The First Dipeptide Mimetic of Neurotrofin-3: Design and Pharmacological Properties.

The dimeric dipeptide mimetic hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301) was created on the basis of the structure of the exposed region of the neurotrophin-3 4th loop. The new compound, as well as the full-length neurotrophin, activated the TrkC and TrkB receptors. GTS-301 showed neuroprotective activity in experiments on HT-22 mouse hippocampal cells under conditions of oxidative stress and glutamate toxicity at concentrations of 10-12 and 10-8 M, respectively, and antidepressant-like activity in the forced swimming test on mice with 7-day intraperitoneal administration in doses of 10-40 mg/kg.

Dipeptide Mimetic of the BDNF Loop 4 Possesses Analgetic Activity.

Previously, we synthesized a dimeric dipeptide mimetic of the brain-derived neurotrophic factor (BDNF) loop 4, GSB-106, which, similarly to BDNF, activated TrkB, PI3K/AKT, and MAPK/ERK. When administered systemically, it exhibited neuroprotective, antidepressant, and antidiabetic activities and stimulated neurogenesis and synaptogenesis. In this study, we established that GSB-106 also exhibits the analgesic activity, typical for BDNF, which was revealed in rats in hot plate and tail flick tests 0.5-48 h after intraperitoneal injection at doses of 0.1 and 1 mg/kg.

A novel dimeric dipeptide mimetic of the BDNF selectively activates the MAPK-Erk signaling pathway.

On the basis of the structure of beta-turn of loop 2 of brain-derived neurotrophic factor (BDNF), its new dimeric dipeptide mimetic bis-(N-hexanoyl-L-seryl-L-lysine) hexamethylenediamide (GTS-201) was created. It activated TrkB and Erk, did not activate Akt, and exhibited neuroprotective activity in vitro at concentrations of 10-5-10-8 M. Unlike the mimetics that activate Erk and Akt, GTS-201 did not exhibit antidepressant properties. For the manifestation of the antidepressant activity of BDNF mimetics, the activation of its both major signaling pathways is required.

Estimation of macro sleep stages from whole night audio analysis.

During routine sleep diagnostic procedure, sleep is broadly divided into three states: rapid eye movement (REM), non-REM (NREM) states, and wake, frequently named macro-sleep stages (MSS). In this study, we present a pioneering attempt for MSS detection using full night audio analysis. Our working hypothesis is that there might be differences in sound properties within each MSS due to breathing efforts (or snores) and body movements in bed. In this study, audio signals of 35 patients referred to a sleep laboratory were recorded and analyzed. An additional 178 subjects were used to train a probabilistic time-series model for MSS staging across the night. The audio-based system was validated on 20 out of the 35 subjects. System accuracy for estimating (detecting) epoch-by-epoch wake/REM/NREM states for a given subject is 74% (69% for wake, 54% for REM, and 79% NREM). Mean error (absolute difference) was 36±34 min for detecting total sleep time, 17±21 min for sleep latency, 5±5% for sleep efficiency, and 7±5% for REM percentage. These encouraging results indicate that audio-based analysis can provide a simple and comfortable alternative method for ambulatory evaluation of sleep and its disorders.

Obstructive sleep apnea severity estimation: Fusion of speech-based systems.

Obstructive sleep apnea (OSA) is a common sleep-related breathing disorder. Previous studies associated OSA with anatomical abnormalities of the upper respiratory tract that may be reflected in the acoustic characteristics of speech. We tested the hypothesis that the speech signal carries essential information that can assist in early assessment of OSA severity by estimating apnea-hypopnea index (AHI). 198 men referred to routine polysomnography (PSG) were recorded shortly prior to sleep onset while reading a one-minute speech protocol. The different parts of the speech recordings, i.e., sustained vowels, short-time frames of fluent speech, and the speech recording as a whole, underwent separate analyses, using sustained vowels features, short-term features, and long-term features, respectively. Applying support vector regression and regression trees, these features were used in order to estimate AHI. The fusion of the outputs of the three subsystems resulted in a diagnostic agreement of 67.3% between the speech-estimated AHI and the PSG-determined AHI, and an absolute error rate of 10.8 events/hr. Speech signal analysis may assist in the estimation of AHI, thus allowing the development of a noninvasive tool for OSA screening.

Can we discriminate between apnea and hypopnea using audio signals?

Obstructive sleep apnea (OSA) affects up to 14% of the population. OSA is characterized by recurrent apneas and hypopneas during sleep. The apnea-hypopnea index (AHI) is frequently used as a measure of OSA severity. In the current study, we explored the acoustic characteristics of hypopnea in order to distinguish it from apnea. We hypothesize that we can find audio-based features that can discriminate between apnea, hypopnea and normal breathing events. Whole night audio recordings were performed using a non-contact microphone on 44 subjects, simultaneously with the polysomnography study (PSG). Recordings were segmented into 2015 apnea, hypopnea, and normal breath events and were divided to design and validation groups. A classification system was built using a 3-class cubic-kernelled support vector machine (SVM) classifier. Its input is a 36-dimensional audio-based feature vector that was extracted from each event. Three-class accuracy rate using the hold-out method was 84.7%. A two-class model to separate apneic events (apneas and hypopneas) from normal breath exhibited accuracy rate of 94.7%. Here we show that it is possible to detect apneas or hypopneas from whole night audio signals. This might provide more insight about a patient's level of upper airway obstruction during sleep. This approach may be used for OSA severity screening and AHI estimation.

Breathing rate estimation during sleep using audio signal analysis.

Sleep is associated with important changes in respiratory rate and ventilation. Currently, breathing rate (BR) is measured during sleep using an array of contact and wearable sensors, including airflow sensors and respiratory belts; there is need for a simplified and more comfortable approach to monitor respiration. Here, we present a new method for BR evaluation during sleep using a non-contact microphone. The basic idea behind this approach is that during sleep the upper airway becomes narrower due to muscle relaxation, which leads to louder breathing sounds that can be captured via ambient microphone. In this study we developed a signal processing algorithm that emphasizes breathing sounds, extracts breathing-related features, and estimates BR during sleep. A comparison between audio-based BR estimation and BR calculated using the traditional (gold-standard) respiratory belts during in-laboratory polysomnography (PSG) study was performed on 204 subjects. Pearson's correlation between subjects' averaged BR of the two approaches was R=0.97. Epoch-by-epoch (30 s) BR comparison revealed a mean relative error of 2.44% and Pearson's correlation of 0.68. This study shows reliable and promising results for non-contact BR estimation.

Breath-by-breath detection of apneic events for OSA severity estimation using non-contact audio recordings.

Obstructive sleep apnea (OSA) is a prevalent sleep disorder, characterized by recurrent episodes of upper airway obstructions during sleep. We hypothesize that breath-by-breath audio analysis of the respiratory cycle (i.e., inspiration and expiration phases) during sleep can reliably estimate the apnea hypopnea index (AHI), a measure of OSA severity. The AHI is calculated as the average number of apnea (A)/hypopnea (H) events per hour of sleep. Audio signals recordings of 186 adults referred to OSA diagnosis were acquired in-laboratory and at-home conditions during polysomnography and WatchPat study, respectively. A/H events were automatically segmented and classified using a binary random forest classifier. Total accuracy rate of 86.3% and an agreement of κ=42.98% were achieved in A/H event detection. Correlation of r=0.87 (r=0.74), diagnostic agreement of 76% (81.7%), and average absolute difference AHI error of 7.4 (7.8) (events/hour) were achieved in in-laboratory (at-home) conditions, respectively. Here we provide evidence that A/H events can be reliably detected at their exact time locations during sleep using non-contact audio approach. This study highlights the potential of this approach to reliably evaluate AHI in at home conditions.

Bioequivalence study of 8 mg ondansetron film-coated tablets in healthy Caucasian volunteers.

The aim of the study was to investigate the bioequivalence of a generic product of 8 mg film-coated tablets (test) to the branded product (reference) at the same strength in order to apply for regulatory approval. The secondary objective of the study was to compare the tolerability of both products. A double blinded, randomized, cross-over, 2-period, comparative study was conducted in healthy Caucasian volunteers under fasting conditions. A single oral dose administration of the test or reference product was followed by a 7-day wash-out period. The ondansetron concentration was determined using a validated high performance liquid chromatography with a UV detection method. The 90% confidence interval of the point estimate (test over reference products) for C(max) and AUC(0-t) fell within the 80.00-125.00% acceptance range. The results of the study indicate that the film-coated tablets of Ondatron 8 mg manufactured by Tarchominskie Zaklady Farmaceutyczne Polfa S.A. (test product) are bioequivalent to those of Zofran manufactured by GlaxoSmithKline Export Ltd (reference product). Both products were well tolerated.

[Study of structure-activity relationship in series of Gsb-106 analogues-dipeptide mimetics of brain-derived neurotrophic factor].

In previous work we have obtained a dimeric dipeptide mimetic of 4th loop of BDNF - hexamethylenediamide bis-(N-monosuccinil-L-seryl-L-lysine) (GSB-106), having a neuroprotective activity in vitro in a concentration range 10(-5)-10(-8) M and an antidepressant activity in vivo at doses 0.1 and 1 mg/kg i.p. in rats. We have investigated the structural and functional relationships among analogues of GSB-106. Glycine scan was performed and a number of appropriate compounds were synthesized: GT-105 (here lysine is replaced by glycine), GT-107 (here serine is replaced by glycine), GT-106Ac (here monosuccinic radical is replaced by acetyl group). We have studied the dependence of activity of following compounds from the configuration of amino acid residues: GT-107D (D-enantiomer of the GT-107), GT-106DL (L-serine was replaced by D-serine), GT-106LD (L-lysine was replaced by D-lysine). The investigation of these compounds using the HT22 cell culture in conditions of oxidative stress has approved only two analogues of GSB-106 to have a neuroprotective effect: in the case of replacement of serine to glycine and of replacment of succinic radical to acetic group. A disappearance of this effect was observed in event of the replacement of lysine residue to glycine in GT-105, L-lysine residue to D-lysine and also by conversion of serine configuration. These results show that lysine residue is crucial for the neuroprotective activity of GSB-106. L-Configuration of the lysine and serine residues required. Configuration of lysine residue becomes critical in absence of serine side group. Thus, the the following fragment is a minimum pharmacophore of beta-turn of 4 loop of BDNF: HOOC-CH2-CH-CO-NH-(S)-CH(CH2OH)-CO-NH-(S)-CH((CH2)4NHz)-CO-NH-(CH2)3-. Only one (GT-106Ac) out of two analogues of GSB-106 with neuroprotective activity possesses antidepressant activity too. This fact indicates about a necessity of more stringent structural requirements for exposure of antidepressant activity. The results obtained can be useful for designing of new active mimetics of BDNE

Detection of breathing sounds during sleep using non-contact audio recordings.

Evaluation of respiratory activity during sleep is essential in order to reliably diagnose sleep disorder breathing (SDB); a condition associated with serious cardio-vascular morbidity and mortality. In the current study, we developed and validated a robust automatic breathing-sounds (i.e. inspiratory and expiratory sounds) detection system of audio signals acquired during sleep. Random forest classifier was trained and tested using inspiratory/expiratory/noise events (episodes), acquired from 84 subjects consecutively and prospectively referred to SDB diagnosis in sleep laboratory and in at-home environment. More than 560,000 events were analyzed, including a variety of recording devices and different environments. The system's overall accuracy rate is 88.8%, with accuracy rate of 91.2% and 83.6% in in-laboratory and at-home environments respectively, when classifying between inspiratory, expiratory, and noise classes. Here, we provide evidence that breathing-sounds can be reliably detected using non-contact audio technology in at-home environment. The proposed approach may improve our understanding of respiratory activity during sleep. This in return, will improve early SDB diagnosis and treatment.

Detection of Obstructive sleep apnea in awake subjects by exploiting body posture effects on the speech signal.

Obstructive sleep apnea (OSA) is a common sleep disorder. OSA is associated with several anatomical and functional abnormalities of the upper airway. It was shown that these abnormalities in the upper airway are also likely to be the reason for increased rate of apneic events in the supine position. Functional and structural changes in the vocal tract can affect the acoustic properties of speech. We hypothesize that acoustic properties of speech that are affected by body position may aid in distinguishing between OSA and non-OSA patients. We aimed to explore the possibility to differentiate OSA and non-OSA patients by analyzing the acoustic properties of their speech signal in upright sitting and supine positions. 35 awake patients were recorded while pronouncing sustained vowels in the upright sitting and supine positions. Using linear discriminant analysis (LDA) classifier, accuracy of 84.6%, sensitivity of 92.7%, and specificity of 80.0% were achieved. This study provides the proof of concept that it is possible to screen for OSA by analyzing and comparing speech properties acquired in upright sitting vs. supine positions. An acoustic-based screening system during wakefulness may address the growing needs for a reliable OSA screening tool; further studies are needed to support these findings.

Neuroprotective effects of dipeptide analogue of brain-derived neurotrophic factor GSB-106 in in vitro experiments.

Dimeric dipeptide GSB-106, a novel structural analogue of neurotrophin BDNF, in doses from 10(-5) to 10(-8) M protected cultured immortalized mouse hippocampal HT-22 neurons from H2O2 or glutamate damage. The neuroprotective effect of GSB-106 was also shown on cultured SH-SY5Y human neuroblastoma cells after treatment with neurotoxin 6-hydroxidopamine. These data attest to functional similarity of GSB-106 and neurotrophin BDNF.

OSA severity assessment based on sleep breathing analysis using ambient microphone.

In this paper, an audio-based system for severity estimation of obstructive sleep apnea (OSA) is proposed. The system estimates the apnea-hypopnea index (AHI), which is the average number of apneic events per hour of sleep. This system is based on a Gaussian mixture regression algorithm that was trained and validated on full-night audio recordings. Feature selection process using a genetic algorithm was applied to select the best features extracted from time and spectra domains. A total of 155 subjects, referred to in-laboratory polysomnography (PSG) study, were recruited. Using the PSG's AHI score as a gold-standard, the performances of the proposed system were evaluated using a Pearson correlation, AHI error, and diagnostic agreement methods. Correlation of R=0.89, AHI error of 7.35 events/hr, and diagnostic agreement of 77.3% were achieved, showing encouraging performances and a reliable non-contact alternative method for OSA severity estimation.

[Design and synthesis of dipeptide mimetics of brain-derived neurotrophic factor].

Low-molecular mimetics of brain-derived neurotrophic factor (BDNF) loops 1 and 4 representing to monomeric and dimeric amides of N-acyldipeptides were constructed and synthesized. The sequence of these dipeptides coinside with the central regions of beta-turns of corresponding loops of neurotrophine sequence, and acyl groups are bioisosters of preceding amino acid residues. Hexa- and heptamethylenediamine were used as spacers linking C-terminus ofdipeptides in BDNF dimeric mimetics. These substances were synthesized by classic peptide synthesis methods in solution and got laboratory codes GSB-104 (HO-Suc-Ser-Lys-NH2), GSB-106 ([HO-Suc-Ser-Lys-NH-(CH2)3-]2), GSB-207 (HO-Suc-Met-Ser-NH2) and GSB-214 ([HO-Suc-Met-Ser-NH-(CH2)7/2-]2). By using the culture of immortalized hippocampal cell line HT-22 on the oxidative stress conditions it was shown that dimeric mimetics of both loops demonstrated neuroprotective activity in the concentration rage of 10(-5)-10(-8) M. Monomeric loop 1 mimetic GSB-207 was inactive in the same concentrations and monomeric loop 4 mimetic GSB-104 in a concentration of 10(-7) M decreased survival of neurons. Presence of neuroprotective activity only for dimeric mimetics correlates with the data that BDNF is active only in homodimeric form. As opposed to dimeric mimetic of loop 1 GSB-214, dimeric mimetic of loop 4 GSB-106 demonstrates specific for BDNF antidepressive activity in Porsolt test on rats in doses 0.1 and 1 mg/kg i.p. It is suggested that antidepressive activity of BDNF is associated with its loop 4. We consider that compounds obtained will be useful for investigation of BDNF action mechanism and can lead to creation of a new group of medicinal substances with antidepressive and neuroprotective activities.

Sleep-quality assessment from full night audio recordings of sleep apnea patients.

In this work, a novel system (method) for sleep quality analysis is proposed. Its purpose is to assist an alternative non-contact method for detecting and diagnosing sleep related disorders based on acoustic signal processing. In this work, audio signals of 145 patients with obstructive sleep apnea were recorded (more than 1000 hours) in a sleep laboratory and analyzed. The method is based on the assumption that during sleep the respiratory efforts are more periodically patterned and consistent relative to a waking state; furthermore, the sound intensity of those efforts is higher, making the pattern more noticeable relative to the background noise level. The system was trained on 50 subjects and validated on 95 subjects. The system accuracy for detecting sleep/wake state is 82.1% (epoch by epoch), resulting in 3.9% error (difference) in detecting sleep latency, 11.4% error in estimating total sleep time, and 11.4% error in estimating sleep efficiency.

Role of growth hormone-releasing hormone in sleep and growth impairments induced by upper airway obstruction in rats.

Upper airway obstruction (UAO) can lead to abnormal growth hormone (GH) homeostasis and growth retardation but the mechanisms are unclear. We explored the effect of UAO on hypothalamic GH-releasing hormone (GHRH), which has a role in both sleep and GH regulation. The tracheae of 22-day-old rats were narrowed; UAO and sham-operated animals were sacrificed 16 days post-surgery. To stimulate slow-wave sleep (SWS) and GH secretion, rats were treated with ritanserin (5-HT(2) receptor antagonist). Sleep was measured with a telemetric system. Hypothalamic GHRH, hypothalamic GHRH receptor (GHRHR) and GH receptor, and orexin were analysed using ELISA, real-time PCR and Western blot. UAO decreased hypothalamic GHRH, GHRHR and GH receptor levels, while orexin mRNA increased (p<0.01). In UAO rats, the duration of wakefulness was elevated and the duration of SWS, paradoxical sleep and slow-wave activity was reduced (p<0.001). Ritanserin alleviated these effects, i.e. normalised hypothalamic GHRH content, decreased wake duration, increased duration and depth of SWS, and attenuated growth impairment (p<0.001). Here, we present evidence that growth retardation in UAO is associated with a reduction in hypothalamic GHRH content. Our findings show that abnormalities in the GHRH/GH axis underlie both growth retardation and SWS-disorder UAO.

Adenotonsillectomy improves slow-wave activity in children with obstructive sleep apnoea.

The aim of the present study was to estimate slow-wave activity (SWA), a marker of sleep homeostasis, in children with obstructive sleep apnoea (OSA) before and after adenotonsillectomy (AT) compared with untreated OSA children (comparison group). 14 children with OSA (mean ± sd age 6.4 ± 2.5 yrs; apnoea-hypopnoea index (AHI) 10.0 ± 10.3 events·h⁻¹) who underwent AT were consecutively recruited to the study. The comparison group comprised six retrospectively recruited children (age 5.4 ± 2.2 yrs; AHI 9.4 ± 7.6 events·h⁻¹) with OSA that did not undergo treatment. Electroencephalogram (derivation C3/A2) was analysed using spectral and waveform analysis to determine SWA energy and slow-wave slope. The same procedure was repeated 5.4 and 19 months later for the AT and comparison groups, respectively. AT improved respiration without a change in duration of sleep stages. Following AT, >50% elevation of SWA during the first two sleep cycles (p<0.01) and a more physiological decay of SWA across the night (p<0.0001) were noted. The slow-wave slope increased by >30% following AT (p<0.03). No significant changes were found in SWA in the comparison group. Sleep homeostasis is considerably impaired in pre-pubescent children with OSA. AT restores more physiological sleep homeostasis in children with OSA. SWA analysis may provide a useful addition to standard sleep-stage analyses in children with OSA.

Elevated healthcare utilisation in young adult males with obstructive sleep apnoea.

The aim of the present study was to explore morbidity and healthcare utilisation among young adult males with obstructive sleep apnoea (OSA) compared with middle-aged OSA patients over the 5-yr period preceding diagnosis. A prospective case-control study was performed; 117 young (22-39-yr-old) males with OSA were matched with 117 middle-aged (40-64-yr-old) OSA males for body mass index, apnoea/hypopnoea index, arterial oxygen saturation, arousal and awakening index, and Epworth Sleepiness Scale score. Each OSA patient was matched with controls by age, geographic area and physician. Young adult males with OSA showed no increase in specific comorbidity compared with controls. Middle-aged OSA patients exhibited increased risk of cardiovascular disease. Healthcare utilisation for the 5-yr period was >or=1.9 times higher among young and middle-aged male OSA patients than among controls. Multiple logistic regression analysis revealed that hyperlipidaemia in young adults and a body mass index of >37 kg x m(-2) and cardiovascular disease in middle-aged adults are the only independent determinants of the upper third, most costly, OSA patients. Compared with middle-aged males with obstructive sleep apnoea, in whom increased expenditure was related to cardiovascular disease and body mass index, utilisation was not related to any specific disease in younger cases.

DNA typeability in liquid urine and urine stains using AmpFISTR SGM Plus.

PURPOSE: Urine specimens are usually collected for biochemical and toxicological tests and for doping control. In forensic casework urine analyses are performed occasionally, however, the authors emphasize their importance in crime scene reconstruction. The objective of the research was to evaluate efficacy of AmpFISTR SGM Plus typing of urine and urine stains which were subject to different temperature conditions. MATERIAL AND METHODS: Urine samples were collected from 10 female and 10 male volunteers. Liquid specimens were stored at room temperature (RT), 4 degrees C and -20 degrees C up to 28 days. Experimental stains were prepared by applying 3 ml urine on sterile cloth 30 x 30 cm, air-dried and stored at RT up to 360 days. The amount of DNA was estimated with use of slot-blot technique (Quantiblot Human DNA Quantitation Kit, Applera). DNA profiles were obtained using AmpFISTR SGM Plus and 310 ABI Prism Genetic Analyzer (Applera). Typing of a experimental sample was considered successful when the full profile was obtained matching that of a reference sample. RESULTS: Significant differences in DNA yield were noted between female and male urine samples. No differences between the extraction methods were found in regard to DNA yield and typeability rate. Different typeability rates were recorded for liquid urine and urine stains depending on storage temperature. CONCLUSIONS: Liquid urine samples and urine stains can be considered as a potential source of DNA in disputable specimen individualization and in forensic casework using the fluorescent multiplex PCR system AmpFLSTR SGM Plus.