Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.
- Klíčová slova
- 3D STED, alcohol dehydration, high refractive index medium, large biological objects, oocyte, sample preparation, super‐resolution,
- MeSH
- alkoholy MeSH
- fluorescenční mikroskopie metody MeSH
- myši MeSH
- odběr biologického vzorku metody MeSH
- oocyty * MeSH
- refraktometrie metody MeSH
- zobrazování trojrozměrné * metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- alkoholy MeSH
The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.
- MeSH
- buněčné jádro * genetika metabolismus MeSH
- editace RNA * MeSH
- genetický kód MeSH
- genom mitochondriální * MeSH
- guide RNA, Kinetoplastida genetika metabolismus MeSH
- kodon genetika MeSH
- messenger RNA genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- otevřené čtecí rámce genetika MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA transferová * genetika metabolismus MeSH
- terminační kodon genetika MeSH
- Trypanosomatina genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- guide RNA, Kinetoplastida MeSH
- kodon MeSH
- messenger RNA MeSH
- protozoální proteiny MeSH
- RNA transferová * MeSH
- terminační kodon MeSH
At the turn of the millennium, the monolithic columns invoked new chances in HPLC. Even more than their organic polymer-based siblings, the inorganic silica-based monoliths targeted the territory of classical fully porous particle-packed columns, promising many benefits. Based on the number of published articles, the monoliths attracted academics just in the first few years after their introduction to the market. Lately, as superficially porous particles and sub-2-micron fully porous particles dominated the market, they stayed in the focus of routine laboratories and those who really appreciated the high porosity of the monolithic bed. The monoliths' practical benefits cannot be easily traced in the literature when they gradually lose academics' interest. Nevertheless, after more than 20 years of our experience, we still favor silica monoliths for their low back pressure and longevity when analyzing samples of clinical, pharmaceutical, and environmental origin. At the same time, the high permeability of monoliths enabled the birth of sequential injection chromatography, the medium-pressure separation technique based on the flexible flow manifold. This minireview aims to check, discuss, and summarize the practical aspects of monolithic silica columns in HPLC and medium-pressure sequential injection chromatography (SIC) that may not be visible at first sight but are evident retrospectively.
A novel virus lytic for Pseudomonas aeruginosa has been purified. Its viral particles have a siphoviral morphology with a head 60 nm in diameter and a noncontractile tail 184 nm long. The dsDNA genome consists of 16,449 bp, has cohesive 3' termini, and encodes 28 putative proteins in a single strain. The peptidoglycan endopeptidase encoded by ORF 16 was found to be the lytic enzyme of this virus. The recombinant, purified enzyme was active up to 55 °C in the pH range 6-9 against all tested isolates of P. aeruginosa, but, surprisingly, also against the distant Gram-positive micrococci Arthrobacter globiformis and A. pascens. Both this virus and its endolysin are further candidates for possible treatment against P. aeruginosa and probably also other bacteria.
- Klíčová slova
- G+ and G− activity, adaiavirus, endolysin, host range,
- Publikační typ
- časopisecké články MeSH
The application-attractive form of TiO2, CeO2 and CuO-based open-cell foam supported catalysts was designed to investigate their catalytic performance in oxidation of two model volatile organic compounds-methanol and dichloromethane. TiO2-CeO2, TiO2-CuO and TiO2-CeO2-CuO catalysts as thin films were deposited on VUKOPOR®A ceramic foam using a reverse micelles-controlled sol-gel method, dip-coating and calcination. Three prepared catalytic foams were investigated via light-off tests in methanol and dichloromethane oxidation in the temperature range of 45-400 °C and 100-500 °C, respectively, at GHSV of 11, 600 h-1, which fits to semi-pilot/industrial conditions. TiO2-CuO@VUKOPOR®A foam showed the best catalytic activity and CO2 yield in methanol oxidation due to its low weak Lewis acidity, high weak basicity and easily reducible CuO species and proved good catalytic stability within 20 h test. TiO2-CeO2-CuO@VUKOPOR®A foam was the best in dichloromethane oxidation. Despite of its lower catalytic activity compared to TiO2-CeO2@VUKOPOR®A foam, its highly-reducible -O-Cu-Ce-O- active surface sites led to the highest CO2 yield and the highest weak Lewis acidity contributed to the highest HCl yield. This foam also showed the lowest amount of chlorine deposits.
- Klíčová slova
- VUKOPOR®A ceramic foam, catalytic oxidation, ceria, copper oxide, dichloromethane, methanol, mixed oxide catalyst, titania, volatile organic compounds,
- Publikační typ
- časopisecké články MeSH
On the basis of previous reports, novel 2-benzoylhydrazine-1-carboxamides were designed as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Inhibitors of these enzymes have many clinical applications. 2-(Substituted benzoyl)hydrazine-1-carboxamides decorated with N-methyl or tridecyl were prepared with three methods from commercially available or self-prepared hydrazides and isocyanates. For methyl derivatives, N-succinimidyl N-methylcarbamate was used or methyl isocyanate was prepared via Curtius rearrangement. Tridecyl isocyanate was synthesized again via Curtius rearrangement or from triphosgene and tridecylamine. The compounds were evaluated for the inhibition of AChE and BChE using Ellman's spectrophotometric method. Most of the derivatives showed the dual inhibition of both enzymes with IC50 values of 44-100 µM for AChE and from 22 µM for BChE. In general, the carboxamides inhibited AChE more strongly. A large number of the compounds showed better or quite comparable inhibition of cholinesterases in vitro than that of the drug rivastigmine. Molecular docking was performed to investigate the possible conformation of the compounds and their interactions with target enzymes. In both AChE and BChE, the compounds occupied the enzyme active cavity, and, especially in the case of BChE, the compounds were placed in close proximity to the catalytic triad.
- Klíčová slova
- acetylcholinesterase, benzohydrazide, butyrylcholinesterase, enzyme inhibition, hydrazine-1-carboxamide, molecular docking,
- Publikační typ
- časopisecké články MeSH
The preparation of anodic TiO2 nanotube layers has been performed using electrochemical anodization of Ti foil for 4 h at different voltages (from 0 V to 80 V). In addition, a TiO2 thin layer has been also prepared using the sol-gel method. All the photocatalysts have been characterized by XRD, SEM, and DRS to investigate the crystalline phase composition, the surface morphology, and the optical properties, respectively. The performance of the photocatalyst has been assessed in versatile photocatalytic reactions including the reduction of N2O gas and the oxidation of aqueous sulfamethoxazole. Due to their high specific surface area and excellent charge carriers transport, anodic TiO2 nanotube layers have exhibited the highest N2O conversion rate (up to 10% after 22 h) and the highest degradation extent of sulfamethoxazole (about 65% after 4 h) under UVA light. The degradation mechanism of sulfamethoxazole has been investigated by analyzing its transformation products by LC-MS and the predominant role of hydroxyl radicals has been confirmed. Finally, the efficiency of the anodic TiO2 nanotube layer has been tested in real wastewater reaching up to 45% of sulfamethoxazole degradation after 4 h.
- Klíčová slova
- N2O, TiO2, air treatment, pharmaceutical, photocatalysis, water treatment,
- MeSH
- katalýza MeSH
- nanotrubičky * chemie MeSH
- odpadní voda * MeSH
- sulfamethoxazol chemie MeSH
- titan chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- odpadní voda * MeSH
- sulfamethoxazol MeSH
- titan MeSH
- titanium dioxide MeSH Prohlížeč
Fertilization is a complex process that requires successive stages and culminates in the adhesion/fusion of gamete membranes. If the question of the involvement of oocyte integrins has been swept away by deletion experiments, that of the involvement of sperm integrins remains to be further characterized. In the present study, we addressed the question of the feasibility of sperm-oocyte adhesion/fusion and early implantation in the absence of sperm β1 integrin. Males and females with β1 integrin-depleted sperm and oocytes were mated, and fertilization outcome was monitored by a gestational ultrasound analysis. Results suggest that although the sperm β1 integrin participates in gamete adhesion/fusion, it is dispensable for fertilization in mice. However, sperm- and/or oocyte-originated integrin β1 is essential for post-implantation development. Redundancy phenomena could be at the origin of a compensatory expression or alternative dimerization pattern.
- Klíčová slova
- Stra8-Cre, fertilization, integrin beta1, sperm, ultrasound,
- MeSH
- antigeny CD29 * genetika metabolismus MeSH
- fertilizace MeSH
- integriny metabolismus MeSH
- interakce spermie a vajíčka * MeSH
- myši MeSH
- oocyty metabolismus MeSH
- sperma metabolismus MeSH
- spermie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antigeny CD29 * MeSH
- integriny MeSH
Rye (Secale cereale) is a climate-resilient cereal grown extensively as grain or forage crop in Northern and Eastern Europe. In addition to being an important crop, it has been used to improve wheat through introgression of genomic regions for improved yield and disease resistance. Understanding the genomic diversity of rye will assist both the improvement of this crop and facilitate the introgression of more valuable traits into wheat. Here, we isolated and sequenced the short arm of rye chromosome 7 (7RS) from Triticale 380SD using flow cytometry and compared it to the public Lo7 rye whole genome reference assembly. We identify 2747 Lo7 genes present on the isolated chromosome arm and two clusters containing seven and sixty-five genes that are present on Triticale 380SD 7RS, but absent from Lo7 7RS. We identified 29 genes that are not assigned to chromosomal locations in the Lo7 assembly but are present on Triticale 380SD 7RS, suggesting a chromosome arm location for these genes. Our study supports the Lo7 reference assembly and provides a repertoire of genes on Triticale 7RS.
- Klíčová slova
- isolated chromosome arm sequencing, presence-absence variation, rye,
- MeSH
- chromozomy rostlin genetika MeSH
- jedlá semena genetika MeSH
- odolnost vůči nemocem genetika MeSH
- pšenice genetika MeSH
- triticale * genetika MeSH
- žito * genetika MeSH
- Publikační typ
- časopisecké články MeSH
Commercial interest in biostimulants as a tool for sustainable green economics and agriculture concepts is on a steep rise, being followed by increasing demand to employ efficient scientific methods to develop new products and understand their mechanisms of action. Biostimulants represent a highly diverse group of agents derived from various natural sources. Regardless of their nutrition content and composition, they are classified by their ability to improve crop performance through enhanced nutrient use efficiency, abiotic stress tolerance, and quality of crops. Numerous reports have described modern, non-invasive sensor-based phenotyping methods in plant research. This review focuses on applying phenotyping approaches in biostimulant research and development, and maps the evolution of interaction of these two intensively growing domains. How phenotyping served to identify new biostimulants, the description of their biological activity, and the mechanism/mode of action are summarized. Special attention is dedicated to the indoor high-throughput methods using model plants suitable for biostimulant screening and developmental pipelines, and high-precision approaches used to determine biostimulant activity. The need for a complex method of testing biostimulants as multicomponent products through integrating other -omic approaches followed by advanced statistical/mathematical tools is emphasized.
- Klíčová slova
- -omics, High-throughput screening, mechanism of action, mode of action, plant biostimulants, plant breeding, plant phenotyping, sensors,
- MeSH
- fyziologický stres * MeSH
- výzkum MeSH
- zemědělské plodiny * MeSH
- zemědělství metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
