An Expert System Approach to the Assessment of Hepatotoxic Potential

Hepatotoxicity is a major cause of pharmaceutical drug attrition and is also a concern within other chemical industries. In silico approaches to the prediction of hepatotoxicity are an important tool in the early identification of adverse effects in the liver associated with exposure to a chemical. Here, we describe work in progress to develop an expert system approach to the prediction of hepatotoxicity, focussing particularly on the identification of structural alerts associated with its occurrence. The development of 74 such structural alerts based on public‐domain literature and proprietary data sets is described. Evaluation results indicate that, whilst these structural alerts are effective in identifying the hepatotoxicity of many chemicals, further research is needed to develop additional structural alerts to account for the hepatotoxicity of a number of chemicals which is not currently predicted. Preliminary results also suggest that the specificity of the structural alerts may be improved by the combined use of applicability domains based on physicochemical properties such as log P and molecular weight. In the longer term, the performance of predictive models is likely to benefit from the further integration of diverse data and prediction model types.     

Building a tree of knowledge: analysis of bitter molecules

A phylogenetic-like tree of structural fragments has been constructed to extract useful insights from a structural database of bitter molecules. The tree of structural fragments summarizes the substructural groups present in the molecules from the bitter database. These structural fragments are compared with a large number of random molecules to highlight substructures specific to bitter molecules. This organization of the structures enabled the detection of structure-activity relationships for the bitter molecules through the construction of R-tables. Key structural groups, able to distinguish between bitter and random molecules, were identified through an analysis of the tree. This information can be used to further understand which structural components are involved in producing a bitter taste.     

Similarity-Based Analysis of Allele Frequency Distribution among Multiple Populations Identifies Adaptive Genomic Structural Variants

Structural variants have a considerable impact on human genomic diversity. However, their evolutionary history remains mostly unexplored. Here, we developed a new method to identify potentially adaptive structural variants based on a similarity-based analysis that incorporates genotype frequency data from 26 populations simultaneously. Using this method, we analyzed 57,629 structural variants and identified 576 structural variants that show unusual population differentiation. Of these putatively adaptive structural variants, we further showed that 24 variants are multiallelic and overlap with coding sequences, and 20 variants are significantly associated with GWAS traits. Closer inspection of the haplotypic variation associated with these putatively adaptive and functional structural variants reveals deviations from neutral expectations due to: 1) population differentiation of rapidly evolving multiallelic variants, 2) incomplete sweeps, and 3) recent population-specific negative selection. Overall, our study provides new methodological insights, documents hundreds of putatively adaptive variants, and introduces evolutionary models that may better explain the complex evolution of structural variants.     

The dependence of all-atom statistical potentials on structural training database

An accurate statistical energy function that is suitable for the prediction of protein structures of all classes should be independent of the structural database used for energy extraction. Here, two high-resolution, low-sequence-identity structural databases of 333 alpha-proteins and 271 beta-proteins were built for examining the database dependence of three all-atom statistical energy functions. They are RAPDF (residue-specific all-atom conditional probability discriminatory function), atomic KBP (atomic knowledge-based potential), and DFIRE (statistical potential based on distance-scaled finite ideal-gas reference state). These energy functions differ in the reference states used for energy derivation. The energy functions extracted from the different structural databases are used to select native structures from multiple decoys of 64 alpha-proteins and 28 beta-proteins. The performance in native structure selections indicates that the DFIRE-based energy function is mostly independent of the structural database whereas RAPDF and KBP have a significant dependence. The construction of two additional structural databases of alpha/beta and alpha + beta-proteins further confirmed the weak dependence of DFIRE on the structural databases of various structural classes. The possible source for the difference between the three all-atom statistical energy functions is that the physical reference state of ideal gas used in the DFIRE-based energy function is least dependent on the structural database.     

The composition of rat liver microsomes. The structural proteins of rat liver microsomes

1. The structural-protein component of microsomal membranes was isolated by three separate methods. Analysis by polyacrylamide-gel electrophoresis indicated that the microsomal structural component is made up of a heterogeneous group of proteins. These proteins were further characterized by their phospholipid-binding capacity. The electrophoretic patterns of microsomal structural proteins were found to differ significantly from those of mitochondrial structural proteins. 2. The reticulosomal fraction was also characterized by electrophoresis with reference to total microsomal proteins, microsomal structural proteins and ribosomal proteins. The reticulosomes gave an electrophoretic pattern significantly different from those of the other three preparations examined. It is suggested that reticulosomes consist largely of enzymic proteins of the endoplasmic reticulum.     

Molecular determinants of the hanatoxin binding in voltage-gated K+-channel drk1

The carboxyl terminus of S3 segment (S3(C)) in voltage-gated potassium channels was proposed to bear the binding site for gating modifier toxins like Hanatoxin and a helical secondary structural arrangement was suggested. Due to the lack of complete structure in high resolution for such a channel molecule, no further direct experimental data to elucidate the mechanism for their binding conformations could thus far be derived. In order to examine the putative three-dimensional structure of S3(C) and to illustrate the residues required for Hanatoxin binding, molecular simulation and docking were performed, based on the solution structure of Hanatoxin and the structural information from lysine-scanning results for S3(C) fragment. From our results, it is indicated that both hydrophobic and electrostatic interactions are utilized to stabilize the toxin binding. Detailed docking residues and appropriate orientation for binding regarding hydrophobic/-philic environments are also described. Compared with the functional data proposed by previous studies, the helical structural arrangement for the C-terminus of S3 segment in voltage-gated potassium channels can therefore be further emphasized.     

基于厚壳贻贝Mytilin-1的抗菌肽设计、固相合成及抗菌谱分析

贻贝抗菌肽Mytilin是贻贝免疫系统的重要组成部分,对其结构与功能的研究表明,其序列中连接两段β-折叠的发夹区域是其抗菌功能的关键所在。为验证该区域是否具有抗菌活性,通过对厚壳贻贝Mytilus coruscus抗菌肽Mytilin进行空间结构模拟,选取其中β-发夹部分肽段,采用了固相化学合成的方法合成了两条10肽,分别命名为Mytilin Derived Peptide-1(MDP-1)和Mytilin Derived Peptide-2(MDP-2)。高效液相色谱以及质谱检测结果表明,合成是成功的。抗菌谱研究表明,MDP-1和MDP-2对革兰氏阳性菌、阴性菌以及真菌均具有明显的抑制作用,同时,合成的MDP由于序列短且有两对二硫键,因此对于温度及人血浆均表现出很强的稳定性。上述研究结果为深入了解厚壳贻贝抗菌肽Mytilin的抗菌机制以及在此基础上开发具有应用价值的新型抗菌肽奠定了基础。     

Phosphorylation of Simian Virus 40 Proteins in a Cell-Free System

We have shown previously that all the structural proteins of simian virus 40 (SV40) are phosphoproteins. Virus phosphorylated in vivo could be further phosphorylated with exogenous cellular protein kinases in a cell-free system containing gamma-(32)P-ATP as phosphate donor. In intact infectious virus only polypeptides 1 and 2 (mol wt 49,000 and 40,800, respectively) were further phosphorylated in vitro. However, when infectious SV40 was partially disrupted, treated with nucleases, and then phosphorylated in vitro, all five structural polypeptides accepted additional phosphate groups. Similarly, all polypeptides of intact empty capsids, derived from infected cells, were further phosphorylated in vitro. Phosphorylation of empty capsids and infectious SV40 in vitro was enhanced from 4- to 11-fold after prior treatment of virus with alkali. The phosphate group was linked only to serine residues of the viral polypeptides phosphorylated both in vitro and in vivo.     

Analysis of the Secondary Structure of ITS1 in Pectinidae: Implications for Phylogenetic Reconstruction and Structural Evolution

It is at present difficult to accurately position gaps in sequence alignment and to determine substructural homology in structure alignment when reconstructing phylogenies based on highly divergent sequences. Therefore, we have developed a new strategy for inferring phylogenies based on highly divergent sequences. In this new strategy, the whole secondary structure presented as a string in bracket notation is used as phylogenetic characters to infer phylogenetic relationships. It is no longer necessary to decompose the secondary structure into homologous substructural components. In this study, reliable phylogenetic relationships of eight species in Pectinidae were inferred from the structure alignment, but not from sequence alignment, even with the aid of structural information. The results suggest that this new strategy should be useful for inferring phylogenetic relationships based on highly divergent sequences. Moreover, the structural evolution of ITS1 in Pectinidae was also investigated. The whole ITS1 structure could be divided into four structural domains. Compensatory changes were found in all four structural domains. Structural motifs in these domains were identified further. These motifs, especially those in D2 and D3, may have important functions in the maturation of rRNAs.     

On the role of structural information in remote homology detection and sequence alignment: new methods using hybrid sequence profiles

Structural alignments often reveal relationships between proteins that cannot be detected using sequence alignment alone. However, profile search methods based entirely on structural alignments alone have not been found to be effective in finding remote homologs. Here, we explore the role of structural information in remote homolog detection and sequence alignment. To this end, we develop a series of hybrid multidimensional alignment profiles that combine sequence, secondary and tertiary structure information into hybrid profiles. Sequence-based profiles are profiles whose position-specific scoring matrix is derived from sequence alignment alone; structure-based profiles are those derived from multiple structure alignments. We compare pure sequence-based profiles to pure structure-based profiles, as well as to hybrid profiles that use combined sequence-and-structure-based profiles, where sequence-based profiles are used in loop/motif regions and structural information is used in core structural regions. All of the hybrid methods offer significant improvement over simple profile-to-profile alignment. We demonstrate that both sequence-based and structure-based profiles contribute to remote homology detection and alignment accuracy, and that each contains some unique information. We discuss the implications of these results for further improvements in amino acid sequence and structural analysis.     

Theoretical study of the partial molar volume change associated with the pressure-induced structural transition of ubiquitin

The partial molar volume (PMV) change associated with the pressure-induced structural transition of ubiquitin is analyzed by the three-dimensional reference interaction site model (3D-RISM) theory of molecular solvation. The theory predicts that the PMV decreases upon the structural transition, which is consistent with the experimental observation. The volume decomposition analysis demonstrates that the PMV reduction is primarily caused by the decrease in the volume of structural voids in the protein, which is partially canceled by the volume expansion due to the hydration effects. It is found from further analysis that the PMV reduction is ascribed substantially to the penetration of water molecules into a specific part of the protein. Based on the thermodynamic relation, this result implies that the water penetration causes the pressure-induced structural transition. It supports the water penetration model of pressure denaturation of proteins proposed earlier.     

Digitally supported public health interventions through the lens of structural injustice: The case of mobile apps responding to violence against women and girls

Mobile applications (apps) have gained significant popularity as a new intervention strategy responding to violence against women and girls. Despite their growing relevance, an assessment from the perspective of public health ethics is still lacking. Here, we base our discussion on the understanding of violence against women and girls as a multidimensional, global public health issue on structural, societal and individual levels and situate it within the theoretical framework of structural injustice, including epistemic injustice. Based on a systematic app review we previously conducted, we evaluate the content and functions of apps through the lens of structural injustice. We argue that technological solutions such as apps may be a useful tool in the fight against violence against women and girls but have to be situated within the broader frame of public health that considers the structural dimensions of such violence. Ultimately, the concerns raised by structural injustice are—alongside key concerns of safety, data privacy, importance of human supportive contact, and so forth—crucial dimensions in the ethical assessment of such apps. However, research on the role and relevance of apps as strategies to address the structural and epistemic dimensions of violence remains scarce. This article aims to provide a foundation for further discussion in this area and could be applicable to other areas in public health policy and practice.     

Tracing surfactant transformation from cellular release to insertion into an air-liquid interface

Pulmonary surfactant is secreted by alveolar type II cells as lipid-rich, densely packed lamellar body-like particles (LBPs). The particulate nature of released LBPs might be the result of structural and/or thermodynamic forces. Thus mechanisms must exist that promote their transformation into functional units. To further define these mechanisms, we developed methods to follow LBPs from their release by cultured cells to insertion in an air-liquid interface. When released, LBPs underwent structural transformation, but did not disperse, and typically preserved a spherical appearance for days. Nevertheless, they were able to modify surface tension and exhibited high surface activity when measured with a capillary surfactometer. When LBPs inserted in an air-liquid interface were analyzed by fluorescence imaging microscopy, they showed remarkable structural transformations. These events were instantaneous but came to a halt when the interface was already occupied by previously transformed material or when surface tension was already low. These results suggest that the driving force for LBP transformation is determined by cohesive and tensile forces acting on these particles. They further suggest that transformation of LBPs is a self-regulated interfacial process that most likely does not require structural intermediates or enzymatic activation.     

Improving structural integrity of cryosections for immunogold labeling

 Cryosections of aldehyde-fixed material prepared according to Tokuyasu are a good substrate for immunocytochemistry. However, structural defects occur that limit the resolution of this approach. We found that the step during which sections are thawed and transferred from the cryochamber to the supporting film on an EM grid is most critical for structural preservation. Surface tension of the transfer medium, on which sections are spread during thawing, can easily damage their structure by overstretching. By substituting a mixture of methylcellulose and sucrose for the conventional sucrose transfer medium, we were able to alleviate the problem of overstretching, thus improving greatly the structural integrity of thin cryosections. Also, material extraction from the sections after thawing causes structural damage, particularly when cross-linking is deficient. Incorporation of uranyl acetate in the transfer medium can then further help to maintain the structural integrity of the sections during the immunolabeling procedure. Excellent ultrastructure was featured in sections picked up and dried directly in methylcellulose/uranyl acetate mixtures. Such preparations can provide new insight into subcellular details and is an efficient back-up for immunolabeled sections in respect of their morphology. Cryosections from fresh frozen tissue can be preserved for immunolabeling by using transfer media that contain fixatives. This approach may have advantages if chemical fixation of tissue is thought to induce morphological artifacts or antigen redistribution. Accepted: 2 May 1996     

Quantifying structure-function uncertainty: a graph theoretical exploration into the origins and limitations of protein annotation

Since the advent of investigations into structural genomics, research has focused on correctly identifying domain boundaries, as well as domain similarities and differences in the context of their evolutionary relationships. As the science of structural genomics ramps up adding more and more information into the databanks, questions about the accuracy and completeness of our classification and annotation systems appear on the forefront of this research. A central question of paramount importance is how structural similarity relates to functional similarity. Here, we begin to rigorously and quantitatively answer these questions by first exploring the consensus between the most common protein domain structure annotation databases CATH, SCOP and FSSP. Each of these databases explores the evolutionary relationships between protein domains using a combination of automatic and manual, structural and functional, continuous and discrete similarity measures. In order to examine the issue of consensus thoroughly, we build a generalized graph out of each of these databases and hierarchically cluster these graphs at interval thresholds. We then employ a distance measure to find regions of greatest overlap. Using this procedure we were able not only to enumerate the level of consensus between the different annotation systems, but also to define the graph-theoretical origins behind the annotation schema of class, family and superfamily by observing that the same thresholds that define the best consensus regions between FSSP, SCOP and CATH correspond to distinct, non-random phase-transitions in the structure comparison graph itself. To investigate the correspondence in divergence between structure and function further, we introduce a measure of functional entropy that calculates divergence in function space. First, we use this measure to calculate the general correlation between structural homology and functional proximity. We extend this analysis further by quantitatively calculating the average amount of functional information gained from our understanding of structural distance and the corollary inherent uncertainty that represents the theoretical limit of our ability to infer function from structural similarity. Finally we show how our measure of functional "entropy" translates into a more intuitive concept of functional annotation into similarity EC classes.     

New opportunities in integrative structural modeling

Integrative structural modeling enables structure determination of macromolecules and their complexes by integrating data from multiple sources. It has been successfully used to characterize macromolecular structures when a single structural biology technique was insufficient. Recent developments in cellular structural biology, including in-cell cryo-electron tomography and artificial intelligence-based structure prediction, have created new opportunities for integrative structural modeling. Here, we will review these opportunities along with the latest developments in integrative modeling methods and their applications. We also highlight open challenges and directions for further development.     

Effect of PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4-dioxane

The employment of enzymes as catalysts within organic media has traditionally been hampered by the reduced enzymatic activities when compared to catalysis in aqueous solution. Although several complementary hypotheses have provided mechanistic insights into the causes of diminished activity, further development of biocatalysts would greatly benefit from effective chemical strategies (e.g., PEGylation) to ameliorate this event. Herein we explore the effects of altering the solvent composition from aqueous buffer to 1,4-dioxane on structural, dynamical, and catalytic properties of the model enzyme subtilisin Carlsberg (SBc). Furthermore, we also investigate the effects of dissolving the enzyme in 1,4-dioxane through chemical modification with poly(ethylene)-glycol (PEG, M(W) = 20 kDa) on these enzyme properties. In 1,4-dioxane a 10(4)-fold decrease in the enzyme's catalytic activity was observed for the hydrolysis reaction of vinyl butyrate with D(2)O and a 50% decrease in enzyme structural dynamics as evidenced by reduced amide H/D exchange kinetics occurred. Attaching increasing amounts of PEG to the enzyme reversed some of the activity loss. Evaluation of the structural dynamic behavior of the PEGylated enzyme within the organic solvent revealed an increase in structural dynamics at increased PEGylation. Correlation analysis between the catalytic and structural dynamic parameters revealed that the enzyme's catalytic activity and enantioselectivity depended on the changes in protein structural dynamics within 1,4-dioxane. These results demonstrate the importance of protein structural dynamics towards regulating the catalytic behavior of enzymes within organic media.     

A hypergraph-based method for unification of existing protein structure- and sequence-families

Classification of proteins is a major challenge in bioinformatics. Here an approach is presented, that unifies different existing classifications of protein structures and sequences. Protein structural domains are represented as nodes in a hypergraph. Shared memberships in sequence families result in hyperedges in the graph. The presented method partitions the hypergraph into clusters of structural domains. Each computed cluster is based on a set of shared sequence family memberships. Thus, the clusters put existing protein sequence families into the context of structural family hierarchies. Conversely, structural domains are related to their sequence family memberships, which can be used to gain further knowledge about the respective structural families.     

Classification of the possible pairs between the first anticodon and the third codon positions based on a simple model assuming two geometries with which the pairing effectively potentiates the decoding complex

Crick's wobble theory states that some specific pairs between the bases at the first position of the anticodon (position 34) and the third position of the codon (position III) are allowed and the others are disallowed during the correct codon recognition. However, later researches have shown that the pairing rule, or the wobble rule, is different from the supposed one. Despite the continuing efforts including computer-aided model building studies and analyses of three-dimensional structures in the crystals of the ribosomes, the structural backgrounds of the wobble rule are still unclear. Here, I classify the possible pairs into 6 classes according to the increases accompanying the formation of the pairs in the potential productivity of the decoding complex on the basis of a simple model that was originally proposed previously and is refined here. In the model, the conformation with the base at position 34 displaced toward the minor groove side from the position for the Watson-Crick pairs is supposed to be equivalent to the conformation with the Watson-Crick pairs. It is also reasoned and supposed that some weak pairs may sometimes be allowed depending on the structural context. It is demonstrated that most of the experimental results reported so far are consistent with the model. I discuss on which experimental facts can be reasoned with the model and which need further explanations. I expect that the model will be a good basis for further understanding of the wobble rule and its structural backgrounds.