The Nicotinic Acetylcholine Receptor and Its Pentameric Homologs: Toward an Allosteric Mechanism of Signal Transduction at the Atomic Level.

The nicotinic acetylcholine receptor has served, since its biochemical identification in the 1970s, as a model of an allosteric ligand-gated ion channel mediating signal transition at the synapse. In recent years, the application of X-ray crystallography and high-resolution cryo-electron microscopy, together with molecular dynamic simulations of nicotinic receptors and homologs, have opened a new era in the understanding of channel gating by the neurotransmitter. They reveal, at atomic resolution, the diversity and flexibility of the multiple ligand-binding sites, including recently discovered allosteric modulatory sites distinct from the neurotransmitter orthosteric site, and the conformational dynamics of the activation process as a molecular switch linking these multiple sites. The model emerging from these studies paves the way for a new pharmacology based, first, upon the occurrence of an original mode of indirect allosteric modulation, distinct from a steric competition for a single and rigid binding site, and second, the design of drugs that specifically interact with privileged conformations of the receptor such as agonists, antagonists, and desensitizers. Research on nicotinic receptors is still at the forefront of understanding the mode of action of drugs on the nervous system.

Structural mechanisms of α7 nicotinic receptor allosteric modulation and activation.

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that plays an important role in cholinergic signaling throughout the nervous system. Its unique physiological characteristics and implications in neurological disorders and inflammation make it a promising but challenging therapeutic target. Positive allosteric modulators overcome limitations of traditional α7 agonists, but their potentiation mechanisms remain unclear. Here, we present high-resolution structures of α7-modulator complexes, revealing partially overlapping binding sites but varying conformational states. Structure-guided functional and computational tests suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. We extend the study using a time-resolved cryoelectron microscopy (cryo-EM) approach to reveal asymmetric state transitions for this homomeric channel and also find that a modulator with allosteric agonist activity exploits a distinct channel-gating mechanism. These results define mechanisms of α7 allosteric modulation and activation with implications across the pentameric receptor superfamily.

A weak coupling mechanism for the early steps of the recovery stroke of myosin VI: A free energy simulation and string method analysis.

Myosin motors use the energy of ATP to produce force and directed movement on actin by a swing of the lever-arm. ATP is hydrolysed during the off-actin re-priming transition termed recovery stroke. To provide an understanding of chemo-mechanical transduction by myosin, it is critical to determine how the reverse swing of the lever-arm and ATP hydrolysis are coupled. Previous studies concluded that the recovery stroke of myosin II is initiated by closure of the Switch II loop in the nucleotide-binding site. Recently, we proposed that the recovery stroke of myosin VI starts with the spontaneous re-priming of the converter domain to a putative pre-transition state (PTS) intermediate that precedes Switch II closing and ATPase activation. Here, we investigate the transition from the pre-recovery, post-rigor (PR) state to PTS in myosin VI using geometric free energy simulations and the string method. First, our calculations rediscover the PTS state agnostically and show that it is accessible from PR via a low free energy transition path. Second, separate path calculations using the string method illuminate the mechanism of the PR to PTS transition with atomic resolution. In this mechanism, the initiating event is a large movement of the converter/lever-arm region that triggers rearrangements in the Relay-SH1 region and the formation of the kink in the Relay helix with no coupling to the active site. Analysis of the free-energy barriers along the path suggests that the converter-initiated mechanism is much faster than the one initiated by Switch II closure, which supports the biological relevance of PTS as a major on-pathway intermediate of the recovery stroke in myosin VI. Our analysis suggests that lever-arm re-priming and ATP hydrolysis are only weakly coupled, so that the myosin recovery stroke is initiated by thermal fluctuations and stabilised by nucleotide consumption via a ratchet-like mechanism.

The effect of probe density coverage on the detection of oenological tannins in quartz crystal microbalance with dissipation monitoring (QCM-D) experiments.

BACKGROUND: Polyphenols are a group of compounds found in grapes, musts, and wines. Their levels are crucial for grape ripening, proper must fermentation, and final wine characteristics. Standard chemical analysis is commonly used to detect these compounds, but it is costly, time consuming, and requires specialized laboratories and operators. To address this, this study explores a functionalized acoustic sensor for detecting oenological polyphenols. RESULTS: The method involves utilizing a quartz crystal microbalance with dissipation monitoring (QCM-D) to detect the target analyte by using a gelatin-based probe layer. The sensor is functionalized by optimizing the probe coverage density to maximize its performance. This is achieved by using 12-mercaptododecanoic acid (12-MCA) to immobilize the probe onto the gold sensor surface, and dithiothreitol (DTT) as a reducing and competitive binding agent. The concentration of 12-MCA and DTT in the solutions is varied to control the probe density. QCM-D measurements demonstrate that the probe density can be effectively adjusted using this approach, ranging from 0.2 × 1013 to 2 × 1013 molecules cm-2 . This study also investigates the interaction between the probe and tannins, confirming the ability of the sensor to detect them. Interestingly, the lower probe coverage achieves higher detection signals when normalized to probe immobilization signals. Moreover, significant changes in mechanical properties of the functionalization layer are observed after the interaction with samples. CONCLUSION: The combination of QCM-D with gelatin functionalization holds great promise for future applications in the wine industry. It offers real-time monitoring capabilities, requires minimal sample preparation, and provides high sensitivity for quality control purposes. © 2024 Society of Chemical Industry.

ChemFlow─From 2D Chemical Libraries to Protein-Ligand Binding Free Energies.

The accurate prediction of protein-ligand binding affinities is a fundamental problem for the rational design of new drug entities. Current computational approaches are either too expensive or inaccurate to be effectively used in virtual high-throughput screening campaigns. In addition, the most sophisticated methods, e.g., those based on configurational sampling by molecular dynamics, require significant pre- and postprocessing to provide a final ranking, which hinders straightforward applications by nonexpert users. We present a novel computational platform named ChemFlow to bridge the gap between 2D chemical libraries and estimated protein-ligand binding affinities. The software is designed to prepare a library of compounds provided in SMILES or SDF format, dock them into the protein binding site, and rescore the poses by simplified free energy calculations. Using a data set of 626 protein-ligand complexes and GPU computing, we demonstrate that ChemFlow provides relative binding free energies with an RMSE < 2 kcal/mol at a rate of 1000 ligands per day on a midsize computer cluster. The software is publicly available at https://github.com/IFMlab/ChemFlow.

ChemFlow_py: a flexible toolkit for docking and rescoring.

The design of accurate virtual screening tools is an open challenge in drug discovery. Several structure-based methods have been developed at different levels of approximation. Among them, molecular docking is an established technique with high efficiency, but typically low accuracy. Moreover, docking performances are known to be target-dependent, which makes the choice of the docking program and corresponding scoring function critical when approaching a new protein target. To compare the performances of different docking protocols, we developed ChemFlow_py, an automated tool to perform docking and rescoring. Using four protein systems extracted from DUD-E with 100 known active compounds and 3000 decoys per target, we compared the performances of several rescoring strategies including consensus scoring. We found that the average docking results can be improved by consensus ranking, which emphasizes the relevance of consensus scoring when little or no chemical information is available for a given target. ChemFlow_py is a free toolkit to optimize the performances of virtual high-throughput screening (vHTS). The software is publicly available at https://github.com/IFMlab/ChemFlow_py .

Crosslinked Chitosan Nanoparticles with Muco-Adhesive Potential for Intranasal Delivery Applications.

Intranasal drug delivery is convenient and provides a high bioavailability but requires the use of mucoadhesive nanocarriers. Chitosan is a well-established polymer for mucoadhesive applications but can suffer from poor cytocompatibility and stability upon administration. In this work, we present a method to obtain stable and cytocompatible crosslinked chitosan nanoparticles. We used 2,6-pyridinedicarboxylic acid as a biocompatible crosslinker and compared the obtained particles with those prepared by ionotropic gelation using sodium tripolyphosphate. Nanoparticles were tested to evaluate the size and the surface charge, as well as their stability in storage conditions (4 °C), at the nasal cavity temperature (32 °C), and at the body temperature (37 °C). The crosslinked chitosan nanoparticles showed a size around 150 nm and a surface charge of 10.3 mV ± 0.9 mV, both compatible with the intranasal drug administration. Size and surface charge parameters did not significantly vary over time, indicating the good stability of these nanoparticles. We finally tested their cytocompatibility in vitro using SHSY5Y human neuroblastoma and RPMI 2650 human nasal epithelial cells, with positive results. In conclusion, the proposed synthetic system shows an interesting potential as a drug carrier for intranasal delivery.

Dual-Readout of the Mechanical Response of a Bis-acridinium [2]Rotaxane.

A [2]rotaxane built around a multi-responsive bis-acridinium macrocycle has been synthesized. Structural investigation has confirmed the interlocked nature of the molecule, and MD simulations illuminated its conformational dynamics with atomic resolution. Both halochromic and redox-switching properties were explored to shed light on the mechanical response and electronic changes that occur in the bis-acridinium [2]rotaxane. The topology of the rotaxane led to different mechanical behaviors upon addition of hydroxide ions or reduction that were easily detected by UV/Vis spectroscopy and electrochemistry.

Capturing the Recognition Dynamics of para-Sulfonato-calix[4]arenes by Cytochrome c: Toward a Quantitative Free Energy Assessment.

Calix[n]arenes' selective recognition of protein surfaces covers a broad range of timely applications, from controlling protein assembly and crystallization to trapping partially disordered proteins. Here, the interaction of para-sulfonated calix-[4]-arenes with cytochrome c is investigated through all-atom, explicit water molecular dynamics simulations which allow characterization of two binding sites in quantitative agreement with experimental evidence. Free energy calculations based on the MM-PBSA and the attach-pull-release (APR) methods highlight key residues implicated in the recognition process and provide binding free energy results in quantitative agreement with isothermal titration calorimetry. Our study emphasizes the role of MD simulations to capture and describe the "walk" of sulfonated calix-[4]-arenes on the cytochrome c surface, with the arginine R13 as a pivotal interacting residue. Our MD investigation allows, through the quasi-harmonic multibasin (QHMB) method, probing an allosteric reinforcement of several per-residue interactions upon calixarene binding, which suggests a more complex mode of action of these supramolecular auxiliaries.

Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking.

Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more detail, Raster Image Correlation Spectroscopy (RICS) performed at fast timescales (i.e., microseconds) reveals the fast motion of fluorescently labeled molecules within two exemplary dynamic subcellular nanostructures of biomedical interest, the lysosome and the insulin secretory granule (ISG). The measurement of molecular diffusion is then used to extract information on the average properties of subcellular nanostructures, such as macromolecular crowding or molecular aggregation. Concerning the lysosome, fast RICS on a fluorescent tracer allowed us to quantitatively assess the increase in organelle viscosity in the pathological condition of Krabbe disease. In the case of ISGs, fast RICS on two ISG-specific secreting peptides unveiled their differential aggregation propensity depending on intragranular concentration. Finally, a combination of fast RICS and feedback-based 3D orbital tracking was used to subtract the slow movement of subcellular nanostructures from the fast diffusion of molecules contained within them and independently validate the results. Results presented here not only demonstrate the acquired ability to address the dynamic behavior of molecules in moving, nanoscopic reference systems, but prove the relevance of this approach to advance our knowledge on cell function at the subcellular scale.

Neural correlates of sex-related differences in attachment dimensions.

The purpose of this study was to investigate sex-related differences in the electrophysiological response to socioemotional stimuli (positive, negative, and ambiguous) depicting couple interactions. The associations between anxiety and avoidance attachment dimensions (measured with the Experiences in Close Relationships-Revised questionnaire) and the strength of cortico-limbic circuit intensity was explored, recorded using a 256-Hydrocel Geodesic Sensor-Net. Event-related potentials (ERPs) and standardized low-resolution electromagnetic tomography (sLORETA) data were analyzed for a total sample of 74 participants. Regression analyses showed that the women presented increased brain intensity compared with that in men, and the avoidance score was positively associated with brain intensity, particularly in response to negative socioemotional stimuli. The interaction sex per avoidance was a significant predictor of intensity in many brain areas, with women displaying significantly more pronounced positive associations between avoidance and brain intensity than men. In conclusion, the findings of the present study showed that women appeared to be more emotionally involved during the socioemotional task. Avoidance was positively associated with intensity of the cingulate and prefrontal regions, and these associations were more pronounced in women than in men. These findings suggested that avoidance seems to represent two different socioemotional strategies, in which women appear to activate an avoidant strategy to modulate increased emotional involvement in relationships, whereas men appear to adopt avoidance with a more intense emotional suppression.

The Glycine Receptor Allosteric Ligands Library (GRALL).

MOTIVATION: Glycine receptors (GlyRs) mediate fast inhibitory neurotransmission in the brain and have been recognized as key pharmacological targets for pain. A large number of chemically diverse compounds that are able to modulate GlyR function both positively and negatively have been reported, which provides useful information for the development of pharmacological strategies and models for the allosteric modulation of these ion channels. RESULTS: Based on existing literature, we have collected 218 unique chemical entities with documented modulatory activities at homomeric GlyR-α1 and -α3 and built a database named GRALL. This collection includes agonists, antagonists, positive and negative allosteric modulators and a number of experimentally inactive compounds. Most importantly, for a large fraction of them a structural annotation based on their putative binding site on the receptor is provided. This type of annotation, which is currently missing in other drug banks, along with the availability of cooperativity factors from radioligand displacement experiments are expected to improve the predictivity of in silico methodologies for allosteric drug discovery and boost the development of conformation-based pharmacological approaches. AVAILABILITY AND IMPLEMENTATION: The GRALL library is distributed as a web-accessible database at the following link: https://ifm.chimie.unistra.fr/grall. For each molecular entry, it provides information on the chemical structure, the ligand-binding site, the direction of modulation, the potency, the 3D molecular structure and quantum-mechanical charges as determined by our in-house pipeline. CONTACT: mcecchini@unistra.fr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ultra-high-frequency (UHF) surface-acoustic-wave (SAW) microfluidics and biosensors.

Surface acoustic waves (SAWs) have the potential to become the basis for a wide gamut of lab-on-a-chips (LoCs). These mechanical waves are among the most promising physics that can be exploited for fulfilling all the requirements of commercially appealing devices that aim to replace-or help-laboratory facilities. These requirements are low processing cost of the devices, scalable production, controllable physics, large flexibility of tasks to perform, easy device miniaturization. To date, SAWs are among the small set of technologies able to both manipulate and analyze biological liquids with high performance. Therefore, they address the main needs of microfluidics and biosensing. To this purpose, the use of high-frequency SAWs is key. In the ultra-high-frequency regime (UHF, 300 MHz-3 GHz) SAWs exhibit large sensitivities to molecule adsorption and unparalleled fluid manipulation capabilities, together with overall device miniaturization. The UHF-SAW technology is expected to be the realm for the development of complex, reliable, fully automated, high-performance LoCs. In this review, we present the most recent works on UHF-SAWs for microfluidics and biosensing, with a particular focus on the LoC application. We derive the relevant scale laws, useful formulas, fabrication guidelines, current limitations of the technology, and future developments.

Acoustic streaming of microparticles using graphene-based interdigital transducers.

Surface acoustic wave (SAW) devices offer many benefits in chemistry and biomedicine, enabling precise manipulation of micro-droplets, mixing of liquids by acoustic streaming and pumping of liquids in enclosed channels, while presenting a cost-effective and easy fabrication and integration with electronic devices. In this work, we present microfluidic devices which use graphene-based interdigital transducers (IDTs) to generate SAWs with a frequency of 100 MHz and an amplitude of up to 200 pm, which allow us to manipulate microparticle solutions by acoustic streaming. Due to the negligible mass loading of the piezoelectric surface by graphene, the SAWs generated by these devices have no frequency shift, typically observed when metal IDTs are used.

Atomistic simulations of gold surface functionalization for nanoscale biosensors applications.

A wide class of biosensors can be built via functionalization of gold surface with proper bio conjugation element capable of interacting with the analyte in solution, and the detection can be performed either optically, mechanically or electrically. Any change in physico-chemical environment or any slight variation in mass localization near the surface of the sensor can cause differences in nature of the transduction mechanism. The optimization of such sensors may require multiple experiments to determine suitable experimental conditions for the immobilization and detection of the analyte. Here, we employ molecular modeling techniques to assist the optimization of a gold-surface biosensor. The gold surface of a quartz-crystal-microbalance sensor is functionalized using polymeric chains of poly(ethylene glycol) (PEG) of 2 KDa molecular weight, which is an inert long chain amphiphilic molecule, supporting biotin molecules (bPEG) as the ligand molecules for streptavidin analyte. The PEG linkers are immobilized onto the gold surface through sulphur chemistry. Four gold surfaces with different PEG linker density and different biotinylation ratio between bPEG and PEG, are investigated by means of state-of-the art atomistic simulations and compared with available experimental data. Results suggest that the amount of biotin molecules accessible for the binding with the protein increases upon increasing the linkers density. At the high density a 1:1 ratio of bPEG/PEG can further improve the accessibility of the biotin ligand due to a strong repulsion between linker chains and different degree of hydrophobicity between bPEG and PEG linkers. The study provides a computaional protocol to model sensors at the level of single molecular interactions, and for optimizing the physical properties of surface conjugated ligand which is crucial to enhance output of the sensor.

Quantitative Microproteomics Based Characterization of the Central and Peripheral Nervous System of a Mouse Model of Krabbe Disease.

Krabbe disease is a rare, childhood lysosomal storage disorder caused by a deficiency of galactosylceramide beta-galactosidase (GALC). The major effect of GALC deficiency is the accumulation of psychosine in the nervous system and widespread degeneration of oligodendrocytes and Schwann cells, causing rapid demyelination. The molecular mechanisms of Krabbe disease are not yet fully elucidated and a definite cure is still missing. Here we report the first in-depth characterization of the proteome of the Twitcher mouse, a spontaneous mouse model of Krabbe disease, to investigate the proteome changes in the Central and Peripheral Nervous System. We applied a TMT-based workflow to compare the proteomes of the corpus callosum, motor cortex and sciatic nerves of littermate homozygous Twitcher and wild-type mice. More than 400 protein groups exhibited differences in expression and included proteins involved in pathways that can be linked to Krabbe disease, such as inflammatory and defense response, lysosomal proteins accumulation, demyelination, reduced nervous system development and cell adhesion. These findings provide new insights on the molecular mechanisms of Krabbe disease, representing a starting point for future functional experiments to study the molecular pathogenesis of Krabbe disease. Data are available via ProteomeXchange with identifier PXD010594.

An intermediate along the recovery stroke of myosin VI revealed by X-ray crystallography and molecular dynamics.

Myosins form a class of actin-based, ATPase motor proteins that mediate important cellular functions such as cargo transport and cell motility. Their functional cycle involves two large-scale swings of the lever arm: the force-generating powerstroke, which takes place on actin, and the recovery stroke during which the lever arm is reprimed into an armed configuration. Previous analyses of the prerecovery (postrigor) and postrecovery (prepowerstroke) states predicted that closure of switch II in the ATP binding site precedes the movement of the converter and the lever arm. Here, we report on a crystal structure of myosin VI, called pretransition state (PTS), which was solved at 2.2 Å resolution. Structural analysis and all-atom molecular dynamics simulations are consistent with PTS being an intermediate along the recovery stroke, where the Relay/SH1 elements adopt a postrecovery conformation, and switch II remains open. In this state, the converter appears to be largely uncoupled from the motor domain and explores an ensemble of partially reprimed configurations through extensive, reversible fluctuations. Moreover, we found that the free energy cost of hydrogen-bonding switch II to ATP is lowered by more than 10 kcal/mol compared with the prerecovery state. These results support the conclusion that closing of switch II does not initiate the recovery stroke transition in myosin VI. Rather, they suggest a mechanism in which lever arm repriming would be mostly driven by thermal fluctuations and eventually stabilized by the switch II interaction with the nucleotide in a ratchet-like fashion.

Beyond the dyad: the role of mother and father in newborns' global DNA methylation during the first month of life-a pilot study.

The study aimed to longitudinally explore the effects of parental prenatal attachment and psychopathological symptomatology on neonatal global DNA methylation (5-mC) variation between birth and the first month of life. Eighteen mothers and thirteen fathers were assessed before childbirth (t0) by Perceived Stress Scale (PSS), Prenatal-Attachment Inventory, and Paternal Antenatal Attachment Scale; 48 hr after childbirth (t1) by SCL-90-R; and one month after childbirth (t2) by PSS. At t1 and t2, buccal swabs from parents and newborns were collected. In newborns' 5-mC and single nucleotide polymorphisms (SNPs) of DAT, MAOA, BDNF, and 5-HTTLPR genes were detected, while in parents only SNPs were measured. At t1, newborns' 5-mC was negatively associated with maternal psychopathological symptoms, while at t2, newborns' 5-mC was positively associated with paternal psychopathological symptoms and negatively with paternal prenatal attachment. The variation of newborns' 5-mC from t1 to t2 was predicted by paternal psychopathological symptoms. No significant correlations among parental SNPs and 5-mC levels were found. Results highlight parent-specific influences on newborn's DNA methylation. At birth, maternal psychological symptoms seem to have an effect on newborns' 5-mC, while after one month of life, paternal psychological characteristics could have a specific role in modulating the newborns' epigenetic responses to the environment.

Chitosan Micro-Grooved Membranes with Increased Asymmetry for the Improvement of the Schwann Cell Response in Nerve Regeneration.

Peripheral nerve injuries are a common condition in which a nerve is damaged, affecting more than one million people every year. There are still no efficient therapeutic treatments for these injuries. Artificial scaffolds can offer new opportunities for nerve regeneration applications; in this framework, chitosan is emerging as a promising biomaterial. Here, we set up a simple and effective method for the production of micro-structured chitosan films by solvent casting, with high fidelity in the micro-pattern reproducibility. Three types of chitosan directional micro-grooved patterns, presenting different levels of symmetricity, were developed for application in nerve regenerative medicine: gratings (GR), isosceles triangles (ISO) and scalene triangles (SCA). The directional patterns were tested with a Schwann cell line. The most asymmetric topography (SCA), although it polarized the cell shaping less efficiently, promoted higher cell proliferation and a faster cell migration, both individually and collectively, with a higher directional persistence of motion. Overall, the use of micro-structured asymmetrical directional topographies may be exploited to enhance the nerve regeneration process mediated by chitosan scaffolds.

A Supramolecular Model for the Co-Catalytic Role of Nitro Compounds in Brønsted Acid Catalyzed Reactions.

Nitro compounds are known to change reaction rates and kinetic concentration dependence of Brønsted-acid-catalyzed reactions. Yet, no mechanistic model exists to account for these observations. In this work, an atomistic model for the catalytically active form for an alcohol dehydroazidation reaction is presented, which is generated by DFT calculations and consists of an H-bonded aggregate of two molecules of Brønsted acid and two molecules of nitro compound. The computed O-H stretching frequencies for the aggregate indicate they are stronger acids than the individual acid molecules and serve as predictors for experimental reaction rates. By applying the model to a chemically diverse set of potential promoters, it was predicted and verified experimentally that sulfate esters induce a similar co-catalytic effect. The important implication is that Brønsted-acid catalysis must be viewed from a supramolecular perspective that accounts for not only the pKa of the acid and the bulk properties of a solvent, but also the weak interactions between all molecules in solution.