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.

Riverine Microplastic Quantification: A Novel Approach Integrating Satellite Images, Neural Network, and Suspended Sediment Data as a Proxy.

Rivers transport terrestrial microplastics (MP) to the marine system, demanding cost-effective and frequent monitoring, which is attainable through remote sensing. This study aims to develop and test microplastic concentration (MPC) models directly by satellite images and indirectly through suspended sediment concentration (SSC) as a proxy employing a neural network algorithm. These models relied upon high spatial (26 sites) and temporal (198 samples) SSC and MPC data in the Tisza River, along with optical and active sensor reflectance/backscattering. A feedforward MLP neural network was used to calibrate and validate the direct models employing k-fold cross-validation (five data folds) and the Optuna library for hyperparameter optimization. The spatiotemporal generalization capability of the developed models was assessed under various hydrological scenarios. The findings revealed that hydrology fundamentally influences the SSC and MPC. The indirect estimation method of MPC using SSC as a proxy demonstrated higher accuracy (R2 = 0.17-0.88) than the direct method (R2 = 0-0.2), due to the limitations of satellite sensors to directly estimate the very low MPCs in rivers. However, the estimation accuracy of the indirect method varied with lower accuracy (R2 = 0.17, RMSE = 12.9 item/m3 and MAE = 9.4 item/m3) during low stages and very high (R2 = 0.88, RMSE = 7.8 item/m3 and MAE = 10.8 item/m3) during floods. The worst estimates were achieved based on Sentinel-1. Although the accuracy of the MPC models is moderate, it still has practical applicability, especially during floods and employing proxy models. This study is one of the very initial attempts towards MPC quantification, thus more studies incorporating denser spatiotemporal data, additional water quality parameters, and surface roughness data are warranted to improve the estimation accuracy.

The effect of ranibizumab and aflibercept treatment on the prevalence of outer retinal tubulation and its influence on retreatment in neovascular age-related macular degeneration.

BACKGROUND: We aimed to analyze the differences in the prevalence of outer retinal tubulation (ORT) in neovascular age-related macular degeneration (AMD) treated with anti-vascular endothelial growth factor (anti-VEGF) agents, either aflibercept or ranibizumab. Our further aim was to examine the changes in the frequency of injections of ranibizumab before and after ORT appearance. METHODS: Two hundred thirty six eyes of 230 patients were included in the study (184 eyes treated with ranibizumab by pro re nata regimen (PRN), 52 eyes with aflibercept bimonthly) and followed for 6-24 months. Using optical coherence tomography (OCT), the first appearance of ORT was documented, and fixed time point evaluations were also made every six months to determine the existence of ORT. The number of injections, the presence or absence of subretinal hyperreflective material (SHRM) at treatment initiation and visual acuity were also noted. RESULTS: The survival analysis with Cox proportional hazard model showed no significant difference between the ranibizumab and aflibercept groups in relation to the development of ORT (p = 0.79, hazard ratio 0.92). In the PRN treated ranibizumab group the number of injections showed significant decrease after ORT development (p = 0.004). When SHRM was present at treatment initiation the chance of developing ORT was 2.75 and 11.14 times higher in the ranibizumab and aflibercept groups, respectively. CONCLUSIONS: The prevalence of ORT increased over time independently from the chosen anti-VEGF drug. Our results suggest that upon the appearance of ORT a decrease in retreatments can be expected.

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.

Assessment of the Physical Activity of Children with Asthma Bronchiale.

Physical activity is an especially important part of everyday life for children with chronic diseases. The aim of the study was to show whether asthma is a barrier to physical activity in our society. The correlations between the severity of the disease, body mass index, and physical activity were analyzed, and parents' opinions on whether children should participate in active sports were assessed. Physical activity of children with asthma was analyzed by questionnaires; 93 parents and their 93 children were involved in the survey. The age of children was 12.6 ± 3.5 years (mean ± SD), 69.9% were boys, 30.1% were girls. A total of 93.4% of the respondents participated in a physical education program and 56.5% also attended sporting activities on a regular basis. In terms of disease severity, 61.2% of the children had mild asthma, 37.6% moderate, and 1.2% severe, and 6.5% of the respondents also stated that their children's illness had been consistently or frequently limiting their performance concerning their school or home duties over the past four weeks. Of the parents surveyed, 12% felt that physical activity was not appropriate in the context of this disease. We concluded that fear of the consequences of physical activity depends largely on education, which should involve parents, teachers, and coaches.

Emerging novel porcine parvoviruses in Europe: origin, evolution, phylodynamics and phylogeography.

To elucidate the spatiotemporal phylodynamics, dispersion and evolutionary processes underlying the emergence of novel porcine parvovirus 2 (PPV2), PPV3 and PPV4 species, we analysed all available complete capsid genes, together with ours, obtained in Europe. Bayesian phylogeography indicates that Romania (PPV2 and PPV4) and Croatia (PPV3) are the most likely ancestral areas from which PPVs have subsequently spread to other European countries and regions. The timescale of our reconstruction supported a relatively recent history of the currently circulating novel PPV species (1920s to 1980s) in the domestic or sylvatic host. While PPV2 strains exhibited a large genetic exchange characterized by significant recombination and gene flow between distinct regions and hosts, PPV3 and PPV4 showed a diversification reflected by the accumulation of geographically structured polymorphisms. The RNA-like evolutionary rates detected inter- and intrahost recombination and the positive selection sites provided evidence that the PPV2-4 capsid gene plays a prominent role in host adaptation.

Capsid protein evolution and comparative phylogeny of novel porcine parvoviruses.

In addition to the well known "classical" porcine parvovirus (PPV1; responsible for reproductive failure of susceptible sows) several new porcine parvoviruses have been recognized (PPV2, PPV3 and PPV4) in recent years. The genetic variation, characteristics and evolutionary factors shaping these novel PPVs were studied by comparing the complete capsid (cap) genes of PPVs from domestic pigs and wild boars. Using Bayesian coalescent methods we estimated the rate of nucleotide substitution for PPV2, PPV3 and PPV4 to be of the order of 3.86 × 10(-4)-8.23 × 10(-4) subs site(-1) year(-1), similar to those commonly measured for RNA viruses, although this rate in case of PPV2 is probably influenced by frequent recombination events. Given such rapid evolutionary dynamics, it is likely that novel PPVs will continue to improve their capacity to spread among Suidae hosts worldwide. The mean time to the most recent common ancestor for the sampled genetic diversity of the newly discovered porcine parvoviruses was estimated. The results indicated that novel PPVs originated within approximately the last 70 years. Incongruent phylogenetic relationships of several strains suggested recombination events supported by several recombination-detecting methods and by split-decomposition phylogenetic networks. Analyses of the selective constraints acting on each codon suggest that some regions of PPV cap genes were under positive selection. This study showed that inter- and intraspecies recombination and diversifying selection pressures are prevalent across the cap genes of novel PPVs, and beside host switching and gene flow are important driving forces of their evolution and may be significant factors in the emergence of new viral variants.

High spatiotemporal resolution analysis on suspended sediment and microplastic transport of a lowland river.

The suspended sediment (SS) and microplastic (MP) transport in rivers is quite a complex process, influenced by several spatially and temporally changing factors (e.g., hydrology, sediment availability, human impact). Researchers usually investigate these factors individually and based on limited repetition in space and time. Therefore, this study aims to compare the driving factors of SS and MP transport by applying dense temporal (72 measurements) and spatial monitoring (at 26 sites). This study was performed on the medium-sized Tisza River, Central Europe. The suspended sediment concentration (SSC) was measured by water sampling and estimated based on Sentinel-2 images, while MP concentration was measured by pumping of water (1 m3). The SSC of the Tisza varied between 12.6 and 322.5 g/m3, whereas the MP concentration ranged 0-129 item/m3. Most of the transported particles were fibers (81-98 %), thus, it was assumed that MPs originated from wastewater. The results reflect that the hydrological conditions basically influence the SS and MP concentrations, as a strong positive correlation was found (ρSSC-MP = 0.6) between them during a year; however, the correlation during floods (minor floods: ρ = 0.63; medium floods: ρ = 0.41) was higher than at low stages (ρ = 0.1). It was assumed that run-off and mobilization of channel materials both contribute to increased SS and MP transport during floods. In contrary, the importance of mobilization of channel materials and wastewater input increase during low stages. The repeated measurements revealed that slope and velocity conditions, proximity of sources, tributaries, and dams influence the longitudinal changes in SS and MP concentrations. However, the effects of tributaries and dams are ambiguous (especially for MP) and require further research. The longitudinal measurements were conducted at low stages; hence, moderate negative correlations (ρ2021 = -0.35; ρ2022 = -0.41) were found between the SS and MP concentrations. Therefore, additional monitoring during (overbank) floods and denser spatial sampling are required to precisely reveal the spatiotemporal changes of SS and MP concentrations in rivers.

Machine learning-based detection and mapping of riverine litter utilizing Sentinel-2 imagery.

Despite the substantial impact of rivers on the global marine litter problem, riverine litter has been accorded inadequate consideration. Therefore, our objective was to detect riverine litter by utilizing middle-scale multispectral satellite images and machine learning (ML), with the Tisza River (Hungary) as a study area. The Very High Resolution (VHR) images obtained from the Google Earth database were employed to recognize some riverine litter spots (a blend of anthropogenic and natural substances). These litter spots served as the basis for training and validating five supervised machine-learning algorithms based on Sentinel-2 images [Artificial Neural Network (ANN), Support Vector Classifier (SVC), Random Forest (RF), Naïve Bays (NB) and Decision Tree (DT)]. To evaluate the generalization capability of the developed models, they were tested on larger unseen data under varying hydrological conditions and with different litter sizes. Besides the best-performing model was used to investigate the spatio-temporal variations of riverine litter in the Middel Tisza. According to the results, almost all the developed models showed favorable metrics based on the validation dataset (e.g., F1-score; SVC: 0.94, ANN: 0.93, RF: 0.91, DT: 0.90, and NB: 0.83); however, during the testing process, they showed medium (e.g., F1-score; RF:0.69, SVC: 0.62; ANN: 0.62) to poor performance (e.g., F1-score; NB: 0.48; DT: 0.45). The capability of all models to detect litter was bounded to the pixel size of the Sentinel-2 images. Based on the spatio-temporal investigation, hydraulic structures (e.g., Kisköre Dam) are the greatest litter accumulation spots. Although the highest transport rate of litter occurs during floods, the largest litter spot area upstream of the Kisköre Dam was observed at low stages in summer. This study represents a preliminary step in the automatic detection of riverine litter; therefore, additional research incorporating a larger dataset with more representative small litter spots, as well as finer spatial resolution images is necessary.

The Importance of Physical Activity in Preventing Fatigue and Burnout in Healthcare Workers.

The workers of the health sector are important to the country's economy in many ways. Healthy and rested workers are highly valuable to the public health sector and give a good perception of their work to patients and society. It is thus important to have a sufficient number of healthy working staff in healthcare institutions who do not have work fatigue and burnout. A total of 987 employees-doctors, professional staff, and others-of a large healthcare institution in Hungary voluntarily participated in a survey regarding their lifestyle and physical activity habits and answered the questions anonymously. Women reported less leisure time (p < 0.02), with 54.9% of female respondents saying that they did not exercise regularly, and fatigue was more common among them (p < 0.001). In this respect, the healthcare workers' responses did not differ from those of the overall population. The most common sports were cycling (17.7%), running (15.4%), and working out in a gym (12.3%). Reasons for not participating in sports included lack of time (70.2%) and fatigue (43.9%) as the most frequent responses. Healthcare workers are exposed to a number of risks that require particular attention to maintain their health. Employers should thus focus on implementing programs that prevent burnout and promote healthy lifestyles.

Half sandwich-type osmium, ruthenium, iridium and rhodium complexes with bidentate glycosyl heterocyclic ligands induce cytostasis in platinum-resistant ovarian cancer cells and bacteriostasis in Gram-positive multiresistant bacteria.

The toxicity of and resistance to platinum complexes as cisplatin, oxaliplatin or carboplatin calls for the replacement of these therapeutic agents in clinical settings. We have previously identified a set of half sandwich-type osmium, ruthenium and iridium complexes with bidentate glycosyl heterocyclic ligands exerting specific cytostatic activity on cancer cells but not on non-transformed primary cells. The apolar nature of the complexes, conferred by large, apolar benzoyl protective groups on the hydroxyl groups of the carbohydrate moiety, was the main molecular feature to induce cytostasis. We exchanged the benzoyl protective groups to straight chain alkanoyl groups with varying length (3 to 7 carbon units) that increased the IC50 value as compared to the benzoyl-protected complexes and rendered the complexes toxic. These results suggest a need for aromatic groups in the molecule. The pyridine moiety of the bidentate ligand was exchanged for a quinoline group to enlarge the apolar surface of the molecule. This modification decreased the IC50 value of the complexes. The complexes containing [(η6-p-cymene)Ru(II)], [(η6-p-cymene)Os(II)] or [(η5-Cp*)Ir(III)] were biologically active unlike the complex containing [(η5-Cp*)Rh(III)]. The complexes with cytostatic activity were active on ovarian cancer (A2780, ID8), pancreatic adenocarcinoma (Capan2), sarcoma (Saos) and lymphoma cell lines (L428), but not on primary dermal fibroblasts and their activity was dependent on reactive oxygen species production. Importantly, these complexes were cytostatic on cisplatin-resistant A2780 ovarian cancer cells with similar IC50 values as on cisplatin-sensitive A2780 cells. In addition, the quinoline-containing Ru and Os complexes and the short chain alkanoyl-modified complexes (C3 and C4) proved to be bacteriostatic in multiresistant Gram-positive Enterococcus and Staphylococcus aureus isolates. Hereby, we identified a set of complexes with submicromolar to low micromolar inhibitory constants against a wide range of cancer cells, including platinum resistant cells and against multiresistant Gram-positive bacteria.

Mountains of plastic: Mismanaged plastic waste along the Carpathian watercourses.

Plastic waste poses numerous risks to mountain river ecosystems due to their high biodiversity and specific physical characteristics. Here, we provide a baseline assessment for future evaluation of such risks in the Carpathians, one of the most biodiverse mountain ranges in East-Central Europe. We used high-resolution river network and mismanaged plastic waste (MPW) databases to map MPW along the 175,675 km of watercourses draining this ecoregion. We explored MPW levels as a function of altitude, stream order, river basin, country, and type of nature conservation in a given area. The Carpathian watercourses below 750 m a.s.l. (142,282 km, 81 % of the stream lengths) are identified as significantly affected by MPW. Most MPW hotspots (>409.7 t/yr/km2) occur along rivers in Romania (6568 km; 56.6 % of all hotspot lengths), Hungary (2679 km; 23.1 %), and Ukraine (1914 km; 16.5 %). The majority of the river sections flowing through the areas with negligible MPW (< 1 t/yr/km2) occur in Romania (31,855 km; 47.8 %), Slovakia (14,577 km; 21.9 %), and Ukraine (7492; 11.2 %). The Carpathian watercourses flowing through the areas protected at national level (3988 km; 2.3 % of all watercourses studied) have significantly higher MPW values (median = 7.7 t/yr/km2) than those protected at regional (51,800 km; 29.5 %) (median MPW = 1.25 t/yrkm2) and international levels (66 km; 0.04 %) (median MPW = 0 t/yr/km2). Rivers within the Black Sea basin (88.3 % of all studied watercourses) have significantly higher MPW (median = 5.1 t/yr/km2, 90th percentile = 381.1 t/yr/km2) than those within the Baltic Sea basin (median = 6.5 t/yr/km2, 90th percentile = 84.8 t/yr/km2) (11.1 % of all studied watercourses). Our study indicates the locations and extent of riverine MPW hotspots in the Carpathian Ecoregion, which can support future collaborations between scientists, engineers, governments, and citizens to better manage plastic pollution in this region.

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.

The effects of environment and physiological cyclicity on the immune system of Viperinae.

One of the important aspects of species' survival is connected with global climate changes, which also conditions the epidemiology of infectious diseases. Poikilotherms are exposed, as other species, to climatic influence, especially due to their physiological peculiarities such as important stages of their life cycle: hibernation, shedding, and active phase. The immune system serves as an accurate indicator of the health status and stress levels in these species. This study aimed to monitor the changes of innate (leukocyte subpopulations and total immune globulins) and adaptive immunity (in vitro leukocyte blast transformation) of two viper species, V. berus berus and V. ammodytes ammodytes, endemic in Europe and spread in different regions of Romania during their three major life cycles, hibernation, shedding, and active phase. The results indicated that seasonal variance and cycle rather than species and regional distribution influence the functionality of the immune system.

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.

Genetic detection and analysis of porcine bocavirus type 1 (PoBoV1) in European wild boar (Sus scrofa).

Novel porcine parvoviruses showing the genetic characteristics of bocaviruses have recently been identified. The first such porcine bocavirus (PoBoV1), described as boca-like virus (PBo-likeV), was discovered in PMWS affected pigs in Sweden. Later, several other bocaviruses with divergent genomes were reported under various names in domestic pigs. This is the first report of the presence of bocaviruses in European wild boars. 842 wild boar samples originating from the Western region of Romania (Transylvania) were collected during the 2006/2007 and the 2010/2011 hunting seasons and tested for the presence of PoBoV1 by polymerase chain reaction and sequencing. The results showed 12.94% (109/842) overall positivity, with an increasing prevalence from the 2006/2007 (9.14%, 43/470) to the 2010/2011 (17.74%, 66/372) season (P < 0.01). Differences between the prevalence of the virus in 6-12-month-old-animal (77.06%, 84/109) and 12-36-month-old-animal (22.94%, 25/109) (P < 0.01) indicated that the infection occurred mainly in younger pigs. Comparative sequence analysis of partial VP1/2 genes from wild boars and those available in the GenBank showed only minor differences, indicating that PoBoV1 circulating within the wild boar populations and domestic pigs from different geographic regions were highly similar.

Spatial distribution of microplastics in the fluvial sediments of a transboundary river - A case study of the Tisza River in Central Europe.

The geographical environment fundamentally influences the transport and deposition of sediments, including microplastics. In addition, the socioeconomic differences inherent in transboundary catchments result in various waste management strategies among the different countries influencing the input of microplastics into rivers. The catchment of the Tisza River in Central Europe is shared by five countries with different economic statuses and wastewater treatment practices. The aim of this research is to study the spatial changes in microplastic debris deposition along the Tisza and its main tributaries. The mean number of microplastic particles in the sediments of the Tisza was 3177 ± 1970 items/kg, while 3808 ± 1605 items/kg were counted in the sediments of the tributaries. Most of the particles were fibres, indicating the dominance of municipal wastewater input; this is especially the case in the upstream sub-catchments, where there are low degrees of wastewater management. The highest amount of microplastics was found in the sediments of the most-upstream section, where a low number of households are connected to wastewater treatment plants. Thus, it is hypothesized that suburban areas where high population densities and improper waste management co-exist may contribute to the direct input of microplastics into river systems and the development of local microplastic contamination hotspots. In addition, a high microplastic concentration was measured at the furthest downstream section, resulting from the decreased flow velocity related to impoundment by a dam. The efficiency of the microplastic trapping of the various depositionary forms varies along the river, suggesting various influencing factors and the complexity of the process. The higher concentration of microplastics observed in the tributaries compared to that observed in sediments of the main stream may reflect the importance of local sources and the complexity of source-to-sink relations for microplastic routes through the fluvial system; these processes do not always reflect progressive downstream increases.

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.