In silico investigation of Alsin RLD conformational dynamics and phosphoinositides binding mechanism

Alsin is a protein known for its major role in neuronal homeostasis and whose mutation is associated with early-onset neurodegenerative diseases. It has been shown that its relocalization from the cytoplasm to the cell membrane is crucial to induce early endosomes maturation. In particular, evidences suggest that the N-terminal regulator of chromosome condensation 1 like domain (RLD) is necessary for membrane association thanks to its affinity to phosphoinositides, membrane lipids involved in the regulation of several signaling processes. Interestingly, this domain showed affinity towards phosphatidylinositol 3-phosphate [PI(3)P], which is highly expressed in endosomes membrane. However, Alsin structure has not been experimentally resolved yet and molecular mechanisms associated with its biological functions are mostly unknown. In this work, Alsin RLD has been investigated through computational molecular modeling techniques to analyze its conformational dynamics and obtain a representative 3D model of this domain. Moreover, a putative phosphoinositide binding site has been proposed and PI(3)P interaction mechanism studied. Results highlight the substantial conformational stability of Alsin RLD secondary structure and suggest the role of one highly flexible region in the phosphoinositides selectivity of this domain

Molecular Dynamics And Binding Mechanisms Of Volatile Anesthetics Targeting Human Tubulin

Anesthesia, despite being the cornerstone of modern surgery, is to this date a biological puzzle. While scientific efforts still have not managed to frame its pharmacology in an exhaustive theoretical framework, microtubules inside neurons are thought to be essential for memory formation and consciousness. The potential ability of volatile anesthetics to alter or dampen the vibrational properties of microtubules justifies the spatiotemporal characterization of the interaction between such molecules and the tubulin dimer through the use of computational molecular modelling

Thermodynamic and kinetic stability of the Josephin Domain closed arrangement: evidences from replica exchange molecular dynamics

Abstract Background Molecular phenomena driving pathological aggregation in neurodegenerative diseases are not completely understood yet. Peculiar is the case of Spinocerebellar Ataxia 3 (SCA3) where the conformational properties of the AT-3 N-terminal region, also called Josephin Domain (JD), play a key role in the first step of aggregation, having the JD an amyloidogenic propensity itself. For this reason, unraveling the intimate relationship between JD structural features and aggregation tendency may lead to a step forward in understanding the pathology and rationally design a cure. In this connection, computational modeling has demonstrated to be helpful in exploring the protein molecular dynamics and mechanism of action. Results Conformational dynamics of the JD is here finely investigated by replica exchange molecular dynamics simulations able to sample the microsecond time scale and to provide both a thermodynamic and kinetic description of the protein conformational changes. Accessible structural conformations of the JD have been identified in: open, intermediate and closed like arrangement. Data indicated the closed JD arrangement as the most likely protein arrangement. The protein transition from closed toward intermediate/open states was characterized by a rate constant higher than 700\ua0ns. This result also explains the inability of classical molecular dynamics to explore transitions from closed to open JD configuration on a time scale of hundreds of nanoseconds. Conclusion This work provides the first kinetic estimation of the JD transition pathway from open-like to closed-like arrangement and vice-versa, indicating the closed-like arrangement as the most likely configuration for a JD in water environment. More widely, the importance of our results is also underscored considering that the ability to provide a kinetic description of the protein conformational changes is a scientific challenge for both experimental and theoretical approaches to date. Reviewers This article was reviewed by Oliviero Carugo, Bojan Zagrovic

Numerical and experimental characterization of a novel modular passive micromixer

This paper reports a new low-cost passive microfluidic mixer design, based on a replication of identical mixing units composed of microchannels with variable curvature (clothoid) geometry. The micromixer presents a compact and modular architecture that can be easily fabricated using a simple and reliable fabrication process. The particular clothoid-based geometry enhances the mixing by inducing transversal secondary flows and recirculation effects. The role of the relevant fluid mechanics mechanisms promoting the mixing in this geometry were analysed using computational fluid dynamics (CFD) for Reynolds numbers ranging from 1 to 110. A measure of mixing potency was quantitatively evaluated by calculating mixing efficiency, while a measure of particle dispersion was assessed through the lacunarity index. The results show that the secondary flow arrangement and recirculation effects are able to provide a mixing efficiency equal to 80 % at Reynolds number above 70. In addition, the analysis of particles distribution promotes the lacunarity as powerful tool to quantify the dispersion of fluid particles and, in turn, the overall mixing. On fabricated micromixer prototypes the microscopic-Laser-Induced-Fluorescence (μLIF) technique was applied to characterize mixing. The experimental results confirmed the mixing potency of the microdevice

Cellular uptake of rose bengal is mediated by OATP1B1/1B3 transporters

Due to its fluorescent properties and high yield of singlet oxygen, rose bengal (RB) is one of the most promising photosensitizers for cancer treatment. However, the negative charge of RB molecule may significantly hamper its intracellular delivery by passive diffusion through the cell membrane. Thus, specific membrane protein transporters may be needed. The organic anion transporting polypeptides (OATPs) family are a well-characterized group of membrane protein transporters, responsible for cellular uptake of a number of drugs. To our knowledge, this is the first study that evaluates cellular transport of RB mediated by the OATP transporter family. First, electrified liquid-liquid interface, together with biophysical analysis and molecular dynamics simulations were used to characterize the interaction of RB with several models of a cellular membranes. These experiments proved that RB interacts only with the membrane’s surface, without spontaneously crossing the lipid bilayer. Evaluation of intracellular uptake of RB by flow cytometry and confocal microscopy showed significant differences in uptake between liver and intestinal cell line models differing in expression of OATP transporters. The use of specific pharmacological inhibitors of OATPs, together with Western blotting and in silico analysis, indicated that OATPs are crucial for cellular uptake of RB

Protein environment : A crucial triggering factor in josephin domain aggregation: The role of 2,2,2-trifluoroethanol

The protein ataxin-3 contains a polyglutamine stretch that triggers amyloid aggregation when it is expanded beyond a critical threshold. This results in the onset of the spinocerebellar ataxia type 3. The protein consists of the globular N-terminal Josephin domain and a disordered C-terminal tail where the polyglutamine stretch is located. Expanded ataxin-3 aggregates via a two-stage mechanism: first, Josephin domain self-association, then polyQ fibrillation. This highlights the intrinsic amyloidogenic potential of Josephin domain. Therefore, much effort has been put into investigating its aggregation mechanism(s). A key issue regards the conformational requirements for triggering amyloid aggregation, as it is believed that, generally, misfolding should precede aggregation. Here, we have assayed the effect of 2,2,2-trifluoroethanol, a co-solvent capable of stabilizing secondary structures, especially α-helices. By combining biophysical methods and molecular dynamics, we demonstrated that both secondary and tertiary JD structures are virtually unchanged in the presence of up to 5% 2,2,2-trifluoroethanol. Despite the preservation of JD structure, 1% of 2,2,2-trifluoroethanol suffices to exacerbate the intrinsic aggregation propensity of this domain, by slightly decreasing its conformational stability. These results indicate that in the case of JD, conformational fluctuations might suffice to promote a transition towards an aggregated state without the need for extensive unfolding, and highlights the important role played by the environment on the aggregation of this globular domain

In Silico Analysis of the Multi-Targeted Mode of Action of Ivermectin and Related Compounds

Some clinical studies have indicated activity of ivermectin, a macrocyclic lactone, against COVID-19, but a biological mechanism initially proposed for this anti-viral effect is not applicable at physiological concentrations. This in silico investigation explores potential modes of action of ivermectin and 14 related compounds, by which the infectivity and morbidity of the SARS-CoV-2 virus may be limited. Binding affinity computations were performed for these agents on several docking sites each for models of (1) the spike glycoprotein of the virus, (2) the CD147 receptor, which has been identified as a secondary attachment point for the virus, and (3) the alpha-7 nicotinic acetylcholine receptor (α7nAChr), an indicated point of viral penetration of neuronal tissue as well as an activation site for the cholinergic anti-inflammatory pathway controlled by the vagus nerve. Binding affinities were calculated for these multiple docking sites and binding modes of each compound. Our results indicate the high affinity of ivermectin, and even higher affinities for some of the other compounds evaluated, for all three of these molecular targets. These results suggest biological mechanisms by which ivermectin may limit the infectivity and morbidity of the SARS-CoV-2 virus and stimulate an α7nAChr-mediated anti-inflammatory pathway that could limit cytokine production by immune cells

Exploration of Spanish olive oil quality with a miniaturized low-cost fluorescence sensor and machine learning techniques

This article belongs to the Special Issue Advanced Analysis Methods for Food Safety, Authenticity and Traceability AssessmentExtra virgin olive oil (EVOO) is the highest quality of olive oil and is characterized by highly beneficial nutritional properties. The large increase in both consumption and fraud, for example through adulteration, creates new challenges and an increasing demand for developing new quality assessment methodologies that are easier and cheaper to perform. As of today, the determination of olive oil quality is performed by producers through chemical analysis and organoleptic evaluation. The chemical analysis requires advanced equipment and chemical knowledge of certified laboratories, and has therefore limited accessibility. In this work a minimalist, portable, and low-cost sensor is presented, which can perform olive oil quality assessment using fluorescence spectroscopy. The potential of the proposed technology is explored by analyzing several olive oils of different quality levels, EVOO, virgin olive oil (VOO), and lampante olive oil (LOO). The spectral data were analyzed using a large number of machine learning methods, including artificial neural networks. The analysis performed in this work demonstrates the possibility of performing the classification of olive oil in the three mentioned classes with an accuracy of 100%. These results confirm that this minimalist low-cost sensor has the potential to substitute expensive and complex chemical analysis