Identification of Plasma Lipid Alterations Associated with Melanoma Metastasis.

The aim of this study was to apply a state-of-the-art quantitative lipidomic profiling platform to uncover lipid alterations predictive of melanoma progression. Our study included 151 melanoma patients; of these, 83 were without metastasis and 68 with metastases. Plasma samples were analyzed using a targeted Lipidyzer™ platform, covering 13 lipid classes and over 1100 lipid species. Following quality control filters, 802 lipid species were included in the subsequent analyses. Total plasma lipid contents were significantly reduced in patients with metastasis. Specifically, levels of two out of the thirteen lipid classes (free fatty acids (FFAs) and lactosylceramides (LCERs)) were significantly decreased in patients with metastasis. Three lipids (CE(12:0), FFA(24:1), and TAG47:2-FA16:1) were identified as more effective predictors of melanoma metastasis than the well-known markers LDH and S100B. Furthermore, the predictive value substantially improved upon combining the lipid markers. We observed an increase in the cumulative levels of five lysophosphatidylcholines (LPC(16:0); LPC(18:0); LPC(18:1); LPC(18:2); LPC(20:4)), each individually associated with an elevated risk of lymph node metastasis but not cutaneous or distant metastasis. Additionally, seventeen lipid molecules were linked to patient survival, four of which (CE(12:0), CE(14:0), CE(15:0), SM(14:0)) overlapped with the lipid panel predicting metastasis. This study represents the first comprehensive investigation of the plasma lipidome of melanoma patients to date. Our findings suggest that plasma lipid profiles may serve as important biomarkers for predicting clinical outcomes of melanoma patients, including the presence of metastasis, and may also serve as indicators of patient survival.

Identification of liquid biopsy-based mutations in colorectal cancer by targeted sequencing assays.

Recently, liquid biopsy, as a promising approach was introduced for the analysis of different tumor-derived circulating markers including tumor DNA and cell free DNA (ct/cfDNA). Identification of mutations in cfDNA may allow the early detection of tumors, as well as predicting and monitoring treatment responses in a minimally invasive way. In the present study, we used commercially available gene panels to verify the mutation overlap between liquid biopsy and abnormalities detected in colorectal tumor tissue. The two panels (Archer®VariantPlex®Solid Tumor and LIQUIDPlexTM ctDNA) overlap in 23 genes, which enables a comprehensive view of tumor-plasma mutational status by next generation sequencing. We successfully analyzed 16 plasma and 16 tumor samples. We found that 87% of tumor tissues contained 44 mutations in 12 genes and 43.8% of cfDNA harbored 13 mutations in 5 genes. To verify whether the mutation pattern of the tumor DNA could be consistently detected in plasma cfDNA, we compared the alterations between cfDNA and matched tissue DNA in nine patients. Six of the 9 tumor tissues harbored mutations in TP53, KRAS or MET genes, those were not detectable by the ctDNA kit, even eventhough the exons of these genes overlap in both panels. Comparing the mutational patterns of the matched samples, we found that only one cfDNA had the same mutations (KRAS, SMAD4 and TP53) in the paired tissue. The results of the comparison between tumor tissue DNA and matched plasma cfDNA underline the importance of studying the paired solid tumor and plasma samples together.

Gene Expression Changes in Cytokine and Chemokine Receptors in Association with Melanoma Liver Metastasis.

Cytokines and chemokines (chemotactic cytokines) are soluble extracellular proteins that bind to specific receptors and play an integral role in the cell-to-cell signaling network. In addition, they can promote the homing of cancer cells into different organs. We investigated the potential relationship between human hepatic sinusoidal endothelial cells (HHSECs) and several melanoma cell lines for the expression of chemokine and cytokine ligands and receptor expression during the invasion of melanoma cells. In order to identify differences in gene expression related to invasion, we selected invasive and non-invasive subpopulations of cells after co-culturing with HHSECs and identified the gene expression patterns of 88 chemokine/cytokine receptors in all cell lines. Cell lines with stable invasiveness and cell lines with increased invasiveness displayed distinct profiles of receptor genes. Cell lines with increased invasive capacity after culturing with conditioned medium showed a set of receptor genes (CXCR1, IL1RL1, IL1RN, IL3RA, IL8RA, IL11RA, IL15RA, IL17RC, and IL17RD) with significantly different expressions. It is very important to emphasize that we detected significantly higher IL11RA gene expression in primary melanoma tissues with liver metastasis as well, compared to those without metastasis. In addition, we assessed protein expression in endothelial cells before and after co-culturing them with melanoma cell lines by applying chemokine and cytokine proteome arrays. This analysis revealed 15 differentially expressed proteins (including CD31, VCAM-1, ANGPT2, CXCL8, and CCL20) in the hepatic endothelial cells after co-culture with melanoma cells. Our results clearly indicate the interaction between liver endothelial and melanoma cells. Furthermore, we assume that overexpression of the IL11RA gene may play a key role in organ-specific metastasis of primary melanoma cells to the liver.

Cytokine and Chemokine Receptor Patterns of Human Malignant Melanoma Cell Lines.

Cytokine and chemokine receptors can promote tumor progression, invasion, and metastasis development by inducing different intracellular signaling pathways. The aim of this study was to determine the cytokine and chemokine receptor gene expression patterns in human melanoma cell lines. We found a large set of cytokine and chemokine receptor genes that were significantly differentially expressed between melanoma cell lines that originated from different subtypes of primary melanomas as well as cell lines that originated from melanoma metastases. The relative expressions of two receptor genes (CCR2 and TNFRSF11B) were positively correlated with the invasive potential of the cell lines, whereas a negative correlation was observed for the TNFRSF14 gene expression. We also found a small set of receptor genes that exhibited a significantly decreased expression in association with a BRAFV600E mutation. Based on our results, we assume that the analyzed cytokine and chemokine receptor collection may provide potential to distinguish the different subtypes of melanomas, helping us to understand the biological behavior of BRAFV600E-mutated melanoma cells.

DNA hypermethylation is associated with invasive phenotype of malignant melanoma.

Tumor cell invasion is one of the key processes during cancer progression, leading to life-threatening metastatic lesions in melanoma. As methylation of cancer-related genes plays a fundamental role during tumorigenesis and may lead to cellular plasticity which promotes invasion, our aim was to identify novel epigenetic markers on selected invasive melanoma cells. Using Illumina BeadChip assays and Affymetrix Human Gene 1.0 microarrays, we explored the DNA methylation landscape of selected invasive melanoma cells and examined the impact of DNA methylation on gene expression patterns. Our data revealed predominantly hypermethylated genes in the invasive cells affecting the neural crest differentiation pathway and regulation of the actin cytoskeleton. Integrative analysis of the methylation and gene expression profiles resulted in a cohort of hypermethylated genes (IL12RB2, LYPD6B, CHL1, SLC9A3, BAALC, FAM213A, SORCS1, GPR158, FBN1 and ADORA2B) with decreased expression. On the other hand, hypermethylation in the gene body of the EGFR and RBP4 genes was positively correlated with overexpression of the genes. We identified several methylation changes that can have role during melanoma progression, including hypermethylation of the promoter regions of the ARHGAP22 and NAV2 genes that are commonly altered in locally invasive primary melanomas as well as during metastasis. Interestingly, the down-regulation of the methylcytosine dioxygenase TET2 gene, which regulates DNA methylation, was associated with hypermethylated promoter region of the gene. This can probably lead to the observed global hypermethylation pattern of invasive cells and might be one of the key changes during the development of malignant melanoma cells.

The role of osteopontin expression in melanoma progression.

It was shown that osteopontin (OPN), a glycophosphoprotein, plays divergent roles in cancer progression. In addition to multiple intra- and extracellular functions, it facilitates migration of tumour cells, has crucial role in cell adhesion and is associated with increased metastasis formation. In previous studies, we performed global gene expression profiling on a series of primary melanoma samples and found that OPN was significantly overexpressed in ulcerated melanomas. The major purpose of this study was to define OPN expression in primary melanomas with differing biological behaviours. OPN mRNA expression was analysed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in primary melanoma tissues. Immunohistochemistry was performed using a tissue microarray. Cox regression tests were used for survival analysis. Greater than 50 % of the tissues exhibited high protein expression that was significantly associated with tumour thickness and metastasis. OPN mRNA expression was significantly increased in thicker melanomas and lesions with an ulcerated surface. Increased expression was primarily detected in advanced-stage tumours. A multivariate Cox regression analysis revealed that high OPN expression, tumour thickness and metastasis were significantly associated with reduced relapse-free survival. In summary, high OPN mRNA and protein expression were associated with a less favourable clinical outcome of primary melanoma patients. We determined that OPN is a significant predictive factor for the survival of primary melanoma patients. Based on our and others data, the high expression of OPN may have a crucial stimulatory role in tumour progression and metastasis formation, which, thus, have been proposed as potential targets for cancer diagnosis and therapy.

[Melanoma research in Hungary: promising results in a previously orphan tumor]. / Hazai melanomakutatások: reményt keltõ eredmények egy korábban reménytelen daganatban.

Melanoma research has a two decade history in Hungary and is based on three groups located at the National Institute of Oncology (NIO), the University of Debrecen (DU) and Semmelweis University (SU). Previously we have summarized the achievements of the NIO group in this Journal, now this paper summarizes the recent results of their collaborations. The research group of DU revealed several novel genetic alterations in the melanoma genome which might have clinical relevance as prognosticators or predictors in light of the novel target therapies. Data indicating unique, perhaps melanoma-specific epigenetic changes during progression might be even more important, identifying novel genes otherwise not detected as genetically altered ones. The research group in Budapest extensively used experimental human melanoma models and demonstrated the host sex as a key factor in progression due to the specific function of NK cells. Identification of functional glucocorticoid receptor in human melanoma might lead to therapeutic exploitation similar to certain leukemias. Studies on extracellular matrix revealed collagen XVII and CD44 splice variants as progression associated factors of melanoma. Since the double wild type genotype of melanoma is lacking effective therapy, data on the use of FGFR2, c-met or cannabinoid receptor as target can be important. On the other hand, experimental data on the antitumoral effects of heparin derivatives or bisphosphonate in melanoma models can also be encouraging.

Expression pattern of osteopontin isoforms in malignant melanoma cell lines.

Osteopontin (OPN) is a secreted integrin-binding protein that plays a role in inflammation, cellular viability, cell adhesion and migration, cancer development, and diabetes through different mechanisms. The splice variants of OPN can play essential roles in cancer development, progression, and metastasis formation; however, limited data are available about the role of OPN isoforms in human malignant melanoma. Our goal was to define the gene expression patterns of five OPN variants (OPN4, OPN5, OPNa, OPNb, and OPNc), integrin, and CD44 receptor genes in primary and metastatic melanoma-originated cell lines (n = 19), and to explore the association of the expression patterns with clinicopathological parameters. We evaluated the invasive property of the cell lines and investigated the potential association between the invasion and gene expression of OPN isoforms. We found a significant rise in the expression of OPNc in the invasive cell lines compared to the noninvasive cells and detected significantly higher expression of the OPN splice variants in melanoma cell lines originating from more advanced stages tumors than cell lines originating from early-stage melanomas. The correlation analysis revealed that all five OPN variants positively correlated with ITGB3 and ITGA9, whereas OPN5 positively correlated with ITGB1, ITGAV, ITGA6, and CD44. OPN can activate extracellular signal-regulated kinase signaling through binding to α9ß1 integrin, promoting melanoma tumor cell migration. It is possible that such associations between OPN splice variants and integrin receptors may play a role in melanoma progression. In conclusion, our findings suggest that high expression of OPNc correlates with the invasive behavior of melanoma cells.

Multi-round recycling of green waste for the production of iron nanoparticles: synthesis, characterization, and prospects in remediation.

Due to the widespread applications of metal nanoparticles (NPs), green synthesis strategies have recently advanced, e.g., methods that utilize extracts made from different plant wastes. A particularly innovative approach to reducing large amounts of available household/agricultural green wastes is their application in nanoparticle generation. Regarding this, the aim of our work was to examine the possibility of upgrading green nanoparticle syntheses from an innovative economic and environmental point of view, namely by investigating the multiple recyclabilities of green tea (GT), coffee arabica (CA), and Virginia creeper (Parthenocissus quinquefolia) (VC) waste residues for iron nanoparticle (FeNPs) synthesis. The plant extracts obtained by each extraction round were analyzed individually to determine the amount of main components anticipated to be involved in NPs synthesis. The synthesized FeNPs were characterized by X-ray powder diffraction and transmission electron microscopy. The activity of the generated FeNPs in degrading chlorinated volatile organic compounds (VOC) and thus their future applicability for remediation purposes were also assessed. We have found that VC and especially GT residues could be reutilized in multiple extraction rounds; however, only the first extract of CA was suitable for FeNPs' generation. All of the obtained FeNPs could degrade VOC with efficiencies GT1-Fe 91.0%, GT2-Fe 83.2%, GT3-Fe 68.5%; CA1-Fe 76.2%; VC1-Fe 88.2%, VC2-Fe 79.7%, respectively, where the number (as in GT3) marked the extraction round. These results indicate that the adequately selected green waste material can be reutilized in multiple rounds for nanoparticle synthesis, thus offering a clean, sustainable, straightforward alternative to chemical methods.

Combining Biomarkers for the Diagnosis of Metastatic Melanoma.

The early detection of melanoma relapse can improve patient survival; thus, there is a great need for easily accessible biomarkers that facilitate the diagnosis of metastatic disease. We investigated the diagnostic effect of blood biomarkers such as lactate dehydrogenase (LDH), S100B, and osteopontin in the detection of metastases. Clinical data and peripheral blood samples of 206 melanoma patients were collected (no metastasis, N = 120; metastasis, N = 86). The discriminative power of blood biomarkers, patient demographics, and clinicopathological parameters of primary melanomas were evaluated using univariate and multivariate logistic regression models and receiver operating characteristic (ROC) analysis to determine the area under the curve (AUC). Plasma osteopontin levels showed a significant and independent effect on the probability of metastasis, similar to serum S100B levels. In addition, the location of the primary tumor on the lower extremities and the American Joint Committee on Cancer (AJCC) categories pT2b-3a, pT3b-4a, and pT4b were associated with the diagnosis of metastasis. Importantly, the combination of the three blood biomarkers and primary tumor localization and AJCC pT category yielded excellent discrimination (AUC: training set: 0.803; validation set: 0.822). In conclusion, plasma osteopontin can be classified as a melanoma biomarker; moreover, by combining clinicopathological prognostic variables, the diagnostic effect of blood biomarkers in the detection of metastatic melanoma can be improved.

The role of CCND1 alterations during the progression of cutaneous malignant melanoma.

It is well demonstrated that CCND1 amplification is a frequent event in the acral subtype of cutaneous malignant melanoma; however, its role in the other subtypes of the disease is still controversial. The objectives of this study were to evaluate genetic and expression alterations of CCND1 with a focus on primary cutaneous melanomas, to define BRAF and NRAS mutation status, and correlate the data with clinical-pathological parameters. CCND1 amplification was associated with ulceration and the localization of the metastasis. After correction for the mutation state of BRAF and NRAS genes, CCND1 amplification in samples without such mutations was associated with ulceration and sun exposure. The cyclin D1 (CCND1) mRNA level decreased in lesions with multiple metastases and was correlated with both the mRNA levels and mutation state of BRAF and NRAS genes. Primary melanomas with BRAF(V600) or NRAS(Q61 ) mutations exhibited lower CCND1 mRNA level. CCND1 protein expression was associated with Breslow thickness, metastasis formation, and shorter survival time. These observations suggest that CCND1 alterations are linked to melanoma progression and are modified by BRAF and NRAS mutations. Our data show that CCND1 amplification could have a prognostic relevance in cutaneous melanoma and highlight that altered CCND1 gene expression may influence the metastatic progression, survival, and the localization of metastases.

PCR-DGGE analysis of the bacterial composition of a kaolin slurry showing altered rheology.

Kaolin is an important industrial raw material and a basis of a range of different products. Microbial spoilage is a detrimental process observed especially in kaolin slurries, leading to low quality products and economic loss. Although the alteration of kaolin slurries in ceramic industry was observed, the process and the microbial background have not been analyzed in details. This study provides the first data using a cultivation independent molecular biological approach (PCR-DGGE) regarding the bacterial composition of an altered kaolin slurry. The results show that potential exopolymer (EPS) producer bacteria (e.g. Acinetobacter, Pseudomonas) appear in the altered kaolin slurry, which may have an important role in the modification of kaolin slurries.

Gene Expression Patterns of Osteopontin Isoforms and Integrins in Malignant Melanoma.

Osteopontin (OPN) is a multifunctional glycoprotein that physiologically interacts with different types of integrins. It is considered to be a possible prognostic biomarker in certain tumor types; however, various splicing isoforms exist, which have not been investigated in melanoma. We aimed to define the relative expression pattern of five OPN isoforms and clarify the prognostic significance of the splice variants in melanoma. We also aimed to investigate the expression pattern of eight integrins in the same tumors. Gene expression analyses revealed that the relative expression of OPNa, OPNb, and OPNc is significantly higher in metastatic tumors compared to primary lesions (p < 0.01), whereas the expression of OPN4 and OPN5 was low in both. The more aggressive nodular melanomas had higher expression levels compared to the superficial spreading subtype (p ≤ 0.05). The relative expression of the eight tested integrins was low, with only the expression of ITGB3 being detectable in nodular melanoma (Medianlog2 = 1.274). A positive correlation was found between Breslow thickness and the expression of OPNc variant, whereby thicker tumors (>4 mm) had significantly higher expression (p ≤ 0.05). The Breslow thickness was negatively correlated with the expression of OPN4, and similarly with ITGA2. OPNc also exhibited significant positive correlation with the presence of metastasis. Our data show that high expression of OPNa, OPNb, and especially OPNc and low expression of OPN4 and ITGA2 are associated with an advanced stage of tumor progression and poor prognosis in melanoma.

Comprehensive analysis of different tumor cell-line produced soluble mediators on the differentiation and functional properties of monocyte-derived dendritic cells.

Developing dendritic cells (DCs) from monocytes is a sensitively regulated process. One possible way for cancers to avoid immune recognition and antitumor response is the modulation of DC differentiation. Although several studies are available on the examination of tumor-associated macrophages, a comprehensive analysis focusing on the effects of tumor-formed DCs is not known to date. We provide a comparative analysis of the tumor-edited-monocyte derived DCs differentiated in the presence of adenocarcinomas (MDA, HT29, HeLa)- and primary (WM278, WM983A) or metastatic (WM1617, WM983B) melanomas. The immunomodulatory effect of tumors is mediated at least partly by secreted mediators. We investigated the impact of tumor cell-derived conditioned media on the differentiation of DCs from CD14+ monocytes, sequentially determining the phenotype, cytokine production, phagocytic, and the T cell polarizing capacity of moDCs. We completed our observations by analyzing our data with bioinformatic tools to provide objective correlations between phenotypical and functional properties of different tumor-educated moDCs. The correlation analysis revealed significant differences in the characteristics of adenocarcinomas- or melanomas-edited moDCs. We highlight the functional differences in the properties of moDCs differentiated in the presence of various cancer cell lines. We offer new information and options for the in vitro differentiation protocols of various tumor-conditioned moDCs. Our results confirm that various immunomodulatory properties of different tumor cell lines result in multiple manipulations of DC differentiation.

[Molecular background of BRAF inhibitor induced resistance in BRAFV600E mutant melanoma cell lines]. / BRAF-inhibitor-kezelést követoen kialakult rezisztencia molekuláris háttere BRAF V600E mutáns melanóma-sejtvonalakban.

Target-specific inhibition of the BRAFV600E mutant protein has been a major breakthrough in the treatment of metastatic cutaneous melanoma. However, the success of therapies is significantly overshadowed by the development of resistance. Understanding the molecular mechanisms associated with acquired resistance is an important step to increase the effectiveness of melanoma treatment. Our aim was to elucidate the molecular differences underlying the development of drug resistance using a mutant BRAF protein inhibitor (vemurafenib analogue: PLX4720) in BRAFV600E mutant melanoma cell lines. We developed four BRAF inhibitor-resistant cell lines and examined the effect of BRAF inhibitor "withdrawal" on cell division. ArrayCGH was used to define genetic, and Affymetrix HumanGene 1.0 microarray to monitor gene expression alterations between the sensitive and resistant cell lines. Protein expression was determined using Proteome Profiler Human XL Oncology Array. We found that withdrawal of the inhibitor reduces cell proliferation in the resistant cells. The invasive potential of the resistant cells increased. Using genomic and proteomic methods we described new molecular alterations associated with acquired resistance.

Molecular Alterations Associated with Acquired Drug Resistance during Combined Treatment with Encorafenib and Binimetinib in Melanoma Cell Lines.

Combination treatment using BRAF/MEK inhibitors is a promising therapy for patients with advanced BRAFV600E/K mutant melanoma. However, acquired resistance largely limits the clinical efficacy of this drug combination. Identifying resistance mechanisms is essential to reach long-term, durable responses. During this study, we developed six melanoma cell lines with acquired resistance for BRAFi/MEKi treatment and defined the molecular alterations associated with drug resistance. We observed that the invasion of three resistant cell lines increased significantly compared to the sensitive cells. RNA-sequencing analysis revealed differentially expressed genes that were functionally linked to a variety of biological functions including epithelial-mesenchymal transition, the ROS pathway, and KRAS-signalling. Using proteome profiler array, several differentially expressed proteins were detected, which clustered into a unique pattern. Galectin showed increased expression in four resistant cell lines, being the highest in the WM1617E+BRes cells. We also observed that the resistant cells behaved differently after the withdrawal of the inhibitors, five were not drug addicted at all and did not exhibit significantly increased lethality; however, the viability of one resistant cell line (WM1617E+BRes) decreased significantly. We have selected three resistant cell lines to investigate the protein expression changes after drug withdrawal. The expression patterns of CapG, Enolase 2, and osteopontin were similar in the resistant cells after ten days of "drug holiday", but the Snail protein was only expressed in the WM1617E+BRes cells, which showed a drug-dependent phenotype, and this might be associated with drug addiction. Our results highlight that melanoma cells use several types of resistance mechanisms involving the altered expression of different proteins to bypass drug treatment.

Cell Proliferation Is Strongly Associated with the Treatment Conditions of an ER Stress Inducer New Anti-Melanoma Drug in Melanoma Cell Lines.

HA15 is a new anti-melanoma drug that triggers endoplasmic reticulum (ER) stress and causes deleterious effects on melanoma cell viability due to autophagy and apoptosis, regardless of driver mutations or drug resistance. In this study, we investigated the effect of HA15 on the viability/proliferation of BRAFV600E-mutant melanoma cells using different culture conditions. In contrast to the published data, we did not detect significant melanoma cell death under normal culture conditions using HA15 treatment. Indeed, only cells that were cultured under long-term starvation conditions were sensitive to the drug. Quantitative measurements of ER stress and autophagy markers showed that the compound HA15 does not trigger stress alone but synergistically enhances ER stress under starvation conditions. Importantly, we observed that the viability of normal melanocytes decreased significantly with treatment, even at low HA15 concentrations. Finally yet importantly, we were able to generate HA15-resistant cell lines, which failed by Cerezo et al. In summary, HA15 only influences the viability of cells that are starved for several hours before and during treatment. However, this in vitro setting is far from the in vivo conditions. In addition, our data clearly show that melanoma cells can acquire HA15 resistance. Further studies are needed to prove that HA15 is an effective anti-cancer agent.

Silencing Osteopontin Expression Inhibits Proliferation, Invasion and Induce Altered Protein Expression in Melanoma Cells.

Osteopontin (OPN) is a multifunctional phosphoprotein that is expressed in different types of cancers, including melanoma. OPN overexpression is associated with tumor progression and metastasis formation; however, the role of OPN in cell invasion and metastasis formation is not completely understood. In this study we aimed to define OPN expression in melanoma tissues and cell lines and investigate the effect of OPN expression on cell proliferation and invasion after inhibiting OPN expression with small interfering RNA (siRNA). OPN gene expression was determined by qRT-PCR, while protein expression was examined using a Proteome Profiler Oncology Array. siRNA-mediated OPN knockdown led to decreased OPN expression in melanoma cell lines, which was associated with decreased cell proliferation and invasion. Proteome profile analysis revealed significantly different protein expression between the original and transfected cell lines. The altered expression of the differently expressed proteins was validated at the mRNA level. Furthermore, OPN-specific siRNA was able to reduce OPN expression and inhibit the invasiveness of melanoma cells. Our results revealed for the first time that silencing the OPN gene influences proliferation and invasion of melanoma cells by effecting EGFR, tenascin C, survivin, galectin-3 and enolase 2 expression. To predict protein-protein interactions along with putative pathways we used STRING analysis for the differentially expressed proteins. These proteins formed multiple clusters, including extracellular matrix organization, regulation of angiogenesis, cell death and cell migration, PI3K-Akt, MAPK and focal adhesion signaling pathways. Taken together these data suggest that OPN might be an ideal target for drug development and therapies.

Characterization of a novel long-chain n-alkane-degrading strain, Dietzia sp. E1.

The newly isolated strain E1, identified as a Dietzia sp., proved to have an excellent ability to degrade n-C12 to n-C38 alkane components of crude oil. The preferred substrate was the very long-chain alkane n-eicosane at an optimal temperature of 37 degrees C and an optimal pH of 8 under aerobic conditions. The growth and substrate uptake kinetics were monitored during the n-alkane fermentation process, and Dietzia sp. E1 cells were found to possess three distinct levels of cell-surface hydrophobicity. Gas chromatographic/mass spectrometric analysis revealed that intracellular substrate mineralization occurred through the conversion of n-alkane to the corresponding n-alkanal. The monoterminal oxidation pathway was presumably initiated by AlkB and CYP153 terminal alkane hydroxylases, both of their partial coding sequences were successfully detected in the genome of strain E1, a novel member of the Dietzia genus.

Degradation of native feathers by a novel keratinase-producing, thermophilic isolate, Brevibacillus thermoruber T1E.

Strain T1E, isolated and identified as Brevibacillus thermoruber, and evolutionally distant from the known keratinolytic isolates, proved to have feather-degrading ability. During the 7-day fermentation period, T1E consumed 10 g/l native goose feathers as the sole source of carbon and energy at 50 degrees C under aerobic conditions. The isolate secreted a thermostable, keratinolytic protease, which exhibited activity optimally at pH 6.5, whilst it was inhibited at alkaline pH. The keratin cleavage and catabolism resulted in the accumulation of free aspartic acid and soluble peptides with maximum values of 31.6 and 720 mg/l, respectively. The majority of the fermentation end-products were found to be small oligopeptides with an average molecular mass of 2275 Da.