A commentary on the inhibition of human TPC2 channel by the natural flavonoid naringenin: methods, experiments, and ideas

: Human endo-lysosomes possess a class of proteins called TPC channels on their membrane, which are essential for proper cell functioning. This protein family can be functionally studied by expressing them in plant vacuoles. Inhibition of hTPC activity by naringenin, one of the main flavonoids present in the human diet, has the potential to be beneficial in severe human diseases such as solid tumor development, melanoma, and viral infections. We attempted to identify the molecular basis of the interaction between hTPC2 and naringenin, using ensemble docking on molecular dynamics (MD) trajectories, but the specific binding site remains elusive, posing a challenge that could potentially be addressed in the future by increased computational power in MD and the combined use of microscopy techniques such as cryo-EM

Comparison and evaluation of the methods for measuring hemolytic activity of <i>Stomolophus meleagris</i> jellyfish tentacle extract

Hemolytic activity assessment is a widely used method to evaluate the toxicity of marine organisms, including jellyfish. However, there are some challenges associated with testing hemolytic activity. In this study, four methods were employed to compare the hemolytic activity of jellyfish tentacle extract (TE). Firstly, a microplate reader was used to compare the mouse hemoglobin solution at three different wavelengths (415 nm, 541 nm, and 576 nm), and the most sensitive wavelength was selected for further experiments. Secondly, photomicrograph counting was used to determine the number of complete red blood cells in the field of view. Thirdly, a microplate reader was used to test hemolytic activity in a 96-well plate at 415 nm. Fourthly, a Bicinchoninic Acid (BCA) kit was used to test the concentration of hemoglobin in the solution. Finally, a UV-Vis Spectrophotometer was used to test hemolytic activity at 415 nm. Among the three wavelengths tested, the absorption value was most sensitive at 415 nm. The photomicrograph counting method was able to reflect changes in the shape of Red Blood Cells (RBCs). The microplate reader method may exhibit deviations when the solution concentration is high, while external factors could influence the BCA kit when the toxin concentration in the experimental group is low. The spectrophotometer method was found to be relatively accurate and sensitive to changes. When optimizing the method, it is important to consider the applicability of the Beer-Lambert law and the concentration of solutions

Potential Biomedical Applications of Collagen Filaments derived from the Marine Demosponges Ircinia oros (Schmidt, 1864) and Sarcotragus foetidus (Schmidt, 1862)

Collagen filaments derived from the two marine demosponges Ircinia oros and Sarcotragus foetidus were for the first time isolated, biochemically characterised and tested for their potential use in regenerative medicine. SDS-PAGE of isolated filaments revealed a main collagen subunit band of 130 kDa in both of the samples under study. DSC analysis on 2D membranes produced with collagenous sponge filaments showed higher thermal stability than commercial mammalian-derived collagen membranes. Dynamic mechanical and thermal analysis attested that the membranes obtained from filaments of S. foetidus were more resistant and stable at the rising temperature, compared to the ones derived from filaments of I. oros. Moreover, the former has higher stability in saline and in collagenase solutions and evident antioxidant activity. Conversely, their water binding capacity results were lower than that of membranes obtained from I. oros. Adhesion and proliferation tests using L929 fibroblasts and HaCaT keratinocytes resulted in a remarkable biocompatibility of both developed membrane models, and gene expression analysis showed an evident up-regulation of ECM-related genes. Finally, membranes from I. oros significantly increased type I collagen gene expression and its release in the culture medium. The findings here reported strongly suggest the biotechnological potential of these collagenous structures of poriferan origin as scaffolds for wound healing

Alginate-Based Electrospun Membranes Containing ZnO Nanoparticles as Potential Wound Healing Patches: Biological, Mechanical, and Physicochemical Characterization

In the present work, alginate-based mats with and without ZnO nanoparticles were prepared via an electrospinning technique and subjected to a washing-cross-linking process to obtain highly stable products characterized by thin and homogeneous nanofibers with a diameter of 100 \ub1 30 nm. Using a commercial collagen product as control, the biological response of the prepared mats was carefully evaluated with particular attention paid to the influence of the used cross-linking agent (Ca2+, Sr2+, or Ba2+ ions) and to the presence of nanofillers. Fibroblast and keratinocyte cultures successfully proved the safety of the prepared alginate-based mats, whereas ZnO nanoparticles were found to provide strong antibacteriostatic and antibacterial properties; above all, the strontium- and barium-cross-linked samples showed performances in terms of cell adhesion and growth very similar to those of the commercial collagen membrane despite them showing a significantly lower protein adsorption. Moreover, the mechanical and water-related properties of the strontium-cross-linked mats embedding ZnO nanoparticles were proven to be similar to those of human skin (i.e., Young modulus of 470 MPa and water vapor permeability of 3.8 7 10-12 g/m Pa s), thus proving the ability of the prepared mats to be able to endure considerable stress, maintaining at the same time the fundamental ability to remove exudates. Taking into account the obtained results, the proposed alginate-based products could lead to harmless and affordable surgical patches and wound dressing membranes with a simpler and safer production procedure than the commonly employed animal collagen-derived systems

Recombinant production of hydroxylated collagen polypeptides derived from the Demospongia C. reniformis: a new biotechnological source of marine collagen

Collagen from marine sources is a very attractive alternative to bovine collagen. In biomedicine, several applications of collagens derived from sponges have been described, many of which from the demospongia Chondrosia reniformis. The production of these molecules in recombinant form would allow to obtain well-defined molecular types and to limit the expensive procedures of sample recovery and purification. Here we report the realization of a yeast strain able to produce hydroxylated collagen polypeptides derived from C. reniformis. First the cDNAs coding for the \uf061 and \uf062\uf020 subunits of the sponge enzyme prolyl-4-hydroxylase (P\uf034\uf048\uf029 were identified by PCR approach, then they were cloned in pPink and in pPIC6\uf061 vectors respectively and finally they were stably integrated in the genome of the methanotrophic yeast Pichia pastoris. The strain with the highest P4H activity was then transformed with a third expression vector (pPICZ) containing the coding region of a sponge collagen polypeptide in frame with an Histidine-TAG at the 3\u2019-end. Recombinant protein expression was then induced and collagen polypeptides were purified by affinity chromatography and analysed by SDS-PAGE followed by Mass Spectrometry, in order to confirm the sequence and the presence of hydroxy-proline residues. The biocompatibility of the recombinant proteins was evaluated by MTT test in human neonatal fibroblasts and furthermore, tissue culture plates were treated with 10 \uf06dg/ml recombinant protein solution to test its cell adhesion properties. In conclusion, a first recombinant hydroxylated marine collagen polypeptide was produced in the Pichia system, resulting biocompatible and suggesting its use as porcine gelatin substitute for cell culture. Research supported by EU (FP7 grant agreement n: 266033 SPonge Enzyme End Cell for Innovative AppLication-SPECIAL

Cell Reactivity to Different Silica

The interaction between mineral structures and living beings is increasingly attracting the interest of research. The formation of skeletons, geomicrobiology, the study of the origin of life, soil biology, benthos biology, human and mammalian diseases generated by the inhalation of dust and biomaterials are some examples of scientific areas where the topic has a relevance. In this chapter we focus on cell reactivity to siliceous rocks and to the various forms of silicon dioxide, in particular. The examples here reported carefully review howsuch minerals may strongly affect different living beings, from simple ones to humans. The biomineralogy concept is explained, focusing on the effects of rocks on cell growth and development. The toxic action of silicon dioxide in mammalian lungs is the oldest evidence of crystalline silica bioactivity. More recently, we could demonstrate that crystalline silica has a deep impact on cell biology throughout the whole animal kingdom. One of the most illustrative case studies is the marine sponge Chondrosia reniformis, which has the amazing ability to incorporate and etch crystalline silica releasing dissolved silicates in the medium. This specific and selective action is due to the chemical reaction of ascorbic acid with quartz surfaces. One consequence of this is an increased production of collagen. The discovery of this mechanism opened the door to a new understanding of silica toxicity for animal cells and mammalian cells in particular. The presence of silica in sea water and substrates also affects processes like the settlement of larvae and the growth of diatoms. The following sections review all such aspects