Determinants of electrical propagation and propagation block in Arrhythmogenic Cardiomyopathy.

Gap junction and ion channel remodeling occur early in Arrhythmogenic Cardiomyopathy (ACM), but their pathogenic consequences have not been elucidated. Here, we identified the arrhythmogenic substrate, consisting of propagation slowing and conduction block, in ACM models expressing two different desmosomal gene variants. Neonatal rat ventricular myocytes were transduced to express variants in genes encoding desmosomal proteins plakoglobin or plakophilin-2. Studies were performed in engineered cells and anisotropic tissues to quantify changes in conduction velocity, formation of unidirectional propagation, cell-cell electrical coupling, and ion currents. Conduction velocity decreased by 71% and 63% in the two ACM models. SB216763, an inhibitor of glycogen synthase kinase-3 beta, restored conduction velocity to near normal levels. Compared to control, both ACM models showed greater propensity for unidirectional conduction block, which increased further at greater stimulation frequencies. Cell-cell electrical conductance measured in cell pairs was reduced by 86% and 87% in the two ACM models. Computer modeling showed close correspondence between simulated and experimentally determined changes in conduction velocity. The simulation identified that reduced cell-cell electrical coupling was the dominant factor leading to slow conduction, while the combination of reduced cell-cell electrical coupling, reduced sodium current and inward rectifier potassium current explained the development of unidirectional block. Expression of two different ACM variants markedly reduced cell-cell electrical coupling and conduction velocity, and greatly increased the likelihood of developing unidirectional block - both key features of arrhythmogenesis. This study provides the first quantitative analysis of cellular electrophysiological changes leading to the substrate of reentrant arrhythmias in early stage ACM.

Nano-scale solution of the Poisson-Nernst-Planck (PNP) equations in a fraction of two neighboring cells reveals the magnitude of intercellular electrochemical waves.

The basic building blocks of the electrophysiology of cardiomyocytes are ion channels integrated in the cell membranes. Close to the ion channels there are very strong electrical and chemical gradients. However, these gradients extend for only a few nano-meters and are therefore commonly ignored in mathematical models. The full complexity of the dynamics is modelled by the Poisson-Nernst-Planck (PNP) equations but these equations must be solved using temporal and spatial scales of nano-seconds and nano-meters. Here we report solutions of the PNP equations in a fraction of two abuttal cells separated by a tiny extracellular space. We show that when only the potassium channels of the two cells are open, a stationary solution is reached with the well-known Debye layer close to the membranes. When the sodium channels of one of the cells are opened, a very strong and brief electrochemical wave emanates from the channels. If the extracellular space is sufficiently small and the number of sodium channels is sufficiently high, the wave extends all the way over to the neighboring cell and may therefore explain cardiac conduction even at very low levels of gap junctional coupling.

A detailed analysis of single-channel Nav 1.5 recordings does not reveal any cooperative gating.

Cardiac voltage-gated sodium (Na+ ) channels (Nav 1.5) are crucial for myocardial electrical excitation. Recent studies based on single-channel recordings have suggested that Na+ channels interact functionally and exhibit coupled gating. However, the analysis of such recordings frequently relies on manual interventions, which can lead to bias. Here, we developed an automated pipeline to de-trend and idealize single-channel currents, and assessed possible functional interactions in cell-attached patch clamp experiments in HEK293 cells expressing human Nav 1.5 channels as well as in adult mouse and rabbit ventricular cardiomyocytes. Our pipeline involved de-trending individual sweeps by linear optimization using a library of predefined functions, followed by digital filtering and baseline offset. Subsequently, the processed sweeps were idealized based on the idea that the ensemble average of the idealized current identified by thresholds between current levels reconstructs at best the ensemble average current from the de-trended sweeps. This reconstruction was achieved by non-linear optimization. To ascertain functional interactions, we examined the distribution of the numbers of open channels at every time point during the activation protocol and compared it to the distribution expected for independent channels. We also examined whether the channels tended to synchronize their openings and closings. However, we did not uncover any solid evidence of such interactions in our recordings. Rather, our results indicate that wild-type Nav 1.5 channels are independent entities or exhibit only very weak functional interactions that are probably irrelevant under physiological conditions. Nevertheless, our unbiased analysis will be important for further studies examining whether auxiliary proteins potentiate functional Na+ channel interactions. KEY POINTS: Nav 1.5 channels are critical for cardiac excitation. They are part of macromolecular interacting complexes, and it was previously suggested that two neighbouring channels may functionally interact and exhibit coupled gating. Manual interventions when processing single-channel recordings can lead to bias and inaccurate data interpretation. We developed an automated pipeline to de-trend and idealize single-channel currents and assessed possible functional interactions between Nav 1.5 channels in HEK293 cells and cardiomyocytes during activation protocols using the cell-attached patch clamp technique. In recordings consisting of up to 1000 sweeps from the same patch, our analysis did not reveal any evidence of functional interactions or coupled gating between wild-type Nav 1.5 channels. Our unbiased analysis may be useful in further studies examining how Na+ channel interactions are affected by mutations and auxiliary proteins.

Unraveling adipose tissue proteomic landscapes in severe obesity: insights into metabolic complications and potential biomarkers.

In this study, we aimed to comprehensively characterize the proteomic landscapes of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in patients with severe obesity, to establish their associations with clinical characteristics, and to identify potential serum protein biomarkers indicative of tissue-specific alterations or metabolic states. We conducted a cross-sectional analysis of 32 patients with severe obesity (16 males and 16 females) of Central European descent who underwent bariatric surgery. Clinical parameters and body composition were assessed using dual-energy X-ray absorptiometry (DXA) and bioelectrical impedance, with 15 patients diagnosed with type 2 diabetes (T2D) and 17 with hypertension. Paired SAT and VAT samples, along with serum samples, were subjected to state-of-the-art proteomics liquid chromatography-mass spectrometry (LC-MS). Our analysis identified 7,284 proteins across SAT and VAT, with 1,249 differentially expressed proteins between the tissues and 1,206 proteins identified in serum. Correlation analyses between differential protein expression and clinical traits suggest a significant role of SAT in the pathogenesis of obesity and related metabolic complications. Specifically, the SAT proteomic profile revealed marked alterations in metabolic pathways and processes contributing to tissue fibrosis and inflammation. Although we do not establish a definitive causal relationship, it appears that VAT might respond to SAT metabolic dysfunction by potentially enhancing mitochondrial activity and expanding its capacity. However, when this adaptive response is exceeded, it could possibly contribute to insulin resistance (IR) and in some cases, it may be associated with the progression to T2D. Our findings provide critical insights into the molecular foundations of SAT and VAT in obesity and may inform the development of targeted therapeutic strategies.NEW & NOTEWORTHY This study provides insights into distinct proteomic profiles of subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and serum in patients with severe obesity and their associations with clinical traits and body composition. It underscores SAT's crucial role in obesity development and related complications, such as insulin resistance (IR) and type 2 diabetes (T2D). Our findings emphasize the importance of understanding the SAT and VAT balance in energy homeostasis, proteostasis, and the potential role of SAT capacity in the development of metabolic disorders.

Status report of the first AMS laboratory in the Czech Republic at the Nuclear Physics Institute, Rez.

The first accelerator mass spectrometry (AMS) laboratory in the Czech Republic has been established and put into routine operation in February 2022. Here we briefly describe the facilities available, namely a 300 kV multi-isotope low-energy AMS system (MILEA) capable of determination 10Be, 14C, 26Al, 41Ca, 129I, isotopes of U, especially 236U, Pu and other actinoids, and accessories for 14C measurements, which include a gas interface system, a preparative gas chromatography system for compound-specific radiocarbon dating analysis, and an isotope-ratio mass spectrometer. The first results achieved for separation and measurement of the above radionuclides (except for 41Ca) are also reported, with the main focus on 14C measurements. A specimen breakdown of 729 graphitised samples analysed for 14C so far is presented, as well as a proof of measurement stability of the MILEA system obtained by analysis of radiocarbon standards and analytical blanks. For the other radionuclides, well proven or novel procedures for sample preparation and measurement are presented.

Active force generation contributes to the complexity of spontaneous activity and to the response to stretch of murine cardiomyocyte cultures.

Cardiomyocyte cultures exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. In such preparations, beat rate variability exhibits features similar to those of heart rate variability in vivo. Mechanical deformations and forces feed back on the electrical properties of cardiomyocytes, but it is not fully elucidated how this mechano-electrical interplay affects beating variability in such preparations. Using stretchable microelectrode arrays, we assessed the effects of the myosin inhibitor blebbistatin and the non-selective stretch-activated channel blocker streptomycin on beating variability and on the response of neonatal or fetal murine ventricular cell cultures against deformation. Spontaneous electrical activity was recorded without stretch and upon predefined deformation protocols (5% uniaxial and 2% equibiaxial strain, applied repeatedly for 1 min every 3 min). Without stretch, spontaneous activity originated from the edge of the preparations, and its site of origin switched frequently in a complex manner across the cultures. Blebbistatin did not change mean beat rate, but it decreased the spatial complexity of spontaneous activity. In contrast, streptomycin did not exert any manifest effects. During the deformation protocols, beat rate increased transiently upon stretch but, paradoxically, also upon release. Blebbistatin attenuated the response to stretch, whereas this response was not affected by streptomycin. Therefore, our data support the notion that in a spontaneously firing network of cardiomyocytes, active force generation, rather than stretch-activated channels, is involved mechanistically in the complexity of the spatiotemporal patterns of spontaneous activity and in the stretch-induced acceleration of beating. KEY POINTS: Monolayer cultures of cardiac cells exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. Beating variability in these preparations recapitulates the power-law behaviour of heart rate variability in vivo. However, the effects of mechano-electrical feedback on beating variability are not yet fully understood. Using stretchable microelectrode arrays, we examined the effects of the contraction uncoupler blebbistatin and the non-specific stretch-activated channel blocker streptomycin on beating variability and on stretch-induced changes of beat rate. Without stretch, blebbistatin decreased the spatial complexity of beating variability, whereas streptomycin had no effects. Both stretch and release increased beat rate transiently; blebbistatin attenuated the increase of beat rate upon stretch, whereas streptomycin had no effects. Active force generation contributes to the complexity of spatiotemporal patterns of beating variability and to the increase of beat rate upon mechanical deformation. Our study contributes to the understanding of how mechano-electrical feedback influences heart rate variability.

Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue.

Bordetella bronchiseptica and Bordetella pertussis are closely related respiratory pathogens that evolved from a common bacterial ancestor. While B. bronchiseptica has an environmental reservoir and mostly establishes chronic infections in a broad range of mammals, B. pertussis is a human-specific pathogen causing acute pulmonary pertussis in infants and whooping cough illness in older humans. Both species employ a type III secretion system (T3SS) to inject a cytotoxic BteA effector protein into host cells. However, compared to the high BteA-mediated cytotoxicity of B. bronchiseptica, the cytotoxicity induced by B. pertussis BteA (Bp BteA) appears to be quite low and this has been attributed to the reduced T3SS gene expression in B. pertussis. We show that the presence of an alanine residue inserted at position 503 (A503) of Bp BteA accounts for its strongly attenuated cytotoxic potency. The deletion of A503 from Bp BteA greatly enhanced the cytotoxic activity of B. pertussis B1917 on mammalian HeLa cells and expression of Bp BteAΔA503 was highly toxic to Saccharomyces cerevisiae cells. Vice versa, insertion of A503 into B. bronchiseptica BteA (Bb BteA) strongly decreased its cytotoxicity to yeast and HeLa cells. Moreover, the production of Bp BteAΔA503 increased virulence of B. pertussis B1917 in the mouse model of intranasal infection (reduced LD50) but yielded less inflammatory pathology in infected mouse lungs at sublethal infectious doses. This suggests that A503 insertion in the T3SS effector Bp BteA may represent an evolutionary adaptation that fine-tunes B. pertussis virulence and host immune response.

Localization of Na+ channel clusters in narrowed perinexi of gap junctions enhances cardiac impulse transmission via ephaptic coupling: a model study.

It has been proposed that when gap junctional coupling is reduced in cardiac tissue, action potential propagation can be supported via ephaptic coupling, a mechanism mediated by negative electric potentials occurring in narrow intercellular clefts of intercalated discs (IDs). Recent studies showed that sodium (Na+ ) channels form clusters near gap junction plaques in nanodomains called perinexi, where the ID cleft is even narrower. To examine the electrophysiological relevance of Na+ channel clusters being located in perinexi, we developed a 3D finite element model of two longitudinally abutting cardiomyocytes, with a central Na+ channel cluster on the ID membranes. When this cluster was located in the perinexus of a closely positioned gap junction plaque, varying perinexal width greatly modulated impulse transmission from one cell to the other, with narrow perinexi potentiating ephaptic coupling. This modulation occurred via the interplay of Na+ currents, extracellular potentials in the cleft and patterns of current flow within the cleft. In contrast, when the Na+ channel cluster was located remotely from the gap junction plaque, this modulation by perinexus width largely disappeared. Interestingly, the Na+ current in the ID membrane of the pre-junctional cell switched from inward to outward during excitation, thus contributing ions to the activating channels on the post-junctional ID membrane. In conclusion, these results indicate that the localization of Na+ channel clusters in the perinexi of gap junction plaques is crucial for ephaptic coupling, which is furthermore greatly modulated by perinexal width. These findings are relevant for a comprehensive understanding of cardiac excitation. KEY POINTS: Ephaptic coupling is a cardiac conduction mechanism involving nanoscale-level interactions between the sodium (Na+ ) current and the extracellular potential in narrow intercalated disc clefts. When gap junctional coupling is reduced, ephaptic coupling acts in conjunction with the classical cardiac conduction mechanism based on gap junctional current flow. In intercalated discs, Na+ channels form clusters that are preferentially located in the periphery of gap junction plaques, in nanodomains known as perinexi, but the electrophysiological role of these perinexi has never been examined. In our new 3D finite element model of two cardiac cells abutting each other with their intercalated discs, a Na+ channel cluster located inside a narrowed perinexus facilitated impulse transmission via ephaptic coupling. Our simulations demonstrate the role of narrowed perinexi as privileged sites for ephaptic coupling in pathological situations when gap junctional coupling is decreased.

The depletion of p38alpha kinase upregulates NADPH oxidase 2/NOX2/gp91 expression and the production of superoxide in mouse embryonic stem cells.

P38alpha kinase plays an important role in the regulation of both cell stress response and cell fate. In this study, we report that p38alpha kinase-deficient embryonic stem cells exhibit a higher production of reactive oxygen species (ROS) in contrast to their wild-type counterpart. Analysis of the expressions of NADPH oxidases (NOXs) and dual oxidases, crucial enzymes involved in intracellular ROS formation, shows NOX2/gp91phox is over-expressed in p38alpha deficient cells. The particular increase in superoxide formation was confirmed by the specific detection of hydroethidine derivate 2-hydroxyethidium. ROS formation decreased when the level of NOX2 was silenced by siRNA in p38alpha deficient cells. These data suggest the importance of p38alpha kinase in the regulation of ROS metabolism in embryonic stem cells and the significance of the observed phenomena of cancer cell-like phenotypes, which is discussed.

Distribution of cardiac sodium channels in clusters potentiates ephaptic interactions in the intercalated disc.

KEY POINTS: It has been proposed that ephaptic conduction, relying on interactions between the sodium (Na+ ) current and the extracellular potential in intercalated discs, might contribute to cardiac conduction when gap junctional coupling is reduced, but this mechanism is still controversial. In intercalated discs, Na+ channels form clusters near gap junction plaques, but the functional significance of these clusters has never been evaluated. In HEK cells expressing cardiac Na+ channels, we show that restricting the extracellular space modulates the Na+ current, as predicted by corresponding simulations accounting for ephaptic effects. In a high-resolution model of the intercalated disc, clusters of Na+ channels that face each other across the intercellular cleft facilitate ephaptic impulse transmission when gap junctional coupling is reduced. Thus, our simulations reveal a functional role for the clustering of Na+ channels in intercalated discs, and suggest that rearrangement of these clusters in disease may influence cardiac conduction. ABSTRACT: It has been proposed that ephaptic interactions in intercalated discs, mediated by extracellular potentials, contribute to cardiac impulse propagation when gap junctional coupling is reduced. However, experiments demonstrating ephaptic effects on the cardiac Na+ current (INa ) are scarce. Furthermore, Na+ channels form clusters around gap junction plaques, but the electrophysiological significance of these clusters has never been investigated. In patch clamp experiments with HEK cells stably expressing human Nav 1.5 channels, we examined how restricting the extracellular space modulates INa elicited by an activation protocol. In parallel, we developed a high-resolution computer model of the intercalated disc to investigate how the distribution of Na+ channels influences ephaptic interactions. Approaching the HEK cells to a non-conducting obstacle always increased peak INa at step potentials near the threshold of INa activation and decreased peak INa at step potentials far above threshold (7 cells, P = 0.0156, Wilcoxon signed rank test). These effects were consistent with corresponding control simulations with a uniform Na+ channel distribution. In the intercalated disc computer model, redistributing the Na+ channels into a central cluster of the disc potentiated ephaptic effects. Moreover, ephaptic impulse transmission from one cell to another was facilitated by clusters of Na+ channels facing each other across the intercellular cleft when gap junctional coupling was reduced. In conclusion, our proof-of-principle experiments demonstrate that confining the extracellular space modulates cardiac INa , and our simulations reveal the functional role of the aggregation of Na+ channels in the perinexus. These findings highlight novel concepts in the physiology of cardiac excitation.

Peripatric speciation associated with genome expansion and female-biased sex ratios in the moss genus Ceratodon.

PREMISE OF THE STUDY: A period of allopatry is widely believed to be essential for the evolution of reproductive isolation. However, strict allopatry may be difficult to achieve in some cosmopolitan, spore-dispersed groups, like mosses. We examined the genetic and genome size diversity in Mediterranean populations of the moss Ceratodon purpureus s.l. to evaluate the role of allopatry and ploidy change in population divergence. METHODS: We sampled populations of the genus Ceratodon from mountainous areas and lowlands of the Mediterranean region, and from Western and Central Europe. We performed phylogenetic and coalescent analyses on sequences from five nuclear introns and a chloroplast locus to reconstruct their evolutionary history. We also estimated genome size using flow cytometry (employing propidium iodide) and determined the sex of samples using a sex-linked PCR marker. KEY RESULTS: Two well-differentiated clades were resolved, discriminating two homogeneous groups: the widespread C. purpureus and a local group mostly restricted to the mountains in Southern Spain. The latter also possessed a genome size 25% larger than the widespread C. purpureus, and the samples of this group consist entirely of females. We also found hybrids, and some of them had a genome size equivalent to the sum of the C. purpureus and Spanish genome, suggesting that they arose by allopolyploidy. CONCLUSIONS: These data suggest that a new species of Ceratodon arose via peripatric speciation, potentially involving a genome size change and a strong female-biased sex ratio. The new species has hybridized in the past with C. purpureus.

A porcine model of skin wound infected with a polybacterial biofilm.

A clinically relevant porcine model of a biofilm-infected wound was established in 10 minipigs. The wounds of six experimental animals were infected with a modified polymicrobial Lubbock chronic wound biofilm consisting of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Bacillus subtilis. Four animals served as uninfected controls. The wounds were monitored until they had healed for 24 days. The biofilm persisted in the wounds up to day 14 and significantly affected healing. The control to infected healed wound area ratios were: 45%/21%, 66%/37%, and 90%/57% on days 7, 10 and 14, respectively. The implanted biofilm prolonged inflammation, increased necrosis, delayed granulation and impaired development of the extracellular matrix as seen in histological and gene expression analyses. This model provides a therapeutic one-week window for testing of anti-biofilm treatments and for research on the pathogenesis of wound infections in pig that is clinically the most relevant animal wound healing model.

Synthetic routes contaminate graphene materials with a whole spectrum of unanticipated metallic elements.

The synthesis of graphene materials is typically carried out by oxidizing graphite to graphite oxide followed by a reduction process. Numerous methods exist for both the oxidation and reduction steps, which causes unpredictable contamination from metallic impurities into the final material. These impurities are known to have considerable impact on the properties of graphene materials. We synthesized several reduced graphene oxides from extremely pure graphite using several popular oxidation and reduction methods and tracked the concentrations of metallic impurities at each stage of synthesis. We show that different combinations of oxidation and reduction introduce varying types as well as amounts of metallic elements into the graphene materials, and their origin can be traced to impurities within the chemical reagents used during synthesis. These metallic impurities are able to alter the graphene materials' electrochemical properties significantly and have wide-reaching implications on the potential applications of graphene materials.

Stochastic pacing reveals the propensity to cardiac action potential alternans and uncovers its underlying dynamics.

KEY POINTS: Beat-to-beat alternation (alternans) of the cardiac action potential duration is known to precipitate life-threatening arrhythmias and can be driven by the kinetics of voltage-gated membrane currents or by instabilities in intracellular calcium fluxes. To prevent alternans and associated arrhythmias, suitable markers must be developed to quantify the susceptibility to alternans; previous theoretical studies showed that the eigenvalue of the alternating eigenmode represents an ideal marker of alternans. Using rabbit ventricular myocytes, we show that this eigenvalue can be estimated in practice by pacing these cells at intervals varying stochastically. We also show that stochastic pacing permits the estimation of further markers distinguishing between voltage-driven and calcium-driven alternans. Our study opens the perspective to use stochastic pacing during clinical investigations and in patients with implanted pacing devices to determine the susceptibility to, and the type of alternans, which are both important to guide preventive or therapeutic measures. ABSTRACT: Alternans of the cardiac action potential (AP) duration (APD) is a well-known arrhythmogenic mechanism. APD depends on several preceding diastolic intervals (DIs) and APDs, which complicates the prediction of alternans. Previous theoretical studies pinpointed a marker called λalt that directly quantifies how an alternating perturbation persists over successive APs. When the propensity to alternans increases, λalt decreases from 0 to -1. Our aim was to quantify λalt experimentally using stochastic pacing and to examine whether stochastic pacing allows discriminating between voltage-driven and Ca(2+) -driven alternans. APs were recorded in rabbit ventricular myocytes paced at cycle lengths (CLs) decreasing progressively and incorporating stochastic variations. Fitting APD with a function of two previous APDs and CLs permitted us to estimate λalt along with additional markers characterizing whether the dependence of APD on previous DIs or CLs is strong (typical for voltage-driven alternans) or weak (Ca(2+) -driven alternans). During the recordings, λalt gradually decreased from around 0 towards -1. Intermittent alternans appeared when λalt reached -0.8 and was followed by sustained alternans. The additional markers detected that alternans was Ca(2+) driven in control experiments and voltage driven in the presence of ryanodine. This distinction could be made even before alternans was manifest (specificity/sensitivity >80% for -0.4 > λalt  > -0.5). These observations were confirmed in a mathematical model of a rabbit ventricular myocyte. In conclusion, stochastic pacing allows the practical estimation of λalt to reveal the onset of alternans and distinguishes between voltage-driven and Ca(2+) -driven mechanisms, which is important since these two mechanisms may precipitate arrhythmias in different manners.

Elemental characterization of single-wall carbon nanotube certified reference material by neutron and prompt γ activation analysis.

Instrumental neutron activation analysis with both relative and k0 standardization was used in four experienced laboratories to determine element mass fractions in single-wall carbon nanotube certified reference material (CRM) SWCNT-1. Results obtained were evaluated using the National Institute of Standards and Technology (NIST) "Type B On Bias" approach and yielded consensus values in agreement with National Research Council Canada (NRCC) certified values for Fe, Co, Ni, and Mo and provided mass fraction values for 13 additional elements, namely, Na, Mg, Al, K, Ca, Ti, V, Cr, Mn, Br, La, W, and Au. In addition, prompt γ neutron activation analysis was employed to determine mass fractions of H, B, Co, Ni, and Mo. Results of this work provide a basis for the establishment of reference values of element mass fractions in CRM SWCNT-1, thus expanding its usability for more accurate characterization and benchmarking of similar nanotechnology materials.

Cancer-associated fibroblasts are not formed from cancer cells by epithelial-to-mesenchymal transition in nu/nu mice.

Cancer-associated fibroblasts are bioactive elements influencing the biological properties of malignant tumors. Their origin from different cell types has been established, and the possibility of their formation by epithelial-to-mesenchymal transition from cancer cells is under debate. This study shows that human cancer cells grafted to nu/nu mice induced formation of tumor stroma with the presence of typical smooth muscle actin-containing cancer-associated fibroblasts. These cells seem to be of the host origin because they are not recognized by an antibody specific for human vimentin, as was also verified in vitro. These results suggest that cancer-associated stromal fibroblasts are not formed by epithelial-to-mesenchymal transition from cancer cells.

Nonlinear behaviour of conduction and block in cardiac tissue with heterogeneous expression of connexin 43.

Altered gap junctional coupling potentiates slow conduction and arrhythmias. To better understand how heterogeneous connexin expression affects conduction at the cellular scale, we investigated conduction in tissue consisting of two cardiomyocyte populations expressing different connexin levels. Conduction was mapped using microelectrode arrays in cultured strands of foetal murine ventricular myocytes with predefined contents of connexin 43 knockout (Cx43KO) cells. Corresponding computer simulations were run in randomly generated two-dimensional tissues mimicking the cellular architecture of the strands. In the cultures, the relationship between conduction velocity (CV) and Cx43KO cell content was nonlinear. CV first decreased significantly when Cx43KO content was increased from 0 to 50%. When the Cx43KO content was ≥60%, CV became comparable to that in 100% Cx43KO strands. Co-culturing Cx43KO and wild-type cells also resulted in significantly more heterogeneous conduction patterns and in frequent conduction blocks. The simulations replicated this behaviour of conduction. For Cx43KO contents of 10-50%, conduction was slowed due to wavefront meandering between Cx43KO cells. For Cx43KO contents ≥60%, clusters of remaining wild-type cells acted as electrical loads that impaired conduction. For Cx43KO contents of 40-60%, conduction exhibited fractal characteristics, was prone to block, and was more sensitive to changes in ion currents compared to homogeneous tissue. In conclusion, conduction velocity and stability behave in a nonlinear manner when cardiomyocytes expressing different connexin amounts are combined. This behaviour results from heterogeneous current-to-load relationships at the cellular level. Such behaviour is likely to be arrhythmogenic in various clinical contexts in which gap junctional coupling is heterogeneous.

Synthetic polyamine BPA-C8 inhibits TGF-ß1-mediated conversion of human dermal fibroblast to myofibroblasts and establishment of galectin-1-rich extracellular matrix in vitro.

Cancer-associated fibroblasts (CAFs) play a role in the progression of malignant tumors. They are formed by conversion of fibroblasts to smooth muscle α-actin-positive (SMA-positive) myofibroblasts. Polyamines are known to change the arrangement of the actin cytoskeleton by binding to the anionic actin. We tested the effect of the synthetic polyamine BPA-C8 on the transition of human dermal fibroblasts to myofibroblasts induced either by TGF-ß1 alone or by TGF-ß1 together with adhesion/growth-regulatory galectin-1. Pre-existing CAFs, myofibroblasts from pancreatitis, and rat smooth muscle cells were also exposed to BPA-C8. BPA-C8 impaired myofibroblast formation from activated fibroblasts, but it had no effect on cells already expressing SMA. BPA-C8 also reduced the occurrence of an extracellular matrix around the activated fibroblasts. The reported data thus extend current insights into polyamine activity, adding interference with tumor progression to the tumor-promoting processes warranting study.

Checklist of colourless euglenoids of the Czech Republic, with several taxonomic additions.

This study presents the results of a complex survey of freshwater heterotrophic euglenoids in the Czech Republic, including both literature data and own field surveys of 469 sites visited in the course of three years. The checklist includes 189 taxa in 28 genera: Anisonema (10), Astasia (26), Atraktomonas (1), Calycimonas (2), Chasmostoma (1), Dinematomonas (3), Distigma (8), Dylakosoma (1), Entosiphon (4), Euglena (1), Gyropaigne (1), Heteronema (19), Jenningsia (11), Khawkinea (1), Lepocinclis (1), Menoidium (7), Neometanema (3), Notosolenus (18), Petalomonas (40), Phacus (1), Ploeotia (2), Pseudoperanema (7), Rhabdomonas (5), Scytomonas (1), Sphenomonas (5), Teloprocta (1) Tropidocyphus (1), Urceolus (4), and 4 species of uncertain identity. In addition, a general description of habitat types in which the taxa were found and a review of the current taxonomy and nomenclature of included taxa are provided. Several taxonomic and nomenclatural novelties are proposed, based on the review of morphological features, mostly applying to the genera Notosolenus and Jenningsia.

Potent synergistic effects of dulaglutide and food restriction in prevention of olanzapine-induced metabolic adverse effects in a rodent model.

BACKGROUND: Antipsychotics are indispensable in the treatment of severe mental illneses, however adverse metabolic effects including diabetes, weight gain, dyslipidemia, and related cardiovascular morbidity are common, and current pharmacological strategies for their management are unsatisfactory. Glucagon-like 1 peptide receptor agonists (GLP-1 RAs) are approved for the treatment of type 2 diabetes and obesity hold promise for the management of antipsychotic-associated adverse metabolic effects. METHODS: To characterize the molecular effects and identify biomarkers for GLP-1 RA preventive treatment, Sprague-Dawley female rats were treated with long-acting formulations of the antipsychotic olanzapine and the GLP-1 RA dulaglutide for 8 days. A pair-feeding protocol evaluated the combined effects of dulaglutide and food restriction on an olanzapine-induced metabolic phenotype. Body weight and food consumption were recorded. Biochemical analysis included a lipid profile, a spectrum of gastrointestinal and adipose tissue-derived hormones, and fibroblast growth factor 21 serum levels. RESULTS: Olanzapine induced hyperphagia, weight gain, increased serum triglycerides and HDL cholesterol. Food restriction affected the OLA-induced phenotype but not serum markers. Dulaglutide led to a modest decrease in food intake, with no effect on weight gain, and did not reverse the OLA-induced changes in serum lipid parameters. Concomitant dulaglutide and food restriction resulted in weight loss, decreased feed efficiency, and lower total and HDL cholesterol. CONCLUSIONS: A combined strategy of dulaglutide and food restriction manifested a massive synergistic benefit. GLP-1RAs represent a promising strategy and deserve thorough future research. Our findings underline the potential importance of lifestyle intervention in addition to GLP-1 RA treatment.