Green extraction of phenolics and flavonoids from black mulberry fruit using natural deep eutectic solvents: optimization and surface morphology.

This study deployed ultrasonic-assisted extraction (UAE), combined with natural deep eutectic solvents (NADES), to extract phenolics and flavonoids from the black mulberry fruit, and the antioxidant activity was examined. The extraction yields of NADES-based UAE were assessed based on the yields of phenolics and flavonoids extracted from the black mulberry fruit. This study selected the molar ratios of hydrogen bond acceptors (HBA) and hydrogen bond donors HBD at 1:2 from previous studies. Choline chloride-lactic acid showed the highest solubility with phenolics and flavonoids among NADES systems. One-factor experiments evaluated the effect of UAE conditions (liquid-to-solid ratio (LSR), water content in NADES, temperature, and time) on TPC, TFC, and antioxidant activity. The suitable NADES-based UAE conditions for extracting phenolics and flavonoids from the black mulberry fruit were 60 ml/g of LSR, 40% water content, 70 °C, and 15 min. Response surface methodology with the Box-Behnken design model optimized the NADES-based UAE process based on response (TPC, TFC, ABTS, OH, and DPPH). The optimal conditions for the NADES-based UAE process were 70 ml/g of LSR, 38.9% water content in NADES, 67.9 °C, and 24.2 min of extraction time. The predicted values of the Box-Behnken design were compatible with the experimental results. Moreover, scanning electron microscopy (SEM) was used to survey the surface of black mulberry fruit with and without sonication. SEM can assist in demonstrating the destructive effect of NADES and ultrasonic waves on material surfaces. SEM findings indicated the high surface destruction capacity of NADES, which partially contributed to a superior extraction yield of NADES than conventional organic solvents. The study proposes an efficient and green method for extracting bioactive compounds from black mulberry fruits. The black mulberry fruit extracts can be applied to meat preservation and beverages with high antioxidants.

Reconstruction of Full Thickness Defects on the Scalp With Artificial Dermal Regeneration Template: Analysis of Long-Term Results in 68 Cases.

BACKGROUND: Artificial skin substitute templates have been shown to be a reliable solution for the reconstruction of large scalp defects with exposed skull bone, but there is a lack of long-term data. OBJECTIVE: The aim of this retrospective study was to investigate the long-term outcome of the procedure in a large cohort of 68 cases. MATERIALS AND METHODS: In total, 58 patients with 68 full thickness scalp defects with exposed skull bone, were included. Mean follow-up time was 24 (±19) months. RESULTS: The mean size of the defects was 63 (±54) cm2. During the follow-up period, no local recurrences occurred. Complications were observed in 13% of the cases including template necrosis (4%), infections (4%), ulcerations (3%), and autograft necrosis (2%). During the final follow-up, 26 patients had died due to internal diseases not associated with the surgery. Cosmetic results were rated good by the patients and an independent observer. CONCLUSION: The use of a dermal regeneration template for the reconstruction of large, full thickness defects of the scalp with exposed skull bone is a reliable method regarding the complication rate, safety of the procedure, and cosmetic outcome. Limitations of this study are the retrospective and single center design.

Melanoma-Derived iPCCs Show Differential Tumorigenicity and Therapy Response.

A point mutation in the BRAF gene, leading to a constitutively active form of the protein, is present in 45%-60% of patients and acts as a key driver in melanoma. Shortly after therapy induction, resistance to MAPK pathway-specific inhibitors develops, indicating that pathway inhibition is circumvented by epigenetic mechanisms. Here, we mimicked epigenetic modifications in melanoma cells by reprogramming them into metastable induced pluripotent cancer cells (iPCCs) with the ability to terminally differentiate into non-tumorigenic lineages. iPCCs and their differentiated progeny were characterized by an increased resistance against targeted therapies, although the cells harbor the same oncogenic mutations and signaling activity as the parental melanoma cells. Furthermore, induction of a pluripotent state allowed the melanoma-derived cells to acquire a non-tumorigenic cell fate, further suggesting that tumorigenicity is influenced by the cell state.

TGF-ß induces SOX2 expression in a time-dependent manner in human melanoma cells.

The sry-related high-mobility box (SOX)-2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF-ß1 stimulation in a time-dependent manner. Moreover, the knockdown of SOX2 impaired TGF-ß-induced invasiveness. This phenotype switch can be explained by SOX2-mediated cross talk between TGF-ß and non-canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF-ß signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF-ß signaling in melanoma, which may have further implications in the clinic.

Directed Dedifferentiation Using Partial Reprogramming Induces Invasive Phenotype in Melanoma Cells.

The combination of cancer-focused studies and research related to nuclear reprogramming has gained increasing importance since both processes-reprogramming towards pluripotency and malignant transformation-share essential features. Studies have revealed that incomplete reprogramming of somatic cells leads to malignant transformation indicating that epigenetic regulation associated with iPSC generation can drive cancer development [J Mol Cell Biol 2011;341-350; Cell 2012;151:1617-1632; Cell 2014;156:663-677]. However, so far it is unclear whether incomplete reprogramming also affects cancer cells and their function. In the context of melanoma, dedifferentiation correlates to therapy resistance in mouse studies and has been documented in melanoma patients [Nature 2012;490:412-416; Clin Cancer Res 2014;20:2498-2499]. Therefore, we sought to investigate directed dedifferentiation using incomplete reprogramming of melanoma cells. Using a murine model we investigated the effects of partial reprogramming on the cellular plasticity of melanoma cells. We demonstrate for the first time that induced partial reprogramming results in a reversible phenotype switch in melanoma cells. Partially reprogrammed cells at day 12 after transgene induction display elevated invasive potential in vitro and increased lung colonization in vivo. Additionally, using global gene expression analysis of partially reprogrammed cells, we identified SNAI3 as a novel invasion-related marker in human melanoma. SNAI3 expression correlates with tumor thickness in primary melanomas and thus, may be of prognostic value. In summary, we show that investigating intermediate states during the process of reprogramming melanoma cells can reveal novel insights into the pathogenesis of melanoma progression. We propose that deeper analysis of partially reprogrammed melanoma cells may contribute to identification of yet unknown signaling pathways that can drive melanoma progression.

NF1 loss induces senescence during human melanocyte differentiation in an iPSC-based model.

Neurofibromatosis type 1 (NF1) is a frequent genetic disease leading to the development of Schwann cell-derived neurofibromas or melanocytic lesions called café-au-lait macules (CALMs). The molecular mechanisms involved in CALMs formation remain largely unknown. In this report, we show for the first time pathophysiological mechanisms of abnormal melanocyte differentiation in a human NF1(+/-) -induced pluripotent stem cell (iPSC)-based model. We demonstrate that NF1 patient-derived fibroblasts can be successfully reprogrammed in NF1(+/-) iPSCs with active RAS signaling and that NF1 loss induces senescence during melanocyte differentiation as well as in patient's-derived CALMs, revealing a new role for NF1 in the melanocyte lineage.

New therapeutic options for advanced non-resectable malignant melanoma.

Melanoma is a malignant tumor which is inclined to metastasize promptly into the lymphatic system and other organs such as lung, liver, brain or bone. Therefore early diagnosis remains crucial for improving clinical outcome for melanoma patients. Current chemotherapy and chemo-immunotherapy regimes have shown little clinical benefit with no improvement in overall survival. However, new advances in melanoma biology such as the discovery of predisposed gene signatures and key somatic events have changed clinical practice. New therapeutic approaches are being tested or have been approved by the FDA/EMA recently including targeted therapies, such as BRAF- and MEK-inhibitors, and novel immunotherapies, such as anti-CTLA4 or anti-PD1 therapies. For these therapies an improvement of progression-free and overall survival has been seen in patients with advanced non-resectable melanoma. The following review summarizes recent therapeutic options after the ASCO and ESMO annual meetings 2014 for the treatment of malignant melanoma.

SOX2 and cancer: current research and its implications in the clinic.

SOX2 is a gene that encodes for a transcription factor belonging to the SOX gene family and contains a high-mobility group (HMG) domain, which permits highly specific DNA binding. Consequently, SOX2 functions as an activator or suppressor of gene transcription. SOX2 has been described as an essential embryonic stem cell gene and moreover, a necessary factor for induced cellular reprogramming. SOX2 research has only recently switched focus from embryogenesis and development to SOX2's function in disease. Particularly, the role of SOX2 in cancer pathogenesis has become of interest in the field. To date, studies have shown SOX2 to be amplified in various cancer types and affect cancer cell physiology via involvement in complicated cell signaling and protein-protein interactions. Recent reviews in this field have highlighted SOX2 in mammalian physiology, development and pathology. In this review, we comprehensively compile what is known to date about SOX2's involvement in cancer biology, focusing on the most recent findings in the fields of cellular signaling and cancer stem cells. Lastly, we underscore the role of SOX2 in the clinic and highlight new findings, which may provide novel clinical applications for SOX2 as a prognostic marker, indicator of metastasis, biomarker or potential therapeutic target in some cancer types.

From skin to other cell types of the body.

Regenerative medicine allows for the customization of tissues and organs which may bring hope to patients with incurable diseases and severe injuries. Therefore, reliable and safe methods for the generation of specific cell types must be established. Recently, different strategies have emerged to convert somatic cells into differentiated cells of interest. One of these strategies is cellular reprogramming, which converts somatic cells into induced pluripotent stem cells (iPSCs). These iPSCs are embryonic stem cell-like cells with almost unlimited developmental potential and can be differentiated into specific lineages. Alternatively, the method of transdifferentiation can be used to directly convert one terminally differentiated cell into another cell type. Both of these methods have proven to have the potential to push the field of cell replacement therapy forward. In this context, the skin is of particular interest because it represents an ideal source of somatic cells for reprogramming to pluripotency as well as transdifferentiation. In this review, we briefly compare both above-mentioned strategies and summarize the latest advances in this highly dynamic field of research.

Delayed closure of complex defects with serial tightening of loop sutures - clinical outcome in 64 consecutive patients.

BACKGROUND: Direct wound closure, flaps or grafts are considered to be gold standards for the reconstruction of defects. However, these techniques may not be applicable in all cases, especially for the challenging closure of large defects. OBJECTIVE: We developed a technique to close large defects, of varying size and shape, using serial tightening loop sutures. In 64 consecutive patients, loop sutures were applied to defects from 3 cm(2) up to 173 cm(2) for a maximum of 42 days with tightening every 2-7 days. RESULTS: The median size of the defects was 20 cm(2). In 58 patients (91%) the defects were closed by direct approximation of the wound edges within a median time of 11 days (range: 4-42 days). Two patients received a skin transplant after a significant reduction of the defect size and four remaining defects healed by secondary intention. The skin stretching of defects located on the trunk was faster compared with defects on the extremities. Defects located on the scalp were closed hair bearing with little scarring. Overall, aesthetic results were satisfying. CONCLUSION: The use of loop sutures allows the delayed closure of large defects avoiding the need for skin flaps or grafts in most cases.

A dominant negative zebrafish Ahr2 partially protects developing zebrafish from dioxin toxicity.

The toxicity by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is thought to be caused by activation of the aryl hydrocarbon receptor (AHR). However, our understanding of how AHR activation by TCDD leads to toxic effects is poor. Ideally we would like to manipulate AHR activity in specific tissues and at specific times. One route to this is expressing dominant negative AHRs (dnAHRs). This work describes the construction and characterization of dominant negative forms of the zebrafish Ahr2 in which the C-terminal transactivation domain was either removed, or replaced with the inhibitory domain from the Drosophila engrailed repressor protein. One of these dnAhr2s was selected for expression from the ubiquitously active e2fα promoter in transgenic zebrafish. We found that these transgenic zebrafish expressing dnAhr2 had reduced TCDD induction of the Ahr2 target gene cyp1a, as measured by 7-ethoxyresorufin-O-deethylase activity. Furthermore, the cardiotoxicity produced by TCDD, pericardial edema, heart malformation, and reduced blood flow, were all mitigated in the zebrafish expressing the dnAhr2. These results provide in vivo proof-of-principle results demonstrating the effectiveness of dnAHRs in manipulating AHR activity in vivo, and demonstrating that this approach can be a means for blocking TCDD toxicity.