Scratch Closure Research Articles

Role of FOXM1 and AURKB in regulating keratinocyte function in psoriasis.

This study investigated the effect of forkhead box M1 (FOXM1) and Aurora kinase B (AURKB) on the epidermal function of keratinocytes. Bioinformatics analysis was used to analyze the co-expression network of FOXM1 and its correlation with AURKB. The expression of FOXM1 and AURKB in tissues and cells was detected by immunofluorescence and real-time quantitative polymerase chain reaction, respectively. HaCaT cells were transfected with si-FOXM1 to knock down FOXM1. Cell proliferation was detected by cell counting kit-8 assay. Cell migration was detected by scratch assay. Cell invasion was detected by the Transwell invasion assay. Cell apoptosis and cell cycle were detected by flow cytometry. FOXM1 and AURKB were positively correlated and highly expressed in psoriatic lesions. After transfection of si-FOXM1, the expression levels of FOXM1 and AURKB genes significantly decreased. The proliferation of HaCaT cells decreased, the apoptosis rate increased significantly, and the proportion of cells in the G1 phase increased significantly, while the proportion of cells in the S phase decreased significantly. The scratch closure of HaCaT cells was reduced, and the number of cell invasions decreased significantly. FOXM1 and AURKB may affect the progression of psoriasis by regulating the proliferation, cell cycle, migration, and invasion of keratinocytes.

In vitro regenerative effects of a pooled pathogen-reduced lyophilized human cord blood platelet lysate for wound healing applications.

Cord blood platelets, easily obtained from blood units not suitable for haematopoietic stem cell transplantation, represent an abundant source of growth factors for use in wound healing. Although several protocols have been described for platelet lysate production, no standard manufacturing protocol is available. The use of pooled cord blood platelets could thus facilitate standardization. In this study, the effect of varying concentrations (up to 20%) of a pooled pathogen-reduced lyophilized cord blood platelet lysate (PRL-CBPL) was investigated in different cell types involved in the wound healing process. The effect of heparin addition was also evaluated. In parallel, a comparison was performed with a single donor cord blood platelet lysate (SD-CBPL). The effect of PRL-CBPL on the viability and proliferation of different cell lines (L929 mouse fibroblasts and HaCaT keratinocytes) and human primary cells (fibroblasts-NHDF, coronary artery smooth muscle cells-HCASMC and coronary artery endothelial cells-HCAEC), on HaCaT migration and the chemotactic effect on human monocytes (THP-1) was evaluated. PRL-CBPL showed a lower PDGF-AB amount compared to SD-CBPL. Differing concentrations of both CBPL were necessary to influence cell viability and proliferation. 3% was the optimal concentration for L929 and HaCaT as well as for NHDF and HCASMC, while HCAEC required 10%. The effect of added heparin was more evident on SD-CBPL and in particular on NHDF and HCASMC proliferation. Keratinocyte scratch closure was obtained with 3 and 5% PRL-CBPL and SD-CBPL respectively. Both CBPLs caused an increase in the number of migrated THP-1 monocytes in a concentration-dependent manner up to 20% with a higher monocyte migration for SD-CBPL with respect to PRL-CBPL and in cells treated with heparin. The data obtained suggest that PRL-CBPL is an effective standardized alternative to SD-CBPL.

The effect of Jordanian essential oil from coriander seeds on antioxidant, anti-inflammatory, and immunostimulatory activities using RAW 246.7 murine macrophages.

Coriander (Coriandrum sativum L.) is a member of the Umbelliferae/Apiaceae family and one of the well-known essential oil-containing plants, in which the seeds are used in traditional medicine, and as flavoring in food preparation. Knowing the diverse chemical components of different parts of the plant, this work aims to investigate the antioxidant, the anti-inflammatory, and the immunostimulatory modulator effects of the Jordanian C. sativum's seed extracted essential oil (JCEO). Coriander oil extract was prepared by hydro-distillation method using the Clevenger apparatus. Different concentrations of coriander oil were examined by using DPPH radical scavenging assay, MTT assay, pro-inflammatory cytokine (Tumor Necrosis Factor-TNF-alpha) production in RAW264.7 murine macrophages in addition, scratch-wound assessment, NO level examination, Th1/Th2 assay, phagocytosis assay, and fluorescence imaging using DAPI stain were conducted. JCEO had a potential metabolic enhancer effect at a concentration of 0.3 mg/mL on cell viability with anti-inflammatory activities via increasing cytokines like IL-10, IL-4, and limiting NO, INF-γ, and TNF-α release into cell supernatant. Antioxidant activity was seen significantly at higher concentrations of JCEO reaching 98.7% when using 100mg/mL and minimally reaching 50% at 12.5mg/mL of the essential oil. Treated macrophages were able to attain full scratch closure after 48-hrs at concentrations below 0.3mg/mL. The seed-extracted JCEO showed significant free radical scavenging activity even at lower dilutions. It also significantly induced an anti-inflammatory effect via an increase in the release of cytokines but reduced the LPS-induced NO and TNF-α production at 0.16-0.3mg/mL. In summary, coriander essential oil demonstrated antioxidant, anti-inflammatory, and immunostimulatory effects, showcasing its therapeutic potential at specific concentrations. The findings underscore its safety and metabolic enhancement properties, emphasizing its promising role in promoting cellular health.

Biocompatibility, Antimicrobial and Wound Healing Activities of Lavandula dentata Leaves Extract

Background: The human skin is a natural barrier that protects against external stress, but its physiological structure can be compromised when it gets damaged. In the field of medical science, wounds are a significant issue that requires immediate attention. Lavandula species are used in the food, perfume and cosmetics industries due to their antimicrobial, anti-inflammatory and wound-healing properties. Methods: The present study was done to assess the biocompatibility, antimicrobial and wound-healing potential of L. dentata leave methanolic extract (LE) that is cultivated in the Taif region of Saudi Arabia. The biocompatibility of LE was analyzed against the normal human skin fibroblast (HSF) cell line using SRB assay. A quarter of LE IC50 was used for wound healing assay. Finally, Escherichia Coli, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes were used to determine LE antibacterial potential through the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) measurement. Result: LE appears safe on HSF cell line with IC50 = 97 mg/mL. LE decreased wound width, wound area and the mean cell migratory rate of wound border cells during scratch closure, while increased the wound closure% in a time-dependent manner compared to negative control. LE shows antimicrobial potential against all four strains at MIC and MBC greater than 3.0 mg/mL, except for Streptococcus pyogenes MIC= 3.0 mg/mL. In conclusion, LE offers therapeutic benefits against wound healing with an antimicrobial effect, however, higher concentrations of LE should be used to assess its effect.

Endothelial cell specific molecule 1 promotes epithelial-mesenchymal transition of cervical cancer via the E-box binding homeobox 1.

To investigate the mechanism of endothelial cell specific molecule 1 (ESM1) promoting cervical cancer cell proliferation and EMT characteristics through zinc finger E-box binding homeobox 1 (ZEB1)/EMT pathway. The correlation between ESM1 expression and prognosis of cervical cancer patients was analyzed by bioinformatics. SiHa, HeLa cell lines and corresponding control cell lines with stable ESM1 expression were obtained. Cell proliferation ability was detected by CCK-8 assay. The invasion and migration ability of Hela and SiHa cells were detected by Transwell assay and scratch closure assay. Expressions of EMT-related markers E-cadherin and Vimentin were detected by real-time PCR. The ability of silenced ESM1 to tumor formation in vivo was detected by tumor formation in nude mice. The effects of aloe-emodin on inhibit ESM1 expression and its inhibitory effect on cervical cancer cells in vitro and in vivo were analyzed by the same method. ESM1 was highly expressed in cervical cancer, and the high expression of ESM1 was associated with poor prognosis of cervical cancer patients. CCK-8 results showed that the proliferation, invasion and migration of Hela and SiHa cells were significantly reduced after siRNA interfered with ESM1 expression. Overexpression of ESM1 promoted the proliferation and migration of cervical cancer cells. Mechanism studies have shown that the oncogenic effect of ESM1 is realized through the ZEB1/PI3K/AKT pathway. High throughput drug screening found that aloe-emodin can target ESM1. Inhibitory effect of aloe emodin on ESM1/ZEB1/EMT signaling pathway and cervical cancer cells. The silencing of ESM1 expression may inhibit the proliferation, invasion, metastasis and epithelial-mesenchymal transformation of cervical cancer cells by inhibiting ZEB1/PI3K/AKT. Aloe-emodin is a potential treatment for cervical cancer, which can play an anti-tumor role by inhibiting ESM1/ZEB1.

Production and comprehensive characterization of PVA/chitosan transdermal composite mats loaded with bioactive curcumin; evaluation of its release kinetics, antioxidant, antimicrobial, and biocompatibility features

AbstractRecently, researchers have shown increasing interest in incorporating bioactive substances with therapeutic properties into fiber‐structured mat biomaterials, which are favored as tissue scaffolds for wound healing applications. In this study, curcumin (Cur)‐loaded polyvinyl alcohol (PVA)/chitosan (CS) composite mats were produced using the electrospinning method and followed by the freeze‐drying method. Scanning electron microscope images proved the homogeneous structure of the composite mats, and Fourier transform infrared spectroscopy analysis showed that the Cur‐loaded composite mats were successfully produced. The antibacterial activity of Cur‐loaded PVA/CS composite mats was evaluated against Escherichia coli and Staphylococcus aureus, and the results showed that the antibacterial activity of the composite mats increased with the addition of Cur. Furthermore, the antioxidant test, release kinetics tests, and in vitro biocompatibility studies such as cytotoxicity, staining, and scratch assay of Cur‐loaded PVA/CS composite mats were carried out. The results showed that adding Cur enhanced the bioactivity of PVA10/CS10 composite mats. Further, the biocompatibility findings indicated that 10Cur‐PVA10/CS10 exhibited the highest viability value throughout all incubation periods compared with the other samples. Moreover, the highest rate of scratch closure on the 10Cur‐PVA/10/CS10 composite mats was observed at the end of 24 h compared with the other composite mats. These findings indicate that the Cur‐loaded PVA10/CS10 composite mats significantly positively impact cell migration and wound healing, making them a promising candidate as transdermal composite mats for tissue engineering and wound care applications.

Development and functional testing of a novel in vitro delayed scratch closure assay

As the development of chronic wound therapeutics continues to expand, the demand for advanced assay systems mimicking the inflammatory wound microenvironment in vivo increases. Currently, this is performed in animal models or in in vitro cell-based models such as cell culture scratch assays that more closely resemble acute wounds. Here, we describe for the first time a delayed scratch closure model that mimics some features of a chronic wound in vitro. Chronic wounds such as those suffered by later stage diabetic patients are characterised by degrees of slowness to heal caused by a combination of continued localised physical trauma and pro-inflammatory signalling at the wound. To recreate this in a cell-based assay, a defined physical scratch was created and stimulated by combinations of pro-inflammatory factors, namely interferon, the phorbol ester PMA, and lipopolysaccharide, to delay scratch closure. The concentrations of these factors were characterised for commonly used human keratinocyte (HaCaT) and dermal fibroblast (HDF) cell lines. These models were then tested for scratch closure responsiveness to a proprietary healing secretome derived from human Wharton’s jelly mesenchymal stem cells (MSCs) previously validated and shown to be highly effective on closure of acute wound models both in vitro and in vivo. The chronically open scratches from HaCaT cells showed closure after exposure to the MSC secretome product. We propose this delayed scratch closure model for academic and industrial researchers studying chronic wounds looking for responsiveness to drugs or biological treatments prior to testing on explanted patient material or in vivo.

Arsenic Impairs Wound Healing Processes in Dermal Fibroblasts and Mice.

Inorganic arsenic (NaAsO2) is a naturally occurring metalloid found in water resources globally and in the United States at concentrations exceeding the U.S. Environmental Protection Agency Maximum Contamination Level of 10 ppb. While exposure to arsenic has been linked to cancer, cardiovascular disease, and skin lesions, the impact of arsenic exposure on wound healing is not fully understood. Cultured dermal fibroblasts exposed to NaAsO2 displayed reduced migration (scratch closure), proliferation, and viability with a lowest observable effect level (LOEL) of 10 µM NaAsO2 following 24 h exposure. An enrichment of Matrix Metalloproteinase 1 (MMP1) transcripts was observed at a LOEL of 1 µM NaAsO2 and 24 h exposure. In vivo, C57BL/6 mice were exposed to 10 µM NaAsO2 in their drinking water for eight weeks, then subjected to two full thickness dorsal wounds. Wounds were evaluated for closure after 6 days. Female mice displayed a significant reduction in wound closure and higher erythema levels, while males showed no effects. Gene expression analysis from skin excised from the wound site revealed significant enrichment in Arsenic 3-Methyltransferase (As3mt) and Estrogen Receptor 2 (Esr2) mRNA in the skin of female mice. These results indicate that arsenic at environmentally relevant concentrations may negatively impact wound healing processes in a sex-specific manner.

Analysis of the potential of the homeopathic eye drops Marine Cineraria 4CH to induce cell migration

The ocular surface comprises diverse components that collaborate to maintain homeostasis, and any change can be termed an ocular surface disorder. One frequently observed change is the development of corneal ulcers, a primary contributor to visual impairment. Cineraria maritima is a botanical species renowned for its therapeutic application in managing diagnosed cataracts and other eye-related ailments. This study evaluated the effect of the homeopathic eye drops containing Cineraria maritima using cell viability assessments, a scratch experiment, and the quantification of IL-8 levels. The in vitro tests revealed the absence of any cytotoxic effects associated with the eye drops, thus demonstrating their safety for use. Furthermore, these eye drops exhibited the capacity to enhance the percentage of scratch closure. The treated group had an average closed area percentage of 57%, while the control group exhibited only 41% area closure, indicating the potential efficacy of eye drops in managing ocular disorders. Finally, IL-8 levels were measured following the induction of an inflammatory response by LPS and subsequent treatment with the eye drops. The outcomes indicate a decrease in the levels of this cytokine in the treated group. Such findings assure the safety and effectiveness of Cineraria maritima 4CH homeopathic eye drops, suggesting a potential avenue for their application to treat corneal ulcers.

Prostaglandin 15d-PGJ2 inhibits proliferation of lung adenocarcinoma cells by inducing ROS production and activation of apoptosis via sirtuin-1

Lung adenocarcinoma (LUADC) belongs to the most prevalent and lethal cancer types. As 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) displays anti-oxidative, -inflammatory, and -cancer properties, we investigated whether this cyclopentenone PG, a stable degradation end-product of cyclooxygenase-generated PGD2, exerts beneficial effects in three LUADC cell lines (A549, H1299, H23). We here report that 15d-PGJ2 had substantial cytotoxic effects in all three LUADC cell lines by promoting early apoptosis and inhibiting the cell cycle, proliferation, and migration. As indicators of cell malignancy, scratch closure and colony formation were significantly inhibited by 15d-PGJ2. 15d-PGJ2 induced generation of ROS and subsequent activation of MAPKs. Expression of Nrf-2, a well-known tumor driver, was markedly diminished by 15d-PGJ2 treatment. Although PPARγ, DP1, and DP2 are expressed in LUADC cells, blocking these receptors with specific inhibitors (SR16832 and BW245C) did not reverse 15d-PGJ2-mediated cytotoxicity, suggesting receptor-independent effects. 15d-PGJ2 decreased SIRT1 expression in LUADC cells and the knockdown of SIRT1 diminished the cytotoxic effects of 15d-PGJ2. Importantly, 15d-PGJ2 significantly reduced tumor growth using the chorioallantoic membrane (CAM) assay. The structural analog of 15d- PGJ2, 9,10-dihydro-15d-PGJ2 (lacking the α,β-unsaturated ketone structural element), did not show any toxic effects in LUADC cells. Altogether, our findings suggest that 15d-PGJ2 led to significantly reduced tumor growth and cell proliferation in three LUADC cell lines. The CAM assay results suggest that 15d-PGJ2 is a suitable endogenous compound to interfere with LUADC tumor progression. We show that SIRT1 modulates the effects of 15d-PGJ2 and may be used as a therapeutic target for LUADC.

A Robust and Standardized Approach to Quantify Wound Closure Using the Scratch Assay.

The scratch assay is an in vitro assay that allows for high-throughput quantification of wound closure by keratinocytes and fibroblasts with relative ease. However, this assay is amenable to experimental variables, which can result in false-positive and false-negative data, making the interpretation of such data difficult. Also, data variability decreases the sensitivity of the scratch assay. Here, we identify important sources of data variation in the scratch assay and provide rational mitigation strategies that enable robust and highly reproducible quantification of scratch width and area, and ultimately the scratch closure rates. By eliminating these sources of variability, the sensitivity of the scratch assay is enhanced, thereby allowing for identification of dependent variables with wide-ranging impacts on wound closure in a robust and standardized manner.

Procedural Promotion of Wound Healing by Graphene-Barium Titanate Nanosystem with White Light Irradiation.

Wound healing is a continuous and complex process that comprises multiple phases including hemostasis, inflammation, multiplication (proliferation) and remodeling. Although a variety of nanomaterials have been developed to control infection and accelerate wound healing, most of them can only promote one phase but not multiple phases, resulting in lower efficient healing. Although various formulations such as nitric oxide releasing wound dressings were developed for dual action, the nanostructure synthesis and the encapsulation process were complex. Here, we report on the design of graphene-barium titanate nanosystem to procedural promote the wound healing process. The antibacterial effect was assessed in Gram-negative Escherichia coli bacteria (E. coli) and Gram-positive Staphylococcus aureus bacteria (S. aureus), the cell proliferation and migration experiment was investigated in mouse embryonic fibroblast (NIH-3T3) cells, and the wound healing effect was analyzed in female BALB/c mice with infected skin wound on the back. Results showed that graphene-barium titanate nanosystem could generate abundant ROS to kill both E. coli and S. aureus. The growth curves, bacterial viability, colony number formation and scanning electron microscopy (SEM) images of E. coli and S. aureus all confirmed the antibacterial effect. Cell Counting Kit-8 (CCK-8) assay displayed that GBT possesses great biocompatibility. EdU assay showed that GBT plus white light irradiation significantly promoted the proliferation and migration of NIH-3T3 cells. Scratch assay found that GBT could achieve a fast scratch closure compared to the control. In vivo wound healing effect indicates that GBT can accelerate wound repair procedure. GBT nanocomposite is capable of programmatically accelerating wound healing through multiple stages, including production of a large amount of ROS after white light exposure to effectively kill E. coli and S. aureus to prevent wound infection and as a scaffold to accelerate fibroblast proliferation and migration to the wound to accelerate wound healing.

Novel quinoxaline-based VEGFR-2 inhibitors to halt angiogenesis

Vascular endothelial growth factor receptor-2 is a dynamic target for therapeutic intervention in various types of cancer. This study was aimed at exploring the VEGFR-2 inhibitory activity of a novel library of quinoxalin-2-one derivatives such as 3-furoquinoxaline carboxamides, 3-pyrazolylquinoxalines, and 3-pyridopyrimidyl-quinoxalines. Among them, 6c, 7a, and 7d-f produced remarkable cytotoxicity against HCT-116 (IC50′s 4.28–9.31 µM) and MCF-7 (IC50′s 3.57–7.57 µM) cell lines using the MTT assay and doxorubicin (DOX) as a reference standard. Interestingly, results of cytotoxicity towards the human fibroblast cell line WI38 revealed that these hits demonstrated higher selectivity indices towards both HCT-116 (SI 8.69–23.19) and MCF-7 (SI 9.48–27.80) than DOX, SI 0.72 and 0.90, respectively. Then, these hits were subjected to a mechanistic study; they showed direct inhibition of VEGFR-2. Impressively, compound 7f displayed 1.2 times the VEGFR-2 inhibitory activity of sorafenib. The antiangiogenic potential of 7f was proved via lowering the level of VEGF-A, than that of control. It as well, exhibited scratch closure percent of 61.8%, compared with 74.5% of control at 48 hrs, indicating the potential anti-migratory effect of the compound 7f. It significantly increased the expression of tumor suppressor gene (p53) on MCF-7 cells by almost 18 folds and upregulated the caspase-3 level by 10.7 folds, compared to the control. Cell cycle analysis revealed cell cycle arrest at G2/M together with a PreG increase which indicated apoptosis induction potential. Annexin V-FITC apoptosis results proposed the two modes of cell death (apoptosis and necrosis) as an inherent mechanism of cytotoxicity of compound 7f. Molecular docking further supported the mechanism showing the affinity of target compounds for VEGFR-2 active site. Moreover, physicochemical and drug-like properties were assessed from the ADME properties.

Assessment of the Efficacy of an LL-37-Encapsulated Keratin Hydrogel for the Treatment of Full-Thickness Wounds.

Wound healing remains a burdensome healthcare problem due to moisture loss and bacterial infection. Advanced hydrogel dressings can help to resolve these issues by assisting and accelerating regenerative processes such as cell migration and angiogenesis because of the similarities between their composition and structure with natural skin. In this study, we aimed to develop a keratin-based hydrogel dressing and investigate the impact of the delivery of LL-37 antimicrobial peptide using this hydrogel in treating full-thickness rat wounds. Therefore, oxidized (keratose) and reduced (kerateine) keratins were utilized to prepare 10% (w/v) hydrogels with different ratios of keratose and kerateine. The mechanical properties of these hydrogels with compressive modulus of 6-32 kPa and tan δ <1 render them suitable for wound healing applications. Also, sustained release of LL-37 from the keratin hydrogel was achieved, which can lead to superior wound healing. In vitro studies confirmed that LL-37 containing 25:75% of keratose/kerateine (L-KO25:KN75) would result in significant fibroblast proliferation (∼85% on day 7), adhesion (∼90 cells/HPF), and migration (73% scratch closure after 12 h and complete closure after 24 h). Also, L-KO25:KN75 is capable of eradicating both Gram-negative and Gram-positive bacteria after 18 h. According to in vivo assessment of L-KO25:KN75, wound closure at day 21 was >98% and microvessel density (>30 vessels/HPF at day 14) was significantly superior in comparison to other treatment groups. The mRNA expression of VEGF and IL-6 was also increased in the L-KO25:KN75-treated group and contributed to proper wound healing. Therefore, the LL-37-containing keratin hydrogel ameliorated wound closure, and also angiogenesis was enhanced as a result of LL-37 delivery. These results suggested that the L-KO25:KN75 hydrogel could be a sustainable substitute for skin tissue regeneration in medical applications.

Balloon Flower Root-Derived Extracellular Vesicles: In Vitro Assessment of Anti-Inflammatory, Proliferative, and Antioxidant Effects for Chronic Wound Healing.

Excessive reactive oxygen species (ROS) in wound lesions can lead to oxidative stress and failure of normal wound healing processes, eventually resulting in chronic skin wounds. A multitude of researchers have investigated various natural products with physiological activities, including antioxidant effects, for healing chronic skin wounds. Balloon flower root (BFR), which contains bioactive components such as platycodins, is known for its anti-inflammatory and antioxidant effects. In this study, we isolated BFR-derived extracellular vesicles (BFR-EVs) that possess anti-inflammatory, proliferative, and antioxidant activities via a combination of polyethylene glycol-based precipitation and ultracentrifugation. Our objective was to investigate the potential of BFR-EVs in treating chronic wounds caused by ROS. Despite efficient intracellular delivery, BFR-EVs showed no significant cytotoxicity. In addition, BFR-EVs inhibited the expression of pro-inflammatory cytokine genes in lipopolysaccharide-stimulated RAW 264.7 cells. Furthermore, water-soluble tetrazolium salt-8 assay showed that BFR-EVs had a proliferation-promoting effect on human dermal fibroblasts (HDFs). Scratch closure and transwell migration assays indicated that BFR-EVs could promote the migration of HDFs. When the antioxidant effect of BFR-EVs was evaluated through 2',7'-dichlorodihydrofluorescein diacetate staining and quantitative real-time polymerase chain reaction, the results revealed that BFR-EVs significantly suppressed ROS generation and oxidative stress induced by H2O2 and ultraviolet irradiation. Our findings suggest that BFR-EVs hold the potential as a natural candidate for healing chronic skin wounds.

Paraprobiotics strains accelerate wound repair by stimulating re-epithelialization of NIH-3T3 cells, decreasing inflammatory response and oxidative stress.

The present study aimed to evaluate the potential and specificity of the inflammatory and antioxidant response of Microbe-Associated Molecular Patterns (MAMPs) in NIH-3T3 fibroblast cells, as well as in the healing process of skin wounds. Cells (NIH-3T3) were cultivated in supplemented specific medium. NIH-3T3 cells were treated with MAMPs (Bifidobacterium lactis or Lactobacillus casei or Lactobacillus gasseri or Lactobacillus paracasei or Streptococcus thermophilus), at two concentrations and insulted with LPS or H2O2. Cell viability, myeloperoxidase activity, nitrite/nitrate, oxidative damage and inflammatory parameters were measured. In addition, scratch assay was performed. Significant scratch closure was observed after 24h and 48h, and the effect of 0.1g/mL MAMPs on wound healing was found to be highly statistically significant. In the viability cellular assay, Lactobacillus showed better response in 0.1g/mL dose, whereas B. lactis and S. thermophilus showed better response in 0.01g/mL dose. There was reduction in IL-6 and IL-1β levels in all treatments insulted with LPS. MAMP's showed preventive efficacy in reducing the effects caused by LPS. The MAMP's action in decreasing the production of ROS, inflammatory activity and increasing cell viability, besides significant cell proliferation during wound healing processes suggests remodeling mechanisms and new possibilities for wound healing.

Bioprospecting Antarctic microalgae as anticancer agent against PC-3 and AGS cell lines

Studies on pharmaceutical and medicine applications of cold adapted polar microalgae are very limited. In this study, it is aimed to evaluate the cytotoxic activity of polar microalgae extracts against different cancer cell lines. In this context, the cultivation of Chlorella variabilis YTU.ANTARCTIC.001 and Auxenochlorella pyrenoidosa OZCIMEN.001 were carried out and their oil extracts were encapsulated in nanoparticle to preserve its bioactive components against oxidation and to increase the penetration of the extract into the cancer cells. To investigate the anticancer effects of microalgae, cell culture studies were performed and then the extracts and nanoparticles were applied to the AGS and PC-3 cell lines via XTT method. In line with the study, it was observed that C. pyrenoidosa extracts showed higher cytotoxicity than C. variabilis against AGS and PC-3 cells. At 500 µg/ml, AGS cells tested with C. variabilis showed a decrease down to 58 % and at the same concentration of C. pyrenoidosa, it was determined as 51 %. As for nanoparticle form, C. variabilis extracts exhibited higher cytotoxic activity and better anticancer properties. It was found that, these potential anticancer effects appeared to be largely concentration-dependent. Moreover, C. variabilis extract was determined as less effective on scratch closure than C. pyrenoidosa. In conclusion, the obtained data will pave the way to produce biotechnological drugs based on polar microalgae.

Extraction Optimization, Antioxidant, Cosmeceutical and Wound Healing Potential of Echinacea purpurea Glycerolic Extracts.

Echinacea purpurea is a plant with immunomodulating properties, often used in topical preparations for treatment of small superficial wounds. In the presented study, the best conditions for ultrasound-assisted extraction of caffeic acid derivatives (caftaric and cichoric acid) (TPA-opt extract), as well as the conditions best suited for preparation of the extract with high radical scavenging activity (RSA-opt extract), from E. purpurea aerial parts were determined. A Box-Behnken design based on glycerol content (%, w/w), temperature (°C), ultrasonication power (W) and time (min) as independent variables was performed. Antioxidant, antiaging and wound healing effects of the two prepared extracts were evaluated. The results demonstrate that glycerol extraction is a fast and efficient method for preparation of the extracts with excellent radical scavenging, Fe2+ chelating and antioxidant abilities. Furthermore, the extracts demonstrated notable collagenase, elastase and tyrosinase inhibitory activity, indicating their antiaging properties. Well-pronounced hyaluronidase-inhibitory activities, with IC50 values lower than 30 μL extract/mL, as well as the ability to promote scratch closure in HaCaT keratinocyte monolayers, even in concentrations as low as 2.5 μL extract/mL (for RSA-opt), demonstrate promising wound healing effects of E. purpurea. The fact that the investigated extracts were prepared using glycerol, a non-toxic and environmentally friendly solvent, widely used in cosmetics, makes them suitable for direct use in specialized cosmeceutical formulations.

Essential Role of Adhesion GPCR, GPR123, for Human Pluripotent Stem Cells and Reprogramming towards Pluripotency.

G-protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. They modulate key physiological functions and are required in diverse developmental processes including embryogenesis, but their role in pluripotency maintenance and acquisition during the reprogramming towards hiPSCs draws little attention. Meanwhile, it is known that more than 106 GPCRs are overexpressed in human pluripotent stem cells (hPSCs). Previously, to identify novel effectors of reprogramming, we performed a high-throughput RNA interference (RNAi) screening assay and identified adhesion GPCR, GPR123, as a potential reprogramming effector. Its role has not been explored before. Herein, by employing GPR123 RNAi we addressed the role of GPR123 for hPSCs. The suppression of GPR123 in hPSCs leads to the loss of pluripotency and differentiation, impacted colony morphology, accumulation of cells at the G2 phase of the cell cycle, and absence of the scratch closure. Application of the GPR123 RNAi at the initiation stage of reprogramming leads to a decrease in the percentage of the "true" hiPSC colonies, a drop in E-cadherin expression, a decrease in the percentage of NANOG+ nuclei, and the absence of actin cytoskeleton remodeling. Together this leads to the absence of the alkaline-phosphatase-positive hiPSCs colonies on the 18th day of the reprogramming process. Overall, these data indicate for the first time the essential role of GPR123 in the maintenance and acquisition of pluripotency.

HuR-dependent expression of Wisp1 is necessary for TGFβ-induced cardiac myofibroblast activity

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGFβ-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGFβ-induced myofibroblast activation upon HuR inhibition. We identified twenty-four mRNA transcripts that were enriched for HuR binding following TGFβ treatment via photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Eleven of these HuR-bound mRNAs also showed significant co-expression correlation with HuR, αSMA, and periostin in primary fibroblasts isolated from the ischemic-zone of infarcted mouse hearts. Of these, WNT1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 partially rescued myofibroblast-induced collagen gel contraction following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGFβ. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGFβ, in part through post-transcriptional regulation of Wisp1.