RESUMO
Robotic milking systems are successful innovations in the development of dairy cattle. The objective of this study was to analyse the milking characteristics and behavior of dairy cows of different calving orders in "milk first" robotic milking systems. The data were collected from a commercial herd located in the Midwest region of Minas Gerais (Brazil), which uses an automatic milking system (AMS TM, DeLaval). Were analysed 26,574 observations of 235 Holstein cows were available. Data were evaluated by multivariate analysis of variance and the Tukey test. - Tthe characteristics milk flow and milking efficiency were more favourable for multiparous cows (p <0.01), while the time in the stall was more favourable for primiparous females (p <0.01). The values of handling time were better in the primiparous cows (p <0.01). Primiparous cows had higher amounts of kick-off (p <0.001), and multiparous cows had higher incomplete milkings (p <0.001). The number of incomplete milkings showed a higher ratio in terms of reduction in milk production in 26.6% in primiparous cows and 26.7% in multiparous cows (p <0.01). Regarding the behavioral characteristics, primiparous cows had higher amounts of kickbacks, while multiparous cows had greater quantities of incomplete milkings.
Assuntos
Comportamento Animal , Indústria de Laticínios , Lactação , Paridade , Robótica , Animais , Bovinos/fisiologia , Feminino , Paridade/fisiologia , Lactação/fisiologia , Indústria de Laticínios/métodos , Comportamento Animal/fisiologia , Gravidez , Leite/química , BrasilRESUMO
Pancreatic ductal adenocarcinomas (PDAC) are the fourth leading cause of death due to neoplasms. In view of the urgent need of effective treatments for PDAC, photodynamic therapy (PDT) appears as a promising alternative. However, its efficacy against PDAC and the mechanisms involved in cell death induction remain unclear. In this study, we set out to evaluate PDT's cytotoxicity using methylene blue (MB) as a photosensitizer (PS) (MB-PDT) and to evaluate the contribution of necroptosis in its effect in human PDAC cells. Our results demonstrated that MB-PDT induced significant death of different human PDAC models presenting two different susceptibility profiles. This effect was independent of MB uptake or its subcellular localization. We found that the ability of triggering necroptosis was determinant to increase the treatment efficiency. Analysis of single cell RNA-seq data from normal and neoplastic human pancreatic tissues showed that specific necroptosis proteins RIPK1, RIPK3 and MLKL presented significant higher expression levels in cells displaying a transformed phenotype providing further support to the use of approaches that activate necroptosis, like MB-PDT, as useful adjunct to surgery of PDAC to tackle the problem of microscopic residual disease as well as to minimize the chance of local and metastatic recurrence.
Assuntos
Adenocarcinoma , Fotoquimioterapia , Humanos , Azul de Metileno/farmacologia , Necroptose , Fármacos Fotossensibilizantes/farmacologia , Fotoquimioterapia/métodos , Apoptose , Neoplasias PancreáticasRESUMO
The epigenetic changes associated with melanoma progression to advanced and metastatic stages are still poorly understood. To shed light on the CpG methylation dynamics during melanoma development, we analyzed the methylome profiles of a four-stage cell line model of melanoma progression: non-tumorigenic melanocytes (melan-a), premalignant melanocytes (4C), non-metastatic melanoma cells (4C11-), and metastatic melanoma cells (4C11+). We identified 540 hypo- and 37 hypermethylated gene promoters that together characterized a malignancy signature, and 646 hypo- and 520 hypermethylated promoters that distinguished a metastasis signature. Differentially methylated genes from these signatures were correlated with overall survival using TCGA-SKCM methylation data. Moreover, multivariate Cox analyses with LASSO regularization identified panels of 33 and 31 CpGs, respectively, from the malignancy and metastasis signatures that predicted poor survival. We found a concordant relationship between DNA methylation and transcriptional levels for genes from the malignancy (Pyroxd2 and Ptgfrn) and metastasis (Arnt2, Igfbp4 and Ptprf) signatures, which were both also correlated with melanoma prognosis. Altogether, this study reveals novel CpGs methylation markers associated with malignancy and metastasis that collectively could improve the survival prediction of melanoma patients.
Assuntos
Metilação de DNA , Melanoma , Ilhas de CpG , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/metabolismo , Prognóstico , Regiões Promotoras GenéticasRESUMO
Cell signaling events triggered by androgen hormone in prostate cells is dependent on activation of the androgen receptor (AR) transcription factor. Androgen hormone binding to AR promotes its displacement from the cytoplasm to the nucleus and AR binding to DNA motifs, thus inducing activatory and inhibitory transcriptional programs through a complex regulatory mechanism not yet fully understood. In this work, we performed RNA-seq deep-sequencing of LNCaP prostate cancer cells and found over 7000 expressed long intergenic non-coding RNAs (lincRNAs), of which â¼4000 are novel lincRNAs, and 258 lincRNAs have their expression activated by androgen. Immunoprecipitation of AR, followed by large-scale sequencing of co-immunoprecipitated RNAs (RIP-Seq) has identified in the LNCaP cell line a total of 619 lincRNAs that were significantly enriched (FDR < 10%, DESeq2) in the anti-Androgen Receptor (antiAR) fraction in relation to the control fraction (non-specific IgG), and we named them Androgen-Receptor-Associated lincRNAs (ARA-lincRNAs). A genome-wide analysis showed that protein-coding gene neighbors to ARA-lincRNAs had a significantly higher androgen-induced change in expression than protein-coding genes neighboring lincRNAs not associated to AR. To find relevant epigenetic signatures enriched at the ARA-lincRNAs' transcription start sites (TSSs) we used a machine learning approach and identified that the ARA-lincRNA genomic loci in LNCaP cells are significantly enriched with epigenetic marks that are characteristic of in cis enhancer RNA regulators, and that the H3K27ac mark of active enhancers is conspicuously enriched at the TSS of ARA-lincRNAs adjacent to androgen-activated protein-coding genes. In addition, LNCaP topologically associating domains (TADs) that comprise chromatin regions with ARA-lincRNAs exhibit transcription factor contents, epigenetic marks and gene transcriptional activities that are significantly different from TADs not containing ARA-lincRNAs. This work highlights the possible involvement of hundreds of lincRNAs working in synergy with the AR on the genome-wide androgen-induced gene regulatory program in prostate cells.
RESUMO
Cell signaling events triggered by androgen hormone in prostate cells is dependent on activation of the androgen receptor (AR) transcription factor. Androgen hormone binding to AR promotes its displacement from the cytoplasm to the nucleus and AR binding to DNA motifs, thus inducing activatory and inhibitory transcriptional programs through a complex regulatory mechanism not yet fully understood. In this work, we performed RNA-seq deep-sequencing of LNCaP prostate cancer cells and found over 7000 expressed long intergenic non-coding RNAs (lincRNAs), of which similar to 4000 are novel lincRNAs, and 258 lincRNAs have their expression activated by androgen. Immunoprecipitation of AR, followed by large-scale sequencing of co-immunoprecipitated RNAs (RIP-Seq) has identified in the LNCaP cell line a total of 619 lincRNAs that were significantly enriched (FDR < 10%, DESeq2) in the anti-Androgen Receptor (antiAR) fraction in relation to the control fraction (non-specific IgG), and we named them Androgen-Receptor-Associated lincRNAs (ARA-lincRNAs). A genome-wide analysis showed that protein-coding gene neighbors to ARA-lincRNAs had a significantly higher androgen-induced change in expression than protein-coding genes neighboring lincRNAs not associated to AR. To find relevant epigenetic signatures enriched at the ARA-lincRNAs' transcription start sites (TSSs) we used a machine learning approach and identified that the ARA-lincRNA genomic loci in LNCaP cells are significantly enriched with epigenetic marks that are characteristic of in cis enhancer RNA regulators, and that the H3K27ac mark of active enhancers is conspicuously enriched at the TSS of ARA-lincRNAs adjacent to androgen-activated protein-coding genes. In addition, LNCaP topologically associating domains (TADs) that comprise chromatin regions with ARA-lincRNAs exhibit transcription factor contents, epigenetic marks and gene transcriptional activities that are significantly different from TADs not containing ARA-lincRNAs. This work highlights the possible involvement of hundreds of lincRNAs working in synergy with the AR on the genome-wide androgen-induced gene regulatory program in prostate cells.
RESUMO
Changes in RNA stability have an important impact in the gene expression regulation. Different methods based on the transcription blockage with RNA polymerase inhibitors or metabolic labeling of newly synthesized RNAs have been developed to evaluate RNA decay rates in cultured cell. Combined with techniques to measure transcript abundance genome-wide, these methods have been used to reveal novel features of the eukaryotic transcriptome. The stability of protein-coding mRNAs is in general closely associated to the physiological function of their encoded proteins, with short-lived mRNAs being significantly enriched among regulatory genes whereas genes associated with housekeeping functions are predominantly stable. Likewise, the stability of noncoding RNAs (ncRNAs) seems to reflect their functional role in the cell. Thus, investigating RNA stability can provide insights regarding the function of yet uncharacterized regulatory ncRNAs. In this chapter, we discuss the methodologies currently used to estimate RNA decay and outline an experimental protocol for genome-wide estimation of RNA stability of protein-coding and lncRNAs. This protocol details the transcriptional blockage of cultured cells with actinomycin D, followed by RNA isolation at different time points, the determination of transcript abundance by qPCR/DNA oligoarray hybridization, and the calculation of individual transcript half-lives.
Assuntos
RNA Mensageiro/química , RNA Mensageiro/isolamento & purificação , RNA não Traduzido/química , RNA não Traduzido/isolamento & purificação , Técnicas de Cultura de Células , Dactinomicina/farmacologia , Perfilação da Expressão Gênica , Genes Essenciais , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Estabilidade de RNA , Transcrição GênicaRESUMO
Breast carcinoma (BC) corresponds to 23 % of all cancers in women, with 1.38 million new cases and 460,000 deaths worldwide annually. Despite the significant advances in the identification of molecular markers and different modalities of treatment for primary BC, the ability to predict its metastatic behavior is still limited. The purpose of this study was to identify novel molecular markers associated with distinct clinical outcomes in a Brazilian cohort of BC patients. We generated global gene expression profiles using tumor samples from 24 patients with invasive ductal BC who were followed for at least 5 years, including a group of 15 patients with favorable outcomes and another with nine patients who developed metastasis. We identified a set of 58 differentially expressed genes (p ≤ 0.01) between the two groups. The prognostic value of this metastasis signature was corroborated by its ability to stratify independent BC patient datasets according to disease-free survival and overall survival. The upregulation of B3GNT7, PPM1D, TNKS2, PHB, and GTSE1 in patients with poor outcomes was confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in an independent sample of patients with BC (47 with good outcomes and eight that presented metastasis). The expression of BCL2-associated agonist of cell death (BAD) protein was determined in 1276 BC tissue samples by immunohistochemistry and was consistent with the reduced BAD mRNA expression levels in metastatic cases, as observed in the oligoarray data. These findings point to novel prognostic markers that can distinguish breast carcinomas with metastatic potential from those with favorable outcomes.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Gradação de Tumores , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Proibitinas , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Tanquirases/genética , Tanquirases/metabolismo , Adulto JovemRESUMO
Long noncoding RNAs (lncRNAs) that map to intragenic regions of the human genome with the same (intronic lncRNAs) or opposite orientation (antisense lncRNAs) relative to protein-coding mRNAs have been largely dismissed from biochemical and functional characterization due to the belief that they are mRNA precursors, byproducts of RNA splicing or simply transcriptional noise. In this work, we used a custom microarray to investigate aspects of the biogenesis, processing, stability, evolutionary conservation, and cellular localization of â¼ 6,000 intronic lncRNAs and â¼ 10,000 antisense lncRNAs. Most intronic (2,903 of 3,427, 85%) and antisense lncRNAs (4,945 of 5,214, 95%) expressed in HeLa cells showed evidence of 5' cap modification, compatible with their transcription by RNAP II. Antisense lncRNAs (median t1/2 = 3.9 h) were significantly (p < 0.0001) more stable than mRNAs (median t1/2 = 3.2 h), whereas intronic lncRNAs (median t1/2 = 2.1 h) comprised a more heterogeneous class that included both stable (t1/2 > 3 h) and unstable (t1/2 < 1 h) transcripts. Intragenic lncRNAs display evidence of evolutionary conservation, have little/no coding potential and were ubiquitously detected in the cytoplasm. Notably, a fraction of the intronic and antisense lncRNAs (13 and 15%, respectively) were expressed from loci at which the corresponding host mRNA was not detected. The abundances of a subset of intronic/antisense lncRNAs were correlated (r ≥ |0.8|) with those of genes encoding proteins involved in cell division and DNA replication. Taken together, the findings of this study contribute novel biochemical and genomic information regarding intronic and antisense lncRNAs, supporting the notion that these classes include independently transcribed RNAs with potentials for exerting regulatory functions in the cell.
Assuntos
Perfilação da Expressão Gênica/métodos , Genoma Humano/genética , Íntrons/genética , RNA Antissenso/genética , RNA Longo não Codificante/genética , Alfa-Amanitina/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Dactinomicina/farmacologia , Decitabina , Células HeLa , Humanos , Células MCF-7 , Inibidores da Síntese de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Estabilidade de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Signaling via epidermal growth factor receptor (EGFR) and Src kinase pathways promote triple-negative breast cancer (TNBC) cell invasion and tumor metastasis. Here, we address the role of Cdc42-interacting protein-4 (CIP4) in TNBC metastasis in vivo, and profile CIP4 expression in human breast cancer patients. In human TNBC cells, CIP4 knock-down (KD) led to less sustained activation of Erk kinase and impaired cell motility compared to control cells. This correlated with significant defects in 3D invasion of surrounding extracellular matrix by CIP4 KD TNBC cells when grown as spheroid colonies. In mammary orthotopic xenograft assays using both human TNBC cells (MDA-MB-231, HCC 1806) and rat MTLn3 cells, CIP4 silencing had no overt effect on tumor growth, but significantly reduced the incidence of lung metastases in each tumor model. In human invasive breast cancers, high CIP4 levels was significantly associated with high tumor stage, TNBC and HER2 subtypes, and risk of progression to metastatic disease. Together, these results implicate CIP4 in promoting metastasis in TNBCs.