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Single-cell RNA-seq (scRNA-seq) analysis of multiple samples separately can be costly and lead to batch effects. Exogenous barcodes or genome-wide RNA mutations can be used to demultiplex pooled scRNA-seq data, but they are experimentally or computationally challenging and limited in scope. Mitochondrial genomes are small but diverse, providing concise genotype information. We developed "mitoSplitter," an algorithm that demultiplexes samples using mitochondrial RNA (mtRNA) variants, and demonstrated that mtRNA variants can be used to demultiplex large-scale scRNA-seq data. Using affordable computational resources, mitoSplitter can accurately analyze 10 samples and 60,000 cells in 6 h. To avoid the batch effects from separated experiments, we applied mitoSplitter to analyze the responses of five non-small cell lung cancer cell lines to BET (Bromodomain and extraterminal) chemical degradation in a multiplexed fashion. We found the synthetic lethality of TOP2A inhibition and BET chemical degradation in BET inhibitor-resistant cells. The result indicates that mitoSplitter can accelerate the application of scRNA-seq assays in biomedical research.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , RNA Mitocondrial , Análise da Expressão Gênica de Célula Única , Mitocôndrias/genéticaRESUMO
Efficient molecular selection is a prerequisite for generating molecular tools used in diagnosis, pathology, vaccinology, and therapeutics. Selection efficiency is thermodynamically highly dependent on the dissociation equilibrium that can be reached in a single round. Extreme shifting of equilibrium towards dissociation favors the retention of high-affinity ligands over those with lower affinity, thus improving the selection efficiency. We propose to synergize dual effects by deterministic lateral-displacement microfluidics, including the collision-based force effect and the two-dimensional (2D) separation-based concentration effect, to greatly shift the equilibrium. Compared with previous approaches, this system can remove more low- or moderate-affinity ligands and maintain most high-affinity ligands, thereby improving affinity discrimination in selection. This strategy is demonstrated on phage display in both experiment and simulation, and two peptides against tumor markers ephrin type-A receptor 2 (EphA2) and CD71 were obtained with high affinity and specificity within a single round of selection, which offers a promising direction for discovery of robust binding ligands for a wide range of biomedical applications.
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Microfluídica , Peptídeos , Biomarcadores Tumorais , Efrinas , Ligantes , Peptídeos/químicaRESUMO
Single-cell RNA-sequencing (scRNA-seq) has become a powerful tool for biomedical research by providing a variety of valuable information with the advancement of computational tools. Lineage analysis based on scRNA-seq provides key insights into the fate of individual cells in various systems. However, such analysis is limited by several technical challenges. On top of the considerable computational expertise and resources, these analyses also require specific types of matching data such as exogenous barcode information or bulk assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) data. To overcome these technical challenges, we developed a user-friendly computational algorithm called "LINEAGE" (label-free identification of endogenous informative single-cell mitochondrial RNA mutation for lineage analysis). Aiming to screen out endogenous markers of lineage located on mitochondrial reads from label-free scRNA-seq data to conduct lineage inference, LINEAGE integrates a marker selection strategy by feature subspace separation and de novo "low cross-entropy subspaces" identification. In this process, the mutation type and subspace-subspace "cross-entropy" of features were both taken into consideration. LINEAGE outperformed three other methods, which were designed for similar tasks as testified with two standard datasets in terms of biological accuracy and computational efficiency. Applied on a label-free scRNA-seq dataset of BRAF-mutated cancer cells, LINEAGE also revealed genes that contribute to BRAF inhibitor resistance. LINEAGE removes most of the technical hurdles of lineage analysis, which will remarkably accelerate the discovery of the important genes or cell-lineage clusters from scRNA-seq data.
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Linhagem da Célula/genética , RNA Mitocondrial/genética , Análise de Sequência de RNA/métodos , Algoritmos , Animais , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação/genética , RNA/análise , Análise de Célula Única/métodos , Sequenciamento do Exoma/métodosRESUMO
Multimodal measurement of single cells provides deep insights into the intricate relationships between individual molecular layers and the regulatory mechanisms underlying intercellular variations. Here, we reported DMF-DM-seq, a highly integrated, sensitive, and automated platform for single-cell mRNA and microRNA (miRNA) co-sequencing based on digital microfluidics. This platform first integrates the processes of single-cell isolation, lysis, component separation, and simultaneous sequencing library preparation of mRNA and miRNA within a single DMF device. Compared with the current half-cell measuring strategy, DMF-DM-seq enables complete separation of single-cell mRNA and miRNA via a magnetic field application, resulting in a higher miRNA detection ability. DMF-DM-seq revealed differential expression patterns of single cells of noncancerous breast cells and noninvasive and aggressive breast cancer cells at both mRNA and miRNA levels. The results demonstrated the anticorrelated relationship between miRNA and their mRNA targets. Further, we unravel the tumor growth and metastasis-associated biological processes enriched by miRNA-targeted genes, along with important miRNA-interaction networks involved in significant signaling pathways. We also deconstruct the miRNA regulatory mechanisms underlying different signaling pathways across different breast cell types. In summary, DMF-DM-seq offers a powerful tool for a comprehensive study of the expression heterogeneity of single-cell mRNA and miRNA, which will be widely applied in basic and clinical research.
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MicroRNAs , RNA Mensageiro , Análise de Célula Única , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/análise , RNA Mensageiro/genética , Automação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Análise de Sequência de RNA , Linhagem Celular Tumoral , Microfluídica/métodosRESUMO
In this work, to promote the separation of photogenerated carriers, prevent the catalyst from photo-corrosion, and improve the photo-Fenton synergistic degradation of organic pollutants, the coating structure of FeOOH/BiO2-x rich in oxygen vacancies was successfully synthesized by a facile and environmentally friendly two-step process of hydrothermal and chemical deposition. Through a series of degradation activity tests of synthesized materials under different conditions, it was found that FeOOH/BiO2-x demonstrated outstanding organic pollutant degradation activity under visible and near-infrared light when hydrogen peroxide was added. After 90 min of reaction under photo-Fenton conditions, the degradation rate of Methylene Blue by FeOOH/BiO2-x was 87.4%, significantly higher than the degradation efficiency under photocatalysis (60.3%) and Fenton (49.0%) conditions. The apparent rate constants of FeOOH/BiO2-x under photo-Fenton conditions were 2.33 times and 3.32 times higher than photocatalysis and Fenton catalysis, respectively. The amorphous FeOOH was tightly coated on the layered BiO2-x, which significantly increased the specific surface area and the number of active sites of the composites, and facilitated the improvement of the separation efficiency of the photogenerated carriers and the prevention of photo-corrosion of BiO2-x. The analysis of the mechanism of photo-Fenton synergistic degradation clarified that ·OH, h+, and ·O2- are the main active substances involved in the degradation of pollutants. The optimal degradation conditions were the addition of the FeOOH/BiO2-x composite catalyst loaded with 20% Fe at a concentration of 0.5 g/L, the addition of hydrogen peroxide at a concentration of 8 mM, and an initial pH of 4. This outstanding catalytic system offers a fresh approach to the creation and processing of iron-based photo-Fenton catalysts by quickly and efficiently degrading various organic contaminants.
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A fluorescence method for the quantitative detection of chloramphenicol (CAP) has been developed using phosphate and fluorescent dye 6-carboxy-x-rhodamine (ROX) double-labeled aptamers of CAP and the bimetallic organic framework nanomaterial Cu/UiO-66. Cu/UiO-66 was prepared by coordinate bonding of metal organic framework (MOF) nanomaterial UiO-66 with copper ions. Cu/UiO-66 contains a large number of metal defect sites, which can be combined with phosphate-modified nucleic acid aptamers through strong coordination between phosphate and zirconium to form "fluorescence turn-on" sensors. In the absence of CAP, all single-stranded aptamers were adsorbed on the surface of Cu/UiO-66 through π-π stacking between single-stranded DNA and Cu/UiO-66, which brings the ROX fluorophores and Cu/UiO-66 into close proximity. The ROX fluorescence of aptamers was then quenched by Cu/UiO-66 through photoinduced electron transfer (PET). In the presence of CAP, however, CAP reacted with nucleic acid aptamers to form a special spatial structure, in which the ROX fluorophores were far away from the MOF surface via a change in the spatial structure of the aptamers, and the fluorescence of ROX was able to be recovered. The quantitative detection of CAP can be achieved by measuring the fluorescence signal of ROX using synchronous scanning fluorescence spectrometry. Under optimum conditions, the fluorescence intensities of ROX exhibit a good linear dependence on the concentration of CAP in the range of 0.2-10 nmol/L, with a detection limit of 0.09 nmol/L. The method has advantages of high sensitivity, good selectivity, and a low limit of detection. Graphical abstract.
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A novel Gram-staining-negative bacterium, designated DH-5T, was isolated from a farmland soil in Chuzhou, Anhui province, China. Cells of strain DH-5T were aerobic, non-motile, non-spore-forming and rod-shaped. The organism grew at 20-37 °C, pH 6.0-9.0 and with 0-5 % NaCl (w/v). The DNA G+C content was 42.8 mol%. The major fatty acids (>5 %) were iso-C15 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C17 : 0 3-OH and C16 : 0. The respiratory quinone was MK-7, and the major polar lipids were phosphatidylethanolamine and phosphoglycolipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DH-5T was a member of the genus Sphingobacterium and shared the highest similarity with Sphingobacterium gobiense H7T (96.0 %), followed by Sphingobacterium arenae H-12T (94.5 %). Strain DH-5T exhibited low DNA-DNA relatedness with S. gobiense H7T (35.1±1.4 %) and S. arenae H-12T (21.4±1.0 %). On the basis of phenotypic, genotypic and phylogenetic evidence, DH-5T is considered to represent a novel species of the genus Sphingobacterium, for which the name Sphingobacterium chuzhouense sp. nov. is proposed. The type strain is DH-5T (=ACCC 19856T=KCTC 42746T).
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Fazendas , Filogenia , Microbiologia do Solo , Sphingobacterium/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Sphingobacterium/genética , Sphingobacterium/isolamento & purificação , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
A yellow-pigmented bacterial strain, designated Y2T, was isolated from farmland soil in Bengbu, Anhui province, China. Cells of strain Y2T were Gram-stain-negative, strictly aerobic, non-motile and rod-shaped. Strain Y2T grew optimally at pH 7.0, 30 °C and in the presence of 2 % (w/v) NaCl. The DNA G+C content was 68.9âmol%. The major fatty acids (>5 %) were iso-C15 : 0, iso-C17 : 0, summed feature 9 (C16 : 0 10-methyl and/or iso-C17 : 1ω9c), iso-C11 : 0 3-OH and iso-C11 : 0. The major respiratory quinone was ubiquinone-8 (Q-8), and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Phylogenetic analysis of the 16S rRNA gene sequences showed that strain Y2T was most closely related to Luteimonas mephitis B1953/27.1T (99.1 % 16S rRNA gene sequence similarity), followed by Luteimonas lutimaris G3T (98.6 %), Luteimonas abyssi XH031T (96.2 %) and Luteimonas aquatica RIB1-20T (96.0 %). Strain Y2T exhibited low DNA-DNA relatedness with Luteimonas mephitis B1953/27.1T (43.6 ± 0.5 %) and Luteimonas lutimaris G3T (43.9 ± 2.1 %). On the basis of phenotypic, genotypic and phylogenetic evidence, strain Y2T represents a novel species of the genus Luteimonas, for which the name Luteimonas soli sp. nov. is proposed. The type strain is Y2T ( = ACCC 19799T = KCTC 42441T).
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Agricultura , Filogenia , Microbiologia do Solo , Xanthomonadaceae/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fosfolipídeos/química , Pigmentação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/química , Xanthomonadaceae/genética , Xanthomonadaceae/isolamento & purificaçãoRESUMO
OBJECTIVE: To explore the relationship between physical fitness index and executive function in Chinese adolescents, and to provide a reference for improving the development of executive function in Chinese adolescents. METHODS: From September to December 2023, 5336 adolescents aged 13 to 18 years were selected by stratified whole cluster random sampling method in six regions of China for physical fitness and executive function tests. The relationship between adolescent physical fitness index and executive function was analyzed using t-test, ANOVA, Pearson's correlation, and logistic regression. RESULTS: The correlation coefficients between adolescents' physical fitness index and inhibitory control reaction time were all 0.00094, p > 0.05; the correlation coefficients between adolescents' physical fitness index and working memory (1-back, 2-back) reaction time were -0.13 and -0.093, respectively, p < .05; the correlation coefficients between adolescents' physical fitness index and cognitive flexibility reaction time were -0.17 and -0.18, p < .05. Logistic regression analyses showed that 1-back, 2-back, and cognitive flexibility were significantly and positively correlated with physical fitness index in Models 1, 2, and 3 (all p values less than.01). The coefficients of inhibitory control were not significant in all three models (p > .05), and there was no significant relationship with physical fitness index. CONCLUSION: The physical fitness index of Chinese adolescents has a significant positive correlation with working memory and cognitive flexibility, but not with inhibitory control, i.e. the higher the physical fitness index, the better the working memory and cognitive flexibility.
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Single-cell microRNA (miRNA) sequencing has allowed for comprehensively studying the abundance and complex networks of miRNAs, which provides insights beyond single-cell heterogeneity into the dynamic regulation of cellular events. Current benchtop-based technologies for single-cell miRNA sequencing are low throughput, limited reaction efficiency, tedious manual operations, and high reagent costs. Here, a highly multiplexed, efficient, integrated, and automated sample preparation platform is introduced for single-cell miRNA sequencing based on digital microfluidics (DMF), named Hiper-seq. The platform integrates major steps and automates the iterative operations of miRNA sequencing library construction by digital control of addressable droplets on the DMF chip. Based on the design of hydrophilic micro-structures and the capability of handling droplets of DMF, multiple single cells can be selectively isolated and subject to sample processing in a highly parallel way, thus increasing the throughput and efficiency for single-cell miRNA measurement. The nanoliter reaction volume of this platform enables a much higher miRNA detection ability and lower reagent cost compared to benchtop methods. It is further applied Hiper-seq to explore miRNAs involved in the ossification of mouse skeletal stem cells after bone fracture and discovered unreported miRNAs that regulate bone repairing.
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MicroRNAs , Microfluídica , Animais , Camundongos , MicroRNAs/genética , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Metabolic RNA labeling-based time-resolved single-cell RNA sequencing (scRNA-seq) has provided unprecedented tools to dissect the temporal dynamics and the complex gene regulatory networks of gene expression. However, this technology fails to reveal the spatial organization of cells in tissues, which also regulates the gene expression by intercellular communication. Herein, it is demonstrated that integrating time-resolved scRNA-seq with spatial transcriptomics is a new paradigm for spatiotemporal analysis. Metabolic RNA labeling-based time-resolved Well-TEMP-seq is first applied to profile the transcriptional dynamics of glioblastoma (GBM) cells and discover two potential pathways of EZH2-mediated mesenchymal transition in GBM. With spatial transcriptomics, it is further revealed that the crosstalk between CCL2+ malignant cells and IL10+ tumor-associated macrophages in the tumor microenvironment through an EZH2-FOSL2-CCL2 axis contributes to the mesenchymal transition in GBM. These discoveries show the power of integrative spatiotemporal scRNA-seq to elucidate the complex gene regulatory mechanism and advance the understanding of cellular processes in disease.
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The spatial transcriptome has enabled researchers to resolve transcriptome expression profiles while preserving information about cell location to better understand the complex biological processes that occur in organisms. Due to technical limitations, the current high-throughput spatial transcriptome sequencing methods (known as next-generation sequencing with spatial barcoding methods or spot-based methods) cannot achieve single-cell resolution. A single measurement site, called a spot, in these technologies frequently contains multiple cells of various types. Computational tools for determining the cellular composition of a spot have emerged as a way to break through these limitations. These tools are known as deconvolution tools. Recently, a couple of deconvolution tools based on different strategies have been developed and have shown promise in different aspects. The resulting single-cell resolution expression profiles and/or single-cell composition of spots will significantly affect downstream data mining; thus, it is crucial to choose a suitable deconvolution tool. In this review, we present a list of currently available tools for spatial transcriptome deconvolution, categorize them based on the strategies they employ, and explain their advantages and limitations in detail in order to guide the selection of these tools in future studies.
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Single-cell RNA sequencing (scRNA-seq) reveals the transcriptional heterogeneity of cells, but the static snapshots fail to reveal the time-resolved dynamics of transcription. Herein, we develop Well-TEMP-seq, a high-throughput, cost-effective, accurate, and efficient method for massively parallel profiling the temporal dynamics of single-cell gene expression. Well-TEMP-seq combines metabolic RNA labeling with scRNA-seq method Well-paired-seq to distinguish newly transcribed RNAs marked by T-to-C substitutions from pre-existing RNAs in each of thousands of single cells. The Well-paired-seq chip ensures a high single cell/barcoded bead pairing rate (~80%) and the improved alkylation chemistry on beads greatly alleviates chemical conversion-induced cell loss (~67.5% recovery). We further apply Well-TEMP-seq to profile the transcriptional dynamics of colorectal cancer cells exposed to 5-AZA-CdR, a DNA-demethylating drug. Well-TEMP-seq unbiasedly captures the RNA dynamics and outperforms the splicing-based RNA velocity method. We anticipate that Well-TEMP-seq will be broadly applicable to unveil the dynamics of single-cell gene expression in diverse biological processes.
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Azacitidina , Alquilação , RNA/genética , Splicing de RNARESUMO
Identification of causative genes or genetic variants associated with phenotype traits benefits the genetic improvement of animals. CISH plays a role in immunity and growth, however, the upstream transcriptional factors of porcine CISH and the genetic variations in these factors remain unclear. In this study, we firstly identified the minimal core promoter of porcine CISH and confirmed the existence of STATx binding sites. Overexpression and RT-qPCR demonstrated STAT5A increased CISH transcriptional activity (P < 0.01) and mRNA expression (P < 0.01), while GATA1 inhibited CISH transcriptional activity (P < 0.01) and the following mRNA expression (P < 0.05 or P < 0.01). Then, the putative functional genetic variations of porcine STAT5A were screened and a PCR-SSCP was established for genotype g.508A>C and g.566C>T. Population genetic analysis showed the A allele frequency of g.508A>C and C allele frequency of g.566C>T was 0.61 and 0.94 in Min pigs, respectively, while these two alleles were fixed in the Landrace population. Statistical analysis showed that Min piglets with CC genotype at g.566C>T or Hap1: AC had higher 28-day body weight, 35-day body weight, and ADG than TC or Hap3: CT animals (P < 0.05, P < 0.05). Further luciferase activity assay demonstrated that the activity of g.508A>C in the C allele was lower than the A allele (P < 0.05). Collectively, the present study demonstrated that STAT5A positively regulated porcine CISH transcription, and SNP g.566C>T in the STAT5A was associated with the Min piglet growth trait.
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Liquid biopsy offers great promise for noninvasive cancer diagnostics, while the lack of adequate target characterization and analysis hinders its wide application. Single-cell RNA sequencing (scRNA-seq) is a powerful technology for cell characterization. Integrating scRNA-seq into a CTC-focused liquid biopsy study can perhaps classify CTCs by their original lesions. However, the lack of CTC scRNA-seq data accumulation and prior knowledge hinders further development. Therefore, we design CTC-Tracer, a transfer learning-based algorithm, to correct the distributional shift between primary cancer cells and CTCs to transfer lesion labels from the primary cancer cell atlas to CTCs. The robustness and accuracy of CTC-Tracer are validated by 8 individual standard datasets. We apply CTC-Tracer on a complex dataset consisting of RNA-seq profiles of single CTCs, CTC clusters from a BRCA patient, and two xenografts, and demonstrate that CTC-Tracer has potential in knowledge transfer between different types of RNA-seq data of lesions and CTCs.
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Células Neoplásicas Circulantes , Humanos , Células Neoplásicas Circulantes/metabolismo , Biópsia Líquida , Aprendizado de MáquinaRESUMO
Human adenoviruses type 26 (HAdV26) and type 35 (HAdV35) have increasingly become the choice of adenovirus vectors for vaccine application. However, the population pre-existing immunity to these two adenoviruses in China, which may reduce vaccine efficacy, remains largely unknown. Here, we established micro-neutralizing (MN) assays to investigate the seroprevalence of neutralizing antibodies (nAbs) against HAdV26 and HAdV35 in the general population of Guangdong and Shandong provinces, China. A total of 1184 serum samples were collected, 47.0% and 15.8% of which showed HAdV26 and HAdV35 nAb activity, respectively. HAdV26-seropositive individuals tended to have more moderate nAbs titers (201-1000), while HAdV35-seropositive individuals appeared to have more low nAbs titers (72-200). The seropositive rates of HAdV26 and HAdV35 in individuals younger than 20 years old were very low. The seropositive rates of HAdV26 increased with age before 70 years old and decreased thereafter, while HAdV35 seropositive rates did not show similar characteristics. Notably, the seropositive rates and nAb levels of both HAdV26 and HAdV35 were higher in Guangdong Province than in Shandong Province, but did not exert significant differences between males and females. The seroprevalence between HAdV26 and HAdV35 showed little correlation, and no significant cross-neutralizing activity was detected. These results clarified the characteristics of the herd immunity against HAdV26 and HAdV35, and provided information for the rational development and application of HAdV26 and HAdV35 as vaccine vectors in China.
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Adenovírus Humanos , Anticorpos Neutralizantes , Adenoviridae , Adulto , Idoso , Anticorpos Antivirais , China/epidemiologia , Feminino , Humanos , Masculino , Estudos Soroepidemiológicos , Adulto JovemRESUMO
Lipidomics is a younger member of the "omics" family. It aims to profile lipidome alterations occurring in biological systems. Similar to the other "omics", lipidomic data is highly dimensional and contains a massive amount of information awaiting deciphering and data mining. Currently, the available bioinformatic tools targeting lipidomic data processing and lipid pathway analysis are limited. A few tools designed for lipidomic analysis perform only basic statistical analyses, and lipid pathway analyses rely heavily on public databases (KEGG, Reactome, and HMDB). Due to the inadequate understanding of lipid signaling and metabolism, the use of public databases for lipid pathway analysis can be biased and misleading. Instead of using public databases to interpret lipidomic ontology, the authors introduce an intra-omic integrative correlation strategy for lipidomic data mining. Such an intra-omic strategy allows researchers to unscramble and predict lipid biological functions from correlated genomic ontological results using statistical approaches. To simplify and improve the lipidomic data processing experience, they designed an interactive web-based tool: LINT-web (http://www.lintwebomics.info/) to perform the intra-omic analysis strategy, and validated the functions of LINT-web using two biological systems. Users without sophisticated statistical experience can easily process lipidomic datasets and predict the potential lipid biological functions using LINT-web.
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Biologia Computacional/métodos , Mineração de Dados/métodos , Lipidômica , Internet , Metabolismo dos Lipídeos , Software , Interface Usuário-ComputadorRESUMO
Biomarkers based on DNA methylation have attracted wide attention in biomedical research due to their potential clinical value. Therefore, a sensitive and accurate method for DNA methylation detection is highly desirable for the discovery and diagnostics of human diseases, especially cancers. Here, an integrated, low-cost, and portable point-of-care (POC) device is presented to analyze DNA methylation, which integrates the process of pyrosequencing in a digital microfluidic chip. Without additional equipment and complicated operation, droplets are manipulated by patterned electrodes with individually programmed control. The system exhibited an excellent sensitivity with a limit of detection (LOD) of 10 pg and a comparable checkout down to 5% methylation level within 30 min, which offered a potential substitute for the detection of DNA methylation. With the advantages of portability, ease of use, high accuracy, and low cost, the POC platform shows great potential for the analysis of tumor-specific circulating DNA.
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Automação , DNA/análise , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Sistemas Automatizados de Assistência Junto ao Leito , Biomarcadores/análise , DNA/genética , Metilação de DNA , Humanos , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Carbamate hydrolase is the initial and key enzyme for degradation of carbamate pesticides. In the present study, we report the isolation of a carbaryl-degrading strain Pseudomonas sp. XWY-1, the cloning of its carbaryl hydrolase gene (mcbA) and the characterization of McbA. Strain XWY-1 was able to utilize carbaryl as a sole carbon source and degrade it using 1-naphthol as an intermediate. Transposon mutagenesis identified a mutant of XWY-1M that was unable to hydrolyze carbaryl. The transposon-disrupted gene mcbA was cloned by self-formed adaptor PCR, then expressed in Escherichia coli BL21(DE3) and purified. McbA was able to hydrolyze carbamate pesticides including carbaryl, isoprocarb, fenobucarb, carbofuran efficiently, while it hydrolyzed aldicarb, and propoxur poorly. The optimal pH of McbA was 7.0 and the optimal temperature was 40°C. The apparent Km and kcat values of McbA for carbaryl were 77.67±12.31µM and 2.12±0.10s-1, respectively. Three amino acid residues (His467, His477 and His504) in the predicted polymerase/histidinol phosphatase-like domain were shown to be closely related to the activity of McbA, with His504 being the most important, as a replacement of His504 led to the complete loss of activity. This is the first study to identify key amino acids in McbA.
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Amidoidrolases/genética , Aminoácidos/metabolismo , Carbaril/metabolismo , Amidoidrolases/química , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Hidrólise , Especificidade por SubstratoRESUMO
A novel carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC) degrading strain SD-4 was isolated and identified preliminarily as Mycobacterium sp. according to its phenotypic features and phylogenetic analysis. This strain could utilize MBC as the sole carbon and nitrogen sources for growth and degrade 50mgL-1 MBC at the average degradation rate of 0.63mgL-1h-1. Strain SD-4 degraded MBC through the typical pathway, in which MBC was first hydrolyzed by MheI to 2-aminobenzimidazole (2-AB) and then converted to 2-hydroxybenzimidazole (2-HB). The MBC hydrolase encoding gene mheI was cloned from strain SD-4 and successfully expressed in Escherichia coli by codon optimization. The sulfhydryl-blocking assay revealed that the activity of MheI was closely related to cysteine, and the site-directed mutation experiment showed that Cys16 and Cys222 played important roles during the hydrolysis of MBC by MheI. Therefore they affected its activity directly and were defined as the key amino acid sites.