RESUMO
Axonal mitochondria defects are early events in the pathogenesis of motoneuron disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. The RNA-binding protein hnRNP R interacts with different motoneuron disease-related proteins such as SMN and TDP-43 and has important roles in axons of motoneurons, including axonal mRNA transport. However, whether hnRNP R also modulates axonal mitochondria is currently unknown. Here, we show that axonal mitochondria exhibit altered function and motility in hnRNP R-deficient motoneurons. Motoneurons lacking hnRNP R show decreased anterograde and increased retrograde transport of mitochondria in axons. Furthermore, hnRNP R-deficiency leads to mitochondrial hyperpolarization, caused by decreased complex I and reversed complex V activity within the respiratory chain. Taken together, our data indicate a role for hnRNP R in regulating transport and maintaining functionality of axonal mitochondria in motoneurons.
Assuntos
Axônios , Neurônios Motores , Potenciais da Membrana , Neurônios Motores/metabolismo , Axônios/patologia , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Mitocôndrias/metabolismoRESUMO
Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length protein and a shorter isoform lacking its N-terminal acidic domain. To investigate functions selectively associated with the full-length hnRNP R isoform, we generated a Hnrnpr knockout mouse (Hnrnprtm1a/tm1a) in which expression of full-length hnRNP R was abolished while production of the truncated hnRNP R isoform was retained. Motoneurons cultured from Hnrnprtm1a/tm1a mice did not show any axonal growth defects but exhibited enhanced accumulation of double-strand breaks and an impaired DNA damage response upon exposure to genotoxic agents. Proteomic analysis of the hnRNP R interactome revealed the multifunctional protein Yb1 as a top interactor. Yb1-depleted motoneurons were defective in DNA damage repair. We show that Yb1 is recruited to chromatin upon DNA damage where it interacts with γ-H2AX, a mechanism that is dependent on full-length hnRNP R. Our findings thus suggest a novel role of hnRNP R in maintaining genomic integrity and highlight the function of its N-terminal acidic domain in this context.
Assuntos
Cromatina/genética , Dano ao DNA , Reparo do DNA/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Neurônios Motores/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Animais , Axônios/metabolismo , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Immunoblotting , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/citologia , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
Long non-coding RNAs (lncRNAs) comprise a vast repertoire of RNAs playing a wide variety of crucial roles in tissue physiology in a cell-specific manner. Despite being engaged in myriads of regulatory mechanisms, many lncRNAs have still remained to be assigned any functions. A constellation of experimental techniques including single-molecule RNA in situ hybridization (sm-RNA FISH), cross-linking and immunoprecipitation (CLIP), RNA interference (RNAi), Clustered regularly interspaced short palindromic repeats (CRISPR) and so forth has been employed to shed light on lncRNA cellular localization, structure, interaction networks and functions. Here, we review these and other experimental approaches in common use for identification and characterization of lncRNAs, particularly those involved in different types of cancer, with focus on merits and demerits of each technique.
Assuntos
Sistemas CRISPR-Cas , Regulação Neoplásica da Expressão Gênica , Biologia Molecular/métodos , Neoplasias/genética , RNA Longo não Codificante/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reagentes de Ligações Cruzadas/química , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoprecipitação/instrumentação , Imunoprecipitação/métodos , Hibridização in Situ Fluorescente , Biologia Molecular/instrumentação , Neoplasias/metabolismo , Neoplasias/patologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Interferência de RNA , RNA Longo não Codificante/metabolismoRESUMO
Streptokinase is a valuable fibrinolytic agent used to cope with myocardial infarction and brain stroke. Despite its high efficiency in dissolving blood clots, streptokinase (SK) has no specificity in binding fibrin, causing some problems such as internal bleedings following its administration. To make streptokinase fibrin specific and limit the fibrinolytic process to the clot location, we engineered a chimeric streptokinase by fusing the fibrin binding Kringle 2 domain of tissue plasminogen activator (TPA) to the streptokinase N-terminal end. The chimeric SK construct (KSK) with inserted Kringle 2 domain was cloned into pET28a expression vector. The expression of recombinant protein was carried out in Escherichia coli origami (DE3) and confirmed by SDS-PAGE and Western blotting analyses. We used the chromogenic substrate S-2251 method to assess the specific activities of the chimeric and control wild-type proteins. Then, the two proteins were added in amounts with equal activity to fibrin clots of identical size. Finally, the supernatant above the fibrin clots was collected and subjected to the chromogenic assay to analyze the specificity of the chimeric protein. The specific activities of the chimeric and wild-type proteins were found to be 0.06 U/mg and 0.07 U/mg, respectively. Because of the binding of the chimeric protein to fibrin, the mean specific activity was significantly lower in the KSK supernatant (0.01) compared with the control (approximately 0.06) (p < 0.05). Our in vitro results indicate that the chimeric streptokinase protein has strong fibrin-specific activity compared to the wild-type protein. However, further in vivo studies are needed to evaluate its potential fibrinolytic effects.
Assuntos
Proteínas de Bactérias , Engenharia de Proteínas , Streptococcus/genética , Estreptoquinase , Ativador de Plasminogênio Tecidual , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Fibrina/química , Fibrina/metabolismo , Fibrinólise , Humanos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Streptococcus/enzimologia , Estreptoquinase/biossíntese , Estreptoquinase/química , Estreptoquinase/genética , Estreptoquinase/isolamento & purificação , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/isolamento & purificaçãoRESUMO
Motoneurons critically depend on precise spatial and temporal control of translation for axon growth and the establishment and maintenance of neuromuscular connections. While defects in local translation have been implicated in the pathogenesis of motoneuron disorders, little is known about the mechanisms regulating axonal protein synthesis. Here, we report that motoneurons derived from Hnrnpr knockout mice show reduced axon growth accompanied by lowered synthesis of cytoskeletal and synaptic components in axons. Mutant mice display denervated neuromuscular junctions and impaired motor behavior. In axons, hnRNP R is a component of translation initiation complexes and, through interaction with O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (Ogt), modulates O-GlcNAcylation of eIF4G. Restoring axonal O-GlcNAc levels rescued local protein synthesis and axon growth defects of hnRNP R knockout motoneurons. Together, these findings demonstrate a function of hnRNP R in controlling the local production of key factors required for axon growth and formation of neuromuscular innervations.
Assuntos
Axônios , Fator de Iniciação Eucariótico 4G , Ribonucleoproteínas Nucleares Heterogêneas , Camundongos Knockout , Neurônios Motores , Biossíntese de Proteínas , Animais , Axônios/metabolismo , Neurônios Motores/metabolismo , Camundongos , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Junção Neuromuscular/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Humanos , Acetilglucosamina/metabolismoRESUMO
The neuronal RNA-binding protein Ptbp2 regulates neuronal differentiation by modulating alternative splicing programs in the nucleus. Such programs contribute to axonogenesis by adjusting the levels of protein isoforms involved in axon growth and branching. While its functions in alternative splicing have been described in detail, cytosolic roles of Ptbp2 for axon growth have remained elusive. Here, we show that Ptbp2 is located in the cytosol including axons and growth cones of motoneurons, and that depletion of cytosolic Ptbp2 affects axon growth. We identify Ptbp2 as a major interactor of the 3' UTR of Hnrnpr mRNA encoding the RNA-binding protein hnRNP R. Axonal localization of Hnrnpr mRNA and local synthesis of hnRNP R protein are strongly reduced when Ptbp2 is depleted, leading to defective axon growth. Ptbp2 regulates hnRNP R translation by mediating the association of Hnrnpr with ribosomes in a manner dependent on the translation factor eIF5A2. Our data thus suggest a mechanism whereby cytosolic Ptbp2 modulates axon growth by fine-tuning the mRNA transport and local synthesis of an RNA-binding protein.
Assuntos
Axônios , Neurônios Motores , Citosol , Regiões 3' não Traduzidas , Ribonucleoproteínas Nucleares Heterogêneas/genética , RNA Mensageiro/genéticaRESUMO
Fragile X syndrome (FXS) is caused by a mutation in the FMR1 gene which can lead to a loss or shortage of the FMR1 protein. This protein interacts with specific miRNAs and can cause a range of neurological disorders. Therefore, miRNAs could act as a novel class of biomarkers for common CNS diseases. This study aimed to test this theory by exploring the expression profiles of various miRNAs in Iranian using deep sequencing-based technologies and validating the miRNAs affecting the expression of the FMR1 gene. Blood samples were taken from 15 patients with FXS (9 males, 6 females) and 12 controls. 25 miRNAs were differentially expressed in individuals with FXS compared to controls. Levels of 9 miRNAs were found to be significantly changed (3 upregulated and 6 downregulated). In Patients, the levels of hsa-miR-532-5p, hsa-miR-652-3p and hsa-miR-4797-3p were significantly upregulated while levels of hsa-miR-191-5p, hsa-miR-181-5p, hsa-miR-26a-5p, hsa-miR-30e-5p, hsa-miR-186-5p, and hsa-miR-4797-5p exhibited significant downregulation; and these dysregulations were confirmed by RT-qPCR. This study presents among the first evidence of altered miRNA expression in blood samples from patients with FXS, which could be used for diagnostic, prognostic, and treatment purposes. Larger studies are required to confirm these preliminary results.
Assuntos
Síndrome do Cromossomo X Frágil , MicroRNAs , Biomarcadores , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Irã (Geográfico) , Masculino , MicroRNAs/metabolismoRESUMO
Purpose: Due to growing concerns about the obesity pandemic as a worldwide phenomenon, a global effort has been made for managing it and associated disorders. Accordingly, metabolomics as a promising field of "OMICS" is presented for investigating different molecular pathways in obesity and related disorders through the evaluation of specific metabolites in both animal and human subjects. Herein, the aim of the present study as the first systematic review is to evaluate all available studies about different mechanisms and their biomarkers discovery using metabolomics approaches. Method: The study was designed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Using a comprehensive search strategy we searched in databases including; Web of Science, PubMed, and Scopus using specific keywords. Based on predefined inclusion/exclusion criteria study selection has been conducted considering the type of studies, participant, and outcome measures. Quality assessment was done using CASP (Critical Appraisal Skills Programme) checklist followed by data extraction according to a predefined data extraction sheet. Results: Among the articles that resulted from electronic search, a total of 74 articles met our inclusion criteria. The most prevalent studied metabolites were amino acids and lipid derivatives and both targeted and non-targeted approaches were applied for metabolomics studies. Conclusion: This systematic review summarized a wide range of studies regardless of the age, history, language, and type of the study. Further studies are needed to compare the application of emerging methods in the treatment of obesity and related disorders. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-021-00917-w.
RESUMO
OBJECTIVES: Human Wharton's Jelly mesenchymal stem cells (hWMSCs) are undifferentiated cells commonly used in regenerative medicine. The aim of this study was to develop a reliable tool for tracking hWMSCs when utilized as therapeutics in burnt disorders and also to optimize the cell-based treatment procedure. MATERIALS AND METHODS: The hWMSCs were first isolated from fresh umbilical cord Wharton's jelly and cultured. The 293LTV cell line was transfected by cGFP containing lentiviral vector and the helper plasmids for production of the viral particle. The viral particles were collected to transduce the hWMSCs. The transduced cells were finally selected based on resistance to puromycin. The burned rats (n=24) were treated with cGFP expressing hWMSCs using the cell spray method, with the cells being tracked 7, 14 and 21 days later. The rats were sacrificed 7, 14 and 21 days following treatment and paraffin embedded sections prepared from the burned area for downstream pathological analyses. RESULTS: The lentiviral particles carrying the cGFP gene were generated and the hWMSCs were transduced. The cGFP-expressing hWMSCs were detected in the burned tissue and the burned injuries were improved dramatically as compared to control. CONCLUSION: Because of the establishment of stably transduced cGFP expressing cells and the ability to detect cGFP for a relatively long-time interval, the method was found to be quite efficient for the purpose of cell tracking. The combination of hWMSC-based cell therapy and sterile Gauze Vaseline (GV) as covering was proven much more efficient than the traditional methods based on GV alone.
RESUMO
BACKGROUND: Infection with hepatitis E virus (HEV) is endemic in developing countries and reveals significant regional differences. Several studies have reported virus transmission via blood transfusion. To date, however, no cases of HEV RNA detection in blood donors have been reported from Iran. OBJECTIVES: The aim of this study was to determine the presence of HEV RNA in plasma samples of blood donors referred to a blood transfusion center in Shiraz in the southwest of Iran. The HEV genotypes were also investigated using nucleotide sequencing. PATIENTS AND METHODS: Blood samples were collected from 700 blood donors who were referred to Fars blood transfusion organization from January to March 2014. Plasma samples were screened for the presence of HEV IgG and IgM antibodies by standard enzyme immunoassay. Samples seroreactive to anti-HEV were further tested for the presence of HEV RNA using nested polymerase chain reaction (PCR) with universal primers for detection of all four HEV genotypes. Positive PCR samples were then subjected to DNA sequencing for further analysis. RESULTS: Fifty (50, 7.1%) out of 700 plasma samples tested positive for anti-HEV antibodies. HEV RNA was detected in 7/50 (12%) of the antibody-positive samples, the majority of which were IgM positive. Sequence analysis of seven isolates of the HEV RNA ORF 2 gene region revealed > 80% similarity with genotype 1. CONCLUSIONS: The analysis indicates that the HEV isolated from blood donors in the southwest of Iran belongs to genotype 1. However, more samples from other geographic regions of Iran are needed to confirm these findings. Because transmission of HEV by administration of blood or blood components is likely to occur, it may be sensible to screen donor blood for HEV to eliminate transfusion-transmitted HEV infection when the recipient is immunocompromised.
RESUMO
BACKGROUND: Viral load measurements are commonly used to monitor HCV infection in patients with chronic diseases or determining the number of HCV-genomes in serum samples of patients after sustained virological response. However, in some patients, HCV viral load in serum samples is too low to be detected by PCR, especially after treatment. OBJECTIVES: The aim of this study was to develop a highly specific, sensitive, and reproducible in-house quantitative PCR using specific primers and probe cited in highly conservative region of HCV genome that allows simultaneous detection of HCV genotypes 1 - 4. MATERIALS AND METHODS: In this study, three sets of primer pairs and a TaqMan probe for amplification and detection of selected region within 5'-non-coding (5'NCR) of four HCV genotypes were used. Using plasmid containing 5'NCR region of HCV, standard curve, threshold, and threshold cycle (CT) values were determined. Real-time and nested PCR were performed on HCV genotypes 1 - 4 extracted from plasma and peripheral blood mononuclear cells (PBMCs) samples collected from patients with chronic HCV infection. RESULTS: The lower limit detection of this in-house HCV real-time RT-PCR was determined as 100 RNA copies/mL. Inter- and intra-assay coefficient of variation (CV) of this in-house HCV real-time RT-PCR ranged from 0.9% to 1.8% and 1.76% to 3.94%, respectively. The viral load of the genotyped samples ranged from 2.0 × 10(6) ± 0.31 to 2.7 × 10(5) ± 0.46 copies/mL in serum samples and 5 × 10(2) ± 0.36 to 4.0 × 10(3) ± 0.51 copies/10(6) cells/mL of PBMCs. CONCLUSIONS: The quite sensitive in-house TaqMan real time RT-PCR assay was able to detect and quantify all four main HCV genotypes prevailing around all geographical regions of Iran.