A nanobody-based strategy for rapid and scalable purification of human protein complexes.

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENPEuB. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.

Exosomes are secreted at similar densities by M21 and PC3 human cancer cells and show paclitaxel solubility.

Exosomes are cell-secreted vesicles less than ≈150 nm in size that contain gene-encoding and gene-silencing RNA and cytosolic proteins with roles in intercellular communication. Interest in the use of exosomes as targeted drug delivery vehicles has grown since it was shown that they can bind specific cells and deliver intact genetic material to the cytosol of target cells. We isolated extracellular vesicles (EVs), consisting of a mixture of exosomes and microvesicles, from prostate (PC3) and melanoma (M21) cancer cell lines using serial ultracentrifugation. Interrogation via western blot analysis confirmed enrichment of CD63, a widely recognized EV surface protein, in the EV pellet from both cell lines. Nanoparticle tracking analysis (NTA) of EV pellets revealed that the two cell lines produced distinct vesicle size profiles in the ≈30 nm to ≈400 nm range. NTA further showed that the fraction of exosomes to all EVs was constant, suggesting cellular mechanisms that control the fraction of secreted vesicles that are exosomes. Transmission electron microscopy (TEM) images of the unmodified PC3 EVs showed vesicles with cup-like (i.e., nanocapsule) and previously unreported prolate morphologies. The observed non-spherical morphologies for dehydrated exosomal vesicles (size ≈30-100 nm) are most likely related to the dense packing of proteins in exosome membranes. Solubility phase diagram data showed that EVs enhanced the solubility of paclitaxel (PTX) in aqueous solution compared to a water-only control. Combined with their inherent targeting and cytosol delivery properties, these findings highlight the potential advantages of using exosomes as chemotherapeutic drug carriers in vivo.

Building Blocks of Psychology: on Remaking the Unkept Promises of Early Schools.

The appeal and popularity of "building blocks", i.e., simple and dissociable elements of behavior and experience, persists in psychological research. We begin our assessment of this research strategy with an historical review of structuralism (as espoused by E. B. Titchener) and behaviorism (espoused by J. B. Watson and B. F. Skinner), two movements that held the assumption in their attempts to provide a systematic and unified discipline. We point out the ways in which the elementism of the two schools selected, framed, and excluded topics of study. After the historical review, we turn to contemporary literature and highlight the persistence of research into building blocks and the associated framing and exclusions in psychological research. The assumption that complex categories of human psychology can be understood in terms of their elementary components and simplest forms seems indefensible. In specific cases, therefore, reliance on the assumption requires justification. Finally, we review alternative strategies that bypass the commitment to building blocks.

Selective attention, diffused attention, and the development of categorization.

How do people learn categories and what changes with development? The current study attempts to address these questions by focusing on the role of attention in the development of categorization. In Experiment 1, participants (adults, 7-year-olds, and 4-year-olds) were trained with novel categories consisting of deterministic and probabilistic features, and their categorization and memory for features were tested. In Experiment 2, participants' attention was directed to the deterministic feature, and in Experiment 3 it was directed to the probabilistic features. Attentional cueing affected categorization and memory in adults and 7-year-olds: these participants relied on the cued features in their categorization and exhibited better memory of cued than of non-cued features. In contrast, in 4-year-olds attentional cueing affected only categorization, but not memory: these participants exhibited equally good memory for both cued and non-cued features. Furthermore, across the experiments, 4-year-olds remembered non-cued features better than adults. These results coupled with computational simulations provide novel evidence (1) pointing to differences in category representation and mechanisms of categorization across development, (2) elucidating the role of attention in the development of categorization, and (3) suggesting an important distinction between representation and decision factors in categorization early in development. These issues are discussed with respect to theories of categorization and its development.

Linguistic labels, dynamic visual features, and attention in infant category learning.

How do words affect categorization? According to some accounts, even early in development words are category markers and are different from other features. According to other accounts, early in development words are part of the input and are akin to other features. The current study addressed this issue by examining the role of words and dynamic visual features in category learning in 8- to 12-month-old infants. Infants were familiarized with exemplars from one category in a label-defined or motion-defined condition and then tested with prototypes from the studied category and from a novel contrast category. Eye-tracking results indicated that infants exhibited better category learning in the motion-defined condition than in the label-defined condition, and their attention was more distributed among different features when there was a dynamic visual feature compared with the label-defined condition. These results provide little evidence for the idea that linguistic labels are category markers that facilitate category learning.

The development of categorization: effects of classification and inference training on category representation.

Does category representation change in the course of development? And if so, how and why? The current study attempted to answer these questions by examining category learning and category representation. In Experiment 1, 4-year-olds, 6-year-olds, and adults were trained with either a classification task or an inference task and their categorization performance and memory for items were tested. Adults and 6-year-olds exhibited an important asymmetry: they relied on a single deterministic feature during classification training, but not during inference training. In contrast, regardless of the training condition, 4-year-olds relied on multiple probabilistic features. In Experiment 2, 4-year-olds were presented with classification training and their attention was explicitly directed to the deterministic feature. Under this condition, their categorization performance was similar to that of older participants in Experiment 1, yet their memory performance pointed to a similarity-based representation, which was similar to that of 4-year-olds in Experiment 1. These results are discussed in relation to theories of categorization and the role of selective attention in the development of category learning.

The role of words in cognitive tasks: what, when, and how?

The current review focuses on how exposure to linguistic input, and count nouns in particular, affect performance on various cognitive tasks, including individuation, categorization and category learning, and inductive inference. We review two theoretical accounts of effects of words. Proponents of one account argue that words have top-down effects on cognitive tasks, and, as such, function as supervisory signals. Proponents of the other account suggest that early in development, words, just like any other perceptual feature, are first and foremost part of the stimulus input and influence cognitive tasks in a bottom-up, non-supervisory fashion. We then review evidence supporting each account. We conclude that, although much research is needed, there is a large body of evidence indicating that words start out like other perceptual features and become supervisory signals in the course of development.