Prasad Dhurjati

Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components

The function of the non‐catalytic, duplicated segment found in C. thermocellum cellulases was investigated. Rabbit antibodies reacting with the duplicated segment of endoglucanase CelD cross‐reacted with a variety of cellulosome components ranging between 50 and 100 kDa. 125I‐labeled forms of CelD and of xylanase XynZ carrying the duplicated segment bound to a set of cellulosome proteins ranging between 66 and 250 kDa, particularly to the 250 kDa SL (or S1) subunit. 125I‐labeled forms of CelD and XynZ devoid of the duplicated segment failed to bind to any cellulosome protein. The duplicated segment appears thus to serve to anchor the various cellulosome subunits to the complex by binding to SL, which may be a scaffolding element of the cellulosome.

Decreased protein expression and intermittent recoveries in BiP levels result from cellular stress during heterologous protein expression in Saccharomyces cerevisiae.

Cells are inherently robust to environmental perturbations and have evolved to recover readily from short‐term exposure to heat, pH changes, and nutrient deprivation during times of stress. The stress of unfolded protein accumulation has been implicated previously in low protein yields during heterologous protein expression. Here we describe the dynamics of the response to this stress, termed the unfolded protein response (UPR), during the expression of the single chain antibody 4–4–20 (scFv) in Saccharomyces cerevisiae. Expression of scFv decreased the growth rate of yeast cells whether the scFv was expressed from single‐copy plasmids or integrated into the chromosome. However, the growth rates recovered at longer expression times, and surprisingly, the recovery occurred more quickly in the high‐copy integration strains. The presence of a functional UPR pathway was necessary for a recovery of normal growth rates. During the growth inhibition, the UPR pathway appeared to be activated, resulting in decreased intracellular scFv levels and intermittent recovery of the chaperone BiP within the endoplasmic reticulum. Intracellular scFv was observed primarily in the endoplasmic reticulum, consistent with activation of the UPR pathway. Although the intracellular scFv levels dropped over the course of the expression, this was not a result of scFv secretion. A functional UPR pathway was necessary for the drop in intracellular scFv, suggesting that the decrease was a direct response of UPR activation. Taken together, these results suggest that control of heterologous gene expression to avoid UPR activation will result in higher production levels.

Characterization of the Stress Response of a Bioluminescent Biological Sensor in Batch and Continuous Cultures

The effects of temperature, growth stage, and inducer (ethanol) concentration on the kinetics and magnitude of the stress response were investigated by using an Escherichia coli strain with the grpE heat shock promoter fused to the Vibrio fischeri lux genes. When stressed, the cells responded by changing the level of specific light emission, which was measured both on‐ and off‐line. These measurements were used to characterize and optimize the sensitivity of the construct by determining the conditions at which the culture exhibited maximum specific bioluminescence and minimum response time to ethanol induction in batch cultivation. The results of the batch study were then applied to continuous cultivation, and the effect of dilution rate was determined. These results are of considerable interest in the development of an on‐line biological sensor system for the detection and toxicity assessment of chemical pollutants.

A Miniature Bioreactor for Sensing Toxicity Using Recombinant Bioluminescent Escherichia coli Cells

A miniature bioreactor was fabricated as a contactor between biosensing cells and toxic materials. This miniature bioreactor (58 mL working volume) showed performance similar to that of a conventional bioreactor, as well as the advantages of easy installation, facile operation, and small medium requirements during long‐term continuous operation. A performance evaluation measured the response to ethanol in continuous operation by using a recombinant bioluminescent Escherichia coli strain. Continuous cultures were repeatedly induced by the ethanol challenge. Steady‐state cell concentrations (OD) were found to be decreased, the induced specific bioluminescence (SBL) peak value was found to be increased, and the peak response time, which is the time constant of this continuous monitoring system, was found to be decreased with increasing dilution rate. Finally, on‐ and off‐line bioluminescence monitoring was shown to be reliable, suggesting that this system is suitable for applications such as monitoring the influent and effluent streams of waste water biotreatment plants.

Hypothesis for a systems connectivity model of Autism Spectrum Disorder pathogenesis: links to gut bacteria, oxidative stress, and intestinal permeability.

Autism Spectrum Disorders are neurodevelopmental disorders with symptoms that include cognitive impairments, stereotyped behaviors, and impairments in social skills. The dramatic increase in incidence of autism in recent years has created an increased need to find effective treatments. This paper proposes a hypothesis for a systems model of the connections between Autism Spectrum Disorder pathogenesis routes observed in recent studies. A combination treatment option is proposed to combat multiple pathogenesis mechanisms at once. Autism has been cited as being linked to gastrointestinal symptoms and is thought to be caused by a combination of genetic predisposition and environmental factors. Neuroinflammation as a result of increased gastrointestinal permeability has been noted as being a likely cause of Autism Spectrum Disorders, with possible primary causes stemming from abnormal intestinal bacteria and/or sulfur metabolic deficiencies. Our pathogenesis model proposes a circular relationship: oxidative stress and sulfur metabolic deficiencies could cause changes in colonic bacterial composition; and environmental bacterial contaminants could lead to elevated oxidative stress in individuals. It would thus be a self-perpetuating process where treatment options with single targets would have short-lived effects. It is believed that bacterial toxins, oxidative stress and dietary allergens such as gluten could all lead to increased epithelial permeability. Therefore, we propose a combination treatment to combat intestinal permeability, abnormal bacteria and/or bacterial overgrowth, and sulfur metabolic deficiencies. It is our hope that the proposed model will inspire new studies in finding effective treatments for individuals with Autism Spectrum Disorders. We suggest possible future studies that may lend more credibility to the proposed model.

Curing Behavior of Thick-Sectioned RTM Composites

The successful manufacture of thick-sectioned composites is challenging, since the highly exothermic nature of thermoset resins and limited temperature control make it difficult to avoid detrimental thermal and cure gradients within the composite. In order to make quality parts, it has been found experimentally that cure temperatures must be lowered as much as 50% from those suggested for thin parts. Differential Scanning Calorimetry (DSC) experiments of a vinyl-ester resin system at these lower temperatures revealed a significant dependence on temperature for the maximum extent of cure. If the resin is cured isothermally at 55°C, the final conversion of the resin was found to only reach 70%. When the maximum extent of cure parameter was incorporated into an empirical autocatalytic kinetic model, it was found to significantly improve the description of the cure kinetics. Inhibitors, added to the resin to improve shelf-life, disappear rapidly at higher cure temperatures but can double the time required to cure a thick composite processed at 55°C. A zeroth order kinetic relationship was developed to estimate the amount of inhibitor in the system during the resins cure. The inhibitor relationship and the improved kinetic model were used in a finite difference cure simulation to successfully predict the thermal gradients during cure of a 2.54 cm thick composite manufactured by resin transfer molding (RTM).

A partial differential equation model predictive control strategy: application to autoclave composite processing

A general framework for a partial differential equation (PDE) model predictive control (MPC) problem is formulated. A first principle model of the system, described by a semi-linear PDE system with boundary control, is employed in a model predictive control (MPC) framework. Here, the aim is to determine, off-line (i.e. without process measurement), the theoretical optimal behavior of the process that will be used during on-line MPC. Input and output constraints are handled in the optimization task using a nonlinear programming method. This strategy is evaluated for the optimization of processing temperatures during the manufacture of thick-sectioned polymer composite laminates. The off-line optimization task consists of determining the optimal temperature profile, otherwise known as the cure cycle. Moreover, for this particular process, the existence of a feasible constrained optimization problem is discussed through the design of a constraint bound.

Cecropins induce the hyperosmotic stress response in Escherichia coli

Cecropin A and B, below or near their minimum inhibitory concentrations in viable Escherichia coli, interfered with the rapid NaCl-induced hyperosmotic shrinkage of the cytoplasmic volume (plasmolysis), and also activated the promoter of the hyperosmotic stress gene osmY. The same promoter was also expressed by hyperosmolar NaCl or sucrose, two of the most commonly used antimicrobial food preservatives. Stress responses were monitored during the logarithmic growth phase of E. coli strains that contain specific promoters fused to a luxCDABE operon on a plasmid. The luminescence assay, developed to monitor the transcriptional response to stresses, is based on the premise that organisms often respond and adapt to sublethal environmental adversities by increased expression of stress proteins to restore homeostasis. The luminescence response from these fusion strains to a specific stress occurs as the transcription at the promoter site is activated. Cecropins induced luminescence response only from the osmY-luxCDABE fusion, but not the corresponding stress promoter activation associated with macromolecular or oxidative damage, or leakage of the cytoplasmic content including the proton gradient. The inhibitory effect of cecropins on plasmolysis is interpreted to suggest that the primary locus of action of these antimicrobial peptides in the periplasmic space is on the coupling between the inner and outer membrane.

eXPatGen: generating dynamic expression patterns for the systematic evaluation of analytical methods

MOTIVATION Experimental gene expression data sets, such as those generated by microarray or gene chip experiments, typically have significant noise and complicated interconnectivities that make understanding even simple regulatory patterns difficult. Given these complications, characterizing the effectiveness of different analysis techniques to uncover network groups and structures remains a challenge. Generating simulated expression patterns with known biological features of expression complexity, diversity and interconnectivities provides a more controlled means of investigating the appropriateness of different analysis methods. A simulation-based approach can systematically evaluate different gene expression analysis techniques and provide a basis for improved methods in dynamic metabolic network reconstruction. RESULTS We have developed an on-line simulator, called eXPatGen, to generate dynamic gene expression patterns typical of microarray experiments. eXPatGen provides a quantitative network structure to represent key biological features, including the induction, repression, and cascade regulation of messenger RNA (mRNA). The simulation is modular such that the expression model can be replaced with other representations, depending on the level of biological detail required by the user. Two example gene networks, of 25 and 100 genes respectively, were simulated. Two standard analysis techniques, clustering and PCA analysis, were performed on the resulting expression patterns in order to demonstrate how the simulator might be used to evaluate different analysis methods and provide experimental guidance for biological studies of gene expression. AVAILABILITY http://www.che.udel.edu/eXPatGen/

A detailed analysis of Saccharomyces cerevisiae growth kinetics in batch, fed-batch, and hollow-fiber bioreactors

Abstract The use of the yeast Saccharomyces cerevisiae has increased greatly over the past few years for the production of pharmaceuticals, specialty chemicals, and other commodities. One reason for this is the many advantages it offers for synthesis and secretion of recombinant DNA products. However, the growth characteristics of this yeast are quite complex and only recently has detailed analysis been available to provide additional insight into such phenomena as biphasic growth and catabolic repression. This paper summarizes and organizes current literature and also details the experimental behavior of on-line and off-line variables of S. cerevisiae in glucose-limited-batch, fed- batch, and hollow-fiber bioreactors. The observed behavior is interpreted using knowledge of yeast catabolism biochemistry and physiology. Special focus is placed on the interpretation of off-gas behavior with respect to TCA enzyme activity and the saturation level of oxygen utilization capacity.