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Alternative glycosylation of immunoglobulin G (IgG) is functionally important in multiple human physiological and pathological states. Our understanding of molecular mechanisms that regulate IgG glycosylation is vague because of the complexity of this process, which involves hundreds of genes. Several genome-wide association (GWA) studies have revealed a network of genes associated with IgG glycosylation that are pleiotropic for a number of diseases. Here, we report a design of a versatile system for IgG production and gene manipulations that can be used for in vitro functional follow-up of GWA hits or any gene of interest. The system is based on CRISPR-dCas9, extended by a piggyBac integrase compatible vector, and drives IgG production in HEK-293F cells. We validated our systems that stably express VPR-dCas9 and KRAB-dCas9 by manipulation of four glyco-genes with a known role in IgG glycosylation, and then functionally validated three GWAS hits for IgG glycosylation with an as-yet-unknown role in this process.

The aim of this study was to develop antimicrobial nanocomposite textile material comprising of Cu-based nanostructures synthesized on oxidized cotton fabric using Arctostaphylos uva-ursi (L.) Spreng., Ericaceae (bearberry leaves) as a green reducing agent for adsorbed Cu2+-ions. In order to provide sufficient number of carboxyl groups for complexation with Cu2+-ions a two-step oxidation process with NaIO4 and NaClO2 was carried out. The influence of NaIO4 concentration on content of carboxyl groups and Cu-based nanoparticles was studied by FTIR and AAS. HPLC analysis identified the gallic acid known as a reducing agent in bearberry leaves extract. FESEM and XRD analyses revealed that using bearberry leaves extract and gallic acid solution as reducing agents led to a formation of spherical Cu2O/CuO nanoparticles and CuO nanosheets, respectively. These nanoparticles and nanosheets provided excellent antibacterial activity against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus. Cytotoxicity on human keratinocyte cells was shown to depend on their copper content.

Seven bis(2-picolyl)amine (bpa) and five iminodiacetamide (imda) ligands were prepared with different modifications in their side chain structure. The coordination properties of the ligands (L) were influenced by changes in the aliphatic linker length (C1, C2, or C3), amide group isomers and type of chiral terminal group. Complexation with Cu(II) afforded two polymorphs of a ML complex which features tetradentate coordination of a ligand with C2 linkers, while crystal structures of three trans–fac ML2 complexes with Cu (II) and Ni(II) show tridentate coordination of ligands with a C3 linker. The stoichiometry and stereochemistry of Zn(II) and Cu(II) complexes was further studied in solution by NMR and UV-Vis spectroscopy. DFT calculations gave an insight into the relative stability of isomers, as well as potential hydrogen bonding between two ligands in a ML2 complex. Furthermore, ML complexes of Cu(II) exhibited DNA cleavage activity.

The demand for medical textiles in various forms with strong antimicrobial activity drastically increased during the COVID19 pandemic. In an attempt to tackle this issue and to develop antimicrobial textiles in more environmentally benign manner, a viscose fabric after coating with biopolymer chitosan has been impregnated with Ag- and Cu-based nanoparticles. Chitosan was applied in the presence and absence of cross-linker 1,2,3,4-butanetetracarboxylic acid (BTCA). In situ green synthesis of nanoparticles was performed using a Punica granatum (pomegranate) peel extract as a reducing and stabilizing agent. Formation of nanoparticles on the fiber surface was confirmed by FESEM. Elemental analysis by XPS showed the synthesized nanoparticles exist as AgCl and a mixture of Cu/CuO/Cu2O in the modified samples. Moreover, these nanoparticles appeared to be present not only on the sample surface but also buried within the fibers, as indicated by XPS mapping and depth profiling measurements. All impregnated fabrics exhibited excellent antifungal activity providing the maximum reduction of yeast Candida albicans colonies. Antibacterial activity was stronger against Gram-negative bacteria Escherichia coli than Gram-positive bacteria Staphylococcus aureus, and it was highly influenced by metal content. The fabrics impregnated with AgCl nanoparticles showed lower cytotoxicity towards human keratinocyte cells.

Wild apple fruit is rich source of bioactive substances, which can be potentially used for a production of dermocosmetic products.The aim of study was in vitro and in vivo investigation of standardized wild apple fruit extracts (obtained by purified water and maceration-EM, percolation-EP, Soxhlet extraction-ES and ultrasonic extraction-EU) and dermocosmetic creams of o/w type containing 6%, 12%, and 15% of extract EU, as extract with the best content of polyphenolic compounds (CE6, CE12, and CE15, respectively), in order to determine the optimal concentration of the extract, which manifests the best effect on the biophysical characteristics of the skin after application.Content of polyphenolic substances-PP was investigated using HPLC analysis, while in vitro biological activity was examined on L929 fibroblasts using MTT viability test. In vivo efficacy included screening and comparison of skin hydration potential, transepidermal water loss-TEWL, and skin pH after 28 days of creams application on healthy volunteers' skin.Content of identified PP was the highest in the extract EU (2001.57 ± 0.87 mg PP/100 g EU) and satisfactory in cream CE6 (53.83 ± 0.23 mg PP/100 g CE6). Procyanidin B2 was the most common PP. Biological activity analysis showed that extract EU could be considered as non-cytotoxic for fibroblasts in examined concentrations. In vivo investigation revealed an increase of skin hydration (after 28 days ΔEC was 21.19 ± 7.59, 26.71 ± 7.94, and 29.60 ± 10.95 for CE6, CE12, and CE15, respectively), with unchanged TEWL and skin pH values.Obtained results indicate that wild apple fruit extract represents a valuable source of bioactive substances, with good hydration effects of creams on human skin (effect of creams increased proportionally to the increase of concentration of incorporated extract), so it might be considered as a great potential for application in dermocosmetic industry.

Immortelle, as rich source of chlorogenic acid and the phloroglucinol alpha-pyrone compound arzanol, possesses anti-inflammatory and antioxidant properties, affects cell regeneration, and has positive effect on many skin conditions. Hemp oil, characterized by a favorable omega-6 to omega-3 ratio, as well as an abundance of essential fatty acids and vitamin E, participates in immunoregulation and also act as an anti-inflammatory. In the present study, we examined the effect on the skin of creams and emulgels with immortelle extract and hemp oil, by comparing them to placebo samples and a non-treated control. A long-term in vivo study of biophysical skin characteristics, which lasted for 30 days, was conducted on 25 healthy human volunteers. Measured parameters were electrical capacitance of the stratum corneum, trans-epidermal water loss (TEWL), and skin pH and erythema index. Further, a sensory study was carried out in which the panelists had to choose descriptive terms for sensory attributes in questionnaire. The results showed that application of all preparations led to increase of skin hydration and TEWL reduction, while the skin was not irritated, and its normal pH was not disrupted. This study also showed importance of the carrier. Not only were emulgels described by panelists as preparations with better sensory properties, there was a significant difference between the skin hydration effect of emulgel with immortelle extract and hemp oil compared to the placebo emulgel, which was not the case with creams. Such findings indicated enhanced delivery of herbal active substances from emulgel compared to the cream.

Supercritical CO2 extraction was applied on fenugreek (Trigonella foenum-graecum L., Fabaceae) seeds with the aim to define optimal process conditions to obtain the maximal extract yields content of steroidal sapogenins. Central composite rotatable design (CCRD) combined with response surface methodology (RSM) was applied to determine optimal process conditions defined by the influence and interaction of pressure, temperature and time of extraction through consumption of SC CO2. Optimization experiments revealed that the pressure of 24.73 MPa, the temperature of 38.°C and the consumption of SC CO2 of 19.24 g/gDM were the best process conditions enabling the maximal yield of extract and gain of the highest content of sapogenins. The optimal values of pressure and temperature defined SC CO2 density of 885.47 kg/m3, which provided the maximal yield of the extract with the highest content of steroidal sapogenins. The achieved yield of extract at these conditions was 0.073g/gDM, with 0.774 mg/gDM diosgenin, 0.477 mg/gDM protodioscin, 0.713 mg/gDM sarsapogenin and 0.205 mg/gDM oleanolic and ursolic acid with a significant quantity of 2.475 mg/gDM in the obtained extract. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. III45017]

Tumour cells develop by accumulating changes in the genome that result in changes of main cellular processes. Aberrations of basic processes such as replication and chromatin reassembly are particularly important for genomic (in)stability. The aim of this study was to analyse the expression of genes whose products are crucial for the regulation of replication and chromatin reassembly during lymphomagenesis (DNMT1, PCNA, MCM2, CDT1, EZH2, GMNN, EP300). Non-tumour B cells were used as a control, and follicular lymphoma (FL) and the two most common groups of diffuse large B cell lymphoma (DLBCL) samples were used as a model for tumour progression. The results showed that there are significant changes in the expression of the analysed genes in lymphomagenesis, but also that these changes do not display linearity when assessed in relation to the degree of tumour aggression. Additionally, an integrated bioinformatics analysis of the difference in the expression of selected genes between tumour and non-tumour samples, and between tumour samples (FL vs. DLBCL) in five GEO datasets, did not show a consistent pattern of difference among the datasets.

Duloxetine hydrochloride (DUL) is effective in treating depression, and was examined for electroanalytical purposes. The DUL standard was investigated by cyclic voltammetry (CV) and determined using differential pulse voltammetry (DPV) via its electro-oxidation at the Au electrode in 0.05 M NaHCO3. DPV showed a linear dependency of the anodic peak currents vs DUL standard concentrations in the range from 0.1 to 3.33 μg ml−1 with the values of the limit of detection (LOD) and the limit of quantification (LOQ): 0.133 and 0.667 μg ml−1, respectively. Using the constructed and validated calibration curve, the values of unknown DUL concentrations in both Taita® tablets and in human serum spiked with the standard were determined. The number of protons and electrons transferred was calculated and possible reaction mechanisms taking place on the surface of both electrodes were proposed. The Au electrode exhibited a better sensitivity and a wider range of current vs concentration linear dependency for DUL quantitative determination than the GC electrode. The study of DUL degradation showed that at the Au and GC electrodes, after 4.5 h of potential cycling, degradation occurs, giving formaldehyde as a product, which was confirmed by high performance liquid chromatography (HPLC).

Bilberry represents a valuable source of antioxidant substances responsible for its application for the treatment of different conditions (such as inflammation, cardiovascular disease, cancer, diabetes, and different age-related diseases) associated with increased oxidative stress. As oxidative stress might cause skin impairments, we aim to evaluate a topical preparation containing bilberry leaves extract and bilberry seeds oil, obtained as a byproduct of the food industry. To obtain the extracts, the conventional maceration technique for leaves, and supercritical carbon dioxide extraction for seeds were employed. The chemical profile of both actives was achieved by HPLC and GC methods, revealing the presence of phenolic acids (chlorogenic being the most abundant), flavonoids (isoquercetin in the highest amount), and resveratrol in leaves extract, while in seeds oil the essential ω-3 and ω-6 fatty acids were determined in favorable ratio, almost being 1. Antioxidant potential of the wild bilberry extract and seed oil was evaluated using in vitro DPPH and FRAP assays. Finally, effects of the oil-in-water creams with mentioned wild bilberry isolates on the skin were investigated in an in vivo study conducted on healthy human volunteers, revealing the significant beneficial effects when topically applied.

Background This study provides an insight into the impact of ultrasound-assisted extraction with water as solvent (UAEW) and extraction by supercritical carbon dioxide (SC-CO2 ) with 5% EtOH on antioxidant and enzyme inhibitory activity in regard to the chemical profile of the edible and medicinal mushroom, Pleurotus pulmonarius. Results Extraction efficiency was between 0.36% and 63.32%, depending on the extraction technique. The main compounds in the extracts were fatty acids. Supercritical CO2 extraction with co-solvent was the most suitable method for obtaining extracts that were rich in ergosterol content, reaching a value of 40.1 mg g-1 . The UAEW of crude mushroom powder ensured the highest yield, as well as the extracts with best antioxidative activity. The measurements of enzyme inhibitory activity revealed that all types of investigated extracts exhibited only tyrosinase and amylase inhibition at a significant level. Conclusion Based on our results, the extraction methods significantly affected the chemical profile and bioactivity of P. pulmonarius. © 2020 Society of Chemical Industry.

This work aimed to obtain a validated model for the prediction of retention times of compounds isolated from Origanum heracleoticum, Origanum vulgare, Thymus vulgaris, and Thymus serpyllum essential oils. In total 68 experimentally obtained retention times of compounds, which were separated and detected by GC-MS were further used to build the prediction models. The quantitative structure–retention relationship was employed to foresee the Kovats retention indices of compounds acquired by GC-MS analysis, using eight molecular descriptors selected by a genetic algorithm. The chosen descriptors were used as inputs for the four artificial neural networks, to construct a Kovats retention indices predictive quantitative structure–retention relationship model. The coefficients of determination in the training cycle were 0.830; 0.852; 0.922 and 0.815 (for compounds found in O. heracleoticum, O. vulgare, T. vulgaris and T. serpyllum essential oils, respectively), demonstrating that these models could be used for prediction of Kovats retention indices, due to low prediction error and high r2. Resumen. El objetivo de este trabajo es la obtención de modelos validados para la predicción del tiempo de retención de los compuestos aislados de aceites esenciales de Origanum heracleoticum, Origanum vulgare, Thymus vulgaris y Thymus serpyllum. Se han obtenido un total de 68 tiempos de retención de compuestos, separándose y detectándose por cromatografía de gases con detección por espectrometría de masas (GC-MS) con posterior desarrollo de modelos de predicción. La relación cuantitativa estructura-retención ha sido utilizada para predecir el índice de retención Kovats de los compuestos obtenidos por análisis de GC-MS, utilizando ocho descriptores moleculares seleccionados mediante algoritmo genético. Los descriptores seleccionados han sido utilizados como entrada para las cuatro redes neuronales artificiales y así elaborar los índices predictivos del modelo de relación cuantitativa estructura-retención. Los coeficientes de determinación en el ciclo de entrenamiento fueron de 0.830; 0.852; 0.922 y 0.815 (para los compuestos identificados en los aceites esenciales del O. heracleoticum, O. vulgare, T. vulgaris y T. serpyllum respectivamente) demostrando así que estos modelos son útiles en la predicción de los índices de retención de Kovats con un error de bajo predicción y alta r2.

An experimental investigation on essential oil of a Montenegrin Helichrysum italicum (Roth) G. Don fil. (Asteraceae) is reported. The essential oil was analysed in both liquid and vapour phases. Gas chromatography-mass spectrometry analysis of the liquid oil showed the predominance of sesquiterpenes with β-eudesmene (21.65%) and β-bisabolene (19.90%) as the major ones. Monoterpene fraction was mainly represented by α-pinene (16.90%) and neryl acetate (10.66%). Head-space technique revealed the vapour phase enriched of monoterpenes with α-pinene (78.76%) predominance. The essential oil was tested against methicillin-sensitive Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Candida albicans (ATCC 14053) and the clinical strains of methicillin-resistant S. aureus, carbapenem-resistant Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas aeruginosa. Interesting fungicidal/bactericidal potency against C. albicans and carbapenem-resistant A. baumannii was revealed at concentration of 5% v/v.

Supercritical CO2 impregnation process was used for the first time to fabricate biodegradable gelatin-chitosan (G/Ch) films containing clove oil (CO) for potential use in active food packaging. All the impregnations were carried out at moderately low temperature (40°C). Aiming to maximize CO loading in the films with acceptable morphological, structural and thermal properties, CO2 pressure, impregnation time and G:Ch mass ratio were varied. Gelatin fraction in the films of 25-50 wt.%, scCO2 pressures of 10-20 MPa and impregnation time of 2-6 h impregnation favoured the CO loading. Processing of the film with equal gelatin to chitosan mass ratio (G/Ch50:50) at 10 MPa for 2 h yielded sufficiently high loading (56 mg CO/gfilm) without an adverse effect on morphological properties. The G/Ch50:50 film was therefore chosen for structural and thermal analyses. ATR-FTIR analysis confirmed successful CO incorporation into the G/Ch50:50 and its interaction with the film. Plasticizing effect of scCO2 and CO on the film was evidenced by DSC. Incorporation of 56 mg CO/gfilm into the G/Ch50:50 didn''t affect thermal stability of the film. Beside environmental benefits, supercritical impregnation process enables fast fabrication of G/Ch bio-composite films containing CO, thermally stable to 110°C, which is suitable for most of food packaging applications. [Projects of the Serbian Ministry of Education, Science and Technological Development, Grant no. III 45017 and Grant no. III45007]

Supercritical CO2 extraction of Helichrysum italicum and impregnation of starch xerogels with the extract by using an integrated scCO2 extraction and impregnation process were performed at 350 bar and 40?C in order to produce biomaterials for possible oral intake of the extract. Xerogels produced by air-drying of acetogels and alcogels were used as carriers in the supercritical impregnation process. The effect of ethanol as a co-solvent, contact time, plant material/carrier mass ratio and xerogel preparation on the impregnation loading was studied. The highest impregnation loading (1.26?0.22%) was achieved after 5 h impregnation of the xerogel obtained from alcogel using pure scCO2 and plant material/ /carrier mass ratio of 10. Chemical analysis of the extracts showed that the addition of ethanol as co-solvent had a positive effect on scCO2 selectivity to terpene fraction and total flavonoids, while it lowered the total phenolic content. Despite the difference in chemical composition, both extracts expressed similar antioxidant activity according to the DPPH and FRAP methods. The integrated process was shown to be a feasible method for isolation and incorporation of bioactive components of H. italicum into starch xerogels.

Lady's mantle (Alchemilla vulgaris L. syn. Alchemilla xanthochlora Rothm., Rosaceae) has been commonly used in folk medicine to heal inflammations in the mouth, bleeding of the nose, furuncules, gynaecological (menorrhagia and dysmenorrhoea), and gastrointestinal disorders. Although therapeutic indications for lady's mantle are non-specific diarrhoea and gastrointestinal complaints, it has been reported to exert, as well, a variety of biological activities, including wound healing, antimicrobial, neuroprotective, gastroprotective, cytotoxic, and antioxidant. Lady's mantle presents a valuable source of natural bioactive compounds, mostly phenolic compounds - a large amount of tannins, phenolcarboxylic acids, and flavonoids, being responsible for the abovementioned effects. In this work, a literature review of biological properties, investigated in in vitro and in vivo experiments in regard to the determined chemical profile is presented. In addition, the data reported are discussed, and the directions for further investigations are proposed.

Supercritical fluid extraction as an environmentally friendly technology was applied to isolate biologically active extracts from celery and parsley fruits for potential applications in the food industry. The extractions were performed under mild temperature conditions of 39.85 &deg, C and at pressures of 10 and 30 MPa. The extracts were analyzed regarding their chemical composition, antibacterial activity, and cytotoxic effect. Sedanolide was the dominant component of the celery fruit extracts, comprising more than 70% of the obtained fraction, while the content of apiole in the parsley fruit SC CO2 extracts exceeded 85%. The celery fruit extracts showed strong and moderately strong antibacterial activity against tested Staphylococcus aureus, Bacillus (B.) cereus, B. subtilis, B. circulans, Listeria (L.) greyi, L. seeligeri and L. welshimeri, with minimal inhibitory concentration (MIC) values between 160 and 640 &micro, g/mL, and weak activity against the selected Salmonella isolates with a MIC of 2560 &micro, g/mL. The parsley extract obtained at 10 MPa showed strong and moderately strong antibacterial effects against Bacillus strains with obtained MICs of 160&ndash, 640 &micro, g/mL, and weak activity against Staphylococcus, Listeria, and Salmonella with a MIC of 2560 &micro, g/mL. Cytotoxicity investigation showed that the extracts with proven antibacterial activity had no cytotoxic effect on rabbit kidney cells at concentrations of up to 640 &micro, g/mL.

Usnic acid is one of the most investigated lichen secondary metabolites, with several proven biological properties with potential medical relevance. However, its unfavorable physico-chemical properties, as well as observed hepatotoxicity, have discouraged wide-range utilization of usnic acid as a promising therapeutic agent. In accordance with the growing research interest in the development of nanotechnology, especially in the arena of preparations based on natural sources of medicinal compounds, usnic acid incorporated into nano- and microsized colloidal carriers has been a subject of a large number of publications. Therefore, this review discusses the overall results of the studies dealing with usnic acid encapsulated into lipid-based, polymeric and nonorganic micro- and/or nanocarriers, as potential drug delivery systems for this natural compound, in an attempt to introduce its usage as a potential antitumor, antimicrobial, wound-healing, antioxidative and anti-inflammatory drug.

Establishing causal relationship between epigenetic marks and gene transcription requires molecular tools, which can precisely modify specific genomic regions. Here, we present a modular and extensible CRISPR/dCas9-based toolbox for epigenetic editing and direct gene regulation. It features a system for expression of orthogonal dCas9 proteins fused to various effector domains and includes a multi-gRNA system for simultaneous targeting dCas9 orthologs to up to six loci. The C- and N-terminal dCas9 fusions with DNMT3A and TET1 catalytic domains were thoroughly characterized. We demonstrated simultaneous use of the DNMT3A-dSpCas9 and TET1-dSaCas9 fusions within the same cells and showed that imposed cytosine hyper- and hypo-methylation altered level of gene transcription if targeted CpG sites were functionally relevant. Dual epigenetic manipulation of the HNF1A and MGAT3 genes, involved in protein N-glycosylation, resulted in change of the glycan phenotype in BG1 cells. Furthermore, simultaneous targeting of the TET1-dSaCas9 and VPR-dSpCas9 fusions to the HNF1A regulatory region revealed strong and persistent synergistic effect on gene transcription, up to 30 days following cell transfection, suggesting involvement of epigenetic mechanisms in maintenance of the reactivated state. Also, modulation of dCas9 expression effectively reduced off-target effects while maintaining the desired effects on target regions.

In recent years, naturally derived tyrosinase inhibitors and antioxidants have become very important, since the synthetic ones are related with several side effects. As wild fruits are rich source of bioactive compounds, this study focused on the examination of Prunus spinosa L. (blackthorn) fruit extracts, widely distributed in our country. However, blackthorn extracts are not enough investigated, and there are only a few published works. Extraction efficiency of P. spinosa ultrasonic fruit extracts obtained by using different solvents (methanol, 70% (v/v) ethanol, 45% (w/w) propylene glycol and water) was evaluated, regarding polyphenolic profile, tyrosinase inhibitory and antioxidant activity. Results suggested that extracts with 45% propylene glycol and water were particularly rich in polyphenolic compounds (especially phenols, flavonoids and anthocyanins). The highest extraction yield was for 45% propylene glycol extract. This extract showed higher tyrosinase inhibitory and antioxidant activity compared to others, while the methanolic extracts had the highest chelating capacity. Therefore, P. spinosa fruit extracts, especially extracts obtained by ultrasonic extraction with 45% propylene glycol, have a great potential to be incorporated in some pharmaceutical formulations for obtaining tyrosinase inhibitory and antioxidant effects. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********

Helichrysum italicum (Roth) G. Don fil., Asteraceae, possesses numerous secondary plant metabolites with a wide range of biological activities. Yet, data on the potential of supercritical fluid extraction (SFE) in their isolation are scarce. This study provides analyses of the chemical profiles of extracts obtained by SFE with or without ethanol as a cosolvent using GC-FID, GC-MS, HPLC, and UHPLC-MS techniques. Among the compounds with proven biological activity identified, the presence of arzanol was confirmed. In the next step, the integrated process of supercritical fluid extraction and impregnation was applied to deliver active compounds to cotton gauze and polypropylene fabric for possible topical applications. The analytical procedures results showed a considerable affinity of both textile materials for incorporating active components present in the H. italicum. The study indicated the high-pressure techniques applied as very efficient in the isolation of bioactive components from H. italicum and their adsorption on selected carriers. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4418]

peer-reviewed, Due to a high content of rutin (2-10%), dried buckwheat leaf and flower (DBLF) formulations were shown to be efficient in the treatment of vascular diseases. In order to find a cost effective way for the extraction of antioxidants, the effects of ethanol/water ratio and temperature on the extraction efficiency of phenolic compounds and the mechanisms of antioxidant action of the extracts were tested. Extraction with ethanol/water mixture (80:20, v/v) for 24 h at room temperature, after the mixture was just brought to boil was demonstrated to be an efficient and cheap way for obtaining a high yield of rutin (49.94±0.623 mg/g DBLF). The most abundant phenolic compounds in DBLF extracts were rutin and chlorogenic acid. Flavonoids, especially rutin, were shown to be the most responsible for the antioxidant activity in all investigated lipid model systems, acting as free radical scavengers, electron-donating substances and chelators of iron ions. In β-carotene bleaching tests, the extracts with the highest activity were as efficient as BHT (butylated hydroxytoluole). Regarding the results of antihemolytic and Schaal oven tests, the extracts demonstrated remarkable ability to inhibit the oxidative destruction of erythrocytes and to prolong the beginning of the oxidation process in sunflower oil., This work is a part of the National Project (TR-31029) financially supported by the Ministry of Education, Science and Technological Development, Republic of Serbia.

Objective The aim of the study was in vitro and in vivo characterization of cosmetic cream with 6% of standardized wild apple fruit extract, stabilized by conventional non-ionic emulsifier-CEW, in order to determine the influence of emulsifiers (conventional vs. biodegradable) on the characteristics of creams and their effects on the skin. Methods Organoleptic and physico-chemical (pH values and electrical conductivity) analysis was performed, determination of fruit acids-FAs content (using HPLC analysis) and estimation of its antioxidant activity-AA (using DPPH test) during 180 days. In vivo study included following examinations: screening of safety profile (after creams application under occlusion during 24 h at human skin); skin moisturizing potential, transepidermal water loss-TEWL, skin pH after 28 days of cream application and hypopigmentation efficacy 7 days of cream application at artificially induced skin hyperpigmentation. Results Investigated cosmetic cream-CEW showed satisfactory organoleptic, physico-chemical characteristics, stability, FAs content (0.13%) and AA (19.25 ± 0.67 %RSC) after preparation, which remained unchanged over the study period. In vivo investigation revealed absence of skin irritation after CEW's application under occlusion. An increase of skin moisturization (after 14 days ΔEC was 18.52 ± 11.51 and after 28 days of applications 16.52 ± 9.36) during 28 day-study, with unchanged TEWL and skin pH values was shown. Decrease of melanin index was revealed, too (after 7 days ΔMI was -31.40 ± 16.50). Conclusion Cosmetic cream stabilized by conventional emulsifier showed better antioxidant potential and weaker moisturizing and hypopigmentation effects related to the cream with same composition but stabilized by biodegradable emulsifiers. Based on all mentioned above, investigated cosmetic cream might be considered for potential use as modern, stable, safe and efficient cosmetic product in the prevention and/or treatment of oxidative stress-related skin changes and/or damages, for moisturization of dry, even irritated skin as well as for lightening of hyperpigmented skin. RESUME: OBJECTIF Le but de l'etude etait la caracterisation in vitro et in vivo d'une creme cosmetique contenant 6 % d'extrait normalise de pomme sauvage, stabilisee par un emulsifiant non ionique conventionnel-CEW, afin de determiner l'influence des emulsifiants (conventionnels vs biodegradables) sur les caracteristiques des cremes et les effets sur la peau. METHODES: Des analyses organoleptiques et physico-chimiques (pH et conductivite electrique) ont ete effectuees, la determination de la teneur en acides de fruits et en acides gras polyinsatures (par CLHP), et l'estimation de sa teneur en activite antioxydante (AA) (a l'aide du test DPPH) pendant 180 jours. L'etude in vivo comprenait les examens suivants : depistage du profil d'innocuite (apres application de cremes sous occlusion pendant 24 h sur la peau humaine); potentiel d'hydratation de la peau, perte d'eau transepidermique (PETE), pH de la peau apres 28 jours d'application de la creme et efficacite de l'hypopigmentation apres 7 jours d'application de la creme sur une hyperpigmentation de la peau induite artificiellement. RESULTATS: La creme cosmetique-CEW etudiee a montre des caracteristiques organoleptiques, physico-chimiques, une stabilite, une teneur en AG (0,13 %) et en AA (19,25 ± 0,67 % RSC) satisfaisantes apres preparation, qui sont demeurees inchangees au cours de la periode de l'etude. L'etude in vivo a revele l'absence d'irritation cutanee apres l'application de CEW sous occlusion. Une augmentation de l'hydratation de la peau (apres 14 jours de DEC etait de 18,52 ± 11,51 et apres 28 jours d'applications de 16,52 ± 9,36) pendant l'etude de 28 jours, avec une PETE et des valeurs de pH cutane inchangees, a ete demontree. Une diminution de l'indice de melanine a ete mise en evidence egalement (apres 7 jours, ΔMI etait de 31,40 ± 16,50). Conclusion La creme cosmetique stabilisee par un emulsifiant conventionnel a montre un meilleur potentiel antioxydant et des effets d'hydratation et d'hypopigmentation plus faibles par rapport a la creme de meme composition mais stabilisee par des emulsifiants biodegradables. D'apres les donnees precedentes, la creme cosmetique etudiee pourrait etre consideree comme un produit cosmetique moderne, stable, sur et efficace dans la prevention et/ou le traitement des changements et/ou dommages cutanes lies au stress oxydatif, pour l'hydratation de la peau seche, voire irritee, ainsi que pour eclaircir la peau hyperpigmentee.

Molecular tools for gene regulation and epigenome editing consist of two main parts: the targeting moiety binding a specific genomic locus and the effector domain performing the editing or regulatory function. The advent of CRISPR-Cas9 technology enabled easy and flexible targeting of almost any locus by co-expression of a small sgRNA molecule, which is complementary to the target sequence and forms a complex with Cas9, directing it to that particular target. Here, we review strategies for recruitment of effector domains, used in gene regulation and epigenome editing, to the dCas9 DNA-targeting protein. To date, the most important CRISPR-Cas9 applications in gene regulation are CRISPR activation or interference, while epigenome editing focuses on targeted changes in DNA methylation and histone modifications. Several strategies for signal amplification by recruitment of multiple effector domains deserve special focus. While some approaches rely on altering the sgRNA molecule and extending it with aptamers for effector domain recruitment, others use modifications to the Cas9 protein by direct fusions with effector domains or by addition of an epitope tag, which also has the ability to bind multiple effector domains. A major barrier to the widespread use of CRISPR-Cas9 technology for therapeutic purposes is its off-target effect. We review efforts to enhance CRISPR-Cas9 specificity by selection of Cas9 orthologs from various bacterial species and their further refinement by introduction of beneficial mutations. The molecular tools available today enable a researcher to choose the best balance of targeting flexibility, activity amplification, delivery method and specificity.

Menopause is characterized by deep metabolic disturbances, including decreased insulin sensitivity, adiposity, and changes in lipid profiles. Estrogen replacement therapy can partially reverse these changes, and while it is safe in most healthy postmenopausal women, there are still existing concerns regarding an increased risk for breast and endometrial cancer as well as a risk for cardiovascular and thromboembolic disease. Therefore, certain natural compounds with positive metabolic effects may be considered as a possible alternative or adjunctive treatment in patients not willing to take estrogens or patients with contraindications for estrogens. The aim of this study was to investigate the influence of Sideritis scardica (mountain tea) extract on metabolic disturbances induced by ovariectomy in rats. The study included 24 rats divided into three groups: ovariectomized rats treated with 200 mg/kg S. scardica extract for 24 weeks (n = 8), ovariectomized non-treated (n = 8), and Sham-operated (n = 8) rats. Food intake, weight gain, body composition, fasting glucose levels, response to oral glucose challenge, liver glycogen content, catalase activity, thiol groups, and malondialdehyde concentrations as well as AMP-activated protein kinase activity in liver cells were studied. Ovariectomized rats treated with S. scardica extract had lower blood triglycerides, reduced fasting glucose levels, as well lower glucose peaks after oral glucose challenge, increased liver glycogen content, and significantly higher catalase activity and thiol group concentration than non-treated ovariectomized rats. The ability of S. scardica extract to attenuate metabolic disturbances associated with ovariectomy was associated with the activation of AMP-activated protein kinase in liver cells.

Paper description: Bilberry ( Vaccinium myrtillus L ., Ericaceae) and its extracts are used in traditional medicine. While research has focused on the health benefits of plant flavonoids, the element content is of potentialy great importance. This study provides information on which part of the bilberry (roots, stems, leaves or fruits) accumulates which elements and on how the metals taken up from the soil might be translocated from the roots to the above-ground parts of the plant. This study provides new findings on element distribution in bilberry and gives insight into the effect of the method of bilberry extract preparation on the element content. Abstract: Bilberry ( Vaccinium myrtillus L., Ericaceae) is a rich source of different mineral elements that are important as essential trace elements, or that can be potentially toxic, depending on their concentration. The contents of sixteen elements: the major (Al, Ca, Fe, K and Mg) and trace elements (As, Ba, Cd, Co, Cu, Cr, Mn, Ni, Pb, Sr and Zn) in roots, stems, leaves and fruits of bilberry and the corresponding soil substrate were determined in order to investigate element mobility and availability in soil. Soil was subjected to sequential extraction procedure as proposed by the Community Bureau of Reference in order to fractionate acid-soluble/exchangeable, reducible, oxidizable and residual fractions. Analysis of water and ethanol extracts of bilberry aerial parts revealed a significant transfer of elements during the extraction procedure, with corresponding extraction coefficient values of up to 95.12%. Results showed that bilberry fruits could serve as a good dietary source of essential elements for humans, especially Mn and Cu. https://doi.org/10.2298/ABS181004056B Received: October 4, 2018; Revised: November 28, 2018; Accepted: November 30, 2018; Published online: December 14, 2018 How to cite this article: Brasanac-Vukanovic S, Tadic VM, Blagojevic N, Vukasinovic-Pesic V, Đurđic S, Stankovic M, Mutic J. Element accumulation capacity of Vaccinium myrtillus from Montenegro: Comparison of element contents in water and ethanol extracts of bilberry plant parts. Arch Biol Sci. 2019;71(1):145-57.

Old man’s beard (Usnea barbata) is one of the most investigated lichens which biological properties (such as antimicrobial, anti-inflammatory and cytotoxic activities) have been proven in several scien- tific studies. Stated properties of old man’s beard have been attributed mainly to its major secondary metabolite - a dibenzofuran derivative, usnic acid. The aim of the current study was to examine cyto- toxic activity of the quantified supercritical CO2 extract and pure usnic acid in parallel against L929 fibrosarcoma cells. Our results indicated the tested extract to be more toxic towards the investigated cell line compared to usnic acid per se, despite the high amount of this compound (81.41% (w/w)) revealed in the extract using HPLC. Such result may be connected to the unidentified compounds present in the supercritical CO2 extract that could be responsible for apoptosis and oxidative stress in the exerted cytotoxicity of the investigated extract, but not usnic acid. Our study supports further in- vestigations of supercritical CO2 extract of U. barbata as a prospective therapeutic agent with potential relevance in the treatment of sarcoma.

The aim of this study was to establish correlation of chemical composition and antioxidant activity of bilberry plants from Montenegro. Total phenolic, tannin, flavonoid, procyanidin and anthocyanin contents were determined in fruits and leaves extracts using spectrophotometric methods, while the measurements of metal content was carried out in an Inductively Coupled Atomic Emission Spectrometer. Qualitative and quantitative analyses of major phenolics were achieved by HPLC. In the investigated extracts, the most abundant phenolic was chlorogenic acid, followed by protocatechuic acid, while resveratrol, isoquercetin, quecetin and hyperoside were also present in significant quantities. Antioxidant potential was evaluated using two in vitro assays&mdash, FRAP and DPPH&mdash, being in the accordance with the cyclic voltammetry tests, performed as well. The results revealed that all the investigated extracts were rich in phenolic and essential mineral constituents, with significant antioxidant activity, depending on the polyphenolic and mineral contents, which was confirmed by principal component analysis.

The objective of this work was to investigate and profoundly characterize low-energy nanoemulsions as multifunctional carriers, with slight reference to dermal administration. An evidence-based approach was offered for deepening the knowledge on their formation via spontaneous emulsification. Curcumin, a compound of natural origin, potentially powerful therapeutic, was chosen as a model API. Due to curcumin's demanding properties (instability, poor solubility, low permeability), its potentials remain unreached. Low-energy nanoemulsions were considered carriers capable of overcoming imposed obstacles. Formulation consisting of Polysorbate 80 and soybean lecithin as stabilizers (9:1, 10%), medium-chain triglycerides as the oil phase (10%) and ultrapure water was selected for curcumin incorporation in 3 different concentrations (1, 2 and 3 mg/mL). Physicochemical stability was demonstrated during 3 months of monitoring (mean droplet size: 111.3-146.8 nm; PDI LT 0.2; pH: 4.73-5.73). Curcumin's release from developed vehicles followed Higuchi's kinetics. DPPH (IC50 = 0.1187 mg/ mL) and FRAP (1.19 +/- 0.02 mmol/g) assays confirmed that curcumin acts as a potent antioxidant through different mechanisms, with no alterations after incorporation in the formulation. High biocompatibility in line with antigenotoxic activity of curcumin-loaded formulations (protective and reparative) was estimated through Comet assay. A multidisciplinary approach is needed to fully characterize developed systems, directing them to more concrete application possibilities. This is the peer-reviewed version of the article: I. Nikolic, D. Jasmin Lunter, D. Randjelovic, et. al., Curcumin-loaded low-energy nanoemulsions as a prototype of multifunctional vehicles for different administration routes: physicochemical and in vitro peculiarities important for dermal application, International, Journal of Pharmaceutics, 2018, 550, 1-2, 333-346, DOI: [https://doi.org/10.1016/j.ijpharm.2018.08.060] The published version: [https://cer.ihtm.bg.ac.rs/handle/123456789/2425]

Objective The aim of this study was in vitro and in vivo characterization of cosmetic cream with 6% of wild apple fruit water extract, containing 3.5% of alpha-hydroxyacids-AHAs and polyphenolic compounds, stabilized by biodegradable alkyl-polyglucoside emulsifiers. Methods In vitro characterization of cream included organoleptic and physico-chemical (pH values and electrical conductivity) analysis, antioxidant activity-AA estimation (using DPPH test) during 180 days of storage at 22±2°C and determination of cosmetic active substances content-AHAs (using HPLC analysis). In vivo estimation of skin irritation potential after creams application under occlusion during 24h was investigated employing the biophysical methods on 12 healthy volunteers; hydration efficiency, transepidermal water loss-TEWL, skin pH, erythema-EI and melanin index-MI on 10 healthy volunteers after 28 days of cream application and bleaching efficiency on 10 healthy volunteers after 7 days of cream application after artificially induced skin hyperpigmentation. Results Investigated cosmetic cream was pale beige color, odorless, semi-solid consistency and homogeneous, with pH values of 6.53±0.14, electrical conductivity above 50 μS/cm and AA of 24.96%RSC after preparation and these characteristics were stable during investigated period. In vivo measurements revealed absence of skin irritation after cream application under occlusion, which was part of it's safety profile. Increase of skin hydration after 14 days of cream application was 17.28±12.23 and after 28 days 21.19±7.59. In addition, in performed experiment TEWL and skin pH values during cream application remained unchanged. Cream application after artificial hyperpigmentation induced decrease of MI (∆MI after 7 days was -45.30±18.55) CONCLUSION: Formulated cosmetic cream with 6% of standardized wild apple fruit water extract and stabilized by biodegradable alkyl-polyglucoside emulsifiers demonstrated good stability, acceptable level of in vitro antioxidant activity, absence of skin irritation after cream application under occlusion and positive effects of cream on human skin after application (hydrating and bleaching effects). All mentioned properties make the cream suitable for possible usage as cosmetic product for preventing the skin damages caused by oxidative stress, for moisturizing the dry skin and bleaching of skin hyperpigmentation. This article is protected by copyright. All rights reserved.

Epigenetic studies relied so far on correlations be- tween epigenetic marks and gene expression pat- tern. Technologies developed for epigenome edit- ing now enable direct study of functional relevance of precise epigenetic modifications and gene reg- ulation. The reversible nature of epigenetic modi- fications, including DNA methylation, has been al- ready exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR- Cas9-based tool for specific DNA methylation con- sisting of deactivated Cas9 (dCas9) nuclease and cat- alytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9- DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the tar- geted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methy- lation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.

The aim of the present work was to examine if hydrogen peroxide treatment of sugar beet fibre that aimed at improving its physicochemical properties would impair its antioxidant potential. Three different sugar beet fibres were obtained from sugar beet - non-treated fibre (NTF) from sugar beet cossettes extracted with sulphurous acid, treated fibre (TF) from NTF treated with hydrogen peroxide in alkaline solution and commercially available Fibrex(®) . The antioxidant activity of extractable and non-extractable fibre fractions in ethanol/water mixture (80:20, v/v) of three fibre samples was estimated. Non-extractable fractions obtained after alkaline treatment of investigated fibres were much higher in phenolic compounds and possessed higher antioxidant potential than extractable fractions. Ferulic acid was proven to be the dominant phenolic acid. Regarding both extractable and non-extractable fractions, Fibrex(®) had the highest antioxidant activity in chemical tests, while NTF was superior in comparison with TF. Based on the results of Caco-2 cells-based test, all non-extractable fractions possessed potential for reactive oxygen species inhibition. Regarding the extractable fractions, only the TF manifested this effect.Copyright © 2016 John Wiley & Sons, Ltd.