The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.
- Klíčová slova
- auxin, auxin metabolism, flow cytometry, nucleus, subcellular fractionation,
- MeSH
- Arabidopsis účinky léků metabolismus ultrastruktura MeSH
- buněčné jádro účinky léků metabolismus ultrastruktura MeSH
- buněčné kultury MeSH
- centrifugace metody MeSH
- frakcionace buněk přístrojové vybavení metody MeSH
- hmotnostní spektrometrie MeSH
- homeostáza fyziologie MeSH
- indoly metabolismus farmakologie MeSH
- kyseliny indoloctové metabolismus MeSH
- protoplasty chemie MeSH
- průtoková cytometrie MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné buňky účinky léků metabolismus ultrastruktura MeSH
- tabák účinky léků metabolismus ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- indole MeSH Prohlížeč
- indoly MeSH
- kyseliny indoloctové MeSH
- regulátory růstu rostlin MeSH
Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.
- Klíčová slova
- cytocentrifugation, microscopy, sample processing, staining,
- MeSH
- barvení a značení metody MeSH
- centrifugace přístrojové vybavení metody MeSH
- cytologické techniky přístrojové vybavení metody MeSH
- lidé MeSH
- mikroskopie metody MeSH
- odběr biologického vzorku metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Physiological aggregation of a recombinant enzyme into enzymatically active inclusion bodies could be an excellent strategy to obtain immobilized enzymes for industrial biotransformation processes. However, it is not convenient to recycle "gelatinous masses" of protein inclusion bodies from one reaction cycle to another, as high centrifugation forces are needed in large volumes. The magnetization of inclusion bodies is a smart solution for large-scale applications, enabling an easier separation process using a magnetic field. RESULTS: Magnetically modified inclusion bodies of UDP-glucose pyrophosphorylase were recycled 50 times, in comparison, inclusion bodies of the same enzyme were inactivated during ten reaction cycles if they were recycled by centrifugation. Inclusion bodies of sialic acid aldolase also showed good performance and operational stability after the magnetization procedure. CONCLUSIONS: It is demonstrated here that inclusion bodies can be easily magnetically modified by magnetic iron oxide particles prepared by microwave-assisted synthesis from ferrous sulphate. The magnetic particles stabilize the repetitive use of the inclusion bodies .
To biochemically and structurally characterize viral intracytoplasmic particles (ICAPs), a sample of high purity and homogeneity is usually required. Production of ICAPs in the system closely related to their natural host cells is crucial for the analysis of host-cell binding proteins involved in ICAPs assembly, transport and budding. However, this approach is often hampered by problems with low yield of the ICAPs due to either low expression or fast release from the host cell. Another obstacle may be a low stability or fragility of the intracellular particles. The published methods for ICAPs isolation often involved several time-consuming centrifugation steps yielding damaged particles. Other papers describe the ICAPs production in non-natural host cells. Here, we optimized the method for purification of unstable Mason-Pfizer monkey virus (M-PMV) ICAPs from non-human primate derived cells, commonly used to study MPMV replication i.e. African green monkey kidney fibroblast cell line (COS-1). Our simple and rapid procedure involved separation of the intracytoplasmic particles from the cell debris and organelles by differential, low-speed centrifugation, their purification using sucrose velocity gradient and final concentrating by low-speed centrifugation. Importantly, the method was established for unstable and fragile M-PMV intracytoplasmic particles. Therefore, it may be suitable for isolation of ICAPs of other viruses.
- Klíčová slova
- Assembly, Intracytoplasmic particles (ICAPs), Mason-Pfizer monkey virus, Retrovirus,
- MeSH
- centrifugace metody MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- cytoplazma virologie MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus izolace a purifikace fyziologie MeSH
- replikace viru MeSH
- sestavení viru MeSH
- virologie metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Global healthcare systems are struggling with the enormous burden associated with infectious diseases, as well as the incessant rise of antimicrobial resistance. In order to adequately address these issues, there is an urgent need for rapid and accurate infectious disease diagnostics. The H2020 project DIAGORAS aims at diagnosing oral and respiratory tract infections using a fully integrated, automated and user-friendly platform for physicians' offices, schools, elderly care units, community settings, etc. Oral diseases (periodontitis, dental caries) will be detected via multiplexed, quantitative analysis of salivary markers (bacterial DNA and host response proteins) for early prevention and personalised monitoring. Respiratory Tract Infections will be diagnosed by means of DNA/RNA differentiation so as to identify their bacterial or viral nature. Together with antibiotic resistance screening on the same platform, a more efficient treatment management is expected at the point-of-care. At the heart of DIAGORAS lies a centrifugal microfluidic platform (LabDisk and associated processing device) integrating all components and assays for a fully automated analysis. The project involves an interface with a clinical algorithm for the comprehensive presentation of results to end-users, thereby increasing the platform's clinical utility. DIAGORAS' performance will be validated at clinical settings and compared with gold standards.
- MeSH
- bakteriální léková rezistence * MeSH
- centrifugace metody MeSH
- DNA bakterií analýza MeSH
- individualizovaná medicína metody MeSH
- infekce dýchací soustavy diagnóza mikrobiologie virologie MeSH
- laboratorní automatizace MeSH
- lidé MeSH
- mikrofluidní analytické techniky MeSH
- parodontitida diagnóza mikrobiologie MeSH
- RNA virová analýza MeSH
- sliny imunologie mikrobiologie MeSH
- zubní kaz diagnóza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA bakterií MeSH
- RNA virová MeSH
BACKGROUND: Breast-conserving treatment (BCT) leads to a progressive and deteriorating breast deformity. Fatgrafting is ideal for breast reconstruction after BCT. The most frequently utilized technique for fat processing is centrifugation. The PureGraft device (Cytori Therapeutics, San Diego, CA, USA) is a new method that involves washing and filtering the fat to prepare the graft. We compared the subjective and objective outcomes of two fat-processing methods, centrifugation and PureGraft filtration. METHODS: Thirty patients underwent breast reconstruction performed by a single surgeon (OM) after BCT in our department between April 2011 and September 2012. The patients were preoperatively divided into two groups randomly: 15 received fatgrafts processed by centrifugation, and 15 received fatgrafts processed by washing in PureGraft bags. The patients were followed up for 12 to 30 months. To measure the subjective outcome, we distributed the BREAST-Q questionnaire to all the patients both preoperatively and 1 year postoperatively. The BCCT.core software evaluated the objective outcome of breast reconstruction by fatgrafting. RESULTS: The Breast-Q results indicated a tremendous improvement in the modules "Satisfaction with Breast" and "Psychosocial Well-being". The "Sexual Well-being" scale also improved. Only the module "Satisfaction with Breasts" significantly differed between groups; patients treated with the PureGraft fat exhibited better outcomes. The BCCT.core results did not significantly differ between the groups. CONCLUSION: One year postoperatively, the outcomes of the use of PureGraft bags or centrifugation to process fat for breast reconstruction after BCT did not differ. The unpredictability of the results following fatgrafting procedures is likely due to interindividual differences with yet-undisclosed causes.
- MeSH
- centrifugace metody MeSH
- dospělí MeSH
- filtrace metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- mamoplastika * MeSH
- nádory prsu chirurgie MeSH
- následné studie MeSH
- podkožní tuk transplantace MeSH
- prognóza MeSH
- průzkumy a dotazníky MeSH
- segmentální mastektomie * MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- randomizované kontrolované studie MeSH
- srovnávací studie MeSH
Concentration of retroviruses from volumes of up to 6 liters of medium by low-speed centrifugation is described. In contrast to pelleting, no damage or aggregation of particles was observed. Surface glycoproteins were also fully preserved. This method enables simple handling of relatively large volumes of medium. Highly purified mouse mammary tumor virus (MMTV) was obtained and its transframe protein p30 in SDS-PAGE was recognized as a double band.
- MeSH
- buněčné linie MeSH
- centrifugace metody MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- molekulová hmotnost MeSH
- Retroviridae izolace a purifikace MeSH
- RNA virová biosyntéza izolace a purifikace MeSH
- virové proteiny izolace a purifikace MeSH
- virus myšího tumoru prsní žlázy izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- RNA virová MeSH
- virové proteiny MeSH
- MeSH
- centrifugace metody MeSH
- lipidy analýza MeSH
- pankreatin analýza MeSH
- teplota MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- Názvy látek
- lipidy MeSH
- pankreatin MeSH
