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1.
BMC Bioinformatics ; 21(1): 332, 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709217

RESUMO

BACKGROUND: In cell biology, increasing focus has been directed to fast events at subcellular space with the advent of fluorescent probes. As an example, voltage sensitive dyes (VSD) have been used to measure membrane potentials. Yet, even the most recently developed genetically encoded voltage sensors have demanded exhausting signal averaging through repeated experiments to quantify action potentials (AP). This analysis may be further hampered in subcellular signals defined by small regions of interest (ROI), where signal-to-noise ratio (SNR) may fall substantially. Signal processing techniques like blind source separation (BSS) are designed to separate a multichannel mixture of signals into uncorrelated or independent sources, whose potential to separate ROI signal from noise has been poorly explored. Our aims are to develop a method capable of retrieving subcellular events with minimal a priori information from noisy cell fluorescence images and to provide it as a computational tool to be readily employed by the scientific community. RESULTS: In this paper, we have developed METROID (Morphological Extraction of Transmembrane potential from Regions Of Interest Device), a new computational tool to filter fluorescence signals from multiple ROIs, whose code and graphical interface are freely available. In this tool, we developed a new ROI definition procedure to automatically generate similar-area ROIs that follow cell shape. In addition, simulations and real data analysis were performed to recover AP and electroporation signals contaminated by noise by means of four types of BSS: Principal Component Analysis (PCA), Independent Component Analysis (ICA), and two versions with discrete wavelet transform (DWT). All these strategies allowed for signal extraction at low SNR (- 10 dB) without apparent signal distortion. CONCLUSIONS: We demonstrate the great capability of our method to filter subcellular signals from noisy fluorescence images in a single trial, avoiding repeated experiments. We provide this novel biomedical application with a graphical user interface at https://doi.org/10.6084/m9.figshare.11344046.v1 , and its code and datasets are available in GitHub at https://github.com/zoccoler/metroid .


Assuntos
Razão Sinal-Ruído , Software , Algoritmos , Animais , Automação , Corantes/química , Simulação por Computador , Fluorescência , Humanos , Potenciais da Membrana , Análise de Componente Principal , Ratos , Processamento de Sinais Assistido por Computador , Frações Subcelulares/metabolismo , Interface Usuário-Computador
2.
Chem Biol Interact ; 328: 109192, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32712081

RESUMO

Many natural products are prodrugs which are biotransformed and activated after oral administration. The investigation of gastrointestinal and hepatic biotransformation can be facilitated by in vitro screening methods. This study compares two widely used in vitro models for hepatic biotransformation: 1) human S9 fractions and 2) human liver microsomes and cytosolic fractions in a two-step sequence, with the purpose of identifying differences in the biotransformation of medicagenic acid, the putative precursor of active metabolites, responsible for the medicinal effects of the herb Herniaria hirsuta. The combination of liquid chromatography coupled to high-resolution mass spectrometry with subsequent suspect and non-target data analysis allowed the identification of thirteen biotransformation products, four of which are reported here for the first time. Eight biotransformation products resulting from oxidative Phase I reactions were identified. Phase II conjugation reactions resulted in the formation of three glucuronidated and two sulfated biotransformation products. No major differences could be observed between incubations with human liver S9 or when utilizing human microsomal and cytosolic fractions. Apart from two metabolites, both methods rendered the same qualitative metabolic profile, with minor quantitative differences. As a result, both protocols applied in this study can be used to study in vitro human liver biotransformation reactions.


Assuntos
Microssomos Hepáticos/metabolismo , Triterpenos/metabolismo , Biotransformação , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Masculino , Frações Subcelulares/metabolismo , Fatores de Tempo , Triterpenos/química
3.
Prostate ; 80(12): 962-976, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32511787

RESUMO

OBJECTIVE: The broad goal of the research described in this study was to investigate the contributions of selenium-binding protein 1 (SBP1) loss in prostate cancer development and outcome. METHODS: SBP1 levels were altered in prostate cancer cell lines and the consequences on oxygen consumption, expression of proteins associated with energy metabolism, and cellular transformation and migration were investigated. The effects of exposing cells to the SBP1 reaction products, H2 O2 and H2 S were also assessed. In silico analyses identified potential HNF4α binding sites within the SBP1 promoter region and this was investigated using an inhibitor specific for that transcription factor. RESULTS: Using in silico analyses, it was determined that the promoter region of SBP1 contains putative binding sites for the HNF4α transcription factor. The potential for HNF4α to regulate SBP1 expression was supported by data indicating that HNF4α inhibition resulted in a dose-response increase in the levels of SBP1 messenger RNA and protein, identifying HNF4α as a novel negative regulator of SBP1 expression in prostate cancer cells. The consequences of altering the levels of SBP1 were investigated by ectopically expressing SBP1 in PC-3 prostate cancer cells, where SBP1 expression attenuated anchorage-independent cellular growth and migration in culture, both properties associated with transformation. SBP1 overexpression reduced oxygen consumption in these cells and increased the activation of AMP-activated protein kinase (AMPK), a major regulator of energy homeostasis. In addition, the reaction products of SBP1, H2 O2 , and H2 S also activated AMPK. CONCLUSIONS: Based on the obtained data, it is hypothesized that SBP1 negatively regulates oxidative phosphorylation (OXPHOS) in the healthy prostate cells by the production of H2 O2 and H2 S and consequential activation of AMPK. The reduction of SBP1 levels in prostate cancer can occur due to increased binding of HNF4α, acting as a transcriptional inhibitor to the SBP1 promoter. Consequently, there is a reduction in H2 O2 and H2 S-mediated signaling, inhibition of AMPK, and stimulation of OXPHOS and building blocks of biomolecules needed for tumor growth and progression. Other effects of SBP1 loss in tumor cells remain to be discovered.


Assuntos
Neoplasias da Próstata/metabolismo , Proteínas de Ligação a Selênio/metabolismo , Linhagem Celular Tumoral , Transformação Celular Viral , Metilação de DNA , Progressão da Doença , Metabolismo Energético , Regulação Neoplásica da Expressão Gênica , Glucose/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/metabolismo , Masculino , Fosforilação Oxidativa , Consumo de Oxigênio , Células PC-3 , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Quinases/metabolismo , Proteínas de Ligação a Selênio/deficiência , Proteínas de Ligação a Selênio/genética , Frações Subcelulares/metabolismo
4.
BMC Bioinformatics ; 21(1): 212, 2020 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-32448129

RESUMO

BACKGROUND: Apoptosis, also called programmed cell death, refers to the spontaneous and orderly death of cells controlled by genes in order to maintain a stable internal environment. Identifying the subcellular location of apoptosis proteins is very helpful in understanding the mechanism of apoptosis and designing drugs. Therefore, the subcellular localization of apoptosis proteins has attracted increased attention in computational biology. Effective feature extraction methods play a critical role in predicting the subcellular location of proteins. RESULTS: In this paper, we proposed two novel feature extraction methods based on evolutionary information. One of the features obtained the evolutionary information via the transition matrix of the consensus sequence (CTM). And the other utilized the evolutionary information from PSSM based on absolute entropy correlation analysis (AECA-PSSM). After fusing the two kinds of features, linear discriminant analysis (LDA) was used to reduce the dimension of the proposed features. Finally, the support vector machine (SVM) was adopted to predict the protein subcellular locations. The proposed CTM-AECA-PSSM-LDA subcellular location prediction method was evaluated using the CL317 dataset and ZW225 dataset. By jackknife test, the overall accuracy was 99.7% (CL317) and 95.6% (ZW225) respectively. CONCLUSIONS: The experimental results show that the proposed method which is hopefully to be a complementary tool for the existing methods of subcellular localization, can effectively extract more abundant features of protein sequence and is feasible in predicting the subcellular location of apoptosis proteins.


Assuntos
Algoritmos , Proteínas Reguladoras de Apoptose/metabolismo , Análise Discriminante , Evolução Molecular , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/química , Sequência Consenso , Bases de Dados de Proteínas , Entropia , Matrizes de Pontuação de Posição Específica , Curva ROC , Frações Subcelulares/metabolismo , Máquina de Vetores de Suporte
5.
Nat Commun ; 11(1): 2099, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350248

RESUMO

Besides pro-inflammatory roles, the ancient cytokine interleukin-17 (IL-17) modulates neural circuit function. We investigate IL-17 signaling in neurons, and the extent it can alter organismal phenotypes. We combine immunoprecipitation and mass spectrometry to biochemically characterize endogenous signaling complexes that function downstream of IL-17 receptors in C. elegans neurons. We identify the paracaspase MALT-1 as a critical output of the pathway. MALT1 mediates signaling from many immune receptors in mammals, but was not previously implicated in IL-17 signaling or nervous system function. C. elegans MALT-1 forms a complex with homologs of Act1 and IRAK and appears to function both as a scaffold and a protease. MALT-1 is expressed broadly in the C. elegans nervous system, and neuronal IL-17-MALT-1 signaling regulates multiple phenotypes, including escape behavior, associative learning, immunity and longevity. Our data suggest MALT1 has an ancient role modulating neural circuit function downstream of IL-17 to remodel physiology and behavior.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/imunologia , Caenorhabditis elegans/fisiologia , Imunidade , Interleucina-17/metabolismo , Longevidade , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Neurônios/metabolismo , Animais , Comportamento Animal , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Imunidade/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Interneurônios/fisiologia , Longevidade/efeitos dos fármacos , Modelos Biológicos , Neurônios/efeitos dos fármacos , Oxigênio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/metabolismo , Transgenes
6.
PLoS One ; 15(5): e0233863, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470053

RESUMO

Adaptive regulation of epithelial transporters to nutrient intake is essential to decrease energy costs of their synthesis and maintenance, however such regulation is understudied. Previously we demonstrated that the transport function of the basolateral amino acid uniporter LAT4 (Slc43a2) is increased by dephosphorylation of serine 274 (S274) and nearly abolished by dephosphorylation of serine 297 (S297) when expressed in Xenopus oocytes. Phosphorylation changes in the jejunum of food-entrained mice suggested an increase in LAT4 transport function during food expectation. Thus, we investigated further how phosphorylation, expression and localization of mouse intestinal LAT4 respond to food-entrained diurnal rhythm and dietary protein content. In mice entrained with 18% protein diet, LAT4 mRNA was not submitted to diurnal regulation, unlike mRNAs of luminal symporters and antiporters. Only in duodenum, LAT4 protein expression increased during food intake. Concurrently, S274 phosphorylation was decreased in all three small intestinal segments, whereas S297 phosphorylation was increased only in jejunum. Interestingly, during food intake, S274 phosphorylation was nearly absent in ileum and accompanied by strong phosphorylation of mTORC1 target S6. Entraining mice with 8% protein diet provoked a shift in jejunal LAT4 localization from the cell surface to intracellular stores and increased S274 phosphorylation in both jejunum and ileum during food anticipation, suggesting decreased transport function. In contrast, 40% dietary protein content led to increased LAT4 expression in jejunum and its internalization in ileum. Ex vivo treatments of isolated intestinal villi fraction demonstrated that S274 phosphorylation was stimulated by protein kinase A. Rapamycin-sensitive insulin treatment and amino acids increased S297 phosphorylation, suggesting that the response to food intake might be regulated via the insulin-mTORC1 pathway. Ghrelin, an oscillating orexigenic hormone, did not affect phosphorylation of intestinal LAT4. Overall, we show that phosphorylation, expression and localization of intestinal mouse LAT4 responds to diurnal and dietary stimuli in location-specific manner.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Ritmo Circadiano , Proteínas na Dieta/farmacologia , Alimentos , Intestinos/fisiologia , Aminoácidos/metabolismo , Animais , Antiporters/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Grelina/administração & dosagem , Grelina/farmacologia , Insulina/metabolismo , Intestino Delgado/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Simportadores/metabolismo , Serina-Treonina Quinases TOR/metabolismo
7.
J Mol Biol ; 432(7): 1901-1909, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32198118

RESUMO

Previous studies with various Src family kinase biosensors showed that the nuclear kinase activities are much suppressed compared to those in the cytosol, suggesting that these kinases are regulated differently in the nucleus and in the cytosol. In this study, using Fyn as an example, we first engineered a Fyn biosensor with a light-inducible nuclear localization signal to demonstrate that the Fyn kinase activity is significantly lower in the nucleus than in the cytosol. To understand how different equilibrium states between Fyn and the corresponding phosphatases are maintained in the cytosol and nucleus, we further engineered a Fyn kinase domain with light-inducible nuclear localization signal. The results revealed that the Fyn kinase can be actively transported into the nucleus upon light activation and upregulate the biosensor signals in the nucleus. Our results suggest that there is limited transport or diffusion of Fyn kinase between the cytosol and nucleus in the cells, which is important for the maintenance of different equilibrium states of Fyn in situ.


Assuntos
Técnicas Biossensoriais/métodos , Núcleo Celular/metabolismo , Citosol/metabolismo , Optogenética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Frações Subcelulares/metabolismo , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Sinais de Localização Nuclear , Transporte Proteico
8.
Gene ; 732: 144370, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31954860

RESUMO

Apoptosis plays a significant role in the cellular immune responses against infections, especially those related to viruses. Across various species, Caspase 3 is a prominent mediator of apoptosis and participates in the cell death signaling cascade. However, its role remains relatively unknown in cod fish. In this study, we aimed to reveal the role of Pacific cod Caspase-3 (GmCasp3) in apoptosis and its evolutionary position. Our results showed that the GmCasp3 cDNA contains an open reading frame of 864 nucleotides; that codes for 287 amino acids long protein with a molecular weight of 32.03 kDa. The sequence alignments and 3-D model indicated that GmCasp3 contained highly conserved domains, such as "QACRG", "GSWFI" and "HG" active sites, however, the phylogenetic tree analysis revealed that both GmCasp3 and Atlantic cod caspase-3 clustered together are far from the high vertebrate branch, indicating they are at a lower position in vertebrate evolution. Red fluorescent labeling vector pDsRed2-C1-GmCasp3 was constructed and it was transfected into EPC cell lines. The result showed that GmCasp3 protein was distribute in the protoplasm and expressed in apoptotic cell debris. Moreover, the GmCasp3 enzyme activity increased with the increased post-transfection analysis time, while the genome DNA was visibly fragmented at 36 h post transfection. Flow cytometry analysis showed that the proportion of apoptosis cells increased from 12 h to 24 h post transfection. In conclusion, the conserved functions of GmCasp3 in apoptosis indicated that Pacific cod has the similar apoptotic characteristics as other animals.


Assuntos
Apoptose/fisiologia , Caspase 3/fisiologia , Proteínas de Peixes/fisiologia , Sequência de Aminoácidos , Animais , Caspase 3/química , Caspase 3/genética , Proteínas de Peixes/química , Proteínas de Peixes/genética , Gadiformes , Filogenia , Alinhamento de Sequência , Frações Subcelulares/metabolismo
9.
Artigo em Inglês | MEDLINE | ID: mdl-31928655

RESUMO

Lysine (K) 63-linked polyubiquitination plays important roles in cellular processes including DNA-damage tolerance (DDT), NF-κB signaling and endocytosis. Compared to yeast and mammals, little is known about K63-linked polyubiquitination in plants. To date, a Uev-Ubc13 complex is the only known Ub-conjugating enzyme to catalyze K63-linked polyubiquitination, in which Uev serves as a regulatory subunit. The Arabidopsis thaliana genome contains four UEV1 genes that can be classified into two subfamilies (UEV1A/B and UEV1C/D), in which Uev1A/B have a C-terminal extension. Database analysis reveals that all higher plant genomes contain both subfamily UEV1s, which were evolved as early as angiosperm plants. Interestingly, all C-terminal tails in the Uev1A/B subfamily contain a putative prenylation motif, CaaX. Combined experimental results using AtUev1B demonstrated that it is most likely farnesylated and that its C-terminal tail, particularly the catalytic Cys residue in the CaaX motif, plays critical roles in protein-protein interaction, nuclear exclusion and membrane association. Using AtUev1B as bait for a yeast-two-hybrid screen, we identified 14 interaction proteins in a prenylation-dependent manner. These results collectively imply that prenylation of AtUev1A/B plays a critical role in its functional differentiation from AtUev1C/D.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição/genética , Enzimas de Conjugação de Ubiquitina/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Prenilação , Domínios e Motivos de Interação entre Proteínas/genética , Alinhamento de Sequência , Frações Subcelulares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo
10.
Nat Methods ; 17(2): 225-231, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907447

RESUMO

Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.


Assuntos
Imagem Óptica , Frações Subcelulares/metabolismo , Animais , Humanos , Organelas/metabolismo
11.
Int J Mol Sci ; 21(1)2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31906564

RESUMO

Multiple isoforms of 14-3-3 proteins exist in different organisms. In mammalian cells, 14-3-3 protein has seven isoforms (α/ß, ε, η, γ, σ, θ/τ, and δ/ζ), with α and δ representing the phosphorylated versions of ß and ζ, respectively. While the existence of multiple isoforms may represent one more level of regulation in 14-3-3 signaling, our knowledge regarding the isoform-specific functions of 14-3-3 proteins is very limited. Determination of the subcellular localization of the different 14-3-3 isoforms could give us important clues of their specific functions. In this study, by using indirect immunofluorescence, subcellular fractionation, and immunoblotting, we studied the subcellular localization of the total 14-3-3 protein and each of the seven 14-3-3 isoforms; their redistribution throughout the cell cycle; and their translocation in response to EGF in Cos-7 cells. We showed that 14-3-3 proteins are broadly distributed throughout the cell and associated with many subcellular structures/organelles, including the plasma membrane (PM), mitochondria, ER, nucleus, microtubules, and actin fibers. This broad distribution underlines the multiple functions identified for 14-3-3 proteins. The different isoforms of 14-3-3 proteins have distinctive subcellular localizations, which suggest their distinctive cellular functions. Most notably, 14-3-3ƞ is almost exclusively localized to the mitochondria, 14-3-3γ is only localized to the nucleus, and 14-3-3σ strongly and specifically associated with the centrosome during mitosis. We also examined the subcellular localization of the seven 14-3-3 isoforms in other cells, including HEK-293, MDA-MB-231, and MCF-7 cells, which largely confirmed our findings with Cos-7 cells.


Assuntos
Proteínas 14-3-3/metabolismo , Ciclo Celular , Fator de Crescimento Epidérmico/farmacologia , Proteínas 14-3-3/genética , Animais , Células COS , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitose , Isoformas de Proteínas/metabolismo , Transporte Proteico , Frações Subcelulares/metabolismo
12.
Development ; 147(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969357

RESUMO

The development and maintenance of the correct morphology of sperm is important for their functions. Cellular morphogenesis of sperm occurs during the post-meiotic developmental stage; however, little is known about what coordinates this process. In the present study, we investigated the role of A-kinase anchoring protein 3 (AKAP3) during mouse spermiogenesis, using both mouse genetics and proteomics. It was found that AKAP3 is essential for the formation of the specific subcellular structure of the sperm flagellum, motility of sperm and male fertility. Additionally, lack of AKAP3 caused global changes of the sperm proteome and mislocalization of sperm proteins, including accumulation of RNA metabolism and translation factors and displacement of PKA subunits in mature sperm, which may underlie misregulated PKA activity and immotility in sperm. Interestingly, sperm lacking a complete fibrous sheath from both Akap3 and Akap4 null mice accumulated F-actin filaments and morphological defects during post-testicular maturation in the epididymis. These results suggest that the subcellular structures of sperm could be formed via independent pathways, and elucidate the roles of AKAP3 during the coordinated synthesis and organization of the sperm proteome and sperm morphology.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Infertilidade Masculina/metabolismo , Espermatozoides/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Epididimo/metabolismo , Deleção de Genes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Proteoma/metabolismo , Transdução de Sinais , Espermatozoides/anormalidades , Espermatozoides/patologia , Espermatozoides/ultraestrutura , Frações Subcelulares/metabolismo
13.
Ecotoxicol Environ Saf ; 187: 109827, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31655413

RESUMO

Earthworms and their biomarkers are considered good indicators for assessing the effects of toxic chemicals. Therefore, in this study, we exposed Eisenia fetida to lethal and sub-lethal concentrations of Cd and Pb nitrate in artificial soil for 14 and 28 days to evaluate the impact on subcellular partitioning, lethal toxicity (LC50), growth, sperm count, morphology and apoptosis (using TUNEL assay). The soluble internal pools of both metals were good predictors of the responses of biomarkers. We found sperm deformation, TUNEL positive sperms and weight loss positively and sperm count negatively correlated with the concentrations of Cd and Pb in the total internal and cytosolic fraction (p < 0.01) and to a lesser extent with Pb concentrations in the granular fraction (p < 0.05). Fourteen days LC50 for Cd and Pb were 2169 ±â€¯322 and 6387 ±â€¯904 µg/g, respectively. Cadmium and Pb caused a significant depression in sperm count after 14 (Cd: up to 46.9%; Pb: up to 36.24%) and 28 (Cd: up to 72.47%; Pb: up to 43.12%) days of exposure relative to the control (p < 0.05). Cadmium induced higher abnormality in sperm heads than Pb. For both metals, TUNEL positive sperms significantly increased after 14 (Cd: up to 14.17%; Pb: up to 16.33%) and 28 (Cd: up to 16.33%; Pb: up to 11.67%) days of exposure compared with the control (p < 0.05). The findings of this study, illustrate the importance of considering sperm parameters as a rapid, easy and sensitive biomarker for the evaluation of metal toxicity.


Assuntos
Cádmio/toxicidade , Chumbo/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Espermatozoides/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Cádmio/metabolismo , Biomarcadores Ambientais/efeitos dos fármacos , Chumbo/metabolismo , Masculino , Oligoquetos/metabolismo , Poluentes do Solo/metabolismo , Espermatozoides/patologia , Frações Subcelulares/metabolismo
14.
Biochem Soc Trans ; 47(6): 1733-1747, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31724693

RESUMO

The second messenger 3',5'-cyclic nucleoside adenosine monophosphate (cAMP) plays a key role in signal transduction across prokaryotes and eukaryotes. Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision. To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited. Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively. In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell. This review provides an overview about optogenetic tools and biosensors to shed light on the subcellular organization of cAMP signaling.


Assuntos
Técnicas Biossensoriais , AMP Cíclico/metabolismo , Optogenética , Transdução de Sinais , Frações Subcelulares/metabolismo , Adenilil Ciclases/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Diester Fosfórico Hidrolases/metabolismo
15.
Anal Bioanal Chem ; 411(30): 7935-7941, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31745610

RESUMO

The use of oversampling in MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry (IMS) to improve lateral resolution is a common practice. However, its application is still controversial and recent studies reported a spot size-dependent change in the relative intensity of the spectra. Previously, using oversampling, we described the lipidome of the human colon epithelium, a 20-30 µm wide cell monolayer; even assessing the changes occurring within this monolayer associated with complex biological processes. Interestingly, the K-means analysis of those experiments unveiled the presence of a third epithelial cluster that anatomically matched the nuclei position. Taking into account the nucleus size (9-12 µm of diameter) and its distinctive lipidome, we decided to test whether this cluster was really of nuclear origin. Hence, the spectra obtained directly from tissue sections were compared with those recorded from the nuclei isolated from colon biopsies. The highest correlation coefficient was obtained when comparing the spectrum of the isolated nuclei with that of the tissue nuclear cluster, demonstrating the successful identification of the nuclear lipidome in the MALDI-IMS experiments run using oversampling and a lateral resolution of 10 µm/pixel. Importantly, it was established that phosphatidylinositol 38:4 nuclear levels remained stable along the colon crypt. That is, it mimicked neither the regular decrease observed in the epithelium nor the regular increase observed in the stroma, eliminating the chance of inter-pixel contamination. Altogether, besides confirming the usefulness of the oversampling technique, these results strongly reinforce the pivotal role IMS may have in promising fields such as single-cell analysis. Graphical abstract.


Assuntos
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Frações Subcelulares/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos
16.
Int J Mol Sci ; 20(22)2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31717404

RESUMO

Rett syndrome (RTT), a neurodevelopmental disorder, is mainly caused by mutations in methyl CpG-binding protein 2 (MECP2), which has multiple functions such as binding to methylated DNA or interacting with a transcriptional co-repressor complex. It has been established that alterations in cyclin-dependent kinase-like 5 (CDKL5) or forkhead box protein G1 (FOXG1) correspond to distinct neurodevelopmental disorders, given that a series of studies have indicated that RTT is also caused by alterations in either one of these genes. We investigated the evolution and molecular features of MeCP2, CDKL5, and FOXG1 and their binding partners using phylogenetic profiling to gain a better understanding of their similarities. We also predicted the structural order-disorder propensity and assessed the evolutionary rates per site of MeCP2, CDKL5, and FOXG1 to investigate the relationships between disordered structure and other related properties with RTT. Here, we provide insight to the structural characteristics, evolution and interaction landscapes of those three proteins. We also uncovered the disordered structure properties and evolution of those proteins which may provide valuable information for the development of therapeutic strategies of RTT.


Assuntos
Simulação por Computador , Evolução Molecular , Fatores de Transcrição Forkhead/genética , Proteína 2 de Ligação a Metil-CpG/genética , Proteínas do Tecido Nervoso/genética , Proteínas Serina-Treonina Quinases/genética , Síndrome de Rett/genética , Animais , Cordados/genética , Ontologia Genética , Humanos , Mutação de Sentido Incorreto/genética , Especificidade de Órgãos , Filogenia , Ligação Proteica , Processamento de Proteína Pós-Traducional , Frações Subcelulares/metabolismo
17.
Curr Top Med Chem ; 19(29): 2708-2717, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31702501

RESUMO

OBJECTIVE: In the present study, an attempt has been made for subtractive proteomic analysis approach for novel drug targets in Salmonella enterica subsp. enterica serover Typhi str.CT18 using computational tools. METHODS: Paralogous, redundant and less than 100 amino acid protein sequences were removed by using CD-HIT. Further detection of bacterial proteins which are non-homologous to host and are essential for the survival of pathogens by using BLASTp against host proteome and DEG`s, respectively. Comparative Metabolic pathways analysis was performed to find unique and common metabolic pathways. The non-redundant, non-homologous and essential proteins were BLAST against approved drug targets for drug targets while Psortb and CELLO were used to predict subcellular localization. RESULTS: There were 4473 protein sequences present in NCBI Database for Salmonella enterica subsp. enterica serover Typhi str. CT18 out of these 327 were essential proteins which were non-homologous to human. Among these essential proteins, 124 proteins were involved in 19 unique metabolic pathways. These proteins were further BLAST against approved drug targets in which 7 cytoplasmic proteins showed druggability and can be used as a therapeutic target. CONCLUSION: Drug targets identification is the prime step towards drug discovery. We identified 7 cytoplasmic druggable proteins which are essential for the pathogen survival and non-homologous to human proteome. Further in vitro and in vivo validation is needed for the evaluation of these targets to combat against salmonellosis.


Assuntos
Farmacorresistência Bacteriana Múltipla , Proteômica , Salmonella enterica/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Simulação por Computador , Salmonella enterica/metabolismo , Frações Subcelulares/metabolismo
18.
Int J Mol Sci ; 20(22)2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31717281

RESUMO

Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. HLB is associated with the non-culturable bacterium, Candidatus Liberibacter asiaticus (CaLas) in the United States. The virulence mechanism of CaLas is largely unknown, partly because of the lack of a mutant library. In this study, Tobacco mosaic virus (TMV) and Nicotiana benthamiana (N. benthamiana) were used for large-scale screening of the virulence factors of CaLas. Agroinfiltration of 60 putative virulence factors in N. benthamiana led to the identification of four candidates that caused severe symptoms in N. benthamiana, such as growth inhibition and cell death. CLIBASIA_05150 and CLIBASIA_04065C (C-terminal of CLIBASIA_04065) could cause cell death in the infiltrated leaves at five days post infiltration. Two low-molecular-weight candidates, CLIBASIA_00470 and CLIBASIA_04025, could inhibit plant growth. By converting start codon to stop codon or frameshifting, the four genes lost their harmful effects to N. benthamiana. It indicated that the four virulence factors functioned at the protein level rather than at the RNA level. The subcellular localization of the four candidates was determined by confocal laser scanning microscope. CLIBASIA_05150 located in the Golgi apparatus; CLIBASIA_04065 located in the mitochondrion; CLIBASIA_00470 and CLIBASIA_04025 distributed in cells as free GFP. The host proteins interacting with the four virulence factors were identified by yeast two-hybrid. The host proteins interacting with CLIBASIA_00470 and CLIBASIA_04025 were overlapping. Based on the phenotypes, the subcellular localization and the host proteins identified by yeast two-hybrid, CLIBASIA_00470 and CLIBASIA_04025, functioned redundantly. The hypothesis of CaLas virulence was proposed. CaLas affects citrus development and suppresses citrus disease resistance, comprehensively, in a complicated manner. Ubiquitin-mediated protein degradation might play a vital role in CaLas virulence. Deep characterization of the interactions between the identified virulence factors and their prey will shed light on HLB. Eventually, it will help in developing HLB-resistant citrus and save the endangered citrus industry worldwide.


Assuntos
Rhizobiaceae/patogenicidade , Vírus do Mosaico do Tabaco/metabolismo , Tabaco/metabolismo , Tabaco/microbiologia , Fatores de Virulência/metabolismo , Proteínas de Bactérias/metabolismo , Morte Celular , Fenótipo , Folhas de Planta/citologia , Folhas de Planta/microbiologia , Frações Subcelulares/metabolismo , Tabaco/virologia
19.
Ecotoxicol Environ Saf ; 185: 109692, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31585391

RESUMO

Canna indica L. is a promising species for heavy metal phytoremediation due to its fast growth rate and large biomass. However, few studies have investigated cadmium (Cd) tolerance mechanisms. In the present study, Canna plants were cultivated under hydroponic conditions with increasing Cd concentrations (0, 5, 10, 15 mg/L). We found that the plants performed well under 5 mg/L Cd2+ stress, but damage was observed under higher Cd exposure, such as leaf chlorosis, growth inhibition, a decreased chlorophyll content, and destruction of the ultrastructure of leaf cells. Additionally, Canna alleviated Cd toxicity to a certain extent. After Canna was exposed to 5, 10 and 15 mg/L Cd2+ for 45 d, the highest Cd concentration was exhibited in roots, which was almost 17-47 times the Cd concentration in leaves and 8-20 times that in stems. At the subcellular level, cellular debris and heat-stable proteins (HSPs) were the main binding sites for Cd, and the proportion of Cd in the two subcellular fractions accounted for 71.4-94.2% of the total Cd. Furthermore, we found that granules could participate in the detoxification process when Cd stress was enhanced. Our results indicated that Canna indica L. can tolerate Cd toxicity by sequestering heavy metals in root tissues, fencing out by cell wall, and binding with biologically detoxified fractions (granules and HSPs).


Assuntos
Cádmio/toxicidade , Poluentes do Solo/toxicidade , Frações Subcelulares/efeitos dos fármacos , Zingiberales/efeitos dos fármacos , Biodegradação Ambiental , Biomassa , Cádmio/metabolismo , Relação Dose-Resposta à Radiação , Tolerância a Medicamentos , Inativação Metabólica , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura , Poluentes do Solo/metabolismo , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Zingiberales/metabolismo , Zingiberales/ultraestrutura
20.
Med Sci Monit ; 25: 7079-7086, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31541070

RESUMO

BACKGROUND Higher fetal hemoglobin (HbF) levels can ameliorate the clinical severity of ß-thalassemia. The use of integrative strategies to combine results from gene microarray expression profiling, experimental evidence, and bioinformatics helps reveal functional long noncoding RNAs (lncRNAs) in ß-thalassemia and HbF induction. MATERIAL AND METHODS In a previous study, a microarray profiling was performed of 7 individuals with high HbF levels and 7 normal individuals. Thirteen paired samples were used for validation. lncRNA NR_001589 and uc002fcj.1 were chosen for further research. The quantitative reverse transcription-PCR was used to detect the expression levels of 2 lncRNAs. The Spearman correlation test was employed. The nuclear and cytoplasmic distribution experiment in K562 cells was used to verify the subcellular localization of 2 lncRNAs. Potential relationships among lncRNAs, predicted microRNAs (miRNAs), and target gene HBG1/2 were based on competitive endogenous RNA theory and bioinformatics analysis. RESULTS Average expression levels of NR_001589 and uc002fcj.1 were significantly higher in the high-HbF group than in the control group. A positive correlation existed between NR_001589, uc002fcj.1, and HbF. The expression of NR_001589 was in both the cytoplasm and the nucleus, mostly (77%) in the cytoplasm. The expression of uc002fcj.1 was in both the cytoplasm and the nucleus; the cytoplasmic proportion was 43% of the total amount. A triple lncRNA-miRNA-mRNA network was established. CONCLUSIONS Novel candidate genetic factors associated with the HBG1/2 expression were identified. Further functional investigation of NR_001589 and uc002fcj.1 can help deepen the understanding of molecular mechanisms in ß-thalassemia.


Assuntos
Hemoglobina Fetal/genética , RNA Longo não Codificante/genética , Talassemia beta/genética , Regulação da Expressão Gênica , Humanos , Células K562 , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Frações Subcelulares/metabolismo
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