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Do all paths lead to Rome? How reliable is umbrella sampling along a single path?
No full textMolecular dynamics (MD) simulations are widely applied to estimate absolute binding free energies of protein-ligand and protein-protein complexes. A routinely used method for binding free energy calculations with MD is umbrella sampling (US), which calculates the potential of mean force (PMF) along a single reaction coordinate. Surprisingly, in spite of its wide-spread use, few validation studies have focused on the convergence of the free energy computed along a single path for specific cases, not addressing the reproducibility of such calculations in general. In this work, we therefore investigate the reproducibility and convergence of US along a standard distance-based reaction coordinate for various protein-protein and protein-ligand complexes, following commonly used guidelines for the setup. We show that repeating the complete US workflow can lead to differences of 2-20~kcal/mol in computed binding free energies. We attribute those discrepancies to small differences in the binding pathways. While these differences are unavoidable in the established US protocol, the popularity of the latter could hint at a lack of awareness of such reproducibility problems. To test if the convergence of PMF profiles can be improved if multiple pathways are sampled simultaneously, we performed additional simulations with an adaptive-biasing method, here the accelerated weight histogram (AWH) approach. Indeed, the PMFs obtained from AHW simulations are consistent and reproducible for the systems tested. To the best of our knowledge, our work is the first to attempt a systematic assessment of the pitfalls in one the most widely used protocols for computing binding affinities. We anticipate therefore that our results will provide an incentive for a critical reassessment of the validity of PMFs computed with US, and make a strong case to further benchmark the performance of adaptive-biasing methods for computing binding affinities
Non-Overlapping Arrangement of Identical Objects: An insight for molecular close packing
No full textIn this study, we investigate the arrangement of identical objects to achieve non-overlapping configurations, a fundamental challenge across various scientific and engineering disciplines, particularly in the context of molecular close-packing. We introduce the concept of "Maximal Boundary Distance", defined as the largest distance between extreme points of an object in a given direction. This length ensures that aligned objects touch without overlapping. Our approach is validated through mathematical proofs and illustrated with both, two-dimensional and three-dimensional examples, emphasizing applications in molecular systems. We further explore the practical implications of this for the packing of molecular crystals by identifying optimal lattice constants. The stability of these configurations is further analyzed using the Density Functional Tight Binding method. This work not only advances our understanding of object arrangement but also has significant implications for the strategic positioning of molecules in close-packed structures
Self-Nanoformulating Poly(2-oxazoline) and Poly(2-oxazine) Copolymers Based Amorphous Solid Dispersions as Microneedle Patch: A Formulation Study
No full textHere, we introduce a pioneering approach in the domain of transdermal drug delivery systems (TDDS) by using microneedles (MNs) fabricated from amorphous solid dispersion comprising only a model drug and an amphiphilic block copolymer to form a drug nanoformulation upon MN dissolution. This approach presents the most minimalistic approach to maximize drug loading in MN, reaching 40 wt.%. Using scanning electron microscopy, we carefully examined the morphology of MNs across a spectrum of drug loading ratios, revealing a remarkable consistency in structure and integrity. Mechanical testing confirms the MNs\u27 proficiency in effective skin penetration. Furthermore, a comparative study on the formation of polymeric micelles underscores the innovative concept of a “nano-in-micro drug delivery system”, offering an approach to drug release. The results demonstrate that MNs manufactured from an amorphous solid dispersion of drug and amphiphilic block copolymer with ultra-high loading, enhancing the availability and release dynamics of hydrophobic drugs, positioning them as a potent tool for enhancing TDDS. This study sets a new benchmark in the utilization of polymer-drug nanoformulations for transdermal applications and underscores the exceptional capacity for high drug loading and the creation of adaptable drug delivery mechanisms for the studied amphiphilic block copolymer
Insights into the Variations of Kinetic and Potential Energies in a Multi-Bond Reaction: The Reaction Electronic Flux Perspective
No full textThe debate over whether kinetic energy (KE) or potential energy (PE) are the fundamental energy components that contribute to forming covalent bonds has been enduring and stimulating over time. However, the supremacy of these energy components in reactions where multiple bonds are simultaneously formed or broken has yet to be explored. In this study, we use the reaction electronic flux (REF), an effective tool for investigating changes in driving electronic activity when bond formation or dissociation occurs in a chemical reaction, to examine the fluctuations in the KE and PE in a multi-bond reaction. To that end, the activation of CO2 by low valent group 14 catalysts through a concerted σ-bond metathesis mechanism is analyzed. The findings of this preliminary study suggest that the REF can be utilized as a tool to rationalize alterations in the KE and PE in a multi-band reaction. Specifically, analyses across the reaction coordinate reveal that changes in the KE and PE precede activation in the REF, stimulating the electronic activity where bond formation or dissociation processes dominate
Hydrogen Economy vs. Hydrogen Embrittlement: Indirect Electrochemical Determination of Hydrogen Diffusion in Steel
No full textHydrogen has reemerged in recent years as a promising environment−friendly energy carrier that can help reduce the world\u27s dependence on fossil fuels. Despite its unique advantages, there are still challenges regarding the storage, packaging, and transportation of hydrogen. Specifically, the phenomenon of hydrogen embrittlement (HE) in metals can hinder the widespread use of hydrogen. This study focuses on the analysis of hydrogen embrittlement and hydrogen permeation through metals, with an emphasis on high−strength and duplex steels. Various steel types were evaluated for their hydrogen permeation properties using a simplified version of the Devanathan−Stachurski permeation cell to measure the diffusion constants und breakthrough times in different steel grades. In combination with Extended X−ray Absorption Fine Structure (EXAFS) analysis, the results indicate that hydrogen embrittlement is dependent on the steel grade and that the manufacturing method plays a key role. Our methodology using indirect electrochemical determination offers rapid and reproducible hydrogen diffusion, providing insights for the development of efficient hydrogen storage systems utilizing steel
Ratio-based indicators for cytosolic Ca2+ with visible light excitation
No full textCalcium ions (Ca2+) play central roles in cellular physiology. Fluorescent indicators for Ca2+ ions revolutionized our ability to make rapid, accurate, and highly parallel measurement of Ca2+ concentrations in living cells. The use of ratio-based imaging with one particular indicator, fura-2, allowed practitioners to correct for a number of experimental confounds, including dye bleaching, variations in sample thickness, and fluctuations in illumination intensity. Ratio-based imaging with fura-2 was the most accurate and reliable method for measuring Ca2+ concentrations. Two drawbacks to fura-2 exist. First, it requires ultraviolet (UV) excitation, which is more toxic to living cells than visible light. Second, our ability to use fura-2 for accurate, stable, ratio-based determinations of Ca2+ concentration in living cells is fast becoming a method of the past. This is due, in part, because modern microscopes are phasing out the use of mercury arc lamps that provide the UV excitation needed for fura-2 imaging. To address this problem, we describe the design, synthesis, and cellular application of benzo[b]phosphole-based fluorescent Ca2+ indicators for ratio-based imaging of Ca2+ in living cells that can be used with modern light emitting diode (LED)-equipped fluorescence microscopes. We show that isoCaRed-1Me has absorbance spectra in the visible range, shows Ca2+ selectivity over Mg2+, and displays a Ca2+-dependent excitation spectra. These unique properties enable ratio-based imaging in immortalized cell lines, primary mammalian hippocampal neurons, and human induced pluripotent stem cell-derived cardiomyocytes. These data show that isoCaRed-1Me will be useful for ratio-based Ca2+ imaging using modern microscopes
Real-Space Imaging of the Conformation and Atomic Structure of Individual β Cyclodextrins with Noncontact AFM
No full textGlycans, consisting of covalently linked sugar units, are a major class of biopolymers essential to all known living organisms. To better understand their biological functions and further applications in fields from biomedicine to materials science, detailed knowledge of their structure is essential. However, due to the extraordinary complexity and conformational flexibility of glycans, state-of-the-art glycan analysis methods often fail to provide structural information with atomic precision. Here, we combine electrospray deposition in ultra-high vacuum with noncontact atomic force microscopy and theoretical calculations to unravel the structure of β-cyclodextrin, a cyclic glucose oligomer, with atomic-scale detail. Our results, established on the single-molecule level, reveal the different adsorption geometries and conformations of β-cyclodextrin. The cyclic arrangement of hydroxy groups on both faces of the molecule and the stabilizing H-bonds are imaged with atomic resolution, enabling the unambiguous assignment of the molecular structure and demonstrating the potential of the method for glycan analysis
Exploiting Decarbonylation and Dehydrogenation of Forma-mides for the Synthesis of Ureas, Polyureas, and Poly(urea-urethanes)
No full textUrea derivatives, polyureas, and poly(urea-urethanes) are materials of great interest. However, their current methods of syn-thesis involve toxic feedstock - isocyanate and phosgene gas. There is a significant interest in developing alternative meth-odologies for their synthesis from safer feedstock. We report here new methods for the synthesis of urea derivatives, polyure-as, and polyurea(urethane) using a ruthenium pincer catalyst. In this approach, urea derivatives and polyureas are synthesized from the self-coupling of formamides and diformamides, respectively, whereas polyurea(urethanes) are synthesized from the coupling of diformamides and diols. CO and H2 gases are eliminated in all these processes. Decarbonylation of formamides using such organometallic catalysts has not been reported before and therefore mechanistic insights have been provided using experiments and DFT computation to shed light on pathways of these processes
Preparation and characterization of liprotides prepared from protein extracts of mung beans (Vigna radiata (L.))
No full textIn recent years, liprotides, which are protein-fatty acid complexes with core-shell structures, have proven to be promising drug carriers for cancer treatment. Due to their novel nature, there are limited studies surrounding liprotides, especially one synthesized from plant-based proteins. Thus, to explore new possibilities for liprotide synthesis, mung bean albumins and globulins were each extracted and combined with oleic acid (OA) at different temperatures (40 °C and 80 °C) to synthesize four variations of plant-based liprotides (Alp40, Alp80, Glp40, and Glp80). These, along with OA and protein controls, were characterized through ATR-FTIR spectroscopy, visual stability testing, particle size analysis, and zeta potential analysis. The IR spectra of the liprotides compared to the controls suggested that the OA molecule was encapsulated within the protein shell as intended. Peak shifts in characteristic absorption bands were also observed, indicating possible structural changes that may or may not be correlated to liprotide formation. Particle size analysis showed that the synthesized liprotides had significantly larger diameters than those indicated in published data and were highly polydispersed (PdI > 0.4). Lastly, zeta-potential analysis of the samples revealed that the oleic acid controls garnered a negative zeta-potential greater than -70 mV. This contrasts the zeta-potential values of both protein controls and liprotides which ranged between -23 mV and -33 mV
Validation of a Rapid GC-MS Method for Forensic Seized Drug and Ignitable Liquid Screening Applications
No full textWith the lack of standardized validation protocols across the forensic chemistry community, validation of instrumentation can be a challenging and time-consuming task. However, this process is crucial to understand the associated capabilities and limitations, especially for nascent technologies. Rapid GC-MS is one such emerging analytical technique being increasingly implemented in forensic laboratories due to its fast and informative screening capabilities. However, a full validation for forensic samples has yet to be published since its debut. This work presents the results of a comprehensive validation of an in-house rapid GC-MS system for seized drug and ignitable liquid analyses through the assessment of nine components: selectivity, matrix effects, precision, accuracy, range, carryover/contamination, robustness, ruggedness, and stability. Single- and/or multi-compound test solutions of commonly encountered seized drug or ignitable liquid compounds were used for each study to assess method and system performance. Results met the designated acceptance criteria for a majority of components. For example, retention time and mass spectral search score % RSDs were ≤ 10 % for precision and robustness studies. Limitations were identified for components that did not meet the acceptance criteria (e.g., inability for isomer differentiation). The study designs are part of a larger validation package developed for rapid GC-MS that includes validation plans and automated workbooks for each forensic application. The template, available for adoption by laboratories as needed, ultimately aims to reduce the barrier of implementation for rapid GC-MS technology