Tph1 (−/−) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system.The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that  Tryptophan hydroxylase (TPH) is the rate-limiting enzyme involved in the biosynthesis of serotonin Tph1 has been linked to nervous system abnormalities in E12.5 mouse embryos removed from Tph1 (−/−) mothers Tph1 (−/−) mothers revealed gross abnormalities such as overall size reduction, altered morphology of the rhombencephale regions and neopallial cortex and decreased mitotic activity in ventricular zone and roof of the neopallial cortex. Defects were not found in Tph1 (−/−) embryos from heterozygous or WT mothers indicating that maternal Tph1 plays a role in development of the CNS. Another potential source of TPH1 during development is from the placenta where it is expressed as early as E10.5 and has been shown to contribute to 5-HT levels in the embryonic forebrain during early development Tph1 (−/−) mothers continue into adulthood, there are studies indicating that altered 5-HT signaling, especially during early postnatal development, can lead to other disorders such as anxiety in the adult Genetic deletion of Tph1 (−/−) mice such as mild anemia Tph1 (−/−) mice.Several phenotypes have been reported in Interestingly, it has recently been reported in humans that maternal use of selective serotonin reuptake inhibitors (SSRIs), which prevent uptake of peripheral serotonin into neurons as well as platelets, is linked to a more pronounced decrease in growth of fetal head circumference when compared to untreated maternal depression Tph1 (−/−) mice born from null mothers could have behavioral abnormalities in adulthood. In the current study, we show that Tph1 (−/−) mice born from knockout mothers have altered gait dynamics throughout life and deficits in rearing behavior with age when compared to age-matched wild-type mice. These data suggest that TPH1 has an impact on nervous system development and possibly maintenance.Taken together, these previous observations led us to hypothesize that Tph1 (−/−) mice were a kind gift, received from M. Bader, Max-Delbruck-Center, Berlin, Germany and were on C57BL/6 background. To avoid genetic drift, Tph1 (−/−) mice were routinely back-crossed to C57BL/6J mice a minimum of five times prior to experimentation and were continued to be back-crossed routinely. All mice used for the behavioral experiments were male, age- and weight-matched. All Tph1 (−/−) mice used for experiments were bred from Tph1 (−/−) pairs.All animal experiments were approved by the Animal Care and Use Committee of the Immune Disease Institute and the Harvard Medical Area standing committee on animals (#04761). Adequate measures were taken to minimize pain and discomfort. C57BL/6J (WT) mice were purchased from Jackson Laboratory . Tph1 (−/−) young n = 10, WT aged n = 10, Tph1 (−/−) aged n = 8. All mice were single-housed and acclimated to the facility for two weeks prior to experimentation.Rotarod, grip strength, open field, metabolic activity, attention performance and social novelty preference tests were performed at the NeuroBehavior Laboratory of the Harvard NeuroDiscovery Center. These paradigms were run in the respective order listed on the same groups of mice (2–3 month old mice termed “young” and 8–9 month old mice termed “aged”). The number of animals used for these studies was: WT young n = 10, Tph1 (−/−) young: n = 8 (gait), n = 7 ; WT aged n = 8 , Tph1 (−/−) aged n = 10 . Different cohorts of mice were used for gait and beam walk for the “young” groups whereas the same cohort of mice was used for the aged group. Mice were housed in the same facility for two weeks prior to experimentation. All experimentation was conducted during the light phase.Gait dynamics and electrocardiogrpahy , and beam walk were performed at the Immune Disease Institute in that order on group-housed mice at two ages (2–3 month old mice termed “young” and an aged group termed “>60 week old mice”) except for electrocardiography which was only performed on the >60 week old mice. The number of animals used for these studies was: WT young: n = 6 (gait), n = 8 ; Tph1 (−/−): n = 4) and female : n = 5) mice that were group-housed.Fasting blood glucose level measurements were performed at the Boston Children's Hospital animal facility on age- and gender-matched young male  to assess sensorimotor ability in each mouse. One hour prior to testing, each mouse was habituated to the rotarod at a fixed speed and was placed back on the rod if it fell off within a 300 second time interval. For testing, the rod was accelerated from 4 to 40 rpm over 180 seconds and time to fall off the apparatus was recorded. Two trials were conducted and times were averaged for between group comparisons.The grip-strength test apparatus consisted of a grasping trapeze connected to a force transducer (Ugo Basile). Each mouse was held by the base of the tail and placed in front of the grasping trapeze. Once the mouse grasped the trapeze, it was slowly pulled back until the pulling force overcame the mouse's grip strength. The grip-strength meter expresses the grip force in grams. Each mouse was submitted to five trials that were separated by an inter-trial interval of 15 minutes. Final score represents the average of the five trials for each mouse.The open field test was used to study general activity in mice confined to a novel arena. The apparatus used was a 27.9×27.9 cm Plexiglas arena with three 16-beam infrared arrays . Mice were acclimated to the testing room for at least 30 minutes prior to testing. At the beginning of each session, mice were placed into the center of the open field and allowed to freely explore for 60 minutes. The total distance traveled (centimeters) and vertical beam breaks (an indication of rearing activity) were automatically recorded.Tph1 (−/−) mice at 8 weeks (young) and >60 weeks (aged) of age as previously described Gait dynamics were assessed using the DigiGait system  in age-matched WT and The beam walk test was set up as previously described A recently developed method for testing visual discrimination learning and attention performance was performed Five days prior to the start of operant conditioning experiments, all subjects were placed on a food restriction diet. On the first day, baseline body weights were recorded and mice were individually placed into clean cages with access to water only. Food rations (regular chow) were calculated as a function of each individual's bodyweight loss/gain from the previous day and delivered daily to maintain a stable 80–85% of free feeding weight. During the 5-day period preceding training, mice were given fifteen 20 mg casein pellets  daily, habituating the mice to the rewards. The food restriction procedure was maintained for the entire training and testing period.In this lever-press learning paradigm, mice were required to discriminate between reinforced and non-reinforced levers in order to obtain a food reward. Previous experiments have demonstrated that learning of the lever/reward association to be dependent on integrity of the hippocampus Mixed-trial attention sessions followed an identical protocol to the discrimination procedure with the exception that the duration of the stimulus light presentation was reduced to 0.5, 1, 2, or 10 seconds. Regardless of the stimulus duration, subjects had 30 seconds to respond before an omission was recorded. Twenty trials at each stimulus duration were presented within a session , and stimulus durations and lever-stimulus pairings were distributed randomly and counterbalanced across trials.A three-chambered rectangular box was used to evaluate sociability and preference for social novelty as previously described Monitoring animals in a familiar environment over an extended period of time is often necessary to detect impairments in circadian rhythms and/or metabolic activity. For that purpose we used the Comprehensive Lab Animal Monitoring System (CLAMS) from Columbus Instruments . Mice were individually placed in CLAMS chambers  for a period of three consecutive days . In these chambers, mice have free access to food (regular chow) and water.Tph1 (−/−) mice and their gender- and age-matched wild-type controls were fasted for 6 hours before measurement of blood glucose. Blood was obtained by tail-nick method and the glucose level was immediately determined using a glucometer .2–3 month old male and female Tph1 (−/−) and WT mice, >60 weeks of age, as previously described using the ECGenie electrocardiography system (Mouse Specifics) Electrocardiograms were recorded non-invasively in awake For vertical rearing counts, grip strength, and rotarod, a 2-way ANOVA was used to examine genotype and age effects as well as the interaction between these factors. For visual discrimination and open field habituation, a repeated measures ANOVA was added across session days and time respectively . During attention testing, the percentage of correct responses was also separated by stimulus duration to provide an accurate measure of performance across varying degrees of attentional demand. When appropriate, Student-Newman-Keuls post hoc tests were used to compare statistical variation between groups. For beam walking, gait analysis, fasting blood glucose levels, heart rate and QRS times a two-tailed, unpaired student's t-test was performed between age groups due to the difference in treadmill speed in young and aged mice. Results were considered significant at P<0.05.Tph1 (−/−) mice were significantly different when compared to age-matched controls. Young mice of both genotypes were made to walk on a treadmill at a speed of 24 cm/sec and >60 week old mice walking at 16 cm/sec. Stride length and swing duration were significantly decreased in the Tph1 (−/−) mice . Consistent with decreased stride length, Tph1 (−/−) mice of both age groups displayed increased stride frequencies , indicating that they were taking more steps in the same period of time  = 2.143; P = 0.152;  = 0.009; P = 0.922; Tph1 (−/−) mice displayed normal locomotor function we utilized the beam walk test (Tph1 (−/−) mice indicating that changes in gait dynamics were not due to problems with fine motor coordination and balance Tph1 (−/−) and WT animals of both sexes  (Tph1 (−/−) and WT mice at >60 weeks of age had similar heart rate (P = 0.56) and QRS values (P = 0.32) indicating that the Tph1 (−/−) mice have normal heart rhythm and ventricular conduction.   A, B. Elduction.  C, D.Tph1 (−/−) mice of both ages (data not shown). Food intake and weight were also similar in WT and Tph1 (−/−) mice of both ages (data not shown).Calorimetry experiments conducted over a 72 hr period demonstrated no differences in metabolic activity as assessed by oxygen consumption, carbon dioxide production and respiratory exchange rate between the WT and  = 21.371; P<0.001). There was no effect of age or genotype on the total distance travelled  = 0.243; P = 0.625)  = 0.150; P = 0.701)  = 5.394; P = 0.027) and genotype  = 7.989; P = 0.008)  and motivated locomotion (rearing activity) were assessed in a single, 60-minute open field session. Our results indicate that all of the mice tested, regardless of age or genotype, demonstrated a significant habituation to the novel environment as reflected by a progressive decrease in activity across session time (F= 0.625)  or the p= 0.701) . However= 0.008) . These dTph1 (−/−) mice were able to learn the association between the stimulus light and reward  = 93.385; P<0.001) however there was a strong trend indicating that aged Tph1 (−/−) mice may acquire this association slower  = 1.398; P = 0.06;  = 10.047; p = 0.003) and 2 seconds.  = 4.212; p = 0.048; Previous work has confirmed that learning the association between an illuminated lever with reward is dependent on the integrity of the hippocampal formation, while attention performance in the same task is dependent on the medial prefrontal cortex Tph1 (−/−) mice and age-matched controls demonstrate increased time spent with a stranger mouse compared to the empty chamber  = 1.243; P = 0.273) or age  = 2.921; P = 0.097) on this performance. These results indicate that Tph1 (−/−) mice demonstrate a preference for social interactions similar to controls. In addition, Tph1 (−/−) mice and controls also displayed increased exploration of a second stranger mouse when compared to one previously investigated  = 0.007; P = 0.933) or age  = 0.149; P = 0.702). This demonstrates both an intact memory for a co-specific odor and the preference for social novelty. Overall these data indicate that Tph1-deficiency does not lead to deficits in social behavior.To determine the extent that Tph1-deficiency played a role in the mouse's preference for social novelty, we used a protocol previously described to assess autistic-like characteristics  chamber  however,stigated  althoughTph1 (−/−) offspring from Tph1 (−/−) mothers results in behavioral abnormalities. The most striking behavioral differences that we report in these Tph1 (−/−) mice are alterations in gait dynamics. Gait dynamics can be used to determine a subject's ability for balance, proprioception and coordination. In addition, these indices commonly reflect motor impairment in diseases such as Parkinson's disease, nerve injury and pain Tph1 (−/−) mice compared to age-matched controls, we did not find any disturbances in general motor and balance abilities. However, in the open field arena aged Tph1 (−/−) mice demonstrated a significant decrease in vertical rearings and a trend towards a decrease in rotarod performance as compared to WT mice of the same age. Vertical rearings have been used in some studies as a means of assessing motor abnormalities and motivated locomotion Tph1 (−/−) mice demonstrate no deficits in basic locomotor activity The goal of this study was to assess whether Tph1 (−/−) is similar to that in a recently published C57BL/6J mouse model of Parkinson's disease using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to induce lesions of the substantia nigra Tph1 (−/−) mice. These mice also exhibited deficiencies in beam walk and rotarod whereas the Tph1 (−/−) mice only displayed deficits in gait.Interestingly, the robust decrease in stride length found in the Tph1 (−/−) during development, and/or during aging. Further studies are necessary to identify whether changes in these systems underlie the decreased swing duration in the Tph1 (−/−) mice.The finding of significantly decreased swing duration in both young and aged Tph1−/− animals compared to WT is intriguing. In contrast to stride length and stance duration, which change in a speed dependent manner and are modified by primary afferent sensory feedback, there is little variation in swing duration Tph1 gene rendering it less active are correlated with an increased prevalence of ADHD in children by 1.5–2.5 fold, suggesting that ADHD may be related to the lack of exposure to maternal serotonin during gestation Tph1 (−/−) mice may acquire the association between light and reward at a slower rate (p = 0.06), each group demonstrated successful learning across daily sessions. This result demonstrates that hippocampal function in Tph1 (−/−) is preserved. This finding is consistent with previous studies showing no changes in learning, memory or motivation in offspring of mothers in which the SSRI paroxetine (Paxil) was administered during gestation Tph1 (−/−) mice showed no overt deficits in attention performance as indicated by performance at the 1 and 2 second stimulus durations. However, it should be noted that in the current study there was a significant decrease in attention performance between 2 to 8 months of age. This overall decrease in performance at 8 months of age may have made it difficult to determine an effect of genotype.Recent clinical studies have shown that mutations of the Tph1 (−/−) mice on the C57BL/6 background have no abnormalities in anxiety-related behaviors tested in either the elevated plus maze or hold board task Tph1 (−/−) mice in a model of social novelty. We found no differences in preference for social novelty in Tph1 (−/−) mice compared to WT mice. It has been shown that anxiety/compulsive behavior and depression-related disorder testing were normal in Tph1 (−/−) mice Previous results show that Tph1 (−/−) mice such as diabetes Tph1 (−/−) mice from our colony demonstrate that the mice used for this work do not have increased blood glucose levels after 6-hour fasting. It is possible that differences in background strains account for this discrepancy. We also did not find any differences between >60 week old Tph1 (−/−) and WT mice in heart rate and QRS interval duration indicating that these mice have normal rhythm and ventricular depolarization, thus it is unlikely that cardiac dysfunction plays a role in gait and rearing differences. Mice used for these studies were not anesthetized during ECG recordings. We have recently confirmed in our Tph1 (−/−) colony that these mice have mild anemia, as previously reported As mentioned earlier, several abnormalities have been reported in Tph1 (−/−) offspring born from knockout mothers. As previous studies did not find developmental CNS abnormalities in Tph1 (−/−) mice from Tph1 (+/−) and Tph1 (+/+) mothers, we speculate that the behavioral differences in these mice are mainly due to CNS abnormalities induced by lack of maternal TPH1 exposure during gestation. In utero use of SSRIs is a very understudied field even though these antidepressants are commonly prescribed during pregnancy Here, we report gait alterations and reduced motivated locomotion in murine Figure S1Tph1 (−/−) mice had normal fasting blood glucose levels , heart rate (C) and QRS interval duration (D).(TIF)Click here for additional data file.


This investigation compared the results of TCs from the cone-section method with those from the cylinder prism and Gaussian quadrature methods using a 9″ × 9″ elevation grid for the inner zone and a 90″ × 90″ elevation grid for the outer zone. The inner and outer radii were set to 20 and 200 km, respectively. The comparisons showed that the cone-section algorithm is consistent with the Gaussian quadrature. Furthermore, the cone-section method is an appropriate approach for TCs in high elevation areas, yielding results that outperform the cylinder prism method.Mt. Jade (or “Yushan” in Chinese) is the highest peak in northeast Asia. The topography is very rugged and complicated. Such terrain makes it difficult to obtain the correct results for terrain corrections (TCs). This paper developed an improved approach, named cone-section method, to compute the TCs of the Mt. Jade area using a high-resolution digital elevation model (DEM) on a 9″ × 9″ grid. The corrections were calculated to the distance of 100 km with an average rock density of 2.57 × 10 The aim2.n−1 and an) derived from DEM to compute the attraction of each cone prism. Obviously, the cone-section method is better than the cylinder prism method that uses the average elevation from the inner and outer elevations of each cylinder prism.As shown in n−1 and an) of each cone prism from a high resolution DEM with a 9″ × 9″ grid; 3) estimate the attraction of each cone prism; 4) obtain the total TCs by summing the contributions from all such cone prisms as The cone-section method yields TCs using the following steps: 1) equally divide the terrain surrounding the surveying site into several small cone prisms ; 2) plotGthe gravitational constantdmthe differential massh0the elevation of surveying site Pρthe distance between surveying site P and differential mass dm as shown in Figure 3brthe horizontal distance between surveying site P and differential mass dmzthe elevation of differential mass dmAs δ3 kg·m−3 [the average rock density kthe intercept parameter of Equation (9)where:The solution form of Assuming the attraction value is positive in a downward direction, the attraction of each cone-section prism  is:(14)Divide the topography surrounding the surveying site P into n concentric circles with k equal cone prisms , then ob3.3 kg·m−3 [Mt. Jade is the highest peak in northeast Asia. The topography of the area is very rugged. Relative gravity measurements of the Mt. Jade area using a LaCoste and Romberg type G gravimeter were obtained in . The abs3 kg·m−3 . The inv3 kg·m−3  and the 3 kg·m−3  for comp3 kg·m−3  recommen3 kg·m−3  develope3 kg·m−3 ). Furthe3 kg·m−3 ) for the3 kg·m−3 . These cThese comparison results demonstrate that the cone-section method is an appropriate approach for TCs in high elevation areas, yielding results that outperform the cylinder prism method. Researchers theoretically regard the Gaussian quadrature formula as the most precise method for estimating TCs . HoweverAs 4.This paper used the cone-section method to compute the TCs for five surveying sites from the Mt. Jade area. This method obtained a significant improvement in TC determination. The cone-section method yielded better TCs than did the cylinder prism method. TC computations only used the information of topography surrounding the surveying sites from 0 to 100 km in the cone-section method. The cylinder prism and Gaussian quadrature methods corrections were calculated to the distance of 200 km. Although the Gaussian quadrature method obtained more high-frequency variations in TCs than do the other two methods, it required more computer time than the above two methods. The results in this paper suggest the cone-section method is an appropriate approach for TCs in high elevation areas. The cone-section method yields results that outperform the cylinder prism method and saves computation time over the Gaussian quadrature method. However, concerns about the precision of topography surrounding the surveying sites, namely Mt. Jade peak S026, limit current study conclusions. In addition, determining the reason for the significant differences in the estimations of TCs on Mt. Jade peak (site ID: S026) remains for finer accuracy of digital elevation model (DEM) than those used in this paper.


Gadus morhua) exploitation back as far as the 16th century, the historical origin of the modern fishery remains obscure. Widespread archaeological evidence for cod consumption around the eastern Baltic littoral emerges around the 13th century, three centuries before systematic documentation, but it is not clear whether this represents (1) development of a substantial eastern Baltic cod fishery, or (2) large-scale importation of preserved cod from elsewhere. To distinguish between these hypotheses we use stable carbon and nitrogen isotope analysis to determine likely catch regions of 74 cod vertebrae and cleithra from 19 Baltic archaeological sites dated from the 8th to the 16th centuries. δ13C and δ15N signatures for six possible catch regions were established using a larger sample of archaeological cod cranial bones (n = 249). The data strongly support the second hypothesis, revealing widespread importation of cod during the 13th to 14th centuries, most of it probably from Arctic Norway. By the 15th century, however, eastern Baltic cod dominate within our sample, indicating the development of a substantial late medieval fishery. Potential human impact on cod stocks in the eastern Baltic must thus be taken into account for at least the last 600 years.Although recent historical ecology studies have extended quantitative knowledge of eastern Baltic cod ( Gadus morhua) populations - particularly within the framework of the History of Marine Animal Populations project - stress the importance of time depth for understanding impacts of interactions between fishing mortality and other factors on stocks Recent studies of eastern Baltic cod  Baltic cod in the area to the east of Bornholm (ICES subdivisions 25–32) comprise a distinct population from that in the western Baltic and Kattegat (ICES subdivisions 21–24) Conversely, around the eastern Baltic littoral - apart from the islands - evidence for cod consumption remains rare until around the 13th century The relative explosion in cod consumption during the 13th to 14th centuries coincides with major historical developments in the region. This period saw the interconnection of much of the eastern Baltic littoral with wider political and economic systems: German and Danish crusaders established settlements in Estonia and Latvia; most of the Polish coast came under the political influence of the Teutonic Order; and the Hanseatic League extended its economic activities throughout the Baltic and beyond. Evidence for cod consumption during the 13th–14th centuries comes precisely from the sites most connected with these developments, including colonial towns such as Tallinn and Pärnu, Polish coastal strongholds that had come under Germanic influence - e.g. Gdańsk, Kołobrzeg - and Teutonic Order castles such as Mała Nieszawka (Poland).inland settlements in the eastern Baltic region indicates that fish were preserved and traded during this period, at least on a local level Medieval evidence for cod consumption at It is historically plausible that cod consumed at medieval Baltic settlements were overwhelmingly imported. Export-driven cod fisheries are known to have developed in Arctic Norway by the 11th to 12th centuries Historical records indicate the existence of taxable commercial cod fisheries off eastern Sweden and south-west Finland (ICES subdivisions 27 and 29) by the 1550s, and in the southern Baltic (ICES subdivision 25) by the start of the 17th century In order to distinguish between local  fishing and importation of preserved cod from, for example, the North or Barents Seas, it is necessary to assign approximate provenances (i.e. catch regions) to cod bone specimens recovered from archaeological sites. We do so using stable carbon and nitrogen isotopic signatures, following a new methodology 13C and δ15N) in fish tissues are likely to vary from region to region since they are known to change with temperature, salinity, nutrient loading, and trophic level Carbon and nitrogen isotopic values  was used to identify isotopic (δ13C and δ15N) signatures for six potential catch regions , specifically vertebrae and cleithra, recovered from Viking Age and medieval sites around the Baltic. Since these elements were typically included in traditional preserved fish products such as stockfish Secondly, the same isotopic values are measured for et al.This approach has previously been used successfully to provenance cod remains from medieval sites around the North Sea littoral 13C values that may reflect the low salinity of the Baltic proper. By contrast the Kattegat/western Baltic sample falls closer to non-Baltic regions, with a large standard deviation in δ13C probably resulting from marked spatial and temporal variability in salinity Group means and 1σ confidence intervals for carbon and nitrogen isotopic values from the 500–1000 mm control sample are shown in 13C between 13th/14th-century (n = 9) and 15th/16th-century (n = 21) specimens  = 15.58, p = 0.0005; mean difference = 1.1‰). The scale of this decrease is insufficient to obscure differences between regions, however, with the eastern Baltic remaining clearly separated from other regions when δ13C and δ15N are taken together . The northeast Atlantic and Arctic Norwegian regions were grouped into a single source (‘northern’) for this purpose. Historical records indicate that dried cod produced both in Norway and in the Scandinavian north-east Atlantic colonies were traded via Bergen during the Middle Ages 13C and δ15N amongst large Arctic Norwegian cod results in substantial overlap with the southern North Sea group. This implies a stronger relationship between fish size (and hence age) and trophic level amongst cod caught off northern Norway  from the resulting centroid. Targets falling within a 95% confidence zone were assumed to be either from non-Baltic sources or the Kattegat/western Baltic. Those falling outside this zone were considered indeterminate but compared with the eastern Baltic data qualitatively in the discussion. The two eastern Baltic control specimens themselves fall well outside the confidence zone - p<0.0001 in both cases - while only one non-Baltic control is a significant outlier (specimen 944: p = 0.0175). Regional temporal comparisons were not carried out for this size class due to the pooling of all non-Baltic regions and the limited sample from the eastern Baltic. The non-Baltic group includes samples spanning the 10th–18th centuries, but remains clearly distinct from the available 14th/15th-century eastern Baltic controls.Given the very limited Baltic data and the overlap between non-Baltic regions, DFA is clearly not applicable for the >1000 mm sample. Instead, all non-Baltic control regions were grouped. Target specimens were then evaluated in terms of Mahalanobis distance . Of 16 target specimens from Haithabu, Stargard Szczeciński, and Lübeck, 14 are attributed non-Baltic provenances by DFA. A vertebra from Stargard Szczeciński with an eastern Baltic signature could nonetheless have been landed at Szczecin - 30 km downstream on the Oder - which lies just west of the boundary between ICES subdivisions 24 and 25  have isotopic ratios similar to the eastern Baltic controls, and it is likely that most or all were imported either from the western Baltic - for which no control data is available - or beyond . Two vermay be from the late 14th century) most sampled specimens were apparently caught locally. Only one of 12 target specimens in the 500–1000 mm class - from Gdańsk - is assigned a non-Baltic provenance, although a cranial specimen from Uppsala also appears to have been imported from Arctic Norway in a departure from the usual stockfish preparation technique  based on bone measurements and comparison with modern standards of known total length (TL). From the total sample of 326, 259 specimens were successfully analysed for stable isotopic data, producing high yields of collagen with acceptable atomic C∶N ratios (see below). Most of these, deriving from fish in the 500–800 mm (n = 87) and 800–1000 mm (n = 90) classes, have previously been used to establish isotopic signatures for the six regions Outliers within the control sample might reflect rare instances of fish imported without prior decapitation et al.13C values are reported relative to the V-PDB scale, and δ15N values relative to the AIR scale. Errors on both the δ13C and δ15N measurements are less than 0.2‰. Amounts of carbon and nitrogen in the collagen extract were measured, and we only report isotope values from those samples with acceptable atomic C∶N ratios, defined as between 2.9 and 3.6 Laboratory methods were identical to those of Barrett 13C and especially δ15N values 13C and δ15N values between control specimens in the 500–800 mm and 800–1000 mm groups within each region revealed only one significant difference, for δ15N in Arctic Norway  Higher trophic levels in older, larger fish may result in elevated δ13C and δ15N values between control specimens in the 800–1000 mm and >1000 mm groups revealed highly significant differences in both carbon and nitrogen isotopic values for Arctic Norway , and a significant increase in δ13C with size class for the southern North Sea . Moreover, inter-regional differences in the relationship between fish size and isotopic values result in significant overlaps between the distributions of certain regions. In addition, the extreme rarity of cod over one metre long in modern populations means that their feeding behaviour is poorly understood Comparison of δ15N values resulting from radical changes in food webs and/or pollutants such as agricultural fertilizers and sewage. The control samples were classified into five two-century groups (9th/10th C to 17th/18th C) and tested for temporal differences within each region using ANOVA. Samples with date ranges spanning more than one two-century block were assigned to chronological group according to the greatest overlap, favouring the later period where overlap with each was equal. This is the same protocol employed for a comparable study of imports to the southern North Sea 13C between the 13th/14th C (n = 9) and 15th/16th C (n = 21) groups in the eastern Baltic  = 15.58, p = 0.0005; mean difference = 1.1‰). No other regions showed significant temporal changes in either δ13C or δ15N  samples from cod with estimated total lengths of more than 1000 mm.(XLS)Click here for additional data file.


Ammodramus nelsoni, A. caudacutus, and A. maritimus, respectively) depend on marsh and wetland habitats – ecosystems in which mercury (Hg) bioavailability is notoriously high. The purpose of the present study was to address the potential impact of Hg on these species using first primary and breast feathers as non-destructive biomonitoring tools.Nelson’s, Saltmarsh, and Seaside Sparrows  were within the range associated with decreased reproduction in other avian species. Hg Dynamics Component analyses demonstrated that Hg increased between first and second captures for Nelson’s (n = 9) and Seaside Sparrows (n = 23). Capture Frequency and Survival Component analyses detected a negative relationship between Hg and capture frequency in Nelson’s Sparrows (n = 315). However, MARK models detected no effect of Hg on apparent survival in any species.Feathers were sampled from wintering sparrows in North Carolina salt marshes (2006–2010). Feather Hg data were used in three risk analysis components (1) Threshold Component – examined feather Hg with regard to published negative effects thresholds; (2) Hg Dynamics Component – examined Hg in sparrows captured multiple times; and (3) Capture Frequency and Survival Component – tested for links between Hg and return frequency and survival. Threshold Component analyses indicated that Hg concentrations in 42–77% of sampled individuals (breast feather This study indicates that current Hg exposure places a considerable proportion of each population at risk. In particular, 52% of all sampled Saltmarsh Sparrows exhibited first primary feather Hg concentrations exceeding those associated with a >60% reduction in reproductive success in other species. This study reports evidence for net annual bioaccumulation, indicating an increased risk in older individuals. These data can be used to inform future population assessments and management for these species. Anthropogenic sources account for approximately two-thirds of all input of mercury (Hg) into the environment Ammodramus nelsoni, A. caudacutus, and A. maritimus, respectively) co-occur in mixed flocks in North Carolina (NC) salt marshes during their non-breeding season . Salt marsh habitats in North America are threatened by anthropogenic impacts such as habitat degradation, sea level rise, and pollution Nelson’s, Saltmarsh, and Seaside Sparrows (Gavia immer) and Carolina Wrens (Thryothorus ludovicianus) In order to assess the impact of Hg exposure on a free-living population, it is necessary to understand several key aspects of that exposure. One such aspect that is commanding the focus of an increasing number of studies is the effect of Hg exposure on reproduction. Two recent studies have provided a solid foundation of reproductive effects threshold data for two avian species, Common Loons  at second capture (6.07±1.08 ppm) compared to first capture (3.90±0.46 ppm). The same was true for Nelson’s Sparrow primary feather Hg . Spearman correlations revealed a significant positive association between breast feather Hg at first and second capture in Nelson’s Sparrows  in Nelson’s Sparrows  such that higher breast feather Hg at first capture was associated with birds captured only once (3.20±0.20 ppm) compared to birds captured multiple times (2.06±0.34 ppm). The proportional odds ratio for breast feather Hg from this statistical test was 0.230 and can be interpreted as follows: every 1 ppm increase in breast feather Hg corresponds to 0.230 times greater odds of being captured only once as opposed to multiple times. In other words, a Nelson’s Sparrow with breast feather Hg at 2 ppm is roughly two times more likely to be captured more than once compared to an individual with breast feather Hg at 11 ppm. No effect of breast feather Hg at first capture on capture frequency was observed in Saltmarsh (P = 0.4070) or Seaside Sparrows (P = 0.2425), and no effect of first primary feather Hg on capture frequency was observed in any species (P≥0.1158).There was a significant effect of breast feather Hg at first capture on capture frequency (multiple . The remaining 26 models were iterations of this model with one or more parameters removed . When an


SCT gene. In vitro studies using mouse hypothalamic cell line N-42 show that ERRα upregulates SCT promoter and gene expression. More importantly, knockdown of endogenous ERRα abolishes SCT promoter activation in response to hypertonic and ANGII stimulations. In mouse brain, ERRα coexpresses with SCT in various osmoregulatory brain regions, including the lamina terminalis and the paraventricular nucleus of the hypothalamus, and its expression is induced by hyperosmotic and ANGII treatments. Based on our data, we propose that both the upregulation of ERRα and/or the increased binding of ERRα to the mouse SCT promoter are two possible mechanisms for the elevated SCT expression upon hyperosmolality and central ANGII stimulation.Osmoregulation via maintenance of water and salt homeostasis is a vital process. In the brain, a functional secretin (SCT) and secretin receptor (SCTR) axis has recently been shown to mediate central actions of angiotensin II (ANGII), including initiation of water intake and stimulation of vasopressin (VP) expression and release. In this report, we provide evidence that estrogen-related receptor α , a transcription factor mainly involved in metabolism, acts as an upstream activator of the  In addition, SCT itself was found as a dipsogenic hormone that controls water intake behavior as well as mediates the dipsogenic effects of ANGII Secretin (SCT) is a classical gastrointestinal hormone VP expression and VP protein release. As ANGII via binding to AT1 receptor could stimulate SCT expression in SFO and OVLT In specific brain regions, both plasma hypertonicity and circulating ANGII are able to stimulate the lamina terminalis, consisting of the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT) and median preoptic nucleus (MnPO), which are critical sites for regulating water homeostasis in rodents SCT promoter have previously been characterized SCT gene by blocking NeuroD-mediated gene activation in response to bile acids in the mouse duodenum The human and rat SCT promoter to control SCT gene expression. After showing that SCT plays an indispensable role in mediating central effects of ANGII (7), the present study, thus, provide evidence to indicate that ERRα is one of the mechanisms that connects SCT with central effects of ANGII and/or plasma osmolality.In the present study, we investigated a possible mechanism for the cross-interaction of SCT and ANGII central pathways in osmoregulation via ERRα. We found that ERRα co-expresses with SCT in various osmocenters in the CNS, and that its own expression is regulated by central ANGII and peripheral hyperosmotic stimulation. Using a hypothalamic cell model, ERRα was then found to interact specificity with an ERE-half site in the proximal region of the mouse Silencer 4.1-CMV vector . The siControl vector provided by the manufacturer was used as a negative control. Western blot analysis The mouse SCT core promoter construct (mSCTP) (from −27 to −399 bp relative to the start codon) was produced by 1) PCR amplification using mouse genomic DNA, 2) sequencing, and 3) subcloning into the pGL2-Basic vector. The mouse ERRα, ERRβ and ERRγ expression vectors were constructed using the pcDNA3 vector . Site-directed mutants of the mSCTP promoter were constructed by a three-step mutagenesis method 2. For transfection, N-42 cells were seeded onto 6-well plates  at a density 2.0×105 cells/well. After 2 d, at about 80% cell confluence, transient transfection was carried out using the GeneJuice transfection reagent  according to the manufacturer's instruction. For over-expression studies, the mouse SCT promoter (mSCTP) 2 for luciferase assay or RNA isolation.The mouse hypothalamic cell line, N-42, was purchased from CELLutions Biosystems Inc . Cells were cultured in DMEM  supplemented with 4.5 mg/ml D-glucose, 10% FBS , and the antibiotics, 100 U/ml penicillin G and 100 µg/ml streptomycin (Invitrogen) at 37°C with 5% CO5 cells/well. After 2 d, at which the cell confluence was about 80%, saline treatment was carried out. Different concentrations of saline were added to the cells so that the final concentrations of the medium were 0, 25, 50, and 100 mM. The plate was then incubated at 37°C with 5% CO2 for various time periods  before harvest. For ANGII treatments, the culture mediums were replaced by the medium with ANGII in various final concentration (ranged from 10−7 to 10−11 M). The cells were collected for 8 h before the RNA isolation and protein extraction. Total RNA were isolated by TRIzol reagent (Invitrogen). The isolated RNA was reverse transcribed with oligo-dT primer and Superscript III reverse-transcriptase (Invitrogen). One-tenth of the first strand cDNAs was then used as the template in qRT-PCR for measuring mouse GAPDH, SCT, ERRα, and S14 mRNA levels by the SYBR Green PCR Master Mix kit  with specific primers  were added for 8 h. After salt treatment, CellTiter 96 Aqueous One Solution Reagent was added for 2 h and absorbance at 490 nm, which is directly proportional to the number of living cells in the culture, was measured. Wells with plain medium with no cells were included as the blank and each sample was repeated at least 5 times in this study.The saline treatments for N-42 cells was performed based on previous studies 32P] ATP by a Ready-To-Go T4-polynucleotide kinase labeling kit  and purified by a Microspin G-25 column (Amersham Biosciences). Binding reactions were carried out at room temperature for 15 min in a 20 µl reaction containing 10 mM Tris (pH 7.5), 50 mM NaCl, 2.5 mM MgCl2, 0.5 mM dithiothretiol, 4% glycerol, 2 µg poly(deoxyinosine∶deoxycytosine), and 1 ρmol radio-labeled probe. Free probes and bound probes were separated by electrophoresis in a 5% polyacrylamide gel Nuclear extracts from mouse hypothalamus were prepared as described before mock– Ctspecific) and normalized with input DNA, where Ctmock and Ctspecific represent the mean threshold cycles (Ct) of qRT-PCR (in triplicates) on purified DNA samples from mock and specific ERRα immunoprecipitation.ChIP assays were performed as described previously ad libitum with standard rodent chow  and water, unless otherwise stated.All animal treatments were in accordance with the guidelines established by the Committee on the Use of Live Animals in Teaching and Research  of the University of Hong Kong with the Cap. 340 animal license issued by the Department of Health of the Hong Kong Government under the Animals Ordinance. All experiments were carried out with adult transgenic mice (20–25 g) of N5 generation, which were kept in a temperature-controlled room with a 12-h light/dark cycle. Mice were fed Mice were anesthetized and surgery was conducted in aseptic conditions. Procedures for ICV were performed as described To induce hypertonic serum in mice, normal drinking water was either removed for 1 d or replaced with 2% saline for 5 d. Treated and control mice were then sacrificed and their brains were collected. For ICV studies, 1 h after the ICV injection, similarly, brains of treated and control mice were isolated. For whole brain studies, total RNA was extracted by the TriPure isolation reagent . For study of the specific brain areas, the brains were embedded in OCT compound  and stored at −20°C. The frozen brain was then sectioned (8-µm thick) using a cryostat microtome  and mounted on plain slices. Brain sections at the level of the lamina terminalis and PVN were stained with hematoxylin and eosin and imaged on the PixCell IIe Laser Capture Microdissection System . Target cells were located according to the mouse brain atlas and were captured on CapSure HS LCM Caps (Arcturus). RNA of captured cells was prepared using the PicoPure RNA Isolation Kit (Arcturus). The isolated RNA was reverse transcribed and real-time PCR were performed as described above.IHC staining was performed as described All data are shown as means ± standard error SEM. The deviations between groups were analyzed using the computer software PRISM . Unpaired t-test was performed when only two groups were under consideration, whereas data from more than two groups were analyzed by one-way ANOVA, followed by Dunnett's test.SCT promoters  was overexpressed with the mouse SCT promoter-luciferase construct. As shown in SCT promoter was significantly  activated only by ERRα, which is a constitutive activator SCT promoter. Consistent with this observation, endogenous SCT mRNA levels were significantly elevated  when ERRα was overexpressed in N-42 cells , resulted in considerable drops in the mouse SCT promoter activities    for osmoregulation romoters , we init42 cells . In ordeecrease) , and alsor 2 µg) . The siRor 2 µg) , in whicanalysis . The proSCT gene, our next task was to study if ERRα was involved also in the osmoregulatory pathway. We have previously revealed that SCT expression was upregulated in the mouse hypothalamus in response to water deprivation and saline drinking SCT gene, N-42 cells were treated with different concentrations of hypertonic salt treatment  for 2, 4 and 8 h  activated mouse SCT promoter (SCT mRNA levels (−10 M was found to produce the greatest stimulation (2.89 folds). Again, ANGII treatment was unable to change the expression of the mouse S14 housekeeping gene, as a control.After showing ERRα as an activator of the  and 8 h  as in a  and 8 h  suggestetivities , in whic pattern . In thiss tested . Consistpromoter  as well A levels . ANGII aSCT, both hypertonic shock and ANGII could also stimulate mouse ERRα mRNA expression (α protein level (ERRα-S1) was employed to silence endogenous ERRα, consistent with findings in SCT expression.Similar to their effects on pression  as well in level  in this in vivo functions of ERRα to upregulate SCT in response to hypertonic treatment and ANGII, we initially investigated expression levels of ERRα in mouse brain in response to hyperosmotic stimuli and central ANGII administration. We found that ERRα transcript levels in the mouse cerebrum were significantly elevated after water deprivation, saline drinking or ICV-ANGII in mice    left. Toobserved  right. Nobserved  middle.in vitro studies  in the mouse hypothalamus  and NeuroD binding with an E-box motif (at −167), SCT promoter is implicated for mediating central effects of ANGII.By reviewing the 5′ sequence  of the mouse  −149 bp . In the There have been studies suggesting estrogen plays functional roles in fluid regulation in rodent and human −/−ERRα) mice have identified the potential of ERRα in regulating blood pressure and water homeostasis, by controlling renal sodium and potassium homeostasis and the RAS −/−ERRα mice have reduced water intake, urine volume, and increased Na+ in blood. These indicate that ERRα is dipsogenic and diuretic. Also, ERRα knockout mice are deficient in renal Na+/K+ handling with a mechanism that favor Na+ retention, leading to the increased blood Na+ and thus increased water reabsorption and decreased urine volume. Using genome-wide ChIP-on-ChIP analysis and expression profile comparison of −/−ERRα and WT mice, the study also identified a number of target genes regulated by ERRα in kidney. Some of these are genes involved in water regulation, including the RAS genes , VP receptor , and tonicity-responsive element (TonE) binding protein (TonEBP). Our findings show that ANGII upregulates ERRα in the lamina terminalis and PVN in the brain; while Tremblay et al. suggested that ERRα could downregulate RAS genes in kidney, which might work as a negative feedback control ERRα, and thus SCT and VP expression; VP receptor genes in kidney were found to be downregulated in the −/−ERRα mice In agreement with its expression patterns in high energy demand tissues, ERRα is generally recognized as a transcription factor that regulates expression of genes involved in bioenergetics


Xiphias gladius was examined based on 2231 individual samples, collected mainly between 2009 and 2010, among three major sampling areas within the Indian Ocean , Atlantic (two sites) and Pacific (one site) Oceans using analysis of nineteen microsatellite loci (n = 2146) and mitochondrial ND2 sequences (n = 2001) data. Sample collection was stratified in time and space in order to investigate the stability of the genetic structure observed with a special focus on the South West Indian Ocean. Significant AMOVA variance was observed for both markers indicating genetic population subdivision was present between oceans. Overall value of F-statistics for ND2 sequences confirmed that Atlantic and Indian Oceans swordfish represent two distinct genetic stocks. Indo-Pacific differentiation was also significant but lower than that observed between Atlantic and Indian Oceans. However, microsatellite F-statistics failed to reveal structure even at the inter-oceanic scale, indicating that resolving power of our microsatellite loci was insufficient for detecting population subdivision. At the scale of the Indian Ocean, results obtained from both markers are consistent with swordfish belonging to a single unique panmictic population. Analyses partitioned by sampling area, season, or sex also failed to identify any clear structure within this ocean. Such large spatial and temporal homogeneity of genetic structure, observed for such a large highly mobile pelagic species, suggests as satisfactory to consider swordfish as a single panmictic population in the Indian Ocean.Genetic population structure of swordfish  Acanthocybium solandriThunnus alalungaMakaira nigricansTetrapturus albidusXiphias gladius with two subdivisions in the Pacific Ocean . Nevertheless, when population structure was considered and when the assessment focused on the southwest Indian Ocean as an independent stock – a case considered by the India Ocean Tuna Commission (IOTC) on the basis of the fishery data The identification of genetic structure in the Indian swordfish should be of importance in term of fish management as one of the challenging issues commonly recognized is to match the artificial spatial scale of stock assessment with the natural spatial structure of the species The present study aims to determine the swordfish genetic population structure in the Indian Ocean. For this purpose, an intensive sampling was conducted over the whole Indian Ocean, at several periods during two consecutive years (2009–2010). We examined genetic variation of more than two thousands swordfish using newly developed genetic markers, supposedly more discriminating than older ones: 19 microsatellite loci Sampling for the present study focused on the Indian Ocean (IO) – as defined by international conventions as the waters delineated from the Atlantic Ocean by the 20° east meridian, from the Pacific by the meridian of 146°55′ east and a southern limit at 60°S Initial sampling strategy was to sample one hundred fish per zone at two targeted seasons  over two consecutive years (2009 and 2010). Initial sampling seasons were defined based on known information of swordfish reproductive condition in IO  following the manufacturer instructions. A 1007 bp fragment of the mitochondrial ND2 gene was amplified by PCR using the primers and recommended conditions defined in Bradman et al. 2, 2 µM of each dNTPs, 0.3 µM of each primer, 0.5 U of SilverStar Polymerase Taq (Eurogentec), 25 ng of genomic DNA. Cycling parameters were 93°C for 3 min, followed by 35 cycles of 93°C for 30 s, 50–62°C for 50 s, and 72°C for 50 s and a final elongation at 72°C for 30 min. PCR were amplified separately and electrophoresed in three multiplex panels. Amplified fragments were separated on an ABI Prism 3100 genetic analyser. Alleles were scored using a co-migrating size standard  and identified using GENE MAPPER v4 (Applied Biosystems Inc.).Nineteen microsatellite loci were also used, three from i.e. swordfish sampled in a given area at a given time; each sample set was named as XXX_00_## .Data analysis was first conducted on the whole dataset to identify global level of structure. We also defined spatially and temporarily stratified sampling sets (h) and nucleotide (π) diversities and Fu’s Nall), and the observed (Ho) and expected (He) heterozygosities Rs) using the rarefaction process of the standArich package  for R isF as estimated by Weir and Cockerham 5 burn-in samples/generations and 5.105 iterations) for K values from 1 to 5.For ND2 sequences, haplotype  for ND2 sequences and SMOGD http://www.geodistance.com website) were plotted against genetic distance (using Фst/(1− Фst) for mitochondrial data or FST/(1− FST) for microsatellite data following the recommendations of Rousset ncf package (available at http://onb.ent.psu.edu/onb1/R).For both ND2 sequence and microsatellite data, the analysis package ARLEQUIN 3.5 a priori grouping based on geographical or temporal proximity, within or between oceans. The software SAMOVA 1.2 a priori group definition and identify geographic partitions that maximize genetic differences between groups and geographic homogeneity within groups; it was tested for K group values ranging from 1 to 4, with 100 annealing replicates each time.ARLEQUIN 3.5 Finally, the same analytical approaches  were processed to assess whether or not sex of individual had an effect on the genetic structure.A total of 2 231 swordfish were sampled from the three major study areas during this study . Sampling details are provided in d) and mean nucleotide diversity (π) were high, respectively 0.886 (±0.04) and 0.0022 (±0.0004), and similarly high within each sample set  of 2 001 swordfish. Approximately 48% of these 282 haplotypes were represented more than once. Mean haplotype diversity  varying from 5.8 for MAD_10_1 to 6.50 for COR_09_1 . FixatioST was 0.006 (p<0.001) when considering all the samples; it decreased to 0.001 and was non-significant (p>0.05) when considering only the swordfish sampled within the IO . Pairwise genetic distance estimates (ФST) between sample sets are summarized by a neighbor-joining (NJ) tree in Appendix F4 in ST values were observed for the NAM sample sets (mean ФST = 0.098), followed by COR and AFS comparisons (0.012 and 0.010 respectively). Consistent with these significant differentiation results, values of Jost’s D were very high when comparisons included NAM samples (D >0.25), even if the highest values of Jost’s D regarded AFS_10_3 (mean D = 0.74). An AMOVA analysis undertaken with grouping made per ocean also demonstrated a small but significant level of structure between oceans . The likelihood decreases when estimates were made with more than one pool (over ten independent simulations: LnP(D) for K = 1 and K = 2 were −109850 and −110440, respectively). When considering two genetic pools, the mean posterior probability per individual is 0.50 (±0.04) providing more evidence against subdivision.ST was 0.0028 (p<0.001) when considering all the sample sets and 0.0026 (p<0.001) when considering only the swordfish sampled within the IO suggesting the same low level of structure within and between oceans. The neighbor-joining tree based on the pairwise FST estimates .Overall FCT and FCT <0.001, p>0.05; see a priori geographic grouping, between-group variance was maximized when one sample set was considered isolated from all the others.We considered only the IO sample sets, excluding the five ones from the Atlantic Ocean (NAM and AFS) and the two from Pacific Ocean (COR). Irrespective of which marker was examined, the AMOVA analysis conducted within the IO showed that more than 99% of the variance was observed within the samples with no variance significantly associated with the partition into any kind of grouping . No significant differences were observed between years or seasons sampled at any site  neither there was significant differences observed between different sites within a season . Looking at microsatellite pairwise FST values (Appendix S2), 139 upon the 240 comparisons appeared significant . However, values of Jost’s D were very low with only 19 values above D = 0.01 (Appendix S2) at the intra IO level and no clear pattern of structure could be detected  were low and not significant, even between the most distant areas  when considering all the samples from this area; it was still not significant and decreased to 0.0009 (p>0.05) when considering only females and increased to 0.0020 (p>0.05) when considering males. A similar trend but significant was observed with microsatellite data as overall FST was 0.0023 (p<0.001) when considering all the samples, decreased to 0.0010 (p<0.05) when considering only the females and increased to 0.0040 (p<0.005) when considering the males. This trend indicated a higher genetic homogeneity between females than between males at the scale of the SWIO.Sex and maturity stage information were only available for localities in the South West Indian Ocean region see . Most ofXiphias gladius within the Indian Ocean (IO). The results obtained from both ND2 sequences and microsatellites on multiple sampling areas over multiple seasons detected some level of genetic heterogeneity but failed to demonstrate evidence that swordfish have multiple discrete populations within the IO. All the analysis focusing on the important spatially and temporally stratified sampling done in the SWIO failed to identify any significant structure. These results are consistent with the existence of a single panmictic population in the Indian Ocean population and thus they contradict previous studies suggesting the presence of at least two potential genetic populations i.e. long distance migration observed for IO swordfish using tag-recapture approach This study aimed as a first step to evaluate genetic structure of the swordfish MONS) in the north and the Indian South Subtropical Gyre Province (ISSG) ALME - which bordered the north west of the MONS province; Appendix F1 in ALME a serious candidate for a discrete feeding area; unfortunately, our sampling scheme did not allow us to identify whether this northwestern area is a specific foraging ground for some IO swordfish. Further exploration into the foraging behaviour of the swordfish, a deep-dwelling predator, with specialization on prey sizes and species The existence of at least two distinct stocks observed for this species in the Atlantic and Pacific Oceans in extenso is another argument suggesting a homogeneous single panmictic population in the IO. In the present case, the more pronounced structure observed with mitochondrial data could also underline the fact that our microsatellite loci were not as discriminating as they were expected to be e.g. SNPs (single nucleotide polymorphisms), or scan genomics approaches and detection of outlier loci Genetic analysis made by sex also failed to reveal any structure, however there was an indication of a higher genetic homogeneity between females than between males at the scale of the SWIO (significant using the nuclear marker but not using the mtDNA one). The fact that the genetic information given by the two genders is not the same could indicate a sex-biased dispersal in which gene flow between populations is accomplished primarily by one gender Analyses of mitochondrial ND2 sequences and microsatellite polymorphism indicated significant isolation between oceans. Both molecular markers showed a significant level of genetic variance associated when comparing samples by Oceans, with a level of differentiation of the IO higher with the Atlantic Ocean (AO) than the Pacific Ocean (PO).ST values observed between PO sample sets and the Indian sample sets are elements that strongly indicated an Indo-Pacific differentiation. Our results also showed that a significant part of genetic variance is associated with the Indo-Pacific differentiation . One of the criteria previously used to discuss the interoceanic differentiation and consider the swordfish from Indo-Pacific as one population was the shared absence of Clade II in both oceans Indo-Pacific swordfish was until now considered to belong to a unique stock ST values observed between NAM and Indian sample sets are elements that strongly indicated an Indo-Atlantic differentiation. Such differentiation observed is consistent with previous studies Thunnus obesusIstiophorus platypterus, and the blue marlin Makaira nigricansSphyrna lewiniChelonia mydasDermochelys coriaceaST and FST). The sample sets AFS_09_1 (July - November 2009) and AFS_10_2 (April 2010) showed a mtDNA signature more related to the AO population while the two others sets (AFS_10_1 and AFS_10_3) were more similar to IO genetic signature  that decreased to less than 5% in the IO and absent in the PO and the consequent high ФFile S1containing: Appendix F1: Geographic location of sampling areas and of the biogeographic provinces . Appendix F2: Swordfish sampling schedule. Appendix F3: Mismatch distribution for swordfish based on 1007 bp of the ND2 sequences. Appendix F4: Neighbor-joining trees showing the relationship between sample sets on the basis of pairwise genetic distances. Appendix S1: ND2 sequences pairwise ФST and D Jost’s values. Appendix S2: Microsatellites pairwise FST and D Jost’s values.(DOCX)Click here for additional data file.


Influenza A (H3N2) offers a well-studied, yet not fully understood, disease in terms of the interactions between pathogen population dynamics, epidemiology and genetics. A major open question is why the virus population is globally dominated by a single and very recently diverged (2–8 years) lineage. Classically, this has been modeled by limiting the generation of new successful antigenic variants, such that only a small subset of progeny acquire the necessary mutations to evade host immunity. An alternative approach was recently suggested by Recker et al. in which a limited number of antigenic variants are continuously generated, but most of these are suppressed by pre-existing host population immunity. Here we develop a framework spanning the regimes described above to explore the impact of rates of mutation and levels of competition on phylodynamic patterns. We find that the evolutionary dynamics of the subtype H3N2 influenza is most easily generated within this framework when it is mutation limited as well as being under strong immune selection at a number of epitope regions of limited diversity. Influenza A (H3N2) has circulated in the human population since 1968 causing considerable annual morbidity and mortality worldwide. Despite the rapid evolution of the hemagglutinin (HA) protein and strong diversifying selection, the global virus population is characterized by a low standing diversity, evident in the serial replacement of antigenic types and in the ‘cactus-like’ structure of its genealogical tree. Elucidating the mechanisms behind these puzzling patterns is key to understanding the evolution of seasonal (H3N2) influenza. One recent epidemiological model proposes a restricted set of antigenic types whose waves of dominance result from frequency-dependent immune selection. Here we develop a model of limited antigenic diversity that explicitly incorporates mutational processes, and use it to address, first, whether this type of antigenic space is capable of generating the characteristic phylogeny of HA sequences, and second, whether the dynamics of (H3N2) influenza are primarily limited by the arrival of mutations or by the opening of antigenic niches. We conclude that a limited antigenic space can explain the observed phylogenetic patterns and that a limited mutation rate is a key property underlying the dynamics of (H3N2) influenza. Our study provides a general framework for assessing the relative roles of selection and mutation in a variety of infectious disease systems. Influenza viruses are classified into types A–C, among which influenza A is the most pathogenic. These viruses cause between a quarter to half a million deaths worldwide The major targets of humoral immunity against influenza A are its envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA); these form the basis of its crude classification into subtypes H1N1, H2N2 and H3N2 etc. Since its emergence in 1968, influenza A (H3N2) has continually circulated in the human population. The phylogeny of its HA protein  shows a The classical view of influenza evolution is one of antigenic drift A competing hypothesis advanced by Recker et al. Here, we have attempted to resolve this question using a large-scale individual-based simulation of epidemiological and evolutionary dynamics that allows the complete phylogenetic tracking of a virus population characterized by defined repertoires of polymorphic epitopes. Our model is based on the multi-locus structure employed by This model structure allows us to make inferences about the roles of mutation and competition in a more general context. Models of antigenic dynamics tend to polarize between those in which the availability of antigenic types dictate the dynamics We use an individual-based SIR model, explicitly tracking the chains of infection of viral lineages as well as the antigenic phenotype of every virus in the population . Thus, oA host population of constant size N is simulated with an equal birth and death rate of μ. Infected hosts randomly contact other hosts in the population at rate β. The risk of infection is based on crossimmunity interactions based on their previous unordered infection history To explore the epidemiological dynamics of our model in isolation, we implemented a parameterization lacking mutation, in which extinction was preempted by maintaining at least one carrier for each antigenic-phenotype. Different colors  represenA mutation-free model can result in different alternative dynamics based on model parameters ranging from stable coexistence of completely discordant antigenic-phenotypes to the successive replacement of strains through chaotic or cyclic behavior We examined the effects of mutation at different rates on the resulting phylodynamic patterns of the virus by seeding the population with a single strain and tracking antigenic and evolutionary changes. We measure diversity π as the average time separating two randomly selected contemporaneous viruses since their divergence from a common ancestor. Because branch lengths in our genealogies are measured in years, the resulting diversity is also measured in years.In the absence of antigenic mutation, only a single strain persists, experiencing transient oscillatory dynamics between near extinction, and endemic equilibrium conditions . As all For low mutation rates  the intrAn increase in mutation rate  leads toAt a relatively high mutation rate , we apprIn general, a threshold exists at which mutation overwhelms selection resulting in a population drifting away from the fittest genotype By comparing fixation versus extinction of antigenic mutations, using a quantity related to the McDonald-Kreitman (MK) index If antigenic mutations are advantageous for long-term virus persistence, an MKR ratio above 1 is expected. In this case, individuals exhibiting these antigenic mutations will be more likely to fix in the population and contribute to substitutions on the trunk of the phylogeny. Similarly, if antigenic mutations are deleterious to the long-term success of the virus, an MKR index of less than 1 is expected. This is because mutant individuals will tend to be lost from the population and side branches will show an excess rate of substitution.We find that, when rare, antigenic mutations show highly increased rates of fixation (MKR = 19±11), and therefore evidence of strong positive selection . Hence, Our model contains a cross-immunity parameter σ which allows us to explore a range of immune selection regimes: when σ = 1, we have full cross-protection (as might arise if each epitope elicited a very strong immune response) and when σ = 0, cross-protection between strains is only high if they share their entire variable repertoire.In general, stronger cross-immunity results in lower prevalence as hosts fail to be re-infected . We find−5). When mutation rate is limiting (ξ<5×10−5) then antigenic mutations are favored by natural selection (MKR>1). However, when mutation rates are higher (5×10−5<ξ<10−3), negative selection by-and-large predominates. The strongest negative selection occurs in a region of moderate cross-immunity (σ = 0.6) corresponding to previously observed discordant dynamics . If we separate results into a regime of positive selection (MKR>1) and a regime of negative selection (MKR<1), we observe similar results within each regime. Stronger positive selection coincides with a decrease in genetic diversity (ρ = −0.85 when MKR>1), and stronger negative selection tends further increase diversity through the persistence of discordant strains and associated deep branches (ρ = −0.28 when MKR<1). As expected from population genetic theory ρ = 0.74 when MKR>1) than under negative selection (ρ = 0.87 when MKR<1) and cannot be trivially dissociated from the effects of selection.There is also a clear relationship between diversity and selection as measured by the MKR index . We obseρ = 0.88 when MKR>1) and a decrease in Shannon diversity (ρ = −0.88 when MKR>1). Negative selection decreases Shannon diversity (ρ = 0.39 when MKR<1) and increases single strain dominance (ρ = −0.23 when MKR<1). While negative selection lowers the number of circulating strains, it increases genetic diversity π through the existence of deep non coalescing branches.Two additional strain diversity measurements based on the ecological dynamics are shown in The patterns described so far suggest that the dynamics of H3N2 influenza within this framework correspond to a regime in which host immune mediated selection is strong and the antigenic mutation rate is low. We now extend the model in order to examine other characteristics relevant to H3N2 in a more detailed epidemiological setting that includes seasonality and a basic global population structure.In this analysis we include three demes representing the northern hemisphere, the southern hemisphere and the tropics. Northern and southern hemisphere demes experience an opposing seasonal modulation (with a 14% amplitude and six months phase difference) while tropical regions experience two weaker seasons annually In this model we use an antigenic repertoire with 4 epitopes differing in the number of alternative variants per epitope. A typical tree for this configuration together with the corresponding diversity skyline is depicted in Average yearly incidence in the northern and southern hemisphere demes is  respectively , while iIn this model the tropics or lower and mixed seasonality populations exhibit a greater role (68%±9) in establishing the trunk of the influenza tree . The souWe find that strong competition and high R0 values generates more regular annual epidemic peaks while maintaining low genetic diversity. In addition we find that within antigenic cluster evolution also contributes to maintain low genetic diversity. An increase (0.005 compared to 0.001 of contacts) in the strength of the metapopulation coupling slightly improves the epidemiology by decreasing the likelihood of long periods without annual epidemics.To establish whether low genetic diversity can be maintained when the number of epitopes or variants per epitope is increased we repeated the same parameterization with double the number of epitopes and with twice the number of variants per epitope and by keeping either the per-epitope or overall mutation rate. The results are summarized in Herein, we implemented an individual-based model that allowed us to track both the ecological and evolutionary dynamics of a pathogen population, in which cross-immunity is orchestrated by a finite set of antigenic loci of limited variability Explicit modeling of evolution, through the introduction of antigenic mutation at different rates, allows us to consider phylogenetic trees in addition to epidemiological dynamics. Resulting phylodynamic patterns range from successive strain turnover, to discordant antigenic sets, to dynamics resembling those of a model lacking explicit evolution and finally to the collapse of antigenic structure. Each of these can be explained by the interplay of selection and mutation, as measured here through the MKR index, and by considering different strengths of immunity generating competition between strains.The dynamics of our individual-based model are generally in good agreement with the epidemic behavior of influenza A . Like obRefinement of the epidemiological model, such as the inclusion of an exposed period, can further improve the comparison to empirical data. In particular, the above properties were obtained with a basic reproduction number of R0≈3.24, on the upper bounds of current estimates for seasonal influenza. This value can possibly be decreased by considering such an extension.−5 per day and a reduction of cross-immunity of 13% per epitope results in phylodynamic patterns broadly consistent with those seen in H3N2 influenza Click here for additional data file.Figure S1Changes in prevalence for carrying strengths of strain competition and antigenic mutation rates. Prevalence increases as crossimmunity between strains decreases, enabling multiple infections. When crossimmunity is 1, all strains are antigenically equal, and one lifetime infection is possible. When no crossimmunity is present, each antigenic-type can independently infect a host once.(PDF)Click here for additional data file.Figure S2Changes in antigenic diversity and the McDonald-Kreitman related index (MKR) for varying strengths of strain competition and antigenic mutation rates.(A) Mean Shannon diversity was measured for 40 years of simulation. Shannon diversity ranges from zero when a single circulating antigenic variant is present at each time point, to approximately 3.5nats when all the possible antigenic variants are continuously present. For a large range of the parameter space, stronger competition and lower mutation rates decrease Shanon diversity as fewer circulating antigenic types co-exist. (B) Single strain dominance based on the quantity ε from (C) Shannon diversity decreases with stronger positive selection (ρ = −0.88 when MKR>1) and with stronger negative selection (ρ = 0.39 when MKR<1). Positive selection roughly corresponds to lower mutation rates (ξ<10−3), while negative selection corresponds to higher mutation rates (ξ>10−4) (D) Single strain dominance increases with stronger positive selection (ρ = 0.88 when MKR>1) and an increases for stronger negative selection (ρ = −0.23 when MKR<1). (see (PDF)Click here for additional data file.Figure S3Seasonal patterns. For simulations including metapopulation sinusoidal seasonal forcing was used (see used see . Contact(PDF)Click here for additional data file.Figure S4Dynamic changes in the percentage of antigenic clusters and the dominance of antigenic variants in different metapopulation demes.(A) Changes in the percentage of the population infected with a specific antigenic cluster variant for the northern hemisphere, the southern hemisphere and the tropics. On average we observe 13±6 antigenic clusters that come to dominate the virus population over the course of the 40 year simulation with an average duration of 4±2 years. One or two clusters usually dominate the deme population. Clusters are defined based on a threshold set when the cumulative change of two or more epitopes between any previous cluster antigenic-type is reached, based on (B) Measurement of the onset time for all antigenic types (prior to cluster subdivision). Onset time was measured as the point where prevalence was estimated to reach 5% of its overall deme prevalence. Antigenic variants are more likely to reach significant prevalence in the tropics: 2±1.5 months earlier in the tropics compared to the northern hemisphere and 3±2 months earlier in the tropics compared to the southern hemisphere (p<0.001 for the combined results).(PDF)Click here for additional data file.Figure S5Antigenic types across 40 years of simulated years. A sample of antigenic types that emerge in the simulation are sampled and numbered sequentially. Red – Antigenic variants sampled from the trunk of the tree (fixed). Black – Antigenic variants sampled from sidebranches of the phylogenetic tree.(PDF)Click here for additional data file.Figure S6Crossimmunity patterns for individual epitopes. The thirteen most prevalent antigenic types from a span of 40 years of simulation were sampled ( sampled  and orde(PDF)Click here for additional data file.Figure S7Full phylogenetic trees following the initial introduction of a virus with an increasing mutation rate. Phylogenetic trees are based on samples of directly measured virus genealogy in the simulation (434 years). (A) Model with no mutation. (B) Model with low mutation rate of ξ = 7.5×10−6. (C) Mutation rate of ξ = 7.5×10−5. (D) Mutation rate of ξ = 7.5×10−4. (E) Mutation rate of ξ = 7.5×10−3.(PDF)Click here for additional data file.Table S1Model results for doubling the number of epitopes or variants per epitope.(PDF)Click here for additional data file.


The aim of this study was to determine the validity of a novel athlete-led protocol for determining maximal aerobic fitness in adults. Twenty-nine participants  from a variety of exercise backgrounds were asked to complete two maximal treadmill running tests (using the standard Bruce or a novel athlete-led protocol [ALP]) to volitional failure in a counter-balanced randomised cross-over trial one week apart. We found no substantial difference in maximal oxygen uptake , evidenced by the Spearman correlation coefficient of 0.93 . However, compared to the Bruce protocol, participants completing the ALP protocol attained a substantially higher maximal heart rate . Additionally, using the Bruce protocol took a longer period of time (23.2 ± 17.0 s) compared to the ALP protocol. It seems that using either treadmill protocol will give you similar maximal oxygen uptake results. We suggest the ALP protocol which is simpler, quicker and probably better at achieving maximal heart rates is a useful alternative to the traditional Bruce protocol.Treadmill tests for maximal oxygen uptake (V̇O Maximal aerobic power is commonly accepted as the best measure of the cardiovascular systems’ functional limits  and has Many measures of aerobic fitness traditionally utilize sub-maximal exercise tests which use algorithms to predict maximal aerobic fitness . A more 2max) test was first standardized by 2max. Subsequently 2max) was able to produce similar V̇O2max values  from a variety of exercise backgrounds volunteered to participate in the present study. Participant characteristics are presented in 2max the participants completed a continuous incremental exercise test to voluntary exhaustion on a calibrated treadmill . Briefly, in the Bruce protocol (Bruce) participants started exercising at a treadmill speed of 2.7 km·h−1 and an incline of 10% gradient for 3 minutes. Workloads (speed and inclination) were subsequently increased each 3-minute period in a simultaneous way until volitional exhaustion was reached  to volitional failure in a randomised cross-over trial 1 week apart. The participants were asked to refrain from intense exercise and alcohol for 24 h prior, and caffeine from 4 h prior to each main trial. Participants also recorded their dietary intake prior to the first trial to allow replication of diet prior to subsequent trials. Participants were asked to drink 500 ml of water approximately 2 hours prior to testing in an attempt to standardise bodily fluid concentrations. To assess V̇O reached .−1) with no incline. This initial speed was set according to the fitness and training status of the individual (8 km·h−1 for more sedentary and 10 km·h−1 for more athletic participants), thereafter, each subsequent minute the speed was increased by 1 km·h−1 until participants reached a pace that was reasonably comfortable and could be maintained for the duration of the test. Once the comfortable running pace was found, the treadmill was increased by a 1% gradient each minute until voluntary exhaustion was reached. Each participant was encouraged to exert a maximal effort. The test was stopped when the participant could not maintain the required pace or had reached voluntary exhaustion. The criterion used to assess V̇O2max included a respiratory exchange ratio ≥ 1.10, a heart rate in excess of 90% of age predicted heart rate maximum (220-age), and identification of a plateau (<150 ml x min−1 increase) in V̇O2max despite a further increase in velocity. In all tests, two of the three criteria were met. To enable direct comparison of V̇O2max data between protocols, participants were asked to maintain their normal level of training for the length of the study.In the athlete-led protocol (ALP) the participants started exercising at an easy running speed (8–10 km·h2 and 5% CO2). Oxygen uptake (V̇O2), minute ventilation (V̇E), end-tidal CO2 (PETCO2) and respiratory exchange ratio (RER) were measured. Face masks  with small dead spaces (approximately 70 ml) were fitted to participants allowing simultaneous breathing at the mouth and nose. To de-emphasise breath-to-breath variation, values for V̇O2 were smoothed by taking the average for every 15-second time period. During the performance, heart rate was recorded continuously by means of a heart rate monitor . Blood lactate concentration was determined from a finger-prick sample at rest and 5 minutes post test and analysed using a portable lactate analyser . Standing height and nude body mass  was determined on arrival at the laboratory.Ventilation and expired gases were measured breath-by-breath using a portable gas exchange system . Before testing, the gas analyser was calibrated for volume  and gas composition  as the smallest worthwhile change  on the y-axis. Correlations between data points on the Bland-Altman plots were used to determine whether there was any form of systematic bias across the range of fitness levels.Data was analysed using a specialised spreadsheet for cross-over trials . We anale change . Uncertae change  guidelin2max is substantially higher than what would be normally expected. However, participant’s blood pressure and BMI levels were normal. Most individual’s were involved in some form of recreational sport or activity and trained approximately 4.5 h·wk−1  compared to the Bruce protocol that produced an overall mean of 46.81 ± 10.7 ml·kg−1·min−1 , however, the 5-minute post-test blood lactate concentrations were similar (ALP = 11.2 ± 3.8 Bruce = 11.7 ± 3.5 mmol·l−1). RER was substantially higher in the Bruce compared to the ALP protocol  compared to the Bruce protocol. Typical (or standard) error of measurement for the V̇O2max tests was 5.7% .The ALP protocol produced a mean V̇O−1·min−1 . There wprotocol . Using t2max) was almost identical whether measured using the Bruce or ALP protocols (r = 0.93). Bland-Altman comparison on the V̇O2max data (−1·min−1).In most cases correlations in the physiological variables at maximal exercise between the two protocols were very large . Maximalmax data  indicate2max scores in individuals with different fitness levels (2max tended to be spread evenly among the two protocols.Type of protocol (Bruce or ALP) had little effect on relative V̇Os levels  and the 2max using progressive treadmill exercise tests to exhaustion. These two tests were highly correlated with V̇O2max being almost identical between the Bruce or ALP protocols and were unaffected by fitness levels of participants, suggesting the two tests are both valid protocols for measuring aerobic capacity.The major aim of this research was to compare two methods of estimating V̇O−1 and an incline of 10% gradient) and therefore requires a longer period of time to get participants to their tolerance limit.As suggested by Buchfuhrer and colleagues both treadmill protocols used in this research brought the participants to their tolerance limit within 10 ± 2 min (10:18 and 10:41 min:sec for the ALP and Bruce protocols respectively) , but the2max was similar between testing protocols the Bruce protocol produced substantially higher RER values compared to the ALP protocol. It is well known that uphill running requires higher energy expenditure than level running .Although the V̇O running . Running running . It is p running  and rece running . However2max test termination may be associated with perceived exertion (The substantially higher maximal heart rate obtained when participants completed the ALP compared to the Bruce protocol is an unexpected finding. We suspect the increase in running incline during the Bruce protocol caused increased feelings of fatigue and leg discomfort resulting in participants reaching exhaustion before maximal heart rate was achieved. There is evidence to suggest that V̇Oexertion , rather 2max values compared to the Bruce protocol, irrespective of age, sex or underlying fitness levels. In addition, the ALP protocol also takes a substantially shorter period of time to complete. We therefore suggest that the ALP protocol which is quicker, simpler and better at achieving maximal heart rate values is a useful alternative to the traditional Bruce protocol for testing maximal aerobic fitness.It seems that using the ALP protocol during an incremental treadmill run to exhaustion in a heterogeneous group of individuals is likely to produce similar V̇O


The TNF SuperFamily (TNFSF) of ligands include 19 molecules some of which play key roles in the immune system. All of these ligands are produced as trimeric Type II membrane molecules that can be released from the cell surface as single trimers. However, more than a decade of research has shown that the receptors for TNFSFs on responding cells require clustering in order to signal. Thus, single trimers are poor stimuli because they cannot produce the necessary clustering. Likewise, studies of agonist anti-TNFSF receptor antibodies  have shown that these antibodies must be mounted via their Fc portion to FcRs on an adjacent cell in order to cluster and signal these receptors. This means that agonistic antibodies to TNFSF receptors only function in microenvironments where there is a directly adjacent FcR-bearing cell. However, we have solved the receptor clustering problem by creating fusion proteins that contain many TNFSF trimers. As scaffolds for these molecules, we used surfactant protein D (SP-D) to make molecules with 4 trimeric arms (“UltraLigands™”) and ACRP30 (A Complement-Related Protein 30 kD or adiponectin) to make molecules with 2 trimeric arms (“MegaLigands™”). Numerous papers from independent labs have reported the ability of UltraCD40L™ and MegaCD40L™ to activate human and mouse cells. UltraOX40L™and MegaOX40L™ have also been studied and most recently Ultra4-1BBL™ has been produced. We are applying these unique materials in the following ways: (1) Using MegaCD40L™ or UltraCD40L™ plus IL-4, human B cells have been grown from small amounts of blood to serve as highly efficient APCs for expanding antigen-specific CD8+ T cells in vitro; (2) Mega4-1BBL™ or Ultra4-1BBL™ are being used to activate TCR-stimulated CD8+ cytotoxic T cells in vitro; and (3) MegaOX40L™ or UltraOX40L™ are being used to stimulate CD4+ T cells in vitro. These cell-free proteins can be used as practical reagents to simplify and improve the generation of immune cells for adoptive immunotherapy.