Recent studies revealed trajectories of mutational events in early melanomagenesis, but the accompanying changes in gene expression are far less understood. Therefore, we performed a comprehensive RNA-seq analysis of laser-microdissected melanocytic nevi (n = 23) and primary melanoma samples (n = 57) and characterized the molecular mechanisms of early melanoma development. Using self-organizing maps, unsupervised clustering, and analysis of pseudotime (PT) dynamics to identify evolutionary trajectories, we describe here two transcriptomic types of melanocytic nevi (N1 and N2) and primary melanomas (M1 and M2). N1/M1 lesions are characterized by pigmentation-type and MITF gene signatures, and a high prevalence of NRAS mutations in M1 melanomas. N2/M2 lesions are characterized by inflammatory-type and AXL gene signatures with an equal distribution of wild-type and mutated BRAF and low prevalence of NRAS mutations in M2 melanomas. Interestingly, N1 nevi and M1 melanomas and N2 nevi and M2 melanomas, respectively, cluster together, but there is no clustering in a stage-dependent manner. Transcriptional signatures of M1 melanomas harbor signatures of BRAF/MEK inhibitor resistance and M2 melanomas harbor signatures of anti-PD-1 antibody treatment resistance. Pseudotime dynamics of nevus and melanoma samples are suggestive for a switch-like immune-escape mechanism in melanoma development with downregulation of immune genes paralleled by an increasing expression of a cell cycle signature in late-stage melanomas. Taken together, the transcriptome analysis identifies gene signatures and mechanisms underlying development of melanoma in early and late stages with relevance for diagnostics and therapy.

The present study identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions of melanomas compared with primary melanomas. miR-638 enhanced the tumorigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling identified new candidate genes including TP53INP2 as miR-638 targets, the majority of which are involved in p53 signalling. Overexpression of TP53INP2 severely attenuated proliferative and invasive capacity of melanoma cells which was reversed by miR-638. Depletion of miR-638 stimulated expression of p53 and p53 downstream target genes and induced apoptosis and autophagy. miR-638 promoter analysis identified the miR-638 target transcription factor associated protein 2α (TFAP2A/AP-2α) as a direct negative regulator of miR-638, suggestive for a double-negative regulatory feedback loop. Taken together, miR-638 supports melanoma progression and suppresses p53-mediated apoptosis pathways, autophagy and expression of the transcriptional repressor TFAP2A/AP-2α.

Background: Regenerative therapies based on autologous mesenchymal stem cells (MSC) as well as stem cells in general are still facing an unmet need for non-invasive sampling, availability, and scalability. The only known adult source of autologous MSCs permanently available with no pain, discomfort, or infection risk is the outer root sheath of the hair follicle (ORS). Methods: This study presents a non-invasively-based method for isolating and expanding MSCs from the ORS (MSCORS) by means of cell migration and expansion in air–liquid culture. Results: The method yielded 5 million cells of pure MSCORS cultured in 35 days, thereby superseding prior art methods of culturing MSCs from hair follicles. MSCORS features corresponded to the International Society for Cell Therapy characterization panel for MSCs: adherence to plastic, proliferation, colony forming, expression of MSC-markers, and adipo-, osteo-, and chondro-differentiation capacity. Additionally, MSCORS displayed facilitated random-oriented migration and high proliferation, pronounced marker expression, extended endothelial and smooth muscle differentiation capacity, as well as a paracrine immunomodulatory effect on monocytes. MSCORS matched or even exceeded control adipose-derived MSCs in most of the assessed qualities. Conclusions: MSCORS qualify for a variety of autologous regenerative treatments of chronic disorders and prophylactic cryopreservation for purposes of acute treatments in personalized medicine.

No abstract

Objective. To identify the shared genetic and epigenetic mechanisms between the osteogenic differentiation of dental pulp stem cells (DPSC) and bone marrow stem cells (BMSC). Materials and Methods. The profiling datasets of miRNA expression in the osteogenic differentiation of mesenchymal stem cells from the dental pulp (DPSC) and bone marrow (BMSC) were searched in the Gene Expression Omnibus (GEO) database. The differential expression analysis was performed to identify differentially expressed miRNAs (DEmiRNAs) dysregulated in DPSC and BMSC osteodifferentiation. The target genes of the DEmiRNAs that were dysregulated in DPSC and BMSC osteodifferentiation were identified, followed by the identification of the signaling pathways and biological processes (BPs) of these target genes. Accordingly, the DEmiRNA-transcription factor (TFs) network and the DEmiRNAs-small molecular drug network involved in the DPSC and BMSC osteodifferentiation were constructed. Results. 16 dysregulated DEmiRNAs were found to be overlapped in the DPSC and BMSC osteodifferentiation, including 8 DEmiRNAs with a common expression pattern (8 upregulated DEmiRNAs (miR-101-3p, miR-143-3p, miR-145-3p/5p, miR-19a-3p, miR-34c-5p, miR-3607-3p, miR-378e, miR-671-3p, and miR-671-5p) and 1 downregulated DEmiRNA (miR-671-3p/5p)), as well as 8 DEmiRNAs with a different expression pattern (i.e., miR-1273g-3p, miR-146a-5p, miR-146b-5p, miR-337-3p, miR-382-3p, miR-4508, miR-4516, and miR-6087). Several signaling pathways (TNF, mTOR, Hippo, neutrophin, and pathways regulating pluripotency of stem cells), transcription factors (RUNX1, FOXA1, HIF1A, and MYC), and small molecule drugs (curcumin, docosahexaenoic acid (DHA), vitamin D3, arsenic trioxide, 5-fluorouracil (5-FU), and naringin) were identified as common regulators of both the DPSC and BMSC osteodifferentiation. Conclusion. Common genetic and epigenetic mechanisms are involved in the osteodifferentiation of DPSCs and BMSCs.

Mutations of mitochondrial (mt)DNA cause a variety of human diseases and are implicated in premature aging syndromes. Here we investigated a single nucleotide exchange (leucine to methionine) at position nt4738 in the mitochondrial NADH dehydrogenase subunit 2 (Nd2) gene of the respiratory chain. Primary fibroblasts derived from the conplastic mouse strain C57BL/6J-mtALR/LTJ with mutant enzyme, possessed high enzyme activity and ATP production and low ROS production. Furthermore, Nd2-mutant fibroblasts expressed lower senescence markers. Transcriptome analysis revealed that the members of the p38MAPK pathway were significantly downregulated in Nd2-mutant mice. In agreement, inhibition of p38MAPK with SB203580 enhanced proliferation and reduced cytokine secretion in fibroblasts. In Nd2-mutant mouse skin, the amount of Ki67-positive cells was significantly higher than in control skin. The higher amount of Ki67-positive cells and the thicker epidermis in Nd2-mutant mice strongly supported the in vitro data. In conclusion, Nd2 is a mitochondrial gene, involved in age-related signaling pathways.

A large-scale RNAi screen was performed for eight different melanoma cell lines using a pooled whole-genome lentiviral shRNA library. shRNAs affecting proliferation of transduced melanoma cells were negatively selected during 10 days of culture. Overall, 617 shRNAs were identified by microarray hybridization. Pathway analyses identified mitogen-activated protein kinase (MAPK) pathway members such as ERK1/2, JNK1/2 and MAP3K7 and protein kinase C β (PKCβ) as candidate genes. Knockdown of PKCβ most consistently reduced cellular proliferation, colony formation and migratory capacity of melanoma cells and was selected for further validation. PKCβ showed enhanced expression in human primary melanomas and distant metastases as compared with benign melanocytic nevi. Moreover, treatment of melanoma cells with PKCβ-specific inhibitor enzastaurin reduced melanoma cell growth but had only small effects on benign fibroblasts. Finally, PKCβ-shRNA significantly reduced lung colonization capacity of stably transduced melanoma cells in mice. Taken together, this study identified new candidate genes for melanoma cell growth and proliferation. PKCβ seems to play an important role in these processes and might serve as a new target for the treatment of metastatic melanoma.

Plagiostomum lemani possesses extremely specialized intraepidermal sensory cells. These obvious photoreceptors, which are not visible with the light microscope, are ciliary aggregations located in an intracellular cavity. The numerous spiralled cilia have the classic 9 × 2 + 2 arrangement at their base and a modified pattern of microtubules apically. The discovered differentiations do not show a connection to the surface. Neither mantle cells nor pigment cells have been found. The structural similarities with other epidermal photoreceptors of species among the different taxa of free-living Plathelminthes are outlined. Besides the larval stages of the taxon Polycladida known so far, the same kind of light-sensing photoreceptive cell has never been described in any other species of the Plathelminthes.& b d y :