Computer simulation of aqueous biomolecular systems

Computer simulation techniques are increasingly being used to predict structural and thermodynamic properties of large heterogeneous macromolecule and solvent assemblies. We discuss, with examples from our own studies, some problems we and others have experienced in using these techniques, which were originally devised for simple liquids. In particular, we consider the problems which arise from the large size and heterogeneity of macromolecule water systems, comparisons with experimental data and equilibrium and sampling procedures.     

The Interaction of DMSO with Model Membranes. II. Direct Evidence of DMSO Binding to Membranes: An NMR Study

Abstract

Modern techniques in nuclear magnetic resonance (NMR) allow investigators to probe molecular interactions with greater sensitivity and speed than ever before. Exploiting the nuclear Overhauser effect (NOE), the intermolecular interactions between dimethylsulfoxide (DMSO) and lipid vesicles were investigated. The DMSO methyl proton signal varies with experimental mixing time suggesting the system behaves in a manner similar to that of a ligand weakly binding to a macromolecule.     

Characterization of a progestin-binding macromolecule in the amphibian oocyte cytosol.

A [³H]-progestin-binding macromolecule has been isolated from $\text{R}\text{.}\text{pipiens}$ oocyte cytosol and characterized using binding assays, gel filtration, DEAE-cellulose chromatography and ultracentrifugal techniques. Macromolecules present in the prophase oocyte cytosol have a high affinity and specificity for the synthetic progestin R5020 and the intact oocyte will concentrate both R5020 and progesterone 20–40 fold from the medium. This may be the first published case of a cytosol steroid binding macromolecule in a cell system in which the steroid appears to act at an extranuclear level.     

Structure prediction and network analysis of chitinases from the Cape sundew,Drosera capensis

BackgroundCarnivorous plants possess diverse sets of enzymes with novel functionalities applicable to biotechnology, proteomics, and bioanalytical research. Chitinases constitute an important class of such enzymes, with future applications including human-safe antifungal agents and pesticides. Here, we compare chitinases from the genome of the carnivorous plant Drosera capensis to those from related carnivorous plants and model organisms.MethodsUsing comparative modeling, in silico maturation, and molecular dynamics simulation, we produce models of the mature enzymes in aqueous solution. We utilize network analytic techniques to identify similarities and differences in chitinase topology.ResultsHere, we report molecular models and functional predictions from protein structure networks for eleven new chitinases from D. capensis, including a novel class IV chitinase with two active domains. This architecture has previously been observed in microorganisms but not in plants. We use a combination of comparative and de novo structure prediction followed by molecular dynamics simulation to produce models of the mature forms of these proteins in aqueous solution. Protein structure network analysis of these and other plant chitinases reveal characteristic features of the two major chitinase families.General significanceThis work demonstrates how computational techniques can facilitate quickly moving from raw sequence data to refined structural models and comparative analysis, and to select promising candidates for subsequent biochemical characterization. This capability is increasingly important given the large and growing body of data from high-throughput genome sequencing, which makes experimental characterization of every target impractical.     

Computational Chemistry in Lead Identification,Library Design and Lead Optimisation

Abstract

We describe a variety of the computational techniques which we use in the drug discovery and design process. Some of these computational methods are designed to support the new experimental technologies of high-throughput screening and combinatorial chemistry. We also consider some new approaches to problems of long-standing interest such as protein-ligand docking and the prediction of free energies of binding.     

Nitrous oxide emissions from agricultural fields: Assessment, measurement and mitigation

In this paper we discuss three topics concerning N₂O emissions from agricultural systems. First, we present an appraisal of N₂O emissions from agricultural soils (Assessment). Secondly, we discuss some recent efforts to improve N₂O flux estimates in agricultural fields (Measurement), and finally, we relate recent studies which use nitrification inhibitors to decrease N₂O emissions from N-fertilized fields (Mitigation).To assess the global emission of N₂O from agricultural soils, the total flux should represent N₂O from all possible sources; native soil N, N from recent atmospheric deposition, past years fertilization, N from crop residues, N₂O from subsurface aquifers below the study area, and current N fertilization. Of these N sources only synthetic fertilizer and animal manures and the area of fields cropped with legumes have sufficient global data to estimate their input for N₂O production. The assessment of direct and indirect N₂O emissions we present was made by multiplying the amount of fertilizer N applied to agricultural lands by 2% and the area of land cropped to legumes by 4 kg N₂O-N ha^-1. No regard to method of N application, type of N, crop, climate or soil was given in these calculations, because the data are not available to include these variables in large scale assessments. Improved assessments should include these variables and should be used to drive process models for field, area, region and global scales.Several N₂O flux measurement techniques have been used in recent field studies which utilize small and ultralarge chambers and micrometeorological along with new analytical techniques to measure N₂O fluxes. These studies reveal that it is not the measurement technique that is providing much of the uncertainty in N₂O flux values found in the literature but rather the diverse combinations of physical and biological factors which control gas fluxes. A careful comparison of published literature narrows the range of observed fluxes as noted in the section on assessment. An array of careful field studies which compare a series of crops, fertilizer sources, and management techniques in controlled parallel experiments throughout the calendar year are needed to improve flux estimates and decrease uncertainty in prediction capability.There are a variety of management techniques which should conserve N and decrease the amount of N application needed to grow crops and to limit N₂O emissions. Using nitrification inhibitors is an option for decreasing fertilizer N use and additionally directly mitigating N₂O emissions. Case studies are presented which demonstrate the potential for using nitrification inhibitors to limit N₂O emissions from agricultural soils. Inhibitors may be selected for climatic conditions and type of cropping system as well as the type of nitrogen (solid mineral N, mineral N in solution, or organic waste materials) and applied with the fertilizers.     

Synthesis of Duplex DNA Containing a Spin Labeled Analog of 2′ Deoxycytidine

Abstract

We report the chemical synthesis of phosphoramidite 8, containing a spin labeled analog of deoxycytidine, C?, and its incorporation into synthetic DNA. The EPR characteristics of the resulting DNAs indicated that the motion of the spin label was well-correlated with the uniform modes of the macromolecule, but that correlation of the spin label with internal motion was less effective than that achieved using a spin labeled quinolone, Q.     

Synthesis,characterisation, optical and nonlinear optical properties of thiazole and benzothiazole derivatives: a dual approach

In the present investigation, we employ a dual approach consisting of experimental and computational techniques to synthesise and characterise the Schiff-base including the moieties of nitrophenyl (3), nitrothiazole (5) and nitrobenzothiazole (7). The synthesised Schiff bases are confirmed by FT-IR, ¹H-NMR and UV-visible spectroscopic techniques. The experimental UV-visible spectroscopic results are compared to the theoretically calculated TD-DFT results. There is a reasonably good agreement between the experimental and the theoretically calculated spectroscopic results. We also calculate the third-order nonlinear optical (NLO) polarizability (γ) of above entitled derivatives using finite field (FF) approach and DFT methods. The compound 7 shows an amplitude of γ as large as 124.15×10⁴ a. u., which is found to be several times larger than that of para-nitroaniline. Moreover, the partial and total density of states (PDOS and TDOS) along with electrostatic potential maps are calculated to get more physical insights into the structure-property relationship and electronic communications between terminal donor and central core acceptor moieties in the synthesised compounds. The present investigation highlights the significance of indigenously synthesised nitrothiazole and nitrobenzothiazole compounds as efficient NLO materials, which may evoke the interest of scientific community in such efficient NLO materials for their potential utilization in device applications.     

Three-Dimensional Trabecular Alignment Model

The Shear-slip Mesh Update Method (SSMUM) is being used in flow simulations involving large but regular displacements of one or more boundaries of the computational domain. We follow up the earlier discussion of the method with notes on practical implementation aspects. In order to establish a benchmark problem for this class of flow problems, we define and report results from a two-dimensional viscous flow around a rotating stirrer in a square chamber. The application potential of the method is demonstrated in the context of biomedical design problem, as we perform an analysis of blood flow in a centrifugal left ventricular assist device, or blood pump, which involves a rotating impeller in a non-axisymmetric housing.     

Revealing of the Spin-Spin Interaction Due to Incorporating a Spin Label in the Carbohydrate Parts of Immuniglobulins M and G

Abstract

The EPR spectra of the preparations produced by spin labeling of the carbohydrate parts in monoclonal IgM and normal IgG with 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl as the spin label indicate the existence of a rapid spin-spin exchange interaction between two spin labels. In the case of spin-labeled IgM, the carrier of such a spectrum is shown to be a glycopeptide noncovalently bound to IgM; it includes two spin labels and may be detached from the macromolecule by a combination of dialysis and gel filtration.     

Intestinal cytosol binders of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3

The binding of 25-hydroxy-[26,27-³H]vitamin D₃ and 1,25-dihydroxy-[26,27-³H]vitamin D₃ to the cytosol of intestinal mucosa of chicks and rats has been studied by sucrose gradient analysis. The cytosol from chick mucosa showed variable binding of 1,25-dihydroxyvitamin D₃ to a 3.0S macromolecule which has high affinity and low capacity for this metabolite. However, when the mucosa was washed extensively before homogenization, a 3.7S macromolecule was consistently observed which showed considerable specificity and affinity for 1,25-dihydroxyvitamin D₃. Although 3.7S binders for 1,25-dihydroxyvitamin D₃ could also be located in other organs, competition experiments with excess nonradioactive 1,25-dihydroxyvitamin D₃ suggested that they were not identical to the 3.7S macromolecule from intestinal mucosal cytosol. As the 3.7S macromolecule was allowed to stand at 4 °C with bound 1,25-dihydroxy-[³H]vitamin D₃, the 1,25-dihydroxy-[³H]vitamin D₃ became increasingly resistant to displacement by non-radioactive 1,25-dihydroxyvitamin D₃. The 1,25-dihydroxy-[³H]vitamin D₃ remained unchanged and easily extractable with lipid solvents through this change, making unlikely the establishment of a covalent bond. Unlike the chick, mucosa from rats yielded cytosol in which no specific binding of 1,25-dihydroxy-[³H]vitamin D₃ was detected. Instead, a 5-6S macromolecule which binds both 1,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃ was found. This protein which was also found in chick mucosa shows preferential binding for 25-hydroxyvitamin D₃. It could be removed by washing the mucosa with buffer prior to homogenization which suggests that it may not be a cytosolic protein. Although the 3.7S protein from chick mucosa has properties consistent with its possible role as a receptor, the 5-6S macromolecule does not appear to have “receptor”-like properties.     

Kinetics of low-frequency excitations in globular macromolecules

The kinetics of low-frequency rotational-vibrational excitations of globular macromolecules is studied. The macromolecule is modelled by a viscous liquid-like deformable body with the thermal motion being generated by the stochastic motion of its surface. The collective excitations are treated in terms of dynamic variables describing the deviations of the macromolecule shape from its equilibrium spherical form. Thermal fluctuations of the variables are described using the kinetic Einstein-Smoluchowski equation. The probability density of the change of the macromolecule dynamical state is found. The Van Hove correlation function determining the spectrum of the Rayleigh scattering of Mössbauer gamma-quanta is calculated. The spectrum is found for large values of the momentum transfer at the scattering, k. The effective width of the spectrum is shown to be proportional to k ³T/, where T is the temperature and is the macromolecule viscosity.     

太赫兹刺激听神经的测试平台搭建

目的 近年来，用于脑功能调控的神经调控技术蓬勃发展，很多方法已在临床上被推广应用，主要包括电极深部脑刺激、经颅磁刺激、光遗传技术、超声深脑刺激等。但是这些调控技术存在刺激靶点改变灵活性差、空间分辨率不足、需要注射病毒转染等问题。与这些技术相比，太赫兹波调控则能以较高的时空分辨率、无需引入外源基因的方式对神经活动进行干预。激光神经刺激是一种具有较明确靶向性的刺激方法，可以通过调整不同激光参数（激光波长、脉冲能量等）控制引起神经兴奋或者抑制。但是由于该研究方向的实验手段和实验平台的缺乏，相关研究开展较少。方法 针对这个问题，从听觉神经入手，在分子、细胞和在体不同层面为相关领域的研究搭建了不同的测试平台。结果 实验结果表明，这些系统在时间和空间上具有良好的耦合性和靶向性，测得的信号受噪音干扰小。结论 这些系统可以有效测试神经系统对太赫兹刺激的响应并精确控制刺激时间和位置。     

Human umbilical cord provides a significant source of unexpanded mesenchymal stromal cells

Background aimsHuman mesenchymal stromal cells (MSC) have considerable potential for cell-based therapies, including applications for regenerative medicine and immune suppression in graft-versus-host disease (GvHD). However, harvesting cells from the human body can cause iatrogenic disorders and in vitro expansion of MSC carries a risk of tumorigenesis and/or expansion of unexpected cell populationsMethodsGiven these problems, we have focused on umbilical cord, a tissue obtained with few ethical problems that contains significant numbers of MSC. We have developed a modified method to isolate MSC from umbilical cord, and investigated their properties using flow cytometry, mRNA analysis and an in vivo GvHD modelResultsOur study demonstrates that, using umbilical cord, large numbers of MSC can be safely obtained using a simple procedure without in vitro expansion, and these non-expanded MSC have the potential to suppress GvHD.ConclusionsOur results suggest that the combined banking of umbilical cord-derived MSC and identical cord blood-derived hematopoietic stem cell banking, where strict inspection of the infectious disease status of donors is performed, as well as further benefits of HLA-matched mesenchymal cells, could become one of the main sources of cells for cell-based therapy against various disorders.     

Thermal Excitations of Hydrated Macromolecules: The Effects of Hydration on the Mössbauer Absorption and Scattering Spectra

Abstract

The spectra of Rayleigh scattering of Mössbauer radiation (RSMR) and Mössbauer absorption by globular macromolecules are calculated. The dependence of the spectra parameters on hydration is modeled with the account for thermal low-frequency vibrations of the particles constituting the globule. Deformational motions of the macromolecule fragments leading to deviations from its equilibrium spherical shape are considered introducing collective dynamical variables governed by Langevin equations with random sources of external forces. The macromolecule is modeled by a double-layered sphere: a rigid (elastic) core is surrounded by a porous hydration shell filled with fluid. The dynamical properties of the bound water inside the shell are described by the Debye-Brinkman equations. The degree of hydration is introduced by means of a combination of the mass coefficients of the porous shell with fluid and the mass coefficients in the limiting cases when the flow inside the shell is “frozen” and in the case of free flow. The hydration-dependent Lamb-Mössbauer factor and the elastic fraction of the RSMR are calculated and compared with experimental data from the literature.     

Deepening our understanding of HDL proteome

ABSTRACT

Introduction: High-density lipoprotein (HDL) particles are heterogeneous and their proteome is complex and distinct from HDL cholesterol. However, it is largely unknown whether HDL proteins are associated with cardiovascular protection.

Areas covered: HDL isolation techniques and proteomic analyses are reviewed. A list of HDL proteins reported in 37 different studies was compiled and the effects of different isolation techniques on proteins attributed to HDL are discussed. Mass spectrometric techniques used for HDL analysis and the need for precise and robust methods for quantification of HDL proteins are discussed.

Expert opinion: Proteins associated with HDL have the potential to be used as biomarkers and/or help to understand HDL functionality. To achieve this, large cohorts must be studied using precise quantification methods. Key factors in HDL proteome quantification are the isolation methodology and the mass spectrometry technique employed. Isolation methodology affects what proteins are identified in HDL and the specificity of association with HDL particles needs to be addressed. Shotgun proteomics yields imprecise quantification, but the majority of HDL studies relied on this approach. Few recent studies used targeted tandem mass spectrometry to quantify HDL proteins, and it is imperative that future studies focus on the application of these precise techniques.     

Genetic counseling and the presenile dementias

Abstract

The clinical application of genetic counseling techniques to the presenile dementing illnesses is discussed, using examples. The problems encountered in adult‐onset illness are very different from those seen in illnesses affecting children. Some general guidelines and some specific ones are presented.     

Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design

BackgroundA natural product analog, 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP) was found to be an effective inhibitor of botulinum neurotoxin type A (BoNT/A).MethodsIn this work, we performed enzyme inhibition kinetics and employed biochemical techniques such as isothermal calorimetry (ITC) and fluorescence spectroscopy as well as molecular modeling to examine the kinetics and binding mechanism of NPP inhibitor with BoNT/A LC.ResultsStudies of inhibition mechanism and binding dynamics of NPP to BoNT/A light chain (BoNT/A LC) showed that NPP is a mixed type inhibitor for the zinc endopeptidase activity, implying that at least part of the inhibitor-enzyme binding site may be different from the substrate-enzyme binding site. By using biochemical techniques, we demonstrated NPP forms a stable complex with BoNT/A LC. These observations were confirmed by Molecular Dynamics (MD) simulation, which demonstrates that NPP binds to the site near the active site.ConclusionThe NPP binding interferes with BoNT/A LC binding to the SNAP-25, hence, inhibits its cleavage. Based on these results, we propose a modified strategy for designing a molecule to enhance the efficiency of the inhibition against the neurotoxic effect of BoNT.General significanceInsights into the interactions of NPP with BoNT/A LC using biochemical and computational approaches will aid in the future development of effective countermeasures and better pharmacological strategies against botulism.     

Big Data to Knowledge: Application of Machine Learning to Predictive Modeling of Therapeutic Response in Cancer

BackgroundIn recent years, the availability of high throughput technologies, establishment of large molecular patient data repositories, and advancement in computing power and storage have allowed elucidation of complex mechanisms implicated in therapeutic response in cancer patients. The breadth and depth of such data, alongside experimental noise and missing values, requires a sophisticated human-machine interaction that would allow effective learning from complex data and accurate forecasting of future outcomes, ideally embedded in the core of machine learning design.ObjectiveIn this review, we will discuss machine learning techniques utilized for modeling of treatment response in cancer, including Random Forests, support vector machines, neural networks, and linear and logistic regression. We will overview their mathematical foundations and discuss their limitations and alternative approaches in light of their application to therapeutic response modeling in cancer.ConclusionWe hypothesize that the increase in the number of patient profiles and potential temporal monitoring of patient data will define even more complex techniques, such as deep learning and causal analysis, as central players in therapeutic response modeling.     

An analogue of the binding polynomial for the case of ligands binding to an aggregating macromolecule

It is known that when a macromolecule exists as a mixture of forms with different molecular weights in equilibrium in dilute solution with ligands then the saturation functions cannot be derived by differentiating a binding polynomial. However, we demonstrate that in such circumstances a saturation function can be expressed in terms of the binding polynomials for each of the polymeric forms of macromolecule and integration of this using the conservation equation affords a function, Q, which is the analogue of the binding polynomial for a non-aggregating system. The existence of Q leads to heterotropic linkage relationships.