Krzysztof Wojcik

Persistent skin colonization with Staphylococcus aureus in atopic dermatitis: relationship to clinical and immunological parameters

Background Staphylococcus aureus has important implications for the pathogenesis of atopic dermatitis (AD). In some patients S. aureus can be eradicated from the skin during anti‐inflammatory treatment, while in others bacterial colonization is persistent. Potential mechanisms and features of these two distinct groups of patients are not known.

Interaction of a DNA intercalator DRAQ5, and a minor groove binder SYTO17, with chromatin in live cells—Influence on chromatin organization and histone—DNA interactions

DNA‐binding dyes are useful in contrasting cell nuclei and chromatin for live cell fluorescence microscopy; however, they may interfere with nuclear and DNA structure and function. We investigated the influence exerted by two DNA dyes on chromatin and nuclear structure, as well as histone–DNA interactions in live HeLa cells. A membrane‐permeant fluorescent DNA intercalator DRAQ5 (anthracycline derivative), at a concentration of 1 μM, caused microscopically detectable changes of nuclear architecture. Following DRAQ5 intercalation into DNA, chromatin aggregated into distinct areas and foci. The loss of 3D chromatin distribution was exerted via interference with a dynamic exchange of a linker histone (H1), which is a known chromatin stabilizing factor. At higher concentrations (3 and 7.5 μM), DRAQ5 interfered with binding of H2B core histones to DNA. Similar effects resulted from intercalation of chemotherapeutic drugs, adriamycin and daunomycin, but were not observed after binding to DNA of a minor groove binder, Syto17.

Daunomycin, an antitumor DNA intercalator, influences histone-DNA interactions.

Although daunomycin and adriamycin are considered effective antitumor drugs and have been used in the clinic for over 40 years, their mechanism of action is still a matter of debate. We investigated the influence of daunomycin on interaction between linker or core histones and DNA in live HeLa cells in vitro, using image and flow cytometry. Exposure to daunomycin at clinically relevant concentrations (25–250 nM) caused dissociation of wild-type H1.1 as well as 4 H1 point mutants from DNA, followed by their accumulation in nucleoli and aggregation of chromatin. A detectable dissociation of H2B core histones occurred only at much higher concentrations of the drug (500 nM). Replication of DNA and synthesis of RNA were not halted by daunomycin (up to 2500 nM); however the characteristic subnuclear distribution of sites of transcription and replication was lost. Dissociation of the H1.1 linker histones and subsequent loss of higher order chromatin structures may constitute an important component of the mechanism of cytotoxicity of daunomycin.

Induced sputum eicosanoids during aspirin bronchial challenge of asthmatic patients with aspirin hypersensitivity

Altered metabolism of eicosanoids is a characteristic finding in aspirin‐exacerbated respiratory disease (AERD). Bronchial challenge with lysyl‐aspirin can be used as a confirmatory diagnostic test for this clinical condition. Induced sputum allows to measure mediators of asthmatic inflammation in bronchial secretions.

Use of Sensitive, Broad-Spectrum Molecular Assays and Human Airway Epithelium Cultures for Detection of Respiratory Pathogens

Rapid and accurate detection and identification of viruses causing respiratory tract infections is important for patient care and disease control. Despite the fact that several assays are available, identification of an etiological agent is not possible in ∼30% of patients suffering from respiratory tract diseases. Therefore, the aim of the current study was to develop a diagnostic set for the detection of respiratory viruses with sensitivity as low as 1–10 copies per reaction. Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ∼76% of cases. To improve assay performance and facilitate the identification of novel species or emerging strains, cultures of fully differentiated human airway epithelium were used to pre-amplify infectious viruses. This additional step resulted in the detection of pathogens in all samples tested. Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

Measurements on MIMO-FRET Nano-Networks Based on Alexa Fluor Dyes

Nanocommunication has gained significant attention in the last few years, as a means to establish information transfer between future nanomachines. Comparing with other communication techniques for nanoscale (calcium ions signaling, molecular or catalytic nanomotors, pheromones propagation, bacteria-based communication), the phenomenon called Förster Resonance Energy Transfer (FRET) offers significantly smaller propagation delays and high channel throughput. In this paper, we report our recent experiments on FRET-based nanonetworks performed in the Laboratory of Cell Biophysics of the Jagiellonian University, Kraków. We propose to use Alexa Fluor dyes as nano transmitters and receivers, as they enable to create multiple-input multioutput (MIMO) FRET communication channels and thus enhance FRET efficiency. We measure FRET efficiency values, calculate bit error rates for the measured scenarios, and extend the calculations to consider a general case of MIMO (n, m) FRET channels.

Aspirin provocation increases 8-iso-PGE2 in exhaled breath condensate of aspirin-hypersensitive asthmatics

BACKGROUND Isoprostanes are bioactive compounds formed by non-enzymatic oxidation of polyunsaturated fatty acids, mostly arachidonic, and markers of free radical generation during inflammation. In aspirin exacerbated respiratory disease (AERD), asthmatic symptoms are precipitated by ingestion of non-steroid anti-inflammatory drugs capable for pharmacologic inhibition of cyclooxygenase-1 isoenzyme. We investigated whether aspirin-provoked bronchoconstriction is accompanied by changes of isoprostanes in exhaled breath condensate (EBC). METHODS EBC was collected from 28 AERD subjects and 25 aspirin-tolerant asthmatics before and after inhalatory aspirin challenge. Concentrations of 8-iso-PGF2α, 8-iso-PGE2, and prostaglandin E2 were measured using gas chromatography/mass spectrometry. Leukotriene E4 was measured by immunoassay in urine samples collected before and after the challenge. RESULTS Before the challenge, exhaled 8-iso-PGF2α, 8-iso-PGE2, and PGE2 levels did not differ between the study groups. 8-iso-PGE2 level increased in AERD group only (p=0.014) as a result of the aspirin challenge. Urinary LTE4 was elevated in AERD, both in baseline and post-challenge samples. Post-challenge airways 8-iso-PGE2 correlated positively with urinary LTE4 level (p=0.046), whereas it correlated negatively with the provocative dose of aspirin (p=0.027). CONCLUSION A significant increase of exhaled 8-iso-PGE2 after inhalatory challenge with aspirin was selective and not present for the other isoprostane measured. This is a novel finding in AERD, suggesting that inhibition of cyclooxygenase may elicit 8-iso-PGE2 production in a specific mechanism, contributing to bronchoconstriction and systemic overproduction of cysteinyl leukotrienes.

Nanocommunication via FRET With DyLight Dyes Using Multiple Donors and Acceptors

The phenomenon of Förster Resonance Energy Transfer, commonly used to measure the distances between fluorophore molecules and to study interactions between fluorescent-tagged proteins in life sciences, can also be applied in nanocommunication networks to transfer information bits. The mechanism offers a relatively large throughput and very small delays, but at the same time the channel bit error rate is too high and the transmission ranges are too limited for communication purposes. In this paper, multiple donors at the transmitter side and multiple acceptors at the receiver side are considered to decrease the bit error rate. As nanoantennas, the DyLight fluorescent dyes, which are very well suited to long range nanocommunication due to their large Förster distances and high degrees of labeling, are proposed. The reported results of the recent laboratory experiments confirm efficient communication on distances over 10 nm.

Induced sputum supernatant bioactive lipid mediators can identify subtypes of asthma.

Induced sputum (IS) allows to measure mediators of asthmatic inflammation in bronchial secretions. The specific role of induced sputum supernatant (ISS) endogenous bioactive lipid mediators in subtypes of asthma is not well understood.

Routing in FRET-Based Nanonetworks

Nanocommunications, understood as communications between nanoscale devices, is commonly regarded as a technology essential for cooperation of large groups of nanomachines and thus crucial for development of the whole area of nanotechnology. While solutions for pointto- point nanocommunications have already been proposed, larger networks cannot function properly without routing. In this article we focus on nanocommunications via FRET, which was found to be a technique with a very high signal propagation speed, and discuss how to route signals through nanonetworks. We introduce five new routing mechanisms, based on biological properties of specific molecules. We experimentally validate one of these mechanisms. Finally, we analyze open issues showing the technical challenges for signal transmission and routing in FRET-based nanocommunications.