Melanoma in the Eyes of Mechanobiology

Skin is the largest organ of the human body with several important functions that can be impaired by injury, genetic or chronic diseases. Among all skin diseases, melanoma is one of the most severe, which can lead to death, due to metastization. Mechanotransduction has a crucial role for motility, invasion, adhesion and metastization processes, since it deals with the response of cells to physical forces. Signaling pathways are important to understand how physical cues produced or mediated by the Extracellular Matrix (ECM), affect healthy and tumor cells. During these processes, several molecules in the nucleus and cytoplasm are activated. Melanocytes, keratinocytes, fibroblasts and the ECM, play a crucial role in melanoma formation. This manuscript will address the synergy among melanocytes, keratinocytes, fibroblasts cells and the ECM considering their mechanical contribution and relevance in this disease. Mechanical properties of melanoma cells can also be influenced by pigmentation, which can be associated with changes in stiffness. Mechanical changes can be related with the adhesion, migration, or invasiveness potential of melanoma cells promoting a high metastization capacity of this cancer. Mechanosensing, mechanotransduction, and mechanoresponse will be highlighted with respect to the motility, invasion, adhesion and metastization in melanoma cancer.The authors acknowledge to Ana Silva ([email protected]) for expert assistance with graphical design, and to Portuguese funds – FCT UID/BIM/04293/2019

Mechanobiology of Colorectal Cancer

In this review, the mechanobiology of colorectal cancer (CRC) are discussed. Mecha-notransduction of CRC is addressed considering the relationship of several biophysical cues and biochemical pathways. Mechanobiology is focused on considering how it may influence epithelial cells in terms of motility, morphometric changes, intravasation, circulation, extravasation, and metastization in CRC development. The roles of the tumor microenvironment, ECM, and stroma are also discussed, taking into account the influence of alterations and surface modifications on mechanical properties and their impact on epithelial cells and CRC progression. The role of cancer-associated fibroblasts and the impact of flow shear stress is addressed in terms of how it affects CRC metastization. Finally, some insights concerning how the knowledge of biophysical mechanisms may contribute to the development of new therapeutic strategies and targeting molecules and how mechanical changes of the microenvironment play a role in CRC disease are presented.The authors are grateful to Ana Silva ([email protected]) for expert assistance with graphical design and to Portuguese Fundação para a Ciência e a Tecnologia (FCT) for funds in the framework of the project Instituto de Investigação e Inovação em Saúde (i3S), ref. ID/BIM/04293/2020

Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35) for Gluco- and Galacto- Substituted mannans and Its calcium induced stability

Articles in International JournalsThis study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×104 M−1), carob galactomannan (Ka = 12.4×104 M−1) and negligible association (Ka = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (Ka = 41×104 M−1) and carob galactomannan (Ka = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (Ka) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C

SLMP53-2 Restores Wild-Type-Like Function to Mutant p53 through Hsp70: Promising Activity in Hepatocellular Carcinoma.

Half of human cancers harbor TP53 mutations that render p53 inactive as a tumor suppressor. In these cancers, reactivation of mutant p53 (mutp53) through restoration of wild-type-like function constitutes a valuable anticancer therapeutic strategy. In order to search for mutp53 reactivators, a small library of tryptophanol-derived oxazoloisoindolinones was synthesized and the potential of these compounds as mutp53 reactivators and anticancer agents was investigated in human tumor cells and xenograft mouse models. By analysis of their anti-proliferative effect on a panel of p53-null NCI-H1299 tumor cells ectopically expressing highly prevalent mutp53, the compound SLMP53-2 was selected based on its potential reactivation of multiple structural mutp53. In mutp53-Y220C-expressing hepatocellular carcinoma (HCC) cells, SLMP53-2-induced growth inhibition was mediated by cell cycle arrest, apoptosis, and endoplasmic reticulum stress response. In these cells, SLMP53-2 restored wild-type-like conformation and DNA-binding ability of mutp53-Y220C by enhancing its interaction with the heat shock protein 70 (Hsp70), leading to the reestablishment of p53 transcriptional activity. Additionally, SLMP53-2 displayed synergistic effect with sorafenib, the only approved therapy for advanced HCC. Notably, it exhibited potent antitumor activity in human HCC xenograft mouse models with a favorable toxicological profile. Collectively, SLMP53-2 is a new mutp53-targeting agent with promising antitumor activity, particularly against HCC

Identification of structural features of condensed tannins that affect protein aggregation

A diverse panel of condensed tannins was used to resolve the confounding effects of size and subunit composition seen previously in tannin-protein interactions. Turbidimetry revealed that size in terms of mean degree of polymerisation (mDP) or average molecular weight (amw) was the most important tannin parameter. The smallest tannin with the relatively largest effect on protein aggregation had an mDP of ~7. The average size was significantly correlated with aggregation of bovine serum albumin, BSA (mDP: r=-0.916; amw: r=-0.925; p<0.01; df=27), and gelatin (mDP: r=-0.961; amw: r=-0.981; p<0.01; df=12). The procyanidin/prodelphinidin and cis-/trans-flavan-3-ol ratios gave no significant correlations. Tryptophan fluorescence quenching indicated that procyanidins and cis-flavan-3-ol units contributed most to the tannin interactions on the BSA surface and in the hydrophobic binding pocket (r=0.677; p<0.05; df=9 and r=0.887; p<0.01; df=9, respectively). Circular dichroism revealed that higher proportions of prodelphinidins decreased the apparent α-helix content (r=-0.941; p<0.01; df=5) and increased the apparent β-sheet content (r=0.916; p<0.05; df=5) of BSA

First Dark Matter Search Results from the LUX-ZEPLIN (LZ) Experiment

The LUX-ZEPLIN experiment is a dark matter detector centered on a dual-phase xenon time projection chamber operating at the Sanford Underground Research Facility in Lead, South Dakota, USA. This Letter reports results from LUX-ZEPLIN's first search for weakly interacting massive particles (WIMPs) with an exposure of 60 live days using a fiducial mass of 5.5 t. A profile-likelihood ratio analysis shows the data to be consistent with a background-only hypothesis, setting new limits on spin-independent WIMP-nucleon, spin-dependent WIMP-neutron, and spin-dependent WIMP-proton cross sections for WIMP masses above 9 GeV/c2. The most stringent limit is set for spin-independent scattering at 36 GeV/c2, rejecting cross sections above 9.2×10-48 cm at the 90% confidence level

Regulation of Oxidative Stress Response by CosR, an Essential Response Regulator in Campylobacter jejuni

CosR (Campylobacter oxidative stress regulator; Cj0355c) is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a leading foodborne pathogen causing human gastroenteritis worldwide. Despite importance, the function of CosR remains completely unknown mainly because of cell death caused by its knockout mutation. To overcome this technical limitation, in this study, antisense technology was used to investigate the regulatory function of CosR by modulating the level of CosR expression. Two-dimensional gel electrophoresis (2DGE) was performed to identify the CosR regulon either by suppressing CosR expression with antisense peptide nucleic acid (PNA) or by overexpressing CosR in C. jejuni. According to the results of 2DGE, CosR regulated 32 proteins involved in various cellular processes. Notably, CosR negatively regulated a few key proteins of the oxidative stress response of C. jejuni, such as SodB, Dps, Rrc and LuxS, whereas CosR positively controlled AhpC. Electrophoretic mobility shift assay showed that CosR directly bound to the promoter region of the oxidative stress genes. DNase I footprinting assays identified 21-bp CosR binding sequences in the sodB and ahpC promoters, suggesting CosR specifically recognizes and binds to the regulated genes. Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide. Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type. Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni

The LUX-ZEPLIN (LZ) Experiment

We describe the design and assembly of the LUX-ZEPLIN experiment, a direct detection search for cosmic WIMP dark matter particles. The centerpiece of the experiment is a large liquid xenon time projection chamber sensitive to low energy nuclear recoils. Rejection of backgrounds is enhanced by a Xe skin veto detector and by a liquid scintillator Outer Detector loaded with gadolinium for efficient neutron capture and tagging. LZ is located in the Davis Cavern at the 4850' level of the Sanford Underground Research Facility in Lead, South Dakota, USA. We describe the major subsystems of the experiment and its key design features and requirements

The LUX-ZEPLIN (LZ) radioactivity and cleanliness control programs

LUX-ZEPLIN (LZ) is a second-generation direct dark matter experiment with spin-independent WIMP-nucleon scattering sensitivity above 1.4×10−48cm2 for a WIMP mass of 40GeV/c2 and a 1000days exposure. LZ achieves this sensitivity through a combination of a large 5.6t fiducial volume, active inner and outer veto systems, and radio-pure construction using materials with inherently low radioactivity content. The LZ collaboration performed an extensive radioassay campaign over a period of six years to inform material selection for construction and provide an input to the experimental background model against which any possible signal excess may be evaluated. The campaign and its results are described in this paper. We present assays of dust and radon daughters depositing on the surface of components as well as cleanliness controls necessary to maintain background expectations through detector construction and assembly. Finally, examples from the campaign to highlight fixed contaminant radioassays for the LZ photomultiplier tubes, quality control and quality assurance procedures through fabrication, radon emanation measurements of major sub-systems, and bespoke detector systems to assay scintillator are presented