A cidade que virou casa: considerações sobre o brincar livre e espontâneo durante o período de isolamento social de 2020 / The city that became home: considerations on free and spontaneous play during the 2020 social isolation period / La ciudad que se convirtió en casa: consideraciones sobre el juego libre y espontáneo durante el período de aislamiento social 2020

Em março de 2020 estávamos nas ruas das cidades observando o brincar livre e espontâneo de crianças em espaços diversos, quando fomos surpreendidos pela pandemia global do novo coronavírus. Medidas variadas de isolamento social foram implementadas. Neste cenário, passamos a indagar sobre como estaria o brincar livre e espontâneo dentro das residências de famílias isoladas. Por meio do olhar fenomenológico, passamos a buscar formas de nos aproximarmos da experiência vivida das crianças de maneira remota e por intermédio de seus responsáveis. Dialogamos com 55 famílias em quatro meses. Percebemos que investigar o brincar durante o isolamento social é investigar um fenômeno que acontece dentro de casa em outros momentos, porém no período de isolamento, com tempos mais amplos e com menos restrições de acesso a objetos e cômodos. Em diálogo com autores da área, evidenciam-se provocações que diferentes cômodos engendraram nas crianças a partir do brincar livre e espontâneo. (AU)

In March 2020 we were on the streets of cities observing the free and spontaneous play of children in different spaces, when we were surprised by the global pandemic of the novel coronavirus. Various measures of isolation and social distancing were implemented. In this new scenario, we started to ask ourselves about what free and spontaneous play would be like inside the homes of isolated families. Through the phenomenological perspective, we started to look for ways to approach the lived experience of children remotely and through their guardians. We followed 55 isolated families for four months. We realized that investigating playing during social isolation allowed the approximation of a phenomenon that happens inside the house at other times, but with longer times and with fewer restrictions on access to objects and rooms. In dialogue with authors in the area, the provocations that the different rooms engendered in children from free and spontaneous play are evident. (AU)

En marzo de 2020 estábamos en las calles de las ciudades observando el juego libre y espontáneo de los niños en diferentes espacios, cuando nos sorprendió la pandemia global del nuevo coronavirus. Se implementaron diversas medidas de aislamiento y distanciamiento social. En este escenario, comenzamos a preguntarnos cómo sería el juego libre y espontáneo dentro de las residencias de familias aisladas. A través de la mirada fenomenológica, comenzamos a buscar formas de acercarnos a la experiencia vivida de los niños de forma remota y a través de sus responsables. Dialogamos con 55 familias a lo largo de cuatro meses. Nos dimos cuenta de que investigar el juego durante el aislamiento social correspondía a investigar un fenómeno que ocurre dentro de las casas en otros momentos, sin embargo, en el periodo de aislamiento, con tiempos más largos y con menos restricciones de acceso a objetos y habitaciones. En diálogo con autores de ese campo, se evidencian las provocaciones que los distintos espacios de la casa generaron en los niños a partir del juego libre y espontáneo. (AU)

Artificial laminin polymers assembled in acidic pH mimic basement membrane organization.

Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 +/- 5 and 86 +/- 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1', which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin alpha1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future.

Heparin-binding sites in granulocyte-macrophage colony-stimulating factor. Localization and regulation by histidine ionization.

The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four alpha-helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His83 and His87) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr83, Gln85, and Tyr87) were replaced by the corresponding basic residues present in hGM-CSF (His83, Lys85, and His87). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His15) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His15 for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF.