PPIG, SFRS-18 és LUC7L3 Splicing regulárok vizsgálata szinkronizált, immortalizált sejtvonalakban és pikkelysömörben

Az mRNS érés (splicing) folyamatának vizsgálata egészen újfajta megközelítésnek számít a pikkelysömör kutatásban: a splicing mechanizmus e betegségben tapasztalt eltéréseiről mindössze néhány közlemény jelent meg napjainkig. Egy nemrég elvégzett microarray vizsgálatban több olyan gént is azonosítottunk, amelyek T-limfokin kezelés hatására eltérő kifejeződés változással reagálnak, majd bioinformatikai módszerekkel tovább elemeztük azokat. A gének között három olyat is találtunk (serine/ arginine-rich splicing factor 18 (SFRS18), peptydilpropyl isomerase G (PPIG), luc-7 like 3 (LUC7L3)) amelyek funkciója az mRNS érés szabályozásához kötődik. A PPIG, SFRS18 és LUC7L3 gének kifejeződését szinkronizált, immortalizált sejtvonalak segítségével követtük. Két független sejtvonal (HaCaT és HPV-KER) esetében is azt tapasztaltuk, hogy a splicing regulátorok kifejeződési mintázata jelentős hasonlóságot mutat. Ezekből az eredményekből arra következtettünk, hogy a gének upstream regulációja közös transzkripciós faktorok útján valósulhat meg. A splicing gének fehérjeszintű expresszióját is meghatároztuk Western blot technika segítségével. Előkísérleteinkben azt találtuk, hogy az SFRS18 és a PPIG expressziója fehérje szinten is hasonlóságot mutat a szinkronizált, immortalizált HPV-KER sejtekben. Az in vitro analízisek mellett egészséges és pikkelysömörös betegekből származó tünetes és tünetmentes mintákon is megvizsgáltuk, miként alakul a három fehérje expressziója. Kísérleteink során megmutattuk, hogy a PPIG fehérje expressziós mintázata és kifejeződésének mennyisége is jelentősen megváltozik: a tünetmentes bőrben a PPIG festődése fokozottnak mutatkozott és eloszlása is eltér az egészséges bőrétől. Eredményeink alapján úgy gondoljuk, hogy a splicing szabályozás eltéréseinek fontos szerepe lehet a pikkelysömör tüneteinek kifejlődésében. A splicing folyamatának további tanulmányozása nagyban segítheti a betegség komplex molekuláris hátterének megértését

Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro

After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments

Statističke analogije između potresa, mikropotresa u metalima i lavina u 1D Burridge-Knopoff modelu

Universalities and intriguing analogies in the statistics of avalanches are revealed for three physical systems defined on largely different length and energy scales. Earthquakes induced by tectonic scale dynamics, micro-scale level quakes observed from slipping crystallographic planes in metals and a one-dimensional, room-scale spring-block type Burridge-Knopoff model is studied from similar statistical viewpoints. The validity of the Gutenberg-Richter law for the probability density of the energies dissipated in the avalanches is proven for all three systems. By analysing data for three different seismic zones and performing acoustic detection for different Zn samples under deformation, universality for the involved scaling exponent is revealed. With proper parameter choices the 1D Burridge-Knopoff model is able to reproduce the same scaling law. The recurrence times of earthquakes and micro-quakes with magnitudes above a given threshold present again similar distributions and striking quantitative similarities. However, the 1D Burridge-Knopoff model cannot account for the correlations observed in such statistics.Univerzalnosti i intrigantne analogije u statistici lavina otkrivene su za tri fizička sustava definirana na uvelike različitim duljinama i energijskim skalama. Potresi uzrokovani dinamikom na tektonskoj skali, mikro-potresi koji nastaju na klizećim kristalografskim ravnina u metalima i jednodimenzionalni Burridge-Knopoffov model opruga i blokova na skali sobe proučeni su sa sličnih statističkih stajališta. Valjanost Gutenberg-Richterove relacije za gustoću vjerojatnosti energija disipirane u lavinama dokazana je za sva tri sustava. Analizom podataka za tri različita seizmički aktivna područja i detekcijom akustičkih valova za različite uzorke Zn pod deformacijom, otkrivena je univerzalnost za uključeni eksponent skaliranja. S pravilnim izborom parametara 1D Burridge-Knopoffov model može reproducirati isti zakon skaliranja. Vremena ponavljanja potresa i mikropotresa s magnitudama iznad zadanog praga opet predstavljaju slične distribucije i zapanjujuće kvantitativne sličnosti. Međutim, 1D Burridge-Knopoffov model ne može objasniti korelacije opažene u takvim statistikama

Melanoma-Derived BRAF(V600E) Mutation in Peritumoral Stromal Cells: Implications for in Vivo Cell Fusion

Melanoma often recurs in patients after the removal of the primary tumor, suggesting the presence of recurrent tumor-initiating cells that are undetectable using standard diagnostic methods. As cell fusion has been implicated to facilitate the alteration of a cell's phenotype, we hypothesized that cells in the peritumoral stroma having a stromal phenotype that initiate recurrent tumors might originate from the fusion of tumor and stromal cells. Here, we show that in patients with BRAF(V600E) melanoma, melanoma antigen recognized by T-cells (MART1)-negative peritumoral stromal cells express BRAF(V600E) protein. To confirm the presence of the oncogene at the genetic level, peritumoral stromal cells were microdissected and screened for the presence of BRAF(V600E) with a mutation-specific polymerase chain reaction. Interestingly, cells carrying the BRAF(V600E) mutation were not only found among cells surrounding the primary tumor but were also present in the stroma of melanoma metastases as well as in a histologically tumor-free re-excision sample from a patient who subsequently developed a local recurrence. We did not detect any BRAF(V600E) mutation or protein in the peritumoral stroma of BRAF(WT) melanoma. Therefore, our results suggest that peritumoral stromal cells contain melanoma-derived oncogenic information, potentially as a result of cell fusion. These hybrid cells display the phenotype of stromal cells and are therefore undetectable using routine histological assessments. Our results highlight the importance of genetic analyses and the application of mutation-specific antibodies in the identification of potentially recurrent-tumor-initiating cells, which may help better predict patient survival and disease outcome

Gene expression and microrna expression analysis in small arteries of spontaneously hypertensive rats. Evidence for er stress

Small arteries are known to develop functional and structural alterations in hypertension. However, the mechanisms of this remodeling are not fully understood.We hypothesized that altered gene expression is associated with the development of hypertension in mesenteric arteries of spontaneously hypertensive rats (SHR). Three sublines of SHR and normotensive Wistar Kyoto rats (WKY) were studied at 6 weeks and 5 months of age. MiRNA and mRNA microarray experiments were performed and analyzed with bioinformatical tools, including Ingenuity Pathway Analysis (IPA). Principal component analysis showed a clear separation in both miRNA and mRNA expression levels between both ages studied, demonstrating strong age-related changes in expression. At the miRNA level, IPA identified differences between SHR and WKY related to metabolic diseases, cellular growth, and proliferation. The mRNAs differentially expressed between SHR and WKY were related to metabolism, cellular movement and proliferation. The most strongly upregulated gene (9.2- fold) was thrombospondin 4 (Thbs4), a protein involved in the endoplasmic reticulum (ER) stress response that activates transcription factor 6α (ATF6α). ATF6α downstream targets were also differentially expressed in SHR vs. WKY. Differential expression of THBS4, the cleaved form of ATF6α, and two of its targets were further confirmed at the protein level by western blot. In summary, these data revealed a number of genes (n = 202) and miRNAs (n = 3) in mesenteric arteries of SHR that had not been related to hypertension previously. The most prominent of these, Thbs4, is related to vascular ER stress that is associated with hypertensionThis work was supported by the European Union, Marie Curie ITN number 606998 and 23571

COMP negatively influences keratinocyte proliferation via α5β1-integrin: Potential relevance of altered COMP expression in psoriasis

In psoriasis, non-lesional skin shows alterations at the dermal–epidermal junction (DEJ) compared to healthy skin. Cartilage oligomeric matrix protein (COMP) is part of the papillary dermis of healthy skin, and its expression has not yet been studied in psoriatic skin. In this study, we found that COMP localization extended deeper into the dermis and formed a more continuous layer in psoriatic non-lesional skin compared to healthy skin, while in psoriatic lesions, COMP showed a partially discontinuous deposition at the DEJ. COMP and β1-integrin showed strong co-localization in non-lesional skin, where the laminin layer within the basement membrane (BM) is discontinuous. In in vitro models, the presence of exogenous COMP decreased the proliferation rate of keratinocytes and this proliferation-suppressing effect was diminished by blocking α5β1-integrin. Our results suggest that COMP can interact with α5β1-integrin of basal keratinocytes through the disrupted BM, and this interaction might stabilize the epidermis in the non-lesional state by contributing to the suppression of keratinocyte proliferation. The antiproliferative effect of COMP is likely to be relevant to other skin diseases in which chronic non-healing wounds are coupled with massive COMP accumulation

Comprehensive Proteomic Analysis Reveals Intermediate Stage of Non-Lesional Psoriatic Skin and Points out the Importance of Proteins Outside this Trend

To better understand the pathomechanism of psoriasis, a comparative proteomic analysis was performed with non-lesional and lesional skin from psoriasis patients and skin from healthy individuals. Strikingly, 79.9% of the proteins that were diferentially expressed in lesional and healthy skin exhibited expression levels in non-lesional skin that were within twofold of the levels observed in healthy and lesional skin, suggesting that non-lesional skin represents an intermediate stage. Proteins outside this trend were categorized into three groups: I. proteins in non-lesional skin exhibiting expression similar to lesional skin, which might be predisposing factors (i.e., CSE1L, GART, MYO18A and UGDH); II. proteins that were diferentially expressed in non-lesional and lesional skin but not in healthy and lesional skin, which might be non-lesional characteristic alteration (i.e., CHCHD6, CHMP5, FLOT2, ITGA7, LEMD2, NOP56, PLVAP and RRAS); and III. proteins with contrasting diferential expression in non-lesional and lesional skin compared to healthy skin, which might contribute to maintaining the non-lesional state (i.e., ITGA7, ITGA8, PLVAP, PSAPL1, SMARCA5 and XP32). Finally, proteins diferentially expressed in lesions may indicate increased sensitivity to stimuli, peripheral nervous system alterations, furthermore MYBBP1A and PRKDC were identifed as potential regulators of key pathomechanisms, including stress and immune response, proliferation and diferentiation