A Simple Method for the Production of Human Skin Equivalent in 3D, Multi-Cell Culture.

An important problem for researchers working in the field of dermatology is the preparation of the human skin equivalent (HSE). Here, we describe a simple and reliable protocol for preparing a skin model from the commercially available cell lines: keratinocytes, fibroblasts, and melanocytes. Importantly, in our 3D model, the keratinocytes are diverse that brings this model closer to the natural skin. For the production of HSE, we used available primary PCS-200-010, PCS-201-010, PCS-200-013, and immortalized CRL-4048 and CRL-4001 cell lines. We used genipin, which is necessary for collagen cross-linking and studied its cytotoxicity for keratinocytes and fibroblasts. The addition of 20 µM genipin reduced the shrinkage of the collagen in the constructs from 59% to 24% on day 12 of the culture of the construct. A higher concentration (80-200 µM) of genipin reduced shrinkage by 14% on average. Genipin in concentration 10 µM and below was not cytotoxic to the keratinocytes, and 150 µM and below to the fibroblasts. Hematoxylin and eosin staining showed that the morphology of HSEs was identical to that of native human skin. The immunohistochemical staining of the constructs showed the presence of vimentin-positive fibroblasts in the skin layer, while the melanocytes were in the epidermis and in the basal layer. We observed that the longer differentiation of constructs led to the higher secretion of GM-CSF, IL-10, IL-15, IL-1α, IL-6, IL-7, IL-8, and MCP-1. We also observed that the longer time of differentiation led to a more stable secretion of all analytes, which was reflected in the coefficient of variation. We described here a simple, reliable, and cost-effective production of the full-thickness human skin equivalents that can be used in the research and industry. With the global trend to decrease animal use for the research and testing, our HSE could be a useful testing tool and an alternative research model.

Vitamin B6 improves blood parameters in rats fed a protein-deficient diet and subjected to moderate, long-term exercise.

Vitamin B6 is necessary for many enzymatic pathways (glucose and lipid metabolism, DNA/RNA synthesis, or modulation of gene expression) and affects immune cell function and blood-forming processes. We hypothesised that supplementing a protein-deficient diet with vitamin B6 may reduce the negative impact of protein malnutrition. Here, we evaluated the effect of moderate, long-term exercise (ninety days) on selected blood parameters in rats fed a normal diet, a protein-deficient diet, or a protein-deficient diet supplemented with vitamin B6. Selected haematological, immunological, and biochemical parameters were examined. A protein-deficient diet lasting 90 days caused significant reduction in body mass, increased activity of aminotransferases (asparagine and alanine), an increased percentage of innate cells in the blood, and decreased haemoglobin concentration in the blood. Adding vitamin B6 significantly increased body and muscle mass, decreased liver parameters, and caused normalisation of haemoglobin concentration and the proportion of white blood cells in the blood. These results indicate that vitamin B6 supplementation significantly improves the health of protein-malnourished rats and paves the way for the development of novel anti-malnutrition therapies.

Mitotic timing is differentially controlled by A- and B-type cyclins and by CDC6 associated with a bona fide CDK inhibitor Xic1 in Xenopus laevis cell-free extract.

The timing of the M-phase is precisely controlled by a CDC6-dependent mechanism inhibiting the mitotic histone H1 kinase. Here, we describe the differential regulation of the dynamics of this mitotic kinase activity by exogenous cyclin A or cyclin B in the Xenopus laevis cycling extracts. We show that the experimental increase in cyclin A modifies only the level of histone H1 kinase activity, while the cyclin B increase modifies two parameters: histone H1 kinase activity and the timing of its full activation, which is accelerated. On the other hand, the cyclin A depletion significantly delays full activation of histone H1 kinase. However, when CDC6 is added to such an extract, it inhibits cyclin B-associated histone H1 kinase, but does not modify the mitotic timing in the absence of cyclin A. Further, we show via p9 co-precipitation with Cyclin-Dependent Kinases (CDKs), that both CDC6 and the bona fide CDK1 inhibitor Xic1 associate with the mitotic CDKs. Finally, we show that the Xic1 temporarily separates from the mitotic CDKs complexes during the peak of histone H1 kinase activity. These data show the differential coordination of the M-phase progression by cyclin A- and cyclin B-dependent CDKs, confirm the critical role of the CDC6-dependent histone H1 kinase inhibition in this process, and show that CDC6 acts differentially through the cyclin B- and cyclin A-associated CDKs. This CDC6- and cyclins-dependent mechanism likely depends on the precisely regulated association of Xic1 with the mitotic CDKs complexes. We postulate that: i. the dissociation of Xic1 from the CDKs complexes allows the maximal activation of CDK1 during the M-phase, ii. the switch between cyclin A- and cyclin B-CDK inhibition upon M-phase initiation may be responsible for the diauxic growth of mitotic histone H1 kinase activity.

In vitro Antioxidant, Anti-inflammatory, Anti-metabolic Syndrome, Antimicrobial, and Anticancer Effect of Phenolic Acids Isolated from Fresh Lovage Leaves [Levisticum officinale Koch] Elicited with Jasmonic Acid and Yeast Extract.

Lovage seedlings were elicited with jasmonic acid (JA) and yeast extract (YE) to induce the synthesis of biologically active compounds. A simulated digestion process was carried out to determine the potential bioavailability of phenolic acids. Buffer extracts were prepared for comparison. The ability to neutralize ABTS radicals was higher in all samples after the in vitro digestion, compared to that in the buffer extracts. However, the elicitation resulted in a significant increase only in the value of the reduction power of the potentially bioavailable fraction of phenolic acids. The effect of the elicitation on the activity of the potentially bioavailable fraction of phenolic acids towards the enzymes involved in the pathogenesis of the metabolic syndrome, i.e., ACE, lipase, amylase, and glucosidase, was analyzed as well. The in vitro digestion caused a significant increase in the ability to inhibit the activity of these enzymes; moreover, the inhibitory activity against alpha-amylase was revealed only after the digestion process. The potential anti-inflammatory effect of the analyzed extracts was defined as the ability to inhibit key pro-inflammatory enzymes, i.e., lipoxygenase and cyclooxygenase 2. The buffer extracts from the YE-elicited lovage inhibited the LOX and COX-2 activity more effectively than the extracts from the control plants. A significant increase in the anti-inflammatory and antimicrobial properties was noted after the simulated digestion.

Different Temperature Treatments of Millet Grains Affect the Biological Activity of Protein Hydrolyzates and Peptide Fractions.

The objective of this study was to analyze millet protein hydrolyzates and peptide fractions with molecular mass under 3.0 kDa obtained from grains treated with different temperature values as inhibitors of angiotensin-converting enzyme (ACE), α-amylase, and α-glucosidase activity. The protein fractions were hydrolyzed in vitro in gastrointestinal conditions and the highest degree of hydrolysis was noted for globulin 7S obtained from control grains (98.33%). All samples were characterized by a high peptide bioaccessibility index, which was 23.89 for peptides obtained from globulin 11S after treatment with 100 °C. The highest peptide bioavailability index was noted for peptides obtained from globulin 11S after the treatment with 65 °C (2.12). The highest potential metabolic syndrome inhibitory effect was determined for peptide fractions obtained from the prolamin control (IC50 for ACE and α-amylase was 0.42 and 0.11 mg/mL, respectively) and after the 100 °C treatment (IC50 for ACE and α-glucosidase was 0.33 and 0.12 mg/mL, respectively) and from globulin 11S after the 65 °C treatment (IC50 0.38 and 0.05 for ACE and α-glucosidase, respectively). The effect of these samples on endothelial cell HECa10 was determined. The sequences of potential inhibitory peptides were identified as GEHGGAGMGGGQFQPV, EQGFLPGPEESGR, RLARAGLAQ, YGNPVGGVGH, and GNPVGGVGHGTTGT.