Biochemical and immunocytochemical characterization of coronins in platelets.

Rapid reorganization of the actin cytoskeleton in response to receptor-mediated signaling cascades allows platelets to transition from a discoid shape to a flat spread shape upon adhesion to damaged vessel walls. Coronins are conserved regulators of the actin cytoskeleton turnover but they also participate in signaling events. To gain a better picture of their functions in platelets we have undertaken a biochemical and immunocytochemical investigation with a focus on Coro1. We found that class I coronins Coro1, 2 and 3 are abundant in human and mouse platelets whereas little Coro7 can be detected. Coro1 is mainly cytosolic, but a significant amount associates with membranes in an actin-independent manner and does not translocate from or to the membrane fraction upon exposure to thrombin, collagen or prostacyclin. Coro1 rapidly translocates to the Triton insoluble cytoskeleton upon platelet stimulation with thrombin or collagen. Coro1, 2 and 3 show a diffuse cytoplasmic localization with discontinuous accumulation at the cell cortex and actin nodules of human platelets, where all three coronins colocalize. Our data are consistent with a role of coronins as integrators of extracellular signals with actin remodeling and suggests a high extent of functional overlap among class I coronins in platelets.

Editorial of Special Issue "Frontiers in the Actin Cytoskeleton".

The actin cytoskeleton is of fundamental importance for eukaryotic cell homeostasis [...].

Coronin 1 Is Required for Integrin ß2 Translocation in Platelets.

Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant coronins 1, 2, and 3 and the less abundant coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of coronin 1. Here we show that the absence of coronin 1 is associated with impaired translocation of integrin ß2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among coronins 1, 2, and 3 in platelets, while aspects like integrin ß2 translocation are specifically or predominantly dependent on coronin 1.

Absence of plastin 1 causes abnormal maintenance of hair cell stereocilia and a moderate form of hearing loss in mice.

Hearing relies on the mechanosensory inner and outer hair cells (OHCs) of the organ of Corti, which convert mechanical deflections of their actin-rich stereociliary bundles into electrochemical signals. Several actin-associated proteins are essential for stereocilia formation and maintenance, and their absence leads to deafness. One of the most abundant actin-bundling proteins of stereocilia is plastin 1, but its function has never been directly assessed. Here, we found that plastin 1 knock-out (Pls1 KO) mice have a moderate and progressive form of hearing loss across all frequencies. Auditory hair cells developed normally in Pls1 KO, but in young adult animals, the stereocilia of inner hair cells were reduced in width and length. The stereocilia of OHCs were comparatively less affected; however, they also showed signs of degeneration in ageing mice. The hair bundle stiffness and the acquisition of the electrophysiological properties of hair cells were unaffected by the absence of plastin 1, except for a significant change in the adaptation properties, but not the size of the mechanoelectrical transducer currents. These results show that in contrast to other actin-bundling proteins such as espin, harmonin or Eps8, plastin 1 is dispensable for the initial formation of stereocilia. However, the progressive hearing loss and morphological defects of hair cells in adult Pls1 KO mice point at a specific role for plastin 1 in the preservation of adult stereocilia and optimal hearing. Hence, mutations in the human PLS1 gene may be associated with relatively mild and progressive forms of hearing loss.

A Cdc42- and Rac-interactive binding (CRIB) domain mediates functions of coronin.

The Cdc42- and Rac-interactive binding motif (CRIB) of coronin binds to Rho GTPases with a preference for GDP-loaded Rac. Mutation of the Cdc42- and Rac-interactive binding motif abrogates Rac binding. This results in increased 1evels of activated Rac in coronin-deficient Dictyostelium cells (corA(-)), which impacts myosin II assembly. corA(-) cells show increased accumulation of myosin II in the cortex of growth-phase cells. Myosin II assembly is regulated by myosin heavy chain kinase-mediated phosphorylation of its tail. Kinase activity depends on the activation state of the p21-activated kinase a. The myosin II defect of corA(-) mutant is alleviated by dominant-negative p21-activated kinase a. It is rescued by wild-type coronin, whereas coronin carrying a mutated Cdc42- and Rac-interactive binding motif failed to rescue the myosin defect in corA(-) mutant cells. Ectopically expressed myosin heavy chain kinases affinity purified from corA(-) cells show reduced kinase activity. We propose that coronin through its affinity for GDP-Rac regulates the availability of GTP-Rac for activation of downstream effectors.

[Electroencephalographic and neurodevelopmental disorders in severe congenital heart disease: A follow-up study]. / Alteraciones electroencefalográficas y del neurodesarrollo en cardiopatías congénitas severas. Estudio de seguimiento.

Congenital heart defects are the most common malformations at birth. Due to the fact that the developmental windows at early stages close rapidly, the aim of this study was to determine the impact of congenital heart defects on the central nervous system at short and medium terms after applying traditional and quantitative electroencephalography techniques and a test of neurodevelopment. Twenty-one patients (8-27 months, x = 14.8) with severe congenital heart defects who had been studied previously, and a control group of 19 healthy children (8-29 months, x = 14.6) were included. In all of them traditional electroencephalography, quantitative electroencephalography, and a test of neurodevelopment were performed. The results between groups (control vs. congenital heart defects) and between congenital heart defects (previous vs. present) were compared. In the second evaluation, congenital heart defect children maintained abnormal quantitative and traditional electroencephalography recordings. Comparing quantitative electroencephalography among congenital heart defects (previous vs. present) and between controls and congenital heart defects, significant differences of theta band activity in frontal, central, and temporal leads were found (p < 0.05). Upon assessing neurodevelopment, 86% of the previously studied congenital heart defect cases kept the same diagnosis of abnormality, of which mild-to-moderate hypotone was the most frequently observed. As hypothesized, congenital heart defect diseases have a very important impact on central nervous system function as determined by neurodevelopmental testing and traditional and quantitative electroencephalography recordings. The alterations observed persisted throughout the period studied.

The Rac GEF ZizB regulates development, cell motility and cytokinesis in Dictyostelium.

Dock (dedicator of cytokinesis) proteins represent a family of guanine nucleotide exchange factors (GEFs) that include the well-studied Dock180 family and the poorly characterised zizimin family. Our current understanding of Dock180 function is that it regulates Rho small GTPases and thus has a role in a number of cell processes, including cell migration, development and division. Here, we use a tractable model for cell motility research, Dictyostelium discoideum, to help elucidate the role of the related zizimin proteins. We show that gene ablation of zizA causes no change in development, whereas ablation of zizB gives rise to an aberrant developmental morphology and a reduction in cell directionality and velocity, and altered cell shape. Fluorescently labelled ZizA protein associates with the microtubule-organising centre (MTOC), whereas ZizB is enriched in the cortex. Overexpression of ZizB also causes an increase in the number of filopodia and a partial inhibition of cytokinesis. Analysis of ZizB protein binding partners shows that it interacts with Rac1a and a range of actin-associated proteins. In conclusion, our work provides insight into the molecular and cellular functions of zizimin GEF proteins, which are shown to have a role in cell movement, filopodia formation and cytokinesis.

Phylogeny-wide analysis of social amoeba genomes highlights ancient origins for complex intercellular communication.

Dictyostelium discoideum (DD), an extensively studied model organism for cell and developmental biology, belongs to the most derived group 4 of social amoebas, a clade of altruistic multicellular organisms. To understand genome evolution over long time periods and the genetic basis of social evolution, we sequenced the genomes of Dictyostelium fasciculatum (DF) and Polysphondylium pallidum (PP), which represent the early diverging groups 1 and 2, respectively. In contrast to DD, PP and DF have conventional telomere organization and strongly reduced numbers of transposable elements. The number of protein-coding genes is similar between species, but only half of them comprise an identifiable set of orthologous genes. In general, genes involved in primary metabolism, cytoskeletal functions and signal transduction are conserved, while genes involved in secondary metabolism, export, and signal perception underwent large differential gene family expansions. This most likely signifies involvement of the conserved set in core cell and developmental mechanisms, and of the diverged set in niche- and species-specific adaptations for defense and food, mate, and kin selection. Phylogenetic dating using a concatenated data set and extensive loss of synteny indicate that DF, PP, and DD split from their last common ancestor at least 0.6 billion years ago.

Expression analysis of mouse Rhobtb3 using a LacZ reporter and preliminary characterization of a knockout strain.

RhoBTB3 is an atypical member of the Rho family of small GTPases. It localizes at the Golgi apparatus and endosomes and is involved in vesicle trafficking and in targeting proteins for degradation in the proteasome. Previous studies using Northern blot analysis showed that Rhobtb3 is ubiquitously expressed in adult mice, but expression is particularly high in brain, heart and uterus. The gene is also expressed between embryonic days 11.5 and 17.5. To investigate the specific cell types that express this gene across tissues, both in the embryo and in the adult organism, we have made use of a gene trap mouse strain that expresses the LacZ gene under the transcriptional control of the endogenous Rhobtb3 promoter. Histochemical detection of ß-galactosidase expression revealed a profile characterized by nearly ubiquitous expression of Rhobtb3 in the embryo, but with particularly high levels in bone, cartilage, all types of muscle, testis and restricted areas of the nervous system. In the adult, expression persists at much lower levels in cardiac muscle, the tunica media of blood vessels and cartilage and at high levels in the seminiferous tubules. A general preliminary characterization of this gene trap mouse strain revealed reduced viability, a postnatal growth defect and reduced testis size. Our results should pave the way for future studies aimed at investigating the roles of RhoBTB3 in tissue development and in cardiac, vascular and testicular function.

GxcC connects Rap and Rac signaling during Dictyostelium development.

BACKGROUND: Rap proteins belong to the Ras family of small G-proteins. Dictyostelium RapA is essential and implicated in processes throughout the life cycle. In early development and chemotaxis competent cells RapA induces pseudopod formation by activating PI3K and it regulates substrate attachment and myosin disassembly via the serine/threonine kinase Phg2. RapA is also important in late development, however so far little is known about the downstream effectors of RapA that play a role in this process. RESULTS: Here we show that cells expressing constitutively active RapA exhibit a high level of Rac activation. With a pull-down screen coupled to mass spectrometry, we identified the Rac specific guanine nucleotide exchange factor, GxcC, as Rap binding partner. GxcC binds directly and specifically to active RapA and binds to a subset of Dictyostelium Rac proteins. Deletion studies revealed that this pathway is involved in regulating Dictyostelium development. CONCLUSIONS: GxcC provides a novel link between Rap and Rac signalling and is one of the Rap effectors regulating the progression of multicellular development.

An attenuating role of a WASP-related protein, WASP-B, in the regulation of F-actin polymerization and pseudopod formation via the regulation of RacC during Dictyostelium chemotaxis.

The WASP family of proteins has emerged as important regulators that connect multiple signaling pathways to regulate the actin cytoskeleton. Dictyostelium cells express WASP, as well as a WASP related protein, WASP-B, endoded by wasB gene. WASP-B contains many of the domains present in WASP. Analysis of wild type, wasB null cells revealed that WASP-B is required for proper control of F-actin polymerization in response to a cAMP gradient. Due to the lack of tight control on actin polymerization, wasB null cells exhibited higher level of F-actin polymerization. wasB(-) cells extend more de novo pseudopods laterally and their average life span is longer than those of wild type cells, causing more turns and inefficient chemotaxis. YFP-WASP-B appears to be uniformly distributed in the cytosol and shows no translocation to cortical membrane upon cAMP stimulation. Active RacC pull-down assay reveals that the level of active RacC in wasB(-) cells is significantly higher than wild type cells. Moreover, the distribution of active RacC is not localized in wasB(-) cells. We conclude that chemotaxis defects of wasB(-) cells are likely to result from the aberrant regulation of RacC activation and localization.

The Use of Intact Fish Skin Grafts in the Treatment of Necrotizing Fasciitis of the Leg: Early Clinical Experience and Literature Review on Indications for Intact Fish Skin Grafts.

BACKGROUND: Necrotizing fasciitis (NF) is a serious infectious disease that can initially place the patient's life in danger and, after successful surgical and antibiotic treatment, leaves extensive wounds with sometimes even exposed bones and tendons. Autologous skin grafts are not always possible or require adequate wound bed preparation. Novel intact fish skin grafts (iFSGs; Kerecis® Omega3 Wound, Kerecis hf, Isafjördur, Iceland) have already shown their potential to promote granulation in many other wound situations. Faster wound healing rates and better functional and cosmetic outcomes were observed due to their additionally postulated anti-inflammatory and analgesic properties. Therefore, iFSGs may also be essential in treating NF. We present our initial experience with iFSGs in treating leg wounds after NF and review the literature for the current spectrum of clinical use of iFSGs. CASE PRESENTATIONS: We present two male patients (aged 60 and 69 years) with chronic or acute postsurgical extensive leg ulcers six weeks and six days after necrotizing fasciitis, respectively. Both suffered from diabetes mellitus without vascular pathologies of the lower limbs. A single application of one pre-meshed (Kerecis® Graftguide) and one self-meshed 300 cm2 iFSG (Kerecis® Surgiclose) was performed in our operation room after extensive surgical debridement and single circles of negative wound pressure therapy. Application and handling were easy. An excellent wound granulation was observed, even in uncovered tibia bone and tendons, accompanied by pain relief in both patients. Neither complications nor allergic reactions occurred. The patients received autologous skin grafting with excellent functional and cosmetic outcomes. CONCLUSIONS: iFSGs have the potential to play a significant role in the future treatment of NF due to the fast promotion of wound granulation and pain relief. Our experience may encourage surgeons to use iFSGs in NF patients, although high-quality, large-sized studies are still required to confirm these results. The observed effects of iFSGs on wounds associated with NF may be transferred to other wound etiologies as well.

Operationalization and measurement of compulsivity across video gaming and gambling behavioral domains.

BACKGROUND: Compulsivity is the hallmark of addiction progression and, as a construct, has played an important role in unveiling the etiological pathways from learning mechanisms underlying addictive behavior to harms resulting from it. However, a sound use of the compulsivity construct in the field of behavioral addictions has been hindered to date by the lack of consensus regarding its definition and measurement. Here we capitalize on a previous systematic review and expert appraisal to develop a compulsivity scale for candidate behavioral addictions (the Granada Assessment for Cross-domain Compulsivity, GRACC). METHODS: The initial scale (GRACC90) consisted of 90 items comprising previously proposed operationalizations of compulsivity, and was validated in two panel samples of individuals regularly engaging in gambling and video gaming, using exploratory structural equation modeling (ESEM) and convergence analyses. RESULTS: The GRACC90 scale is unidimensional and structurally invariant across samples, and predicted severity of symptoms, lower quality of life, and negative affect, to similar degrees in the two samples. Additionally, poorer quality of life and negative affect were comparably predicted by compulsivity and by severity of symptoms. A shorter version of the scale (GRACC18) is proposed, based on selecting the 18 items with highest factor loadings. CONCLUSIONS: Results support the proposal that core symptoms of behavioral addictions strongly overlap with compulsivity, and peripheral symptoms are not essential for their conceptualization. Further research should clarify the etiology of compulsive behavior, and whether pathways to compulsivity in behavioral addictions could be common or different across domains.

Ectopic expression of cyclase associated protein CAP restores the streaming and aggregation defects of adenylyl cyclase a deficient Dictyostelium discoideum cells.

BACKGROUND: Cell adhesion, an integral part of D. discoideum development, is important for morphogenesis and regulated gene expression in the multicellular context and is required to trigger cell-differentiation. G-protein linked adenylyl cyclase pathways are crucially involved and a mutant lacking the aggregation specific adenylyl cyclase ACA does not undergo multicellular development. RESULTS: Here, we have investigated the role of cyclase-associated protein (CAP), an important regulator of cell polarity and F-actin/G-actin ratio in the aca- mutant. We show that ectopic expression of GFP-CAP improves cell polarization, streaming and aggregation in aca- cells, but it fails to completely restore development. Our studies indicate a requirement of CAP in the ACA dependent signal transduction for progression of the development of unicellular amoebae into multicellular structures. The reduced expression of the cell adhesion molecule DdCAD1 together with csA is responsible for the defects in aca- cells to initiate multicellular development. Early development was restored by the expression of GFP-CAP that enhanced the DdCAD1 transcript levels and to a lesser extent the csA mRNA levels. CONCLUSIONS: Collectively, our data shows a novel role of CAP in regulating cell adhesion mechanisms during development that might be envisioned to unravel the functions of mammalian CAP during animal embryogenesis.

Neuronal Activity during Exposure to Specific Phobia through fMRI: Comparing Therapeutic Components of Cognitive Behavioral Therapy.

Cognitive behavioral therapy (CBT) packages for anxiety disorders, such as phobias, usually include gradual exposure to anxious contexts, positive self-verbalizations, and relaxation breathing. The objective of this research was to analyze the specific neural activation produced by the self-verbalizations (S) and breathing (B) included in CBT. Thirty participants with clinical levels of a specific phobia to small animals were randomly assigned to three fMRI conditions in which individuals were exposed to phobic stimuli in real images: a group underwent S as a technique to reduce anxiety; a second group underwent B; and a control group underwent exposure only (E). Simple effects showed higher brain activation comparing E > S, E > B, and S > B. In particular, in the E group, compared to the experimental conditions, an activation was observed in sensory-perceptive and prefrontal and in other regions involved in the triggering of emotion (i.e., amygdala, supplementary motor area, and cingulate gyrus) as well as an activation associated with interoceptive sensitivity (i.e., insula and cingulate cortex). According to the specific tool used, discrepancies in the neural changes of CBT efficacy were observed. We discuss the theoretical implications according to the dual model of CBT as a set of therapeutic tools that activate different processes.

Complex Transcriptional Profiles of the PPP1R12A Gene in Cells of the Circulatory System as Revealed by In Silico Analysis and Reverse Transcription PCR.

The myosin light chain phosphatase target subunit 1 (MYPT1), encoded by the PPP1R12A gene, is a key component of the myosin light chain phosphatase (MLCP) protein complex. MYPT1 isoforms have been described as products of the cassette-type alternative splicing of exons E13, E14, E22, and E24. Through in silico analysis of the publicly available EST and mRNA databases, we established that PPP1R12A contains 32 exons (6 more than the 26 previously reported), of which 29 are used in 11 protein-coding transcripts. An in silico analysis of publicly available RNAseq data combined with validation by reverse transcription (RT)-PCR allowed us to determine the relative abundance of each transcript in three cell types of the circulatory system where MYPT1 plays important roles: human umbilical vein endothelial cells (HUVEC), human saphenous vein smooth muscle cells (HSVSMC), and platelets. All three cell types express up to 10 transcripts at variable frequencies. HUVECs and HSVSMCs predominantly express the full-length variant (58.3% and 64.3%, respectively) followed by the variant skipping E13 (33.7% and 23.1%, respectively), whereas in platelets the predominant variants are those skipping E14 (51.4%) and E13 (19.9%), followed by the full-length variant (14.4%). Variants including E24 account for 5.4% of transcripts in platelets but are rare (<1%) in HUVECs and HSVSMCs. Complex transcriptional profiles were also found across organs using in silico analysis of RNAseq data from the GTEx project. Our findings provide a platform for future studies investigating the specific (patho)physiological roles of understudied MYPT1 isoforms.

Interactome and evolutionary conservation of Dictyostelid small GTPases and their direct regulators.

GTP binding proteins known as small GTPases make up one of the largest groups of regulatory proteins and control almost all functions of living cells. Their activity is under, respectively, positive and negative regulation by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which together with their upstream regulators and the downstream targets of the small GTPases form formidable signalling networks. While genomics has revealed the large size of the GTPase, GEF and GAP repertoires, only a small fraction of their interactions and functions have yet been experimentally explored. Dictyostelid social amoebas have been particularly useful in unravelling the roles of many proteins in the Rac-Rho and Ras-Rap families of GTPases in directional cell migration and regulation of the actin cytoskeleton. Genomes and cell-type specific and developmental transcriptomes are available for Dictyostelium species that span the 0.5 billion years of evolution of the group from their unicellular ancestors. In this work, we identified all GTPases, GEFs and GAPs from genomes representative of the four major taxon groups and investigated their phylogenetic relationships and evolutionary conservation and changes in their functional domain architecture and in their developmental and cell-type specific expression. We performed a hierarchical cluster analysis of the expression profiles of the ~2000 analysed genes to identify putative interacting sets of GTPases, GEFs and GAPs, which highlight sets known to interact experimentally and many novel combinations. This work represents a valuable resource for research into all fields of cellular regulation.

A Voxel-Based Morphometric Study of Gray Matter in Specific Phobia.

The objective of this study was to analyze the neurostructural abnormalities of brain areas responsible for the acquisition and maintenance of fear in small animal phobia by comparing gray matter volume (GMV) in individuals with phobia and non-fearful controls. Structural magnetic resonance imaging was obtained from 62 adults (79% female) assigned to one of two groups: 31 were diagnosed with small animal phobia and 31 were non-fearful controls. To investigate structural alterations, a whole-brain voxel-based morphometry analysis was conducted to compare the GMV of the brain areas involved in fear between both groups. The results indicated that individuals with a small animal specific phobia showed smaller GMV in cortical regions, such as the orbitofrontal (OFC) and medial frontal cortex, and greater GMV in the putamen than non-fearful controls. These brain areas are responsible for avoidant behavior (putamen) and emotional regulation processes or inhibitory control (prefrontal cortex (PFC)), which might suggest a greater vulnerability of phobic individuals to acquiring non-adaptive conditioned responses and emotional dysregulation. The findings provide preliminary support for the involvement of structural deficits in OFC and medial frontal cortex in phobia, contributing to clarify the neurobiological substrates for phobias.

Linguistic perception: neural processing of a whistled language.

Silbo Gomero is a whistled language that is a rare and endangered surrogate of Spanish, used by shepherds on the island of La Gomera in the Canary Islands for communication over long distances on difficult terrain. Here we show that areas of the brain normally associated with spoken-language function are also activated in proficient whistlers, but not in controls, when they are listening to Silbo Gomero. Our findings demonstrate that the language-processing regions of the human brain can adapt to a surprisingly wide range of signalling forms.

Changes in Brain Activation through Cognitive-Behavioral Therapy with Exposure to Virtual Reality: A Neuroimaging Study of Specific Phobia.

BACKGROUND: Cognitive-behavioral therapy (CBT) with exposure is the treatment of choice for specific phobia. Virtual reality exposure therapy (VRET) has shown benefits for the treatment and prevention of the return of fear in specific phobias by addressing the therapeutic limitations of exposure to real images. METHOD: Thirty-one participants with specific phobias to small animals were included: 14 were treated with CBT + VRET (intervention group), and 17 were treated with CBT + exposure to real images (active control group). Participants' scores in anxiety and phobia levels were measured at baseline, post-treatment, and 3-month follow-up, and brain activation was measured through functional magnetic resonance imaging (fMRI) baseline and post-treatment. RESULTS: Both groups showed a significant decrease in anxiety and phobia scores after the therapy and were maintained until follow-up. There were no significant differences between both groups. Overall, fMRI tests showed a significant decrease in brain activity after treatment in some structures (e.g., prefrontal and frontal cortex) and other structures (e.g., precuneus) showed an increasing activity after therapy. However, structures such as the amygdala remained active in both groups. CONCLUSIONS: The efficacy of CBT + VRET was observed in the significant decrease in anxiety responses. However, the results of brain activity observed suggest that there was still a fear response in the brain, despite the significant decrease in subjective anxiety levels.