Using a time series of skin tissue samples as a biological archive, isotopic records from individual amino acids (AAs) can reveal the proximate factors driving a temporal decline we observed in bulk isotope values (a decline of ≥1 ‰) by decoupling changes in primary producer isotope values from those linked to the trophic position of this toothed whale. A continuous decline in baseline  δ15N and δ13C values was observed from 1993 to 2005 , while the trophic position of whales was variable over time and it did not exhibit directional trends. The baseline δ15N and δ13C shifts suggest rapid ongoing changes in the carbon and nitrogen biogeochemical cycling in the offshore CCS, potentially occurring at faster rates than long-term shifts observed elsewhere in the Pacific. While the mechanisms forcing these biogeochemical shifts remain to be determined, our data suggest possible links to natural climate variability, and also corresponding shifts in surface nutrient availability. Our study demonstrates that isotopic analysis of individual amino acids from a top marine mammal predator can be a powerful new approach to reconstructing temporal variation in both biochemical cycling and trophic structure.Climatic variation alters biochemical and ecological processes, but it is difficult both to quantify the magnitude of such changes, and to differentiate long-term shifts from inter-annual variability. Here, we simultaneously quantify decade-scale isotopic variability at the lowest and highest trophic positions in the offshore California Current System (CCS) by measuring δ Given the lack of detailed proxy records to trace simultaneously biochemical baselines and length of food webs, assessing the extent to which biogeochemical cycling and community structure in pelagic ecosystems have changed over the past century is difficult, as is attributing change to natural cycles versus anthropogenic disturbances.The California Current System (CCS) contains one of the five major coastal upwelling zones in the world’s oceans, and hosts a great diversity and abundance of marine life 2 or nitrate concentrations, as well as biological differences such as physiology and growth rate 13C and δ15N values, respectively) of primary producers, also known as “baseline isotope values”, vary in space and time as a function of these fundamental ecosystem properties 15N) in consumers 13C and δ15N values from a resident animal can potentially provide an integrated record of the biogeochemical characteristics of its habitat, as well as its trophic position 13C and δ 15N values ultimately recorded in consumer tissues, it is often difficult to disentangle the effects of changing trophic position from shifts in baseline values.The isotopic values of marine primary producers are sensitive to environmental variation, such as change in temperature, and CO13C or δ15N values have declined over the last century, but interpretations of these trends have varied widely  15N values are sometimes attributed to a drop in consumer trophic level  15N values in the offshore Central Pacific 15N values from three zooplakton species have exhibited no long-term trends, whereas data for a specialized zooplankton feeder decreased by approximately 3‰ 13C values over the 20th century are expected due to the combustion of fossil fuels , and have been observed in many records and ecosystems 13C declines has suggested that other factors, such as declining primary productivity, could also contribute in some regions 13C time series for organic or inorganic material.Studies in different ocean basins have shown that bulk tissue δ15N and δ13C values from primary producers are relatively well preserved within the group of ‘source-AAs’ for nitrogen Isotopic analysis of individual amino acids (AAs) can effectively separate trophic effects from shifts in baseline isotope values 13C and 1.2‰ for δ15N) is similar to the variation observed in other sperm whale populations that are considered to be resident   = 10.9‰ (0.9)) are relatively consistent with expected nitrate and particulate organic matter δ15N values from the oceanic northern CCS (∼6 to 10 ‰) 15N values from muscle of the jumbo squid  15N values are a proxy for primary producer values 15N-enrichment with trophic transfer 15N values versus baseline inorganic N sources. Lastly, because latitudinal trends in the δ15N values from predator source-AAs can indicate their geographic residency 15N values  strongly suggests that the individual sperm whales sampled here were not foraging in different localized regions, but rather foraged over a broad latitudinal range within the northern and central CCS. While the isotopic incorporation rate for extremely large animals like whales is not well known, the thick skin of sperm whales likely integrates information for at least three and possibly more than six months prior to sampling In the CCS off the US west coast, sperm whales are found in oceanic waters from California to Washington 13C and δ15N values in whale skin decreased from 1993 to 2005 by 1.1‰ and 1.7‰, respectively. These decreases were statistically significant at the alpha = 0.05 level. Inclusion of a single sample available from 1972 further suggests possible longer-term temporal declines for both δ13C and δ15N values by ≥4‰ and >3‰, that are also statistically significant  15N record from southern California (∼3‰ in 50 years) Bulk δnificant . Togetheurbation . In part13C values, source AA for δ15N values). Linear regression models for average δ13C and δ15N values from the most accurately measured essential- and source-AAs both exhibited strong negative temporal trends across all the data  could have also changed, since phytoplankton preferentially assimilate 14NO−3−3 concentrations are higher −3 utilization during seasonal upwelling might also be expected to depress the δ15N values of primary producers, propagating the 15N-depleted signal into food webs during their most productive periods. At present, there simply are not enough detailed data on nutrient concentrations and other oceanographic factors in the outer CCS to deduce a mechanism. However, the observed declining baseline values revealed by sperm whales do indicate a recently progressive shift in primary producer dynamics, likely associated with changes in SST, average state of surface nutrients and/or primary production.In particular, shifts in offshore and onshore oceanographic conditions appear to be decoupled. Coastal upwelling has recently increased, as expected for enhanced alongshore winds 15N and δ13C values in the outer CCS from 1992 to 2005, likely indicative of major recent shifts in biochemical cycling. At the same time, however, the overall similarity in whale trophic position signifies that the broad trophic structure is realtively unaffected. We note that in comparison with the recent deep sea coral data from the gyre offshore of this region 15N annual decrease of ∼0.015‰ over the last 150 years with a total drop of 2.3 ‰ in exported primary production δ15N values over that period. In contrast, our molecular-level proxies for δ15N values at the base of the food chain (the source AAs) indicate more rapid annual declines of 0.35 ‰ since the 1990’s. The independent molecular proxies for primary production δ13C values  indicate relatively similar declines.Although our time series data are limited for both elements, the compound-specific AA data identify a parallel decline in both baseline δ15N declines are observed in both data sets, different biogeochemical mechanisms may underlie the changes in these very different oceanographic regions. Climate variability likely affects the biochemistry of ecosystems differently depending on the oceanographic properties, microbial and phytoplankton communities, and species assemblages. In the eastern Pacific Ocean, the structure of the pycnocline varies strongly among the known biogeochemical provinces Together with the CCS observations discussed above, the contrast with the NPSG coral data , suggests that despite the fact that baseline δOur work highlights that detailed time-series of biochemical baseline and trophic structure records among different ecosystems will be crucial to identify rapid ecosystem shifts in response to climate change. In particular, in the face of uncertain coupling of natural and anthropogenic climate forcing, understanding the timing, extent and especially the mechanistic basis for baseline shifts now represents an urgent challenge. However, despite many efforts to unravel the linkage and feedback controls between the carbon and nitrogen cycles, and the effect of their variability on primary production and food-web dynamics, they are still not well understood. This study has demonstrated the great potential in coupling molecular isotopic tools with the unique bioarchive of sperm whales (or other top predators), as sentinels of offshore ecosystems. This may allow, for the first time, decoding of the factors that underlie temporal trends in bulk isotopic records, while simultaneosly monitoring changes at both the highest and lowest trophic levels. We suggest that integrating this approach with detailed oceanographic data will be a major new tool to identify the effects of natural climate variability versus anthropogenic global warming on ecosystem biochemistry and primary production. Elucidating such patterns from this and other ocean margin regions, in particular their relationships with oceanographic and climatic variations and shifts in primary production, will be an essential part of the critical task of predicting future trends in both ecosystem biochemistry and trophic dynamics.15N values, and 9 samples for δ13C values. The Southwest Fisheries Science Center/Pacific Islands Fisheries Science Center Institutional Animal Care and Use Committee (IACUC) approved the original animal work that produced the samples. Sex was determined genetically using qPCR sexing assay by the PRD-Genetic Lab at NOAA 2 for nitrogen. Results are expressed in part per thousand (‰) and standard notation: δHX = [(Rsample/Rstandard)−1]×1000, where H is the mass number of the heavy isotope, X is either C or N, and Rsample and Rstandard are the ratio of 13C/12C or 15N/14N in the sample and standard, respectively.A total of 18 skin samples  were ana15N values from source- and trophic-AAs and δ13C values from essential- and non-essential-AAs. All derivatives were injected with an AA control, N-leucine, to verify accuracy during each run, and analyzed via gas chromatography-IRMS to obtain δ15N and δ13C values from individual AAs. Each sample was run 3–4 times to maximize accuracy among chromatograms. The associated analytical error among replicates was <1.0 ‰. For all samples, δ15N values were obtained from a total of four source-AAs , and five trophic-AAs   proline) . For δ13tyrosine .15N and δ13C values was highly consistent with past work from other organisms and tissues 15N values, and essential- or non-essential-AAs for δ13C values to increase power in the analysis and evaluate temporal variation. We calculated average values for each AA group and they are reported in 15N and δ13C values , indicating that trophic-AA δ15N values could not be predicted by the variability in source-AAs, and vice versa. However, the correlation between average essential-AA and non-essential-AA δ13C values was moderate . Since the controls on isotopic patterns for non-essential-AA δ13C values are complex and dependent on diet quality and quantity, including de novo synthesis and routing of AAs from diet-to-tissue, this group was not considered in the linear regression analysis.There was a weak correlation between average source-AA and trophic-AA  in skin samples of sperm whales . (A) Four δ15N Source-AAs: phenylalanine (phe), glycine (gly), lysine (lys), tyrosine (tyr), and five Trophic-AAs: glutamic acid (glx), alanine , isoleucine (ile), leucine (leu), proline (Pro); and (B) Three δ13C essential-AAs: phe, leu, and valine .(TIF)Click here for additional data file.Table S1Average values and one standard deviations (SD) were calculated for Source-AAs , Trophic-AAs  and Essential-AAs .(DOCX)Click here for additional data file.


Objective: Hemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. A wide range of mutations, showing extensive molecular heterogeneity, have been described in hemophilia B patients. Our study was aimed at genetic analysis and prenatal diagnosis of hemophilia B in order to further elucidate the pathogenesis of the hemophilia B pedigree in China.Materials and Methods: Polymerase chain reaction amplification and direct sequencing of all the coding regions was conducted in hemophilia B patients and carriers. Prenatal diagnosis of the proband was conducted at 20 weeks.Results: We identified the novel point mutation 10.389 A>G, located upstream of the intron 3 acceptor site in hemophilia B patients. The fetus of the proband’s cousin was identified as a carrier.Conclusion: Our identification of a novel mutation in the F9 gene associated with hemophilia B provides novel insight into the pathogenesis of this genetically inherited disorder and also represents the basis of prenatal diagnosis.  Amaç: Hemofili B, X kromozomu üzerinde Xq27,1’e lokalize faktör IX genindeki koagülasyon defektleri nedeniyle oluşur. Hemofili B hastalarında yaygın moleküler heterojenite gösteren ve geniş bir dağılımı olan mutasyonlar tanımlanmıştır. Çalışmamız Çin’deki bir hemofili B ailesindeki patogenezi açığa kavuşturulmak için hemofili B’nin genetik analizi ve prenatal tanısını amaçlamıştır. Gereç ve Yöntemler: Hemofili B hastalarında ve taşıyıcılarda tüm kodlanan bölgelerin polimeraz zincir reaksiyonu ile amplifikasyonu ve direkt dizileme yapılmıştır. Probandın prenatal tanısı 20. haftada yapılmıştır. Bulgular: Hemofili B hastalarında yeni bir nokta mutasyonu olan 10,389 A>G’nin intron 3’ün alıcı bölgesinin yukarı akımında bulunduğunu tanımladık. Probanın kuzeninin cenininin de taşıyıcı olduğu bulundu. Sonuç: F9 geninde hemofili B ile ilişkili yeni bir mutasyonu tanımlamamız genetik olarak kalıtılan bu hastalığın patogenezine yeni bir açıklama getirmiştir ve prenatal tanının temelini temsil etmektedir.Hemophilia B (HB) is an X-linked inherited disorder resulting from deficiency in the blood coagulation factor IX (F9) caused by mutation of the factor IX gene . F9 is located at Xq27.1 and comprises 8 exons encoding 6 functional domains: prepropeptide, the Gla domain, epidermal growth factor (EGF)-1 and EGF-2 domains, the activation domain, and the catalytic domain [In 2011, we ascertained a HB pedigree from Liaoning Province, China. In this study, we conducted genetic analysis and prenatal diagnosis of HB in order to further elucidate the pathogenesis of the HB pedigree and help a pregnant woman deliver a healthy baby.ProbandAt 6 months of age, the proband  presented with pelioma, cyanotic bumps, subcutaneous bleeding, and knee swelling following crawling movement. The child was diagnosed as factor IX-deficient (test results: F9 coagulum activity 1%) by the Department of Hematology of Beijing Union Hospital in May 2011. Currently the child exhibits swelling of both knee joints and limited mobility, and he has been treated with monthly doses of factor IX.PedigreeThe identified HB pedigree comprises 4 patients ,2. I 8, DNA Isolation and DNA Analysis with PCRGenomic DNA samples of patients , 4 obligate carriers , and 100 healthy adults from our center were isolated from peripheral blood leukocytes using a DNA extraction kit . The F9 exons were amplified in a total of 7 reactions using the primers and polymerase chain reaction (PCR) conditions shown in Table 1. Sequencing was performed using the BigDye® Terminator v3.1 cycle sequencing kit  and analyzed with the ABI 3130 Genetic Analyzer. Exons 1 to 8 of the F9 gene were sequenced in all patients. The F9 sequence was used as a reference and mutations were numbered accordingly [Amniotic Fluid Culture Conditions and TreatmentA third-generation member of the pedigree (III 4), a 25-year-old female, was pregnant (20 weeks). She was identified as a carrier by sequencing analysis and the couple received genetic counseling at the Medical Center of the No. 202 Hospital of the People’s Liberation Army, China. The genetic development of HB and the probability of the presence of this gene in the fetus, as well as the consequences if the child was affected by the disorder or identified as a carrier, were explained in detail. Amniocentesis and amniotic fluid culture were then carried out at the parents’ request. DNA analysis was performed as described.FIX mRNA AnalysisThe F9 gene is expressed as a 2.8-kb mRNA. Elucidation of mutations in the F9 gene is important in understanding how such mutations affect the function of the F9 protein. To investigate this issue, RNA was extracted from the peripheral blood of patients and normal controls using the Blood RNA Kit (Tiangen) according to the manufacturer’s protocol. Using the Reverse Transcription Kit , cDNA was generated according to the manufacturer’s instructions. Intron 3 was amplified with 2 pairs of primers containing the 5’-end GT donor splice site and the 3’-end AG acceptor splice site, which were designed using Primer5 software. The products were 438 bp and 213 bp in length, respectively. Amplification of the β-actin gene (the β-actin is specific to the cDNA) was included as a reference (626 bp). Details of the primers and PCR conditions are shown in Table 2. The PCR products were ligated into the TA cloning vector for sequencing as described.Sequencing of Intron 3A pair of primers for amplification were designed using Primer5 software to amplify intron 3. The upstream primer (5’-TTGAAGAAGCACGAGAA-3’) is located in exon 2 while the downstream primer (5’-AACAACCCGAGTGAAGT-3’) is located in exon 6. The target fragment size in normal human cDNA is 473 bp. PCR amplification was performed using patient and control cDNA as templates and sequencing was conducted as described.Genetic AnalysisScreening for molecular differences in the F9 gene was conducted by sequencing of genomic DNA in 3 patients, 4 carriers, and 100 healthy adults. We identified a novel point mutation at position 10,389 (A>G), located upstream of the AG splice site between intron 3 and exon 4. All carriers were identified as heterozygotes and the mutation was not detected in the 100 healthy adults.Prenatal DiagnosisThe fetal DNA was screened for prenatal diagnosis at the request of the mother (carrier) using the same method. The results identified the fetus (IV 2) as a heterozygote for this mutation, thus confirming the child to be a carrier for HB. The prenatal protocol included 2 counseling sessions, psychosocial evaluation of the couple, and an obstetric assessment.Study of HB PathogenesisThe target fragment (213 bp) containing the 3’-end AG acceptor splice site at the intersection of intron 3 and exon 4 was successfully amplified from normal human DNA  and patient cDNA templates . However, the target fragment was not amplified from the control cDNA template , suggesting that intron 3 and exon 4 were correctly spliced and there was no residue of intron 3 in the cDNA of the control cDNA. The correct sequences of PCR products confirmed the presence of a residual part of intron 3 in the cDNA of the HB patient.The target fragment (438 bp) containing the 5’-end GT donor splice site of intron 3 at the intron 3/exon 3 intersection of the F9 gene was not amplified from the patient  or control  cDNA templates. However, the target fragment was successfully amplified from normal human DNA  and the correct sequences were confirmed. This demonstrated the functional splicing activity of the 5’-end GT donor splice site of intron 3.Sequencing of Intron 3The target fragment was successfully amplified from normal human cDNA templates and the correct PCR products were confirmed by sequencing. The target fragment was not amplified from HB patient cDNA templates.HB occurs in approximately 1 in 30,000 male live births  and thisThe third base of the upstream 3’ acceptor splice site of the F9 gene intron 3 (-3 A>G) was identified as a heterozygous mutation locus in the proband’s mother (III 1) as well as the maternal grandmother (II 1), great-grandmother (I 2), and maternal first cousin (III 4), all of whom were carriers. The fetus (IV 2) of III 4 was identified as a carrier by prenatal diagnosis. The baby girl was delivered normally and normal F9 activity was confirmed.Intronic splice sites are critical for maturation of pre-mRNA. Therefore, we investigated the presence of splice site mutations in the F9 gene at the mRNA level by RT-PCR analysis. It can be speculated that the (-3 A>G) mutation may lead to failure of normal intron 3 splicing. We propose that the mutated splicing site leads to abnormal transcription of DNA to mRNA. Consequently, the abnormal mRNA results in the synthesis of abnormal F9 protein, thus causing patient morbidity. It is also possible that abnormally spliced mRNA cannot enter the cytoplasm, resulting in the absence of the translated protein. Thus, we hypothesize that the (-3 A>G) mutation at the base upstream of the intron 3/exon 4 AG splice site in the patient in the pedigree results in aberrant splicing of the F9 mRNA. This accounts for the presence of partial residual sequences of intron 3 in patient cDNA, although it is unclear how long these sequences are maintained in patient cDNA. Amplification of this region was not achieved from patient cDNA even with the use of multiple primer pairs. Several factors may account for this: 1) no target fragment was amplified by PCR in the presence of excessive residual intron 3; 2) amplification is hindered owing to a high GC content and repetitive sequences in the introns; 3) the F9 gene in the patient cDNA is cleaved into 2 fragments such that exon 3 is separated from the residual portion of intron 3.Point mutations (single-nucleotide substitutions) were the most common gene defects. Few mutations occur in the introns of the F9 gene, while most insertions and deletions in the exons cause frame shifts, leading to severe HB in almost all patients . The finAcknowledgmentsWe thank all the patients and their families who participated in this study. This study was supported by the National Foundation of China (No. 30973140), the Doctor Starting Funding of Liaoning (No. 20111123), and the Natural Science Foundation of Liaoning (No. 201202230).Conflict of Interest StatementThe authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.


Weed control is generally considered to be essential for crop production and herbicides have become the main method used for weed control in developed countries. However, concerns about harmful environmental consequences have led to strong pressure on farmers to reduce the use of herbicides. As food demand is forecast to increase by 50% over the next century, an in-depth quantitative analysis of crop yields, weeds and herbicides is required to balance economic and environmental issues. This study analysed the relationship between weeds, herbicides and winter wheat yields using data from 150 winter wheat fields in western France. A Bayesian hierarchical model was built to take account of farmers’ behaviour, including implicitly their perception of weeds and weed control practices, on the effectiveness of treatment. No relationship was detected between crop yields and herbicide use. Herbicides were found to be more effective at controlling rare plant species than abundant weed species. These results suggest that reducing the use of herbicides by up to 50% could maintain crop production, a result confirmed by previous studies, while encouraging weed biodiversity. Food security and biodiversity conservation may, therefore, be achieved simultaneously in intensive agriculture simply by reducing the use of herbicides. Human food sources depend, directly or indirectly, on four main annual crops: wheat, barley, corn and rice12There has recently been a general call to limit the use of herbicides at European and national levels19242728Experimental and modelling studies usually ignore one further aspect: the farmers’ decisions and practices302330sensu lato) as a parameter influencing the latent variable, λ, of the expected number of weeds per unit area. This parameter quantifies the farmers’ impact on the pairwise ‘crop yield-herbicides-weeds’ relationships. This method differed from the conventional statistical approach by assuming that a farmer’s behaviour  affect the crop yield-herbicide relationship through his own perception of weeds and weed control management strategies (e.g. timing of treatment). To include more realistic conditions in the model, a framework was developed to take account of the adaptive management by a given farmer to deal with the specific conditions encountered in his fields, by allowing a nested effect of field within farmer  and also taking account of the differential effectiveness of herbicide treatments depending on the weed species, ηRFfs (ηAFfs). We then analysed the interactive effects of farmers’s behaviour, for both ηF (ηAF) and ηFf (ηAFf), and the herbicide application rate on weeds, testing the hypothesis that herbicide treatment affected the abundance of weeds rather than species richness and targeted species (those thought to reduce the yields) rather than non-targeted species, using estimated values of ηRFfs (ηAFfs).This study used empirical data on weeds, herbicide practices and winter wheat yields from 150 fields belonging to 30 farmers, to determine whether the use of herbicides improved yields and/or decreased weed abundance. As no clear relationships between herbicide use and weeds nor between yields and weeds were detected using standard statistical models, we modelled these relationships taking into account implicitly the effects of farmers’ behaviour and of environmental conditions on the effectiveness of weed management. Although farmers’ behaviour is usually taken into account in decision support systems313032Polygonum aviculare L., Veronica persica Poir., Mercurialis annua L., Fallopia convolvulus L. and Galium aparine L., with the exception of Poa sp. . We first attempted to determine a positive relationship between the crop yield and the herbicide application rates, expressed as the total application rate over the cultivation period, using linear mixed models (with field nested within farmer as a random effect). The relationship between the crop yield and the herbicide application rate was actually negative (LMM: estimate = −0.0034 (SE = 0.0016), F1,103.86 = 4.933, P = 0.028; F1,116.66 = 0.889, P = 0.347; 1,131.62 = 0.0006, P = 0.939; F1,132.31 = 0.112, P = 0.738). There was a slight negative relationship between crop yield and weed frequency for the highest weed frequency, but the relationship was far from significant . Similar results (108 species were found over the 150 fields with an average of 9.46 species per field (range 0–25). All but one species of the six most commonly found were annual dicotyledons, i.e.  results  were fou results .No evidence was found for any relationship between weeds, herbicide application rates and crop yield. One reason could be that farmers adapt their treatment strategy in order to keep the weed risk below a given threshold and guarantee a minimum yieldRF and ηAF) assuming a similar effect across the five fields farmed by the farmer, or with variability between fields for a given farmer, which was modelled as a nested effect at field scale within a farm (ηRFf and ηAFf) (Ff) explained the variability in weed species richness much better . This model also explained the weed species richness much better than the model without any farmer effect (Rich_base: DIC = 34200). Similar results were found for weed estimated abundance  and ηRFf (ηAFf) are surrogates for the effectiveness of treatment and vary between 0 and 1, a value of 1 being the effectiveness expected if weed control were complete. There was a strong farmer identity effect on the effectiveness of the weed control treatment (AFf over the five fields farmed by each farmer in 32RFf (ηAFf) and the farmers’ weeding strategies, described by the diversity and date of introduction of products applied as well as the number of tank-mixed commercial herbicides  were generally lower than ηAFf estimates for the abundance model  and wheat yield . Finally, although in previous studies of weeds, herbicides and yields the sample size was often limited . Despite these clear advantages, farmers are reluctant to reduce herbicide use: for instance, integrated pest management (IPM) has long been promoted by experts2258To ensure food security while conserving biodiversity in intensive agriculture, government policies have often targeted a combination of changes in herbicide use with increased diversification in crop rotations, as well as the use of IPM or organic farming13a priori excluded  and general information about the farm  was collected by means of a questionnaire sent out to all participating farmers. The response was 98% representing 30 farms. Herbicide use was described by the name and the concentration of each of the active ingredient and the day or week of application. Herbicides were further classified as monocotyledon specific, dicotyledon specific or broad spectrum. Crop yields were not available for 3 of the 30 farms.2) at 10 m intervals in line from the border of the field toward the centre, perpendicular to the tracks made by farm machinery within the field. The first quadrat was 20 meters from the edge of the field. For each quadrat, weed species were recorded as either present or absent, irrespective of the number of individual plants, giving a list of species present in each quadrat.Botanical surveys were carried out once during the flowering to milk-ripening stage of winter wheat, in spring/summer 2007SM Materials and Methods). LMM were analysed with a type III analysis of variance with Satterthwaite approximation for degrees of freedom. A model selection procedure based on Akaïke Criterion  with the farmer as random effect and with and without nitrogen input as a co-variable. Two indicators were used for the amount of herbicide applied: the total application rate and the treatment frequency index b where D is the amount of herbicide applied, a is a scale factor and b is a shape factor describing the concavity of the reduction after the application of the herbicide. The non-linear function of D allows the species richness to tend to zero as D becomes large, and to equal μ when no herbicide is applied (D = 0). A second model took account of farmers’ behaviour on the effectiveness of chemical weed control. A parameter ηRF was used to describe the effectiveness of the treatment as a function of the farmer, λ being modelled as a Poisson distribution with mean μ/(1 + aηRFD)b. All fields of a given farm, F, shared a common farmer effect, ηRF. A third model included the farmer effect at field scale with a factor ηRFf for field f belonging to a farm F as ηRFf = ηRF ηRf. The best of the three models  was selected based on the deviance information criterion (DIC) which is a hierarchical modelling generalization of the AICIn order to account for the high variability in the herbicide-weed richness relationship (LMM described above)Estimating weed abundance). The initial model assumed that herbicides had a similar effect on all weed species in the field and two models were built: one considered the same effect of the farmer’s decision in all his fields λs = μs/(1 + aηAFD)b and the other modelled the effect of the farmer’s behaviour at field scale λs = μs/(1 + aηAFfD)b. In both models, λs was the average number of plants of a given weed species s in a given area. The second step was to build a more realistic model, λs = μs/(1 + asηAFfsD)bs, which considered that the effect of the herbicide depended on the weed species s.No relationship was observed between herbicide use and abundance using LMM. Consequently, hierarchical models similar to those for the herbicide-weed species richness were built but, at the last step the species abundance was estimated using the presence-absence data (see details in 2) was: 1 − exp(μsW/(1 + aηAFD)b) . We measured the survival of a species as the probability to observed one individual in W. For a species s with abundance intensity λs, its survival rate was therefore e−Wλs under the assumption of Poisson distribution of the individuals of this species.The weed abundance at field scale was estimated by assuming that weed abundance follows a Poisson distribution and that the probability of finding at least one plant in an area W(4000 mRF/ηRFf/ηRFfs (ηAF/ηAFf/ηAFfs)). The following priors were used: a Gaussian distribution N for log(μ), log(a) and log(b), so that a, b and μ follow log-Gaussian distributions, ensuring that μ, a and b were strictly positive and a non-informative uniform distribution U for ηRF (ηAF), ηRFf(ηAFf) and ηRFfs(ηAFfs). 20,000 iterations were run with three independent chains in the MCMC procedure. For each chain, the first 10,000 iterations were discarded. After this “burn-in” period, inferences were derived from a sample of 20,000 iterations. Modelling was performed using WinbugsThe Bayesian posterior distributions for each of the model parameters, including uncertainty due to variability in the data and the uncertainty of prior information, were approximated using Monte Carlo–Markov chain (MCMC) methods with prior information for the parameters .


Artemether dose adjustments were then simulated in order to predict optimal dosing in co-infected patients and inform future interaction study design.To treat malaria, HIV-infected patients normally receive artemether (80 mg twice daily) concurrently with antiretroviral therapy and drug-drug interactions can potentially occur. Artemether is a substrate of CYP3A4 and CYP2B6, antiretrovirals such as efavirenz induce these enzymes and have the potential to reduce artemether pharmacokinetic exposure. The aim of this study was to develop an In vitro data describing the chemical properties, absorption, distribution, metabolism and elimination of efavirenz and artemether were obtained from published literature and included in a physiologically based pharmacokinetic model (PBPK) to predict drug disposition simulating virtual clinical trials. Administration of efavirenz and artemether, alone or in combination, were simulated to mirror previous clinical studies and facilitate validation of the model and realistic interpretation of the simulation. Efavirenz (600 mg once daily) was administered to 50 virtual subjects for 14 days. This was followed by concomitant administration of artemether (80 mg eight hourly) for the first two doses and 80 mg (twice daily) for another two days.max by 60% and AUC by 80%. Dose increases of artemether, to correct for the interaction, were simulated and a dose of 240 mg was predicted to be sufficient to overcome the interaction and allow therapeutic plasma concentrations of artemether.Simulated pharmacokinetics and the drug-drug interaction were in concordance with available clinical data. Efavirenz induced first pass metabolism and hepatic clearance, reducing artemether CThe model presented here provides a rational platform to inform the design for a clinical drug interaction study that may save time and resource while the optimal dose is determined empirically. Wider application of IVIVE could help researchers gain a better understanding of the molecular mechanisms underpinning variability in drug disposition. The geographical overlap in the prevalence, morbidity and mortality of malaria and HIV constitutes a major public health burden in low- and middle-income countries. In 2010 alone, more than 34 million people were living with HIV worldwide (68% in Sub-Saharan Africa) and approximately 1.8 million died of AIDS-related illnesses UNAIDS . ApproxiLimited access to highly active antiretroviral therapy (HAART) in low and middle income countries constitutes a major barrier that substantially diminishes the number of therapeutic options available. Therefore, protection of currently available first-line antiretroviral and antimalarial drugs against resistance is of paramount importance  for treatment of malaria, is currently administered concomitantly with HAART in co-infected patients in several African countries. A six-dose regimen administered over three days has excellent efficacy against ions WHO . Gonzaleet al. reported 59% and 79% reductions in artemether Cmax and plasma AUC, respectively, when co-administered with efavirenz in HIV-infected adults  and CYP2B6, and is quickly absorbed in the first two to three hours after oral administration. Bioavailability is low due to intestinal and first-pass hepatic metabolism  is a bottom up technique which aims to simulate pharmacokinetics using in vitro data, such as the physicochemical characteristics and intrinsic clearance (CLint) through physiologically-based pharmacokinetic (PBPK) models, which mathematically describe absorption, distribution, metabolism and elimination (ADME). Therapeutic agents can be administered via different routes and absorption can be simulated taking into account the dynamic interplay between dissolution, passive permeability and affinity/activity of metabolic enzymes and transporters. For instance, oral bioavailability  can be influenced by tablet dissolution and solubility, impacting the fraction of dose available for absorption (Fa), whereas intestinal  and first-pass hepatic metabolism (Fh = fraction of dose available following hepatic metabolism) can reduce the amount of drug reaching the systemic circulation  models estimating the fraction of dose absorbed and the rate of drug absorption based on transit models. Volume of distribution is simulated by evaluating tissue volumes and the diffusion of drugs into tissues, which is influenced by physicochemical properties defining the plasma to tissue ratio (Pt:p) as described by Poulin and Theil , which provides a statistical description of the physiological and anatomical characteristics in the human population, focusing on parameters that are essential in the PBPK approach  and subsequently the constant of absorption (ka) , and intestinal (Fh) and first-pass hepatic metabolism (Fg) can reduce the amount of drug reaching the systemic circulation. Tablet dissolution for artemether was taken into account using a first-order dissolution constant as previously described  and induction of enzyme expression in liver (E) were determined using the following equations . in vitro intrinsic clearance (CLint) for the two compounds were included in the model: efavirenz is hydroxylated to 7-hydroxy efavirenz by CYP2A6, to 8-hydroxy efavirenz by CYP2B6, CYP2A6, CYP1A2, CYP3A4 and CYP3A5, and glucuronidated by UGT2B7 while artemether is metabolised by CYP2B6 and CYP3A4. After correction with fraction unbound in microsomes  vs 29 (10–247) ng/ml. Simulation of artemether drug disposition was characterised by low bioavalability , with around 14% of the dose not absorbed (Fa = 0.86), high intestinal metabolism (Fg = 0.21) and high first pass metabolism (Fh = 0.60). High apparent volume of distribution (V/F = 1640 L) was predicted, in accordance to clinical studies. Systemic metabolism mediated by CYP2B6 and CYP3A4 was characterised by a high CL/F of 268 L/h.Simulated pharmacokinetics of artemether and efavirenz were in good accordance with previously described clinical data, as represented in Table trough was equal to 2341 ± 2196 (mean ± SD) vs 1752 ± 1001 ng/ml , AUC 76282 ± 57204 vs 57592 ± 22849 ng/ml•h, Cmax 3520 ± 2567 vs 4037 ± 1158 ng/ml , low intestinal metabolism (Fg = 0.95) and low first-pass metabolism (Fh = 0.93). Apparent volume of ditribution was 150 L and systemic metabolism, mainly mediated by CYP2B6, CYP1A2, CYP2A6 and CYP3A4, gave a CL/F of 9.7 L/h.For efavirenz (600 mg once daily), the simulated Cmax of 6.5 and EC50 of 3.9 μM for CYP3A4 and a Indmax of 5.7 and IndC50 of 0.8 μM for CYP2B6. In the IVIVE model, efavirenz caused an increase in CYP3A4 and CYP2B6 expression, inducing artemether rate of metabolism with a substantial effect on first pass-metabolism and systemic clearence. Artemether Fh was therefore reduced by 60% (from 0.6 to 0.23) and CL/F was increseasd by 6-fold. This had a major effect on artemether pharmacokinetics reducing Cmax by 60% and AUC by 80%.Efavirenz is an inducer of CYP3A4 and CYP2B6 expression with an EA dose increase of artemether was simulated and a dose of 240 mg artemether was predicted to be sufficient to overcome the effect of efavirenz on artemether drug disposition and consequently achieve therapeutic artemether plasma concentrations. At the standard regimen (80 mg twice daily) the simulated AUC of artemether was equal to 166 (55–678) ng.h/mL and the interaction with efavirenz caused a reduction to 41 (13–228) ng.h/mL. A simulated dose increase to 160 mg twice daily gave a median (range) artemether AUC of 77 (25–620) ng.h/ml and a dose increase to 240 mg twice daily resulted in a median (range) artemether AUC of 115 (53–650) ng.h/ml. Artemether is currently coadministered with lumefantrine and EFV has been shown to decrease lumefantrine exposure inducing its metabolism  in our simulations. The induction by efavirenz was predicted to have a major impact on the systemic clearance of artemether, increasing plasma CL/F by 6 fold as shown in Table Plasmodium falciparum with comparable EC50 in vitro, then it may be necessary to consider plasma concentrations of DHA to get a complete picture of antimalarial activity (Alin et al. in vitro study aimed to characterise DHA metabolism and distribution may be necessary to develop IVIVE models for the prediction of its pharmacokinetics. Moreover the effect of efavirenz on expression UGTs in the kidney should be investigated to further clarify the effect of EFV on DHA elimination. Efflux and influx transporters can have a big impact upon drug absorption and diffusion into tissues, and might help to explain part of the variability observed in efavirenz and artemether pharmacokinetics. Efavirenz is a strong inducer of ABCB1 and other transporters. There are currently too many gaps in knowledge of these processes to have incorporated them into the current model. However, in vitro investigation of artemether could clarify the role of transporters in this or similar drug-drug interactions (Weiss et al. Numerous clinical studies have described poor artemether bioavailability following oral administration but to date a comprehensive measure of its oral bioavailability has not been completed. In our simulation we estimated a FThe developed IVIVE model accurately predicted the pharmacokinetics of efavirenz and artemether and their interaction. The main pharmacokinetic variables for different dosing strategies were simulated and the effect of efavirenz on artemether clearance and bioavailability has been quantified. The clarification of the magnitude of drug-drug interaction between efavirenz and artemether is clinically relevant since sub-therapeutic artemether concentrations can lead to therapeutic failure. The IVIVE approach can be applied to not only to predict the utility of potential dose adjustments but also for the management of drug-drug interactions in special populations such as paediatric, elderly and patients affected by multiple morbidities.This simulation approach can be viewed as a paradigmatic example demonstrating that IVIVE can be used to investigate clinically relevant ‘what-if’ questions and to inform the design of prospective clinical trials. As previously demonstrated in numerous studies, IVIVE can be used to predict pharmacokinetics in numerous diseases, investigating molecular mechanisms which can impact drug disposition and to inform future clinical studies. Wider application of IVIVE could help researchers gain a better understanding of the molecular mechanisms underpinning variability in drug disposition, an essential condition to further improve the quality of future research projects. Future application of this approach may include other drug-drug interaction simulations, dose optimisation in special populations, prediction of pharmacogenetic effects and optimisation of treatment strategies.


The efficient coupling of incoherent, spectrally broad sunlight in small gain volumes should allow the generation of coherent laser light from intensities of less than 100 suns.The optical conversion of incoherent solar radiation into a bright, coherent laser beam enables the application of nonlinear optics to solar energy conversion and storage. Here, we present an architecture for solar pumped lasers that uses a luminescent solar concentrator to decouple the conventional trade-off between solar absorption efficiency and the mode volume of the optical gain material. We report a 750-μm-thick Nd Despite these favorable optical gain properties, neodymium is not an ideal solar absorber due to poor spectral overlap with sunlight and low peak absorption coefficients, of <10 cm−1 at λ ~ 800 nm that necessitate large optical gain media volumes. While larger gain volumes result in increased solar pump absorption, the equivalent increase in mode volume cancels out the increase in excitation density of the mode that is essential for reaching population inversion. Consequently, state-of-the-art solar pumped lasers operate above 1000 suns. Nd3+:YAG laser rods, for example, operate at power thresholds of 275 W-cm−2 utilizing large Fresnel lenses to concentrate sunlight3+:YAG ceramics sensitized by Cr3+ ions have broad absorption bands in the visible spectrum, and solar lasing thresholds of 160 W-cm−2 et al. demonstrated passive cooling of a solar pumped laser through the use of a small volume Nd3+-doped glass fiber operating at a solar lasing threshold of 370 W-cm−2 5 cm−1 above an exciton absorption line that is tunable through size and material composition13The conversion of broadband solar radiation into coherent and narrow-band laser radiation has attracted attention in recent years due to its potential applicability in solar energy conversion. Solar pumped lasers (SPL) can be used to drive reactions that store solar energy in chemical formThe semiconductor nanocrystals are chosen such that their photoluminescence wavelength matches an absorption peak of the optical gain medium . As the 3+ doped Yittrium Aluminum Garnet (YAG) gain medium that we ground and polished to form a planar waveguide with length, width and thickness of 43 mm × 2 mm × 750 μm, respectively. The Nd3+:YAG waveguide was coated with a 15-μm-thick poly (PVB-CVA-CVAc) thin film containing 10% wt. colloidal CdSe/CdZnS nanocrystals, provided by QD Vision, Inc. The colloidal nanocrystal quantum efficiency (QE), defined as the ratio between the number of emitted and absorbed photons, is 75% when doped into PVB-CVA-CVAc, and the film is photostable in air. The overlap between the solar spectrum and the absorption of the CdSe/CdZnS:PVB-CVA-CVAc thin film can be seen in 3+:YAG. Indeed, as shown in 1018As a proof of principle, we constructed a solar-pumped laser based on a conventional 1% atomic Nd3+ ions, the Nd3+:YAG waveguide is illuminated by monochromatic light at various wavelengths (see METHODS) with and without a 15-μm-thick 10% wt. CdSe/CdZnS:PVB-CVA-CVAc thin film. To increase the absorption of the incident light within the sample, a simple backing silver mirror is employed. Luminescence from the sample is collected with an integrating sphere and 850 nm long pass wavelength filter is used to discriminate between CdSe/CdZnS and Nd3+:YAG emission. The key parameter is the efficiency of cascade energy transfer, which we define as the fraction of pump photon rate ultimately transferred to Nd3+. Cascade energy transfer depends on the nanocrystal absorption, photoluminescent quantum yield, the fraction of nanocrystal photoluminescence trapped in the waveguide, and the competition between photoluminescence reabsorption losses in the nanocrystal film and waveguided pumping of the optical gain media. As can be seen by the blue line in 3+:YAG sample in the visible spectrum leads to very little energy transfer from the pump to the Nd3+. With the addition of the thin film of CdSe/CdZnS:PVB-CVA-CVAc on the Nd3+:YAG waveguide, cascade energy transfer generates a broad visible excitation spectrum with peak value of 14% that follows the absorption profile of the nanocrystal (gray dotted line). Averaged over the solar spectrum out to the nanocrystal absorption cutoff at λ = 600 nm, the waveguide pump efficiency due to the nanocrystal is 8.3%, resulting in total pump efficiency of 9.7%; see grey dashed line and red line in 3+ within the waveguide. The inputs to the simulation are the sample geometry, the material’s spectral absorption coefficients and refractive indices, photoluminescence spectrum, and photoluminescence quantum yield. An input elliptical Gaussian beamshape is used to simulate concentrated input light, with full width half max of FWHMW = 1 mm and FWHML = 26 mm along the width and length of the sample respectively. In 3+:YAG waveguide, and determine the density of excited Nd3+ ions per 100 μm width across the sample. Normalization of this parameter with the pump photon flux results in the relative distribution of Nd3+ excited states. This pump distribution analysis is done at three different pump wavelengths. Incident light at a wavelength of λ = 735 nm is beyond the absorption edge of the nanocrystals and only excites neodymium ions. The resultant profile of Nd3+ excited states mirrors the non-uniformity of the pump intensity distribution. Absorption of incident light at λ = 405 nm, however, is dominated by the nanocrystals. Subsequent cascade energy transfer produces a near uniform Nd3+ population despite the non-uniformity in incident intensity. The improved uniformity is a result of the relatively long absorption path length of waveguided photoluminescence compared to the sample thickness. Modelling predicts that 15.3% of incident photons at λ = 405 nm are transferred to Nd3+ as compared to the measured 14%. The slight discrepancy between simulation and experiment may be due to scattering at surfaces which is not included in the model. Finally, for pump wavelengths such as λ = 531 nm, that excite neodymium and the nanocrystals, the resultant excited state profile is a weighted average of direct absorption and cascade energy transfer. The modelled total integrated Nd3+ excited states for pump wavelength of λ = 531 nm is 7.4% compared to the measured 6.3%. These simulations indicate that cascade energy transfer results in improved uniformity of the excited state population and thermal dissipation within the optical gain media.To measure the efficiency of cascade energy transfer from input light to excited Nd−2 without active cooling, as seen in the inset of −2 is due to the low glass transition temperature Tg ~ 75 °C 3+ transition at λ = 1064 nm, as depicted in −2. t = 8.45 × 103+-m−3. Because the excitation distribution occupies only part of the sample, a conservative estimation of the 531 nm pump that is required for lasing at the entire sample would be equal to maximal photon flux of 14.54 W-cm−2. Calculating the solar concentration required to induce the same maximal flux must take into account the difference between the pumping efficiency at 531 nm (6.3%) and the average pumping efficiency over the solar spectrum between 350 nm and 825 nm  enhanced solar absorption with near-infrared (NIR) rather than visible spectrum colloidal nanocrystals183+ and Tm3+ allows the decoupling of solar absorption from mode volume and enables solar laser operation from the visible to the near infrared. Solar powered Tm3+ lasers are especially notable because they potentially enable the efficient up-conversion of sub-bandgap infrared sunlight for silicon solar cells.In conclusion, cascade energy transfer presents a path to efficient solar powered lasers operating under passive cooling, and potentially in the non-tracking regime3+:YAG slab waveguide was formed through conventional polishing techniques from commercial laser rods of diameter 3 mm and length of 43 mm with λ = 1064 nm antireflection coatings on both faces. Organic soluble CdSe/CdZnS colloidal semiconductor nanocrystals that emit at 585 nm were provided by QD Vision, Inc. The PVB-CVA-CVAc host was purchased from Sigma Aldrich. A solution of 100 mg/mL 10% wt. CdSe/CdZnS:PVB-CVA-CVAc in chloroform was drop cast onto the Nd3+:YAG waveguide producing a film of 15 μm thickness. The nanocrystal doped polymer film on glass was excited at λ = 405 nm and the photoluminescence spectrum was recorded using a calibrated Ocean Optics spectrometer positioned perpendicular to the substrate. The Nd3+:YAG and Nd3+:YAG with CdSe/CdZnS: PVB-CVA-CVAc film was excited with monochromatic light. An evaporated silver mirror was used behind the sample to double the optical path length of incident light. The monochromatic light was generated using a tungsten bulb, monochromator with a spectral resolution of 3.5 nm full width at half maximum, and a mechanical chopper. The sample photoluminescence was measured with a lock-in amplifier, spectrally calibrated photodetector, and long wavelength filters with a cutoff at λ = 850 nm were used to discriminate between Nd3+:YAG and CdSe/CdZnS emission in the integrating sphere. Although the fine structure of the Nd3+ absorption is partly obscured by the resolution of the monochromator, its spectral resolution does not affect calculations of the equivalent solar threshold obtained by integrating the product of solar and device excitation spectra. The laser resonator is a hemispherical cavity with a planar mirror of reflectivity 99.8% and an output mirror with a radius of curvature of 15 cm and reflectivity of 98%. A 18 W λ = 531 nm CW laser was used to optically excite the NC film coated Nd3+:YAG slab in air. An Acton spectrometer with a grating of 1000 grooves per mm and a calibrated Newport photodetector and neutral density filter were used to measure the lasing spectrum and power threshold of the laser respectively.The NdHow to cite this article: Reusswig, P. D. et al. A path to practical Solar Pumped Lasers via Radiative Energy Transfer. Sci. Rep.5, 14758; doi: 10.1038/srep14758 (2015).


Shewanella oneidensis at ambient (0.1 MPa) and high (200 MPa) pressure using H/D isotopic contrast experiments for normal and perdeuterated bacteria and buffer solutions to distinguish intracellular and transmembrane processes. The results indicate that intracellular water dynamics are comparable with bulk diffusion rates in aqueous fluids at ambient conditions but a significant reduction occurs in high pressure mobility. We interpret this as due to enhanced interactions with macromolecules in the nanoconfined environment. Overall diffusion rates across the cell envelope also occur at similar rates but unexpected narrowing of the QENS signal appears between momentum transfer values Q = 0.7–1.1 Å−1 corresponding to real space dimensions of 6–9 Å. The relaxation time increase can be explained by correlated dynamics of molecules passing through Aquaporin water transport complexes located within the inner or outer membrane structures.Quasielastic neutron scattering (QENS) is an ideal technique for studying water transport and relaxation dynamics at pico- to nanosecond timescales and at length scales relevant to cellular dimensions. Studies of high pressure dynamic effects in live organisms are needed to understand Earth’s deep biosphere and biotechnology applications. Here we applied QENS to study water transport in  Q) and energy (ħω) transfers at nano- to picosecond timescales relevant to cellular biology7810Shewanella oneidensis bacteria at ambient (0.1 MPa) and high (200 MPa) pressures relevant to subsurface biological processes.Water dynamics inside cells and across the cell envelope are key to biological processes1235S. oneidensis as a model for our study. This prokaryotic organism exists under both aerobic and anaerobic conditions and its genus contains several piezophilic (“pressure-loving”) species151618in situ spectroscopy under HP conditions2021S. oneidensis has also been investigated to study its pressure adaptation and survival under conditions extending into the GigaPascal (P > 1 GPa) range20Observations of live bacteria at >10 km below the ocean surface as well as samples recovered from continental and suboceanic drilling projects have revealed microbial lifeforms existing at up to at least P = 110 MPa12Q range (0.2–1.8 Å−1) corresponding to real space correlation lengths ~3–30 Å  ~3–30 Å . A detai+ and Cl− with structure-breaking to borderline properties along with structure-making phosphate anionsT(Q2) relations to a Singwi-Sjölander (SS) jump modelT = 2.70±0.20 × 10−5 and 2.00±0.11 × 10−5 cm2s−1 for Hb and Db, respectively, at ambient conditions  buffer Db) that served as the bacterial growth and suspension media for our experiments. The main electrolyte species present are Nanditions , Table 1T(Q2) properties of each dataset, we also carried out a complementary examination of the susceptibility function χ′ normalized to the frequency (energy transfer) scale28Q, ω) is asymmetric with respect to the energy loss (ω) axis due to the Bose-Einstein thermal population factor. Studying the form of χ′ reveals the proper distribution of spectral weight among the different dynamic processes, along with additional useful information concerning the static structure factor S(Q). Furthermore, using a stretched energy scale with respect to the corresponding S spectrum allows a clear separation between and determination of the number of relaxation mechanisms contained within the density of states responsible for the diffusive dynamics. Similar interpretations can also be achieved by developing a sophisticated analysis of relationships between fitting parameters used to reproduce the QENS linewidth result; however, we preferred to crosscheck the fit analyses by using the χ′ results as an independent method. For simplicity we show the details only for the case of the bulk aqueous solutions, even though its analysis has supported the interpretation of all the reported results and also provided an in situ marker of the hydrostatic conditions achieved during the experiments. In Q values representative of long (Q = 0.6 Å−1) and short (Q = 1.8 Å−1) range dynamics. In the case of the Hb data (Q).As we investigated the Γ Hb data , corresp Hb data , where tt(Q2) data by the SS jump model led to an intracellular diffusion coefficient DT = 2.43±0.14 × 10−5 cm2s−1, intermediate between the values for Hb and Db buffer solutions. This result indicates that water diffusion in the intracellular medium is indistinguishable from the bulk over the examined Q-range corresponding to correlated motions extending between approximately 3–30 Å in real space. Jasnin et al. likewise observed no differences in E. coli cytoplasm dynamics from diffusion in bulk aqueous media examined by QENS at ambient pressure678S. oneidensis became reduced by 18%, nearly twice that observed for the Hb or Db aqueous buffer solutions  (T(Q2) is already obvious from initial inspection of the QENS data sets that clearly show an enhanced signal at large Q values for the Im compared with bulk Hb, Db buffer solutions  using isotopic contrasts derived from QENS datasets for: (i) unlabelled bacterial cells (Hc) suspended in deuterated buffer solution (Hc/Db); (ii) perdeuterated cells (Dc) suspended in deuterated buffer (Dc/Db) and (iii) perdeuterated cells in unlabelled buffer (Dc/Hb), to construct the isotopic contrast [(Dc/Hb–Dc/Db)–Hb] , Table 11%, −9%) , Table 1olutions . This dyTo investigate water transport across the cell envelope we first examined the isotopic contrasts (Hc/Db–Db) and (Dc/Hb–Hb) assuming that water diffusion rates inside the cell remained the same as in bulk aqueous media at high pressure. In a second refined version of the model we then constructed the isotopic contrasts [((H/D)–Im)–Db] and [((D/H)–Im)–Hb] that take into account the differential effect on Im diffusion at pressure compared with the Hb and Db buffer solutions noted above . Both apT(Q2) data present a similar picture of water dynamics to the bulk and Im media but an unexpected narrowing of the QENS linewidths occurs between Q2 = 0.5–1.2 Å−2. A small downwards deviation in Γ(Q2) does appear to be present for the Im data set  might have been slightly affected by transmembrane water diffusion processes, but to a much smaller extent than observed for the datasets presented in The Γdata set  between Q < 0.7 Å−1) and short (Q > 1.1 Å−1) correlation lengths we obtained DT = 2.43–2.64 × 10−5 and 1.95–2.10 × 10−5 cm2s−1 , depending on the isotopic contrast model used (dashed line fit illustrated in Q < 1 Å−1) had been considered in the QENS analysis  (Fitting the SS model to the ensemble of data points at long (ctively) , Table 1T(Q2) relations. An initial downwards deviation of the data from the initial progression of the curve near 0.7 Å−1 is followed by a rapid upturn as the data regains the “normal” diffusion relation at approximately 1.1 Å−1. In an early analysis of the data, we considered the possibility that this might be associated with two superposed motions . However, it quickly became clear that the jump model, or any superposition of jump models, could not describe the water dynamics in any meaningful or justifiable way in terms of likely relaxation or nanoconfinement scenarios. However, a simpler model can be advanced to interpret the transmembrane ΓT(Q2) relations by examining the relaxation timescales implied by the QENS data, along with models that are becoming established for molecular water transport across the cell membrane structures via Aquaporin protein complex channels.We now examine the implications of these results and provide an initial interpretation of the unusual form of the ΓT(Q2) relation expected for a jump diffusion model applied to a single diffusion process, between Q = 0.7–1.1 Å−1. Each data point within this range represents an unexpected narrowing of the QENS linewidth and hence a correspondingly enhanced relaxation time for those correlated water transport motions occurring with correlation lengths between 6–9 Å in real space  datasets had been due to a systematic instrumental artefact, this would have been removed by the subtraction procedures. However, if such an artefact had been present in only one of the datasets, this would have given rise to anomalous slope changes occurring in opposite directions for different contrast combinations. The data in First we note that the unexpected slope changes can be interpreted as a “dip” relative to the Γal space . This sp2O molecules traverse the Aqp channel in single file in a spatially correlated motion, with a reversal of the molecular orientation occurring midway through the channel that ensures transport via intact molecular units2O transport dynamics can then explain the unusually long lifetime recorded by our QENS data, that correspond to 6.3 ps at 1 Å−1, compared with 5.3 ps expected by interpolating between the bulk diffusion dynamics occurring at longer and shorter Q values  mobility is indistinguishable from bulk water dynamics over correlation lengths extending between 3–30 Å. However, the reduction in diffusion coefficients with pressure is significantly greater than for the bulk aqueous solutions indicating the presence of interactions with macromolecules within the nanoconfined cellular environment. The overall water diffusion rate across the cell envelope is similar to that in the intracellular medium at ambient and high pressure. However, an unusual narrowing of the QENS line occurs for Q = 0.7–1.1 Å−1 corresponding to an enhanced relaxation time for correlated water transport over a real space distance ~6–9 Å. This corresponds to H2O molecules passing through the transmembrane Aqp channels embedded within the outer or inner cell membrane structures. Our HP-QENS studies do not reveal remarkable effects on H2O diffusion either inside the cells or across the cell membrane at pressures up to 200 MPa, that are relevant to the range in which subsurface microbial life has been shown to exist. In situ studies of both aerobic and anaerobic metabolic processes enabled by monitoring the rates of formate oxidation and Se4+ or Fe3+ reduction have demonstrated a >3-fold decrease in respiration rates within the same pressure range21We used QENS at ambient (0.1 MPa) and high pressure 200 MPa) combined with a range of H/D contrast experiments on isotopically labelled samples to reveal details of water dynamics occurring within the intracellular medium and across the cell membrane for 0 MPa comS. oneidensis samples (CIP 106686) obtained from the Collection Institut Pasteur  were grown at 30 °C in LB broth at 180 rpm after selecting a single colony from a LB agar plate and harvested at stationary phase (108 cells/mL). A perdeuterated strain was grown at the Deuteration Laboratory . The D-silantes medium used to grow and deuterate the bacteria prior to the neutron scattering experiments led to >98% labelingBecause H or D incorporated within the macromolecular cellular structures are primarily non-labileThe dataset subtractions made to construct the isotopic contrasts were enabled by the large difference in incoherent scattering cross section between H and D , taking account of the fact that D is almost invisible relative to H when both are present. The D-labelled growth/suspension medium was not 100% deuterated and the resulting perdeuterated cells also retained a small quantity of hydrogenated component. Because of the scattering cross sections involved, the isotopic contrast Dc/Db could be used to remove most of the coherent scattering contribution that is mainly present in the elastic line, as well as incoherent scattering from the H-component that is essentially located in the inelastic part of the scattering pattern, so that the results we report are truly QENS data sets. The presence of the isotopic impurity also helped ensure that an incoherent signal was observed in these Dc/Db experiments.via exchangeable protons when D-cells are placed in H-buffer, and vice versa. Each sample was resuspended three times in the relevant isotopic medium and left to equilibrate for at least 30 minutes before any data acquisition in order to ensure complete re-equilibration among any chemically exchangeable protons and deuterons in the bacterial sample37Some D/H exchange occurs naturally −1) Q range. TOFTOF is a direct geometry spectrometer: the Q resolution is ensured by the large solid angle covered by the detector area, whereas the energy of the scattered neutrons is measured after the time-of-flight for travelling from the sample to the detectors. The instrumental energy resolution (0.06 meV) defined by the incident neutron wavelength (λ = 6 Å) and chopper frequency (12000 rpm) produced a constant Gaussian lineshape that was perfectly suited to the characterization of the QENS signals of interest.QENS experiments were carried out at the time-of-flight spectrometer TOFTOF Our experiments that present a new approach using neutron scattering techniques to investigate important biological and biophysical dynamics required about 18 months of dedicated technical and conceptual development. It is important to evaluate the reproducibility and reliability of our findings. We repeated the experiments twice over a period of several months to allow an assessment of all potential sources of uncertainty ranging from sample growth, deuteration and resuspension protocols, through possible changes in samples occurring during shipping and handling, to instrumental issues associated with neutron flux, humidity accumulation on detectors, cell alignment, etc., and testing of software and protocols for data handling. The two independent datasets provided fully consistent results, and they could be averaged to provide error bars that we associated with each experimental data point. These error bars were so minor that they appear included within the size of the data points shown in Q, ω) of interest. Data analysis was performed on spectra at different constant Q values. Further details are given below. HP-QENS data were acquired using an Al-body cell with an internal gauge used to continuously monitor the pressure. In contrast to many high pressure investigations that are affected by a huge background from the sample environment, the measurement of a liquid solution in such an Al-cell represents a favorable condition for QENS studies. At an incident wavelength of 6 Å the pressure cell is almost transparent: i.e., it exhibits 97% neutron transmission overall, no incoherent scattering in the QENS region, Bragg peaks outside the accessible Q-range, and phonons well separated in energy from any liquid features. Moreover, we chose a scattering geometry with the high pressure cell rotated by 45° clockwise with respect to the propagation axis of the incident neutron beam, thus allowing a continuous signal acquisition over the entire detector area . Further details concerning the high pressure cell and the scattering geometry are provided inThe neutron scattering data were handled and treated using the Large Array Manipulation Program (LAMP)Q resolution is provided by the distinct angular coordinate 2Θ assigned to each detector. Time-of-flight and angular units were then converted into the energy and momentum transfer scale according to standard LAMP routines, developed to provide the exact transformation of the double differential cross section into the corresponding dynamic structure factor S. The intensity data from scattered neutrons were either grouped according to a regular  grid (linear binning) to provide information on S, or in constant Q spectra by grouping the signal from neighboring detectors. Additional normalization steps and data analysis were carried out directly on the S spectra using Microcal OriginPro 8.6. These normalizations considered A) the total quantity of material present in the beam and B) the correct quantity of buffer to be subtracted. With respect to point B), we had to take into account the volume fraction (ϕ = 5%) occupied by the bacterial cells. For step A) we note that the scattered intensity I, and therefore S, is proportional to the amount of material in the beam and is already weighted for the corresponding coherent/incoherent cross section. Each set of S spectra generated by the same S matrix was normalized to the integral of S over the entire Q range and the quasielastic ω range. The resolution of the time-of-flight experiment excluded any phononic contribution from the empty can.Raw data files were normalized to the neutron flux and the acquisition time, thus removing any dependence on the neutron source and the experimental duration. The global detector efficiency was calibrated to the energy of the incoming neutron beam. Further detector-specific efficiency fluctuations were corrected individually after comparison with the purely incoherent signal scattered from an elemental vanadium (V) reference sample. This constituted a 1.1 mm thick flat plate that also provided a calibrant for the experimental elastic resolution function. Indeed to guarantee a uniform normalization over all spectra contained within a workspace dataset, the time-resolved scattered intensity recorded by each detector was normalized to the integrated elastic intensity scattered by the vanadium reference standard at the same 2Θ angular position.We note that in the case of direct geometry TOF spectrometers, the Q.The spectra were analyzed using a built-in least squares algorithm that takes into account a central Gaussian feature (to account for the energy resolution) along with up to three Lorentzian functions. The elastic component was removed to reveal the quasi-elastic line broadening of interest for each dataset as a function of the scattering vector Q, once it is converted into inverse timescale units (s−1) via ΔE = ħω. The translational linewidth (ΓT) is analyzed as a function of Q using different models for the transport process678Following data acquisition and discrimination of the QENS signal from the pure elastic, Gaussian scattering component determined by the instrument resolution function we proceeded with construction of the isotopic contrasts and comparison between 0.1 and 200 MPa profiles, followed by data fitting using a hierarchy of models developed to describe the relaxation dynamics and diffusion processes67891011Q, ω) for the different cell/buffer combinations:The following protocols were applied to establish S [bacterial cells in buffer] = S [bacterial cells in buffer within the Al HP container] – S [empty container](i) S and aqueous buffer media  were hydrogenated (H) or perdeuterated (D) varieties. A similar subtraction procedure was used for the buffer solutions alone:Q, ω) [buffer] = S [buffer within the Al HP container] – S [empty container](ii) S [bacterial cells] = S [bacterial cells in buffer] – α × S [buffer](iii) S(−1), where pure coherent scattering was expected to occur.Here the factor α = (1–ϕ) accounts for the macromolecular volume fraction (5%) occupied by the bacterial cells in solution. In contrast to macromolecules such as proteins, with a composition that can be determined readily by sequencing and other assay techniques, the exact molecular composition of the live cells is not known precisely. However, because the buffer does not produce in any intense peak in the elastic region, the elastic scattering from the cells gives a reliable estimate of the macromolecular concentration. We thus estimated ϕ and α by normalizing the elastic intensity of (Dc/Db) to that of (Dc/Hb) at the first diffraction maximum (1.8 ÅThe relationshipT (cm2s−1) applies when interactions between molecules are weak, and in general as DT → 0. Typically plots of ΓT versus Q2 are analyzed to provide estimates of DT that are generally found to be comparable with NMR and bulk transport measurements678that introduces the diffusion coefficient D0, during which the molecules undergo oscillatory motions:As intramolecular interactions become significant the diffusion processes deviate from Fick’s law and the dynamics are described using two (or more) characteristic timescales. A typical approach involves the use of molecular jump models between sites separated by average length, l, and mean site residence time, τSS) jump model, in which each water molecule executes oscillations during a mean residence time (τ0) then diffuses during time τ1 to a second site67891011If the jump lengths are distributed randomly, the diffusion is usefully described by a Singwi-Sjölander .


TP53, IKBKB and TGFB1 were up-regulated. In addition to cell cycle regulators, upstream regulators that were predicted to be inhibited included Retinoblastoma 1, E2 transcription factor 1, and hepatocyte growth factor. Our study suggests that during antral atresia of small follicles in the theca interna, arrest of cell cycle and DNA replication occurs rather than up- regulation of apoptosis-associated genes as occurs in granulosa cells.The theca interna is a specialized stromal layer that envelops each growing ovarian follicle. It contains capillaries, fibroblasts, immune cells and the steroidogenic cells that synthesize androgens for conversion to estradiol by the neighboring granulosa cells. During reproductive life only a small number of follicles will grow to a sufficient size to ovulate, whereas the majority of follicles will undergo regression/atresia and phagocytosis by macrophages. To identify genes which are differentially regulated in the theca interna during follicular atresia, we undertook transcriptome profiling of the theca interna from healthy (n = 10) and antral atretic (n = 5) bovine follicles at early antral stages (<5 mm). Principal Component Analyses and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. A total of 543 probe sets were differentially expressed between the atretic and healthy theca interna. Further analyses of these genes by Ingenuity Pathway Analysis and Gene Ontology Enrichment Analysis Toolkit software found most of the genes being expressed were related to cytokines, hormones and receptors as well as the cell cycle and DNA replication. Cell cycle genes which encode components of the replicating chromosome complex and mitotic spindle were down-regulated in atretic theca interna, whereas stress response and inflammation-related genes such as  Mature ovarian follicles consist of an oocyte surrounded by epithelial granulosa cells all enclosed by a basal lamina which separates these cells from the surrounding stromal thecal layers. The thecal layers can be divided into the theca interna, which is closest to the follicular basal lamina and contains steroidogenic cells, fibroblastic cells, immune cells and capillaries, and the theca externa which is composed mostly of fibroblastic cells and smooth muscle-like cells. The main function of the theca interna is to produce androgens which serve as precursors for estradiol synthesis by the granulosa cells In bovine ovaries two morphological phenotypes of healthy follicles have been observed at sizes <5 mm in vitro, led to a decline in steroidogenesis as well as cell cycle progression and increased fragmentation of granulosa cell DNA During each cycle the majority of growing follicles undergo follicular atresia potentially as a means to limit the numbers of follicles that ovulate and to assist in co-ordination of the timing of ovulation. During atresia, cell death and phagocytosis by macrophages is observed Basal atresia affects the granulosa cells closest to the basal lamina and can lead to disruptions in the basal lamina and entry of macrophages, fibroblasts and endothelial cells into the damaged granulosa cell layers In contrast to basal atresia, during antral atresia the granulosa cells closest to the follicular antrum undergo cell death first, whereas the cells close to the basal lamina and the basal lamina itself remain intact until later in the process Bos taurus heifers at an abattoir . Follicles with an external diameter of 3–5 mm as measured by callipers were dissected for classification and analysis. Granulosa cells were aspirated and scraped from each follicle with a Pasteur pipette, whose tip had been blunted previously by heating, and the granulosa cells were discarded. The theca interna was then dissected from the follicle wall under a Zeiss Stemi D4 stereomicroscope  in cold Hank's balanced-salt solution with Mg2+ and Ca2+  and stored at −80°C prior to RNA extraction. An excised portion of the follicle wall (2×2×2 mm) was taken prior to granulosa and thecal cell removal and fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer for subsequent histological assessment. Follicles were classified as healthy or atretic based upon the morphology of the membrana granulosa and the presence or absence of apoptotic cells as previously described Pairs of ovaries were collected from non-pregnant cycling RNA was extracted from theca interna by the Trizol method . Briefly, each sample was homogenized in 1 ml of Trizol with 1.4 mm ceramic beads in a Precellys 24 Bead Mill Homogenizer  with two 10 s cycles of 6,000 rpm each. The samples were then extracted with 200 µl of chloroform and the aqueous phase was purified through a Qiagen RNEasy mini-preparative column  according to the manufacturer's instructions. Five µg of RNA was treated to remove genomic DNA contamination with 2 units of DNAse 1 (Ambion Life Technologies) prior to labeling for microarray analyses. The integrity of all the RNA samples was assessed by microfluidic analyses on a 2000 BioAnalyzer  and were all found to have RNA integrity numbers (RIN)≥8.In vitro transcription reactions were performed in batches to generate biotinylated cRNA targets, which were subsequently chemically fragmented at 95°C for 35 min. Ten µg of the fragmented biotinylated cRNA was hybridized at 45°C for 16 h to Affymetrix GeneChip Bovine Genome Arrays, which contain 24,128 probe sets representing over 23,000 transcripts and variants, including 19,000 UniGene clusters. The arrays were then washed and stained with streptavidin-phycoerythrin . Signal amplification was achieved by using a biotinylated anti-streptavidin antibody. The array was then scanned according to the manufacturer's instructions . The arrays were inspected for defects or artefacts. The array data were converted to CEL file format for analyses.DNAse-treated thecal RNA of 100 ng from each individual follicle was labeled using the 3′IVT Express labeling kit . In brief, the RNA was reverse transcribed using a T7 oligo dT primer followed by second-strand synthesis. Quality control for the cDNA labeling was determined by the use of internal array controls. All the arrays passed these controls. The array data was subjected to Robust Multi Array Average (RMA) summarization 2 signal intensity of >3.0 for ≥50% of the arrays of one follicle type were considered to be above the detection threshold. Before statistical analyses, the data were first subjected to PCA GSE49505).The 15 arrays were analyzed as part of a larger set of CEL files  These differentially expressed genes were uploaded to the Ingenuity database  for pathway and functional analysis as described previously −Δ Ct for the target gene to PPIA and GAPDH.Synthesis of cDNA was performed as previously P<0.05, between the two groups of healthy follicles. Therefore these were treated as one group for further analyses (n = 10).Theca interna from a total of 15 small antral follicles  were classified on the basis of follicle phenotype as described in the methods and then examined by microarray analyses of gene expression. These consisted of three groups each of n = 5. One group contained antral atretic follicles To exclude the possibility of contamination of the theca interna with granulosa cells, the array intensities of a granulosa cell marker, FSH receptor, was examined. All healthy thecal samples had signal intensities less than 3.0, compared with a mean ± SEM of 8.6±0.05 for granulosa cells (n = 10) from small healthy follicles of a similar size in an analogous study PCA mapped the overall differences in gene expression between the individual arrays as shown in P<0.05) by one-way ANOVA analyses in Partek , was determined to be differentially expressed between the atretic and healthy theca interna  and glutathione peroxidase 3 (GPX3). Differential expression of CD68, ADM and GPX3 were also validated by qRT-PCR as shown in IL-6 (interleukin-6), interleukin receptors IL1R1 and IL10RA, prostaglandin receptor PTGER4, Toll-like receptor 7 (TLR7), and the high affinity receptors for the Fc fragment of IgG and IgE, FCGR1A and FCER1A, respectively. Other genes of interest from this group which may mediate extracellular ligand actions were NTRK2 , cell cycle: G2/M DNA damage checkpoint regulation (P = 4.96×10−5), pyrimidine deoxyribonucleotides de novo biosynthesis I (P = 1.63×10−3), mitotic roles of polo-like kinase (P = 2.90×10−3), DNA damage-induced 14-3-3δ signaling (P = 3.34×10−3) and GADD45 signaling (P = 1.24×10−3). The genes which mapped to these pathways were all down regulated, indicating inhibition of these processes. Additionally, the interleukin-10 (P = 2.08×10−2) and complement signaling pathways (P = 1.63×10−3) which are related to stress response and inflammation appeared to be activated. The GO enrichment analysis of our differentially expressed genes (P = 2.54×10−3) and pyrimidine deoxyribonucleotide metabolic process (P = 4.74×10−3), and some inflammation-related associations through complement (P = 3.94×10−2) and TGFβ pathways (P = 1.27×10−2).IPA determined that six of the top ten canonical pathways which were associated with our data set were involved with cell division and DNA replication . These ied genes  also shoNCAPH, NCAPD3 and NCAPG2 and the mini-chromosome complex which initiates DNA replication MCM2, MCM4 and MCM6 were among these. This connection with cell division was further strengthened by the fact that the top canonical pathway in IPA associated with the differentially regulated data set was cell cycle control of chromosomal replication , estrogens and the transcription factors E2F1, EP400 and TBX2  CYP17A1CEBPD is a transcription factor which is involved with regulation of cell division and appears to be playing a similar role here FOSL2 and JUN encode components of the AP-1 transcription complex which participates in the terminal differentiation of granulosa cells to luteal cells RB1 controls E2F1 transcription and that RB1 plays a role in regulating follicular development in mice There were several important transcription factors which were differentially regulated between small healthy and atretic follicle theca interna in our analyses. Some of these mentioned below were up regulated in atresia and are known to play a role in the control of steroidogenic pathways in thecal cells and other ovarian tissues. Interestingly c-Fos and EGR1 are key effectors of the Protein Kinase C pathway for modulation of androstenedione production via the action of VEGF and FGFR2, as reviewed in A few of the genes which were down regulated in atretic follicles have a positive role in angiogenesis such as It is obvious that the main effect of atresia in the theca interna of small follicles at the transcriptome level, was the considerable inhibition of the processes of cell division and DNA replication. This may be due to the predicted activation of p53, revealed by the upstream regulator analyses in There were very few indicators of cell death observed in the theca interna of antral atretic follicles in our array analyses. This prompts the question as to whether significant cell death actually occurs early in the theca interna layer in antral atretic follicles. In the literature there is ample evidence of cell death in the theca interna occurring early in basal atretic follicles Figure S1Unsupervised hierarchical clustering across all probe sets and arrays. Probe sets  and arrays (n = 15) were clustered using the Euclidian dissimilarity algorithm with the average linkage method in Partek Genomics Suite. The heatmap represents the distribution of normalized signal intensity, grouping by pattern similarity for both probe set and array. Abbreviations are explained in (TIF)Click here for additional data file.Figure S2Scatter plot of fold-changes in microarray intensity versus expression determined by qRT-PCR. Values represent 10 selected genes as presented in 2R = 0.93, P<0.001).(PDF)Click here for additional data file.Figure S3The canonical pathway of cell cycle control of chromosomal replication in IPA. Genes which were down regulated in small atretic follicles are in green and the degree of fold difference is commensurate with the color intensity. Benjamini-Hochberg FDR P value of gene association with pathway = 3.36×10−6.(TIF)Click here for additional data file.Table S1Probe sets which were 2-fold or more up regulated in atretic with respect to healthy follicles. Analyses were carried out by ANOVA in Partek with P<0.05 (n = 543) and assignations are presented in alphabetical order. Probe sets which did not have gene assignations are placed at the end of the list. The P value for multiple corrections was determined by the step-up FDR method.(PDF)Click here for additional data file.