Thermal Conductivity Gas Sensors for High-Temperature Applications.

This paper describes a fast and flexible microfabrication method for thermal conductivity gas sensors useful in high-temperature applications. The key parts of the sensor, the microheater and the package, were fabricated from glass-coated platinum wire and the combination of laser micromilling (ablation) of already-sintered monolithic ceramic materials and thick-film screen-printing technologies. The final thermal conductivity gas sensor was fabricated in the form of a complete MEMS device in a metal ceramic package, which could be used as a compact miniaturized surface-mounted device for soldering to standard PCB. Functional test results of the manufactured sensor are presented, demonstrating their full suitability for gas sensing applications and indicating that the obtained parameters are at a level comparable to those of standard industrially produced sensors. The results of the design and optimization principles of applied methods are discussed with regard to possible wider applications in thermal gas sensor prototyping in the future. The advantage of the developed sensors is their ability to operate in air environments under high temperatures of 900 °C and above. The sensor element material and package metallization were insensitive to oxidation compared with classical sensor-solution-based metal-glass packages and silicone MEMS membranes, which exhibit mechanical stress at temperatures above 700 °C.

Different Structures-Similar Effect: Do Substituted 5-(4-Methoxyphenyl)-1H-indoles and 5-(4-Methoxyphenyl)-1H-imidazoles Represent a Common Pharmacophore for Substrate Selective Inhibition of Linoleate Oxygenase Activity of ALOX15?

Mammalian 15-lipoxygenases (ALOX15) are lipid peroxidizing enzymes that exhibit variable functionality in different cancer and inflammation models. The pathophysiological role of linoleic acid- and arachidonic acid-derived ALOX15 metabolites rendered this enzyme a target for pharmacological research. Several indole and imidazole derivatives inhibit the catalytic activity of rabbit ALOX15 in a substrate-specific manner, but the molecular basis for this allosteric inhibition remains unclear. Here, we attempt to define a common pharmacophore, which is critical for this allosteric inhibition. We found that substituted imidazoles induce weaker inhibitory effects when compared with the indole derivatives. In silico docking studies and molecular dynamics simulations using a dimeric allosteric enzyme model, in which the inhibitor occupies the substrate-binding pocket of one monomer, whereas the substrate fatty acid is bound at the catalytic center of another monomer within the ALOX15 dimer, indicated that chemical modification of the core pharmacophore alters the enzyme-inhibitor interactions, inducing a reduced inhibitory potency. In our dimeric ALOX15 model, the structural differences induced by inhibitor binding are translated to the hydrophobic dimerization cluster and affect the structures of enzyme-substrate complexes. These data are of particular importance since substrate-specific inhibition may contribute to elucidation of the putative roles of ALOX15 metabolites derived from different polyunsaturated fatty acids in mammalian pathophysiology.

Red light-emitting short Mango-based system enables tracking a mycobacterial small noncoding RNA in infected macrophages.

Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.

Dynamics of endogenous levels and subcellular localization of ABA and cytokinins during pollen germination in spruce and tobacco.

We used the enzyme-linked immunosorbent assay (ELISA) to assess the level of endogenous hormones in spruce pollen, and immunolocalization and confocal microscopy to study hormone localization in spruce and tobacco pollen. During pollen activation, the levels of ABA, zeatin, and its riboside significantly decreased. After the initiation of polar growth, the levels of all cytokinins increased sharply; ABA level also increased. In dormant spruce pollen grains, zeatin and ABA were localized uniformly throughout the cytoplasm. Zeatin was not detected in the nuclei, and the antheridial cell showed higher levels than the vegetative cell; ABA signal was detected in the cytoplasm and the nuclei. In germinating pollen, both hormones were detected mainly in plastids. The similar pattern was found in growing pollen tubes; signal from ABA also had a noticeable level in the cytosol of the tube cell, and was weaker in the antheridial cell. Zeatin fluorescence, on the other hand, was more pronounced in the antheridial cell. In non-germinated grains of tobacco, zeatin was localized mainly in organelles. ABA in dormant pollen grains demonstrated uniform localization, including the nuclei and cytoplasm of both cells. After germination, zeatin was accumulated in the plasmalemma or cell wall. ABA signal in the cytoplasm decreased; in the nuclei, it remained high. In growing tubes, the strongest zeatin and ABA signals were observed at the plasma membrane. The differences in ABA and cytokinin localization between species and dynamic changes in their level in spruce pollen highlight the key spatial and temporal parameters of hormonal regulation of gymnosperm pollen germination.

Transgenerational changes in Daphnia magna under radio frequency radiation in the juvenile and puberty period.

PURPOSE: To analyze the results of direct and transgenerational effects of radio frequency electromagnetic fields (RF-EMF) on the model organism of crustaceans Daphnia magna. MATERIALS AND METHODS: D. magna were chronically exposed at 900 GHz EMF with an energy flux density (EFD) of about 1 mW/cm2 in the juvenile and pubertal periods of their ontogenesis. The cytotoxicity of exposure as well as survival, fertility and teratogenic effect of directly exposed daphnids and their progeny across three generations were analyzed. RESULTS AND CONCLUSIONS: The results of our study show that exposure of RF-EMF at juvenile period can significantly affect the fertility and size of irradiated daphnids and their offspring of the first generation. The decrease in fertility may be associated with a cytotoxic effect on the cells of irradiated animals. The reduction in the size of the terminal spine and the body of individuals is an indicator of the negative impact of radiation on the protective strategy of the crustacean population. The reproductive process is restored by the second generation. The results of our study provide further insights into the possible mechanisms underlying the in vivo effects of RF-EMF.

Novel characterization discoveries of ferroptosis-associated molecules in COAD microenvironment based TCGA data.

Background and Objective: One of the most recent forms of programmed cell death, ferroptosis, is crucial in tumorigenesis. Ferroptosis is characterized by iron-dependent oxidative destruction of cellular membranes following the antioxidant system's failure. However, it is unknown whether ferroptosis-related genes (FRGs) are associated with colon adenocarcinoma (COAD) metastasis, immune cell infiltration, and oxidative stress in COAD. The current study concentrated on FRGs expression in colon cancer metastasis, their relationship to immune cell infiltration (ICI), and potential pathological pathways in COAD. Methods and Results: Clinical information and mRNA expression patterns for patients with COAD metastasis were obtained from the public TCGA database. Patients with low mRNA levels showed good overall survival than patients with high mRNA levels. The genomic-clinicopathologic nomogram was subsequently created by combining risk score and clinicopathological features. Absolute Shrinkage and Selection Operator have shown a 4 gene signature that can stratify cancer patients into high-risk versus low-risk. These four FRGs were found to be significantly linked to the overall survival of COAD patients and predicted high risk score. Next, age, stage, and PTNM were combined in univariate and multivariate cox regression models to perform a filtering procedure. The receiver operating characteristic (ROC) and calibration curves indicated that constructed signature model exhibited high prediction accuracy and clinical relevance in COAD. ARID3A showed a strong negative correlation with a wide range of immune tumour-infiltrating cells in COAD microenvironment. According to the single sample gene set enrichment analysis (ssGSEA) results, FRGs are involved in variety of pathological pathways including PI3K-AKT-mTOR pathway, reactive oxygen species (ROS) pathway, response to hypoxia pathway, and other inflammation related pathways. Moreover, dysregulation of FRGs in COAD patients showed a significance correlation with wide range of miRNAs and transcription factors (TFs). Conclusion: We identified new diagnostic biomarkers and established prognostic models for ferroptosis related programmed cell death in COAD metastasis. FRGs may improve tumor cell survival by activating the TGFB pathway, which can stimulate ROS production, accelerates ECM breakdown, and promote tumor progression and invasion. Genes implicated in ferroptosis, as revealed by the Kaplan Meier and a genomic-clinicopathologic nomogram, are potential therapeutic targets and prognosis indications for metastasis COAD patients.

Synthesis of Novel Lipophilic Polyamines via Ugi Reaction and Evaluation of Their Anticancer Activity.

Natural polyamines (PAs) are involved in the processes of proliferation and differentiation of cancer cells. Lipophilic synthetic polyamines (LPAs) induce the cell death of various cancer cell lines. In the current paper, we have demonstrated a new method for synthesis of LPAs via the multicomponent Ugi reaction and subsequent reduction of amide groups by PhSiH3. The anticancer activity of the obtained compounds was evaluated in the A-549, MCF7, and HCT116 cancer cell lines. For the first time, it was shown that the anticancer activity of LPAs with piperazine fragments is comparable with that of aliphatic LPAs. The presence of a diglyceride fragment in the structure of LPAs appears to be a key factor for the manifestation of high anticancer activity. The findings of the study strongly support further research in the field of LPAs and their derivatives.

Aberrantly hypermethylated ARID1B is a novel biomarker and potential therapeutic target of colon adenocarcinoma.

Background and Objective: Understanding the tumor microenvironment (TME) and immune cell infiltration (ICI) may help guide immunotherapy efforts for colon cancer (COAD). However, whether ARID1B is truly regulated by hypermethylation or linked to immune infiltration remains unknown. The current work focused on the ARID1B gene expression and methylation in COAD, as well as its relation with ICI. Methods and Results: Multiple tools based on TCGA were used to analyze the differences in the expression of the ARID1B gene, DNA methylation, and its association with various clinicopathological features, somatic mutations, copy number variation, and the prognosis of patients with COAD. According to the analysis results, patients with high mRNA, low methylation levels showed better overall survival than patients with low mRNA, high methylation levels. The correlation analysis of immune cell infiltration and immune checkpoint gene expression showed that the infiltration rates of the main ICI subtypes, cancer-associated fibroblast, and myeloid cells were significantly enriched and correlated with ARID1B in COAD. An association between ARID1B expression and immune infiltration in COAD was found by correlating ICI indicators with ARID1B expression in the immune cell composition of the COAD microenvironment. Notably, M2 chemokines were related to ARID1B expression, while M1 chemokines were not. Conclusion: This study provided evidence that ARID1B may have a role in the pathogenesis of COAD. The specific underlying mechanisms that could be responsible for ARID1B's downregulation in COAD will need to be investigated in the future.

Differentiation of human macrophages with anaphylatoxin C3a impairs alternative M2 polarization and decreases lipopolysaccharide-induced cytokine secretion.

Anaphylatoxin C3a is a small signaling polypeptide that is generated during complement activation. C3a is involved in the regulation of various innate and adaptive immune system processes; however, the role of C3a in macrophage differentiation and polarization is poorly elucidated. Here we showed that C3a impairs alternative M2 polarization of human macrophages and suppressed CD206, IL1Ra and CCL22 expression. C3a leads to a decrease of nuclear receptor PPARγ expression via the ERK1/2 signaling pathway, resulting in repressed PPARγ-dependent activation of CD36, FABP4 and LXRα genes and blunted response to an LXR ligand TO901317. Using small interfering RNA and agonist/antagonist approaches we showed that C3a decreases CD206, IL1Ra and CCL22 transcription at least partly in a PPARγ-dependent manner in M2 macrophages. Moreover, C3a impairs efferocytosis by M2 macrophages and inhibits their migratory activity. By contrast, macrophages treated with C3a during differentiation show blunted response to lipopolysaccharide stimulation owing to downregulation of TLR4 and lipid raft content. At the same time, differentiation of macrophages with C3a does not change M1 polarization in interferon gamma (IFNγ) and IFNγ + lipopolysaccharide-treated macrophages. These data provide a novel role of complement system and C3a in the regulation of M2 macrophage polarizations and suggest crosstalk between C3a, TLR4, PPARγ and LXR signaling pathways.

Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach.

Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.

Scope and limitations of pseudoprolines as individual amino acids in peptide synthesis.

Protected 4-carboxyoxazolidines and thiazolidines (pseudoprolines) are derivatives of serine, threonine or cysteine amino acids. Such compounds are used in peptide synthesis among the other protected amino acids. They are usually practiced when a peptide sequence is readily aggregating during synthesis due to their ability to disrupt secondary structure formation. Such compounds are usually applied as dipeptides. In present work Fmoc-protected pseudoprolines were synthesized and applied in peptide synthesis not as dipeptides but as individual amino acids. Different acylation protocols and amino acids were tested to acylate pseudoprolines. Several "difficult" peptides were synthesized to confirm the efficacy of such constructions. It was shown that pseudoprolines could be easily synthesized and used in automated or manual synthesis not as dipeptides but as ordinary amino acids.

The Emericellipsins A-E from an Alkalophilic Fungus Emericellopsis alkalina Show Potent Activity against Multidrug-Resistant Pathogenic Fungi.

Novel antimicrobial peptides with antifungal and cytotoxic activity were derived from the alkalophilic fungus Emericellopsis alkalina VKPM F1428. We previously reported that this strain produced emericellipsin A (EmiA), which has strong antifungal and cytotoxic properties. Further analyses of the metabolites obtained under a special alkaline medium resulted in the isolation of four new homologous (Emi B-E). In this work, we report the complete primary structure and detailed biological activity for the newly synthesized nonribosomal antimicrobial peptides called emericellipsins B-E. The inhibitory activity of themajor compound, EmiA, against drug-resistant pathogenic fungi was similar to that of amphotericin B (AmpB). At the same time, EmiA had no hemolytic activity towards human erythrocytes. In addition, EmiA demonstrated low cytotoxic activity towards the normal HPF line, but possessed cancer selectivity to the K-562 and HCT-116 cell lines. Emericillipsins from the alkalophilic fungus Emericellopsis alkaline are promising treatment alternatives to licensed antifungal drugs for invasive mycosis therapy, especially for multidrug-resistant aspergillosis and cryptococcosis.

The Monoacylglycerol Lipase Inhibitor JZL184 Inhibits Lung Cancer Cell Invasion and Metastasis via the CB1 Cannabinoid Receptor.

A targeted modulation of the endocannabinoid system is currently discussed as a promising strategy for cancer treatment. An important enzyme for the endocannabinoid metabolism is the monoacylglycerol lipase (MAGL), which catalyzes the degradation of 2-arachidonoylglycerol (2-AG) to glycerol and free fatty acids. In this study, we investigated the influence of MAGL inhibition on lung cancer cell invasion and metastasis. Using LC-MS, significantly increased 2-AG levels were detected in A549 cells treated with the MAGL inhibitor JZL184. In athymic nude mice, JZL184 suppressed metastasis of A549 cells in a dose-dependent manner, whereby the antimetastatic effect was cancelled by the CB1 receptor antagonist AM-251. In vitro, JZL184 induced a time- and concentration-dependent reduction of A549 cell invasion through Matrigel-coated membranes, which was likewise reversed by AM-251. An MAGL inhibition-associated reduction of free fatty acids as a cause of the anti-invasive effect could be excluded by add-back experiments with palmitic acid. Both JZL184 and the MAGL substrate 2-AG led to an increased formation of the tissue inhibitor of metalloproteinase-1 (TIMP-1), whereby a TIMP-1 knockdown using siRNA significantly attenuated the anti-invasive effects of both substances. Decreased invasion and TIMP-1 upregulation was also caused by the MAGL inhibitors JW651 and MJN110 or transfection with MAGL siRNA. A CB1- and TIMP-1-dependent anti-invasive effect was further confirmed for JZL184 in H358 lung cancer cells. In conclusion, MAGL inhibition led to a CB1-dependent decrease in human lung cancer cell invasion and metastasis via inhibition of 2-AG degradation, with TIMP-1 identified as a mediator of the anti-invasive effect.

Identification of isocyclosporins by collision-induced dissociation of doubly protonated species.

Nonribosomal cyclopeptide cyclosporin A (CsA), produced by fungus Tolypocladium inflatum, is an extremely important immunosuppressive drug used in organ transplantations and for therapy of autoimmune diseases. Here we report for the first time production of CsA, along with related cyclosporins B and C, by Tolypocladium inflatum strains of marine origin (White Sea). Cyclosporins A-C contain an unusual amino acid, (4R)-4-((E)-2-butenyl)-4,N-dimethyl-l-threonine (MeBmt), and are prone to isomerization to non-active isocyclosporin by N→O acyl shift of valine connected to MeBmt in acidic conditions. CsA and isoCsA are not distinguishable in MS analysis of [M+H]+ ions due to rapid [CsA + H]+→[isoCsA + H]+ conversion. We found that the N→O acyl shift is completely suppressed in cyclosporine [M+2H]2+ ions, and their collision-induced dissociation (CID) can be used for rapid and unambiguous analysis of cyclosporins and isocylosporins. Fragmentation patterns of [CsA+2H]2+ and [isoCsA+2H]2+ ions were analyzed and explained. The developed approach could be useful for MS analysis of other peptides containing ß-hydroxy-α-amino acids.

10-(4-Phenylpiperazine-1-carbonyl)acridin-9(10H)-ones and related compounds: Synthesis, antiproliferative activity and inhibition of tubulin polymerization.

As part of our continuing search for potent inhibitors of tubulin polymerization, two novel series of 42 10-(4-phenylpiperazine-1-carbonyl)acridin-9(10H)-ones and N-benzoylated acridones were synthesized on the basis of a retrosynthetic approach. All newly synthesized compounds were tested for antiproliferative activity and interaction with tubulin. Several analogs potently inhibited tumor cell growth. Among the compounds tested, 10-(4-(3-methoxyphenyl)piperazine-1-carbonyl)acridin-9(10H)-one (17c) exhibited excellent growth inhibitory effects on 93 tumor cell lines, with an average GI50 value of 5.4 nM. We were able to show that the strong cytotoxic effects are caused by disruption of tubulin polymerization, as supported by the EBI (N,N'-Ethylenebis(iodoacetamide)) assay and the fact that the most potent inhibitors of cancer cell growth turned out to be the most efficacious tubulin polymerization inhibitors. Potencies were nearly comparable or superior to those of the antimitotic reference compounds. Closely related to this, the most active analogs inhibited cell cycling at the G2/M phase at concentrations down to 30 nM and induced apoptosis in K562 leukemia cells. We believe that our work not only proves the excellent suitability of the acridone scaffold for the design of potent tubulin polymerization inhibitors but also enables synthetic access to further potentially interesting N-acylated acridones.

Hybrides of Alkaloid Lappaconitine with Pyrimidine Motif on the Anthranilic Acid Moiety: Design, Synthesis, and Investigation of Antinociceptive Potency.

Convenient and efficient routes to construct hybrid molecules containing diterpene alkaloid lappaconitine and pyrimidine fragments are reported. One route takes place via first converting of lappaconitine to 1-ethynyl-lappaconitine, followed by the Sonogashira cross-coupling-cyclocondensation sequences. The other involves the palladium-catalyzed carbonylative Sonogashira reaction of 5'-iodolappaconitine with aryl acetylene and Mo (CO)6 as the CO source in acetonitrile and subsequent cyclocondensation reaction of the generated alkynone with amidines. The reaction proceeded cleanly in the presence of the PdCl2-(1-Ad)2PBn∙HBr catalytic system. The protocol provides mild reaction conditions, high yields, and high atom and step-economy. Pharmacological screening of lappaconitine-pyrimidine hybrids for antinociceptive activity in vivo revealed that these compounds possessed high activity in experimental pain models, which was dependent on the nature of the substituent in the 2 and 6 positions of the pyrimidine nucleus. Docking studies were undertaken to gain insight into the possible binding mode of these compounds with the voltage-gated sodium channel 1.7. The moderate toxicity of the leading compound 12 (50% lethal dose (LD50) value was more than 600 mg/kg in vivo) and cytotoxicity to cancer cell lines in vitro encouraged the further design of therapeutically relevant analogues based on this novel type of lappaconitine-pyrimidine hybrids.

Spatial Structure and Activity of Synthetic Fragments of Lynx1 and of Nicotinic Receptor Loop C Models.

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 ß-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 1H-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.

Phenoxazine-based scaffold for designing G4-interacting agents.

G-quadruplexes (G4) represent one class of non-canonical secondary nucleic acid structures that are currently regarded as promising and attractive targets for anti-cancer, anti-viral and antibacterial therapy. Herein, we probe a new i-clamp-inspired phenoxazine scaffold for designing G4-stabilizing ligands. The length of the protonated aminoalkyl tethers ('arms') of the phenoxazine-based ligand was optimized in silico. Two double-armed ligands differing in the relative orientation of their arms and one single-armed ligand were synthesized. The two-armed ligands significantly enhanced the thermal stability of the G-quadruplex structures (increasing the melting temperature by up to 20 °C) and displayed G4 selectivity over duplex DNA. The ligands look promising for biological studies and the phenoxazine scaffold could be a starting point for designing new G4-interacting compounds.

Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide.

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.

Oocyte quality in women with infertility associated endometriosis.

The mechanisms of endometriosis-related infertility remain still unknown. Endometriosis and clinical markers of oocyte quality are a very important problem of reproduction. The purpose of the study is to assess the quality of oocytes in women with infertility associated with endometriosis. The study included infertile reproductive aged women, between 29 and 40 years who underwent IVF and ICSI procedures. The patients were divided into three groups: group I involved 50 (n = 50) patients with recurrent unilateral endometriomas, group II included 50 patients (n = 50) unilateral endometriomas after surgical treatment and control group with 30 (n = 30) patients with tubal factor infertility. Clinical and morphological assessment of oocyte quality was performed in all IVF/ICSI cycles. The results of the study demonstrate a statistically significant increase in the number of immature oocytes of metaphase MI and immature oocytes at the GV germinal vesicle stage in patients with infertility associated with endometriosis, compared with the control group (p<.005). There is deterioration in the quality of the obtained oocytes in patients with the presence of endometrioma more than 3 cm in diameter. The results of this study allow to conclude that endometriomas negatively affect quality of oocyte and ovarian reserve, whereas endometriomas after cystectomy, have a deleterious and sustained effect on ovarian reserve.