Analysis of Alternative mRNA Splicing in Vemurafenib-Resistant Melanoma Cells

Alternative splicing (AS) is one of the hallmarks of human cancer. One of the most common mechanisms of vemurafenib resistance in malignant melanoma is AS of BRAF, occurring in 15-30% of patients. The aim of our study was to investigate the transcriptome and AS D04landscape in the isogenic BRAF V600E cell line pair SK-MEL-239, where the vemurafenib-resistant derivative expresses a truncated BRAF transcript that lacks the RAS-binding domain. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. AS analysis, by four different tools, DEXSeq, rMATS, ASpli, and LeafCutter, has identified genes enriched for cell motility and melanin synthesis in vemurafenib-resistant cells. Overlapping predictions for all four tools have been experimentally validated. Our study expands the understanding of melanoma drug resistance from a new perspective and supports the need to investigate in detail the aberrant AS landscape in patients with malignant melanoma. Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses.Peer reviewe

Analysis of Alternative mRNA Splicing in Vemurafenib-Resistant Melanoma Cells

Alternative splicing (AS) is one of the hallmarks of human cancer. One of the most common mechanisms of vemurafenib resistance in malignant melanoma is AS of BRAF, occurring in 15-30% of patients. The aim of our study was to investigate the transcriptome and AS D04landscape in the isogenic BRAF V600E cell line pair SK-MEL-239, where the vemurafenib-resistant derivative expresses a truncated BRAF transcript that lacks the RAS-binding domain. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. AS analysis, by four different tools, DEXSeq, rMATS, ASpli, and LeafCutter, has identified genes enriched for cell motility and melanin synthesis in vemurafenib-resistant cells. Overlapping predictions for all four tools have been experimentally validated. Our study expands the understanding of melanoma drug resistance from a new perspective and supports the need to investigate in detail the aberrant AS landscape in patients with malignant melanoma. Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses.Peer reviewe

Prediction of Solar Particle Events with SRAM-Based Soft Error Rate Monitor and Supervised Machine Learning

This work introduces an embedded approach for the prediction of Solar Particle Events (SPEs) in space applications by combining the real-time Soft Error Rate (SER) measurement with SRAM-based detector and the offline trained machine learning model. The proposed approach is intended for the self-adaptive fault-tolerant multiprocessing systems employed in space applications. With respect to the state-of-the-art, our solution allows for predicting the SER 1 h in advance and fine-grained hourly tracking of SER variations during SPEs as well as under normal conditions. Therefore, the target system can activate the appropriate mechanisms for radiation hardening before the onset of high radiation levels. Based on the comparison of five different machine learning algorithms trained with the public space flux database, the preliminary results indicate that the best prediction accuracy is achieved with the recurrent neural network (RNN) with long short-term memory (LSTM)

Solar Particle Event and Single Event Upset Prediction from SRAM-based Monitor and Supervised Machine Learning

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Multihazard susceptibility assessment: A case study – Municipality of Štrpce (Southern Serbia)

The municipality of Štrpce (Southern Serbia) is an area located within Šar Mountain National Park, which is of great ecological importance. Due to the vicinity of settlements, it is necessary to analyze the terrain's susceptibility to natural hazards. The main goal of this research was to determine locations that are highly vulnerable at times of natural hazards (such as earthquakes, erosion, torrential flooding, snow avalanches, and forest fires). The first step in this research was to analyze seismic hazards for a 475 years return period (VII–VIII MCS for the observed area), which was possible by means of Geographic Information Systems. The second step was to determine the intensity of erosion and total sediment production using the Erosion Potential Model. The third step was related to the analysis of the potential of torrential floods using the Flash Flood Potential Index. The Avalanches Potential Index method was used as the fourth step. The fifth step included the analysis of a terrain susceptibility to the occurrence of forest fires. Following the five criteria analysis, weight coefficients were assigned to each of the analyzed parameters by using the Analytical Hierarchy Process (AHP), which provided results of the total susceptibility to natural hazards of the territory of Štrpce. Results indicated that over 45% of the municipality is highly or very highly susceptible to various natural hazards. This article represents a significant step toward a better understanding of natural hazards and it provides a unique knowledge basis for establishing the management and mitigation guidelines and measures, not only within the researched area but at regional and national levels as well

Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial

Background Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. Methods In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH,non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2–F3, or F1 with at least oneaccompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpointsfor the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2–F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. Findings Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1–F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2–F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1–F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). Interpretation Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes