Freezing and lyophilization have been utilized for decades to stabilize pharmaceutical and food products. Freezing a solution that contains dissolved salt and/or organic matter produces pure primary ice crystal grains separated by freeze-concentrated solutions (FCS). The microscopic size of the primary ice crystals depends on the cooling conditions and the concentration of the solutes. It is generally accepted that primary ice crystals size influences the rate of sublimation and also can impact physico-chemical behaviour of the species in the FCS. This article, however, presents a case where the secondary ice formed inside the FCS plays a critical role. We microscoped the structures of ice-cast FCS with an environmental scanning electron microscope and applied the aggregation-sensitive spectroscopic probe methylene blue to determine how the microstructure affects the molecular arrangement. We show that slow cooling at -50 °C produces large salt crystals with a small specific surface, resulting in a high degree of molecular aggregation within the FCS. In contrast, fast liquid nitrogen cooling yields an ultrafine structure of salt crystals having a large specific surface area and, therefore, inducing smaller aggregation. The study highlights a critical role of secondary ice in solute aggregation and introduces methylene blue as a molecular probe to investigate freezing behaviour of aqueous systems with crystalline solute.
- MeSH
- led * MeSH
- lyofilizace MeSH
- methylenová modř * MeSH
- roztoky MeSH
- voda chemie MeSH
- zmrazování MeSH
- Publikační typ
- časopisecké články MeSH
Infusing pancreatic islets into the portal vein currently represents the preferred approach for islet transplantation, despite considerable loss of islet mass almost immediately after implantation. Therefore, approaches that obviate direct intravascular placement are urgently needed. A promising candidate for extrahepatic placement is the omentum. We aimed to develop an extracellular matrix skeleton from the native pancreas that could provide a microenvironment for islet survival in an omental flap. To that end, we compared different decellularization approaches, including perfusion through the pancreatic duct, gastric artery, portal vein, and a novel method through the splenic vein. Decellularized skeletons were compared for size, residual DNA content, protein composition, histology, electron microscopy, and MR imaging after repopulation with isolated islets. Compared to the other approaches, pancreatic perfusion via the splenic vein provided smaller extracellular matrix skeletons, which facilitated transplantation into the omentum, without compromising other requirements, such as the complete depletion of cellular components and the preservation of pancreatic extracellular proteins. Repeated MR imaging of iron-oxide-labeled pancreatic islets showed that islets maintained their position in vivo for 49 days. Advanced environmental scanning electron microscopy demonstrated that islets remained integrated with the pancreatic skeleton. This novel approach represents a proof-of-concept for long-term transplantation experiments.
- Publikační typ
- časopisecké články MeSH
Frozen aqueous solutions are an important subject of study in numerous scientific branches including the pharmaceutical and food industry, atmospheric chemistry, biology, and medicine. Here, we present an advanced environmental scanning electron microscope methodology for research of ice samples at environmentally relevant subzero temperatures, thus under conditions in which it is extremely challenging to maintain the thermodynamic equilibrium of the specimen. The methodology opens possibilities to observe intact ice samples at close to natural conditions. Based on the results of ANSYS software simulations of the surface temperature of a frozen sample, and knowledge of the partial pressure of water vapor in the gas mixture near the sample, we monitored static ice samples over several minutes. We also discuss possible artifacts that can arise from unwanted surface ice formation on, or ice sublimation from, the sample, as a consequence of shifting conditions away from thermodynamic equilibrium in the specimen chamber. To demonstrate the applicability of the methodology, we characterized how the true morphology of ice spheres containing salt changed upon aging and the morphology of ice spheres containing bovine serum albumin. After combining static observations with the dynamic process of ice sublimation from the sample, we can attain images with nanometer resolution.
Pyrrhocoroidea represents an important group of true bugs (Insecta: Hemiptera: Heteroptera) which includes fire bugs, cotton stainers and other taxa widely used in experimental studies or known as pests. However, the morphology and phylogeny of Pyrrhocoroidea have been only poorly studied so far. Here, structures of the external scent efferent system of the metathoracic scent glands are examined in 64 out of 71 currently valid genera of Pyrrhocoroidea and scanning electron micrographs are provided for most taxa. Several characters are revealed which define each of the three higher taxa within Pyrrhocoroidea: Larginae (small auriculate peritreme lacking manubrium and median furrow; metathoracic spiracle never surrounded by evaporatorium), Physopeltinae (large, widely open ostiole; large peritremal disc with manubrium [new term], lacking median furrow; mace-like mycoid filter processes of equal shape and size on both anterior and posterior margins of metathoracic spiracle), and Pyrrhocoridae (elongate auriculate peritreme with deep median furrow). Within Pyrrhocoridae, three main types (A, B and C) of the external scent efferent system are distinguished, differring in the amount of reductions. The findings are interpreted in the context of phylogenetic hypotheses available for Pyrrhocoroidea and their close relatives, Coreoidea and Lygaeoidea. An updated identification key to the families and subfamilies of Pyrrhocoroidea applicable for both sexes is provided.
- MeSH
- feromony MeSH
- fylogeneze MeSH
- Heteroptera * MeSH
- pachové žlázy MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The paper discusses the real-time monitoring of the changing sample morphology during the entire lyophilization (freeze-drying) and vacuum-drying processes of model biopharmaceutical solutions by using an environmental scanning electron microscope (ESEM); the device's micromanipulators were used to study the interior of the samples in-situ without exposing the samples to atmospheric water vapor. The individual collapse temperatures (Tc) of the formulations, pure bovine serum albumin (BSA) and BSA/sucrose mixtures, ranged from -5 to -29 °C. We evaluated the impact of the freezing method (spontaneous freezing, controlled ice nucleation, and spray freezing) on the morphologies of the lyophiles at the constant drying temperature of -20 °C. The formulations with Tc above -20 °C resulted in the lyophiles' morphologies significantly dependent on the freezing method. We interpret the observations as an interplay of the freezing rates and directionalities, both of which markedly influence the morphologies of the frozen formulations, and, subsequently, the drying process and the mechanical stability of the freeze-dried cake. The formulation with Tc below -20 °C yielded a collapsed cake with features independent of the freezing method. The vacuum-drying produced a material with a smooth and pore-free surface, where deep cracks developed at the end of the process.
Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50-500 μM and 10-50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce.
- MeSH
- aktivní transport MeSH
- arsen farmakokinetika toxicita MeSH
- biotechnologie MeSH
- buněčné linie MeSH
- fyziologický stres MeSH
- klíčení účinky léků MeSH
- látky znečišťující životní prostředí farmakokinetika toxicita MeSH
- měď farmakokinetika toxicita MeSH
- mikroskopie elektronová rastrovací MeSH
- smrk účinky léků embryologie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The Extended Low Temperature Method (ELTM) for the in-situ preparation of plant samples in an environmental scanning electron microscope enables carrying out repetitive topographical and material analysis at a higher resolution in the vacuum conditions of a scanning electron microscope or in the low gas pressure conditions of an environmental scanning electron microscope. The method does not require any chemical intervention and is thus suitable for imaging delicate structures rarely observable with common treatment methods. The method enables both sample stabilization as close to their native state as possible, as well as the transfer of the same sample from a low vacuum to an atmospheric condition for sample storage or later study. It is impossible for wet samples in the environmental scanning electron microscope. Our studies illustrate the high applicability of the ELTM for different types of plant tissue, from imaging of plant waxes at higher resolution, the morphological study of highly susceptible early somatic embryos to the elemental microanalysis of root cells. The method established here provides a very fast, universal and inexpensive solution for plant sample treatment usable in a commercial environmental scanning electron microscope equipped with a cooling Peltier stage.
The discovery and exploration of cryptic species have been profoundly expedited thanks to developments in molecular biology and phylogenetics. In this study, we apply a reverse taxonomy approach to the Brachionus calyciflorus species complex, a commonly studied freshwater monogonont rotifer. By combining phylogenetic, morphometric and morphological analyses, we confirm the existence of four cryptic species that have been recently suggested by a molecular study. Based on these results and according to an exhaustive review of the taxonomic literature, we name each of these four species and provide their taxonomic description alongside a diagnostic key.
- MeSH
- DNA helmintů genetika MeSH
- druhová specificita MeSH
- ekosystém MeSH
- fylogeneze MeSH
- mikroskopie elektronová rastrovací MeSH
- molekulární biologie MeSH
- sekvenční analýza DNA MeSH
- sladká voda MeSH
- vířníci anatomie a histologie klasifikace genetika MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Nizozemsko MeSH
Biogenesis of the plant secondary cell wall involves many important aspects, such as phenolic compound deposition and often silica encrustation. Previously, we demonstrated the importance of the exocyst subunit EXO70H4 for biogenesis of the trichome secondary cell wall, namely for deposition of the autofluorescent and callose-rich cell wall layer. Here, we reveal that EXO70H4-driven cell wall biogenesis is constitutively active in the mature trichome, but also can be activated elsewhere upon pathogen attack, giving this study a broader significance with an overlap into phytopathology. To address the specificity of EXO70H4 among the EXO70 family, we complemented the exo70H4-1 mutant by 18 different Arabidopsis (Arabidopsis thaliana) EXO70 paralogs subcloned under the EXO70H4 promoter. Only EXO70H4 had the capacity to rescue the exo70H4-1 trichome phenotype. Callose deposition phenotype of exo70H4-1 mutant is caused by impaired secretion of PMR4, a callose synthase responsible for the synthesis of callose in the trichome. PMR4 colocalizes with EXO70H4 on plasma membrane microdomains that do not develop in the exo70H4-1 mutant. Using energy-dispersive x-ray microanalysis, we show that both EXO70H4- and PMR4-dependent callose deposition in the trichome are essential for cell wall silicification.
- MeSH
- Arabidopsis účinky léků genetika metabolismus MeSH
- buněčná membrána účinky léků metabolismus MeSH
- buněčná stěna účinky léků metabolismus MeSH
- epidermis rostlin cytologie účinky léků metabolismus MeSH
- fenotyp MeSH
- flagelin farmakologie MeSH
- glukany MeSH
- glukosyltransferasy metabolismus MeSH
- mutace genetika MeSH
- oxid křemičitý metabolismus MeSH
- podjednotky proteinů chemie metabolismus MeSH
- proteinové domény MeSH
- proteiny huseníčku chemie metabolismus MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- trichomy metabolismus MeSH
- upregulace účinky léků MeSH
- vezikulární transportní proteiny chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viable microbial cells are important biocatalysts in the production of fine chemicals and biofuels, in environmental applications and also in emerging applications such as biosensors or medicine. Their increasing significance is driven mainly by the intensive development of high performance recombinant strains supplying multienzyme cascade reaction pathways, and by advances in preservation of the native state and stability of whole-cell biocatalysts throughout their application. In many cases, the stability and performance of whole-cell biocatalysts can be highly improved by controlled immobilization techniques. This review summarizes the current progress in the development of immobilized whole-cell biocatalysts, the immobilization methods as well as in the bioreaction engineering aspects and economical aspects of their biocatalytic applications.
- MeSH
- bioinženýrství * MeSH
- biokatalýza * MeSH
- bioreaktory * MeSH
- imobilizované buňky * MeSH
- lidé MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
