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1.
BMC Genomics ; 25(1): 186, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365592

RESUMO

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.


Assuntos
Crotalus , MicroRNAs , Toxinas Biológicas , Serpentes Peçonhentas , Viperidae , Humanos , Animais , Elapidae/genética , Venenos de Serpentes/química , Venenos de Serpentes/genética , Venenos de Serpentes/metabolismo , Venenos Elapídicos/química , Venenos Elapídicos/genética , Venenos Elapídicos/metabolismo , Viperidae/genética , Viperidae/metabolismo , Transcriptoma , Fatores de Transcrição/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo
3.
Poult Sci ; 100(2): 998-1003, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33518154

RESUMO

Antibiotics have played a critical role in sustaining and improving livestock production in the past decades, but the emergence of antimicrobial resistance has led several countries to ban or limit their use. Since then, in-feed alternatives have gained a lot of attention but the development of efficacious alternatives implies a better understanding of the mode of action of antibiotic growth promoters (AGP) when administered at subtherapeutic concentrations. In the present study, 120 broiler chickens per group (8 pens/group) were fed for 35 d with either basal feed (control group) or feed supplemented with avilamycin (AGP group; 10 g/1,000 kg of feed). At the end of the trial, the ileum from the small intestine of 5 birds per group was sampled, and RNA were isolated for profiling their transcriptome via RNA sequencing (RNA-Seq). As expected, the growth of chickens in the AGP group was significantly higher than in the control group. Overall, 66 differentially expressed genes (false discovery rate ≤ 0.05 and fold change ≥ 2 or ≤ -2) were found in the ileum of chickens fed avilamycin in comparison with the control group. The functional analysis showed reduced activity of genes related to signaling by interleukins, with IL-22, SOCS3, and certain antimicrobial peptides found multiple times in these pathways in the AGP group at day 35. In addition, higher activity was predicted in a module of genes related to lipid metabolism and transport in the avilamycin group. The use of RNA-Seq allowed a snapshot of the whole transcriptome at day 35 and aimed at delivering additional data on the host-centric hypothesis regarding the mode of action of AGP (i.e. immunomodulation, reduction of the immunological stress).


Assuntos
Ração Animal , Antibacterianos/administração & dosagem , Galinhas , Íleo/química , Oligossacarídeos/administração & dosagem , Transcriptoma , Ração Animal/análise , Animais , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , Análise de Sequência de RNA/veterinária
4.
Eur J Epidemiol ; 36(1): 129-142, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33222050

RESUMO

The Singapore Preconception Study of Long-Term Maternal and Child Outcomes (S-PRESTO) is a preconception, longitudinal cohort study that aims to study the effects of nutrition, lifestyle, and maternal mood prior to and during pregnancy on the epigenome of the offspring and clinically important outcomes including duration of gestation, fetal growth, metabolic and neural phenotypes in the offspring. Between February 2015 and October 2017, the S-PRESTO study recruited 1039 Chinese, Malay or Indian (or any combinations thereof) women aged 18-45 years and who intended to get pregnant and deliver in Singapore, resulting in 1032 unique participants and 373 children born in the cohort. The participants were followed up for 3 visits during the preconception phase and censored at 12 months of follow up if pregnancy was not achieved (N = 557 censored). Women who successfully conceived (N = 475) were characterised at gestational weeks 6-8, 11-13, 18-21, 24-26, 27-28 and 34-36. Follow up of their index offspring (N = 373 singletons) is on-going at birth, 1, 3 and 6 weeks, 3, 6, 12, 18, 24 and 36 months and beyond. Women are also being followed up post-delivery. Data is collected via interviewer-administered questionnaires, metabolic imaging (magnetic resonance imaging), standardized anthropometric measurements and collection of diverse specimens, i.e. blood, urine, buccal smear, stool, skin tapes, epithelial swabs at numerous timepoints. S-PRESTO has extensive repeated data collected which include genetic and epigenetic sampling from preconception which is unique in mother-offspring epidemiological cohorts. This enables prospective assessment of a wide array of potential determinants of future health outcomes in women from preconception to post-delivery and in their offspring across the earliest development from embryonic stages into early childhood. In addition, the S-PRESTO study draws from the three major Asian ethnic groups that represent 50% of the global population, increasing the relevance of its findings to global efforts to address non-communicable diseases.


Assuntos
Estilo de Vida , Comportamento Materno , Estado Nutricional , Vigilância da População/métodos , Cuidado Pré-Concepcional/estatística & dados numéricos , Cuidado Pré-Natal/estatística & dados numéricos , Adolescente , Adulto , Afeto , Feminino , Humanos , Estudos Longitudinais , Fenômenos Fisiológicos da Nutrição Materna , Pessoa de Meia-Idade , Gravidez , Resultado da Gravidez/epidemiologia , Medição de Risco , Singapura/epidemiologia , Adulto Jovem
5.
Cytokine ; 125: 154815, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31476685

RESUMO

BACKGROUND: TNF-α, a pro-inflammatory cytokine is one of the major contributors for metabolic syndromes including insulin resistance, obesity, type II diabetes etc. The role of alternative splicing, a post-transcriptional regulation of gene expression on the onset of these syndromes is poorly understood. However, the role of alternative splicing, which more than 95% of all exons in eukaryotic cells undergo in several other diseases including cancer and muscle dystrophy, has been elucidated. In this study we aim to investigate the role of alternative splicing in pathways leading to metabolic syndromes mediated by TNF-α. METHODS: A genome wide transcriptome analysis was carried out using Illumina platform. Results were validated using RT-PCR analysis. Various bioinformatics tools and databases (for example IPA, KEGG, STRING etc) were used for the pathway and interactome analysis. CURRENT FINDINGS: Transcriptome wide analysis revealed that TNF-α treatment in vitro causes a significant change in expression of 228 genes at the level of alternative splicing. Regulation of some of these genes was validated in different cell lines. Pathway analysis showed at least 15% of the alternatively spliced genes fall under the contributory pathways leading to different metabolic syndromes, among which the maximally interconnected genes were transcription regulators. CONCLUSION: These findings suggest that TNF-α.-mediated alternative splicing plays a crucial role in regulating various genes involved in pathways connected to metabolic syndromes.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Síndrome Metabólica/metabolismo , Transcriptoma/genética , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular , Biologia Computacional , Bases de Dados Genéticas , Éxons , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Síndrome Metabólica/genética , Camundongos , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Nat Commun ; 10(1): 5808, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862890

RESUMO

The causes of impaired skeletal muscle mass and strength during aging are well-studied in healthy populations. Less is known on pathological age-related muscle wasting and weakness termed sarcopenia, which directly impacts physical autonomy and survival. Here, we compare genome-wide transcriptional changes of sarcopenia versus age-matched controls in muscle biopsies from 119 older men from Singapore, Hertfordshire UK and Jamaica. Individuals with sarcopenia reproducibly demonstrate a prominent transcriptional signature of mitochondrial bioenergetic dysfunction in skeletal muscle, with low PGC-1α/ERRα signalling, and downregulation of oxidative phosphorylation and mitochondrial proteostasis genes. These changes translate functionally into fewer mitochondria, reduced mitochondrial respiratory complex expression and activity, and low NAD+ levels through perturbed NAD+ biosynthesis and salvage in sarcopenic muscle. We provide an integrated molecular profile of human sarcopenia across ethnicities, demonstrating a fundamental role of altered mitochondrial metabolism in the pathological loss of skeletal muscle mass and function in older people.


Assuntos
Envelhecimento/fisiologia , Mitocôndrias/patologia , Músculo Esquelético/patologia , NAD/biossíntese , Sarcopenia/patologia , Idoso , Idoso de 80 Anos ou mais , Biópsia , Estudos de Casos e Controles , Metabolismo Energético/fisiologia , Humanos , Jamaica , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Oxirredução , Fosforilação Oxidativa , Estresse Oxidativo/fisiologia , Proteostase , Sarcopenia/etnologia , Singapura , Reino Unido
7.
Sci Rep ; 9(1): 8771, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217429

RESUMO

Current opinion views androgens as the pathogenic driver in the miniaturization of hair follicles of androgenetic alopecia by interfering with the dermal papilla. This cannot be the sole cause and therefore it is important for therapeutic and diagnostic purposes to identify additional pathways. Comparative full transcriptome profile analysis of the hair bulb region of normal and miniaturized hair follicles from vertex and occipital region in males with and without androgenetic alopecia revealed that next to the androgen receptor as well the retinoid receptor and particularly the PPAR pathway is involved in progressive hair miniaturization. We demonstrate the concurrent up-regulation of PPARGC1a in the epithelial compartment and androgen receptor in the dermal papilla of miniaturized hair. Dynamic Ppargc1a expression in the mouse hair cycle suggests a possible role in regulating hair growth and differentiation. This is supported by reduced proliferation of human dermal papilla and predominantly epithelial keratinocytes after incubation with AICAR, the agonist for AMPK signaling which activates PPARGC1a and serves as co-activator of PPARγ. In addition, miRNA profiling shows enrichment of miRNA-targeted genes in retinoid receptors and PPARGC1α/PPARγ signaling, and antigen presentation pathways.


Assuntos
Alopecia/metabolismo , Regulação da Expressão Gênica , Folículo Piloso/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Alopecia/genética , Alopecia/patologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animais , Linhagem Celular Transformada , Folículo Piloso/patologia , Humanos , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo
8.
J Chem Inf Model ; 59(4): 1529-1546, 2019 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-30794402

RESUMO

Small molecule drugs bind to a pocket in disease causing target proteins based on complementarity in shape and physicochemical properties. There is a likelihood that other proteins could have binding sites that are structurally similar to the target protein. Binding to these other proteins could alter their activities leading to off target effects of the drug. One such small molecule drug Nutlin binds the protein MDM2, which is upregulated in several types of cancer and is a negative regulator of the tumor suppressor protein p53. To investigate the off target effects of Nutlin, we present here a shape-based data mining effort. We extracted the binding pocket of Nutlin from the crystal structure of Nutlin bound MDM2. We next mined the protein structural database (PDB) for putative binding pockets in other human protein structures that were similar in shape to the Nutlin pocket in MDM2 using our topology-independent structural superimposition tool CLICK. We detected 49 proteins which have binding pockets that were structurally similar to the Nutlin binding site of MDM2. All of the potential complexes were evaluated using molecular mechanics and AutoDock Vina docking scores. Further, molecular dynamics simulations were carried out on four of the predicted Nutlin-protein complexes. The binding of Nutlin to one of these proteins, gamma glutamyl hydrolase, was also experimentally validated by a thermal shift assay. These findings provide a platform for identifying potential off-target effects of existing/new drugs and also opens the possibilities for repurposing drugs/ligands.


Assuntos
Imidazóis/farmacologia , Terapia de Alvo Molecular , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Temperatura , Proteína Supressora de Tumor p53/química
9.
Development ; 145(1)2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29229769

RESUMO

In the earliest stages of animal development following fertilization, maternally deposited mRNAs direct biological processes to the point of zygotic genome activation (ZGA). These maternal mRNAs undergo cytoplasmic polyadenylation (CPA), suggesting translational control of their activation. To elucidate the biological role of CPA during embryogenesis, we performed genome-wide polysome profiling at several stages of zebrafish development. Our analysis revealed a correlation between CPA and polysome-association dynamics, demonstrating a coupling of translation to the CPA of maternal mRNAs. Pan-embryonic CPA inhibition disrupted the maternal-to-zygotic transition (MZT), causing a failure of developmental progression beyond the mid-blastula transition and changes in global gene expression that indicated a failure of ZGA and maternal mRNA clearance. Among the genes that were differentially expressed were those encoding chromatin modifiers and key transcription factors involved in ZGA, including nanog, pou5f3 and sox19b, which have distinct CPA dynamics. Our results establish the necessity of CPA for ensuring progression of the MZT. The RNA-seq data generated in this study represent a valuable zebrafish resource for the discovery of novel elements of the early embryonic transcriptome.


Assuntos
Citoplasma/metabolismo , Poliadenilação/fisiologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Zigoto/metabolismo , Animais , Feminino , RNA Mensageiro/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Zigoto/citologia
10.
J Exp Med ; 214(10): 2889-2900, 2017 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-28827448

RESUMO

Epithelial carcinomas are well known to activate a prolonged wound-healing program that promotes malignant transformation. Wound closure requires the activation of keratinocyte migration via a dual-state molecular switch. This switch involves production of either the anti-migratory microRNA miR-198 or the pro-migratory follistatin-like 1 (FSTL1) protein from a single transcript; miR-198 expression in healthy skin is down-regulated in favor of FSTL1 upon wounding, which enhances keratinocyte migration and promotes re-epithelialization. Here, we reveal a defective molecular switch in head and neck squamous cell carcinoma (HNSCC). This defect shuts off miR-198 expression in favor of sustained FSTL1 translation, driving metastasis through dual parallel pathways involving DIAPH1 and FSTL1. DIAPH1, a miR-198 target, enhances directional migration through sequestration of Arpin, a competitive inhibitor of Arp2/3 complex. FSTL1 blocks Wnt7a-mediated repression of extracellular signal-regulated kinase phosphorylation, enabling production of MMP9, which degrades the extracellular matrix and facilitates metastasis. The prognostic significance of the FSTL1-DIAPH1 gene pair makes it an attractive target for therapeutic intervention.


Assuntos
Transformação Celular Neoplásica/metabolismo , Fator de Crescimento Epidérmico/fisiologia , Proteínas Relacionadas à Folistatina/fisiologia , MicroRNAs/fisiologia , Cicatrização/fisiologia , Animais , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Proliferação de Células , Feminino , Genes de Troca/fisiologia , Neoplasias de Cabeça e Pescoço/metabolismo , Imunoprecipitação , Espectrometria de Massas , Camundongos Endogâmicos NOD
11.
Sci Rep ; 7(1): 5805, 2017 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-28724991

RESUMO

Several available online tools provide network growing functions where an algorithm utilizing different data sources suggests additional genes/proteins that should connect an input gene set into functionally meaningful networks. Using the well-studied system of influenza host interactions, we compare the network growing function of two free tools GeneMANIA and STRING and the commercial IPA for their performance of recovering known influenza A virus host factors previously identified from siRNA screens. The result showed that given small (~30 genes) or medium (~150 genes) input sets all three network growing tools detect significantly more known host factors than random human genes with STRING overall performing strongest. Extending the networks with all the three tools significantly improved the detection of GO biological processes of known host factors compared to not growing networks. Interestingly, the rate of identification of true host factors using computational network growing is equal or better to doing another experimental siRNA screening study which could also be true and applied to other biological pathways/processes.


Assuntos
Benchmarking , Biologia Computacional/métodos , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno/genética , Vírus/metabolismo , Algoritmos , Ontologia Genética , Humanos , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
12.
PLoS One ; 11(9): e0162541, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27622715

RESUMO

Development of myopia is associated with large-scale changes in ocular tissue gene expression. Although differential expression of coding genes underlying development of myopia has been a subject of intense investigation, the role of non-coding genes such as microRNAs in the development of myopia is largely unknown. In this study, we explored myopia-associated miRNA expression profiles in the retina and sclera of C57Bl/6J mice with experimentally induced myopia using microarray technology. We found a total of 53 differentially expressed miRNAs in the retina and no differences in miRNA expression in the sclera of C57BL/6J mice after 10 days of visual form deprivation, which induced -6.93 ± 2.44 D (p < 0.000001, n = 12) of myopia. We also identified their putative mRNA targets among mRNAs found to be differentially expressed in myopic retina and potential signaling pathways involved in the development of form-deprivation myopia using miRNA-mRNA interaction network analysis. Analysis of myopia-associated signaling pathways revealed that myopic response to visual form deprivation in the retina is regulated by a small number of highly integrated signaling pathways. Our findings highlighted that changes in microRNA expression are involved in the regulation of refractive eye development and predicted how they may be involved in the development of myopia by regulating retinal gene expression.


Assuntos
MicroRNAs/genética , Miopia/genética , RNA Mensageiro/genética , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miopia/etiologia , Miopia/metabolismo , Estimulação Luminosa , RNA Mensageiro/metabolismo , Retina/metabolismo , Esclera/metabolismo , Privação Sensorial , Transdução de Sinais/genética
14.
Diabetes ; 65(5): 1164-78, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26936961

RESUMO

Increased visceral fat, rather than subcutaneous fat, during the onset of obesity is associated with a higher risk of developing metabolic diseases. The inherent adipogenic properties of human adipose-derived stem cells (ASCs) from visceral depots are compromised compared with those of ASCs from subcutaneous depots, but little is known about the underlying mechanisms. Using ontological analysis of global gene expression studies, we demonstrate that many genes involved in retinoic acid (RA) synthesis or regulated by RA are differentially expressed in human tissues and ASCs from subcutaneous and visceral fat. The endogenous level of RA is higher in visceral ASCs; this is associated with upregulation of the RA synthesis gene through the visceral-specific developmental factor WT1. Excessive RA-mediated activity impedes the adipogenic capability of ASCs at early but not late stages of adipogenesis, which can be reversed by antagonism of RA receptors or knockdown of WT1. Our results reveal the developmental origin of adipocytic properties and the pathophysiological contributions of visceral fat depots.


Assuntos
Adipogenia , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Gordura Intra-Abdominal/metabolismo , Receptores do Ácido Retinoico/agonistas , Transdução de Sinais , Tretinoína/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Cirurgia Bariátrica , Benzoatos/farmacologia , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ontologia Genética , Humanos , Gordura Intra-Abdominal/citologia , Gordura Intra-Abdominal/efeitos dos fármacos , Gordura Intra-Abdominal/patologia , Pessoa de Meia-Idade , Naftalenos/farmacologia , Obesidade Mórbida/metabolismo , Obesidade Mórbida/patologia , Obesidade Mórbida/cirurgia , Interferência de RNA , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/metabolismo , Elementos de Resposta/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Gordura Subcutânea Abdominal/citologia , Gordura Subcutânea Abdominal/efeitos dos fármacos , Gordura Subcutânea Abdominal/metabolismo , Gordura Subcutânea Abdominal/patologia , Regulação para Cima/efeitos dos fármacos , Proteínas WT1/antagonistas & inibidores , Proteínas WT1/genética , Proteínas WT1/metabolismo
15.
PLoS One ; 11(1): e0143235, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26799392

RESUMO

The presence of multiple variants for many mRNAs is a major contributor to protein diversity. The processing of these variants is tightly controlled in a cell-type specific manner and has a significant impact on gene expression control. Here we investigate the differential translation rates of individual mRNA variants in embryonic stem cells (ESCs) and in ESC derived Neural Precursor Cells (NPCs) using polysome profiling coupled to RNA sequencing. We show that there are a significant number of detectable mRNA variants in ESCs and NPCs and that many of them show variant specific translation rates. This is correlated with differences in the UTRs of the variants with the 5'UTR playing a predominant role. We suggest that mRNA variants that contain alternate UTRs are under different post-transcriptional controls. This is likely due to the presence or absence of miRNA and protein binding sites that regulate translation rate. This highlights the importance of addressing translation rate when using mRNA levels as a read out of protein abundance. Additional analysis shows that many annotated non-coding mRNAs are present on the polysome fractions in ESCs and NPCs. We believe that the use of polysome fractionation coupled to RNA sequencing is a useful method for analysis of the translation state of many different RNAs in the cell.


Assuntos
Células-Tronco Embrionárias/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento Insulin-Like II/genética , Camundongos , Células-Tronco Neurais/fisiologia , Polirribossomos/genética , Polirribossomos/metabolismo , Splicing de RNA , Ribonucleoproteínas/genética , Análise de Sequência de RNA
16.
BMC Genomics ; 16: 950, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26574018

RESUMO

BACKGROUND: The role of microRNAs in gene regulation has been well established. The extent of miRNA regulation also increases with increasing genome complexity. Though the number of genes appear to be equal between human and zebrafish, substantially less microRNAs have been discovered in zebrafish compared to human (miRBase Release 19). It appears that most of the miRNAs in zebrafish are yet to be discovered. RESULTS: We sequenced small RNAs from brain, gut, liver, ovary, testis, eye, heart and embryo of zebrafish. In brain, gut and liver sequencing was done sex specifically. Majority of the sequenced reads (16-62 %) mapped to known miRNAs, with the exception of ovary (5.7 %) and testis (7.8 %). Using the miRNA discovery tool (miRDeep2), we discovered novel miRNAs from the unannotated reads that ranged from 7.6 to 23.0 %, with exceptions of ovary (51.4 %) and testis (55.2 %). The prediction tool identified a total of 459 novel pre-miRNAs. We compared expression of miRNAs between different tissues and between males and females to identify tissue associated and sex associated miRNAs respectively. These miRNAs could serve as putative biomarkers for these tissues. The brain and liver had highest number of tissue associated (22) and sex associated (34) miRNAs, respectively. CONCLUSIONS: This study comprehensively identifies tissue and sex associated miRNAs in zebrafish. Further, we have discovered 459 novel pre-miRNAs (~30 % seed homology to human miRNA) as a genomic resource which can facilitate further investigations to understand miRNA-mRNA gene regulatory networks in zebrafish which will have implications in understanding the function of human homologs.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/genética , Análise de Sequência de RNA , Caracteres Sexuais , Peixe-Zebra/genética , Animais , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Especificidade de Órgãos , Peixe-Zebra/fisiologia
17.
Cytotherapy ; 17(9): 1169-77, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26276001

RESUMO

Mesenchymal stem/stromal cells (MSCs) have been extensively investigated for their regenerative, immune-modulatory, and wound healing properties. While the laboratory studies have suggested that MSC's have a unique potential for modulating the etiopathology of multiple diseases, the results from clinical trials have not been encouraging or reproducible. One of the explanations for such variability is explained by the "art" of isolating and propagating MSCs. Therefore, establishing more than minimal criteria to define MSC would help understand best protocols to isolate, propagate and deliver MSCs. Developing a calibration standard, a database and a set of functional tests would be a better quality metric for MSCs. In this review, we discuss the importance of selecting a standard, issues associated with coming up with such a standard and how these issues can be mitigated.


Assuntos
Separação Celular/normas , Células-Tronco Mesenquimais/citologia , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Padrões de Referência
18.
Sci Rep ; 5: 9737, 2015 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-26024509

RESUMO

Oxidative stress (OS) is caused by an imbalance between pro- and anti-oxidant reactions leading to accumulation of reactive oxygen species within cells. We here investigate the effect of OS on the transcriptome of human fibroblasts. OS causes a rapid and transient global induction of transcription characterized by pausing of RNA polymerase II (PolII) in both directions, at specific promoters, within 30 minutes of the OS response. In contrast to protein-coding genes, which are commonly down-regulated, this novel divergent, PolII pausing-phenomenon leads to the generation of thousands of long noncoding RNAs (lncRNAs) with promoter-associated antisense lncRNAs transcripts (si-paancRNAs) representing the major group of stress-induced transcripts. OS causes transient dynamics of si-lncRNAs in nucleus and cytosol, leading to their accumulation at polysomes, in contrast to mRNAs, which get depleted from polysomes. We propose that si-lncRNAs represent a novel component of the transcriptional stress that is known to determine the outcome of immediate-early and later cellular stress responses and we provide insights on the fate of those novel mature lncRNA transcripts by showing that their association with polysomal complexes is significantly increased in OS.


Assuntos
Genoma Humano , Estresse Oxidativo , RNA Mensageiro/genética , RNA não Traduzido/genética , Transcriptoma , Sítios de Ligação , Linhagem Celular , Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Regiões Promotoras Genéticas , Biossíntese de Proteínas , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , RNA Antissenso/genética , RNA Longo não Codificante/classificação , RNA Longo não Codificante/genética , Fatores de Transcrição/metabolismo
19.
Proc Natl Acad Sci U S A ; 111(52): E5688-96, 2014 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-25512551

RESUMO

Interactions between natural killer (NK) cells and dendritic cells (DCs) aid DC maturation and promote T-cell responses. Here, we have analyzed the response of human NK cells to tumor cells, and we identify a pathway by which NK-DC interactions occur. Gene expression profiling of tumor-responsive NK cells identified the very rapid induction of TNF superfamily member 14 [TNFSF14; also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT)], a cytokine implicated in the enhancement of antitumor responses. TNFSF14 protein expression was induced by three primary mechanisms of NK cell activation, namely, via the engagement of CD16, by the synergistic activity of multiple target cell-sensing NK-cell activation receptors, and by the cytokines IL-2 and IL-15. For antitumor responses, TNFSF14 was preferentially produced by the licensed NK-cell population, defined by the expression of inhibitory receptors specific for self-MHC class I molecules. In contrast, IL-2 and IL-15 treatment induced TNFSF14 production by both licensed and unlicensed NK cells, reflecting the ability of proinflammatory conditions to override the licensing mechanism. Importantly, both tumor- and cytokine-activated NK cells induced DC maturation in a TNFSF14-dependent manner. The coupling of TNFSF14 production to tumor-sensing NK-cell activation receptors links the tumor immune surveillance function of NK cells to DC maturation and adaptive immunity. Furthermore, regulation by NK cell licensing helps to safeguard against TNFSF14 production in response to healthy tissues.


Assuntos
Comunicação Celular/imunologia , Células Dendríticas/imunologia , Vigilância Imunológica , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Técnicas de Cocultura , Células Dendríticas/citologia , Feminino , Proteínas Ligadas por GPI/imunologia , Humanos , Interleucina-15/imunologia , Interleucina-2/imunologia , Células K562 , Células Matadoras Naturais/citologia , Masculino , Neoplasias/imunologia , Receptores de IgG/imunologia
20.
PLoS One ; 9(1): e85419, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24416407

RESUMO

Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones.


Assuntos
Reprogramação Celular , Fibroblastos/metabolismo , Receptores de Hialuronatos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Transcriptoma , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Células Clonais , Células Alimentadoras/citologia , Fibroblastos/citologia , Citometria de Fluxo , Humanos , Receptores de Hialuronatos/genética , Imuno-Histoquímica , Imunofenotipagem , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos
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