[The effectiveness of suggestive techniques to improve sleep is enhanced by combining with sound stimulus based on binaural beats]. / Effektivnost' suggestivnykh metodik dlya uluchsheniya sna povyshaetsya pri sochetanii ikh so zvukovym stimulom na osnove binaural'nykh bienii.

OBJECTIVE: To test the hypothesis of the difference between 3 means of sleep latency (SL) during falling asleep: accompanied by audio stimulus embedded with binaural beats (BB); after listening to suggestive body relaxation instructions; accompanied by audio stimulus embedded with BB after listening to suggestive body relaxation instructions (that is the combination of 1 and 2). MATERIAL AND METHODS: For the purpose of the study, a special Android application was developed and installed on the subjects' individual smartphones. The application assumed screen tapping test to control for fall-asleep process. The data of 63 subjects presented with the 3 types of sound stimuli mentioned above in a counterbalanced scheme were analyzed. RESULTS: Statistical analysis confirmed the initial hypothesis about the dependence of LS on the type of sound stimulus (p<0.05). Pairwise SL comparison showed reliable difference between stimuli (3) - 1149±113 s, and (1) - 1469±89 s (p<0.01). SL for the stimulus (2) had an intermediate value of 1269±112 s (difference from (1) at a trend level). CONCLUSION: The use of background sound embedded with BBs enhances the effect of suggestive instructions to improve sleep. But it is the suggestion as a psychotherapeutic technique that is determinant.

[Monotonous sound stimuli embedded with either binaural or monaural beats have the same effect on sleep latency: study on a large subject group]. / Vliyanie binaural'nykh i monaural'nykh bienii v strukture monotonnogo zvukovogo stimula na latentnost' sna.

OBJECTIVE: The aim of this study was to test on a large group of subjects the hypothesis that sleep latency (SL) does not depend on the nature of low-frequency beats embedded into monotonous sound stimulus supplied through the fall-asleep process. Specifically, it does not depend on whether those beats are monaural (MB) or binaural (BB). MATERIAL AND METHODS: A special application for Android OS was developed and installed on the individual smartphones of 221 subjects for the purpose of the study. Three attempts were performed with each of them using 3 different kinds of monotonous sound supplied according to counterbalanced design. Three kinds of sound were identical in pitch but differed in the beat presence and type: BB, MB or sham (sound without beats). RESULTS: Repeated measures analysis of variance (rANOVA) revealed no significant statistical effect of stimulus type on SL (p=0.21). A pairwise comparison of SL for different stimulation conditions showed the null hypothesis significance level adjusted according to multiple comparison correction to be p=1.0. Thus, in this experiment SL did not significantly depend on the monotonous sound stimulus type: MB, BB, or sham. CONCLUSION: The software application developed is useful as universal platform to assess at home conditions the impact of various external factors on fall-asleep process.

IPSC-Derived Human Neurons with GCaMP6s Expression Allow In Vitro Study of Neurophysiological Responses to Neurochemicals.

The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON-NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [Ca2+]i which are caused by ionotropic receptors for Glu, predominantly of the NMDA-type. Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.

[The effect of music embedded with binaural beats on heart rate parameters during nap]. / Vliyanie muzyki s effektom binaural'nykh bienii na parametry serdechnogo ritma cheloveka v protsesse dnevnogo sna.

OBJECTIVE: To test hypothesis that music embedded with binaural beats can boost activity of parasympathetic part of autonomic nervous system (PPANS) with the development of nap. MATERIAL AND METHODS: The power of high-frequency component of heart rate variability spectrum computed on successive 2-minute intervals during 20-minute nap was a comparison criterion. The criterion was compared during nap accompanied by music with embedded binaural beats (stimulus condition) and nap in silence (control condition). RESULTS AND CONCLUSION: Statistical comparison revealed the increase of PPANS activity during nap in stimulus condition vs. control condition. It is consistent with conclusions of other papers about positive effect of sound stimuli embedded with binaural beats on PPANS.

Neuroprotective Potential of Peptides HFRWPGP (ACTH6-9PGP), KKRRPGP, and PyrRP in Cultured Cortical Neurons at Glutamate Excitotoxicity.

Glutamate (Glu) excitotoxicity, which accompanies brain ischemia or traumatic brain injury, is the leading mechanism of neuronal death. In the present work, we studied the effects of the peptides HFRWPGP (ACTH6-9PGP), KKRRPG, and PyrRP on the survival of cultured cortical neurons on the background of excitotoxic effect of Glu (100 µM). Biochemical (MTT/WST) and morphometric analyzes showed that, depending on the dose, ACTH6-9PGP and KKRRPGP protect neurons from the cells death, while PyrRP, conversely, enhances it. The neuroprotective effect of ACTH6-9PGP is accompanied by a slowdown in the development of delayed calcium dysregulation and synchronous mitochondrial depolarization. Among the studied peptides, only ACTH6-9PGP significantly increased the number of neurons that restored Ca2+ homeostasis after Glu was abolished. The influence of KKRRPGP was less pronounced, whereas PyrRP, on the contrary, reduced the number of neurons with low [Ca2+]i. Thus, this study revealed the high therapeutic significance of ACTH6-9PGP and allowed assessing the prospects for its possible clinical use.

[The effect of music containing binaural beats on daytime fall-asleep dynamics]. / Vliianie muzyki, soderzhashchei binaural'nye bieniia, na dinamiku dnevnogo zasypaniia.

AIM: To test the hypothesis that listening to the music with the effect of binaural beats of theta and delta range during nap decreases sleep latency defined by 2nd slow wave sleep stage appearance, as well as improves its stability. MATERIAL AND METHODS: The experimental set of 20 min duration was established according to the counterbalanced scheme with 21 subjects. Each subject participated in two attempts: one attempt included sound stimulation (music) and another one was sham (silence). RESULTS AND CONCLUSION: The decrease in sleep latency during stimulation is not confirmed reliably. The increase in sleep stability has been confirmed reliably using nonlinear regression model. The findings can be used in the development of non-pharmacologic ways of sleep treatment.

Chitosan-g-oligo(L,L-lactide) copolymer hydrogel for nervous tissue regeneration in glutamate excitotoxicity: in vitro feasibility evaluation.

Over the last decade, a number of hydrogels attracted great attention in the area of brain tissue engineering. The hydrogels are composed of hydrophilic polymers forming 3D network in water. Their function is promoting structural and functional restoration of damaged brain tissues by providing mechanical support and navigating cell fate. This paper reports on the neurocompatibility of chitosan-g-oligo(L,L-lactide) copolymer hydrogel with primary rat cortical neuron culture. The hydrogel was produced by a molding technique on the base of photocurable composition consisting of chitosan-g-oligo(L,L-lactide) copolymer, poly(ethylene glycol) diacrylate and photosensitizer Irgacure 2959. The influence of the hydrogel on cell viability, phenotype and calcium homeostasis, mitochondrial potential and oxygen consumption rate in glutamate excitotoxicity was analyzed using primary neuron cultures obtained from a neonatal rat cortex. This study revealed that the hydrogel is non-cytotoxic. Dissociated neonatal rat cortical cells were actively attaching to the hydrogel surface and exhibited the phenotype, calcium homeostasis and mitochondrial function in both standard conditions and glutamate excitotoxicity (100 µM) similar to the control cells cultured without the hydrogel. To conclude, in this study we assessed the feasibility of the application of chitosan-g-oligo(L,L-lactide) copolymer hydrogel for tissue engineering therapy of brain injury in an in vitro model. The results support that the hydrogel is able to sustain realization of the functional metabolic activity of neonatal rat cortical cells in response to glutamate excitotoxicity.

Collagen Hydrolysis Products Reduce the Formation of Stress-Induced Ulcers by Regulating Stress-Associated Activation of the Neuroendocrine and Immune Systems.

We studied the effect of collagen fragments (PGP and AcPGP) on serum content of epinephrine, corticosterone, and IL-1ß in rats subjected to water-immersion stress. The degree of local inflammation accompanying ulceration was assessed by IL-1ß production by ln. gastricus caudalis cells. In 1 h, the sharp increase in hormone concentrations in the blood of stressed animals reflected the high stress intensity. Intranasal administration of PGP reduced the area of stress-induced ulcers by 63%, prevented the increase in the levels of stress hormones and the main proinflammatory cytokine in rat blood. The concentrations of IL-1ß in cell culture from regional lymph node of experimental animals returned to normal in 24 and 48 h after the stress. Acetylation of PGP prevents with gastroprotection, but does not abrogate other properties of the peptide.

Glyprolines exert protective and repair-promoting effects in the rat stomach: potential role of the cytokine GRO/CINC-1.

Glyprolines have been reported to exert protective effects in the stomach. In this study, we examined the potential effects of intranasal administration of Pro-Gly-Pro (PGP) and N-acetyl-Pro-Gly-Pro (AcPGP) on experimental gastric ulcer formation and healing. We also studied gastric release of the cytokine GRO/CINC-1, and its potential role in ulcer development and healing. Gastric ulcers were induced in rats by applying acetic acid to the serosa of the stomach. PGP and AcPGP were then administered at a dose of 3.7 µmol/kg once daily on either days 1 - 3 (ulcer formation) or days 4 - 6 (ulcer healing). Measurement of ulcer area and histological examination of gastric tissue were carried out on days 4 and 7 after application of acetic acid. In vitro studies involved addition of the glyprolines to cultured rat gastric epithelial cells with or without lipopolysaccharide. Reverse transcription PCR, real-time PCR and ELISA were used for cytokine analysis. PGP and AcPGP significantly reduced ulcer areas on the 4(th) day and accelerated the healing on the 7(th) day compared with the control. After acetic acid-induced ulceration, the expression of GRO/CINC-1 mRNA in gastric tissue was increased 9-fold versus the sham-operated group. Treatment with PGP or AcPGP both significantly suppressed the expression of GRO/CINC-1 mRNA in gastric tissue. However, the glyprolines did not alter LPS-induced mRNA expression or release of GRO/CINC-1 from cultured rat gastric epithelial cells, even though those cells were harvested from rats subjected to the ulcer-induction procedure. The results of this study show that intranasal administration of PGP and AcPGP significantly increased resistance against acetic acid-induced ulceration and accelerated healing in the rats. These effects may be due, at least in part, to their ability to reduce the acetic acid-induced GRO/CINC-1 expression and production in gastric tissue.

Correlation analysis of cytokine gene expression changes and antiulcer effects of Pro-Gly-Pro and N-Acetyl-Pro-Gly-Pro in experimental models of ulceration.

Direct correlation of the cytokine gene expression level in peripheral blood mononuclear cells (BMNCs) and gastric mucosa (GM) cells with the development of gastric ulcers of various etiologies was shown for the first time. Ethanol-induced ulceration causes an increased transcription of IFNa, IL-8, and IL-12 mRNA in BMNCs. GM damages caused by water immersion stress were accompanied by an increased transcription of TNFa. The sizes of acetate-induced damages were positively correlated with the expression of IL-10 and IL-8 genes in BMNCs and with the expression of IFNa, IL-2, IL-12, and TNF genes in GM cells. Intranasal administration of Pro-Gly-Pro (PGP) reduced ethanol-induced ulceration, activating the transcription of IFNγ, IL-2, and IL-4 mRNA in BMNCs and prevents the formation of stress- and acetateinduced ulcers by inhibiting the expression of IL-8 and IL-10 genes, respectively.

Protective and therapeutic effects of glyprolines in psychoemotional stress induced by cholecystokinin-4 injection.

Experiments on outbred albino male rats showed that psychoemotional stress induced by intraperitoneal injection of cholecystokinin-4 (100 microg/kg) increased anxiety, impaired orientation and exploration activities in the elevated plus-maze and hole-board tests, and increased the level of depression of Porsolt test. Preliminary intranasal administration of glyprolines (15 min before cholecystokinin) in a dose of 3.7 micromol/kg prevented the development of stress-induced behavioral disturbances. Administration of peptides 30 min after cholecystokinin-4, i.e., to rats with developed behavioral disturbances, almost completely abolished these disturbances.

Therapeutic effects of glyprolines (PGP, GP, and PG) in rats with stress-induced behavioral disorders.

Experiments on male outbred albino rats showed that stress (10-min swimming) increased anxiety and inhibited orientation and exploratory activities. Poststress (15 min after the end of swimming) intranasal administration of peptides Pro-Gly-Pro and Gly-Pro in a dose of 3.7 micromol/kg prevented stress-induced behavioral disorders. This effect persisted for 3 h.

Selank and its metabolites maintain homeostasis in the gastric mucosa.

Antiulcer properties of a synthetic anxiolytic Selank and in vivo formed metabolites of this compound were studied on 3 experimental models of ulceration. The test peptides decreased the area of experimental gastric ulcers.

Histomorphological characteristics of accelerated healing of acetate ulcers under the effect of glyprolines.

Glyprolines (PGP, GPG, GPGP, PGPGP, and GPGPGP) modulated histomorphological characteristics of acetate ulcers. They accelerated healing of acetate ulcers, promote complete differentiation of the surface epithelium and glands in the gastric mucosa, contributed to the appearance of a considerable number of fibroblasts at the site of the regenerating mucosa, and significantly decreased the count of macrophages.

Peptide correction of disorders in rat mesenteric microcirculatory system during inflammation.

PGP peptide had a protective effect in contractile dysfunction of the rat mesenteric lymph vessels under conditions of inflammation, irrespective of the time of its injection (before or after inflammatory agent). The preventive effect of this peptide is largely determined by its capacity to prevent mast cells activation. PGP injected 2 h after induction of inflammation did not inhibit secretory activity of mast cells, which suggests other mechanisms of its therapeutic action.

[Reculatory fragments of collagen in gastric mucosa homeostasis].

The new glyproline family was distinguished from the regulatory peptides recently. It includes the simplest proline- and glycine-containing peptides: PG, GP, PGP, and respective peptides with hydroxylated proline residues. Glyproline's bioactivity covers many important systems of the body including suppression of some reaction in the blood coagulation and platelet aggregation and gastric mucosal maintenance. It was shown that PGP, PG and GP have a wide spectrum of antiulcer activity with respect to gastric mucosal damages of various aetiology. GHyp and HypGP show also antiulcer action. In vivo glyprolines being fragments of collagen may be generated during synthesis and catabolism of collagen. It is well known that approximately 10-60% of newly synthesized collagen degrade intracellularly with succeeding secretion of small peptides composed of less than 5 aminoacid residues out of cells. Different simplest proline and hydroxyproline fragments of glyprolines are revealed in various type of collagen: GP, GHyp, PG, PPG, PGP, PHypG., GPHyp, GPP, GPG, GHypP, HypGP. It is possible that these fragments may be also secreted out of cells during the stage of degradation of newly synthesized collagen. We showed that the intragastric (per oral) introduction of hydrolyzed gelatin, having 20 small peptide fragments, including PGP and HypGP, also increase gastric stability showing protective and therapeutic antiulcer effect. The corresponding receptors for glyprolines are not completely identified yet but it may be supposed that PGP, GP and other glyprolines interact with the same receptors with which the III type collagen is binding with platelet's receptors. It is supposed that octapeptide sequence KPGGluPGPK of collagen is rather important for binding with receptor. When this sequence in the structure of collagen's molecule binds with the receptor, platelet aggregation is induced. Free octapeptide blocks the receptor and inhibits platelet aggregations. Qualitatve characteristics of parameters of inhibition with intact octapeptide and glyproline, as well as the receptor's structure--that's our concern for the nearest future.

[Evenly tritium-labeled peptides and their in vivo and in vitro biodegradation].

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50-150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium-labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a tg scale of biological samples both in vitro and in vivo.

[Short peptide fragments with antiulcer activity from a collagen hydrolysate].

A peptide acidic hydrolysate of collagen (PHC) was obtained under conditions (4 N HCl) ensuring the predominant formation of short peptides, glyprolines. They were separated and their antiulcer activity was studied. Thirty individual peptides with molecular masses of 174-420 amu were isolated from the PHC by HPLC. The PHC was shown to predominantly contain 2- to 4-aa peptides, including PG, GP, and PGP. Experiments on rats demonstrated that, on intragastric administration at a dose of 1 mg/kg, PHC enhances the stability of the gastric mucosa to the action of ulcerogenic factors, such as ethanol and stress, and exhibits a protecting antiulcer effect. Even a lesser dose (0.1 mg/kg), which reduced ulcer area twofold, was effective in the stress model of ulcer formation. The intraperitoneal and intragastric administration of PHC at a dose of 1 mg/kg was found to exhibit a therapeutic effect in the acetate model of ulcer formation.

The role of protective effects of proline-containing peptides (PGP, PG, and GP) in contractile dysfunction of mesenteric lymphatic vessels in rats with experimental acute peritonitis.

The development of acute peritonitis in rats induced by intraperitoneal injection of thioglycollate was accompanied by a decrease in contractile function of mesenteric lymphatic vessels and impaired response to norepinephrine. Administration of proline-containing peptides after induction of inflammation significantly decreased the severity of these disorders. Our results attest to the possibility of using peptides for the correction of mesenteric microcirculatory disturbances during inflammation.

[Effects of glyprolines on development and healing of acetate ulcers in rats].

We studied glyprolines influence on development and healing of acetate ulcers in rats. A maximal ulcer area has developed four days after application of acetic acid to gastric serous mucosa. Estimation of ulcer area in the control and experimental animals receiving a physiological solution or peptides was carried out on day 4 or 7 after ulcerogenesis. Single administration of PGP, PG or GP in an equimolar dose of 3.7 mcmol/kg (i/p) during 1-3 days after the application significantly reduced ulcers by 88%, 93% and 94%, respectively, compared to the controls. Administration of these peptides for 4-6 days after application led to a significant reduction of the lesions by 46,50 and 96%, respectively, vs controls. Thus, glyprolines inhibit development of acetic ulcers and enhance their healing.