Decomposition of cell activities revealing the role of the cell cycle in driving biofunctional heterogeneity.

Heterogeneity of cell phenotypes remains a barrier in progressing cell research and a challenge in conquering cancer-related drug resistance. Cell morphology, the most direct property of cell phenotype, evolves along the progression of the cell cycle; meanwhile, cell motility, the dynamic property of cell phenotype, also alters over the cell cycle. However, a quantifiable research understanding the relationship between the cell cycle and cell migration is missing. Herein, we coordinate the migratory behaviours of NIH 3T3 fibroblasts to their corresponding phases of the cell cycle, the G1, the S, and the G2 phases, and explain the relationship through the spatiotemporal arrangements between the Rho GTPases' signals and cyclin-dependent kinase inhibitors, p21Cip1, and p27Kip1. Taken together, we demonstrate that both cell morphology and the dynamic subcellular behaviour are homogenous within each stage of the cell cycle phases but heterogenous between phases through quantitative cell analyses and an interactive molecular mechanism between the cell cycle and cell migration, posing potential implications in countering drug resistance.

The Effect of Z-Ligustilide on the Mobility of Human Glioblastoma T98G Cells.

Z-ligustilide (LIG), an essential oil extract from Radix Angelica sinensis, has broad pharmaceutical applications in treating cardio-vascular diseases and ischemic brain injury. Recently, LIG has been connected to Glioblastoma multiforme (GBM) because of its structural similarity to 3-n-alkyphthalide (NBP), which is specifically cytotoxic to GBM cells. Hence, we investigated LIG's effect on GBM T98G cells. The study shows that LIG can significantly reduce T98G cells' migration in a dose-dependent manner. Furthermore, the attenuation of cellular mobility can be linked to the activity of the Rho GTPases (RhoA, Rac1 and Cdc42), the three critical molecular switches governing cytoskeleton remodeling; thus, regulating cell migration. LIG significantly reduces the expression of RhoA and affects in a milder manner the expression of Cdc42 and Rac1.

Optimizing cellulase usage for improved mixing and rheological properties of acid-pretreated sugarcane bagasse.

Consolidation of bioprocessing steps with lignocellulose is limited by hydrolysate toxicity, the fibrous nature of suspensions, and low activity of cellulase enzymes. Combinations of enzyme dose and treatment conditions improved the flow properties and pumping of acid-pretreated sugarcane bagasse slurries (10% dry weight). Low levels of cellulase enzyme (0.1 and 0.5 FPU/g dry weight acid-pretreated bagasse) were found to reduce viscosities by 77-95% after 6 h, solubilizing 3.5% of the bagasse dry weight. Flow of slurries through small funnels was a useful predictor of success with centrifugal and diaphragm pumps. Equations were derived that describe viscosity and solubilized carbohydrates as a function of time and cellulase dosage. Blending of acid-pretreated bagasse (10% dry weight) with suspensions of acid-pretreated bagasse (10% dry weight) that had been previously digested with cellulase enzymes (low viscosity) did not increase viscosity in a linear fashion. Viscosity of these mixtures remained relatively constant until a threshold level of new fiber was reached, followed by a rapid increase with further additions. Up to 35% fresh acid-pretreated bagasse could be blended with enzyme-digested fiber (5.0 FPU/g dry weight acid-pretreated fiber; 6 h) with only a modest increase in viscosity. The smooth surfaces of enzyme-treated fiber are proposed to hinder the frequency and extent of interactions between fibrils of fresh fiber particles (acid-pretreated) until a threshold concentration is achieved, after which fiber interactions and viscosity increase dramatically. These results were used to model the viscosity in an ideal continuous stirred tank reactor (liquefaction) as a function of residence time and enzyme dosage.

Influence of alternating oxic/anoxic conditions on growth of denitrifying bacteria.

Denitrifying bacteria that are switched from oxic to anoxic conditions can experience diauxic lag, which is the time required for re-synthesis of nitrate reductase and other denitrifying enzymes. Pseudomonas denitrificans were exposed to alternating oxic/anoxic phases in a continuous flow reactor with either 4-h or 8-h anoxic phase lengths, in comparison to a measured diauxic lag of 9.5h following steady-state oxic conditions. The P. denitrificans were unable to sustain anoxic growth at either of the anoxic phase lengths tested. Diauxic lag observed after several cycles of alternating oxic/anoxic phases was significantly longer than the diauxic lag measured after steady-state oxic conditions. This may be attributed to increase of cell maintenance energy requirements due to substrate accumulation during anoxic phases and concomitant high specific growth rates during oxic phases.

Effect of periplasmic nitrate reductase on diauxic lag of Paracoccus pantotrophus.

Paracoccus pantotrophus expresses two nitrate reductases-membrane bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap). In growth experiments with two denitrifying species (Paracoccus pantotrophus and Alcaligenes eutrophus) that have both Nap and Nar and two species (Pseudomonas denitrificans and Pseudomonas fluorescens) with Nar only, it was found that diauxic lag is shorter for bacteria that express Nap. In P. pantotrophus, napEDABC encodes the periplasmic nitrate reductase. To analyze the effect of Nap on diauxic lag, the nap operon was deleted from P. pantotrophus. The growth experiments with nap(-) mutant resulted in increased diauxic lag when switched from aerobic to anoxic respiration, suggesting Nap is responsible for shorter lags and helps in adaptation to anoxic metabolism after transition from aerobic conditions.

Determination of diauxic lag in continuous culture.

A procedure was developed to characterize diauxic lag of bacteria switching between electron acceptors in continuous culture. In this procedure, a virtual batch growth curve is developed by integrating the time-dependent net specific growth rates of bacteria observed under continuous flow conditions. The length of diauxic lag and the highest net specific growth rate following lag are conveniently estimated from the virtual batch curve. The procedure was found to give reproducible diauxic lag lengths and highest net specific growth rates when applied to experimental data from replicate continuous culture trials.

Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate.

Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes. This was done by a combination of gene deletions (genetic engineering) and metabolic evolution with over 2,000 generations of growth-based selection. After deletion of the central anaerobic fermentation genes (ldhA, adhE, ackA), the pathway for malate and succinate production remained as the primary route for the regeneration of NAD+. Under anaerobic conditions, ATP production for growth was obligately coupled to malate dehydrogenase and fumarate reductase by the requirement for NADH oxidation. Selecting strains for improved growth co-selected increased production of these dicarboxylic acids. Additional deletions were introduced as further improvements (focA, pflB, poxB, mgsA). The best succinate biocatalysts, strains KJ060(ldhA, adhE, ackA, focA, pflB) and KJ073(ldhA, adhE, ackA, focA, pflB, mgsA, poxB), produce 622-733 mM of succinate with molar yields of 1.2-1.6 per mole of metabolized glucose. The best malate biocatalyst, strain KJ071(ldhA, adhE, ackA, focA, pflB, mgsA), produced 516 mM malate with molar yields of 1.4 per mole of glucose metabolized.

Estimation of nitrate reductase enzyme parameters in activated sludge using an extended Kalman filter algorithm.

This paper demonstrates an application of the extended Kalman filter to a wastewater plant using real process data to estimate parameters in a semi-mechanistic model. This technique for parameter identification allows a semi-mechanistic model developed for pure cultures to be applied to a mixed culture population where isolation of enzyme kinetic parameters is not practical. This paper demonstrates an application in which a wastewater treatment facility with three physical unit operations and an unusual operating profile is modeled as 11 reactor units with recirculation streams. On-line nutrient probe data from two locations in the aerobic reactor are combined with operational data for flows and feed composition. This paper demonstrates the successful application of a biochemical model developed at the bench scale to this facility through the use of an extended Kalman filter. The estimated biological model parameters and the covariance matrix diagonal converge to stable values, indicating a successful implementation.

Effect of carbon substrate on electron acceptor diauxic lag and anoxic maximum specific growth rate in species with and without periplasmic enzyme.

The effect of oxidation state of carbon substrate on the diauxic lag of facultative anaerobic denitrifying bacteria growing aerobically upon switching to anoxic growth was studied. Also studied was the effect on the anoxic maximum specific growth rate. Two pure bacteria cultures were used, Paracoccus pantotrophus, denitrifying bacteria containing a periplasmic nitrate reductase (Nap), and Pseudomonas denitrificans, denitrifying bacteria lacking the periplasmic nitrate reductase. The anoxic maximum specific growth rate of both cultures following a period of aerobic growth with identical dilution up to steady-state was indeed affected by the oxidation state of the carbon, with the most oxidized substrate yielding the highest anoxic maximum specific growth rate. The diauxic lags for Paracoccus pantotrophus were considerably shorter than those for Pseudomonas denitrificans, something expected due to the presence of Nap, an enzyme not affected by aerobiosis. Since the activity of Nap in Paracoccus pantotrophus under aerobic conditions has been shown to increase with the extent of reduction of the carbon substrate, it was also expected that the diauxic lag length for these bacteria would decrease as the reduction state of the carbon substrate increased. This could not be demonstrated, as no significant lags were observed for this species. Pseudomonas denitrificans exhibited a shorter diauxic lag with the more oxidized carbon source.

A simple model for diauxic growth of denitrifying bacteria.

A simple model has been formulated to simulate diauxic growth of denitrifying bacteria. It is capable of fitting the experimental results of batch growth experiments with Pseudomonas denitrificans under various conditions. It successfully predicts the observed lags when a pure culture of this bacterium switches from oxygen to nitrate as terminal electron acceptor. The model includes the effect of carbon substrate limitation and length of aerobic phase and does not run into problems when switching from anoxic to aerobic conditions, unlike prior models of diauxic growth.

Structured model for denitrifier diauxic growth.

We present a model for diauxic growth of denitrifying bacteria in which nitrate reductase synthesis kinetics dominate the overall growth kinetics. The model is based on the assumption of the existence of a nitrate respiration operon, thereby linking the rate of nitrate uptake to the activity of nitrate reductase. We show that this approach can model diauxic growth of Pseudomonas denitrificans by conducting experiments in which nitrate reductase activity was measured during both lag and ensuing exponential growth phases. We consistently observed the pattern of low nitrate reductase enzyme activity during the lag phase, increasing before the onset of growth. By fitting model parameters we were able to successfully match experimental data for growth, nitrate uptake, and enzyme activity level.