Stabilizing Scaffold for Short Peptides Based on Knottins.

BACKGROUND: Bombesin (BBN) is a short peptide with a high affinity for receptors that are expressed on the surface of various types of cancer cells. However, a full length BBN molecule has low in vivo stability. OBJECTIVE: In our study, we propose the use of peptide toxins, derived from animal and plant toxins, as scaffold molecules to enhance the bioavailability and stability of bombesin. These peptides possess a unique structure known as an inhibitory cystine knot. METHODS: We synthesized structures in which short bombesin was incorporated into various domains of arthropod and plant toxins using solid-phase peptide synthesis. The stability under different conditions was assessed through high-performance liquid chromatography, and binding to cell cultures expressing the bombesin receptor was analyzed. Additionally, toxicity to cell cultures was evaluated using fluorescence microscopy. RESULTS: The data obtained demonstrated that placing the short peptide between the first and second cysteine residues in arachnid toxins results in increased in vitro stability and bioavailability, as well as low cytotoxicity. CONCLUSION: Arachnid toxins with an inhibitory cystine knot can be considered as a scaffold for increasing the stability of therapeutic peptides.

Protective Effect of Peptide Calcium Channel Blocker Omega-Hexatoxin-Hv1a on Epithelial Cell during Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is a common phenomenon that develops both from natural causes and during major operations. Many intracellular processes mediated by calcium ions are involved in the development of IRI. Currently, chemical calcium channel blockers are used but they have a number of limitations. In this article, we study the effect of the omega-hexatoxin-Hv1a peptide toxin, an alternative to chemical calcium channel blockers, on the mechanisms of IRI development in epithelial cell culture. The toxin was produced using solid phase peptide synthesis. IRI was caused by deprivation of glucose, serum and oxygen. The data obtained demonstrate that the omega-hexatoxin-Hv1a toxin in nanomolar concentrations is able to prevent the development of apoptosis and necrosis in epithelial cells by reducing the concentration of calcium, sodium and potassium ions, as well as by delaying rapid normalization of the pH level, affecting the mitochondrial potential and oxidative stress. This toxin can be used as an alternative to chemical calcium channel blockers for preventing tissue and organ IRI due to its low-dose requirement and high bioavailability.

Peptide Sodium Channels Modulator Mu-Agatoxin-Aa1a Prevents Ischemia-Reperfusion Injury of Cells.

Ischemia-reperfusion injury (IRI) is an irreversible functional and structural injury. Restoration of normal oxygen concentration exacerbates the emergence and development of deadly cells. One of the possible moments of reperfusion damage to cells is an increase in the intracellular concentration of sodium ions. In this article, we study the mu-agatoxin-Aa1a, a modulator of sodium channels, on the processes of IRI cells damage. The toxin was synthesized using an automatic peptide synthesizer. Hypoxia was induced by reducing the content of serum and oxygen in the CHO-K1 culture. The influence of the toxin on the level of apoptosis; intracellular concentration of sodium, calcium, and potassium ions; intracellular pH; totality of reactive oxygen species (ROS), nitric oxide (NO), and ATP; and changes in the mitochondrial potential were studied. The experiments performed show that mu-agatoxin-Aa1a effectively prevents IRI of cells. Toxin reduces the level of apoptosis and prevents a decrease in the intracellular concentration of sodium and calcium ions during IRI. Mu-agatoxin-Aa1a contributes to the maintenance of elevated intracellular pH, reduces the intracellular concentration of ROS, and prevents the decrease in intracellular NO concentration and mitochondrial potential under conditions of reoxygenation/reperfusion. An analysis of experimental data shows that the mu-agatoxin-Aa1a peptide has adaptogenic properties. In the future, this peptide can be used to prevent ischemia/reperfusion tissue damage different genesis.

The effect of chromosome abnormalities on expression of SnoRNA in radioresistant and radiosensitive cell lines after irradiation.

In this paper, we have studied the role of chromosomal abnormalities in the expression of small nucleolar RNAs (snoRNAs) of radioresistant (K562) and radiosensitive (HL-60) leukemia cell line. Cells were exposed to an X-ray dose of 4 Gy. SnoRNA expression was investigated using NGS sequencing. The distribution of expressed snoRNAs on chromosomes has been found to be different for two cell lines. The most significant differences in the expression of snoRNAs were found in the K562 cell line based on the analysis of the dynamics of log2fc values. The type of clustering, the number and type of snoRNAs slightly differed in the chromosomes with trisomy and monosomy and had a pronounced difference in pairs with marker chromosomes in both cell lines. In this study, we have demonstrated that chromosomal abnormalities alter the expression of snoRNA after irradiation. Trisomies and monosomies do not have such a noticeable effect on the expression of snoRNAs as the presence of marker chromosomes.

Arthropod toxins inhibiting Ca2+ and Na+ channels prevent AC-1001 H3 peptide-induced apoptosis.

Peptide toxins of arthropods are one of the potential sources of bioactive substances. Toxins are able to bind to calcium channels and block them. Ca2+ ions play an important role in many cell processes, in particular, in apoptosis. In this work, we study the effect of some arthropod toxins on intracellular processes associated with the induction of apoptosis. Synthetic analogs of U5 -scytotoxin-Sth1a, ω-hexatoxin-Hv1a, ω-theraphotoxin-Hhn2a, and µ-agatoxin-Aa1a toxins-inhibitors of calcium L, P, and Q channels and sodium channels were used in the study. Apoptosis was induced by AC-1001 H3 peptide. We study the effect of toxins on the level of apoptosis, ROS, mitochondrial potential, GSH, and ATP in CHO-K1 cells. We show that all the tested toxins are able to dose dependently block the induction of apoptosis triggered by AC-1001 H3 and reduce the level of natural apoptosis in CHO-K1 cells. Cell incubation with apoptosis inducer AC-1001 H3 in the presence and absence of toxins causes an increase in the intracellular concentrations of ROS, ATP, and mitochondrial potential and decreases the GSH concentration. The present study reveals the antiapoptotic effect of a number of arthropod peptide toxins. The toxins studied can represent a novel approach used in the treatment of pathologies associated with the activation of apoptotic mechanisms.

Author Correction: The light-oxygen effect in biological cells enhanced by highly localized surface plasmon-polaritons.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The light-oxygen effect in biological cells enhanced by highly localized surface plasmon-polaritons.

Here at the first time we suggested that the surface plasmon-polariton phenomenon which it is well described in metallic nanostructures could also be used for explanation of the unexpectedly strong oxidative effects of the low-intensity laser irradiation in living matters (cells, tissues, organism). We demonstrated that the narrow-band laser emitting at 1265 nm could generate significant amount of the reactive oxygen species (ROS) in both HCT116 and CHO-K1 cell cultures. Such cellular ROS effects could be explained through the generation of highly localized plasmon-polaritons on the surface of mitochondrial crista. Our experimental conditions, the low-intensity irradiation, the narrow spectrum band (<4 nm) of the laser and comparably small size bio-structures (~10 µm) were shown to be sufficient for the plasmon-polariton generation and strong laser field confinement enabling the oxidative stress observed.

Radiation-Induced Changes of microRNA Expression Profiles in Radiosensitive and Radioresistant Leukemia Cell Lines with Different Levels of Chromosome Abnormalities.

In our study, we estimate an effect from chromosome aberrations and genome mutations on changes in microRNA expression profiles in cancer cell lines demonstrating different radiosensitivity. Here, cell viability and microRNA spectrum have been estimated 1, 4, and 24 h after irradiation. MiSeq high-throughput sequencing system (Illumina, San Diego, CA, USA) is employed to perform microRNA spectrum estimation. In the K562 cell line, the number of expressed microRNAs in chromosomes demonstrates a more pronounced variation. An analysis of microRNA effects on signaling pathway activity demonstrates differences in post-transcriptional regulation of the expression of genes included into 40 signaling pathways. In the K562 cell line, microRNA dynamics analyzed for their dependence on chromosome localization show a wider scattering of microRNA expression values for a pair of chromosomes compared to the HL-60 cell line. An analysis of microRNAs expression in the K562 and HL-60 cell lines after irradiation has shown that chromosome abnormalities can affect microRNA expression changes. A study of radiation-induced changes of microRNA expression profiles in the K562 and HL-60 cell lines has revealed a dependence of microRNA expression changes on the number of chromosome aberrations and genome mutations.

Data aggregation at the level of molecular pathways improves stability of experimental transcriptomic and proteomic data.

High throughput technologies opened a new era in biomedicine by enabling massive analysis of gene expression at both RNA and protein levels. Unfortunately, expression data obtained in different experiments are often poorly compatible, even for the same biologic samples. Here, using experimental and bioinformatic investigation of major experimental platforms, we show that aggregation of gene expression data at the level of molecular pathways helps to diminish cross- and intra-platform bias otherwise clearly seen at the level of individual genes. We created a mathematical model of cumulative suppression of data variation that predicts the ideal parameters and the optimal size of a molecular pathway. We compared the abilities to aggregate experimental molecular data for the 5 alternative methods, also evaluated by their capacity to retain meaningful features of biologic samples. The bioinformatic method OncoFinder showed optimal performance in both tests and should be very useful for future cross-platform data analyses.

Mitochondrial dependent oxidative stress in cell culture induced by laser radiation at 1265 nm.

Photodynamic therapy is the main technique applied for surface carcinoma treatment. This technique employs singlet oxygen generated via a laser excited photosensitizer as a main damaging agent. However, prolonged sensitivity to intensive light, relatively low tissue penetration by activating light the cost of photosensitizer (PS) administration can limit photodynamic therapy applications. Early was reported singlet oxygen generation without photosensitizer induced by a laser irradiation at the wavelength of 1250-1270 nm. Here, we study the dynamics of oxidative stress, DNA damage, changes of mitochondrial potential, and mitochondrial mass induced by a laser at 1265 nm have been studied in HCT-116 and CHO-K cells. Laser irradiation of HCT-116 and CHO-K cells has induced a dose-dependent cell death via increasing intracellular reactive oxygen species (ROS) concentration, increase of DNA damage, decrease of mitochondrial potential, and reduced glutathione. It has been shown that, along with singlet oxygen generation, the increase of the intracellular ROS concentration induced by mitochondrial damage contributes to the damaging effect of the laser irradiation at 1265 nm.

Application of the fractional-stable distributions for approximation of the gene expression profiles.

Nowadays, there are reliable scientific data highlighting that the probability density function of the gene expression demonstrates a number of universal features commonly observed in the microarray experiments. First of all, these distributions demonstrate the power-law asymptotics and, secondly, the shape of these distributions is inherent for all organisms and tissues. This fact leads to appearance of a number of works where authors investigate various probability distributions for an approximation of the empirical distributions of the gene expression. Nevertheless, all these distributions are not a limit distribution and are not a solution of any equation. These facts, in our opinion, are essential shortcoming of these probability laws. Besides, the expression of the individual gene is not an accidental phenomenon and it depends on the expression of the other genes. This suggests an existence of the genic regulatory net in a cell. The present work describes the class of the fractional-stable distributions. This class of the distributions is a limit distribution of the sums of independent identically distributed random variables. Due to the power-law asymptotics, these distributions are applicable for the approximation of the experimental densities of the gene expression for microarray experiments. The parameters of the fractional stable distributions are statistically estimated by experimental data and the functions of the empirical and theoretical densities are compared. Here we describe algorithms for simulation of the fractional-stable variables and estimation of the parameters of the the fractional stable densities. The results of such a comparison allow to conclude that the empirical densities of the gene expression can be approximated by the fractional-stable distributions.