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1.
PLoS Comput Biol ; 15(8): e1007274, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31465436

RESUMO

The popularity of CRISPR-based gene editing has resulted in an abundance of tools to design CRISPR-Cas9 guides. This is also driven by the fact that designing highly specific and efficient guides is a crucial, but not trivial, task in using CRISPR for gene editing. Here, we thoroughly analyse the performance of 18 design tools. They are evaluated based on runtime performance, compute requirements, and guides generated. To achieve this, we implemented a method for auditing system resources while a given tool executes, and tested each tool on datasets of increasing size, derived from the mouse genome. We found that only five tools had a computational performance that would allow them to analyse an entire genome in a reasonable time, and without exhausting computing resources. There was wide variation in the guides identified, with some tools reporting every possible guide while others filtered for predicted efficiency. Some tools also failed to exclude guides that would target multiple positions in the genome. We also considered two collections with over a thousand guides each, for which experimental data is available. There is a lot of variation in performance between the datasets, but the relative order of the tools is partially conserved. Importantly, the most striking result is a lack of consensus between the tools. Our results show that CRISPR-Cas9 guide design tools need further work in order to achieve rapid whole-genome analysis and that improvements in guide design will likely require combining multiple approaches.

2.
Nucleic Acids Res ; 47(8): 3846-3861, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30864654

RESUMO

HepG2 is one of the most widely used human cancer cell lines in biomedical research and one of the main cell lines of ENCODE. Although the functional genomic and epigenomic characteristics of HepG2 are extensively studied, its genome sequence has never been comprehensively analyzed and higher order genomic structural features are largely unknown. The high degree of aneuploidy in HepG2 renders traditional genome variant analysis methods challenging and partially ineffective. Correct and complete interpretation of the extensive functional genomics data from HepG2 requires an understanding of the cell line's genome sequence and genome structure. Using a variety of sequencing and analysis methods, we identified a wide spectrum of genome characteristics in HepG2: copy numbers of chromosomal segments at high resolution, SNVs and Indels (corrected for aneuploidy), regions with loss of heterozygosity, phased haplotypes extending to entire chromosome arms, retrotransposon insertions and structural variants (SVs) including complex and somatic genomic rearrangements. A large number of SVs were phased, sequence assembled and experimentally validated. We re-analyzed published HepG2 datasets for allele-specific expression and DNA methylation and assembled an allele-specific CRISPR/Cas9 targeting map. We demonstrate how deeper insights into genomic regulatory complexity are gained by adopting a genome-integrated framework.

3.
Genome Res ; 29(3): 472-484, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30737237

RESUMO

K562 is widely used in biomedical research. It is one of three tier-one cell lines of ENCODE and also most commonly used for large-scale CRISPR/Cas9 screens. Although its functional genomic and epigenomic characteristics have been extensively studied, its genome sequence and genomic structural features have never been comprehensively analyzed. Such information is essential for the correct interpretation and understanding of the vast troves of existing functional genomics and epigenomics data for K562. We performed and integrated deep-coverage whole-genome (short-insert), mate-pair, and linked-read sequencing as well as karyotyping and array CGH analysis to identify a wide spectrum of genome characteristics in K562: copy numbers (CN) of aneuploid chromosome segments at high-resolution, SNVs and indels (both corrected for CN in aneuploid regions), loss of heterozygosity, megabase-scale phased haplotypes often spanning entire chromosome arms, structural variants (SVs), including small and large-scale complex SVs and nonreference retrotransposon insertions. Many SVs were phased, assembled, and experimentally validated. We identified multiple allele-specific deletions and duplications within the tumor suppressor gene FHIT Taking aneuploidy into account, we reanalyzed K562 RNA-seq and whole-genome bisulfite sequencing data for allele-specific expression and allele-specific DNA methylation. We also show examples of how deeper insights into regulatory complexity are gained by integrating genomic variant information and structural context with functional genomics and epigenomics data. Furthermore, using K562 haplotype information, we produced an allele-specific CRISPR targeting map. This comprehensive whole-genome analysis serves as a resource for future studies that utilize K562 as well as a framework for the analysis of other cancer genomes.


Assuntos
Genoma Humano , Humanos , Células K562 , Cariótipo , Polimorfismo Genético , Sequenciamento Completo do Genoma
4.
Nat Commun ; 9(1): 5229, 2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30523329

RESUMO

Analysis of sleep for the diagnosis of sleep disorders such as Type-1 Narcolepsy (T1N) currently requires visual inspection of polysomnography records by trained scoring technicians. Here, we used neural networks in approximately 3,000 normal and abnormal sleep recordings to automate sleep stage scoring, producing a hypnodensity graph-a probability distribution conveying more information than classical hypnograms. Accuracy of sleep stage scoring was validated in 70 subjects assessed by six scorers. The best model performed better than any individual scorer (87% versus consensus). It also reliably scores sleep down to 5 s instead of 30 s scoring epochs. A T1N marker based on unusual sleep stage overlaps achieved a specificity of 96% and a sensitivity of 91%, validated in independent datasets. Addition of HLA-DQB1*06:02 typing increased specificity to 99%. Our method can reduce time spent in sleep clinics and automates T1N diagnosis. It also opens the possibility of diagnosing T1N using home sleep studies.

5.
BMC Bioinformatics ; 19(Suppl 20): 509, 2018 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-30577803

RESUMO

BACKGROUND: Sequencing highly-variable 16S regions is a common and often effective approach to the study of microbial communities, and next-generation sequencing (NGS) technologies provide abundant quantities of data for analysis. However, the speed of existing analysis pipelines may limit our ability to work with these quantities of data. Furthermore, the limited coverage of existing 16S databases may hamper our ability to characterise these communities, particularly in the context of complex or poorly studied environments. RESULTS: In this article we present the SigClust algorithm, a novel clustering method involving the transformation of sequence reads into binary signatures. When compared to other published methods, SigClust yields superior cluster coherence and separation of metagenomic read data, while operating within substantially reduced timeframes. We demonstrate its utility on published Illumina datasets and on a large collection of labelled wound reads sourced from patients in a wound clinic. The temporal analysis is based on tracking the dominant clusters of wound samples over time. The analysis can identify markers of both healing and non-healing wounds in response to treatment. Prominent clusters are found, corresponding to bacterial species known to be associated with unfavourable healing outcomes, including a number of strains of Staphylococcus aureus. CONCLUSIONS: SigClust identifies clusters rapidly and supports an improved understanding of the wound microbiome without reliance on a reference database. The results indicate a promising use for a SigClust-based pipeline in wound analysis and prediction, and a possible novel method for wound management and treatment.


Assuntos
Análise de Dados , Metagenômica/métodos , Algoritmos , Análise por Conglomerados , Humanos , Microbiota/genética
6.
Conf Proc IEEE Eng Med Biol Soc ; 2018: 566-569, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30440460

RESUMO

Recent progress in tissue clearing allows the imaging of entire organs at single-cell resolution. A necessary step in analysing these images is registration across samples. Existing methods of registration were developed for lower resolution image modalities (e.g., MRI) and it is unclear whether their performance and accuracy is satisfactory at this larger scale (several gigabytes for a whole mouse brain). In this study, we evaluated five freely available image registration tools. We used several performance metrics to assess accuracy, and completion time as a measure of efficiency. The results of this evaluation suggest that ANTS provides the best registration accuracy, while Elastix has the highest computational efficiency among the methods with an acceptable accuracy. The results also highlight the need to develop new registration methods optimised for these high-resolution 3D images.

7.
F1000Res ; 7: 1286, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30271588

RESUMO

Biological networks are highly modular and contain a large number of clusters, which are often associated with a specific biological function or disease. Identifying these clusters, or modules, is therefore valuable, but it is not trivial. In this article we propose a recursive method based on the Louvain algorithm for community detection and the PageRank algorithm for authoritativeness weighting in networks. PageRank is used to initialise the weights of nodes in the biological network; the Louvain algorithm with the Newman-Girvan criterion for modularity is then applied to the network to identify modules. Any identified module with more than k nodes is further processed by recursively applying PageRank and Louvain, until no module contains more than k nodes (where k is a parameter of the method, no greater than 100). This method is evaluated on a heterogeneous set of six biological networks from the Disease Module Identification DREAM Challenge. Empirical findings suggest that the method is effective in identifying a large number of significant modules, although with substantial variability across restarts of the method.

8.
Cell Rep ; 24(9): 2231-2247.e7, 2018 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-30157420

RESUMO

Sleep regulation involves interdependent signaling among specialized neurons in distributed brain regions. Although acetylcholine promotes wakefulness and rapid eye movement (REM) sleep, it is unclear whether the cholinergic pathway is essential (i.e., absolutely required) for REM sleep because of redundancy from neural circuits to molecules. First, we demonstrate that synaptic inhibition of TrkA+ cholinergic neurons causes a severe short-sleep phenotype and that sleep reduction is mostly attributable to a shortened sleep duration in the dark phase. Subsequent comprehensive knockout of acetylcholine receptor genes by the triple-target CRISPR method reveals that a similar short-sleep phenotype appears in the knockout of two Gq-type acetylcholine receptors Chrm1 and Chrm3. Strikingly, Chrm1 and Chrm3 double knockout chronically diminishes REM sleep to an almost undetectable level. These results suggest that muscarinic acetylcholine receptors, Chrm1 and Chrm3, are essential for REM sleep.

9.
IEEE Rev Biomed Eng ; 11: 53-67, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29993607

RESUMO

The market for smartphones, smartwatches, and wearable devices is booming. In recent years, individuals and researchers have used these devices as additional tools to monitor and track sleep, physical activity, and behavior. Their use in sleep research and clinical applications could address the difficulties in scaling up studies that rely on polysomnography, the gold-standard. However, the use of commercial devices for large-scale sleep studies is not without challenges. With this in mind, this paper presents an extensive review of sleep monitoring systems and the techniques used in their development. We also discuss their performance in terms of reliability and validity, and consider the needs and expectations of users, whether they are experts, patients, or the general public. Through this review, we highlight a number of challenges with current studies: a lack of standard evaluation methods for consumer-grade devices (e.g., reliability and validity assessment); limitations in the populations studied; consumer expectations of monitoring devices; constraints on the resources of consumer-grade devices (e.g., power consumption).

10.
Neuron ; 90(1): 70-85, 2016 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-26996081

RESUMO

The detailed molecular mechanisms underlying the regulation of sleep duration in mammals are still elusive. To address this challenge, we constructed a simple computational model, which recapitulates the electrophysiological characteristics of the slow-wave sleep and awake states. Comprehensive bifurcation analysis predicted that a Ca(2+)-dependent hyperpolarization pathway may play a role in slow-wave sleep and hence in the regulation of sleep duration. To experimentally validate the prediction, we generate and analyze 21 KO mice. Here we found that impaired Ca(2+)-dependent K(+) channels (Kcnn2 and Kcnn3), voltage-gated Ca(2+) channels (Cacna1g and Cacna1h), or Ca(2+)/calmodulin-dependent kinases (Camk2a and Camk2b) decrease sleep duration, while impaired plasma membrane Ca(2+) ATPase (Atp2b3) increases sleep duration. Pharmacological intervention and whole-brain imaging validated that impaired NMDA receptors reduce sleep duration and directly increase the excitability of cells. Based on these results, we propose a hypothesis that a Ca(2+)-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals.


Assuntos
Sinalização do Cálcio/genética , Cálcio/metabolismo , Sono/genética , Animais , Canais de Cálcio Tipo T/genética , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Simulação por Computador , Maleato de Dizocilpina/farmacologia , Eletroencefalografia , Eletromiografia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Fenciclidina/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Sono/efeitos dos fármacos , Sono REM/efeitos dos fármacos , Sono REM/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Fatores de Tempo
11.
Cell Rep ; 14(3): 662-677, 2016 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-26774482

RESUMO

The identification of molecular networks at the system level in mammals is accelerated by next-generation mammalian genetics without crossing, which requires both the efficient production of whole-body biallelic knockout (KO) mice in a single generation and high-performance phenotype analyses. Here, we show that the triple targeting of a single gene using the CRISPR/Cas9 system achieves almost perfect KO efficiency (96%-100%). In addition, we developed a respiration-based fully automated non-invasive sleep phenotyping system, the Snappy Sleep Stager (SSS), for high-performance (95.3% accuracy) sleep/wake staging. Using the triple-target CRISPR and SSS in tandem, we reliably obtained sleep/wake phenotypes, even in double-KO mice. By using this system to comprehensively analyze all of the N-methyl-D-aspartate (NMDA) receptor family members, we found Nr3a as a short-sleeper gene, which is verified by an independent set of triple-target CRISPR. These results demonstrate the application of mammalian reverse genetics without crossing to organism-level systems biology in sleep research.


Assuntos
Receptores de N-Metil-D-Aspartato/genética , Genética Reversa , Sono/fisiologia , Vigília/fisiologia , Animais , Sistemas CRISPR-Cas/genética , Eletroencefalografia , Eletromiografia , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monofenol Mono-Oxigenase/deficiência , Monofenol Mono-Oxigenase/genética , Fenótipo , Receptores de N-Metil-D-Aspartato/metabolismo
12.
Nat Protoc ; 10(11): 1709-27, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26448360

RESUMO

Here we describe a protocol for advanced CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis). The CUBIC protocol enables simple and efficient organ clearing, rapid imaging by light-sheet microscopy and quantitative imaging analysis of multiple samples. The organ or body is cleared by immersion for 1-14 d, with the exact time required dependent on the sample type and the experimental purposes. A single imaging set can be completed in 30-60 min. Image processing and analysis can take <1 d, but it is dependent on the number of samples in the data set. The CUBIC clearing protocol can process multiple samples simultaneously. We previously used CUBIC to image whole-brain neural activities at single-cell resolution using Arc-dVenus transgenic (Tg) mice. CUBIC informatics calculated the Venus signal subtraction, comparing different brains at a whole-organ scale. These protocols provide a platform for organism-level systems biology by comprehensively detecting cells in a whole organ or body.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Imagem Óptica/métodos , Patologia/métodos , Animais , Encéfalo/patologia , Camundongos
13.
Cell ; 159(4): 911-24, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25417165

RESUMO

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.


Assuntos
Amino Álcoois/análise , Análise de Célula Única/métodos , Imagem Corporal Total/métodos , Animais , Diabetes Mellitus/patologia , Imagem Tridimensional/métodos , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
14.
Biophys J ; 107(6): 1462-73, 2014 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-25229153

RESUMO

Models of the mammalian clock have traditionally been based around two feedback loops-the self-repression of Per/Cry by interfering with activation by BMAL/CLOCK, and the repression of Bmal/Clock by the REV-ERB proteins. Recent experimental evidence suggests that the D-box, a transcription factor binding site associated with daytime expression, plays a larger role in clock function than has previously been understood. We present a simplified clock model that highlights the role of the D-box and illustrate an approach for finding maximum-entropy ensembles of model parameters, given experimentally imposed constraints. Parameter variability can be mitigated using prior probability distributions derived from genome-wide studies of cellular kinetics. Our model reproduces predictions concerning the dual regulation of Cry1 by the D-box and Rev-ErbA/ROR response element (RRE) promoter elements and allows for ensemble-based predictions of phase response curves (PRCs). Nonphotic signals such as Neuropeptide Y (NPY) may act by promoting Cry1 expression, whereas photic signals likely act by stimulating expression from the E/E' box. Ensemble generation with parameter probability restraints reveals more about a model's behavior than a single optimal parameter set.


Assuntos
Relógios Circadianos , Regulação da Expressão Gênica , Modelos Biológicos , Animais , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/deficiência , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Biologia de Sistemas
15.
Cell ; 157(3): 726-39, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24746791

RESUMO

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.


Assuntos
Neuroimagem/métodos , Animais , Encéfalo/citologia , Callithrix , Indicadores e Reagentes/química , Camundongos , Microscopia/métodos
16.
Artigo em Inglês | MEDLINE | ID: mdl-24109756

RESUMO

In providing simultaneous information on expression profiles for thousands of genes, microarray technologies have, in recent years, been largely used to investigate mechanisms of gene expression. Clustering and classification of such data can, indeed, highlight patterns and provide insight on biological processes. A common approach is to consider genes and samples of microarray datasets as nodes in a bipartite graphs, where edges are weighted e.g. based on the expression levels. In this paper, using a previously-evaluated weighting scheme, we focus on search algorithms and evaluate, in the context of biclustering, several variations of Genetic Algorithms. We also introduce a new heuristic "Propagate", which consists in recursively evaluating neighbour solutions with one more or one less active conditions. The results obtained on three well-known datasets show that, for a given weighting scheme, optimal or near-optimal solutions can be identified.


Assuntos
Algoritmos , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Análise em Microsséries , Inteligência Artificial , Bases de Dados Genéticas , Modelos Teóricos
17.
Adv Exp Med Biol ; 696: 377-84, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21431578

RESUMO

Biomedical systems involve a large number of entities and intricate interactions between these. Their direct analysis is, therefore, difficult, and it is often necessary to rely on computational models. These models require significant resources and parallel computing solutions. These approaches are particularly suited, given parallel aspects in the nature of biomedical systems. Model hybridisation also permits the integration and simultaneous study of multiple aspects and scales of these systems, thus providing an efficient platform for multidisciplinary research.


Assuntos
Biologia Computacional/métodos , Animais , Biologia Computacional/estatística & dados numéricos , Simulação por Computador , Mineração de Dados , Humanos , Modelos Biológicos , Modelos Imunológicos , Modelos Estatísticos , Software , Biologia de Sistemas , Integração de Sistemas
18.
PLoS One ; 5(11): e14031, 2010 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-21152421

RESUMO

Characterization of the epigenetic profile of humans since the initial breakthrough on the human genome project has strongly established the key role of histone modifications and DNA methylation. These dynamic elements interact to determine the normal level of expression or methylation status of the constituent genes in the genome. Recently, considerable evidence has been put forward to demonstrate that environmental stress implicitly alters epigenetic patterns causing imbalance that can lead to cancer initiation. This chain of consequences has motivated attempts to computationally model the influence of histone modification and DNA methylation in gene expression and investigate their intrinsic interdependency. In this paper, we explore the relation between DNA methylation and transcription and characterize in detail the histone modifications for specific DNA methylation levels using a stochastic approach.


Assuntos
Biologia Computacional/métodos , Metilação de DNA , Histonas/metabolismo , Modelos Genéticos , Acetilação , Algoritmos , Epigênese Genética , Epigenômica/métodos , Humanos , Metilação , Fosforilação , Processamento de Proteína Pós-Traducional , Transcrição Genética
19.
Immunome Res ; 6 Suppl 1: S3, 2010 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-20875154

RESUMO

BACKGROUND: Recent advances in Immunology highlighted the importance of local properties on the overall progression of HIV infection. In particular, the gastrointestinal tract is seen as a key area during early infection, and the massive cell depletion associated with it may influence subsequent disease progression. This motivated the development of a large-scale agent-based model. RESULTS: Lymph nodes are explicitly implemented, and considerations on parallel computing permit large simulations and the inclusion of local features. The results obtained show that GI tract inclusion in the model leads to an accelerated disease progression, during both the early stages and the long-term evolution, compared to a theoretical, uniform model. CONCLUSIONS: These results confirm the potential of treatment policies currently under investigation, which focus on this region. They also highlight the potential of this modelling framework, incorporating both agent-based and network-based components, in the context of complex systems where scaling-up alone does not result in models providing additional insights.

20.
J Theor Biol ; 264(2): 570-7, 2010 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-20219476

RESUMO

Epigenetic changes correspond to heritable modifications of the chromatin structure, which do not involve any alteration of the DNA sequence but nonetheless affect gene expression. These mechanisms play an important role in cell differentiation, but aberrant occurrences are also associated with a number of diseases, including cancer and neural development disorders. In particular, aberrant DNA methylation induced by H. Pylori has been found to be a significant risk factor in gastric cancer. To investigate the sensitivity of different genes and cell types to this infection, a computational model of methylation in gastric crypts is developed. In this article, we review existing results from physical experiments and outline their limitations, before presenting the computational model and investigating the influence of its parameters.


Assuntos
Metilação de DNA , Infecções por Helicobacter/complicações , Modelos Genéticos , Neoplasias Gástricas/genética , Simulação por Computador , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Helicobacter pylori/fisiologia , Interações Hospedeiro-Patógeno , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/microbiologia , Neoplasias Gástricas/complicações , Neoplasias Gástricas/patologia
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