Projecting kelp (Ecklonia radiata) gametophyte thermal adaptation and persistence under climate change.

BACKGROUND AND AIMS: Kelp forests underpin temperate marine ecosystems but are declining due to ocean warming, causing loss of associated ecosystem services. Projections suggest significant future decline but often only consider the persistence of adult sporophytes. Kelps have a biphasic life cycle, and the haploid gametophyte can be more thermally tolerant than the sporophyte. Therefore, projections may be altered when considering the thermal tolerance of gametophytes. METHODS: We undertook thermal tolerance experiments to quantify the effect of temperature on gametophyte survival, relative growth rate (RGR) and sex ratio for three genetically distinct populations of Ecklonia radiata gametophytes from comparatively high, mid- and low latitudes (43°, 33° and 30°S). We then used these data to project the likely consequences of climate-induced thermal change on gametophyte persistence and performance across its eastern Australian range, using generalized additive and linear models. KEY RESULTS: All populations were adapted to local temperatures and their thermal maximum was 2-3 °C above current maximum in situ temperatures. The lowest latitude population was most thermally tolerant (~70 % survival up to 27 °C), while survival and RGR decreased beyond 25.5 and 20.5 °C for the mid- and low-latitude populations, respectively. Sex ratios were skewed towards females with increased temperature in the low- and high-latitude populations. Spatially explicit model projections under future ocean warming (2050-centred) revealed a minimal decline in survival (0-30 %) across populations, relative to present-day predictions. RGRs were also projected to decline minimally (0-2 % d-1). CONCLUSIONS: Our results contrast with projections for the sporophyte stage of E. radiata, which suggest a 257-km range contraction concurrent with loss of the low-latitude population by 2100. Thermal adaptation in E. radiata gametophytes suggests this life stage is likely resilient to future ocean warming and is unlikely to be a bottleneck for the future persistence of kelp.

Loss of a globally unique kelp forest from Oman.

Kelp forests are declining in many regions globally with climatic perturbations causing shifts to alternate communities and significant ecological and economic loss. Range edge populations are often at most risk and are often only sustained through localised areas of upwelling or on deeper reefs. Here we document the loss of kelp forests (Ecklonia radiata) from the Sultanate of Oman, the only confirmed northern hemisphere population of this species. Contemporary surveys failed to find any kelp in its only known historical northern hemisphere location, Sadah on the Dhofar coast. Genetic analyses of historical herbarium specimens from Oman confirmed the species to be E. radiata and revealed the lost population contained a common CO1 haplotype found across South Africa, Australia and New Zealand suggesting it once established through rapid colonisation throughout its range. However, the Omani population also contained a haplotype that is found nowhere else in the extant southern hemisphere distribution of E. radiata. The loss of the Oman population could be due to significant increases in the Arabian Sea temperature over the past 40 years punctuated by suppression of coastal upwelling. Climate-mediated warming is threatening the persistence of temperate species and precipitating loss of unique genetic diversity at lower latitudes.

Climate refugia for kelp within an ocean warming hotspot revealed by stacked species distribution modelling.

Canopy forming macroalgae are declining globally due to climate change and the identification of refuges for these habitats is crucial for their conservation. This is particularly pertinent in ocean warming hotspots where significant range contractions of kelp have occurred and are projected to continue. We developed a stacked urchin-kelp species distribution model (SDM) to predict climate refugia for kelp (Ecklonia radiata) in an ocean warming hotspot, south-eastern Australia. The optimal stacked-SDM incorporated biotic and abiotic explanatory covariates and was validated using an independent dataset. Density of the urchin Centrostephanus rodgersii, summer bottom temperature and photosynthetically available radiation at the seabed were significant predictors of kelp cover, highlighting the physiological and ecological influence of these variables on the distribution of kelp. Our optimal stacked-SDM predicted three spatially distinct refuge areas, where kelp occurs in deeper waters than surrounding seascapes. The presence of kelp at two of these refuge areas was confirmed using independent data. The identification of these refuge areas is crucial for conservation, as they are likely to facilitate the persistence of ecologically and economically important kelp forests as waters warm in shallow areas and kelp retreat to depth under climate change. Furthermore, identification of refugia will enable proactive spatial planning that prioritises new locations for protection to ensure that key kelp habitats can persist in a future of increasing stress.

The Silver Lining of Extreme Events.

Extreme climatic events cause devastating impacts to species and ecosystems, precipitating significant mortality. However, emerging empirical evidence is revealing that such mortality can drive directional selection and result in increased tolerance. We discuss the novel opportunities for promoting climate resilience presented by this 'silver lining' of extreme events.

A fixed-target platform for serial femtosecond crystallography in a hydrated environment.

For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24 kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering.

Crystallization of ApoA1 and ApoE4 nanolipoprotein particles and initial XFEL-based structural studies.

Nanolipoprotein particles (NLPs), also called "nanodiscs", are discoidal particles with a patch of lipid bilayer corralled by apolipoproteins. NLPs have long been of interest due to both their utility as membrane-model systems into which membrane proteins can be inserted and solubilized and their physiological role in lipid and cholesterol transport via HDL and LDL maturation, which are important for human health. Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a powerful approach for structural biology of membrane proteins, which are traditionally difficult to crystallize as large single crystals capable of producing high-quality diffraction suitable for structure determination. To facilitate understanding of the specific role of two apolipoprotein/lipid complexes, ApoA1 and ApoE4, in lipid binding and HDL/LDL particle maturation dynamics and develop new SFX methods involving NLP membrane protein encapsulation, we have prepared and crystallized homogeneous populations of ApoA1 and ApoE4 NLPs. Crystallization of empty NLPs yields semi-ordered objects that appear crystalline and give highly anisotropic and diffuse X-ray diffraction, similar in characteristics to fiber diffraction. Several unit cell parameters were approximately determined for both NLPs from these measurements. Thus, low-background, sample conservative methods of delivery are critical. Here we implemented a fixed target sample delivery scheme utilizing the Roadrunner fast-scanning system and ultra-thin polymer/graphene support films, providing a low-volume, low-background approach to membrane protein SFX. This study represents initial steps in obtaining structural information for ApoA1 and ApoE4 NLPs and developing this system as a supporting scaffold for future structural studies of membrane proteins crystalized in a native lipid environment.

Low dose radiation response curves, networks and pathways in human lymphoblastoid cells exposed from 1 to 10cGy of acute gamma radiation.

We investigated the low dose dependency of the transcriptional response of human cells to characterize the shape and biological functions associated with the dose-response curve and to identify common and conserved functions of low dose expressed genes across cells and tissues. Human lymphoblastoid (HL) cells from two unrelated individuals were exposed to graded doses of radiation spanning the range of 1-10cGy were analyzed by transcriptome profiling, qPCR and bioinformatics, in comparison to sham irradiated samples. A set of ∼80 genes showed consistent responses in both cell lines; these genes were associated with homeostasis mechanisms (e.g., membrane signaling, molecule transport), subcellular locations (e.g., Golgi, and endoplasmic reticulum), and involved diverse signal transduction pathways. The majority of radiation-modulated genes had plateau-like responses across 1-10cGy, some with suggestive evidence that transcription was modulated at doses below 1cGy. MYC, FOS and TP53 were the major network nodes of the low-dose-response in HL cells. Comparison our low dose expression findings in HL cells with those of prior studies in mouse brain after whole body exposure, in human keratinocyte cultures, and in endothelial cells cultures, indicates that certain components of the low dose radiation response are broadly conserved across cell types and tissues, independent of proliferation status.

The effectiveness of glibenclamide in women with gestational diabetes.

BACKGROUND: Several studies have suggested that glibenclamide may be used safely and effectively in women with gestational diabetes mellitus (GDM). The aim of our study was to assess effectiveness and safety of glibenclamide for GDM in UK clinical practice. METHODS: Women with GDM requiring pharmacological therapy were offered a choice of insulin or glibenclamide. Maternal and foetal outcomes were assessed in women treated with insulin (45) or glibenclamide (44) and also compared with women treated with diet alone (55). RESULTS: Thirty-four (77%) achieved adequate glycaemic control with glibenclamide. Women choosing glibenclamide were more likely to be Asian and had higher fasting and 2-h glucose at diagnosis than those choosing insulin. There was no difference in maternal age or parity. Ten women treated with glibenclamide switched to insulin [inadequate control (7), unpredictable hypoglycaemia (1) and other reason (2)]. There was no difference in mode of birth, birth weight or birth weight centile between groups. One stillbirth occurred with glibenclamide. Glibenclamide treatment was associated with lower Apgar scores and increased neonatal jaundice. Neonatal hypoglycaemia occurred more frequently in babies of women treated with either glibenclamide or insulin. CONCLUSION: The use of glibenclamide in pregnancy is associated with adequate glycaemic control in 77% of women and achieved similar foetal outcomes to women treated with insulin.

Acute fatty liver of pregnancy; three cases and discussion of analgesia and anaesthesia.

Acute fatty liver of pregnancy is a rare, potentially fatal, complication of late pregnancy. The incidence is estimated at 1:7000-1:15000 pregnancies. Presentation is classically with malaise, nausea and vomiting, abdominal pain and rarely encephalopathy. Prolongation of laboratory clotting tests is an early feature. Ultrasound examination of the liver is performed to exclude biliary stasis. Rapid clinical deterioration may occur and urgent delivery should be organised. Anaesthetists form part of a multidisciplinary approach before, during and after delivery but there are few reports of anaesthetic involvement. One dilemma facing an anaesthetist called to assist in these cases is the potentially negative effect of general anaesthesia on hepatic encephalopathy versus the risks associated with regional anaesthesia in the presence of coagulopathy. Postoperative analgesia may also be complicated by impaired renal and hepatic function. We present three cases that occurred in our unit in a 6-month period illustrating the spectrum of disease severity and the successful use of different anaesthetic techniques to facilitate management including delivery.

High-throughput, fluorescence-based screening for soluble protein expression.

Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.

Gene expression changes in mouse brain after exposure to low-dose ionizing radiation.

PURPOSE: To characterize the cellular functions associated with the altered transcript profiles of mouse brain exposed to low-dose in vivo gamma-irradiation. MATERIALS AND METHODS: Cerebral RNA was isolated at 30 min and 4 h after whole-body irradiation at 0.1 or 2 Gy, hybridized to random oligonucleotide arrays, and evaluated for time and dose-response patterns by multifactorial analyses. RESULTS: Brain irradiation modulated the expression patterns of 1574 genes, of which 855 showed more than 1.5-fold variation. about 30% of genes showed dose-dependent variations, including genes exclusively affected by 0.1 Gy. About 60% of genes showed time-dependent variation with more genes affected at 30 min than at 4 h. Early changes involved signal transduction, ion regulation and synaptic signalling. Later changes involved metabolic functions including myelin and protein synthesis. Low-dose radiation also modulated the expression of genes involved in stress response, cell-cycle control and DNA synthesis/repair. CONCLUSIONS: Doses of 0.1 Gy induced changes in gene expression that were qualitatively different from those at 2 Gy. The findings suggest that low-dose irradiation of the brain induces the expression of genes involved in protective and reparative functions, while down-modulating genes involved in neural signalling activity.

A postnatal fasting plasma glucose is useful in determining which women with gestational diabetes should undergo a postnatal oral glucose tolerance test.

AIMS: It is recommended that women with gestational diabetes (GDM) should have a 6-week postnatal oral glucose tolerance test (OGTT). As this test may be unpleasant, time-consuming and has resource implications, we evaluated whether the 6-week postnatal fasting glucose could be used to determine which women should undergo an OGTT. METHODS: All women with GDM, diagnosed according to the World Health Organization criteria, who were delivered at the Princess Anne Hospital, Southampton between May 2000 and May 2002, were recommended to have an OGTT. The results of the fasting plasma glucose concentration were assessed in relation to the 2-h glucose value. RESULTS: One-hundred and fifty-two women with GDM were delivered. Thirty (19.7%) women refused an OGTT or failed to attend. In the 122 OGTTs, three (2.4%; 95% confidence interval 0.8, 7) women had diabetes, three had impaired glucose tolerance and four had impaired fasting glycaemia. No woman with a normal test had fasting glucose of > or =6.0 mmol/l. Fasting glucose was correlated with the 2-h glucose (r=0.62, P<0.0001). Only 10 (8.1%) of the OGTTs would have been performed if only women with fasting glucose of > or =6.0 mmol/l underwent the test. The sensitivity and specificity of this approach for the diagnosis of postnatal diabetes is 100% and 94%, respectively. Linear regression methods indicate that it would miss fewer than three in 10 000 cases. CONCLUSIONS: In our population, a 6-week postnatal fasting plasma glucose is useful in determining which women with gestational diabetes should undergo an OGTT. Consequently we now perform OGTT only in women whose postnatal fasting plasma glucose is > or =6.0 mmol/l.

Predicting preterm delivery: comparison of cervicovaginal interleukin (IL)-1beta, IL-6 and IL-8 with fetal fibronectin and cervical dilatation.

OBJECTIVE: To compare the cervicovaginal cytokines IL-1beta, IL-6 and IL-8 with fetal fibronectin (fFN) and cervical dilatation in the prediction of preterm delivery. STUDY DESIGN: Cervicovaginal cytokine concentration and fFN status were measured in 104 women with symptoms of preterm labour and intact membranes between 24(0) and 33(6) weeks and related to delivery within 2 and 7 days. RESULTS: A group of 18% had cervical dilatation > or = 1cm and 18% were positive for fFN. Preterm delivery within 2 and 7 days occurred in 5 and 12%, respectively. Only IL-6 demonstrated any ability to predict delivery within 2 and 7 days (area under the ROC curve = 0.63 and 0.75, respectively). Using 35pg/ml (75th centile) as a cut-off, IL-6 had a sensitivity and specificity of 60 and 77% for predicting delivery within 2 days, and 62 and 80% for predicting delivery within 7 days. This is similar to the performance of cervical dilatation or fFN status. CONCLUSIONS: Measurement of cervicovaginal cytokines has limited ability to predict imminent delivery apart from cervicovaginal IL-6 concentrations, which, in this population, is equivalent to that of fFN status and cervical dilatation > or = 1cm.

Functional characterization of Ape1 variants identified in the human population.

Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions. Ape1 is the major human repair enzyme for abasic sites and incises the phosphodiester backbone 5' to the lesion to initiate a cascade of events aimed at removing the AP moiety and maintaining genetic integrity. Through resequencing of genomic DNA from 128 unrelated individuals, and searching published reports and sequence databases, seven amino acid substitution variants were identified in the repair domain of human Ape1. Functional characterization revealed that three of the variants, L104R, E126D and R237A, exhibited approximately 40-60% reductions in specific incision activity. A fourth variant, D283G, is similar to the previously characterized mutant D283A found to exhibit approximately 10% repair capacity. The most common substitution (D148E; observed at an allele frequency of 0.38) had no impact on endonuclease and DNA binding activities, nor did a G306A substitution. A G241R variant showed slightly enhanced endonuclease activity relative to wild-type. In total, four of seven substitutions in the repair domain of Ape1 imparted reduced function. These reduced function variants may represent low penetrance human polymorphisms that associate with increased disease susceptibility.

Corticotropin-releasing hormone, corticotropin-releasing hormone-binding protein, and activin A in maternal serum: prediction of preterm delivery and response to glucocorticoids in women with symptoms of preterm labor.

OBJECTIVE: The aim of this study was to determine prospectively whether serum concentrations of corticotropin-releasing hormone, corticotropin-releasing hormone-binding protein, and activin A (1) predict preterm birth within 10 days of hospital admission or at <37 weeks' gestation among women with symptoms of preterm labor and (2) are affected by glucocorticoid therapy. STUDY DESIGN: Serum concentrations of corticotropin-releasing hormone and activin A were measured in 94 women with symptoms of preterm labor between 24 and 34 weeks' gestation, and delivery outcomes were monitored. Corticotropin-releasing hormone-binding protein concentrations were measured in 71 of these women. In a subgroup of 15 women the serum analytes were assayed in conjunction with estriol before and 12 to 24 hours after administration of dexamethasone. RESULTS: Forty-six percent (6/13) of the women who were delivered within 10 days of hospital admission had a raised serum corticotropin-releasing hormone level, but the predictive relationship was not significant (chi(2) = 1.7; P =.2). Among the 31 women (including the 6 previously mentioned) who were delivered at <37 weeks' gestation, 39% (12/31) had a raised corticotropin-releasing hormone level. Although a raised corticotropin-releasing hormone concentration was positively associated with delivery at <37 weeks' gestation (chi(2) = 9; P =.003), the predictive diagnostic value was poor, with sensitivity, specificity, and positive and negative predictive values of 39%, 90%, 67%, and 75%, respectively. The serum concentrations of corticotropin-releasing hormone-binding protein and activin A were unrelated to gestational age at delivery. Dexamethasone markedly lowered the serum estriol level (P <.001) but had no effect on concentrations of corticotropinreleasing hormone, corticotropin-releasing hormone-binding protein, and activin A. CONCLUSION: Serum concentrations of corticotropin-releasing hormone, corticotropin-releasing hormone-binding protein, and activin A are not clinically useful for the prediction of preterm delivery among women with symptoms of preterm labor and are not affected by administration of glucocorticoids.