Methods of Expression, Purification, and Preparation of the c-Myc b-HLH-LZ for Its Biophysical Characterization

AbstractMelanoma is the most fatal form of skin cancer, with increasing prevalence worldwide. The most common melanoma genetic driver is mutation of the proto-oncogene serine/threonine kinase BRAF; thus, the inhibition of its MAP kinase pathway by specific inhibitors is a commonly applied therapy. However, many patients are resistant, or develop resistance to this type of monotherapy, and therefore combined therapies which target other signaling pathways through various molecular mechanisms are required. A possible strategy may involve targeting cellular energy metabolism, which has been recognized as crucial for cancer development and progression and which connects through glycolysis to cell surface glycan biosynthetic pathways. Protein glycosylation is a hallmark of more than 50% of the human proteome and it has been recognized that altered glycosylation occurs during the metastatic progression of melanoma cells which, in turn facilitates their migration. This review provides a description of recent advances in the search for factors able to remodel cell metabolism between glycolysis and oxidative phosphorylation, and of changes in specific markers and in the biophysical properties of cells during melanoma development from a nevus to metastasis. This development is accompanied by changes in the expression of surface glycans, with corresponding changes in ligand-receptor affinity, giving rise to structural features and viscoelastic parameters particularly well suited to study by label-free biophysical methods.

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Remote Sensing Methods for the Biophysical Characterization of Protected Areas Globally: Challenges and Opportunities

ISPRS International Journal of Geo-Information ◽

10.3390/ijgi10060384 ◽

2021 ◽

Vol 10 (6) ◽

pp. 384

Author(s):

Javier Martínez-López ◽

Bastian Bertzky ◽

Simon Willcock ◽

Marine Robuchon ◽

María Almagro ◽

...

Keyword(s):

Remote Sensing ◽

Protected Areas ◽

Large Scale ◽

Anthropogenic Activities ◽

Global Scale ◽

Area Network ◽

Biophysical Characterization ◽

Challenges And Opportunities ◽

Scale Characterization

Protected areas (PAs) are a key strategy to reverse global biodiversity declines, but they are under increasing pressure from anthropogenic activities and concomitant effects. Thus, the heterogeneous landscapes within PAs, containing a number of different habitats and ecosystem types, are in various degrees of disturbance. Characterizing habitats and ecosystems within the global protected area network requires large-scale monitoring over long time scales. This study reviews methods for the biophysical characterization of terrestrial PAs at a global scale by means of remote sensing (RS) and provides further recommendations. To this end, we first discuss the importance of taking into account the structural and functional attributes, as well as integrating a broad spectrum of variables, to account for the different ecosystem and habitat types within PAs, considering examples at local and regional scales. We then discuss potential variables, challenges and limitations of existing global environmental stratifications, as well as the biophysical characterization of PAs, and finally offer some recommendations. Computational and interoperability issues are also discussed, as well as the potential of cloud-based platforms linked to earth observations to support large-scale characterization of PAs. Using RS to characterize PAs globally is a crucial approach to help ensure sustainable development, but it requires further work before such studies are able to inform large-scale conservation actions. This study proposes 14 recommendations in order to improve existing initiatives to biophysically characterize PAs at a global scale.

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Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry

Scientific Reports ◽

10.1038/s41598-021-90002-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Giulia Tedeschi ◽

Lorenzo Scipioni ◽

Maria Papanikolaou ◽

Geoffrey W. Abbott ◽

Michelle A. Digman

Keyword(s):

Plasma Membrane ◽

Protein Diffusion ◽

Ion Diffusion ◽

Fluorescence Fluctuation Spectroscopy ◽

Biophysical Characterization ◽

Kv Channels ◽

Subunit Stoichiometry ◽

Spatio Temporal ◽

Voltage Sensing Domain

AbstractVoltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

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