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Sequencing the tyrosine phosphoproteome using MS-based proteomics is challenging due to the low abundance of tyrosine phosphorylation in cells, a challenge compounded in scarce samples like primary cells or clinical samples. The broad-spectrum optimisation of selective triggering (BOOST) method was recently developed to increase phosphotyrosine sequencing in low protein input samples by leveraging tandem mass tags (TMT), phosphotyrosine enrichment, and a phosphotyrosine-loaded carrier channel. Here, we demonstrate the viability of BOOST in T cell receptor (TCR)-stimulated primary murine T cells by benchmarking the accuracy and precision of the BOOST method and discerning significant alterations in the phosphoproteome associated with receptor stimulation. Using 1 mg of protein input (about 20 million cells) and BOOST, we identify and precisely quantify more than 2000 unique pY sites compared to about 300 unique pY sites in non-BOOST control samples. We show that although replicate variation increases when using the BOOST method, BOOST does not jeopardise quantitative precision or the ability to determine statistical significance for peptides measured in triplicate. Many pY previously uncharacterised sites on important T cell signalling proteins are quantified using BOOST, and we identify new TCR responsive pY sites observable only with BOOST. Finally, we determine that the phase-spectrum deconvolution method on Orbitrap instruments can impair pY quantitation in BOOST experiments.
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In patients with breast cancer, metastases of cancer cells to the axial skeleton may cause excruciating pain, particularly in the advanced stages. The current drug treatments available to alleviate this debilitating pain condition often lack efficacy and/or produce undesirable side effects. Preclinical animal models of cancer-induced bone pain are key to studying the mechanisms that cause this pain and for the success of drug discovery programs. In a previous study conducted in our laboratory, we validated and characterised the rat model of Walker 256 cell-induced bone pain, which displayed several key resemblances to the human pain condition. However, gene level changes that occur in the pathophysiology of cancer-induced bone pain in this preclinical model are unknown. Hence, in this study, we performed the transcriptomic characterisation of the Walker 256 cell line cultured in vitro to predict the molecular genetic profile of this cell line. We also performed transcriptomic characterisation of the Walker 256 cell-induced bone pain model in rats using the lumbar spinal cord and lumbar dorsal root ganglia tissues. Here we show that the Walker 256 cell line resembles the basal-B molecular subtype of human breast cancer cell lines. We also identify several genes that may underpin the progression of pain hypersensitivities in this condition, however, this needs further confirmatory studies. These transcriptomic insights have the potential to direct future studies aimed at identifying various mechanisms underpinning pain hypersensitivities in this model that may also assist in discovery of novel pain therapeutics for breast cancer-induced bone pain.
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Neoplasias Óseas/genética , Neoplasias Óseas/secundario , Dolor en Cáncer/genética , Carcinoma 256 de Walker/genética , Carcinoma 256 de Walker/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Transcriptoma , Animales , Biomarcadores de Tumor/genética , Neoplasias Óseas/complicaciones , Dolor en Cáncer/etiología , Dolor en Cáncer/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Hiperalgesia/etiología , Hiperalgesia/genética , Hiperalgesia/patología , Dolor/etiología , Dolor/genética , Dolor/patología , Ratas , Ratas Wistar , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
INTRODUCTION: The aim of the present study was to evaluate the effects of the novel kinin B1 receptor antagonist BI113823 on postinfarction cardiac remodeling and heart failure, and to determine whether B1 receptor blockade alters the cardiovascular effects of an angiotensin 1 converting enzyme (ACE) inhibitor in rats. METHODS AND RESULTS: Sprague Dawley rats were subjected to permanent occlusion of the left coronary artery. Cardiovascular function was determined at 6weeks postinfarction. Treatment with either B1 receptor antagonist (BI113823) or an ACE inhibitor (lisinopril) alone or in combination significantly reduced the heart weight-to-body weight and lung weight-to-body weight ratios, and improved postinfarction cardiac function as evidenced by greater cardiac output, the maximum rate of left ventricular pressure rise (±dP/dtmax), left ventricle ejection fraction, fractional shorting, better wall motion, and attenuation of elevated left ventricular end diastolic pressure (LVEDP). Furthermore, all three treatment groups exhibited significant reduction in cardiac interstitial fibrosis, collagen deposition, CD68 positive macrophages, neutrophils, and proinflammatory cytokine production (TNF-α and IL-1ß), compared to vehicle controls. CONCLUSION: The present study shows that treatment with the novel kinin B1 receptor antagonist, BI113823, reduces postinfarction cardiac remodeling and heart failure, and does not influence the cardiovascular effects of the ACE inhibitor.
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Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Antagonistas del Receptor de Bradiquinina B1/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Lisinopril/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Remodelación Ventricular/efectos de los fármacos , Animales , Cardiomegalia/tratamiento farmacológico , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Masculino , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Ratas Sprague-Dawley , Receptor de Bradiquinina B1/genéticaRESUMEN
Viral vectors have been applied successfully to generate disease-related animal models and to functionally characterize target genes in vivo. However, broader application is still limited by complex vector production, biosafety requirements, and vector-mediated immunogenic responses, possibly interfering with disease-relevant pathways. Here, we describe adeno-associated virus (AAV) variant 6.2 as an ideal vector for lung delivery in mice, overcoming most of the aforementioned limitations. In a proof-of-concept study using AAV6.2 vectors expressing IL-13 and transforming growth factor-ß1 (TGF-ß1), we were able to induce hallmarks of severe asthma and pulmonary fibrosis, respectively. Phenotypic characterization and deep sequencing analysis of the AAV-IL-13 asthma model revealed a characteristic disease signature. Furthermore, suitability of the model for compound testing was also demonstrated by pharmacological intervention studies using an anti-IL-13 antibody and dexamethasone. Similarly, the AAV-TGF-ß1 fibrosis model showed several disease-like pathophenotypes monitored by micro-computed tomography imaging and lung function measurement. Most importantly, analyses using stuffer control vectors demonstrated that in contrast to a common adenovirus-5 vector, AAV6.2 vectors did not induce any measurable inflammation and therefore carry a lower risk of altering relevant readouts. In conclusion, we propose AAV6.2 as an ideal vector system for the functional characterization of target genes in the context of pulmonary diseases in mice.
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Asma/inmunología , Dependovirus/genética , Fibrosis Pulmonar Idiopática/inmunología , Animales , Asma/genética , Asma/metabolismo , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Interleucina-13/biosíntesis , Interleucina-13/genética , Ratones Endogámicos BALB C , Transducción Genética , Factor de Crecimiento Transformador beta1/biosíntesis , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Fully differentiated pancreatic ß cells are essential for normal glucose homeostasis in mammals. Dedifferentiation of these cells has been suggested to occur in type 2 diabetes, impairing insulin production. Since chronic fuel excess ("glucotoxicity") is implicated in this process, we sought here to identify the potential roles in ß-cell identity of the tumor suppressor liver kinase B1 (LKB1/STK11) and the downstream fuel-sensitive kinase, AMP-activated protein kinase (AMPK). Highly ß-cell-restricted deletion of each kinase in mice, using an Ins1-controlled Cre, was therefore followed by physiological, morphometric, and massive parallel sequencing analysis. Loss of LKB1 strikingly (2.0-12-fold, E<0.01) increased the expression of subsets of hepatic (Alb, Iyd, Elovl2) and neuronal (Nptx2, Dlgap2, Cartpt, Pdyn) genes, enhancing glutamate signaling. These changes were partially recapitulated by the loss of AMPK, which also up-regulated ß-cell "disallowed" genes (Slc16a1, Ldha, Mgst1, Pdgfra) 1.8- to 3.4-fold (E < 0.01). Correspondingly, targeted promoters were enriched for neuronal (Zfp206; P = 1.3 × 10(-33)) and hypoxia-regulated (HIF1; P = 2.5 × 10(-16)) transcription factors. In summary, LKB1 and AMPK, through only partly overlapping mechanisms, maintain ß-cell identity by suppressing alternate pathways leading to neuronal, hepatic, and other characteristics. Selective targeting of these enzymes may provide a new approach to maintaining ß-cell function in some forms of diabetes.
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Proteínas Quinasas Activadas por AMP/metabolismo , Células Secretoras de Insulina/enzimología , Insulina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Ratones Endogámicos C57BL , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: The past decade has seen an abundance of transcriptional profiling studies of preclinical models of persistent pain, predominantly employing microarray technology. In this study we directly compare exon microarrays to RNA-seq and investigate the ability of both platforms to detect differentially expressed genes following nerve injury using the L5 spinal nerve transection model of neuropathic pain. We also investigate the effects of increasing RNA-seq sequencing depth. Finally we take advantage of the "agnostic" approach of RNA-seq to discover areas of expression outside of annotated exons that show marked changes in expression following nerve injury. RESULTS: RNA-seq and microarrays largely agree in terms of the genes called as differentially expressed. However, RNA-seq is able to interrogate a much larger proportion of the genome. It can also detect a greater number of differentially expressed genes than microarrays, across a wider range of fold changes and is able to assign a larger range of expression values to the genes it measures. The number of differentially expressed genes detected increases with sequencing depth. RNA-seq also allows the discovery of a number of genes displaying unusual and interesting patterns of non-exonic expression following nerve injury, an effect that cannot be detected using microarrays. CONCLUSION: We recommend the use of RNA-seq for future high-throughput transcriptomic experiments in pain studies. RNA-seq allowed the identification of a larger number of putative candidate pain genes than microarrays and can also detect a wider range of expression values in a neuropathic pain model. In addition, RNA-seq can interrogate the whole genome regardless of prior annotations, being able to detect transcription from areas of the genome not currently annotated as exons. Some of these areas are differentially expressed following nerve injury, and may represent novel genes or isoforms. We also recommend the use of a high sequencing depth in order to detect differential expression for genes with low levels of expression.
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Regulación de la Expresión Génica/fisiología , Neuralgia/metabolismo , Neuralgia/patología , Células Receptoras Sensoriales/metabolismo , Análisis de Secuencia de ARN , Transcripción Genética/fisiología , Animales , Mapeo Cromosómico , Modelos Animales de Enfermedad , Ganglios Espinales/patología , Perfilación de la Expresión Génica , Genoma/fisiología , Masculino , Análisis por Micromatrices/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Wistar , Médula Espinal/patología , Nervios Espinales/lesionesRESUMEN
This study examined responses of isolated pig coronary arteries after kinin B1 receptor induction by endotoxin. Des-Arg9-bradykinin (DBK) induced concentration-dependent, endothelium-independent contractions in lipopolysaccharide (LPS)-treated but not untreated arterial rings. The B1-receptor antagonist SSR240612, but not the B2-receptor antagonist HOE140, prevented the endothelium-independent contractions to DBK. The DBK-induced contractions were blocked by indomethacin (nonselective cyclooxygenase [COX] inhibitor), celecoxib (selective COX-2 inhibitor), and terbogrel (thromboxane-prostanoid [TP] receptor antagonist) but not valeryl salicylate (selective COX-1 inhibitor), AH6809 (an E prostanoid [EP] and PGD2 receptor [DP1] receptor antagonist), AL 8810 (a selective PGF2α [FP] receptor antagonist), or RO1138452 (a selective I prostanoid [IP] receptor antagonist). They were attenuated by N-(p-amylcinnamoyl) anthranilic acid (ACA), and by DETCA plus tiron but not by l-NAME. Quantitative RT-PCR revealed excessive up-regulations of mRNA expressions of B1 receptors, COX-2, and thromboxane A synthase 1 (TBXAS1) following LPS incubation, but not of B2 receptors or COX-1. The present data demonstrate that B1 receptors are coupled to COX-2 in causing endothelium-independent contractions in endotoxin-treated pig coronary arteries. Accordingly, kinin B1 receptor induction during inflammation may have a pathological significance in the vasculature, particular in coronary arteries with dysfunctional endothelial cells.
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Vasos Coronarios/fisiología , Ciclooxigenasa 2/fisiología , Receptor de Bradiquinina B1/fisiología , Vasoconstricción/fisiología , Animales , Bradiquinina/análogos & derivados , Bradiquinina/farmacología , Antagonistas del Receptor de Bradiquinina B1/farmacología , Vasos Coronarios/efectos de los fármacos , Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Dioxoles/farmacología , Endotelio Vascular , Técnicas In Vitro , Lipopolisacáridos/farmacología , ARN Mensajero/biosíntesis , Receptor de Bradiquinina B1/genética , Receptor de Bradiquinina B2/genética , Sulfonamidas/farmacología , Porcinos , Tromboxano-A Sintasa/genética , Vasoconstricción/efectos de los fármacosRESUMEN
This study examined the vascular effects of bradykinin in health and vascular inflammation comparing responses of isolated pig coronary arteries in the absence and presence of endotoxins. Bradykinin induced contractions in lipopolysaccharide-treated, but not untreated, arterial rings without endothelium. The B2-receptor antagonist HOE140, but not the B1-receptor inhibitor SSR240612, blocked these endothelium-independent contractions in response to bradykinin. The bradykinin-induced contractions were blocked by indomethacin, celecoxib, and terbogrel but not valeryl salicylate, AH6809, AL 8810, or RO1138452. They were attenuated by N-(p-amylcinnamoyl) anthranilic acid, and by diethyldithiocarbamate plus tiron but not by L-NAME. Quantitative reverse-transcription polymerase chain reaction revealed significant upregulations of messenger RNA expressions of B1 receptors, COX-2, and thromboxane A synthase 1 (TBXAS1) following lipopolysaccharide incubation but not of B2 receptors or COX-1. The present data demonstrate that bradykinin induces contractions mediated by the COX-2 pathway in endotoxin-treated pig coronary arteries. These results support differential roles of bradykinin in health and disease.
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Bradiquinina/metabolismo , Vasos Coronarios/metabolismo , Ciclooxigenasa 2/metabolismo , Inflamación/patología , Animales , Bradiquinina/farmacología , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/patología , Ciclooxigenasa 2/genética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotoxinas/farmacología , Lipopolisacáridos/farmacología , Contracción Muscular/efectos de los fármacos , ARN Mensajero , Receptor de Bradiquinina B1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Tromboxano-A Sintasa/genética , Regulación hacia ArribaRESUMEN
Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4(+) Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4(+) T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4(+) Th cells upon activation, whereas the 'standard' proteasome is up-regulated in Tregs only upon activation.
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Factores de Transcripción Forkhead/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Transcriptoma , Empalme Alternativo , Animales , Sitios de Unión , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Humanos , Ratones , ARN Mensajero/química , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Análisis de Secuencia de ARNRESUMEN
Alongside cell lines such as 3T3-L1 cells, primary cell culture models of adipogenesis have helped in developing an understanding of the process of adipocyte recruitment and maintenance, which may lead to therapeutic advances to treat the growing epidemic of obesity. Recently, it has been demonstrated that fat cell progenitors (DFAT) established through ceiling culture of adipocytes retain an enhanced ability to undergo adipocyte differentiation compared to preadipocytes isolated from the stromal vascular fraction of adipose tissue. Clonal expansion of rat DFAT cells identified differentiation capable and incapable cell strains. To understand the mechanisms underlying these differences, comparison of their transcriptomes by next generation sequencing was performed. Two hundred seventy-eight genes with a significant fold change of 1.4 were detected as being consistently deregulated between differentiating and non-differentiating strains. Bioinformatic network analyses identified components of the extra-cellular matrix and PPARγ as important genes in this process, suggesting crosstalk between ECM and transcription factors influences differentiation. Analyses of the transcriptomes of human DFAT cells in early and late passage (non-differentiating) confirmed the importance of these pathways in maintaining an adipogenic potential.
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Adipocitos , Diferenciación Celular , Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis , Animales , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , PPAR gamma/metabolismo , Ratas , Factores de Transcripción/metabolismoRESUMEN
The arrival of next-generation sequencing (NGS) technologies has led to novel opportunities for expression profiling and genome analysis by utilizing vast amounts of short read sequence data. Here, we demonstrate that expression profiling in organisms lacking any genome or transcriptome sequence information is feasible by combining Illumina's mRNA-seq technology with a novel bioinformatics pipeline that integrates assembled and annotated Chinese hamster ovary (CHO) sequences with information derived from related organisms. We applied this pipeline to the analysis of CHO cells which were chosen as a model system owing to its relevance in the production of therapeutic proteins. Specifically, we analysed CHO cells undergoing butyrate treatment which is known to affect cell cycle regulation and to increase the specific productivity of recombinant proteins. By this means, we identified sequences for >13,000 CHO genes which added sequence information of approximately 5000 novel genes to the CHO model. More than 6000 transcript sequences are predicted to be complete, as they covered >95% of the corresponding mouse orthologs. Detailed analysis of selected biological functions such as DNA replication and cell cycle control, demonstrated the potential of NGS expression profiling in organisms without extended genome sequence to improve both data quantity and quality.
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Perfilación de la Expresión Génica , Análisis de Secuencia de ARN , Animales , Butiratos/farmacología , Células CHO , Cricetinae , Cricetulus , Reparación del ADN , Replicación del ADN , Expresión Génica/efectos de los fármacos , Genes cdc , Genómica , Ratones , Ratas , Recombinación Genética , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Hox genes are key regulators of appendage development in the insect body plan. The body plan of mayfly (Ephemeroptera) nymphs differs due to the presence of abdominal appendages called gills. Despite mayflies' phylogenetic position in Paleoptera and novel morphology amongst insects, little is known of their developmental genetics, such as the appendage-regulating Hox genes. To address this issue we present an annotated, early instar transcriptome and embryonic expression profiles for Antennapedia, Ultrabithorax, and Abdominal A proteins in the mayfly Hexagenia limbata, identify putative Hox protein sequences in the mayflies H. limbata, Cloeon dipterum, and Ephemera danica, and describe the genomic organization of the Hox gene cluster in E. danica. RESULTS: Transcriptomic sequencing of early instar H. limbata nymphs yielded a high-quality assembly of 83,795 contigs, of which 22,975 were annotated against Folsomia candida, Nilaparvata lugens, Zootermopsis nevadensis and UniRef90 protein databases. Homeodomain protein phylogeny and peptide annotations identified coding sequences for eight of the ten canonical Hox genes (excluding zerknüllt/Hox3 and fushi tarazu) in H. limbata and C. dipterum, and all ten in E. danica. Mayfly Hox protein sequences and embryonic expression patterns of Antp, Ubx, and Abd-A appear highly conserved with those seen in other non-holometabolan insects. Similarly, the genomic organization of the Hox cluster in E. danica resembles that seen in most insects. CONCLUSIONS: We present evidence that mayfly Hox peptide sequences and the embryonic expression patterns for Antp, Ubx, and Abd-A are extensively conserved with other insects, as is organization of the mayfly Hox gene cluster. The protein data suggest mayfly Antp, Ubx, and Abd-A play appendage promoting and repressing roles during embryogenesis in the thorax and abdomen, respectively, as in other insects. The identified expression of eight Hox genes, including Ubx and abd-A, in early instar nymphs further indicates a post-embryonic role, possibly in gill development. These data provide a basis for H. limbata as a complementary Ephemeridae model to the growing repertoire of mayfly model species and molecular techniques.
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Increase in both productivity and product yields in biopharmaceutical process development with recombinant protein producing mammalian cells can be mainly attributed to the advancements in cell line development, media, and process optimization. Only recently, genome-scale technologies enable a system-level analysis to elucidate the complex biomolecular basis of protein production in mammalian cells promising an increased process understanding and the deduction of knowledge-based approaches for further process optimization. Here, the use of gene expression profiling for the analysis of a low titer (LT) and high titer (HT) fed batch process using the same IgG producing CHO cell line was investigated. We found that gene expression (i) significantly differed in HT versus LT process conditions due to differences in applied chemically defined, serum-free media, (ii) changed over the time course of the fed batch processes, and that (iii) both metabolic pathways and 14 biological functions such as cellular growth or cell death were affected. Furthermore, detailed analysis of metabolism in a standard process format revealed the potential use of transcriptomics for rational media design as is shown for the case of lipid metabolism where the product titer could be increased by about 20% based on a lipid modified basal medium. The results demonstrate that gene expression profiling can be an important tool for mammalian biopharmaceutical process analysis and optimization.
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Biotecnología/métodos , Técnicas de Cultivo de Célula/métodos , Cricetulus/genética , Perfilación de la Expresión Génica , Animales , Células CHO , Cricetinae , Cricetulus/metabolismoRESUMEN
INTRODUCTION: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are being evaluated for their use in pharmacological and toxicological testing, particularly for electrophysiological side effects. However, little is known about the composition of the commercially available iCell cardiomyocyte (Fuijifilm Cellular Dynamics) cultures and the transcriptomic phenotype of individual cells. METHODS: We characterized iCell cardiomyocytes (assumed to be a mixture of nodal-, atrial-, and ventricular-like cardiomyocytes together with potential residual non-myocytes) using bulk RNA-sequencing, followed by investigation of cellular heterogeneity using two different single-cell RNA-sequencing platforms. RESULTS: Bulk RNA-sequencing identified key cardiac markers (TNNT2, MYL7) as well as fibroblast associated genes (P4HB, VIM), and cardiac ion channels in the iCell cardiomyocyte culture. High-resolution single cell RNA-sequencing demonstrated that both, cardiac and fibroblast-related genes were co-expressed throughout the cell population. This approach resolved two cell clusters within iCell cardiomyocytes. Interestingly, these clusters could not be associated with known cardiac subtypes. However, transcripts of ion channels potentially useful as functional markers for cardiac subtypes were below the detection limits of the single-cell approaches used. Instead, one cluster (10.8% of the cells) is defined by co-expression of cardiac and cell cycle-related genes (e.g. TOP2A). Incorporation of bromodeoxyuridine further confirmed the capability of iCell cardiomyocytes to enter cell cycle. DISCUSSION: The co-expression of cardiac related genes with cell cycle or fibroblast related genes may be interpreted either as aberrant or as an immature feature. However, this excludes the presence of a non-cardiomyocyte sub-population and indicates that some cardiomyocytes themselves enter cell cycle.
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Miocitos Cardíacos/fisiología , RNA-Seq/métodos , Análisis de la Célula Individual/métodos , Biomarcadores/análisis , Ciclo Celular/genética , Diferenciación Celular/genética , Línea Celular , Separación Celular , Evaluación Preclínica de Medicamentos/métodos , Fibroblastos/fisiología , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Transcriptoma/fisiologíaRESUMEN
The human KCTD13 gene is located within the 16p11.2 locus and copy number variants of this locus are associated with a high risk for neuropsychiatric diseases including autism spectrum disorder and schizophrenia. Studies in zebrafish point to a role of KCTD13 in proliferation of neural precursor cells which may contribute to macrocephaly in 16p11.2 deletion carriers. KCTD13 is highly expressed in the fetal human brain and in mouse cortical neurons, but its contribution to the development and function of mammalian neurons is not completely understood. In the present study, we deleted the KCTD13 gene in human-induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 nickase. Following neural differentiation of KCTD13 deficient and isogenic control iPSC lines, we detected a moderate but significant inhibition of DNA synthesis and proliferation in KCTD13 deficient human neural precursor cells. KCTD13 deficient cortical neurons derived from iPSCs showed decreased neurite formation and reduced spontaneous network activity. RNA-sequencing and pathway analysis pointed to a role for ERBB signaling in these phenotypic changes. Consistently, activating and inhibiting ERBB kinases rescued and aggravated, respectively, impaired neurite formation. In contrast to findings in non-neuronal human HeLa cells, we did not detect an accumulation of the putative KCTD13/Cullin-3 substrate RhoA, and treatment with inhibitors of RhoA signaling did not rescue decreased neurite formation in human KCTD13 knockout neurons. Taken together, our data provide insight into the role of KCTD13 in neurodevelopmental disorders, and point to ERBB signaling as a potential target for neuropsychiatric disorders associated with KCTD13 deficiency.
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Sistemas CRISPR-Cas/genética , Corteza Cerebral/patología , Técnicas de Inactivación de Genes , Predisposición Genética a la Enfermedad , Células Madre Pluripotentes Inducidas/patología , Trastornos Mentales/genética , Neuronas/patología , Proteínas Nucleares/genética , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Diferenciación Celular , Proliferación Celular , ADN/biosíntesis , Humanos , Células-Madre Neurales/metabolismo , Neuritas/metabolismo , Proteínas Nucleares/deficiencia , Receptor ErbB-2/metabolismo , Factores de Riesgo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has recently become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomic changes associated with cell preservation and how they may bias further downstream analysis remain unknown. Here, we present a side-by-side droplet-based scRNA-seq analysis, comparing the gold standard - fresh cells - to three different cell preservation workflows: dimethyl sulfoxide based cryopreservation, methanol fixation and CellCover reagent. Cryopreservation proved to be the most robust protocol, maximizing both cell integrity and low background ambient RNA. Importantly, gene expression profiles from fresh cells correlated most with those of cryopreserved cells. Such similarities were consistently observed across the tested cell lines (R ≥ 0.97), monocyte-derived macrophages (R = 0.97) and immune cells (R = 0.99). In contrast, both methanol fixation and CellCover preservation showed an increased ambient RNA background and an overall lower gene expression correlation to fresh cells. Thus, our results demonstrate the superiority of cryopreservation over other cell preservation methods. We expect our comparative study to provide single-cell omics researchers invaluable support when integrating cell preservation into their scRNA-seq studies.
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Criopreservación/métodos , Dimetilsulfóxido , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Perfilación de la Expresión Génica/métodos , HumanosRESUMEN
Chronic administration of L-DOPA, the first-line treatment of dystonic symptoms in childhood or in Parkinson's disease, often leads to the development of abnormal involuntary movements (AIMs), which represent an important clinical problem. Although it is known that Riluzole attenuates L-DOPA-induced AIMs, the molecular mechanisms underlying this effect are not understood. Therefore, we studied the behavior and performed RNA sequencing of the striatum in three groups of rats that all received a unilateral lesion with 6-hydroxydopamine in their medial forebrain bundle, followed by the administration of saline, L-DOPA, or L-DOPA combined with Riluzole. First, we provide evidence that Riluzole attenuates AIMs in this rat model. Subsequently, analysis of the transcriptomics data revealed that Riluzole is predicted to reduce the activity of CREB1, a transcription factor that regulates the expression of multiple proteins that interact in a molecular landscape involved in apoptosis. Although this mechanism underlying the beneficial effect of Riluzole on AIMs needs to be confirmed, it provides clues towards novel or existing compounds for the treatment of AIMs that modulate the activity of CREB1 and, hence, its downstream targets.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Discinesia Inducida por Medicamentos/metabolismo , Discinesia Inducida por Medicamentos/prevención & control , Levodopa/toxicidad , Riluzol/uso terapéutico , Animales , Modelos Animales de Enfermedad , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Masculino , Oxidopamina/toxicidad , Mapas de Interacción de Proteínas/efectos de los fármacos , Mapas de Interacción de Proteínas/fisiología , Distribución Aleatoria , Ratas , Ratas Wistar , Riluzol/farmacologíaRESUMEN
BACKGROUND: The ability to generate recombinant drug target proteins is important for drug discovery research as it facilitates the investigation of drug-target-interactions in vitro. To accomplish this, the target's exact protein sequence is required. Public databases, such as Ensembl, UniProt and RefSeq, are extensive protein and nucleotide sequence repositories. However, many sequences for non-human organisms are predicted by computational pipelines and may thus be incomplete or incorrect. This could lead to misinterpreted experimental outcomes due to gaps or errors in orthologous drug target sequences. Transcriptome analysis by RNA-Seq has been established as a standard method for gene expression analysis. Apart from this common application, paired-end RNA-Seq data can also be used to obtain full coverage cDNA sequences via de novo transcriptome assembly. METHODS: To assess whether de novo transcriptome assemblies can be used to determine a protein's sequence by searching the assembly for a known orthologous sequence, we generated 3 × 6 = 18 tissue specific assemblies (three organs: brain, kidney and liver; six species: human, mouse, rat, dog, pig and cynomolgus monkey). These assemblies and the manually curated human protein sequences from UniProtKB/Swiss-Prot were used in a reciprocal BLAST search to identify best matching hits. We automated and generalised our approach and present the a&o-tool, a workflow which exploits de novo assemblies of paired-end RNA-Seq data and orthology information for target sequence validation and refinement across related species. Furthermore, the a&o-tool extracts best hits' sequences from a reciprocal BLAST search, translates them into protein sequences, computes a multiple sequence alignment and quantifies the refinement. RESULTS: For the three human assemblies we observed a hit rate greater than 60% with 100% sequence coverage and identity. For assemblies from the other species we observed similar hit rates and coverage with highest identities for cynomolgus monkey. CONCLUSIONS: In summary, we show how to refine protein sequences using RNA-Seq data and sequence information from closely related species. With the a&o-tool we provide a fully automated pipeline to perform refinement including cDNA translation and multiple sequence alignment for visual inspection. The major prerequisite for applying the a&o-tool is high quality sequencing data.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Homología de Secuencia de Ácido Nucleico , Animales , Genómica , Humanos , Análisis de Secuencia de ARNRESUMEN
Efficient transcytosis across the blood-brain-barrier (BBB) is an important strategy for accessing drug targets within the central nervous system (CNS). Despite extensive research the number of studies reporting successful delivery of macromolecules or macromolecular complexes to the CNS has remained very low. In order to expand current research it is important to know which receptors are selective and abundant on the BBB so that novel CNS-targeting antibodies or other ligands could be developed, targeting those receptors for transcytosis. To do that, we have set up a proteomics- and transcriptomics-based workflow within the COMPACT project (Collaboration on the Optimization of Macromolecular Pharmaceutical Access to Cellular Targets) of the Innovative Medicines Initiative (IMI) of the EU. Here we summarise our overall strategy in endothelial transcytosis research, describe in detail the related challenges, and discuss future perspectives of these studies. This article is part of the Special Issue entitled "Beyond small molecules for neurological disorders".
Asunto(s)
Transporte Biológico/fisiología , Barrera Hematoencefálica/fisiología , Sistemas de Liberación de Medicamentos , Transcitosis/fisiología , Animales , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteómica , Transcitosis/efectos de los fármacosRESUMEN
Gene functionality is closely connected to its expression specificity across tissues and cell types. RNA-Seq is a powerful quantitative tool to explore genome wide expression. The aim of this study is to provide a comprehensive RNA-Seq dataset across the same 13 tissues for mouse and rat, two of the most relevant species for biomedical research. The dataset provides the transcriptome across tissues from three male C57BL6 mice and three male Han Wistar rats. We also describe our bioinformatics pipeline to process and technically validate the data. Principal component analysis shows that tissue samples from both species cluster similarly. We show by comparative genomics that many genes with high sequence identity with respect to their human orthologues also have a highly correlated tissue distribution profile and are in agreement with manually curated literature data for human. In summary, the present study provides a unique resource for comparative genomics and will facilitate the analysis of tissue specificity and cross-species conservation in higher organisms.