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排序方式: 共有1324条查询结果,搜索用时 15 毫秒
51.
Monika Radvánszka Evan D. Paul Roman Hajdu Kristína Boršová Viera Kováčová Piotr Putaj Stanislava Bírová Ivana Čirková Martin Čarnecký Katarína Buranovská Adrián Szobi Nina Vojtaššáková Diana Drobná Viktória Čabanová Monika Sláviková Martina Ličková Veronika Vaňová Sabína Fumačová Havlíková Ľubomíra Lukáčiková Ivana Kajanová Juraj Koči Diana Rusňáková Tatiana Sedláčková Klaas E. A. Max Thomas Tuschl Tomáš Szemes Boris Klempa Pavol Čekan 《Microbial biotechnology》2022,15(7):1995-2021
Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic. 相似文献
52.
Proteases were, for a long time, mainly considered as protein degrading enzymes. However, in the last decade this view has changed dramatically, and the focus is now on proteases as signalling molecules. One of the best examples is apoptosis, the major mechanism used by eukaryotes to remove superfluous, damaged and potentially dangerous cells, in which a number of proteases have been found to play a central role. Of these the caspases have been considered to be the major players. However, more recently, other proteases have been increasingly suggested as being important in apoptosis, in particular the cysteine cathepsins. In this review the roles of caspases and cysteine cathepsins in apoptosis signalling are compared and discussed. 相似文献
53.
Fiser R Masín J Basler M Krusek J Spuláková V Konopásek I Sebo P 《The Journal of biological chemistry》2007,282(5):2808-2820
The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner. 相似文献
54.
Beinrohr L Harmat V Dobó J Lörincz Z Gál P Závodszky P 《The Journal of biological chemistry》2007,282(29):21100-21109
C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded beta-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack. 相似文献
55.
Sorg UR Kleff V Fanaei S Schumann A Moellmann M Opalka B Thomale J Moritz T 《DNA Repair》2007,6(8):1197-1209
As haematopoietic stem cell gene therapy utilizing O(6)-methylguanine-DNA-methyltransferase has reached the clinical stage, safety-related questions become increasingly important. These issues concern insertional mutagenesis of viral vectors, the acute toxicity of pre-transplant conditioning protocols and in vivo selection regimens as well as potential genotoxic side effects of the alkylating drugs administered in this context. To address these questions, we have investigated toxicity-reduced conditioning regimens combining low-dose alkylator application with sublethal irradiation and have analysed their influence on engraftment and subsequent selectability of transduced haematopoietic stem cells. In addition, a strategy to monitor the acute and long-term genotoxic effects of drugs with high guanine-O(6) alkylating potential, such as chloroethylnitrosoureas or temozolomide is introduced. For this purpose, assays were implemented which allow an assessment of the generation and fate of primary drug-induced adducts as well as their long-term effect on chromosomal integrity at the single cell level. 相似文献
56.
This study tested whether potash tailing piles can be restored with biological soil crusts using an additive of the company upi. A biocrust community consists of different organisms, such as microalgae, lichens, and mosses. An established biocrust stabilizes the ground and traps rain water, which could in return reduce salt leachate into the environment. This pioneer community promotes formation of a new habitat that can be recolonized by higher plants. For this study microalgae were isolated from biological soil crusts collected at potash tailing pile sites. We characterized their salt tolerance and established first artificial biocrusts on the heap material. Upcoming experiments will focus on the establishment of artificial biocrusts in selected heap areas. 相似文献
57.
58.
Christelle Monteil Caroline Leclere Franoise Dantzer Veronika Elkaz Jean-Paul Fillastre Jean-Paul Morin 《Cell biology international》1993,17(10):953-960
We have assessed the impact of increasing oxygen availability on cellular phenotype expression of rabbit proximal tubule cells in primary culture developed with variable glucose and/or insulin contents. To mitigate hypoxia at the cell/medium interface, cells were shaken for the whole culture duration and their expressed phenotype was compared with those expressed by static cultures. O2 and CO2 tensions were kept constant in the incubator atmosphere. Glycolysis and gluconeogenesis pathways, detoxication system, and mitochondrial, apical and basolateral membrane marker enzyme activities were assessed. This study showed that the induction of glycolysis which appear in primary cultures of proximal tubule cells may be partially prevented by continuously shaking the cultures. This effect was more marked in the presence of glucose, suggesting better substrate oxidation in shaken cultures. 相似文献
59.
Hägerstrand H Kralj-Iglic V Bobrowska-Hägerstrand M Iglic A 《Bulletin of mathematical biology》1999,61(6):1019-1030
We observed that amphiphile-induced microexovesicles may be spherical or cylindrical, depending on the species of the added
amphiphile. The spherical microexovesicle corresponds to an extreme local difference between the two monolayer areas of the
membrane segment with a fixed area, while the cylindrical microexovesicle corresponds to an extreme local area difference
if the area of the budding segment is increased due to lateral influx of anisotropic membrane constituents. Protein analysis
showed that both types of vesicles are highly depleted in the membrane skeleton. It is suggested that a partial detachment
of the skeleton in the budding region is favoured due to accumulated skeleton shear deformations in this region. 相似文献
60.
Purine nucleosides support the neurite outgrowth of primary rat cerebellar granule cells after hypoxia 总被引:2,自引:0,他引:2
Böcklinger K Tomaselli B Heftberger V Podhraski V Bandtlow C Baier-Bitterlich G 《European journal of cell biology》2004,83(2):51-54
Mammalian neurons require a constant supply of oxygen to maintain adequate cellular functions and survival. Following sustained hypoxia during ischemic events in brain, the energy status of neurons and glia is compromised, which may subsequently lead to cell death by apoptosis and necrosis. Concomitant with energy depletion is the formation of the purine nucleoside adenosine, a powerful endogenous neuroprotectant. In this paper the effect of chemical hypoxia on cell survival and neurite outgrowth of primary cerebellar granule cells was investigated. Rotenone, a mitochondrial complex I inhibitor, induced a 30.4 +/- 3.6% loss of viable cells and a 35.0 +/- 4.4% loss of neurite formation of cerebellar granule cells, which was partially restored by the addition of purine nucleosides adenosine, inosine and guanosine. Inosine had the most striking effect of 37.7 +/- 2.9% rescue of viability and 71.2 +/- 18.4% rescue of neurite outgrowth. Data confirm the suggested role of purine nucleosides for the neuronal regeneration of primary brain cells following hypoxic insult. 相似文献