One-pot detection
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BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).
- MeSH
- Brevibacillus * genetika MeSH
- COVID-19 * diagnóza MeSH
- guide RNA, Kinetoplastida MeSH
- lidé MeSH
- SARS-CoV-2 genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Metal-organic frameworks (MOFs) have been widely used as porous nanomaterials for different applications ranging from industrial to biomedicals. An unpredictable one-pot method is introduced to synthesize NH2-MIL-53 assisted by high-gravity in a greener media for the first time. Then, porphyrins were deployed to adorn the surface of MOF to increase the sensitivity of the prepared nanocomposite to the genetic materials and in-situ cellular protein structures. The hydrogen bond formation between genetic domains and the porphyrin' nitrogen as well as the surface hydroxyl groups is equally probable and could be considered a milestone in chemical physics and physical chemistry for biomedical applications. In this context, the role of incorporating different forms of porphyrins, their relationship with the final surface morphology, and their drug/gene loading efficiency were investigated to provide a predictable pattern in regard to the previous works. The conceptual phenomenon was optimized to increase the interactions between the biomolecules and the substrate by reaching the limit of detection to 10 pM for the Anti-cas9 protein, 20 pM for the single-stranded DNA (ssDNA), below 10 pM for the single guide RNA (sgRNA) and also around 10 nM for recombinant SARS-CoV-2 spike antigen. Also, the MTT assay showed acceptable relative cell viability of more than 85% in most cases, even by increasing the dose of the prepared nanostructures.
- MeSH
- buňky Hep G2 MeSH
- buňky PC12 MeSH
- COVID-19 diagnóza MeSH
- CRISPR-Cas systémy MeSH
- dusík chemie MeSH
- guide RNA, Kinetoplastida MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- jednovláknová DNA MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- limita detekce MeSH
- nanokompozity MeSH
- nanostruktury MeSH
- porézní koordinační polymery chemie MeSH
- poréznost MeSH
- porfyriny chemie MeSH
- povrchové vlastnosti MeSH
- RNA virová metabolismus MeSH
- SARS-CoV-2 MeSH
- senzitivita a specificita MeSH
- testování na COVID-19 MeSH
- vodíková vazba MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Je známo, že tromboembolická nemoc se může vyskytovat současně s nádorovým one-mocněním, často ho také může předcházet, a to i o několik měsíců. Z tohoto důvodujsou při zjištění žilní trombózy nebo plicní embolie často prováděna vyšetření k vylou-čení konkomitantní neoplazie. Na otázku, kdy a v jakém rozsahu onkoscreening pro-vádět, stále neexistuje jednoznačná odpověď. Jsou však situace, kdy by podrobnější vy-šetření mělo být provedeno. Jednou z nich je případ recidivujících tromboembolickýchpříhod v krátkém časovém odstupu a při účinné antikoagulační léčbě. Autoři prezentují dvě kazuistiky. První je případ 59letého muže, který byl vyšetřovánpro recidivující povrchní a hluboké žilní trombózy a plicní embolii při účinné antikoa -gulační léčbě. Stav byl poté komplikován opakovanou myokardiální ischemií (STEMIspodní stěny) na podkladě trombotického uzávěru koronárních tepen. V rámci dalšídiagnostiky byl prokázán uzlinový syndrom. Bylo uvažováno o hematoonkologickémstonání, nakonec se potvrdil plicní tumor s metastázami do uzlin, mozku, mozečkua skeletu. Stav rychle progredoval a nemocný zemřel. Druhou nemocnou byla 46letážena s opakovanými žilními trombózami i přes účinnou léčbu. Dodatečně byl prokázánplicní nádor s metastázami do uzlin a skeletu. Krátce po stanovení diagnózy pacientkazemřela na masivní plicní embolii při účinné antikoagulační léčbě.
It is well known that thromboembolic disease can occur concurrently with a malignancy,often it can also precede tumour even of a number of months. For this reason, when ve-nous thrombosis or pulmonary embolism is detected, examinations to exclude conco-mitant neoplasia are often indicated. There are different opinions on the benefits of a rou-tine cancer screening. However, there are situations where a more detailed examinationshould be done. One of these is the case of recurrent thromboembolic events withina short period of time, despite an effective anticoagulant therapy. The authors present two case reports. The first case is a 59-year-old man who had beenexamined for recurrent superficial and deep vein thrombosis and pulmonary embolismdespite an effective treatment with warfarin (coumarin anticoagulant). The health con-dition was then complicated by recurrent myocardial ischemia (STEMI of the dia -phragmatic wall) on the basis of thrombotic occlusion of the coronary artery. In pursuanceof the next diagnostics the nodal syndrome was detected. A haematological malignancywas considered at first and finally the lung tumour with metastases to the nodes, brain,cerebellum and skeleton was confirmed. The patient ́s condition quickly progressed, andhe died. The second patient was a 46-year-old woman with recurrent venous thrombosisdespite an effective anticoagulant therapy. Subsequently a lung tumour with metastasesto the nodes and skeleton was proven. Shortly after the diagnosis, the woman died ofa massive pulmonary embolism, although according to laboratory tests she had beenadequately treated.
- MeSH
- antikoagulancia terapeutické užití MeSH
- dabigatran terapeutické užití MeSH
- fatální výsledek MeSH
- ischemická choroba srdeční etiologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfatické uzliny patologie MeSH
- metastázy nádorů MeSH
- nádory plic * diagnostické zobrazování MeSH
- plicní embolie diagnostické zobrazování etiologie MeSH
- recidiva MeSH
- tromboembolie * diagnostické zobrazování farmakoterapie komplikace MeSH
- žilní trombóza diagnostické zobrazování etiologie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
RATIONALE: Silver tellurides find applications in the development of infrared detection, imaging, magnetics, sensors, memory devices, and optic materials. However, only a limited number of silver tellurides have been described to date. Laser ablation synthesis (LAS) was selected to generate new Ag-Te clusters. METHODS: Isothermal adsorption was used to study the formation of silver nano-particles-tellurium aggregates. Laser desorption ionization quadrupole ion trap time-of-flight mass spectrometry (LDI-QIT-TOFMS) was used for the generation and analysis of Agm Ten clusters. Scanning electron microscopy and energy-dispersive X-ray spectroscopy were used to visualize the structure of materials. The stoichiometry of the generated clusters was determined by computer modeling of isotopic patterns. RESULTS: A simple, one-pot method for the preparation of Ag-Te nano-composite was developed and found suitable for LAS of silver tellurides. The LDI of Ag-Te nano-composite leads to the formation of 11 unary and 52 binary clusters. The stoichiometry of the 34 novel Agm Ten clusters is reported here for the first time. CONCLUSIONS: LAS with TOFMS detection was proven to be a powerful technique for the generation of silver telluride clusters. Knowledge of the stoichiometry of the generated clusters might facilitate the further development of novel high-tech silver tellurium nano-materials.
Polybrominated diphenyl ethers (PBDEs) are used as additive flame retardants. PBDEs are persistent, bioaccumulative and toxic compounds. They are often detected in sewage sludge which is applied on agricultural soils as fertilizer. The objective of this study was to find out whether plants are able to accumulate and translocate PBDEs. Tobacco (Nicotiana tabacum) and nightshade (Solanum nigrum) were planted in pots containing contaminated sewage sludge and uncontaminated substrate. After 6 months of plant cultivation in sewage sludge up to 15.4 ng g(-1) dw and 76.6 ng g(-1) dw of PBDE congeners--BDE 47, BDE 99 and BDE 100---were accumulated in the nightshade and tobacco tissue, respectively. Corresponding values in plants vegetated in the control garden substrate were 10 times lower. The bioconcentration factors (BCFs) of accumulated congeners were calculated. Tobacco exhibited higher BCFs values and for both plants BCFs values of BDE 47, BDE 99, BDE 100 and BDE 209 negatively correlated with their octanol-water partition coefficients (logK(ow)). The exception was decaBDE (BDE 209) which was accumulated only in tobacco tissue in the concentration of 116.8 ng g(-1) dw. The majority of PBDEs was detected in above-ground plant biomass indicating that both plants have the ability to translocate PBDEs. To our knowledge this is one of the first studies reporting the accumulation of both lower PBDEs and BDE 209 in plants. Our results suggest that absorption, accumulation and translocation of PBDEs by plants and their transfer to the food chain could represent another possible risk for human exposure.
- MeSH
- biodegradace MeSH
- halogenované difenylethery analýza metabolismus MeSH
- látky znečišťující půdu analýza metabolismus MeSH
- odpadní vody chemie MeSH
- retardanty hoření analýza metabolismus MeSH
- rostliny metabolismus MeSH
- Solanum růst a vývoj metabolismus MeSH
- tabák růst a vývoj metabolismus MeSH
- vývoj rostlin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
