The interplay between nitric oxide (NO) and reactive oxygen species can lead to an induction of cell death in plants. The aim of our work was to find out if cyanide released from sodium nitroprusside (SNP; a donor of NO) could be involved in the cell death induction, which is triggered by SNP and H 2 O 2 . Cell suspension of Nicotiana tabacum L. (line BY-2) was treated with 0.5 mM SNP, 0.5 mM potassium ferricyanide (PFC; analogue of sodium nitroprusside which can not release NO) and/or by 0.5 mM glucose with 0.5 U cm -3 glucose oxidase (GGO; a donor system of H 2 O 2 ). The cell death was induced only by combination of SNP and GGO. Thus cyanide released was not involved in the induction of cell death. However, SNP showed toxic effect because of decrease in activities of intracellular oxidoreductases and esterases. The cell death caused by SNP and GGO occurred within 12 h. During cell death either length or width of the cell increased. Central vacuole was formed in 20 to 40 % of cells. Most of the dead cells showed a condensed cytoplasm. Two hallmarks of programmed cell death (PCD), chromatin condensation and blebbing of nuclear periphery, were observed. However, oligonucleosomal fragmentation of DNA, another hallmark of PCD, was not detected.Additional key words: Nicotiana tabacum, nitric oxide, potassium ferricyanide, programmed cell death.

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bβ chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

On-chip vascular microfluidic models provide a great tool to study aspects of cardiovascular diseases in vitro. To produce such models, polydimethylsiloxane (PDMS) has been the most widely used material. For biological applications, its hydrophobic surface has to be modified. The major approach has been plasma-based surface oxidation, which has been very challenging in the case of channels enclosed within a microfluidic chip. The preparation of the chip combined a 3D-printed mold with soft lithography and commonly available materials. We have introduced the high-frequency low-pressure air-plasma surface modification of seamless channels enclosed within a PDMS microfluidic chip. The plasma treatment modified the luminal surface more uniformly than in previous works. Such a setup enabled a higher degree of design freedom and a possibility of rapid prototyping. Further, plasma treatment in combination with collagen IV coating created a biomimetic surface for efficient adhesion of vascular endothelial cells as well as promoted long-term cell culture stability under flow. The cells within the channels were highly viable and showed physiological behavior, confirming the benefit of the presented surface modification.

Diseases with the highest burden for society such as stroke, myocardial infarction, pulmonary embolism, and others are due to blood clots. Preclinical and clinical techniques to study blood clots are important tools for translational research of new diagnostic and therapeutic modalities that target blood clots. In this study, we employed a three-dimensional (3D) printed middle cerebral artery model to image clots under flow conditions using preclinical imaging techniques including fluorescent whole-body imaging, magnetic resonance imaging (MRI), and computed X-ray microtomography (microCT). Both liposome-based, fibrin-targeted, and non-targeted contrast agents were proven to provide a sufficient signal for clot imaging within the model under flow conditions. The application of the model for clot targeting studies and thrombolytic studies using preclinical imaging techniques is shown here. For the first time, a novel method of thrombus labeling utilizing barium sulphate (Micropaque®) is presented here as an example of successfully employed contrast agents for in vitro experiments evaluating the time-course of thrombolysis and thus the efficacy of a thrombolytic drug, recombinant tissue plasminogen activator (rtPA). Finally, the proof-of-concept of in vivo clot imaging in a middle cerebral artery occlusion (MCAO) rat model using barium sulphate-labelled clots is presented, confirming the great potential of such an approach to make experiments comparable between in vitro and in vivo models, finally leading to a reduction in animals needed.

Introduction: Direct clot targeting represents attractive concept for clot imaging as well as targeted delivery of drugs, e.g. thrombolytics. Small protein binders attached to nanoliposomes may target thrombi and deliver drugs although selective affinity to fibrin and not fibrinogen is the main challenge. Methods: For identification and preparation of fibrin-specific artificial protein binders derived from scaffolds of albumin-binding domain (ABD) of streptococcal protein G, a highly complex ABD-derived combinatorial library in combination with ribosome display selection was used. In vitro models were used to document delivery of nanoliposomes to human thrombi. Results: A recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptides of the Bβ chain fused to TolA protein carrying polyhistidylated tag and Avitag was constructed. Ribosome display was followed by large-scale ELISA screening of protein binders. Only four protein variants had selective affinity to human fibrin - see figure 1A. The most selective, variant D7, was modified by C-terminal FLAG/His 6 or His 6 /His 6 tag in order to be attached onto the surface of nanoliposomes. The electron microscopy then confirmed the structure of nanoliposome-binder particles. Isothermal titration calorimetry provided dissociation constant for liposome-binder metallochelating bond in the range 10 -7 to 10 -9 for mono- and double-HisTag forms. In vitro, in silicone replica of small diameter artery, the confocal and scanning electron microscopy confirmed a successful binding of D7-attached- to-nanoliposomes to fibrin fibres, see figure 1B. Conclusions: We developed binders relatively selective to fibrin, attached them to nanoliposomes, and documented targeting of fibrin in vitro. As the next step, selectivity needs to be now documented in animal studies.