Alcoholic fermentation: modelling based on sole substrate and product measurement

Optimal automatic bioreactor control requires a mathematical model adapted to the potency of reliable sensors. A new relationship describing the kinetic behavior of alcoholic fermentation is discussed. By analogy with chemical kinetics, the biological rate of substrate consumption is related to substrate and product concentration by the following equation: \documentclass{article}\pagestyle{empty}\begin{document}$$r_s = kS;\alpha P;\beta$$\end{document} Using the well known yield relation between product and substrate, it is possible to describe in both batch and continuous cultures the ethanol and sugar concentrations versus time. This pattern has been successfully tested on several fermentations performed by yeasts (S. cerevisiae, S. bayanus, and S. cerevisiae sake) and a bacterium (Z. mobilis). This simple relationship is proposed as a tool for process control alcoholic fermentation.     

A study of long-term effects on plasmid-containing Escherichia coli in carbon-limited chemostat using 2D-fluorescence spectrofluorimetry

Strain stability of plasmid-containing recombinant organisms is clearly important for industrial applications. Stability is normally assessed by methods such as selective colony forming units or by simply measuring the recombinant product. These methods are typically performed off-line, are time-consuming, and do not give detailed information on the changes in the metabolism. In the current work, long-term stability of a plasmid-containing strain of Escherichia coli (W3110.shik1) capable of shikimic acid overproduction was studied by means of a 2D-fluorescence sensor (BioView) able to emit and detect light in ranges of 260-560 nm and 300-600 nm, respectively. Long-term carbon-limited chemostat experiments were made under both selective (tetracycline-containing medium) and nonselective conditions. It is shown that the fluorescence spectra provide information about metabolic changes at an earlier stage, thereby giving a noninvasive method for monitoring of strain stability. Further, the fluorescence measurements showed that (i) the metabolic changes in the strain W3110.shik1 with time were qualitatively different in selective and nonselective environment, (ii) plasmid recombination resulted primarily in increased biomass yield, and (iii) a change in metabolism probably involving FAD/FMN and pyridoxal-5-P occurred in all experiments. It was concluded that the strain was not stable in any growth condition for more than about 25 growth generations and even less if plasmid recombination took place.     

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes

OBJECTIVE: To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. STUDY DESIGN: Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared. RESULTS: Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements. CONCLUSION: Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.     

Correcting for measurement error in fractional polynomial models using Bayesian modelling and regression calibration,with an application to alcohol and mortality

Exposure measurement error can result in a biased estimate of the association between an exposure and outcome. When the exposure–outcome relationship is linear on the appropriate scale (e.g. linear, logistic) and the measurement error is classical, that is the result of random noise, the result is attenuation of the effect. When the relationship is non‐linear, measurement error distorts the true shape of the association. Regression calibration is a commonly used method for correcting for measurement error, in which each individual's unknown true exposure in the outcome regression model is replaced by its expectation conditional on the error‐prone measure and any fully measured covariates. Regression calibration is simple to execute when the exposure is untransformed in the linear predictor of the outcome regression model, but less straightforward when non‐linear transformations of the exposure are used. We describe a method for applying regression calibration in models in which a non‐linear association is modelled by transforming the exposure using a fractional polynomial model. It is shown that taking a Bayesian estimation approach is advantageous. By use of Markov chain Monte Carlo algorithms, one can sample from the distribution of the true exposure for each individual. Transformations of the sampled values can then be performed directly and used to find the expectation of the transformed exposure required for regression calibration. A simulation study shows that the proposed approach performs well. We apply the method to investigate the relationship between usual alcohol intake and subsequent all‐cause mortality using an error model that adjusts for the episodic nature of alcohol consumption.     

Quantitative FRET measurement based on spectral unmixing of donor,acceptor and spontaneous excitation‐emission spectra

The spontaneous excitation‐emission (ExEm) spectrum is introduced to the quantitative mExEm‐spFRET methodology we recently developed as a spectral unmixing component for quantitative fluorescence resonance energy transfer measurement, named as SPEES‐FRET method. The spectral fingerprints of both donor and acceptor were measured in HepG2 cells with low autofluorescence separately expressing donor and acceptor, and the spontaneous spectral fingerprint of HEK293 cells with strong autofluoresence was measured from blank cells. SPEES‐FRET was performed on improved spectrometer‐microscope system to measure the FRET efficiency (E) and concentration ratio (R _C) of acceptor to donor vales of FRET tandem plasmids in HEK293 cells, and obtained stable and consistent results with the expected values. Moreover, SPEES‐FRET always obtained stable results for the bright and dim cells coexpressing Cerulean and Venus or Cyan Fluorescent Protein (CFP)‐Bax and Yellow fluorescent protein (YFP)‐Bax, and the E values between CFP‐Bax and YFP‐Bax were 0.02 for healthy cells and 0.14 for the staurosporine (STS)‐treated apoptotic cells. Collectively, SPEES‐FRET has very strong robustness against cellular autofluorescence, and thus is applicable to quantitative evaluation on the protein‐protein interaction in living cells with strong autofluoresence.      

Development and evaluation of the measurement system for the human shoulder joint based on the 6 DOF kinematic modelling

Although numerical models on the shoulder complex joint are currently available, many are impractical because of the procedural complexity coupled with limited and mere simple simulations. The present study defined the clavicle-scapula system as the "base of the humerus" in determining the position of proximal head of humerus, rendering conclusive innovation of a six degree of freedom (DOF) shoulder complex joint model. Furthermore, a complete measurement system where evaluation by calibrating the actual values via the use of an electromagnetic tracking device (ETD) was developed based on the innovated model. The special calibration method using optimizing calculation to work out the rotational center of humerus was employed and actually tested if the theoretical consideration was practically available. As a result of accuracy check experiments, the measurement error was defined within 2-3 mm, indicating sufficient accuracy in studies for human movement. Our findings strongly advocate that the benefit of this novel measurement system would contribute to studies related to shoulder movements in physiological anthropology.     

In situ measurement of degranulation as a biosensor based on RBL-2H3 mast cells

A direct degranulation assay has been developed to enable the use of RBL mast cells as a biosensor for screening chemical libraries for drug discovery and environmental toxicity evaluation. Release of beta-hexosaminidase into the extracellular milleu is widely used to characterize cellular components and mechanisms involved in stimulated exocytosis, including those initiated by crosslinking of IgE receptors on mast cells. To adapt this versatile assay for high throughput screening, we developed a direct, in situ method in which beta-hexosaminidase detection is carried out in a single step, convenient for multi-sample processing and thus for biosensor applications. This direct assay is efficient for measuring exocytosis in antigen-stimulated RBL mast cells, detecting antigen concentrations as low as 1 pM. We also demonstrate its utility in detecting inhibition of degranulation by a known pharmacologic inhibitor that blocks Syk tyrosine kinase activity critical for cell activation.     

Abnormal spectra alteration observed in Triton calibration method for measuring [Ca2+]i with fluorescence indicator, fura-2

We compared two commonly used calibration methods for measuring the concentration of intracellular free calcium ([Ca2+]i) by ratiometric fluorescence dye, fura-2 in mouse neuroblastoma-rat glioma hybrid cells (NG108-15). One calibration method, the Triton method, employs detergent Triton X-100, while the other, the Ionomycin method, uses a calcium-specific ionophore, Ionomycin. In the Triton method, we observed that at excitation 380 nm, the fura-2 fluorescence intensity of steady-state cells abnormally situated beyond the limiting intensity for calibration. By excitation scan, we demonstrated that this abnormality was caused by the change of fura-2 isosbestic points, which in turn was due to cell lysis after the addition of Triton X-100. This problem was resolved in the Ionomycin method by avoidance of cell lysis. Our results showed the correlation between inconsistent isosbestic points and cell lysis. As the basis for [Ca2+]i calibration, the proportionality between the fluorescence intensity and the concentration of dye species was impaired because of inconsistent isosbestic points. This inconsistency can be eliminated by a preliminary experiment of excitation scan to test the feasibility of different calibration methods.     

Integrated electrodes on a silicon based ion channel measurement platform

We demonstrate the microfabrication of a low-noise silicon based device with integrated silver/silver chloride electrodes used for the measurement of single ion channel proteins. An aperture of 150 microm diameter was etched in a silicon substrate using a deep silicon reactive ion etcher and passivated with 30 nm of polytetrafluoroethylene via chemical vapor deposition. The average recorded noise in measurements of lipid bilayers was reduced by a factor of four through patterning of a 75 microm thick SU-8 layer around the aperture. Integrated electrodes were fabricated on both sides of the device and used for repeatable, stable, giga-seal bilayer formations as well as characteristic measurements of the transmembrane protein OmpF porin.     

Automated diagnostics of a magnetron discharge plasma based on atomic molecular emission spectra

A software-hardware complex intended for investigating spatial distributions of the plasma spectral emissivity is described. It allows us to record and identify the lines and systems of molecular bands in an automatic mode and to perform computer processing of spectra. Molecular bands of deuterium for different electronic-vibrational-rotational transitions are identified. The excitation temperatures of atomic levels, translational, rotational and vibrational temperatures are estimated for a discharge in a planar magnetron.     

A Bayesian adjustment for multiplicative measurement errors for a calibration problem with application to a stem cell study

We develop a Bayesian approach to a calibration problem with one interested covariate subject to multiplicative measurement errors. Our work is motivated by a stem cell study with the objective of establishing the recommended minimum doses for stem cell engraftment after a blood transplant. When determining a safe stem cell dose based on the prefreeze samples, the postcryopreservation recovery rate enters in the model as a multiplicative measurement error term, as shown in the model. We examine the impact of ignoring measurement errors in terms of asymptotic bias in the regression coefficient. According to the general structure of data available in practice, we propose a two-stage Bayesian method to perform model estimation via R2WinBUGS (Sturtz, Ligges, and Gelman, 2005, Journal of Statistical Software 12, 1-16). We illustrate this method by the aforementioned motivating example. The results of this study allow routine peripheral blood stem cell processing laboratories to establish recommended minimum stem cell doses for transplant and develop a systematic approach for further deciding whether the postthaw analysis is warranted.     

Calibration-curve-free quantitative PCR: a quantitative method for specific nucleic acid sequences without using calibration curves

We have developed a simple quantitative method for specific nucleic acid sequences without using calibration curves. This method is based on the combined use of competitive polymerase chain reaction (PCR) and fluorescence quenching. We amplified a gene of interest (target) from DNA samples and an internal standard (competitor) with a sequence-specific fluorescent probe using PCR and measured the fluorescence intensities before and after PCR. The fluorescence of the probe is quenched on hybridization with the target by guanine bases, whereas the fluorescence is not quenched on hybridization with the competitor. Therefore, quench rate (i.e., fluorescence intensity after PCR divided by fluorescence intensity before PCR) is always proportional to the ratio of the target to the competitor. Consequently, we can calculate the ratio from quench rate without using a calibration curve and then calculate the initial copy number of the target from the ratio and the initial copy number of the competitor. We successfully quantified the copy number of a recombinant DNA of genetically modified (GM) soybean and estimated the GM soybean contents. This method will be particularly useful for rapid field tests of the specific gene contamination in samples.     

Simplified measurement of insulin sensitivity with the minimal model procedure in type 2 diabetic patients without measurement of insulinemia.

This study aimed to evaluate a simplified minimal model protocol for measuring insulin sensitivity in mild and severe type 2 diabetes, considering that changes in serum insulin during an insulin-modified intravenous glucose tolerance test almost only reflect the insulin injection. Two groups of diabetics treated with high doses of antidiabetic agents were recruited. Mean insulin responses were calculated in group 1 (n = 30). In group 2 (n = 38), we compared insulin sensitivity (SI) obtained with reference protocol with SI calculated by a minimal model procedure including the theoretical average insulin profile determined in group 1, and with Homeostasis Model Assessment (HOMA-R). Additionally, the cost of each procedure was calculated. SI measured by the reference method strongly correlated with SI determined by the simplified protocol (r = 0.966, p < 0.0001), while no correlation was found with HOMA-R (r = - 0.349, NS). Reduction of cost for HOMA-R and simplified minimal model procedure were - 92 and - 81 %, respectively. This simplified and relative inexpensive protocol, using minimal model procedure without insulin measurement, accurately measures SI regardless of beta-cell defect degree. This approach could be of interest when limits of validity of simple indexes are reached.     

Protein structure modelling and evaluation based on a 4-distance description of side-chain interactions

Background  

Accurate evaluation and modelling of residue-residue interactions within and between proteins is a key aspect of computational structure prediction including homology modelling, protein-protein docking, refinement of low-resolution structures, and computational protein design.     

Acute effects of different light spectra on simulated night-shift work without circadian alignment

Short-wavelength and short-wavelength-enhanced light have a strong impact on night-time working performance, subjective feelings of alertness and circadian physiology. In the present study, we investigated acute effects of white light sources with varied reduced portions of short wavelengths on cognitive and visual performance, mood and cardiac output.Thirty-one healthy subjects were investigated in a balanced cross-over design under three light spectra in a simulated night-shift paradigm without circadian adaptation.Exposure to the light spectrum with the largest attenuation of short wavelengths reduced heart rate and increased vagal cardiac parameters during the night compared to the other two light spectra without deleterious effects on sustained attention, working memory and subjective alertness. In addition, colour discrimination capability was significantly decreased under this light source.To our knowledge, the present study for the first time demonstrates that polychromatic white light with reduced short wavelengths, fulfilling current lighting standards for indoor illumination, may have a positive impact on cardiac physiology of night-shift workers without detrimental consequences for cognitive performance and alertness.     

Evaluating the efficiency of enzyme accelerated CO2 capture: chemical kinetics modelling for interpreting measurement results

In this paper, the efficiency of the carbonic anhydrase (CA) enzyme in accelerating the hydration of CO₂ is evaluated using a measurement system which consists of a vessel in which a gaseous flow of mixtures of nitrogen and CO₂ is bubbled into water or water solutions containing a known quantity of CA enzyme. The pH value of the solution and the CO₂ concentration at the measurement system gas exhaust are continuously monitored. The measured CO₂ level allows for assessing the quantity of CO₂, which, subtracted from the gaseous phase, is dissolved into the liquid phase and/or hydrated to bicarbonate. The measurement procedure consists of inducing a transient and observing and modelling the different kinetics involved in the steady-state recovery with and without CA. The main contribution of this work is exploiting dynamical system theory and chemical kinetics modelling for interpreting measurement results for characterising the activity of CA enzymes. The data for model fitting are obtained from a standard bioreactor, in principle equal to standard two-phase bioreactors described in the literature, in which two different techniques can be used to move the process itself away from the steady-state, inducing transients.