL), the Reed-Sternberg (RS) cells of Hodgkin's lymphoma and scattered large parafollicular cells in normal lymphoid tissues. More recent reports have described CD30 expression in non-hematopoietic and malignant cells such as cultured human macrophages, human decidual cells, histiocytic neoplastic cells, mesothelioma cells, embryonal carcinoma and seminoma cells.     

A meta-analytic review of the association between cortisol reactivity in response to a stressor and attention-deficit hyperactivity disorder

A substantial literature suggests that abnormal cortisol reactivity may be a vulnerability for deleterious mental health outcomes, including ADHD. ADHD has been linked with difficulty in emotion regulation and increased risk of experiencing stressors, both of which may be related to psychobiological abnormalities (e.g., abnormal cortisol reactivity). Research has been mixed regarding the association between cortisol reactivity and ADHD. Therefore, the present meta-analytic review (k = 12) sought to quantify this association and review the relevant methodological issues and theoretical implications of this area of research. Overall, no effect was found between cortisol reactivity and ADHD (r = 0), although significant heterogeneity in the analyses suggested that there might be moderators of this association, if one does exist. Results highlight the importance of addressing limitations of the current literature on cortisol reactivity and ADHD and exploring additional indices of emotion regulation that may be associated with ADHD. Implications for future research efforts are discussed.     

Age-dependent strategies related to lionfish activities in the Mexican Caribbean

Lionfish are successful invasive predators in the Caribbean region and inhabit a large range of habitats. Our study in the Caribbean has focused on the relationships between the biological characteristics of lionfish particularly their size, their activities and use of those different habitats. In this study, we observed a high number of lionfish individuals, focusing on the behavioural activities and biological traits in relation to different habitats and environmental characteristics. We monitored 793 individuals, recording their activities, biological traits, and habitat characteristics. Our results report that lionfish are not solitary, but frequently form groups for many activities. We provide evidence of differences between lionfish habitat use according to activity, and the size of individual fish. Considering the size is correlated with age, coral reefs appear to be the preferred habitat of older individuals, whereas the youngest lionfish use a diversity of habitats, ranging from mangroves to coral reefs. In addition, this study suggests that predation of lionfish is age-dependent strategy, and depends on time and the tone of the environment. Lionfish do not only use the head-down posture to catch prey but also horizontal and head-up postures. The youngest lionfish hunt mainly in dark areas and during the night while the older fish were observed hunting mostly during the day and in clear areas. These new aspects of lionfish ecology and behaviour are discussed in light of their invasive success.     

Biochemical characterization of the interaction between HspA1A and phospholipids

Seventy-kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis. Apart from their indispensable roles in protein homeostasis, specific Hsp70s localize at the plasma membrane and bind to specific lipids. The interaction of Hsp70s with lipids has direct physiological outcomes including lysosomal rescue, microautophagy, and promotion of cell apoptosis. Despite these essential functions, the Hsp70-lipid interactions remain largely uncharacterized. In this study, we characterized the interaction of HspA1A, an inducible Hsp70, with five phospholipids. We first used high concentrations of potassium and established that HspA1A embeds in membranes when bound to all anionic lipids tested. Furthermore, we found that protein insertion is enhanced by increasing the saturation level of the lipids. Next, we determined that the nucleotide-binding domain (NBD) of the protein binds to lipids quantitatively more than the substrate-binding domain (SBD). However, for all lipids tested, the full-length protein is necessary for embedding. We also used calcium and reaction buffers equilibrated at different pH values and determined that electrostatic interactions alone may not fully explain the association of HspA1A with lipids. We then determined that lipid binding is inhibited by nucleotide-binding, but it is unaffected by protein-substrate binding. These results suggest that the HspA1A lipid-association is specific, depends on the physicochemical properties of the lipid, and is mediated by multiple molecular forces. These mechanistic details of the Hsp70-lipid interactions establish a framework of possible physiological functions as they relate to chaperone regulation and localization.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0636-6) contains supplementary material, which is available to authorized users.