Water regulates the residence time of Benzamidine in Trypsin

We simulate with state-of-the-art enhanced sampling techniques the binding of Benzamidine to Trypsin which is a much studied and paradigmatic ligand-protein system. We use machine learning methods and in particular Time-lagged Independent Component Analysis to determine efficient collective coordinates. These coordinates are used to perform On-the-fly Probability Enhanced Sampling simulations, which we adapt to calculate also the ligand residence time. Our results, both static and dynamic, are in good agreement with experiments. We underline the role of water in the unbinding process and find that the presence of a water molecule located at the bottom of the binding pocket allows via a network of hydrogen bonds the ligand to be released into the solution. On a finer scale, even when unbinding is allowed, another water molecule further modulates the exit time

The role of water in host-guest interaction

One of the main applications of atomistic computer simulations is the calculation of ligand binding energies. The accuracy of these calculations depends on the force field quality and on the thoroughness of configuration sampling. Sampling is an obstacle in modern simulations due to the frequent appearance of kinetic bottlenecks in the free energy landscape. Very often this difficulty is circumvented by enhanced sampling techniques. Typically, these techniques depend on the introduction of appropriate collective variables that are meant to capture the system's degrees of freedom. In ligand binding, water has long been known to play a key role, but its complex behaviour has proven difficult to fully capture. In this paper we combine machine learning with physical intuition to build a non-local and highly efficient water-describing collective variable. We use it to study a set of of host-guest systems from the SAMPL5 challenge. We obtain highly accurate binding energies and good agreement with experiments. The role of water during the binding process is then analysed in some detail

OneOPES, a Combined Enhanced Sampling Method to Rule Them All

Enhanced sampling techniques have revolutionized molecular dynamics (MD) simulations, enabling the study of rare events and the calculation of free energy differences in complex systems. One of the main families of enhanced sampling techniques uses physical degrees of freedom called collective variables (CVs) to accelerate a system’s dynamics and recover the original system’s statistics. However, encoding all the relevant degrees of freedom in a limited number of CVs is challenging, particularly in large biophysical systems. Another category of techniques, such as parallel tempering, simulates multiple replicas of the system in parallel, without requiring CVs. However, these methods may explore less relevant high-energy portions of the phase space and become computationally expensive for large systems. To overcome the limitations of both approaches, we propose a replica exchange method called OneOPES that combines the power of multireplica simulations and CV-based enhanced sampling. This method efficiently accelerates the phase space sampling without the need for ideal CVs, extensive parameters fine tuning nor the use of a large number of replicas, as demonstrated by its successful applications to protein–ligand binding and protein folding benchmark systems. Our approach shows promise as a new direction in the development of enhanced sampling techniques for molecular dynamics simulations, providing an efficient and robust framework for the study of complex and unexplored problems

Rare Event Kinetics from Adaptive Bias Enhanced Sampling

We introduce a novel enhanced sampling approach named OPES flooding for calculating the kinetics of rare events from atomistic molecular dynamics simulation. This method is derived from the On-the-fly-Probability-Enhanced-Sampling (OPES) approach [Invernizzi and Parrinello, JPC Lett. 2020], which has been recently developed for calculating converged free energy surfaces for complex systems. In this paper, we describe the theoretical details of the OPES flooding technique and demonstrate the application on three systems of increasing complexity: barrier crossing in a two-dimensional double well potential, conformational transition in the alanine dipeptide in gas phase, and the folding and unfolding of the chignolin polypeptide in aqueous environment. From extensive tests, we show that the calculation of accurate kinetics not only requires the transition state to be bias-free, but the amount of bias deposited should also not exceed the effective barrier height measured along the chosen collective variables. In this vein, the possibility of computing rates from biasing suboptimal order parameters has also been explored. Furthermore, we describe the choice of optimum parameter combinations for obtaining accurate results from limited computational effort

Orexin-A exerts equivocal role in atherosclerosis process depending on the duration of exposure : in vitro study

Orexin-A is a peptide hormone that plays a crucial role in feeding regulation and energy homeostasis. Diurnal intermittent fasting (DIF) has been found to increase orexin-A plasma levels during fasting hours, while Ramadan fasting which resembles DIF, has led to beneficial effects on endothelial function. Herein, we aimed to investigate the effects of orexin-A on the expression of molecules involved in the atherogenesis process: Monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 and 2 (TIMP-1 and TIMP-2), in human aortic endothelial cells (HAECs). HAECs were incubated with orexin-A at concentrations of 40 ng/mL, 200 ng/mL and 400 ng/mL for 6, 12 and 24 h. The mRNA levels of MCP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 and orexin-1 receptor were measured by real-time qPCR. We also evaluated the MMP-2, p38, phospho-p38, NF-κΒ/p65 as well as TIMP-1 protein levels by Western blot and ELISA, respectively. MMP-2 activity was measured by gelatin zymography. Short-term 6-h incubation of HAECs with orexin-A at a high concentration (400 ng/mL) decreased MCP-1, MMP-2 expression, MMP-2/TIMP-1 ratio (p < 0.05), and MMP-2 activity, while incubation for 24 h increased MCP-1, MMP-2 expression (p < 0.05), MMP-2/TIMP-1 and MMP-2/TIMP-2 ratio (p < 0.01 and p < 0.05, respectively) as well as MMP-2 activity. The dual effects of orexin-A are mediated, at least in part, via regulation of p38 and NF-κΒ pathway. Orexin-A may have an equivocal role in atherosclerosis process with its effects depending on the duration of exposure

Insights into the emerging networks of voids in simulated supercooled water

The structural evolution of supercooled liquid water as we approach the glass transition temperature continues to be an active area of research. Here, we use molecular dynamics simulations of TIP4P/ice water to study the changes in the connected regions of empty space within the liquid, which we investigate using the Voronoi-voids network. We observe two important features: supercooling enhances the fraction of nonspherical voids and different sizes of voids tend to cluster forming a percolating network. By examining order parameters such as the local structure index (LSI), tetrahedrality and topological defects, we show that water molecules near large void clusters tend to be slightly more tetrahedral than those near small voids, with a lower population of under- and overcoordinated defects. We show further that the distribution of closed rings of water molecules around small and large void clusters maintain a balance between 6 and 7 membered rings. Our results highlight the changes of the dual voids and water network as a structural hallmark of supercooling and provide insights into the molecular origins of cooperative effects underlying density fluctuations on the subnanometer and nanometer length scale. In addition, the percolation of the voids and the hydrogen bond network around the voids may serve as useful order parameters to investigate density fluctuations in supercooled water

Squeezing Oil into Water under Pressure: Inverting the Hydrophobic Effect

The molecular structure of dense homogeneous fluid water-methane mixtures has been determined for the first time using high-pressure neutron-scattering techniques at 1.7 and 2.2 GPa. A mixed state with a fully H-bonded water network is revealed. The hydration shell of the methane molecules is, however, revealed to be pressure-dependent with an increase in the water coordination between 1.7 and 2.2 GPa. In parallel, ab initio molecular dynamics simulations have been performed to provide insight into the microscopic mechanisms associated with the phenomenon of mixing. These calculations reproduce the observed phase change from phase separation to mixing with increasing pressure. The calculations also reproduce the experimentally observed structural properties. Unexpectedly, the simulations show mixing is accompanied by a subtle enhancement of the polarization of methane. Our results highlight the key role played by fine electronic effects on miscibility and the need to readjust our fundamental understanding of hydrophobicity to account for these

Estrogen Receptor Subtypes Elicit a Distinct Gene Expression Profile of Endothelial-Derived Factors Implicated in Atherosclerotic Plaque Vulnerability

In the presence of established atherosclerosis, estrogens are potentially harmful. MMP-2 and MMP-9, their inhibitors (TIMP-2 and TIMP-1), RANK, RANKL, OPG, MCP-1, lysyl oxidase (LOX), PDGF-β, and ADAMTS-4 play critical roles in plaque instability/rupture. We aimed to investigate (i) the effect of estradiol on the expression of the abovementioned molecules in endothelial cells, (ii) which type(s) of estrogen receptors mediate these effects, and (iii) the role of p21 in the estrogen-mediated regulation of the aforementioned factors. Human aortic endothelial cells (HAECs) were cultured with estradiol in the presence or absence of TNF-α. The expression of the aforementioned molecules was assessed by qRT-PCR and ELISA. Zymography was also performed. The experiments were repeated in either ERα- or ERβ-transfected HAECs and after silencing p21. HAECs expressed only the GPR-30 estrogen receptor. Estradiol, at low concentrations, decreased MMP-2 activity by 15-fold, increased LOX expression by 2-fold via GPR-30, and reduced MCP-1 expression by 3.5-fold via ERβ. The overexpression of ERα increased MCP-1 mRNA expression by 2.5-fold. In a low-grade inflammation state, lower concentrations of estradiol induced the mRNA expression of MCP-1 (3.4-fold) and MMP-9 (7.5-fold) and increased the activity of MMP-2 (1.7-fold) via GPR-30. Moreover, p21 silencing resulted in equivocal effects on the expression of the abovementioned molecules. Estradiol induced different effects regarding atherogenic plaque instability through different ERs. The balance of the expression of the various ER subtypes may play an important role in the paradoxical characterization of estrogens as both beneficial and harmful

SGLT-2 Inhibitors in NAFLD: Expanding Their Role beyond Diabetes and Cardioprotection?

Non-alcoholic fatty liver disease (NAFLD) is an ‘umbrella’ term, comprising a spectrum ranging from benign, liver steatosis to non-alcoholic steatohepatitis, liver fibrosis and eventually cirrhosis and hepatocellular carcinoma. NAFLD has evolved as a major health problem in recent years. Discovering ways to prevent or delay the progression of NAFLD has become a global focus. Lifestyle modifications remain the cornerstone of NAFLD treatment, even though various pharmaceutical interventions are currently under clinical trial. Among them, sodium-glucose co-transporter type-2 inhibitors (SGLT-2i) are emerging as promising agents. Processes regulated by SGLT-2i, such as endoplasmic reticulum (ER) and oxidative stress, low-grade inflammation, autophagy and apoptosis are all implicated in NAFLD pathogenesis. In this review, we summarize the current understanding of the NAFLD pathophysiology, and specifically focus on the potential impact of SGLT-2i in NAFLD development and progression, providing current evidence from in vitro, animal and human studies. Given this evidence, further mechanistic studies would advance our understanding of the exact mechanisms underlying the pathogenesis of NAFLD and the potential beneficial actions of SGLT-2i in the context of NAFLD treatment

Upregulation of interleukin-19 in severe asthma: a potential saliva biomarker for asthma severity

Interleukin (IL)-19, a designated IL-20 subfamily cytokine, has been implicated in inflammatory disorders including rheumatoid arthritis, psoriasis and, lately, asthma. Here, through the analysis of transcriptomic datasets of lung tissue of large asthma cohorts, we report that IL-19 expression is upregulated in asthma and correlates with disease severity. The gene expression of IL-19 was significantly higher in lung tissue from patients with severe and mild/moderate asthma compared to healthy controls. IL-19 protein level, however, was significantly higher in the blood and saliva of patients with severe asthma compared to mild/ moderate subgroups as measured by ELISA assay. IL-19 protein level was not affected by corticosteroid treatment in plasma. Our data provide insights into the potential use of IL-19 as a saliva marker for asthma severity and a potential therapeutic target