Currently, the normative evaluation of a new persistent luminescent material is focused on the light emission spectrum, the afterglow decay curve and the total duration time of the persistent luminescence. In this paper, we investigate the temperature dependent persistent luminescence in some well-known persistent phosphors and relate this to their thermoluminescence properties. The concept of the optimum working temperature is proposed as a new means for evaluating persistent phosphors. It is shown that there is a clear relation between the efficient temperature range of the afterglow output and the thermoluminescence glow curve. The experimental work is supported by simulations of thermoluminescence and afterglow characteristics. The concept of the optimum working temperature for persistent phosphors can be used as an evaluative criterion for applications in various working environments.Development of persistent luminescent materials has drawn continuous attention in recent years in view of their potential applications in the fields of security night-vision signage,  As one of the commercial LPPs for emergency lighting, the SrAl2O4:Eu2+, Dy3+ phosphor can give bright emission for more than 16 h  at room temperature (RT) after removal of the excitation source5. Significant achievements have been made in the visible region and some representative LPPs emitting primary colors are SrAl2O4:Eu2+, Dy3+ with green emission6, CaAl2O4:Eu2+, Nd3+ with violet emission8, Sr2MgSi2O7:Eu2+, Dy3+ with blue emission9 and both CaS:Eu2+, Dy3+  10 and Y2O2S:Eu3+, Mg2+, Ti4+ with red emission11. Near-infrared (NIR) persistent luminescent nano-particles are of particular interest for in vivo imaging, due to their merits of autofluorescence-free detection and high sign-to-noise ratio3. As a result, much effort has been spent to develop LPPs from the NIR emitting family. Up to now, there are growing numbers of persistent phosphors for NIR emission, with the representative ones including Zn3Ga2Ge2O10:Cr3+ 2, LiGa5O8:Cr3+ 14, La3Ga5GeO14:Cr3+ 16, LaAlO3:Mn4+ 18. The experimental material research has allowed the improvement of the LPPs in order to obtain a sufficiently strong and long lasting luminescence.Persistent luminescent materials or long persistent phosphors (LPPs) are a group of luminescent materials possessing extraordinary energy storage ability and long-lasting emission after stopping the excitation. Development of persistent luminescent materials has drawn continuous attention because of their potential applications in the fields of emergency lighting signs, dials and security displays, night-vision signage, 20. The afterglow duration time can range from minutes to several hours after excitation mostly by ultraviolet or blue light, and is typically governed by co-dopants, intrinsic defects, or dopant-related defects22. These trapping defects can store charge carriers that are released from the excited state of the activator and gradually deliver these charge carriers over minutes to days, depending on the nature of the defects and the ambient temperature. In general, both emitters and traps play an important role in the persistent luminescence. Basic mechanisms for persistent luminescence have been suggested. However, the exact trapping and detrapping mechanism of the persistent luminescence is still under discussion22.Persistent phosphors consist of an inorganic host matrix and activating dopant ions. The activator or dopant inside the host provides luminescence upon excitation and the emission color can be tuned as a function of the dopants and the host materialin vivo imaging, while green emission, close to the maximum eye sensitivity, is ideally suited for emergency lighting24. The afterglow decay curve is commonly applied to evaluate and compare LPPs. Useful information of traps can be obtained from the shape of the afterglow profile and it has been suggested that absolute intensity units should be used for specifying the afterglow instead of arbitrary units, in order to be able to make comparison between measurements and samples. For instance, units cd/m2 can be used for visible emission, whereby 0.32 mcd/m2 is often used as threshold for defining the afterglow time. For IR emission, this should be replaced by the equivalent radiometric unit mW/m2/sr. Hereby, 10−3 mW/m2/sr is roughly equivalent to 0.32 mcd/m2 25. The duration time of persistent luminescence is a straightforward and standardized parameter to evaluate the new LPPs. It needs to be mentioned that all these standardized parameters are typically measured at room temperature, while the afterglow performance is temperature dependent and there could be a different optimum working temperature for a certain phosphor. As far as we know, a systematic exploration of the temperature dependence of persistent luminescence and its optimum working temperature has not been reported26. Insights into the relation between afterglow output and temperature yield very important information on the application potential of certain compounds and the way their performance can be optimized. In this paper, we propose the concept of the optimum working temperature as a new criterion for evaluating persistent phosphors.Currently, the normative evaluation of a new persistent luminescent material is focused on the emission spectra, the afterglow decay curves and the duration time of persistent luminescence. The spectral distribution during the afterglow process is an important evaluation criterion for a particular application and it determines the practical application of the LPPs. For example, the phosphors with persistent NIR emission after stopping excitation are very promising for advanced 27. In this work, TL glow curves are investigated for the prediction of the efficient afterglow output. The temperature dependent persistent luminescence is studied in some well-known persistent phosphors with thermoluminescence analysis. The persistent phosphors are Sr2MgSi2O7:Eu2+, Dy3+ (SMSO), SrAl2O4:Eu2+, Dy3+ (SAO), Sr4Al14O25:Eu2+, Dy3+ (SAO25), CaAl2O4:Eu2+, Nd3+ (CAO), Y2O2S:Eu3+, Mg2+, Ti4+ (YOS), CaS:Eu2+, Dy3+ (CaS) and LaAlO3:Mn4+, Na+ (LAO) phosphors. We show that there is a clear relation between the optimum afterglow temperature range and its corresponding thermoluminescence glow curve in commonly used persistent phosphors. A strategy for evaluation of the optimum working temperature for persistent phosphors is thus proposed through the TL measurements. The experiments are supported by simulations of thermoluminescence and afterglow characteristics. The experimental TL data can be described and afterglow properties at various temperatures can be predicted using the well-known Randall-Wilkins first-order expression of a single glow peak model. Realizing the optimum working temperature of persistent phosphors, appropriate candidates can be chosen for a certain temperature environment. The approach used in this paper is easily transferable to other new persistent phosphors for better practical applications.Thermoluminescence (TL) measurements are effective to investigate the trapping behavior in persistent phosphors. Information on the amount of charge carriers and insights into the trap depth distribution inside these persistent phosphors can be obtained from TL glow curves28, YOS-ICSD No.067503 29, SMSO-ICSD No.031308 30, SAO-ICSD No.026466 31, SAO25-ICSD No.027744 32, CaS-ICSD No.619527 33 and LAO-ICSD No.153821 34), without traces of other crystalline phases.The measured XRD patterns and their corresponding reference patterns of LAO, YOS, CAO, SAO, SAO25, SMSO and CaS phosphors are shown in Fig. 6. Therefore, the intensity of the TL glow peak increases gradually. Charge carriers trapped inside the phosphor release more energy with a longer fading time. In the temperature range from −40 °C to 20 °C, the afterglow output keeps increasing monotonically as shown in Fig. 6, largely influenced by its working temperature.In order to investigate the temperature dependence of the persistent luminescence of these phosphors, a dedicated TL experiment was conducted as shown in Fig. Afterglow measurements at various temperatures were performed with the aim to compare with TL experiments. The afterglow intensity of SMSO phosphor as a function of time, measured at lower temperatures, from −60 °C to 20 °C, and at elevated higher temperatures, from 20 °C to 120 °C, are illustrated in Fig. 35.To evaluate the optimum working temperature for other compounds, this TL experimental procedure as described above was also applied to other persistent materials . The same color codes were used in Fig. The predicted afterglow output of the different LPPs is given as a function of temperature and their corresponding normalized TL glow curves are shown in Fig. 36. The Randall-Wilkins first-order expression of a single glow peak was applied as described in the methods section38. Computerized TL glow curve fitting was used to extract the relevant trapping parameters from the experimental TL measurements. Extracted fit parameters of these phosphors are given in Table s and linear heating rate were kept constant at 1012 s−1 and 0.5 K s−1 respectively. The TL glow curve of the CAO phosphor and its corresponding fitting curve are illustrated as an example in Fig. To further verify the ideal temperature range of the persistent luminescence proposed by the experimental procedures, a simulation of the TL glow curve and the afterglow profile at various temperatures was made. The detrapping behavior was assumed to follow the first-order kinetics, thus negligible retrapping during the heating stage was assumedopt.) and the temperature of the TL glow peak (Tmax.) is presented for all the phosphors in Fig. A comparison of the optimum afterglow temperature , SrAl2O4:Eu2+, Dy3+ (SAO), Sr4Al14O25:Eu2+, Dy3+ (SAO25), CaAl2O4:Eu2+, Nd3+ (CAO), Y2O2S:Eu3+, Mg2+, Ti4+ (YOS), CaS:Eu2+, Dy3+ (CaS) were used as well as the LaAlO3:Mn4+, Na+ (LAO) phosphor synthesized in our lab35.Several widely studied and commercialized persistent luminescent compounds (Glo.Tech.Intl.), namely Sr◦ to 80◦ at room temperature with a step time of 1.5 second and step size of 0.02◦. The measured XRD patterns were compared with the standard data from the structures of the corresponding host lattices.The crystal structures of the studied powders were verified using powder X-ray diffraction (XRD) measurements on a Siemens D5000 diffractometer  using Cu Kα1 radiation (λ = 0.154 nm). The XRD data were collected in the range 2θ from 106. Identical size thin pellet samples  were used and these samples were in good thermal contact with the heat exchanger by using thermally conductive adhesive. The excitation source for the charging was a monochromated 300 W Xenon arc lamp . The fiber-coupled monochromator in combination with light source allows strong and direct monochromatic excitation light towards the sample. The light emitted from the samples was guided by an optical fiber and collected using a ProEM1600 EMCCD camera connected to a 300 mm monochromator (Princeton Instruments). At a certain ambient temperature TAmbient, the sample was charged by constant irradiation intensity with monochromatic light of an appropriate wavelength  using a constant heating rate β (β = 30 °C min−1). This moderate heating rate was chosen and tested to improve the signal-to-noise ratio and prevent temperature differences between the heating plate and the phosphor studied, which is beneficial for the precision and repeatability of the measurements. A fixed cooling rate of 50 °C min−1 was employed for each TL cooling process. In addition, the two starting temperatures  for TL recording were used to limit the loss of energy from a long cooling process and improve the precision of the TL readout. The maximum 225 °C was fixed and no higher temperature was applied in order to restrain the thermal quenching (TQ) effects which may suppress the TL behavior at higher temperatures, but these trapped charges could still contribute to the afterglow which occurs at lower temperatures39. A series of these TL experiments were performed, each one having a different fading time (waiting 1 second or 30 minutes) and a different temperature TAmbient in the range from −60 °C to 120 °C with an interval of 20 °C. Prior to each TL experiment, a thermal cleaning was conducted by heating up to 225 °C, to assure all relevant traps were empty at the start of experiment. A schematic view of the TL experimental procedure is shown in Fig. A small lab-built vacuum chamber with a well-characterized cooling and heating stage was used for TL measurements43.Afterglow decay curves at each temperature were collected with a ProEM1600 EMCCD camera attached to an Acton SP2300 monochromator (Princeton Instruments). The excitation source was a monochromated 300 W Xenon arc lamp  at a certain wavelength for 10 min at each temperature. Different characteristic emission spectra were collected and integrated according to the corresponding emission wavelength ranges of the different phosphors. The excitation wavelengths and integrated emission ranges were applied as shown in Table 38 was applied.In order to simulate the thermoluminescence glow curves, the first-order expression of a trap depth distribution from the Randall-Wilkins methods is the frequency factor or attempt-to-escape factor, which was considered as a constant value independent of temperature. The value of s was kept constant at 1012 s−1 of the order of the lattice vibration frequency. β is the constant linear heating rate (K s−1), and 0.5 K s−1 (30 °C min−1) was employed for all the TL measurements. N(E) is a continuous distribution in trap depths and several distribution shapes can be considered. In this work, we chose a Gaussian shape where the mean energy and the width of the Gaussian function are dependent on the material studied. f0(T) is the filling factor of the distribution and saturated filling of traps was assumed with f0   = 1. E is the trap depth or activation energy which is needed to release the charge carrier (an electron or a hole) from the trap into the conduction band or the valence band. k is the Boltzmann constant with a value of 8.6173303 × 10−5 eV K−1. T is the absolute temperature in Kelvin. The expression (1) gives the TL intensity as function of temperature and describes the TL data of a single glow peak with the choice of expression for the trap distribution. The relevant Gaussian function with mean energy EG and width σG can be extracted by utilizing this expression. It is worth noting that some simplifying assumptions are made to develop the analytical equation for first-order kinetics, such as negligible retrapping during the heating stage  and the quasi equilibrium assumption by Chen and McKeever44. Gaussian distributions were chosen as a reasonable assumption for a random incorporation of trap states45. Some other possible trap depth distributions have been investigated such as exponential47, uniform49 or binomial50 trap depth distribution models in literature, but for all selected materials in this work, the approximation used was found to yield good results.The term 48.The obtained parameters for the trap distribution from the TL glow curves were utilized as input for the simulation of the afterglow profile. The expected afterglow profile follows the equation of the first-order kinetics in the presence of a trap distributionThe simulation of the afterglow at different temperatures was made for all the investigated phosphors. The effective working temperature region from the simulation results was further compared with experimental data.Supporting information


Astyanax mexicanus and identified several differences in heart morphology between the surface (epigean morph) and cave-dwelling (troglomorph) morphs. Examination of the adult revealed that the troglomorph possesses a smaller heart with a rounder ventricle in comparison to the epigean morph. The size differences identified appear to arise early in development, as early as 24 h post-fertilisation (hpf), while shape differences begin to appear at 2 days post-fertilisation. The heart of the first-generation cross between the cave-dwelling and river-dwelling morph shows uncoupling of different phenotypes observed in the parental populations and indicates that the cardiac differences have become embedded in the genome during evolution. The differences in heart morphology are accompanied by functional changes between the two morphs, with the cave-dwelling morph exhibiting a slower heart rate than the river-dwelling morph. The identification of morphological and functional differences in the A. mexicanus heart could allow us to gain more insight into how such parameters are regulated during cardiac development, with potential relevance to cardiac pathologies in humans.Regulation of heart size and shape is one of the least understood processes in developmental biology. We have for the first time analysed the hearts of  •Differences in heart size, shape and tissue structure between Astyanax populations.•Furthermore, differences in cardiac melanophore and adipocyte numbers.•Heart size and shape differences are apparent early in development.•Surface and Pachón show differences in heart rate during development and adulthood.•F1 hybrids show uncoupling of features observed in surface and Pachón populations. Many human congenital defects affecting heart size (hypertrophic cardiomyopathy), shape (single-ventricle defects) and tissue structure (non-compaction cardiomyopathy) are known to exist , with geAstyanax mexicanus (Mexican cavefish) as a new model for understanding the regulation of heart size and shape. A. mexicanus is a single fish species comprising troglomorph (cave-dwelling) and epigean (surface) river populations. This species possesses unique traits that make it a useful model organism to study various aspects of adaptation to a food-scarce environment in perpetual darkness. Several cavefish populations are known to have derived from a surface fish ancestor, suggested from a couple of thousand to several million years ago  Act 1986 and institutional guidelines. Laboratory strain of surface fish were originally collected at Nacimiento Del Rio Choi, San Luis Potosí, Mexico. The three laboratory strains of cavefish population are originally from Cueva de El Pachón in Tamaulipas, Mexico (Pachón cavefish), El Sótano de la Tinaja (Tinaja cavefish) in San Luis Potosi, Mexico, and La Cueva Chica (Chica cavefish) originally provided by the Steinhart Aquarium . The fish used in this study have been bred for multiple generations at the Yamamoto laboratory after having been obtained from University of Maryland (Jeffery laboratory). All the fish were maintained and fed once a day at 20 °C in pH 7.2 tank water with a 14-h light and 10-h-dark light cycle. Embryos were obtained by temperature induced spawning after increasing the tank water temperature to 25 °C.The experiments were performed on laboratory stock of teleost 2.22HPO4, 0.15 mM KH2PO4, 1 mM CaCl2, 1 mM MgSO4·7H2O, 0.7 mM NaHCO3). At specific time stages, embryos were fixed in 4% paraformaldehyde in PBS overnight at 4 °C. Embryos were then dehydrated gradually by increasing gradients of methanol/PBS and stored at − 20 °C.Embryos were collected and incubated in petri dishes at 25 °C in zebrafish embryo medium 2 , after which the standard body length from snout to tail base and the body weight were measured. Then the fish were placed on a cut sponge in a ventral-side-up orientation. Spring scissors were used to make an incision to penetrate the thorax and open the pericardial sac. The ventricle was exposed and hearts were isolated by cutting the artery superior to the bulbus arteriosus and the veins inferior to the atrium. The isolated hearts were directly fixed in 4% paraformaldehyde in PBS at 4 °C overnight, followed by washing with PBS and stored in PBS containing 0.05% sodium azide at 4 °C or processed for paraffin embedding.2.4To visualise the transparent embryos in paraffin, the embryos were transferred from 100% methanol to 0.1% eosin staining in 100% ethanol to stain the embryos for 30 s, followed by 3 washes with 100% ethanol. The ethanol solution was replaced by 1-butanol, the samples transferred to liquid paraffin wax and kept at 65 °C for 30 min. After washing twice with liquid paraffin wax, embryos were embedded. For embedding of adult hearts, after 4% paraformaldehyde fixation, the hearts were dehydrated by increasing ethanol gradients , kept in 1-butanol overnight, after which the hearts were transferred to liquid paraffin wax at 65 °C. After washing twice for 1 h with liquid paraffin wax, the hearts were embedded. 7–12 µm sections were cut using a microtome (AO Spencer 820) and mounted onto Superfrost slides (Thermo Scientific), then dried overnight at 37 °C.2.5Sections were deparaffinised by xylene and rehydrated by decreasing ethanol gradients  to PBS, or distilled water in case of AFOG staining. For AFOG staining, the sections were placed in Bouin’s fixative at 60 °C for at least 2 h and washed in distilled water until sections were clear. The sections were then placed in aqueous 1% phosphomolybdic acid for 5 min, again rinsed in distilled water, before stained with AFOG solution for 5 min , pH1.09). The sections were quickly rinsed in distilled water before dehydration and mounting using DPX (Sigma).For fluorescence immunohistochemistry, 30 min of 3% (w/w) hydrogen peroxide was followed by high-pressure antigen retrieval with antigen unmasking solution . Sections were then blocked for 30 min with TNB . The primary antibody against monoclonal mouse anti-myosin heavy chain 2 (MF20)  was incubated with samples overnight at room temperature. AlexaFluor 488 goat anti-mouse IgG secondary antibody (1:200 in TNB) and DAPI  were incubated for 2 h at room temperature. Immunohistological stained sections were mounted with hydromounting solution (Applied Genetic Technologies Corporation) or Mowiol 4-88 (Applichem).2.62 dpf embryos were rehydrated from 100% methanol to PBS followed by 10 min of proteinase K treatment at 65 °C (10 µg/ml in PBS). After washing in PBS-T, the embryos were fixed in 4% paraformaldehyde for 10 min and blocked for 2 h in TNB followed by primary antibody (MF20) overnight at room temperature. After washing in PBS-T, the embryos were incubated with secondary antibody and DAPI  for 2 h . After washing in PBS-T, the embryos were embedded in 0.5% agarose/PBS in glass capillaries for light sheet microscopy (Zeiss Z1).2.7Pictures of stained sections were taken by fluorescent microscope (Leica DM4500 LED). Three-dimensional (3D) reconstructions of hearts were performed using Amira (Visage Imaging). Pictures of sections were aligned automatically, followed by manual adjustment. Light sheet data was loaded directly. Different regions of the heart were labelled by the brush tool with the MF20 or AFOG staining as a reference. The 3D reconstructions were made by this programme according to the labelling.2.8Embryos were selected at the specific stages and anaesthetised with 0.16 mg/ml M MS-222 for 5 min, in separate 6-well culture plates. The working MS-222 solution was made by diluting the stock 4 mg/ml MS-222 with E2 medium (for 3 dpf and 5 pf embryos) or in tank water (for 10 dpf embryos). The embryos were then moved to 24-well culture plates and incubated at 25 °C for 25 min. Following incubation, the embryos (that had been immersed in MS-222 for approximately 30 min) were recorded under an inverted microscope  for 30 s.For adult heart rate measurements, Pachón were anaesthetised in 0.16 mg/ml MS222 for 20 min, while surface and F1 generation fish were anaesthetised for 10 min. This difference was needed as Pachón fish were not fully anaesthetised until 20 min, whereas surface fish and F1 took 10 min. Additional experiments with few Pachón at 10 min and surface fish and F1 at 20 min only enhanced observed differences . When fully anaesthetised, the fish were placed ventral side up in a cut sponge and the heart rate was filmed for 1 min . The heart beat was visible through the body wall and did not require opening of the thorax.2.9myh6 forward TTTCTGCGTTCTGAAAGGGC, reverse GAAACAGCGTGGCTTTGACA, myl7 forward CAAGCTGACTGGTGCCATTAT and reverse Reverse GAAGAGCCCTTCTTCTTTCCA. PCR fragments were purified using QIAquick Purification Kit (Qiagen). The blunt PCR fragments were subject to A-tailing with GoTaq Flexi DNA polymerase and cloned into pGEM-T vector (Promega) and RNA probes were generated using DIG RNA Labelling Kit (Roche).RNA from 5 dpf, 6 dpf surface and 24 h Pachón cave fish was extracted using TRIzol Reagent (Thermo Fisher) following the RNA isolation TRI Reagent protocol (Sigma-Aldrich). cDNA was synthesised according using RT-PCR. The probes were made using the following primers: 2.10In situ hybridisation was performed as described , with se2.11Brightfield images were taken with the Nikon Digital Sight stereo microscope  using the NIS Elements F 2.30 software. Measurements of the adult hearts were done using Nikon ATC-2U software. A triangle surrounding the ventricle in lateral view was drawn onto the images of each heart and the blank area of the triangle, which was not covered by the ventricle, was measured to analyse ventricle shape. Measurements as shown in the main figures were corrected for body length. As an additional correction factor we used bulbus arteriosus area, which gave similar results to body length . In addi2.12To test for statistical significance of measurement differences, one-way ANOVA, Dunnett’s Multiple Comparison Test or independent 2-tailed, Student’s T-test for unpaired samples was used, as indicated in the figure legends. P < 0.05 is denoted by *, P < 0.01 is denoted by **, P < 0.001 by *** and< 0.0001 by ****.33.1A. mexicanus hearts fall within the type II category. Both Pachón and surface fish have a compact wall with coronary vessels present within this wall, especially surface fish have clearly visible intramural coronary vessels  than its Pachón counterpart. In order to characterise the morphology of the adult heart of the epigean morph and other cave populations of 3.3Once the morphological differences in the adult heart were established, our next aim was to determine if these differences are already present during development. Embryos stained for myocardial marker MF20 already show differences in morphology at 2 dpf, when the heart is still merely a linear tube, with the ventricle showing a larger bend in the outer curvature towards the outflow tract E. AdditiA. mexicanus cardiac muscle gene myosin light chain 7 (myl7) and examined its expression pattern by in situ hybridisation. In all the A. mexicanus populations examined, myl7 is uniformly expressed in the heart tube at 24 hpf but at 48 hpf, its expression is more concentrated in the ventricular than the atrial region  revealed significant differences between the surface and cave populations except for the Pachón at 24 hpf , a gene that is specific for atrial myocardium in zebrafish (myh6 is expressed in a sub-region of the heart tube in both the epigean morph and troglomorphs (myh6 (myh6 expression shows that the surface morph has a larger area than the all troglomorph populations examined. While the area of myh6 expression is smaller in cavefish, the level of myh6 expression seems increased. These differences are detectable from 24 hpf and are maintained at 48 hpf (The volume and area of the ebrafish . At 24 hlomorphs A–D. At 4hs (myh6 E–H. The t 48 hpf I–J.Fig. 3.4As our data suggested that the shape and size differences of the heart have a developmental origin, we hypothesised that such traits are genetically heritable. We examined the adult heart of the F1 hybrid generation of surface and Pachón fish at 1 year of age. Interestingly, the measurement of ventricle area, width and height showed that F1 hybrid fish have a ventricle size more similar to Pachón than surface fish A–D. The 3.5A. mexicanus is higher than in the adult stages and significantly different heart rates are observed from 5 dpf onwards (After the characterisation of numerous morphological differences of the heart of the surface fish, cavefish and the F1 hybrid, we postulated whether this correlated to any functional differences. We therefore measured the heart rate of anesthetised surface and Pachón fish in vivo. At early developmental stages, the heart rate of  onwards A. At 5 a onwards B.Fig. 7(4Astyanax mexicanus has been a valuable research model for different research fields for decades, however, its heart has been ignored so far. Here, we have identified differences in heart size, morphology, melanophore and adipocyte cell populations, and beating frequency. These differences already occur very early in development, but become increasingly apparent over life. The ventricle of the Pachón fish heart is not only smaller than the surface fish ventricle, it is also spongier and much less triangular in shape. The same holds true for the other cavefish populations examined. As these differences already occur very early during heart development, before external factors start to play a role, this suggests that the differences in morphology are genetically regulated. Moreover, the heart of the first-generation cross between the cave-dwelling and river-dwelling morph displays a mixture of phenotypes similar to either of the two parental populations, further indicating that the genetic determinants are quantitatively inherited. Morphological differences partially uncouple with functional differences. As such, A. mexicanus could be a valuable new model for identifying the molecular and cellular mechanisms underlying heart cell populations, and size and shape regulation.5Astyanax mexicanus. This has also been observed in the mouse and human heart (L-tyrosine into melanin in the melanosome, but the transportation of L-tyrosine from cytoplasm to the melanosome is blocked (While the Pachón heart does not have pigmented melanophores, these cells can be found in varying degrees on surface fish hearts. It is not clear yet why there are pigment cells present on the heart, as the organ is located within the body and thus not exposed to sunlight. The presence of melanophores on the heart is not unique to an heart , though an heart . Pachón  blocked . It is nIn addition, surface fish have a large amount of fat cells on the surface of the heart, mainly located in the atrioventricular groove and between the bulbus arteriosus and ventricle. It is interesting that surface fish store less adipose cells in the body but have more adipocytes on the heart and vice versa in cavefish . The fun6A. mexicanus. Surface fish that have a more triangular shaped heart, also have a less spongy heart, which means a larger ratio of compact wall to trabeculae. A thicker outer compact layer of myocardium has been shown to be an adaptation required by fish which use large amounts of metabolic energy for activities such as long migrations, constant fast swimming against a strong current or hunting. For example, teleost flatfish like the Plaice have a spongy rounder heart, while highly migratory teleost Salmon have a triangular shaped heart (While both the Pachón and surface fish hearts are comparable to the zebrafish heart and follow the general pattern of a ‘modern teleost’ type II heart , there aed heart . A hearted heart . As wateed heart , a sponged heart , in conted heart . The faced heart . We confed heart . This ined heart .7In addition to the factors mentioned above, exercise and hypoxia, also differences in temperature, food deprivation and light cycle have been shown to influence heart size and beating frequency . The cavThe developmental processes that lead to a change in heart size are likely due to the regulation of cell specification, proliferation and apoptosis, or a combination of these mechanisms. The Hippo signalling pathway is known to negatively regulate cell proliferation, survival and to some extent cell growth . Hippo smyh6 expression in cavefish, which suggest further advanced myocardial differentiation compared to the surface fish, as less new diluting progenitor cells are added. If sinus node development is altered in cavefish, this would be a likely explanation of heart rate reduction.The reduced size of both the atrium and ventricle in cavefish during early heart development suggests decreased contribution of second heart field progenitors to both the arterial and venous poles of the heart tube. While the initial heart tube is derived from the first heart field, the cells added to the heart after initial tube formation are called the second heart field progenitors . The sinA. mexicanus will be a valuable model to understand how hearts size control is regulated in the mononucleated proliferating fish heart. The zebrafish has been a popular model organism within the fields of cardiovascular development and heart regeneration. Our identification of the morphological and functional differences in the A.mexicanus heart demonstrates its potential as a new model system for studying cardiovascular development and pathologies. Further genetic and molecular characterisation of the phenotypes shown may enable us to elucidate the mechanisms behind organ shape and size, as well as their relationship with function. Moreover, large-scale drug testing that has been done with zebrafish (A. mexicanus, with potential relevance to human cardiac defects and disease.Most cardiomyocytes in the fish heart not only remain mononucleated throughout life, they also retain the capacity for proliferation . This caebrafish  may also


Amphibalanus eburneus, a dominant suspension feeder within the Indian River Lagoon (IRL) of central Florida, we compared the stable isotopes ratios (δ13C and δ15N) of barnacle tissue to those of particulate organic matter (POM). Collections were carried out quarterly for a year from 29 permanent sites and at sites impacted by an Aureoumbra lagunensis bloom. δ13C and δ15N values of Amphibalanus eburneus varied across sites, but δ15N was more stable over time. There was a range of δ15N values of Amphibalanus eburneus tissue from 6.0‰ to 10.5‰ across sites. Because land-based sources such as sewage are generally enriched in 15N, this suggests a continuum of anthropogenic influence across sites in the IRL. Over 70% of the variation in δ15N values of Amphibalanus eburneus across sites was driven by the δ15N values of POM, supporting a generalist feeding strategy on available sources of suspended organic matter. The dominance of this generalist consumer in the IRL may be linked to its ability to consume spatially and temporally variable food resources derived from natural and anthropogenic sources, as well as Aureoumbra lagunensis cells. Generalist consumers such as Amphibalanus eburneus serve an important ecological role in this ecosystem and act as a sentinel species and recorder of local, site-specific isotopic baselines.The success of many sessile invertebrates in marine benthic communities is linked to their ability to efficiently remove suspended organic matter from the surrounding water column. To investigate the diet of the barnacle  Organisms that can exploit suspended organic matter fill a unique niche in aquatic ecosystems . By effiAlthough suspension feeders appear to be ecologically similar and can occupy overlapping niches within a habitat, there is substantial variation in the feeding structures and mechanisms of coexisting species . This vaCoastal ecosystems are subject to seasonal shifts in environmental conditions and strong resource gradients from a combination of inputs from marine and terrestrial sources . In addiAureoumbra lagunensis (4-5 μm) in 2012, 2013, and the winter of 2016 (The Indian River Lagoon (IRL) of central Florida is a subtropical, shallow (mean depth of <1 m) estuary that spans 250 km of the east coast of central Florida. The IRL supports a high diversity of marine species due to a variety of habitats  and its location in a tropical/temperate transition zone in close proximity to the Gulf Stream . Like ma of 2016  were par of 2016 .Amphibalanus eburneus, with a mean percent cover of 30% to 40% across sites . To investigate this, we compared the stable isotope ratios (δ13C and δ15N) of Amphibalanus eburneus tissue to that of POM from the water column. Collections were carried out quarterly for a year and also during an Aureoumbra lagunensis bloom. We tested the following hypotheses: (1) the δ13C and δ15N values of Amphibalanus eburneus vary over space and time and will be closely tied to the δ13C and δ15N values of POM, and (2) Amphibalanus eburneus will demonstrate shifts in δ13C and/or δ15N values that indicate consumption of the brown tide Aureoumbra lagunensis.Surveys of epifauna communities at 90 sites in the NIRL have found high species diversity (175 taxa in 11 phyla) and a dominance of the barnacle Amphibalanus eburneus  were carried out at each of these monitoring sites on a quarterly basis . Permits for species collections were provided by the Florida Fish and Wildlife Conservation Commission (SAL-14-1567-SR). To test whether a bloom of Aureoumbra lagunensis influenced the diet of Amphibalanus eburneus, samples were also collected opportunistically at sites within the Banana River and IRL that were influenced by Aureoumbra lagunensis from December of 2015 to March of 2016  of the northern region of the greater IRL NIRL; . Collect of 2016 . BarnaclAureoumbra lagunensis collections, a sample of seawater (20 L) for POM was also taken at each site to monitor the δ13C and δ15N values of general sources of particulate carbon and nitrogen available to Amphibalanus eburneus. In the laboratory, this seawater was prefiltered through 105 μm mesh and then filtered through a Millepore quartz fiber filter (2 μm porosity) stacked on top of a Whatman glass fiber (GF) filter (0.7 μm porosity) to obtain a single POM sample for each time point at each site. The organic matter content on GF filters was below detection limits, so POM δ13C and δ15N values are based on the results from organic matter on quartz fiber filters (2 μm porosity) that were stacked on top of the GF/F.During seasonal and Amphibalanus eburneus were kept separate and held overnight in flowing, sand-filtered seawater to allow for gut evacuation and then frozen at −20 °C. Once thawed, the shell diameter was measured for each individual (n = 10 per site for each sampling period) and, using forceps, all tissue within the shell of a barnacle was placed into an individual glass vial  isotopic space, we calculated isotopic dissimilarity (measured as Euclidean distance) among samples and analyzed dissimilarity using a permutational multivariate analysis of variance (PERMANOVA) (Primer 6 with PERMANOVA+ add-on). Seasonal variation in individual isotope values was assessed using a Kruskal–Wallis One-Way Analysis of Variance (ANOVA). Linear regressions were used to investigate the relationship between the δ13C and δ15N values of Amphibalanus eburneus tissue and the δ13C and δ15N values of POM at each site. These analyses were carried out using Systat.Isotope values provide a time-integrated record of an organism’s diet, with δimilated . To test15N and δ13C values of Amphibalanus eburneus tissue over space and time  = 0.001] and season )  = 0.001)  δ15N values in the northern region of the IRL (site #s I1–5) and in the Mosquito Lagoon (site #s M1–6) compared to sites in the Banana River and the southern IRL (site #s B and I6–13) that had δ15N values between 8.3‰ and 10.5‰  than δ15N values  (There was significant variation in the δ= 0.001) . Annual nd 10.5‰ . Annual N values ; Fig. S3ctively) ; Fig. S313C and δ15N values of POM varied across space and time    ; Fig. S415N of Amphibalanus eburneus tissue was between 9.1‰ and 13.1‰ during the Aureoumbra lagunensis bloom, with an average enrichment of 1.8‰ (range of 0.7 to 2.7‰) compared to annual mean values  compared to the annual mean at a site  relative to the annual mean  and +1.91 ± 0.75 SE (range −1.59 to +4.83) for δ15N and δ13C, respectively  and detritus from terrestrial C3 plants such as mangroves (−35‰ to −25‰) and seagrasses (−13.5‰ and −5.2‰)  are suggestive of nitrogen derived from 15N-enriched sources such as wastewater  are more impacted by these 15N-enriched sources than those in the more sparsely populated Mosquito Lagoon and northern IRL sites; our POM values are in general agreement with the magnitude of 15N enrichment found in macroalgae from this region of the IRL (The δd −5.2‰) . Likewisstewater . POM δ15ic tanks . Our dat the IRL .Amphibalanus eburneus tissue was coupled to the temporal and spatial dynamics of POM δ15N and, to a lesser extent, δ13C values, supporting the contention that barnacles are generalist suspension feeders utilizing predominant components of the organic matter pool in the water column , the trend may have been even stronger if we had finer temporal resolution instead of a single POM isotope “snapshot” for each season from a site. The tissue of Amphibalanus eburneus was generally enriched in 13C and 15N  relative to POM . Because consumers are generally enriched in both 13C and 15N due to the process of trophic enrichment, this implies the presence of a “trophic step” between particulate matter and Amphibalanus eburneus  and Amphibalanus eburneus tissue isotope values. Enriched δ15N values are thus likely the result of the passage of anthropogenically-derived nitrogen assimilated by phytoplankton into the epifaunal food web. Enriched δ15N values in POM and barnacle tissue at sites impacted by anthropogenically-derived nutrients has been reported before in other systems, with up to a 5‰ enrichment in barnacles from impacted sites and strong linear correlations between POM and barnacle δ15N values  in the more urbanized regions of the NIRL (Banana River and southern sites in the Indian River) and relatively stable δ15N values across seasons therefore suggest chronic exposure to nitrogen from anthropogenic sources at some sites. In contrast, δ13C values within a site were more variable over both space and time, implying shifts in carbon sources over small spatial scales and the potential for seasonal fluctuations in phytoplankton productivity or growth rates  revealed that δ15N and, to a lesser extent, δ13C values of Amphibalanus eburneus were enriched during the Aureoumbra bloom compared to annual means. For δ13C, this shift likely reflects a combination of source homogenization (predominately Aureoumbra) and isotopic fractionation associated with algal growth and high productivity that alters the δ13C signal at the base of the food web  during the bloom, substantial variation in this enrichment (range −1.59‰ to 4.83‰) suggests that direct reliance of Amphibalanus eburneus on phytoplankton may be reduced during blooms.The IRL has been exposed to multiple, acute algal blooms over the past decade, with a particularly detrimental superbloom in 2011 and recurring and 2016 . The Aurfood web . Althoug15N (up to 13‰) may reflect increased Amphibalanus eburneus consumption of phytoplankton that are relying on enriched sources of anthropogenically-derived N. Although blooms of Aureoumbra in the IRL have previously been shown to elicit lower POM15N values than under non-bloom conditions  compared to POM during the bloom) as Amphibalanus eburneus is feeding more heavily on zooplankton than phytoplankton during an algal bloom. These data provide initial evidence of nutritional shifts in Amphibalanus eburneus under bloom conditions, but additional work in the laboratory is needed to verify the role of this epifaunal species in bloom mitigation . Data are shown for each season from January to October of 2015 and during an Click here for additional data file.10.7717/peerj.5485/supp-6Supplemental Information 6Aureoumbra lagunensis bloom in the January of 2016.Data include 29 sites within the sublagoons of the North Indian River Lagoon. Tissue and POM samples were taken each season from January to October of 2015 and during an Click here for additional data file.


Diatomic nitrogen is an archetypal molecular system known for its exceptional stability and complex behavior at high pressures and temperatures, including rich solid polymorphism, formation of energetic states, and an insulator-to-metal transformation coupled to a change in chemical bonding. However, the thermobaric conditions of the fluid molecular–polymer phase boundary and associated metallization have not been experimentally established. Here, by applying dynamic laser heating of compressed nitrogen and using fast optical spectroscopy to study electronic properties, we observe a transformation from insulating (molecular) to conducting dense fluid nitrogen at temperatures that decrease with pressure and establish that metallization, and presumably fluid polymerization, occurs above 125 GPa at 2500 K. Our observations create a better understanding of the interplay between molecular dissociation, melting, and metallization revealing features that are common in simple molecular systems. Nitrogen is a model system still presenting unknown behaviors at the pressures and temperatures typical of deep planets’ interiors. Here the authors explore, by pulsed laser heating in a diamond anvil cell and optical measurements, the metallization and non-molecular states of nitrogen in a previously unexplored domain above 1 Mbar and at 2000-7000K. P) and temperatures (T) including the stability of the molecular state and related formation of electrically conducting phases offer fundamental constraints on how simple molecular systems related to energetic materials respond to extremes. Moreover, these properties are central for understanding how nitrogen, the primary constituent of Earth’s atmosphere, behaves in the deep interiors of planets, where it can appear as a molecule due to ammonia dissociation1, in subduction zones at oxidizing conditions2, or as an impurity in iron-rich cores at reduced conditions3. Exploring nitrogen under extremes is especially relevant given the challenges of reliably predicting4 and measuring9 the high P–T properties of another important diatomic element, hydrogen. In fact, condensed molecular nitrogen under high P and T behaves similar in many respects to hydrogen: each exhibits melting temperature maxima12 and a fluid insulator-to-metal, molecular-to-nonmolecular transformation that is predicted to be continuous at high temperatures and first order at low temperatures14 and progressive molecular breakdown in the solid state17. While such thermodynamic and electronic characteristics are similar in the limit of molecular breakdown into a metallic liquid18, nitrogen differs from hydrogen in its detailed chemical behavior forming a variety of metastable polynitrogen molecules with a reduced bond order19 due to its ability to adopt differing bonding types (single to triple).The physical and chemical properties of pure nitrogen at high pressures 27, cubic-gauche (cg)28, and layered (LP)30 structures, with the onset of dissociation shifting to lower T at higher P31. Probing fluid nitrogen in single-shock experiments revealed anomalous decreases in volume, temperature, and electrical resistivity at 30–60 GPa and 7000–12,000 K32, interpreted as signatures of molecular dissociation. In contrast, dynamic multiple compression experiments at ~7000 K reported similar cooling effects at ~90 GPa and a transition to a metallic state above 120 GPa33, leaving, however, intermediate temperatures of 2000–6000 K unexplored above ~50 GPa. The onset of dissociation with increasing P and T has also been linked to an increase of optical absorption35, manifesting a connection between dissociation and electronic transformation at optical wavelengths. First principles theoretical calculations37 predict a first-order transformation from molecular insulating fluid to polymeric conducting liquid ending in a critical point near 75 GPa and 4500 K with a continuous dissociation into a semiconducting atomic liquid at higher T and lower P.Solid nitrogen at high pressures shows a rich variety of stable and metastable diatomic (triple-bonded) molecular phases33 and static compression heating experiments to below ~2500 K38  using a streak camera39. We present the experimental observation of a transition line that delineates insulating and conducting fluid nitrogen phases (plasma line) and find the conditions of the insulator-to-metal transition that is associated with a long-sought fluid polymeric nitrogen state. These conditions are in a broad agreement with the previous shock wave experiments albeit our experiments show that nitrogen reaches the metallic state at much lower temperatures (<2500 vs ~7000 K). The plasma transition is at higher pressures than theoretically predicted posing a challenge for theory.The previous gap in experimental data probing nitrogen’s states and properties at higher pressure owed to restrictions on dynamic compression pathways to above ~6000 KK38 Fig. . Here we32 and are sufficient for reaching thermodynamic equilibrium in fluid samples39. However, we find this timescale sufficiently short to avoid crystallization of the polymeric solids29  in the quenched samples. The P–T paths along which we probed the material properties  can be considered nearly isobaric, with the maximum added thermal pressure <5 GPa at 3000 K40 or even smaller given the local nature of the pulsed heating6.The microseconds (μs) long experiments reported here   Fig.  and in tΩP and the mean free time between the electron collisions τ. The fits yield ΩP = 5.6 (3) eV and τ = 0.9 (3) fs, which are characteristic of a good metal. The large experimental uncertainty in τ is due to the narrowness of the spectral range of observations. The electrical direct current (DC) conductivity we obtained in the saturation regime is σ0 = 5600 (1700) S/cm in a good agreement with shock wave measurements (≥2000 S/cm)33 and theoretical predictions (≈3000 S/cm)35 for the metallic fluid, with the small differences likely due to low-frequency deviation from the Drude model39; moreover, the predicted reflectance spectra35 are only in a slight variance with our measurements. A large experimental error in the value of conductivity includes uncertainty in the correction for absorption of semiconducting nitrogen and in the contribution of bound electrons (Methods section).Above 121 GPa, we observed a transition to a state with a drastically increased transient reflectivity Fig. . Precedi31. Transient optical absorption at these extreme P–T conditions is not related to formation of cg/LP states as these quench to ambient temperature after heating31, which was not observed here. The absence of cg or LP nitrogen in our experiments is attributed to short heating times: even ~100 heating cycles (our maximum at each pressure) only yields about 0.5 ms of total heating, much less than that required to crystallize cg- or LP-N in prior studies31.All our observations of transient changes in optical properties during heating events are nearly reversible, with small, permanent enhancement of sample absorption and changes to Raman spectra .Our measurements show that fluid Nope Fig. . In the P–T line along which this transition occurs has a nearly flat slope in a marked contrast to the steep negative slope of the transition to the absorptive state. The pressure at which nitrogen becomes reflective is very close to the conditions where cg-N starts to form17, suggesting that the molecular dissociation energy is close to zero42. At these P conditions, the T difference between the appearance of the absorptive and reflective states is very small, suggesting that T-induced dissociation is abrupt18. In fact, the onset of reflection is close to the conditions where the dense phases of nonmolecular (insulating and semiconducting) solid nitrogen are expected to melt43, suggesting that the metallization transition may occur upon melting, similar to carbon44. We suggest PC = 120 GPa and TC = 3000 K as the critical point delineating continuous and discontinuous molecular dissociation transitions, as predicted theoretically albeit at much lower pressures and higher temperatures36. Thus the fluid fully dissociated state of nitrogen occurs above 120 GPa  and is sufficiently dense to support metallic conductivity12, which may explain a small negative slope of the transition to a metallic state. Recent X-ray diffraction experiments to just lower temperatures and pressures than the critical point38 found no evidence for expected liquid–liquid transformations and require that these occur at higher pressure and temperature consistent with our observations . Even though N2 and H2 molecules are different in chemical bonding, we confirm that major features of their phase diagrams related to molecular dissociation and metallization are very similar. Our observations of two distinct boundaries for the semiconducting and metallic fluid states, and a higher pressure for sudden metallization than theoretically predicted, resonate with recent reports on hydrogen isotopes7. As observed here in nitrogen, the pressures at which dissociated fluid metallic states occur in hydrogen are close to the conditions of the formation of solid phases with the features of nonmolecular chemical bonding such as in phases IV and V above 200 GPa45. With respect to planetary interiors, although molecular nitrogen in an enriched state remains stable throughout the conditions of Earth’s mantle, it dissociates into a fluid metal at the P–T conditions of the Earth’s core  using a streak camera, as has been described in our previous publications46 have been used to model the temperature distribution in the high-pressure cavity to estimate the necessary corrections.Time-resolved (with the resolution down to 0.5 μs) sample temperature was obtained from fitting thermal radiation spectra emitted by the coupler and hot sample to a Planck function 2/(nN + nD)2, where nN and nD are the refractive indices of nitrogen and diamond, respectively, in the high-pressure chamber. While we assumed that the refractive index of diamond is the same as at ambient pressure, we have performed a separate experiment in molecular nitrogen at 75 GPa to determine its refractive index  of the diamond–sample interface at room temperature measured just before the laser heating shot as the reference −1, where ω is the angular frequency. The DC conductivity σ0 = Ωp2τε0, in which Ωp is the plasma frequency, τ the scattering time, and ε0 the permittivity of free space. The dielectric constant is ε* = εb + iσ(ωε0)−1 where εb is the bound electron contribution to the dielectric constant. A range of εb from 1 to nN2 are examined when assessing uncertainty; we generally found εb = nN2 provided a better fit to the data. The corresponding index of refraction is R = |(n* − nN)/(n* + nN)|2, where the refractive index of the semiconducting state is assumed to be the same as for molecular nitrogen determined in our separate experiment (Supplementary Fig. Reflectivity spectra are well fitted by a Drude model having conductivity of the form All relevant numerical data are available from the authors upon request.Supplementary InformationPeer Review File


Ovarian cancer is often diagnosed in advanced stages, when survival is poor. Treatment advances have been made, but are inconsistently implemented. Our purpose was to project the maximum life expectancy gains that could be achieved in women with stage IIIC epithelial ovarian cancer if the implementation of available chemotherapy regimens could be optimized.We used a microsimulation model to estimate life expectancy benefits associated with “optimized” implementation of four post-operative chemotherapy options: standard intravenous chemotherapy; intraperitoneal + intravenous chemotherapy; bevacizumab + intravenous chemotherapy; and hyperthermic intraperitoneal chemotherapy + intravenous chemotherapy. Optimized implementation was defined as follows. Patients triaged to primary cytoreductive surgery received intraperitoneal + intravenous chemotherapy if optimally or completely cytoreduced, and bevacizumab + intravenous chemotherapy if suboptimally cytoreduced. Patients triaged to neoadjuvant chemotherapy received hyperthermic intraperitoneal chemotherapy at interval cytoreductive surgery if optimally or completely cytoreduced, and standard IV chemotherapy if suboptimally cytoreduced. Life expectancy associated with optimized implementation was compared with that of current utilization practices, estimated using published literature and the National Cancer Database. Effects of model uncertainty were evaluated in sensitivity analyses.Life expectancy associated with optimized implementation vs. current practice was 76.7 vs. 64.5 months (life expectancy gain = 12.2 months). Providing intraperitoneal + intravenous chemotherapy to all eligible patients was the largest driver of life expectancy gains, due to both the potential benefit conferred by intraperitoneal + intravenous chemotherapy and the proportion of eligible women who do not receive intraperitoneal + intravenous chemotherapy in current practice.Population-level life expectancy in stage IIIC epithelial ovarian cancer could be substantially improved through greater uptake of available chemotherapy regimens. Most pain women , when thin women . The proin women .Treatment for advanced EOC typically includes both cytoreductive surgery and chemotherapy . The decRandomized control trials (RCTs) have shown that after complete or optimal cytoreduction at PCS, intraperitoneal (IP) + intravenous (IV) chemotherapy lengthens progression-free and overall survival (OS) compared to treatment with standard IV chemotherapy (carboplatin/cisplatin and paclitaxel) , 8. In pIn this study, we evaluated the potential life expectancy (LE) benefits of optimized implementation of standard, IP, bevacizumab, and HIPEC chemotherapy in a population of women with stage IIIC EOC. To accomplish this, we utilized a previously-developed microsimulation model for ovarian cancer treatment  and assiWe modified a previously described microsimulation model for ovarian cancer treatment  to studyUsing the model, we compared outcomes from “optimized implementation” vs. “current practice” strategies. The “optimized implementation” strategy was recognized to be hypothetical and actively evolving. Specifically, in real-world settings, some patients are ineligible for a given chemotherapy option for reasons that cannot be changed, e.g. comorbidities. Moreover, data that inform OS associated with certain therapies in our analysis are still new  . Other pTo project the associated LE for each strategy of interest, we modified a previously-defined standard-of-care treatment strategy within our original model . In the For patients triaged to PCS, three post-operative chemotherapy regimens were possible: standard IV chemotherapy; IP+IV chemotherapy; or bevacizumab+IV chemotherapy. IP+IV chemotherapy was reserved for patients who were completely (no visible disease) or optimally (visible disease <1 cm in diameter) cytoreduced . Bevaciz2-1hr infusion), compared to one dose per cycle (180 mg/m2-3hr infusion) in standard IV chemotherapy–has also been used after a Japanese RCT demonstrated increased progression-free and OS when administered after PCS [In some practices, dose-dense chemotherapy–defined by three doses of paclitaxel per cycle  [11–2014 . Once a Optimized chemotherapy implementation–and additional scenarios of chemotherapy selection–are described below. Using a one-month cycle length and a lifetime horizon, we predicted the mean LE and median survival among a simulated cohort of women with newly diagnosed stage IIIC EOC, under assumptions of different chemotherapy strategies. A more comprehensive description of the model and its parameters are in the published article and its online supplemental materials .We compared several hypothetical, population-level treatment strategies to current clinical practice. Descriptions of each strategy and corresponding model inputs can be found below and in First, we compared “current practice” to “optimized implementation” . In the In the “optimized implementation” strategy, after PCS, all eligible patients  received IP+IV chemotherapy. The remaining PCS patients  received bevacizumab+IV chemotherapy. All eligible NACT+ICS patients  received HIPEC+IV chemotherapy.Next, we evaluated three strategies in which we isolated each component of optimized chemotherapy implementation. In the “optimized IP+IV chemotherapy” strategy, we assumed all individuals who were completely or optimally cytoreduced at PCS received IP+IV chemotherapy. In the “optimized bevacizumab+IV chemotherapy” strategy, we assumed that after PCS, all suboptimally reduced patients received bevacizumab+IV chemotherapy. In the “optimized HIPEC” strategy, we assumed that after ICS, all completely or optimally cytoreduced NACT+ICS patients received HIPEC+IV chemotherapy . In eachPrimary outcome measures, per strategy, were median survival and LE. LE was calculated by taking the average length of survival, in months, among simulated patients; median survival represented the median number of months alive among simulated patients. LE gains and improvements in median survival for “optimized” strategies were calculated by comparing with the “current practice” strategy.We conducted deterministic sensitivity analyses to evaluate the effects of uncertainty in the survival benefits associated with each chemotherapy on LE gains. We analyzed the model over the reported ranges of uncertainty (elicited from the literature) for the OS hazard ratios for IP+IV, bevacizumab+IV, and HIPEC+IV chemotherapy , 10, 12.Our estimate for IP+IV chemotherapy utilization among optimally and completely cytoreduced patients was obtained from a study among patients treated at NCCN centers , which mRecognizing that 100% adherence with the “optimized implementation” strategy was unlikely, we evaluated additional scenarios in which such implementation was imperfect. Specifically, we considered 60%, 70%, 80%, and 90% implementation of “optimized” chemotherapy. We assumed that all patients not receiving “optimized” chemotherapy would receive standard IV chemotherapy.Lastly, we re-ran analyses of “current practice” and “optimized implementation” assuming that the survival benefit of dose-dense chemotherapy was as observed in a Japanese RCT (hazard ratio = 0.79) . In the We found that patients in the “current practice” strategy had a LE of 64.5 months and a median survival of 41.5 months. Patients in the “optimized implementation” strategy had a LE of 76.7 months and a median survival of 47.5 months, outperforming “current practice” by 12.2 and 6.0 months, respectively .For patients in the “optimized IP+IV chemotherapy” strategy, the projected LE and median survival were 71.7 months and 44.5 months, respectively, outperforming “current practice” by 7.2 and 3.0 months, respectively.For patients in the “optimized bevacizumab+IV chemotherapy” strategy, the projected LE and median survival were 65.0 months and 41.5 months, respectively, increasing LE by 0.5 months over “current practice”; median survival did not increase.For patients in the “optimized HIPEC” strategy, the projected LE and median survival were 69.0 months and 43.5 months, respectively, outperforming “current practice” by 4.5 and 2.0 months, respectively.Results of sensitivity analyses pertaining to survival benefits  are included in LE gains associated with the “optimized implementation” strategy were relatively insensitive to the survival benefit of bevacizumab. When we varied the hazard ratio from the lower to the upper bound of the reported 95% confidence interval, 0.74 to 0.96 , LE gainAs the proportion of eligible patients who were subject to “optimized implementation” increased, LE gains increased . For exaRCTs studying IP+IV, bevacizumab, and HIPEC chemotherapy options have demonstrated a survival benefit–relative to standard IV chemotherapy–for specific subpopulations of women with advanced EOC –9, 12. EThe gap between who is eligible and who receives IP chemotherapy is not well understood , 26. In Our study has additional limitations that merit mention. First, the literature regarding the clinical benefit of dose-dense IV chemotherapy administration is conflicting , 23, 28.Lastly, due to the absence of robust data, we did not simulate the impact of the various chemotherapy options on patients’ quality-of-life. While there is a difference in overall quality-of-life scores between IP+IV vs. standard IV chemotherapy during and in the few months after treatment (IP+IV scores are lower), after one year there is no reported difference . SimilarDespite these limitations, our results provide valuable context for clinicians and researchers in this field. We found that survival in a population of women diagnosed with stage IIIC EOC may be substantially improved when chemotherapy receipt is optimized, underscoring the importance of overcoming barriers to the use of optimal therapies. Equally, in an era of new and evolving treatment approaches for ovarian cancer, our study provides an example of how simulation models can be used to rapidly integrate new data on treatment effectiveness as it emerges, providing unique insight into the population-level impact of new therapies in the coming years.S1 FileModel output for life expectancy under “current practice” strategy assuming baseline proportion of eligible patients recieving IP chemotherapy is 0.615.(TXT)Click here for additional data file.S2 FileModel output for life expectancy under “current practice” strategy assuming baseline proportion of eligible patients recieving IP chemotherapy is 0.205.(TXT)Click here for additional data file.S3 FileModel output for life expectancy under “current practice” strategy assuming that patients are cured after surviving 16 years from diagnosis.(TXT)Click here for additional data file.S4 FileModel output for life expectancy under “current practice” strategy assuming that patients are cured after surviving 8 years from diagnosis.(TXT)Click here for additional data file.S5 FileModel output for life expectancy under the “current practice” strategy.(TXT)Click here for additional data file.S6 FileModel output for life expectancy under the “current practice” strategy with a hazard ratio of 0.96 on bevacizumab+IV.(TXT)Click here for additional data file.S7 FileModel output for life expectancy under the “current practice” strategy with a hazard ratio of 0.94 on HIPEC+IV.(TXT)Click here for additional data file.S8 FileModel output for life expectancy under the “current practice” strategy with a hazard ratio of 0.97 on IP+IV.(TXT)Click here for additional data file.S9 FileModel output for life expectancy under the “current practice” strategy with a hazard ratio of 0.74 on bevacizumab+IV.(TXT)Click here for additional data file.S10 FileModel output for life expectancy under the “current practice” strategy with a hazard ratio of 0.48 on HIPEC+IV.(TXT)Click here for additional data file.S11 FileModel output for life expectancy under the “current practice” strategy with a hazard ratio of 0.59 on IP+IV.(TXT)Click here for additional data file.S12 FileModel output for life expectancy under the “current practice” strategy assuming that bevacizumab has no additional survivial benefit (HR = 1.0) compared to standard IV chemotherapy.(TXT)Click here for additional data file.S13 FileModel output for life expectancy under the “current practice” strategy assuming that dose dense is used on 15.2% of all PCS patients and that it confers a survival benefit (HR = 0.79).(TXT)Click here for additional data file.S14 FileModel output for life expectancy under the “optimized implementation” strategy assuming that only 60% of eligible patients receive the optimized therapy according to their cytoreductive status. Patients who do not receive the optimal therapy are assumed to receive standard IV chemotherapy.(TXT)Click here for additional data file.S15 FileModel output for life expectancy under the “optimized implementation” strategy assuming that only 70% of eligible patients receive the optimal therapy according to their cytoreductive status. Patients who do not receive the optimal therapy are assumed to receive standard IV chemotherapy.(TXT)Click here for additional data file.S16 FileModel output for life expectancy under the “optimized implementation” strategy assuming that only 80% of eligible patients receive the optimal therapy according to their cytoreductive status. Patients who do not receive the optimal therapy are assumed to receive standard IV chemotherapy.(TXT)Click here for additional data file.S17 FileModel output for life expectancy under the “optimized implementaion” strategy assuming that only 90% of eligible patients receive the optimal therapy according to their cytoreductive status. Patients who do not receive the optimal therapy are assumed to receive standard IV chemotherapy.(TXT)Click here for additional data file.S18 FileLife expectancy output for strategy in which all primary cytoreductive sugery patients who are suboptimally cytoreduced receive bevacizumab+IV chemotherapy. All other patients receive chemotherapy according to “current practice”.(TXT)Click here for additional data file.S19 FileModel output for life expectancy under “optimized implementation” strategy assuming that patients are cured after surviving 16 years from diagnosis.(TXT)Click here for additional data file.S20 FileModel output for life expectancy under the “optimized implementation” strategy assuming that patients are cured 8 years from diagnosis.(TXT)Click here for additional data file.S21 FileModel output for life expectancy under the “optimized implementation” strategy with a hazard ratio of 0.96 on bevacizumab+IV.(TXT)Click here for additional data file.S22 FileModel output for life expectancy under the “optimized implementation” strategy with a hazard ratio of 0.94 on HIPEC+IV.(TXT)Click here for additional data file.S23 FileModel output for life expectancy under the “optimized implementation” strategy with a hazard ratio of 0.97 on IP+IV.(TXT)Click here for additional data file.S24 FileLife expectancy output assuming that 100% of complete or optimally cytoreduced patients who received neoadjuvant chemotherapy and interval cytoreductive surgery are given HIPEC+IV.(TXT)Click here for additional data file.S25 FileLife expectancy output under the “optimized implementation” strategy.(TXT)Click here for additional data file.S26 FileLife expectancy output assuming that 100% of all primary cytoreductive surgery patients who are complete or optimally cyroreduced receive IP+IV chemotherapy.(TXT)Click here for additional data file.S27 FileModel output for life expectancy under the “optimized implementation” strategy with a hazard ratio of 0.74 on bevacizumab+IV.(TXT)Click here for additional data file.S28 FileModel output for life expectancy under the “optimized implementation” strategy with a hazard ratio of 0.48 on HIPEC.(TXT)Click here for additional data file.S29 FileModel output for life expectancy under the “optimized implementation” strategy with a hazard ratio of 0.59 on IP+IV.(TXT)Click here for additional data file.S30 FileModel output for life expectancy under the “optimized implementation” strategy assuming that bevacizumab has no additional survivial benefit (HR = 1.0) compared to standard IV chemotherapy.(TXT)Click here for additional data file.S31 FileModel output for life expectancy under the “optimized implementation” strategy assuming that dose dense is used on all primary cytoreductive surgery patients who were suboptimally cytoreduced and that it confers a survival benefit (HR = 0.79).(TXT)Click here for additional data file.


A negative carbon isotope excursion recorded in terrestrial and marine archives reflects massive carbon emissions into the exogenic carbon reservoir during the Paleocene-Eocene Thermal Maximum. Yet, discrepancies in carbon isotope excursion estimates from different sample types lead to substantial uncertainties in the source, scale, and timing of carbon emissions. Here we show that membrane lipids of marine planktonic archaea reliably record both the carbon isotope excursion and surface ocean warming during the Paleocene-Eocene Thermal Maximum. Novel records of the isotopic composition of crenarchaeol constrain the global carbon isotope excursion magnitude to −4.0 ± 0.4‰, consistent with emission of >3000 Pg C from methane hydrate dissociation or >4400 Pg C for scenarios involving emissions from geothermal heating or oxidation of sedimentary organic matter. A pre-onset excursion in the isotopic composition of crenarchaeol and ocean temperature highlights the susceptibility of the late Paleocene carbon cycle to perturbations and suggests that climate instability preceded the Paleocene-Eocene Thermal Maximum. The Paleocene-Eocene Thermal Maximum (c. 55 million years ago) was a period associated with massive carbon injection into the atmosphere, yet discrepancies in carbon isotope proxy records have led to substantial uncertainties in the source, scale, and timing of carbon emissions. Here, the authors propose that membrane lipids of marine planktonic archaea can reliably record the carbon isotope excursion and surface ocean warming, giving a new constraint for the source and size of the PETM carbon emissions. The most intense hyperthermal, the Paleocene-Eocene Thermal Maximum (PETM), was a ~200 ka global climate perturbation2 associated with increased atmospheric CO2 levels4, ocean warming5, and ocean acidification6. Widespread observations of a negative carbon isotopic excursion (CIE) in various geological substrates indicate substantial emissions of 13C-depleted carbon into the biosphere-ocean-atmosphere (exogenic) reservoir during the PETM9. Due to the fossil nature of the carbon and the potentially rapid release, the PETM represents a close albeit imperfect analog for modern anthropogenic climate change12. Accurately reconstructing the amount, origin, and timing of carbon emissions during the PETM14 is a critical step towards understanding the associated climatic forcing and feedback mechanisms, and thus the impacts of carbon cycle perturbations on the past and future Earth system15.The early Paleogene witnessed frequent intervals of transient warming, so-called hyperthermals, superimposed on a greenhouse background climate16. The magnitude of the CIE can in principle be reconstructed through δ13C measurements of carbon-bearing substrates in geologic records, and is thus commonly used for mass balance calculations of PETM emission16  and > 10,000 Pg C (ref. 17), respectively. However, reconstructions of the isotopic composition of the carbon source from these models still depend on assumptions about the CIE magnitude18.The duration and magnitude of the CIE are important constraints on the amount and source of the emitted carbon13C values of the substrate. These factors include diagenetic overprints, source heterogeneity, and vital effects of the source organisms22. For instance, the carbon isotopic composition of soil bulk organic matter is affected by variations in higher plant assemblages23 as well as by variable fractionation imparted by changes in atmospheric CO2 concentrations and associated growth rates3. These factors similarly affect the carbon isotopic composition of marine phytoplankton biomass24 and thus bias reconstructions from bulk sediment organic matter (often towards larger CIEs), in addition to offsets resulting from varying admixtures of terrigenous organic matter into marine sediments19. By contrast, δ13C records of bulk carbonate and foraminifera may underestimate the magnitude of the CIE due to biases imposed by carbonate dissolution25, diagenesis26, and vital effects27. Collectively, these uncertainties confound not only the quantification of carbon emissions during the PETM16, but also our understanding of temporal release patterns28, climate sensitivity15, and ecosystem recovery10.The discrepancy in the magnitude of the CIE between sample substrates and across sampling locations likely originates from changes in geochemical and biological factors across the PETM that influence δ29, a group of marine planktonic archaea that fix dissolved inorganic carbon (DIC)30. δ13Ccren should thus primarily reflect δ13CDIC at the habitat depth of Thaumarcheaota32, which predominantly reside at the base of the photic and upper sub-photic zones34. Analysis of thaumarchaeal lipids additionally provides coupled ocean temperature estimates through the TEX86 proxy35, such that both δ13CDIC and ocean temperature can be obtained on the same substrate, circumventing stratigraphic uncertainties inherent to CIE–temperature correlations based on different substrates.A proxy for the CIE that is insensitive to mineral dissolution and has negligible or readily quantifiable vital effects would help constrain carbon emissions during hyperthermals. The archaeal lipid biomarker crenarchaeol may satisfy these criteria for the marine environment of the Paleogene. This organic compound originates from Thaumarchaeota13Ccren (described in detail in the Methods section and in ref. 32) to reconstruct the PETM CIE in thaumarchaeal lipids from three globally distributed sites. Values of δ13Ccren are then compared to the CIE recorded in newly generated and previously published δ13C records of total organic carbon (TOC)36, benthic foraminifera37, dinocysts38, and n-alkanes39. We argue that under PETM boundary conditions  δ13Ccren is offset by a predictable fractionation factor from δ13CDIC (refs. 41) and that the change in δ13Ccren during the PETM closely approximates the CIE in the global exogenic carbon reservoir. We further couple δ13Ccren to TEX86 analyses which allows simultaneous reconstruction of CIE and temperature change in low-carbonate sediments, thereby circumventing confounding effects of carbonate dissolution prevalent during the PETM and other climate warming events. Our independent CIE estimate derived from δ13Ccren represents an important step towards constraining PETM carbon emissions.Here we apply spooling-wire microcombustion–isotope ratio mass spectrometry of δ42. The Arctic Ocean site was located on the submerged northern flank of the Lomonosov Ridge43. Records of δ13Ccren were generated for all three sites and were compared against δ13C changes in other substrates at these sites. Newly generated (New Jersey) and existing  TEX86 records were used to compare changes in ocean temperature and δ13Ccren across the PETM. To assess the potential influence of terrigenous input of crenarchaeol on δ13Ccren, records of BIT (branched-over-isoprenoid tetraether index), a semi-quantitative tracer for input of soil organic matter44, were generated for the New Jersey core, while literature data were used for the Tasman Sea36 and Arctic Ocean43 cores. δ13CTOC records were generated for the Ancora and Arctic Ocean cores and literature data were used for the Tasman Sea site36.We sampled the Paleocene-Eocene boundary in three marine sediment cores Fig.  retrieve13Ccren data allow an assessment of the magnitude of the crenarchaeol CIE (CIEcren) at each location. For the Ancora and Tasman Sea datasets, we estimate CIEcren as the difference between mean pre-PETM and mean peak-PETM values 16. The intervals of the pre- and peak-PETM were determined through ramp function fitting45 , and −6.67 ± 0.22‰ in the Arctic Ocean. These values are significantly more heterogeneous than our δ13Ccren-based estimates, possibly due to variable proportions of terrigenous and marine input influencing the δ13CTOC signal, which leads to an overestimation of the CIE at least for the Arctic Ocean record19. At the New Jersey shelf, the only site studied here with sufficiently preserved carbonates, benthic foraminifera46 indicate a CIE of −3.54 ± 0.34‰ at Ancora and −3.76 ± 0.44‰ at Bass River  is −4.31 ± 0.39‰ at Ancora, −3.06 ± 0.37‰ in the Tasman Sea  throughout the Ancora record, which is similar to previous records from Bass River86 values increase by ~0.2 in the Ancora record, indicating ocean warming, and return to pre-CIE levels after the PETM , which has previously been attributed to unusually high abundances of tricyclic GDGT at this site relative to modern core-top compilations43. However, the modified TEX86’ index, which omits tricyclic GDGT but is still well-correlated to SST in modern core-top sediments43, shows an increase of 0.07 in parallel with the CIE, and returns to pre-CIE levels after the PETM . For comparison, TEX86’ values calculated for the Ancora and Tasman Sea records also increase in parallel with TEX86  and δ13Ccren signals at the PETM onset, potentially indicating synchronous change in temperature and CIE. However, incomplete recovery in the Arctic Ocean record43 and potential condensation in the Tasman Sea record47 could obscure potential lead-lag relationships.At the depth of the CIE, TEX3−) rather than from CO2 (ref. 30). Flux balance modeling41 and environmental data40 suggest that carbon isotopic fractionation in marine Thaumarchaeota originates from passive CO2 transport into the cell combined with slow conversion to HCO3-, resulting in a dependence of the expressed fractionation factor on the dissolved CO2 concentration. Importantly, the effect is inverse to the influence of pCO2 on carbon isotopic fractionation in phytoplankton, resulting in a smaller fractionation factor (εDIC-Cren) at high dissolved CO2 concentrations41. Due to the pCO2 increase during the PETM εDIC-Cren may have been smaller by 0.75 ± 0.15‰ (range 0.6–0.9‰) during the peak-PETM compared to the pre-PETM  of −3.87 ± 0.25‰ at Ancora, −4.03 ± 0.27‰ in the Tasman Sea, −4.06 ± 0.27‰ in the Arctic Ocean, yielding an average of −4.0 ± 0.4‰. Tighter constraints on Paleocene-Eocene pCO2 will allow reducing the uncertainty in εDIC-Cren estimates. Regardless, we expect the pCO2-dependent sensitivity of εDIC-Cren to be relatively constant across archaeal species, as cell morphology and physiology are highly similar among marine planktonic Thaumarchaeota50 and the 3-hydroxypropionate/4-hydroxybutyrate pathway is phylogenetically conserved30. Therefore, minimal effects are expected from changes in the diversity or distribution of thaumarchaeal clades between the modern ocean and past ecosystems. Studies with additional thaumarchaeal cultures will be needed to fully assess potential impacts of changes in community composition on εDIC-Cren and δ13Ccren records.The distinctive biology of Thaumarchaeota yields important constraints on the carbon isotope systematics of their lipids. Thaumarchaeota fix carbon using the 3-hydroxypropionate/4-hydroxybutyrate pathway exclusively from bicarbonate , where significant changes in water column hydrography and changing contributions from deep-water Thaumarchaeota did not lead to significant changes in sediment δ13Ccren. The consistency of δ13Ccren data through the sapropel events  imply that strong changes in ecosystem states, such as eutrophication, habitat depth changes, and anoxic/euxinic conditions do not significantly impact δ13Ccren values53. This is explained by recognizing that the depth habitat of Thaumarchaeota is linked to oceanographic parameters such as the deep chlorophyll maximum and the depth of maximum remineralization. The thaumarchaeal population should thus maintain a similar relative position along the δ13C gradient, rather than being fixed to a specific water depth. Although it is possible that warming-induced changes in remineralization rates during the PETM could have impacted δ13Ccren values by changing δ13CDIC gradients through the thaumarchaeal depth habitat, the extent of these changes likely was minimal given the warm greenhouse background climate of the Paleocene-Eocene54. Overall a primary advantage of estimating the CIEDIC from δ13Ccren is the apparent niche stability of marine Thaumarchaeota.Due to their ecological niche as chemolithoautotrophic ammonia oxidizers, Thaumarchaeota are most abundant in a narrow interval at the base of the photic zone13Ccren, assuming autotrophic δ13Ccren is > −20‰ and δ13C of the organic substrate would be < −20‰56. However, close examination of earlier studies demonstrates that there is currently no direct evidence for mixotrophy among epipelagic Thaumarchaeota57. Previous estimates indicating 17 ± 11% heterotrophic carbon assimilation were based on suspended particulate matter samples from 700 m water depth56 and thus were not representative of the major habitat and export depth of Thaumarchaeota, which is centered around ~150 m water depth58. Second, previous reports of amino acid utilization55 solely demonstrated uptake of exogenous amino acids into thaumarchaeal cells but not contribution to biomass carbon. Finally, recent 13C-label experiments demonstrated that all cultivated Thaumarchaeota are strictly autotrophic57. In summary, present knowledge supports the idea that values of δ13Ccren reflect autotrophic growth and reliably record δ13CDIC.Putative mixotrophy  by marine archaeal consortia could affect values of δ13Ccren values at our three study sites additionally corroborate an autotrophic origin of crenarchaeol. Preceding the CIE, absolute values of δ13Ccren differ by up to 3‰ between sites, with the most positive δ13Ccren values found in the Tasman Sea and the most negative values observed in the Arctic Ocean, with an intermediate gradient of ~1 to 1.5‰ in δ13Ccren values between the Tasman Sea and New Jersey shelf; the latter gradient is similar in magnitude but opposite in direction to the modern gradient between the North Pacific and North Atlantic59. Although lower δ13Ccren values in the northern Atlantic and Arctic compared to the Pacific Ocean support previous arguments for distinct, and potentially reversed, ocean circulation patterns during the Paleocene-Eocene63, similar CIE magnitudes at all sites suggest that the effect of potential deep water circulation reversals on the CIE was minor in comparison to whole-ocean δ13CDIC changes. Calculating pre-PETM δ13CDIC from an expected εDIC-Cren of −19.7 ± 0.1‰ , −0.5 ± 0.2‰ (New Jersey shelf), and −2.2 ± 0.3‰ (Arctic Ocean). These δ13CDIC reconstructions may prove useful as paleoclimate model targets.Systematic offsets between δ13Ccren values in the Arctic Ocean, when compared to the Tasman Sea and New Jersey shelf, indicate distinct environmental conditions for the Arctic basin. Lower absolute δ13Ccren values may reflect accumulation of 13C-depleted DIC in the chemocline and anoxic zone of the Arctic Ocean due to reduced mixing under enclosed, euxinic conditions similar to the modern Black Sea or Cariaco Basin64. Further, post-PETM δ13Ccren values in the Arctic Ocean are equal to or higher than pre-PETM δ13Ccren values, while they are lower than pre-PETM values in the Tasman Sea and New Jersey records , enclosed basins (Arctic Ocean), and deep open ocean environments, additional records from deep ocean environments could help constrain the influence of local effects on δ13CDIC and δ13Ccren. Terrigenous input of crenarchaeol into the proximal Arctic Ocean also could yield depleted δ13Ccren values and a biased CIEcren, given that terrigenous crenarchaeol would likely be more depleted in 13C than marine-derived crenarchaeol32. The BIT record at our deep Arctic Ocean site indicates significant soil input only during the pre-PETM interval  using crenarchaeol degradation products from a North Sea record31, although this previous record is too sparsely resolved to allow rampfit estimates.The lack of biases from source heterogeneity and the agreement between all three marine sediment cores yields confidence in our δ13Ccren may circumvent problems that confound other types of δ13C records. Among the most commonly used proxies, bulk marine, and soil organic matter as well as plant and algal biomarkers tend to overestimate the CIE due to increased isotopic fractionation at higher CO2 levels67. Bulk measurements are additionally prone to biases resulting from shifting plant/algal community composition or from varying admixtures of terrigenous and marine organic carbon to the bulk marine TOC pool19. These effects are reflected in variable CIE magnitudes of −3.1‰ to −6.7‰ in δ13CTOC at our three study sites . CIEs reconstructed from more specific algal and plant lipid biomarkers likely suffer from similar biases, yielding diverging results of ~ −6.0 ± 0.2‰ for plant-derived C27n-alkanes but no discernable CIE for putatively algal C17n-alkanes at our Arctic Ocean study site39  CIE of −4.6‰ (ref. 23). However, this estimate does not account for changes in pCO2-dependent fractionation and thus may overestimate the true CIE.Comparison with established proxies suggests that δ6; e.g., below ~1500 m water depth in the South Atlantic25), and/or post-depositional carbonate recrystallization26. In addition, vital effects in foraminifera may result in δ13C carbonate-seawater disequilibria70. These confounding effects are reflected in a large spread of foraminifera-based CIE estimates between −0.6‰ and −5.1‰ for benthic species (median −2.6‰)16 and −0.7‰ to −4.4‰ for planktonic species (median −2.6‰)16. At the New Jersey shelf, the only site studied here with well-preserved (glassy) foraminifera, benthic foraminifera indicate a CIE of −3.5 ± 0.3‰ (Ancora37) and −3.8 ± 0.4‰  likely is biased by generally poor carbonate preservation. Additional biases from vital effects70 may have amplified  or dampened  the CIE in foraminifera. However, it is currently not possible to quantify the net bias resulting from these effects.In contrast to bulk organic matter, bulk marine carbonate and foraminiferal carbonate may underestimate the CIE due to carbonate dissolution in the global deep ocean during the PETM . Equilibration of the surface and deep ocean carbon pools within < 4 ka is supported by model simulations across a wide range of carbon emission rates, magnitudes, and isotopic compositions13Ccren can be used to refine plausible carbon emission scenarios for the PETM. To this end, we compiled previous estimates of CIE magnitude as well as the mass and isotopic composition of emitted carbon 20, to 3000–3700 Pg C if the source is only methane 71, and to 8200–10,300 Pg C if the source is organic matter (δ13C = −22‰ to −26‰)21. These ranges of δ13Ccren-based emissions estimates are not only narrower than previous mass balance estimates but also more precise, with an uncertainty of ~10% compared to the much larger uncertainties resulting from the wide scatter of other substrate-specific CIEs16 , with a CIE of −3‰ in foraminifera used to independently constrain the δ13C of the carbon source to −50‰. The -4.0 ± 0.4‰ CIE in crenarchaeol instead implies that the carbon isotopic composition of the emitted carbon would be required to be more negative (< −60‰) for this scenario. Similarly, ocean pH data-model comparisons17 suggesting larger emissions  used a CIE of −2.6‰ derived from planktonic foraminifera to constrain the δ13C of the carbon source to −11‰ (ref. 17), implying emission of mostly mantle-derived CO2. Our larger δ13Ccren-based CIE again would imply a more 13C-depleted carbon source; specifically, it would scale the mean emission δ13C value to −17‰ . A revised emission δ13C value of −17‰ suggests larger contributions from organic matter, such as from thermogenic methane generated by North Atlantic sill emplacement72, sedimentary organic matter oxidation73, or methane hydrate dissociation8. In summary, the tighter constraints on the CIE from δ13Ccren suggest that previous estimates based on smaller CIEs may have pointed to carbon source δ13C values that are too enriched in 13C and/or underestimated the magnitude of carbon emissions. Carbon emissions must have been larger than 3000 Pg C for a release from methane hydrates and larger than 4400 Pg C for any scenario involving contributions from sedimentary organic matter oxidation and/or geothermal heating. Building on our refinement of the CIE magnitude and uncertainty, emerging highly resolved records of pCO2 or carbonate system parameters  will allow closer evaluation of the rates and mechanisms of PETM carbon emissions.Although a mass-balance approach assumes a single emission pulse, our δ86-paleothermometer, which can be used to evaluate the role of pre-CIE warming as a trigger for carbon emissions. This approach avoids biases from diagenetic carbonate overgrowth and dissolution affecting foraminiferal δ13C values26 and stratigraphic ambiguities that arise when separate substrates are used for the CIE and warming. Although incompleteness of the PETM onset in the Arctic Ocean record, and potentially the Tasman core, as well as a lack of well-constrained age models limit the determination of lead-lag relationships, the parallel changes in δ13Ccren and TEX86 , recorded in a terrestrial section from the Bighorn Basin 12. If these events were indeed coeval, the POE must have been a global event. The POE was an ephemeral event that lasted < 2 ka before recovery to pre-POE conditions12, as recorded in the decrease of both TEX86 and δ13Ccren values in the Tasman Sea immediately prior to the PETM onset  purification and carbon stable isotopic analysis of crenarchaeol and (ii) analysis of lipid abundances for determination of TEX86 and BIT.Sediment samples (10–20 cm³) were freeze dried, homogenized, and subsequently extracted in teflon vessels in three steps  using a MARS5 microwave-assisted extraction system . The microwave program consisted of 30 min heating to 100 °C and 20 min hold time, after which the solvent was decanted and fresh solvent was added. The extracts from the successive extraction steps were combined into a total lipid extract (TLE), dried under N2 (130–270 mesh) to yield a GDGT-containing fraction32. The GDGT-containing fraction was further purified by orthogonal semi-preparative high performance liquid chromatography (HPLC) on an Agilent 1200 series HPLC equipped with a fraction collector, following previously described protocols53.TLE aliquots were separated over pre-combusted SiO32. The fractions were dissolved in ethyl acetate and 30–100 ng were manually injected onto the wire . The raw data were corrected for blanks and absolute offsets using dilution series of the C46-GTGT standard. The 1σ precision averaged over all samples was 0.15‰. Purity of the crenarchaeol fraction was assessed using the ratio of SWiM–IRMS F2/F1 peak areas 53.Relative abundances of GDGTs (Supplementary Data 86 and BIT values were calculated from relative abundances of GDGTs for the Ancora record. Published TEX86 and BIT data from adjacent samples were used for the Arctic Ocean43 and Tasman Sea records36. TEX86 values were corrected for differences in peak separation between the traditional single-column HPLC-MS method and the newer protocol76 employed here by using a cross-calibration of 27 Arabian Sea and Atlantic Ocean sediments. Due to the ambiguity associated with choosing appropriate deep-time TEX86 sea surface temperature calibrations, all data are presented as TEX86 or TEX86’ ratios rather than putative sea surface temperatures, with the understanding that higher ratios correspond to relatively warmer temperatures.TEX13CTOC as determined by replicate analysis of reference standards was 0.04‰.Content and carbon isotopic composition of TOC at sites ODP 174AX and IODP 302 (Supplementary Data DIC-Cren was calculated for pre-PETM and peak-PETM boundary conditions (Supplementary Fig. 41. Boundary conditions17 were set as: 25 °C seawater temperature, salinity 35, [DIC] 2000–3000 µmol kg-1, pH 7.75–7.45. εDIC-Cren depends on assumed pCO2 change across the PETM, following results from Gutjahr et al.17, εDIC-Cren changed by 0.9‰, from of 19.7 ± 0.1‰ for the pre-PETM to 18.8 ± 0.05‰ for the peak-PETM. However, using the smaller pCO2 change suggested by Zeebe et al.13, εDIC-cren changed by only 0.6‰. Uncertainty in pCO2 change across the PETM thus leads to an uncertainty in εDIC-cren of ± 0.15‰ from an average of 0.75‰. CIEcren was calculated from pre-PETM and peak-PETM δ13Ccren data using the rampfit software45. To derive CIEDIC, pre-PETM and peak-PETM εDIC-cren were applied to CIEcren to derive CIEDIC and uncertainty was determined by propagating the measurement uncertainty, the rampfit estimated error of CIEcren, and the uncertainty in εDIC-cren.εSupplementary InformationPeer Review FileDescription of Additional Supplementary filesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4