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Purpose: Conditions and thresholds applied for evidence weighting of within-codon concordance (PM5) for pathogenicity vary widely between laboratories and expert groups. Because of the sparseness of available clinical classifications, there is little evidence for variation in practice. Methods: We used as a truthset 7541 dichotomous functional classifications of BRCA1 and MSH2, spanning 311 codons of BRCA1 and 918 codons of MSH2, generated from large-scale functional assays that have been shown to correlate excellently with clinical classifications. We assessed PM5 at 5 stringencies with incorporation of 8 in silico tools. For each analysis, we quantified a positive likelihood ratio (pLR, true positive rate/false positive rate), the predictive value of PM5-lookup in ClinVar compared with the functional truthset. Results: pLR was 16.3 (10.6-24.9) for variants for which there was exactly 1 additional colocated deleterious variant on ClinVar, and the variant under examination was equally or more damaging when analyzed using BLOSUM62. pLR was 71.5 (37.8-135.3) for variants for which there were 2 or more colocated deleterious ClinVar variants, and the variant under examination was equally or more damaging than at least 1 colocated variant when analyzed using BLOSUM62. Conclusion: These analyses support the graded use of PM5, with potential to use it at higher evidence weighting where more stringent criteria are met.

Exercise duration may influence the acute effects on cognition. However, only one study to date has explored the dose-response relationship between exercise duration and cognition in adolescents. Thus, the purpose of this study was to investigate the effect of differing durations of high-intensity intermittent running on cognition in adolescents. Thirty-eight adolescents (23 girls) completed three trials separated by 7 d: 30 min exercise, 60 min exercise, and rest, in a randomised crossover design. The exercise was a modified version of the Loughborough Intermittent Shuttle Test (LIST), which elicited high-intensity intermittent exercise. Cognitive function tests (Stroop test, Sternberg paradigm, Flanker task) were completed 30 min pre, immediately post, and 45 min post exercise. Response times on the incongruent level of the Flanker task improved to a greater extent 45 min following the 30 min LIST, compared to rest (p = 0.009). Moreover, response times improved to a greater extent on the three-item level of Sternberg paradigm 45 min following the 30 min LIST, compared to the 60 min LIST (p = 0.002) and rest (p = 0.013), as well as on the five-item level 45 min following the 30 min LIST, compared to the 60 min LIST (p = 0.002). In conclusion, acute exercise enhanced subsequent cognition in adolescents, but overall, 30 min of high-intensity intermittent running is more favourable to adolescents’ cognition, compared to 60 min.

The Daily Mile is a popular physical activity initiative in primary schools, yet little is known regarding the activity patterns of children during The Daily Mile. Eighty children (10.4 ± 0.7 years) participated in The Daily Mile (20-min). Activity patterns were assessed using global positioning systems (total distance and age-specific speed zones), alongside heart rate. Cardiorespiratory fitness was assessed using the Multi-Stage Fitness Test. Participants covered a distance of 2511 ± 550 m during The Daily Mile, and heart rate was 163 ± 27 beats.min−1. Participants travelled the furthest distance, and were most intermittent, during the first 5 min (main effect of time, both p p p

The present study examined the metabolic responses to an acute bout of football and the overall and moderating role of physical fitness on these responses, in adolescents. Thirty-six adolescents (16 girls, 20 boys; 12.6±0.5 y) completed two trials (60-min football and 60-min seated rest) separated by 7-d. Capillary blood samples were taken at baseline (60-min prior to exercise/rest), immediately, 30- and 60-min post-exercise and 30-, 60- and 120-min following a standardised lunch (1.5-, 2- and 3-h post-exercise), for the determination of blood glucose and plasma insulin concentrations. The median split of distance covered on the multi-stage fitness test was used to define high- and low-fit groups. Overall plasma insulin tAUC following lunch was lower in high-fit participants compared to low-fit (high-fit: 3784.2±1653.1 pmol·L−1x120min, low-fit: 6457.3±3290.7 pmol·L−1x120min; p0.05). Football reduced blood glucose concentration 1-h post-exercise compared to control (exercise: 3.8±0.6 mmol·L−1, rest: 4.6±0.8 mmol·L−1; p0.05). Blood glucose tAUC was not affected by exercise or physical fitness (p>0.05). These data emphasise the importance of physical fitness for metabolic health in adolescents, as well as the utility of football as a popular form of games-based activity for improving glucose regulation.

Postprandial glycaemia and insulinaemia are important risk factors for type 2 diabetes. The prevalence of insulin resistance in adolescents is increasing, but it is unknown how adolescent participant characteristics such as BMI, waist circumference, fitness and maturity offset may explain responses to a standard meal. The aim of the present study was to examine how such participant characteristics affect the postprandial glycaemic and insulinaemic responses to an ecologically valid mixed meal. Data from the control trials of three separate randomised, crossover experiments were pooled, resulting in a total of 108 participants (fifty-two boys, fifty-six girls; aged 12·5 (SD 0·6) years; BMI 19·05 (SD 2·66) kg/m2). A fasting blood sample was taken for the calculation of fasting insulin resistance, using the homoeostatic model assessment of insulin resistance (HOMA-IR). Further capillary blood samples were taken before and 30, 60 and 120 min after a standardised lunch, providing 1·5 g/kg body mass of carbohydrate, for the quantification of blood glucose and plasma insulin total AUC (tAUC). Hierarchical multiple linear regression demonstrated significant predictors for plasma insulin tAUC were waist circumference, physical fitness and HOMA-IR (F(3,98) = 36·78, P < 0·001, adjusted R2 = 0·515). The variance in blood glucose tAUC was not significantly explained by the predictors used (F(7,94) = 1·44, P = 0·198). Significant predictors for HOMA-IR were BMI and maturity offset (F(2,102) = 14·06, P < 0·001, adjusted R2 = 0·021). In summary, the key findings of the study are that waist circumference, followed by physical fitness, best explained the insulinaemic response to an ecologically valid standardised meal in adolescents. This has important behavioural consequences because these variables can be modified.

High-intensity intermittent exercise (HIIE) is a potential intervention to manage hyperglycaemia and insulin resistance in adolescents. The aim of this study was to determine the optimum duration of HIIE to reduce postprandial glycaemic and insulinaemic responses in adolescents and the longevity of the response. Thirty-nine participants (12.4 &plusmn, 0.4 year) completed a 30- and 60-min exercise trial (Loughborough Intermittent Shuttle Test) and a rested control trial in a randomised crossover design. Capillary blood samples were taken at baseline, immediately and 1-h post‐exercise, and 30, 60 and 120 min following a standardised lunch (day one) and a standardised breakfast 24-h post-exercise. Plasma insulin total area under the curve (tAUC) following lunch was lower following 60-min HIIE (21754 &plusmn, 16861 pmol&middot, L&minus, 1 &times, 120 min, p = 0.032) and tended to be lower following 30-min HIIE (24273 &plusmn, 16131 pmol&middot, 120 min, p = 0.080), when compared with the resting condition (26931 &plusmn, 21634 pmol&middot, 120 min). Blood glucose concentration was lower 1-h post-exercise following 30-min HIIE (3.6 &plusmn, 0.6 mmol&middot, 1) when compared to resting (4.1 &plusmn, 0.9 mmol&middot, 1, p = 0.001). Blood glucose and plasma insulin concentration did not differ across trials on day two. Shorter bouts of HIIE (30-min), as well as a 60-min bout, reduced the postprandial insulinaemic response to lunch, an ecologically valid marker of insulin sensitivity. As the beneficial effects of HIIE were limited to 3 h post-exercise, adolescents are recommended to engage daily HIIE to enhance metabolic health.

Additional file 1. A visual representation of the testing procedures for the Stroop Test and Sternberg Paradigm. Description of Data: These figures show exactly what participants saw on their laptop screens and outlines the instructions that were provided to them; i.e. what to do, how to do it. Copyright: these images were developed by the research team and have no copyright associated with them.

Additional file 2. Main effects (Trial, time and fitness), two-way interactions (trial by time) and three-way interactions (trial by time by fitness) for response times and accuracy across all test levels of the Stroop and Sternberg tests. Description of data: This table summarises the statistical outputs (p values) for the cognitive function tests to facilitate interpretation.

Background: An acute bout of exercise elicits a beneficial effect on subsequent cognitive function in adolescents. The effect of games-based activity, an ecologically valid and attractive exercise model for young people, remains unknown; as does the moderating effect of fitness on the acute exercise-cognition relationship. Therefore, the aim of the present study was to examine the effect of games-based activity on subsequent cognition in adolescents, and the moderating effect of fitness on this relationship.\ud \ud Methods: Following ethical approval, 39 adolescents (12.3 ± 0.7 year) completed an exercise and resting trial in a counterbalanced, randomised crossover design. During familiarisation, participants completed a multi-stage fitness test to predict VO2 peak. The exercise trial consisted of 60-min games-based activity (basketball), during which heart rate was 158 ± 11 beats∙min−1. A battery of cognitive function tests (Stroop test, Sternberg paradigm, trail making and d2 tests) were completed 30-min before, immediately following and 45-min following the basketball.\ud \ud Results: Response times on the complex level of the Stroop test were enhanced both immediately (p = 0.021) and 45-min (p = 0.035) post-exercise, and response times on the five item level of the Sternberg paradigm were enhanced immediately post-exercise (p = 0.023). There were no effects on the time taken to complete the trail making test or any outcome of the d2 test. In particular, response times were enhanced in the fitter adolescents 45-min post-exercise on both levels of the Stroop test (simple, p = 0.005; complex, p = 0.040) and on the three item level of the Sternberg paradigm immediately (p = 0.017) and 45-min (p = 0.008) post-exercise.\ud \ud Conclusions: Games-based activity enhanced executive function and working memory scanning speed in adolescents, an effect particularly evident in fitter adolescents, whilst the high intensity intermittent nature of games-based activity may be too demanding for less fit children.

During puberty young people undergo significant hormonal changes which affect metabolism and, subsequently, health. Evidence suggests there is a period of transient pubertal insulin resistance, with this effect greater in girls than boys. However, the response to everyday high and low glycaemic index (GI) meals remains unknown. Following ethical approval, forty adolescents consumed a high GI or low GI breakfast, in a randomised cross-over design. Capillary blood samples were taken during a 2-h postprandial period, examining the glycaemic and insulinaemic responses. Maturity offset and homoeostatic model assessment (HOMA) were also calculated. The glycaemic response to the breakfasts was similar between boys and girls, as shown by similar peak blood glucose concentrations and incremental AUC (IAUC) following both high and low GI breakfasts (all P>0·05). Girls exhibited a higher peak plasma insulin concentration 30 min post-breakfast following both high GI (P=0·043, g=0·69) and low GI (P=0·010, g=0·84) breakfasts, as well as a greater IAUC following high GI (P=0·041, g=0·66) and low GI (P=0·041, g=0·66) breakfasts. HOMA was positively correlated with the insulinaemic responses (all PP=0·037). The findings of the present study suggest that pubertal insulin resistance affects the postprandial insulinaemic responses to both high and low GI meals. Specifically, girls exhibit a greater insulinaemic response than boys to both meals, despite similar glycaemic responses. This study is the first to report the glycaemic and insulinaemic responses to everyday meals in boys and girls, supporting the recommendation for young people to base their diet on low GI carbohydrates.

Paroxysmal Nocturnal Hemoglobinuria is a stem cell disorder characterized by an acquired somatic mutation in the PIG-A gene. This results in a loss of GPI-linked complement inhibitors, CD55 and CD59, on the surface of red blood cells. Untreated patients experience chronic intravascular hemolysis and hemoglobinuria mediated by complement, as well as acute attacks. The lack of these complement inhibitors on platelets may lead to their activation and may explain the marked hypercoagulable state in this disorder. Eculizumab, a humanized monoclonal antibody targeting the C5 component of complement, prevents the formation of the C5b-9 complex and results in a major reduction in red cell transfusion requirements, a marked decrease in thrombotic events, and an improvement in quality of life. It was recognized soon after the introduction of this therapy that patients would often maintain an elevation in the reticulocyte count despite marked improvements in the LDH, and that some patients would have only a partial normalization of the hemoglobin--in some cases accompanied by an increase in bilirubin levels. It is now understood that in patients treated with C5 inhibition, C3 degradation products can accumulate on the red cell surface, resulting in opsonization and extravascular hemolysis. Based on this model, it would be expected that treated patients with PNH would now resemble those with other forms of extravascular hemolysis, with some of the same complications. Indeed, a rising ferritin has been reported in some patients, but until now, there have been only a few case reports of gallstone formation, and only one report commented on whether the stones were comprised of bilirubin. Here we review a set of 51 patients with PNH (31 women and 20 men) treated with eculizumab for a total of 280 patient years. There have been 7 cases of symptomatic cholelithiasis requiring surgery (5 women and 2 men), as well as one woman with a radiologically abnormal gallbladder removed at the time of a hemicolectomy for colon cancer. In four of these cases we have photographs of the surgical specimen: 3 patients had dark stones and/or debris, and one patient had stones with a mixed bilirubin/cholesterol appearance. For patients who had cholecystectomies at outside institutions, the pathology report indicated small bilirubin stones in one case, there was a verbal report of dark gallstones in another, and in two cases the nature of the stones is not known. Based on a Kaplan-Meier curve, the risk of gallstones leading to a cholecystectomy after 10 years of treatment was estimated to be 22.5% (95% CI 10%-46%) and did not differ based on gender. We then considered that markers of hemolysis might predict gallstone disease. The median bilirubin was only slightly higher in those with gallstones (2.1 vs 1.6 mg/dl, p = 0.06, one sided rank sum test), the median reticulocyte count was somewhat higher (303,500 versus 206,000 per μl, p=0.04, one sided rank sum test), and the median ferritin levels were approximately the same in both groups (361 versus 291, normal 10-143 ng/ml p=0.16). The median age at the time of the event or last follow-up was 46 years in both groups. All of the patients have recovered after their surgery. However, one of these patients had a complicated intraoperative course due to significant varices surrounding the gallbladder associated with cavernous transformation of the portal vein, due to prior thrombotic complications of PNH. This patient had presented with severe symptoms of cholelithiasis, including bacteremia from a biliary source. Another patient required a cholecystostomy tube due to severe gallbladder wall inflammation several weeks before the operation was performed. Another patient developed biochemical evidence of pancreatitis shortly before the procedure. We believe that bilirubin stones, a predictable consequence of extravascular hemolysis, represent, in addition to meningococcal infection and iron overload, a potentially significant side effect of inhibition of C5 in patients with PNH. This should be discussed with patients prior to initiation of therapy, and physicians should have a low threshold for obtaining appropriate imaging if a patient on a C5 inhibitor experiences complaints or laboratory evidence suggestive of gallstone disease. Disclosures No relevant conflicts of interest to declare.

Growth of seaweeds in the vicinity of fish farm cages in northwest Scotland was investigated as a means of extracting, from the surrounding water, nutrients added via fish feed and excretory products. Enhanced growth and yields of Palmaria palmata and Saccharina latissima cultures were found when grown adjacent to fish farm cages. Growth rates in summer for P. palmata and S. latissima were enhanced by up to 48% and 61%, respectively, and biomass yields over a growth season were enhanced by 63% and 27%, respectively. The nitrogen content of the macroalgae grown close to the fish cages was greater than for those grown at reference sites away from the cages. Extrapolation show that under optimal conditions, a hectare of P. palmata could yield up to 180 tonnes wet weight per annum and a hectare of S. latissima 220 tonnes wet weight per annum. Conservative estimates of yields show that P. palmata could be expected to remove up to 12% and S. latissima 5% of the waste nitrogen released during the growth of 500 tonnes of salmon in the sea over 2 years. The practicalities and logistics of culturing macroalgae near fish cages are considered and, because of the wide distribution of nitrogen emanating from fish culture and the need to optimise growth conditions for cultured macroalgae, it is recommended that macroalgal culture for bioremediation should be considered at wider geographical scales i.e., bay wide at least.

Cultivation of Palmaria palmata was performed by a technique introduced in this paper of tetraspore release and attachment to culture string. Optimal conditions for laboratory growth were investigated with respect to irradiance and nutrient media. The length of laboratory culture required for the growth and development of attached sporelings was also investigated, prior to deployment of seeded strings on a longline structure in Strangford Lough, Northern Ireland. Of sporelings grown in 5, 10, 25 and 50 μmol m − 2 s − 1 , those at the lower irradiances (5 and 10 μmol m − 2 s − 1 ) were significantly longer than those at higher irradiances after 12 weeks. Sporeling length was also significantly greater at lower irradiances when in F/2 nutrient media with additional vitamin solution, compared with F/2 medium without the vitamin solution. Seeded string was held in the laboratory for 2, 4, 6, or 8 weeks. After 3 months at sea, the 2-week strings had fewer visible plants and had shorter plants than the 4, 6 and 8-week strings, but plants on all strings were similar in length after 7 months at sea. The demand for P. palmata as a snack food in Northern Ireland regularly outstrips the supply obtainable from natural populations. The technique of seeding string with tetraspores is proposed as one method of commercially cultivating P. palmata on a large scale.

In the present study, the use of seaweeds for phosphate absorption was examined as a tertiary treatment in sewage treatment plants, to improve the water quality and reduce eutrophication risks. The data came from both laboratory and field experiments that took place on Ios Island sewage treatment plant. Three different macroalgae were tested and Ulva lactuca was finally chosen thanks to its high survivability in low salinity waters. Since the main restrictive factor was low salinity, we initially established the ratio of seawater:effluent that combined satisfactory viability with maximum phosphate absorption. The biomass growth under these conditions was also examined. Based on the above results, we designed a continuous-flow system with a 1/4 volume per hour water turnover, in a mixture of 60% sewage effluent: 40% sea water and 30 g L-1 initial biomass of U. lactuca that must be renewed every 10 days. Under these conditions and time frame, the phosphate content of the effluent was reduced by about 50%.

Intertidal macroalgae Fucus and Laminaria experience seasonally fluctuating inorganic N supply. This study examined the effects of long-term N deprivation, recovery following N resupply, and effects of elevated ammonium and nitrate exposure on N acquisition in intertidal algae using manipulations of N supply in tank culture. Over 15 weeks of N deprivation, internal N and nitrate reductase activity (NRA) declined, but maximum quantum yield of PSII was unaffected in Fucus serratus and Fucus vesiculosus. Low NRA was maintained despite no external nitrate availability and depletion of internal pools, suggesting a constitutive NRA, insensitive to N supply. Nitrate resupplied to N-starved thalli was rapidly taken up and internal nitrate pools and NRA increased. Exposure to elevated (50 microM) nitrate over 4 days stimulated nitrate uptake and NRA in Laminaria digitata and F. serratus. Exposure to elevated ammonium suppressed NRA in L. digitata but not in F. serratus. This novel insensitivity of NRA to ammonium in Fucus contrasts with regulation of NRA in other algae and higher plants. Ammonium suppression of NRA in L. digitata was not via inhibition of nitrate uptake and was independent of nitrate availability. L. digitata showed a higher capacity for internal nitrate storage when exposed to elevated ambient nitrate, but NRA was lower than in Fucus. All species maintained nitrate assimilation capacity in excess of nitrate uptake capacity. N uptake and storage strategies of these intertidal macroalgae are adaptive to life in fluctuating N supply, and distinct regulation of N metabolism in Fucus vs Laminaria may relate to position in the intertidal zone.

Fucus vesiculosus from the northern Baltic Sea (5 psu) and from the Irish Sea (35 psu) were cultivated at different temperatures, salinities and dissolved inorganic carbon (DIC) concentrations with the addition of different nutrient concentrations. The influence of these abiotic factors was assessed by measuring photosynthesis as electron transport rate (ETR) and growth as relative growth rate (RGR). The maximal ETR and the RGR of the Irish Sea plants in their natural seawater (50.8 µmol electrons m−2 s−1; 0.024 g g−1 day−1) were significantly higher than those of the Baltic plants in their natural seawater (21.9 µmol electrons m−2 s−1; 0.007 g g−1 day−1). When Baltic F. vesiculosus was cultivated at a DIC concentration similar to that of the Irish Sea, the ETR as well as RGR increased, but never equalled the rates of the marine F. vesiculosus from the Irish Sea. Cultivation at different salinities showed that F. vesiculosus from the Baltic has a higher ETRmax and RGR at low salinities (5–10 psu) than F. ...

The aim of this study was to determine nutrient concentrations in the vicinity of fish farm cages to maximise potential utilization by cultured macroalgae. At 4 m depth, nitrate and phosphate concentrations did not vary significantly with distance from fish farm cages. Elevated levels of ammonium were detected for extended periods of time during the day for distances that exceed 200 m. Principal enhancements were within 50 m in the direction of currents, and no increases were detected in the direction from which the current impacted the cages. Ammonium concentrations measured at 4 m depth within 50 m of the cages reached a maximum of 8 μM but most ranged from 2 to 4 μM and 2–3 μM above background. While current measurements gave an indication of ammonium distributions, the geography of the site and the physical presence of the fish farm cages were influencing factors. The highest concentrations of ammonium found at these fish farm sites were on the lee sides of cages in shallow water. Concentrations of ammonium throughout a relatively small-enclosed bay with fish farms were higher than in a similar bay without fish farms consistent with broader scale effects of nutrient enhancement. Multiple drogues used to follow currents were found to be a valuable tool for prediction of enhanced ammonium concentrations. The data suggest that there are elevated levels of ammonium at appropriate depths for potential utilization by marine macroalgae (i.e. in the photic zone) for an extended period of time at distances from the fish farm cages greater than 50 m. Careful assessment of current patterns around farm cages should be made if the objective is to maximise exposure of cultured algae to farm-derived nutrients.

The aims of this work were to investigate the conversion of a marine alga into hydroxyapatite (HA), and furthermore to design a composite bone tissue engineering scaffold comprising the synthesised HA within a porous bioresorbable polymer. The marine alga, Phymatolithon calcareum , which exhibits a calcium carbonate honeycomb structure, with a natural architecture of interconnecting permeable pores (microporosity 4–11 μm), provided the initial raw material for this study. The objective was to convert the alga into hydroxyapatite while maintaining its porous morphology using a sequential pyrolysis and chemical synthesis processes. Semi-quantitative XRD analysis of the post-hydrothermal material (pyrolised at 700–750 °C), indicated that the calcium phosphate (CaP) ceramic most likely consisted of a calcium carbonate macroporous lattice, with hydroxyapatite crystals on the surface of the macropores. Cell visibility (cytotoxicity) investigations of osteogenic cells were conducted on the CaP ceramic (i.e., the material post-hydrothermal analysis) which was found to be non-cytotoxic and displayed good biocompatibility when seeded with MG63 cells. Furthermore, a hot press scaffold fabrication technique was developed to produce a composite scaffold of CaP (derived from the marine alga) in a polycaprolactone (PCL) matrix. A salt leaching technique was further explored to introduce macroporosity to the structure (50–200 μm). Analysis indicated that the scaffold contained both micro/macroporosity and mechanical strength, considered necessary for bone tissue engineering applications.

There is a great need to design functional bioactive substitute materials capable of surviving harsh and diverse conditions within the human body. Calcium-phosphate ceramics, in particular hydroxyapatite are well established substitute materials for orthopaedic and dental applications. The aim of this study was to develop a bioceramic from alga origins suitable for bone tissue application. This was achieved by a novel synthesis technique using ambient pressure at a low temperature of 100 °C in a highly alkaline environment. The algae was characterised using SEM, BET, XRD and Raman Spectroscopy to determine its physiochemical properties at each stage. The results confirmed the successful conversion of mineralised red alga to hydroxyapatite, by way of this low-pressure hydrothermal process. Furthermore, the synthesised hydroxyapatite maintained the unique micro-porous structure of the original algae, which is considered beneficial in bone repair applications.

Fucus and Laminaria species, dominant seaweeds in the intertidal and subtidal zones of the temperate North Atlantic, experience tidal cycles that are not synchronized with light:dark (L:D) cycles. To investigate how nutrient assimilation is affected by light cycles, the activity of nitrate reductase (NR) was examined in thalli incubated in outdoor tanks with flowing seawater and natural L:D cycles. NR activity in Laminaria digitata (Huds.) Lamour. showed strong diel patterns with low activities in darkness and peak activities near midday. This diel pattern was controlled by light but not by a circadian rhythm. In contrast, there was no diel variation in NR activity in Fucus serratus L., F. vesiculosus (L.) Lamour., and F. spiralis L. either collected directly from the shore or maintained in the outdoor tanks. In laboratory cultures, transfer to continuous darkness suppressed NR activity in L. digitata, but not in F. vesiculosus; continuous light increased NR activity in L. digitata but decreased activity in F. vesiculosus. Furthermore, 4 d enrichment with ammonium (50 μmol · L⁻¹ pulses), resulted in NR activity declining by >80% in L. digitata, but no significant changes in F. serratus. Seasonal differences in maximum NR activity were present in both genera with activities highest in late winter and lowest in summer. This is the first report of NR activity in any alga that is not strongly regulated by light and ammonium. Because light and tidal emersion do not always coincide, Fucus species may have lost the regulation of NR by light that has been observed in other algae and higher plants.

Nitrogen metabolism was examined in the intertidal seaweeds Fucus vesiculosus, Fucus serratus, Fucus spiralis and Laminaria digitata in a temperate Irish sea lough. Internal NO(3) (-) storage, total N content and nitrate reductase activity (NRA) were most affected by ambient NO(3) (-), with highest values in winter, when ambient NO(3) (-) was maximum, and declined with NO(3) (-) during summer. In all species, NRA was six times higher in winter than in summer, and was markedly higher in Fucus species (e.g. 256 +/- 33 nmol NO(3) (-) min(-1) g(-1) in F. vesiculosus versus 55 +/- 17 nmol NO(3) (-) min(-1) g(-1) in L. digitata). Temperature and light were less important factors for N metabolism, but influenced in situ photosynthesis and respiration rates. NO(3) (-) assimilating capacity (calculated from NRA) exceeded N demand (calculated from net photosynthesis rates and C : N ratios) by a factor of 0.7-50.0, yet seaweeds stored significant NO(3) (-) (up to 40-86 micromol g(-1)). C : N ratio also increased with height in the intertidal zone (lowest in L. digitata and highest in F. spiralis), indicating that tidal emersion also significantly constrained N metabolism. These results suggest that, in contrast to the tight relationship between N and C metabolism in many microalgae, N and C metabolism could be uncoupled in marine macroalgae, which might be an important adaptation to the intertidal environment.

Sargassum muticum is an invasive brown macroalga that originates from Japan. In the introduced range, thalli can grow in soft substratum habitats attached to embedded rock fragments and shells. Within Strangford Lough, Northern Ireland, S. muticum has rapidly colonised large areas of soft substrata, where dispersal by peripatetic or 'stone-walking' plants is very effective. Sediment cores were collected under and outside canopies of S. muticum in Strangford Lough (S. muticum first recorded there in 1995) and Langstone Harbour, English Channel (S. muticum first found there in 1974) to investigate modification of the infaunal assemblages. At both study sites, community analy- ses highlighted significant differences between the assemblages under the canopies and those in adjacent unvegetated areas. In Strangford Lough, the invertebrate community under the canopy con- tained a higher abundance of smaller, opportunistic, r-selected species than outside the canopy. By contrast, the communities under and outside the canopy at Langstone Harbour were similar in spe- cies composition, diversity and dominance, but overall faunal abundance was greater under the canopy. Sediment characteristics were not affected by S. muticum canopies, but the infaunal changes may be related to environmental modification; shading, flow suppression and temperature stratifica- tion were also investigated. The differences between these 2 sites indicate that localised conditions and/or the duration of colonisation of S. muticum are important in determining the nature of habitat modification.

Macroalgal epiphytes within seagrass meadows make a significant contribution to total primary production by assimilating water column N and transferring organic N to sediments. Assim- ilation of NO3 - requires nitrate reductase (NR, EC 1.6.6.1); NR activity represents the capacity for NO3 - assimilation. An optimised in vitro assay for determining NR activity in algal extracts was applied to a wide range of macroalgae and detected NR activity in all 22 species tested with activity 2 to 290 nmol NO3 - min -1 g -1 frozen thallus. With liquid-N2 freezing immediately after sample collec- tion, this method was practical for estimating NR activity in field samples. Vertical distribution of NR activity in macroalgal epiphytes was compared in contrasting Posidonia sinuosa and Amphibolis antarctica seagrass meadows. Epiphytes on P. sinuosa had higher mass-specific NR activity than those on A. antarctica. In P. sinuosa canopies, NR activity increased with distance from the sediment surface and was negatively correlated with (NH4 + ) in the water but uncorrelated with (NO3 - ). This supported the hypothesis that NH4 + released from the sediment suppresses NR in epiphytic algae. In contrast, the vertical variation in NR activity in macroalgae on A. antarctica was not statistically sig- nificant although there was a weak correlation with (NO3 - ), which increased with distance from the sediment. Estimated capacities for NO3 - assimilation in macroalgae epiphytic on seagrasses during summer (24 and 46 mmol N m -2 d -1 for P. sinuosa and A. antarctica, respectively) were more than twice the estimated N assimilation rates in similar seagrasses. When the estimates were based on annual average epiphyte loads for seagrass meadows in other locations, they were comparable to those of seagrasses. We conclude that epiphytic algae represent a potentially important sink for water-column nitrate within seagrass meadows.

Nitrate and phosphate uptake mechanisms have been characterised under conditions of 100 and 50% seawater in 3 common brown algae of NW Europe: Fucus vesiculosus, F. serratus and Laminaria digitata. Under low salinity, the growth rate and internal nitrate accumulation of F. serra- tus significantly increased (20 and 48%, respectively), but no significant changes were observed for F. vesiculosus and L. digitata. However, nitrate uptake rates were reduced in L. digitata, so that this species was less adaptable to low salinity than the Fucus species. Both F. vesiculosus and F. serratus reached a steady-state uptake rate after acclimation regardless of the salinity treatment. All 3 species had a high capacity for storing inorganic N and P intracellularly. The results for F. serratus pointed to a dual mechanism of adaptation to the special characteristics of the intertidal environment where it grows. Non-saturating (low affinity) nitrate uptake and biphasic (double Michaelis-Menten curve) phosphate uptake are adaptations to high nutrient concentrations. Temporal partition of cellular energy for carbon metabolism and nutrient uptake is also suggested as an adaptation to the transient nutrient inputs occurring in these environments.

Field-collected specimens of three species of Laminaria and three species of subtidal red algae (Delesseria sanguinea, Plocamium cartilagineum and Phyllophora pseudoceranoides) were exposed to natural summer sunlight on Helgoland (southern North Sea) for up to 4 h at 15 °C. Dark-adapted variable fluorescence (F, : F m ) was measured immediately after these treatments, and following 6, 24 and 48 h of recovery in moderate irradiances of white light. The response of plants to the full spectrum of natural sunlight was compared with that to PAR alone, UV-A + visible, UV-A + UV-B, or UV-A alone. The F, : F m values of all species were reduced to minimal values after 4 h in all of these treatments, but those of the more resistant species (Laminaria spp. and P. pseudoceranoides) were higher after shorter exposures to UV radiation alone than to PAR with or without UV. The recovery of F, : F m in all species was also more rapid in the two treatments that contained UV radiation alone than in those that included PAR. These results suggest that it is the high irradiances of PAR in natural sunlight which are responsible for the photoinhibition of photosynthesis of subtidal seaweeds and that the current ambient irradiances of UV radiation (either UV-B or UV-A) in northern temperate latitudes would not contribute significantly to this photoinhibition.

In laboratory experiments, copper concentrations in plants of Ascophyllum nodosum (L.) Le Jolis (Fucales, Phaeophyta) increased with the concentrations in the culture media and were highest in younger, meristematic thallus parts. After initial accumulation in high-copper medium and subsequent transfer to clean seawater for 5 days, no release of copper could be detected. Iron concentrations in A. nodosum tissue were not related to concentrations in the culture medium. Differences between copper concentrations in plants from different sites in areas with high yachting activity in Strangford Lough, Northern Ireland, could be explained by differences in water motion and human activity, in particular the application of copper-releasing antifouling paints to leisure boats. Iron concentrations were also highest in plants from the sheltered, polluted site but did not differ significantly between the other two sites. No differences in copper nor iron concentrations were found between different-aged thallus parts of plants from any site. X-ray microanalysis revealed that most of the iron detected was located in epiphytic pennate diatoms on the A. nodosum surface. In thallus areas without diatoms, iron levels were below the detection limit for X-ray microanalysis. Mapping for copper indicated that most of the accumulated copper was located in cells near and immediately below the thallus surface. “Epidermis”-shedding occurred in plants from the culture experiments and also in freshly-collected material and may have resulted in a loss of metal ions accumulated by surface cells and by epiphytic diatoms. The results suggest that A. nodosum could be used as a biological indicator for copper but not for iron, and that young, apical plant parts are most sensitive to changes in metal concentrations in the water.

Volatile halocarbons released by 12 Falkenbergia phase isolates of Asparagopsis taxiformis and Asparagopsis armata collected from widely scattered geographical locations were identified using a purge and trap technique and gas chro-matography/mass spectrometry. Production of the compounds under normal and high irradiance was quantified where possible. Bromoform (CHBr3), dibromomethane (CH2Br2), 1,2-di-bromoethylene (C2H2Br2), dibromochloromethane (CHBr2Cl), and tribromoethylene (C2HBr3) were identified as algal products. C2H2Br2 and C2HBr3 have not previously been recorded as natural products. Considerable qualitative and quantitative differences existed between isolates in the halocarbons released. The rates of CHBr3 and C2H2Br2 production were negatively correlated. Increased irradiance invariably resulted in higher rates of release of halocarbons. Initial rates of production ranged between

Pigment contents and photosynthetic parameters of different regions of thalli of Ascophyllum nodosum (L.) Le Jolis collected from Strangford Lough, Irish Sea, and Helgoland, North Sea, were examined on a seasonal basis and after exposure to various irradiance and nutrient treatments in the laboratory. In culture, pigment contents of young plant parts acclimated rapidly to changes in irradiance and increased after nutrient enrichment in all irradiances. Chlorophyll a levels in plant tips in situ increased during autumn and winter and were reduced by 50% during summer. In older plant parts, which were subject to less seasonal variation in irradiance and were more shaded at both high and low tide, chlorophyll a levels were higher than in the tips at all times of the year. Older plant material from the lower shore contained higher pigment levels than corresponding material from the upper shore. Fucoxanthin : chlorophyll a ratios peaked during summer in apical plant parts from the upper shore and incr...

Thirteen species from the red algal flora of Helgoland (southern North Sea) were exposed to UVA + UVB radiation for various periods in the laboratory, and dark-adapted variable fluorescence (F v:F m) was measured immediately after the UV treatment and again after various recovery times in white light. With the exception of Porphyra umbilicalis, all species showed a decrease in F v:F m on exposure to UV radiation, followed by recovery towards the initial values during the next 24–48 h in white light. The rate of the initial decrease was greater, and the extent of recovery was less, in deep subtidal species (e.g. Delesseria sanguinea, Plocamium cartilagineum) than in intertidal or shallow subtidal species, although there was no direct correlation between these indicators of sensitivity to UV radiation and the depth range of a species, and only slight differences were detected between populations of a single species collected from different depths. There was also little evidence of a seasonal change in sensi...

Zoospores, gametophytes, young sporophytes and discs cut from mature sporophytes of Laminaria digitata, L. hyperborea and L. saccharina were exposed in the laboratory to UV-radiation, with a spectral composition and irradiance similar to natural sunlight, for periods ranging from 15 min to 8 d, and were then returned to white light. Germination of zoospores and the growth of gametophytes were reduced after exposures to UV longer than 1 h, whereas UV had little effect on the growth of young or mature sporophytes unless exposure continued for more than 48 h. The variable fluorescence (F v:Fm) of all stages was strongly reduced immediately after short exposures to UV, but recovered almost completely within 24 h. However, exposure of gametophytes to UV for >4 h resulted in little or no recovery of F v:Fm, whereas >16 h of UV were required to produce this result in young sporophytes, and >48 h in mature sporophytes. Thus, sensitivity to UV-radiation decreased from gametophytes to sporophytes, and with increasing age of sporophytes, but, in gametophytes, growth appeared to be a more sensitive indicator of UV-damage than F v:Fm after 24 h recovery. The responses to UV of the zoospores and gametophytes of all three species were similar, but both growth and fluorescence measurements suggested that the sporophytes of L. saccharina were more sensitive to UV than those of the other two species.

Stimulation or light-saturated rates of photosynthesis in Ectocarpus siliculosus (Dillwyn) Lyngb. by blue light was eliminated by increasing dissolved inorganic carbon (DIC) or by lowering pH in natural seawater. The amplitude of the circadian rhythm of photosynthesis was also diminished under these conditions, and the pH compensation points in a closed system were higher in the presence of blue light and during the circadian day. These observations suggest that blue light and the circadian clock regulate the activity of a carbon acquisition system in these plants. The inhibitor of external carbonic anhydrase, acetazolamide, reduced overall rates of photosynthesis by only about 30%, but ethoxyzolamide suppressed the circadian rhythm of photosynthesis almost completely and markedly reduced the duration of responses to blue light pulses. Similar patterns were obtained when photosynthesis was measured in strongly limiting DIC concentrations (0–0.5 mol m−3). Since blue light stimulated photosynthesis under these conditions of strong carbon limitation, we suggest that blue light activates the release of CO2 from an internal CO2 store. We propose a metabolic pathway with similarities to that of CAM plants. Non-photosynthetic fixation leads to the accumulation of a storage metabolite. The circadian clock and blue light control the mobilization of CO2 at the site of decarboxylation of this metabolite. In the presence of continuous blue light the pathway is proposed to cycle and act as a pump for CO2 into the chloroplasts. This hypothesis helps to explain a number of previously reported peculiarities of brown algal photosynthesis.

In saturating irradiances of red light, photosynthesis of Laminaria saccharina (L.) Lamouroux was stimulated by low irradiances of continuous blue light only when the supply of dissolved inorganic carbon (DIC) was limiting. The degree of this stimulation was inversely proportional to the logarithm of the concentration of free CO 2 , whether this was adjusted by varying the total DIC or the pH at a given DIC concentration. The final pH reached in a closed system was higher in blue light than in red light. Both acetazolamide and ethoxyzolamide suppressed the responses to blue light almost completely, but reduced photosynthesis in red light by only 30%. Buffering the pH of the seawater also suppressed the stimulation of photosynthesis by blue light without affecting the photosynthetic rate in red light. The transient stimulation of O 2 evolution by a blue light pulse was not accompanied by a corresponding increase in CO 2 consumption. These observations could be explained if, in analogy to the mechanism proposed for Ectocarpus (Schmid, Mills & Dring 1996, Plant Cell and Environment 19, 373-382, this issue, accompanying paper), photosynthesis was supported by a blue-light-activated release of CO 2 from an internal store. We suggest that the store is located in the vacuoles of the cortical tissue of the blades. The main photosynthetic tissue, however, is in the overlying meristoderm, and blue-light-activated mobilization of the store could stimulate O 2 evolution only if periplasmic carbonic anhydrase was available to facilitate CO 2 uptake from the cortex.