3D RNA-seq:A powerful and flexible tool for rapid and accurate differential expression and alternative splicing analysis of RNA-seq data for biologists

RNA-sequencing (RNA-seq) analysis of gene expression and alternative splicing should be routine and robust but is often a bottleneck for biologists because of different and complex analysis programs and reliance on specialized bioinformatics skills. We have developed the ‘3D RNA-seq’ App, an R shiny App and web-based pipeline for the comprehensive analysis of RNA-seq data from any organism. It represents an easy-to-use, flexible and powerful tool for analysis of both gene and transcript-level gene expression to identify differential gene/transcript expression, differential alternative splicing and differential transcript usage (3D) as well as isoform switching from RNA-seq data. 3D RNA-seq integrates state-of-the-art differential expression analysis tools and adopts best practice for RNA-seq analysis. The program is designed to be run by biologists with minimal bioinformatics experience (or by bioinformaticians) allowing lab scientists to analyse their RNA-seq data. It achieves this by operating through a user-friendly graphical interface which automates the data flow through the programs in the pipeline. The comprehensive analysis performed by 3D RNA-seq is extremely rapid and accurate, can handle complex experimental designs, allows user setting of statistical parameters, visualizes the results through graphics and tables, and generates publication quality figures such as heat-maps, expression profiles and GO enrichment plots. The utility of 3D RNA-seq is illustrated by analysis of data from a time-series of cold-treated Arabidopsis plants and from dexamethasone-treated male and female mouse cortex and hypothalamus data identifying dexamethasone-induced sex- and brain region-specific differential gene expression and alternative splicing

Determination of levetiracetam in human plasma by dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry

Levetiracetam (LEV) is an antiepileptic drug that is clinically effective in generalized and partial epilepsy syndromes. The use of this drug has been increasing in clinical practice and intra- or -interindividual variability has been exhibited for special population. For this reason, bioanalytical methods are required for drug monitoring in biological matrices. So this work presents a dispersive liquid-liquid microextraction method followed by gas chromatography-mass spectrometry (DLLME-GC-MS) for LEV quantification in human plasma. However, due to the matrix complexity a previous purification step is required. Unlike other pretreatment techniques presented in the literature, for the first time, a procedure employing ultrafiltration tubes Amicon (R) (10 kDa porous size) without organic solvent consumption was developed. GC-MS analyses were carried out using a linear temperature program, capillary fused silica column, and helium as the carrier gas. DLLME optimized parameters were type and volume of extraction and dispersing solvents, salt addition, and vortex agitation time. Under chosen parameters (extraction solvent: chloroform, 130 mu Ldispersing solvent: isopropyl alcohol, 400 mu Lno salt addition and no vortex agitation time), the method was completely validated and all parameters were in agreement with the literature recommendations. LEV was quantified in patient's plasma sample using less than 550 mu L of organic solvent.Sao Paulo Research Foundation (FAPESP) [2012/07210-8]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 14040-903 Ribeirão Preto, SP, BrazilDepartment of Exact and Earth Sciences, Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo, 09972-270 Diadema, SP, BrazilDepartament of Chemistry, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, 14040-901 Ribeirão Preto, SP, BrazilDepartment of Clinical Analysis, Toxicology and Food Science, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 14040-903 Ribeirão Preto, SP, BrazilDepartment of Exact and Earth Sciences, Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo, 09972-270 Diadema, SP, BrazilFAPESP: 2012/07210-8Web of Scienc

Vitamin A oral supplementation induces oxidative stress and suppresses IL-10 and HSP70 in skeletal muscle of trained rats

Exercise training intensity is the major variant that influences the relationship between exercise, redox balance, and immune response. Supplement intake is a common practice for oxidative stress prevention; the effects of vitamin A (VA) on exercise training are not yet described, even though this molecule exhibits antioxidant properties. We investigated the role of VA supplementation on redox and immune responses of adult Wistar rats subjected to swimming training. Animals were divided into four groups: sedentary, sedentary + VA, exercise training, and exercise training + VA. Over eight weeks, animals were submitted to intense swimming 5 times/week and a VA daily intake of 450 retinol equivalents/day. VA impaired the total serum antioxidant capacity acquired by exercise, with no change in interleukin-1 and tumor necrosis factor- levels. In skeletal muscle, VA caused lipid peroxidation and protein damage without differences in antioxidant enzyme activities; however, Western blot analysis showed that expression of superoxide dismutase-1 was downregulated, and upregulation of superoxide dismutase-2 induced by exercise was blunted by VA. Furthermore, VA supplementation decreased anti-inflammatory interleukin-10 and heat shock protein 70 expression, important factors for positive exercise adaptations and tissue damage prevention. Our data showed that VA supplementation did not confer any antioxidative and/or protective effects, attenuating exercise-acquired benefits in the skeletal muscle

How does temperature affect splicing events? Isoform switching of splicing factors regulates splicing of <i>LATE ELONGATED HYPOCOTYL</i> (<i>LHY</i>)

One of the ways in which plants can respond to temperature is via alternative splicing (AS). Previous work showed that temperature changes affected the splicing of several circadian clock gene transcripts. Here we investigated the role of RNA‐binding splicing factors (SFs) in temperature‐sensitive alternative splicing (AS) of the clock gene LATE ELONGATED HYPOCOTYL (LHY). We characterised, in wild type plants, temperature‐associated isoform switching and expression patterns for SF transcripts from a high‐resolution temperature and time series RNA‐seq experiment. In addition we employed quantitative RT‐PCR of SF mutant plants to explore the role of the SFs in cooling‐associated AS of LHY. We show that the splicing and expression of several SFs responds sufficiently rapidly and sensitively to temperature changes to contribute to the splicing of the 5’UTR of LHY. Moreover the choice of splice site in LHY was altered in some SF mutants. The splicing of the 5’UTR region of LHY has characteristics of a molecular thermostat, where the ratio of transcript isoforms is sensitive to temperature changes as modest as 2°C and is scalable over a wide dynamic range of temperature. Our work provides novel insight into SF‐mediated coupling of the perception of temperature to post‐transcriptional regulation of the clock

Cold-Dependent Expression and Alternative Splicing of Arabidopsis Long Non-coding RNAs

Plants re-program their gene expression when responding to changing environmental conditions. Besides differential gene expression, extensive alternative splicing (AS) of pre-mRNAs and changes in expression of long non-coding RNAs (lncRNAs) are associated with stress responses. RNA-sequencing of a diel time-series of the initial response of Arabidopsis thaliana rosettes to low temperature showed massive and rapid waves of both transcriptional and AS activity in protein-coding genes. We exploited the high diversity of transcript isoforms in AtRTD2 to examine regulation and post-transcriptional regulation of lncRNA gene expression in response to cold stress. We identified 135 lncRNA genes with cold-dependent differential expression (DE) and/or differential alternative splicing (DAS) of lncRNAs including natural antisense RNAs, sORF lncRNAs, and precursors of microRNAs (miRNAs) and trans-acting small-interfering RNAs (tasiRNAs). The high resolution (HR) of the time-series allowed the dynamics of changes in transcription and AS to be determined and identified early and adaptive transcriptional and AS changes in the cold response. Some lncRNA genes were regulated only at the level of AS and using plants grown at different temperatures and a HR time-course of the first 3 h of temperature reduction, we demonstrated that the AS of some lncRNAs is highly sensitive to small temperature changes suggesting tight regulation of expression. In particular, a splicing event in TAS1a which removed an intron that contained the miR173 processing and phased siRNAs generation sites was differentially alternatively spliced in response to cold. The cold-induced reduction of the spliced form of TAS1a and of the tasiRNAs suggests that splicing may enhance production of the siRNAs. Our results identify candidate lncRNAs that may contribute to the regulation of expression that determines the physiological processes essential for acclimation and freezing tolerance

Adenoma de hipófise em uma gata com hiperadrenocorticismo

Background:  :  :  : Feline Cushing’s syndrome (FCS) is a disorder of excessive cortisol secretion by the adrenal glands and is rare in cats. The most frequently observed clinical signs are polyuria, polydipsia, and polyphagia which are also consistent with diabetes mellitus. These diabetic cats are often insulin resistants. The dexamethasone suppression test is considered the test of choice for the diagnosis of hyperadrenocorticism. The majority of cats with naturally occurring Cushing’s syndrome have pituitary-dependent hyperadrenocorticism and it is caused by functional microadenoma or macroadenoma pituitary. Computed tomography or magnetic resonance imaging is helpful in diagnosis of pituitary tumors. Treatments of pituitary-dependent hyperadrenocorticism include surgery of the pituitary or adrenals, radiation of the pituitary, and medical therapies. Bilateral adrenalectomy continues to represent the best long-term therapeutic strategy until hypophysectomy becomes more widely available. This paper reports a cat with macroadenoma pituitary causing hiperadrenocorticism and insulin resistance. Case: A 12-year-old female castrated Brazilian shorthair cat was referred to the veterinary due to polyuria, polydipsia, weight loss and polyphagia. The presence of hyperglycemia (blood glucose >250 mg/dl), glucosuria and elevated fructosamine concentration revealed diabetes mellitus. Insulin therapy was introduced but the glycemia was poorly controlled despite the high dose of insulin. Concomitant disease was suspected. Abdominal ultrasonography revealed bilaterally enlarged adrenals. The dexamethasone suppression test showed pituitary-dependent hyperadrenocorticism. Computed tomography or hypophysectomy wasn‘t available. Medical therapy with mitotane was introduced but anorexia and vomiting developed. Bilateral adrenalectomy was performed without complications and histological evaluation of adrenal revealed hyperplasia. After surgery, treatment with mineralcorticoids and glucocorticoids was introduced. The cat had resolution of clinical signs and insulin requirements were decreased. According to the owner, three weeks after surgery, the cat showed abnormal behavior, compulsive walking and circling. The cat died eight months after bilateral adrenalectomy. A complete necropsy was performed and histopathological examination confirmed the pituitary macroadenoma. Discussion: Insulin resistance should be suspected in diabetic cat if control of glycemia is poor despite the high insulin dosage. Clinical signs related to poorly controlled diabetes mellitus are common in cats with hyperadrenocorticism. Hyperadrenocorticism can cause severe insulin resistance and it is often associated with a pituitary macrotumor. Pituitary tumors may lead to hypercotisolism. Bilateral adrenalectomy is a viable alternative to transphenoidal hypophysectomy for treatment of feline pituitary-dependent hyperadrenocorticism when hypophysectomy is not available. Neurological signs can be a result of pituitary tumors and they can get worse after the adrenalectomy because of the enlargement of the tumor. Despite of clinical signs, the cat had improved in response to the bilateral adrenalectomy and had a good quality of life during eight months after surgery

AtRTD - a comprehensive reference transcript dataset resource for accurate quantification of transcript - specific expression in <i>Arabidopsis thaliana</i>

RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression.</p