The genus Boechera, a prominent member of the tribe Boechereae within the family Brassicaceae, has emerged as an exceptional model for apomixis research, owing to its unique evolutionary, reproductive, and genomic characteristics. With over 480 genetically distinct taxa and a distribution spanning North America, Greenland, and parts of Asia, Boechera exemplifies remarkable ecological and genetic diversity. A hallmark of the genus is its high frequency of gametophytic apomixis, including diplospory and apospory, which, combined with hybridization, drive genetic innovation and environmental adaptability. Moreover, Boechera is notable for its diploid expression of apomixis, independent of polyploidy, providing an unparalleled system for dissecting asexual reproduction. Genomic adaptations, such as heterochromatic supernumerary chromosomes, further highlight its evolutionary complexity. Recent advances, including the discovery of critical regulatory genes like APOLLO and UPGRADE2, have deepened our understanding of the genetic basis of apomixis. These insights position Boechera as a cornerstone for elucidating apomictic pathways and leveraging these mechanisms for crop improvement. The genus not only enhances our understanding of apomixis but also offers transformative potential for agriculture.
- Klíčová slova
- Apomixis, Boechera, Brassicaceae, Chromosome dynamics, Evolutionary biology, Gametophytic reproduction, Genomic architecture, Heterochromatic chromosomes, Hybridization,
- MeSH
- apomixie * genetika MeSH
- Brassicaceae * genetika fyziologie MeSH
- genetická variace MeSH
- genom rostlinný MeSH
- regulace genové exprese u rostlin MeSH
- Publikační typ
- časopisecké články MeSH
Chromosome preparation and fluorescent in situ hybridization (FISH) are indispensable methodologies in cytogenetics, serving as a vital link between molecular biology and genome visualization. In the era of next-generation sequencing and near-complete genome assemblies, these techniques remain crucial for elucidating chromosomal architecture, dynamics, and evolutionary relationships. This chapter presents robust protocols for preparing mitotic and meiotic chromosomes from root tips and floral tissues, optimized for diverse plant species. Detailed procedures for enzymatic digestion, chromosome squashing and spreading, and FISH are described, ensuring reproducible and high-quality results. Furthermore, protocols for probe labeling and fluorescence detection highlight the adaptability of FISH for various cytogenetic applications, including chromosome painting or genomic in situ hybridization. By mastering these approaches, researchers can explore chromosomal organization with unprecedented resolution. These step-by-step protocols are designed to be accessible to both beginners and experienced researchers, ensuring wide applicability across plant species and cytogenetic contexts.
- Klíčová slova
- Cytogenetics, Fluorescent in situ hybridization, Mitotic and meiotic chromosomes, Plant genomes,
- MeSH
- chromozomy rostlin * genetika MeSH
- hybridizace in situ fluorescenční * metody MeSH
- malování chromozomů metody MeSH
- meióza MeSH
- meristém genetika MeSH
- mitóza MeSH
- rostliny genetika MeSH
- Publikační typ
- časopisecké články MeSH
Flow cytometry is widely used to estimate ploidy levels and genome sizes in plants and animals. It is also a common tool for determining the reproductive mode in flowering plants through flow cytometric seed screening (FCSS), which can differentiate sexual and apomictic pathways. However, FCSS has limitations, as it is not universally applicable to all apomictic taxa and cannot resolve certain reproductive pathways. These challenges can be addressed by germinating seeds and genotyping seedlings and their parents. However, this approach can be time- and space-intensive and carry other drawbacks (e.g., the ploidy level of the endosperm remains undetected). Here, we present a streamlined method that combines flow cytometry with genotyping of the same seeds. Applying both methods to a single seed enables comprehensive insights into reproductive modes, detecting various forms of apomixis and automixis, assessment of the level of selfing, regardless of the mode of reproduction mode, and identifying pollen donors in the endosperm of apomictic seeds. Each protocol-FCSS and seed genotyping-can also be used independently to determine basic reproductive modes.
- Klíčová slova
- Apomixis, FCSS, Mode of reproduction, SSR-seq, Seed genotyping,
- MeSH
- apomixie genetika MeSH
- endosperm genetika MeSH
- genotyp MeSH
- genotypizační techniky * metody MeSH
- Magnoliopsida * genetika fyziologie MeSH
- průtoková cytometrie * metody MeSH
- pyl genetika MeSH
- rozmnožování genetika MeSH
- semena rostlinná * genetika MeSH
- Publikační typ
- časopisecké články MeSH
Fed-batch is the most commonly used cultivation mode for industrial production of recombinant proteins with Pichia pastoris. On a laboratory scale, fed-batch culture provides a way to control the specific biomass growth rates at any pre-set value, allowing the conditions of biomass growth and recombinant product formation to be systematically studied.In this chapter, we present an accessible and versatile approach for designing, performing, and evaluating a fed-batch cultivation in laboratory-scale stirred tank bioreactors.
- Klíčová slova
- Bioprocess design and development, Bioreactor, Data evaluation, Fed-batch culture, Feed rate, Fermentation, Komagataella phaffii, Pichia pastoris, Specific growth rate, Specific productivity,
- MeSH
- biomasa MeSH
- bioreaktory mikrobiologie MeSH
- fermentace MeSH
- kultivační média MeSH
- Pichia * růst a vývoj MeSH
- rekombinantní proteiny genetika biosyntéza MeSH
- Saccharomycetales * růst a vývoj metabolismus MeSH
- techniky vsádkové kultivace * metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kultivační média MeSH
- rekombinantní proteiny MeSH
The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) system has become a commonly used tool for genome editing and metabolic engineering. For Komagataella phaffii, commercialized as Pichia pastoris, the CRISPR/Cas9 protocol for genome editing was established in 2016 and since then has been employed to facilitate genetic modifications such as markerless gene disruptions and deletions as well as to enhance the efficiency of homologous recombination.In this chapter, we describe a robust basic protocol for CRISPR-based genome editing, demonstrating near 100% targeting efficiency for gene inactivation via a frameshift mutation. As described in other chapters of this volume, CRISPR/Cas9 technologies for use in P. pastoris have been further optimized for various specific purposes.
- Klíčová slova
- CRISPR/Cas9, Gene knockout, Genome editing, Genome engineering, Guide RNA, Homologous recombination, Komagataella phaffii, Pichia pastoris, Synthetic biology,
- MeSH
- CRISPR-Cas systémy * genetika MeSH
- editace genu * metody MeSH
- genom fungální MeSH
- metabolické inženýrství * metody MeSH
- Pichia * genetika MeSH
- Saccharomycetales * genetika MeSH
- vodící RNA, systémy CRISPR-Cas genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- vodící RNA, systémy CRISPR-Cas MeSH
The use of giant vesicles as microreactors presents a novel approach to control biochemical reactions in confined spaces, offering advantages such as compartmentalization, tunable permeability, and potential for biomimetic applications. These constructs can serve as versatile platforms for catalysis, drug delivery, and synthetic biology by providing confined environments that mimic natural cellular compartments. We have successfully produced microvesicles (also referred to as giant vesicles) by means of the simple double emulsification method using five amphiphilic block copolymers comprising poly(ethylene oxide) (PEO) as hydrophilic segment and five disparate hydrophobic blocks: poly(caprolactone) (PCL), poly(methyl methacrylate) (PMMA), poly(lactic acid) (PLA), poly[2-(diisopropylamino)ethyl methacrylate] (PDPA), and poly[2-(heptamethyleneimino)ethyl methacrylate] (PHIA). The last two blocks are pH-responsive (PDPA, PHIA), while the first ones are not (PCL, PMMA, PLA). The resulting vesicles have average size ranging from 2.9 to 9.3 μm, with the pH-responsive vesicles exhibiting larger diameters, likely due to partial protonation of the hydrophobic blocks. The formation of the giant vesicles was confirmed via optical and fluorescence microscopy using Nile red as a hydrophobic marker. The ability of the vesicles to encapsulate larger molecules was demonstrated by loading Alexa-labeled bovine serum albumin (BSA-Alexa). Furthermore, the potential of these vesicles as microreactors was explored by encapsulating horseradish peroxidase enzyme (HRP) and evaluating the catalytic oxidation of o-dianisidine in the presence of hydrogen peroxide (H₂O₂), a reaction catalyzed by the HRP enzyme. The experimental data confirm that the pH-responsive vesicles are permeable to the reactants, as evidenced by colored product formation, whereas the permeability of the nonresponsive assemblies is negligible. The non-responsive vesicles exhibited particularly low permeability, even at the pH where the catalytic activity of the enzyme is optimum. These findings highlight the potential of pH-responsive vesicles for controlled molecular transport and catalytic applications, paving the way for their use in biocatalysis as microreactors.
- Klíčová slova
- Block copolymers, Giant polymer vesicles, Microreactors, Self-assembly,
- MeSH
- hydrofobní a hydrofilní interakce MeSH
- koncentrace vodíkových iontů MeSH
- křenová peroxidasa chemie metabolismus MeSH
- permeabilita MeSH
- polyestery chemie MeSH
- polymery * chemie MeSH
- povrchové vlastnosti MeSH
- velikost částic MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- křenová peroxidasa MeSH
- polyestery MeSH
- polymery * MeSH
Colorectal cancer remains a major health burden, and its early detection is crucial for effective treatment. This study investigates the use of a handheld Raman spectrometer in combination with machine learning to classify colorectal tissue samples collected during colonoscopy. A dataset of 330 spectra from 155 participants was preprocessed using a standardized pipeline, and multiple classification models were trained to distinguish between healthy and pathological tissue. Due to the strong class imbalance and limited data size, a custom grid search approach was implemented to optimize both model hyperparameters and preprocessing parameters. Unlike standard GridSearchCV, our method prioritized balanced accuracy on the test set to reduce bias toward the dominant class. Among the tested classifiers, the Decision Tree (DT) and Support Vector Classifier (SVC) achieved the highest balanced accuracy (71.77% for DT and 70.77% for SVC), outperforming models trained using traditional methods. These results demonstrate the potential of Raman spectroscopy as a rapid, non-destructive screening tool and highlight the importance of tailored model selection strategies in biomedical applications. While this study is based on a limited dataset, it serves as a promising step toward more robust classification models and supports the feasibility of this approach for future clinical validation.
- Klíčová slova
- Balanced accuracy, Colorectal cancer, Machine learning, Preprocessing pipeline, Raman spectroscopy, Spectral classification,
- MeSH
- kolorektální nádory * diagnóza diagnostické zobrazování MeSH
- lidé středního věku MeSH
- lidé MeSH
- Ramanova spektroskopie * metody MeSH
- rozhodovací stromy MeSH
- strojové učení * MeSH
- support vector machine MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
