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This study investigates the potential role of Glycosyltransferases (GTs) in the glycosylation process and their association with malignant tumors. Specifically, the study focuses on PARP14, a member of GTs, and its potential as a target for tumors in the diagnosis and treatment of cervical cancer. To gather data, the study used somatic mutation data, gene expression data and clinical information from TCGA-CESE dataset as well as tissue samples from cervical cancer patients. Further verification was conducted through RT-qPCR and immunohistochemistry staining on cervical cancer tissues to confirm the expression of PARP14. The study utilized Kaplan-Meier for survival analysis of cervical cancer patient and found significant mutational abnormalities in GTs. The high frequency mutated gene was identified as PARP14. RT-qPCR revealed significantly higher mRNA expression of PARP14 compared to precancerous tissue. Using IHC combined with Kaplan-Meier,patients in the PARP14 high expression group had a better prognosis than the low expression group. The study identified PARP14 as a frequently mutated gene in cervical cancer and proposed its potential role in diagnosis and treatment.
Assuntos
Poli(ADP-Ribose) Polimerases , Neoplasias do Colo do Útero , Feminino , Humanos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Glicosiltransferases/genética , Prognóstico , MutaçãoRESUMO
BACKGROUND: Oligodendrocytes, responsible for axon ensheathment, are critical for central nervous system (CNS) development, function, and diseases. OLIG2 is an important transcription factor (TF) that acts during oligodendrocyte development and performs distinct functions at different stages. Previous studies have shown that lncRNAs (long non-coding RNAs; > 200 bp) have important functions during oligodendrocyte development, but their roles have not been systematically characterized and their regulation is not yet clear. RESULTS: We performed an integrated study of genome-wide OLIG2 binding and the epigenetic modification status of both coding and non-coding genes during three stages of oligodendrocyte differentiation in vivo: neural stem cells (NSCs), oligodendrocyte progenitor cells (OPCs), and newly formed oligodendrocytes (NFOs). We found that 613 lncRNAs have OLIG2 binding sites and are expressed in at least one cell type, which can potentially be activated or repressed by OLIG2. Forty-eight of them have increased expression in oligodendrocyte lineage cells. Predicting lncRNA functions by using a "guilt-by-association" approach revealed that the functions of these 48 lncRNAs were enriched in "oligodendrocyte development and differentiation." Additionally, bivalent genes are known to play essential roles during embryonic stem cell differentiation. We identified bivalent genes in NSCs, OPCs, and NFOs and found that some bivalent genes bound by OLIG2 are dynamically regulated during oligodendrocyte development. Importantly, we unveiled a previously unknown mechanism that, in addition to transcriptional regulation via DNA binding, OLIG2 could self-regulate through the 3' UTR of its own mRNA. CONCLUSIONS: Our studies have revealed the missing links in the mechanisms regulating oligodendrocyte development at the transcriptional level and after transcription. The results of our research have improved the understanding of fundamental cell fate decisions during oligodendrocyte lineage formation, which can enable insights into demyelination diseases and regenerative medicine.
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Oligodendroglia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Proteínas do Tecido Nervoso/genética , RNA Longo não Codificante/genéticaRESUMO
Micro RNAs (miRNAs) are small non-coding RNAs which are 19-24 nucleotides in length. MiRNAs play a vital role in the whole process of tumour development, but how they influence the tumourigenecity of epithelial ovarian cancer (EOC)cells is rarely researched. In our study, it was verified that miR-628-5p decreased the stem like cell percentage of EOC cells by inducing their apoptosis. The animal experiments showed that miR-628-5p decreased the tumourigenecity of EOC cells. Besides, we found miR-628-5p targeted at and down-regulated the expression of fibroblast growth factor receptor 2 (FGFR2). FGFR2 expressed higher in ovarian cancer tissues and was correlated with worse prognosis. Our findings indicated that miR-628-5pplays an important role in ovarian cancer stem cell driven tumorigenesis.
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Carcinogênese/metabolismo , MicroRNAs/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Apoptose , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , HumanosRESUMO
Long non-coding RNAs (lncRNAs) (> 200 bp) play crucial roles in transcriptional regulation during numerous biological processes. However, it is challenging to comprehensively identify lncRNAs, because they are often expressed at low levels and with more cell-type specificity than are protein-coding genes. In the present study, we performed ab initio transcriptome reconstruction using eight purified cell populations from mouse cortex and detected more than 5000 lncRNAs. Predicting the functions of lncRNAs using cell-type specific data revealed their potential functional roles in Central Nervous System (CNS) development. We performed motif searches in ENCODE DNase I digital footprint data and Mouse ENCODE promoters to infer transcription factor (TF) occupancy. By integrating TF binding and cell-type specific transcriptomic data, we constructed a novel framework that is useful for systematically identifying lncRNAs that are potentially essential for brain cell fate determination. Based on this integrative analysis, we identified lncRNAs that are regulated during Oligodendrocyte Precursor Cell (OPC) differentiation from Neural Stem Cells (NSCs) and that are likely to be involved in oligodendrogenesis. The top candidate, lnc-OPC, shows highly specific expression in OPCs and remarkable sequence conservation among placental mammals. Interestingly, lnc-OPC is significantly up-regulated in glial progenitors from experimental autoimmune encephalomyelitis (EAE) mouse models compared to wild-type mice. OLIG2-binding sites in the upstream regulatory region of lnc-OPC were identified by ChIP (chromatin immunoprecipitation)-Sequencing and validated by luciferase assays. Loss-of-function experiments confirmed that lnc-OPC plays a functional role in OPC genesis. Overall, our results substantiated the role of lncRNA in OPC fate determination and provided an unprecedented data source for future functional investigations in CNS cell types. We present our datasets and analysis results via the interactive genome browser at our laboratory website that is freely accessible to the research community. This is the first lncRNA expression database of collective populations of glia, vascular cells, and neurons. We anticipate that these studies will advance the knowledge of this major class of non-coding genes and their potential roles in neurological development and diseases.
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Diferenciação Celular/genética , Linhagem da Célula/genética , Córtex Cerebral/crescimento & desenvolvimento , RNA Longo não Codificante/genética , Transcriptoma/genética , Animais , Córtex Cerebral/metabolismo , Sequência Conservada/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma , Camundongos , Neurônios/metabolismo , Oligodendroglia/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/classificaçãoRESUMO
The lineage transition between epithelium and mesenchyme is a process known as epithelial-mesenchymal transition (EMT), by which polarized epithelial cells lose their adhesion property and obtain mesenchymal cell phenotypes. EMT is a biological process that is often involved in embryogenesis and diseases, such as cancer invasion and metastasis. The EMT and the reverse process, mesenchymal-epithelial transition (MET), also play important roles in stem cell differentiation and de-differentiation (or reprogramming). In this review, we will discuss current research progress of EMT in embryonic development, cellular differentiation and reprogramming, and cancer progression, all of which are representative models for researches of stem cell biology in normal and in diseases. Understanding of EMT and MET may help to identify specific markers to distinguish normal stem cells from cancer stem cells in future.
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Diferenciação Celular , Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal , Células-Tronco/citologia , Animais , Reprogramação Celular , HumanosRESUMO
The quality of an interferogram, which is limited by various phase noise, will greatly affect the further processes of InSAR, such as phase unwrapping. Interferometric SAR (InSAR) geophysical measurements', such as height or displacement, phase filtering is therefore an essential step. In this work, an improved Goldstein interferogram filter is proposed to suppress the phase noise while preserving the fringe edges. First, the proposed adaptive filter step, performed before frequency estimation, is employed to improve the estimation accuracy. Subsequently, to preserve the fringe characteristics, the estimated fringe frequency in each fixed filtering patch is removed from the original noisy phase. Then, the residual phase is smoothed based on the modified Goldstein filter with its parameter alpha dependent on both the coherence map and the residual phase frequency. Finally, the filtered residual phase and the removed fringe frequency are combined to generate the filtered interferogram, with the loss of signal minimized while reducing the noise level. The effectiveness of the proposed method is verified by experimental results based on both simulated and real data.
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Reinforced biofilm structures and dysfunctional neutrophils induced by excessive oxidative stress contribute to the refractoriness of diabetes-related biofilm infections (DRBIs). Herein, in contrast to traditional antibacterial therapies, an immune switchpoint-driven neutrophil immune function conversion strategy based on a deoxyribonuclease I loaded vanadium carbide MXene (DNase-I@V2 C) nanoregulator is proposed to treat DRBIs via biofilm lysis and redirecting neutrophil functions from NETosis to phagocytosis in diabetes. Owing to its intrinsic superoxide dismutase/catalase-like activities, DNase-I@V2 C effectively scavenges reactive oxygen species (ROS) in a high oxidative stress microenvironment to maintain the biological activity of DNase-I. By increasing the depth of biofilm penetration of DNase-I, DNase-I@V2 C thoroughly degrades extracellular DNA and neutrophil extracellular traps (NETs) in extracellular polymeric substances, thus breaking the physical barrier of biofilms. More importantly, as an immune switchpoint regulator, DNase-I@V2 C can skew neutrophil functions from NETosis toward phagocytosis by intercepting ROS-NE/MPO-PAD4 and activating ROS-PI3K-AKT-mTOR pathways in diabetic microenvironment, thereby eliminating biofilm infections. Biofilm lysis and synergistic neutrophil function conversion exert favorable therapeutic effects on biofilm infections in vitro and in vivo. This study serves as a proof-of-principle demonstration of effectively achieving DRBIs with high therapeutic efficacy by regulating immune switchpoint to reverse neutrophil functions.
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Diabetes Mellitus , Neutrófilos , Humanos , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Biofilmes , Diabetes Mellitus/metabolismo , Desoxirribonucleases/metabolismoRESUMO
Chronic, often intractable, pain is caused by neuropathic conditions such as traumatic peripheral nerve injury (PNI) and spinal cord injury (SCI). These conditions are associated with alterations in gene and protein expression correlated with functional changes in somatosensory neurons having cell bodies in dorsal root ganglia (DRGs). Most studies of DRG transcriptional alterations have utilized PNI models where axotomy-induced changes important for neural regeneration may overshadow changes that drive neuropathic pain. Both PNI and SCI produce DRG neuron hyperexcitability linked to pain, but contusive SCI produces little peripheral axotomy or peripheral nerve inflammation. Thus, comparison of transcriptional signatures of DRGs across PNI and SCI models may highlight pain-associated transcriptional alterations in sensory ganglia that do not depend on peripheral axotomy or associated effects such as peripheral Wallerian degeneration. Data from our rat thoracic SCI experiments were combined with meta-analysis of published whole-DRG RNA-seq datasets from prominent rat PNI models. Striking differences were found between transcriptional responses to PNI and SCI, especially in regeneration-associated genes (RAGs) and long noncoding RNAs (lncRNAs). Many transcriptomic changes after SCI also were found after corresponding sham surgery, indicating they were caused by injury to surrounding tissue, including bone and muscle, rather than to the spinal cord itself. Another unexpected finding was of few transcriptomic similarities between rat neuropathic pain models and the only reported transcriptional analysis of human DRGs linked to neuropathic pain. These findings show that DRGs exhibit complex transcriptional responses to central and peripheral neural injury and associated tissue damage. Although only a few genes in DRG cells exhibited similar changes in expression across all the painful conditions examined here, these genes may represent a core set whose transcription in various DRG cell types is sensitive to significant bodily injury, and which may play a fundamental role in promoting neuropathic pain.
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Neuralgia , Traumatismos da Medula Espinal , Ratos , Humanos , Animais , Gânglios Espinais/metabolismo , Neuralgia/genética , Neuralgia/metabolismo , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Neurônios/metabolismoRESUMO
INTRODUCTION: Adoptive cellular therapy with tumor-infiltrating lymphocytes (TIL) has demonstrated promising clinical benefits in several solid tumors, but the efficacy of this therapy might be compromised by the "prone-to-exhaustion" phenotype of TIL and poor persistence in vivo. This calls for a robust expansion process to produce a large number of cells for clinical usage while at the same time maintaining favorable anti-tumor function and memory phenotype. Previous studies showed that the PI3K-AKT signaling pathway plays a key role in the regulation of T cell activation, differentiation and memory formation. METHOD: We modulated the PI3K-AKT pathway in TIL isolated from cervical and ovarian cancer by application of AKT or PI3K inhibitors or CRISPR knockout of AKT1 and/or AKT2, and characterized their effects on TIL phenotype and effector function. Mechanistic study was further performed with RNA-seq analysis of AKT1/2 KO TIL in comparison to control TIL. RESULT: The inhibition of either PI3K or AKT led to an increase in the population of effector CD8+ T cells with upregulation of activation markers, elevated CD39- CD69- memory T cells, and significantly enhanced cytotoxicity when cocultured with tumor cell lines and patient-derived tumor samples. Moreover, dual knockout of AKT1 and AKT2 largely phenocopies the functional impact of AKT or PI3K inhibition on TIL. This result was further validated by RNA-seq analysis indicating that AKT1/2 ablation primarily regulates T cell differentiation and function-related programs. CONCLUSION: Modulation of PI3K-AKT signaling represents a promising strategy to enhance TIL stemness and cytotoxicity and improve the clinical outcome of current TIL-based therapy to treat solid tumors.
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Linfócitos do Interstício Tumoral , Neoplasias Ovarianas , Humanos , Feminino , Linfócitos do Interstício Tumoral/metabolismo , Imunoterapia Adotiva , Linfócitos T CD8-Positivos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Neoplasias Ovarianas/patologiaRESUMO
RNA interference (RNAi) is an effective approach for gene function analysis, which is well developed in mammal cell lines. However, RNAi has rarely been reported in marine bivalve species. To provide support on functional analysis of bivalve genes, for the first time to our knowledge, we conducted RNAi assay on primary cell of clam Meretrix meretrix in this study. Firstly we explored the method of culturing primary cells of M. meretrix to ensure the cells to live at high activity for at least 2 weeks. Ferritin gene was chosen as the target gene and RNAi assay was conducted through soaking the primary cells of M. meretrix digestive gland in medium containing dsRNA of ferritin gene. Realtime PCR, western blot and immunocytochemistry analysis were used to analyze the inhibition of gene expression after RNAi. Results showed the ferritin mRNA was significantly down-regulated by 66.11% after RNAi. Western blot result showed that the expression level of ferritin protein was also depressed post RNAi. The method developed in this study proved to be reliable and effective for RNAi assay on marine bivalve cells. It would be an efficient tool for gene function analysis in marine bivalves and more studies based on primary cells of marine bivalves can be expected.
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Bivalves/genética , Bivalves/metabolismo , Interferência de RNA , Animais , Anticorpos/imunologia , Ferritinas/genética , Ferritinas/imunologia , Ferritinas/metabolismo , Expressão Gênica , Cultura Primária de Células , RNA de Cadeia Dupla/síntese química , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The sulfotransferase (SULT) is an important factor in phase II metabolism. Three single nucleotide polymorphisms (SNPs; MmeSUl-696, MmeSUl-714 and MmeSUl-806) were discovered in a SULT-like gene of Meretrix meretrix. A modified allele-specific PCR detection assay was developed to perform polymorphism analysis of the three SNPs in two families and two populations of M. meretrix. Results showed that the genotype of the three SNPs is accordance to the expected Mendelian segregation ratios and conforms to Hardy-Weinberg Equilibrium (HWE) in families and populations, respectively. Moreover, sampled clams (n = 151) from the two populations were tested for the associations between SNPs and growth traits. The single SNP association analysis indicated MmeSUl-806 significantly (P < 0.05) correlated with growth traits of M. meretrix. A further haplotype-trait association analysis confirmed that the three SNPs were in linkage disequilibrium and showed that the haplotype TTG and TCG associated with low growth in the two populations, respectively, which supported that MmeSUl-806 was a growth related marker. This study suggested SULT gene might be a candidate gene related to growth in bivalves and MmeSUl-806 could be a gene marker in linkage map construction and breeding programs of M. meretrix.
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Bivalves/genética , Variação Genética , Modelos Moleculares , Polimorfismo de Nucleotídeo Único/genética , Sulfotransferases/genética , Animais , Bivalves/crescimento & desenvolvimento , Primers do DNA/genética , Fluorescência , Genética Populacional , Genótipo , Haplótipos/genética , Desequilíbrio de Ligação , Reação em Cadeia da Polimerase/métodos , Sulfotransferases/químicaRESUMO
Macrophages are essential in innate immunity and are involved in a variety of biological functions. Due to high plasticity, macrophages are polarized in different phenotypes depending on different microenvironments to perform specific functions. Although many studies have focused on macrophage polarization, few have explored the polarization characteristics of macrophages at the subcellular level, even at nanoscale resolution. Here, we utilize AFM-based infrared spectroscopy (AFM-IR) to investigate the influence of an inducer on the expressed proteins of M1/M2 macrophages (induced by LPS and IL-13, respectively). The results from AFM-IR combined with principal component analysis revealed that the characteristic proteins within M1 contain about 35% antiparallel ß-sheets (due to the high expression of TNF-α), while the proteins within M2 are made up of approximately 38.8% α-helices. The corresponding nanoscale chemical mapping demonstrates a remarkably heterogeneous distribution of expressed proteins inside single macrophages. Beside the biochemical properties, the biomechanical properties of macrophages were found to be softened in response to the polarization process.
Assuntos
Interleucina-13/genética , Lipopolissacarídeos/genética , Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Fenômenos Biomecânicos , Microambiente Celular , Regulação da Expressão Gênica , Interleucina-13/metabolismo , Lipopolissacarídeos/metabolismo , Ativação de Macrófagos , Macrófagos/ultraestrutura , Camundongos , Microscopia de Força Atômica , Fenótipo , Análise de Componente Principal , Conformação Proteica , Células RAW 264.7 , Espectrofotometria Infravermelho , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BMAL1 is a core component of the mammalian circadian clockwork. Removal of BMAL1 from the retina significantly affects visual information processing in both rod and cone pathways. To identify potential pathways and/or molecules through which BMAL1 alters signal transmission at the cone pedicle, we performed an RNA-seq differential expression analysis between cone-specific Bmal1 knockout cones (cone-Bmal1-/- ) and wild-type (WT) cones. We found 88 genes differentially expressed. Among these, Complexin3 (Cplx3), a SNARE regulator at ribbon synapses, was downregulated fivefold in the mutant cones. The purpose of this work was to determine whether BMAL1 and/or the cone clock controls CPLX3 protein expression at cone pedicles. We found that CPLX3 expression level was decreased twofold in cone-Bmal1-/- cones. Furthermore, CPLX3 expression was downregulated at night compared to the day in WT cones but remained constitutively low in mutant cones both day and night. The transcript and protein expression levels of Cplx4-the other complexin expressed in cones-were similar in WT and mutant cones; in WT cones, CPLX4 protein level did not change with the time of day. In silico analysis revealed four potential BMAL1:CLOCK binding sites upstream from exon one of Cplx3 and none upstream of exon one of Cplx4. Our results suggest that CPLX3 expression is regulated at the transcriptional level by the cone clock. The modulation of CPLX3 may be a mechanism by which the clock controls the cone synaptic transfer function to second-order cells and thereby impacts retinal signal processing during the day/night cycle.
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Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Relógios Circadianos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Proteínas SNARE/fisiologia , Fatores de Transcrição ARNTL/deficiência , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Regulação para Baixo , Feminino , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA-Seq , Pigmentos da Retina/genética , Rodopsinas Sensoriais/genética , Transdução de Sinais/fisiologiaRESUMO
Peritoneal metastasis is a common issue in the progression of high-grade serous ovarian cancers (HGSOCs), yet the underlying mechanism remains unconfirmed. We demonstrated that ZEB2, the transcription factor of epithelial-mesenchymal transition (EMT), was upregulated in ascites cells from HGSOC patients and in CD133+ cancer stem-like cells (CSLCs) from epithelial ovarian cancer (EOC) cell lines. SiRNA-mediated knockdown of ZEB2 in EOC cells decreased the percentage of CSLCs and reduced the colony forming potential, cell invasion capacity and expression of pluripotent genes Oct4 and Nanog. Inhibition of ZEB2 also induced cellular apoptosis and impacted the tumorigenicity of ovarian CSLCs. The mesenchymal markers N-cadherin and vimentin were downregulated, while the epithelial marker E-cadherin was upregulated after ZEB2 knockdown. MiR-200a, a molecule that downregulates ZEB2, had the opposite effect of ZEB2 expression in EOC-CSLCs. A retrospective study of 98 HGSOC patients on the relationship of ascites volume, pelvic and abdominal metastasis, International Federation of Gynecology and Obstetrics (FIGO) stage and the malignant involvement of abdominal organs and lymph nodes was performed. Patients with high expression of ZEB2 in tumour tissues had a higher metastasis rate and a poorer prognosis than those with low expression. The parameters of ZEB2 expression and ascites volume were strongly linked with the prognostic outcome of HGSOC patients and had higher hazard ratios. These findings illustrated that ZEB2 facilitates the invasive metastasis of EOC-CSLCs and can predict peritoneal metastasis and a poor prognosis in HGSOC patients.
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Transformação Celular Neoplásica/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/secundário , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Biomarcadores , Transformação Celular Neoplásica/metabolismo , Cistadenocarcinoma Seroso/mortalidade , Feminino , Imunofluorescência , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunofenotipagem , Estimativa de Kaplan-Meier , MicroRNAs/genética , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/mortalidade , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/mortalidade , Prognóstico , Modelos de Riscos Proporcionais , Interferência de RNA , Curva ROC , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismoRESUMO
Spinal cord injury (SCI) is one of the most devastating neural injuries without effective therapeutic solutions. Astrocytes are the predominant component of the scar. Understanding the complex contributions of reactive astrocytes to SCI pathophysiologies is fundamentally important for developing therapeutic strategies. We have studied the molecular changes in the injury environment and the astrocyte-specific responses by astrocyte purification from injured spinal cords from acute to chronic stages. In addition to protein-coding genes, we have systematically analyzed the expression profiles of long non-coding RNAs (lncRNAs) (>200 bp), which are regulatory RNAs that play important roles in the CNS. We have identified a highly conserved lncRNA, Zeb2os, and demonstrated using functional assays that it plays an important role in reactive astrogliosis through the Zeb2os/Zeb2/Stat3 axis. These studies provide valuable insights into the molecular basis of reactive astrogliosis and fill the knowledge gap regarding the function(s) of lncRNAs in astrogliosis and SCI.
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Astrócitos/metabolismo , Gliose/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Humanos , Camundongos , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Mouse photoreceptors are electrically coupled via gap junctions, but the relative importance of rod/rod, cone/cone, or rod/cone coupling is unknown. Furthermore, while connexin36 (Cx36) is expressed by cones, the identity of the rod connexin has been controversial. We report that FACS-sorted rods and cones both express Cx36 but no other connexins. We created rod- and cone-specific Cx36 knockout mice to dissect the photoreceptor network. In the wild type, Cx36 plaques at rod/cone contacts accounted for more than 95% of photoreceptor labeling and paired recordings showed the transjunctional conductance between rods and cones was ~300 pS. When Cx36 was eliminated on one side of the gap junction, in either conditional knockout, Cx36 labeling and rod/cone coupling were almost abolished. We could not detect direct rod/rod coupling, and cone/cone coupling was minor. Rod/cone coupling is so prevalent that indirect rod/cone/rod coupling via the network may account for previous reports of rod coupling.
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Enolase (EC 4.2.1.11) acts as a multifunctional enzyme in many organisms, being involved in metabolism, transcription regulation and pathogenesis. In the current study, the recombinant α-enolase from Taenia solium (His-Tseno) was prepared and antiserum against His-Tseno was generated in rabbits. Consequently, we analyzed the enzymatic characteristics, plasminogen binding activity, tissue localization and expression patterns of Tseno. The study demonstrated that the enzymatic activity of His-Tseno was enhanced at pH around 7.0-7.5 and affected by addition of metal ions. Kinetic measurements using 2-phospho-d-glycerate (2-PGA) substrates gave a specific activity of 60.72⯱â¯0.84â¯U/mg and 1.1â¯mM of Km2-PGA value. Plasminogen binding assay showed that His-Tseno could bind to human plasminogen and generate plasmin activated by a tissue-type plasminogen activator (t-PA). In addition, the lysine analogue 6-aminocaproic acid (ε-ACA) could inhibit the binding of plasminogen to His-Tseno. Quantitative real-time PCR confirmed that Tseno was expressed 2.38 folds higher in the adult worms (pâ¯<â¯0.05) than in the cysticerci. Further, an immunolocalization assay indicated that native Tseno was mainly distributed in the tegument and eggs of gravid proglottis from adult T. solium. In conclusion, Tseno executes the innate glycolytic function to supply energy for the growth, egg production, and even invasion of T. solium.
Assuntos
Regulação da Expressão Gênica , Proteínas de Helminto/genética , Fosfopiruvato Hidratase/genética , Taenia solium/genética , Sequência de Aminoácidos , Animais , Cysticercus/enzimologia , Cysticercus/genética , Cysticercus/crescimento & desenvolvimento , Perfilação da Expressão Gênica/veterinária , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/metabolismo , Filogenia , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/veterinária , Taenia solium/enzimologia , Taenia solium/crescimento & desenvolvimentoRESUMO
In mammalian cells, gene transcription is regulated in a cell type specific manner by the interactions of transcriptional factors with genomic DNA. Lineage-specific transcription factors are considered to play essential roles in cell specification and differentiation during development. ChIP coupled with high-throughput DNA sequencing (ChIP-seq) is widely used to analyze genome-wide binding sites of transcription factors (or its associated complex) to genomic DNA. However, a large number of cells are required for one standard ChIP reaction, which makes it difficult to study the limited number of isolated primary cells or rare cell populations. In order to understand the regulatory mechanism of oligodendrocyte lineage-specific transcription factor Olig2 in acutely purified mouse OPCs, a detailed method using ChIP-seq to identify the genome-wide binding sites of Olig2 (or Olig2 complex) is shown. First, the protocol explains how to purify the platelet-derived growth factor receptor alpha (PDGFRα) positive OPCs from mouse brains. Next, Olig2 antibody mediated ChIP and library construction are performed. The last part describes the bioinformatic software and procedures used for Olig2 ChIP-seq analysis. In summary, this paper reports a method to analyze the genome-wide bindings of transcriptional factor Olig2 in acutely purified brain OPCs.
Assuntos
Imunoprecipitação da Cromatina/métodos , Genômica/métodos , Fator de Transcrição 2 de Oligodendrócitos/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Sítios de Ligação , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Ligação Proteica , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Systematic study of the regulatory mechanisms of Hematopoietic Stem Cell and Progenitor Cell (HSPC) self-renewal is fundamentally important for understanding hematopoiesis and for manipulating HSPCs for therapeutic purposes. Previously, we have characterized gene expression and identified important transcription factors (TFs) regulating the switch between self-renewal and differentiation in a multipotent Hematopoietic Progenitor Cell (HPC) line, EML (Erythroid, Myeloid, and Lymphoid) cells. Herein, we report binding maps for additional TFs (SOX4 and STAT3) by using chromatin immunoprecipitation (ChIP)-Sequencing, to address the underlying mechanisms regulating self-renewal properties of lineage-CD34+ subpopulation (Lin-CD34+ EML cells). Furthermore, we applied the Assay for Transposase Accessible Chromatin (ATAC)-Sequencing to globally identify the open chromatin regions associated with TF binding in the self-renewing Lin-CD34+ EML cells. Mass spectrometry (MS) was also used to quantify protein relative expression levels. Finally, by integrating the protein-protein interaction database, we built an expanded transcriptional regulatory and interaction network. We found that MAPK (Mitogen-activated protein kinase) pathway and TGF-ß/SMAD signaling pathway components were highly enriched among the binding targets of these TFs in Lin-CD34+ EML cells. The present study integrates regulatory information at multiple levels to paint a more comprehensive picture of the HSPC self-renewal mechanisms.
Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34/metabolismo , Linhagem Celular , Linhagem da Célula , Cromatina/química , Imunoprecipitação da Cromatina , Análise por Conglomerados , Células Eritroides/citologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Hematopoese , Humanos , Linfócitos/citologia , Espectrometria de Massas , Camundongos , Células Mieloides/citologia , Mapeamento de Interação de Proteínas , Fatores de Transcrição SOXC/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Periprosthetic joint infection (PJI) is a devastating condition and Staphylococcus spp. are the predominant pathogens responsible, particularly coagulase-negative staphylococci (CoNS) and Staphylococcus aureus. The aim of the present systematic review was to evaluate the distribution characteristics of specific Staphylococcus spp. in different PJI phases, reveal the effect of pathogens' feature on their distribution and suggest recommendations for antibiotic treatment of Staphylococcal PJI. The present systematic review was performed using PubMed and EMBASE databases with the aim to identify existing literature that presented the spectrum of Staphylococcus spp. that occur in PJI. Once inclusion and exclusion criteria were applied, 20 cohort studies involving 3,344 cases in 3,199 patients were included. The predominant pathogen involved in PJI was indicated to be CoNS (31.2%), followed by S. aureus (28.8%). This trend was more apparent in hip replacement procedures. In addition, almost equal proportions of CoNS and S. aureus (28.6 and 30.0%, respectively) were indicated in the delayed phase. CoNS (36.6%) were the predominant identified organism in the early phase, whereas S. aureus (38.3%) occurred primarily in the late phase. In PJI caused by S. aureus, the number of cases of methicillin-sensitive Staphylococcus aureus (MSSA) was ~2.5-fold greater than that of methicillin-resistant Staphylococcus aureus (MRSA). MRSA occurred predominantly in the early phase, whereas MSSA was largely observed in the delayed and late phases. With regards to antibiotic treatment, the feature of various pathogens and the phases of PJI were the primary considerations. The present review provides useful information for clinical practice and scientific research of PJI.