Control of yeast fed-batch process through regulation of extracellular ethanol concentration

At high growth rates, the biomass yield of baker's yeast (Saccharomyces cerevisiae) decreases due to the production of ethanol. For this reason, it is standard industrial practice to use a fed-batch process whereby the specific growth rate,μ, is fixed at a level below the point of ethanol production, i.e.,μcrit. Optimally, growth should be maintained atμcrit, but in practice, this is difficult becauseμcrit is dependent upon strain and culture conditions. In this work, growth was maintained at a point just aboveμcrit by regulating ethanol concentration in the bioreactor. The models used for control design are shown, as are the experimental results obtained when this strategy was implemented. This technique should be applicable to all microorganisms that exhibit an "overflow” type metabolis

Microfluidic Device Generating Stable Concentration Gradients for Long-Term Cell Culture: Application to Wnt3a Regulation of B-catenin signaling

In developing tissues, proteins and signaling molecules present themselves in the form of concentration gradients, which determine the fate specification and behavior of the sensing cells. To mimic these conditions in vitro, we developed a microfluidic device designed to generate stable concentration gradients at low hydrodynamic shear and allowing long term culture of adhering cells. The gradient forms in a culture space between two parallel laminar flow streams of culture medium at two different concentrations of a given morphogen. The exact algorithm for defining the concentration gradients was established with the aid of mathematical modeling of flow and mass transport. Wnt3a regulation of B-catenin signaling was chosen as a case study. The highly conserved Wnt-activated B-catenin pathway plays major roles in embryonic development, stem cell proliferation and differentiation. Wnt3a stimulates the activity of B-catenin pathway, leading to translocation of B-catenin to the nucleus where it activates a series of target genes. We cultured A375 cells stably expressing a Wnt/B-catenin reporter driving the expression of Venus, pBARVS, inside the microfluidic device. The extent to which the B-catenin pathway was activated in response to a gradient of Wnt3a was assessed in real time using the BARVS reporter gene. On a single cell level, the B-catenin signaling was proportionate to the concentration gradient of Wnt3a; we thus propose that the modulation of Wnt3a gradients in real time can provide new insights into the dynamics of B-catenin pathway, under conditions that replicate some aspects of the actual cell-tissue milieu. Our device thus offers a highly controllable platform for exploring the effects of concentration gradients on cultured cells

Micro-Bioreactor Array for Controlling Cellular Microenvironments

High throughput experiments can be used to spatially and temporally investigate the many factors that regulate cell differentiation. We have developed a micro-bioreactor array (MBA) that is fabricated using soft lithography and contains twelve independent micro-bioreactors perfused with culture medium. The MBA enables cultivation of cells that are either attached to substrates or encapsulated in hydrogels, at variable levels of hydrodynamic shear, and with automated image analysis of the expression of cell differentiation markers. The flow and mass transport in the MBA were characterized by computational fluid dynamic (CFD) modeling. The representative MBA configurations were validated using the C2C12 cell line, primary rat cardiac myocytes and human embryonic stem cells (hESCs) (lines H09 and H13). To illustrate the utility of the MBA for controlled studies of hESCs, we established correlations between the expression of smooth muscle actin and cell density for three different flow configurations

Surface-Patterned Electrode Bioreactor for Electrical Stimulation

We present a microscale cell culture system with an interdigitated microarray of excimer-laser-ablated indium tin oxide electrodes for electrical stimulation of cultured cells. The system has been characterized in a range of geometeries and stimulation regimes via electrochemical impedance spectroscopy and used to culture primary cardiomyocytes and human adipose derived stem cells. Over 6 days of culture with electrical stimulation (2 ms duration, 1 Hz, 180 μm wide electrodes with 200 μm spacing), both cell types exhibited enhanced proliferation, elongation and alignment, and adipose derived stem cells exhibited higher numbers of Connexin-43-composed gap junctions

Functional interdependence of BRD4 and DOT1L in MLL leukemia.

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis

The Helicobacter pylori Genome Project : insights into H. pylori population structure from analysis of a worldwide collection of complete genomes

Helicobacter pylori, a dominant member of the gastric microbiota, shares co-evolutionary history with humans. This has led to the development of genetically distinct H. pylori subpopulations associated with the geographic origin of the host and with differential gastric disease risk. Here, we provide insights into H. pylori population structure as a part of the Helicobacter pylori Genome Project (HpGP), a multi-disciplinary initiative aimed at elucidating H. pylori pathogenesis and identifying new therapeutic targets. We collected 1011 well-characterized clinical strains from 50 countries and generated high-quality genome sequences. We analysed core genome diversity and population structure of the HpGP dataset and 255 worldwide reference genomes to outline the ancestral contribution to Eurasian, African, and American populations. We found evidence of substantial contribution of population hpNorthAsia and subpopulation hspUral in Northern European H. pylori. The genomes of H. pylori isolated from northern and southern Indigenous Americans differed in that bacteria isolated in northern Indigenous communities were more similar to North Asian H. pylori while the southern had higher relatedness to hpEastAsia. Notably, we also found a highly clonal yet geographically dispersed North American subpopulation, which is negative for the cag pathogenicity island, and present in 7% of sequenced US genomes. We expect the HpGP dataset and the corresponding strains to become a major asset for H. pylori genomics

Standard setting and carrier differentiation at seaports

Abstract The deployment of mega container ships with a capacity for 18,000 + TEU on major trade lanes is a recent trend within the ocean shipping industry. Larger ships pose multiple challenges to ports and hinterland connections as well as to the beneficial cargo owners. To achieve maximum utilization of their larger vessels, carriers have entered cooperative global alliances on predetermined routes, resulting in new discharge patterns at U.S. ports. These multi-partner networks involve several competing firms which voluntarily and interactively engage in service delivery. Container carriage is increasingly competitive, and requires continuing cost reductions. There is increasing evidence, however, that cargo shippers are less satisfied with the service their supply chains are receiving. Standardizing process performance through supply chain integration and removing inefficiency will be needed to stabilize the international shipping market, but the question remains how ocean carriers will be able differentiate themselves and create improved supply chain performance. This paper suggests an answer through a simple standard performance measurement model. We will suggest that old systems of carrier competition could evolve to greater cooperation and coordination between business competitors, a state sometimes called “coopetition”, by development of standard setting processes for sharing information while retaining specific service delivery structures to provide differentiated value to customers

On-line monitoring of Phaffia rhodozyma fed-batch process with in situ dispersive raman spectroscopy

Since the yeast Phaffia rhodozyma was first described some 35 yr ago, there has been significant interest in the development of com. processes to exploit its ability to produce carotenoids (.apprx.80% astaxanthin). However, the optimal conditions for carotenoid prodn. are not well understood. A key limitation has been the lack of an appropriate sensor for online carotenoid quantification. In this study, an in situ Raman spectroscopy probe was used to monitor intracellular carotenoid prodn. for three consecutive P. rhodozyma fed-batch expts. Raman spectroscopy is particularly well suited to the study of carotenoids due to a resonance effect, which greatly enhances the intensity of the three fundamental carotenoid bands, n1 (1513 cm-1, C=C stretch), n2 (1154 cm-1, C-C stretch), and n3 (1003 cm-1, CH3 rock). For all three cultures, the peak height of these bands was linearly correlated with intracellular carotenoid content (1 to 45 mg/L) to a precision of better than 5%, and the correlation from one expt. was directly applicable to others. [on SciFinder (R)

Metabolic network analysis on Phaffia rhodozyma yeast using 13C-labeled glucose and gas chromatography-mass spectrometry

Carotenoid prodn. by microorganisms, as opposed to chem. synthesis, could fulfill an ever-increasing demand for 'all natural' products. The yeast Phaffia rhodozyma has received considerable attention because it produces the red pigment astaxanthin, commonly used as an animal feed supplement. In order to have a better understanding of its metab., labeling expts. with [1-13C]glucose were conducted with the wildtype strain (CBS5905 T) and a hyper-producing carotenoid strain (J4-3) in order to det. their metabolic network structure and est. intracellular fluxes. Amino acid labeling patterns, as detd. by GC-MS, were in accordance with a metabolic network consisting of the Embden-Meyerhof-Parnas pathway, the pentose phosphate pathway, and the TCA cycle. Glucose was mainly consumed along the pentose phosphate pathway (.apprx.65% for wildtype strain), which reflected high NADPH requirements for lipid biosynthesis. Although common to other oleaginous yeast, there was no, or very little, malic enzyme activity for carbon-limited growth. In addn., there was no evidence of phosphoketolase activity. The central carbon metab. of the mutant strain was similar to that of the wildtype strain, though the relative pentose phosphate flux was lower and the TCA cycle flux in accordance with the biomass yield being lower. [on SciFinder (R)