Recombination Can Lead to Spurious Results in Retroviral Transduction with Dually Fluorescent Reporter Genes

Fluorescent proteins are routinely employed as reporters in retroviral vectors. Here, we demonstrate that transduction with retroviral vectors carrying a tandem-dimer Tomato (TdTom) reporter produces two distinct fluorescent cell populations following template jumping due to a single nucleotide polymorphism between the first and second Tomato genes. Template jumping also occurs between repeated sequences in the Tomato and green fluorescent protein (GFP) genes. Thus, proper interpretation of the fluorescence intensity of transduced cells requires caution.     

Phylogeny and taxonomy of European funnel‐web spiders of the Tegenaria−Malthonica complex (Araneae: Agelenidae) based upon morphological and molecular data

The taxonomy and systematics of European house spiders, currently constituting the ill‐defined Tegenaria?Malthonica complex (including Aterigena) in the family Agelenidae, are revised. In Europe four monophyletic genera and 81 species are defined. One genus, Eratigena gen. nov. , and seven species are described as new; at species level 17 new synonyms and 20 new combinations are proposed, and the original combination of 14 species is reinstated. Five species could not be placed (incertae sedis) because of insufficient material and one taxon is regarded as ‘nomen dubium’. On the basis of a detailed morphological assessment, 88 characters were chosen for a cladistic analysis. Phylogenetically informative characters include mostly spination patterns as well as spinneret and genital structures. In addition to morphology, three gene sections [cytochrome c oxidase subunit 1 (CO1), nicotinamide adenine dinucleotide dehydrogenase subunit 1 (NADH1) 28S] were analysed. Morphological and molecular analyses were performed individually and in combination applying maximum parsimony and Bayesian tree search methods. In all resulting trees Malthonica and Tegenaria in their present composition are either polyphyletic or paraphyletic. Consequently, we redefined the two genera and erected a new genus, Eratigena gen. nov. Identification keys are provided for the European agelenid genera as well as for the European species of Tegenaria and Eratigena gen. nov. The genera and most of the constituent species are described and illustrated. The new classification has also been applied to some extra European members of the Tegenaria‐Malthonica complex resulting in additional three new synonyms, seven reversals to the original combination, and four new combinations. © 2013 The Linnean Society of London     

STATES OF DELINQUENCY: RACE AND SCIENCE IN THE MAKING OF CALIFORNIA'S JUVENILE JUSTICE SYSTEM

Literature which debates the degree of liberation or oppression associated with wearing the hijab is frequently focused on the Middle East. Two strands of feminist writing have either condemned the veil as oppressive or reframed it as liberating and as a sign of resistance. I argue that the significance of wearing the hijab in Britain is frequently missed. In Britain far from being a sign of conformity and fulfilling the function of making the wearer invisible the converse is true. The hijab is, of itself, neither liberating nor oppressive; the power relations invested in it are situational and contextual. The white Muslim women who participated in this study experience various kinds of responses from non-Muslims. At times they find themselves on the receiving end of what amounts to racial abuse. In this article I suggest that although neither “race” nor racism are fixed, they tend, nevertheless, to shift as reinventions of the same phenomena.     

Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Replicative polymerase stalling is coordinated with replicative helicase stalling in eukaryotes, but the mechanism underlying this coordination is not known. Cdc45 activates the Mcm2-7 helicase. We report here that Cdc45 from budding yeast binds tightly to long (≥ 40 nucleotides) genomic single-stranded DNA (ssDNA) and that 60mer ssDNA specifically disrupts the interaction between Cdc45 and Mcm2-7. We identified a mutant of Cdc45 that does not bind to ssDNA. When this mutant of cdc45 is expressed in budding yeast cells exposed to hydroxyurea, cell growth is severely inhibited, and excess RPA accumulates at or near an origin. Chromatin immunoprecipitation suggests that helicase movement is uncoupled from polymerase movement for mutant cells exposed to hydroxyurea. These data suggest that Cdc45-ssDNA interaction is important for stalling the helicase during replication stress.     

Ganglioside GM1-mediated Transcytosis of Cholera Toxin Bypasses the Retrograde Pathway and Depends on the Structure of the Ceramide Domain

Cholera toxin causes diarrheal disease by binding ganglioside GM1 on the apical membrane of polarized intestinal epithelial cells and trafficking retrograde through sorting endosomes, the trans-Golgi network (TGN), and into the endoplasmic reticulum. A fraction of toxin also moves from endosomes across the cell to the basolateral plasma membrane by transcytosis, thus breeching the intestinal barrier. Here we find that sorting of cholera toxin into this transcytotic pathway bypasses retrograde transport to the TGN. We also find that GM1 sphingolipids can traffic from apical to basolateral membranes by transcytosis in the absence of toxin binding but only if the GM1 species contain cis-unsaturated or short acyl chains in the ceramide domain. We found previously that the same GM1 species are needed to efficiently traffic retrograde into the TGN and endoplasmic reticulum and into the recycling endosome, implicating a shared mechanism of action for sorting by lipid shape among these pathways.     

CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777–788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer.     

Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.     

Good student questions in inquiry learning

Acquisition of scientific reasoning is one of the big challenges in education. A popular educational strategy advocated for acquiring deep knowledge is inquiry-based learning, which is driven by emerging ‘good questions’. This study will address the question: ‘Which design features allow learners to refine questions while preserving student ownership of the inquiry process?’ This design-based research has been conducted over several years with advanced high-school biology classes. The results confirm the central role of question elaboration as an interactive process that leads from vague to complex and adequate. To make this happen, the inquiry process must extend over a long time, learners and teachers should share a knowledge improvement goal, and text produced by students should be structured by question–answer pairs addressing a single concept using authentic resources. Further features are discussion with peers, teacher feedback with respect to answer elaboration and conceptual differentiation, and finally, teacher guidance that should fade out in successive inquiry cycles to ensure student responsibility.     

The Superfast Human Extraocular Myosin Is Kinetically Distinct from the Fast Skeletal IIa,IIb, and IId Isoforms

Humans express five distinct myosin isoforms in the sarcomeres of adult striated muscle (fast IIa, IId, the slow/cardiac isoform I/β, the cardiac specific isoform α, and the specialized extraocular muscle isoform). An additional isoform, IIb, is present in the genome but is not normally expressed in healthy human muscles. Muscle fibers expressing each isoform have distinct characteristics including shortening velocity. Defining the properties of the isoforms in detail has been limited by the availability of pure samples of the individual proteins. Here we study purified recombinant human myosin motor domains expressed in mouse C₂C₁₂ muscle cells. The results of kinetic analysis show that among the closely related adult skeletal isoforms, the affinity of ADP for actin·myosin (K_AD) is the characteristic that most readily distinguishes the isoforms. The three fast muscle myosins have K_AD values of 118, 80, and 55 μm for IId, IIa, and IIb, respectively, which follows the speed in motility assays from fastest to slowest. Extraocular muscle is unusually fast with a far weaker K_AD = 352 μm. Sequence comparisons and homology modeling of the structures identify a few key areas of sequence that may define the differences between the isoforms, including a region of the upper 50-kDa domain important in signaling between the nucleotide pocket and the actin-binding site.