Carbene footprinting accurately maps binding sites in protein–ligand and protein–protein interactions

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation

The ‘heritagisation’ of the British seaside resort: The rise of the ‘old penny’ arcade.

Amusement arcades have long been a key component of the British seaside resort. For almost a century, they enjoyed popularity and success and became established as a quintessential feature of the British seaside holiday. However, the advent of home-based video games along with recent gambling legislation has led to a decline of the seaside amusement arcade sector. Arcades gained a reputation as unsavoury places and their appearance and fortunes often mirrored those of the resorts in which they were located. However, over the past decade, a new variant of the seaside amusement arcade has appeared, featuring mechanical machines working on pre-decimal currency. Such ‘old penny arcades’ frequently describe themselves as museums or heritage centres and they offer an experience based on a nostalgic affection for the ‘traditional’ seaside holiday. They have appeared in the context of an increasing interest in the heritage of the British seaside resort and constitute one element of the ‘heritagisation’ of such resorts. This paper argues that such arcades can be important elements of strategies to reposition and rebrand resorts for the heritage tourism market

Growth landscape formed by perception and import of glucose in yeast

An important challenge in systems biology is to quantitatively describe microbial growth using a few measurable parameters that capture the essence of this complex phenomenon. Two key events at the cell membrane—extracellular glucose sensing and uptake—initiate the budding yeast’s growth on glucose. However, conventional growth models focus almost exclusively on glucose uptake. Here we present results from growth-rate experiments that cannot be explained by focusing on glucose uptake alone. By imposing a glucose uptake rate independent of the sensed extracellular glucose level, we show that despite increasing both the sensed glucose concentration and uptake rate, the cell’s growth rate can decrease or even approach zero. We resolve this puzzle by showing that the interaction between glucose perception and import, not their individual actions, determines the central features of growth, and characterize this interaction using a quantitative model. Disrupting this interaction by knocking out two key glucose sensors significantly changes the cell’s growth rate, yet uptake rates are unchanged. This is due to a decrease in burden that glucose perception places on the cells. Our work shows that glucose perception and import are separate and pivotal modules of yeast growth, the interaction of which can be precisely tuned and measured.National Institutes of Health (U.S.). Pioneer AwardNatural Sciences and Engineering Research Council of Canada (NSERC). Graduate Fellowshi

Polyamorous Families – Parenting Practice, Stigma and Social Regulation

As a response to the greater visibility of alternative relationship and family forms, polyamory (i.e. the practice of consensual multi-partner relationships) has recently moved to the centre of public media attention. Questions of polyamory have emerged as a major concern within law, social policy, family sociology, gender and sexuality studies. Yet certain core issues have remained underexplored. This includes the distinctive nature of polyamorous intimacy, the structure of poly household formations and the dynamics of care work within poly families. In particular, poly parenting has been subject to tabooisation and scandalisation. Governing bodies, the judiciary and educational institutions have remained largely ignorant of polyamorous relationships. Research documents the exclusions of poly families (and individuals) from access to legal provisions and protections and their common discrimination in the courts, namely in custody cases. It further highlights the discrimination of polyidentified adolescents in school and college settings and the predicament that poly families face when interacting with public institutions (including schools and kindergardens). Insights into parenting practices and the organisation of childcare is vital for understanding the transformative potential of polyamorous ways of relating. It is also important for challenging the common demonisation and stigmatisation of polyamory within conservative family politics that perceives polyamory exclusively from a harm perspective. This paper will review and critically analyse existing research on poly parenting focussing on three dimensions: (a) parenting practices, (b) social and legal discrimination, and (c) parental response to stigmatisation. The paper argues for a stronger incorporation of queer perspectives within the guiding frameworks of research into parenting in consensually non-monogamous and polyamorous relationships to highlight the transformative potential of the ‘queer bonds’ that sustain many of these practices

A synthetic circuit for selectively arresting daughter cells to create aging populations

The ability to engineer genetic programs governing cell fate will permit new safeguards for engineered organisms and will further the biological understanding of differentiation and aging. Here, we have designed, built and implemented a genetic device in the budding yeast Saccharomyces cerevisiae that controls cell-cycle progression selectively in daughter cells. The synthetic device was built in a modular fashion by combining timing elements that are coupled to the cell cycle, i.e. cell-cycle specific promoters and protein degradation domains, and an enzymatic domain which conditionally confers cell arrest. Thus, in the presence of a drug, the device is designed to arrest growth of only newly-divided daughter cells in the population. Indeed, while the engineered cells grow normally in the absence of drug, with the drug the engineered cells display reduced, linear growth on the population level. Fluorescence microscopy of single cells shows that the device induces cell arrest exclusively in daughter cells and radically shifts the age distribution of the resulting population towards older cells. This device, termed the ‘daughter arrester’, provides a blueprint for more advanced devices that mimic developmental processes by having control over cell growth and death

Receptor Sorting within Endosomal Trafficking Pathway Is Facilitated by Dynamic Actin Filaments

Early endosomes (EEs) are known to be a sorting station for internalized molecules destined for degradation, recycling, or other intracellular organelles. Segregation is an essential step in such sorting, but the molecular mechanism of this process remains to be elucidated. Here, we show that actin is required for efficient recycling and endosomal maturation by producing a motile force. Perturbation of actin dynamics by drugs induced a few enlarged EEs containing several degradative vacuoles and also interfered with their transporting ability. Actin repolymerization induced by washout of the drug caused the vacuoles to dissociate and individually translocate toward the perinuclear region. We further elucidated that cortactin, an actin-nucleating factor, was required for transporting contents from within EEs. Actin filaments regulated by cortactin may provide a motile force for efficient sorting within early endosomes. These data suggest that actin filaments coordinate with microtubules to mediate segregation in EEs

A Novel Genetic Screen Implicates Elm1 in the Inactivation of the Yeast Transcription Factor SBF

BACKGROUND: Despite extensive large scale analyses of expression and protein-protein interactions (PPI) in the model organism Saccharomyces cerevisiae, over a thousand yeast genes remain uncharacterized. We have developed a novel strategy in yeast that directly combines genetics with proteomics in the same screen to assign function to proteins based on the observation of genetic perturbations of sentinel protein interactions (GePPI). As proof of principle of the GePPI screen, we applied it to identify proteins involved in the regulation of an important yeast cell cycle transcription factor, SBF that activates gene expression during G1 and S phase. METHODOLOGY/PRINCIPLE FINDINGS: The principle of GePPI is that if a protein is involved in a pathway of interest, deletion of the corresponding gene will result in perturbation of sentinel PPIs that report on the activity of the pathway. We created a fluorescent protein-fragment complementation assay (PCA) to detect the interaction between Cdc28 and Swi4, which leads to the inactivation of SBF. The PCA signal was quantified by microscopy and image analysis in deletion strains corresponding to 25 candidate genes that are periodically expressed during the cell cycle and are substrates of Cdc28. We showed that the serine-threonine kinase Elm1 plays a role in the inactivation of SBF and that phosphorylation of Elm1 by Cdc28 may be a mechanism to inactivate Elm1 upon completion of mitosis. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that GePPI is an effective strategy to directly link proteins of known or unknown function to a specific biological pathway of interest. The ease in generating PCA assays for any protein interaction and the availability of the yeast deletion strain collection allows GePPI to be applied to any cellular network. In addition, the high degree of conservation between yeast and mammalian proteins and pathways suggest GePPI could be used to generate insight into human disease

Poly Economics-Capitalism, Class, and Polyamory

Academic research and popular writing on nonmonogamy and polyamory has so far paid insufficient attention to class divisions and questions of political economy. This is striking since research indicates the significance of class and race privilege within many polyamorous communities. This structure of privilege is mirrored in the exclusivist construction of these communities. The article aims to fill the gap created by the silence on class by suggesting a research agenda which is attentive to class and socioeconomic inequality. The paper addresses relevant research questions in the areas of intimacy and care, household formation, and spaces and institutions and advances an intersectional perspective which incorporates class as nondispensable core category. The author suggests that critical research in the field can stimulate critical self-reflexive practice on the level of community relations and activism. He further points to the critical relevance of Marxist and Postmarxist theories as important resources for the study of polyamory and calls for the study of the contradictions within poly culture from a materialist point of view. © 2013 Springer Science+Business Media New York

Facing polyamorous lives: translation and validation of the attitudes towards polyamory scale in a Portuguese sample

© 2018, © 2018 College of Sexual and Relationship Therapists. Consensual Non-Monogamies (CNMs) have been receiving the attention of academics, however attitudes towards polyamory (ATP) are still a new field of research. We aim at studying the reliability and validity of the ATP scale in a Portuguese sample. The present analysis utilized cross-sectional data from 609 volunteers that completed the online survey with a socio-demographic questionnaire; the ATP scale; the Modern Heterosexism subscale of the Multidimensional Scale of Attitudes Toward Lesbians and Gay Men; the Rosenberg Self-Esteem Scale; and the Willingness to Engage in Non-Monogamy Scale. After randomly splitting the sample (1:1) for cross-validation purposes, Exploratory (EFA) and Confirmatory factor analysis (CFA) were conducted using Principal Axis Factoring (PAF) and Maximum Likelihood Estimation, respectively. Cronbach's alpha and item-total correlations were used to determine the internal consistency of the scale. Feasibility and acceptability were examined in terms of missing values, floor and ceiling effects. The ATP proved to be reliable (Cronbach’s alpha >.80). In the current study, the final structure of the scale, proved through CFA, included 6 items aggregated in a single factor. This final 6-item measure proved to have convergent, divergent and concurrent criterion validity. Implications for research in polyamory and CNMs are discussed

Sexual Priming, Gender Stereotyping, and Likelihood to Sexually Harass: Examining the Cognitive Effects of Playing a Sexually-Explicit Video Game

The present study examines the short-term cognitive effects of playing a sexually explicit video game with female “objectification” content on male players. Seventy-four male students from a university in California, U.S. participated in a laboratory experiment. They were randomly assigned to play either a sexually-explicit game or one of two control games. Participants’ cognitive accessibility to sexual and sexually objectifying thoughts was measured in a lexical decision task. A likelihood-to-sexually-harass scale was also administered. Results show that playing a video game with the theme of female “objectification” may prime thoughts related to sex, encourage men to view women as sex objects, and lead to self-reported tendencies to behave inappropriately towards women in social situations