First Synthesis of DBU-Conjugated Cationic Carbohydrate Derivatives and Investigation of Their Antibacterial and Antifungal Activity.

The emergence of drug-resistant bacteria and fungi represents a serious health problem worldwide. It has long been known that cationic compounds can inhibit the growth of bacteria and fungi by disrupting the cell membrane. The advantage of using such cationic compounds is that the microorganisms would not become resistant to cationic agents, since this type of adaptation would mean significantly altering the structure of their cell walls. We designed novel, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene)-derived amidinium salts of carbohydrates, which may be suitable for disturbing the cell walls of bacteria and fungi due to their quaternary ammonium moiety. A series of saccharide-DBU conjugates were prepared from 6-iodo derivatives of d-glucose, d-mannose, d-altrose and d-allose by nucleophilic substitution reactions. We optimized the synthesis of a d-glucose derivative, and studied the protecting group free synthesis of the glucose-DBU conjugates. The effect of the obtained quaternary amidinium salts against Escherichia coli and Staphylococcus aureus bacterial strains and Candida albicans yeast was investigated, and the impact of the used protecting groups and the sugar configuration on the antimicrobial activity was analyzed. Some of the novel sugar quaternary ammonium compounds with lipophilic aromatic groups (benzyl and 2-napthylmethyl) showed particularly good antifungal and antibacterial activity.

The multitargeted receptor tyrosine kinase inhibitor sunitinib induces resistance of HER2 positive breast cancer cells to trastuzumab-mediated ADCC.

Despite recent advances in the development of novel personalized therapies, breast cancer continues to challenge physicians with resistance to various advanced therapies. The anticancer action of the anti-HER2 antibody, trastuzumab, involves antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) cells. Here, we report a repurposing screen of 774 clinically used compounds on NK-cell + trastuzumab-induced killing of JIMT-1 breast cancer cells. Using a calcein-based high-content screening (HCS) assay for the image-based quantitation of ADCC that we have developed and optimized for this purpose, we have found that the multitargeted tyrosine kinase inhibitor sunitinib inhibits ADCC in this model. The cytoprotective effect of sunitinib was also confirmed with two other assays (lactate dehydrogenase release, and electric cell substrate impedance sensing, ECIS). The drug suppressed NK cell activation as indicated by reduced granzyme B deposition on to the target cells and inhibition of interferon-γ production by the NK cells. Moreover, sunitinib induced downregulation of HER2 on the target cells' surface, changed the morphology and increased adherence of the target cells. Moreover, sunitinib also triggered the autophagy pathway (speckled LC3b) as an additional potential underlying mechanism of the cytoprotective effect of the drug. Sunitinib-induced ADCC resistance has been confirmed in a 3D tumor model revealing the prevention of apoptotic cell death (Annexin V staining) in JIMT-1 spheroids co-incubated with NK cells and trastuzumab. In summary, our HCS assay may be suitable for the facile identification of ADCC boosting compounds. Our data urge caution concerning potential combinations of ADCC-based immunotherapies and sunitinib.

Synthesis and Anticancer and Antiviral Activities of C-2'-Branched Arabinonucleosides.

d-Arabinofuranosyl-pyrimidine and -purine nucleoside analogues containing alkylthio-, acetylthio- or 1-thiosugar substituents at the C2' position were prepared from the corresponding 3',5'-O-silylene acetal-protected nucleoside 2'-exomethylenes by photoinitiated, radical-mediated hydrothiolation reactions. Although the stereochemical outcome of the hydrothiolation depended on the structure of both the thiol and the furanoside aglycone, in general, high d-arabino selectivity was obtained. The cytotoxic effect of the arabinonucleosides was studied on tumorous SCC (mouse squamous cell) and immortalized control HaCaT (human keratinocyte) cell lines by MTT assay. Three pyrimidine nucleosides containing C2'-butylsulfanylmethyl or -acetylthiomethyl groups showed promising cytotoxicity at low micromolar concentrations with good selectivity towards tumor cells. SAR analysis using a methyl ß-d-arabinofuranoside reference compound showed that the silyl-protecting group, the nucleobase and the corresponding C2' substituent are crucial for the cell growth inhibitory activity. The effects of the three most active nucleoside analogues on parameters indicative of cytotoxicity, such as cell size, division time and cell generation time, were investigated by near-infrared live cell imaging, which showed that the 2'-acetylthiomethyluridine derivative induced the most significant functional and morphological changes. Some nucleoside analogues also exerted anti-SARS-CoV-2 and/or anti-HCoV-229E activity with low micromolar EC50 values; however, the antiviral activity was always accompanied by significant cytotoxicity.

NMNAT1 Is a Survival Factor in Actinomycin D-Induced Osteosarcoma Cell Death.

Osteosarcoma is a frequent and extremely aggressive type of pediatric cancer. New therapeutic approaches are needed to improve the overall survival of osteosarcoma patients. Our previous results suggest that NMNAT1, a key enzyme in nuclear NAD+ synthesis, facilitates the survival of cisplatin-treated osteosarcoma cells. A high-throughput cytotoxicity screening was performed to identify novel pathways or compounds linked to the cancer-promoting role of NMNAT1. Nine compounds caused higher toxicity in the NMNAT1 KO U2OS cells compared to their wild type counterparts, and actinomycin D (ActD) was the most potent. ActD-treatment of NMNAT1 KO cells increased caspase activity and secondary necrosis. The reduced NAD+ content in NMNAT1 KO cells was further decreased by ActD, which partially inhibited NAD+-dependent enzymes, including the DNA nick sensor enzyme PARP1 and the NAD+-dependent deacetylase SIRT1. Impaired PARP1 activity increased DNA damage in ActD-treated NMNAT1 knockout cells, while SIRT1 impairment increased acetylation of the p53 protein, causing the upregulation of pro-apoptotic proteins (NOXA, BAX). Proliferation was decreased through both PARP- and SIRT-dependent pathways. On the one hand, PARP inhibitors sensitized wild type but not NMNAT1 KO cells to ActD-induced anti-clonogenic effects; on the other hand, over-acetylated p53 induced the expression of the anti-proliferative p21 protein leading to cell cycle arrest. Based on our results, NMNAT1 acts as a survival factor in ActD-treated osteosarcoma cells. By inhibiting both PARP1- and SIRT1-dependent cellular pathways, NMNAT1 inhibition can be a promising new tool in osteosarcoma chemotherapy.

Coping and Adaptation in Response to Environmental and Climatic Stressors in Caribbean Coastal Communities.

Cumulative and synergistic impacts from environmental pressures, particularly in low-lying tropical coastal regions, present challenges for the governance of ecosystems, which provide natural resource-based livelihoods for communities. Here, we seek to understand the relationship between responses to the impacts of El Niño and La Niña events and the vulnerability of mangrove-dependent communities in the Caribbean region of Colombia. Using two case study sites, we show how communities are impacted by, and undertake reactive short-term responses to, El Niño and La Niña events, and how such responses can affect their adaptive capacity to progressive environmental deterioration. We show that certain coping measures to climate variability currently deliver maladaptive outcomes, resulting in circumstances that could contribute to system 'lock-in' and engender undesirable ecological states, exacerbating future livelihood vulnerabilities. We highlight the significant role of social barriers on vulnerabilities within the region, including perceptions of state abandonment, mistrust and conflicts with authorities. Opportunities to reduce vulnerability include enhancing the communities' capacity to adopt more positive and preventative responses based on demonstrable experiential learning capacity. However, these will require close cooperation between formal and informal organisations at different levels, and the development of shared coherent adaptation strategies to manage the complexity of multiple interacting environmental and climatic pressures.

Antifungal Activity of an Original Amino-Isocyanonaphthalene (ICAN) Compound Family: Promising Broad Spectrum Antifungals.

: Multiple drug resistant fungi pose a serious threat to human health, therefore the development of completely new antimycotics is of paramount importance. The in vitro antifungal activity of the original, 1-amino-5-isocyanonaphthalenes (ICANs) was evaluated against reference strains of clinically important Candida species. Structure-activity studies revealed that the naphthalene core and the isocyano- together with the amino moieties are all necessary to exert antifungal activity. 1,1-N-dimethylamino-5-isocyanonaphthalene (DIMICAN), the most promising candidate, was tested further in vitro against clinical isolates of Candida species, yielding a minimum inhibitory concentration (MIC) of 0.04-1.25 µg/mL. DIMICAN was found to be effective against intrinsically fluconazole resistant Candida krusei isolates, too. In vivo experiments were performed in a severly neutropenic murine model inoculated with a clinical strain of Candida albicans. Daily administration of 5 mg/kg DIMICAN intraperitoneally resulted in 80% survival even at day 13, whereas 100% of the control group died within six days. Based on these results, ICANs may become an effective clinical lead compound family against fungal pathogens.

Synthesis and Cytostatic Effect of 3'-deoxy-3'-C-Sulfanylmethyl Nucleoside Derivatives with d-xylo Configuration.

A small library of 3'-deoxy-C3'-substituted xylofuranosyl-pyrimidine nucleoside analogues were prepared by photoinduced thiol-ene addition of various thiols, including normal and branched alkyl-, 2-hydroxyethyl, benzyl-, and sugar thiols, to 3'-exomethylene derivatives of 2',5'-di-O-tert-butyldimethylsilyl-protected ribothymidine and uridine. The bioactivity of these derivatives was studied on tumorous SCC (mouse squamous carcinoma cell) and immortalized control HaCaT (human keratinocyte) cell lines. Several alkyl-substituted analogues elicited promising cytostatic activity in low micromolar concentrations with a slight selectivity toward tumor cells. Near-infrared live-cell imaging revealed SCC tumor cell-specific mitotic blockade via genotoxicity of analogue 10, bearing an n-butyl side chain. This analogue essentially affects the chromatin structure of SCC tumor cells, inducing a condensed nuclear material and micronuclei as also supported by fluorescent microscopy. The results highlight that thiol-ene chemistry represents an efficient strategy to discover novel nucleoside analogues with non-natural sugar structures as anticancer agents.

Gadolinium induced effects on mammalian cell motility, adherence and chromatin structure.

The toxicity of gadolinium is reduced by chelating agents that render this heavy metal into contrast complexes used for medical magnetic resonance imaging. However, the dissociation of gadolinium chelates is known to generate Gd3+ ions, the cellular toxicity of which has not been tested in details. The cytotoxic effects of Gd(III) ions were evaluated by monitoring the proliferation, measuring the cellular motility and following chromatin changes in various cell lines upon Gd3+ treatment. Measurements applied long-term scanning microscopy and a perfusion platform that replaced the medium with test solutions, bypassed physical contact with the cell culture during experiments, and provided uninterrupted high time-resolution time-lapse photomicrography for an extended period of time. Genotoxicity specific chromatin changes characteristic to Gd(III) were distinguished in human skin keratinocytes (HaCaT), human limbal stem cells (HuLi), colorectal adenocarcinoma (CaCO2), murine squamous carcinoma (SCC) and Indian muntjac (IM) cell lines. Characteristic features of Gd(III) toxicity were: loss of cellular motility, irreversible attachment of cells to the growth surface and cell death. Injury-specific chromatin changes manifested at micromolar Gd3+ concentrations as premature chromatin condensation and highly condensed sticky chromatin patches. Gd(III) concentration- and cell type-dependent reduction of normal adherence, as well as premature chromatin condensation confirmed apoptosis. The risk related to the release of toxic Gd3+ ions from gadolinium complexes and their effects on mono- and multi-layer cellular barriers have to be reconsidered when these chelated complexes are used as contrasting agents especially in relation to possible blood-brain barrier damages.

Predictors of cerebral microembolization during phased radiofrequency ablation of atrial fibrillation: role of the ongoing rhythm and the site of energy delivery.

BACKGROUND: Pulmonary vein isolation with phased radiofrequency current and use of a pulmonary vein ablation catheter (PVAC) has recently been associated with a high incidence of clinically silent brain infarcts on diffusion-weighted magnetic resonance imaging, and a high microembolic signal (MES) count detected by transcranial Doppler. We investigated the potential effects of the ongoing rhythm and the target vein during energy delivery (ED) on MES generation during PVAC ablations. METHODS AND RESULTS: A total of 735 EDs during 48 PVAC ablations were analyzed. MES counts were recorded for each ED and time-stamped for correlation with the ongoing rhythm and the target vein for each ED. Significantly higher MES counts were observed during ablations of the left-sided as compared with the right-sided pulmonary veins (P = 0.0003). Similarly, higher MES counts were detected during EDs in atrial fibrillation as compared with sinus rhythm when the temperature was >56°C (P < 0.0001). The ongoing rhythm had no effect on the number of MESs at lower temperatures during ablation. CONCLUSIONS: Both the ongoing rhythm during ED and the site of ablation influence microembolus generation during PVAC ablation procedures.

[Primary squamous cell carcinoma of the thyroid gland]. / A pajzsmirigy primer laphámcarcinomája.

Primary squamous cell carcinoma (SCC) of the thyroid gland is now considered as a member of the anaplastic thyroid carcinoma group based on the latest version of the WHO tumor classification. It is a very rare entity, the prognosis is adverse with a short survival time. The aim of this article is to emphasize the therapeutic complexity of this disease. A 68-year-old woman presented with rapidly growing right-sided neck mass with hoarseness and compressive symptoms. Physical examination revealed a hard fixed tumor with right-sided vocal cord palsy. Fine-needle aspiration cytology revealed a case of SCC in the location of the thyroid gland, imaging studies excluded the possibility of other primary malignancies. Surgical intervention was performed aiming the total removal of the tumor. Histopathological result confirmed the diagnosis of SCC of the thyroid. Finally the patient died during the palliative radiation therapy. SCC of the thyroid gland is a great challenge for both the surgeon and the multidisciplinary team to come up with the best treatment option which is suitable for the patient due to its unfavorable prognosis. Because of the poor response to the radiation and chemotherapy, complete surgical removal and the identification of any possible targetable molecular pathological change play a unique role in the therapy. Orv Hetil. 2023; 164(39): 1556-1559.

Potential Original Drug for Aspergillosis: In Vitro and In Vivo Effects of 1-N,N-Dimethylamino-5-Isocyanonaphthalene (DIMICAN) on Aspergillus fumigatus.

As the recent outbreak of coronavirus disease 2019 (COVID-19) has shown, viral infections are prone to secondary complications like invasive aspergillosis with a high mortality rate, and therefore the development of novel, effective antifungals is of paramount importance. We have previously demonstrated that 1-amino-5-isocyanonaphthalene (ICAN) derivatives are promising original drug candidates against Candida strains (Patent pending), even against fluconazole resistant C. albicans. Consequently, in this study ICANs were tested on Aspergillus fumigatus, an opportunistic pathogen, which is the leading cause of invasive and systematic pulmonary aspergillosis in immunosuppressed, transplanted and cancer- or COVID-19 treated patients. We have tested several N-alkylated ICANs, a well as 1,5-naphthalene-diisocyanide (DIN) with the microdilution method against Aspergillus fumigatus strains. The results revealed that the diisocyanide (DIN) was the most effective with a minimum inhibitory concentration (MIC) value as low as 0.6 µg mL-1 (3.4 µM); however, its practical applicability is limited by its poor water solubility, which needs to be overcome by proper formulation. The other alkylated derivatives also have in vitro and in vivo anti-Aspergillus fumigatus effects. For animal experiments the second most effective derivative 1-N, N-dimethylamino-5-isocyanonaphthalene (DIMICAN, MIC: 7-8 µg mL-1, 36-41 µM) was selected, toxicity tests were made with mice, and then the antifungal effect of DIMICAN was tested in a neutropenic aspergillosis murine model. Compared to amphotericin B (AMB), a well-known antifungal, the antifungal effect of DIMICAN in vivo turned out to be much better (40% vs. 90% survival after eight days), indicating its potential as a clinical drug candidate.

In situ remediation efficacy of hybrid aerogel adsorbent in model aquatic culture of Paramecium caudatum exposed to Hg(II).

Silica-gelatin hybrid aerogel of 24 wt% gelatin content is an advanced functional material suitable for the high performance selective adsorption of aqueous Hg(II). The remediation efficacy of this adsorbent was tested under realistic aquatic conditions by exposing cultures of Paramecium caudatum to Hg(II) and monitoring the model cultures by time-lapse video microscopy. The viability of Paramecium was quantified by analyzing the pixel differences of the sequential images caused by the persistent movement (motility) of the cells. The viability of Paramecium displays a clear exposure-response relationship with Hg(II) concentration. Viability decreases with increasing Hg(II) concentration when the latter is higher than 125 µg L-1. In the presence of 0.1 mg mL-1 aerogel adsorbent, the viability of the cells decreases only at Hg(II) concentrations higher than 500 µg L-1, and 220 min survival time was measured even at 1000 µg L-1 Hg(II). The effective toxicity of Hg(II) is lower in the presence of the aerogel, because the equilibrium concentration of aqueous Hg(II) is low due to adsorption, thus Paramecium cells do not uptake as much Hg(II) as in the un-remediated cultures. Video imaging of Paramecium cultures offers a simple, robust and flexible method for providing quantitative information on the effectiveness of advanced materials used in adsorption processes for water treatment.

MTT Test and Time-lapse Microscopy to Evaluate the Antitumor Potential of Nucleoside Analogues.

BACKGROUND/AIM: Conventional viability tests, help to screen the cellular effects of candidate molecules, but the endpoint of these measurements lacks sufficient information regarding the molecular aspects. A non-invasive, easy-to-setup live-cell microscopic method served to in-depth analysis of mechanisms of potential anticancer drugs. MATERIALS AND METHODS: The proposed method combining the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test with time-lapse scanning microscopy (TLS), provided additional data related to the cell-cycle and the dynamic properties of cell morphology. Apoptotic and necrotic events became detectable with these methods. RESULTS: Quantification of the results was assisted by image analysis of the acquired image sequences. After demonstrating the potential of the TLS method, a series of experiments compared the in vitro effect of a known and a newly synthesized nucleoside analogue. CONCLUSION: The proposed approach provided a more in-depth insight into the cellular processes that can be affected by known chemotherapeutic agents including nucleoside analogues rather than applying repeated individual treatments.

High-content screening identifies inhibitors of oxidative stress-induced parthanatos: cytoprotective and anti-inflammatory effects of ciclopirox.

BACKGROUND AND PURPOSE: Excessive oxidative stress can induce PARP1-mediated programmed necrotic cell death, termed parthanatos. Inhibition of parthanatos may be therapeutically beneficial in a wide array of diseases associated with tissue injury and inflammation. Our goal was to identify novel molecules inhibiting parthanatos. EXPERIMENTAL APPROACH: A small library of 774 pharmacologically active compounds was screened in a Sytox Green uptake assay, which identified 20 hits that reduced hydrogen-peroxide-induced parthanatos with an efficiency comparable to the benchmark PARP inhibitor, PJ34. KEY RESULTS: Of these hits, two compounds, antifungal ciclopirox and dopamine receptor agonist apomorphine, inhibited PAR polymer synthesis. These two compounds prevented the binding of PARP1 to oxidatively damaged DNA but did not directly interfere with the interaction between DNA and PARP1. Both compounds inhibited mitochondrial superoxide and H2 O2 production and suppressed DNA breakage. Since H2 O2 -induced damage is dependent on Fe2+ -catalysed hydroxyl radical production (Fenton chemistry), we determined the iron chelation activity of the two test compounds and found that ciclopirox and, to a lesser extent, apomorphine act as iron chelators. We also show that the Fe2+ chelation and indirect PARP inhibitory effects of ciclopirox translate to anti-inflammatory actions as demonstrated in a mouse dermatitis model, where ciclopirox reduced ear swelling, inflammatory cell recruitment and poly(ADP-ribosyl)ation. CONCLUSION AND IMPLICATIONS: Our findings indicate that the antimycotic drug, ciclopirox, acts as an iron chelator and thus targets an early event in hydrogen-peroxide-induced parthanatos. Ciclopirox has the potential to be repurposed as a cytoprotective and anti-inflammatory agent.

Targeting Nuclear NAD+ Synthesis Inhibits DNA Repair, Impairs Metabolic Adaptation and Increases Chemosensitivity of U-2OS Osteosarcoma Cells.

Osteosarcoma (OS) is the most common bone tumor in children and adolescents. Modern OS treatment, based on the combination of neoadjuvant chemotherapy (cisplatin + doxorubicin + methotrexate) with subsequent surgical removal of the primary tumor and metastases, has dramatically improved overall survival of OS patients. However, further research is needed to identify new therapeutic targets. Here we report that expression level of the nuclear NAD synthesis enzyme, nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1), increases in U-2OS cells upon exposure to DNA damaging agents, suggesting the involvement of the enzyme in the DNA damage response. Moreover, genetic inactivation of NMNAT1 sensitizes U-2OS osteosarcoma cells to cisplatin, doxorubicin, or a combination of these two treatments. Increased cisplatin-induced cell death of NMNAT1-/- cells showed features of both apoptosis and necroptosis, as indicated by the protective effect of the caspase-3 inhibitor z-DEVD-FMK and the necroptosis inhibitor necrostatin-1. Activation of the DNA damage sensor enzyme poly(ADP-ribose) polymerase 1 (PARP1), a major consumer of NAD+ in the nucleus, was fully blocked by NMNAT1 inactivation, leading to increased DNA damage (phospho-H2AX foci). The PARP inhibitor, olaparib, sensitized wild type but not NMNAT1-/- cells to cisplatin-induced anti-clonogenic effects, suggesting that impaired PARP1 activity is important for chemosensitization. Cisplatin-induced cell death of NMNAT1-/- cells was also characterized by a marked drop in cellular ATP levels and impaired mitochondrial respiratory reserve capacity, highlighting the central role of compromised cellular bioenergetics in chemosensitization by NMNAT1 inactivation. Moreover, NMNAT1 cells also displayed markedly higher sensitivity to cisplatin when grown as spheroids in 3D culture. In summary, our work provides the first evidence that NMNAT1 is a promising therapeutic target for osteosarcoma and possibly other tumors as well.

Uninterrupted Dabigatran Administration Provides Greater Inhibition against Intracardiac Activation of Hemostasis as Compared to Vitamin K Antagonists during Cryoballoon Catheter Ablation of Atrial Fibrillation.

BACKGROUND: Cerebral thromboembolism is a rare but feared complication of transcatheter ablation in patients with atrial fibrillation (AF). Here, we aimed to test which pre-procedural anticoagulation strategy results in less intracardiac activation of hemostasis during ablation. PATIENTS AND METHODS: In this observational study, 54 paroxysmal/persistent AF patients undergoing cryoballoon ablation were grouped according to their periprocedural anticoagulation strategy: no anticoagulation (oral anticoagulation (OAC) free; n = 24), uninterrupted vitamin K antagonists (VKA) (n = 11), uninterrupted dabigatran (n = 17). Blood was drawn from the left atrium before and immediately after the ablation procedure. Cryoablations were performed according to standard protocols, during which heparin was administered. Heparin-insensitive markers of hemostasis and endothelial damage were tested from intracardiac samples: D-dimer, quantitative fibrin monomer (FM), plasmin-antiplasmin complex (PAP), von Willebrand factor (VWF) antigen, chromogenic factor VIII (FVIII) activity. RESULTS: D-dimer increased significantly in all groups post-ablation, with lowest levels in the dabigatran group (median [interquartile range]: 0.27 [0.36] vs. 1.09 [1.30] and 0.74 [0.26] mg/L in OAC free and uninterrupted VKA groups, respectively, p < 0.001). PAP levels were parallel to this observation. Post-ablation FM levels were elevated in OAC free (26.34 [30.04] mg/L) and VKA groups (10.12 [16.01] mg/L), but remained below cut-off in all patients on dabigatran (3.98 [2.0] mg/L; p < 0.001). VWF antigen and FVIII activity increased similarly post-ablation in all groups, suggesting comparable procedure-related endothelial damage. CONCLUSION: Dabigatran provides greater inhibition against intracardiac activation of hemostasis as compared to VKAs during cryoballoon catheter ablation.

Intracardiac Fibrinolysis and Endothelium Activation Related to Atrial Fibrillation Ablation with Different Techniques.

OBJECTIVE: The effect of pulmonary vein isolation (PVI) on fibrinolytic and endothelial activation with currently applied periprocedural anticoagulation has not been explored. We measured markers of fibrinolysis and endothelium activation before and after PVI with the second-generation cryoballoon (Cryo), pulmonary vein ablation catheter (PVAC-Gold), and irrigated radiofrequency (IRF). METHODS: Markers of fibrinolysis and endothelium activation in left atrial (LA) blood samples were measured in 31 patients before and after PVI (Cryo:10, PVAC-Gold: 7, IRF: 14). Periprocedural anticoagulation included uninterrupted vitamin K antagonist and iv heparin (ACT≥300 sec) during LA dwelling. RESULTS: Levels of D-dimer (median; interquartile range, mgFEU/L) increased with all techniques (PVAC: 0.34; 0.24-0.50 versus 0.70; 0.61-1.31; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; PAP complex level (ng/ml) increased after Cryo (247.3, 199.9-331.6 versus 270.9, 227.9-346.7; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; PAI-1 activity (%) decreased with the PVAC (1.931; 0.508-3.859 versus 0.735, 0.240-2.707; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; VWF antigen levels and FVIII activity increased after PVI with all the 3 techniques. The levels of soluble VCAM-1 (ng/ml) did not change after PVAC procedures, but increased after Cryo (542, 6; 428.5-753.1 versus 619.2; 499.8-799.0; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93; p=0.0313, Cryo: 0.33; 0.28-0.49 versus 0.79; 0.65-0.93. CONCLUSION: PVI with contemporary ablation techniques and periprocedural antithrombotic treatment induces coagulation and endothelium activation of similar magnitude with different ablation methods.

Amino-isocyanoacridines: Novel, Tunable Solvatochromic Fluorophores as Physiological pH Probes.

Amino-isocyanoacridines (ICAAcs), as first members of their class, turned out to be a novel, multifunctional acridine orange (AO) type dye family with a number of additional favorable properties. They have enhanced solvatochromic emission range, low quantum yields (ΦF = 2.9-0.4%) in water, reduced basicity (pKa = 7.05-7.58), and their optical behavior could be fine-tuned by complexation with Ag(I) ions, too. Based on both their vibronic absorption and the charge transfer bands, ICAAcs can be applied as stable pH-probes with great precision (2-3% error) in the physiological pH range of 6-8 using UV-vis and fluorescence detection. The dyes are also able to sense pH change in different microenvironments, such as the Stern layer, as it was demonstrated on sodium lauryl sulfate micelles. The optical behavior of the ICAAc derivatives is discussed based on high-level quantum chemical calculations. All three dyes are well-applicable with conventional epifluorescence imaging. Furthermore, at the blue excitation, diMICAAc is optimally suited as a whole-cell probe for both the conventional microscopic and the laser-illumination studies, like flow- and imaging cytometric, or confocal laser-scanning microscopic examinations.

MICAN, a new fluorophore for vital and non-vital staining of human cells.

Fluorescence time-lapse microscopy is in connection with the invasive properties of fluorochrome applied, and with the toxicity of the excitation energy and wavelength of the dye itself. Experiments with the newly synthesized fluorescent dye 1-N-methylamino-5-isocyanonaphthalene (MICAN) served to test its cytotoxicity on human HaCaT keratinocyte cell cultures. Experiments related to staining capability were performed with paraformaldehyde (PFA) fixed cells and observed with fluorescence microscope. It was assumed that the fluorophore 1-amino-5-isocyanonaphthalene (ICAN) and especially its N-methylamino derivative MICAN, containing condensed aromatic rings could serve as a nonselective fluorescent dye capable to stain cellular structures of fixed, living, damaged and dead cells. This notion was confirmed by the MICAN staining of cytoplasmic proteins primarily rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SEM) and less efficiently nuclear proteins suggesting the involvement of staining of subcellular structures involved in protein synthesis. MICAN was not only well tolerated by living cells but turned out to be a strong heterochromatin and RER staining agent. This led to the development of a MICAN staining protocol for native and living samples. Relative to other fluorescent dyes, MICAN is not only useful but also cost-effective. Toxicology tests were performed using 30, 10, 5, 0.5 µg/ml MICAN concentrations. Time-lapse videomicroscopy at near-infrared (NIR) illumination has been used for the examination of MICAN effect on cell division. It was found that MICAN as a vital stain had no significant harmful effect on HaCaT cells. MICAN turned out to be a non-toxic, highly quantum-efficient vital stain with minimal, or no photobleaching, and can be applied to co-stain with propidium-iodide due the strong spectral separation.