A novel algorithm for ranking RNA structure candidates

RNA research is advancing at an ever increasing pace. The newest and most state-of-the-art instruments and techniques have made possible the discoveries of new RNAs, and they have carried the field to new frontiers of disease research, vaccine development, therapeutics, and architectonics. Like proteins, RNAs show a marked relationship between structure and function. A deeper grasp of RNAs requires a finer understanding of their elaborate structures. In pursuit of this, cutting-edge experimental and computational structure-probing techniques output several candidate geometries for a given RNA, each of which is perfectly aligned with experimentally determined parameters. Identifying which structure is the most accurate, however, remains a major obstacle. In recent years, several algorithms have been developed for ranking candidate RNA structures in order from most to least probable, though their levels of accuracy and transparency leave room for improvement. Most recently, advances in both areas are demonstrated by rsRNASP, a novel algorithm proposed by Tan et al. rsRNASP is a residue-separation-based statistical potential for three-dimensional structure evaluation, and it outperforms the leading algorithms in the field.     

In vivo versus in vitro screening or selection for catalytic activity in enzymes and abzymes

The recent development of catalytic antibodies and the introduction of new techniques to generate huge libraries of random mutants of existing enzymes have created the need for powerful tools for finding in large populations of cells those producing the catalytically most active proteins. Several approaches have been developed and used to reach this goal. The screening techniques aim at easily detecting the clones producing active enzymes or abzymes; the selection techniques are designed to extract these clones from mixtures. These techniques have been applied both in vivo and in vitro. This review describes the advantages and limitations of the various methods in terms of ease of use, sensitivity, and convenience for handling large libraries. Examples are analyzed and tentative rules proposed. These techniques prove to be quite powerful to study the relationship between structure and function and to alter the properties of enzymes.     

Characterization of novel proteins based on known protein structures

The genome sciences face the challenge to characterize structure and function of a vast number of novel genes. Sequence search techniques are used to infer functional and structural information from similarities to experimentally characterized genes or proteins. The persistent goal is to refine these techniques and to develop alternative and complementary methods to increase the range of reliable inference.Here, we focus on the structural and functional assignments that can be inferred from the known three-dimensional structures of proteins. The study uses all structures in the Protein Data Bank that were known by the end of 1997. The protein structures released in 1998 were then characterized in terms of functional and structural similarity to the previously known structures, yielding an estimate of the maximum amount of information on novel protein sequences that can be obtained from inference techniques.The 147 globular proteins corresponding to 196 domains released in 1998 have no clear sequence similarity to previously known structures. However, 75 % of the domains have extensive structure similarity to previously known folds, and most importantly, in two out of three cases similarity in structure coincides with related function. In view of this analysis, full utilization of existing structure data bases would provide information for many new targets even if the relationship is not accessible from sequence information alone. Currently, the most sophisticated techniques detect of the order of one-third of these relationships.     

Genes and genomes: Chromosome bands – flavours to savour

The mammalian chromosome is longitudinally heterogeneous in structure and function and this is the basis for the specific banding patterns produced by various chromosome staining techniques. The two most frequently used techniques are G, or Giemsa banding and R, or reverse banding. Each type of stained band is characterised by variations in gene density, time of replication, base composition, density of repeat sequences, and chromatin packaging. It is increasingly apparent that R and G bands, which are complementary to each other, represent separate compartments of the euchromatic human genome, with R bands containing the vast majority of genes. R bands are also more GC-rich, contain a higher density of Alu repeats, and replicate earlier in S phase, than G bands. These properties may be interdependent and may have coevolved.     

Chromatin structure and specificity revealed by immunological techniques

The relation between the structure and function of chromatin is complex and not fully understood; therefore, a variety of experimental techniques have to be employed to elucidate the structural basis of the regulation of the genetic message encoded in DNA. The present review attempts to point out and summarize the use of antibodies as probes to study the structure and specificity of chromatin, chromosomes and their components.

The applicability of immunological techniques requires two major steps. The first step requires elucidation of a large repertoire of antibodies specific for the various forms and components of chromatin and chromosomes. The usefulness of antibodies as probes for chromatin structure is directly dependent on the number of specific, well characterized antisera. The available immunochemical techniques allow elucidation and purification of antibodies against almost any conceivable antigen. Indeed, as indicated in this review, a large number of antisera is already available. However the list of potentially useful antisera is almost unlimited. The second step involves adaptation of various aerological and immunochemical techniques to the study of chromatin and chromosomes. Unfortunately most of the techniques used are not quantitative. However, the information obtainable on both the gross and fine arrangement of individual components in chromatin and chromosomes is not easily available by other techniques.

As the techniques for preparing ‘clean’, well-defined nuclei, soluble chromatin, well-defined nucleosomes or polynucleosomes, and unfixed chromosomes improve the information obtainable by immunological techniques will be more exact and defined. Table 1 lists some uses of aerological techniques in the study of chromatin, chromosomes and their components.     

Integrative Structural Biology in the Era of Accurate Structure Prediction

Characterizing the three-dimensional structure of macromolecules is central to understanding their function. Traditionally, structures of proteins and their complexes have been determined using experimental techniques such as X-ray crystallography, NMR, or cryo-electron microscopy—applied individually or in an integrative manner. Meanwhile, however, computational methods for protein structure prediction have been improving their accuracy, gradually, then suddenly, with the breakthrough advance by AlphaFold2, whose models of monomeric proteins are often as accurate as experimental structures. This breakthrough foreshadows a new era of computational methods that can build accurate models for most monomeric proteins. Here, we envision how such accurate modeling methods can combine with experimental structural biology techniques, enhancing integrative structural biology. We highlight the challenges that arise when considering multiple structural conformations, protein complexes, and polymorphic assemblies. These challenges will motivate further developments, both in modeling programs and in methods to solve experimental structures, towards better and quicker investigation of structure–function relationships.     

Mini review on the structure and supramolecular assembly of VDAC

The Voltage Dependent Anion Channel (VDAC) is the most abundant protein in the outer membrane of mitochondria. This strategic localization puts it at the heart of a great number of phenomena. Its recent implication in apoptosis is an example of the major importance of this protein and has created a surge of interest in VDAC. There is no atomic-resolution structure allowing a better understanding of the function of VDAC, so alternative techniques to X-ray diffraction have been used to study VDAC. Here we discuss structural models from folding predictions and review data acquired by Atomic Force Microscopy (AFM) imaging that allowed to observe VDAC’s structure and supramolecular organization in the mitochondrial outer membrane.     

Applying microscopy to the analysis of nuclear structure and function

One of the ultimate goals of biological research is to understand mechanisms of cell function within living organisms. With this in mind, many sophisticated technologies that allow us to inspect macromolecular structure in exquisite detail have been developed. Although knowledge of structure derived from techniques such as X-ray crystallography and nuclear magnetic resonance is of vital importance, these approaches cannot reveal the remarkable complexity of molecular interactions that exists in vivo. With this in mind, this review focuses on the use of microscopy techniques to analyze cell structure and function. We describe the different basic microscopic methodologies and how the routine techniques are best applied to particular biological problems. We also emphasize the specific capabilities and uses of light and electron microscopy and highlight their individual advantages and disadvantages. For completion, we also comment on the alternative possibilities provided by a variety of advanced imaging technologies. We hope that this brief analysis of the undoubted power of microscopy techniques will be enough to stimulate a wider participation in this rapidly developing area of biological discovery.     

A novel method for the purification of recombinant subunit I of theDolichos biflorus seed lectin

Lectins are carbohydrate-binding proteins that are ubiquitous in nature. Their ability to specifically bind carbohydrates has been used as a means of purification mainly through affinity chromatography techniques. Plant lectins are one of the most thoroughly studied class of lectins, however, details of theirin situ function remains elusive. Recent advances in recombinant DNA techniques have been used in several laboratories to study the function of these lectins by heterologous over-expression. The larger subunit of theDolichos biflorus seed lectin was described by Chao et al. in 1994 and purification through affinity chromatography techniques was described. Here we report on a new method for the purification of this recombinant protein with techniques that are not dependent on the ability of the lectin to bind sugars. This method may have uses in the purification of mutant proteins that may not bind carbohydrates. Characterization of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy shows that the lectin is over 99% pure with a molecular weight of 27,090±16.17 Da, and hemagglutination assays confirm that the lectin retains its biological activity.     

Structure of slowly adapting pulmonary stretch receptors in the lung periphery.

Pulmonary sensory receptors are the initiating sites for lung reflexes; however, little is known about their structure, especially the relationship between the structure and function of these receptors. Using a novel approach (combining electrophysiological and morphological techniques), we examined the structures of the typical slowly adapting pulmonary stretch receptors (SARs) located in the lung periphery. We recorded SAR activities in the cervical vagus nerve, identified the receptive field, dissected the SARs in blocks, fixed and processed these blocks for immunohistochemical staining using anti-Na+/K+-ATPase, and examined the blocks under a confocal microscope. These SAR structures have multiple endings that have terminal knobs. Some structures that are located in the airway walls have terminal knobs buried in smooth muscle. Others are in the most peripheral part of the lung, and their terminal knobs have no obvious relation to smooth muscle, suggesting that muscle contraction may not be a direct factor for SAR activation.     

Secondary structure of ovalbumin messenger RNA.

The secondary structure of highly purified ovalbumin mRNA was studied by automated thermal denaturation techniques and the data were subjected to computer processing. Comparative studies with 20 natural and synthetic model nucleic acids suggested that the secondary structure of ovalbumin mRNA possesses the following features: the extent of base pairing of ovalbumin mRNA is similar to that found in tRNAs or ribosomal RNAs; the secondary structure of ovalbumin mRNA is more thermolabile than any of the model compounds tested, including the copolymer poly(A-U); ovalbumin mRNA does not have extensive G-C rich stems as found in tRNAs or ribosomal RNAs; the base composition of the double-stranded regions reveals 54% G-C residues which was significantly higher than that noted in the whole molecule (approximately 41.5% G-C). The presence of 46% A-U pairs in short stems of about five base pairs would have a very large destabilizing effect on the secondary structure of ovalbumin mRNA. However, at 0.175 M monovalent cations and 36 degrees C most of the secondary structure of ovalbumin mRNA is preserved. These data suggest that the double-stranded regions in ovalbumin mRNA are of sufficient length to provide the necessary stability for maintaining the open loop regions in an appropriate conformation which may be required for the biological function of ovalbumin mRNA. Furthermore, the lability of the double-stranded regions in ovalbumin mRNA may also be important for the biological function of this mRNA.     

A QCAR-approach to materials modeling

Little is known about the relationship between the function and structure of materials. Materials (solids with a function) are complex entities and a better knowledge of the parameters that contribute to function is desirable. Here, we present modeling approaches that correlate chemical composition with function of heterogeneous catalysts. The complete composition space of the mixed oxides of Ni–Cr–Mn and of Ni–Co–Mo–Mn (10% spacing) have been measured for the oxidation of propene to acroleine. The data have been collected, visualized and modeled. Different mathematical approaches such as Support Vector Machines, multilevel B-splines approximation and Kriging have been applied to model this relationship. High-throughput screening data of ternary and quaternary composition spreads are approximated to locate catalysts of high activity within the search space. For quaternary systems, slice plots offer a good tool for visualization of the results. Using these approximation techniques, the composition of the most active catalysts can be predicted. The study documents that distinct relationships between chemical composition and catalytic function exist and can be described by mathematical models. Visualization of a ternary catalyst system and its approximation using slice plots     

杂交酶及其相关技术研究进展

根据杂交酶研究目的以及构建杂交酶方法,文中总结近年来杂交酶研究的成果,将杂交酶的应用分为以下几个主要的方面改变酶的非催化特性;创造新活性的酶;研究蛋白质的结构和功能的关系等.还介绍了DNA序列改组、表达克隆、分子筛选、人造细胞样区室筛选基因等技术的进展以及这些技术在构建杂交酶方面的应用.     

TeloTool: a new tool for telomere length measurement from terminal restriction fragment analysis with improved probe intensity correction

Telomeres comprise the protective caps of natural chromosome ends and function in the suppression of DNA damage signaling and cellular senescence. Therefore, techniques used to determine telomere length are important in a number of studies, ranging from those investigating telomeric structure to effects on human disease. Terminal restriction fragment (TRF) analysis has for a long time shown to be one of the most accurate methods for quantification of absolute telomere length and range from a number of species. As this technique centers on standard Southern blotting, telomeric DNA is observed on resulting autoradiograms as a heterogeneous smear. Methods to accurately determine telomere length from telomeric smears have proven problematic, and no reliable technique has been suggested to obtain mean telomere length values. Here, we present TeloTool, a new program allowing thorough statistical analysis of TRF data. Using this new method, a number of methodical biases are removed from previously stated techniques, including assumptions based on probe intensity corrections. This program provides a standardized mean for quick and reliable extraction of quantitative data from TRF autoradiograms; its wide application will allow accurate comparison between datasets generated in different laboratories.     

Structural characteristics of the mature embryo sac of Camellia oleifera

The ovule is the most important reproductive organ in the pistil of phanerogamae. Camellia oleifera (Theaceae) is an important woody plant producing edible oil in southern China, and its embryo sac structure has a positive effect on seed breeding. In this study, the microstructure, ultrastructure and three‐dimensional structure of the ovule and embryo sac of C. oleifera were observed and described based on a combination of advanced microscopy techniques (SEM, TEM, CLSM). The ovule comprises the inner and outer integument. Large quantities of secretions in the micropylar canal exit and may participate in the guidance of the entry of the pollen tube into the embryo sac. The synergids have a dense cytoplasm, abundant organelles, and strong polarity. Little cytoplasm is present in the egg cell, yet there are many vacuoles. The center of the cell is taken up by a large vacuole, and the cytoplasm is pushed towards the edges to form obvious cytoplasmic cords. The two polar nuclei are large and conspicuous. The antipodal cells degenerate to fulfill a nutritional function.     

Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx,GPx4) in mammalian cells

Reactive oxygen species (ROS) are known mediators of intracellular signal cascades. Excessive production of ROS may lead to oxidative stress, loss of cell function, and cell death by apoptosis or necrosis. Lipid hydroperoxides are one type of ROS whose biological function has not yet been clarified. Phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxide in mammalian cells. This contrasts with most antioxidant enzymes, which cannot reduce intracellular phospholipid hydroperoxides directly. In this review, we focus on the structure and biological functions of PHGPx in mammalian cells. Recently, molecular techniques have allowed overexpression of PHGPx in mammalian cell lines, from which it has become clear that lipid hydroperoxides also have an important function as activators of lipoxygenase and cyclooxygenase, participate in inflammation, and act as signal molecules for apoptotic cell death and receptor-mediated signal transduction at the cellular level.     

Unravelling the Leishmania genome

The past few years have seen significant advances in our understanding of eukaryotic genomes. In the field of parasitology, this is best exemplified by the application of genome mapping techniques to the study of genome structure and function in the protozoan parasite, Leishmania. Although much is known about the organism and the diseases it causes, molecular genetics has only recently begun to play a major part in elucidating some of the unusual characteristics of this interesting parasite. Mapping of the small (35 Mb) genome and determination of the functional role of genes by the application of in vitro homologous gene targeting techniques are revealing novel avenues for the development of prophylactic measures.     

Characterization of measurement errors using structure‐from‐motion and photogrammetry to measure marine habitat structural complexity

Habitat structural complexity is one of the most important factors in determining the makeup of biological communities. Recent advances in structure‐from‐motion and photogrammetry have resulted in a proliferation of 3D digital representations of habitats from which structural complexity can be measured. Little attention has been paid to quantifying the measurement errors associated with these techniques, including the variability of results under different surveying and environmental conditions. Such errors have the potential to confound studies that compare habitat complexity over space and time. This study evaluated the accuracy, precision, and bias in measurements of marine habitat structural complexity derived from structure‐from‐motion and photogrammetric measurements using repeated surveys of artificial reefs (with known structure) as well as natural coral reefs. We quantified measurement errors as a function of survey image coverage, actual surface rugosity, and the morphological community composition of the habitat‐forming organisms (reef corals). Our results indicated that measurements could be biased by up to 7.5% of the total observed ranges of structural complexity based on the environmental conditions present during any particular survey. Positive relationships were found between measurement errors and actual complexity, and the strength of these relationships was increased when coral morphology and abundance were also used as predictors. The numerous advantages of structure‐from‐motion and photogrammetry techniques for quantifying and investigating marine habitats will mean that they are likely to replace traditional measurement techniques (e.g., chain‐and‐tape). To this end, our results have important implications for data collection and the interpretation of measurements when examining changes in habitat complexity using structure‐from‐motion and photogrammetry.     

Molecular evolution and secondary structural conservation in the B-cell lymphoma leukemia 2 (bcl-2) family of proto-oncogene products

The nature of the bcl-2 family of protooncogenes was analyzed by sequence alignment, secondary structure prediction, and phylogenetic techniques. Phylogenies were inferred from both the nucleic acid and amino acid sequences of the human, murine, rat, and chicken sequences for BCL-2 and BCL-X, human MCL1, murine A1, the nematode Caenorhabditis elegans and Caenorhabditis briggsiae ced-9 proteins, and the sequences BHRF1 from Epstein-Barr and LMW5-HL from African swine fever viruses. Both sequence alignment and secondary structure prediction techniques supported the conservation of both the overall secondary structure and the carboxy-terminal transmembrane domain in all members of the family. All the treeing methods employed (distance matrix, maximum likelihood, and parsimony) supported a tree in which the proapoptotic proteins BCL-2 and BCL-X represent the most recent additions to the group. All the trees also indicated that the viral proteins BHRF1 and LMW-HL arose from a common ancestor, an ancestor they shared in common with the pro-apoptotic control protein BAX, indicating that this function of BAX evolved only recently. The most ancient branches are represented by the nematode ced-9 protein and by the control genes MCL1 and A1, which in the treeing methods employed represent separate lineages within the most ancient grouping. These results demonstrate the evolution of a highly conserved family of developmental control genes from nematode to man—genes that encode proteins essential for normal development but which are highly conserved in terms of predicted structure and possible cellular localization. The evolutionary analysis also indicates that the family may be even larger than originally predicted and that other members are waiting to be discovered. Correspondence to: D. Lloyd Evans