RESUMO
Rab11 family-interacting protein 5 (Rab11fip5) is an adaptor protein that binds to the small GTPase Rab11, which has an important function in endosome recycling and trafficking of cellular proteins to the plasma membrane. Rab11fip5 is involved in many cellular processes, such as cytoskeleton rearrangement, iron uptake and exocytosis in neuroendocrine cells, and is also known as a candidate gene for autism-spectrum disorder. However, the role of Rab11fip5 during early embryonic development is not clearly understood. In this study, we identified Rab11fip5 as a protein that interacts with ephrinB1, a transmembrane ligand for Eph receptors. The PDZ binding motif in ephrinB1 and the Rab-binding domain in Rab11fip5 are necessary for their interaction in a complex. EphrinB1 and Rab11fip5 display overlapping expression in the telencephalon of developing amphibian embryos. The loss of Rab11fip5 function causes a reduction in telencephalon size and a decrease in the expression level of ephrinB1. Moreover, morpholino oligonucleotide-mediated knockdown of Rab11fip5 decreases cell proliferation in the telencephalon. The overexpression of ephrinB1 rescues these defects, suggesting that ephrinB1 recycling by the Rab11/Rab11fip5 complex is crucial for proper telencephalon development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Efrina-B1/metabolismo , Telencéfalo/crescimento & desenvolvimento , Telencéfalo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proliferação de Células , Citoesqueleto , Endossomos/metabolismo , Efrina-B1/genética , Exocitose , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Neurogênese , Telencéfalo/citologia , Xenopus laevisRESUMO
Fine control of Wnt signaling is essential for various cellular and developmental decision-making processes. However, deregulation of Wnt signaling leads to pathological consequences, one of which is cancer. Here, we identify a function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells and is required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing a secondary axis with ß-catenin target gene upregulation. Upon Wnt signaling activation, PAF dissociates from PCNA and binds directly to ß-catenin. Then, PAF recruits EZH2 to the ß-catenin transcriptional complex and specifically enhances Wnt target gene transactivation, independently of EZH2's methyltransferase activity. In mice, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling and propose a regulatory mechanism of Wnt signaling during tumorigenesis.
Assuntos
Proteínas de Transporte/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteínas de Ligação a DNA , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação da Expressão Gênica no Desenvolvimento , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Repressor Polycomb 2/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xenopus laevis , beta Catenina/genéticaRESUMO
Although the canonical Wnt pathway and ß-catenin have been extensively studied, less is known about the role of p120-catenin (also known as δ1-catenin) in the nuclear compartment. Here, we report that p120-catenin binds and negatively regulates REST and CoREST (also known as Rcor1), a repressive transcriptional complex that has diverse developmental and pathological roles. Using mouse embryonic stem cells (mESCs), mammalian cell lines, Xenopus embryos and in vitro systems, we find that p120-catenin directly binds the REST-CoREST complex, displacing it from established gene targets to permit their transcriptional activation. Importantly, p120-catenin levels further modulate the mRNA and protein levels of Oct4 (also known as POU5F1), Nanog and Sox2, and have an impact upon the differentiation of mESCs towards neural fates. In assessing potential upstream inputs to this new p120-catenin-REST-CoREST pathway, REST gene targets were found to respond to the level of E-cadherin, with evidence suggesting that p120-catenin transduces signals between E-cadherin and the nucleus. In summary, we provide the first evidence for a direct upstream modulator and/or pathway regulating REST-CoREST, and reveal a substantial role for p120-catenin in the modulation of stem cell differentiation.
Assuntos
Cateninas/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Animais , Cateninas/genética , Proteínas Correpressoras , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Repressoras/genética , Xenopus laevis , delta CateninaRESUMO
The Wnt pathways contribute to many processes in cancer and development, with ß-catenin being a key canonical component. p120-catenin, which is structurally similar to ß-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ- and zinc-finger-domain-containing transcription factor Kaiso. We have identified the kinase Dyrk1A as a component of the p120-catenin-Kaiso trajectory of the Wnt pathway. Using rescue and other approaches in Xenopus laevis embryos and mammalian cells, we found that Dyrk1A positively and selectively modulates p120-catenin protein levels, thus having an impact on p120-catenin and Kaiso (and canonical Wnt) gene targets such as siamois and wnt11. The Dyrk1A gene resides within the Down's syndrome critical region, which is amplified in Down's syndrome. A consensus Dyrk phosphorylation site in p120-catenin was identified, with a mutant mimicking phosphorylation exhibiting the predicted enhanced capacity to promote endogenous Wnt-11 and Siamois expression, and gastrulation defects. In summary, we report the biochemical and functional relationship of Dyrk1A with the p120-catenin-Kaiso signaling trajectory, with a linkage to canonical Wnt target genes. Conceivably, this work might also prove relevant to understanding the contribution of Dyrk1A dosage imbalance in Down's syndrome.
Assuntos
Cateninas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cateninas/genética , Primers do DNA/genética , Síndrome de Down/genética , Síndrome de Down/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismo , delta CateninaRESUMO
Modified asphalt binders are still considered important in asphalt pavement. However, the comprehensive use of various modifiers is limited due to storage stability issues. Moreover, there is a scarcity of detailed analyses regarding the degree of separation for asphalt binders among each method despite the utilization of various methods to assess the storage stability of binders. Therefore, a comprehensive analysis was conducted to assess the storage stability of asphalt binder modified with a crumb rubber modifier (CRM) and styrene-isoprene-styrene (SIS), utilizing five evaluation factors following the ASTM D7173 guidelines based on four mixing methods (A: high-shear mixing method, B: low-speed agitating method, C: high-shear mixing method + low mixing method, D: low-speed agitating method + low mixing method). To produce the modified asphalt binder, the proportions of the CRM were 5% and 10% for each binder, and 10% SIS was added to all binders. The results in this study convey that (1) the addition of the modifier led to an increase in G*/sin δ with different mixing methods, but using mixing methods (C and D) for a relatively long time resulted in a lower G*/sin δ, indicating suboptimal performance; (2) through the multiple stress creep recovery (MSCR), rheological properties of Jnr and % rec exhibited trends similar to G*/sin δ evaluation, highlighting an improved elastic recovery with a higher modifier content; (3) storage stability assessment revealed consistent trends in high-shear mixing groups (A and C), while low-speed mixing groups (B and D) exhibited an elevated separation index (SI), suggesting a sensitivity to modification conditions; (4) evaluation using the MSCR method indicated that % rec with a 3.2 kPa load is effective for the sensitive assessment of binder storage stability and Jnr showed a limited sensitivity across varying loads, advocating for % rec for precise evaluation; and (5) despite permitting various tests, achieving consistent results remains challenging. Future research should explore diverse modifiers and optimal evaluation methods to enhance knowledge of binder behavior and separation dynamics.
RESUMO
Apical constriction is a cell shape change critical to vertebrate neural tube closure, and the contractile force required for this process is generated by actin-myosin networks. The signaling cue that instructs this process has remained elusive. Here, we identify Wnt4 and the transmembrane ephrinB2 protein as playing an instructive role in neural tube closure as members of a signaling complex we termed WERDS (Wnt4, EphrinB2, Ror2, Dishevelled (Dsh2), and Shroom3). Disruption of function or interaction among members of the WERDS complex results in defects of apical constriction and neural tube closure. The mechanism of action involves an interaction of ephrinB2 with the Dsh2 scaffold protein that enhances the formation of the WERDS complex, which in turn, activates Rho-associated kinase to induce apical constriction. Moreover, the ephrinB2/Dsh2 interaction promotes non-canonical Wnt signaling and shows how cross-talk between two major signal transduction pathways, Eph/ephrin and Wnt, coordinate morphogenesis of the neural tube.
Assuntos
Efrina-B2 , Transdução de Sinais , Efrina-B2/genética , Constrição , Transdução de Sinais/fisiologia , Morfogênese/fisiologia , Tubo NeuralRESUMO
Crumb rubber modifier (CRM) binders easily suffer from instability at high temperatures, with many suggestions being developed to evaluate their storage stability. However, much uncertainty around CRM binders still exists regarding the relationship between mixing methods and experiments in order to calculate the separation index. In this study, a laboratory investigation into how CRM binders behave regarding storage stability using different mixing methods and experiments was conducted. The CRM asphalt binder in this study was prepared through a wet mixing process with the addition of 5% and 10% crumb rubber modifier (by weight) at 200 °C. The three main modification methods were method A: high-shear mixing (8000 rpm) for 2 h, method B: low mixing (300 rpm) for 8 h, and method C: high-shear mixing (8000 rpm) for 2 h + low mixing (300 rpm) for 6 h. In addition, the effect of separation index (SI) on storage stability was assessed, measuring viscosity, G*/sin δ, and percentage recovery. In general, the results of this study revealed that method C appeared to have the most prominent effect on decreasing the difference between the values of the top and bottom parts; the results for 5% CRM using method C showed that there were no differences among the values for the top, middle, and bottom parts; from the MSCR test, method C was effective in decreasing the difference between the values of the top and bottom parts. It was observed that method C improved storage stability. However, the results for percentage recovery were relatively higher than the separation index when using rotational viscosity and G*/sin δ.
RESUMO
This study aimed to evaluate the impact of a two-step modification process involving kaolinite and cloisite Na+ on the storage stability of rubberized binders. The process involved the manual combination of virgin binder PG 64-22 with crumb rubber modifier (CRM), which was heated to condition it. The preconditioned rubberized binder was then modified for two hours at a high speed of 8000 rpm using wet mixing. The second stage modification was performed in two parts, with part 1 using only crumb rubber as the modifier and part 2 involving the use of kaolinite and montmorillonite nano clays at a replacement percentage of 3% to the original weight of the binder along with the crumb rubber modifier. The Superpave and multiple shear creep recovery (MSCR) test methods were used to calculate the performance characteristics and separation index % of each modified binder. The results showed that the viscosity properties of kaolinite and montmorillonite improved the performance class of the binder, with montmorillonite demonstrating greater viscosity values than kaolinite even at high temperatures. Additionally, kaolinite with rubberized binders showed higher resistance to rutting, and the % recovery value from multiple shear creep recovery testing indicated that kaolinite with rubberized binders was more effective than montmorillonite with rubberized binders, even at higher load cycles. The use of kaolinite and montmorillonite reduced phase separation between the asphaltene phase and rubber-rich phase at higher temperatures, but the performance of the rubber binder was affected by higher temperatures. Overall, kaolinite with the rubber binder generally demonstrated greater binder performance.
RESUMO
The study presents an experimental evaluation to improve the resistivity of binders with "Styrene-Butadiene-Styrene" (SBS) and "Processed oil" by studying the physical properties, rheology, and cracking. For this experiment, PG 64-22 was mixed with SBS at different percentages of 5%, 10%, and 15% by weight of the original binder with two processed oil contents of 6% and 12% by weight of the binder. Laboratory tests have been conducted at various high, medium, and low temperature ranges to evaluate their properties. The processed oil polymer modified asphalt (PMA) binder is artificially aged in both the short and long-term using a Rolling Thin Film Oven (RTFO) and a Pressure Aging Vessel (PAV). The Superpave testing method was performed on modified binders using a Rotational Viscometer (RV), Dynamic Shear Rheometer (DSR), and Bending Beam Rheometer (BBR). The results of this study illustrate (1) The addition of SBS leads to higher viscosity, but the co-modification of asphalt binder with the processed oil shows a significant modulation of the viscosity value. (2) In addition, processed oil reduced the resistance to rutting, but the addition of SBS significantly improved the rutting resistance of the asphalt binder. (3) The addition of SBS and processed oil improved the value of G sin δ, notably. (4) According to BBR, it has been shown that the addition of SBS in addition to the processed oil improves the stiffness values of modified asphalt binders.
RESUMO
Despite the great potential of using positively charged gold nanoparticles (AuNPs) in nanomedicine, no systematic studies have been reported on their synthesis optimization or colloidal stability under physiological conditions until a group at the National Institute of Standards and Technology recently succeeded in producing remarkably stable polyethyleneimine (PEI)-coated AuNPs (Au-PEI). This improved version of Au-PEI (Au-PEI25kB) has increased the demand for toxicity and teratogenicity information for applications in nanomedicine and nanotoxicology. In vitro assays for Au-PEI25kB in various cell lines showed substantial active cytotoxicity. For advanced toxicity research, the frog embryo teratogenesis assay-Xenopus (FETAX) method was employed in this study. We observed that positively-charged Au-PEI25kB exhibited significant toxicity and teratogenicity, whereas polyethylene glycol conjugated AuNPs (Au-PEG) used as comparable negative controls did not. There is a characteristic avidity of Au-PEI25kB for the jelly coat, the chorionic envelope (also known as vitelline membrane) and the cytoplasmic membrane, as well as a barrier effect of the chorionic envelope observed with Au-PEG. To circumvent these characteristics, an injection-mediated FETAX approach was utilized. Like treatment with the FETAX method, the injection of Au-PEI25kB severely impaired embryo development. Notably, the survival/concentration curve that was steep when the standard FETAX approach was employed became gradual in the injection-mediated FETAX. These results suggest that Au-PEI25kB may be a good candidate as a nanoscale positive control material for nanoparticle analysis in toxicology and teratology.
Assuntos
Nanopartículas Metálicas , Teratogênese , Animais , Ouro/toxicidade , Polietilenoimina/toxicidade , Polietilenoglicóis/toxicidade , Xenopus laevis , Nanopartículas Metálicas/toxicidade , Embrião não Mamífero , Teratogênicos/toxicidade , MamíferosRESUMO
δ-Catenin is an Armadillo protein of the p120-catenin subfamily capable of modulating cadherin stability, small GTPase activity, and nuclear transcription. From yeast two-hybrid screening of a human embryonic stem cell cDNA library, we identified δ-catenin as a potential interacting partner of the caspase-3 protease, which plays essential roles in apoptotic as well as non-apoptotic processes. Interaction of δ-catenin with caspase-3 was confirmed using cleavage assays conducted in vitro, in Xenopus apoptotic extracts, and in cell line chemically induced contexts. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo repeat 6 of δ-catenin, was identified through peptide sequencing. Cleavage thus generates an amino-terminal (residues 1-816) and carboxyl-terminal (residues 817-1314) fragment, each containing about half of the central Armadillo domain. We found that cleavage of δ-catenin both abolishes its association with cadherins and impairs its ability to modulate small GTPases. Interestingly, 817-1314 possesses a conserved putative nuclear localization signal that may facilitate the nuclear targeting of δ-catenin in defined contexts. To probe for novel nuclear roles of δ-catenin, we performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating interaction with an uncharacterized KRAB family zinc finger protein, ZIFCAT. Our results indicate that ZIFCAT is nuclear and suggest that it may associate with DNA as a transcriptional repressor. We further determined that other p120 subfamily catenins are similarly cleaved by caspase-3 and likewise bind ZIFCAT. Our findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120-catenin subfamily members, facilitating the coordinate modulation of cadherins, small GTPases, and nuclear functions.
Assuntos
Apoptose/fisiologia , Caspase 3/metabolismo , Cateninas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Animais , Caderinas/genética , Caderinas/metabolismo , Caspase 3/genética , Cateninas/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Saccharomyces cerevisiae , Técnicas do Sistema de Duplo-Híbrido , Xenopus laevis , delta CateninaRESUMO
In common with other p120-catenin subfamily members, Xenopus ARVCF (xARVCF) binds cadherin cytoplasmic domains to enhance cadherin metabolic stability or, when dissociated, modulates Rho-family GTPases. We report here that xARVCF binds and is stabilized by Xenopus KazrinA (xKazrinA), a widely expressed conserved protein that bears little homology to established protein families, and which is known to influence keratinocyte proliferation and differentiation and cytoskeletal activity. Although we found that xKazrinA binds directly to xARVCF, we did not resolve xKazrinA within a larger ternary complex with cadherin, nor did it co-precipitate with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin, suggesting a potential means by which xKazrinA localizes to cell-cell borders. This was supported by the resolution of a ternary biochemical complex of xARVCF-xKazrinA-xß2-spectrin and, in vivo, by the finding that ectodermal shedding followed depletion of xKazrin in Xenopus embryos, a phenotype partially rescued with exogenous xARVCF. Cell shedding appeared to be the consequence of RhoA activation, and thereby altered actin organization and cadherin function. Indeed, we also revealed that xKazrinA binds p190B RhoGAP, which was likewise capable of rescuing Kazrin depletion. Finally, xKazrinA was found to associate with δ-catenins and p0071-catenins but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin subfamily. Taken together, our study supports the essential role of Kazrin in development, and reveals the biochemical and functional association of KazrinA with ARVCF-catenin, spectrin and p190B RhoGAP.
Assuntos
Proteínas do Domínio Armadillo/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Epiteliais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Espectrina/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/genética , Caderinas/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Linhagem Celular , Células Epiteliais/química , Células Epiteliais/enzimologia , Proteínas Ativadoras de GTPase/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Espectrina/genética , Técnicas do Sistema de Duplo-Híbrido , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The novel adaptor protein Kazrin associates with multifunctional entities including p120-subfamily members (ARVCF-, delta-, and p0071-catenin). Critical contributions of Kazrin to development or homeostasis are indicated with respect to ectoderm formation, integrity and keratinocyte differentiation, whereas its presence in varied tissues suggests broader roles. We find that Kazrin is maternally loaded, is expressed across development and becomes enriched in the forming head. Kazrin's potential contributions to craniofacial development were probed by means of knockdown in the prospective anterior neural region. Cartilaginous head structures as well as eyes on injected sides were reduced in size, with molecular markers suggesting an impact upon neural crest cell establishment and migration. Similar effects followed the depletion of ARVCF (or delta-catenin), with Kazrin:ARVCF functional interplay supported upon ARVCF partial rescue of Kazrin knockdown phenotypes. Thus, Kazrin and its associating ARVCF- and delta-catenins, are required to form craniofacial tissues originating from cranial neural crest and precordal plate.
Assuntos
Proteínas do Domínio Armadillo/metabolismo , Cateninas/metabolismo , Moléculas de Adesão Celular/metabolismo , Movimento Celular/fisiologia , Proteínas de Membrana/metabolismo , Crista Neural/embriologia , Organogênese/fisiologia , Fosfoproteínas/metabolismo , Crânio/embriologia , Proteínas de Xenopus/metabolismo , Animais , Proteínas do Domínio Armadillo/genética , Cartilagem/embriologia , Cateninas/genética , Moléculas de Adesão Celular/genética , Olho/embriologia , Técnicas de Silenciamento de Genes , Proteínas de Membrana/genética , Fosfoproteínas/genética , Crânio/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis , delta CateninaRESUMO
This study investigates the effectiveness of processed oil in the modification of PG 64-22 and PG 76-22 by assessing their physical and rheological properties, and multiple comparison was conducted between the two binders. The base binders PG 64-22 and PG 76-22 were blended with processed oil at four different percentages of contents (3%, 6%, 9% and 12% by the weight of the binder) and compared with the control binder in each test. The base and modified binders were artificially short-term and long-term aged using a rolling thin film oven (RTFO) and pressure aging vessel (PAV) procedures. Superpave binder tests were performed on the modified binders by applying a rotational viscometer (RV), dynamic shear rheometer (DSR), and bending beam rheometer (BBR). The comparisons and results presented in this study indicate that (1) the processed oil has a significant effect on the binders' viscosity, which changes with respect to the increment of processed oil content. The viscosity of both modified binders decreased with the addition of 3, 6, 9 and 12% processed oil; (2) the performed DSR test showed that the addition of processed oil had a negative effect on the rutting resistance for both binders, since in PG 64-22, G*/Sin δ values decreased by 55, 65, 75 and 83% with the addition of 3, 6, 9 and 12% processed oil, respectively, while a decrement of G*/Sin δ of 24, 45, 58 and 65% with the addition of 3, 6, 9 and 12% processed oil was observed in PG 76-22; meanwhile, the fatigue cracking performance was improved and was found to be effective, while G* Sin δ in PG76-22 decreased by 9, 30, 36, and 52% and in PG 64-22 by 27, 44, 53, and 67% with the addition of 3, 6, 9 and 12% processed oil; (3) the results from the BBR test indicate significant improvement in the thermal cracking properties of the binders. The addition of 3, 6, 9 and 12% processed oil resulted in a decrease in the stiffness of both the PG 64-22 and PG 76-22 binders, with a positive effect consequently being observed on the m-values of the binders.
RESUMO
Crumb rubber binder with thermoplastic polyurethane (TPU) has been experimented with to characterize the performance properties considering the workability, rutting, fatigue cracking and cracking resistance at low temperatures depending on the temperatures and aging states. Physical and rheological properties were evaluated to proceed with the study by applying Superpave asphalt binder testing and multi-stress creep recovery (MSCR). Based on the targeted experiments, the binder samples were produced at three aging states (original, short-term aged and long-term aged) using a rolling thin film oven (RTFO) and pressure aging vessel (PAV). The results revealed that (i) the addition of TPU into CRM binders has a potential effect on increasing viscoelasticity at the original condition, (ii) CRM binders containing TPU showed improved anti-aging performance based on results of RTFO residues and (iii) the inclusion of TPU made it possible for CRM asphalt binder to improve its fatigue and cracking resistance at low temperature.
RESUMO
The Zic family of zinc finger transcription factors plays a critical role in multiple developmental processes. Using loss-of-function studies, we find that Zic5 is important for the differentiation of retinal pigmented epithelium (RPE) and the rod photoreceptor layer through suppressing Hedgehog (Hh) signaling. Further, Zic5 interacts with the critical Hh signaling molecule, Gli3, through the zinc finger domains of both proteins. This Zic5-Gli3 interaction disrupts Gli3/Gli3 homodimerization, resulting in Gli3 protein stabilization via a reduction in Gli3 ubiquitination. During embryonic Hh signaling, the activator form of Gli is normally converted to a repressor form through proteosome-mediated processing of Gli3, and the ratio of Gli3 repressor to full-length (activator) form of Gli3 determines the Gli3 repressor output required for normal eye development. Our results suggest Zic5 is a critical player in regulating Gli3 stability for the proper differentiation of RPE and rod photoreceptor layer during Xenopus eye development.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Retina/crescimento & desenvolvimento , Proteínas de Xenopus/metabolismo , Proteína Gli3 com Dedos de Zinco/metabolismo , Animais , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , XenopusRESUMO
Proper cilia formation in multiciliated cells (MCCs) is necessary for appropriate embryonic development and homeostasis. Multicilia share many structural characteristics with monocilia and primary cilia, but there are still significant gaps in our understanding of the regulation of multiciliogenesis. Using the Xenopus embryo, we show that CEP97, which is known as a negative regulator of primary cilia formation, interacts with dual specificity tyrosine phosphorylation regulated kinase 1A (Dyrk1a) to modulate multiciliogenesis. We show that Dyrk1a phosphorylates CEP97, which in turn promotes the recruitment of Polo-like kinase 1 (Plk1), which is a critical regulator of MCC maturation that functions to enhance centriole disengagement in cooperation with the enzyme Separase. Knockdown of either CEP97 or Dyrk1a disrupts cilia formation and centriole disengagement in MCCs, but this defect is rescued by overexpression of Separase. Thus, our study reveals that Dyrk1a and CEP97 coordinate with Plk1 to promote Separase function to properly form multicilia in vertebrate MCCs.
Assuntos
Centríolos/metabolismo , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Organogênese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/química , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Humanos , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas/metabolismo , Especificidade por Substrato , Xenopus , Proteínas de Xenopus/química , Quinase 1 Polo-LikeRESUMO
The study describes the laboratory assessment (physical and rheological properties) of the binders (PG 64-22 and PG 76-22) modified with Styrene Butadiene Rubber (SBR), and a comprehensive comparison between these two modified binder types. PG 64-22 and PG 76-22 were used as base binders. Both of the base binders were blended with SBR at four different percentages of content (0%, 4%, 6%, and 8% by the weight of the binder). The base and modified binders were artificially short-term and long-term aged using a rolling thin film oven (RTFO) and pressure aging vessel (PAV) procedures. Superpave binder tests were conducted on the SBR modified binder using rotational viscometer (RV), dynamic shear rheometer (DSR), and bending beam rheometer (BBR). In depth rutting performance was investigated using Multiple Stress Creep Recovery (MSCR). The results of this study indicated that (1) the addition of SBR into both binders increased the viscosity and polymer modified asphalt (PMA) binders observed to have more significant effect on its viscosity property; (2) the higher the SBR content, the better the rutting resistance of the binder and it is observed that the effect is prominent on the control binder; (3) MSCR test results showed that the SBR modified binders improved the binder percentage recovery and found to have a more significant effect on the PG 76-22 binder compared to PG 64-22; and (4) both the control PG 64-22 and PMA PG 76-22 binders resulted in similar trends on the cracking properties and were found to have insignificant effects due to the addition of an SBR modifier.
RESUMO
The engineering properties of asphalt binders depend on the types and amounts of additives. However, measuring engineering properties is time-consuming, requires technical expertise, specialized equipment, and effort. This study develops a deep regression model for predicting the engineering property of asphalt binders based on analysis of atomic force microscopy (AFM) image analysis to test the feasibility of replacing traditional measuring estimate techniques. The base asphalt binder PG 64-22 and styrene-isoprene-styrene (SIS) modifier were blended with four different polymer additive contents (0%, 5%, 10%, and 15%) and then tested with a dynamic shear rheometer (DSR) to evaluate the rheological data, which indicate the rutting properties of the asphalt binders. Different deep regression models are trained for predicting engineering property using AFM images of SIS binders. The mean absolute percentage error is decisive for the selection of the best deep regression architecture. This study's results indicate the deep regression architecture is found to be effective in predicting the G*/sin δ value after the training and validation process. The deep regression model can be an alternative way to measure the asphalt binder's engineering property quickly. This study would encourage applying a deep regression model for predicting the engineering properties of the asphalt binder.
RESUMO
Sproutys are negative regulators of the Ras/Raf/MAPK signaling pathway and involved in regulation of organogenesis, differentiation, cell migration and proliferation. Although the function of Sproutys have been extensively studied during embryonic development, their role and mode of action during eye formation in vertebrate embryonic development is still unknown. Here we show that Xenopus sprouty2 is expressed in the optic vesicle at late neurula stage and knockdown of Sprouty2 prevents retinal progenitors from populating the retina, which in turn gives rise to small eyes. In the absence of Sprouty2, progenitor cell population of the retina can be restored by blocking the MAPK signaling pathway through overexpression of DN-Ras or DN-Raf. In contrast, activation of the MAPK pathway through overexpression of a constitutively active form of c-Raf (ca-Raf) inhibits progenitor population of the retina, similar to the Sprouty2 loss-of-function phenotype. Moreover, we present evidence that the retinal defect observed in Sprouty2 morphants is attributed to the failure of proper movement of retinal progenitors into the optic vesicle, rather than an effect on progenitor cell survival. These results suggest that Sprouty2 is required for the positioning of retinal progenitors within the optic vesicle through suppressing Ras/Raf/MAPK signaling pathway.