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1.
Front Cell Infect Microbiol ; 11: 716592, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368019

RESUMO

Bacteria modify their morphology in response to various factors including growth stage, nutrient availability, predation, motility and long-term survival strategies. Morphological changes may also be associated with specific physiological phenotypes such as the formation of dormant or persister cells in a "viable but non-culturable" (VBNC) state which frequently display different shapes and size compared to their active counterparts. Such dormancy phenotypes can display various degrees of tolerance to antibiotics and therefore a detailed understanding of these phenotypes is crucial for combatting chronic infections and associated diseases. Cell shape and size are therefore more than simple phenotypic characteristics; they are important physiological properties for understanding bacterial life-strategies and pathologies. However, quantitative studies on the changes to cell morphologies during bacterial growth, persister cell formation and the VBNC state are few and severely constrained by current limitations in the most used investigative techniques of flow cytometry (FC) and light or electron microscopy. In this study, we applied high-throughput Imaging Flow Cytometry (IFC) to characterise and quantify, at single-cell level and over time, the phenotypic heterogeneity and morphological changes in cultured populations of four bacterial species, Bacillus subtilis, Lactiplantibacillus plantarum, Pediococcus acidilactici and Escherichia coli. Morphologies in relation to growth stage and stress responses, cell integrity and metabolic activity were analysed. Additionally, we were able to identify and morphologically classify dormant cell phenotypes such as VBNC cells and monitor the resuscitation of persister cells in Escherichia coli following antibiotic treatment. We therefore demonstrate that IFC, with its high-throughput data collection and image capture capabilities, provides a platform by which a detailed understanding of changes in bacterial phenotypes and their physiological implications may be accurately monitored and quantified, leading to a better understanding of the role of phenotypic heterogeneity in the dynamic microbiome.


Assuntos
Bactérias , Escherichia coli , Escherichia coli/genética , Citometria de Fluxo , Viabilidade Microbiana , Fenótipo
2.
Arterioscler Thromb Vasc Biol ; 41(9): 2454-2468, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34261327

RESUMO

Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.


Assuntos
Sistema Cardiovascular/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sistema Cardiovascular/embriologia , Separação Celular , Microscopia Crioeletrônica , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Vesículas Extracelulares/genética , Vesículas Extracelulares/ultraestrutura , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
3.
Heart Rhythm ; 18(7): 1142-1150, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33781980

RESUMO

BACKGROUND: Cardiac device procedures require tissue dissection to free existing device lead(s). Common techniques include blunt dissection, standard electrocautery, and low-temperature electrocautery (PlasmaBlade, Medtronic); however, data on the type of electrosurgical tool used and the development of procedure- or lead-related adverse events are limited. OBJECTIVE: The purpose of this study was to determine whether standard or low-temperature electrocautery impacts the development of an adverse event. METHODS: We evaluated patients enrolled in WRAP-IT (Worldwide Randomized Antibiotic EnveloPe Infection PrevenTion Trial) undergoing cardiac implantable electronic device (CIED) revision, upgrade, or replacement. All adverse events were adjudicated by an independent physician committee. Data were analyzed using Cox proportional hazard regression modeling. RESULTS: In total, 5641 patients underwent device revision/upgrade/replacement. Electrocautery was used in 5205 patients (92.3%) (mean age 70.6 ± 12.7 years; 28.8% female), and low-temperature electrocautery was used in 1866 patients (35.9%). Compared to standard electrocautery, low-temperature electrocautery was associated with a 23% reduction in the incidence of a procedure- or lead-related adverse event through 3 years of follow up (hazard ratio [HR] 0.77; 95% confidence interval [CI] 0.65-0.91; P = .002). After controlling for the number of active leads, degree of capsulectomy, degree of lead dissection, and renal dysfunction, low-temperature electrocautery was associated with a 32% lower risk of lead-related adverse events (HR 0.68; 95% CI 0.52-0.89; P = .004). These effects were consistent across a spectrum of lead-related adverse event types. CONCLUSION: This study represents one of the largest assessments of electrocautery use in patients undergoing CIED revision, upgrade, or replacement procedures. Compared to standard electrocautery, low-temperature electrocautery significantly reduces adverse effects from these procedures.

4.
Microbiol Resour Announc ; 10(7)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602733

RESUMO

Weissella paramesenteroides has potential as an industrial biocatalyst due to its ability to produce lactic acid. A novel strain of W. paramesenteroides was isolated from ensiled sorghum. The genome was sequenced using a hybrid assembly of Oxford Nanopore and Illumina data to produce a 2-Mbp genome and 22-kbp plasmid sequence.

5.
PLoS Pathog ; 17(1): e1009194, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33439894

RESUMO

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.


Assuntos
Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Viabilidade Microbiana , Proteoma/metabolismo , Vibrio parahaemolyticus/crescimento & desenvolvimento , Vibrio parahaemolyticus/patogenicidade , Virulência , Células Cultivadas , Meios de Cultura , Regulação Bacteriana da Expressão Gênica , Proteoma/análise , Vibrioses/metabolismo , Vibrioses/microbiologia , Vibrio parahaemolyticus/metabolismo
6.
Sci Rep ; 10(1): 20727, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33244023

RESUMO

Size is a fundamental cellular trait that is important in determining phytoplankton physiological and ecological processes. Fossil coccospheres, the external calcite structure produced by the excretion of interlocking plates by the phytoplankton coccolithophores, can provide a rare window into cell size in the past. Coccospheres are delicate however and are therefore poorly preserved in sediment. We demonstrate a novel technique combining imaging flow cytometry and cross-polarised light (ISX+PL) to rapidly and reliably visually isolate and quantify the morphological characteristics of coccospheres from marine sediment by exploiting their unique optical and morphological properties. Imaging flow cytometry combines the morphological information provided by microscopy with high sample numbers associated with flow cytometry. High throughput imaging overcomes the constraints of labour-intensive manual microscopy and allows statistically robust analysis of morphological features and coccosphere concentration despite low coccosphere concentrations in sediments. Applying this technique to the fine-fraction of sediments, hundreds of coccospheres can be visually isolated quickly with minimal sample preparation. This approach has the potential to enable rapid processing of down-core sediment records and/or high spatial coverage from surface sediments and may prove valuable in investigating the interplay between climate change and coccolithophore physiological/ecological response.


Assuntos
Citometria de Fluxo/métodos , Sedimentos Geológicos/análise , Microscopia/métodos , Fitoplâncton/isolamento & purificação , Fitoplâncton/fisiologia , Carbonato de Cálcio/química , Fósseis
7.
Front Cell Infect Microbiol ; 10: 565975, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33194805

RESUMO

The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10-3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.


Assuntos
Campylobacter jejuni , Antibacterianos/farmacologia , Células Epiteliais , Oxirredução , Penicilinas/farmacologia
8.
Chem Commun (Camb) ; 56(52): 7108-7111, 2020 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-32458833

RESUMO

We report a method for embedding cell-free protein synthesis reactions in macro-scale hydrogel materials without a free liquid phase. This paper focuses on methods of preparation for a variety of hydrogels and an investigation of the impact that the hydrogel material has on cell-free protein synthesis.


Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Hidrogéis/metabolismo , Biossíntese de Proteínas/genética , Proteínas/genética , Tecidos Suporte/química , Extratos Celulares , Linhagem Celular , DNA/metabolismo , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polímeros/química , Polímeros/metabolismo , Sefarose/química , Sefarose/metabolismo
9.
Circ Arrhythm Electrophysiol ; 13(5): e008280, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32281393

RESUMO

BACKGROUND: Current understanding of the impact of cardiac implantable electronic device (CIED) infection is based on retrospective analyses from medical records or administrative claims data. The WRAP-IT (Worldwide Randomized Antibiotic Envelope Infection Prevention Trial) offers an opportunity to evaluate the clinical and economic impacts of CIED infection from the hospital, payer, and patient perspectives in the US healthcare system. METHODS: This was a prespecified, as-treated analysis evaluating outcomes related to major CIED infections: mortality, quality of life, disruption of CIED therapy, healthcare utilization, and costs. Payer costs were assigned using medicare fee for service national payments, while medicare advantage, hospital, and patient costs were derived from similar hospital admissions in administrative datasets. RESULTS: Major CIED infection was associated with increased all-cause mortality (12-month risk-adjusted hazard ratio, 3.41 [95% CI, 1.81-6.41]; P<0.001), an effect that sustained beyond 12 months (hazard ratio through all follow-up, 2.30 [95% CI, 1.29-4.07]; P=0.004). Quality of life was reduced (P=0.004) and did not normalize for 6 months. Disruptions in CIED therapy were experienced in 36% of infections for a median duration of 184 days. Mean costs were $55 547±$45 802 for the hospital, $26 867±$14 893, for medicare fee for service and $57 978±$29 431 for Medicare Advantage (mean hospital margin of -$30 828±$39 757 for medicare fee for service and -$6055±$45 033 for medicare advantage). Mean out-of-pocket costs for patients were $2156±$1999 for medicare fee for service, and $1658±$1250 for medicare advantage. CONCLUSIONS: This large, prospective analysis corroborates and extends understanding of the impact of CIED infections as seen in real-world datasets. CIED infections severely impact mortality, quality of life, healthcare utilization, and cost in the US healthcare system. Registration: URL: https://www.clinicaltrials.gov Unique Identifier: NCT02277990.


Assuntos
Antibacterianos/economia , Antibacterianos/uso terapêutico , Antibioticoprofilaxia/economia , Desfibriladores Implantáveis/economia , Custos de Cuidados de Saúde , Recursos em Saúde/economia , Marca-Passo Artificial/economia , Infecções Relacionadas à Prótese/economia , Infecções Relacionadas à Prótese/prevenção & controle , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/efeitos adversos , Antibioticoprofilaxia/efeitos adversos , Causas de Morte , Desfibriladores Implantáveis/efeitos adversos , Remoção de Dispositivo/economia , Custos de Medicamentos , Planos de Pagamento por Serviço Prestado/economia , Feminino , Gastos em Saúde , Custos Hospitalares , Humanos , Tempo de Internação/economia , Masculino , Medicare/economia , Pessoa de Meia-Idade , Marca-Passo Artificial/efeitos adversos , Readmissão do Paciente/economia , Estudos Prospectivos , Infecções Relacionadas à Prótese/microbiologia , Infecções Relacionadas à Prótese/mortalidade , Qualidade de Vida , Método Simples-Cego , Fatores de Tempo , Resultado do Tratamento , Estados Unidos
10.
EMBO Rep ; 21(4): e49493, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32147905

RESUMO

Science is key to developing sustainable products and solutions. But scientists also need to work more with governments, industry and society to help implement those solutions.


Assuntos
Governo
11.
Nat Commun ; 10(1): 4902, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653841

RESUMO

Genome-wide association studies (GWAS) have identified genetic variants associated with age-related macular degeneration (AMD), one of the leading causes of blindness in the elderly. However, it has been challenging to identify the cell types associated with AMD given the genetic complexity of the disease. Here we perform massively parallel single-cell RNA sequencing (scRNA-seq) of human retinas using two independent platforms, and report the first single-cell transcriptomic atlas of the human retina. Using a multi-resolution network-based analysis, we identify all major retinal cell types, and their corresponding gene expression signatures. Heterogeneity is observed within macroglia, suggesting that human retinal glia are more diverse than previously thought. Finally, GWAS-based enrichment analysis identifies glia, vascular cells, and cone photoreceptors to be associated with the risk of AMD. These data provide a detailed analysis of the human retina, and show how scRNA-seq can provide insight into cell types involved in complex, inflammatory genetic diseases.


Assuntos
Expressão Gênica , Degeneração Macular/genética , Neuroglia/metabolismo , Retina/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Neurônios Retinianos/metabolismo , Vasos Retinianos/citologia , Células Amácrinas/metabolismo , Astrócitos/metabolismo , Vasos Sanguíneos , Células Ependimogliais/metabolismo , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Microglia/metabolismo , Retina/metabolismo , Células Bipolares da Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Células Horizontais da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Vasos Retinianos/metabolismo , Análise de Sequência de RNA , Análise de Célula Única
12.
PLoS One ; 14(6): e0218208, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31188885

RESUMO

Geobacillus thermoglucosidans DSM2542 is an industrially important microbe, however the complex nutritional requirements of Geobacilli confound metabolic engineering efforts. Previous studies have utilised semi-defined media recipes that contain complex, undefined, biologically derived nutrients which have unknown ingredients that cannot be quantified during metabolic profiling. This study used design of experiments to investigate how individual nutrients and interactions between these nutrients contribute to growth. A mathematically derived defined medium has been formulated that has been shown to robustly support growth of G. thermoglucosidans in two different environmental conditions (96-well plate and shake flask) and with a variety of lignocellulose-based carbohydrates. This enabled the catabolism of industrially relevant carbohydrates to be investigated.


Assuntos
Meios de Cultura/metabolismo , Geobacillus/crescimento & desenvolvimento , Geobacillus/metabolismo , Metaboloma/fisiologia , Metabolômica/métodos
13.
ACS Synth Biol ; 8(5): 1175-1186, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30995831

RESUMO

Well-characterized promoter collections for synthetic biology applications are not always available in industrially relevant hosts. We developed a broadly applicable method for promoter identification in atypical microbial hosts that requires no a priori understanding of cis-regulatory element structure. This novel approach combines bioinformatic filtering with rapid empirical characterization to expand the promoter toolkit and uses machine learning to improve the understanding of the relationship between DNA sequence and function. Here, we apply the method in Geobacillus thermoglucosidasius, a thermophilic organism with high potential as a synthetic biology chassis for industrial applications. Bioinformatic screening of G. kaustophilus, G. stearothermophilus, G. thermodenitrificans, and G. thermoglucosidasius resulted in the identification of 636 100 bp putative promoters, encompassing the genome-wide design space and lacking known transcription factor binding sites. Eighty of these sequences were characterized in vivo, and activities covered a 2-log range of predictable expression levels. Seven sequences were shown to function consistently regardless of the downstream coding sequence. Partition modeling identified sequence positions upstream of the canonical -35 and -10 consensus motifs that were predicted to strongly influence regulatory activity in Geobacillus, and artificial neural network and partial least squares regression models were derived to assess if there were a simple, forward, quantitative method for in silico prediction of promoter function. However, the models were insufficiently general to predict pre hoc promoter activity in vivo, most probably as a result of the relatively small size of the training data set compared to the size of the modeled design space.


Assuntos
Geobacillus/genética , Biologia Sintética/métodos , Análise dos Mínimos Quadrados , Redes Neurais de Computação , Regiões Promotoras Genéticas
14.
Biochemistry ; 58(17): 2199-2207, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30938154

RESUMO

The ability to precisely control protein complex formation has high utility in the expanding field of biomaterials. Driving protein-protein binding through metal-ligand bridging interactions is a promising method of achieving this goal. Furthermore, the capacity to precisely regulate both complex formation and dissociation enables additional control not available with constitutive protein complexes. Here we describe the design of three metal-controlled protein dimers that are completely monomeric in the absence of metal yet form high-affinity symmetric homodimers in the presence of zinc sulfate. The scaffold used for the designed dimers is the ß1 domain of streptococcal protein G. In addition to forming high-affinity dimers in the presence of metal, the complexes also dissociate upon addition of EDTA. Biophysical characterization revealed that the proteins maintain relatively high thermal stability, bind with high affinity, and are completely monodisperse in the monomeric and dimeric states. High-resolution crystal structures revealed that the dimers adopt the target structure and that the designed metal-binding histidine residues successfully bind zinc and function to drive dimer formation.


Assuntos
Proteínas de Bactérias/química , Metais/química , Domínios Proteicos , Multimerização Proteica , Proteínas de Bactérias/metabolismo , Ligação Competitiva , Dicroísmo Circular , Cristalografia por Raios X , Desenho de Fármacos , Metais/metabolismo , Modelos Moleculares , Ligação Proteica , Sulfato de Zinco/química , Sulfato de Zinco/metabolismo
15.
J Phys Chem Lett ; 10(6): 1374-1381, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30829040

RESUMO

We employed bias-assisted charge extraction techniques to investigate the transient and steady-state recombination of photogenerated charge carriers in complete devices of a disordered polymer-fullerene blend. Charge recombination is shown to be dispersive, with a significant slowdown of the recombination rate over time, consistent with the results from kinetic Monte Carlo simulations. Surprisingly, our experiments reveal little to no contributions from early time recombination of nonequilibrated charge carriers to the steady-state recombination properties. We conclude that energetic relaxation of photogenerated carriers outpaces any significant nongeminate recombination under application-relevant illumination conditions. With equilibrated charges dominating the steady-state recombination, quasi-equilibrium concepts appear suited for describing the open-circuit voltage of organic solar cells despite pronounced energetic disorder.

16.
Immunology ; 157(1): 86-91, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30768794

RESUMO

Designing improved vaccines against mutable viruses such as dengue and influenza would be helped by a better understanding of how the B-cell memory compartment responds to variant antigens. Towards this we have recently shown, after secondary immunization of mice with a widely variant dengue virus envelope protein with only 63% amino acid identity, that IgM+ memory B cells with few mutations supported an efficient secondary germinal centre (GC) and serum response, superior to a primary response to the same protein. Here, further investigation of memory responses to variant proteins, using more closely related influenza virus haemagglutinins (HA) that were 82% identical, produced a variant-induced boost response in the GC dominated by highly mutated B cells that failed, not efficiently improving serum avidity even in the presence of extra adjuvant, and that was worse than a primary response. This supports a hypothesis that over a certain level of antigenic differences, cross-reactive memory B-cell populations have reduced competency for affinity maturation. Combined with our previous observations, these findings also provide new parameters of success and failure in antibody memory responses.


Assuntos
Linfócitos B/fisiologia , Centro Germinativo/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Receptores de Antígenos de Linfócitos B/genética , Animais , Afinidade de Anticorpos , Reações Cruzadas , Feminino , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Humanos , Imunidade Humoral , Imunização Secundária , Memória Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Polimorfismo Genético
17.
Sci Rep ; 9(1): 1776, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30742038

RESUMO

Botryococcus braunii is a colonial microalga that appears early in the fossil record and is a sensitive proxy of environmental and hydroclimatic conditions. Palaeozoic Botryococcus fossils which contribute up to 90% of oil shales and approximately 1% of crude oil, co-localise with diagnostic geolipids from the degradation of source-signature hydrocarbons. However more recent Holocene sediments demonstrate no such association. Consequently, Botryococcus are identified in younger sediments by morphology alone, where potential misclassifications could lead to inaccurate paleoenvironmental reconstructions. Here we show that a combination of flow cytometry and ancient DNA (aDNA) sequencing can unambiguously identify Botryococcus microfossils in Holocene sediments with hitherto unparalleled accuracy and rapidity. The application of aDNA sequencing to microfossils offers a far-reaching opportunity for understanding environmental change in the recent geological record. When allied with other high-resolution palaeoenvironmental information such as aDNA sequencing of humans and megafauna, aDNA from microfossils may allow a deeper and more precise understanding of past environments, ecologies and migrations.


Assuntos
Fósseis , Genoma de Planta , Hidrocarbonetos/metabolismo , Microalgas/genética , Microalgas/metabolismo , DNA de Plantas/genética
18.
Artigo em Inglês | MEDLINE | ID: mdl-30687838

RESUMO

Thermoanaerobacter ethanolicus JW 200 has been identified as a potential sustainable biofuel producer due to its ability to readily ferment carbohydrates to ethanol. A hybrid sequencing approach, combining Oxford Nanopore and Illumina DNA sequence reads, was applied to produce a single contiguous genome sequence of 2,911,280 bp.

19.
Bioresour Technol ; 271: 1-8, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30253273

RESUMO

Yeast propagation using 50% diluted hydrolysate in water was utilized for the fermentation of hydrolysate derived from pre-treated ensiled sweet sorghum. The purpose was to condition the yeast to the inhibitors generated during the ensiling of sweet sorghum. The conditioned seed cultures exhibited similar fermentation performance and superior kinetics than the inoculum prepared in YPD medium. Furthermore, the conditioned yeast showed increased tolerance to the increased levels of these inhibitors, including ethanol, acetic and lactic acids, demonstrating an effective way to increase the robustness of yeast fermentation for ethanol production.


Assuntos
Etanol/metabolismo , Saccharomyces cerevisiae/metabolismo , Fermentação , Hidrólise , Técnicas de Diluição do Indicador , Sorghum
20.
ACS Synth Biol ; 7(7): 1676-1684, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29976056

RESUMO

Multifactorial approaches can quickly and efficiently model complex, interacting natural or engineered biological systems in a way that traditional one-factor-at-a-time experimentation can fail to do. We applied a Design of Experiments (DOE) approach to model ethanol biosynthesis in yeast, which is well-understood and genetically tractable, yet complex. Six alcohol dehydrogenase (ADH) isozymes catalyze ethanol synthesis, differing in their transcriptional and post-translational regulation, subcellular localization, and enzyme kinetics. We generated a combinatorial library of all ADH gene deletions and measured the impact of gene deletion(s) and environmental context on ethanol production of a subset of this library. The data were used to build a statistical model that described known behaviors of ADH isozymes and identified novel interactions. Importantly, the model described features of ADH metabolic behavior without explicit a priori knowledge. The method is therefore highly suited to understanding and optimizing metabolic pathways in less well-understood systems.


Assuntos
Álcool Desidrogenase/metabolismo , Etanol/metabolismo , Isoenzimas/metabolismo , Modelos Estatísticos , Álcool Desidrogenase/genética , Isoenzimas/genética , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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