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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

[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.

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.

Prevalence of Insulin Resistance in the Hungarian General and Roma Populations as Defined by Using Data Generated in a Complex Health (Interview and Examination) Survey.

Data mainly from one-off surveys clearly show that the health of Roma, the largest ethnic minority of Europe, is much worse than that of the general population. However, results from comprehensive exploratory studies are missing. The aim of our study was to create a complex database for comparative and association studies to better understand the background of the very unfavourable health of Roma, especially the high burden of cardiometabolic diseases. A three-pillar (questionnaire-based, physical and laboratory examinations) health survey was carried out on randomly selected samples of the Hungarian general (HG, n = 417) and Roma (HR, n = 415) populations, and a database consisting of more than half a million datapoints was created. Using selected data, the prevalence rates of metabolic syndrome (MetS) and of its components were determined, and to estimate the risk of insulin resistance (IR), surrogate measures (the homeostasis model assessment of insulin resistance index, quantitative insulin sensitivity check index, McAuley and TyG indices and the TG/HDL-C ratio) were calculated. Receiver operating characteristic curve analysis and Youden's method were used to define the optimal cut-off values of each IR index. The prevalence of MetS was very high in both study populations (HG: 39.8%, HR: 44.0%) with no statistically significant difference between the two groups in females or males. The prevalence of MetS showed a very marked increase in the HR 35-49 years age group. Among surrogate measures, the TyG index showed the greatest power for predicting IR/MetS at a cut-off value of 4.69 (77% sensitivity, 84% specificity) and indicated a 42.3% (HG) and 40.5% (HR) prevalence of IR. The prevalence of MetS and IR is almost equally very unfavourable in both groups; thus, the factors underlying the high premature mortality burden of Roma should be further clarified by investigating the full spectrum of risk factors available in the database, with a special focus on the access of Roma people to preventive and curative health services.

Gene Expression Signature of BRAF Inhibitor Resistant Melanoma Spheroids.

In vitro cell cultures are frequently used to define the molecular background of drug resistance. The majority of currently available data have been obtained from 2D in vitro cultures, however, 3D cell culture systems (spheroids) are more likely to behave similarly to in vivo conditions. Our major aim was to compare the gene expression signature of 2D and 3D cultured BRAFV600E mutant melanoma cell lines. We successfully developed BRAF-drug resistant cell lines from paired primary/metastatic melanoma cell lines in both 2D and 3D in vitro cultures. Using Affymetrix Human Gene 1.0 ST arrays, we determined the gene expression pattern of all cell lines. Our analysis revealed 1049 genes (562 upregulated and 487 downregulated) that were differentially expressed between drug-sensitive cells grown under different cell cultures. Pathway analysis showed that the differently expressed genes were mainly associated with the cell cycle, p53, and other cancer-related pathways. The number of upregulated genes (72 genes) was remarkably fewer when comparing the resistant adherent cells to cells that grow in 3D, and were associated with cell adhesion molecules and IGF1R signalling. Only 1% of the upregulated and 5.6% of the downregulated genes were commonly altered between the sensitive and the resistant spheroids. Interestingly, we found several genes (BNIP3, RING1 and ABHD4) with inverse expression signature between sensitive and resistant spheroids, which are involved in anoikis resistance and cell cycle regulation. In summary, our study highlights gene expression alterations that might help to understand the development of acquired resistance in melanoma cells in tumour tissue.

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.

Chronic responses of aerobic granules to the presence of graphene oxide in sequencing batch reactors.

The chronic responses of aerobic granular sludge (AGS) to the presence of graphene oxide nanoparticles (GO NPs) (5, 15, 25, 35, 45, 55, 65, 75, 85, and 95 mg/L of GO NPs for 7 days) during biological wastewater treatment processes were investigated. Bioreactor performance, extracellular polymeric substance (EPS) secretion, and microbial community characteristics were assessed. The results showed that the effects of GO NPs on bioreactor performances were dependent on the dose applied and the duration for which it was applied. At concentrations of 55, 75, and 95 mg/L, GO NPs considerably inhibited the efficiency of organic matter and ammonia removal; however, nitrite and nitrate removal rates were unchanged. Biological phosphorus removal decreased even when only low concentrations of GO NPs were used. The secretion of EPS, which could alleviate the toxicity of GO NPs, also changed. The increased amount of nanoparticles also resulted in significant changes to the bacterial community structure. Based on the amplicon sequencing of 16S rRNA genes, Paracoccus sp., Klebsiella sp., and Acidovorax species were identified as the most tolerant strains.

Molecular alterations associated with acquired resistance to BRAFV600E targeted therapy in melanoma cells.

Selective inhibition of the mutant BRAF protein is a highly promising therapeutic approach for melanoma patients carrying the BRAF mutation. Despite the remarkable clinical response, most patients develop resistance and experience tumour regrowth. To clarify the molecular background of BRAF inhibitor resistance, we generated four drug-resistant melanoma cell lines from paired primary/metastatic cell lines using a vemurafenib analogue PLX4720. Three of the resistant cell lines showed decreased proliferation after drug withdrawal, but the proliferation of one cell line (WM278) increased notably. Furthermore, we observed opposite phenomena in which a 'drug holiday' could not only be beneficial but also contribute to tumour progression. Using genomic and proteomic approaches, we found significantly different alterations between the sensitive and resistant cell lines, some of which have not been reported previously. In addition to several other changes, copy number gains were observed in all resistant cell lines on 8q24.11-q24.12 and 8q21.2. Gene expression analysis showed that most genes upregulated in the resistant cell lines were associated with cell motility and angiogenesis. Increased expression of six proteins (ANGPLT4, EGFR, Endoglin, FGF2, SerpinE1 and VCAM-1) and decreased expression of two proteins (osteopontin and survivin) were observed consistently in all resistant cell lines. In summary, we identified new genomic alterations and characterized the protein expression patterns associated with the resistant phenotype. Although several proteins have been shown to be associated with BRAF resistance, our study is the first to describe the association of VCAM-1 and osteopontin with BRAF resistance.

Detection of CCND1 Locus Amplification by Fluorescence In Situ Hybridization.

It is well known that chromosomal aberrations of tumors are associated with the initiation and progression of malignancy. Fluorescence in situ hybridization (FISH) is a powerful, rapid method to detect chromosome copy number and structural alterations in tissue sections, chromosome, or interphase cellular preparations via hybridization of complementary probe sequences. The technique is based on the complementary nature of DNA double strands, which allows fluorescently labeled DNA probes to be used as probes to label the complementary sequences of target cells, chromosomes, and tissues. FISH technique has many applications, including basic gene mapping, used in pathological diagnosis to detect chromosome and gene copy number aberrations, translocations, microdeletions, and duplications. For the recognition of gene amplifications and deletions, locus-specific probes that are collections of one or a few cloned DNA sequences are routinely used. Multiplex-FISH (M-FISH) technique visualizes all chromosomes with different colors using spectrally distinct fluorophores for each chromosome in one experiment to detect numerical and structural alterations of chromosomes obtained from tumor cells. Recently many of the gene-specific probes are commercially available.

Altered integrin expression patterns shown by microarray in human cutaneous melanoma.

A large variety of molecular pathways in melanoma progression suggests that no individual molecular alteration is crucial in itself. Our aim was to define the molecular alterations underlying metastasis formation. Gene expression profiling was performed using microarray and qRT-PCR to define alterations between matched primary and metastatic melanoma cell lines. These data were integrated with publicly available unmatched tissue data. The invasiveness of cell lines was determined by Matrigel invasion assays and invasive clones from primary melanoma-derived cell lines were also selected. Two metastatic cell line models were created: the regional lymph node WM983A-WM983A-WM983B and the distant lung WM793B-WM793B-1205Lu metastatic models. The majority of metastasis genes were downregulated and enriched in adhesion and ITGA6-B4 pathways. Upregulation of immune pathways was characteristic of distant metastases, whereas increased Rap1 signaling was specific for regional (sub)cutaneous metastases. qRT-PCR analysis of selected integrins (A2, A3, A4, A9, B5, B8, A6, B1, and B3) highlighted the possible importance of ITGA3/4 and B8 in the metastatic process, distinguishing regional and distant metastases. We identified functionally relevant gene clusters that influenced metastasis formation. Our data provide further evidence that integrin expression patterns may be important in distant metastasis formation.

Impact of the morphology and reactivity of nanoscale zero-valent iron (NZVI) on dechlorinating bacteria.

Nanoscale zero-valent iron (NZVI) is increasingly used for reducing chlorinated organic contaminants in soil or groundwater. However, little is known about what impact the particles will have on the biochemical processes and the indigenous microbial communities. Nanoiron reactivity is affected by the structure and morphology of nanoparticles that complicates the applicability in bioremediation. In this study, the effect of precursors (ferrous sulfate and ferric chloride) and reducing agents (sodium dithionite and sodium borohydride) on the morphology and the reactivity of NZVIs was investigated. We also studied the impact of differently synthesized NZVIs on microbial community, which take part in reductive dechlorination. We demonstrated that both the applied iron precursor and the reducing agent had influence on the structure of the nanoparticles. Spherical nanoparticles with higher Fe(0) content (>90%) was observed by using sodium borohydride as reducing agent, while application of sodium dithionite as reducing agent resulted nanostructures with lower Fe(0) content (between 68,7 and 85,5%). To determine the influence of differently synthesized NZVIs on cell viability anaerobic enriched microcosm were used. NVZI was used in 0.1 g/L concentration in all batch experiments. Relative amount of Dehalococcoides, sulfate reducers (SRBs) and methanogens were measured by quantitative PCR. We found that the relative amount of Dehalococcoides slowly decreased in all experiments independently from the precursor and reducing agent, whereas the total amount of microbes increased. The only clear distinction was in relative amount of sulfate reducers which were higher in the presence of NZVIs synthesized from sodium dithionite.