Biomolecular condensates in membrane receptor signaling

Clustering is a prominent feature of receptors at the plasma membrane (PM). It plays an important role in signaling. Liquid–liquid phase separation (LLPS) of proteins is emerging as a novel mechanism underlying the observed clustering. Receptors/transmembrane signaling proteins can be core components essential for LLPS (such as LAT or nephrin) or clients enriched at the phase-separated condensates (for example, at the postsynaptic density or at tight junctions). Condensate formation has been shown to regulate signaling in multiple ways, including by increasing protein binding avidity and by modulating the local biochemical environment. In moving forward, it is important to study protein LLPS at the PM of living cells, its interplay with other factors underlying receptor clustering, and its signaling and functional consequences.     

Xylem traits of peatland Scots pines reveal a complex climatic signal: A study in the Eastern Italian Alps

Long-term climate reconstructions are frequently based on tree-ring high-resolution proxies extracted from subfossil peatland trees. Peatlands are peculiar ecosystems characterized by high moisture in the upper soil part which creates a harsh living environment for trees. The climate mostly indirectly influences tree growth determining seasonal variations in the water table level. Within this framework, the aim of this study was to investigate climate responses of trees (Pinus sylvestris L.) growing inside and outside a Southern Alpine peat bog, by using tree-ring and wood anatomical traits (e.g. tracheid number and dimension, cell-wall thickness). Our results showed differences in the xylem structure and climate signal recorded by peatland and mineral soil trees. Peatland trees were characterized by narrow rings and tracheids with thinner cell wall. Summer temperature and precipitation were the major drivers of xylem formation in peatland trees. At intra-annual level wood anatomical traits revealed a complex within-ring signal during the growing season. The multi-parameters approach together with the high-resolution gained by using tree-ring sectors allowed us to obtain new detailed information on the xylem development of peatland trees and climate drivers that influenced it.     

CPOP: An open source C++ cell POPulation modeler for radiation biology applications

PurposeMulticellular tumor spheroids are realistic in-vitro systems used in radiation biology research to study the effect of anticancer drugs or to evaluate the resistance of cancer cells under specific conditions. When combining the modeling of spheroids together with the simulation of radiation using Monte Carlo methods, one could estimate cell and DNA damage to be compared with experimental data. We developed a Cell Population (CPOP) modeler combined to Geant4 simulations in order to tackle how energy depositions are allocated to cells, especially when enhancing radiation outcomes using high-Z nanoparticles. CPOP manages to model large three-dimensional cell populations with independent deformable cells described with their nucleus, cytoplasm and membranes together with force law systems to manage cell–cell interactions.MethodsCPOP is an opensource platform written in C++. It is divided into two main libraries: a “Modeler” library, for cell geometry modeling using meshes, and a Multi Agent System (MAS) library, simulating all agent (cell) interactions among the population. CPOP is fully interfaced with the Geant4 Monte Carlo toolkit and is able to directly launch Geant4 simulations after compilation.We modeled a full and realistic 3D cell population from SK-MEL28 melanoma cell population cultured experimentally. The spheroid diameter of 550 ± 40 µm corresponds to a population of approximately 1000 cells having a diameter of 17.2 ± 2.5 µm and a nucleus diameter of 11.2 ± 2.0 µm. We decided to reproduce cell irradiations performed with a X-RAD 320 Biological Irradiator (Precision XRay Inc., North Branford, CT).ResultsWe simulated the energy spectrum of secondary particles generated in the vicinity of the spheroid and plotted the different energy spectra recovered internally to the spheroid. We evaluated also the impact of AGuIX (Gadolinium) nanoparticles modeled into the spheroid with their corresponding secondary energy spectra.ConclusionsWe succeeded into modeling cell populations and combined them with Geant4 simulations. The next step will be to integrate DNA geometrical models into cell nuclei and to use the Geant4-DNA physics and radiolysis modeling capabilities in order to evaluate early strand breaks induced on DNA.     

The Modular Organisation and Stability of a Thermostable Family 10 Xylanase

The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca²⁺ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.     

The advantage of using a starch based non-Newtonian fluid to prepare micro sections

The breakage or distortion of cellular structures is one of the biggest problems in creating micro-sections for wood anatomical analyses in tree-ring as well as other branches of anatomical research. These broken or distorted structures cause artifacts in photomicrographs that require time consuming image manipulation or corrections prior to further analyses. The simple application of a cornstarch, water, and glycerol (CWG) solution (10:8:7 ratio), a so called non-Newtonian fluid to the surface of wooden specimen before sectioning improves the overall quality of the resulting micro-sections. In particular the problem of secondary cell walls splitting off the primary wall while sectioning is drastically reduced. The quality of the sections using this solution is comparable to that obtained from the more laborious and expensive paraffin embedding.     

Inhibition of NF-κB signaling interferes with phorbol ester-induced growth arrest of keratinocytes in a TNFR1-independent manner

A skin-specific block in NF-κB signaling leads to hyperproliferation of the keratinocytes, inflammation, and spontaneous development of squamous cell carcinoma (SCC). Here we show that an inhibition of NF-κB signaling in keratinocytes, via the expression of the super-repressor/ degradation-resistant form of the IκBα protein (IκBαDN), interferes with the growth arrest induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). The IκBαDN cells are able to overcome the TPA-induced cell cycle block. Although SCC development as well as hyperproliferation due to IκBαDN expression in keratinocytes is known to require TNFR1 signaling, the effect of IκBαDN on phorbol ester signaling is downstream/independent of TNFR1. These data thus identify an interaction between IκBαDN and the tumor promoter TPA in the growth regulation of keratinocytes. The proposed mechanism is also likely to be significant in the process of cancer development due to NF-κB inhibition.     

Development of rapid and highly sensitive HSPA1A promoter-driven luciferase reporter system for assessing oxidative stress associated with low-dose photodynamic therapy

Photodynamic therapy (PDT) is a regulatory-approved modality for treating a variety of malignant tumors. It induces tumor tissue damage via photosensitizer-mediated oxidative cytotoxicity. The heat shock protein 70 (HSP70-1) is a stress protein encoded by the HSPA1A gene and is significantly induced by oxidative stress associated with PDT. The aim of this study was to identify the functional region of the HSPA1A promoter that responds to PDT-induced oxidative stress and uses the stress responsiveness of HSPA1A expression to establish a rapid and cost-effective photocytotoxic assessment bioassay to evaluate the photodynamic potential of photosensitizers. By constructing luciferase vectors with a variety of hspa1a promoter fractions and examining their relative luciferase activity, we demonstrated that the DNA sequence from −218 to +87 of the HSPA1A gene could be used as a functional promoter to detect the PDT-induced oxidative stress. The maximal relative luciferase activity level of HSPA1A (HSP70-1) induced by hypericin-PDT was nearly nine times that of the control. Our results suggest that the novel reporter gene assay using a functional region of the HSP70A1A promoter has significant advantages for the detection of photoactivity in terms of both speed and sensitivity, when compared with a cell viability test based on ATP quantification and ROS levels. Furthermore, phthalocyanine zinc and methylene blue both induced significantly elevated levels of relative luciferase activity in a dose-dependent manner.