Isolation,Culture and Comprehensive Characterization of Biological Properties of Human Urine-Derived Stem Cells

Mesenchymal stem cells (MSCs) represent an attractive source within the field of tissue engineering. However, their harvesting often requires invasive medical procedures. Urine-derived stem cells (UDSCs) display similar properties to MSCs, and their obtention and further processing is non-invasive for the donors as well as low cost. Here, we offer a comprehensive analysis of their biological properties. The goal of this study was to analyze their morphology, stemness, differentiation potential and cytokine profile. We have successfully isolated UDSCs from 25 urine samples. First colonies emerged up to 9 days after the initial seeding. Cell doubling time was 45 ± 0.24 SD, and when seeded at the density of 100 cells/cm², they formed 42 ± 6.5 SD colonies within 10 days. Morphological analyzes revealed that two different types of the cell populations have been present. The first type had a rice-grain shape and the second one was characterized by a polyhedral shape. In several cell cultures, dome-shaped cells were observed as well. All examined UDSCs expressed typical MSC-like surface markers, CD73, CD90 and CD105. Moreover, conditioned media from UDSCs were harvested, and cytokine profile has been evaluated showing a significantly higher secretory rate of IL-8, IL-6 and chemokines MCP-1 and GM-CSF. We have also successfully induced human UDSCs into chondrogenic, osteogenic and myogenic cell lineages. Our findings indicate that UDSCs might have immense potential in the regeneration of the damaged tissues.     

Alanine minimises hepatocyte injury after ischemia-reperfusion in an <i>ex vivo</i> rat liver model

Ischemia-reperfusion injury is a determinant in liver injury occurring during surgery, ischemic states and multiple organ failure. The pre-existing nutritional status of the liver, i.e., fasting, might contribute to the extent of tissue injury. This study investigated whether alanine, an amino acid precursor of glucose, could protect ex vivo perfused livers of fasting rats from reperfusion injury. The portal vein was cannulated, the liver removed and perfused in a closed ex vivo system. Isolated livers were perfused either with glucose 1 g/L and 10 g/L, or with equal concentrations of alanine (n = 10 in each group). The experiment consisted of perfusion for 15 min, ischemia for 60 min, and reoxygenation during 60 min. Enzymes, glucose, lactate and bilirubin were analysed in perfusate samples. The proportion of glycogen as well as activation of caspase 3 was determined in biopsies. Alanine at a concentration of 10 g/L attenuated enzymes release in the perfusate during reoxygenation when compared to glucose-treated groups. Lactate level in the perfusate was lowest in alanine groups. Ischemia-reperfusion and mainly alanine activated apoptosis, specifically in Kupffer and endothelial cells. Alanine presents a protective effect on normothermic ischemia-reperfusion injury of the fasting rat liver when compared to glucose     

The Instrumented Indentation Study of HVOF-Sprayed Hardmetal Coatings

Elastic-plastic properties, namely, hardness and Young’s modulus, of four HVOF-sprayed hardmetal coatings were measured by instrumented indentation using Oliver-Pharr method Nanoindenter XP MTS with a continuous stiffness measurement (CSM) module. The results show that with sufficient number of CSM measurements, one can distinguish between indents made in the hard particles and indents made in the binder material. This can be accomplished by analyzing the plots of hardness and Young’s modulus versus load (or versus indentation depth). Further development of the dependence curves enables the load (or indentation depth) to be set to correspond to the point of transition from a single structure component to the composite material and to determine the properties of both. Comparison of results of CSM measurement with the results of single indentation measurement at a defined load reveals a new perspective on the origin of the indentation size effect in hardmetal coatings. The measurements show that the increase in both the hardness and Young’s modulus with decreasing load is caused mainly by the predominant influence of hard particles in the coatings.     

Bioactive components in forced sweet cherry fruit (Prunus avium L.), antioxidative capacity and allergenic potential as dependent on cultivation under cover

Five early maturing varieties of Prunus avium L. on dwarfing rootstock were forced at Bonn, Germany in spring under transparent plastic cover without additional heating to ripen at a time before field-grown German fruit become available, with better fruit quality than imported sweet cherries; trees grown outside without cover served as control. The cover reduced the incident radiation by up to 54% PAR, UV by up to 22% UV-A and UV-B up to 2% and increased day temperature indoors. Fruit were as firm in the early varieties grown under cover as those in the field and slightly softer than in the late harvesting cultivars. Sugar content was slightly less in four of the five varieties; no differences in acidity and sugar/acid ratio as a taste indicator were found. Fruits were up to 3 mm larger when grown under cover in cvs. ‘Burlat’ and ‘Souvenir’, but no differences were observed in cvs. ‘Earlise’ and ‘Prime Giant’. Fruit of three cultivars, ‘Burlat’, ‘Samba’ and ‘Prime Giant’ can be classified as premium quality with 28 mm–30 mm diameter, when grown under cover. Overall, all cherry fruit were of the market, i.e. consumer- preferred, dark red colouration in line with enhanced anthocyanin contents. In two cultivars, the cover induced healthier fruit. Antioxidative capacity was larger in cv. ‘Samba’, while ascorbic acid (Vitamin C) was increased in cv. ‘Souvenir’; phenols were enhanced in all cultivars and attributed to heat stress. Allergenic protein (Pru av 1) in cherry fruits of both cultivation types were below the detection level at harvest time, indicating that cultivation under cover had no adverse effects on this particular health component.Overall, forcing successfully resulted in 2 weeks earlier ripening, thereby providing the first fresh and healthy (low allergen, high phenolic compounds and high anthocyanin) German cherries of high fruit quality on the market in spring.     

Preparation of immobilized Trametes pubescens laccase on a cryogel-type polymeric carrier and application of the biocatalyst to apple juice phenolic compounds oxidation

A new biocatalyst was prepared by the immobilization of a Trametes pubescens laccase, into a wide-pore poly(vinyl alcohol) cryogel. The known enzyme was produced and purified by the previously described procedure. The resulted laccase (yield 40%) has an activity of 46.4 U mg⁻¹ and 12.51 mg mL⁻¹ protein content. The enzyme was subsequently immobilized in a functionalized macroporous cryogel beads by a covalent immobilization technique. The time dependence of the immobilization process and the enzyme loading of the carrier material (5.2 mg g⁻¹ cryogel) were determined by measuring the decrease of protein amount in the enzyme solution. In conversion experiments, a higher stability of the immobilized biocatalyst compared to the free enzyme was evidenced. Steady-state kinetic characterization of four phenols (catechol, caffeic and chlorogenic acids, and catechin) has been performed with free and immobilized laccase, the catalytic parameters being determined and compared. The effect of both laccases (free and immobilized) on the phenol content of retailed apple juice samples, having the same initial composition, was also investigated by working in batch conversion. The variation in phenolic compound content has been compared with that of an untreated apple juice sample having initially the same content of phenolic compounds. A number of advantages resulted in using the immobilized laccase for the apple juice treatment (conservation to some extent of enzyme activity, higher content of phenols preserved, easy separation of the enzyme from the apple juice, therefore avoiding the possible unhealthy effects due to the remaining protein, etc.).     

A variant human IgG1-Fc mediates improved ADCC

Ribosome display was applied to the Fc region of human immunoglobulin G (IgG1) to select for improved binding to human FcγRIIIa, the receptor expressed on human natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC). A library of human Fcγ1 variants was generated using error-prone polymerase chain reaction, and subjected to multiple rounds of ribosome display selection against progressively decreasing concentrations of soluble human FcγRIIIa, to enrich for improved binders. Radioimmunoassay and alphascreen analyses of the aglycosylated IgG-Fc output revealed variants with improved binding to FcγRIIIa relative to wild-type IgG-Fc. Subsequent expression in human (HEK-EBNA) cells generated glycosylated IgGs with modified activity in ADCC assays. One particular variant, 125_B01 triggered enhanced ADCC (EC(50) up to four-fold reduced with increased maximal lysis) relative to wild-type antibody, having more equal levels of ADCC for each allotype (V158/F158) of FcγRIIIa. Deconvolution of individual replacements within the variant showed that improved function arose from the Phe243Leu replacement within the CH2 domain, rather than the CH3 domain replacements Thr393Ala or His433Pro. Surprisingly, the oligosaccharide profiles of 125_B01 indicated more oligosaccharide chains lacking fucose, or with bisecting N-acetylglucosamine relative to wild-type IgG1, which correlates with improved function and the replacement Phe243Leu that is a carbohydrate contact residue within the C(H)2 domain.     

A transparent hybrid of nanocrystalline cellulose and amorphous calcium carbonate nanoparticles

Nanocellulose hybrids are promising candidates for biodegradable multifunctional materials. Hybrids of nanocrystalline cellulose (NCC) and amorphous calcium carbonate (ACC) nanoparticles were obtained through a facile chemical approach over a wide range of compositions. Controlling the interactions between NCC and ACC results in hard, transparent structures with tunable composition, homogeneity and anisotropy.