In animals, collective cell migration is critical during development and adult life for repairing organs. It remains, however, poorly understood compared with single-cell migration. The polymerization of branched actin by the RAC1-WAVE-Arp2/3 pathway is established to power membrane protrusions at the front of migrating cells, but also to maintain cell junctions in epithelial monolayers. Here, novel regulators of collective cell migration are identified using a two-pronged approach: candidates are extracted from publicly available RAC1-WAVE-Arp2/3 dependency maps and screened in a second step using CRISPR/Cas9 genetic inactivation. In a wound healing assay, PKN2 knockout (KO) MCF10A cells display decreased collective migration due to destabilization of adherens junctions, whereas MOB4 KO cells display increased collective migration with a loss of migration orientation. Upon wound healing, PKN2 relocalizes to lateral junctions and maintains coordinated migration in the monolayer, whereas MOB4 relocalizes to the front edge of leader and follower cells collectively migrating toward the wound. The role of MOB4 in controlling collective migration requires YAP1, since MOB4 KO cells fail to activate YAP1, and their phenotype is rescued by constitutively active YAP1. Together, these findings reveal two complementary activities required for coordinating cells in collective migration.

Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here, we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI-P878A) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin-Arp2/3 interaction participates in cell-cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density.

The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knockdown (KD) or knockout (KO), we combine the inactivation of the Arp2/3 inhibitory protein arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit CYFIP2, all of which enhance the polymerization of cortical branched actin. Inactivation of the three negative regulators of cortical branched actin increases migration persistence of human breast MCF10A cells and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO cells, but not in triple KD cells, the 'super-migrator' phenotype was associated with a heterogenous downregulation of vimentin (VIM) expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells largely restored normal persistence of single cell migration, suggesting that vimentin downregulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression.

Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin-Arp2/3 interaction participates in cell-cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density.

Whole exome sequencing of invasive mammary carcinomas revealed the association of mutations in PTEN and ZFHX3 tumor suppressor genes (TSGs). We generated single and combined PTEN and ZFHX3 knock-outs (KOs) in the immortalized mammary epithelial cell line MCF10A to study the role of these genes and their potential synergy in migration regulation. Inactivation of PTEN, but not ZFHX3, induced the formation of large colonies in soft agar. ZFHX3 inactivation in PTEN KO, however, increased colony numbers and normalized their size. Cell migration was affected in different ways upon PTEN and ZFHX3 KO. Inactivation of PTEN enhanced coordinated cell motility and thus, the collective migration of epithelial islets and wound healing. In contrast, ZFHX3 knockout resulted in the acquisition of uncoordinated cell movement associated with the appearance of immature adhesive junctions (AJs) and the increased expression of the mesenchymal marker vimentin. Inactivation of the two TSGs thus induces different stages of partial epithelial-to-mesenchymal transitions (EMT). Upon double KO (DKO), cells displayed still another motile state, characterized by a decreased coordination in collective migration and high levels of vimentin but a restoration of mature linear AJs. This study illustrates the plasticity of migration modes of mammary cells transformed by a combination of cancer-associated genes.

Dynamic contacts between cells within the developing neuroepithelium are poorly understood but play important roles in cell and tissue morphology and cell signalling. Here, using live-cell imaging and electron microscopy we reveal multiple protrusive structures in neuroepithelial apical endfeet of the chick embryonic spinal cord, including sub-apical protrusions that extend laterally within the tissue, and observe similar structures in human neuroepithelium. We characterise the dynamics, shape and cytoskeleton of these lateral protrusions and distinguish them from cytonemes, filopodia and tunnelling nanotubes. We demonstrate that lateral protrusions form a latticework of membrane contacts between non-adjacent cells, depend on actin but not microtubule dynamics, and provide a lamellipodial-like platform for further extending fine actin-dependent filipodia. We find that lateral protrusions depend on the actin-binding protein WAVE1 (also known as WASF1): misexpression of mutant WAVE1 attenuated protrusion and generated a round-ended apical endfoot morphology. However, this did not alter apico basal cell polarity or tissue integrity. During normal neuronal delamination, lateral protrusions were withdrawn, but precocious protrusion loss induced by mutant WAVE1 was insufficient to trigger neurogenesis. This study uncovers a new form of cell-cell contact within the developing neuroepithelium, regulation of which prefigures neuronal delamination. This article has an associated First Person interview with the first author of the paper.

A model of practice is a professional framework that guides practitioners and is informed by and integrates the core concepts, theories, tools, interventions and elements of work in a profession. A multi-disciplinary team contributed to the development of an Extension Model of Practice. Using a mixed methods research design, data were gathered from a) semistructured interviews (n=206) and a survey (n=90) with extension practitioners, b) six codesign workshops with extension agents (n=88), c) three semi-structured interviews with farmers (n=60) and extension leaders. The resultant model conceptualises the helping process of extension and the way extension professionals engage with and support farmers. A suitable support system comprising targeted awareness sessions and training modules is required to sustain its implementation. The model supports early-career professionals and guides servicedelivery and the helping process for all extension practitioners to better address the imperative for greater practice change in agriculture in partnership with farmers.

Unicellular protozoa that encyst individually upon starvation evolved at least eight times into organisms that instead form multicellular fruiting bodies with spores. The Dictyostelia are the largest and most complex group of such organisms. They can be subdivided into 4 major groups, with many species in groups 1–3 having additionally retained encystment. To understand fitness differences between spores and cysts, we measured long-term survival of spores and cysts under climate-mimicking conditions, investigated spore and cyst ultrastructure, and related fitness characteristics to species ecology. We found that spores and cysts survived 22 °C equally well, but that spores survived wet and dry frost better than cysts, with group 4 spores being most resilient. Spore walls consist of three layers and those of cysts of maximally two, while spores were also more compacted than cysts, with group 4 spores being the most compacted. Group 4 species were frequently isolated from arctic and alpine zones, which was rarely the case for group 1–3 species. We inferred a fossil-calibrated phylogeny of Dictyostelia, which showed that its two major branches diverged 0.52 billion years ago, following several global glaciations. Our results suggest that Dictyostelium multicellular sporulation was a likely adaptation to a cold climate.

In 1918, R.A.Fisher published, in Transactions of the Royal Society of Edinburgh (vol 52: 399–433), his great paper on correlations between relatives on the supposition of Mendelian inheritance. He had completed the paper a couple of years earlier and tried to have it published by the Royal Society of London Unfortunately, the referees, the leading biometrician and the leading Mendelian in England were quite unimpressed by the paper, so Fisher had to find another publisher (and financial support) in order for the paper to appear. Background can be found in J.H.Bennett's 1983 book of Fisher's correspondence, Natural selection, heredity and eugenics. What did Fisher set out to do? As a member of the Eugenics Society, he was concerned that apparently less able people had more children than average, and that this might be inherited. Biometricians had estimated correlations between several classes of relatives. Fisher wanted to show that the correlations between relatives which had been found in human data could be readily accounted for by assuming that they were controlled by a large number of genes, each of small effect, inherited in Mendelian fashion. He made definitions of additive, dominance and epistatic effects and introduced the variance as the appropriate measure of the amount of variability in a population. He then proposed the analysis of the variance into components of different origin, and showed how the correlations between relatives depended on different combinations of these components. He also took into account the way in which assortative mating affected the correlation, using a particular model of assortative mating which allowed for possible different causes of correlation between mates. His concept of the analysis of variance introduced in this paper was of major importance in areas of statistics outside of genetics. Given that this was such a classic paper, why did Pearson and Bateson find it of so little value? From my reading, it seems that they were both so immersed in their own ways of thinking that they did not realize how fundamentally Fisher had rebased the study of the inheritance of quantitative traits, but looked at it as a small complication of what had been performed before. The paper was not only long, but also quite difficult to read, and Bateson could make nothing of the mathematics. From a biometrical viewpoint, Pearson would have thought that he and his colleagues had performed the basic work, and he was not prepared to put in the effort to understand Fisher's advances, suggesting that it was a frill for Mendelians to consider. In fact, the paper was so difficult to understand in detail that the Galton Laboratory published in 1966, as Eugenics Laboratory Memoirs XLI, a thorough commentary on the paper by P.A.P.Moran and C.A.B.Smith, which helps greatly in the comprehension of what Fisher achieved. In fact, Fisher had set up a method on the basis of which he was able to propose in his book The Genetical Theory of Natural Selection his Fundamental Theorem of Natural Selection, equating the rate of gain in fitness with the additive genetic variance in fitness (another advantage of his choice of variance as the basic metric for variation). Fisher's partition of genetic variance remained as the high point of the problem until 1954, when Cockerham, and Anderson and Kempthorne in successive papers in Genetics (vol 39: 859–882, and 883–898, respectively) extended Fisher's method somewhat further by partitioning the epistatic variance. This enabled the correlations between relatives to include the appropriate epistatic components with their correct weights. In 1960, Bruce Griffing (Austr. J. Biol. Sci. 13: 307–343) extended selection theory to include the contribution of epistatic effects to response. Some years later, in 1982, Bill Hill took the definitions further to cross-breeding (J. Anim. Breed. Genet. 99: 161–168). Prior to these developments, there had been much activity (perhaps more in animal breeding than elsewhere) on estimating correlations between relatives as a step in estimating heritability. This was largely based on Sewall Wright's method of path coefficients, which essentially dealt with additive variance components, although it was recognized that dominance variance affected correlations between full sibs, and that differences in shared environment also affected correlations between relatives. When I started to learn genetics, the quantitative genetics literature was full of path diagrams. Largely through the influence of J. L. Lush, Sewall Wright's way of dealing with correlations dominated work in animal breeding. Essentially that began to change in 1960 when Douglas Falconer's Introduction to Quantitative Genetics appeared and made things much simpler, and not just because one no longer had to draw path diagrams. There was also a shift in emphasis from correlations to variance components and regressions, although correlations remained important. It is now not easy to see how poorly understood the problems of genetic control of quantitative traits were before Fisher made his huge contribution. Prior to his 1918 paper, Fisher had already published pioneering work in statistics, including his groundbreaking study of the distribution of the correlation coefficient, where he represented a sample as a point in multidimensional space, and deduced the exact sampling distribution of the sample correlation. This approach enabled him to develop a great deal of statistical theory and to incorporate his advances in Statistical Methods for Research Workers, a revolutionary text of major importance in guiding scientific research. Even if Fisher had made no contribution to genetics and evolution, his role as a major developer of statistical methods would have guaranteed his position as a giant of 20th century science. That position is raised higher by his contributions to quantitative genetics and evolution, as frequently recognized by the regular mention of him as one of the big three (Fisher, Wright, Haldane) who were responsible for the creation of quantitative genetics. Throughout his career, he continued to maintain his interest in both statistical and genetical developments. It is therefore appropriate to remember his fundamental reshaping of the study of correlations between relatives shortly after his fundamental reshaping of the study of sampling distributions of estimated statistics.

The Enabling change and innovation webinar series commenced in 2012. While initially targeting 30 FutureBeef extension officers, it was expanded to include anyone interested in the topic. It eventually attracted over 2000 people from 38 countries. Twentytwo webinars were delivered, receiving 6739 registrations of which 3152 (48%) attended live and the webinar recordings received over 60,000 views. A post-event survey cumulatively collected 1255 responses. The results indicated that the attendees found it relatively easy to join (average rating 9.4/10), use the audio (9.4) and interact (9.1). Attendees' knowledge levels moved from 4.7 to 7.2, a cumulative change of 25%. Very few FutureBeef staff actually attended the webinars, so the webinar series concluded in 2016, as it was deemed a low priority activity. So no matter how apparently successful an activity might be, it must still achieve the initial aims of the funder to be deemed a success.

The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formationThe Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation' Development, vol 136, no. 4, pp. 655-64. General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. INTRODUCTION The chicken talpid 3 mutant has a complex phenotype, including polydactylous limbs with many unpatterned digits, vascular defects, hypoteleorism, abnormal dorsoventral patterning of the neural tube, loss of endochondral bone formation and embryonic lethality (Davey et al. Development of all the regions affected in the talpid 3 mutant embryo requires Hedgehog (Hh) signalling and analysis of the developing mutant limb bud and neural tube has shown that it is the response to Hh signalling that is defective (Lewis et al., 1999). Expression of some downstream Shh target genes in talpid 3 mutant limb buds, head, neural tube and somites is lost, whereas other genes are expressed more widely, suggesting that there is failure both to activate gene expression in response to Shh and to repress gene expression in its absence (Buxton et al., 2004; Davey et al., 2006; Lewis et al., 1999). These opposing effects can be understood in terms of the bifunctionality of the Gli proteins, the transcriptional effectors of vertebrate Hedgehog (Hh) signalling, which function as activators (mainly Gli1, Gli2) or are processed to short forms (mainly Gli3) that function as repressors (Aza-Blanc et al.

Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.

To examine the concordance between spirometry and asthma symptoms in assessing asthma severity and beginning therapy by the general pediatrician.All children with asthma who were 5 to 19 years of age and were referred to the Spirometry and Aeroinhalant Allergy Testing Clinic at Connecticut Children’s Medical Center between 2008 and 2012 by a clinician participating in a study were eligible for inclusion.Spirometry testing was satisfactorily performed in 894 children whose asthma severity had been determined by their pediatrician using asthma guideline–based clinical criteria. Spirometry-determined asthma severity using national asthma guidelines and clinician-determined asthma severity were compared for concordance using weighted κ coefficients.Thirty percent of participants had clinically determined intermittent asthma; 32%, 33%, and 5% had mild, moderate, and severe, persistent asthma, respectively. Increasing disease severity was associated with decreases in the forced expiratory volume in 1 second/forced vital capacity (FVC) ratio (P < .001), the forced expiratory volume in 1 second/FVC% predicted (P < .0001), and the FVC% predicted (P < .01). In 319 children (36%), clinically determined asthma severity was lower than spirometry-determined severity. Concordance was 0.16 (95% confidence interval 0.10, 0.23) and, when adjusted for bias and prevalence, was 0.20 (95% confidence interval 0.17, 0.23). When accounting for age, sex, exposure to smoke, and insurance type, only spirometry-determined asthma severity was a significant predictor of agreement (P < .0001), with worse agreement as spirometry-determined severity increased.Concordance between spirometry and asthma symptoms in determining asthma severity is low even when guideline-based clinical assessment tools are used. Because appropriate therapy reduces asthma morbidity and is guided by disease severity, results from spirometry testing could better guide pediatricians in determining appropriate therapy for their patients with asthma.Yogi Berra once said, “It’s like déjà vu all over again.” I really think that this quote is pertinent here. For years, national asthma guidelines have recommended the use of spirometry in pediatric patients with asthma who can perform this maneuver. This recommendation is not always followed appropriately. In this current investigation, the authors report that even when clinical criteria for the determination of asthma are used correctly, spirometry testing has added value in management. Specifically, they demonstrated that the use of spirometry testing would have resulted in an assignment of a higher asthma severity category in 36% of the children in their study. This information can certainly affect the overall management of asthma in the pediatric population. So I hope I am preaching to the choir here in emphasizing that increasing access to spirometry testing, whether in the pediatrician’s office or through a consultant to a specialist, should continue to be a realistic goal to improve the overall asthma care for children.

To examine the effects of food avoidance on the growth of children with food allergies.A retrospective chart review was performed for children with and without food allergies followed at 2 New York City general pediatric practices. The practices differed by the primary type of insurance accepted without overlap, state insurance versus commercial insurance.Charts were selected on the basis of codes from the International Classification of Diseases, 9th Revision, for well-child visit, food allergy, anaphylaxis, and/or epinephrine autoinjector prescriptions. Heights and weights were obtained to calculate BMI, height, and weight z scores.Of the 9938 children seen, 439 (4.4%) were avoiding ≥1 foods. Of those with commercial insurance, children with food allergies were significantly shorter (mean height z score = 0.06; P = .01) and weighed less (mean weight z score −0.1; P = .006) than children without food allergies (mean height z score = 0.42; mean weight z score = 0.07). In contrast, children with food allergies and state insurance were not smaller in height or weight compared with children without food allergies. Among white subjects, there was a significant effect of food allergies on height and weight (analysis of variance for height P = .012, for weight P = .0036) that was not observed for Hispanic/Latino, black, or Asian subjects. Children with allergies to milk weighed significantly less than children without milk allergies (P = .0006).Children with food allergies and commercial insurance have significant impairment in growth compared with those without food allergies. Additionally, children avoiding all forms of milk are shorter and weigh less than matched counterparts. Therefore, height and weight measurements should be assessed routinely in children with food allergies because there is risk for growth impairment in this population.There were 2 interesting observations in this investigation, which apparently is the largest population of children to be studied for growth impairment as the result of avoidance diets for food allergy. First, although a difference in growth between children with and without food allergies was not seen in the overall population, patients with food allergies and commercial insurance compared with state insurance were found to be shorter and weigh less than patients without food allergies. I do not know how to completely explain this finding. Second, and as has been reported previously, children who were avoiding all forms of cow’s milk were significantly shorter and weighed less compared with healthy control children. As acknowledged by the authors, major limitations of this investigation included the following: (1) its retrospective design, (2) the reliance on physician diagnosis of food allergy without the use of specific IgE testing to foods (eg, prick skin test and/or serum IgE), and (3) no oral food challenges were performed to confirm the diagnosis. I think the real take-home message here should be that physicians caring for children with food allergies should be aware of the risk of growth impairment (ie, height and weight) from specific eliminations diets, and appropriate nutritional counseling should be provided to the families of these children to help prevent and avoid nutritional deficiencies.

JA Bird, M Crain, P Varshney. J Pediatr. 2015;166(1):97–100 To determine the utility of food allergy panel testing among patients referred to a pediatric food allergy center. New patients seen between September 2011 and December 2012 by 1 provider in a tertiary referral pediatric food allergy center at Southwestern Medical Center in Dallas, Texas. This was a retrospective chart review. An analysis was performed to calculate the estimated cost of evaluation for patients who have received a food allergy panel. Of …

The adoption and use of three Web 2.0 technologies (web conferencing, eSurveys, and YouTube videos) were studied using the following four adoption models: the Diffusion of Innovations (DoI), the Unified Theory of Acceptance and Use of Technology (UTAUT), the Adoption and Diffusion Outcome Prediction Tool (ADOPT), and Switch: How to change things when change is hard. A web-based survey was used to identify the factors that encouraged the adoption and use of the new technologies. The study was conducted within an organisational context of a state government agriculture department. Unlike previous studies which focused on individuals in an organisational setting with factors such as perceived use, this study considered the actual usage of the technologies by government staff. A new model for the adoption and use of Web 2.0 technologies, the User benefits model, was developed for an organisational setting. It comprises four factors related to user benefits: contagious benefits, supporting benefits, working smarter benefits and noticeable, trialable benefits.

1 Time-resolved quantitative proteomics implicates the core snRNP protein, SmB, together with the Survival of Motor Neuron protein, in neural trafficking. Alan R Prescott, Alexandra Bales, John James, Laura Trinkle-Mulcahy and Judith E. Sleeman. School of Biology, University of St Andrews, BMS, North Haugh, St Andrews, Fife, KY16 9ST, UK. College of Life Sciences, University of Dundee, DD1 5EH, UK. 3 Department of Cellular and Molecular Medicine and Ottawa Institute of Systems Biology, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada.

This parameter was developed by the Joint Task Force on Practice Parameters, representing the American Academy of Allergy, Asthma & Immunology (AAAAI); the American College of Allergy, Asthma & Immunology (ACAAI); and the Joint Council of Allergy, Asthma & Immunology (JCAAI). The AAAAI and the ACAAI have jointly accepted responsibility for establishing ‘‘Food Allergy: A practice parameter update— 2014.’’ This is a complete and comprehensive document at the current time. The medical environment is a changing one, and not all recommendations will be appropriate for all patients.