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.
In screening biocontrol strains with broad-spectrum and high-efficiency herbicidal activities, a strain with strong pathogenicity, HY-021, was isolated from the leaves of Rumex acetosa, which was identified as Botrytis fabiopsis based on morphology and molecular biology. The herbicidal activities of the fermentation filtrate of strain HY-021 against nine weeds, including Chenopodium album L., Elsholtzia densa Benth., Malva verticillata L. var. Crispa, Polygonum lapathifolium L., Amaranthus retroflexus L., Avena fatua L., Thlaspi arvense L., Polygonum aviculare L., and Galium spurium L., were determined in vitro and in vivo. The results showed that the pathogenicity of strain HY-021 to the different weeds in vitro was as follows: E. densa > A. retroflexus > P. aviculare > P. lapathifolium > M. verticillata > T. arvense > G. spurium > A. fatua > C. album. Seven days after inoculation with the HY-021 strain, the incidences in nine weeds were in the range of 32.9-87.23%, and the disease index values of the nine weeds were 41.73-94.57%. The pathogenic effects from high to low were A. retroflexus > E. densa > A. fatua > G. spurium > C. album > M. verticillata > T. arvense > P. aviculare > P. lapathifolium. The crop safety test showed that the biocontrol strain HY-021 was safe to V. faba, P. sativum, H. vulgare, and T. aestivum, but had a slight effect on B. napus. Scanning electron microscopy showed that the mycelium of strain HY-021 invaded the tissue through the stomata of C. album leaves, parasitized and reproduced in the tissue, and gradually destroyed the tissue. The results of this study provide a basis for the development and utilization of new and efficient microbial source herbicides.
- MeSH
- Pest Control, Biological * methods MeSH
- Biological Control Agents * MeSH
- Botrytis * isolation & purification physiology genetics pathogenicity MeSH
- Herbicides metabolism pharmacology MeSH
- Weed Control * methods MeSH
- Plant Leaves microbiology MeSH
- Plant Diseases * microbiology prevention & control MeSH
- Plant Weeds * microbiology MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- China MeSH
Acinetobacter baumannii thrives within eukaryotic cells, influencing persistence, treatment approaches, and progression of disease. We probed epithelial cell invasion by A. baumannii and the influence of antibodies raised to outer membrane protein 34 (Omp34) on epithelial interactions. We expressed and purified recombinant Omp34 and induced anti-Omp34 antibodies in Bagg albino or BALB/c mice. Omp34 was evaluated for acute toxicity in mice through histological analysis of six organs. The host cell line, A549, was exposed to both A. baumannii 19606 and a clinical isolate. The study also investigated serum resistance, adherence, internalization, and proliferation of A. baumannii in A549 cells, with and without anti-Omp34 sera, utilizing cell culture techniques and light microscopy. A549 cell viability was evaluated by A. baumannii challenge and exposure to anti-Omp34 sera. Actin disruption experiments using cytochalasin D probed microfilament and microtubule roles in A. baumannii invasion. Omp34 prompted antibody production without toxicity in mice. The serum showed bactericidal effects on both strains. Additionally, both A. baumannii strains were found to form biofilms. Omp34 serum was observed to decrease biofilm formation, bacterial adherence, internalization, and proliferation in A549 cells. Furthermore, the use of anti-Omp34 serum enhanced the post-infection survival of the host cell. Pre-exposure of A549 cells to cytochalasin D reduced bacterial internalization, highlighting the role of actin polymerization in the invasion process. Microscopic analysis revealed various interactions, such as adherence, membrane alterations, vacuolization, apoptosis, and cellular damage. Anti-Omp34 serum-exposed A549 cells were protected and showed reduced damage. The findings reveal that A. baumannii can significantly multiply intracellularly within host cells. This suggests the bacterium's ability to establish an environment conducive to its replication by preventing fusion with degradative lysosomes and inhibiting acidification. This finding contributes to the understanding of A. baumannii's intracellular persistence and highlights the role of Omp34 in influencing apoptosis, autophagy, and bacterial adherence, which may impact the development of effective treatments against A. baumannii infections.
- MeSH
- Acinetobacter baumannii * physiology immunology pathogenicity MeSH
- Bacterial Adhesion * MeSH
- Biofilms growth & development MeSH
- A549 Cells MeSH
- Epithelial Cells microbiology MeSH
- Acinetobacter Infections * microbiology immunology MeSH
- Humans MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Antibodies, Bacterial * immunology MeSH
- Cell Survival MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
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.
G protein-coupled receptors (GPCRs) play a crucial role in cell function by transducing signals from the extracellular environment to the inside of the cell. They mediate the effects of various stimuli, including hormones, neurotransmitters, ions, photons, food tastants and odorants, and are renowned drug targets. Advancements in structural biology techniques, including X-ray crystallography and cryo-electron microscopy (cryo-EM), have driven the elucidation of an increasing number of GPCR structures. These structures reveal novel features that shed light on receptor activation, dimerization and oligomerization, dichotomy between orthosteric and allosteric modulation, and the intricate interactions underlying signal transduction, providing insights into diverse ligand-binding modes and signalling pathways. However, a substantial portion of the GPCR repertoire and their activation states remain structurally unexplored. Future efforts should prioritize capturing the full structural diversity of GPCRs across multiple dimensions. To do so, the integration of structural biology with biophysical and computational techniques will be essential. We describe in this review the progress of nuclear magnetic resonance (NMR) to examine GPCR plasticity and conformational dynamics, of atomic force microscopy (AFM) to explore the spatial-temporal dynamics and kinetic aspects of GPCRs, and the recent breakthroughs in artificial intelligence for protein structure prediction to characterize the structures of the entire GPCRome. In summary, the journey through GPCR structural biology provided in this review illustrates how far we have come in decoding these essential proteins architecture and function. Looking ahead, integrating cutting-edge biophysics and computational tools offers a path to navigating the GPCR structural landscape, ultimately advancing GPCR-based applications. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.
- MeSH
- Protein Conformation MeSH
- Humans MeSH
- Receptors, G-Protein-Coupled * chemistry metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Cíl: Používání filtroventrilačních systémů v městských autobusech ve vyspělých zemích zvyšuje komfort a kvalitu vnitřního ovzduší v prostředcích pozemní dopravy. Mikrobiální kontaminace byla studována na výstupních a vstupních plochách 5 vzduchových filtrů vyjmutých z klimatizačního systému městských autobusů při pravidelné údržbě. Materiál a metodika: K získání vzorků z výstupní i vstupní strany filtrů byla použita technika suchého stěru. Kultivace byla provedena na různých selektivních nebo selektivně-diagnostických půdách pro kultivaci životaschopných bakterií. K identifikaci bakteriálních druhů bylo použito barvení podle Grama a imerzní mikroskopie. Vybrané kolonie byly rovněž podrobeny proteomické studii. Po identifikaci byly bakterie kvantifikovány. Výsledky: Na vstupním i výstupním povrchu filtrů převažovaly bakterie rodu Bacillus – Bacillus cereus, Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus flexus. Identifikovány byli také bakterie rodů Staphylococcus, Brevibacillus, Peribacillus a Paenibacillus. Kvantifikace ukázala nízkou kontaminaci výstupních povrchů filtrů 1 a 2. Kontaminace vstupní a výstupní strany filtrů 3, 4 a 5 a odhalila téměř stejnou kontaminaci vstupních a výstupních ploch. Závěry: Podle nalezených výsledků doporučujeme buď častější výměnu filtrů, nebo volbu filtrů s nižší porozitou.
The use of HVAC in urban buses in developed countries increases the comfort and indoor air quality in the means of ground transportation. The microbial contamination was studied on outlet and inlet surfaces of 5 air filters removed from the urban buses HVAC during regular maintenance. To acquire samples from both the outlet and the inlet sides of the filters, dry swabbing technique was used. Cultivation was performed on different selective or selective-diagnostic agars, to cultivate viable bacteria. To identify the bacterial species, Gram stain and immerse microscopy was used. Selected colonies underwent the proteomic study (MALDI-TOF) as well. After identification, bacteria were quantified. The bacteria of the genus Bacillus – Bacillus cereus, Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus flexus prevailed on both inlet and outlet surfaces of the filters. The members of genera Staphylococcus, Brevibacillus, Peribacillus or Paenibacillus were also identified. The quantification of colony forming units showed low contamination of the outlet surfaces of filters 1 and 2. The contamination of inlet and outlet sides of filters 3, 4, and 5 was comparable, revealed nearly the same contamination of inlet and outlet surfaces. In the case of filters 3, 4 and 5 we recommend more frequent filter changing or more efficient filter choice.
Autoři popisují případ 62leté ženy, která několik měsíců pozorovala na zadní straně pravého stehna asymptomatický kožní projev sestávající z mnohočetných tmavých drsných papul seskupených na malé ploše. Dermatoskopické a histologické vyšetření potvrdilo diagnózu naevus comedonicus. Práce uvádí přehled současných poznatků o tomto onemocnění.
Stationary papular keratotic plaque on the thigh – Nevus Comedonicus An asymptomatic keratotic skin lesion lasting several months developed on the thigh of a 62-year-old female. The plaque was composed of multiple dark rough papules grouped in a small area. Clinical, dermoscopic and histopathologic diagnosis of naevus comedonicus was established. The article presents an overview of current knowledge about this disability.
- Keywords
- naevus comedonicus,
- MeSH
- Dermoscopy methods MeSH
- Diagnosis, Differential MeSH
- Hamartoma * diagnosis classification pathology MeSH
- Skin Diseases diagnosis classification pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Nevus * diagnosis classification pathology MeSH
- Hair Follicle pathology MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Case Reports MeSH
Laboratorní diagnostika infekčního agens je založena na metodách přímého průkazu (kultivace, mikroskopie, PCR, antigenní test) a na metodách nepřímého průkazu (detekce protilátek). Sérologie zůstává důležitou součástí laboratorní diagnostiky infekčních onemocnění, přestože význam některých sérologických testů klesá s rozvojem molekulárně biologických metod. Pro pochopení, jakou roli hrají protilátky v procesu imunitní odpovědi na infekční agens, je nutné znát obecné mechanismy, které jsou s tvorbou protilátek spojené. Interpretace sérologických výsledků je složitá a závisí na fázi imunitní odpovědi, antigenních vlastnostech patogenu, použité metodě, senzitivitě a specificitě testů a klinickém kontextu konkrétního pacienta. Správná interpretace sérologických testů vyžaduje hlubokou znalost patogeneze infekce (specifická reakce na bakterie, viry nebo prvoky), metodických limitací a klinických souvislostí, což je zásadní pro efektivní diagnostiku a léčbu pacientů v klinické praxi.
Martinek J, Lochmanová A, Maďar R. Interpretation of serological results in the diagnosis of infectious diseases Laboratory diagnosis of infectious agents is based on direct detection methods (culture, microscopy, PCR, antigen test) and indirect detection methods (antibody detection). Serology remains an important part of the laboratory diagnosis of infectious diseases, although the importance of some serological tests is declining with the development of molecular biological methods. To understand the role of antibodies in the immune response to infectious agents, it is necessary to know the general mechanisms involved in antibody production. Interpretation of serological results is complex and depends on the phase of the immune response, the antigenic properties of the pathogen, the method used, the sensitivity and specificity of the tests, and the clinical context of the individual patient. Correct interpretation of serological tests requires an in-depth knowledge of the pathogenesis of infection (specific response to bacteria, viruses or protozoa), methodological limitations and clinical context, which is essential for effective diagnosis and treatment of patients in clinical practice.
