Исследовано влияние различных концентраций сивушного масла в составе 40 %-ного раствора этилового спирта на проявление синдрома отмены у крыс. Симптомы отмены этилового спирта регистрировали спустя 13, 15 и 17 ч после последнего введения крысам исследуемых растворов. В диапазоне концентраций сивушного масла от 0 до 1600 мг/дм3 острота симптоматики постинтоксикации снижается. При этих значениях снижается также концентрация ацетальдегида в крови животных. Методом корреляционного анализа (метод Спирмена) определена зависимость между концентрацией сивушного масла в исследуемых растворах и степенью проявления синдрома отмены этанола в диапазоне снижения значений синдрома отмены – от 0 до 1600 мг/дм3, что соответствует 1000–4000 мг/дм3 в пересчете на безводный спирт. Коэффициент корреляции составил 0,9, что соответствует высокой силе связи при отрицательной направленности и значимости корреляционной связи P<0,05. Коэффициент корреляции между содержанием ацетальдегида в крови животных и усредненными значениями проявления отмены этилового спирта за весь период наблюдения составил 0,66, что является существенным уровнем связи. При дальнейшем возрастании концентрации сивушного масла возрастают проявление синдрома отмены и концентрация ацетальдегида в крови крыс. Полученные результаты соответствуют сведениям о способности сивушного масла снижать остроту проявлений сидрома отмены этанола. Это происходит за счет конкуренции поступления этилового спирта и сивушного масла в кровеносное русло и, соответственно, в печень. Сивушное масло выступает в роли ингибитора всасывания этилового спирта, следовательно, продлевается состояние опьянения, а образование ацетальдегида и проявление похмелья замедляются. При этом полученные результаты относятся строго к условиям настоящего исследования – различные концентрации сивушного масла вносились в 40 %-ный раствор этилового спирта. При замене раствора этилового спирта на какой-либо напиток, полученный методом дистилляции, например виски, результаты могут отличаться ввиду его многокомпонентности. The effect of various concentrations of fusel oil in the composition of a 40 % solution of ethyl alcohol on the manifestation of withdrawal syndrome in rats was studied. Ethyl alcohol withdrawal symptoms were recorded 13, 15 and 17 hours after the last administration of the test solutions to rats. In the concentration range of fusel oil from 0 to 1600 mg/dm3, the severity of postintoxication symptoms decreases. At these values, the concentration of acetaldehyde in the blood of animals also decreases. The correlation analysis method (Spearman method) determined the relationship between the concentration of fusel oil in the studied solutions and the degree of manifestation of the ethanol withdrawal syndrome in the range of decreasing withdrawal syndrome values – from 0 to 1600 mg/dm3, which corresponds to 1000–4000 mg/dm3 in terms of anhydrous alcohol. The correlation coefficient was 0.9, which corresponds to a high strength of the connection with a negative direction and the significance of the correlation P<0.05. The correlation coefficient between the content of acetaldehyde in the blood of animals and the average values of the manifestation of ethyl alcohol withdrawal for the entire observation period was 0.66, which is a significant level of connection. With a further increase in the concentration of fusel oil, the manifestation of the withdrawal syndrome and the concentration of acetaldehyde in the blood of rats increase. The obtained results are consistent with the information on the ability of fusel oil to reduce the severity of manifestations of ethanol withdrawal syndrome. This occurs due to the competition between the entry of ethyl alcohol and fusel oil into the bloodstream and, accordingly, into the liver. Fusel oil acts as an inhibitor of the absorption of ethyl alcohol, therefore, the state of intoxication is prolonged, and the formation of acetaldehyde and the manifestation of a hangover slows down. At the same time, the results obtained relate strictly to the conditions of this study – various concentrations of fusel oil were introduced into a 40 % solution of ethyl alcohol. When replacing an ethyl alcohol solution with a distilled drink, such as whiskey, the results may differ due to its multicomponent nature.

Technological advances in Next-Generation Sequencing dramatically increased clinical efficiency of genetic testing, allowing detection of a wide variety of variants, from single nucleotide events to large structural aberrations. Whole Genome Sequencing (WGS) has allowed exploration of areas of the genome that might not have been targeted by other approaches, such as intergenic regions. A single technique detecting all genetic variants at once is intended to expedite the diagnostic process while making it more comprehensive and efficient. Nevertheless, there are still several shortcomings that cannot be effectively addressed by short read sequencing, such as determination of the precise size of short tandem repeat (STR) expansions, phasing of potentially compound recessive variants, resolution of some structural variants and exact determination of their boundaries, etc. Therefore, in some cases variants can only be tentatively detected by short reads sequencing and require orthogonal confirmation, particularly for clinical reporting purposes. Moreover, certain regulatory authorities, for example, New York state CLIA, require orthogonal confirmation of every reportable variant. Such orthogonal confirmations often involve numerous different techniques, not necessarily available in the same laboratory and not always performed in an expedited manner, thus negating the advantages of "one-technique-for-all" approach, and making the process lengthy, prone to logistical and analytical faults, and financially inefficient. Fortunately, those weak spots of short read sequencing can be compensated by long read technology that have comparable or better detection of some types of variants while lacking the mentioned above limitations of short read sequencing. At Variantyx we have developed an integrated clinical genetic testing approach, augmenting short read WGS-based variant detection with Oxford Nanopore Technologies (ONT) long read sequencing, providing simultaneous orthogonal confirmation of all types of variants with the additional benefit of improved identification of exact size and position of the detected aberrations. The validation study of this augmented test has demonstrated that Oxford Nanopore Technologies sequencing can efficiently verify multiple types of reportable variants, thus ensuring highly reliable detection and a quick turnaround time for WGS-based clinical genetic testing.

Supplementary Figure 1 - Maspin expression in PTEN cells

Supplementary Figure 3 - Docetexel and Salinomycin effect on normal prostate cells

Supplementary Figure 2 - Maspin expression modulates HDAC enzymatic activity

<p>Table 1S - Sequence of Real-Time PCR Primers</p>

Investigation of the antioxidant activity (AOA) induction by selenium compounds (Na2SeO3, diacetophenonyl selenide (DAPS-25), L-selenocystine) in various organisms is interesting as a protection from stress induced cell membranes damages. A comparative analysis of the antioxidant activity of 23 amino acids was performed by coulometric titration with electrogenerated bromine and iodine. The activity decreases in the order: cystine tryptophan selenocystine tyrosine 3,3'-dimethyl-L-selenocystine methionine. Only amino acids with sulfhydryl and selenol groups as more active reductants can interact with the electrogenerated iodine: cysteine selenocysteine threo-3-methyl-L-selenocysteine. Probably the correction of the antioxidant status at the amino acids level is based on the sulfhydryl and selenol groups in radicals. In case they are not enough, cystine, selenocystin, tryptophan, tyrosine, and methionine will act as scavenger-reductants. It was found that selenium compounds dose-dependently induce the total antioxidant activity of the A. niger mycelium and affect indicators of the antioxidant status (amino acid composition and catalase activity), which in turn stimulates the biomass accumulation. DAPS-25 and sodium selenite treatment at the high doses (0.025 mg Se/L) caused the greatest effect on the total AOA induction (3.4–5.5 times). Lower concentrations (0.0025–0.00025 mg Se/L) had a lesser effect (25.8–41.7%). Activity in samples with L-selenocystin increased by 1.6–43.3%. It is noted that the iodine antioxidant activity in the mycelium was generally lower than the bromine one.

Sulfur and selenium containing amino acids - enantiomers of cysteine and selenocystine,-are of increasing interest as active centers of enzymes, antioxidants and starting compounds in peptide synthesis. The paper presents a convenient synthesis of cystine and selenocystine enantiomers from serine and threonine and also new enantiomers of selenocystine: 3,3 '-dimethyl-L-selenocystine; 3,3'-dimethyl-D-selenocystine. Commercially available L and D isomers of the amino acids serine and threonine, as well as their racemic mixtures, were used as starting compounds. A direct synthesis of sulfur and selenium containing amino acids from hydroxyl containing amino acids is energetically unprofitable but may be achieved via intermediate halogen containing amino acids: beta-chloro-L-alanine, beta-chloor-D-alanine, and also (alpha S, beta R)-alpha-amino-beta-chlorobutiric and (alpha R, beta S)-alpha-amino-beta-chlorobutiric acid Cystine enanthiomers synthesis was carried out in ethanol via reduction of elemental sulfur to Na2S by borohydride and sulfur attachment to form Na2S2, and also alkaline solution of beta-chloralanine. Synthesis of selenocystine and 3,3'-dimethylselenocystine enanthiomers was carried out in water solution, using a reduction of elemental selenium by sodium borohydride with a formation of Na2Se and selenium attachment to form Na2Se2, and also alkaline solution of beta-chloralanine or alpha-amino-beta-chlorobutiric acid NMR spectra was obtained on spectrophotometer Varian 400, with instrument operating frequencies 400 and 100 MHz, respectively. Amino acids were dissolved in a mixture of D2O and 60 mu L 50 % ortho-phosphoric acid. Elemental analysis of compounds was carried out on Vario El cube. Selenocystine and 3,3'-dimethylselenocystibe L and D enanthiomers may serve as conveniate starting compounds for the synthesis of appropriate methylated derivatives: L and D - methylselenocysteine and 2-amino-3-(methylselanyl)butyric acid. The advantages of the developed synthesis of selenium containing amino acids enanthiomers include the availability of the starting compounds and reagents, their low cost and low energetic profitability, high yields, and the possibility to synthetize concrete enanthiomer of selenium containing amino acids, simplicity of purification (filtration), storage stability, relatively uncomplicated equipment used All these facts allow to substantiate the feasibility of industrial implementation of the process.

Objectives. While organic and inorganic derivatives of selenium like thiol poisons are known to activate enzymes in cells of different organisms, the mechanism of enzyme activity induction is poorly studied. Therefore, the aim of the study was to investigate the effect of selenium compounds on peroxidase activity induction in maize tissues.Methods. Mechanism of sulfhydryl groups blocking in selenium derivatives was studied on maize in comparison with fungicide tolylfluanid—a typical thiol poison. Electrolytes leakage was determined using conductometry and capillary electrophoresis, protein fractions—by the Ermakov–Durinina method, protein concentration—according to Bradford protein essay, and peroxidase activity—by the Boyarkin method.Results. Diacetophenolylselenide (DAPS-25) was shown to react with SH-groups similarly with tolylfluanid fungicide. DAPS-25 increased K+ and leakage by 58 and 14 times, while appropriate increases for tolylfluanid were 4.4 and 1.5 times as compared to control. Increased total protein content—especially albumins—was due to electrolyte leakage from maize cells. DAPS-25 increased albumins concentration by 2.4–4.5 times, and tolylfluanid application by 2 times. Similar increase of peroxidase activity in maize roots and sprouts as a result of DAPS-25 (by 63% and 112%) and tolylfluanid (by 73% and 63%) application indicates close mechanism of their effect. Under DAPS-25 loading L-cysteine decreases peroxidase activity, which records the removal of SH-groups blockage. A less intensive effect was registered for sodium selenite and L-selenocystin, also capable of reacting with SH-groups. L-cysteine supplementation to DAPS-25 solution decreases selenium concentration in maize, indicating the decrease of selenium bioavailability.Conclusions. The results indicated that selenium containing compounds react with SH-groups of maize cells increasing electrolytes leakage, protein content and especially albumins resulting in the increase of peroxidase activity.

BACKGROUND:The clinical characteristics of symptomatic and asymptomatic carriers of early- onset autosomal dominant Alzheimer's (EOADAD) due to a yet-undescribed chromosomal rearrangement may add to the available body of knowledge about Alzheimer's disease and may enlighten novel and modifier genes. We report the clinical and genetic characteristics of asymptomatic and symptomatic individuals carrying a novel APP duplication rearrangement. METHODS:Individuals belonging to a seven-generation pedigree with familial cognitive decline or intracerebral hemorrhages were recruited. Participants underwent medical, neurological, and neuropsychological evaluations. The genetic analysis included chromosomal microarray, Karyotype, fluorescence in situ hybridization, and whole genome sequencing. RESULTS:Of 68 individuals, six females presented with dementia, and four males presented with intracerebral hemorrhage. Of these, nine were found to carry Chromosome 21 copy number gain (chr21:27,224,097-27,871,284, GRCh37/hg19) including the APP locus (APP-dup). In seven, Chromosome 5 copy number gain (Chr5: 24,786,234-29,446,070, GRCh37/hg19) (Chr5-CNG) cosegregated with the APP-dup. Both duplications co-localized to chromosome 18q21.1 and segregated in 25 pre-symptomatic carriers. Compared to non-carriers, asymptomatic carriers manifested cognitive decline in their mid-thirties. A third of the affected individuals carried a diagnosis of a dis-immune condition. CONCLUSION:APP extra dosage, even in isolation and when located outside chromosome 21, is pathogenic. The clinical presentation of APP duplication varies and may be gender specific, i.e., ICH in males and cognitive-behavioral deterioration in females. The association with immune disorders is presently unclear but may prove relevant. The implication of Chr5-CNG co-segregation and the surrounding chromosome 18 genetic sequence needs further clarification.

Objectives. To develop a method for the microfluidic synthesis of oligohexamethylene guanidine salts in a flow-type reactor and to evaluate its effectiveness in relation to the synthesis in a traditional capacitive reactor and compare the purities of products obtained by these methods.Methods. The synthesis of oligohexamethylene guanidine bihydrocarbonate (OHMG-BHC) was done using microfluidic hardware and the classical approach in volume. The purity and structure of the resulting product were confirmed by 13C NMR spectroscopy and high-performance liquid chromatography (HPLC).Results. The 13C NMR spectrum of OHMG-BHC in classical bulk synthesis demonstrates that the product is unbranched and contains additionally unidentifiable impurities, in contrast to the sample obtained by the microfluidic method. Furthermore, the HPLC analysis showed that the OHMG-BHC sample synthesized using microfluidic technology has a 1.5-fold lower content than the initial monomers.Conclusions. The advantage of synthesizing OHMG-BHC in a flow-type reactor compared to the traditional method of synthesis in volume is demonstrated since a product with a higher degree of purity is obtained.

Autism spectrum disorder (ASD) is a heterogeneous condition with a complex genetic etiology. The objective of this study is to identify the complex genetic factors that underlie the ASD phenotype and other clinical features of Professor Temple Grandin, an animal scientist and woman with high-functioning ASD. Identifying the underlying genetic cause for ASD can impact medical management, personalize services and treatment, and uncover other medical risks that are associated with the genetic diagnosis. Prof. Grandin underwent chromosomal microarray analysis, whole exome sequencing, and whole genome sequencing, as well as a comprehensive clinical and family history intake. The raw data were analyzed in order to identify possible genotype-phenotype correlations. Genetic testing identified variants in three genes (SHANK2, ALX1, and RELN) that are candidate risk factors for ASD. We identified variants in MEFV and WNT10A, reported to be disease-associated in previous studies, which are likely to contribute to some of her additional clinical features. Moreover, candidate variants in genes encoding metabolic enzymes and transporters were identified, some of which suggest potential therapies. This case report describes the genomic findings in Prof. Grandin and it serves as an example to discuss state-of-the-art clinical diagnostics for individuals with ASD, as well as the medical, logistical, and economic hurdles that are involved in clinical genetic testing for an individual on the autism spectrum.

Inactivated (whole-virion, split, subunit, and adjuvanted) vaccines and live attenuated vaccine were tested in parallel to compare their immunogenicity and protective efficacy. Homologous and heterosubtypic protection against the challenge with influenza H5N1 and H1N1 viruses in a mouse model were studied. Single immunization with live or inactivated whole-virion H5N1 vaccine elicited a high level of serum antibodies and provided complete protection against the challenge with the lethal A/Chicken/Kurgan/3/05 (H5N1) virus, whereas application of a single dose of the split vaccine was much less effective. Adjuvants increased the antibody levels. Addition of the Iso-SANP adjuvant to the split vaccine led to a paradoxical outcome: it increased the antibody levels but reduced the protective effect of the vaccine. All tested adjuvants shifted the ratio between IgG1 and IgG2a antibodies. Immunization with any of the tested heterosubtypic live viruses provided partial protection against the H5N1 challenge and significantly reduced mouse mortality, while inactivated H1N1 vaccine offered no protection at all. More severe course of illness and earlier death were observed in mice after immunization with adjuvanted subunit vaccines followed by the challenge with the heterosubtypic virus compared to challenged unvaccinated animals.

Alimentary atherosclerosis is associated with a significant decrease in the content of phosphatidylcholine, the phospholipid that provides antioxidant protection, in the plasma membrane of liver cells, while the level of phosphatidic acid that initiates generation of superoxides, on the contrary, increases. The level of membrane phosphatidylserine, a target of the scavenger receptors, which initiates removal of damaged cells and modified lipoproteins from the circulation was also elevated. In the blood serum of rabbits receiving an atherogenic diet, the content of cardiolipin involved in the immune mechanisms of atherosclerosis development and a risk factor for thrombosis, sharply increased. The level of lysophosphatidylcholine that mediates initiation and progression of atherosclerosis increased. The content of phosphatidylinositol that is involved in the mechanisms protecting from exposure to excess cholesterol was significantly reduced. Treatment of alimentary atherosclerosis with “empty” phosphatidylcholine nanosomes eliminates the key factors initiating atherosclerosis development.

The physicochemical parameters of spherical amorphous nanoparticles (SANPs), consisting of a mixture of triterpenoids of birch bark (MTB) with 20-hydroxyecdysone (20E), are studied. The nanoparticles are obtained by injection and investigated by electron microscopy, dynamic light scattering, and UV spectroscopy. In the studied series of concentrations of 20E from 5 to 20 wt %, in relation to the mass of the MTB, the optimal concentration is 20E 10 wt %; at this concentration, the most stable nanodispersions are formed in water, 20E is fully included in the SANPs, their average size is 193.1 nm, they are characterized by a narrow polydispersity interval, and they have the greatest absolute value of the ζ-potential. Ecdysteroid-containing SANPs of this composition can be recommended for use in medicine as prolonged forms of ecdysteroids as radioprotective and other pharmacological agents.

Triterpenoids from birch bark are valuable biologically active substances. They exhibit important biological properties. However, triterpenoids are insoluble in water due to their hydrophobicity. In order to increase their bioavailability we developed a technique for obtaining spherical amorphous nanoparticles (SANP) by their precipitation with water from a solution in THF. In the form of nanoparticles, triterpenoids show additional useful properties: they can be loaded with hydrophobic substances. SANP were also shown to be effective immunological adjuvants. This article is devoted to the study of conditions that could optimize their production. As a result, it was shown that among the three investigated solvents miscible with water - acetone, dioxane, THF -the latter gives the best results. Lowering temperature decreases the size of SANP. An increased temperature leads to the formation of betulin crystals. When the concentration of triterpenoids in THF is higher than 5 mg/ml, crystals of betulin also begin to form. The results of this study lead to the assumption that the main parameter of the process determining the formation of SANP is the rate of the solvent diffusion into water.

BACKGROUND:With the continuing decrease in cost of whole genome sequencing (WGS), we have already approached the point of inflection where WGS testing has become economically feasible, facilitating broader access to the benefits that are helping to define WGS as the new diagnostic standard. WGS provides unique opportunities for detection of structural variants; however, such analyses, despite being recognized by the research community, have not previously made their way into routine clinical practice.RESULTS:We have developed a clinically validated pipeline for highly specific and sensitive detection of structural variants basing on 30X PCR-free WGS. Using a combination of breakpoint analysis of split and discordant reads, and read depth analysis, the pipeline identifies structural variants down to single base pair resolution. False positives are minimized using calculations for loss of heterozygosity and bi-modal heterozygous variant allele frequencies to enhance heterozygous deletion and duplication detection respectively. Compound and potential compound combinations of structural variants and small sequence changes are automatically detected. To facilitate clinical interpretation, identified variants are annotated with phenotype information derived from HGMD Professional and population allele frequencies derived from public and Variantyx allele frequency databases. Single base pair resolution enables easy visual inspection of potentially causal variants using the IGV genome browser as well as easy biochemical validation via PCR. Analytical and clinical sensitivity and specificity of the pipeline has been validated using analysis of Genome in a Bottle reference genomes and known positive samples confirmed by orthogonal sequencing technologies.CONCLUSION:Consistent read depth of PCR-free WGS enables reliable detection of structural variants of any size. Annotation both on gene and variant level allows clinicians to match reported patient phenotype with detected variants and confidently report causative finding in all clinical cases used for validation.