This article describes a novel method to identify as yet undiscovered telomere sequences, which combines next generation sequencing (NGS) with BAL31 digestion of high molecular weight DNA. The method was applied to two groups of plants: i) dicots, genus Cestrum, and ii) monocots, Allium species (e.g. A. ursinum and A. cepa). Both groups consist of species with large genomes (tens of Gb) and a low number of chromosomes (2n=14-16), full of repeat elements. Both genera lack typical telomeric repeats and multiple studies have attempted to characterize alternative telomeric sequences. However, despite interesting hypotheses and suggestions of alternative candidate telomeres (retrotransposons, rDNA, satellite repeats) these studies have not resolved the question. In a novel approach based on the two most general features of eukaryotic telomeres, their repetitive character and sensitivity to BAL31 nuclease digestion, we have taken advantage of the capacity and current affordability of NGS in combination with the robustness of classical BAL31 nuclease digestion of chromosomal termini. While representative samples of most repeat elements were ensured by low-coverage (less than 5%) genomic shot-gun NGS, candidate telomeres were identified as under-represented sequences in BAL31-treated samples.
Cell culture process monitoring in monoclonal antibody (mAb) production is essential for efficient process development and process optimization. Currently employed online, at line and offline methods for monitoring productivity as well as process reproducibility have their individual strengths and limitations. Here, we describe a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based on a microarray for mass spectrometry (MAMS) technology to rapidly monitor a broad panel of analytes, including metabolites and proteins directly from the unpurified cell supernatant or from host cell culture lysates. The antibody titer is determined from the intact antibody mass spectra signal intensity relative to an internal protein standard spiked into the supernatant. The method allows a semi-quantitative determination of light and heavy chains. Intracellular mass profiles for metabolites and proteins can be used to track cellular growth and cell productivity.
Advances in fluorescent probe design and synthesis have allowed the uniform in situ labeling of individual RNA molecules. In a technique referred to as single molecule RNA FISH (smRNA FISH), the labeled RNA molecules can be imaged as diffraction-limited spots and counted using image analysis algorithms. Single RNA counting has provided valuable insights into the process of gene regulation. This microscopy-based method has often revealed a high cell-to-cell variability in expression levels, which has in turn led to a growing interest in investigating the biological significance of gene expression noise. Here we describe the application of the smRNA FISH technique to samples of Caenorhabditis elegans, a well-characterized model organism.
- MeSH
- algoritmy MeSH
- Caenorhabditis elegans genetika růst a vývoj metabolismus MeSH
- embryo nesavčí metabolismus ultrastruktura MeSH
- fixace tkání metody MeSH
- fluorescenční barviva chemie MeSH
- genetická transkripce MeSH
- hybridizace in situ fluorescenční metody MeSH
- messenger RNA chemie genetika metabolismus MeSH
- poměr signál - šum MeSH
- vývojová regulace genové exprese MeSH
- zobrazení jednotlivé molekuly metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
As the amount of genome information increases rapidly, there is a correspondingly greater need for methods that provide accurate and automated annotation of gene function. For example, many high-throughput technologies--e.g., next-generation sequencing--are being used today to generate lists of genes associated with specific conditions. However, their functional interpretation remains a challenge and many tools exist trying to characterize the function of gene-lists. Such systems rely typically in enrichment analysis and aim to give a quick insight into the underlying biology by presenting it in a form of a summary-report. While the load of annotation may be alleviated by such computational approaches, the main challenge in modern annotation remains to develop a systems form of analysis in which a pipeline can effectively analyze gene-lists quickly and identify aggregated annotations through computerized resources. In this article we survey some of the many such tools and methods that have been developed to automatically interpret the biological functions underlying gene-lists. We overview current functional annotation aspects from the perspective of their epistemology (i.e., the underlying theories used to organize information about gene function into a body of verified and documented knowledge) and find that most of the currently used functional annotation methods fall broadly into one of two categories: they are based either on 'known' formally-structured ontology annotations created by 'experts' (e.g., the GO terms used to describe the function of Entrez Gene entries), or--perhaps more adventurously--on annotations inferred from literature (e.g., many text-mining methods use computer-aided reasoning to acquire knowledge represented in natural languages). Overall however, deriving detailed and accurate insight from such gene lists remains a challenging task, and improved methods are called for. In particular, future methods need to (1) provide more holistic insight into the underlying molecular systems; (2) provide better follow-up experimental testing and treatment options, and (3) better manage gene lists derived from organisms that are not well-studied. We discuss some promising approaches that may help achieve these advances, especially the use of extended dictionaries of biomedical concepts and molecular mechanisms, as well as greater use of annotation benchmarks.
Protein-protein interactions play a central role in the regulation of many biochemical processes (e.g. the system participating in enzyme catalysis). Therefore, a deeper understanding of protein-protein interactions may contribute to the elucidation of many biologically important mechanisms. For this purpose, it is necessary to establish the composition and stoichiometry of supramolecular complexes and to identify the crucial portions of the interacting molecules. This study is devoted to structure-functional relationships in the microsomal Mixed Function Oxidase (MFO) complex, which is responsible for biotransformation of many hydrophobic endogenous compounds and xenobiotics. In particular, the cytochrome b5 interaction with MFO terminal oxygenase cytochrome P-450 (P450) was studied. To create photolabile probes suitable for this purpose, we prepared cytochrome b5 which had a photolabile diazirine analog of methionine (pMet) incorporated into the protein sequence, employing recombinant expression in Escherichia coli. In addition to wild-type cytochrome b5, where three methionines (Met) are located at positions 96, 126, and 131, six mutants containing only one Met in the sequence were designed and expressed (see Table 1). In these mutants, a single Met was engineered into the catalytic domain (at positions 23, 41, or 46), into the linker between the protein domains (at position 96), or into the membrane region (at positions 126 or 131). These mutants should confirm or exclude these portions of cytochrome b5 which are involved in the interaction with P450. After UV irradiation, the pMet group(s) in the photolabile cytochrome b5 probe was(were) activated, producing covalent crosslinks with the interacting parts of P450 2B4 in the close vicinity. The covalent complexes were analyzed by the "bottom up" approach with high-accuracy mass spectrometry. The analysis provided an identification of the contacts in the supramolecular complex with low structural resolution. We found that all the above-mentioned cytochrome b5 Met residues can form intermolecular crosslinks and thus participate in the interaction. In addition, our results indicate the existence of at least two P450:cytochrome b5 complexes which differ in the orientation of individual proteins. The results demonstrate the advantages of the photo-initiated crosslinking technique which is able to map the protein-protein interfaces not only in the solvent exposed regions, but also in the membrane-embedded segments (compared to a typical crosslinking approach which generally only identifies crosslinks in solvent exposed regions).
- MeSH
- aromatické hydroxylasy analýza chemie metabolismus MeSH
- cytochromy b5 analýza chemie metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- králíci MeSH
- mapy interakcí proteinů fyziologie MeSH
- reagencia zkříženě vázaná chemie metabolismus MeSH
- světelná stimulace metody MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chemical cross-linking is a promising technology for protein tertiary structure determination. Though the data has low spatial resolution, it is possible to obtain it at physiological conditions on proteins that are not amenable to standard high resolution techniques such as X-ray, NMR analysis and cryo-EM. Here we demonstrate the utilization of isotopically labeled chemical cross-linking to visualize protein conformation rearrangements. Since calmodulin exists in two distinct conformations (calcium-free and calcium-containing forms), we selected this protein for testing the potential and the limits of a new technique. After cross-linking of both calmodulin forms, the calcium-free and calcium-containing forms were mixed together and digested under different conditions and the products of proteolysis were monitored using high resolution mass spectrometry. Finally, the ratios of heavy/light cross-links were calculated by mMass open source platform.
- MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- kalmodulin analýza chemie MeSH
- konformace proteinů MeSH
- mapování interakce mezi proteiny metody MeSH
- reagencia zkříženě vázaná chemie MeSH
- sekundární struktura proteinů MeSH
- skot MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MOTIVATION: Metallothionein-III (MT-III) displays neuro-inhibitory activity and is involved in the repair of neuronal damage. An altered expression level of MT-III suggests that it could be a mitigating factor in Alzheimer's disease (AD) neuronal dysfunction. Currently there are limited marketed drugs available against MT-III. The inhibitors are mostly pseudo-peptide based with limited ADMET. In our present study, available database InterBioScreen (natural compounds) was screened out for MT-III. Pharmacodynamics and pharmacokinetic studies were performed. Molecular docking and simulations of top hit molecules were performed to study complex stability. RESULTS: Study reveals potent selective molecules that interact and form hydrogen bonds with amino acids Ser-6 and Lys-22 are common to established melatonin inhibitors for MT-III. These include DMHMIO, MCA B and s27533 derivatives. The ADMET profiling was better with comparable interaction energy values. It includes properties like blood brain barrier, hepatotoxicity, druggability, mutagenicity and carcinogenicity. Molecular dynamics studies were performed to validate our findings.
- MeSH
- Alzheimerova nemoc metabolismus patologie MeSH
- biofyzikální jevy MeSH
- lidé MeSH
- proteiny nervové tkáně antagonisté a inhibitory chemie MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Contemporary molecular biology deals with wide and heterogeneous sets of measurements to model and understand underlying biological processes including complex diseases. Machine learning provides a frequent approach to build such models. However, the models built solely from measured data often suffer from overfitting, as the sample size is typically much smaller than the number of measured features. In this paper, we propose a random forest-based classifier that reduces this overfitting with the aid of prior knowledge in the form of a feature interaction network. We illustrate the proposed method in the task of disease classification based on measured mRNA and miRNA profiles complemented by the interaction network composed of the miRNA-mRNA target relations and mRNA-mRNA interactions corresponding to the interactions between their encoded proteins. We demonstrate that the proposed network-constrained forest employs prior knowledge to increase learning bias and consequently to improve classification accuracy, stability and comprehensibility of the resulting model. The experiments are carried out in the domain of myelodysplastic syndrome that we are concerned about in the long term. We validate our approach in the public domain of ovarian carcinoma, with the same data form. We believe that the idea of a network-constrained forest can straightforwardly be generalized towards arbitrary omics data with an available and non-trivial feature interaction network. The proposed method is publicly available in terms of miXGENE system (http://mixgene.felk.cvut.cz), the workflow that implements the myelodysplastic syndrome experiments is presented as a dedicated case study.
- MeSH
- genové regulační sítě MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- mikro RNA genetika MeSH
- umělá inteligence MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects. We will describe here a simple TALEN-based mutagenesis protocol suitable for the generation of germline mutations in Bombyx mori and Drosophila melanogaster. The protocol includes assembly of specific TAL modules, in vitro synthesis of TALEN RNAs, egg microinjection and mutation detection using PCR analysis. Our procedure allows a high frequency induction of NHEJ mutations, which often allows the reception of homozygous mutants already in the G1.
- MeSH
- bourec genetika MeSH
- deoxyribonukleasy genetika MeSH
- Drosophila melanogaster genetika MeSH
- dvouřetězcové zlomy DNA MeSH
- mikroinjekce přístrojové vybavení MeSH
- molekulární sekvence - údaje MeSH
- mutageneze cílená metody MeSH
- oprava DNA spojením konců * MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Raman spectroscopy is a powerful tool for the elucidation of qualitative and quantitative information from biological systems and has huge potential in areas such as biotechnologies, drug discovery, agro-chemical research and clinical diagnostics. This report summarises the principal Raman techniques applied to biomedical systems and discusses the challenges that exist to the wide spread adoption of Raman spectroscopy.
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- lidé MeSH
- nelineární dynamika MeSH
- optika a fotonika * MeSH
- Ramanova spektroskopie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
