High-level living cell production of cytidine-5′-diphosphocholine in metabolically engineered yeast

Scientists have traditionally studied complex biologic systems by reducing them to simple building blocks. Genome sequencing, high-throughput screening, and proteomics have, however, generated large datasets, revealing a high level of complexity in components and interactions. Systems biology embraces this complexity with a combination of mathematical, engineering, and computational tools for constructing and validating models of biologic phenomena. The validity of mathematical modeling in hematopoiesis was established early by the pioneering work of Till and McCulloch. In reviewing more recent papers, we highlight deterministic, stochastic, statistical, and network-based models that have been used to better understand a range of topics in hematopoiesis, including blood cell production, the periodicity of cyclical neutropenia, stem cell production in response to cytokine administration, and the emergence of imatinib resistance in chronic myeloid leukemia. Future advances require technologic improvements in computing power, imaging, and proteomics as well as greater collaboration between experimentalists and modelers. Altogether, systems biology will improve our understanding of normal and abnormal hematopoiesis, better define stem cells and their daughter cells, and potentially lead to more effective therapies.

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Engineering a Saccharomyces cerevisiae Wine Yeast That Exhibits Reduced Ethanol Production during Fermentation under Controlled Microoxygenation Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.00750-06 ◽

2006 ◽

Vol 72 (9) ◽

pp. 5822-5828 ◽

Cited By ~ 39

Author(s):

Stï¿½phanie Heux ◽

Jean-Marie Sablayrolles ◽

Rï¿½my Cachon ◽

Sylvie Dequin

Keyword(s):

Saccharomyces Cerevisiae ◽

Nadh Oxidase ◽

Ethanol Yield ◽

Oxygen Transfer Rate ◽

Wine Yeast ◽

Glycolytic Flux ◽

Fermentation Performance ◽

Engineered Yeast ◽

High Level ◽

The Impact

ABSTRACT We recently showed that expressing an H2O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD+ reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.

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Engineering substrate and energy metabolism for living cell production of cytidine‐5′‐diphosphocholine

Biotechnology and Bioengineering ◽

10.1002/bit.27291 ◽

2020 ◽

Vol 117 (5) ◽

pp. 1426-1435

Author(s):

Yanna Ren ◽

Qi Liu ◽

Haifeng Liu ◽

Xiangshan Zhou ◽

Yuanxing Zhang ◽

...

Keyword(s):

Energy Metabolism ◽

Living Cell ◽

Cell Production

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Solving the mechanisms responsible for organelle behavior in vivo by same-cell correlative light and electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120473 ◽

1992 ◽

Vol 50 (1) ◽

pp. 14-15

Author(s):

Conly L. Rieder

Keyword(s):

Electron Microscopy ◽

Quantitative Analysis ◽

Light Microscopy ◽

Living Cell ◽

Light And Electron Microscopy ◽

Time Behavior ◽

Dynamic Interactions ◽

Positive Identification ◽

Cellular Components

The behavior of many cellular components, and their dynamic interactions, can be characterized in the living cell with considerable spatial and temporal resolution by video-enhanced light microscopy (video-LM). Indeed, under the appropriate conditions video-LM can be used to determine the real-time behavior of organelles ≤ 25-nm in diameter (e.g., individual microtubules—see). However, when pushed to its limit the structures and components observed within the cell by video-LM cannot be resolved nor necessarily even identified, only detected. Positive identification and a quantitative analysis often requires the corresponding electron microcopy (EM).

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Transcription factor/DNA interactions visualized by electron spectroscopic imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122654 ◽

1992 ◽

Vol 50 (1) ◽

pp. 450-451

Author(s):

David P. Bazett-Jones ◽

Mark L. Brown

Keyword(s):

Transcription Factor ◽

Dna Sequences ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Phosphorus Content ◽

Spectroscopic Imaging ◽

Electron Spectroscopic Imaging ◽

Dna Backbone ◽

Two Parameters ◽

High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

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Advances in Stem Instrumentation for Biology

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180562 ◽

1990 ◽

Vol 48 (1) ◽

pp. 362-363

Author(s):

J. S. Wall

Keyword(s):

Scanning Transmission Electron Microscope ◽

Carbon Films ◽

Specimen Preparation ◽

Material Deposition ◽

Active User ◽

Percent Improvement ◽

Scanning Transmission ◽

The Moment ◽

Film Substrate ◽

High Level

The forte of the Scanning transmission Electron Microscope (STEM) is high resolution imaging with high contrast on thin specimens, as demonstrated by visualization of single heavy atoms. of equal importance for biology is the efficient utilization of all available signals, permitting low dose imaging of unstained single molecules such as DNA.Our work at Brookhaven has concentrated on: 1) design and construction of instruments optimized for a narrow range of biological applications and 2) use of such instruments in a very active user/collaborator program. Therefore our program is highly interactive with a strong emphasis on producing results which are interpretable with a high level of confidence.The major challenge we face at the moment is specimen preparation. The resolution of the STEM is better than 2.5 A, but measurements of resolution vs. dose level off at a resolution of 20 A at a dose of 10 el/A2 on a well-behaved biological specimen such as TMV (tobacco mosaic virus). To track down this problem we are examining all aspects of specimen preparation: purification of biological material, deposition on the thin film substrate, washing, fast freezing and freeze drying. As we attempt to improve our equipment/technique, we use image analysis of TMV internal controls included in all STEM samples as a monitor sensitive enough to detect even a few percent improvement. For delicate specimens, carbon films can be very harsh-leading to disruption of the sample. Therefore we are developing conducting polymer films as alternative substrates, as described elsewhere in these Proceedings. For specimen preparation studies, we have identified (from our user/collaborator program ) a variety of “canary” specimens, each uniquely sensitive to one particular aspect of sample preparation, so we can attempt to separate the variables involved.

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A Questionnaire Survey of Current Rehabilitation Practices for Adults With Normal Hearing Sensitivity Who Experience Auditory Difficulties

American Journal of Audiology ◽

10.1044/2020_aja-20-00027 ◽

2020 ◽

Vol 29 (4) ◽

pp. 738-761

Author(s):

Tess K. Koerner ◽

Melissa A. Papesh ◽

Frederick J. Gallun

Keyword(s):

Hearing Aids ◽

Questionnaire Survey ◽

Patient Population ◽

Clinical Care ◽

Normal Hearing ◽

Auditory Training ◽

Training Methods ◽

Hearing Sensitivity ◽

Considerable Benefit ◽

High Level

Purpose A questionnaire survey was conducted to collect information from clinical audiologists about rehabilitation options for adult patients who report significant auditory difficulties despite having normal or near-normal hearing sensitivity. This work aimed to provide more information about what audiologists are currently doing in the clinic to manage auditory difficulties in this patient population and their views on the efficacy of recommended rehabilitation methods. Method A questionnaire survey containing multiple-choice and open-ended questions was developed and disseminated online. Invitations to participate were delivered via e-mail listservs and through business cards provided at annual audiology conferences. All responses were anonymous at the time of data collection. Results Responses were collected from 209 participants. The majority of participants reported seeing at least one normal-hearing patient per month who reported significant communication difficulties. However, few respondents indicated that their location had specific protocols for the treatment of these patients. Counseling was reported as the most frequent rehabilitation method, but results revealed that audiologists across various work settings are also successfully starting to fit patients with mild-gain hearing aids. Responses indicated that patient compliance with computer-based auditory training methods was regarded as low, with patients generally preferring device-based rehabilitation options. Conclusions Results from this questionnaire survey strongly suggest that audiologists frequently see normal-hearing patients who report auditory difficulties, but that few clinicians are equipped with established protocols for diagnosis and management. While many feel that mild-gain hearing aids provide considerable benefit for these patients, very little research has been conducted to date to support the use of hearing aids or other rehabilitation options for this unique patient population. This study reveals the critical need for additional research to establish evidence-based practice guidelines that will empower clinicians to provide a high level of clinical care and effective rehabilitation strategies to these patients.

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