Dry-Milling and Fractionation of Transgenic Maize Seed Tissues with Green Fluorescent Protein as a Tissue Marker

The efficiency of fractionating cereal grains (e.g., dry corn milling) can be evaluated and monitored by quantifying the proportions of seed tissues in each of the recovered fractions. The quantities of individual tissues are typically estimated using indirect methods such as quantifying fiber or ash to indicate pericarp and tip cap contents, and oil to indicate germ content. More direct and reliable methods are possible with tissue-specific markers. We used two transgenic maize lines, one containing the fluorescent protein green fluorescent protein (GFP) variant S65T expressed in endosperm, and the other containing GFP expressed in germ to determine the fate of each tissue in the dry-milling fractionation process. The two lines were dry-milled to produce three fractions (bran-, endosperm-, and germ-rich fractions) and GFP fluorescence was quantified in each fraction to estimate the tissue composition. Using a simplified laboratory dry-milling procedure and our GFP-containing grain, we determined that the endosperm-rich fraction contained 4% germ tissue, the germ-rich fraction contained 28% germ, 20% endosperm, and 52% nonendosperm and nonembryo tissues, and the bran-rich fraction contained 44% endosperm, 13% germ, and 43% nonendosperm and nonembryo tissues. GFP-containing grain can be used to optimize existing fractionation methods and to develop improved processing strategies

Green Fluorescent Protein as a Tissue Marker in Transgenic Maize Seed

Seed tissues (endosperm, embryo, and pericarp) are often separated into tissue-enriched fractions by wet- or dry-milling methods for use in food, feed, and industrial products. Seed tissue markers that are sensitive and readily quantifiable would be useful to optimize fractionation processes. To meet this need for tissue markers, we set out to produce and characterize different transgenic maize lines, each containing green fluorescent protein (GFP) in either endosperm or embryo. We examined mRNA transcripts using expressed sequence tag (EST) profiles of several major seed proteins and selected several with strong seed tissue preferences. Stably transformed maize lines were produced, and visual observation of fluorescence confirmed the presence of GFP in the desired tissues. To establish the utility of this grain for evaluating the effectiveness or separation efficiencies of fractionation processes, transgenic kernels were hand-dissected into pericarp, endosperm, and embryo fractions and the GFP concentration in each fraction was determined. The GFP distribution between fractions of each transgenic event was calculated from GFP concentration and mass balance, which enabled the determination of GFP yield based on the hand-dissection fractionation data and the amount of tissue contamination in each fraction. Our transgenic lines exhibited strong tissue preference for either embryo or endosperm. These lines should be useful for assessing separation efficiencies in maize fractionation processes

Absence of inspiratory laryngeal constrictor muscle activity during nasal neurally adjusted ventilatory assist in newborn lambs

It has been demonstrated that a progressive increase in nasal pressure support ventilation (nPSV) leads to an active inspiratory glottal closure in non-sedated newborn lambs, which limits lung ventilation (24, 33). Unlike nPSV, the pressure delivered during nasal Neurally Adjusted Ventilatory Assist (nNAVA) is synchronized to the diaphragm electrical activity on inspiration (36). Given the tight neural integration of the glottal dilators and constrictors with diaphragm activity on inspiration and expiration respectively, the aim of the present study was to test the hypothesis that inspiratory glottal closure does not develop during nNAVA. Polysomnographic recordings were performed in eight non-sedated, chronically instrumented lambs, which were ventilated with progressively increasing levels of nPSV and nNAVA, in random order. States of alertness, diaphragm and glottal muscle electrical activity, tracheal pressure, SpO2, tracheal PETCO2 and respiratory inductive plethysmography were continuously recorded. While phasic inspiratory glottal constrictor electrical activity appeared with increasing levels of nPSV in 5 out of 8 lambs, it was never observed at any nNAVA level in any lamb, even at maximal achievable nNAVA levels. In addition, a decrease in arterial PCO2 was neither necessary nor sufficient for the development of phasic inspiratory glottal constrictor activity. In conclusion, nNAVA does not induce active glottal closure in non-sedated newborn lambs at high-pressure levels, in contrast to nPSV

Effect of dynamic random leaks on the monitoring accuracy of home mechanical ventilators: a bench study

BACKGROUND: So far, the accuracy of tidal volume (VT) and leak measures provided by the built-in software of commercial home ventilators has only been tested using bench linear models with fixed calibrated and continuous leaks. The objective was to assess the reliability of the estimation of tidal volume (VT) and unintentional leaks in a single tubing bench model which introduces random dynamic leaks during inspiratory or expiratory phases. METHODS: The built-in software of four commercial home ventilators and a fifth ventilator-independent ad hoc designed external software tool were tested with two levels of leaks and two different models with excess leaks (inspiration or expiration). The external software analyzed separately the inspiratory and expiratory unintentional leaks. RESULTS: In basal condition, all ventilators but one underestimated tidal volume with values ranging between -1.5 ± 3.3% to -8.7% ± 3.27%. In the model with excess of inspiratory leaks, VT was overestimated by all four commercial software tools, with values ranging from 18.27 ± 7.05% to 35.92 ± 17.7%, whereas the ventilator independent-software gave a smaller difference (3.03 ± 2.6%). Leaks were underestimated by two applications with values of -11.47 ± 6.32 and -5.9 ± 0.52 L/min. With expiratory leaks, VT was overestimated by the software of one ventilator and the ventilator-independent software and significantly underestimated by the other three, with deviations ranging from +10.94 ± 7.1 to -48 ± 23.08%. The four commercial tools tested overestimated unintentional leaks, with values between 2.19 ± 0.85 to 3.08 ± 0.43 L/min. CONCLUSIONS: In a bench model, the presence of unintentional random leaks may be a source of error in the measurement of VT and leaks provided by the software of home ventilators. Analyzing leaks during inspiration and expiration separately may reduce this source of error