Biomedical subjects

Mark R Marten

Publications and source records attributed to Mark R Marten.

18 recordsLinked to original sources

PubMed2006

Optical analysis of liquid mixing in a minibioreactor.

A novel optical sensor was used to study mixing and mean circulation time in a model minibioreactor (12.5 mL stirred vessel, equipped with a paddle impeller). Rotational rates in the range of 10-1,000 rpm corresponding to Reynolds number between 14 and 1,350 were studied. Results suggest that depending on the impeller rotational speed, mixing times up to 214 +/- 87 s can be reproducibly achieved. The minibioreactor was operated in the transitional regime, and it was determined that the non-dimensional form for mixing time, NTheta(M) was linearly dependent on Reynolds number. A linear correlation between mean circulation time and the inverse of rotational speed was also determined. The mean circulation time dependence on rotational speed in the 12.5 mL stirred vessel is similar to those found in large-scale stirred vessels. These results suggest that mixing and circulation times found in large-scale reactors can be replicated in minibioreactors.

Algorithms↗

PubMed2006

Proteomic analysis of extracellular proteins from Escherichia coli W3110.

While numerous proteomic analyses have been carried out on Escherichia coli, the vast majority have focused on expression of intracellular proteins. Yet, recent literature reports imply that even in laboratory strains, significant proteins may be found outside the cell. Here, we identify extracellular proteins associated with nonpathogenic E. coli strain W3110. Two-dimensional gel electrophoresis (2DE) revealed approximately 66 prominent protein spots during exponential growth (4 and 8 h shake flask culture) in minimal medium. The absence of detectable nucleic acids in the culture supernatant implies these proteins did not result from cell lysis. MALDI-TOF MS was used to identify 44 proteins, most of which have been previously identified as either outer membrane or extracellular proteins. In addition, 2DE protease zymogram analysis was carried out which facilitated identification of three extracellular proteases, one of which was not observed during standard 2DE. Our results are consistent with previous findings which imply outer membrane proteins are shed during growth.

Carrier Proteins↗

PubMed2006

Protein image alignment via piecewise affine transformations.

We present a new approach for aligning families of 2D gels. Instead of choosing one of the gels as reference and performing a pairwise alignment, we construct an ideal gel that is representative of the entire family and obtain a set of piecewise affine transformations that optimally align each gel of the family to the ideal gel. The coefficients defining the transformations as well as the ideal landmarks are obtained as the solution of a large-scale quadratic programming problem that can be solved efficiently by interior-point methods.

Animals↗

PubMed2005

Proteome analysis to assess physiological changes in Escherichia coli grown under glucose-limited fed-batch conditions.

Proteome analysis was used to compare global protein expression changes in Escherichia coli fermentation between exponential and glucose-limited fed-batch phase. Two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry were used to separate and identify 49 proteins showing >2-fold difference in expression. Proteins upregulated during exponential phase include ribonucleotide biosynthesis enzymes and ribosomal recycling factor. Proteins upregulated during fed-batch phase include those involved in high-affinity glucose uptake, transport and degradation of alternate carbon sources and TCA cycle, suggesting an enhanced role of the cycle under glucose- and energy-limited conditions. We report the upregulation of several putative proteins (ytfQ, ygiS, ynaF, yggX, yfeX), not identified in any previous study under carbon-limited conditions.

Adaptation, Physiological↗

PubMed2005

Effect of cycle time on fungal morphology, broth rheology, and recombinant enzyme productivity during pulsed addition of limiting carbon source.

For many years, high broth viscosity has remained a key challenge in large-scale filamentous fungal fermentations. In previous studies, we showed that broth viscosity could be reduced by pulsed addition of limiting carbon during fed-batch fermentation. The objective in this study was to determine how changing the frequency of pulsed substrate addition affects fungal morphology, broth rheology, and recombinant enzyme productivity. To accomplish this, a series of duplicate fed-batch fermentations were performed in 20-L fermentors with a recombinant glucoamylase producing strain of Aspergillus oryzae. The total cycle time for substrate pulsing was varied over a wide range (30-2,700 s), with substrate added only during the first 30% of each cycle. As a control, a fermentation was conducted with continuous substrate feeding, and in all fermentations the same total amount of substrate was added. Results show that the total biomass concentration remained relatively unaltered, while a substantial decrease in the mean projected area of fungal elements (i.e., average size) was observed with increasing cycle time. This led to reduced broth viscosity and increased oxygen uptake rate. However, high values of cycle time (i.e., 900-2,700 s) showed a significant increase in fungal conidia formation and significantly reduced recombinant enzyme productivity, suggesting that the fungi channeled substrate to storage compounds rather than to recombinant protein. In addition to explaining the effect of cycle time on fermentation performance, these results may aid in explaining the discrepancies observed on scale-up to larger fermentors.

Aspergillus oryzae↗

PubMed2005

Assessment of elasticity and topography of Aspergillus nidulans spores via atomic force microscopy.

Previous studies have described both surface morphology and adhesive properties of fungal spores, but little information is currently available on their mechanical properties. In this study, atomic force microscopy (AFM) was used to investigate both surface topography and micromechanical properties of Aspergillus nidulans spores. To assess the influence of proteins covering the spore surface, wild-type spores were compared with spores from isogenic rodA(+) and rodA(-) strains. Tapping-mode AFM images of wild-type and rodA(+) spores in air showed characteristic "rodlet" protein structures covering a granular spore surface. In comparison, rodA(-) spores were rodlet free but showed a granular surface structure similar to that of the wild-type and rodA(+) spores. Rodlets were removed from rodA(+) spores by sonication, uncovering the underlying granular layer. Both rodlet-covered and rodlet-free spores were subjected to nanoindentation measurements, conducted in air, which showed the stiffnesses to be 110 +/- 10, 120 +/- 10, and 300 +/- 20 N/m and the elastic moduli to be 6.6 +/- 0.4, 7.0 +/- 0.7, and 22 +/- 2 GPa for wild-type, rodA(+) and rodA(-) spores, respectively. These results imply the rodlet layer is significantly softer than the underlying portion of the cell wall.

Aspergillus nidulans↗

PubMed2004

In silico reconstruction of nutrient-sensing signal transduction pathways in Aspergillus nidulans.

We report here probable nutrient-sensing signal transduction pathways in Aspergillus nidulans, a model filamentous fungus, based on sequence homology studies with known Saccharomyces cerevisiae and Schizosaccharomyces pombe proteins. Specifically, we identified A. nidulans homologs for yeast proteins involved in (1) filamentation-invasion, (2) cAMP-PKA, (3) pheromone response, (4) cell integrity and (5) TOR signaling pathways. We have also studied autophagy, one of the most important cellular responses regulated by TOR signaling. The Basic Local Alignment Search Tool program "blastp" was used to assess the homology of proteins. We note that by using a highly conservative approach, 70% of the S. cerevisiae signal transduction proteins (107 proteins out of 153 proteins studied) have significant homologs in A. nidulans. Using a slightly less conservative approach, we are able to identify homologs for as high as 91% of the S. cerevisiae signal transduction proteins (139 proteins out of 153 proteins studied). The filamentation-invasion, cell integrity and TOR signaling pathways showed greatest similarity with S. cerevisiae, while the cAMP-PKA and pheromone response pathways showed greater similarity with S. pombe. Based on these results, probable pathways in A. nidulans were constructed using well-established S. cerevisiae and S. pombe models.

Aspergillus nidulans↗

PubMed2003

Pulsed addition of limiting-carbon during Aspergillus oryzae fermentation leads to improved productivity of a recombinant enzyme.

Fungal morphology in many filamentous fungal fermentations leads to high broth viscosity which limits oxygen mass transfer, and often results in reduced productivity. The objective in this study was to determine if a simple, fed-batch, process strategy-pulsed addition of limiting-carbon source-could be used to reduce fungal broth viscosity, and increase productivity of an industrially relevant recombinant enzyme (glucoamylase). As a control, three Aspergillus oryzae fed-batch fermentations were carried out with continuous addition of limiting-carbon. To determine the effect of pulse-feeding, three additional fermentations were carried out with limiting-carbon added in 90-second pulses, during repeated five-minute cycles. In both cases, overall carbon feed-rate was used to control dissolved oxygen concentration, such that increased oxygen availability led to increased addition of limiting-carbon. Pulse-fed fermentations were found to have smaller fungal mycelia, lower broth viscosity, and improved oxygen mass transfer. As a result, more carbon was added to pulse-fed fermentations that led to increased enzyme productivity by as much as 75%. This finding has significant implications for the bioprocessing industry, as a simple process modification which is likely to cost very little to implement in most production facilities, has the potential to substantially increase productivity.

Aspergillus oryzae↗

PubMed2003

Pulsed feeding during fed-batch fungal fermentation leads to reduced viscosity without detrimentally affecting protein expression.

The goal in this study was to determine if pulsed addition of substrate could be used to alter filamentous fungal morphology during fermentation, to result in reduced broth viscosity. In all experiments, an industrially relevant strain of Aspergillus oryzae was grown in 20-liter fermentors. As a control, cultures were fed limiting substrate (glucose) continuously. Tests were performed by altering the feeding strategy so that the same total amount of glucose was fed in repeated 300-s cycles, with the feed pump on for either 30 or 150 s during each cycle. Variables indicative of cellular metabolic activity (biomass concentration, oxygen uptake rate, base consumed for pH control) showed no significant difference between continuous and pulse-fed fermentations. In addition, there was no significant difference between total extracellular protein expression or the apparent distribution of these proteins. In contrast, fungal mycelia during the second half of pulse-fed fermentations were approximately half the size (average projected area) of fungi during fermentations with continuous addition of glucose. As a result, broth viscosity during the second half of pulse-fed fermentations was approximately half that during the second half of continuous fermentations. If these results prove to be applicable for other fungal strains and processes, then this method will represent a simple and inexpensive means to reduce viscosity during filamentous fungal fermentation.

Aspergillus oryzae↗

PubMed2002

Quantitative comparison and evaluation of two commercially available, two-dimensional electrophoresis image analysis software packages, Z3 and Melanie.

While a variety of software packages are available for analyzing two-dimensional electrophoresis (2-DE) gel images, no comparisons between these packages have been published, making it difficult for end users to determine which package would best meet their needs. The goal here was to develop a set of tests to quantitatively evaluate and then compare two software packages, Melanie 3.0 and Z3, in three of the fundamental steps involved in 2-DE image analysis: (i) spot detection, (ii) gel matching, and (iii) spot quantitation. To test spot detection capability, automatically detected protein spots were compared to manually counted, "real" protein spots. Spot matching efficiency was determined by comparing distorted (both geometrically and nongeometrically) gel images with undistorted original images, and quantitation tests were performed on artificial gels with spots of varying Gaussian volumes. In spot detection tests, Z3 performed better than Melanie 3.0 and required minimal user intervention to detect approximately 89% of the actual protein spots and relatively few extraneous spots. Results from gel matching tests depended on the type of image distortion used. For geometric distortions, Z3 performed better than Melanie 3.0, matching 99% of the spots, even for extreme distortions. For nongeometrical distortions, both Z3 and Melanie 3.0 required user intervention and performed comparably, matching 95% of the spots. In spot quantitation tests, both Z3 and Melanie 3.0 predicted spot volumes relatively well for spot ratios less than 1:6. For higher ratios, Melanie 3.0 did much better. In summary, results suggest Z3 requires less user intervention than Melanie 3.0, thus simplifying differential comparison of 2-DE gel images. Melanie 3.0, however, offers many more optional tools for image editing, spot detection, data reporting and statistical analysis than Z3. All image files used for these tests and updated information on the software are available on the internet (http://www.umbc.edu/proteome), allowing similar testing of other 2-DE image analysis software packages.

Electronic Data Processing↗

PubMed2002

Comparison of lysis methods and preparation protocols for one- and two-dimensional electrophoresis of Aspergillus oryzae intracellular proteins.

Filamentous fungal fermentations are used to produce billions of dollars of biochemical and pharmaceutical products annually, yet are plagued by a number of poorly understood problems that would benefit from proteomic analysis. Unfortunately, few publications are available which describe extraction of filamentous fungal proteins for two-dimensional electrophoresis. The goal here was to develop protocols for extraction of fungal proteins, from both wild-type and a recombinant strain of the industrially important filamentous fungi Aspergillus oryzae, to be used for both one- and two-dimensional electrophoresis (1-DE and 2-DE). Because fungal cell walls are exceptionally resistant to fragmentation, four lysis protocols were tested: (i) boiling in strong alkali solution, (ii) boiling in Sodium dodecyl surfate (SDS), (iii) chemical lysis in Y-PER(R) reagent, and (iv) mechanical lysis via rapid agitation with glass beads in a Mini-BeadBeater(R). For both 1-DE and 2-DE, rapid agitation with glass beads was found to be the most efficient extraction method, yielding both mini- and large-format gels with little streaking or spot tailing, and proteins comprising a broad range of molecular weights and pI values.

Aspergillus oryzae↗

PubMedDate not supplied

Effects of increased impeller power in a production-scale Aspergillus oryzae fermentation.

The goal in this study was to determine how increased impeller power affects enzyme expression in large-scale (80 m(3)), fed-batch Aspergillus oryzae fermentations. An approximate 50% increase in average impeller power was achieved by increasing impeller diameter approximately 10%, while operating at slightly reduced speed. Measured decreases in terminal (95%) mixing time show increased power improved bulk mixing. However, batches operated at increased power had lower recombinant enzyme productivity. Biomass assays and image analysis tests showed no significant difference between "high power" and control batches, suggesting that slower growth, altered morphology, or increased hyphal fragmentation were not the cause of reduced productivity. Off-line tests on the shear-thinning, highly viscous broth show oxygen limitation occurred after transport through the air-liquid interface and imply the limitation may involve bulk mixing. Specifically, oxygen transfer may be limited to a small zone surrounding each impeller. When this is the case, oxygen mass transfer will be determined by both impeller shear and fluid circulation, which have been characterized with the energy dissipation/circulation function (EDCF). EDCF values during control fermentations were approximately constant at 25 kW m (-3) s(-1), while EDCF values during "high power" batches fell linearly from 40 to 15 kW m (-3) s(-1). The point at which "high power" EDCF values drop below those in control fermentations corresponds almost exactly with the point at which product titer stops increasing. Thus, our findings suggest oxygen mass transfer was less efficient during the latter half of "high power" fermentations because of reductions in impeller speed and subsequent decreases in EDCF values. This observation has clear implications during the scale-up of viscous fungal fermentations, implying that not only is the level of impeller power important, but also relevant is how this power is applied.

Aspergillus oryzae↗

PubMedDate not supplied

Using computational fluid dynamics software to estimate circulation time distributions in bioreactors.

Nonideal mixing in many fermentation processes can lead to concentration gradients in nutrients, oxygen, and pH, among others. These gradients are likely to influence cellular behavior, growth, or yield of the fermentation process. Frequency of exposure to these gradients can be defined by the circulation time distribution (CTD). There are few examples of CTDs in the literature, and experimental determination of CTD is at best a challenging task. The goal in this study was to determine whether computational fluid dynamics (CFD) software (FLUENT 4 and MixSim) could be used to characterize the CTD in a single-impeller mixing tank. To accomplish this, CFD software was used to simulate flow fields in three different mixing tanks by meshing the tanks with a grid of elements and solving the Navier-Stokes equations using the kappa-epsilon turbulence model. Tracer particles were released from a reference zone within the simulated flow fields, particle trajectories were simulated for 30 s, and the time taken for these tracer particles to return to the reference zone was calculated. CTDs determined by experimental measurement, which showed distinct features (log-normal, bimodal, and unimodal), were compared with CTDs determined using CFD simulation. Reproducing the signal processing procedures used in each of the experiments, CFD simulations captured the characteristic features of the experimentally measured CTDs. The CFD data suggests new signal processing procedures that predict unimodal CTDs for all three tanks.

Algorithms↗

PubMedDate not supplied

Pulsed feeding during fed-batch Aspergillus oryzae fermentation leads to improved oxygen mass transfer.

Productivity in many fungal fermentations is detrimentally affected by high broth viscosity and consequent reduced oxygen mass transfer capacity. The goal here was to determine whether pulsed feeding of limiting carbon in a fungal fermentation could lead to reduced viscosity and improved oxygen mass transfer. As a model, an industrially relevant recombinant strain of Aspergillus oryzae was grown in carbon-limited, fed-batch mode. Maltodextrin was used as a carbon source and was added either continuously or in 1.5-min pulses, 3.5 min apart. In both feeding modes the same total amount of carbon was added, and carbon feed rate was at sufficiently low levels to ensure cultures were always carbon-limited. Compared to continuous feeding, pulsed addition of substrate led to smaller fungal elements, which resulted in a significant reduction in broth viscosity. This in turn led to higher dissolved oxygen concentrations and increased oxygen uptake rates during pulsed feeding.

Aspergillus oryzae↗

PubMedDate not supplied

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy.

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.

Aspergillus nidulans↗

PubMedDate not supplied

Confocal optical system: a novel noninvasive sensor to study mixing.

A novel confocal optical system to study mixing time in small-scale bioreactors is presented. The system is designed to monitor fluorescence upon tracer addition from a localized confocal volume of 0.21 mL within a glass vessel. The key elements of the fluorescence-based confocal system are a pinhole, a lens, an APD (Avalanche photodiode) detector, and light filters. The optical technique was validated by comparison with a pH-based technique. Finally, the optical sensor was tested and a real cultivation media (i.e., spent mammalian cell media) was used to measure mixing time in a 12.5-mL stirred transparent vessel. High accuracy, easy results interpretation, and low costs are the three most attractive characteristics of the sensor. Because of its noninvasive nature and versatility, the results suggest that the confocal system is a promising tool to perform mixing time studies in stirred vessels.

Cell Culture Techniques↗

PubMedDate not supplied

Solubilization of trichloroacetic acid (TCA) precipitated microbial proteins via naOH for two-dimensional electrophoresis.

In preparing intracellular microbial samples for one- or two-dimensional electrophoresis, trichloroacetic acid (TCA) precipitation is frequently used to remove interfering compounds. Solubilization of TCA precipitate typically requires the addition of a number of chaotropes or detergents, in a multistep process, that requires hours to carry out. In this study, a simple, rapid, one-step method to solubilize TCA precipitated proteins is presented. Precipitated proteins are pretreated with 0.2 M NaOH for less than 5 min, followed by addition of standard sample solubilization buffer (SSSB). When compared to solubilization with SSSB alone, NaOH pretreatment of TCA-precipitated intracellular protein from Aspergillus oryzae and Escherichia coli shows an approximate 5-fold increase in soluble protein. In addition, two-dimensional gel electrophoresis on resolubilized proteins shows an equivalent number of proteins in samples with and without NaOH pretreatment.

Aspergillus oryzae↗

PubMedDate not supplied

Proteome analysis of membrane and cell wall associated proteins from Staphylococcus aureus.

Pathogenesis of Staphylococcus aureus, an opportunistic human pathogen, is complex and involves many virulence factors including an array of surface proteins (adhesins) that promote bacterial interactions with extracellular matrix components. A better understanding of these interactions can be achieved by studying the expression of membrane and cell wall associated proteins using a proteome analysis approach. To accomplish this, our goal here was to construct a reference map of membrane and cell wall associated proteins for S. aureus. Various lytic and solubilization methods have been tested to identify a suitable methodology for detection of these proteins in two-dimensional electrophoresis (2DE). Results demonstrate that cell lysis with lysostaphin, which lyses staphylococcal peptidoglycan, followed by solubilization with urea, thiourea, amidosulfobetaine 14 (ASB 14) and dithiothreitol (DTT) is an effective method, yielding a sample comprising proteins of wide molecular ranges and isoelectric points with minimum contamination from cytosolic proteins. Mass spectrometric analysis was employed to identify the membrane and cell surface proteins present in the sample and consequently an initial proteomic map of membrane and cell wall associated proteins for S. aureus is presented.

Bacterial Proteins↗