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
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.
- Klíčová slova
- process control virus,
- MeSH
- infekce RNA viry * diagnóza MeSH
- Levivirus * izolace a purifikace MeSH
- lidé MeSH
- polymerázová řetězová reakce s reverzní transkripcí * metody MeSH
- RNA virová genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
In this study we reviewed the use of viral vectors for delivery of drugs and genes. Viruses are monouniformic and they can be easily produced in high yield. The drug encapsulation into viral capsid differs based on the viral species used. Some viruses undergo pH dependent swelling due to the change in pH or ions concentration of surrounding solution. In others, drug can intercalate into viral nucleic acid. This work was focused on often studied viruses, adenoviruses, plant viruses (CPMV, CCMV and RCNMV) or bacteriophages (MS2 or M13).
- Klíčová slova
- enkapsulace léčiv,
- MeSH
- Adenoviridae MeSH
- bakteriofág M13 MeSH
- Bromovirus MeSH
- Comovirus MeSH
- lékové transportní systémy * MeSH
- Levivirus MeSH
- lidé MeSH
- nanočástice MeSH
- Tombusviridae MeSH
- virům podobné částice * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy 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.
- 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
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.
- 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
