Analysis of Array {CGH} Data for the Estimation of Genetic Tumor Progression

In cancer research, prediction of time to death or relapse is important for a meaningful tumor classification and selecting appropriate therapies. The accumulation of genetic alterations during tumor progression can be used for the assessment of the genetic status of the tumor. ArrayCGH technology is used to measure genomic amplifications and deletions, with a high resolution that allows the detection of down to single genes copy number changes. \\\\We propose an automated method for analysis of cancer mutations accumulation based on statistical analysis of arrayCGH data. The method consists of the four steps: arrayCGH smoothing, aberrations detection, consensus analysis and oncogenetic tree models estimation. For the second and third steps, we propose new algorithmic solutions. First, we use the adaptive weights smoothing-based algorithm GLAD for identifying regions of constant copy number. Then, in order to select regions of gain and loss, we fit robust normals to the smoothed Log$_2$Ratios of each CGH array and choose appropriate significance cutoffs. The consensus analysis step consists of an automated selection of recurrent aberrant regions when multiple CGH experiments on the same tumor type are available. We propose to associate $p$-values to each measured genomic position and to select the regions where the $p$-value is sufficiently small. \\\\The aberrant regions computed by our method can be further used to estimate evolutionary trees, which model the dependencies between genetic mutations and can help to predict tumor progression stages and survival times. \\\\We applied our method to two arrayCGH data sets obtained from prostate cancer and glioblastoma patients, respectively. The results confirm previous knowledge on the genetic mutations specific to these types of cancer, but also bring out new regions, often reducing to single genes, due to the high resolution of arrayCGH measurements. An oncogenetic tree mixture model fitted to the Prostate Cancer data set shows two distinct evolutionary patterns discriminating between two different cell lines. Moreover, when used as clustering features, the genetic mutations our algorithm outputs separate well arrays representing 4 different cell lines, proving that we extract meaningful information.

Detection by real-time PCR of colistin resistance genes (mcr-1 to mcr-3) in animal manure and agricultural soil in Northern Italy

Introduction: The presence of antimicrobials, such as colistin, in animal manure may promote the dissemination of plasmid-mediated colistin resistance (mcr genes) into the environment. Objectives: To assess resistance to colistin mediated by mcr genes in soil fertilized with manure from intensive animal farms. Aim: To provide qualitative data for mcr-1 to mcr-3 genes in manure and soil using a real-time PCR approach. Methods: A total of 84 samples (1 sample of manure/slurry; 1 sample of soil before fertilization, T0; and 1 sample of soil about 30 days after fertilization, T30 per farm) collected from dairy (n = 11), swine (n = 10) and chicken (n = 7) farms were analysed by real-time PCR. Results: Mcr-1, mcr-2 and mcr-3 genes were detected in all three farms and samples. In swine farms, all 3 mcr genes (i.e. 5 slurry, 2 T0 and 3 T30 for mcr-1; 3 slurry for mcr-2; 3 slurry, 2 T0 and 2 T30 for mcr-3) were detected. In chicken and dairy farms, only mcr-1 (i.e. 3 manure in dairy, and 3 T0 and 1 T30 in chicken) and mcr-3 genes (i.e. 2 manure in dairy, and 1 manure and 1 T0 in chicken) were found. Mcr-1 was the most prevalent gene (20%), followed by mcr-3 (13%) and mcr-2 (3.5%). Conclusions: The presence of mcr genes into the environment, following fertilization with animal manure, may be of concern for public health since colistin is considered as last therapeutic option for treating severe human infections

Colistin resistance genes (mcr-1 to mcr-5) in agricultural soil fertilized with animal manure in Northern Italy

Fertilization with animal manure is a common agricultural practice in Italy. Waste products from intensive animal farming, such as cattle, swine and poultry farms, are the most frequently utilized. However, manure may contain residues of antimicrobials used during rearing that may have a negative impact on the environment, including the dissemination of antimicrobial resistance genes [1, 2]. Colistin is an antimicrobial commonly used in animals, and recent studies showed that resistance can be induced by plasmid-mediated mcr genes [3]. The presence of antimicrobials, such as colistin, in animal manure used as fertilizer of agricultural soil may promote the dissemination of mcr genes into the environment. This study aimed at investigating the presence of colistin residues and mcr-1 to mcr-5 genes in agricultural soil before and after fertilization with manure/slurry from intensive animal (i.e. dairy, swine and chicken) farms in Northern Italy. A total of 84 samples (1 sample of manure/slurry per farm; 1 sample of soil before fertilization, T0, per farm; and 1 sample of soil about 30 days after fertilization, T30, per farm) were collected from dairy (n = 11), swine (n = 10) and chicken (n = 7) farms. All samples were analysed by HPLC-MS/MS and by SYBR\uae Green Real-Time PCR for the detection of colistin and mcr-1 to mcr-5 genes, respectively. None of the samples contained colistin residues, whereas all the mcr genes were found. In swine farms, all 5 mcr genes (i.e. 5 slurry, 2 T0 and 3 T30 samples for mcr-1; 3 slurry samples for mcr-2; 3 slurry, 2 T0 and 2 T30 samples for mcr-3; 2 slurry and 1 T30 samples for mcr-4; 1 slurry and 1 T30 samples for mcr-5) were detected. In chicken and dairy farms, mcr-1 (i.e. 3 manure samples in dairy, and 3 T0 and 1 T30 samples in chicken), mcr-3 (i.e. 2 manure in dairy samples, and 1 manure and 1 T0 samples in chicken), mcr-4 (i.e. 1 manure sample in dairy) and mcr-5 (i.e. 1 T0 sample in dairy) genes were found. Overall, mcr-1 was the prevalent gene (20%), followed by mcr-3 (13%), mcr-4 (4.8%), mcr-5 (3.6%), and mcr-2 (3.5%). This study showed that all the mcr genes were present in all types of animal (i.e. dairy, swine and chicken) manure and related fertilized soils regardless of the presence of colistin residues. Furthermore, a higher prevalence and diversity of mcr genes was found in swine manure and related fertilized soils compared to those from dairy and chicken farms. In conclusion, the presence of plasmid-mediated mcr genes both in manure and in agricultural soil is concerning for environmental and public health and further research is needed to understand their ecology into the environment

Protective effects of quercetin towards aflatoxin B1-induced hepatotoxicity in cattle: a whole transcriptomic in vitro study

Introduction: Aflatoxin B1 (AFB1) is a natural feed and food contaminant. In the dairy industry, AFB1 and its derivatives are of concern for the related economic losses and their possible presence in milk and dairy food products. Oxidative stress is one of the prominent toxic effects of AFB1, thus, dietary supplementation with natural antioxidants is a valid strategy to mitigate AFB1 toxicity. In this respect, quercetin (QUE) is a promising bioactive compound. Here we assessed the protective role of QUE in bovine foetal hepatocyte-derived cells (BFH12) exposed to AFB1, by measuring the QUE impact on AFB1-induced cytotoxicity and related whole- transcriptional changes. Methods: to increase cells responsiveness to AFB1, monolayers were pre-treated with 1 nM PCB126 for 24h and then exposed to 3.6 μM AFB1, alone or in combination with sub-cytotoxic concentrations of QUE (10, 20, 30 μM). Cytotoxicity was then assessed. For the whole-transcriptome study, we exposed cells to 30 μM QUE, 3.6 μM AFB1, or their combination. Cells pre-treated with PCB126 and exposed to DMSO were used as control (CTRL). Total RNA was isolated, 12 tagged RNA-seq libraries were prepared and sequenced (50 bp single-end). Reads were quality checked, trimmed and mapped to cow reference genome using validated pipelines. EdgeR and clusterProfiler were used to identify differentially expressed genes (DEGs) and perform the functional enrichment analysis, respectively. Results: 30 μM QUE significantly reduced AFB1-induced cytotoxicity by 13.39%. As to RNA-seq, the differential expression analysis comparing QUE vs CTRL and QUE+AFB1 vs AFB1 identified 1028 and 1890 DEGs, respectively. QUE alone affected the expression of genes related to steroids metabolism, inflammation, immunity, and P450-mediated drug metabolism. When comparing QUE+AFB1 vs AFB1, top upregulated genes possess antiapoptotic and antioxidant activity (e.g. SEMA5A, TF), while top downregulated genes play a role in inflammation and cancer progression (e.g. CXCL5, IL6). If comparing these results with the mRNA changes induced in cells exposed to AFB1 alone, all the top-10 genes upregulated by the cotreatment were inhibited by AFB1; on the opposite, all the top-10 downregulated genes were induced by AFB1 alone. Conclusions: we showed the QUE potential to mitigate AFB1-induced toxicity and the underneath molecular mechanisms. If the beneficial effects will be confirmed in vivo, QUE-supplemented diets might be adopted to improve health status of cattle exposed to AFB1-contaminated feed

Colistin resistance genes (mer-1 to mer-5) in agricultural soil fertilized with animal manure in Northern Italy

Fertilization with animal manure is a common agricultural practice in Italy. Waste products from intensive animal farming, such as cattle, swine and poultry farms, are the most frequently utilized. However, manure may contain residues of antimicrobials used during rearing that may have a negative impact on the environment, including the dissemination of antimicrobial resistance genes [1, 2]. Colistin is an antimicrobial commonly used in animals, and recent studies showed that resistance can be induced by plasmid-mediated mcr genes [3]. The presence of antimicrobials, such as colistin, in animal manure used as fertilizer of agricultural soil may promote the dissemination of mcr genes into the environment. This study aimed at investigating the presence of colistin residues and mer-1 to mcer-5 genes in agricultural soil before and after fertilization with manure/slurry from intensive animal (i.e. dairy, swine and chicken) farms in Northern Italy. A total of 84 samples (1 sample of manure/slurry per farm; 1 sample of soil before fertilization, TO, per farm; and 1 sample of soil about 30 days after fertilization, T30, per farm) were collected from dairy (n = 11), swine (n = 10) and chicken (n = 7) farms. All samples were analysed by HPLC- MS/MS and by SYBR\uae Green Real-Time PCR for the detection of colistin and mer-1 to mer-5 genes, respectively. None of the samples contained colistin residues, whereas all the mcr genes were found. In swine farms, all 5 mer genes (i.e. 5 slurry, 2 TO and 3 T30 samples for mer-1; 3 slurry samples for mer-2; 3 slurry, 2 TO and 2 T30 samples for mer-3; 2 slurry and 1 T30 samples for mcr-4; 1 slurry and 1 T30 samples for mcr-5) were detected. In chicken and dairy farms, mer- 1 (i.e. 3 manure samples in dairy,-and 3 TO and 1 T30 samples in chicken), mcr-3 (i.e. 2 manure in dairy samples, and 1 manure and 1 TO samples in chicken), mcr-4 (i.e. 1 manure sample in dairy) and mcr-S (i.e. 1 TO sample in dairy) genes were found. Overall, mcr-1 was the prevalent gene (20%), followed by mcer-3 (13%), mer-4 (4.8%), mer-5 (3.6%), and mer-2 (3.5%). This study showed that all the mcr genes were present in all types of animal (i.e. dairy, swine and chicken) manure and related fertilized soils regardless of the presence of colistin residues. Furthermore, a higher prevalence and diversity of mer genes was found in swine manure and related fertilized soils compared to those from dairy and chicken farms. In conclusion, the presence of plasmid- mediated mcr genes both in manure and in agricultural soil is concerning for environmental and public health and further research is needed to understand their ecology into the environment