Primary cilia and the regulation of hedgehog signaling: link to cardiac development

RESUMO: As células eucarióticas evoluíram um sistema de sinalização complexo que lhes permite responder aos sinais extracelulares e intracelulares. Desta forma, as vias de sinalização são essenciais para a sobrevivência da célula e do organismo, uma vez que regulam processos fundamentais, tais como o desenvolvimento, o crescimento, a imunidade, e a homeostase dos tecidos. A via de transdução de sinal Hedgehog (Hh) envolve o receptor Patched1 (Ptch1), que tem um efeito inibidor sobre a proteína Smoothened (Smo) na ausência dos seus ligandos, as proteínas Sonic hedgehog (Shh). Estas proteínas são reguladores fundamentais do desenvolvimento embrionário, como ilustrado pelas malformações drásticas observadas em embriões humanos e de murganho com perturbações da transdução de sinal da via Hh e que incluem polidactilia, defeitos craniofaciais e malformações ósseas. Igualmente importantes são as consequências da ativação inapropriada da via de sinalização Hh na formação de tumores. Curiosamente, os componentes desta via localizam-se nos cílios primários. Além disso, demonstrou-se que esta localização é crucial para a sinalização através da via Hh. Na presença dos ligandos, Ptch1 é internalizado e destinado a degradação ou sequestrado num compartimento da célula de onde não pode desempenhar o seu papel inibitório. A proteína Arl13b é uma pequena GTPase pertencente à família Arf/Arl da superfamília Ras de pequenas GTPases e foi implicada no síndrome de Joubert, uma ciliopatia caracterizada por ataxia congénita cerebelar, hipotonia, atrso mental e cardiopatia congénita. Murganhos deficientes para Arl13b, chamado hennin (hnn) morrem morrem prematuramente ao dia 13,5 de gestação (E13,5) e exibem anomalias morfológicas nos cílios que levam à interrupção da sinalização Hh. Além disso, a Arl13b está diretamente envolvida na regulação da via Hh, controlando a localização de vários componentes desta via nos cílios primários. Neste trabalho, mostramos que a Arl13b se localiza em circular dorsal ruffles (CDRs), que são estruturas de actina envolvidas em macropinocitose e internalização de recetores, e que regula a sua formação. Além disso, aprofundámos o conhecimento do processo de ativação da via de sinalização Hh, mostrando que as CDRs sequestram seletivamente e internalizam o recetor Ptch1. As CDRs formam-se minutos após ativação da via por ligandos Shh ou pelo agonista de Smo SAG e continuam a ser formadas a partir daí, sugerindo uma indução contínua da reorganização do citoesqueleto de actina quando a via está ativada. Observámos ainda que a inibição da formação de CDRs através do silenciamento de WAVE1, uma proteína necessária para a formação destas estruturas, resulta na diminuição da ativação da via de sinalização Hh. Além disso, o bloqueio da macropinocitose, que se segue ao fecho das CDRs, através do silenciamento de uma proteína necessária para a cisão de macropinossomas, nomeadamente a proteína BARS, tem um efeito semelhante. Estes resultados sugerem que as CDRs e a macropinocitose são necessárias para a ativação da via de sinalização Hh e indicam que esta via de internalização controla os níveis de sinal Hh. Durante o desenvolvimento, as células proliferativas dependem do cílio primário para a transdução de várias vias de sinalização. A via Hh induz a diferenciação do músculo cardíaco. Por conseguinte, os murganhos deficientes na via de sinalização Hh exibem uma variedade de defeitos de lateralidade, incluindo alteração do looping do coração, como pode ser visto em murganhos deficientes para Arl13b. Por conseguinte, investigámos o papel da Arl13b no desenvolvimento do coração. Mostramos que a Arl13b é altamente expressa no coração de embriões de murganho e de murganhos adultos ao nível do mRNA e da proteína. Além disso, o perfil de distribuição da Arl13b no coração segue o dos cílios primários, que são essenciais para o desenvolvimento cardíaco. Corações de murganhos hnn no estadio E12,5 mostram um canal átrio-ventricular aberto, espessamento da camada compacta ventricular e aumento do índice mitótico no ventrículo esquerdo. Além disso, um atraso de 1 a 2 dias no desenvolvimento é observado em corações de murganhos hnn, quando comparados com controlos selvagens no estadio E13,5. Assim, estes resultados sugerem que a Arl13b é necessária para o desenvolvimento embrionário do coração e que defeitos cardíacos podem contribuir para a letalidade embrionária de murganhos hnn. Em suma, foi estabelecido um novo mecanismo para a regulação dos níveis de superfície do recetor Ptch1, que envolve a remodelação do citoesqueleto de actina e a formação de CDRs após a ativação da via de sinalização Hh. Este mecanismo permite um feedback negativo que evita a repressão excessiva da via através da remoção de Ptch1 da superfície da célula. Além disso, determinou-se que uma mutação de perda de função na Arl13b causa defeitos cardíacos durante o desenvolvimento, possivelmente relacionados com a associação dos defeitos em cílios primários e na sinalização Hh, existentes em murganhos deficientes para Arl13b. A via de sinalização Hh tem tido um papel central entre as vias de sinalização, uma vez que a sua regulação é crucial para o funcionamento apropriada da célula. Assim, a descoberta de um novo mecanismo de tráfego através de macropinocitose e CDRs que controla a ativação e repressão da via de sinalização Hh traz novas perspetivas de como esta via pode ser regulada e pode ainda conduzir à identificação de novos alvos e estratégias terapêuticas. --------------------ABSTRACT: Eukaryotic cells have evolved a complex signaling system that allows them to respond to extracellular and intracellular cues. Signaling pathways are essential for cell and organism survival, since they regulate fundamental processes such as development, growth, immunity, and tissue homeostasis. The Hedgehog (Hh) pathway of signal transduction involves the receptor Patched1 (Ptch1), which has an inhibitory effect on Smoothened (Smo) in the absence of its ligands, the Sonic hedgehog (Shh) proteins. These proteins are fundamental regulators of embryonic development, as illustrated by the dramatic malformations seen in human and mouse embryos with perturbed Hh signal transduction that include polydactyly, craniofacial defects and skeletal malformations. Equally important are the consequences of inappropriate activation of the Hh signaling response in tumor formation. Interestingly, the components of this pathway localize to primary cilia. Moreover, it has been shown that this localization is crucial for Hh signaling. However, in the presence of the ligands, Ptch1 is internalized and destined for degradation or sequestered in a cell compartment where it no longer can play its inhibitory role. ADP-ribosylation factor-like (Arl) 13b, a small GTPase belonging to Arf/Arl family of the Ras superfamily of small GTPases has been implicated in Joubert syndrome, a ciliopathy characterized by congenital cerebellar ataxia, hypotonia, intellectual disability and congenital heart disease. Arl13b-deficient mice, called hennin (hnn) die at embryonic day 13.5 (E13.5) and display morphological abnormalities in primary cilia that lead to the disruption of Hh signaling. Furthermore, Arl13b is directly involved in the regulation of Hh signaling by controlling the localization of several components of this pathway to primary cilia. Here, we show that Arl13b localizes to and regulates the formation of circular dorsal rufles (CDRs), which are actin-basedstructures known to be involved in macropinocytosis and receptor internalization. Additionally, we extended the knowledge of the Hh signaling activation process by showing that CDRs selectively sequester and internalize Ptch1 receptors. CDRs are formed minutes after Hh activation by Shh ligands or the Smo agonist SAG and keep being formed thereafter, suggesting a continuous induction of actin reorganization when the pathway is switched on. Importantly, we observed that disruption of CDRs by silencing WAVE1, a protein required for CDR formation, results in down-regulation of Hh signaling activation. Moreover, the blockade of macropinocytosis, which follows CDR closure, through silencing of a protein necessary for the fission of macropinosomes, namely BARS has a similar effect. These results suggest that CDRs and macropinocytosis are necessary for activation of Hh signaling and indicate that this pathway of internalization controls Hh signal levels. During development, proliferating cells rely on the primary cilium for the transduction of several signaling pathways. Hh induces the differentiation of cardiac muscle. Accordingly, Hh-deficient mice display a variety of laterality defects, including alteration of heart looping, as seen in Arl13b-deficient mice. Therefore, we investigated the role of Arl13b in heart development. We show that Arl13b is highly expressed in the heart of both embryonic and adult mice at mRNA and protein levels. Also, Arl13b localization profile mimics that of primary cilia, which have been shown to be essential to early heart development. E12.5 hnn hearts show an open atrioventricular channel, increased thickening of the ventricular compact layer and increased mitotic index in the left ventricle. Moreover, a delay of 1 to 2 days in development is observed in hnn hearts, when compared to wild-type controls at E13.5. Hence, these results suggest that Arl13b is necessary for embryonic heart development and that cardiac defects might contribute to the embryonic lethality of hnn mice. Altogether, we established a novel mechanism for the regulation of Ptch1 surface levels, involving cytoskeleton remodeling and CDR formation upon Hh signaling activation. This mechanism allows a negative feedback loop that prevents excessive repression of the pathway by removing Ptch1 from the cell surface. Additionally, we determined that the Arl13b loss-offunction mutation causes cardiac defects during development, possibly related to the associated ciliary and Hh signaling defects found in Arl13b-deficient mice. Hh signaling has taken a center stage among the signaling pathways since its regulation is crucial for the appropriate output and function of the cell. Hence, the finding of a novel trafficking mechanism through CDRs and macropinocytosis that controls Hh signaling activation and repression brings new insights to how this pathway can be regulated and can lead to the discovery of novel therapeutic targets and strategies.

Burkitt-like lymphoma in an infant: a case report

Childhood non-Hodgkin's lymphomas, including Burkitt and Burkitt-like, are rarely diagnosed in infants. A case of B-cell lymphoma in a 13-month-old girl with extensive abdominal disease, ascites, pleural effusion, and tumor lysis syndrome is reported. Phenotypic analysis showed a germinal center B-cell phenotype, and a B-cell clonality was confirmed by polymerase chain reaction. There was no evidence of Epstein-Barr and HIV infection. The case herein reported emphasizes the need for considering the diagnosis of lymphoma even in very young children.

Epidermis recreation in spongy-like hydrogels: New opportunities to explore epidermis-like analogues

[Excerpt] On the road to successfully achieving skin regeneration, 3D matrices/scaffolds that provide the adequate physico-chemical and biological cues to recreate the ideal healing environment are believed to be a key element [1], [2] and [3]. Numerous polymeric matrices derived from both natural [4] and [5] and synthetic [6], [7] and [8] sources have been used as cellular supports; nowadays, fewer matrices are simple carriers, and more and more are ECM analogues that can actively participate in the healing process. Therefore, the attractive characteristics of hydrogels, such as high water content, tunable elasticity and facilitated mass transportation, have made them excellent materials to mimic cells’ native environment [9]. Moreover, their hygroscopic nature [10] and possibility of attaining soft tissues-like mechanical properties mean they have potential for exploitation as wound healing promoters [11], [12], [13] and [14]. Nonetheless, hydrogels lack natural cell adhesion sites [15], which limits the maximization of their potential in the recreation of the cell niche. This issue has been tackled through the use of a range of sophisticated approaches to decorate the hydrogels with adhesion sequences such as arginine-glycine-aspartic acid (RGD) derived from fibronectin [16], [17] and [18], and tyrosine-isoleucine-glycine-serine-arginine (YIGSR) derived from laminin [18] and [19], which not only aim to modulate cell adhesion, but also influencing cell fate and survival [18]. Nonetheless, its widespread use is still limited by significant costs associated with the use of recombinant bioactive molecules.

Search for vector-like B quarks in events with one isolated lepton, missing transverse momentum and jets at s√= 8 TeV with the ATLAS detector

A search has been performed for pair production of heavy vector-like down-type (B) quarks. The analysis explores the lepton-plus-jets final state, characterized by events with one isolated charged lepton (electron or muon), significant missing transverse momentum and multiple jets. One or more jets are required to be tagged as arising from b-quarks, and at least one pair of jets must be tagged as arising from the hadronic decay of an electroweak boson. The analysis uses the full data sample of pp collisions recorded in 2012 by the ATLAS detector at the LHC, operating at a center-of-mass energy of 8 TeV, corresponding to an integrated luminosity of 20.3 fb−1. No significant excess of events is observed above the expected background. Limits are set on vector-like B production, as a function of the B branching ratios, assuming the allowable decay modes are B→Wt/Zb/Hb. In the chiral limit with a branching ratio of 100% for the decay B→Wt, the observed (expected) 95% CL lower limit on the vector-like B mass is 810 GeV (760 GeV). In the case where the vector-like B quark has branching ratio values corresponding to those of an SU(2) singlet state, the observed (expected) 95% CL lower limit on the vector-like B mass is 640 GeV (505 GeV). The same analysis, when used to investigate pair production of a colored, charge 5/3 exotic fermion T5/3, with subsequent decay T5/3→Wt, sets an observed (expected) 95% CL lower limit on the T5/3 mass of 840 GeV (780 GeV).

Search for production of vector-like quark pairs and of four top quarks in the lepton-plus-jets final state in pp collisions at root 8=8 TeV with the ATLAS detector

A search for pair production of vector-like quarks, both up-type (T) and down-type (B), as well as for four-top-quark production, is presented. The search is based on pp collisions at s√=8 TeV recorded in 2012 with the ATLAS detector at the CERN Large Hadron Collider and corresponding to an integrated luminosity of 20.3 fb−1. Data are analysed in the lepton-plus-jets final state, characterised by an isolated electron or muon with high transverse momentum, large missing transverse momentum and multiple jets. Dedicated analyses are performed targeting three cases: a T quark with significant branching ratio to a W boson and a b-quark (TT¯→Wb+X), and both a T quark and a B quark with significant branching ratio to a Higgs boson and a third-generation quark (TT¯→Ht+X and BB¯→Hb+X respectively). No significant excess of events above the Standard Model expectation is observed, and 95% CL lower limits are derived on the masses of the vector-like T and B quarks under several branching ratio hypotheses assuming contributions from T→Wb, Zt, Ht and B→Wt, Zb, Hb decays. The 95% CL observed lower limits on the T quark mass range between 715 GeV and 950 GeV for all possible values of the branching ratios into the three decay modes, and are the most stringent constraints to date. Additionally, the most restrictive upper bounds on four-top-quark production are set in a number of new physics scenarios.

Global vs. local nonlinear optimization techniques for human-like movement of an anthropomorphic robot

In this paper a comparison between using global and local optimization techniques for solving the problem of generating human-like arm and hand movements for an anthropomorphic dual arm robot is made. Although the objective function involved in each optimization problem is convex, there is no evidence that the admissible regions of these problems are convex sets. For the sequence of movements for which the numerical tests were done there were no significant differences between the optimal solutions obtained using the global and the local techniques. This suggests that the optimal solution obtained using the local solver is indeed a global solution.

Nonlinear optimization for human-like synchronous movements of a dual arm-hand robotic system

In previous work we have presented a model capable of generating human-like movements for a dual arm-hand robot involved in human-robot cooperative tasks. However, the focus was on the generation of reach-to-grasp and reach-to-regrasp bimanual movements and no synchrony in timing was taken into account. In this paper we extend the previous model in order to accomplish bimanual manipulation tasks by synchronously moving both arms and hands of an anthropomorphic robotic system. Specifically, the new extended model has been designed for two different tasks with different degrees of difficulty. Numerical results were obtained by the implementation of the IPOPT solver embedded in our MATLAB simulator.

Towards human-like bimanual movements in anthropomorphic robots: a nonlinear optimization approach

Previously we have presented a model for generating human-like arm and hand movements on an unimanual anthropomorphic robot involved in human-robot collaboration tasks. The present paper aims to extend our model in order to address the generation of human-like bimanual movement sequences which are challenged by scenarios cluttered with obstacles. Movement planning involves large scale nonlinear constrained optimization problems which are solved using the IPOPT solver. Simulation studies show that the model generates feasible and realistic hand trajectories for action sequences involving the two hands. The computational costs involved in the planning allow for real-time human robot-interaction. A qualitative analysis reveals that the movements of the robot exhibit basic characteristics of human movements.

A new competitive implementation of the electromagnetism-like algorithm for global optimization

The Electromagnetism-like (EM) algorithm is a population- based stochastic global optimization algorithm that uses an attraction- repulsion mechanism to move sample points towards the optimal. In this paper, an implementation of the EM algorithm in the Matlab en- vironment as a useful function for practitioners and for those who want to experiment a new global optimization solver is proposed. A set of benchmark problems are solved in order to evaluate the performance of the implemented method when compared with other stochastic methods available in the Matlab environment. The results con rm that our imple- mentation is a competitive alternative both in term of numerical results and performance. Finally, a case study based on a parameter estimation problem of a biology system shows that the EM implementation could be applied with promising results in the control optimization area.

Exploring the properties of genetically engineered silk-elastin-like protein films

Free standing films of a genetically engineered silk-elastin-like protein (SELP) were prepared using water and formic acid as solvents. Exposure to methanol-saturated air promoted the formation of aggregated β-strands rendering aqueous insolubility and improved the mechanical properties leading to a 10-fold increase in strain-to-failure. The films were optically clear with resistivity values similar to natural rubber and thermally stable up to 180 °C. Addition of glycerol showed to enhance the flexibility of SELP/glycerol films by interacting with SELP molecules through hydrogen bonding, interpenetrating between the polymer chains and granting more conformational freedom. This detailed characterization provides cues for future and unique applications using SELP based biopolymers.

Development of elastin-like recombinamer films with antimicrobial activity

In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4-A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.

Pneumomediastino espontâneo como apresentação de infecção por Mycoplasma pneumoniae

Introdução: O pneumomediastino espontâneo (PE) define-se pela presença de ar livre no mediastino, sem causa traumática. É raro em idade pediátrica e exige elevada suspeição diagnóstica. Caso Clínico: Adolescente de 17 anos, longilíneo, fumador ocasional, sem história traumática nem patologia pulmonar, observado na Urgência por dor retrosternal intensa acompanhada por dispneia ligeira. Referência a acessos de tosse seca violenta horas antes. O diagnóstico de PE suspeitou-se pela palpação de enfisema subcutâneo supraclavicular e confirmou-se por telerradiografia do tórax mostrando ar livre mediastínico. A investigação revelou infeção por Mycoplasma pneumoniae pelo que foi medicado com macrólido para além do repouso e analgesia propostos para o PE, evoluindo favoravelmente. Discussão/Conclusôes: O PE é um diagnóstico a não esquecer perante dor torácica aguda no adolescente. A presença de sintomas, por vezes, frustres e a evolução habitualmente benigna contribuem para um provável subdiagnóstico. O tabagismo é um fator predisponente. A associação a infeção por Mycoplasma pneumoniae só raramente foi descrita.

Gellan gum-hyaluronate spongy-like hydrogels promote angiogenesis in hindlimb ischemia

"Tissue engineering: part A", vol. 21, suppl. 1 (2015)

Coinfection with Mycoplasma Pneumoniae and Chlamydia Pneumoniae in ruptured plaques associated with acute myocardial infarction

OBJECTIVE: To study atheromas, Mycoplasma pneumoniae (M. pneumoniae), and Chlamydia pneumoniae (C. pneumoniae). METHODS: C. pneumoniae was studied with immunohistochemistry and M. pneumoniae with in situ hybridization (ISH), in segments of coronary arteries (SCA) as follows: group A - thrombosed ruptured plaques (TRP) of 23 patients who died due to acute myocardial infarction (AMI); group B - 23 nonruptured plaques (NRP) of group A patients; group C - NRP of 11 coronary patients who did not die due to AMI; and group D - 11 SCA from patients with dilated cardiomyopathy or Chagas' disease without atherosclerosis. RESULTS: The mean number of C. pneumoniae+ cells/400x in groups A, B, C, and D was, respectively, 3.3±3.6; 1.0±1.3; 1.2±2.4; and 0.4±0.3; and the percentage of M. pneumoniae area was, respectively, 3.9±3.5; 1.5± 1.6; 0.9±0.9; and 0.4±0.2. More M. pneumoniae and C. pneumoniae were found in of group A than in group B (P<0.01). Good correlation was seen between the area of the vessel and the M. pneumoniae area in the plaque (r = 0.46; P=0.001) and between C. pneumoniae+ cells and CD4+ T lymphocytes (r = 0.42; P<0.01). The number of C. pneumoniae+ cells correlated with CD20+ B cells (r=0.48; P<0.01). CONCLUSION: M. pneumoniae and C. pneumoniae are more frequently found in TRP correlate with the intensity of the inflammation and diameter of the vessel (positive remodeling).