AF microscopy
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In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.
The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell-free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell-free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (next-generation sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high-quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained. However, the choice of blood collection tubes is a critical pre-analytical variable.
- MeSH
- cirkulující nádorová DNA krev MeSH
- extracelulární vezikuly genetika metabolismus ultrastruktura MeSH
- lidé MeSH
- melanom krev patologie MeSH
- mikro RNA krev genetika MeSH
- mutace MeSH
- nádorové biomarkery krev genetika MeSH
- nádorové buněčné linie MeSH
- nádorové cirkulující buňky metabolismus MeSH
- senioři MeSH
- staging nádorů MeSH
- tekutá biopsie přístrojové vybavení metody MeSH
- transmisní elektronová mikroskopie MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mechanical interaction of cell with extracellular environment affects its function. The mechanisms by which mechanical stimuli are sensed and transduced into biochemical responses are still not well understood. Considering this, two finite element (FE) bendo-tensegrity models of a cell in different states are proposed with the aim to characterize cell deformation under different mechanical loading conditions: a suspended cell model elucidating the global response of cell in tensile test simulation and an adherent cell model explicating its local response in atomic force microscopy (AFM) indentation simulation. The force-elongation curve obtained from tensile test simulation lies within the range of experimentally obtained characteristics of smooth muscle cells (SMCs) and illustrates a nonlinear increase in reaction force with cell stretching. The force-indentation curves obtained from indentation simulations lie within the range of experimentally obtained curves of embryonic stem cells (ESCs) and exhibit the influence of indentation site on the overall reaction force of cell. Simulation results have demonstrated that actin filaments (AFs) and microtubules (MTs) play a crucial role in the cell stiffness during stretching, whereas actin cortex (AC) along with actin bundles (ABs) and MTs are essential for the cell rigidity during indentation. The proposed models quantify the mechanical contribution of individual cytoskeletal components to cell mechanics and the deformation of nucleus under different mechanical loading conditions. These results can aid in better understanding of structure-function relationships in living cells.
- MeSH
- analýza metodou konečných prvků * MeSH
- biologické modely * MeSH
- biomechanika MeSH
- cytoskelet metabolismus MeSH
- eukaryotické buňky cytologie metabolismus MeSH
- mechanické jevy * MeSH
- mikrofilamenta metabolismus MeSH
- mikrotubuly metabolismus MeSH
- pevnost v tahu MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: The aim of this work was to demonstrate an immunostimulatory and adjuvant effect of new apyrogenic lipophilic derivatives of norAbuMDP and norAbuGMDP formulated in nanoliposomes. METHODS: Nanoliposomes and metallochelating nanoliposomes were prepared by lipid film hydration and extrusion methods. The structure of the liposomal formulation was studied by electron microscopy, AF microscopy, and dynamic light scattering. Sublethal and lethal γ-irradiation mice models were used to demonstrate stimulation of innate immune system. Recombinant Hsp90 antigen (Candida albicans) bound onto metallochelating nanoliposomes was used for immunisation of mice to demonstrate adjuvant activities of tested compounds. RESULTS: Safety and stimulation of innate and adaptive immunity were demonstrated on rabbits and mice. The liposomal formulation of norAbuMDP/GMDP was apyrogenic in rabbit test and lacking any side effect in vivo. Recovery of bone marrow after sublethal γ-irradiation as well as increased survival of mice after lethal irradiation was demonstrated. Enhancement of specific immune response was demonstrated for some derivatives incorporated in metallochelating nanoliposomes with recombinant Hsp90 protein antigen. CONCLUSIONS: Liposomal formulations of new lipophilic derivatives of norAbuMDP/GMDP proved themselves as promising adjuvants for recombinant vaccines as well as immunomodulators for stimulation of innate immunity and bone-marrow recovery after chemo/radio therapy of cancer.
- MeSH
- acetylmuramyl-alanyl-isoglutamin aplikace a dávkování analogy a deriváty chemie farmakologie terapeutické užití MeSH
- adaptivní imunita účinky léků MeSH
- adjuvancia imunologická aplikace a dávkování chemie farmakologie terapeutické užití MeSH
- analýza přežití MeSH
- antigeny fungální imunologie MeSH
- experimentální radiační poranění imunologie prevence a kontrola MeSH
- králíci MeSH
- liposomy MeSH
- mikroskopie atomárních sil MeSH
- mikroskopie elektronová rastrovací MeSH
- molekulární struktura MeSH
- myši inbrední ICR MeSH
- myši MeSH
- nanočástice MeSH
- nosiče léků chemie MeSH
- přirozená imunita účinky léků MeSH
- proteiny tepelného šoku HSP90 imunologie MeSH
- protilátky fungální krev MeSH
- rekombinantní proteiny imunologie MeSH
- transmisní elektronová mikroskopie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The present investigation deals with preparation of three different adsorbent materials namely; potassium hydroxide activated carbon based apricot stone (C), calcium alginate beads (G) and calcium alginate/activated carbon composite beads (GC) were used for the removal of arsenic. The prepared adsorbent materials were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), N2-adsorption at -196°C and point of zero charge. From the obtained results, it was found that the porosity, surface area and total pore volume of the adsorbent material C>GC>G respectively, however, the G adsorbent has more acidic function group than the other adsorbents. The influence of pH, time, temperature and initial concentration of arsenic(V) were studied and optimized. GC exhibits the maximum As(V) adsorption (66.7mg/g at 30°C). The adsorption of arsenic ions was observed to follow pseudo-second order mechanism as well as the thermodynamic parameters confirm also the endothermic spontaneous and a physisorption process.
- MeSH
- adsorpce MeSH
- algináty chemie MeSH
- arsen izolace a purifikace MeSH
- časové faktory MeSH
- dřevěné a živočišné uhlí chemie MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- kyselina glukuronová chemie MeSH
- kyseliny hexuronové chemie MeSH
- mikrosféry * MeSH
- mikroskopie elektronová rastrovací MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Autofluorescence (primary fluorescence (AF)) of fruiting bodies and stems of the fungus Morchella conica var. rigida was studied by fluorescence microscopy including sporangia and ascospores. The ascospores were characterized by a weak green-yellow AF at blue excitation. Using a green excitation, no AF was observed. The hyphae located under the layer of asci with ascospores exhibited a higher primary fluorescence, namely their walls that had green-yellow color at blue excitation. Also, their red AF observed when a green excitation was used was significant. Similarly, the hyphae located in the fungal stem exhibited a significant AF, especially their walls when the blue light was used for excitation. In addition, large, yellow-to-yellow/green, oval-to-round bodies with strong fluorescence were detected whose morphological equivalents were not clearly visible in the white halogen light. The AF of the fungus M. conica var. rigida was lower compared with the other higher fungi studied so far.
Hsp90-CA is present in cell wall of Candida pseudohyphae or hyphae-typical pathogenic morphotype for both systemic and mucosal Candida infections. Heat shock protein from Candida albicans (hsp90-CA) is an important target for protective antibodies during disseminated candidiasis of experimental mice and human. His-tagged protein rHsp90 was prepared and used as the antigen for preparation of experimental recombinant liposomal vaccine. Nickel-chelating liposomes (the size around 100nm, PDI≤0.1) were prepared from the mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (molar ratio 95:5%) by hydration of lipid film and extrusion methods. New non-pyrogenic hydrophobised derivative of MDP (C18-O-6-norAbuMDP) was incorporated into liposomes as adjuvans. rHsp90 was attached onto the surface of metallochelating liposomes by metallochelating bond and the structure of these proteoliposomes was studied by dynamic light scattering, AF microscopy, TEM and GPC. The liposomes with surface-exposed C18-O-6-norAbuMDP were well recognised and phagocyted by human dendritic cells in vitro. In vivo the immune response towards this experimental vaccine applied in mice (i.d.) demonstrated both TH1 and TH2 response comparable to FCA, but without any side effects. Metallochelating liposomes with lipophilic derivatives of muramyl dipeptide represent a new biocompatible platform for construction of experimental recombinant vaccines and drug-targeting systems.
- MeSH
- antigeny fungální imunologie metabolismus MeSH
- biokompatibilní potahované materiály metabolismus MeSH
- buněčná imunita MeSH
- Candida imunologie MeSH
- chelátory chemie metabolismus MeSH
- dendritické buňky imunologie metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- liposomy MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nikl imunologie metabolismus MeSH
- proteiny tepelného šoku HSP90 imunologie metabolismus MeSH
- syntetické vakcíny imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The autofluorescence (primary fluorescence, AF) of agar cultures of the brown-rot fungus Piptoporus betulinus was investigated in Zeiss Jenalumar and Nikon Eclipse 8201 fluorescence microscopes at various excitations. The strongest AF of hyphae was found in minimal medium with glucose, where the hyphae exhibited green AF at violet (450 nm) excitation and red AF at green (570 nm) excitation. Addition of metals to cultivation media led to enhanced white-blue AF in the presence of Co (at 450 nm) and yellow to yellow-brown AF at 510 nm. When cultivated with Mn and Zn, enhanced AF of intracellular content was observed. Only a weak signal was found in the presence of Cu and Fe.
The autofluorescence (primary fluorescence, AF) of the freshly collected fruiting bodies of the fungus Macrolepiota rhacodes was studied in a Zeiss Jenalumar fluorescence microscope at a blue and a green excitation. The strongest yellow AF at blue excitation was displayed by irregular granules on the surface of the fungal pileus. A weaker yellow-green AF was exhibited by spherical cells and hyphae in the central part of the pileus while basidiospores emitted somewhat stronger AF. At green excitation, a considerable red AF was emitted only by basidiospores, other parts of the pileus showing a very weak red AF. M. rhacodes AF is much weaker than the AF of wood-rotting fungi, such as Fomes fomentarius, Daedalea quercina, Piptoporus betulinus, Fomitopsis pinicola and others.
... Green: Scanning electron microscopy of collecting lymphatics, the thoracic duct and their valves 18 - ... ... Lennert: Morphologic alterations in lymph nodes af ter experimental lymphos ta sis and venostasis 79 ...
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