OBJECTIVE: Transgenic mice with fluorescent protein (FP) reporters take full advantage of new in vivo imaging technologies. Therefore, we generated a TRPC5- and a TRPA1-reporter mouse based on FP C-terminal fusion, providing us with better alternatives for studying the physiology, interaction and coeffectors of these two TRP channels at the cellular and tissue level. METHODS: We generated transgenic constructs of the murine TRPC5- and TRPA1-gene with a 3*GGGGS linker and C-terminal fusion to mCherry and mTagBFP, respectively. We microinjected zygotes to generate reporter mice. Reporter mice were examined for visible fluorescence in trigeminal ganglia with two-photon microscopy, immunohistochemistry and calcium imaging. RESULTS: Both TRPC5-mCherry and TRPA1-mTagBFP knock-in mouse models were successful at the DNA and RNA level. However, at the protein level, TRPC5 resulted in no mCherry fluorescence. In contrast, sensory neurons derived from the TRPA1-reporter mice exhibited visible mTag-BFP fluorescence, although TRPA1 had apparently lost its ion channel function. CONCLUSIONS: Creating transgenic mice with a TRP channel tagged at the C-terminus with a FP requires detailed investigation of the structural and functional consequences in a given cellular context and fine-tuning the design of specific constructs for a given TRP channel subtype. Different degrees of functional impairment of TRPA1 and TRPC5 constructs suggest a specific importance of the distal C-terminus for the regulation of these two channels in trigeminal neurons.
- MeSH
- Red Fluorescent Protein MeSH
- Trigeminal Ganglion metabolism MeSH
- Gene Knock-In Techniques * MeSH
- TRPC Cation Channels * genetics metabolism MeSH
- TRPA1 Cation Channel * genetics metabolism MeSH
- Luminescent Proteins * genetics metabolism MeSH
- Mice, Transgenic * MeSH
- Mice MeSH
- Recombinant Fusion Proteins metabolism genetics MeSH
- Calcium metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Glucocorticoids are potent anti-inflammatory drugs, although their use is associated with severe side effects. Loading glucocorticoids into suitable nanocarriers can significantly reduce these undesirable effects. Macrophages play a crucial role in inflammation, making them strategic targets for glucocorticoid-loaded nanocarriers. The main objective of this study is to develop a glucocorticoid-loaded PLGA nanocarrier specifically targeting liver macrophages, thereby enabling the localized release of glucocorticoids at the site of inflammation. Dexamethasone acetate (DA)-loaded PLGA nanospheres designed for passive macrophage targeting are synthesized using the nanoprecipitation method. Two types of PLGA NSs in the size range of 100-300 nm are prepared, achieving a DA-loading efficiency of 19 %. Sustained DA release from nanospheres over 3 days is demonstrated. Flow cytometry analysis using murine bone marrow-derived macrophages demonstrates the efficient internalization of fluorescent dye-labeled PLGA nanospheres, particularly into pro-inflammatory macrophages. Significant down-regulation in pro-inflammatory cytokine genes mRNA is observed without apparent cytotoxicity after treatment with DA-loaded PLGA nanospheres. Subsequent experiments in mice confirm liver macrophage-specific nanospheres accumulation following intravenous administration using in vivo imaging, flow cytometry, and fluorescence microscopy. Taken together, the data show that the DA-loaded PLGA nanospheres are a promising drug-delivery system for the treatment of inflammatory liver diseases.
- MeSH
- Anti-Inflammatory Agents pharmacology chemistry MeSH
- Dexamethasone * pharmacology chemistry analogs & derivatives MeSH
- Liver * drug effects metabolism MeSH
- Polylactic Acid-Polyglycolic Acid Copolymer * chemistry MeSH
- Macrophages * drug effects metabolism MeSH
- Mice MeSH
- Nanospheres * chemistry MeSH
- Drug Carriers chemistry pharmacology MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Tailocins are nano-scale phage tail-like protein complexes that can mediate antagonistic interactions between closely related bacterial species. While the capacity to produce R-type tailocin was found widely across Gammaproteobacteria, the production of F-type tailocins seems comparatively rare. In this study, we examined the freshwater isolate, Pragia fontium 24613, which can produce both R- and F-type tailocins. We investigated their inhibition spectrum, focusing on clinically relevant enterobacteria, and identified the associated tailocin gene cluster. Transmission electron microscopy confirmed that inactivation of the tape measure protein within the tailocin cluster disrupted R-tailocin production. Comparative analysis of Budviciaceae gene clusters showed high conservation of R-type tailocin genes, whereas F-type tailocin genes were found in only a few species, with little conservation. Our findings indicate a high prevalence of bacteriocin production among underexplored Enterobacteriales species. Detected tailocins showed potential as antimicrobials targeting clinically significant pathogens.
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.
There is increasing pressure on meat producers worldwide due to the need for higher yields and improved meat quality. This is why anabolic androgenic steroids (AAS) have been widely used in most countries, due to their ability to accelerate animal muscle growth. However, out of concern for their side effects, EU states have banned their use and implemented control mechanisms. But they are reaching their limits, and therefore, it is necessary to look for new ways and investigate the mechanism of action of AAS on muscle tissue. This study replicated the administration of banned AAS (testosterone, nandrolone and their combination) and observed their effect on pig muscle. The pig model was purposely chosen for the study, as no such research has been carried out on this species. At the same time, pork is one of the most consumed meats in Europe. It focused on histological changes in muscle structure, specifically the size of muscle fibres and the number of satellite cells per muscle fibre. Furthermore, ultrastructural changes in muscle fibres, the diameter of myofibrils, the number of myofibrils per area, the distance between myofibrils and the size of sarcomeres were examined. The results using the techniques of histology, fluorescent labelling and transmission electron microscopy showed that, after the application of AAS, there is an increase in the diameter of muscle fibres, an increase in the diameter of myofibrils, a decrease in the number of myofibrils per surface area and, in the case of testosterone, an increase in the distance between myofibrils and an increase in the length of sarcomeres. There was also a significant increase in the number of satellite cells per muscle fibre. The detected statistically significant differences between control and experimental groups provide evidence that selected histological parameters could be additional mechanisms for detecting the presence of AAS in pork meat in the future.
- MeSH
- Anabolic Agents * pharmacology MeSH
- Muscle Fibers, Skeletal * drug effects ultrastructure MeSH
- Muscle, Skeletal drug effects anatomy & histology ultrastructure MeSH
- Myofibrils * drug effects ultrastructure MeSH
- Nandrolone * pharmacology MeSH
- Swine anatomy & histology MeSH
- Sarcomeres drug effects ultrastructure MeSH
- Satellite Cells, Skeletal Muscle drug effects ultrastructure MeSH
- Testosterone * pharmacology MeSH
- Microscopy, Electron, Transmission veterinary MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an RNA virus responsible for coronavirus disease 2019 (COVID-19). While SARS-CoV-2 primarily targets the lungs and airways, it can also infect other organs, including the central nervous system (CNS). The aim of this study was to investigate whether the choroid plexus could serve as a potential entry site for SARS-CoV-2 into the brain. Tissue samples from 24 deceased COVID-19-positive individuals were analyzed. Reverse transcription real-time PCR (RT-qPCR) was performed on selected brain regions, including the choroid plexus, to detect SARS-CoV-2 viral RNA. Additionally, immunofluorescence staining and confocal microscopy were used to detect and localize two characteristic proteins of SARS-CoV-2: the spike protein S1 and the nucleocapsid protein. RT-qPCR analysis confirmed the presence of SARS-CoV-2 viral RNA in the choroid plexus. Immunohistochemical staining revealed viral particles localized in the epithelial cells of the choroid plexus, with the spike protein S1 detected in the late endosomes. Our findings suggest that the blood-cerebrospinal fluid (B-CSF) barrier in the choroid plexus serves as a route of entry for SARS-CoV-2 into the CNS. This study contributes to the understanding of the mechanisms underlying CNS involvement in COVID-19 and highlights the importance of further research to explore potential therapeutic strategies targeting this entry pathway.
- MeSH
- COVID-19 * virology MeSH
- Adult MeSH
- Phosphoproteins * metabolism MeSH
- Spike Glycoprotein, Coronavirus * genetics metabolism MeSH
- Blood-Brain Barrier * virology MeSH
- Virus Internalization MeSH
- Coronavirus Nucleocapsid Proteins MeSH
- Middle Aged MeSH
- Humans MeSH
- Choroid Plexus * virology MeSH
- RNA, Viral * genetics MeSH
- SARS-CoV-2 * physiology MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
160 stran : ilustrace ; 28 cm
Atlas, který se zaměřuje na anatomii lidského mozku, zobrazenou mikroskopicky, makroskopicky a pomocí magnetické rezonance. Určeno odborné veřejnosti.
- MeSH
- Magnetic Resonance Imaging MeSH
- Microscopy MeSH
- Brain anatomy & histology diagnostic imaging MeSH
- Neuroanatomy MeSH
- Publication type
- Atlas MeSH
Využití digitální patologie a umělé inteligence v anatomické patologii představuje revoluční krok směrem k modernizaci diagnostických procesů. Digitalizace, postavená zejména na využívání tzv. whole slide imaging, umožňuje vytvářet celoplošné digitální obrazy histologických preparátů, což přináší potenciální benefity v oblasti přesnosti a dostupnosti diagnostiky. Na rozdíl od tradiční mikroskopie poskytuje digitální patologie též možnost telemedicíny a vzdálené konzultace, čímž otevírá nové možnosti spolupráce a sdílení odborných znalostí na národní i mezinárodní úrovni. Implementace digitálního pracovního postupu nicméně vyžaduje rozsáhlé investice do skenerů, softwarových platforem, vysokokapacitních úložišť a IT infrastruktury. Navzdory nemalým nákladům na implementaci však přináší řadu výhod, včetně časových úspor, možnosti centralizace diagnostiky a snížení nákladů na transport vzorků. Tento příspěvek se zaměřuje na praktické aspekty implementace digitální patologie v patologických laboratořích s důrazem na přínosy, rizika a technologické požadavky spojené s digitalizací a diskutuje i zásadní role validace a verifikace celého nového pracovního procesu. Článek představuje digitální patologii jako dynamicky se rozvíjející obor s vysokým potenciálem pro personalizovanou medicínu, zlepšení diagnostické přesnosti a podporu vzdálené spolupráce, čímž reaguje na rostoucí nároky moderní medicíny.
The application of digital pathology and artificial intelligence in anatomical pathology represents a revolutionary step towards the modernization of diagnostic processes. Digitalization, primarily based on creation and subsequent use of whole slide imaging, enables generating of full digital images of histological slides, offering potential benefits in diagnostic accuracy and accessibility. Unlike traditional microscopy, digital pathology also facilitates telemedicine and remote consultation, opening new possibilities for collaboration and sharing of expertise at both national and international levels. However, implementing a digital workflow requires substantial investments in scanners, software platforms, high-capacity storage, and IT infrastructure. Despite considerable costs of implementation, it brings numerous advantages, including time savings, opportunities for centralized diagnostics, and a reduction in sample transport costs. This paper focuses on the practical aspects of implementing digital pathology in pathology laboratories, emphasizing the benefits, risks, and technological requirements associated with digitalized workflows. It also discusses crucial roles of validation and verification, which are essential for ensuring a diagnostic accuracy of digital images compared to conventional microscopy. The article presents digital pathology as a dynamically evolving field with high potential for personalized medicine, improved diagnostic accuracy, and support for remote collaboration, addressing the growing demands of modern medicine.
- MeSH
- Humans MeSH
- Pathology * trends MeSH
- Machine Learning * trends MeSH
- Telemedicine trends MeSH
- Check Tag
- Humans MeSH
Rentgenová výpočetní mikrotomografie (mikroCT) představuje moderní zobrazovací technologii s vysokým rozlišením umožňující detailní analýzu zobrazovaného vzorku. Nabízí jedinečný pohled na trojrozměrnou architekturu díky rozlišení na pomezí makroskopického a histologického zobrazení. V oblasti anatomické patologie mikroCT nachází uplatnění zejména při morfometrické analýze nádorů, hodnocení resekčních okrajů chirurgických vzorků či detekci metastáz v lymfatických uzlinách. Kombinace mikroCT s tradičními histopatologickými technikami a s využitím digitální 3D rekonstrukce otevírá nové možnosti při analýze komplexních patologických procesů. Přestože je tato metoda zatím převážně využívána ve výzkumu, její klinický potenciál je značný. Mezi hlavní přednosti patří neinvazivní zobrazení a možnost integrace s digitální patologií a nástroji umělé inteligence. Hlavními limitacemi v současné době zůstávají potřeba kontrastování vzorků, monochromatická povaha obrazu a vysoká radiační zátěž. Pokrok v technologickém vývoji však může tyto překážky překonat a umožnit širší využití mikroCT v rutinní klinické diagnostice. Tento článek představuje technologii mikroCT a její diagnostický potenciál v patologii, přibližuje její aplikace, výhody a omezení, a nabízí vhled do budoucí perspektivy jejího využití.
X-ray microtomography (microCT) represents a modern high-resolution imaging technology enabling detailed analysis of the tissue. It offers a unique perspective on three-dimensional architecture, bridging the gap between macroscopic and histological imaging. In anatomical pathology, microCT is particularly utilized for morphometric tumor analysis, evaluation of surgical specimen resection margins, and detection of metastases in lymph nodes. The combination of microCT with traditional histopathological techniques, and with digital 3D reconstructions, opens new avenues for analyzing complex pathological processes. Although this method is currently used in research, its clinical potential is significant. Key advantages include non-invasive imaging and the ability to be integrated with digital pathology and artificial intelligence tools. Current limitations include the need for sample contrast enhancement, the monochromatic nature of the images, and high radiation exposure. Advances in technological development, however, may overcome these barriers and enable the broader adoption of microCT in routine clinical diagnostics. This article explores the diagnostic potential of microCT in pathology, highlighting its applications, advantages, and limitations, while offering insights into current capabilities and future perspectives of this technology.
Severe acute pancreatitis (SAP) is associated with metabolic disorders, hypocalcemia, and multiple organ failure. The objective of this study was to investigate changes in thyroid ultrastructure and function in rats with SAP and to provide a theoretical basis for the clinical treatment of thyroid injury in patients with SAP. 64 male SPF Wistar rats were randomly divided into the SAP group and the control group. Pancreatic enzymatic indicators and thyroid hormones were detected, pathology scores were evaluated, and morphological changes were observed under light microscopy and transmission electron microscopy (TEM) in both groups. The serum levels of triiodothyronine (T3), tetraiodothyronine (T4) and Ca2+ were significantly lower in the SAP group than in the control group (P<0.05), whereas the level of calcitonin (CT) was significantly higher than that in the control group (P<0.05). The thyroid structure (pathology and electron microscopy) of the SAP rats was seriously damaged and worsened over time. SAP can cause thyroid injury through a variety of mechanisms, which can also retroact to pancreatitis to aggravate the inflammatory response. This study may have theoretical significance for basic research on SAP. Key words Severe acute pancreatitis, Thyroid, Structure and functional changes, Transmission electron microscopy.
- MeSH
- Acute Disease MeSH
- Thyroid Hormones blood MeSH
- Rats MeSH
- Disease Models, Animal MeSH
- Pancreatitis * pathology MeSH
- Rats, Wistar * MeSH
- Thyroid Gland * pathology ultrastructure metabolism MeSH
- Severity of Illness Index MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
