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The strong interaction of blood with the foreign surface of membrane oxygenators during ECMO therapy leads to adhesion of immune cells on the oxygenator membranes, which can be visualized in the form of image sequences using confocal laser scanning microscopy. The segmentation and quantification of these image sequences is a demanding task, but it is essential to understanding the significance of adhering cells during extracorporeal circulation. The aim of this work was to develop and test a deep learning-supported image processing tool (Deetect), suitable for the analysis of confocal image sequences of cell deposits on oxygenator membranes at certain predilection sites. Deetect was tested using confocal image sequences of stained (DAPI) blood cells that adhered to specific predilection sites (junctional warps and hollow fibers) of a phosphorylcholine-coated polymethylpentene membrane oxygenator after patient support (>24 h). Deetect comprises various functions to overcome difficulties that occur during quantification (segmentation, elimination of artifacts). To evaluate Deetects performance, images were counted and segmented manually as a reference and compared with the analysis by a traditional segmentation approach in Fiji and the newly developed tool. Deetect outperformed conventional segmentation in clustered areas. In sections where cell boundaries were difficult to distinguish visually, previously defined post-processing steps of Deetect were applied, resulting in a more objective approach for the resolution of these areas.

A family of three cDNAs, designated TaSUT1A, 1B and 1D, encoding sucrose transporter (SUT) proteins was isolated from a hexaploid wheat (Triticum aestivum) endosperm library. The cDNA sequences are 96% identical but are distinguishable from one another by virtue of a size polymorphism in the 3'-untranslated region (UTR). The predicted amino acid sequences are 98% identical and are highly similar to the sucrose transporters from rice, maize and barley. A gene for TaSUT1 was isolated from genomic libraries of Aegilops tauschii (the donor of the D genome of wheat) and the coding sequence found to be identical to that of TaSUT1D cDNA. There is only one copy of each TaSUT1 gene in hexaploid wheat and it is located on chromosome 4. Genomic Southern analysis and PCR analysis across the 3' polymorphic region of hexaploid, tetraploid and progenitor diploid wheat DNAs established that the TaSUT1A gene was present in the putative A-genome progenitor, T. monococcum, and that the TaSUT1B gene was present in the putative B-genome progenitor, T. searsii. All three TaSUT1 genes are expressed at high levels in filling grain, showing a good correlation with the developmental time course of growth. This reinforces the view that in cereals a major role of SUT1 is in the post-phloem sugar transport pathway associated with seed filling.

Plants, like animals, are obligate aerobes, but due to their inability to move, have evolved adaptation mechanisms that enable them to survive short periods of low oxygen supply, such as those occurring after heavy rain or flooding. Crop plants are often grown on soils subject to waterlogging and many are sensitive to waterlogging of the root zone. The combination of unfavourable weather conditions and suboptimal soil and irrigation techniques can result in severe yield losses. The molecular basis of the adaptation to transient low oxygen conditions has not been completely characterized, but progress has been made towards identifying genes and gene products induced during low oxygen conditions. Promoter elements and transcription factors involved in the regulation of anaerobically induced genes have been characterized. In this paper an account is presented of the molecular strategies that have been used in an attempt to increase flooding tolerance of crop plants.

The transcription factor AtMYB2 binds to two sequence motifs in the promoter of the Arabidopsis ADH1 gene. The binding to the GT-motif (5'-TGGTTT-3') is essential for induction of ADH1 by low oxygen, while binding to the second motif, MBS-2, is not essential for induction. We show that AtMYB2 is induced by hypoxia with kinetics compatible with a role in the regulation of ADH1. Like ADH1, AtMYB2 has root-limited expression. When driven by a constitutive promoter, AtMYB2 is able to transactivate ADH1 expression in transient assays in both Arabidopsis and Nicotiana plumbaginifolia protoplasts, and in particle bombardment of Pisum sativum leaves. Mutation of the GT-motif abolished binding of AtMYB2 and caused loss of activity of the ADH1 promoter in both transient assays and transgenic Arabidopsis plants. These results are consistent with AtMYB2 being a key regulatory factor in the induction of the ADH1 promoter by low oxygen.

The structural gene for the respiratory nitrous-oxide reductase from Paracoccus denitrificans has been cloned using a probe derived from the structural gene, nosZ, for this enzyme from Pseudomonas stutzeri. The cloned gene could be expressed surprisingly well (presumably yielding an apo-protein) using an expression vector in Escherichia coli. Sequencing the nosZ gene from P. denitrificans has shown that the periplasmic nitrous-oxide reductase of this organism is highly similar in sequence to previously derived primary sequences for the enzyme from three other organisms. As with the other reductases, an unusually long signal sequence is deduced and a common motif of GXXRRXXLG near the beginning of this sequence is present. The results of N-terminal sequencing of the mature nitrous-oxide reductase from the closely related organism Thiosphaera pantotropha indicate that processing of the P. denitrificans precursor occurs between amino acids at positions 57 and 58. The predicted signal peptide is therefore of the same length and of similar overall structure to that previously described for the P. denitrificans methylamine dehydrogenase small subunit (MauA). The P. denitrificans sequence for the mature nitrous-oxide reductase reduces from 14 to 11 and 6 to 4, respectively, the number of conserved histidine and methionine residues compared to previous sequences. Three cysteine and four tryptophan residues, previously identified as conserved amongst nitrous-oxide reductases, are found in the Paracoccus enzyme. A comparison of the sequence of the C-terminal region of the nitrous-oxide-reductase sequence with that for the CuA region of subunit II of the cytochrome aa3 from P. denitrificans reveals considerable sequence similarities. Upstream of the structural gene for nosZ are sequences TTGAAGCTTAACCAG (centred at position -21 with respect to the start codon) and CCCGGTGGTCATCAAG (centred at position -126). Although both could be FNR (ANR) boxes, the latter is far more probable to have this role because only it is likely to be upstream of a promoter site. This is the first indication at the DNA sequence level for the existence of this regulatory system in P. denitrificans. Analysis of the flanking DNA sequences revealed reading frames upstream and downstream of the nosZ gene showing similarity to the nosR and nosD genes, respectively, of Pseudomonas species. An S30 in vitro transcription/translation system was developed for P. denitrificans which permitted the expression of the cloned gene for nitrous-oxide reductase and which will be of general value in other studies of this organism.