The marine nitrogen cycle is dominated by redox-controlled biogeochemical processes and, therefore, is likely to have been revolutionised in response to Earth-surface oxygenation. The details, timing, and trajectory of nitrogen cycle evolution, however, remain elusive. Here we couple nitrogen and carbon isotope records from multiple drillcores through the Rooihoogte–Timeball Hill Formations from across the Carletonville area of the Kaapvaal Craton where the Great Oxygenation Event (GOE) and its aftermath are recorded. Our data reveal that aerobic nitrogen cycling, featuring metabolisms involving nitrogen oxyanions, was well established prior to the GOE and that ammonium may have dominated the dissolved nitrogen inventory. Pronounced signals of diazotrophy imply a stepwise evolution, with a temporary intermediate stage where both ammonium and nitrate may have been scarce. We suggest that the emergence of the modern nitrogen cycle, with metabolic processes that approximate their contemporary balance, was retarded by low environmental oxygen availability. The response of biogeochemical nitrogen cycle to Earth-surface oxygenation remains poorly known. Here, the authors show that aerobic nitrogen cycling was pervasive prior to the Great Oxygenation Event (GOE), but its evolution was complex, with diazotrophy prevailing and sustaining productivity after the GOE. Thus, bioavailable nitrogen is a key nutrient influencing primary productivity, thereby linking global biogeochemical cycles and the Earth’s climate2.Nitrogen is an essential component of amino acids, nucleotides and other compounds central to the cellular metabolism. Although nitrogen is one of the most abundant terrestrial elements—comprising 78% of the Earth’s present atmosphere as N3. Microbial nitrogen fixation by diazotrophs, reducing atmospheric N2 to ammonia, is the primary source of bioavailable nitrogen and likely had its roots in the Mesoarchaean4. In the presence of oxygen, ammonium formed via organic matter remineralisation undergoes a stepwise oxidation to nitrite (NO2−) and ultimately to nitrate , mediated by aerobic ammonium/ammonia-oxidising bacteria (AOB), archaea (AOA) or methanotrophs6. In the largely oxygenated contemporary ocean, nitrification is quantitative3. Once oxidised, however, bioavailable nitrogen can then be lost in oxygen-deficient settings by denitrification (dissimilatory nitrate reduction) and/or anaerobic ammonium oxidation . These are the principal sinks for bioavailable nitrogen in the present-day ocean7.The contemporary marine nitrogen cycle, in which nitrate is the dominant bioavailable nitrogen species, is overwhelmingly governed by biological processes8. It follows, therefore, that in the oxygen-free early Archaean10, the nitrogen cycle would have been relatively simple and vastly different from its contemporary counterpart3. Ammonium would have been the predominant form of fixed nitrogen and would have been both assimilated directly and recycled within the water column11. Given this intrinsic link between environmental oxygen availability and nitrogen cycling, the timing of the transition from an anaerobic to an aerobic nitrogen cycle  has been used as a line of evidence to constrain the minimum age of the emergence of oxygenic photosynthesis13.In the absence of oxygen, ammonium remains stable and can accumulate in deep waters, as exemplified by the anoxic water column of Framvaren Fjord, Norway, which supports ammonium concentrations in excess of 1 mM14. This model, however, is not universally accepted, with others favouring a more protracted evolution. Specifically, Fennel et al.15, and others since16, proposed that the early anaerobic nitrogen cycle evolved to today’s aerobic equivalent in a more protracted or stepwise manner. Beginning with an ammonium-dominant stage, extremely oxygen-deficient (<11 μM) conditions are thought to have resulted in less efficient nitrification (versus denitrification), stabilising ammonium as the dominant bioavailable nitrogen species. Ushered in by moderate increases in environmental oxygen availability, the second stage is thought to have featured nitrification in more oxygen-replete surface waters and quantitative denitrification in adjacent oxygen-deficient deep waters. During this stage, both ammonium and nitrogen oxyanions (nitrate and nitrite) were destabilised, instigating a hypothetical nutrient deficiency—a ‘nitrogen famine’. Finally, in the third stage, progressive ventilation of the deep oceans led to the establishment of a nitrogen cycle where both nitrate and nitrite were stabilised as dominant bioavailable nitrogen species.Although the early Earth’s marine nitrogen cycle was almost certainly different from today’s, the timing and trajectory of the transition towards a modern nitrogen cycle (the relative importance of the metabolisms in the nitrogen cycle are similar to those in the modern ocean) remains poorly understood. For example, a recent study proposed that the aerobic nitrogen cycle emerged and stabilised in the immediate aftermath of the Great Oxygenation Event (GOE)18. Biologically mediated redox cycling exerts the dominant control on the δ15N of dissolved nitrogen species, which is ultimately communicated to the geological record after biological assimilation18. These principles were recently applied to reveal how the GOE impacted nitrogen cycling ~2300 million years ago (Ma) through the upper Rooihoogte and lowermost Timeball Hill Formations14.The transition between successive stages in the evolution of the nitrogen cycle was undoubtedly complex and remains poorly known. Despite these difficulties, nitrogen cycling can be tracked by determining the nitrogen isotopic composition of sedimentary organic phases, which can be applied to illuminate processes active in both recent and ancient oceans alike15Nkerogen, δ15Nbulk and δ13Corg from an extended stratigraphic succession spanning the upper Rooihoogte to upper Timeball Hill formations in three drillcores  from the Carltonville area of South Africa 20. Geochronological constraints 26. Thus, the investigated succession spans the first increase in atmospheric pO2 from near zero to potentially significantly elevated levels23, providing a unique opportunity to examine the evolution of the marine nitrogen cycle in response to evolving atmospheric chemistry.Building on this initial study, we determined the δOur study reveals that aerobic nitrogen cycling was established prior to the disappearance of sulphur mass-independent fractionation (S-MIF); however, the evolution towards its modern counterpart was far from simple, negotiating an ephemeral interval of bioavailable nitrogen scarcity. Rather than instigating a nitrogen famine, promoted by rising environmental oxygen availability, diazotrophy was sufficient to replenish bioavailable nitrogen losses and sustain primary productivity in the wake of planetary oxygenation.13Corg values from all three of the studied cores range from ‒36 to ‒32‰  and kerogen (δ15Nkerogen) nitrogen isotope profiles show similar intra- and inter-core stratigraphic trends . In the uppermost part of the Timeball Hill Formation, a small increase in δ15Nkerogen is observed in cores EBA-2 and EBA-4 but not in core KEA-4, which may reflect sampling biases given the different resolutions of the records.The bulk   Fig.  suggests15Nkerogen values17. It has been shown that the δ15N of surface sediments approximates that of sinking organic particles in areas with high sediment accumulation rates, high organic matter content and reducing bottom-water conditions31. The expected high sedimentation rates in a deltaic sedimentary environment, and the relatively high TOC content  of the studied samples, suggest that early diagenesis probably had a limited effect on δ15Nkerogen. The high C/Nkerogen ratios  are similar to those from other Archaean and lower Palaeoproterozoic successions and are consistent with nitrogen loss during mild post-depositional thermal alteration32. Given that the Carletonville strata experienced only lower greenschist metamorphism28, thermal alteration is not expected to have increased δ15N by more than~1 to 2‰34. This inference is braced by the lack of significant correlations between δ15Nkerogen and [N kerogen]  values vary around −1.3 ± 1.3‰. This close agreement between δ15Nbulk and δ15Nkerogen 40. While microbial culture experiments indicate that trace metal availability can alter the magnitude of the nitrogen isotopic fractionation associated with diazotrophy, they demonstrate that nitrogen fixation is unlikely to produce biomass with a δ15N much higher than 0‰42. Likewise, remineralisation of diazotrophic biomass carries no appreciable isotope effect (~−1‰)18, and simply serves to shuttle 15N-depleted ammonium to the deep ocean. By contrast, the large fractionation associated with ammonium assimilation (>15‰)43 produces strongly 15N-depleted biomass, with δ15N values lower than the precursor ammonium, as is sometimes suggested for the Archaean record14. Although ammonium dissociation to ammonia and degassing could conceivably produce positive δ15N values under an anoxic atmosphere44, it requires a high pH and is therefore unlikely to have operated outside of atypical and isolated evaporative lacustrine settings45. Consequently, the emergence of positive δ15N values (>3‰) in well-preserved and unaltered sedimentary rocks from open-marine settings is generally interpreted as the isotopic fingerprint of an aerobic nitrogen cycle, constraining when nitrate first formed part of the dissolved nitrogen inventory14.Prior to the advent of oxygenic photosynthesis, the Earth’s nitrogen cycle operated in an almost entirely anaerobic mode, dominated by diazotrophy and ammonification46, Archaean sequences commonly feature positive δ15N values18. Although ammonium oxidation to N2 by ferric oxyhydroxides may produce positive δ15N values, the magnitude of this fractionation remains uncertain47. Moreover, it is unlikely to have been of significance in the examined cores given their low ferric oxyhydroxide contents14. Therefore, despite the anoxic backdrop, the preferred explanation of these positive δ15N values commonly invokes the intermittent operation of an aerobic nitrogen cycle32. The sustained positive δ15Nbulk and δ15Nkerogen values preserved in the lower part of all three examined cores 10, and the correspondingly short half-life of oxygen in the highly reducing pre-GOE atmosphere (~9 h)49, aerobic nitrogen cycling, and thus dissolved nitrate availability, must have been spatially variable and confined to sites of nutrient availability and active oxygenic photosynthesis—so-called ‘oxygen oases’50. Thus, the advent of oxygenic photosynthesis must have significantly predated the GOE, and the positive δ15N values in the Archaean record might constitute some of the earliest evidence for intermittent biogenic oxygen production13.It is striking that while S-MIF persists into the early Palaeoproterozoic15N-enriched biomass. Nitrification, for example, produces 15N-depleted nitrate while simultaneously driving the residual ammonium pool to more positive δ15N values53. Similarly, denitrification and anammox preferentially shuttle 14N to the atmosphere as N2, concomitantly increasing the δ15N of the residual dissolved nitrogen oxyanion pool54. In present-day marine settings, the fractionation accompanying nitrification is typically not expressed owing to rapid and quantitative ammonium oxidation to nitrate7, leaving nitrogen losses, via either denitrification and/or anammox, as the dominant control on the δ15N of dissolved inorganic nitrogen7. By analogy, the positive δ15N values observed in the prelude to the GOE, reflected in the basal part of our record, have been ascribed to complete nitrification coupled with incomplete denitrification14. While we acknowledge that most of the processes central to the modern nitrogen cycle  had almost certainly evolved by the late Archaean14, their relative importance remains unresolved. This, in turn, leaves an open question: How did the nitrogen cycle evolve in the aftermath of the GOE?In detail, multiple metabolic processes within the aerobic nitrogen cycle produce pO2 and a ferruginous and ammonium-rich deep ocean55. At low oxygen concentrations denitrification outpaces nitrification resulting in quantitative loss of nitrite and nitrate. Modelling approaches suggest denitrification outpaced nitrification when dissolved oxygen concentrations were <~11 μm15, yet the threshold value where ammonium concentrations exceed nitrate concentrations can be substantially higher in contemporary anoxic settings (~35 μm)8. Admittedly, evaluating the absolute oxygen contents of the Palaeoproterozoic surface ocean remains difficult; however, dissolved oxygen concentrations were certainly low before and in the immediate aftermath of the GOE, encroaching on the threshold where nitrate/nitrite losses exceeded their replenishment58. Given these boundary conditions, with a potentially large ammonium reservoir at depth, localised rapid and quantitative denitrification of a relatively small standing nitrate reservoir may have minimised denitrification’s characteristic isotope effect.Even directly after the termination of S-MIF, the chemical composition of the early-Palaeoproterozoic ocean–atmosphere system would have remained vastly different from its present-day counterpart, with low atmospheric 15N values observed in the Carletonville records. In regions where the chemocline was relatively shallow, ammonium may have been the preferred bioavailable nitrogen species, especially given that ammonium suppresses the expression of nitrate reductase and the genes that regulate nitrate assimilation59. This explanation, although not universally accepted45, has been proposed previously to explain the extreme positive δ15N values recorded in the 2.7 billion-year-old Tumbiana Formation32. Given this oxygen-poor backdrop, the nitrogen cycle would have been subtly different and could have been both spatially and diurnally variable. Despite the prevalence of ammonium, in regions of active photosynthetic activity and O2 production, nitrate may have been stable and utilised by phytoplankton as their preferred nitrogen-substrate. Taken together, we argue that, at least initially, the positive δ15N values seen at and around the termination of S-MIF could have originated from a combination of incomplete nitrification, as well as incomplete denitrification as others have proposed14. This represents a subtle but distinct difference in the operation of the Palaeoproterozoic nitrogen cycle relative to its present-day counterpart , as well as an increase in the marine sulphate inventory20. These coeval isotopic changes argue for a strong yet evolving connection between the biosphere and the chemical composition of the atmosphere and ocean, whereby the chemical evolution of Earth’s surficial environments influenced the emergence and distribution of ecological niches, not to mention the metabolisms that filled them. Previously, a negative correlation between δ13Corg and δ15Nkerogen was used to argue that aerobic methanotrophy mediated nitrification in the 2.7 billion-year-old Tumbiana Formation32. Aerobic methanotrophy, however, is unlikely to have been important in the Carletonville area over the examined time interval because the δ13Corg values are incompatibly high (>−37‰), and there is no significant correlation between δ13Corg and δ15Nkerogen  depositional conditions, with occasional relaxations toward oxic deposition14. Consequently, the decimetre-scale 6–8‰ negative δ15Nkerogen excursions can be explained by the occasional injection of ammonium-rich anoxic deep-water into the photic zone. Alternatively, denitrification and concomitant nitrogen limitation could have also periodically stimulated diazotrophy, whose isotope effect would have been more pronounced if nitrogen-deficient and Fe2+-replete waters were emplaced from depth41. Following the initial variability, δ15Nkerogen values stabilise around 5.6 ± 1.5‰ in the lower Timeball Hill Formation 58 and modelling approaches indicate that the global ocean remained stratified with anoxic water beneath oxygen-deficient surface waters (≤10 µM O2)57. These data, therefore, require the operation of nitrification, but we are unable to unequivocally decipher whether the positive δ15N values were a product of an ammonium precursor.Punctuating the increase in δion Fig. . In this15N values to around 0‰ in the upper Timeball Hill Formation 66, which approximate those predicted for Proterozoic oceans67. Given that molybdenum concentrations encroached on levels where co-limitation could have occurred, the Mo-N co-limitation hypothesis cannot be unequivocally dismissed. Nevertheless, several lines of evidence suggest that diazotrophy could keep pace with bioavailable nitrogen losses, sustaining primary productivity in this part of the Palaeoproterozoic ocean.The global expression of nitrogen limitation, and the feasibility of a widespread nitrogen famine, is dependent on the global balance between nitrogen losses via canonical denitrification, its retention as ammonia following dissimilatory nitrate reduction2 necessitated by the Lomagundi carbon isotope excursion23, requires quantitative recycling of organic nitrogen and/or pervasive diazotrophy. Consequently, extrapolating our observations outside of the Carletonville area, we hypothesise that enhanced phosphorous availability—derived from enhanced weathering68 coupled with intensified redox-promoted recycling70—associated with evolving ocean–atmospheric chemistry61, was capable of sustaining widespread, and potentially more efficient diazotrophy16, replenishing the lost nitrate and preventing any long-lived nitrogen famine. The poorly defined positive shift in the uppermost part of the upper Timeball Hill Formation , which tentatively suggest increasing denitrification, consistent with contraction of anoxia and partial oxygenation of the deep ocean58. Consequently, we argue that any hypothetical nitrogen famine would have been a transient Earth-system state, and would have most likely been terminated by the onset of the Lomagundi Event (~2220 Ma). Therefore, assuming the decrease in δ15N values to around 0‰ represents a global nitrogen cycle response, constrains any potential nitrogen famine to ~2250–2220 Ma.The continued and prodigious release of OpO2, during the Lomagundi interval and beyond, would have ultimately stabilised dissolved nitrogen oxyanions and ushered in a nitrogen cycle more reminiscent of the present day 26.The samples from the upper Rooihoogte to uppermost Timeball Hill formations were collected from cores KEA-4, EBA-4 and EBA-2, which were drilled in the Carltonville area of South Africa Fig. . Detailshed Fig. . The Roo20. Aliquots of samples used previously for sulphur isotope analysis20 were quantitatively decalcified using 6 M HCl before being rinsed to neutrality using ultra-pure water and dried at 60 °C. Kerogens were isolated at Massachusetts Institute of Technology using a method modified from Zerkle et al.14. Briefly, the carbonate fraction was removed from ~4 g aliquots of rock powder via an overnight 6 M HCl dissolution. The residues were then rinsed to neutrality using successive ultra-pure water, decanting the supernatant following centrifugation. The silicate fraction was then volatilised via duplicate overnight treatments with 20 ml of a mixed HF and HCl (3:2) solution. Each acid treatment was followed by multiple (n = 4) 1 M HCl rinses and then dried for mass spectrometric analysis. The low carbonate abundances and the high thermal maturity negated the need for replicate 6 M HCl and dichloromethane treatments. Heavy minerals such as pyrite were not removed as they do not affect nitrogen isotopic compositions.Core samples were cut into small pieces, removing all exterior surfaces. Fresh chips were ultrasonically cleaned with successive deionised water, methanol, and dichloromethane rinses prior to homogenisation (<100 mesh) using a precleaned stainless steel puck mill15Nbulk) and carbon (δ13Corg) isotope values were determined by flash combustion using an elemental-analyser (EA) coupled to a continuous-flow isotope ratio mass spectrometer (IRMS) at Massachusetts Institute of Technology. The low nitrogen content prevented simultaneous δ15Nbulk and δ13Corg determinations. For δ15Nbulk analysis, the decalcified residues (~40 to 80 mg) were combusted at 1040 °C, and the resulting nitrogen oxides were reduced to N2 by Cu at 650 °C. The analyte gas stream was then stripped of CO2 and water via contact with Carbosorb® and anhydrous magnesium perchlorate, respectively. For δ13Corg analysis, smaller aliquots of decarbonated residues (~1–8 mg) were combusted and dried as detailed for δ15Nbulk determination. Most samples were analysed in triplicate, yielding standard deviations (1σ) for δ15Nbulk and δ13Corg better than 0.3‰ (1σ) and 0.2‰ (1σ), respectively. All isotope data are reported in standard delta-notation, reflecting per mille variations from their respective international reference standards:aX/bX is the ratio between the heavier  and the lighter isotope  of element X (C or N), and the standard is either air for δ15N or VPDB for δ13C. Nitrogen isotope data were calibrated using IAEA-N-2 (δ15N = +20.3 ± 0.2‰), IAEA-NO-3 (δ15N = +4.7 ± 0.2‰) and urea (δ15N = +0.3 ± 0.25‰), and C-isotope data were calibrated using urea (δ13C = −34.1‰), sucrose (δ13C = −10.5‰), Arndt acetanilide (δ13C = −29.5‰), and an internal laboratory standard . Nitrogen and TOC abundances were calculated from the integrated N2 and CO2 peak areas, respectively. Replicate analysis of samples and standards demonstrates a relative precision of better than 10%.Bulk nitrogen (δ15Nkerogen) was determined in the GAPP Lab  using an automated Nano-EA system similar to that described by Polissar et al.72 The use of a Nano-EA system facilitates determination of δ15N in nitrogen-lean Precambrian kerogens, permitting analysis of 1–3 mg of kerogen equating to 25–100 nanomoles of nitrogen. Sample capsules were evacuated and purged with argon prior to introduction into the EA (Elementar Isotope Cube) to remove interstitial atmospheric nitrogen. A 90-s O2 pulse ensured complete combustion after an initial 45-s He purge. The combustion products were then carried in a He stream (150 ml per min), via oxidation (1100 °C) and reduction (650 °C) reactors, to a silica gel cryotrap immersed in liquid nitrogen. Using a reduced He carrier flow (~2 ml per min), the analyte N2 was introduced to the Isoprime 100 IRMS through an Agilent CarboBond column (25 m × 0.53 mm × 5 μm). Given the high C/N ratios, the CO2 generated during combustion was retained in a molecular sieve trap, eliminating CO2 carryover and preventing the generation of neoformed isobaric CO in the ion source. Additionally, the use of small sample sizes permitted by the Nano-EA technique eliminates the potential for incomplete oxidation and generation of CO during combustion.The isotopic composition of kerogen-bound nitrogen (δ73 Reproducibility for samples and standards (±0.25‰) approaches that reported for reference materials (±0.2‰) approximating standard EA-IRMS techniques.Individual kerogen samples were run in triplicate using sequentially larger samples and blank-corrected using Keeling-style plots. International  and in-house  reference materials were run in a similar manner to samples, using equivalent quantities of nitrogen. The resulting blank-corrected sample and standard data were expressed in delta-notation (Eq. 1) and corrected to the certified values of the reference materials using the correction scheme described in Coplen et al.The data that support the findings of this study are available from the corresponding authors upon reasonable request.Supplementary InformationPeer Review File


Functional neuroimaging, applied to pre-clinical models of chronic pain, offers unique advantages in the drive to discover new treatments for this prevalent and oppressive condition. The high spatial and temporal resolution of fMRI affords detailed mapping of regional pharmacodynamics that underlie mechanisms of pain suppression by new analgesics. Despite evidence supporting the translational relevance of this approach, relatively few studies have investigated fMRI abnormalities in rodent models of chronic pain. In this study, we used fMRI to map the BOLD response in a recently developed putative rat model of fibromyalgia to innocuous and acute nociceptive stimuli by applying a step-wise graded electrical forepaw stimulation paradigm, with comparison to healthy controls. We observed discriminatory functional signatures (p < 0.001) to 2 mA electrical forepaw stimulation, found to be innocuous in the control group. As such, this translational approach provides sensitive and quantitative neural correlates of the underlying chronic disease. The regional patterns of functional augmentation were found to be concordant with previous studies of nociception in the anaesthetised rat brain, supporting the specificity of this approach in the study of altered central pain processing in reserpine induced myalgia. The methodology introduced in this work represents a novel platform for emerging treatment evaluation in highly experimentally controlled conditions. Fibromyalgia (FM) is a prevalent disorder with as yet undefined aetiology1236767Animal models of chronic pain that faithfully recapture properties of the human condition are highly useful for mechanistic study and therapeutic evaluation11121314151617181920212223241218272829We have developed a non-surgical putative rat model of FM which reflects key symptoms of the human condition, including tactile allodynia and depression2 = 38 (±2) mmHg; pO2 = 109 (±5) mmHg; RIM mean (SD): pCO2 = 33 (±3) mmHg; pO2 = 118 (±9) mmHg. Blood pressure was measured throughout functional imaging: Control mean (SD) = 108/73 (±8/±5) mmHg; RIM mean (SD) = 97/69 (±10/±6) mmHg. In addition the average blood pressure change during “mild”, “moderate” and “high” electrical forepaw stimulation was recorded: Control mean (SD): “mild” = 0 mmHg (±0), “moderate” = 1 (±1), “high” = 7 (±4) mmHg; RIM mean (SD): “mild” = 0 mmHg (±0), “moderate” = 0 mmHg (±0), “high” = 3 (±1) mmHg. The von Frey tests in the RIM subjects returned a mean (SD) of 1.73 (±0.83) g, concordant with our previous characterisation of the RIM modelBlood gasses were sampled directly before and after fMRI acquisitions and were maintained within physiological range: Control mean (SD): pCOIn the same animals, “moderate” stimulation (2 mA) evoked widespread, regionally specific BOLD signals in the RIM cohort with activation still confined to the fpss in the control group . Visual 16As the electrical stimulus was further increased to 3.8 mA, widespread bilateral activation was observed in both control and RIM groups . HoweverA two-group random effect design was employed to investigate voxel-wise differences in BOLD contrast changes due to forepaw stimulation between control and RIM animals at each of the applied electrical forepaw stimulus intensities. After more than 10 years since the first fMRI study in FM patients711Despite prior clinical investigations, the mechanisms that trigger and sustain FM are poorly understoodTo our knowledge, this is the first report of functional activation of the nociceptive pathway during 2 mA electrical stimulus, normally considered to be innocuous in anaesthetised ratset al.,fMRI studies of FM patients have found significant increases in the BOLD response to the same acute painful stimuli , in the SI/SII, anterior cingulate cortex, insula, posterior cingulate cortex, superior temporal gyrus, cerebellum and inferior parietal lobule2 is slightly higher in the control group relative to the RIM group (38 vs 33 mmHg). However, the pCO2 in both groups is still within physiological range and is unlikely to account for the observed differences in BOLD response between the groups. Indeed we observed no correlation between the pCO2 and BOLD response across the 15 animals studied in this work within the ssfp regions at any of the stimulation strengths (data not shown). Furthermore the BOLD response to innocuous “mild” stimulation were highly similar between the RIM and control groups suggesting that possible respiratory differences do not account for the between-group differences observed at “moderate” stimulation.The fMRI measurements in this study were performed in the anaesthetized brain (1.0% isoflurane), which may confound the physiological relevance of data interpretation. Indeed a fMRI study in the awake rat revealed more widespread patterns of BOLD signal changes in response to noxious heat stimuli (though contributions of motion and stress cannot be easily dissected)To conclude, in this study we investigated the functional response to acute innocuous/noxious stimuli in the reserpine induced rat model of FM using fMRI. We observed discriminatory patterns of BOLD activations to “moderate” stimulation (found to be innocuous in the control group). This finding reveals the regional functional augmentation that underlies behavioural measures of tactile alloydinia in the RIM model, and mirrors observations in FM patients. Given the sensitivity and reproducibility of the fMRI measures applied to the non-surgical model, we suggest that this method may be highly advantageous for drug development prior to clinical assessment.All animal work included carefully applied endpoint definition (see below) and was approved by the Institutional Animal Care and Use Committee of the National Institute of Radiological Sciences . All the experiments in this study were carried out in accordance with the Institutional Animal Care and Use Committee of the National Institute of Radiological Sciences . The endpoint definition was as follows: i) observation of difficulty in breathing, spasm, self-mutilation or abnormal movement; ii) more than 20% body-weight loss per week; iii) the RIM model must be euthanized within 3 weeks after the final administration of reserpine or directly after fMRI experiments (non-recovery). All animal models were carefully bred and observed by animal technologists and no animal deaths were observed during the study. The RIM model was generated as described previouslyPrior to imaging, anaesthesia was induced and maintained on 2.0% isoflurane . The femoral vein and artery were cannulated using polyethylene catheters  for drug administration, monitoring of blood pressure and blood gas sampling. The rat was intubated to allow ventilation  during image acquisition. The animal was then transferred to the scanner bed where 2 needle electrodes were implanted subcutaneously between the second and fourth digit of the right forepaw. Forepaw muscle twitching was visually confirmed prior to subsequent infusion of muscle relaxant. Once the animal was transferred to the centre of the MRI scanner bore, isoflurane concentration was reduced to 1.0% in air/oxygen and the muscle relaxant gallamine  was infused into the femoral vein. During imaging, animal temperature was maintained at 36.5–37.5 °C using heated water tubing and a hot air fan.Images were acquired using a small animal 7 T MRI scanner . Signal transmission and reception was achieved using a single loop surface coil positioned dorsal to the head. Following acquisition of scout images, an automated field-mapping based shimming procedure was performed over the imaging volume . Functional images were acquired using a 4 shot segmented GE-EPI sequence with the following parameters: TE = 15 ms, TR = 500 ms, 2 averages [temporal resolution = 4 s ], matrix size = 96 × 96, field of view = 38.4 × 38.4 mm, 13 slices, slice thickness = 1 mm, slice gap = 0 mm. The 13 coronal slices (acquired in an interleaved manner) were manually positioned to contain the rostral edge of the pre-frontal cortex to immediately rostral to the cerebellum. Blood gasses were sampled prior to fMRI acquisitions  and ventilation parameters were adjusted until normal physiological range was achieved. Blood gasses were additionally sampled at the end of fMRI data collection. Blood pressure and core temperature were monitored continuously throughout imaging . Typical ventilation parameters were 70 breaths/min and 2.8 ml tidal volume.In order to investigate BOLD changes in response to electrical forepaw stimulation, a standard ‘task-based’ block design approach was used that consisted of 20 s forepaw stimulation  with 2 minutes inter-stimulus-interval, repeated 5 times for a total imaging time of ~11 minutes. The current of the electrical forepaw stimulation was modulated  for each fMRI timeseries. Each of these fMRI time-series (consisting of the block design described above) at constant electrical forepaw stimulation current were run three times in an interleaved manner .http://www.fil.ion.ucl.ac.uk/spm/ -described immediately below).In a single control animal, artefacts were present in the GE-EPI images in one of the three fMRI experiments at 2 mA stimulation current, due to RF interference. These data were excluded from the analysis. No other acquired data in the 15 subjects was excluded and data analysis was performed using an automated pipeline in the statistical parametric mapping (SPM) software (http://fmri.wfubmc.edu/software/pickatlas) and the marsbar toolbox (http://marsbar.sourceforge.net/) were used to extract the BOLD timeseries data from anatomically defined ROIs that were delineated from the parcellated rat brain atlas46For each subject, fMRI timeseries data at each of the three different applied electrical forepaw stimulus intensity values were averaged and registered to a rat brain atlas46To investigate possible differences in the magnitude of the BOLD signal within the ROIs , the mean BOLD signal % change from baseline during 20 s stimulation was calculated for each subject/forepaw stimulation intensity. Possible differences in the mean BOLD % change between the RIM and control subjects were investigated using Mann-Whitney (a non-parametric test was chosen given the sample size [8 control and 7 RIM rats]).http://warwick.ac.uk/snpm) with a minimum threshold of p < 0.001 and a cluster size of >5 voxels. After the MRI acquisitions, all animals were euthanized with a pentobarbital injection via femoral vein.A two group random effects voxel-wise analysis was performed to examine differences between the BOLD activation to forepaw stimulation in the control vs RIM cohort. The statistical non-parametric mapping toolbox was used ; glial fibrillary acidic protein (GFAP); caspase-3; ionized calcium-binding adapter molecule (Iba1). All histological data are reported in How to cite this article: Wells, J. A. et al. Functional MRI of the Reserpine-Induced Putative Rat Model of Fibromyalgia Reveals Discriminatory Patterns of Functional Augmentation to Acute Nociceptive Stimuli. Sci. Rep.7, 38325; doi: 10.1038/srep38325 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Within the concentration range of 5 × 10−7 ~50 × 10−7mol/L, the linear regression equation of IPL = 1226.3-13.6[CCu2+] (R = 0.99) was established for Cu2+ quantitative detection. And finally, Cu2+ fluorescence quenching mechanism of NPSi prober was proposed by studying the surface chemistry change of NPSi and metal ions immersed-NPSi using XPS characterization. The results indicate that SiHx species obviously contribute to the PL emission of NPSi, and the introduce of oxidization state and the nonradiative recombination center are responsible for the PL quenching. These results demonstrate how virgin NPSi wafer can serve as Cu2+ sensor. This work is of great significant to promote the development of simple instruments that could realize rapid, visible and real-time detection of various toxic metal ions.Developing an innovative “Test Paper” based on virgin nanoporous silicon (NPSi) which shows intense visible emission and excellent fluorescence stability. The visual fluorescence quenching “Test Paper” was highly selective and sensitive recognizing Cu Palestino et al.2+ detection. However, until now, no study about solid virgin NPSi as fluorescence sensor for the detection of Cu2+ had been reported. Based on virgin NPSi with intense and stable luminescent emission, in the present work, we have proposed a solid sensor that can monitor Cu2+ with the naked-eye by fluorescence quenching. The effect of surface morphology on chemistry structure and luminescent stability of NPSi also have been investigated. The Cu2+ sensing mechanism was researched based on the evolution of NPSi surface chemistry.Nanoporous Si (NPSi) has been recognized as a versatile material due to its unique physical and chemical characteristics, such as large surface area, strong fluorescence, tunable porous nanostructure and surface chemistry, biocompatibility, which has widely application in photoelectron devices151720After the etching, the color of samples show uniform greenyellow and becomes gradually shallow with the decreasing HF concentration. The SEM images show that numerous “ditches” with the depth of ~2 μm are present on entire sample surfaces , they caTEM characterization was employed to investigate more detailed morphology and structure of NPSi samples. The typical TEM images and corresponding (001) orientation electron diffraction patterns were shown in Moreover, the fluorescence stability of NPSi was tested in air ambient. The fluorescence emission of NPSi sample was excitated by ultraviolet lamp at the wavelength of 365 nm. Both fresh NPSi glow with the visible orange-red light , this lu252O3, and SiO2 (x (x = 1~3) species, which may be oxidized by O2 in ambientx completely disappeared according to the deconvoluted Si 2p peak of sample II, which means that the sample surface is totally covered by the oxidizing species of Si2O3 and SiO2  of sample I and II were characterized after aging under air ambient for one week. For sample I, the Si 2p peak can be deconvoluted into four bands at 99.5 eV, 100.4 eV, 102.4 eV, 103.5 eV, and 104.4 eV, which can be assigned to the Si, SiH, SiO, Siand SiO2 . As knowand SiO2 . The res2+. Various heavy metal ions solution  were prepared. The concentrations of interference metal ions and target metal ions are respectively 500 μmol/L and 10 μmol/L. To evaluate the selectivity of NPSi sensor, the freshly prepared NPSi sample was immersed into different metal ions solution for 30 min. After that, the samples were removed and dried under nitrogen for fluorescence characterization. The corresponding ultraviolet lamp excited fluorescence photos and the photoluminescence spectra were shown in 2+. Due to the capture of excited carriers and the interruption of the radiative recombination, Cu2+ could dramatically reduce the quantum efficiency of NPSi2+.The resultant sample I with excellent fluorescence stability was selected as fluorescence probes to detect Cu2+ was carried out to investigate the working principle. The PL intensity (IPL) at ~575 nm shows continuous quenching with the increasing Cu2+ concentration  of 0.99.Furthermore, the fluorescent sensing of NPSi toward Cuntration . The intx groups are the most distinctive species on the fresh NPSi surface and benefit for the intense luminescence emission33x groups of fresh and immersion-treated sample were characterized in −1 are assigned to SiH and SiH2 stretching modes, and the signal of SiH3 species at the band of ~2140 cm−1 is overlapped in the wide band regionxgroups decrease when NPSi sample is immersed into aqueous solution, especially for the Cu2+ immersed sample it has a dramatic intensity reduction, which may be attributed to the oxidization introduction caused by Cu2+ species, and the corresponding reaction can be described as follow. Meanwhile, a slight peak shift can be observed for all of the aqueous solution immersed samples, which can be attributed to the higher polar effect of aqueous solution than that of alcohol.Numerous research has confirmed that the surface chemical species has a crucial effect on luminescence properties of NPSix peak, the slight decay of PL intensity may be mainly caused by the oxidization of H2O in the solution.We can also note that the spectra intensity for those NPSi immersed into solution containing various metal ions species all show a slight reduction, and the difference between of them is not evident, thus, we can deduce that the selected interference ions species has no obvious effect on intensity reduce of SiH2+ on NPSi surface, the fresh and Cu2+-immersed NPSi sample were characterized by XPS spectroscopy, and the results were shown in 2+-immersion, several new peaks present between the range of 550~730 eV can be assigned to metal copper (Cu) species. It indicates that copper ions are reduced during the immersion step, and leading to the further oxidation of NPSi surface, this is confirmed by the increasing intensity of oxygen peaks. Furthermore, the corresponding Si 2p peaks were deconvoluted and analyzed. For the un-immersed NPSi sample, the Si 2p peak mainly locate at 99.8 eV and has a broadened peak at the higher binding energy, which is attributed to that the bind between Si and H, O. This Si 2p peak can be deconvoluted into six peaks which can be respectively assigned to Si, SiH, SiH2, SiH3, Si2O, and SiO  has the most relatively positive potential, its electroless deposition is the most likely to occur on NPSi surface . By comparing the surface chemistry structure evolution of NPSi sample, fluorescence quenching mechanism of NPSi probe was investigated and we concluded that SiHx species obviously contribute to the PL emission of NPSi. Formation of oxidization state and the nonradiative recombination center caused by the preferential deposition of Cu2+ions are responsible for the PL quenching, and it is mainly attributed to its more positive redox potential of copper ions species.An innovative “test paper” based on the solid flake-like nanoporous silicon has been developed and which shows excellent fluorescence stability. Fluorescence quenching in as-prepared NPSi probe was highly selective and sensitive with copper ions in solution, it exhibits the visual qualitative recognition capability for Cu2, and then respectively cleaned in ethanol and distilled water for 10 min under sonication. The native oxidation layer was removed by immersing the specimens in 5 wt.% HF aqueous for 20 min. The treated wafers were electrochemically etched in the double-tanks electrochemical cell. The mixture solutions of HF (with concentration of 20 wt.%) and ethanol with volume ratio 1:4 and 1:5 were selected as etchant, etching current density of 25 mA/cm2 was imposed by dc power supply, etching time was 60 min, and the resulting samples were respectively marked by sample I and sample II. After etching, the NPSi samples were rinsed with absolute ethanol and dried under dry N2 stream. The stock solutions of Cd, Co, Pb, Mn, Zn, Ni, Cr and Cu were prepared by dissolving metal salts in ultrapure water, which contains CdCl2, CoCl2·6H2O, PbCl2, MnCl2·4H2O, MnCl2·4H2O, NiCl2·6H2O, CrCl3·6H2O and CuCl2·2H2O, they were all purchased from China National Medicines Corporation Ltd. The test processes were implemented by immersing NPSi sensor into 100 ml solution with a fixed metal ions concentrations for 10 min, the immersion solution can be prepared by diluting the stock solution without any pH adjustion. After the test, the NPSi was rinsed with DI water and dried under N2 stream.Silicon wafers were purchased from 46th institute of semiconductor of China. P(100)-type single crystal Si wafers with resistivity of 0.01~0.09 Ω·cm were cut into small pieces with a size of ~1 × 1 cmNPSi layer morphology was characterized by scanning electron microscopy . The detail characterization were performed by the JEM-2100 high-resolution transmission electron microscope . The samples were prepared by spreading NPSi layer into ethanol and then salvaging with copper grids. The HRTEM characterization was operated at 200 kV. The HRTEM images were analyzed to obtain interplanar spacing (d) and the diffraction pattern. The photoluminescence (PL) spectra of solid NPSi wafer were measured by F-320 fluorescence spectrophotometer at the room temperature, and the excitation wave length was 365 nm. The FTIR characterization was performed by fourier transform infrared spectroscopy (Bruker EQUINOX 55). XPS analysis was performed by the PHI5600 X-ray photoelectron spectroscopy .How to cite this article: Li, S. et al. An Innovative Metal Ions Sensitive ‘‘Test Paper’’ Based on Virgin Nanoporous Silicon Wafer: Highly Selective to Copper(II). Sci. Rep.6, 36654; doi: 10.1038/srep36654 (2016).


There is today no gold standard method to accurately define the time passed since infection at HIV diagnosis. Infection timing and incidence measurement is however essential to better monitor the dynamics of local epidemics and the effect of prevention initiatives.Three methods for infection timing were evaluated using 237 serial samples from documented seroconversions and 566 cross sectional samples from newly diagnosed patients: identification of antibodies against the HIV p31 protein in INNO-LIA, SediaTM BED CEIA and SediaTM LAg-Avidity EIA. A multi-assay decision tree for infection timing was developed.Clear differences in recency window between BED CEIA, LAg-Avidity EIA and p31 antibody presence were observed with a switch from recent to long term infection a median of 169.5, 108.0 and 64.5 days after collection of the pre-seroconversion sample respectively. BED showed high reliability for identification of long term infections while LAg-Avidity is highly accurate for identification of recent infections. Using BED as initial assay to identify the long term infections and LAg-Avidity as a confirmatory assay for those classified as recent infection by BED, explores the strengths of both while reduces the workload. The short recency window of p31 antibodies allows to discriminate very early from early infections based on this marker. BED recent infection results not confirmed by LAg-Avidity are considered to reflect a period more distant from the infection time. False recency predictions in this group can be minimized by elimination of patients with a CD4 count of less than 100 cells/mm3 or without no p31 antibodies. For 566 cross sectional sample the outcome of the decision tree confirmed the infection timing based on the results of all 3 markers but reduced the overall cost from 13.2 USD to 5.2 USD per sample.A step-wise multi assay decision tree allows accurate timing of the HIV infection at diagnosis at affordable effort and cost and can be an important new tool in studies analyzing the dynamics of local epidemics or the effects of prevention strategies. Measurement of HIV incidence is an essential instrument for reliable monitoring of the effect of preventive interventions like generalized treatment, early treatment, pre-exposure prophylaxis or vaccines. HIV surveillance in Western-European countries is mostly based on case reporting of new cases but defining the infection prevalence is less well suited for tracking trends in HIV transmission as it is prone to biases resulting from shifts in testing rates or changes in populations at risk. UNAIDS provides guidelines for incidence measurement  and manySeveral assays have been developed that rely on characteristics of the humoral immune response after infection to estimate recency of infection, such as the increase in anti-HIV IgG antibody concentration relative to total IgG concentration and the increase in anti-HIV antibody affinity. Ideally, these assays should have a false recency rate of less than 2% . The strSystematic incidence analysis also has logistic and financial implications especially when multiple assays are involved. Serial testing approaches or decision trees maintain accuracy but control costs by reducing the number of samples for extensive testing . They arThe following markers of infection time were evaluated: absence of antibodies against the HIV-1 p31 antigen as visualized on the immunoblot confirmation test , low HIV IgG concentration as defined by Sedia™ BED HIV-1 Incidence Enzyme Immuno Assay  and low HIV antibody avidity as defined by Sedia™ HIV-1 Limiting Antigen Avidity Enzyme Immuno Assay . Strengths and weaknesses of these markers alone and in combination were defined on serial samples from seroconverters, on samples from patients with particular conditions and on cross-sectional samples from the target population. Based on the obtained results a decision tree for future infection timing was developed.Table The longitudinal sample series comprised 237 serial plasma samples from 42 patients with a documented seroconversion. The mean number of samples tested per patient was 7 (range 4 to 15) and the mean total follow-up period was 696 days (range 67 to 2364 days). The first sample collected from each patient (day 0) showed a reactive p24 antigen test but no detectable antibodies in the INNO-LIA HIV I/II Score.n = 74), F (n = 63), A (n = 33), C (n = 26), CRF01_AE (n = 17), G (n = 11), other CRF (n = 9) and D (n = 7).The cross-sectional sample series consisted of single samples collected at diagnosis or a maximum of 6 weeks thereafter from 566 individuals diagnosed with HIV-1 in Belgium between 2012 and 2014. All patients were therapy naïve and had a detectable viral load (>20 c/ml) at the time of sample collection. The HIV subtype was available for 511 individuals, the majority (53.0%) were infected with a subtype B virus. The other subtypes and circulating recombinant forms (CRF) represented were CRF02_AG (3).The sample series of patients with particular conditions known to be associated with a higher likelihood for false recency predictions contained single samples from 12 patients on antiretroviral therapy (ART) ), from 11 elite controllers , from 6 slow progressors  and from 19 advanced infections .– or ± was given and present when the score was 1+ or more. For the longitudinal samples, INNO-LIA was performed on serial samples until presence of p31 antibodies was documented. For the samples from selected populations an INNO-LIA was performed in the context of this study, for the cross-sectional samples from newly diagnosed patients the results of the diagnostic assay were used.The INNO-LIA HIV I/II Score assay (Fujirebio) was performed according to the manufacturers’ instructions. Using the band intensity of the three internal standards, antibody reaction to the individual HIV antigens was scored as -, ±, 1+, 2+ or 3+ either visually or by the automated LIRAS reading system (Fujirebio). Antibodies against the integrase protein p31 were considered absent when a score of n > 1.2 for BED CEIA or >2.0 for LAg-Avidity EIA. For samples with OD-n ≤ these respective cut-offs, retesting in triplicate was performed as recommended and discrimination between recent and long term infection was based on median OD-n > 0.8 for BED CEIA and >1.5 for LAg-Avidity EIA.Sedia™ BED CEIA  and Sedia™ HIV-1 LAg-Avidity EIA (Sedia Biosciences Corporation), were performed according to the manufacturers’ instructions. Results are normalized using an internal calibrator and reported as normalized optical densities (OD-n). Attribution of long term infections was based on a single measurement and OD-protease and reverse transcriptase genes gathered for the purpose of baseline resistance testing were used for subtype assignment using Rega v3, http://dbpartners.stanford.edu:8080/RegaSubtyping/stanford-hiv/typingtool/  [http://comet.retrovirology.lu/  [Sequences of the HIV-1 Belgium)  and Comeembourg) . The subhttps://www.medcalc.org/manual/outliers.php; last accessed on November, 29th 2017). Fisher’s exact test was used to compare the representation of patients with low CD4 count amongst the congruent and incongruent predictions.Rosner’s Extreme Studentized Deviate (ESD) test with a significance level set at 0.05 was used to identify outliers  sample was considered as day 0. Antibodies against the p31 antigen were absent in 28.7% of the samples and were first detected in a sample collected at day 32. The median time point between the last sample without p31 antibodies and the first sample with p31 antibodies was day 64.5 (IQR 33.5 – 89.0) Fig. . With thFigure The 5 patients with outlier results were all infected with a subtype B virus. Three of the 4 patients with an extended recency window for either both EIAs and p31 antibodies .High rates of false recent predictions were observed in patients on ART and in elite controllers Table , with BEAn overview of the results for the 566 cross-sectional samples from newly diagnosed patients is shown in Table n = 18, 3.2%) with the highest number resulting from false recent predictions, 17 for p31 antibodies, 8 for BED CEIA and 1 for LAg-Avidity EIA.For subsequent analysis, results that followed the expectations considering the differences in recency window between the assays were called congruent and results that could not be explained by the recency window were called incongruent Table . The tot3; p < 0.01) but influence of viral load or subtype was not significant. Of note, 7 of the 9 patients classified as long term infections by both EIAs but in whom p31 antibodies were absent were subtype B infections.To further identify potential reasons for incongruences, viral load and subtype distribution were compared between the classification groups. The results showed that incongruences were observed significantly more frequently in patients with a low CD4 count  may serve this purpose needs to be examined.The choice for BED CEIA in first line is justified by its high reliability for the identification of long term infections and its lower price (approximately 450 USD per kit versus 650 USD for LAg-Avidity EIA). The use of BED CEIA in first line also allows to profit from the longer recency window of this assay and provides a way to discriminate between ‘early’ and ‘recent’ infections. The classifications ‘very early’ or ‘early’ infection will always be based on a predicted recent infection by both EIAs and is therefore highly reliable. The classification ‘recent’ infection will be only based on a predicted recent infection by the BED CEIA, an assay with a higher false recent rate. Despite a correction for false receny predictions based on the CD4 count and the p31 antibody presence or absence this category may suffer from some erroneous classifications. For this study the information on the presence or absence of p31 antibodies is deduced from the INNO-LIA confirmatory assay. Whether the more recently developed immunochromatographic assay, GeeniusThis study was performed in a country with a heterogeneous HIV epidemic , 32. TheAny incidence measurement that is based on specific features of the humoral immune response will be prone to errors because of the natural variability in antibody production and maturation. Quantification of the genetic variability of the virus has been suggested as an alternative marker of infection time  but the Serial testing algorithms for HIV incidence measurement have been presented before , 36. UsiTaken into account the strengths and weaknesses of different markers of infection time, a stepwise decision tree was set up that allows to classify, at diagnosis, an HIV infection as very early (infection of less than 3 months before), early (less than 4 months before) and recent (less than 6 months before). Being able to discriminate these early infection phases will be an important asset in studies aimed at analyzing the dynamics of local epidemics and the effects of prevention strategies.


Objective: Because of functional impairment caused by generalized anxiety disorder and due to cognitive side effects of many anti-anxiety agents, in this study we aimed to evaluate the influence of Passion flower standardized extract on reaction time in patients with generalized anxiety disorder.Method: Thirty patients aged 18 to 50 years of age, who were diagnosed with generalized anxiety disorder and fulfilled the study criteria, entered this double-blind placebo-controlled study. Reaction time was measured at baseline and after one month of treatment using computerized software. Correct responses, omission and substitution errors and the mean time of correct responses (reaction time) in both visual and auditory tests were collected. The analysis was performed between the two groups and within each group utilizing SPSS PASW- statics, Version 18. P-value less than 0.05 was considered statistically significant.Results: All the participants were initiated on Sertraline 50 mg/day, and the dosage was increased to 100 mg / day after two weeks. Fourteen patients received Pasipy (Passion Flower) 15 drops three times daily and 16 received placebo concurrently. Inter-group comparison proved no significant difference in any of the test items between assortments while a significant decline was observed in auditory omission errors in passion flower group after on month of treatment using intra-group analysis.Conclusion: This study noted that passion flower might be suitable as an add-on in the treatment of generalized anxiety disorder with low side effects. Further studies with longer duration are recommended to confirm the results of this study. The Generalized Anxiety Disorder (GAD) is defined as the basic anxiety disorder, which may reflect the fundamental process of all emotional disorders and significant degree of functional impairment . GAD is Response procedure is directly based on circumstances . Many faPassion flower symbolizes the passion of Jesus in Christian theology because of its unique structure .Traditionally its extract has been used as an herbal remedy for nervous anxiety  and insResearch ParticipantsThirty outpatients entered this randomized double-blind placebo-controlled study . The participants were included in the study from Roozbeh and Baharloo hospitals and private psychiatric offices during 2010- 2012. Patients were diagnosed with Generalized Anxiety Disorder (GAD) based on DSM-IV criteria and clinical interviews. Their family history was considered as well. They were tested using Hamilton Anxiety Rating Scale Form A (HARS). Hamilton Rating Scale for Depression (HRSD) was utilized to determine the patients’ comorbid depression. The Hamilton Scales were standardized for Iranian patients.Patients between 18 to 24 years of age were included. In addition, sertraline consumption was considered the best treatment for their current disease per decision of the psychiatrist. All patients were initiated on Sertraline. The exclusion criteria were as follows: Having difficulty including allergic reactions to sertraline or active ingredients of passion flower, renal or hepatic impairment, age under 18, pregnancy and lactation, consuming Warfarin, Hexobarbital, Pantobarbital, Levothyroxine or other thyroid medications, using alcohol or hallucinogens and history of tachycardia. The patients with a history of kidney or liver dysfunction were excluded. An informed consent was obtained from the patients prior to the initiation of the examination. MedicationThe first-line treatment for GAD patients was 50 mg Sertraline tablet for both groups. Pasipy® Drop - Iran Darouk Co. was the standardized hydroalcoholic extract of passion flower as an add-on therapy. Placebo consisted of 20% aqueous solution of absolute edible alcohol and natural coloring agents. The placebo mixture was filled in amber glass bottles with dropper identical to the drug container.AssortmentThe participants were randomly assigned into two groups to receive either Sertraline + Pasipy (S-drug group), or Sertraline + placebo (S-placebo group) for one month. All patients were initiated on Sertraline 50 mg/day; the dosage was increased to 100 mg/day after two weeks. Pasipy and its placebo were given at 15 drops three times daily.Data Collection ToolsThe Reaction Time (RT) test was utilized as the standard computerized software. These process measured psycho-neural responses toward visual and auditory stimuli. The input variables were the number of correct responses, omission and substitution errors and the mean time of correct responses (mean reaction time) . After rStatistical AnalysisDemographic characteristics were compared between the two groups. The RT test outputs were analyzed once in comparison between S-drug and S-placebo groups using independent sample t-test (inter-group comparison); then reaction time changes after one month was determined in each group using a paired sample t-test (intra-group comparison). Scores from the Hamilton anxiety scale form A (HAM-A) were compared between S-drug and S-placebo groups using an independent sample t-test. The aim was to reconfirm the positive effect of passion flower on GAD and the possible improvement of the add-on therapy encountered with the SSRI monotherapy. All the comparisons were performed utilizing SPSS software (PASW – statistics 18). A p-value of less than 0.05 was considered as the minimal level of statistical significance in all measures.Seventy patients were selected for the study; of whom, 24 were excluded as they did not meet our criteria, and 16 did not follow the medication protocol because of low compliance and drug incompatibility. The patients who met the inclusion criteria were randomized by permeated block randomization .Fourteen patients  were initiated on Sertraline (50 mg/day and the dosage was increased to 100 mg/day after two weeks) + Pasipy (15 drops three times daily). The mean age ± standard deviation (SD) of these patients was 29.07 ± 8.60. Sixteen patients  were initiated on Sertraline (50 mg/day and the dosage was increased to 100 mg/day after two weeks) + placebo (15 drops three times daily). The mean age in this group was 32.19 ± 11.43.Inter-group analysisAfter one month, independent sample t-test did not demonstrate any significant difference in any of visual or auditory items. Baseline scores were proved not to be statistically different, but they are not displayed in the tables. In the visual test for the drug group, the omission errors were less than the placebo consumers, but this difference was not statistically significant (P = 0.666). However, the mean reaction time was slightly longer in this group (P = 0.720). In auditory analysis for the drug group, omission errors were less than the placebo group, but the difference was not significant (P = 0.476). However, the mean reaction time toward sound threats improved slightly after one month of taking Pasipy in the drug group compared to the placebo group (P = 0.467) .Intra-group AnalysisIn the drug group, a significant decline in auditory omission errors was observed after one month of treatment (P = 0.045). The mean reaction time had a non-significant increase in both visual (P = 0.288) and auditory tests (P = 0.484) in drug intra-group analysis. None of the changes in test variables in placebo consumers reached the significant level. The mean reaction time was a bit longer in the visual test (P = 0.549), but had a non-significant improvement toward auditory  stimuli in this group (P = 0.312) .Hamilton TestHamilton Anxiety Rating Scale Form A (HARS) questionnaires were ranged between 18 to 24 (mild to moderate). A significant improvement to relieve anxiety symptoms was observed in the add-on therapy group compared to the Sertraline + placebo after one month of administration (P = 0.039) .Adverse ReactionsBased on data from ‘Fear appeal’ is a brain message against threatening situations . It is a. PassioThe limitations of the present study were as following: Firstly, the sample size was relatively small. Secondly, one month may not be considered long enough to precisely evaluate the effects of Passion flower extract. Thirdly, "structured interview", a more precise mean of evaluation of the patients, was not utilized in this study.This study noted that passion flower might be consumed as a safe (low side effects) add-on in the treatment of generalized anxiety disorder. Further studies with longer duration are recommended to confirm the results of this study.


Here we test how expression of the cyanobacterial, bifunctional fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) affects carbon assimilation and seed yield (SY) in a major crop . For three growing seasons, wild-type (WT) and FBP/SBPase-expressing (FS) plants were grown in the field under ambient (400 μmol mol−1) and elevated (600 μmol mol−1) CO2 concentrations [CO2] and under ambient and elevated temperatures  at the SoyFACE research site. Across treatments, FS plants had significantly higher carbon assimilation (4–14%), Vc,max (5–8%), and Jmax (4–8%). Under ambient [CO2], elevated temperature led to significant reductions of SY of both genotypes by 19–31%. However, under elevated [CO2] and elevated temperature, FS plants maintained SY levels, while the WT showed significant reductions between 11% and 22% compared with plants under elevated [CO2] alone. These results show that the manipulation of the photosynthetic carbon reduction cycle can mitigate the effects of future high CO2 and high temperature environments on soybean yield.Predictions suggest that current crop production needs to double by 2050 to meet global food and energy demands. Based on theory and experimental studies, overexpression of the photosynthetic enzyme sedoheptulose-1,7-bisphosphatase (SBPase) is expected to enhance C SBPase is unique to the PCR cycle and catalyzes the dephosphorylation of sedoheptulose-1,7-bisphosphate to sedoheptulose-7-phosphate at the branch point of RuBP regeneration and carbon export  cycle. Using a complete dynamic model of photosynthetic carbon metabolism coupled to an evolutionary algorithm, A  was expressed in tobacco , a major crop species and the most widely grown legume worldwide  and is predicted to increase to 371 Mt by 2030 . Given the limited availability of farmland and the negative environmental impacts of conversion of natural ecosystems, increases in soybean yield per area are a necessity for future sustainable increases in production. Experiments with soybean under elevated [CO2] and elevated temperatures indicate that CO2 alone will not increase yields in the future. In a field study, 2] was not able to mitigate the yield losses attributed to warming. This observation further emphasizes the importance of adapting crops to warmer and CO2-enriched environmental conditions.The interaction between increased SBPase activity and future growth conditions was tested in the present study using soybean  combined with canopy heating of +2.7 °C during the day and +3.4 °C at night. The full factorial experiment was replicated over three growing seasons (2013–2015) at the SoyFACE research facility in central Illinois, USA. We hypothesized that expression of the cyanobacterial FBP/SBPase bifunctional enzyme would increase RuBP regeneration capacity (Jmax) and thus A under environmental conditions that favor RuBP regeneration-limited A , and that the increases in A would be reflected in higher yields.To investigate if increased SBPase activity will increase yield in soybean under future climate, we grew WT and FBP/SBPase-expressing (FS) plants side by side under ambient and elevated [COGlycine max (L.) Merr. cv. ‘Thorne’] WT and FS plants derived from the same cultivar were grown in a complete block design (n=4) in the Soybean Temperature by Free Air CO2 Enrichment (Soy-T-FACE) experiment at the SoyFACE field site near Urbana-Champaign, IL, USA  during the 2013, 2014, and 2015 growing seasons. The experiment consisted of four blocks, each containing one ambient and one elevated [CO2] plot. Within each plot was nested an unheated and a heated subplot. Each subplot was further divided with plantings of the WT and FS lines. Seeds were planted by hand at 5 cm intervals in 38 cm rows. Eight 11 m long rows, four of WT and four of FS, were planted next to each other in each of the eight plots. Planting and harvest dates are given in 2] plots were at ~400 μmol mol−1 and the elevated plots were fumigated to ~600 μmol mol−1 using free air CO2 enrichment (FACE) technology  connected to data-loggers . Canopy temperature measurements were collected every 5 s to control the heater output and 10 min mean values were stored. The actual mean season canopy temperatures for the four treatments in the three seasons are given in Ac’ , ‘Ah’ (ambient [CO2]+heated), ‘Ec’ , and ‘Eh’ (elevated [CO2]+heated).During the day (6:00 h to 18:00 h), and with rainy days excluded, mean temperature differences between the subplots were between 0.5 °C and 1.0 °C lower than the target set point , resultihttp://mrcc.isws.illinois.edu/CLIMATE/, last accessed 2 August 2016), 1 km west of the site, and the photosynthetically active radiation  data from the University of Illinois Energy Farm , 2 km to the east. Air temperature during the June–October growing season was close to the 20 year average (19.9 °C) in all three years. In comparison with the 20 year averages of the annual sum of precipitation (922 mm) and the sum of precipitation during the June–October growing season (446 mm), precipitation was slightly lower in 2013 and higher in 2014 and 2015 (−2 s−1 (2013), 2388/721 μmol m−2 s−1 (2014), and 2236/724 μmol m−2 s−1 (2015).Weather data for all three seasons were available from nearby weather stations. The air temperature and precipitation data  were obtand 2015 . PAR wasGlycine max (L.) Merr. cv. ‘Thorne’ was previously transformed  using the Agrobacterium-mediated method  promoter, coupled with the tobacco etch translational enhancer element, and attached to the pea Rubisco small subunit transit peptide for transport of the enzyme across the plastid membrane . The transgene cassette was subcloned into the binary vector pPTN200 which harbors a Pnos bar cassette for selection. Greenhouse and field phenotyping and molecular characterizations are described in nsformed  with thed method  to test 7, T8, and T9 populations derived from homozygous T3 and T4 populations of this line. T5 and T6 populations were multiplied in the field in 2011 and 2012 to provide enough seed for the start of the 3 year experiment. WT and FS plants were grown side by side, and only seeds harvested from the control treatment were used for planting in the following growing season. The presence of the transgene was reconfirmed in these subsequent generations using quantitative reverse transcription–PCR (data not shown) coupled with western blotting to test for the encoded protein.Due to spatial constraints of the experimental area and statistical considerations, we compared a single transgenic line with the WT. We used the same homozygous line (480-8) selected previously , as it sin situ gas exchange measurements were collected. Additionally, the levels of transketolase were assessed as a loading control, and levels of native SBPase were assessed to check if expression of the bifunctional enzyme affected levels of the native enzyme. Three leaflets from three different plants in each treatment were cut and immediately plunged into liquid nitrogen. Leaflets were taken from the most recently fully expanded leaves at the top of the canopy.The level of bifunctional FBP/SBPase protein was assessed in fully expanded leaves that were sampled in the field at noon on the same days when 2; 1 mM EDTA; 1 mM EGTA; 10% glycerol; 0.1% Triton X-100; 2 mM benzamidine; 2 mM aminocaproic acid; 0.5 mM phenylmethylsulphonyl fluoride; 10 mM DTT] using a pre-cooled mortar and pestle at 4 °C. The resulting suspension was clarified at 10000 g for 2 min at 4 °C, and the supernatant was transferred to separate tubes for protein quantification and western blotting. Protein quantification was assessed by Bradford Assay . A 200 μl aliquot of 313 mM Tris–HCl (pH 6.8), 10% SDS, 25% glycerol, 25% 2-mercaptoethanol was added to the protein samples for western blotting, boiled, and diluted to a uniform protein concentration. Samples were loaded on an equal protein basis (10 μg of soluble protein) on a 4% stacking gel, separated using 10% (w/v) SDS–PAGE, and transferred using a Mini Trans-Blot cell (Bio-Rad) to a nitrocellulose blotting membrane (Amersham Protran). The membrane was blocked in 6% (w/v) skim milk  in phosphate-buffered saline (PBS) and incubated with a primary antibody , dilution 1:5000) in 3% skim milk in PBS at 4 °C overnight. SBPase polyclonal antibodies were raised in rabbits against Arabidopsis SBPase, and TK antibodies were raised against tobacco plastid TK as described in Samples were stored at −80 °C before grinding in liquid nitrogen, and the resulting powder was subsequently stored at −80 °C until analysis. Subsamples of 100 μl of powder were mixed with 600 μl of protein extraction buffer  was set to 410 μmol mol−1 and 610 μmol mol−1 for the ambient and elevated [CO2] plots in order to match plot treatment conditions within the leaf chamber after the leaves lowered the [CO2]. Ambient PAR was measured immediately before each time point using the LI-190 installed on the LI-6400. The output of the chamber light-emitting diodes (LEDs) was set to deliver the same PAR and was maintained at these values throughout the course of all measurements at that time point. Relative humidity (RH) in the leaf chamber was not controlled directly, but was targeted to be kept between 50% and 70% during the measurement, which was achieved for 90% of the data. Environmental conditions during the in situ measurements are presented in Gas exchange measurements were conducted on 2 (2013), 5 (2014), and 4 (2015) d during the growing seasons , coverinIn 2013 each of the four LI-6400 systems was randomly assigned to one block and the measurements conducted on the subplots in randomized order. In 2014 and 2015, two teams, each equipped with two LI-6400 gas exchange systems assigned at random, conducted the measurements in parallel per block, with one instrument assigned to the heated subplot and the other to the unheated subplot. At each time point, instruments stayed assigned to the same temperature treatment within a block but were switched on transition to the next block to avoid confounding any undetected instrument difference with a treatment. All measurements for a given time point were completed within 1.5–2 h.Vc,max) and Jmax were determined from A–Ci curve measurements of A at varying intercellular [CO2] following the protocol of A–Ci curve measurements were conducted within 2 d before or after the in situ gas exchange measurements. Measurements on DOY 255 in 2014 were excluded from the analysis as leaves were starting to senesce. The petiole of the youngest fully expanded leaf from two plants per subplot was cut close to the stem and then immediately re-cut in water. Leaves were transported to the lab in an opaque box and were kept at 20 °C and under low-light conditions (PAR <10 μmol photons m−2 s−1) until measurement. Leaves were exposed to high light (1000 μmol m−2 s−1 PAR) for 10–15 min before clamping the leaf cuvette onto the middle leaflet. Conditions in the leaf cuvette were set to reflect the treatment [CO2] (410 μmol mol−1 or 610 μmol mol−1), the light level was set to 1500 μmol m−2 s−1, leaf temperature was controlled at 25 °C (±0.3 °C SD), and mean vapor pressure deficit during the measurements was 1.4 kPa (±0.2 kPa SD). After ~5 min, the leaf had reached steady-state A and the A–Ci curve autoprogram was initiated. [CO2] was decreased stepwise to 50 μmol mol−1 or 100 μmol mol−1, then increased back to the starting value (410 μmol mol−1 or 610 μmol mol−1) and then stepwise up to 1100 μmol mol−1 or 1500 μmol mol−1, depending on the CO2 treatment and developmental stage. Measurements of A were recorded at each [CO2] set point after stability was reached. Vc,max@25 °C and Jmax@25 °C were derived from a curve-fitting procedure of the underlying biochemical models , temperature , genotype , and day of the measurement , which was included as a repeated measure. Block was included as a random factor. The model for end of season yield data included the fixed factors [CO2], temperature, and genotype, and block as random factor. Significant differences between least square means for a priori determined comparisons were analyzed using post-hoc tests . Probability for statistical significance was set at P<0.1 a priori to reduce the possibility of type II errors.Gas exchange and yield data were analyzed using a mixed-model analysis of variance , taking into account the split-plot design of the experiment. The analyses were conducted separately for individual years. The model for the diurnal gas exchange data was analyzed separately by time of day  and included the fixed factors treatment, but it is unclear why this proposed mechanism, if it occurred, was not beneficial in the heat treatment under ambient [CO2].In addition to the expected increase in S plants , indicatA in our experiment did not always differ under high light and high temperature conditions, indicating that factors beyond temperature and light may play a role. A during the vegetative stage and not during the reproductive stage in the field. Under controlled environment conditions, A with maturation. We observed no differences in A during the early vegetative developmental stages (V3–V5) even under high light levels of up to 2000 μmol m−2 s−1. Differences here compared with previous work might result from the use of different species (tobacco versus soybean), the type of promoter used [Cauliflower mosaic virus (CaMV) 35S versus Peanut chlorotic streak caulimovirus (PCISV)], differences between the SBPase and FBP/SBPase enzyme, and/or the growth conditions (field versus controlled environment).The measured rates of A in the tobacco plants (over)expressing either SBPase or the bifunctional FBP/SBPase enzyme translated into higher biomass in all three previous studies , FS plants maintained SY at the same level as under the Ec (control temperature and elevated [CO2]) treatment, while WT SY was reduced by 11% to 22% in the three years. This difference was related to a higher number of pods of FS plants in 2013 and 2015 and a higher seed weight in 2014, indicating differences in allocation between the WT and FS plants. Accordingly, HI was also increased significantly for the FS plants. The observed consistent reduction of WT SY under heat treatment in our three season long experiment was similar to the effect reported by These differences in the responses of A in field-grown soybean and prevents yield losses under high [CO2] and high temperature, conditions that are expected for the near future. These findings are the first to show with a major food crop under open-air field conditions that manipulation of the PCR cycle can be used to mitigate the effects of global increases in temperature on yield under future elevated CO2 conditions.In conclusion, we show that expression of the cyanobacterial, bifunctional FBP/SBPase generally leads to higher Supplementary DataClick here for additional data file.


MTNR1B, HMGA2, SH2B3, KCNAB1, L3MBTL3, GCK, EBF1, TCF7L2, ACTL9, CYP3A7) were associated with offspring birth weight at P < 5 × 10−8. In SEM analyses, at least 7 of the 10 associations were consistent with effects of the maternal genotype acting via the intrauterine environment, rather than via effects of shared alleles with the fetus. Variants, or correlated proxies, at many of the loci had been previously associated with adult traits, including fasting glucose  and sex hormone levels (CYP3A7), and one (EBF1) with gestational duration. The identified associations indicate that genetic effects on maternal glucose, cytochrome P450 activity and gestational duration, and potentially on maternal blood pressure and immune function, are relevant for fetal growth. Further characterization of these associations in mechanistic and causal analyses will enhance understanding of the potentially modifiable maternal determinants of fetal growth, with the goal of reducing the morbidity and mortality associated with low and high birth weights.Genome-wide association studies of birth weight have focused on fetal genetics, whereas relatively little is known about the role of maternal genetic variation. We aimed to identify maternal genetic variants associated with birth weight that could highlight potentially relevant maternal determinants of fetal growth. We meta-analysed data on up to 8.7 million SNPs in up to 86 577 women of European descent from the Early Growth Genetics (EGG) Consortium and the UK Biobank. We used structural equation modelling (SEM) and analyses of mother–child pairs to quantify the separate maternal and fetal genetic effects. Maternal SNPs at 10 loci ( S1The basic characteristics of study participants in the EGG Consortium discovery, EGG follow-up and UK Biobank GWAS analyses are presented in P < 5 × 10−8  were identified in UK Biobank data only, and the index SNPs were unavailable in the EGG Consortium data. Consideration of results for proxy SNPs at these three loci from the EGG meta-analysis is in the next section. For the index SNPs at the other seven loci, we observed no strong evidence of heterogeneity in allelic effects between the EGG Consortium and UK Biobank components of the meta-analysis (r2 < 0.95), but the index SNP in SH2B3, rs3184504, is a non-synonymous coding variant (R262W). Approximate conditional analysis  showed no evidence of secondary signals at any locus at P < 5 × 10−8. In combination, the 10 loci explained 1.4% [standard error (SE) = 1.2%] of variance in birth weight, whereas the variance in birth weight captured by all autosomal genotyped variants on the UK Biobank array was considerably greater: 11.1% (SE = 0.6%).We identified 10 autosomal loci that were associated with offspring birth weight at 5 × 10−8 . The lin5 × 10−8  from theanalysis . The majKCNAB1, EBF1 and CYP3A7 resulted from analysis of UK Biobank data only, and index SNPs were unavailable in the EGG Consortium meta-analysis (imputed to HapMap Phase 2). To investigate further the evidence for association at these loci, we identified proxy SNPs (r2 = 1) for KCNAB1 (rs9872556) and EBF1 (rs2964484) that were available in HapMap Phase 2 (no proxy SNP was available at r2 > 0.5 at the CYP3A7 locus). Meta-analysis of the EGG Consortium and UK Biobank data showed weaker evidence of association overall, with some evidence of heterogeneity between the EGG meta-analysis and UK Biobank  and KCNAB1 , mapped >2 Mb away from, and were statistically independent of any index SNPs previously associated with birth weight at P < 5x10−8 in a fetal GWAS (r2 < 0.05) , and the directions of association were consistent, suggesting that they were tagging the same causal variant. At the L3MBTL3 locus, the maternal and fetal directions of association were consistent, but the index SNPs were weakly correlated (r2 = 0.13), and conditional analyses in UK Biobank suggested weakening of the association when the L3MBTL3 SNP identified in the fetal GWAS was accounted for  to allow comparison of data across studies. We excluded multiple births, stillbirths, congenital anomalies (where known) and births before 37 weeks of gestation (where known). We assessed the association between each SNP and offspring birth weight using linear regression of the birth weight z-score against maternal genotype (additive genetic model), with sex and gestational duration as covariables . Ancestry principal components were included as covariables where necessary in the individual studies. Genome-wide association analyses were conducted using PLINK  to a ng PLINK , SNPTESTng PLINK , Mach2qtng PLINK  or Beaglng PLINK  . Data annotation, exchange and storage were facilitated by the SIMBioMS platform  < 0.01 and poorly imputed SNPs  were excluded. To adjust for inflation in test statistics generated in each cohort, genomic control  was applplatform . Qualityplatform . We obtaP-value threshold of P < 1 × 10−5 for follow-up in additional, independent studies. Of these, one SNP (rs11020124) was in LD  with SNP rs10830963 at the MTNR1B locus known to be associated with fasting glucose and Type 2 diabetes (P < 5 × 10−8) of association with other phenotypes, and therefore higher prior odds of association with birth weight: rs2971669 near GCK (r2 = 0.73 with rs4607517 associated with fasting glucose) (LMO1 (r2 = 0.90 with rs110419 associated with neuroblastoma) (RAD51AP1 (r2 = 0.27 with rs2970818 associated with serum phosphorus concentration) (We selected 15 SNPs that surpassed a diabetes . We assulastoma)  and rs79n = 18 319 individuals), 9 studies (n = 15 288) provided custom genotyping of between 4 and 18 SNPs, whereas 4 studies  had in silico genome-wide or exome-wide SNP genotypes available. Where SNPs were imputed, we included only those with quality scores (r2hat or proper_info) >0.8. We excluded directly genotyped SNPs showing evidence of deviation from Hardy–Weinberg Equilibrium at P < 0.0028 (Bonferroni corrected for 18 tests). Where genotypes were unavailable for the index SNP, we used r2 > 0.8 proxies  were excluded from the analysis along with those reporting offspring birth weights of <2.5 or > 4.5 kg (n = 5479). ‘British descent’ was defined as individuals who both self-identified as white British and were confirmed as ancestrally Caucasian using principal components analyses . A total of 1976 of the women were asked to repeat the questionnaire at a follow-up assessment and therefore had two reports of birth weight of first child. Those with values differing by ≥1 lb (0.45 kg) were excluded (n = 280). This resulted in n = 48 632 women with both genotype data and a valid offspring birth weight value, which was z-score transformed for analysis (P < 1 × 10−6), MAF < 0.01 or overall missing rate > 0.015, resulting in 17 352 SNPs for analysis in PLINK v.1.07, with the first 5 ancestry principal components as covariates.UK Biobank data were available for 502 655 participants, of whom 273 463 were women , and of analysis  to accouanalysis . We addiIn both the full UK Biobank sample and our refined sample, birth weight of first child was associated with mother’s smoking status, maternal BMI and maternal height in the expected directions  using METAL  using a sample of 10 000 individuals selected at random from the UK Biobank to approximate patterns of LD between variants in these regions.At each of the identified loci, we looked for the presence of multiple distinct association signals in the region 1 Mb up- and down-stream from the lead SNP through approximate conditional analysis. Conditional and joint analysis in the analysis program, genome-wide complex trait analysis  was applr2 > 0.8) reported from GTEx v4, GEUVADIS, and 11 other studies using Haploreg v4.1 .To search for candidate genes at the four loci not already covered by the previous fetal GWAS of birth weight , we idenBecause of the small number of cohorts with both maternal and offspring genotype data available to conduct conditional analysis, we developed a novel method using SEM to estimate the conditional maternal and fetal genetic effects on birth weight, which we subsequently applied to the maternal and offspring birth weight data in the UK Biobank. SEM is a flexible multivariable statistical approach that allows investigators to model the covariance between an observed set of variables  as a function of several latent unobserved variables . The full details of the SEM method for estimating the conditional fetal and maternal effects are described elsewhere . BrieflyP-value for the fetal and maternal paths was calculated using a Wald test. We fitted a second SEM without the child and maternal path to conduct a 2 degree of freedom test for the effect of the SNP on birth weight.To fit the SEM, we used OpenMx  in R ve with thez-scores for analysis.Genotype data from the UK Biobank May 2015 release was used for analysis. We included 57 711 participants who reported their own birth weight and 45 436 women who reported the birth weight of their first child, giving a total of 78 674 unique individuals in the analysis (24 473 women had both their own and their offspring’s birth weight). Individuals who were not of ‘British descent’ (as defined earlier), or were related to others in the sample, or who were part of multiple births, were excluded. The birth weight of offspring phenotype was prepared as described earlier, whereas own birth weight was prepared as described previously , becausez-score and maternal genotype conditional on fetal genotype and vice versa . We combined the results from the individual studies using inverse variance meta-analysis with fixed effects. We performed a further meta-analysis to combine the overall estimates with those from the SEM using UK Biobank data.We analysed up to 8705 mother–child pairs from 4 studies with both maternal and fetal genotypes available . We used linear regression to test the association between birth weight KCNAB1, EBF1 and CYP3A7 were unavailable, we used proxies . Because GWAS summary statistics for blood pressure were not publicly available, we used the UK Biobank May 2015 genetic data release and tested associations between the SNPs and systolic and diastolic blood pressure (SBP and DBP) in 127 968 and 127 776 British descent participants, respectively. Two blood pressure readings were taken approximately 5 min apart using an automated Omron blood pressure monitor. Two valid measurements were available for most participants, and the average was taken. Individuals were excluded if the two readings differed by more than 4.56 SD , and blood pressure measurements more than 4.56 SD away from the mean were excluded. We accounted for blood pressure medication use by adding 15 mmHg to the SBP measure and 10 mmHg to the DBP measure in those reporting regular use of any antihypertensive. Blood pressure was adjusted for age, sex and centre location and then inverse normalized before analysis.We looked up associations between the 10 identified loci and various anthropometric and cardiometabolic traits in available GWAS result sets. The traits and sources are presented in http://www.ebi.ac.uk/gwas/home, last accessed 2 August 2017) for associations P < 5 × 10−8 between any additional traits or diseases and SNPs within 500 kb of, and in LD with, the index SNP at each locus.We additionally queried the NHGRI-EBI catalog of published GWAS  was corrected for confounding factors such as gene size, SNP density and LD-related properties in a regression model. Genes within the HLA-region were excluded. Genes were then ranked by their adjusted gene scores. The observed number of gene scores in a given pathway with a ranked score above a given threshold (95th and 75th percentiles) was calculated and this statistic was compared with 1 000 000 randomly permuted pathways of the same size. This generated an empirical P value for each pathway, and we considered pathways reaching FDR < 0.05 to be of interest. The 3230 biological pathways tested were from the BIOCARTA, Gene Ontology, Ingenuity, KEGG, PANTHER and REACTOME databases, with a small number of additional custom pathway.We used MAGENTA to test for pathway-based associations using summary statistics from the overall meta-analysis . The sofHMG online. Summary statistics from the meta-analysis are available at http://egg-consortium.org/.Supplementary InformationClick here for additional data file.Supplementary TablesClick here for additional data file.