A High Redox Potential Laccase from Pycnoporus sanguineus RP15: Potential Application for Dye Decolorization

Laccase production by Pycnoporus sanguineus RP15 grown in wheat bran and corncob under solid-state fermentation was optimized by response surface methodology using a Central Composite Rotational Design. A laccase (Lacps1) was purified and characterized and the potential of the pure Lacps1 and the crude culture extract for synthetic dye decolorization was evaluated. At optimal conditions (eight days, 26 °C, 18% (w/w) milled corncob, 0.8% (w/w) NH₄Cl and 50 mmol·L⁻¹ CuSO₄, initial moisture 4.1 mL·g⁻¹), the laccase activity reached 138.6 ± 13.2 U·g⁻¹. Lacps1 was a monomeric glycoprotein (67 kDa, 24% carbohydrate). Optimum pH and temperature for the oxidation of 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were 4.4 and 74.4 °C, respectively. Lacps1 was stable at pH 3.0–8.0, and after two hours at 55–60 °C, presenting high redox potential (0.747 V vs. NHE). ABTS was oxidized with an apparent affinity constant of 147.0 ± 6.4 μmol·L⁻¹, maximum velocity of 413.4 ± 21.2 U·mg⁻¹ and catalytic efficiency of 3140.1 ± 149.6 L·mmol⁻¹·s⁻¹. The maximum decolorization percentages of bromophenol blue (BPB), remazol brilliant blue R and reactive blue 4 (RB4), at 25 or 40 °C without redox mediators, reached 90%, 80% and 60%, respectively, using either pure Lacps1 or the crude extract. This is the first study of the decolorization of BPB and RB4 by a P. sanguineus laccase. The data suggested good potential for treatment of industrial dye-containing effluents.     

Citrange Fruit Extracts Alleviate Obesity-Associated Metabolic Disorder in High-Fat Diet-Induced Obese C57BL/6 Mouse

Obesity is becoming one of the global epidemics of the 21st century. In this study, the effects of citrange (Citrus sinensis × Poncirus trifoliata) fruit extracts in high-fat (HF) diet-induced obesity mice were studied. Female C57BL/6 mice were fed respectively a chow diet (control), an HF diet, HF diet supplemented with 1% w/w citrange peel extract (CPE) or 1% w/w citrange flesh and seed extract (CFSE) for 8 weeks. Our results showed that both CPE and CFSE regulated the glucose metabolic disorders of obese mice. In CPE and CFSE-treated groups, the body weight gain, blood glucose, serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-c) levels were significantly (p < 0.05) reduced relative to those in the HF group. To explore the mechanisms of action of CPE and CFSE on the metabolism of glucose and lipid, related genes’ expressions in liver were assayed. In liver tissue, the expression level of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes were down-regulated by CPE and CFSE supplementation as revealed by qPCR tests. In addition, both CPE and CFSE decreased the expression level of liver X receptor (LXR) α and β, which are involved in lipid and glucose metabolism. Taken together, these results suggest that CPE and CFSE administration could ameliorate obesity and related metabolic disorders in HF diet-induced obesity mice probably through the inhibition of PPARγ and LXRs gene expressions.     

Silver Nanoparticles from Oregano Leaves’ Extracts as Antimicrobial Components for Non-Infected Hydrogel Contact Lenses

The oregano leaves’ extract (ORLE) was used for the formation of silver nanoparticles (AgNPs(ORLE)). ORLE and AgNPs(ORLE) (2 mg/mL) were dispersed in polymer hydrogels to give the pHEMA@ORLE_2 and pHEMA@AgNPs(ORLE)_2 using hydroxyethyl–methacrylate (HEMA). The materials were characterized by X-ray fluorescence (XRF) spectroscopy, X-ray powder diffraction analysis (XRPD), thermogravimetric differential thermal analysis (TG-DTA), derivative thermogravimetry/differential scanning calorimetry (DTG/DSC), ultraviolet (UV-Vis), and attenuated total reflection mode (ATR-FTIR) spectroscopies in solid state and UV–Vis in solution. The crystallite size value, analyzed with XRPD, was determined at 20 nm. The antimicrobial activity of the materials was investigated against Gram-negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The Gram-positive ones of the genus of Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) are known to be involved in microbial keratitis by the means of inhibitory zone (IZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The IZs, which developed upon incubation of P. aeruginosa, E. coli, S. epidermidis, and S. aureus with paper discs soaked in 2 mg/mL of AgNPs(ORLE), were 11.7 ± 0.7, 13.5 ± 1.9, 12.7 ± 1.7, and 14.3 ± 1.7 mm. When the same dose of ORLE was administrated, the IZs were 10.2 ± 0.7, 9.2 ± 0.5, 9.0 ± 0.0, and 9.0 ± 0.0 mm. The percent of bacterial viability when they were incubated over the polymeric hydrogel discs of pHEMA@AgNPs(ORLE)_2 was interestingly low (66.5, 88.3, 77.7, and 59.6%, respectively, against of P. aeruginosa, E. coli, S. epidermidis, and S. aureus) and those of pHEMA@ORLE_2 were 89.3, 88.1, 92.8, and 84.6%, respectively. Consequently, pHEMA@AgNPs(ORLE)_2 could be an efficient candidate toward the development of non-infectious contact lenses.     

The Beneficial Effects of Combined Grape Pomace and Omija Fruit Extracts on Hyperglycemia,Adiposity and Hepatic Steatosis in db/db Mice: A Comparison with Major Index Compounds

This study investigated the effects of combined grape pomace and omija fruit extracts (GO) on diabetes-related metabolic changes in type 2 diabetic db/db mice. The effects of GO were compared with those of a resveratrol and schizandrin mixture (RS), which is a mixture of major components of GO. Mice were fed a normal diet with RS (0.005% resveratrol and 0.02% schizandrin in diet, w/w) or GO (0.3% grape pomace ethanol extract and 0.05% omija fruit ethanol extract in diet, w/w) for seven weeks. RS and GO not only lowered the levels of blood and plasma glucose, HbA_1c, insulin and homeostasis model assessment of insulin resistance (HOMA-IR) with a simultaneous decrease in hepatic gluconeogenic enzymes activities and adiposity, but also improved preservation of the pancreatic β-cells. Plasma leptin and resistin levels were lower while the plasma adiponectin level was higher in the RS and GO groups than in the control group. Especially, GO increased hepatic glucokinase activity and gene expression and improved hepatic steatosis by elevating fatty acid oxidation compared to RS. These findings suggest that GO ameliorates hyperglycemia, adiposity and hepatic steatosis in type 2 diabetic mice.     

Tuning Physicochemical Properties of a Macroporous Polysaccharide-Based Scaffold for 3D Neuronal Culture

Central nervous system (CNS) lesions are a leading cause of death and disability worldwide. Three-dimensional neural cultures in biomaterials offer more physiologically relevant models for disease studies, toxicity screenings or in vivo transplantations. Herein, we describe the development and use of pullulan/dextran polysaccharide-based scaffolds for 3D neuronal culture. We first assessed scaffolding properties upon variation of the concentration (1%, 1.5%, 3% w/w) of the cross-linking agent, sodium trimetaphosphate (STMP). The lower STMP concentration (1%) allowed us to generate scaffolds with higher porosity (59.9 ± 4.6%), faster degradation rate (5.11 ± 0.14 mg/min) and lower elastic modulus (384 ± 26 Pa) compared with 3% STMP scaffolds (47 ± 2.1%, 1.39 ± 0.03 mg/min, 916 ± 44 Pa, respectively). Using primary cultures of embryonic neurons from PGK^Cre, Rosa26^tdTomato embryos, we observed that in 3D culture, embryonic neurons remained in aggregates within the scaffolds and did not attach, spread or differentiate. To enhance neuronal adhesion and neurite outgrowth, we then functionalized the 1% STMP scaffolds with laminin. We found that treatment of the scaffold with a 100 μg/mL solution of laminin, combined with a subsequent freeze-drying step, created a laminin mesh network that significantly enhanced embryonic neuron adhesion, neurite outgrowth and survival. Such scaffold therefore constitutes a promising neuron-compatible and biodegradable biomaterial.     

Preparation and Characterization of Nanoliposomes Entrapping Medium-Chain Fatty Acids and Vitamin C by Lyophilization

The complex nanoliposomes encapsulating both a hydrophilic drug vitamin C (vit C) and hydrophobic drug medium-chain fatty acids (MCFAs) was prepared by combining double emulsion method with dynamic high pressure microfluidization. The complex nanoliposomes was further freeze-dried under −86 °C for 48 h with sucrose at the sucrose/lipids ratio of 2:1(w/w) in order to enhance its stability. The freeze-dried complex nanoliposomes under the suitable conditions exhibited high entrapment efficiency of MCFAs (44.26 ± 3.34)%, relatively high entrapment efficiency of vit C (62.25 ± 3.43)%, low average size diameter (110.4 ± 7.28) nm and good storage stability at 4 °C for 60 days with slight changes in mean particle diameter and drug entrapment efficiencies. The results of transmission electron microscopy of freeze-dried complex nanoliposomes also showed that the freeze-dried samples with sucrose were stable without great increase in their particle sizes and without destroying their spherical shape. The results indicated that sucrose presented well protection effects in MCFAs-vit C complex nanoliposomes, suggesting the possibility of further usage in commercial liposomes.     

Linoleic Acid Upregulates Microrna-494 to Induce Quiescence in Colorectal Cancer

Cancer dormancy is a state characterized by the quiescence of disseminated cancer cells, and tumor recurrence occurs when such cells re-proliferate after a long incubation period. These cancer cells tend to be treatment resistant and one of the barriers to successful therapeutic intervention. We have previously reported that long-term treatment of cancer cells with linoleic acid (LA) induces a dormancy-like phenotype. However, the mechanism underpinning this effect has not yet been clarified. Here, we investigate the mechanism of LA-induced quiescence in cancer cells. We first confirmed that long-term treatment of the mouse colorectal cancer cell line CT26 with LA induced quiescence. When these cells were inoculated subcutaneously into a syngeneic mouse and fed with an LA diet, the inoculated cancer cells maintained the quiescent state and exhibited markers of dormancy. LA-treated CT26 cells showed reduced oxidative phosphorylation, glycolysis, and energy production as well as reduced expression of the regulatory factors Pgc1α and MycC. MicroRNA expression profiling revealed that LA induced an upregulation in miR-494. The expression of Pgc1α and MycC were both induced by an miR-494 mimic, and the LA-induced decrease in gene expression was abrogated by an miR-494 inhibitor. The expression of miR-494 was enhanced by the mitochondrial oxidative stress produced by LA. In a syngeneic mouse subcutaneous tumor model, growth suppression by an LA diet and growth delay by LA pretreatment + LA diet were found to have similar effects as administration of an miR-494 mimic. In contrast, the effects of LA were abrogated by an miR-494 inhibitor. Analysis of human colorectal cancer tissue revealed that miR-494 was present at low levels in non-metastatic cases and cases with simultaneous liver metastases but was expressed at high levels in cases with delayed liver metastases, which also exhibited reduced expression of PGC1α and MYCC. These results suggest that miR-494 is involved in cancer dormancy induced by high levels of LA intake and that this microRNA may be valuable in targeting dormant cancer cells.     

Tobramycin Supplemented Small-Diameter Vascular Grafts for Local Antibiotic Delivery: A Preliminary Formulation Study

Peripheral artery occlusive disease is an emerging cardiovascular disease characterized by the blockage of blood vessels in the limbs and is associated with dysfunction, gangrene, amputation, and a high mortality risk. Possible treatments involve by-pass surgery using autologous vessel grafts, because of the lack of suitable synthetic small-diameter vascular prosthesis. One to five percent of patients experience vascular graft infection, with a high risk of haemorrhage, spreading of the infection, amputation and even death. In this work, an infection-proof vascular graft prototype was designed and manufactured by electrospinning 12.5% w/v poly-L-lactic-co-glycolic acid solution in 75% v/v dichloromethane, 23.8% v/v dimethylformamide and 1.2% v/v water, loaded with 0.2% w/w_PLGA. Polymer and tobramycin concentrations were selected after viscosity and surface tension and after HPLC-UV encapsulation efficiency (EE%) evaluation, respectively. The final drug-loaded prototype had an EE% of 95.58% ± 3.14%, with smooth fibres in the nanometer range and good porosity; graft wall thickness was 291 ± 20.82 μm and its internal diameter was 2.61 ± 0.05 mm. The graft’s antimicrobic activity evaluation through time-kill assays demonstrated a significant and strong antibacterial activity over 5 days against Staphylococcus aureus and Escherichia coli. An indirect cell viability assay on Normal Human Dermal Fibroblasts (NHDF) confirmed the cytocompatibility of the grafts.     

Palmitic Acid Impairs Myogenesis and Alters Temporal Expression of miR-133a and miR-206 in C2C12 Myoblasts

Palmitic acid (PA), a saturated fatty acid enriched in high-fat diet, has been implicated in the development of sarcopenic obesity. Herein, we chose two non-cytotoxic concentrations to better understand how excess PA could impact myotube formation or diameter without inducing cell death. Forty-eight hours of 100 µM PA induced a reduction of myotube diameter and increased the number of type I fibers, which was associated with increased miR-206 expression. Next, C2C12 myotube growth in the presence of PA was evaluated. Compared to control cells, 150 µM PA reduces myoblast proliferation and the expression of MyoD and miR-206 and miR-133a expression, leading to a reduced number and diameter of myotubes. PA (100 µM), despite not affecting proliferation, impairs myotube formation by reducing the expression of Myf5 and miR-206 and decreasing protein synthesis. Interestingly, 100 and 150 µM PA-treated myotubes had a higher number of type II fibers than control cells. In conclusion, PA affects negatively myotube diameter, fusion, and metabolism, which may be related to myomiRs. By providing new insights into the mechanisms by which PA affects negatively skeletal muscle, our data may help in the discovery of new targets to treat sarcopenic obesity.     

Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis

The periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli is a molybdenum enzyme involved in detoxification of aldehydes in the cell. It is an example of an αβγ heterotrimeric enzyme of the xanthine oxidase family of enzymes which does not dimerize via its molybdenum cofactor binding domain. In order to structurally characterize PaoABC, X-ray crystallography and small angle X-ray scattering (SAXS) have been carried out. The protein crystallizes in the presence of 20% (w/v) polyethylene glycol 3350 using the hanging-drop vapour diffusion method. Although crystals were initially twinned, several experiments were done to overcome twinning and lowering the crystallization temperature (293 K to 277 K) was the solution to the problem. The non-twinned crystals used to solve the structure diffract X-rays to beyond 1.80 Å and belong to the C2 space group, with cell parameters a = 109.42 Å, b = 78.08 Å, c = 151.77 Å, β = 99.77°, and one molecule in the asymmetric unit. A molecular replacement solution was found for each subunit separately, using several proteins as search models. SAXS data of PaoABC were also collected showing that, in solution, the protein is also an αβγ heterotrimer.     

Rhus coriaria L. (Sumac) Demonstrates Oncostatic Activity in the Therapeutic and Preventive Model of Breast Carcinoma

Comprehensive scientific data provide evidence that isolated phytochemicals or whole plant foods may beneficially modify carcinogenesis. The aim of this study was to evaluate the oncostatic activities of Rhus coriaria L. (sumac) using animal models (rat and mouse), and cell lines of breast carcinoma. R. coriaria (as a powder) was administered through the diet at two concentrations (low dose: 0.1% (w/w) and high dose: 1 % (w/w)) for the duration of the experiment in a syngeneic 4T1 mouse and chemically-induced rat mammary carcinoma models. After autopsy, histopathological and molecular analyses of tumor samples in rodents were performed. Moreover, in vitro analyses using MCF-7 and MDA-MB-231 cells were conducted. The dominant metabolites present in tested R. coriaria methanolic extract were glycosides of gallic acid (possible gallotannins). In the mouse model, R. coriaria at a higher dose (1%) significantly decreased tumor volume by 27% when compared to controls. In addition, treated tumors showed significant dose-dependent decrease in mitotic activity index by 36.5% and 51% in comparison with the control group. In the chemoprevention study using rats, R. coriaria at a higher dose significantly reduced the tumor incidence by 20% and in lower dose non-significantly reduced tumor frequency by 29% when compared to controls. Evaluations of the mechanism of oncostatic action using valid clinical markers demonstrated several positive alterations in rat tumor cells after the treatment with R. coriaria. In this regard, histopathological analysis of treated tumor specimens showed robust dose-dependent decrease in the ratio of high-/low-grade carcinomas by 66% and 73% compared to controls. In treated rat carcinomas, we found significant caspase-3, Bax, and Bax/Bcl-2 expression increases; on the other side, a significant down-regulation of Bcl-2, Ki67, CD24, ALDH1, and EpCam expressions and MDA levels. When compared to control specimens, evaluation of epigenetic alterations in rat tumor cells in vivo showed significant dose-dependent decrease in lysine methylation status of H3K4m3 and H3K9m3 and dose-dependent increase in lysine acetylation in H4K16ac levels (H4K20m3 was not changed) in treated groups. However, only in lower dose of sumac were significant decreases in the expression of oncogenic miR210 and increase of tumor-suppressive miR145 (miR21, miR22, and miR155 were not changed) observed. Finally, only in lower sumac dose, significant decreases in methylation status of three out of five gene promoters–ATM, PTEN, and TIMP3 (PITX2 and RASSF1 promoters were not changed). In vitro evaluations using methanolic extract of R. coriaria showed significant anticancer efficacy in MCF-7 and MDA-MB-231 cells (using Resazurin, cell cycle, annexin V/PI, caspase-3/7, Bcl-2, PARP, and mitochondrial membrane potential analyses). In conclusion, sumac demonstrated significant oncostatic activities in rodent models of breast carcinoma that were validated by mechanistic studies in vivo and in vitro.     

Selected Tea and Tea Pomace Extracts Inhibit Intestinal α-Glucosidase Activity in Vitro and Postprandial Hyperglycemia in Vivo

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by postprandial hyperglycemia, which is an early defect of T2DM and thus a primary target for anti-diabetic drugs. A therapeutic approach is to inhibit intestinal α-glucosidase, the key enzyme for dietary carbohydrate digestion, resulting in delayed rate of glucose absorption. Although tea extracts have been reported to have anti-diabetic effects, the potential bioactivity of tea pomace, the main bio waste of tea beverage processing, is largely unknown. We evaluated the anti-diabetic effects of three selected tea water extracts (TWE) and tea pomace extracts (TPE) by determining the relative potency of extracts on rat intestinal α-glucosidase activity in vitro as well as hypoglycemic effects in vivo. Green, oolong, and black tea bags were extracted in hot water and the remaining tea pomace were dried and further extracted in 70% ethanol. The extracts were determined for intestinal rat α-glucosidases activity, radical scavenging activity, and total phenolic content. The postprandial glucose-lowering effects of TWE and TPE of green and black tea were assessed in male Sprague-Dawley (SD) rats and compared to acarbose, a known pharmacological α-glucosidase inhibitor. The IC₅₀ values of all three tea extracts against mammalian α-glucosidase were lower or similar in TPE groups than those of TWE groups. TWE and TPE of green tea exhibited the highest inhibitory effects against α-glucosidase activity with the IC₅₀ of 2.04 ± 0.31 and 1.95 ± 0.37 mg/mL respectively. Among the specific enzymes tested, the IC₅₀ values for TWE (0.16 ± 0.01 mg/mL) and TPE (0.13 ± 0.01 mg/mL) of green tea against sucrase activity were the lowest compared to those on maltase and glucoamylase activities. In the animal study, the blood glucose level at 30 min after oral intake (0.5 g/kg body wt) of TPE and TWE of both green and black tea was significantly reduced compared to the control in sucrose-loaded SD rats. The TPE of all three teas had significantly higher phenolic content than those of the TWE groups, which correlated strongly with the DPPH radical scavenging activity. This is the first report of tea pomace extract significantly inhibits intestinal α-glucosidase, resulting in delayed glucose absorption and thereby suppressed postprandial hyperglycemia. Our data suggest that tea pomace-derived bioactives may have great potential for further development as nutraceutical products and the reuse of otherwise biowaste as valuable bioresources for the industry.     

miR-16-5p Is a Stably-Expressed Housekeeping MicroRNA in Breast Cancer Tissues from Primary Tumors and from Metastatic Sites

For quantitative microRNA analyses in formalin-fixed paraffin-embedded (FFPE) tissue, expression levels have to be normalized to endogenous controls. To investigate the most stably-expressed microRNAs in breast cancer and its surrounding tissue, we used tumor samples from primary tumors and from metastatic sites. MiRNA profiling using TaqMan^® Array Human MicroRNA Cards, enabling quantification of 754 unique human miRNAs, was performed in FFPE specimens from 58 patients with metastatic breast cancer. Forty-two (72%) samples were collected from primary tumors and 16 (28%) from metastases. In a cross-platform analysis of a validation cohort of 32 FFPE samples from patients with early breast cancer genome-wide microRNA expression analysis using SurePrintG3 miRNA (8 × 60 K)^® microarrays from Agilent^® was performed. Eleven microRNAs could be detected in all samples analyzed. Based on NormFinder and geNorm stability values and the high correlation (rho ≥ 0.8) with the median of all measured microRNAs, miR-16-5p, miR-29a-3p, miR-126-3p, and miR-222-3p are suitable single gene housekeeper candidates. In the cross-platform validation, 29 human microRNAs were strongly expressed (mean log2-intensity > 10) and 21 of these microRNAs including miR-16-5p and miR-29a-3p were also stably expressed (CV < 5%). Thus, miR-16-5p and miR-29a-3p are both strong housekeeper candidates. Their Normfinder stability values calculated across the primary tumor and metastases subgroup indicate that miR-29a-3p can be considered as the strongest housekeeper in a cohort with mainly samples from primary tumors, whereas miR-16-5p might perform better in a metastatic sample enriched cohort.     

Functional Properties of Gelatin/Polyvinyl Alcohol Films Containing Black Cumin Cake Extract and Zinc Oxide Nanoparticles Produced via Casting Technique

This study aimed to develop and characterize gelatin/polyvinyl alcohol (G/PVA) films loaded with black cumin cake extract (BCCE) and zinc oxide nanoparticles (ZnONPs). The BCCE was also applied for the green synthesis of ZnONPs with an average size of less than 100 nm. The active films were produced by a solvent-casting technique, and their physicochemical and antibacterial properties were investigated. Supplementation of G/PVA film in ZnONPs decreased the tensile strength (TS) from 2.97 MPa to 1.69 MPa. The addition of BCCE and ZnONPs increased the elongation at the break (EAB) of the enriched film by about 3%. The G/PVA/BCCE/ZnONPs film revealed the lowest water vapor permeability (WVP = 1.14 × 10⁻⁹ g·mm·Pa⁻¹·h⁻¹·mm⁻²) and the highest opacity (3.41 mm⁻¹). The QUick, Easy, New, CHEap and Reproducible (QUENCHER) methodologies using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS) and cupric ion reducing antioxidant capacity (CUPRAC) were applied to measure antioxidant capacity (AC) of the prepared films. The incorporation of BCCE and ZnONPs into G/PVA films enhanced the AC by 8–144%. The films containing ZnONPs and a mixture of BCCE and ZnONPs inhibited the growth of three Gram-positive bacterial strains. These nanocomposite films with desired functional properties can be recommended to inhibit microbial spoilage and oxidative rancidity of packaged food.     

Flavonoid and Phenolic Acids Content and In Vitro Study of the Potential Anti-Aging Properties of Eutrema japonicum (Miq.) Koidz Cultivated in Wasabi Farm Poland

Skin aging is a natural, unavoidable, and complex process caused by oxidative stress. As a consequence, it leads to an increase in the activation of extracellular matrix disruption enzymes and DNA damage. The search for natural sources that inhibit these mechanisms can be a good approach to prevent skin aging. The purpose of our study was to evaluate the composition of flavonoids and phenolic acids in the extracts obtained from the flowers, roots, and leaves of Eutrema japonicum cultivated in Poland. Then, the resultant extracts were subjected to an assessment of antioxidant, anti-collagenase, anti-elastase, anti-hyaluronidase, antibacterial, and cytotoxic properties. It was demonstrated that the extract from the flowers had the highest content of flavonoid glycosides (17.15 mg/g DE). This extract showed the greatest antioxidant, anti-collagenase, anti-elastase, and anti-hyaluronidase activities compared to the other samples. Importantly, the collagenase inhibitory activity of this extract (93.34% ± 0.77%) was better than that of positive control epigallocatechin gallate (88.49% ± 0.45%). An undeniable advantage of this extract was also to possess moderate antibacterial properties and no cytotoxicity towards normal human skin fibroblasts. Our results suggest that extracts from E. japonicum flowers may be considered as a promising antiaging compound for applications in cosmetic formulations.     

Biocatalyzed Synthesis of Flavor Esters and Polyesters: A Design of Experiments (DoE) Approach

In the present work, different hydrolases were adsorbed onto polypropylene beads to investigate their activity both in short-esters and polyesters synthesis. The software MODDE^® Pro 13 (Sartorius) was used to develop a full-factorial design of experiments (DoE) to analyse the thermostability and selectivity of the immobilized enzyme towards alcohols and acids with different chain lengths in short-esters synthesis reactions. The temperature optima of Candida antarctica lipase B (CaLB), Humicola insolens cutinase (HiC), and Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) were 85 °C, 70 °C, and 50 °C. CaLB and HiC preferred long-chain alcohols and acids as substrate in contrast to Thc_Cut1, which was more active on short-chain monomers. Polymerization of different esters as building blocks was carried out to confirm the applicability of the obtained model on larger macromolecules. The selectivity of both CaLB and HiC was investigated and best results were obtained for dimethyl sebacate (DMSe), leading to polyesters with a M_w of 18 kDa and 6 kDa. For the polymerization of dimethyl adipate (DMA) with BDO and ODO, higher molecular masses were obtained when using CaLB onto polypropylene beads (CaLB_PP) as compared with CaLB immobilized on macroporous acrylic resin beads (i.e., Novozym 435). Namely, for BDO the M_n were 7500 and 4300 Da and for ODO 8100 and 5000 Da for CaLB_PP and for the commercial enzymes, respectively. Thc_Cut1 led to polymers with lower molecular masses, with M_n < 1 kDa. This enzyme showed a temperature optimum of 50 °C with 63% of DMA and BDO when compared to 54% and 27%, at 70 °C and at 85 °C, respectively.     

Does Melatonin Homeostasis Play a Role in Continuous Epigastric Pain Syndrome?

Two clinical forms of functional dyspepsia (FD) are listed in the Rome III criteria: postprandial distress syndrome (PDS) and epigastric pain syndrome (EPS), differing in the recurrence of ailments depending on the diet. Continuous EPS (CEPS) is observed in some EPS patients, also at night, but its cause is still unknown. We showed previously that melatonin (MEL) homeostasis may be associated with FD. In the present work we evaluated selected components of melatonin homeostasis in patients with CEPS. The study included 30 patients with CEPS, 21 women and nine men, aged 21–49 years and 30 control subjects (EPS excluded); organic and mental diseases, as well as Helicobacter pylori infection, were excluded in both groups. The average severity of abdominal pain in the last three months was estimated in a 10-point scale (Visual Analog Scale). The levels of mRNA expression of arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), the main components of MEL homeostasis, were determined in gastric mucosa with real time PCR. The fasting serum level of MEL (at 09:00 a.m.) and circadian urine excretion of 6-sulfatoxymelatonin (6-HMS) were determined with ELISA. AANAT expression in antral mucosa of control subjects was 1.76 ± 0.41, in the gastric body 1.35 ± 0.38, and in the dyspeptic group 1.42 ± 0.38 (p < 0.05) and 0.92 ± 0.55 (p < 0.05), respectively. HIOMT expression in the control was 2.05 ± 0.70 in the antrum and 1.57 ± 0.69 in the body and in the CEPS group, it was: 1.51 ± 0.57 (p < 0.05) and 0.74 ± 0.31 (p < 0.001), respectively. MEL concentration (pg/mL) was 9.41 ± 3.09 in the control group and 5.62 ± 1.34 (p < 0.01) in the CEPS group. Urinary 6-HMS excretion (μg/24 h) was 11.40 ± 4.46 in the controls and 7.68 ± 2.88 (p < 0.05) in the CEPS. Moreover, a negative correlation was found between the tested parameters and severity of epigastric pain. These results indicate that patients with CEPS may display low level of AANAT and HIOMT expression in gastric mucosa, resulting in decreased MEL synthesis.     

Angiotensin II Regulates microRNA-132/-212 in Hypertensive Rats and Humans

MicroRNAs (miRNAs), a group of small non-coding RNAs that fine tune translation of multiple target mRNAs, are emerging as key regulators in cardiovascular development and disease. MiRNAs are involved in cardiac hypertrophy, heart failure and remodeling following cardiac infarction; however, miRNAs involved in hypertension have not been thoroughly investigated. We have recently reported that specific miRNAs play an integral role in Angiotensin II receptor (AT₁R) signaling, especially after activation of the Gαq signaling pathway. Since AT₁R blockers are widely used to treat hypertension, we undertook a detailed analysis of potential miRNAs involved in Angiotensin II (AngII) mediated hypertension in rats and hypertensive patients, using miRNA microarray and qPCR analysis. The miR-132 and miR-212 are highly increased in the heart, aortic wall and kidney of rats with hypertension (159 ± 12 mm Hg) and cardiac hypertrophy following chronic AngII infusion. In addition, activation of the endothelin receptor, another Gαq coupled receptor, also increased miR-132 and miR-212. We sought to extend these observations using human samples by reasoning that AT₁R blockers may decrease miR-132 and miR-212. We analyzed tissue samples of mammary artery obtained from surplus arterial tissue after coronary bypass operations. Indeed, we found a decrease in expression levels of miR-132 and miR-212 in human arteries from bypass-operated patients treated with AT₁R blockers, whereas treatment with β-blockers had no effect. Taken together, these data suggest that miR-132 and miR-212 are involved in AngII induced hypertension, providing a new perspective in hypertensive disease mechanisms.