Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.     

Mitochondrial rate variation among lineages of passerine birds

The order Passeriformes comprises the majority of extant avian species. Analyses of molecular data have provided important insights into the evolution of this diverse order. However, molecular estimates of the evolutionary and demographic timescales of passerine species have been hindered by a lack of reliable calibrations. This has led to a reliance on the application of standard substitution rates to mitochondrial DNA data, particularly rates estimated from analyses of the gene encoding cytochrome b (CYTB). To investigate patterns of rate variation across passerine lineages, we used a Bayesian phylogenetic approach to analyse the protein‐coding genes of 183 mitochondrial genomes. We found that the most commonly used mitochondrial marker, CYTB, has low variation in rates across passerine lineages. This lends support to its widespread use as a molecular clock in birds. However, we also found that the patterns of among‐lineage rate variation in CYTB are only weakly related to the evolutionary rate of the mitochondrial genome as a whole. Our analyses confirmed the presence of mutational saturation at third codon positions across the protein‐coding genes of the mitochondrial genome, reinforcing the view that these sites should be excluded in studies of deep passerine relationships. The results of our analyses have provided information that will be useful for molecular‐clock studies of passerine evolution.     

Sequence analysis and functional characterization of the promoter of the PiceaglaucaCinnamylAlcoholDehydrogenase gene in transgenic white spruce plants

The enzyme Cinnamyl Alcohol Dehydrogenase (CAD) catalyses the last step of lignin monomer synthesis, and is considered as a molecular marker of cell wall lignification in different plants species. Here, we report the isolation and analysis of 5′ flanking genomic DNA regions upstream to the CAD gene, from two conifers, i.e. white spruce (Picea glauca (Moench) Voss) and loblolly pine (Pinus taeda L.). Sequence comparisons with available CAD gene promoters from angiosperms highlighted the conservation of cis-elements matching MYB, WRKY and bHLH binding sites. Functional characterization of the P. glauca CAD promoter used P. glauca seedlings stably transformed with a DNA fragment of 1,163 base pairs (PgCAD) fused to the β-glucuronidase (GUS) gene. Histochemical observations of different vegetative organs of the transgenic trees showed that this sequence was sufficient to drive GUS expression in lignifying tissues, and more specifically in differentiating xylem cells. Quantitative RT-PCR experiments also indicated that the native CAD gene was preferentially expressed in differentiating xylem both in stems and roots. In addition, GUS expression driven by the PgCAD promoter was wound-inducible which was consistent with the accumulation of CAD mRNA in response to jasmonate application and mechanical wounding. The spruce CAD promoter represents a valuable tool for research and biotechnology applications related to xylem and wood. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Human impact on vegetation at the Alpine tree-line ecotone during the last millennium: lessons from high temporal and palynological resolution

Three mires and a small lake in the Swiss and Austrian Alps were studied palynologically at high resolution, covering the last 1,000, 400, 50 and 1,200 years, respectively. Methodological lessons include: (1) Sub-decadal resolution in upper, little-decomposed peat layers reveals recurrent marked fluctuations in both percentages and influx of regional tree-pollen types, reflecting variations in pollen production rather than in plant-population sizes. (2) Intermittent, single-spectrum pollen maxima in samples of sub-decadal resolution indicate pollen transport in clumps. This type of pollen transport may remain unrecognized in sections with lower sampling resolution, which may then lead to inappropriate interpretation in terms of plant-population sizes. (3) The detection of short-lived phases of human impact in decomposed peat requires sampling intervals as close as 0.2 cm. (4) PAR (pollen influx) may reflect vegetation dynamics more faithfully than percentages. Reliable PAR, however, is difficult to achieve in Alpine mires due to past human impact on peat growth, even when complex depth–age modelling techniques are used. Critical comparison of PAR with percentages is therefore essential. (5) Careful consideration of spatial scales in pollen signals (local–regional and subdivisions) is essential for a realistic palaeo-ecological interpretation. Results in terms of past human impact on vegetation are summarized as follows: (1) Trends in pollen types reflecting regional human action are in general agreement with earlier findings for the western Swiss Alps, allowing for regional differences. (2) All mires in the Alps investigated here and in an earlier study experienced human impact during the last millennium. The studied small lake, lying in sub-alpine pasture, records forest dynamics at a lower elevation since a.d. 800.     

A pigtailed macaque model of Kyasanur Forest disease virus and Alkhurma hemorrhagic disease virus pathogenesis

Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.     

Genome-Based Identification of Active Prophage Regions by Next Generation Sequencing in Bacillus licheniformis DSM13

Prophages are viruses, which have integrated their genomes into the genome of a bacterial host. The status of the prophage genome can vary from fully intact with the potential to form infective particles to a remnant state where only a few phage genes persist. Prophages have impact on the properties of their host and are therefore of great interest for genomic research and strain design. Here we present a genome- and next generation sequencing (NGS)-based approach for identification and activity evaluation of prophage regions. Seven prophage or prophage-like regions were identified in the genome of Bacillus licheniformis DSM13. Six of these regions show similarity to members of the Siphoviridae phage family. The remaining region encodes the B. licheniformis orthologue of the PBSX prophage from Bacillus subtilis. Analysis of isolated phage particles (induced by mitomycin C) from the wild-type strain and prophage deletion mutant strains revealed activity of the prophage regions BLi_Pp2 (PBSX-like), BLi_Pp3 and BLi_Pp6. In contrast to BLi_Pp2 and BLi_Pp3, neither phage DNA nor phage particles of BLi_Pp6 could be visualized. However, the ability of prophage BLi_Pp6 to generate particles could be confirmed by sequencing of particle-protected DNA mapping to prophage locus BLi_Pp6. The introduced NGS-based approach allows the investigation of prophage regions and their ability to form particles. Our results show that this approach increases the sensitivity of prophage activity analysis and can complement more conventional approaches such as transmission electron microscopy (TEM).     

Ultrafiltration-based techniques for rapid and simultaneous concentration of multiple microbe classes from 100-L tap water samples

This study focused on ultrafiltration as a technique for simultaneously concentrating and recovering viruses, bacteria and parasites in 100-L drinking water samples. A chemical dispersant, sodium polyphosphate, and Tween 80 were used to increase microbial recovery efficiencies. Secondary concentration was performed to reduce sample volumes to 3-5 mL for analysis using tissue culture, microscopy, and real-time PCR and RT-PCR. At seeding levels of 100-1000 (CFU, PFU, oocysts, or particles), a "high-flux" ultrafiltration procedure was found to achieve mean recoveries of 51-94% of simultaneously seeded MS2 bacteriophage, echovirus 1, Salmonella enterica subsp. enterica serovar Typhimurium, Bacillus atrophaeus subsp. globigii endospores, Cryptosporidium parvum oocysts, and 4.5-mum microspheres. When 4-7% of the final sample concentrate volume was assayed using real-time PCR and RT-PCR, overall method sensitivities were <100 C. parvum oocysts, <240 PFU echovirus 1, <100 CFU Salmonella and approximately 160 CFU B. atrophaeus spores in 100-L drinking water samples. The "high-flux" ultrafiltration procedure required approximately 2 h, including time required for backflushing. Secondary concentration procedures required an additional 1-3 h, while nucleic acid extraction and real-time PCR procedures required an additional 2-2.5 h. Thus, this study demonstrated that efficient recovery and sensitive detection of diverse microbes in 100-L drinking water samples could be achieved within 5-8 h using ultrafiltration, rapid secondary processing techniques, and real-time PCR.     

Carbon catabolite regulation of rebeccamycin production inSaccharothrix aerocolonigenes

Summary A new antitumor antibiotic named rebeccamycin was isolated from fermentations of an actinomycete,Saccharothrix aerocolonigenes. A defined medium was developed to study the regulation of synthesis of rebeccamycin byS. aerocolonigenes. In glucose medium formation of rebeccamycin was detected only after glucose was depleted. Examination of eleven different carbon sources revealed that carbon catabolite regulation is a major control mechanism for rebeccamycin production.     

Cell division systems that account for the various arrangements and types of differentiated cells within the secondary phloem of conifers

There are two main types of arrangement of differentiated cells within the radial cell files of secondary phloem in conifer trees. In the C-type arrangement, characteristic of the Cupressaceae, fibre (F), parenchyma (P) and sieve (S) cells are arranged in recurrent groups, such as the “standard” cellular quartet (FSPS). In the P-type arrangement, characteristic of the Pinaceae, there are no fibres and one of the characteristic recurrent arrangements is the cellular sextet (PSSSSS). In addition, in both C-type and P-type arrangements, similar cell types are often organised into tangential bands. A simulation model, based on the theory of L-systems, was devised to account for the determination of these two types of regular and recurrent patterns of differentiated phloem cells. It was based on the supposition that, in the meristematic portion of the phloem domain, there are specific spatio-temporal patterns of periclinal cell division. When new cells are produced, those already present are displaced along the cell file, occupying a predictable number of cellular positions as a result of each round of cell division. Each cellular position is assumed to be associated with a specific value of a morphogen, such as the auxin, indole acetic acid, relevant for vascular differentiation. Using published quantitative data on the distribution auxin across the phloem, and assuming specific threshold values of auxin necessary for the determination of each cell type, it was found that sequences of F, S or P cells developed in accordance with the specific pattern of cell division and the related positional values of auxin experienced by the cells during their displacement through the immediately post-mitotic zone of cell determination. The model accounts not only for the typical C-type and P-type cellular arrangements, but also for certain variant arrangements. It provides a working example of the concepts of positional information and positional value for patterned differentiation within a developing plant tissue. There are similarities between the way groups of phloem cells develop and the differentiation of somites in the embryos of vertebrates.