Oxylipin profiling for clinical research: Current status and future perspectives.

Oxylipins are potent lipid mediators with increasing interest in clinical research. They are usually measured in systemic circulation and can provide a wealth of information regarding key biological processes such as inflammation, vascular tone, or blood coagulation. Although procedures still require harmonization to generate comparable oxylipin datasets, performing comprehensive profiling of circulating oxylipins in large studies is feasible and no longer restricted by technical barriers. However, it is essential to improve and facilitate the biological interpretation of complex oxylipin profiles to truly leverage their potential in clinical research. This requires regular updating of our knowledge about the metabolism and the mode of action of oxylipins, and consideration of all factors that may influence circulating oxylipin profiles independently of the studied disease or condition. This review aims to provide the readers with updated and necessary information regarding oxylipin metabolism, their different forms in systemic circulation, the current limitations in deducing oxylipin cellular effects from in vitro bioactivity studies, the biological and technical confounding factors needed to consider for a proper interpretation of oxylipin profiles.

Effect of Peptides on the Synthesis, Properties and Wound Healing Capacity of Silver Nanoparticles.

The aim of this study is the synthesis of novel peptide-silver nanoparticle conjugates with enhanced wound healing capacity. Peptide-silver nanoparticle conjugates were synthesized using myristoyl tetrapeptide 6 (MT6) or copper tripeptide 1 (CuTP1). Peptide-free silver nanoparticles (AgNP) were synthesized using NaBH4 and sodium citrate and were used as control. The addition of the peptides during or after the synthesis of nanoparticles and its impact on the properties of the synthesized peptide-silver nanoparticle conjugates were assessed. The monitoring of the synthesis of nanoparticles was achieved using ultraviolet-visible spectrophotometry (UV-/Vis). The characteristics and colloidal stability of the nanoparticles (size and ζ-potential distribution, morphology, composition and structure) were monitored using dynamic laser scattering (DLS), transmission electron microscopy (TEM), atomic absorption spectroscopy (AAS) and X-ray diffraction (XRD). The wound healing capacity of the peptide-silver nanoparticle conjugates was assessed using scratch test assay on fibroblasts (NIH/3T3). The results indicated that the addition of the peptides during the synthesis of nanoparticles lead to better yield of the reaction and more effective capping while the size distribution and ζ-potential of the conjugates indicated long-term colloidal stability. The MT6-AgNP conjugate exhibited 71.97 ± 4.35% wound closure, which was about 5.48-fold higher (p < 0.05) than the corresponding free MT6. The CuTP1-AgNP conjugate exhibited 62.37 ± 18.33% wound closure that was better by 2.82 fold (p < 0.05) compared to the corresponding free CuTP1. Both peptides led to the synthesis of silver nanoparticle conjugates with enhanced wound healing capacity compared to the respective free peptide or to the peptide-free AgNP (29.53 ± 4.71% wound closure, p < 0.05). Our findings demonstrated that the synthetized peptide-silver nanoparticle conjugates are promising ingredients for wound care formulation.

Selenium, Selenoproteins and 10-year Cardiovascular Risk: Results from the ATTICA Study.

BACKGROUND: Selenium (Se) is an essential trace element that is involved in several pathophysiological functions. The relationship of Se with cardiovascular disease remains inconclusive, especially regarding the role of different selenospecies. OBJECTIVE: The present study assessed the levels of Se distribution in plasma selenoproteins, namely glutathione peroxidase 3 (GPx3), selenoprotein P (SelP) and selenoalbumin (SeAlb) and total Se in selenoproteins in relation to 10-year cardiovascular risk in the ATTICA prospective study. METHODS: A sub-sample from the ATTICA Study's database, consisting of 278 subjects (114 women and 164 men) with data on Se and selenoproteins levels, was considered. SeGPx3, SelP, and SeAlb in human plasma were simultaneously determined by high-performance liquid chromatography (HPLC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) at baseline. The duration of the follow-up was 8.74 ±2.36 years (mean± standard deviation) and cardiovascular outcomes were recorded. Cox proportional hazards models were applied with total Se or selenoprotein Se as independent variables adjusted for several covariates. RESULTS: Total Se in selenoproteins was positively related to 10-year relative risk of cardiovascular disease (Hazard Ratios of 3rd vs 2nd tertile 10.02, 95% CI:1.15, 92.34). Subjects with high Se but low SeGPx3, as identified by discordant percentiles in the distribution of SeGPx3 and Se, had a higher cardiovascular risk. CONCLUSION: The differentiated effects of circulating selenoproteins on cardiovascular disease risk in the present study, suggest the importance of redox regulation by specific selenoproteins.

A simple and robust LC-ESI single quadrupole MS-based method to analyze polyphenols in plant extracts using deep eutectic solvents.

Currently, the interest in polyphenols is increasing due to their significant properties in health. Polyphenols exist in a range of natural products, however their extraction as well as their characterization are important issues as they are mainly present in complex matrices. Therefore, sensitive and selective analytical methods based on liquid chromatography coupled to tandem mass spectrometry are essential. Nevertheless, access to such high-resolution techniques is quite rare. Thus, in this work we present a simple, selective and robust method based on a single-quadrupole (Q) MS technique) for the analysis of a wide range of polyphenols such as flavonoids, phenolic acids and anthocyanins. Specifically, we present:•A simple liquid chromatography electro-spray ionization (LC-ESI) single-quadrupole mass selective (MS) method for the analysis of 18 different polyphenols.•Application of the method to three plant-based extracts that are derived after green extraction methods.

Probing the effects of sweet cherry (Prunus avium L.) extract on 2D and 3D human skin models.

BACKGROUND: Natural products are not only positioned in the heart of traditional medicine but also in modern medicine as many current drugs are coming from natural sources. Apart from the field of medicine and therapeutics, natural products are broadly used in other industrial fields such as nutrition, skincare products and nanotechnology. METHODS AND RESULTS: The aim of this study was to assess the effects of sweet cherry (Prunus avium L.) fruit extract from the Greek native cultivar 'Vasiliadi', on the human 2D and 3D in vitro models in order to investigate its potential impact on skin. We focused on 2D culture of primary normal human epidermal keratinocytes (NHEK) that were treated with sweet cherry fruit extract. In the first place, we targeted fruit extract potential cytotoxicity by determining ATP intracellular levels. Furthermore, we assessed its potential skin irritability by using 3D skin model. To better understand the bioactivity of sweet cherry fruit. extract, we used qPCR to study the expression of various genes that are implicated in the skin functions. Our experiments showed that sweet cherry fruit extract is non-toxic in 2D keratinocytes culture as well as non-irritant in 3D skin model. Our results revealed that the extract mediated important pathways for the optimum epidermis function such as cell proliferation, immune and inflammatory response. CONCLUSION: The sweet cherry fruit extracts possesses significant activity in epidermis function without any potential of cytotoxicity or skin irritability, which makes it a rather promising active agent for skincare.

Research Techniques Made Simple: Lipidomic Analysis in Skin Research.

Although lipids are crucial molecules for cell structure, metabolism, and signaling in most organs, they have additional specific functions in the skin. Lipids are required for the maintenance and regulation of the epidermal barrier, physical properties of the skin, and defense against microbes. Analysis of the lipidome-the totality of lipids-is of similar complexity to those of proteomics or other omics, with lipid structures ranging from simple, linear, to highly complex structures. In addition, the ordering and chemical modifications of lipids have consequences on their biological function, especially in the skin. Recent advances in analytic capability (usually with mass spectrometry), bioinformatic processing, and integration with other dermatological big data have allowed researchers to increasingly understand the roles of specific lipid species in skin biology. In this paper, we review the techniques used to analyze skin lipidomics and epilipidomics.

Cartilage Acidic Protein a Novel Therapeutic Factor to Improve Skin Damage Repair?

Fish skin has been gaining attention due to its efficacy as a human-wound-treatment product and to identify factors promoting its enhanced action. Skin fibroblasts have a central role in maintaining skin integrity and secrete extra cellular matrix (ECM) proteins, growth factors and cytokines to rapidly repair lesions and prevent further damage or infection. The effects on scratch repair of the ubiquitous but poorly characterized ECM protein, cartilage acidic protein 1 (CRTAC1), from piscine and human sources were compared using a zebrafish SJD.1 primary fibroblast cell line. A classic in vitro cell scratch assay, immunofluorescence, biosensor and gene expression analysis were used. Our results demonstrated that the duplicate sea bass Crtac1a and Crtac1b proteins and human CRTAC-1A all promoted SJD.1 primary fibroblast migration in a classic scratch assay and in an electric cell impedance sensing assay. The immunofluorescence analysis revealed that CRTAC1 enhanced cell migration was most likely caused by actin-driven cytoskeletal changes and the cellular transcriptional response was most affected in the early stage (6 h) of scratch repair. In summary, our results suggest that CRTAC1 may be an important factor in fish skin promoting damage repair.

Drought induces variation in the DNA methylation status of the barley HvDME promoter.

Cytosine methylation is an epigenetic modification with essential roles in diverse plant biological processes including vegetative and reproductive development and responsiveness to environmental stimuli. A dynamic process involving DNA methyltransferases and DNA demethylases establishes cytosine DNA methylation levels and distribution along the genome. A DNA demethylase gene from barley (Hordeum vulgare), DEMETER (HvDME), the homologue of the Arabidopsis thaliana DME (AtDME), has been characterized previously and found to respond to drought conditions. Here, the promoter of the HvDME gene was analysed further by in silico and DNA methylation analysis. The effect of drought conditions on the DNA methylation status of HvDME was investigated at single-cytosine resolution using bisulfite sequencing. It was demonstrated that the HvDME promoter can be divided into two discrete regions, in terms of DNA methylation level and density; a relatively unmethylated region proximal to the translational start site that is depleted of non-CG (CHG, CHH) methylation and another distal region, approximately 1500 bp upstream of the translational start site, enriched in CG, as well as non-CG methylation. Drought stress provoked alterations in the methylation status of the HvDME promoter distal region, whereas the DNA methylation of the proximal region remained unaffected. Computational analysis of the HvDME promoter revealed the presence of several putative regulatory elements related to drought responsiveness, as well as transposable elements (TEs) that may affect DNA methylation. Overall, our results expand our investigations of the epigenetic regulation of the HvDME gene in response to drought stress in barley and may contribute to further understanding of the epigenetic mechanisms underlying abiotic stress responses in barley and other cereals.

Deciphering the role of cartilage protein 1 in human dermal fibroblasts: a transcriptomic approach.

Cartilage acidic protein 1A (hCRTAC1-A) is an extracellular matrix protein (ECM) of human hard and soft tissue that is associated with matrix disorders. The central role of fibroblasts in tissue integrity and ECM health made primary human dermal fibroblasts (NHDF) the model for the present study, which aimed to provide new insight into the molecular function of hCRTAC1-A. Specifically, we explored the differential expression patterns of specific genes associated with the presence of hCRTAC1-A by RNA-seq and RT-qPCR analysis. Functional enrichment analysis demonstrated, for the very first time, that hCRTAC1-A is involved in extracellular matrix organization and development, through its regulatory effect on asporin, decorin, and complement activity, in cell proliferation, regeneration, wound healing, and collagen degradation. This work provides a better understanding of putative hCRTAC1-A actions in human fibroblasts and a fundamental insight into its function in tissue biology.

The pleiotropic effects of Prunus avium L. extract against oxidative stress on human fibroblasts. An in vitro approach.

There is a persistent interest in innovative and multifunctional ingredients in biology research. With regards to this, natural sources have an important role due to their multiple benefits. Thus, this study aims to present the pleiotropic activity of Prunus avium L. extract on human primary fibroblasts for proving its efficacy in dermis-related processes. We focused on the safety and efficacy assessments based on cytotoxicity and gene expression analysis under oxidative stress. Specifically, Prunus avium L. extract was proved non-cytotoxic in human fibroblasts. The gene expression analysis unveiled that this extract has in vitro protective properties on human dermal fibroblasts under oxidative stress related to antioxidant activity, anti-inflammatory response, cell proliferation and cell- aging. Our study demonstrated for the very first time that the Prunus avium L. extract is a multifunctional ingredient as it mediates several human dermis-related in vitro processes highlighting its potential to be used as an active ingredient in skin care products.

Tracing skin aging process: a mini- review of in vitro approaches.

Skin is a rather complex, yet useful organ of our body. Besides, skin aging is a complicated process that gains a growing interest as mediates many molecular processes in our body. Thus, an efficient skin model is important to understand skin aging function as well as to develop an effective innovative product for skin aging treatment. In this mini review, we present in vitro methods for assessments of skin aging in an attempt to pinpoint basic molecular mechanisms behind this process achieving both a better understanding of aging function and an effective  evaluation of potential products or ingredients that counteract aging. Specifically, this study presents in vitro assays such as 2D or 3D skin models, to evaluate skin aging-related processes such as skin moisturization, photoaging, wound healing, menopause, and skin microbiome as novel efforts in the designing of efficacy assessments in the development of skincare products.

A New Controlled Release System for Propolis Polyphenols and Its Biochemical Activity for Skin Applications.

Propolis is a resinous substance produced by bees that exhibits antimicrobial, immunostimulatory and antioxidant activity. Its use is common in functional foods, cosmetics and traditional medicine despite the fact that it demonstrates low extraction yields and inconsistency in non-toxic solvents. In this work, a new encapsulation and delivery system consisting of liposomes and cyclodextrins incorporating propolis polyphenols has been developed and characterized. The antioxidant, antimutagenic and antiaging properties of the system under normal and UVB-induced oxidative stress conditions were investigated in cultured skin cells and/or reconstituted skin model. Furthermore, the transcript accumulation for an array of genes involved in many skin-related processes was studied. The system exhibits significant polyphenol encapsulation efficiency, physicochemical stability as well as controlled release rate in appropriate conditions. The delivery system can retain the anti-mutagenic, anti-oxidative and anti-ageing effects of propolis polyphenols to levels similar and comparable to those of propolis methanolic extracts, making the system ideal for applications where non-toxic solvents are required and controlled release of the polyphenol content is desired.

In vitro protective effects of Paeonia mascula subsp. hellenica callus extract on human keratinocytes.

Natural ingredients have been used to improve the state of health in humans. The genus Paeonia has been studied only limited yet it's reported to have many activities such as antioxidant and anti-inflammatory. To this context, here we focused on an endemic Paeonia species in Attica. This study aims to present the development of the Paeonia mascula subsp. hellenica callus extract and its pleiotropic bioactivity on human primary keratinocytes exploring its potential application as an active agent in skin-related products. This extract showed a high scavenging activity with high phenolic content and an interesting metabolic profile. At a molecular level, the study on the transcript accumulation of genes revealed that this extract exhibits in vitro skin-related protection properties by mediating mitochondrial energy, cell proliferation, immune and inflammatory response and positively regulates genes involved in epidermal and in stratum corneum function. Besides, the extract is proven not skin irritant on reconstructed human skin model. These findings indicate that the specific P. mascula subsp. hellenica extract possesses significant in vitro protection activity on human epidermis and provides new insights into its beneficial role in skin confirming that the advent of biotechnology contribution the past few decades.

Marine Fungus Aspergillus chevalieri TM2-S6 Extract Protects Skin Fibroblasts from Oxidative Stress.

The strain Aspergillus chevalieri TM2-S6 was isolated from the sponge Axinella and identified according to internal transcribed spacer (ITS) molecular sequence homology with Aspergillus species from the section Restricti. The strain was cultivated 9 days on potato dextrose broth (PDB), and the medium evaluated as antioxidant on primary normal human dermal fibroblasts (NHDF). The cultivation broth was submitted to sterile filtration, lyophilized and used without any further processing to give the Aspergillus chevalieri TM2-S6 cultivation broth ingredient named ACBB. ACCB contains two main compounds: tetrahydroauroglaucin and flavoglaucin. Under oxidative stress, ACCB showed a significant promotion of cell viability. To elucidate the mechanism of action, the impact on a panel of hundreds of genes involved in fibroblast physiology was evaluated. Thus, ACCB stimulates cell proliferation (VEGFA, TGFB3), antioxidant response (GPX1, SOD1, NRF2), and extracellular matrix organization (COL1A1, COL3A1, CD44, MMP14). ACCD also reduced aging (SIRT1, SIRT2, FOXO3). These findings indicate that Aspergillus chevalieri TM2-S6 cultivation broth exhibits significant in vitro skin protection of human fibroblasts under oxidative stress, making it a potential cosmetic ingredient.

Transcriptional and epigenetic effects of Vitis vinifera L. leaf extract on UV-stressed human dermal fibroblasts.

Adverse environmental conditions such as UV radiation induce oxidative and aging events leading to severe damage to human skin cells. Natural products such as plant extracts have been implicated in the skin anti-oxidant and anti-aging cellular protection against environmental stress. Moreover, environmental factors have been shown to impact chromatin structure leading to altered gene expression programs with profound changes in cellular functions. In this study, we assessed the in vitro effect of a leaf extract from Vitis vinifera L. on UV-stressed primary human dermal fibroblasts, focusing on gene expression and DNA methylation as an epigenetic factor. Expression analysis of two genes known to be implicated in skin anti-aging, SIRT1and HSP4, demonstrated significant induction in the presence of the extract under normal or UVA conditions. In addition, DNA methylation profiling of SIRT1 and HSP47 promoters showed that the V. vinifera L. extract induced changes in the DNA methylation pattern of both genes that may be associated with SIRT1 and HSP47 gene expression. Our study shows for the first time transcriptional and DNA methylation alterations on human skin fibroblasts exposed to UV stress and suggest a protective effect of a V. vinifera extract possibly through transcriptional regulation of critical skin anti-aging genes.

In vitro protective effects of marine-derived Aspergillus puulaauensis TM124-S4 extract on H2O2-stressed primary human fibroblasts.

Nowadays, there is a huge interest in natural products obtained from marine organisms that can promote human health.The aim of the present study is to evaluate for the first time, the in vitro effects of marine Aspergillus puulaauensis TM124-S4 extract against oxidative stress in human fibroblasts, and its potential as a cosmetic ingredient. The strain was isolated from the Mediterranean Sea star, Echinaster sepositus, and identified according to ITS molecular sequence homology as a member of Aspergillus section versicolores.To gain insight on the bioactivity underpinning the effects of TM124-S4 extract on oxidative stress, we examined a panel of a hundred genes as well as cell viability. Initially, Aspergillus puulaauensis TM124-S4 promoted cell viability.The change in gene transcripts revealed that Aspergillus puulaauensis TM124-S4 extracts exhibited skin protection properties by mediating cell proliferation (EPS8, GDF15, CASP7, VEGFA), antioxidant response (CAT, SOD1, TXN, GPX1), skin hydration (CD44, CRABP2, SERPINE) and DNA repair (PCNA, P21). The extract also modulated the expression of genes involved in skin pigmentation and aging (TYR, FOXO3).These findings indicate that Aspergillus puulaauensis TM124-S4 extract possesses significant in-vitro skin protection activity against induced oxidative stress.Furthermore, new insights are provided into the beneficial role of fungal bioactive compounds in skin related research.

Cartilage acidic protein 1 promotes increased cell viability, cell proliferation and energy metabolism in primary human dermal fibroblasts.

Cartilage acidic protein 1 (CRTAC1) is an extracellular matrix protein of human chondrogenic tissue that is also present in other vertebrates, non-vertebrate eukaryotes and in some prokaryotes. The function of CRTAC1 remains unknown but the protein's structure indicates a role in cell-cell or cell-matrix interactions and calcium-binding. The aim of the present study was to evaluate the in vitro effects of hCRTAC1-A on normal human dermal fibroblasts (NHDF). A battery of in vitro assays (biochemical and PCR), immunofluorescence and a biosensor approach were used to characterize the protein's biological activities on NHDF cells in a scratch assay. Gene expression analysis revealed that hCRTAC1-A protein is associated with altered levels of expression for genes involved in the processes of cell proliferation (CXCL12 and NOS2), cell migration (AQP3 and TNC), and extracellular matrix-ECM regeneration and remodeling (FMOD, TIMP1, FN1) indicating a role for hCRTAC1-A in promoting these activities in a scratch assay. In parallel, the candidate processes identified by differential gene transcription were substantiated and extended using Electric cell-substrate impedance sensing (ECIS) technology, immunofluorescence and cell viability assays. Our findings indicate that hCRTAC1-A stimulated cell proliferation, migration and ECM production in primary human fibroblasts in vitro.

Genomic and phylogenetic analysis of choriolysins, and biological activity of hatching liquid in the flatfish Senegalese sole.

The hatching enzymes or choriolysins are key proteases in fish life cycle controlling the release of larvae to surrounding environment that have been suggested as target for novel biotechnological uses. Due to the large amounts of eggs released by the flatfish Solea senegalensis, during the spawning season, the hatching liquid properties and choriolysin-encoding genes were investigated in this species. A genomic analysis identified four putative genes referred to as SseHCEa, SseHCEb, SseLCE and SseHE. The phylogenetic analysis classified these paralogs into two clades, the clade I containing SseHCE paralogs and the clade II containing two well-supported subclades named as HE and LCE. The two SseHCE paralogs were intron-less and both genes were tandemly arrayed very close in the genome. The synteny and gene rearrangement identified in the flatfish lineage indicated that the duplication of these two paralogs occurred recently and they are under divergent evolution. The genes SseHE and SseLCE were structured in 8 exons and 7 introns and the synteny was conserved in teleosts. Expression studies confirmed that the four genes were expressed in the hatching gland cells and they migrate co-ordinately from the head to around the yolk sac close to the hatch with specific temporal and intensity expression profiles. Although the mRNA levels of the four genes peaked in the hours previous to larval hatching, the SseHCE and SseLCE paralogs kept a longer expression than SseHE after hatching. These expression patterns were consistent even when larvae were incubated at different temperatures that modified hatching times. The analysis of hatching-liquid using SDS-PAGE and zymography analyses of hatching liquid identified a major band of expected choriolysin size. The optimal pH for protease activity was 8.5 and inhibition assays using EDTA demonstrated that most of the activity in the hatching liquid was due to metalloproteases with Ca2+ ions acting as the most effective metal to restore the activity. All these data provide new clues about the choriolysin evolution and function in flatfish with impact in the aquaculture and the blue cosmetic industry.