Molecular Dynamics Simulation Study of the Self-Assembly of Phenylalanine Peptide Nanotubes.

In this paper, we propose and use a new approach for a relatively simple technique for conducting MD simulation (MDS) of various molecular nanostructures, determining the trajectory of the MD run and forming the final structure using external force actions. A molecular dynamics manipulator (MD manipulator) is a controlled MDS type. As an example, the applicability of the developed algorithm for assembling peptide nanotubes (PNT) from linear phenylalanine (F or Phe) chains of different chirality is presented. The most adequate regimes for the formation of nanotubes of right chirality D from the initial L-F and nanotubes of left chirality L of their initial dipeptides D-F modes were determined. We use the method of a mixed (vector-scalar) product of the vectors of the sequence of dipole moments of phenylalanine molecules located along the nanotube helix to calculate the magnitude and sign of chirality of self-assembled helical phenylalanine nanotubes, which shows the validity of the proposed approach. As result, all data obtained correspond to the regularity of the chirality sign change of the molecular structures with a hierarchical complication of their organization.

Quantitative Assessment of Chirality of Protein Secondary Structures and Phenylalanine Peptide Nanotubes.

In this study we consider the features of spatial-structure formation in proteins and their application in bioengineering. Methods for the quantitative assessment of the chirality of regular helical and irregular structures of proteins are presented. The features of self-assembly of phenylalanine (F) into peptide nanotubes (PNT), which form helices of different chirality, are also analyzed. A method is proposed for calculating the magnitude and sign of the chirality of helix-like peptide nanotubes using a sequence of vectors for the dipole moments of individual peptides.

Modeling of Self-Assembled Peptide Nanotubes and Determination of Their Chirality Sign Based on Dipole Moment Calculations.

The chirality quantification is of great importance in structural biology, where the differences in proteins twisting can provide essentially different physiological effects. However, this aspect of the chirality is still poorly studied for helix-like supramolecular structures. In this work, a method for chirality quantification based on the calculation of scalar triple products of dipole moments is suggested. As a model structure, self-assembled nanotubes of diphenylalanine (FF) made of L- and D-enantiomers were considered. The dipole moments of FF molecules were calculated using semi-empirical quantum-chemical method PM3 and the Amber force field method. The obtained results do not depend on the used simulation and calculation method, and show that the D-FF nanotubes are twisted tighter than L-FF. Moreover, the type of chirality of the helix-like nanotube is opposite to that of the initial individual molecule that is in line with the chirality alternation rule general for different levels of hierarchical organization of molecular systems. The proposed method can be applied to study other helix-like supramolecular structures.

Hydrophilic interaction liquid chromatography method for eremomycin determination in pre-clinical study.

A complex of hydrophilic interaction liquid chromatography (HILIC) methods for simple and efficient determination of eremomycin (ERM) as an active pharmaceutical ingredient (API) of a novel drug is proposed for preclinical study, which includes the dissolution test and pharmacokinetic study on the animals. A home-made HILIC silica-based stationary phase (SP) containing diol functionalities and positively charged nitrogen atoms in its structure was synthesized for this research and applied for the first time for performing the first step of preclinical study (dissolution test) of the novel ERM-containing drug. HILIC method developed using novel home-made SP allowed us to avoid any interferences from polyethylene glycol (PEG) contained in the drug matrix thus providing a unique advantage of the proposed approach over RP HPLC. The home-made SP demonstrated better chromatographic performance as compared to the tested commercially available columns with various functionalities. Different retention behaviour and mechanisms with various electrostatic impact were demonstrated for two glycopeptide antibiotics, namely, ERM and its analogue vancomycin (VAN), on the home-made SP. For the second step of the preclinical study HILIC-MS/MS method for ERM determination in rabbit plasma was developed and validated in accordance with the EMA requirements and successfully applied to the preclinical study on rabbits after intravenous and intraperitoneal drug administration. The results of dissolution test and pharmacokinetic study revealed similar in vitro solubility of ERM and VAN and low ERM bioavailability, which proved the potential safety and efficiency of the novel drug.

A model of autowave self-organization as a hierarchy of active media in the biological evolution.

Within the framework of the active media concept, we develop a biophysical model of autowave self-organization which is treated as a hierarchy of active media in the evolution of the biosphere. We also propose a mathematical model of the autowave process of speciation in a flow of mutations for the three main taxonometric groups (prokaryotes, unicellular and multicellular eukaryotes) with a naturally determined lower boundary of living matter (the appearance of prokaryotes) and an open upper boundary for the formation of new species. It is shown that the fluctuation-bifurcation description of the evolution for the formation of new taxonometric groups as a trajectory of transformation of small fluctuations into giant ones adequately reflects the process of self-organization during the formation of taxa. The major concepts of biological evolution, conditions of hierarchy formation as a fundamental manifestation of self-organization and complexity in the evolution of biological systems are considered.

Prognostic indices of pathological tooth wear development.

OBJECTIVE: The aim: To study possibilities of prognosis of pathological wear of tooth hard tissues development depending on the functional activity of masticatory muscles. PATIENTS AND METHODS: Materials and methods: In order to achieve the goal of the study, a survey of patients at the age of 19 to 69 years was conducted for precision of number of persons who have increased tooth wear. During the examination of all patients, three groups were selected: control and two researched ones. Patients in the control group (30 patients between the age of 18 and 60 years old) had an intact dentition without any visible sign of increased tooth wear. The researched groups included 25 patients aged from 18 to 60 years old. The secondresearch group included patients who had increased tooth wear of I-III level. The children of the patients of the II group with or without any sign of increased tooth wear were referred to the third research group. RESULTS: Results: The method of electromyography was performed for the study of peculiarities of the muscular activity of the maxillofacial area of the patients of the control and research groups. In our opinion, all manifestations of increased tooth wear are related to changes in the muscle system, the motor apparatus and the nervous activity of an organism. CONCLUSION: Conclusions: The study of direct muscular apparatus and related bone system may predict the possibility of developing of an increased tooth wear of young patients in the future and prevent its development, as well as to justify the application of a prevention and treatment plan of the studied pathology.

4-Methylamino antipyrine determination in human plasma by high-performance liquid chromatography tandem mass spectrometry.

In the present study, a simple and rapid method for metamizole metabolite 4-methylamino antipyrine (MAA) determination in human plasma was developed, validated and successfully applied to a clinical trial. Chromatographic separation was achieved in HILIC mode on a YMC-Pack SIL column (100 × 2.0 mm; S-5 µm, 30 nm), with a mobile phase consisting of acetonitrile, water and formic acid. Protein precipitation of a small plasma volume using acetonitrile was selected for sample preparation. The multiple reaction monitoring transitions in the positive ionization mode were m/z 218.2 → 56.2 for MAA and m/z 221.2 → 56.2 for MAA-d3 (IS, internal standard). Concentration levels of MAA calibration standards were in the range of 0.100-20 µg/ml. Metamizole conversion into MAA in both water and organic media was investigated, and the level of the conversion in commercially available injection solutions was estimated.

A percolation model of natural selection.

A new approach has been proposed and developed: the selection of optimal variants in the evolutionary mutation flow is considered as an analogue of a percolation filter. Interaction of mutations in a series of generations and random processes of drift determine the collective behavior of nodes (individuals - carriers and converters of mutations) and bonds (mutations) in the space of percolation lattice. It is shown that the choice of the development trajectory at the population level depends on the spectrum of supporting and prohibiting mutations under the influence of conjugate deterministic and random factors. From the point of view of the fluctuation-bifurcation process, new concepts of the lower and upper thresholds of the percolation selection grid are defined in the hierarchical structure of speciation. The upper threshold determines the state of self-organized criticality, which, when overcome, leads to irreversible self-organization processes in the population caused by the accumulation of mutations.

Changes in the functional condition of the masticatory muscles with increased teeth abrasion.

OBJECTIVE: Introduction: The term "pathological abrasion" (increased abrasion) can be described as a loss of tooth hard tissue on the occlusal, oral, vestibular surfaces of teeth or in the occlusal area. Treatment of increased abrasion of teeth is a difficult task for the practical work of a dentist. The choice of the optimal treatment plan provides the greatest likelihood of long-term success and minimal probability of complication appearance. The aim of our work was to study the functional activity of the masticatory muscles in the pathogenesis of the development of increased abrasion of tooth hard tissues. PATIENTS AND METHODS: Materials and methods: In order to achieve the goal of the study, an examination of patients aged between 19 and 69 years old was carried out in order to identify the number of persons with increased abrasion of teeth. During the examination of all patients one control and one researched group were formed. The patients of the control group (30 patients aged from 18 to 60 years) which had intact dental rows with any signs of increased tooth wear. The investigated group consisted of 25 patients, aged from 18 to 60 years. Depending on the complex treatment, the patients of the second group were divided into two subgroups. Subgroup 2A - (10 patients) with increased abrasion of tooth hard tissues with planned complex treatment of the disease without additional use of mouthguards after the end of treatment. For the patients from the 2B subgroup (15 people with increased abrasion of tooth hard tissues) a mouthguard was prescribed after the end of the complex treatment. In order to study some peculiarities of the muscular activity of the maxillofacial region an electromyogram was taken from all participants before treatment and repeated in a six- and twelve-months term. RESULTS: Results and conclusions: In our opinion, all manifestations of increased tooth abrasion are associated with changes in the muscular system, the motor apparatus and the nervous activity of the body. Regarding to this, the study of the propria muscular system and the related bone system can justify the usage of one or another prevention or treatment of the pathology. Based on the results of our research, we can conclude that the study of the functional state of the muscle complex is a fairly reliable prognostic sign in the study of such a disease as an increased tooth abrasion.

[Clinical and laboratory grounding of using medication based on the antiseptic troklosene to treat patients with chronic generalized periodontitis of the ii and iii degrees of severity].

OBJECTIVE: Introduction: Periodontal diseases take second place in the world among dental diseases and tend to be ubiquitous regardless of age, sex and place of residence. According to the modern point of view inflammatory periodontal diseases refer to infectious chronic inflammatory diseases, therefore, the normalization of the microflora of the oral cavity is an essential condition for their rational therapy. The aim: To increase the effectiveness of conservative treatment of chronic generalized periodontitis of the II-nd and III-rd severity using the medication based on the antiseptic Troklosene. PATIENTS AND METHODS: Materials and methods: 34 patients of age 45 to 55 years who were diagnosed with chronic generalized periodontitis of the IIInd and IIIrd degree of severity were examined and treated. All patients reported signs of the disease for 3-15 years. Patients of the study group by simple randomization were divided into two subgroups, depending on the therapeutic treatment performed. The I-st subgroup -16 patients treated with the additional use of the medication based on the antiseptic Troklosene, the IInd subgroup - 18 patients who were treated without using this drug. RESULTS: Results: Analyzing the results after the treatment in the patients of the I -st and II -nd subgroup, they noted a decrease in PMA index, Green-Vermillion index, bleeding index. Pathological mobility of teeth decreased to the I degree. The hygienic condition of the oral cavity was characterized in patients of both subgroups after treatment as satisfactory. In patients of the Ist subgroup (with the additional use of the medication based on the antiseptic Troklosene), after the 5th visit there were signs of remission, and in the IInd subgroup (using standard base therapy), after 7 visits, there were signs of clinical remission. With the additional use of the drug based on the antiseptic Troklosene, in contrast to the traditional therapy, a change in the bacterial ratio of microorganisms occurred, which influenced a significant decrease in the total bacterial mass. CONCLUSION: Conclusion: The best results were obtained in the Ist subgroup, where along with the standard base therapy, the supplement of based on the antiseptic Troklosene was used additionally. Introduction to the treatment regimen for patients with chronic generalized periodontitis with II and III severity of the drug based on the antiseptic Troklosene contributed to a stable clinical effect, as evidenced by the dynamics of clinical indicators and the reduction in the number of visits. Also, a change in the bacterial ratio of microorganisms with a significant decrease due to Enterobacterium spp. is noted. To maintain this result, it is recommended to continue the use of the drug based on the antiseptic Troklosene with a frequency of 4 times per day within 7 days after the end of treatment.

HPLC-MS/MS method for troventol determination in human plasma and its application to biological samples.

For the first time, an HPLC-MS/MS method for the determination pmol/l levels of troventol (TRV) and clenbuterol as an internal standard (IS) in human plasma was developed, validated and tested on biological samples. The method included solid phase extraction by Waters Oasis WCX cartridges and chromatographic separation on a YMC-Pack SIL (100mm×2.1mm, 5µm, 12nm) analytical column with acetonitrile-water-formic acid (50:50:0.1, v/v/v) as the mobile phase; the selected ion transitions were m/z 332.2→138.2 and m/z 277.0→203.1 for TRV and IS, respectively, in positive ionization mode. The calibration curve for TRV showed good linearity in the concentration range of 35-500pg/ml. The method was applied to real samples taken from healthy subjects after inhalation of an aerosol containing 640µg of TRV.

Development of a HPLC-MS/MS method for the simultaneous determination of nifedipine and lidocaine in human plasma.

The method for simultaneous determination of nifedipine (NIF) and lidocaine (LID) in human plasma by one-step sample preparation has been developed for the first time. Due to the photosensitivity of nifedipine and its low plasma concentrations a precise and reliable method was required. The method involved liquid-liquid extraction (methyl tert-butyl ether, MTBE), and 10µL of the resulting sample was analyzed by HPLC-MS/MS. Chromatographic separation was achieved on an YMC-Triart C18 HPLC column (100×2.0mm; S-5µm 12nm). The mobile phase was methanol:water, 60:40 (v/v) and contained 0.15% acetic acid. The linearity of the method was established in the concentration ranges of 0.5-50ng/mL for NIF and 1.0-500ng/mL for LID. Photodestruction of NIF under ambient light was evaluated. The validated method was successfully applied to analyze human plasma samples after rectal application of the drug (1g) containing 2.0% LID and 0.3% NIF.

In vitro and in vivo stability of oseltamivir within a bioequivalence trial.

A simple, precise, and rapid method to simultaneously determine the levels of oseltamivir (OS) and oseltamivir carboxylate (OSC) in human plasma was developed. Additionally, the stability of both substances in plasma was investigated under different conditions. The method involved protein precipitation (0.01 % HCl in acetonitrile), and then the supernatant was injected into the high-performance liquid chromatography (HPLC)-MS/MS. The chromatographic separation was achieved on a YMC-Triart C18 (100 × 2.0 mm, 5 µm) column using acetonitrile/water (30:70, v/v) containing 0.1 % formic acid as the mobile phase. Sample volume was 5 µl. The linearity of the method was established in the concentration range of 0.5-100 ng/mL for OS and 1.0-1000 ng/mL for OSC. The intra-day precision and accuracy for oseltamivir were 1.5-8.9 and 94.4-101.0 %, respectively. For oseltamivir carboxylate, the intra-day precision and accuracy were 3.2-12.7 and 92.8-108.8 %, respectively, whereas the inter-day precision and accuracy were 5.5-11.5 and 94.6-104.0 % for oseltamivir and 4.7-11.5 and 99.9-103.9 % for oseltamivir carboxylate, respectively. The application of this method was demonstrated by a bioequivalence study in 28 healthy humans with 75 mg oseltamivir phosphate capsules (Tamiflu®). Sodium fluoride (2.4 mg/mL) with potassium oxalate (3 mg/mL) was used as anticoagulant within sampling of trial. The assay reproducibility was established by reanalysis of 80 incurred samples.

Exploring bitterness of traditional Chinese medicine samples by potentiometric electronic tongue and by capillary electrophoresis and liquid chromatography coupled to UV detection.

Instrumental bitterness assessment of traditional Chinese herbal medicine (TCM) preparations was addressed in this study. Three different approaches were evaluated, high-performance liquid chromatography coupled to UV detector (HPLC), capillary electrophoresis coupled to UV detector (CE) and a potentiometric multisensor system - electronic tongue (ET). Most studies involving HPLC and CE separations use these as selective instruments for quantification of individual substances. However we employed these techniques to provide chromatographic or electrophoretic sample profiles. These profiles are somewhat analogous to the profiles produced by the ET. Profiles from all instruments were then related to professional sensory panel evaluations using projections on latent structures (PLS) regression. It was found that all three methods allow for bitterness assessment in TCM samples in terms of human sensory panel with root mean squared errors of prediction ca. 0.9 within bitterness scale from 0 (no bitterness) to 6 (maximal bitterness).

Two low-cost digital camera-based platforms for quantitative creatinine analysis in urine.

In clinical analysis creatinine is a routine biomarker for the assessment of renal and muscular dysfunctions. Although several techniques have been proposed for a fast and accurate quantification of creatinine in human serum or urine, most of them require expensive or complex apparatus, advanced sample preparation or skilled operators. To circumvent these issues, we propose two home-made platforms based on a CD Spectroscope (CDS) and Computer Screen Photo-assisted Technique (CSPT) for the rapid assessment of creatinine level in human urine. Both systems display a linear range (r(2) = 0.9967 and 0.9972, respectively) from 160 µmol L(-1) to 1.6 mmol L(-1) for standard creatinine solutions (n = 15) with respective detection limits of 89 µmol L(-1) and 111 µmol L(-1). Good repeatability was observed for intra-day (1.7-2.9%) and inter-day (3.6-6.5%) measurements evaluated on three consecutive days. The performance of CDS and CSPT was also validated in real human urine samples (n = 26) using capillary electrophoresis data as reference. Corresponding Partial Least-Squares (PLS) regression models provided for mean relative errors below 10% in creatinine quantification.

Determination of urine ionic composition with potentiometric multisensor system.

The ionic composition of urine is a good indicator of patient's general condition and allows for diagnostics of certain medical problems such as e.g., urolithiasis. Due to environmental factors and malnutrition the number of registered urinary tract cases continuously increases. Most of the methods currently used for urine analysis are expensive, quite laborious and require skilled personnel. The present work deals with feasibility study of potentiometric multisensor system of 18 ion-selective and cross-sensitive sensors as an analytical tool for determination of urine ionic composition. In total 136 samples from patients of Urolithiasis Laboratory and healthy people were analyzed by the multisensor system as well as by capillary electrophoresis as a reference method. Various chemometric approaches were implemented to relate the data from electrochemical measurements with the reference data. Logistic regression (LR) was applied for classification of samples into healthy and unhealthy producing reasonable misclassification rates. Projection on Latent Structures (PLS) regression was applied for quantitative analysis of ionic composition from potentiometric data. Mean relative errors of simultaneous prediction of sodium, potassium, ammonium, calcium, magnesium, chloride, sulfate, phosphate, urate and creatinine from multisensor system response were in the range 3-13% for independent test sets. This shows a good promise for development of a fast and inexpensive alternative method for urine analysis.

Development and validation of a LC-MS/MS method for D-cycloserine determination in human plasma for bioequivalence study.

A reliable and high throughput high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for determining levels of the antitubercular drug-D -cycloserine in human plasma. Plasma samples analyte with an internal standard (IS) (niacin) were prepared by solid-phase extraction using Waters Oasis MCX cartridges. The chromatographic separation was performed using the HILIC mode on a YMC-Pack SIL-06 column (150 × 4.6 mm; 3 µm) under isocratic conditions. The run time of analysis was 5 min. The mobile phase consisted of methanol, propanol-2 and 0.075 % trifluoroacetic acid (66.5:28.5:5, v/v/v). Protonated ions formed by turbo ion spray in positive mode were used to detect the analyte and the IS. MS/MS detection was used to monitor the fragmentation of 103-75 m/z for cycloserine and 124 to 80 m/z for niacin (IS) on an API 4000 (AB Sciex) triple quadrupole mass spectrometer. A linear dynamic range of 0.3-30 µg/mL was established for cycloserine using 0.2 mL human plasma and a 1 µL injection volume. The mean relative recovery of cycloserine and niacin were 77.2 and 82.4 %, respectively. The procedure of sample preparation was consistent and reproducible (precision, 0.8-3.4 %; accuracy, 93.8-104.9 %). The method was validated in accordance with requirements of the European Medicines Agency and successfully applied to a bioequivalence study of 250 mg tablet formulations in 23 healthy human subjects.