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1.
Microbiology ; 2019 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-31585061

RESUMO

Microbial biofilms are ubiquitous in drinking water systems, yet our understanding of drinking water biofilms lags behind our understanding of those in other environments. Here, a six-member model bacterial community was used to identify the interactions and individual contributions of each species to community biofilm formation. These bacteria were isolated from the International Space Station potable water system and include Cupriavidus metallidurans, Chryseobacterium gleum, Ralstonia insidiosa, Ralstonia pickettii, Methylorubrum (Methylobacterium) populi and Sphingomonas paucimobilis, but all six species are common members of terrestrial potable water systems. Using reconstituted assemblages, from pairs to all 6 members, community biofilm formation was observed to be robust to the absence of any single species and only removal of the C. gleum/S. paucimobilis pair, out of all 15 possible 2-species subtractions, led to loss of community biofilm formation. In conjunction with these findings, dual-species biofilm formation assays supported the view that the contribution of C. gleum to community biofilm formation was dependent on synergistic biofilm formation with either R. insidiosa or C. metallidurans. These data support a model of multiple, partially redundant species interactions to generate robustness in biofilm formation. A bacteriophage and multiple predatory bacteria were used to test the resilience of the community to the removal of individual members in situ, but the combination of precise and substantial depletion of a single target species was not achievable. We propose that this assemblage can be used as a tractable model to understand the molecular bases of the interactions described here and to decipher other functions of drinking water biofilms.

3.
J Biomol Tech ; 30(Suppl): S55, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31897010

RESUMO

As microbiome and metagenomic studies span nearly all aspects of science, the need to characterize the limitations and strengths of the full laboratory process is critical. This includes DNA and RNA extraction efficiencies, type of sequencing, and bioinformatics, to name a few. Presently, a commercial accurately enumerated whole cell microbial standard does not exist, except for the MGRG WCM Standard that is near completion. The minimum design characteristics included 1) accuracy cellular enumeration of each species, 2) intact cellular integrity and morphology that came be confirmed by microscopy and staining, 3) preserved, 4) fully sequenced genomes, 5) various Gram reaction and GC content, and 6) able to maintain cell wall digestion with lytic enzymes. In this study, we have performed initial experiments to characterize this standard using various kits and sequencing approaches. We will present both RNA and DNA extractions and sequence results as well as open up the discussion to the audience.

6.
Genome Biol ; 18(1): 182, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28934964

RESUMO

BACKGROUND: One of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole-genome shotgun sequencing data, comprehensive comparisons of these methods are limited. RESULTS: In this study, we use the largest-to-date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of 11 metagenomic classifiers. Tools were characterized on the basis of their ability to identify taxa at the genus, species, and strain levels, quantify relative abundances of taxa, and classify individual reads to the species level. Strikingly, the number of species identified by the 11 tools can differ by over three orders of magnitude on the same datasets. Various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species. Overall, pairing tools with different classification strategies (k-mer, alignment, marker) can combine their respective advantages. CONCLUSIONS: This study provides positive and negative controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision, accuracy, and recall. We show that proper experimental design and analysis parameters can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.


Assuntos
Benchmarking/métodos , Mapeamento de Sequências Contíguas/métodos , Código de Barras de DNA Taxonômico/métodos , Metagenoma , Análise de Sequência de DNA/métodos , Software , Benchmarking/normas , Mapeamento de Sequências Contíguas/normas , Código de Barras de DNA Taxonômico/normas , Humanos , Microbiota , Filogenia , Análise de Sequência de DNA/normas
7.
Nat Biotechnol ; 35(8): 725-731, 2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-28787424

RESUMO

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.


Assuntos
Genoma Arqueal/genética , Genômica/métodos , Metagenômica/métodos , Genoma Bacteriano/genética , Genômica/normas , Metagenômica/normas , Análise de Sequência de DNA
8.
Scientifica (Cairo) ; 2017: 1964087, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28409052

RESUMO

Background. Township hospitals in China provide rural communities with basic but much needed critical health care services. The doctors working in these hospitals often feel unsatisfied when considering their work schedules and financial rewards. Method. To explore job satisfaction of health workers in a township hospital, a qualitative study was conducted of 39 doctors from five township hospitals in Guangxi Zhuang Autonomous Region. The goal was to understand the level of job satisfaction of doctors and to make recommendations for improvements. Results. About 75% (28/39) of the doctors expressed negative attitudes related to their work conditions. Slightly more than half (22/39) mentioned they should receive greater compensation for their work and more than one were seriously considering other options. Many participants (35/39) showed their satisfaction about the achievement of serving as a doctor. Conclusion. Their main concerns related to job satisfaction included working conditions, financial rewards, and the doctor's relationships with patients. Increasing the incomes and fringe benefits of healthcare workers, improving their work conditions, and providing training and continuing education opportunities would help rural clinics retain doctors and eliminate the current unsatisfactory conditions. The findings also highlight the need for the government to increase financial support of township hospitals.

10.
J Biomol Tech ; 28(1): 31-39, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28337070

RESUMO

The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the world with the intent of discovering new species, genes, and gene clusters. Once a project site is complete, the resulting data will be publically available. Sites include Lake Hillier in Western Australia, the "Door to Hell" crater in Turkmenistan, deep ocean brine lakes of the Gulf of Mexico, deep ocean sediments from Greenland, permafrost tunnels in Alaska, ancient microbial biofilms from Antarctica, Blue Lagoon Iceland, Ethiopian toxic hot springs, and the acidic hypersaline ponds in Western Australia.


Assuntos
Microbiologia Ambiental , Microbiota/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Ambientes Extremos , Metagenoma , Tipagem Molecular/normas , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Padrões de Referência , Análise de Sequência de DNA/normas
11.
J Biomol Tech ; 28(1): 8-18, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28337071

RESUMO

Challenges and biases in preparing, characterizing, and sequencing DNA and RNA can have significant impacts on research in genomics across all kingdoms of life, including experiments in single-cells, RNA profiling, and metagenomics (across multiple genomes). Technical artifacts and contamination can arise at each point of sample manipulation, extraction, sequencing, and analysis. Thus, the measurement and benchmarking of these potential sources of error are of paramount importance as next-generation sequencing (NGS) projects become more global and ubiquitous. Fortunately, a variety of methods, standards, and technologies have recently emerged that improve measurements in genomics and sequencing, from the initial input material to the computational pipelines that process and annotate the data. Here we review current standards and their applications in genomics, including whole genomes, transcriptomes, mixed genomic samples (metagenomes), and the modified bases within each (epigenomes and epitranscriptomes). These standards, tools, and metrics are critical for quantifying the accuracy of NGS methods, which will be essential for robust approaches in clinical genomics and precision medicine.


Assuntos
Perfilação da Expressão Gênica/normas , Animais , DNA/genética , DNA/isolamento & purificação , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Metagenoma , Padrões de Referência , Análise de Sequência de DNA/normas , Software , Transcriptoma
12.
J Biomol Tech ; 28(1): 2-7, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28337073

RESUMO

The ability to sequence DNA outside of the laboratory setting has enabled novel research questions to be addressed in the field in diverse areas, ranging from environmental microbiology to viral epidemics. Here, we demonstrate the application of offline DNA sequencing of environmental samples using a hand-held nanopore sequencer in a remote field location: the McMurdo Dry Valleys, Antarctica. Sequencing was performed using a MK1B MinION sequencer from Oxford Nanopore Technologies (ONT; Oxford, United Kingdom) that was equipped with software to operate without internet connectivity. One-direction (1D) genomic libraries were prepared using portable field techniques on DNA isolated from desiccated microbial mats. By adequately insulating the sequencer and laptop, it was possible to run the sequencing protocol for up to 2½ h under arduous conditions.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Tipagem Molecular/instrumentação , Análise de Sequência de DNA/instrumentação , Regiões Antárticas , Bacteriófago lambda/genética , Clima Desértico , Microbiologia Ambiental , Padrões de Referência
13.
Cell Syst ; 1(1): 72-87, 2015 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26594662

RESUMO

The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station's history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.

14.
J Cell Physiol ; 230(8): 1929-43, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25573156

RESUMO

Glioblastoma (GBM), the most common primary adult malignant brain tumor, is associated with a poor prognosis due, in part, to tumor recurrence mediated by chemotherapy and radiation resistant glioma stem-like cells (GSCs). The metabolic and epigenetic state of GSCs differs from their non-GSC counterparts, with GSCs exhibiting greater glycolytic metabolism and global hypoacetylation. However, little attention has been focused on the potential use of acetate supplementation as a therapeutic approach. N-acetyl-l-aspartate (NAA), the primary storage form of brain acetate, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis, are significantly reduced in GBM tumors. We recently demonstrated that NAA supplementation is not an appropriate therapeutic approach since it increases GSC proliferation and pursued an alternative acetate source. The FDA approved food additive Triacetin (glyceryl triacetate, GTA) has been safely used for acetate supplementation therapy in Canavan disease, a leukodystrophy due to ASPA mutation. This study characterized the effects of GTA on the proliferation and differentiation of six primary GBM-derived GSCs relative to established U87 and U251 GBM cell lines, normal human cerebral cortical astrocytes, and murine neural stem cells. GTA reduced proliferation of GSCs greater than established GBM lines. Moreover, GTA reduced growth of the more aggressive mesenchymal GSCs greater than proneural GSCs. Although sodium acetate induced a dose-dependent reduction of GSC growth, it also reduced cell viability. GTA-mediated growth inhibition was not associated with differentiation, but increased protein acetylation. These data suggest that GTA-mediated acetate supplementation is a novel therapeutic strategy to inhibit GSC growth.


Assuntos
Antineoplásicos/farmacologia , Glioblastoma/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Triacetina/farmacologia , Adulto , Idoso , Animais , Astrócitos/efeitos dos fármacos , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Células-Tronco Neurais/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Cancer Cell ; 27(1): 41-56, 2015 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-25584893

RESUMO

Braf(V600E) induces benign, growth-arrested melanocytic nevus development, but also drives melanoma formation. Cdkn2a loss in Braf(V600E) melanocytes in mice results in rare progression to melanoma, but only after stable growth arrest as nevi. Immediate progression to melanoma is prevented by upregulation of miR-99/100, which downregulates mTOR and IGF1R signaling. mTORC1 activation through Stk11 (Lkb1) loss abrogates growth arrest of Braf(V600E) melanocytic nevi, but is insufficient for complete progression to melanoma. Cdkn2a loss is associated with mTORC2 and Akt activation in human and murine melanocytic neoplasms. Simultaneous Cdkn2a and Lkb1 inactivation in Braf(V600E) melanocytes results in activation of both mTORC1 and mTORC2/Akt, inducing rapid melanoma formation in mice. In this model, activation of both mTORC1/2 is required for Braf-induced melanomagenesis.


Assuntos
Melanoma Experimental/patologia , Complexos Multiproteicos/metabolismo , Nevo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Melanócitos/metabolismo , Melanoma Experimental/metabolismo , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Mutação , Nevo/patologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Neoplasias Cutâneas/patologia
18.
Nat Biotechnol ; 32(9): 915-925, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25150835

RESUMO

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.


Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transcriptoma
19.
PLoS One ; 8(11): e80714, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24278309

RESUMO

Cancer is associated with globally hypoacetylated chromatin and considerable attention has recently been focused on epigenetic therapies. N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate and ultimately acetyl-Coenzyme A for histone acetylation, are reduced in oligodendroglioma. The short chain triglyceride glyceryl triacetate (GTA), which increases histone acetylation and inhibits histone deacetylase expression, has been safely used for acetate supplementation in Canavan disease, a leukodystrophy due to ASPA mutation. We demonstrate that GTA induces cytostatic G0 growth arrest of oligodendroglioma-derived cells in vitro, without affecting normal cells. Sodium acetate, at doses comparable to that generated by complete GTA catalysis, but not glycerol also promoted growth arrest, whereas long chain triglycerides promoted cell growth. To begin to elucidate its mechanism of action, the effects of GTA on ASPA and acetyl-CoA synthetase protein levels and differentiation of established human oligodendroglioma cells (HOG and Hs683) and primary tumor-derived oligodendroglioma cells that exhibit some features of cancer stem cells (grade II OG33 and grade III OG35) relative to an oligodendrocyte progenitor line (Oli-Neu) were examined. The nuclear localization of ASPA and acetyl-CoA synthetase-1 in untreated cells was regulated during the cell cycle. GTA-mediated growth arrest was not associated with apoptosis or differentiation, but increased expression of acetylated proteins. Thus, GTA-mediated acetate supplementation may provide a safe, novel epigenetic therapy to reduce the growth of oligodendroglioma cells without affecting normal neural stem or oligodendrocyte progenitor cell proliferation or differentiation.


Assuntos
Acetatos/farmacologia , Antígenos/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Oligodendroglioma/patologia , Proteoglicanas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Acetilação/efeitos dos fármacos , Amidoidrolases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Humanos , Mesoderma/efeitos dos fármacos , Mesoderma/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Oligodendroglioma/enzimologia , Fenótipo , Transporte Proteico/efeitos dos fármacos
20.
J Gastrointest Surg ; 16(6): 1136-43, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22450950

RESUMO

BACKGROUND: To investigate the inhibitory role of pterostilbene in pancreatic cancer, we conducted a genomic analysis of pterostilbene-treated pancreatic cancer cells. We also investigated the effect of pterostilbene upon the carcinogenic markers, manganese superoxide dismutase, cytochrome C, Smac/DIABLO, and STAT3 phosphorylation in vitro. The antiproliferative effects of pterostilbene were further evaluated in an in vivo model. METHODS: Pancreatic cancer cells were treated with pterostilbene and evaluated with DNA microarray analysis. Pterostilbene-treated cells were analyzed for cytochrome C, Smac/DIABLO, manganese superoxide dismutase (MnSOD)/antioxidant activity, and STAT3 phosphorylation using ELISA. Data were statistically analyzed using ANOVA. Pterostilbene was then administered to nude mice for 8 weeks, and tumor growth rates were recorded and statistically analyzed. RESULTS: Microarray analysis of pterostilbene-treated cells revealed upregulation of pro-apoptosis genes. In vitro, pterostilbene treatment altered levels of phosphorylated STAT3, MnSOD/antioxidant activity, cytochrome C, and Smac/DIABLO. In nude mice, oral pterostilbene inhibited tumor growth rates. CONCLUSION: Pterostilbene alters gene expression in pancreatic cancer and increases the antiproliferative markers cytochrome C, Smac/DIABLO, and MnSOD/antioxidant activity. It was also shown to inhibit phosphorylated STAT3, a marker of accelerated tumorigenesis, and decrease pancreatic tumor growth in vivo. Further studies are warranted to elucidate the effects of pterostilbene in humans.


Assuntos
Proliferação de Células/efeitos dos fármacos , DNA de Neoplasias/análise , Genoma/efeitos dos fármacos , Neoplasias Experimentais , Neoplasias Pancreáticas/tratamento farmacológico , Estilbenos/uso terapêutico , Animais , Apoptose , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Pterocarpus , Regulação para Cima/efeitos dos fármacos
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