Alternative method to visualize receptor dynamics in cell membranes

There is a close relation between membrane receptor dynamics and their behavior. Several microscopy techniques have been developed to study protein dynamics in live cells such as the Fluorescence Recovery After Photobleaching (FRAP) or the Single Particle Tracking (SPT). These methodologies require expensive instruments, are time consuming, allow the analysis of a small portion of the cell or an extremely small number of receptors at a time. Here we propose a time-saving approach that allows to visualize the entire receptor pool and its localization in time. This protocol requires an epifluorescence microscope equipped for structured illuminated sectioning and for live cell imaging. It can be applied to characterize membrane receptor and multi-protein complex and their response to activators or inhibitors. Image acquisition and analysis can be performed in two days, while cells and substratum preparation require a few minutes a day for three days

For “a positive and feasible architecture”. The contribution of Mario Chiattone to the avant-garde movements of the early 20th century

The aim of this study is to highlight the contribution made by the Ticino-native architect, Mario Chiattone (1891–1957) to the development of modern language. His architectural designs, made in his studio in Milan between 1914 and 1915, highlight his artistic sensibility and demonstrate how he was particularly receptive to the needs of a rapidly evolving society. The working method employed by the architect, his extreme attention to architectural construction and the practicality of the solutions developed for his “Modern Metropolis” emerged clearly through the analysis carried out directly on the works using 3D imaging and reconstruction techniques. At the same time, the results of the archival-bibliographic research carried out simultaneously made it possible to contextualize Chiattone’s work in the light of historical and artistic developments in early-20th century Milan, including the numerous facets of his architectural output and the multiple sources of inspiration that characterized its development. A portrait emerged with unprecedented clarity of an extremely cultured, impassioned architect, of a tireless designer, open to experimentation and at the same time, respectful of tradition, far removed from propagandistic Futurist rhetoric, an advocate for a new, “positive and feasible” architecture

Hot spot mutation in receptor tyrosine kinases elicits pro-oncogenic effects

Tyrosine kinase receptors are frequently altered both in expression and activity in cancer. We performed a pan-cancer analysis to identify novel putative cancer driver mutations or/and therapeutically actionable mutations of the kinase domain of different tyrosine kinase receptors (RTK). To pinpoint those mutations that may be clinically relevant, we exploited the recurrence of alterations on analogous amino acid residues within the kinase domain (PK_Tyr_Ser-Thr) across different kinases as a predictor of functional impact. By exploiting MutationAligner and LowMACA bioinformatics resources, we highlighted novel uncharacterized mutations in position 256 of the consensus sequences of KD. This alteration is located in the A-loop of TKD and found in FGFR1-4 , FLT3, FLT4, PFGFRA, EGFR , VEGFR2 receptors, possibly leading to constitutive activation of the RTK. These amino acid residues correspond to the well-known activating V600E mutation of the oncogene B-Raf. Our analyses demonstrate that the previously uncharacterized VEGFR2 R1051Q and FGFR1 D647N mutations lead to receptor phosphorylation and metabolic changes which might be worth comparing and investigating. For this purpose, residue-substitutions were introduced by point mutation using the Quikchange lightning site-directed mutagenesis kit. Receptors and their mutants were expressed in HEK293T cells and assessed for ATP affinity using an ADP-glo kinase assay and phosphorylation rate through WB analysis. The results confirm a similar altered ATP affinity as well as a great phosphorylation for both mutant receptors. When expressed in tumoral cells both mutants lead to a strong increase in the cell migratory capacity, compared to WT receptor, possibly suggesting an aggressive and metastatic cell phenotype both in vitro and in vivo invasion assay (i.e. CAM). Finally, it was evaluated the activity of tyrosine kinase inhibitors (TKi) including Erdafitinib, Lenvatinib, Sunitinib and Linifanib following receptor phosphorylation by WB. Both mutated receptors exhibit higher sensitivity to TKi. These data were confirmed in vivo for VEGFR2 R1051Q mutant. Indeed, Sk-Mel-31 expressing VEGFR2 R1051Q, injected s.c. n NOD/SCID mice, are more sensitive to the VEGFR2-targeted TKI Linifanib. Our data confirm that similar alterations in the tyrosine kinase domain modulate similar biological responses and druggability

VEGFR2(R1051Q) and FGFR1(D647N) correspondent mutations exhibit pro-oncogenic effects both in vitro and in vivo

Background Tyrosine kinase receptors(RTK) are frequently altered both in expression and activity in cancer. We performed a pan-cancer analysis to identify novel putative cancer driver or/and therapeutically actionable mutations of the kinase domain of different RTK(1). To pinpoint those mutations that may be clinically relevant, we exploited the recurrence of alterations on analogous amino acid residues within the kinase domain (PK_Tyr_Ser-Thr) across different kinases as a predictor of functional impact(2). By exploiting MutationAligner and LowMACA bioinformatics resources, we highlighted novel uncharacterized mutations in position 256 of the consensus sequences of KD. This alteration is located in the A-loop of TKD of FGFR1-4, FLT3, FLT4, PDGFRA, EGFR, VEGFR2 receptors, possibly leading to constitutive activation of the RTK(3). Methods Our previous results demonstrate that the substitution R1051Q in VEGFR2 leads to an increase of kinase activity and phosphorylation of receptor and supports metabolic changes. In order to evaluate if similar alterations in the tyrosine kinase domain modulate similar biological responses and druggability, we introduced the R1051Q correspondent substitution in FGFR1 (FGFR1 D647N) using the Quickchange lightning site-directed mutagenesis kit. Receptors and their mutants were expressed in HEK293T cells and assessed for ATP affinity using an ADP-glo kinase assay and phosphorylation rate through WB analysis. Cell migratory capacity and metastatic cell phenotype were assessed with both in vitro and in vivo invasion assay (i.e., CAM). Results The results confirm a similar altered ATP affinity as well as a great phosphorylation for both mutant receptors. When expressed in tumoral cells both mutants lead to a strong increase in the cell migratory capacity, compared to WT receptor. Finally, it was evaluated the activity of tyrosine kinase inhibitors (TKi) including Erdafitinib, Lenvatinib, Sunitinib and Linifanib, following receptor phosphorylation by WB. Both mutated receptors exhibit higher sensitivity to TKi. Conclusions Our data confirm our previous hypotheses concerning the biological effect of these mutations. Future studies will allow to extend this knowledge to other significant mutations in the same hotspot position. This concept is particularly important for therapeutically actionable mutations. Indeed, PD-based analyses have the potential to accelerate the choice of patient-specific targeted drugs

Role of gremlin-1 in the pathophysiology of the adipose tissues

Gremlin-1 is a secreted bone morphogenetic protein (BMP) antagonist playing a pivotal role in the regulation of tissue formation and embryonic development. Since its first identification in 1997, gremlin-1 has been shown to be a multifunctional factor involved in wound healing, inflammation, cancer and tissue fibrosis. Among others, the activity of gremlin-1 is mediated by its interaction with BMPs or with membrane receptors such as the vascular endothelial growth factor receptor 2 (VEGFR2) or heparan sulfate proteoglycans (HSPGs). Growing evidence has highlighted a central role of gremlin-1 in the homeostasis of the adipose tissue (AT). Of note, gremlin-1 is involved in AT dysfunction during type 2 diabetes, obesity and non-alcoholic fatty liver disease (NAFLD) metabolic disorders. In this review we discuss recent findings on gremlin-1 involvement in AT biology, with particular attention to its role in metabolic diseases, to highlight its potential as a prognostic marker and therapeutic target

Digital Modernism Heritage Lexicon

The book investigates the theme of Modernism (1920-1960 and its epigones) as an integral part of tangible and intangible cultural heritage which contains the result of a whole range of disciplines whose aim is to identify, document and preserve the memory of the past and the value of the future. Including several chapters, it contains research results relating to cultural heritage, more specifically Modernism, and current digital technologies. This makes it possible to record and evaluate the changes that both undergo: the first one, from a material point of view, the second one from the research point of view, which integrates the traditional approach with an innovative one. The purpose of the publication is to show the most recent studies on the modernist lexicon 100 years after its birth, moving through different fields of cultural heritage: from different forms of art to architecture, from design to engineering, from literature to history, representation and restoration. The book appeals to scholars and professionals who are involved in the process of understanding, reading and comprehension the transformation that the places have undergone within the period under examination. It will certainly foster the international exchange of knowledge that characterized Modernism

Effects of pre‐operative isolation on postoperative pulmonary complications after elective surgery: an international prospective cohort study

We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05-1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4-7 days or >= 8 days of 1.25 (1.04-1.48), p = 0.015 and 1.31 (1.11-1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care