A new set of reference genes for comparative gene expression analyses in Yarrowia lipolytica

ABSTRACT Accurate quantitation of gene expression levels require sensitive, precise and reproducible measurements of specific transcripts. Normalization to a reference gene is the most common practice to minimize the impact of the uncontrolled variation. The fundamental prerequisite for an accurate reference gene is to be stably expressed amongst all the samples included in the analysis. In the present study we aimed to assess the expression level and stability of a panel of 21 genes in Yarrowia lipolytica throughout varying conditions, covering composition of the culturing medium, growth phase and strain—wild type and recombinant burdened with heterologous protein overexpression. The panel of the selected candidate genes covered those essential for growth and maintenance of metabolism and homologs of commonly used internal references in RT-qPCR. The candidate genes expression level and stability were assessed and the data were processed using dedicated computational tools (geNorm and NormFinder). The results obtained here indicated genes unaffected by the burden of overexpression (TEF1, TPI1, UBC2, SRPN2, ALG9-like, RYL1) or by the culture medium used (ACT1, TPI1, UBC2, SEC61, ODC, CLA4, FKS1, TPS1), as well as those the least (SSDH, ODC, GPD) and the most (SEC62, TPI1, IPP1) suitable for normalization of RT-qPCR data in Y. lipolytica.

On Deposition and Build-Up Detachment in Compressor Fouling

Compressor fouling is a severe problem for both heavy-duty and aero-propulsion gas turbines. Particles can impinge on the blade and annulus surfaces, sticking there. The consequences of particle deposition are the increase of the roughness and an uncontrolled variation in the surface shape. These problems have a major effect on the performance of the compressor over time. Variations in the flow field can make the flow quantities close to the deposit to change, and it may happen that the conditions for the sticking do not hold any longer. If this is the case, the build-up detachment may happen. This occurrence can mitigate the fouling effects and may be exploited for keeping the performance of the compressor as high as possible over the operating period. In this work, an innovative model is proposed in order to evaluate the adhesion forces and the possible detachment. Particularly, the same forces that keep a gecko stuck to a surface are considered: the van der Waals forces (due to the proximity of the two bodies) and the Laplace force (due to the curvature of the liquid film related to the humidity). The so formulated model, named gecko-like for such a reason, is used for the numerical analyses of a deposition problem. Both the sticking and possible build-up detachment are considered. The outcome of this work can be regarded as an a-priori estimate of the forces to be kept into account when dealing with compressor fouling problems.

Sensitivity of a VCSEL threshold performance to inaccuracies in its manufacturing

The paper describes an impact of various possible inaccuracies in manufacturing of verticalcavity surface-emitting diode lasers (VCSELs), like thicknesses and compositions of their layers different from assumed ones, on VCSEL room-temperature (RT) continuous-wave (CW) threshold performance. To this end, the fully self-consistent comprehensive optical-electrical-thermal-recombination VCSEL model has been applied. While the analysis has been carried out for the 1.3-μm oxide-confined intra-cavity contacted GaInNAs/GaAs VCSEL, its conclusions are believed to be more general and concern most of modern VCSEL designs. As expected, the VCSEL active region has been found to require the most scrupulous care in its fabrication, any uncontrolled variation in compositions and/or thicknesses of its layers is followed by unaccepted RT CW lasing threshold increase. Also spacer thicknesses should be manufactured with care to ensure a proper overlapping of the optical standing wave and both the gain and lossy areas within the cavity. On the contrary, less than 5% thickness changes in distributed-Bragg-reflectors are followed by nearly insignificant changes in VCSEL RT CW threshold. However, exceeding the above limit causes a rapid increase in lasing thresholds. As expected, in all the above cases, VCSELs equipped with larger active regions have been confirmed to require more careful technology. The above results should enable easier organization of VCSEL manufacturing.

Accuracy of Fourier transform infrared spectrometry in determination of casein in dairy cows' milk

For practical purposes the casein content of milk is often estimated from the crude protein content using a general conversion factor. This is done even though it is known that several factors can influence the percentage of casein nitrogen in the total nitrogen (casein number). The uncontrolled variation in casein number affects optimization of cheese production because casein is a limiting factor for cheese yield. Fourier transform infrared spectrometry has recently been introduced for casein determination. Using this technique we obtained a standard error of prediction (SEP) of 0·033% for casein concentrations in the range 2·1–4·0%. The true prediction error was estimated as 0·025%. The correlation between casein numbers obtained by reference analysis and infrared spectrometry could be expressed by an R2 of 0·73 and an SEP of 0·89 for casein numbers in the range 70·7–81·0.

Effect of frequency of ovine ruminal sampling on microbial activity and substrate fermentation

Food eaten by a ruminant firstly undergoes microbial fermentation within the rumen. Nutritionally important characteristics of the food are the rate and extent of fermentation of its carbohydrate fraction, which can both be estimated using the in vitro gas production technique. The single greatest source of uncontrolled variation in any in vitro rumen fermentation system is the rumen fluid; curves produced from gas production data were influenced significantly by the variation in microbial activity between days (Menke and Steingass, 1988; Beuvink et al, 1992). A more reliable measure of rumen fluid activity is needed. The objective of this study was to determine whether the frequency of sampling of rumen fluid affected the microbial activity and subsequent fermentation.

Effects of the level of concentrate in the diet of the donor animal on the in vitro digestibility of hay

The inoculum represents one of the greatest sources of uncontrolled variation in digestibility estimates via in vitro rumen fermentation methods (Marten and Barnes 1980). Microbial activity of rumen inoculum may be influenced by animal and diet factors. The objective of this work was to evaluate the effects of the level of inclusion of concentrate in the diet of the donor animal on the measurement of the in vitro digestibility of hay.

Effects of the level of concentrate in the diet of the donor animal on the in vitro digestibility of hay

The inoculum represents one of the greatest sources of uncontrolled variation in digestibility estimates via in vitro rumen fermentation methods (Marten and Barnes 1980). Microbial activity of rumen inoculum may be influenced by animal and diet factors. The objective of this work was to evaluate the effects of the level of inclusion of concentrate in the diet of the donor animal on the measurement of the in vitro digestibility of hay.

Clinical Pathology: Preanalytical Variation in Preclinical Safety Assessment Studies-Effect on Predictive Value of Analyte Tests

Significant differences in concentrations of analytes in samples may be introduced before samples enter analyzers. These differences are known as preanalytical variation and are part of the overall variation in analytical data. Preanalytical variation is caused by factors that operate during animal preparation prior to sampling, sample collection, sample processing, and sample storage prior to measurement. Preanalytical variation is important because it detracts from the predictive value of analyte measurements. Preanalytical variation may permanently damage data. Because its effects are difficult to quantitate it should be minimized in safety assessment studies. Sources of preanalytical variation are actions performed on animals prior to sample collection and actions performed on the specimen prior to analysis. Preanalytical variation produces a range of artefacts in experimental data. Consequences of preanalytical variation are loss of confidence in the data, obfuscation of real test article effects, false effects, and possibly the expense of repeating a study. To limit preanalytical variation, its sources must be identified, the effects documented, and measures devised to eliminate its sources. Predictive value (likelihood of actual disease) of appropriate clinical pathology tests in toxicology is inversely dependent on preanalytical variation: uncontrolled variation produces data with low predictive values, and controlled variation produces data with high predictive values.

Slope-Controlled Performance Testing

Cognitive-ability tests, though promising in other respects, generally show pronounced practice effects and have weak test-retest reliabilities. One reason for the low reliabilities appears to be that practice effects themselves vary from individual to individual, so that subjects differ not only in the levels at which they are performing when testing ends but also in the slopes leading up to those levels. Since slope of the performance curve late in practice has been shown to affect performance at reacquisition (retest), uncontrolled variation in slope may lower test-retest reliability. A possible approach to this problem is experimentally to control slope during testing so that all subjects are improving at roughly the same rates when testing ends. The expected effect is that, with inter-subject differences in slope controlled, the temporal stability of cognitive-ability tests will improve. If temporal stability improves, however, predictive validities ought also to improve.

Bond Angle Disorder in Tetrahedrally Bonded Amorphous Silicon

ABSTRACTThere have been many studies of the vibrational and optical properties of a- Si and a-Ge that have correlated systematic variations of these properties with films grown and/or annealing temperatures, and have attributed these variations to changes the local atomic structure and intrinsic network disorder that are correlated with the thermal history of a particular sample. The most frequently proposed atomic structure parameter associated with this intrinsic disorder has been the width of the bond angle distribution. We attempted to isolate the effects of bond angle disorder on the vibrational and electronic properties of a-Si using a Bethe Lattice structure that avoids some of the uncertainties introduced by the uncontrolled variation in other atom structure parameters. Using this approach, we show that increases in the width of the bond angle distribution can account for trends in the calculated vibrational and optical properties wth thermal hisrory that are in good qualitative agreement with trends reported in the experiments.