MS2 phage-like particles (MS2 PLP) are artificially constructed pseudo-viral particles derived from bacteriophage MS2. They are able to carry a specific single stranded RNA (ssRNA) sequence of choice inside their capsid, thus protecting it against the effects of ubiquitous nucleases. Such particles are able to mimic ssRNA viruses and, thus, may serve as the process control for molecular detection and quantification of such agents in several kinds of matrices, vaccines and vaccine candidates, drug delivery systems, and systems for the display of immunologically active peptides or nanomachines. Currently, there are several different in vivo plasmid-driven packaging systems for production of MS2 PLP. In order to combine all the advantages of the available systems and to upgrade and simplify the production and purification of MS2 PLP, a one-plasmid double-expression His-tag system was designed. The described system utilizes a unique fusion insertional mutation enabling purification of particles using His-tag affinity. Using this new production system, highly pure MS2 PLP can be quickly produced and purified by a fast performance liquid chromatography (FPLC) approach. The system can be easily adapted to produce other MS2 PLP with different properties.
- MeSH
- Levivirus * chemie genetika metabolismus MeSH
- plazmidy * genetika metabolismus MeSH
- rekombinantní fúzní proteiny * biosyntéza chemie genetika izolace a purifikace MeSH
- virion * chemie genetika izolace a purifikace metabolismus MeSH
- virové plášťové proteiny * biosyntéza chemie genetika izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- rekombinantní fúzní proteiny * MeSH
- virové plášťové proteiny * MeSH
The detection and quantification of enteric RNA viruses is based on isolation of viral RNA from the sample followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). To control the whole process of analysis and in order to guarantee the validity and reliability of results, process control viruses (PCV) are used. The present article describes the process of preparation and use of such PCV- MS2 phage-like particles (MS2 PLP) - in RT-qPCR detection and quantification of enteric RNA viruses. The MS2 PLP were derived from bacteriophage MS2 carrying a unique and specific de novo-constructed RNA target sequence originating from the DNA of two extinct species. The amount of prepared MS2 particles was quantified using four independent methods - UV spectrophotometry, fluorimetry, transmission electron microscopy and a specifically developed duplex RT-qPCR. To evaluate the usefulness of MS2 PLP in routine diagnostics different matrices known to harbor enteric RNA viruses (swab samples, liver tissue, serum, feces, and vegetables) were artificially contaminated with specific amounts of MS2 PLP. The extraction efficiencies were calculated for each individual matrix. The prepared particles fulfill all requirements for PCV - they are very stable, non-infectious, and are genetically distinct from the target RNA viruses. Due to these properties they represent a good morphological and physiochemical model. The use of MS2 PLP as a PCV in detection and quantification of enteric RNA viruses was evaluated in different types of matrices.
- Klíčová slova
- MS2 phage-like particle, RNA virus, RT-qPCR, detection, extraction efficiency calculation, isolation, process control virus, quantification,
- Publikační typ
- časopisecké články MeSH
Trapped ion mobility spectrometry (TIMS) adds an additional separation dimension to mass spectrometry (MS) imaging, however, the lack of fragmentation spectra (MS2) impedes confident compound annotation in spatial metabolomics. Here, we describe spatial ion mobility-scheduled exhaustive fragmentation (SIMSEF), a dataset-dependent acquisition strategy that augments TIMS-MS imaging datasets with MS2 spectra. The fragmentation experiments are systematically distributed across the sample and scheduled for multiple collision energies per precursor ion. Extendable data processing and evaluation workflows are implemented into the open source software MZmine. The workflow and annotation capabilities are demonstrated on rat brain tissue thin sections, measured by matrix-assisted laser desorption/ionisation (MALDI)-TIMS-MS, where SIMSEF enables on-tissue compound annotation through spectral library matching and rule-based lipid annotation within MZmine and maps the (un)known chemical space by molecular networking. The SIMSEF algorithm and data analysis pipelines are open source and modular to provide a community resource.
- MeSH
- algoritmy MeSH
- iontová mobilní spektrometrie * MeSH
- krysa rodu Rattus MeSH
- metabolomika * metody MeSH
- software MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ethylene oxide (EO), an industrial intermediate and gaseous sterilant for medical devices, is carcinogenic to humans, which warrants minimization of exposure in the workplaces. The principal analytical strategy currently used in biomonitoring of exposure to EO consists in the conversion of N-(2-hydroxyethyl) adduct at the N-terminal valine (HEV) in globin to a specific thiohydantoin derivative accessible to GC-MS analysis (modified Edman degradation, MED). Though highly sensitive, the method is laborious and, at least in our hands, not sufficiently robust. Here we developed an alternative strategy of HEV determination based on acidic hydrolysis (AH) of globin followed directly by HPLC-ESI-MS2 analysis. Limit of quantitation is ca. 25 pmol HEV/g globin. Comparative analyses of globin samples from EO-exposed workers by both the AH-based and MED-based methods provided results that correlated well with each other (R2 > 0.95) but those obtained with AH were significantly more accurate (according to external quality control programme G-EQUAS) and repeatible (5% and 6% for intra-day and between-day analyses, respectively). In conclusion, the new AH-based method surpassed MED being similarly sensitive, much less laborious and more reliable, thus applicable as an effective tool for biomonitoring of EO in exposure control and risk assessment.
- Klíčová slova
- Biological monitoring, Ethylene oxide, Globin adducts, N-(2-Hydroxyethyl)valine, Protein hydrolysis,
- MeSH
- bioindikátory MeSH
- ethylenoxid škodlivé účinky krev MeSH
- globiny analýza MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací * MeSH
- hodnocení rizik MeSH
- hydrolýza MeSH
- hygiena práce * MeSH
- inhalační expozice * škodlivé účinky MeSH
- kyseliny chemie MeSH
- lidé MeSH
- monitorování životního prostředí metody MeSH
- pracovní expozice * škodlivé účinky MeSH
- reprodukovatelnost výsledků MeSH
- valin analogy a deriváty krev MeSH
- vysokoúčinná kapalinová chromatografie * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 2-hydroxyethylvaline MeSH Prohlížeč
- bioindikátory MeSH
- ethylenoxid MeSH
- globiny MeSH
- kyseliny MeSH
- valin MeSH
RNA viruses are pathogenic agents of many serious infectious diseases affecting humans and animals. The detection of pathogenic RNA viruses is based on modern molecular methods, of which the most widely used methods are the reverse transcription polymerase chain reaction (RT-PCR) and the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). All steps of RT-PCR and qRT-PCR should be strictly controlled to ensure the validity of obtained results. False-negative results may be caused not only by inhibition of RT or/and PCR steps but also by failure of the nucleic acid extraction step, particularly in the case of viral RNA extraction. The control of nucleic acid extraction generally involves the utilization of a non-pathogenic virus (process control virus) of similar structural properties to those of the target virus. Although in clinical samples the use of such process control virus is only recommended, in other kinds of settings such as food matrices its use is necessary. Currently, several different process control viruses are used for these purposes. Process control viruses can also be constructed artificially using technology for production of MS2 phage-like particles, which have many advantages in comparison with other used controls and are especially suited for controlling the detection and quantification of certain types of RNA viruses. The technology for production of MS2 phage-like particles is theoretically well established, uses the knowledge gained from the study of the familiar bacteriophage MS2 and utilizes many different approaches for the construction of the various process control viruses. Nevertheless, the practical use of MS2 phage-like particles in routine diagnostics is relatively uncommon. The current situation with regard to the use of MS2 phage-like particles as process control viruses in detection of RNA viruses and different methods of their construction, purification and use are summarized and discussed in this review.
- Publikační typ
- časopisecké články MeSH
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
- Publikační typ
- tisková chyba MeSH
Double and triple bonds have significant effects on the biological activities of lipids. Determining multiple bond positions in their molecules by mass spectrometry usually requires chemical derivatization. This work presents an HPLC/MS method for pinpointing the double and triple bonds in fatty acids. Fatty acid methyl esters were separated by reversed-phase HPLC with an acetonitrile mobile phase. In the APCI source, acetonitrile formed reactive species, which added to double and triple bonds to form [M + C3H5N]+• ions. Their collisional activation in an ion trap provided fragments helpful in localizing the multiple bond positions. This approach was applied to fatty acids with isolated, cumulated, and conjugated double bonds and triple bonds. The fatty acids were isolated from the fat body of early-nesting bumblebee Bombus pratorum and seeds or seed oils of Punicum granatum, Marrubium vulgare, and Santalum album. Using the method, the presence of the known fatty acids was confirmed, and new ones were discovered.
- Klíčová slova
- acetonitrile-related adducts, acetylenic lipids, double and triple bond localization, in-source derivatization, mass spectrometry,
- MeSH
- acetonitrily chemie MeSH
- estery chemie izolace a purifikace MeSH
- hmotnostní spektrometrie MeSH
- mastné kyseliny chemie izolace a purifikace MeSH
- molekulární struktura MeSH
- včely chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetonitrile MeSH Prohlížeč
- acetonitrily MeSH
- estery MeSH
- mastné kyseliny MeSH
RNA interference (RNAi) designates sequence-specific mRNA degradation mediated by small RNAs generated from long double-stranded RNA (dsRNA) by RNase III Dicer. RNAi appears inactive in mammalian cells except for mouse oocytes, where high RNAi activity exists because of an N-terminally truncated Dicer isoform, denoted DicerO. DicerO processes dsRNA into small RNAs more efficiently than the full-length Dicer expressed in somatic cells. DicerO is expressed from an oocyte-specific promoter of retrotransposon origin, which is silenced in other cell types. In this work, we evaluated CRISPR-based strategies for epigenetic targeting of the endogenous Dicer gene to restore DicerO expression and, consequently, RNAi. We show that reactivation of DicerO expression can be achieved in mouse embryonic stem cells, but it is not sufficient to establish a robust canonical RNAi response.
- Klíčová slova
- CRISPR, Dicer, MS2, RNAi, VP64, dCas9, sgRNA,
- MeSH
- buňky 3T3 MeSH
- DEAD-box RNA-helikasy antagonisté a inhibitory genetika MeSH
- embryonální kmenové buňky cytologie metabolismus MeSH
- malá interferující RNA genetika MeSH
- myši MeSH
- promotorové oblasti (genetika) * MeSH
- ribonukleasa III antagonisté a inhibitory genetika MeSH
- RNA interference MeSH
- sekvence CRISPR * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DEAD-box RNA-helikasy MeSH
- Dicer1 protein, mouse MeSH Prohlížeč
- malá interferující RNA MeSH
- ribonukleasa III MeSH
Faecal samples were collected from 352 horses on 23 farms operating under six different management systems in the Czech Republic and Poland during 2011 and 2012. Farms were selected without previous knowledge of parasitological status. All faecal samples were screened for Cryptosporidium spp. presence using microscopy, following aniline-carbol-methyl violet staining and PCR analysis of the small-subunit (SSU) rRNA and the 60-kDa glycoprotein (gp60) genes. Cryptosporidium muris-positive samples were additionally genotyped at four minisatellite markers: MS1 (encoding a hypothetical protein), MS2 (encoding a 90-kDa heat shock protein), MS3 (encoding a hypothetical protein) and MS16 (encoding a leucine-rich repeat family protein). Cryptosporidium spp. was detected by PCR in 12/352 (3.4%) samples from 4 out of 13 farms. None of the samples tested by microscopy was positive. There was no relationship between Cryptosporidium prevalence and age, sex, diarrhoea or management system; however, Cryptosporidium was found only on farms where horses were kept on pasture during the day and in a stable overnight. Sequence analyses of SSU and gp60 genes revealed the presence of C. muris RN66 (n = 9), Cryptosporidium parvum IIaA15G2R1 (n = 1), Cryptosporidium tyzzeri IXbA22R9 (n = 1), and Cryptosporidium horse genotype VIaA15G4 (n = 1). The C. muris subtypes were identified as MS1-M1, MS2-M4, novel MS2-M7 and MS16-M1 by multilocus sequence of three minisatellite loci. The MS3 locus was not amplified from any isolate. This is the first report of C. tyzzeri and C. muris subtypes from horses.
- MeSH
- Cryptosporidium klasifikace genetika izolace a purifikace MeSH
- feces parazitologie MeSH
- genetická variace * MeSH
- genotyp MeSH
- koně MeSH
- kryptosporidióza epidemiologie MeSH
- nemoci koní parazitologie MeSH
- prevalence MeSH
- průjem parazitologie veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
- Polsko epidemiologie MeSH
Dietary composition and metabolism of fatty acids (FA) influence insulin resistance, atherogenic dyslipidemia and other components of the metabolic syndrome (MS). It is known that patients with MS exhibit a heterogeneous phenotype; however, the relationships of individual FA to MS components have not yet been consistently studied. We examined the plasma phosphatidylcholine FA composition of 166 individuals (68F/98M) with MS and of 188 (87F/101M) controls. Cluster analysis of FA divided the groups into two clusters. In cluster 1, there were 65.7 % of MS patients and 37.8 % of controls, cluster 2 contained 34.3 % of patients and 62.2 % of controls (P<0.001). Those with MS within cluster 1 (MS1) differed from individuals with MS in cluster 2 (MS2) by concentrations of glucose (P<0.05), NEFA (P<0.001), HOMA-IR (P<0.05), and levels of conjugated dienes in LDL (P<0.05). The FA composition in MS1 group differed from MS2 by higher contents of palmitoleic (+30 %), gamma-linolenic (+22 %), dihomo-gamma-linolenic (+9 %) acids and by a lower content of linoleic acid (-25 %) (all P<0.01). These FA patterns are supposed to be connected with the progression and/or impaired biochemical measures of MS (lipolysis, oxidative stress, dysglycidemia, and insulin resistance).
- MeSH
- fosfatidylcholiny krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- mastné kyseliny krev MeSH
- metabolický syndrom krev klasifikace diagnóza MeSH
- senioři MeSH
- shluková analýza MeSH
- stravovací zvyklosti fyziologie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fosfatidylcholiny MeSH
- mastné kyseliny MeSH
