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Introduction: The recently developed platelet aggregation technique based on low-angle light scattering (LaSca) in diluted platelet-rich plasma (PRP) requires only a small sample volume and provides information about platelet aggregation and shape change. This study aimed to investigate the influence of preanalytical and analytical variables and to validate the method in a real-life pediatric hematology hospital setting., Methods: Platelet aggregation was induced by ADP in diluted PRP in the presence of 2 mM calcium at 23 °C. The study included healthy adults (n = 30), healthy children (n = 20), and pediatric patients with suspected or diagnosed platelet function abnormalities (n = 25)., Results: The assay parameters were stable for at least 3 h after isolation of PRP and were sensitive to plasma dilution in the range of 2-8%. The initial aggregation velocity was significantly reduced in pediatric patients compared with healthy children (p < 0.05). ADP-induced light transmission amplitude was moderately correlated with LaSca amplitude of aggregation in healthy children (p = 0.52, p < 0.05) but not in pediatric patients., Conclusions: We standardized the protocol for platelet aggregation assessment by LaSca and characterized the influence of preanalytical and analytical variables on it., (© 2024. Japanese Society of Hematology.)

Objectives: Flow cytometry with adenosine diphosphate (ADP) allows to characterize molecular changes of platelet function caused by this physiologically important activation, but the methodology has not been thoroughly investigated, standardized and characterized yet. We analyzed the influence of several major variables and chose optimal conditions for platelet function assessment., Methods: For activation, 2.5 μM CaCl 2 , 5 μM ADP and antibodies were added to diluted blood and incubated for 15 min. We analyzed kinetics of antibody binding and effects of their addition sequence, agonist concentration, blood dilution, exogenous calcium addition and platelet fixation., Results: We tested our protocol on 11 healthy children, 22 healthy adult volunteers, 9 patients after a month on dual antiplatelet therapy after percutaneous coronary intervention (PCI), 7 adult patients and 14 children with immune thrombocytopenia (ITP). We found that our protocol is highly sensitive to ADP stimulation with low percentage of aggregates formation. The assay is also sensitive to platelet function inhibition in post-PCI patients. Finally, platelet preactivation with ITP plasma was stronger and caused increase in activation response to ADP stimulation compared to preactivation with low dose of ADP., Conclusions: Our assay is sensitive to antiplatelet therapy and platelet preactivation in ITP patients under physiological conditions with minimal percentage of aggregates formation., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Platelets are blood cells responsible for the prevention of blood loss upon vessel wall disruption. It has been demonstrated that platelet functioning differs significantly between adult and pediatric donors. This study aimed to identify potential differences between the protein composition of platelets of pediatric, adolescent, and adult donors., Methods: Platelet functional testing was conducted with live cell flow cytometry. Using a straightforward approach to platelet washing based on the sequential platelets centrifugation-resuspension, we were able to obtain stable and robust proteomics results, which corresponded to previously published data., Results: We have identified that pediatric donors' platelets have increased amounts of proteins, responsible for mitochondrial activity, proteasome activity, and vesicle transport. Flow cytometry analysis of platelet intracellular signaling and functional responses revealed that platelets of the pediatric donors have diminished granule secretion and increased quiescent platelet calcium concentration and decreased calcium mobilization in response to ADP. We could explain the observed changes in calcium responses by the increased mitochondria protein content, and the changes in granule secretion could be explained by the differences in vesicle transport protein content., Conclusions: Therefore, we can conclude that the age-dependence of platelet functional responses originates from the difference in platelet protein content., Impact: Platelets of infants are known to functionally differ from the platelet of adult donors, although the longevity and persistivity of these differences are debatable. Pediatric donor platelets have enhanced amounts of mitochondrial, proteasomal, and vesicle transport proteins. Platelets of the pediatric donors had increased cytosolic calcium in the resting state, what is explained by the increased numbers of mitochondrial proteins. Infants had decreased platelet granule release, which resolved upon adolescence. Thus, platelets of the infants should be assessed differently from adult platelets. Differences in platelet proteomic contents persisted in adolescent groups, yet, no significant differences in platelet function were observed., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Activated platelets provide phospholipid surface and secrete coagulation factors, enhancing blood clotting. We investigated the role of platelets in the regulation of blood coagulation spatial dynamics. We activated blood clotting with tissue factor-bearing (TF) surface in platelet-rich plasma (PRP) or platelet-free plasma (PFP). When blood coagulation was initiated by high TF density, clot growth rate (V) in PRP (2 × 10 5 /μL platelets) was only 15 % greater than in PFP. Spatial distribution of thrombin in PRP had a peak-like shape in the area of the fibrin clot edge, while in PFP thrombin was distributed in the shape of descending plateau. Platelet inhibition with prostaglandin E1 or cytochalasin D made spatial thrombin distribution look like in the case of PFP. Inhibition of blood coagulation by natural endogenous inhibitor heparin was diminished in PRP, while the effect of the exogenous or artificial inhibitors (rivaroxaban, nitrophorin, hirudin) remained undisturbed in the presence of platelets. Ten times decrease of the TF surface density greatly depressed blood coagulation in PFP. In PRP only clotting initiation phase was, while the propagation phase remained intact. Coagulation factor deficiency greatly reduced amount of thrombin and decreased V in PFP rather than in PPR. Thus, platelets were redundant for clotting in normal plasma under physiological conditions but provided robustness of the coagulation system to the changes in initial conditions., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: FIA and MAP are former employees and cofounders of Hemacore Labs LLC, OAF is employee of Hemacore Labs LLC. Hemacore Labs LLC holds several patents and patent applications that are related to the diagnostic use of spatial clot growth and thrombin generation and which has developed the assay under the trade name of Thrombodynamics®. Hemacore Labs LLC provided support in the form of reagents for Thrombodynamics-4D but did not have any additional role in the design of the study, collection and analysis of data, decision to publish or preparation of the manuscript. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. All other authors declare no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and ex vivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The growing interest in potassium channels as pharmacological targets has stimulated the development of their fluorescent ligands (including genetically encoded peptide toxins fused with fluorescent proteins) for analytical and imaging applications. We report on the properties of agitoxin 2 C-terminally fused with enhanced GFP (AgTx2-GFP) as one of the most active genetically encoded fluorescent ligands of potassium voltage-gated K v 1.x (x = 1, 3, 6) channels. AgTx2-GFP possesses subnanomolar affinities for hybrid KcsA-K v 1.x (x = 3, 6) channels and a low nanomolar affinity to KcsA-K v 1.1 with moderate dependence on pH in the 7.0-8.0 range. Electrophysiological studies on oocytes showed a pore-blocking activity of AgTx2-GFP at low nanomolar concentrations for K v 1.x (x = 1, 3, 6) channels and at micromolar concentrations for K v 1.2. AgTx2-GFP bound to K v 1.3 at the membranes of mammalian cells with a dissociation constant of 3.4 ± 0.8 nM, providing fluorescent imaging of the channel membranous distribution, and this binding depended weakly on the channel state (open or closed). AgTx2-GFP can be used in combination with hybrid KcsA-K v 1.x (x = 1, 3, 6) channels on the membranes of E. coli spheroplasts or with K v 1.3 channels on the membranes of mammalian cells for the search and study of nonlabeled peptide pore blockers, including measurement of their affinity.

In aqueous solutions, cobra cytotoxins (CTX), three-finger folded proteins, exhibit conformational equilibrium between conformers with either cis or trans peptide bonds in the N-terminal loop (loop-I). The equilibrium is shifted to the cis form in toxins with a pair of adjacent Pro residues in this loop. It is known that CTX with a single Pro residue in loop-I and a cis peptide bond do not interact with lipid membranes. Thus, if a cis peptide bond is present in loop-I, as in a Pro-Pro containing CTX, this should weaken its lipid interactions and likely cytotoxic activities. To test this, we have isolated seven CTX from Naja naja and N. haje cobra venoms. Antibacterial and cytotoxic activities of these CTX, as well as their capability to induce calcein leakage from phospholipid liposomes, were evaluated. We have found that CTX with a Pro-Pro peptide bond indeed exhibit attenuated membrane-perturbing activity in model membranes and lower cytotoxic/antibacterial activity compared to their counterparts with a single Pro residue in loop-I., Competing Interests: The authors declare no conflicts of interest.

Herein, we report a new conjugate BChl-S-S-NI based on the second-generation photosensitizer bacteriochlorin e6 (BChl) and a 4-styrylnaphthalimide fluorophore (NI), which is cleaved into individual functional fragments in the intracellular medium. The chromophores in the conjugate were cross-linked by click chemistry via a bis(azidoethyl)disulfide bridge which is reductively cleaved by the intracellular enzyme glutathione (GSH). A photophysical investigation of the conjugate in solution by using optical spectroscopy revealed that the energy transfer process is realized with high efficiency in the conjugated system, leading to the quenching of the emission of the fluorophore fragment. It was shown that the conjugate is cleaved by GSH in solution, which eliminates the possibility of energy transfer and restores the fluorescence of 4-styrylnaphthalimide. The photoinduced activity of the conjugate and its imaging properties were investigated on the mouse soft tissue sarcoma cell line S37. Phototoxicity studies in vitro show that the BChl-S-S-NI conjugate has insignificant dark cytotoxicity in the concentration range from 15 to 20,000 nM. At the same time, upon photoexcitation, it exhibits high photoinduced activity.

The voltage-gated potassium Kv1.3 channel is an essential component of vital cellular processes which is also involved in the pathogenesis of some autoimmune, neuroinflammatory and oncological diseases. Pore blockers of the Kv1.3 channel are considered as potential drugs and are used to study Kv1 channels' structure and functions. Screening and study of the blockers require the assessment of their ability to bind the channel. Expanding the variety of methods used for this, we report on the development of the fluorescent competitive binding assay for measuring affinities of pore blockers to Kv1.3 at the membrane of mammalian cells. The assay constituents are hongotoxin 1 conjugated with Atto488, fluorescent mKate2-tagged Kv1.3 channel, which was designed to improve membrane expression of the channel in mammalian cells, confocal microscopy, and a special protocol of image processing. The assay is implemented in the "mix and measure", format and allows the screening of Kv1.3 blockers, such as peptide toxins, that bind to the extracellular vestibule of the K + -conducting pore, and analyzing their affinity.

The aim of this work was to design and characterize peptides based on the α-helices h1 and h2 of the ACE2 receptor, forming the interaction interface between the receptor-binding domain (RBD) of the SARS-CoV-2 S protein and the cellular ACE2 receptor. Monomeric and heterodimeric peptides connected by disulfide bonds at different positions were synthesized. Solubility, RBD-binding affinity, and peptide helicity were experimentally measured, and molecular dynamics simulation was performed in various solvents. It was established that the preservation of the helical conformation is a necessary condition for the binding of peptides to RBD. The peptides have a low degree of helicity and low affinity for RBD in water. Dimeric peptides have a higher degree of helicity than monomeric ones, probably due to the mutual influence of helices. The degree of helicity of the peptides in trifluoroethanol is the highest; however, for in vitro studies, the most suitable solvent is a water-ethanol mixture., (© 2022. The Author(s).)

Among voltage-gated potassium channel (K V ) isoforms, K V 1.6 is one of the most widespread in the nervous system. However, there are little data concerning its physiological significance, in part due to the scarcity of specific ligands. The known high-affinity ligands of K V 1.6 lack selectivity, and conversely, its selective ligands show low affinity. Here, we present a designer peptide with both high affinity and selectivity to K V 1.6. Previously, we have demonstrated that K V isoform-selective peptides can be constructed based on the simplistic α-hairpinin scaffold, and we obtained a number of artificial Tk-hefu peptides showing selective blockage of K V 1.3 in the submicromolar range. We have now proposed amino acid substitutions to enhance their activity. As a result, we have been able to produce Tk-hefu-11 that shows an EC 50 of ≈70 nM against K V 1.3. Quite surprisingly, Tk-hefu-11 turns out to block K V 1.6 with even higher potency, presenting an EC 50 of ≈10 nM. Furthermore, we have solved the peptide structure and used molecular dynamics to investigate the determinants of selective interactions between artificial α-hairpinins and K V channels to explain the dramatic increase in K V 1.6 affinity. Since K V 1.3 is not highly expressed in the nervous system, we hope that Tk-hefu-11 will be useful in studies of K V 1.6 and its functions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The properties of a lysozyme solution under laser-induced breakdown were studied. An optical breakdown under laser action in protein solutions proceeds with high efficiency: the formation of plasma and acoustic oscillations is observed. The concentration of protein molecules has very little effect on the physicochemical characteristics of optical breakdown. After exposure to optical breakdown, changes were observed in the enzymatic activity of lysozyme, absorption and fluorescence spectra, viscosity, and the sizes of molecules and aggregates of lysozyme measured by dynamic light scattering. However, the refractive index of the solution and the Raman spectrum did not change. The appearance of a new fluorescence peak was observed upon excitation at 350 nm and emission at 434 nm at exposure for 30 min. Previously, a peak in this range was associated with the fluorescence of amyloid fibrils. However, neither the ThT assay nor the circular dichroism dispersion confirmed the formation of amyloid fibrils. Probably, under the influence of optical breakdown, a small part of the protein degraded, and a part changed its native state and aggregated, forming functional dimers or "native aggregates".

An increase in the frequency of mycoses and spreading of multidrug-resistant fungal pathogens necessitates the search for new antifungal agents. Earlier, we isolated the novel defensin from lentil Lens culinaris seeds, designated as Lc-def, which inhibited the growth of phytopathogenic fungi. Here, we studied an antifungal activity of Lc-def against human pathogenic Candida species, structural stability of the defensin, and its immunomodulatory effects that may help to prevent fungal infection. We showed that Lc-def caused 50% growth inhibition of clinical isolates of Candida albicans , C. krusei , and C. glabrata at concentrations of 25-50 μM, but was not toxic to different human cells. The lentil defensin was resistant to proteolysis by C. albicans and was not cleaved during simulated gastroduodenal digestion. By using the multiplex xMAP assay, we showed for the first time for plant defensins that Lc-def increased the production of such essential for immunity to candidiasis pro-inflammatory cytokines as IL-12 and IL-17 at the concentration of 2 μM. Thus, we hypothesized that the lentil Lc-def and plant defensins in general may be effective in suppressing of mucocutaneous candidiasis due to their antifungal activity, high structural stability, and ability to activate a protective immune response.

Studies on platelet function in children older than neonatal period are few and their results are controversial. The pediatric platelets were alternatively reported to be more active or less active than adults' ones. We compared platelet function in the several age groups of children to adults and evaluated the age when platelet function reaches the adults' status. The study included 76 healthy children and 49 healthy adult volunteers. Types of platelet activation used included: collagen-related peptide (CRP) and PAR-1 activating peptide SFLLRN; SFLLRN, PAR-4 activating peptide AYPGKF and adenosine diphosphate (ADP); ADP. The parameters determined included forward (FSC) and side scatter (SSC), CD42b, CD61, CD62P, PAC-1, annexin V binding and mepacrine release levels. Resting pediatric platelets were similar to adults' platelets except for 1.2-fold decreased FSC and dense granules volume in youngest children, and 2.5-fold increased annexin V level in children aged 1-10 years. After CRP+SFLLRN stimulation, pediatric platelets had a 1.2-fold lower alpha- and 1.1-fold lower dense granule release than adults. For SFLLRN+AYPGKF+ADP stimulation, this was observed only for youngest children. The response to ADP stimulation was identical for pediatric platelets and adults. Pediatric platelets have lower granular release than adults' platelets, which persists until the age of 18.

Background: Coagulation system is heavily involved into the process of infective endocarditis (IE) vegetation formation and can facilitate further embolization. In this study we aimed to assess the coagulation and platelet state in IE implementing a wide range of standard and global laboratory assays. We also aim to determine whether prothrombotic genetic polymorphisms play any role in embolization and mortality in IE patients., Methods: 37 patients with IE were enrolled into the study. Coagulation was assessed using standard coagulation assays (activated partial thromboplastin time (APTT), prothrombin, fibrinogen, D-dimer concentrations) and integral assays (thromboelastography (TEG) and thrombodynamics (TD)). Platelet functional activity was estimated by flow cytometry. Single nuclear polymorphisms of coagulation system genes were studied., Results: Fibrinogen concentration and fibrinogen-dependent parameters of TEG and TD were increased in patients indicating systemic inflammation. In majority of patients clot growth rate in thrombodynamics was significantly shifted towards hypercoagulation in consistency with D-dimers elevation. However, in some patients prothrombin, thromboelastography and thrombodynamics were shifted towards hypocoagulation. Resting platelets were characterized by glycoprotein IIb-IIIa activation and degranulation. In patients with fatal IE, we observed a significant decrease in fibrinogen and thrombodynamics. In patients with embolism, we observed a significant decrease in the TEG R parameter. No association of embolism or mortality with genetic polymorphisms was found in our cohort., Conclusions: Our findings suggest that coagulation in patients with infective endocarditis is characterized by general hypercoagulability and platelet pre-activation. Some patients, however, have hypocoagulant coagulation profile, which presumably can indicate progressing of hypercoagulation into consumption coagulopathy., Competing Interests: We disclose a conflict of interest for Prof. Fazoil Ataullakhanov, who is a co-founder of HemaCore LLC, which holds several patents and patent applications that are related to the diagnostic use of spatial clot growth and which has developed the assay under the trade name of Thrombodynamics® (Ataullakhanov FI, international patent applications PCT/CH2007/000543 filing date 02.11.2007 and РСТ/RU2012/000570 filing date 16.07.2012). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Plant lipid transfer proteins (LTPs) are known to be clinically significant allergens capable of binding various lipid ligands. Recent data showed that lipid ligands affected the allergenic properties of plant LTPs. In this work, we checked the assumption that specific amino acid residues in the Len c 3 structure can play a key role both in the interaction with lipid ligands and IgE-binding capacity of the allergen. The recombinant analogues of Len c 3 with the single or double substitutions of Thr41, Arg45 and/or Tyr80 were obtained by site-directed mutagenesis. All these amino acid residues are located near the "bottom" entrance to the hydrophobic cavity of Len c 3 and are likely included in the IgE-binding epitope of the allergen. Using a bioinformatic approach, circular dichroism and fluorescence spectroscopies, ELISA, and experiments mimicking the allergen Len c 3 gastroduodenal digestion we showed that the substitution of all the three amino acid residues significantly affected structural organization of this region and led both to a change of the ligand-binding capacity and the allergenic potential of Len c 3.

Previously, we have demonstrated that Gly m 4, one of the major soybean allergens, could pass through the Caco-2 epithelial barrier and have proposed a mechanism of sensitization. However, it is not known yet whether Gly m 4 can reach the intestine in its intact form after digestion in stomach. In the present work, we studied an influence of various factors including lipids (fatty acids and lysolipids) on digestibility of Gly m 4. Using fluorescent and CD spectroscopies, we showed that Gly m 4 interacted with oleic acid and LPPG (lyso-palmitoyl phosphatidylglycerol), but its binding affinity greatly decreased under acidic conditions, probably due to the protein denaturation. The mimicking of gastric digestion revealed that Gly m 4 digestibility could be significantly reduced with the change of pH value and pepsin-to-allergen ratio, as well as by the presence of LPPG. We suggested that the protective effect of LPPG was unlikely associated with the allergen binding, but rather connected to the pepsin inhibition due to the lipid interaction with its catalytic site. As a result, we assumed that, under certain conditions, the intact Gly m 4 might be able to reach the human intestine and thereby could be responsible for allergic sensitization.

Immune thrombocytopenia (ITP) is believed to be associated with platelet function defects. However, their mechanisms are poorly understood, in particular with regard to differences between ITP phases, patient age, and therapy. We investigated platelet function and bleeding in children with either persistent or chronic ITP, with or without romiplostim therapy. The study included 151 children with ITP, of whom 56 had disease duration less than 12 months (grouped together as acute/persistent) and 95 were chronic. Samples of 57 healthy children were used as controls, while 5 patients with leukemia, 5 with aplastic anemia, 4 with MYH9-associated thrombocytopenia, and 7 with Wiskott-Aldrich syndrome were used as non-ITP thrombocytopenia controls. Whole blood flow cytometry revealed that platelets in both acute/persistent and chronic ITP were increased in size compared with healthy donors. They were also pre-activated as assessed by PAC1, CD62p, cytosolic calcium, and procoagulant platelet levels. This pattern was not observed in other childhood thrombocytopenias. Pre-activation by CD62p was higher in the bleeding group in the chronic ITP cohort only. Romiplostim treatment decreased size and pre-activation of the patient platelets, but not calcium. Our data suggest that increased size, pre-activation, and cytosolic calcium are common for all ITP platelets, but their association with bleeding could depend on the disease phase.

During the past decades, it has been increasingly recognized that the major function of accelerating membrane-dependent reactions of blood coagulation is predominantly implemented by a subset of activated platelets. These procoagulant platelets (also called collagen- and thrombin-activated or COAT, coated, necrotic, although there could be subtle differences between these definitions) are uniquely characterized by both procoagulant activity and, at the same time, inactivated integrins and profibrinolytic properties. The mechanisms of their generation both in vitro and in situ have been increasingly becoming clear, suggesting unique and multidirectional roles in hemostasis and thrombosis. In this mini-review, we shall highlight the existing concepts and challenges in this field., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three β-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea stagnalis AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on Torpedo nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the 1 H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.

Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

Previously, we isolated the lentil allergen Len c 3, belonging to the class of lipid transfer proteins, cross-reacting with the major peach allergen Pru p 3 and binding lipid ligands. In this work, the allergenic capacity of Len c 3 and effects of different lipid ligands on the protein stability and IgE-binding capacity were investigated. Impacts of pH and heat treating on ligand binding with Len c 3 were also studied. It was shown that the recombinant Len c 3 (rLen c 3) IgE-binding capacity is sensitive to heating and simulating of gastroduodenal digestion. While being heated or digested, the protein showed a considerably lower capacity to bind specific IgE in sera of allergic patients. The presence of lipid ligands increased the thermostability and resistance of rLen c 3 to digestion, but the level of these effects was dependent upon the ligand's nature. The anionic lysolipid LPPG showed the most pronounced protective effect which correlated well with experimental data on ligand binding. Thus, the Len c 3 stability and allergenic capacity can be retained in the conditions of food heat cooking and gastroduodenal digestion due to the presence of certain lipid ligands.

Childhood essential thrombocythemia (ET) is a rare chronic myeloproliferative disorder. The quality of life of ET patients may decrease as a result of ischemic and hemorrhagic complications of unclear origin. Our goal was to characterize the hemostatic system in children with ET. We genotyped and investigated blood samples from 20 children with ET in a prospective case series study using platelet aggregation, functional flow cytometry (FC) assay and standard clotting assays. Three children had a JAK2V617F mutation, 4 had mutations in CALR and 13 were triple-negative. Myelofibrosis in stage 1-2 was detected in 3 children. Three patients had bleeding episodes and seven had ischemic events. Aggregation in response to collagen, adenosine diphosphate, and ristomycin was decreased in all patients. In FC, significant changes in the whole patient group compared to the healthy children control group were decrease in the resting forward scatter and PAC1 binding (activated GPIIb/IIIa) level. For the activated platelets, dense granules release (by mepacrine), PAC1, and GPIIb/IIIa levels were significantly decreased. GPIb/V/IX, P -selectin, and phosphatidylserine levels manifested only moderate differences. Forward and side scatter changes in response to stimulation (representing shape change) and dense granules release were significantly lower in the 3 patients with bleeding than in the 17 patients without hemorrhage. Activated partial thromboplastin time was slightly prolonged, prothrombin index was slightly shortened and thrombin time was normal, while fibrinogen was mildly decreased in the ET patients. It could be concluded that the observed platelet function defects could be related to bleeding in ET, and be potentially used as a marker.

Programmed cell death of non-nucleated blood cells - platelets - could be associated with pathophysiology of oncologic and oncohematologic diseases. It contributes to both bleedings (caused by the thrombocytopenia, which is induced by elimination of the platelets) and thrombosis (caused by the processes of blood coagulation on the surface of phosphatidylserine exposing platelets). Here we characterized functional responses of platelets from the patients with various oncological disorders undergoing chemotherapy and compared them to the platelets from the healthy donors and platelets pre-incubated with apoptosis inducer ABT-737. Some patients exhibited diminished capability of platelets to aggregate. Immunophenotyping of these platelets revealed their pre-activation in comparison to the platelets from the healthy donors. Calcium signaling analysis revealed that in the patient-derived platelets, as well as in the apoptotic platelets, intracellular calcium levels were increased in resting cells. However, moderate level of this increase together with weak expression of phosphatidylserine allows us to assume that apoptotic processes in the circulating platelets from the patients are limited.

Background: Therapy with irreversible Bruton's tyrosine kinase inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) is associated with bleeding., Objectives: To propose the predictive markers of such bleeding, as well as mechanisms responsible for decreased bleeding at later therapy stages., Patients/methods: We investigate platelet functional activity in 50 CLL and 16 MCL patients on ibrutinib using flow cytometry and light transmission aggregometry., Results: Prior to treatment, both patient groups had decreased platelet counts; impaired aggregation with adenosine diphosphate (ADP); and decreased binding of CD62P, PAC1, and annexin V upon stimulation. Bleeding in patients treated with ibrutinib was observed in 28 (56%) CLL patients, who had decreased aggregation with ADP and platelet count before therapy. Their platelet count on therapy did not change, platelet aggregation with ADP steadily improved, and aggregation with collagen first decreased and then increased in anticorrellation with bleeding. Bleeding in MCL was observed in 10 (62%) patients, who had decreased dense granule release before therapy. ADP and ristocetin induced platelet aggregation in ibrutinib-treated MCL patients increased on therapy, while collagen-induced aggregation evolved similarly to CLL patients., Conclusions: Our results suggest that ibrutinib-dependent bleeding in CLL patients involves three mechanisms: decreased platelet count (the most important discriminator between bleeding and non-bleeding patients), impaired platelet response to ADP caused by CLL, and inhibition by ibrutinib. Initially, ibrutinib shifts the balance to bleeding, but then it is restored because of the improved response to ADP., (© 2020 International Society on Thrombosis and Haemostasis.)

Lipid transfer proteins (LTPs) are an important class of plant proteins containing an internal cavity and binding hydrophobic ligands. Although LTP structures and functions are well studied, mechanisms of ligand binding remain unclear. Earlier, we discovered the lentil lipid transfer protein Lc-LTP2 capable of binding and transfer various ligands. We have shown that the "bottom" entrance of the Lc-LTP2 cavity takes part in attachment to the micelle surface and in lipids uptake. Here, we studied the role of Arg45 and Tyr80, located at the "bottom" entrance, in Lc-LTP2 ligand binding. We obtained recombinant mutant analogs of Lc-LTP2 (R45A, Y80A, R45A/Y80A), investigated their ability to bind fatty acids and lysolipids, as well as performed molecular modeling of the protein-ligand complexes. We showed that replacement of one or both residues led to a change of the internal hydrophobic cavity dimensions. As a result, lipids may change their orientation into the protein cavity, and thereby binding ability of mutant analogs may be affected as well. In the present work, we revealed an important role of Arg45 and Tyr80 in stabilization of the Lc-LTP2 complexes with both fatty acids and lysolipids with different ligand orientation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Efficiency of both photodynamic and boron-neutron capture anticancer therapies (BNCT) depends on the properties of the used photo- and neutronsensitizer. We report on the synthesis and properties of the advanced photo- and neutronsensitizer designed as a conjugate of chlorin e 6 with iron bis(dicarbollide) nanocluster. Results: The conjugate is shown to accumulate efficiently in rat glioblastoma C6 cells delivering >10 9 boron atoms per cell and thus meeting requirements for BNCT agents, to provide photoinduced 50% death of C6 cells at 35 ± 3 nM, to be not toxic for cells without activating stimulus. Conclusions: The conjugate is a prospective theranostic agent for photodynamic, BNCT and fluorescent diagnostics of tumors.

Wiskott-Aldrich syndrome (WAS) is associated with thrombocytopenia of unclear origin. We investigated real-time cytosolic calcium dynamics, mitochondrial membrane potential and phoszphatidylserine (PS) exposure in single fibrinogen-bound platelets using confocal microscopy. The WAS platelets had higher resting calcium levels, more frequent spikes, and their mitochondria more frequently lost membrane potential followed by PS exposure (in 22.9% of platelets vs 3.9% in controls; P <0.001) after the collapse of the last mitochondria. This phenomenon was inhibited by the mitochondrial permeability transition pore inhibitor cyclosporine A, as well by xestospongin C and lack of extracellular calcium. Thapsigargin by itself caused accelerated cell death in the WAS platelets. The number of mitochondria was predictive of PS exposure: 33% of platelets from WAS patients with fewer than five mitochondria exposed PS, while only 12% did among those that had five or more mitochondria. Interestingly, healthy donor platelets with fewer mitochondria also more readily became procoagulant upon PAR1/PAR4 stimulation. Collapse of single mitochondria led to greater cytosolic calcium increase in WAS platelets if they had one to three mitochondria compared with platelets containing higher numbers. A computer systems biology model of platelet calcium homeostasis showed that smaller platelets with fewer mitochondria could have impaired calcium homeostasis because of higher surface-to-volume ratio and greater metabolic load, respectively. There was a correlation (C=0.81, P <0.02) between the mean platelet size and platelet count in the WAS patients. We conclude that WAS platelets readily expose PS via a mitochondria-dependent necrotic mechanism caused by their smaller size, which could contribute to the development of thrombocytopenia., (Copyright© 2020 Ferrata Storti Foundation.)

Antibacterial activity of the three-finger toxins from cobra venom, including cytotoxin 3 from N. kaouthia, cardiotoxin-like basic polypeptide A5 from N. naja (CLBP), and alpha-neurotoxin from N. oxiana venom, was investigated. All toxins failed to influence Gram-negative bacteria. The most pronounced activity against Bacillus subtilis was demonstrated by CLBP. The latter is ascribed to the presence of additional Lys-residues within the membrane-binding motif of this toxin., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Preterm newborns are at thrombohemorrhagic risk during the early neonatal period. Taking into account the lack of informative tools for the laboratory diagnosis of hemostasis disorders in newborns, our goal was to determine the baseline values of thrombodynamics and platelet functional activity in healthy term and moderately preterm newborns during the early neonatal period future potential clinical use of these tests., Methods: Coagulation was assessed using an integral assay of thrombodynamics and standard coagulation assays, and platelet functional activity was estimated by flow cytometry., Results: Hypercoagulation of newborns, represented by a significantly higher clot growth velocity and the presence of spontaneous clots in the thrombodynamics, was combined with platelet hypoactivity. Granule release, phosphatidylserine exposure, and the ability to change shape upon activation were decreased in the platelets of moderately preterm newborns. The platelet function remained at the same level over the first four days of life, whereas the hypercoagulation became less pronounced., Conclusions: The hemostasis of newborns is characterized by hypercoagulation combined with reduced platelet functional activity. Moderately preterm and term newborns do not differ in the parameters of coagulation, while some of the functional responses of platelets are lower in moderately preterm newborns than in term.

The ability of platelets to carry out their hemostatic function can be impaired in a wide range of inherited and acquired conditions: trauma, surgery, inflammation, pre-term birth, sepsis, hematological malignancies, solid tumors, chemotherapy, autoimmune disorders, and many others. Evaluation of this impairment is vitally important for research and clinical purposes. This problem is particularly pronounced in pediatric patients, where these conditions occur frequently, while blood volume and the choice of blood collection methods could be limited. Here we describe a simple flow cytometry-based screening method of comprehensive whole blood platelet function testing that was validated for a range of pediatric and adult samples (n = 31) in the hematology hospital setting including but not limited to: classic inherited platelet function disorders (Glanzmann's thrombasthenia; Bernard-Soulier, Wiscott-Aldrich, and Hermasky-Pudlak syndromes, MYH9-dependent thrombocytopenia), healthy and pre-term newborns, acute and chronic immune thrombocytopenia, chronic lympholeukemia, effects of therapy on platelet function, etc. The method output includes levels of forward and side scatter, levels of major adhesion and aggregation glycoproteins Ib and IIb-IIIa, active integrins' level based on PAC-1 binding, major alpha-granule component P-selectin, dense granule function based on mepacrine uptake and release, and procoagulant activity quantified as a percentage of annexin V-positive platelets. This analysis is performed for both resting and dual-agonist-stimulated platelets. Preanalytical and analytical variables are provided and discussed. Parameter distribution within the healthy donor population for adults (n = 72) and children (n = 17) is analyzed.

Aim: Spider venom is a rich source of antibacterial peptides, whose hemolytic activity is often excessive., Methodology: How to get rid of it? Using latarcins from Lachesana tarabaevi and oxyopinin Oxt 4a from Oxyopes takobius spider venoms we performed coarse-grained molecular dynamics simulations of these peptides in the presence of lipid bilayers, mimicking erythrocyte membranes. This identified hemolytically active fragments within Oxt 4a and latarcins. Then, we synthesized five 20-residue peptides, containing different parts of the Oxt 4a and latarcin-1 sequence, carrying mutations within the identified regions., Conclusion: The antibacterial and hemolytic tests suggested that the three of the synthesized peptides demonstrated substantial decrease in hemolytic activity, retaining, or even exceeding antibacterial potential of the parent peptides.

Background: Recent advances in nanomedicine have shown the great interest of active targeting associated to nanoparticles. Single chain variable fragments (scFv) of disease-specific antibodies are very promising targeting entities because they are small, not immunogenic and able to bind their specific antigens. The present paper is devoted to biological properties in vitro and in vivo of fluorescent and pegylated iron oxide nanoparticles (SPIONs-Cy-PEG-scFv) functionalized with scFv targeting Human Epithelial growth Receptor 2 (HER2)., Results: Thanks to a site-selective scFv conjugation, the resultant nanoprobes demonstrated high affinity and specific binding to HER2 breast cancer cells. The cellular uptake of SPIONs-Cy-PEG-scFv was threefold higher than that for untargeted PEGylated iron oxide nanoparticles (SPIONs-Cy-PEG) and is correlated to the expression of HER2 on cells. In vivo, the decrease of MR signals in HER2+ xenograft tumor is about 30% at 24 h after the injection., Conclusions: These results all indicate that SPIONs-Cy-PEG-scFv are relevant tumor-targeting magnetic resonance imaging agents, suitable for diagnosis of HER2 overexpressing breast tumor.

Novel hybrid molecule containing 2-mercaptoethylamine was synthesized starting from O-propyloxime-N-propoxy bacteriopurpurinimide (dipropoxy-BPI), which was readily oxidized in oxygen atmosphere yielding the corresponding disulfide analogue (disulfide-BPI). Spectral, photophysical, photodynamic, and biological properties of compound were properly evaluated. Compounds bearing disulfide moiety can directly interact with glutathione (GSH), thereby reducing its intracellular concentration. Indeed, mice sarcoma S37 cell line was treated in vitro with disulfide-BPI, yielding a CC 50 value of 0.05 ± 0.005 μM. A relatively high level of singlet oxygen was detected. It was demonstrated (by fluorescence) that the PS was rapidly accumulated in a cancer nest (S37) at a relatively high level after 2 h upon intravenous administration. After 24 h, no traces of the molecule were detected in the tumor mass. Moreover, high photodynamic efficiency was demonstrated at doses of 150-300 J/cm 2 against two different in vivo tumor models, achieving 100% regression of cancer growth.

Propargyl-15 2 ,17 3 -dimethoxy-13 1 -amide of bacteriochlorin e (BChl) and a 4-(4-N,N-dimethylaminostyryl)-N-alkyl-1,8-naphthalimide bearing azide group in the N-alkyl fragment were conjugated by the copper(i)-catalyzed 1,3-dipolar cycloaddition to produce a novel dyad compound BChl-NI for anticancer photodynamic therapy (PDT) combining the modalities of a photosensitizer (PS) and a fluorescence imaging agent. A precise photophysical investigation of the conjugate in solution using steady-state and time-resolved optical spectroscopy revealed that the presence of the naphthalimide (NI) fragment does not decrease the photosensitizing ability of the bacteriochlorin (BChl) core as compared with BChl; however, the fluorescence of naphthalimide is completely quenched due to resonance energy transfer (RET) to BChl. It has been shown that the BChl-NI conjugate penetrates into human lung adenocarcinoma A549 cells, and accumulates in the cytoplasm where it has a mixed granular-diffuse distribution. Both NI and BChl fluorescence in vitro provides registration of bright images showing perfectly intracellular distribution of BChl-NI. The ability of NI to emit light upon excitation in imaging experiments has been found to be due to hampering of RET as a result of photodestruction of the energy acceptor BChl unit. Phototoxicity studies have shown that the BChl-NI conjugate is not toxic for A549 cells at tested concentrations (<8 μM) without light-induced activation. At the same time, the concentration-dependent killing of cells is observed upon the excitation of the bacteriochlorin moiety with red light that occurs due to reactive oxygen species formation. The presented data demonstrate that the BChl-NI conjugate is a promissing dual function agent for cancer diagnostics and therapy.

Background: This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA., Methods: SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS., Results: The SFP-FA have zeta potential below -6mW and stable hydrodynamic diameter close to 100nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH 3 and 22-27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells., Conclusions: The present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis., General Significance: Composition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

It has been suggested that platelet function in chronic immune thrombocytopenic purpura (ITP) may be abnormal. Thrombopoietin mimetics used for treatment can affect it, but the data remain limited. We investigated platelet function of 20 children diagnosed with severe ITP (aged 1-16 years, 12 females and eight males). Platelet functional activity in whole blood was characterized by flow cytometry before and after stimulation with SFLLRN plus collagen-related peptide. Levels of CD42b, PAC1, and CD62P, but not CD61 or annexin V, were significantly increased (P < 0.05) in resting platelets of patients before treatment compared with healthy donors. On average, PAC1 and CD62P in patients after activation were also significantly elevated, although some patients failed to activate integrins. Romiplostim (1-15 μg/kg/week s.c.) was prescribed to seven patients, with clinical improvement in six. Interestingly, one patient had clinical improvement without platelet count increase. Eltrombopag (25-75 mg/day p.o.) was given to four patients, with positive response in one. Others switched to romiplostim, with one stable positive response, one unstable positive response, and one non-responding. Platelet quality improved with romiplostim treatment, and their parameters approached the normal values. Our results suggest that platelets in children with severe ITP are pre-activated and abnormal, but improve with treatment.

Background and Objectives: Pathogen reduction technologies may affect platelet quality during storage. We studied functional characteristics and clinical effectiveness of platelet concentrates (PCs) treated with Mirasol in plasma and in platelet-additive solution SSP+., Materials and Methods: Mirasol-treated, gamma-irradiated and untreated apheresis PCs were examined on days 0, 1, 3 and 5 of storage. Phosphatidylserine, P-selectin and active glycoprotein IIb/IIIa were analysed using flow cytometry before and after platelet stimulation. Platelet count increments, the numbers of inefficient transfusions and post-transfusion reactions were analysed to estimate clinical effectiveness., Results: A significant increase in all platelet activation markers occurred during storage in all PC groups. Activation markers in Mirasol-treated samples were already significantly higher compared with the control ones on the day of harvesting, and continued to grow during the storage. Mirasol treatment increased the number of platelets with a mitochondrial membrane potential loss. On the 3rd day of storage, 50% of Mirasol-treated platelets did not respond to activation; on the 5th day, none did. This agreed well with a decrease (approximately twofold) in the effectiveness of Mirasol-treated PC transfusions. Transfusions of PCs stored in SSP+ were accompanied by fewer inefficient transfusions and post-transfusion reactions than of PCs stored in plasma., Conclusion: Treatment with Mirasol decreased platelet function, particularly profoundly on the 5th day of storage, and led to a decrease in the effectiveness of transfusions. SSP+ did not affect laboratory parameters significantly compared with plasma, but decreased the percentage of transfusion complications., (© 2015 International Society of Blood Transfusion.)

Further development of boron neutron capture therapy (BNCT) requires new neutronsensitizers with improved ability to deliver (10)B isotopes in cancer cells. Conjugation of boron nanoparticles with porphyrin derivatives is an attractive and recognized strategy to solve this task. We report on breakthroughs in the structural optimization of conjugates of chlorin e6 derivative with cobalt-bis(dicarbollide) nanoparticles resulting in the creation of dimethyl ester 13-carbomoylchlorin e6 [N-hexylamine-N'-ethoxyethoxy]-cobalt-bis(dicarbollide) (conjugate 1). Conjugate 1 is able to accumulate quickly and efficiently (distribution factor of 80) in cancer cells, thus delivering more than 10(9) boron atoms per cell when its extracellular concentration is more than 1 μmol L(-1). Also 1 is an active photosensitizer and is phototoxic towards human lung adenocarcinoma A549 cells at 80 nmol L(-1) (50% cell death). Photoinduced cytotoxicity of 1 is associated with lipid peroxidation, lysosome rupture and protease activity enhancement. Conjugate 1 fluoresces in the red region (670 nm), which is useful to monitor its accumulation and distribution in vivo. It is not toxic to cells without activation by neutrons or photons. Structural features that improve the functional properties of 1 are discussed. The properties of 1 warrant its preclinical evaluation as a multifunctional agent for BNCT, photodynamic therapy and fluorescent tumor diagnosis.

In addition to the conventional neurotoxins and cytotoxins, venom of the lynx spider Oxyopes takobius was found to contain two-domain modular toxins named spiderines: OtTx1a, 1b, 2a and 2b. These toxins show both insecticidal activity (a median lethal dose against flesh fly larvae of 75 μg·g(-1)) and potent antimicrobial effects (minimal inhibitory concentrations in the range 0.1-10 μm). Full sequences of the purified spiderines were established by a combination of Edman degradation, mass spectrometry and cDNA cloning. They are relatively large molecules (~ 110 residues, 12.0-12.5 kDa) and consist of two distinct modules separated by a short linker. The N-terminal part (~ 40 residues) contains no cysteine residues, is highly cationic, forms amphipathic α-helical structures in a membrane-mimicking environment, and shows potent cytolytic effects on cells of various origins. The C-terminal part (~ 60 residues) is disulfide-rich (five S-S bonds), and contains the inhibitor cystine knot (ICK/knottin) signature. The N-terminal part of spiderines is very similar to linear cytotoxic peptides found in various organisms, whereas the C-terminal part corresponds to the usual spider neurotoxins. We synthesized the modules of OtTx1a and compared their activity to that of full-length mature toxin produced recombinantly, highlighting the importance of the N-terminal part, which retained full-length toxin activity in both insecticidal and antimicrobial assays. The unique structure of spiderines completes the range of two-domain spider toxins., (© 2013 FEBS.)

Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.

Conjugation of boron nanoparticles with porphyrins is an attractive way to create dual agents for anticancer boron neutron capture therapy (BNCT) and photodynamic therapy (PDT). Properties of chlorin e(6) conjugated with two cobalt bis(dicarbollide) nanoparticles (1) or with a closo-dodecaborate nanoparticle (2) are reported. Fluorescent dianionic conjugates 1 and 2 penetrate in A549 human lung adenocarcinoma cells, stain cytoplasm diffusely and accumulate highly in lysosomes but are not toxic themselves for cells. Average cytoplasmic concentration of boron atoms (B) achieves 270 μM (ca. 2 × 10(8) B/cell) and 27 μM (ca. 2 × 10(7) B/cell) at the 1.5 μM extracellular concentration of 1 and 2, respectively, that makes conjugate 1 especially suitable for BNCT. Conjugate 2 causes photoinduced cell death at micromolar concentrations and can be considered also as a photosensitizer for PDT. Conjugates 1 and 2 have high quantum yields of singlet oxygen generation (0.55 and 0.85 in solution, respectively), identical intracellular localization and similar lipid-like microenvironment but conjugate 1 possesses no photoinduced cytotoxicity. A presence of cobalt complexes in conjugate 1 is supposed to be a reason of the observed antioxidative effect in cellular environment, but an exact mechanism of this intriguing phenomenon is unclear. Due to increased intracellular accumulation and absence of photoinduced cytotoxicity conjugate 1 is promising for fluorescence diagnostics of cancer.

The interaction of 13,15-N-(3'-hydroxypropyl)cycloimide of chlorin p(6) (CIC) with normal blood cells and human K562 and HL60 myeloid leukemia cells was studied. CIC was found to be bound by the erythrocyte membrane but did not penetrate into the cytoplasm. It is characterized by a diffuse distribution in the cytoplasm of normal leukocytes, whereas its diffuse distribution in K562 and HL60 cells is accompanied by perinuclear accumulation and binding to the plasma membrane. The average cytoplasmic concentration corresponding to the CIC accumulation in leukemic cells at saturation is 2.2 to 2.6 times higher than that in normal leukocytes. CIC is more intensely accumulated in granulocytes than in lymphocytes. The kinetics of the cellular uptake and efflux was characterized. The normal leukocytes and erythrocytes were found to be 1.5 times and 3 to 4 times less sensitive, respectively, to the photodynamic action of CIC than the K562 and HL60 cells.

Potassium voltage-gated channels (Kv) are considered as molecular targets in a number of serious neuronal, immune, and cardiac disorders. Search for efficient low-molecular weight modulators of Kv channel function provides a basis for the development of an appropriate therapy for various Kv-mediated diseases. We report here on a new bacterial cell-based system, which is suitable for study of interactions between ligands and ligand-binding sites of eukaryotic Kv1.3 and Kv1.1 channels. To create this system, high-level expression of KcsA-Kv1.3 and KcsA-Kv1.1 hybrid proteins (ligand-binding sites of Kv1.3 or Kv1.1 fused with prokaryotic KcsA potassium channel) was achieved in the plasma membrane of Escherichia coli. An efficient procedure of E. coli conversion to intact spheroplasts was developed. We demonstrate that fluorescently labeled agitoxin 2 binds specifically to high-affinity and lower-affinity sites of KcsA-Kv1.3 and KcsA-Kv1.1, respectively, at the membrane of spheroplasts. Number of binding sites per cell is estimated to be (1.0 +/- 0.6) x 10(5) and (0.3 +/- 0.2) x 10(5) for KcsA-Kv1.3- and KcsA-Kv1.1-presenting cells, respectively, that allows reliable detection of ligand-receptor interactions by confocal laser scanning microscopy. This bacterial cell-based system is intended for screening of ligands to membrane-embedded pharmaceutical targets.

The ability of microbial degraders of polycyclic aromatic hydrocarbons to grow at 24 degrees C in liquid mineral medium supplemented with oil as the sole source of carbon and energy was studied. Growth characteristics (CFU) and the level of oil destruction by plasmid-bearing and plasmid-free strains were determined after seven days of cultivation. The presence of catabolic plasmids in the degrader strains, including rhizosphere pseudomonads, was shown to increase cell growth and enhance the level of oil degradation. Strain Pseudomonas chlororaphis BS 1391 bearing plasmid pBS216 was found to be the most effective oil degrader.