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.
Bioethanol production from lignocellulosic materials is hindered by the high costs of pretreatment and the enzymes. The present study aimed to evaluate whether co-cultivation of four selected cellulolytic fungi yields higher cellulase and xylanase activities compared to the monocultures and to investigate whether the enzymes from the co-cultures yield higher saccharification on selected plant materials without thermo-chemical pretreatment. The fungal isolates, Trichoderma reesei F118, Penicillium javanicum FS7, Talaromyces sp. F113, and Talaromyces pinophilus FM9, were grown as monocultures and binary co-cultures under submerged conditions for 7 days. The cellulase and xylanase activities of the culture filtrates were measured, and the culture filtrates were employed for the saccharification of sugarcane leaves, Guinea grass leaves, and water hyacinth stems and leaves. Total reducing sugars and individual sugars released from each plant material were quantified. The co-culture of Talaromyces sp. F113 with Penicillium javanicum FS7 and of T. reesei F118 with T. pinophilus FM9 produced significantly higher cellulase activities compared to the corresponding monocultures whereas no effect was observed on xylanase activities. Overall, the highest amounts of total reducing sugars and individual sugars were obtained from Guinea grass leaves saccharified with the co-culture of T. reesei F118 with T. pinophilus FM9, yielding 63.5% saccharification. Guinea grass leaves were found to be the most susceptible to enzymatic saccharification without pre-treatment, while water hyacinth stems and leaves were the least. Accordingly, the study suggests that fungal co-cultivation could be a promising approach for the saccharification of lignocellulosic materials for bioethanol production.
- MeSH
- Cellulase * metabolism MeSH
- Endo-1,4-beta Xylanases metabolism MeSH
- Ethanol metabolism MeSH
- Hypocreales enzymology metabolism growth & development MeSH
- Coculture Techniques * MeSH
- Lignin * metabolism MeSH
- Plant Leaves microbiology MeSH
- Penicillium * enzymology metabolism growth & development MeSH
- Saccharum * microbiology metabolism MeSH
- Talaromyces * enzymology metabolism growth & development MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Leukemia is driven by complex interactions within the inherently hypoxic bone marrow microenvironment, impacting both disease progression and therapeutic resistance. Co-cultivation of leukemic cells with feeder cells has emerged as a valuable tool to mimic the bone marrow niche. This study explores the interplay between human commercial SD-1 and patient-derived UPF26K leukemic cell lines with feeders - human fibroblasts (NHDF) and mesenchymal stem cells (hMSCs) under normoxic and hypoxic conditions. RESULTS: Co-cultivation with feeders significantly enhances proliferation and glycolytic activity in the SD-1 cells, improving their viability, while this interaction inhibits the growth and glucose metabolism of the feeders, particularly NHDF. In contrast, UPF26K cells show reduced proliferation when co-cultivated with the feeders while this interaction stimulates NHDF and hMSCs proliferation and glycolysis but reduce their mitochondrial metabolism with hypoxia amplifying these effects. CONCLUSIONS: Cells that switch to glycolysis during co-cultivation, particularly under hypoxia, benefit most from these low oxygen conditions. Due to this leukemic cells' response heterogeneity, targeting microenvironmental interactions and oxygen levels is crucial for personalized leukemia therapy. Advancing co-cultivation models, particularly through innovations like spheroids, can further enhance in vitro studies of primary leukemic cells and support the testing of novel therapies.
- MeSH
- Fibroblasts * metabolism MeSH
- Glycolysis MeSH
- Cell Hypoxia MeSH
- Coculture Techniques methods MeSH
- Leukemia * pathology metabolism MeSH
- Humans MeSH
- Mesenchymal Stem Cells * metabolism MeSH
- Mitochondria metabolism MeSH
- Cell Line, Tumor MeSH
- Tumor Microenvironment MeSH
- Cell Proliferation MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
OBJECTIVES: Treponema pallidum subsp. pallidum (T. pallidum) is the etiological agent of syphilis, a sexually transmitted disease of global public health importance. The objective of this study was to introduce a novel in vitro protocol for isolation of T. pallidum directly from patients' clinical samples, eliminating the need for rabbit propagation. METHODS: Four oral and five genital swabs were collected from nine epidemiologically unrelated patients at two hospitals in Brno, Czech Republic. Swabs were submerged in TpCM-2 medium for transport. Samples were then placed on a 0.4 μm filters and incubated for 2.5 hours. During this period, spiral T. pallidum cells passed through the filter pores to the well containing TpCM-2 medium and rabbit feeder cells (Sf1Ep). Stable T. pallidum cultures (containing >1 × 107 treponemes) were achieved by subculturing every 7 days into fresh well. RESULTS: A successful protocol for in vitro isolation of T. pallidum was established. From the nine clinical specimens processed, six T. pallidum cultures (MU1-MU6) were derived after 14 to 112 days of cultivation. Five of these strains (MU1-MU5) belonged to SS14-like cluster and shared the same allelic profile 1.3.1. The remaining strain (MU6) was identified as a Nichols-like strain with an allelic profile 9.16.3. DISCUSSION: The introduced in vitro protocol enables isolation of T. pallidum from clinical material, including frozen samples, without the need for experimental rabbits. This method facilitates the isolation of contemporary, clinically relevant treponemal strains.
- MeSH
- Bacteriological Techniques * methods MeSH
- Rabbits MeSH
- Humans MeSH
- Genitalia microbiology MeSH
- Syphilis * microbiology diagnosis MeSH
- Treponema pallidum * isolation & purification genetics classification MeSH
- Mouth microbiology MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
For the treatment of bilateral limbal stem cell deficiency (LSCD), cell therapy with transplantation of cultivated oral mucosa epithelial cells (COMET) is a promising alternative. Although not yet established, current protocols on the cultivation of oral mucosal epithelial cell (OMECs) sheets are based mainly on substrates and xenobiotic additives that may lead to variable outcomes and undesirable immune responses by the patient. The aim of this study was to characterize OMECs cultivated in xenobiotic-free media (XF) seeded on fibrin gel, in comparison to conventional complex (COM) medium. Oral mucosal biopsies were retrieved from 31 donors. After cultivation in COM or XF medium, OMECs were compared based on growth kinetics, morphology, cell size and viability. Using immunofluorescence and gene expression analyses, the degree of stemness, proliferation and differentiation was evaluated in OMEC cultures. Our findings showed that although OMECs showed a similar morphology and viability, and comparable growth kinetics, immunofluorescence revealed the preservation of stemness (p63 + p40 positivity in cells ≤11 μm) and proliferation in both COM and XF. Gene expression analyses showed that keratin (K)13 and K15 expression levels were significantly higher in XF (adj. p < 0.001), but otherwise COM and XF-treated OMECs had comparable transcriptional profiles in a panel of stemness, proliferation and differentiation genes. These results demonstrate the feasibility of culturing OMECs on fibrin gel without xenogeneic additives, while maintaining their undifferentiated state and preserving stemness. In conclusion, both in terms of results and methodology, the procedures presented here are suitable for implementation in clinical practice.
- MeSH
- Cell Differentiation MeSH
- Cell Culture Techniques * MeSH
- Limbal Stem Cell Deficiency MeSH
- Adult MeSH
- Epithelial Cells * metabolism drug effects MeSH
- Fibrin * MeSH
- Gels MeSH
- Stem Cells * metabolism cytology MeSH
- Culture Media MeSH
- Cells, Cultured MeSH
- Middle Aged MeSH
- Humans MeSH
- Limbus Corneae * cytology pathology metabolism MeSH
- Corneal Diseases pathology drug therapy metabolism MeSH
- Cell Proliferation drug effects MeSH
- Epithelium, Corneal metabolism cytology drug effects pathology MeSH
- Aged MeSH
- Stem Cell Transplantation methods MeSH
- Mouth Mucosa * cytology MeSH
- Cell Survival MeSH
- Xenobiotics pharmacology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Psilocybe cubensis, a widely recognized psychoactive mushroom species, has played a significant role in both historical and modern therapeutic practices. This review explores the complex interplay between genetic diversity, strain variability and environmental factors that shape the biosynthesis of key psychoactive compounds, including psilocybin and psilocin. With many strains exhibiting substantial variability in their phenotypic characteristics and biochemical content, understanding and documenting this diversity is crucial for optimizing therapeutic applications. The review also highlights advances in cultivation techniques, such as submerged fermentation of the mycelium, and innovative analytical methodologies that have improved the precision of compound quantification and extraction. Although there is limited scientific information on P. cubensis due to nearly four decades of regulatory restrictions on psychedelic research, recent developments in genetic and biochemical studies are beginning to provide valuable insights into its therapeutic potential. Furthermore, this review emphasizes key knowledge gaps and offers insights into future research directions to advance the cultivation, scientific documentation of strain diversity, regulatory considerations and therapeutic use of P. cubensis.
- Publication type
- Journal Article MeSH
- Review MeSH
The oviduct provides an optimal environment for the final preparation, transport, and survival of gametes, the fertilization process, and early embryonic development. Most of the studies on reproduction are based on in vitro cell culture models because of the cell's accessibility. It creates opportunities to explore the complexity of directly linked processes between cells. Previous studies showed a significant expression of genes responsible for cell differentiation, maturation, and development during long-term porcine oviduct epithelial cells (POECs) in vitro culture. This study aimed at establishing the transcriptomic profile and comprehensive characteristics of porcine oviduct epithelial cell in vitro cultures, to compare changes in gene expression over time and deliver information about the expression pattern of genes highlighted in specific GO groups. The oviduct cells were collected after 7, 15, and 30 days of in vitro cultivation. The transcriptomic profile of gene expression was compared to the control group (cells collected after the first day). The expression of COL1A2 and LOX was enhanced, while FGFBP1, SERPINB2, and OVGP1 were downregulated at all selected intervals of cell culture in comparison to the 24-h control (p-value < 0.05). Adding new detailed information to the reproductive biology field about the diversified transcriptome profile in POECs may create new future possibilities in infertility treatments, including assisted reproductive technique (ART) programmes, and may be a valuable tool to investigate the potential role of oviduct cells in post-ovulation events.
- MeSH
- Cell Culture Techniques methods MeSH
- Epithelial Cells * metabolism cytology MeSH
- Cells, Cultured MeSH
- Swine MeSH
- Gene Expression Regulation MeSH
- Gene Expression Profiling MeSH
- Transcriptome * MeSH
- Oviducts metabolism cytology MeSH
- Fallopian Tubes metabolism cytology MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 μm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, β-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.
- MeSH
- Cell Differentiation * physiology MeSH
- Cell Culture Techniques methods MeSH
- Cell Line MeSH
- Spheroids, Cellular * metabolism MeSH
- Mice MeSH
- Neurons metabolism MeSH
- Prion Diseases * metabolism pathology MeSH
- Prions metabolism MeSH
- Cell Culture Techniques, Three Dimensional methods MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Drug delivery to central nervous pathologies is compromised by the blood-brain barrier (BBB). A clinically explored strategy to promote drug delivery across the BBB is sonopermeation, which relies on the combined use of ultrasound (US) and microbubbles (MB) to induce temporally and spatially controlled opening of the BBB. We developed an advanced in vitro BBB model to study the impact of sonopermeation on the delivery of the prototypic polymeric drug carrier pHPMA as a larger molecule and the small molecule antiviral drug ribavirin. This was done under standard and under inflammatory conditions, employing both untargeted and RGD peptide-coated MB. The BBB model is based on human cerebral capillary endothelial cells and human placental pericytes, which are co-cultivated in transwell inserts and which present with proper transendothelial electrical resistance (TEER). Sonopermeation induced a significant decrease in TEER values and facilitated the trans-BBB delivery of fluorescently labeled pHPMA (Atto488-pHPMA). To study drug delivery under inflamed endothelial conditions, which are typical for e.g. tumors, neurodegenerative diseases and CNS infections, tumor necrosis factor (TNF) was employed to induce inflammation in the BBB model. RGD-coated MB bound to and permeabilized the inflamed endothelium-pericyte co-culture model, and potently improved Atto488-pHPMA and ribavirin delivery. Taken together, our work combines in vitro BBB bioengineering with MB-mediated drug delivery enhancement, thereby providing a framework for future studies on optimization of US-mediated drug delivery to the brain.
- MeSH
- Antiviral Agents administration & dosage chemistry pharmacology pharmacokinetics MeSH
- Endothelial Cells * drug effects metabolism MeSH
- Blood-Brain Barrier * metabolism MeSH
- Coculture Techniques * MeSH
- Drug Delivery Systems methods MeSH
- Humans MeSH
- Microbubbles * MeSH
- Oligopeptides * chemistry administration & dosage pharmacokinetics MeSH
- Pericytes * metabolism drug effects MeSH
- Polymers chemistry administration & dosage MeSH
- Ribavirin administration & dosage chemistry pharmacokinetics MeSH
- Ultrasonic Waves MeSH
- Inflammation drug therapy MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Bartonelloses are neglected emerging infectious diseases caused by facultatively intracellular bacteria transmitted between vertebrate hosts by various arthropod vectors. The highest diversity of Bartonella species has been identified in rodents. Within this study we focused on the edible dormouse (Glis glis), a rodent with unique life-history traits that often enters households and whose possible role in the epidemiology of Bartonella infections had been previously unknown. We identified and cultivated two distinct Bartonella sub(species) significantly diverging from previously described species, which were characterized using growth characteristics, biochemical tests, and various molecular techniques including also proteomics. Two novel (sub)species were described: Bartonella grahamii subsp. shimonis subsp. nov. and Bartonella gliris sp. nov. We sequenced two individual strains per each described (sub)species. During exploratory genomic analyses comparing two genotypes ultimately belonging to the same species, both factually and most importantly even spatiotemporally, we noticed unexpectedly significant structural variation between them. We found that most of the detected structural variants could be explained either by prophage excision or integration. Based on a detailed study of one such event, we argue that prophage deletion represents the most probable explanation of the observed phenomena. Moreover, in one strain of Bartonella grahamii subsp. shimonis subsp. nov. we identified a deletion related to Bartonella Adhesin A, a major pathogenicity factor that modulates bacteria-host interactions. Altogether, our results suggest that even a limited number of passages induced sufficient selective pressure to promote significant changes at the level of the genome.
- Publication type
- Journal Article MeSH
