Bordetella pertussis is the causative agent of whooping cough in humans, a disease that has recently experienced a resurgence. In contrast, Bordetella bronchiseptica infects the respiratory tract of various mammalian species, causing a range of symptoms from asymptomatic chronic carriage to acute illness. Both pathogens utilize type III secretion system (T3SS) to deliver the effector protein BteA into host cells. Once injected, BteA triggers a cascade of events leading to caspase 1-independent necrosis through a mechanism that remains incompletely understood. We demonstrate that BteA-induced cell death is characterized by the fragmentation of the cellular endoplasmic reticulum and mitochondria, the formation of necrotic balloon-like protrusions, and plasma membrane permeabilization. Importantly, genome-wide CRISPR-Cas9 screen targeting 19,050 genes failed to identify any host factors required for BteA cytotoxicity, suggesting that BteA does not require a single nonessential host factor for its cytotoxicity. We further reveal that BteA triggers a rapid and sustained influx of calcium ions, which is associated with organelle fragmentation and plasma membrane permeabilization. The sustained elevation of cytosolic Ca2+ levels results in mitochondrial calcium overload, mitochondrial swelling, cristolysis, and loss of mitochondrial membrane potential. Inhibition of calcium channels with 2-APB delays both the Ca2+ influx and BteA-induced cell death. Our findings indicate that BteA exploits essential host processes and/or redundant pathways to disrupt calcium homeostasis and mitochondrial function, ultimately leading to host cell death.IMPORTANCEThe respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica exhibit cytotoxicity toward a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of B. bronchiseptica is associated with enhanced expression of T3SS and BteA. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.

• MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• Bordetella bronchiseptica genetika   metabolismus   účinky léků   MeSH
• Bordetella pertussis genetika   patogenita   metabolismus   účinky léků   MeSH
• buněčná smrt * účinky léků   MeSH
• endoplazmatické retikulum metabolismus   účinky léků   MeSH
• homeostáza * MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• sekreční systém typu III metabolismus   genetika   MeSH
• vápník * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Perineuronal nets (PNNs) enwrap mature neurons, playing a role in the control of plasticity and synapse dynamics. PNNs have been shown to have effects on memory formation, retention and extinction in a variety of animal models. It has been proposed that the cavities in PNNs, which contain synapses, can act as a memory store and that they remain stable after events that cause synaptic withdrawal such as anoxia or hibernation. We examine this idea by monitoring place memory before and after synaptic withdrawal caused by acute hibernation-like state (HLS). Animals lacking hippocampal PNNs due to enzymatic digestion by chondroitinase ABC or knockout of the PNN component aggrecan were compared with wild type controls. HLS-induced synapse withdrawal caused a memory deficit, but not to the level of untreated naïve animals and not worsened by PNN attenuation. After HLS, only animals lacking PNNs showed memory restoration or relearning. Absence of PNNs affected the restoration of excitatory synapses on PNN-bearing neurons. The results support a role for hippocampal PNNs in learning, but not in long-term memory storage for correction of deficits.

• MeSH
• extracelulární matrix - proteiny MeSH
• extracelulární matrix * MeSH
• neurony fyziologie   MeSH
• synapse * MeSH
• učení MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

• MeSH
• alografty diagnostické zobrazování   účinky léků   MeSH
• apoptóza účinky léků   MeSH
• dárci tkání MeSH
• dimethylsulfoxid farmakologie   MeSH
• fluorescenční barviva * MeSH
• konfokální mikroskopie metody   MeSH
• kryoprezervace metody   MeSH
• kryoprotektivní látky farmakologie   MeSH
• lidé MeSH
• optické zobrazování metody   MeSH
• transplantace cév metody   MeSH
• vena femoralis diagnostické zobrazování   účinky léků   MeSH
• vena saphena diagnostické zobrazování   účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• zmrazování * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork.

• MeSH
• blízká infračervená spektroskopie metody   MeSH
• fibrosarkom patologie   MeSH
• invazivní růst nádoru MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• nádorové buňky kultivované MeSH
• nádory prsu patologie   MeSH
• počítačové zpracování obrazu MeSH
• podozomy patologie   MeSH
• zobrazování trojrozměrné metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The aim of our study was to assess the impact of different thawing protocols on morphological changes arising in cryopreserved human saphenous vein grafts. METHODS: The study was performed in 12 saphenous vein grafts harvested in brain death donors. Storage in the vapor phase of liquid nitrogen for 3 or 5 years followed. Two thawing protocols were tested: slow thawing in a refrigerator at temperature +4°C for 2 hr and rapid thawing-in a water bath at +37°C. Grafts were processed for scanning electron microscopy. Comparisons of continuous parameters under study between experimental groups were performed using the t-test (age, cold ischemia time, exposure to cryoprotectant, time of storage, total thawing time, mean thawing rate, morphology scoring of thawed HSVG) and the median test (HSVG length). Categorical parameters (sex and blood group) were formally tested using the chi-square test. RESULTS: All samples were evaluated according to morphological changes and scored in terms of morphologically intact endothelium, confluent endothelium with structural inhomogeneity, disruption of the intercellular contacts, separation of the endothelial cells, complete loss of the endothelium, and damage of the subendothelial layers. There is no statistically significant difference between the sample sets at the significance level of 0.05. There was no association with donors' age, sex, and time of storage. CONCLUSIONS: Human cryopreserved saphenous vein grafts in our experimental work showed no difference in terms of structural deterioration of the endothelial surface and basal membrane depending on different thawing protocols used.

• MeSH
• časové faktory MeSH
• dospělí MeSH
• endoteliální buňky účinky léků   transplantace   ultrastruktura   MeSH
• kryoprezervace * MeSH
• kryoprotektivní látky farmakologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• odběr tkání a orgánů MeSH
• přežití tkáně MeSH
• vena saphena účinky léků   transplantace   ultrastruktura   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Plants from the Amaryllidaceae family have been shown to be a promising source of biologically active natural compounds of which some selected are currently in pre-clinical development. Regardless of interesting pioneer works, little is known about Amaryllidaceae alkaloids that have shown promising anti-cancer activities. The crinane group of the Amaryllidaceae, including haemanthamine and haemanthidine, was amongst the first of these compounds to exhibit an interesting cytotoxic potential against cancer cell lines. However, the mechanism of cytotoxic and anti-proliferative activity is not yet entirely clear. The primary objectives of the current study were to investigate the effects of haemanthamine and haemanthidine on the induction of apoptosis and the cell cycle regulatory pathway in p53-null Jurkat cells. Results indicate that haemanthamine and haemanthidine treatment decreases cell viability and mitochondrial membrane potential, leads to a decline in the percentage of cells in the S phase of the cell cycle, induces apoptosis detected by Annexin V staining and increases caspase activity. Dose dependent apoptosis was cross verified by fluorescence and bright field microscopy through Annexin V/propidium iodine staining and morphological changes which characteristically attend programmed cell death. The apoptotic effect of haemanthamine and haemanthidine on leukemia cells is more pronounced than that of gamma radiation. Contrary to gamma radiation, Jurkat cells do not completely halt the cell cycle 24h upon haemanthamine and haemanthidine exposure. Both Amaryllidaceae alkaloids accumulate cells preferentially at G1 and G2 stages of the cell cycle with increased p16 expression and Chk1 Ser345 phosphorylation. Concerning the pro-apoptotic effect, haemanthidine was more active than haemanthamine in the Jurkat leukemia cell line.

• MeSH
• alkaloidy amarylkovitých farmakologie   terapeutické užití   MeSH
• apoptóza účinky léků   MeSH
• fenantridiny farmakologie   terapeutické užití   MeSH
• fytogenní protinádorové látky analýza   MeSH
• fytoterapie * MeSH
• geny p53 MeSH
• inhibitor p16 cyklin-dependentní kinasy metabolismus   MeSH
• Jurkat buňky MeSH
• kaspasy metabolismus   MeSH
• kontrolní body buněčného cyklu účinky léků   MeSH
• leukemie farmakoterapie   MeSH
• lidé MeSH
• liliovité chemie   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• proteinkinasy metabolismus   MeSH
• rostlinné extrakty farmakologie   terapeutické užití   MeSH
• screeningové testy protinádorových léčiv MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This article summarizes basic information about the Amaryllidaceae alkaloids with cytotoxic and apoptosisinducing potential in diverse cancer cell lines and tumour xenografts. The review focuses on molecular mechanisms leading to cell death mediated by Amaryllidaceae alkaloids. Since some of them possess high cytotoxic efficiency in cancer cell lines study of Amaryllidaceae alkaloids could generate chemical structures for potential drugs. The biological activities that could be useful especially in topical cancer treatment are indicated. We have tested cytotoxic and antiproliferative activities of lycorine, galanthine and tazzetine in breast adenocarcinoma cells SK-BR-3 using label-free and real-time monitoring of the xCELLigence cell viability system.

• MeSH
• alkaloidy amarylkovitých * farmakokinetika   farmakologie   klasifikace   terapeutické užití   MeSH
• Alzheimerova nemoc farmakoterapie   MeSH
• analgetika MeSH
• antibakteriální látky MeSH
• antifungální látky MeSH
• antivirové látky MeSH
• apoptóza MeSH
• cholinesterasové inhibitory MeSH
• cytotoxické testy imunologické MeSH
• fytogenní protinádorové látky izolace a purifikace   terapeutické užití   MeSH
• galantamin analogy a deriváty   biosyntéza   MeSH
• intramolekulární oxidoreduktasy MeSH
• lidé MeSH
• nádory * farmakoterapie   MeSH
• preklinické hodnocení léčiv * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH