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Extinction rates in the modern world are currently at their highest in 66 million years and are likely to increase with projections of future climate change. Our knowledge of modern-day extinction risk is largely limited to decadal-centennial terrestrial records, while data from the marine realm is typically applied to high-order (> 1 million year) timescales. At present, it is unclear whether fossil organisms with common ancestry and ecological niche exhibit consistent indicators of ecological stress prior to extinction. The marine microfossil record, specifically that of the planktonic foraminifera, allows for high-resolution analyses of large numbers of fossil individuals with incredibly well-established ecological and phylogenetic history. Here, analysis of the isochronous extinction of two members of the planktonic foraminiferal genus Dentoglobigerina shows disruptive selection differentially compounded by permanent ecological niche migration, "pre-extinction gigantism", and photosymbiont bleaching prior to extinction. Despite shared ecological and phylogenetic affinity, and timing of extinction, the marked discrepancies observed within the pre-extinction phenotypic responses are species-specific. These behaviours may provide insights into the nature of evolution and extinction in the open ocean and can potentially assist in the recognition and understanding of marine extinction risk in response to global climate change.

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The end-Permian mass extinction, ∼252 million years ago, is notable for a complex recovery period of ∼5 Myr. Widespread euxinic (anoxic and sulfidic) oceanic conditions have been proposed as both extinction mechanism and explanation for the protracted recovery period, yet the vertical distribution of anoxia in the water column and its temporal dynamics through this time period are poorly constrained. Here we utilize Fe-S-C systematics integrated with palaeontological observations to reconstruct a complete ocean redox history for the Late Permian to Early Triassic, using multiple sections across a shelf-to-basin transect on the Arabian Margin (Neo-Tethyan Ocean). In contrast to elsewhere, we show that anoxic non-sulfidic (ferruginous), rather than euxinic, conditions were prevalent in the Neo-Tethys. The Arabian Margin record demonstrates the repeated expansion of ferruginous conditions with the distal slope being the focus of anoxia at these times, as well as short-lived episodes of oxia that supported diverse biota.

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To describe the signalment, clinicopathological findings and outcome in dogs presenting with acute kidney injury (AKI) and skin lesions between November 2012 and March 2014, in whom cutaneous and renal glomerular vasculopathy (CRGV) was suspected and renal thrombotic microangiopathy (TMA) was histopathologically confirmed. The medical records of dogs with skin lesions and AKI, with histopathologically confirmed renal TMA, were retrospectively reviewed. Thirty dogs from across the UK were identified with clinicopathological findings compatible with CRGV. These findings included the following: skin lesions, predominantly affecting the distal extremities; AKI; and variably, anaemia, thrombocytopaenia and hyperbilirubinaemia. Known causes of AKI were excluded. The major renal histopathological finding was TMA. All thirty dogs died or were euthanised. Shiga toxin was not identified in the kidneys of affected dogs. Escherichia coli genes encoding shiga toxin were not identified in faeces from affected dogs. CRGV has previously been reported in greyhounds in the USA, a greyhound in the UK, without renal involvement, and a Great Dane in Germany. This is the first report of a series of non-greyhound dogs with CRGV and AKI in the UK. CRGV is a disease of unknown aetiology carrying a poor prognosis when azotaemia develops.

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Concentrations of dissolved organic carbon have increased in many, but not all, surface waters across acid impacted areas of Europe and North America over the last two decades. Over the last eight years several hypotheses have been put forward to explain these increases, but none are yet accepted universally. Research in this area appears to have reached a stalemate between those favouring declining atmospheric deposition, climate change or land management as the key driver of long-term DOC trends. While it is clear that many of these factors influence DOC dynamics in soil and stream waters, their effect varies over different temporal and spatial scales. We argue that regional differences in acid deposition loading may account for the apparent discrepancies between studies. DOC has shown strong monotonic increases in areas which have experienced strong downward trends in pollutant sulphur and/or seasalt deposition. Elsewhere climatic factors, that strongly influence seasonality, have also dominated inter-annual variability, and here long-term monotonic DOC trends are often difficult to detect. Furthermore, in areas receiving similar acid loadings, different catchment characteristics could have affected the site specific sensitivity to changes in acidity and therefore the magnitude of DOC release in response to changes in sulphur deposition. We suggest that confusion over these temporal and spatial scales of investigation has contributed unnecessarily to the disagreement over the main regional driver(s) of DOC trends, and that the data behind the majority of these studies is more compatible than is often conveyed.

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A pilot-scale primary maturation pond was spiked with (15)N-labelled ammonia ((15)NH(4)Cl) and (15)N-labelled nitrite (Na(15)NO(2)), in order to improve current understanding of the dynamics of inorganic nitrogen transformations and removal in WSP systems. Stable isotope analysis of delta(15)N showed that nitrification could be considered as an intermediate step in WSP, which is masked by simultaneous denitrification, under conditions of low algal activity. Molecular microbiology analysis showed that denitrification can be considered a feasible mechanism for permanent nitrogen removal in WSP, which may be supported either by ammonia-oxidising bacteria (AOB) or by methanotrophs, in addition to nitrite-oxidising bacteria (NOB). However, the relative supremacy of the denitrification process over other nitrogen removal mechanisms (e.g., biological uptake) depends upon phytoplanktonic activity.

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In this work a set of experiments was undertaken in a pilot-scale WSP system to determine the importance of organic nitrogen sedimentation on ammonium and total nitrogen removals in maturation ponds and its seasonal variation under British weather conditions, from September 2004 to May 2007. The nitrogen content in collected sediment samples varied from 4.17% to 6.78% (dry weight) and calculated nitrogen sedimentation rates ranged from 273 to 2868 g N/ha d. High ammonium removals were observed together with high concentrations of chlorophyll-a in the pond effluent. Moreover, chlorophyll-a had a very good correlation with the corresponding increment of VSS (algal biomass) and suspended organic nitrogen (biological nitrogen uptake) in the maturation pond effluents. Therefore, when ammonium removal reached its maximum, total nitrogen removal was very poor as most of the ammonia taken up by algae was washed out in the pond effluent in the form of suspended solids. After sedimentation of the dead algal biomass, it was clear that algal-cell nitrogen was recycled from the sludge layer into the pond water column. Recycled nitrogen can either be taken up by algae or washed out in the pond effluent. Biological (mainly algal) uptake of inorganic nitrogen species and further sedimentation of dead biomass (together with its subsequent mineralization) is one of the major mechanisms controlling in-pond nitrogen recycling in maturation WSP, particularly when environmental and operational conditions are favourable for algal growth.

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ERF/AP2 transcription factors play an important role in plant stress tolerance. However, little is known about the functional significance of ERF/AP2 genes in pine, compared to the model plant species Arabidopsis. Capsicum annuum pathogen and freezing tolerance-related protein 1 (CaPF1) is an ERF/AP2 transcription factor. We show here that overexpression of CaPF1 resulted in a dramatic increase in tolerance to drought, freezing, and salt stress in a gymnosperm species, eastern white pine (Pinus strobus L.). Measurement of polyamines demonstrated that the levels of putrescine (Put), spermidine (Spd), and spermine (Spm) did not increase but remain constant in CaPF1-overexpressed eastern white pine, whereas the levels decreased in the controls, probably increasing the ability of transgenic callus cultures and plants to stress tolerance. These results demonstrated that enhanced stress tolerance in transgenic pine expressing the pepper CaPF1 gene is associated with the polyamine biosynthesis and this pepper transcription factor may be used to engineer pine species for multiple stress tolerance.

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We report on a rapid method for reagentless identification and discrimination of single bacterial cells in aqueous solutions using a combination of laser tweezers and confocal Raman spectroscopy (LTRS). The optical trapping enables capturing of individual bacteria in aqueous solution in the focus of the laser beam and levitating the captured cell well off the cover plate, thus maximizing the excitation and collection of Raman scattering from the cell and minimizing the unwanted background from the cover plate and environment. Raman spectral patterns excited by a near-infrared laser beam provide intrinsic molecular information for reagentless analysis of the optically isolated bacterium. In our experiments, six species of bacteria were used to demonstrate the capability of the confocal LTRS in the identification and discrimination between the diverse bacterial species at various growth conditions. We show that synchronized bacterial cells can be well-discriminated among the six species using principal component analyses (PCA). Unsynchronized bacterial cells that are cultured at stationary phases can also be well-discriminated by the PCA, as well as by a hierarchical cluster analysis (HCA) of their Raman spectra. We also show that unsynchronized bacteria selected from random growth phases can be classified with the help of a generalized discriminant analysis (GDA). These findings demonstrate that the LTRS may find valuable applications in rapid sensing of microbial cells in diverse aqueous media.

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Genetic modification of conifers through gene transfer technology is now an important field in forest biotechnology. Two basic methodologies, particle bombardment and Agrobacterium-mediated transformation, have been used on conifers. The use of particle bombardment has produced stable transgenic plants in Picea abies, P. glauca, P. mariana, and Pinus radiata. Transgenic plants have been produced from Larix decidua, Picea abies, P. glauca, P. mariana, Pinus strobus, P. taeda, and P. radiata via Agrobacterium-mediated transformation. Agrobacterium-mediated transformation has advantages over particle bombardment such as a simpler integration pattern and a limited rearrangement in the introduced DNA. At present, genetic transformation of conifers has been directed toward improving growth rate, wood properties and quality, pest resistance, stress tolerance, and herbicide resistance, which will drive forestry to enter a new era of productivity and quality.

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The Dc8 gene of carrot (Daucus carota L.) shows differential expression during embryo development. Changes in methylation patterns of a segment of about 500 bp (from base +120 to base -446) of Dc8 allele 6 were investigated by treating genomic DNA, extracted from embryogenic callus at different stages of development, with sodium bisulfite to modify nonmethylated cytosines. Following asymmetric (strand-specific) amplification, base sequences for samples from each developmental stage were determined for each strand directly from the PCR products or from cloned PCR products. Different methylation patterns were detected in the two strands. The 5' to 3' sense (coding) strand was almost completely nonmethylated, whereas almost all the cytosines in the 3' to 5' (template) strand were methylated. By 71 days after transfer to embryo-inducing medium, few methylcytosines remained; those that were present were generally near the TATA box or in a region beyond -300. The cytosines that were methylated were not limited to CG or CNG sequences. The difference in the extent of methylation between the two complementary strands implies either that there is a mechanism for strand-specific methylation, or that complementary sequences can differ greatly in sensitivity to bisulfite treatment or PCR amplification.

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A cDNA clone (lp3) from loblolly pine induced by water deficit stress (WDS) has been isolated. It is preferentially induced in roots with a constitutive basal level of expression also observed in stems and needles. Northern blot analysis with well irrigated ABA-treated seedlings indicated that the overall accumulation of lp3 transcripts in the roots was lower than that of water deficit-stressed seedlings. However, within roots, lp3 was induced by ABA indicating that the expression of lp3 in roots under WDS conditions was partly mediated by ABA. The lp3 clone is similar to a group of genes called asr (ABA stress and ripening) genes identified in several species. A genomic clone (lp3-1) was identified and its putative protein has the hydrophylicity profile similar to that of lp3 except for two deletions in the 5' region. The genomic Southern and RT-PCR (reverse transcriptase-polymerase chain reaction) analyses indicate that the lp3 gene belongs to a small multigene family of at least four members with a distinct pattern of expression during WDS.

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A cDNA clone (pLP6) of a gene which is repressed under water deficit was isolated from a loblolly pine (Pinus taeda L.) cDNA library and characterized. The predicted polypeptide encoded by pLP6 bears strong resemblance to a number of Class I chitinases. However, LP6 lacks most of the amino-terminal and, consequently the signal peptide, cysteine-rich chitin-binding domain and glycine/proline-rich "hinge' region, diagnostic of Class I chitinases, are absent. Although the cDNA is similar in size to its mRNA, the long open reading frame encoding the LP6 protein commences halfway through the mRNA, implying a 5'-untranslated region of over 700 nucleotides. Subfragments from the 5' end of pLP6 hybridize to the same mRNA as do probes consisting of the entire cDNA. Reverse transcription(RT)-PCR experiments confirm that the cDNA derives from a single mRNA molecule. Analysis of the 5'-UTR revealed six upstream open reading frames and four inverted repeat structures. Expression of the pLP6 gene is repressed by water deficit stress and wounding. Possible functions and origin of this gene are discussed.

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DNA methylation has been associated with gene activity in differentiating and developing plant tissues. The objective of this study was to determine the involvement of methylation in the expression of a gene transferred into carrot (Daucus carota L.) tissues by particle bombardment. Expression of the Dc8-GUS gene construct in response to treatments with 5-azacytidine (S-azaC) and to in vitro methylation by methylases was investigated by histochemical assay of GUS activity. The 5-azaC treatment increased the frequency of Dc8-driven GUS expression in both calli and somatic embryos. The increase occurred with treatment either to E. coli containing the plasmid insert or to the carrot tissues before bombardment. GUS expression, increased by the 5-azaC treatment, was enhanced by ABA treatment of both calli and somatic embryos and was more prominent in the latter. Increased digestion of the 5-azaC-treated plasmid DNA with EcoRII suggested that demethylation had occurred. In vitro methylation of Dc8-GUS by methylases generally resulted in a lower frequency of GUS expression. SssI methylase completely inhibited GUS expression. The level of GUS expression was correlated with the extent of methylation of the plasmid.

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The 1.5 kilobase promoter sequence upstream of Dc8, a late embryo abundant gene of Daucus, fused to the reporter ß-glucuronidase gene was introduced into several tissues of Picea abies via a custom-made electric-discharge particle accelerator. Transient expression was measured histochemically as spot number 2 d after bombardment. Embryogenic suspensions gave higher levels of expression depending upon cell line than embryogenic callus or zygotic embryos. Expression was enhanced when cultures were treated with abscisic acid for 3 d before bombardment. A mean and maximum of 17 and 34 spots/disk, respectively, were observed with the best cell line, which was comparable with the level of expression driven by an enhanced 35S promoter.

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Twenty-two week-old Pinus taeda L. (loblolly pine) seedlings of 30 open-pollinated and five full-sib families, representing a wide range in geographic origin, were grown in charcoal-filtered (CF) air or CF-air supplemented with 160 or 320 nl liter(-1) ozone for 8 h day(-1), 4 days week(-1), for 9 weeks. Visible foliar injury (banded chlorosis, tip burn and premature senescence) was apparent in many families after 3 weeks in 320 nl liter(-1) and 6 weeks in 160 nl liter(-1) ozone. Decreases in relative height and root collar diameter growth rates, total dry weight, root dry weight, shoot dry weight, and root/shoot ratios were evident after 9 weeks of treatment with both 160 and 320 nl liter(-1) ozone. For relative height growth rates, family differences in response to ozone were observed. By the study's end, net photosynthesis rates were 15% less for the 320 nl liter(-1) ozone treatment as compared to the CF-air treatment. Total soluble sugar and total starch content of roots were not changed after 9 weeks of ozone exposure.

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Clonal propagation of Virginia Pine (Pinus virginiana Mill.) was achieved by organogenesis on cotyledon explants. The influence of several cytokinins and abscisic acid on adventitious shoot production from cotyledon explants was investigated. Benzyladenine was more effective in shoot induction than three other cytokinins tested. Benzyladenine (22.2 µM) in combination with naphthaleneacetic acid(0.05 µM) in a Gresshoff and Doy (1972) medium was found to increase shoot bud production. Abscisic acid (7.6 µM) in combination with benzyladenine and naphthaleneacetic acid enhanced shoot formation by an additional 65%. Root initiation was achieved with 0.5 strength Gresshoff and Doy media amended with naphthaleneacetic acid (1.3 µM), indole-3-butyric acid (1.2 µM) and benzyladenine (0.4 µM). Over 2400 plantlets from 2 families survived and were transferred to a greenhouse in preparation for field planting. After ten months, the maximum number of surviving plantlets/seed explant from these two sources was 57 for family ALPV-38 and 41 for family ALPV-78, respectively.

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Enhancement of shoot regeneration from loblolly pine (Pinus taeda L.) cotyledon explants was studied by addition of abscisic acid (ABA) to Gresshoff-Doy (GD) shoot induction medium containing benzylaminopurine (BA) and naphthaleneacetic acid (NAA). Addition of ABA (10(-7) M) doubled the morphogenic area of cotyledons and increased the fresh weight of cotyledon explants by 40 to 45% after 4 weeks. A 4-week exposure to ABA resulted in a larger morphogenic area per cotyledon than 3, 2, or 1 week(s) respectively. The enhancement by ABA was related to the explant seed source and was not increased by prolonged exposure. Compared to controls, shoot number was enhanced by 31% and 56% with 2 and 4 weeks of ABA (10(-7) M) exposure, respectively. Abscisic acid has a role in enhancing shoot morphogenesis in loblolly pine.