RESUMO
Insertions are one of the major types of structural variations and are defined as the addition of 50 nucleotides or more into a DNA sequence. Several methods exist to detect insertions from next-generation sequencing short read data, but they generally have low sensitivity. Our contribution is two-fold. First, we introduce INSurVeyor, a fast, sensitive and precise method that detects insertions from next-generation sequencing paired-end data. Using publicly available benchmark datasets (both human and non-human), we show that INSurVeyor is not only more sensitive than any individual caller we tested, but also more sensitive than all of them combined. Furthermore, for most types of insertions, INSurVeyor is almost as sensitive as long reads callers. Second, we provide state-of-the-art catalogues of insertions for 1047 Arabidopsis Thaliana genomes from the 1001 Genomes Project and 3202 human genomes from the 1000 Genomes Project, both generated with INSurVeyor. We show that they are more complete and precise than existing resources, and important insertions are missed by existing methods.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodosRESUMO
Pressure ulcer (PU) is a chronic wound often seen in patients with spinal cord injury and other bed-bound individuals, particularly in the elderly population. Despite its association with high mortality, the pathophysiology of PU remains poorly understood. In this study, we compared single-cell transcriptomic profiles of human epidermal cells from PU wound edges with those from uninjured skin and acute wounds in healthy donors. We identified significant shifts in the cell composition and gene expression patterns in PU. In particular, we found that major histocompatibility complex class IIâexpressing keratinocytes were enriched in patients with worse healing outcomes. Furthermore, we showed that the IFN-γ in PU-derived wound fluid could induce major histocompatibility complex II expression in keratinocytes and that these wound fluidâtreated keratinocytes inhibited autologous T-cell activation. In line with this observation, we found that T cells from PUs enriched with major histocompatibility complex II+ keratinocytes produced fewer inflammatory cytokines. Overall, our study provides a high-resolution molecular map of human PU compared with that of acute wounds and intact skin, providing insights into PU pathology and the future development of tailored wound therapy.
Assuntos
Úlcera por Pressão , Idoso , Humanos , Queratinócitos/metabolismo , Complexo Principal de Histocompatibilidade , Análise de Célula Única , Cicatrização/genéticaRESUMO
Alzheimer's disease (AD) is an aging-related progressive neurodegenerative disorder. Previous studies suggested that various soluble Aß species are neurotoxic and able to activate apoptosis and autophagy, the type I and type II programmed cell death, respectively. However, the sequential and functional relationships between these two cellular events remain elusive. Here we report that low molecular weight Aß triggered cleavage of caspase 3 and poly (ADP-ribose) polymerase to cause neuronal apoptosis in rat cortical neurons. On the other hand, Aß activated autophagy by inducing autophagic vesicle formation and autophagy related gene 12 (ATG12), and up-regulated the lysoso-mal machinery for the degradation of autophagosomes. Moreover, we demonstrated that activation of autophagy by Aß preceded that of apoptosis, with death associated protein kinase phosphorylation as the potential molecular link. More importantly, under Aß toxicity, neurons exhibiting high level of autophagosome formation were absent of apoptotic features, and inhibition of autophagy by 3-methylade-nine advanced neuronal apoptosis, suggesting that autophagy can protect neurons from Aß-induced apoptosis.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apoptose , Autofagia , Neurônios/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Doença de Alzheimer , Peptídeos beta-Amiloides/química , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Proteínas Quinases Associadas com Morte Celular , Neurônios/citologia , Neurônios/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinson's disease research.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Tretinoína/farmacologia , 1-Metil-4-fenilpiridínio/toxicidade , Biomarcadores/análise , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Modelos Neurológicos , Neuroblastoma , Neurônios/efeitos dos fármacos , Oxidopamina/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) system plays a causal role in the development and course of depression. Clinically effective antidepressant drugs normalize the disturbed activity of the HPA axis by inhibition of corticotrophin releasing factor gene promoter activity. Furocoumarins from Psoralea corylifolia have been demonstrated to possess potent antidepressant properties. In order to ascertain whether these coumarin components directly regulate corticotrophin releasing factor (CRF) gene transcription, we studied their effect on CRF promoter activity using the luciferase reporter assay in Neuro-2A cells. CRF promoter was cloned into firefly luciferase reporter vector and co-transfected into Neuro-2A cells with Renilla luciferase plasmid as internal control. CRF promoter transcription activity was induced by forskolin. We found that one of the components of P. corylifolia, psoralidin, strongly inhibited forskolin-induced CRF promoter activity. We further confirmed that psoralidin suppressed CRF gene transcription by quantitative reverse transcription polymerase chain reaction. Hence, down-regulation of CRF gene transcription by psoralidin may be involved in the molecular mechanism underlying its potent antidepressant effect.
Assuntos
Hormônio Liberador da Corticotropina/genética , Furocumarinas/farmacologia , Psoralea/química , Animais , Antidepressivos de Segunda Geração/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colforsina/antagonistas & inibidores , Colforsina/farmacologia , Hormônio Liberador da Corticotropina/biossíntese , Fluoxetina/farmacologia , Furocumarinas/isolamento & purificação , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/química , Sais de Tetrazólio , TiazóisRESUMO
Mitochondrial fragmentation due to fission/fusion imbalance has often been linked to mitochondrial dysfunction and apoptosis in neurodegeneration. Conventionally, it is believed that once mitochondrial morphology shifts away from its physiological tubular form, mitochondria become defective and downstream apoptotic signaling pathways are triggered. However, our study shows that beta-amyloid (Aß) induces morphological changes in mitochondria where they become granular-shaped and are distinct from fragmented mitochondria in terms of both morphology and functions. Accumulation of mitochondrial reactive oxygen species triggers granular mitochondria formation, while mitoTEMPO (a mitochondria-targeted superoxide scavenger) restores tubular mitochondrial morphology within Aß-treated neurons. Interestingly, modulations of mitochondria fission and fusion by genetic and pharmacological tools attenuated not only the induction of granular mitochondria, but also mitochondrial superoxide levels in Aß-treated neurons. Our study shows a reciprocal relationship between mitochondrial dynamics and reactive oxygen species and provides a new potential therapeutic target at early stages of neurodegenerative disease pathogenesis.
Assuntos
Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Caspase 3/metabolismo , Células Cultivadas , Feminino , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Peróxido de Hidrogênio/toxicidade , Microscopia Confocal , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Rotenona/toxicidadeRESUMO
Although Wnt7a has been implicated in axon guidance and synapse formation, investigations of its role in the early steps of neurogenesis have just begun. We show here that Wnt7a is essential for neural stem cell self-renewal and neural progenitor cell cycle progression in adult mouse brains. Loss of Wnt7a expression dramatically reduced the neural stem cell population and increased the rate of cell cycle exit in neural progenitors in the hippocampal dentate gyrus of adult mice. Furthermore, Wnt7a is important for neuronal differentiation and maturation. Loss of Wnt7a expression led to a substantial decrease in the number of newborn neurons in the hippocampal dentate gyrus. Wnt7a(-/-) dentate granule neurons exhibited dramatically impaired dendritic development. Moreover, Wnt7a activated ß-catenin and its downstream target genes to regulate neural stem cell proliferation and differentiation. Wnt7a stimulated neural stem cell proliferation by activating the ß-catenin-cyclin D1 pathway and promoted neuronal differentiation and maturation by inducing the ß-catenin-neurogenin 2 pathway. Thus, Wnt7a exercised critical control over multiple steps of neurogenesis by regulating genes involved in both cell cycle control and neuronal differentiation.
Assuntos
Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Proteínas Wnt/metabolismo , Fatores Etários , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Giro Denteado/citologia , Giro Denteado/fisiologia , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Mutantes , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Prosencéfalo/citologia , Prosencéfalo/crescimento & desenvolvimento , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: Median survival for patients with glioblastoma multiforme, the most aggressive glioma, is only 12-15 months, despite multimodal treatment that includes surgery, chemotherapy, and radiotherapy. Thus, identification of genes that control the progression of glioblastoma multiforme is crucial for devising new therapies. We investigated the involvement of cell cycle-related kinase (CCRK), a novel protein kinase that is homologous to cyclin-dependent kinase 7, in glioblastoma multiforme carcinogenesis. METHODS: We analyzed the expression levels of CCRK in 26 glioma patient samples (19 high-grade and seven low-grade) and normal brain by semiquantitative reverse transcription-polymerase chain reaction assays. CCRK expression was knocked down in human glioma U-373 MG and U-87 MG cells with small-interfering RNAs and short hairpin RNAs (siCCRK and shCCRK, respectively), and cell proliferation, cell cycle distribution, and cyclin-dependent kinase 2 (CDK2) phosphorylation were examined. A subcutaneous nude mouse xenograft model (n = 4 mice per group) was used to study the effect of CCRK knockdown and overexpression on tumorigenicity and growth of glioblastoma multiforme cells in vivo. All statistical tests were two-sided. RESULTS: CCRK mRNA was elevated at least 1.5-fold and as much as 3.7-fold in 14 (74%) of 19 high-grade glioblastoma multiforme patient samples and in four (80%) of five glioma cell lines examined compared with normal brain tissue. Suppression of CCRK by siCCRK inhibited the proliferation of U-373 MG and U-87 MG glioblastoma cells in a time- and dose-dependent manner. The growth-inhibiting effect of siCCRK was mediated via G1- to S-phase cell cycle arrest and reduced CDK2 phosphorylation. CCRK knockdown statistically significantly suppressed glioma cell growth in vivo as indicated by the mean tumor volumes at week 6 after tumor cell injection (U-373-control = 1352 mm3, U-373-shCCRK = 294 mm3, difference = 1058 mm3, 95% confidence interval [CI] = 677 to 1439 mm3, P<.001; U-87-control = 1910 mm3, U-87-shCCRK = 552 mm3, difference = 1358 mm3, 95% CI = 977 to 1739 mm3, P<.001). CONCLUSIONS: CCRK is a candidate oncogene in glioblastoma multiforme tumorigenesis.