We first examined alterations in the expression of uterine contraction-associated genes in uteri of oxytocin receptor–deficient mice (Oxtr−/−) during parturition. We found that induction of cyclooxygenase (COX)-2 and connexin 43 expression was impaired in Oxtr−/−, whereas that of PGFα2 receptor expression was not. We next generated mice with double knockout of genes for the oxytocin receptor/oxytocin and PGFα2 receptor  and evaluated their parturition with Oxtr−/−, Oxt−/−, Ptgfr−/−, and wild-type mice. In Oxtr−/−;Ptgfr−/− and Oxt−/−;Ptgfr−/−, pregnancy rates were similar to those of other genotypes. However, normal parturition was not observed in Oxtr−/−;Ptgfr−/− or Oxt−/−;Ptgfr−/− because of persistent progesterone from the corpus luteum, as observed in Ptgfr−/−. We administered RU486, a progesterone antagonist, to Ptgfr−/−, Oxtr−/−;Ptgfr−/−, and Oxt−/−;Ptgfr−/− on gestation day 19. These mice were able to deliver a living first pup and the parturition onset was similar to that in Ptgfr−/−. Meanwhile, unlike Ptgfr−/−, ∼75% of Oxtr−/−;Ptgfr−/− and Oxt−/−;Ptgfr−/− administered RU486 remained in labor at 24 hours after the onset of parturition. All of the pups that experienced prolonged labor died. We thus revealed that the oxytocin receptor is an upstream regulator of COX-2 and connexin 43 in the uterus during parturition and that both oxytocin/oxytocin receptor and PGFα2 receptor are major components for successful parturition.Parturition is an essential process in placental mammals for giving birth to offspring. However, the molecular machineries of parturition are not fully understood. We investigated whether oxytocin plays a crucial role in the progress of parturition in cooperation with the prostaglandin F Oxt−/−) and oxytocin receptor–deficient mice (Oxtr−/−) showed normal parturition, and roles of the oxytocin system in parturition remain unclear  and prostaglandin E2 (PGE2) are known as uterotonic factors and have been clinically used to augment labor (α2 receptor–deficient mice (Ptgfr−/−), progesterone withdrawal in blood during late pregnancy and the subsequent onset of parturition did not occur. A reduction of blood progesterone level with ovariectomy at day 19 of pregnancy induced expression of the oxytocin receptor in the uteri and enabled successful parturition in Ptgfr−/−, although uterine contractile activity via the PGFα2 receptor was lost  and evaluated their parturition.To clarify the hierarchy of these molecules for preparation, onset, and progress of parturition, we first examined the expression of mRNA for uterine contraction-associated genes in the uterus of Animal experiments were carried out after receiving approval from the Animal Experiment Committee of Tohoku University and were conducted in accordance with the Institutional Regulations for Animal Experiments and Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.Oxtr−/−, Oxt−/−, and Ptgfr−/− with a chimeric background (129 × C57BL/6J), generated previously , 5:26:15:19:39:28:10:26:5; , 16:48:21:41:62:51:17:42:20]. This result indicated that double deletion of oxytocin or the oxytocin receptor and the PGFα2 receptor did not affect embryonic lethality. Body weights of wild-type (WT), Oxtr−/−;Ptgfr−/−, and Oxt−/−;Ptgfr−/− females were 20.5 ± 1.4 g, 20.2 ± 1.1 g, and 20.7 ± 0.6 g, respectively .We used eviously , 9. Oxtram to 7:30 pm). Females (8 to 29 weeks old) were mated with male C57BL/6J mice  during the dark phase. Females were separated from males the next morning. The morning when a vaginal plug was observed was defined as gestation day (GD) 0.5. The pregnant females were individually housed from GD 17.5 on. From the day before expected birth (GD 18.5), pregnant females were observed every 6 hours and the number of pups at each time was recorded. Initiation of parturition was determined as the time that a female delivered the first pup. The uterus of each female was checked by opening the abdominal cavity 24 hours after birth of the first pup. All of the pups that remained in the uterus of Oxtr−/−;Ptgfr+/−, Oxtr−/−;Ptgfr−/−, and Oxt−/−;Ptgfr−/− died at that time.Mice were housed in rooms with controlled temperature (25 ± 2°C) under a 10-hour light/14-hour dark cycle  and digested with DNase I  to prevent genomic DNA contamination. Ten micrograms of DNase I–treated RNA was then reverse transcribed by using SuperScript II reverse transcription  and oligo(dT) primer in a 97-μL reaction volume according to the manufacturer’s instructions. Nondiluted first-strand cDNA solution was used for quantitative real-time PCR (qPCR).Total RNA from uterine horns of α2 receptor, connexin 43, COX-1, COX-2, and PGE2 receptors 1 to 4 using a DNA Engine Opticon system . Mouse ribosomal protein large P0 was used as an internal control. The assay was performed in a 20-μL reaction volume containing DyNAmo SYBR Green qPCR master mix , 1 μL of cDNA solution, and 12.5 pmol of each of gene-specific primer. The primers used are shown in qPCR was conducted to measure the relative mRNA expression levels of the oxytocin receptor, PGFPtgfr−/−, Oxtr−/−;Ptgfr−/−, and Oxt−/−;Ptgfr−/− were injected subcutaneously with the progesterone receptor antagonist RU486  (225 μg in 100 μL of peanut oil) or a vehicle on GD 19.0.Trunk blood was collected by decapitation on GD 17.0 and GD 19.0. Plasma samples were obtained by centrifugation. The plasma concentration of progesterone was measured in duplicate by an RIA   and stored at −80°C. Coronal cervix sections were cut at 10 μm with a cryostat. The sections were assessed for collagen fibers by Elastica-Masson staining (blue stain). Muscle tissues and nuclei are stained red and deep red, respectively.Uterine cervixes of WT, P < 0.05 was considered statistically significant.Data are expressed as means + SEM. Data were analyzed by one-way ANOVA followed by a Tukey–Kramer posttest and Fisher exact probability test. Oxtr−/−. We first confirmed the expression of mRNA for the oxytocin receptor in the WT uterus on GD 17.0 and GD 19.0 and during parturition. Oxytocin receptor expression during parturition was significantly increased compared with that on GD 17.0 and GD 19.0    and indual cells . The proal cells . Alternaal cells . There iregnancy , 29. It in vitro . A studye uterus . Oxytociin vitro . It is lα2 receptor gene or COX-1 gene (a major gene responsible for the synthesis of PGFα2 in luteolysis) in mice resulted in a deficit of luteolysis at term, and progesterone concentration did not decline during late pregnancy in these deficient mice  was lower than the percentages of Oxtr−/−;Ptgfr−/− and Oxt−/−;Ptgfr−/− . These findings suggested that both oxytocin/oxytocin receptor signaling and PGFα2 receptor signaling play an important role in the progress of parturition. Sensitivity of uterine contraction to vasopressin increased at late pregnancy (Oxt−/−;Ptgfr−/− administered RU486 were similar to those of Oxtr−/−;Ptgfr−/− administered RU486. These findings suggested that vasopressin did not compensate for oxytocin function via the oxytocin receptor and did not have a major contribution to the progress of parturition in mice.regnancy , 6. In tPtgfr−/− studies clearly indicated that a decline of maternal blood progesterone concentration via luteolysis in late pregnancy is essential for initiation of labor (α2 receptor, COX-2, and connexin 43 is induced during parturition in mice. In humans, oxytocin and PGFα2 have been used to induce or augment labor in clinical practice (α2 receptor, COX-2, and connexin 43 was upregulated at term in labor in the human myometrium (in vitro (In humans, maternal serum progesterone concentrations do not vary significantly in the late third trimester of pregnancy, and there is no evidence of a fall in maternal plasma or uterine tissue progesterone at labor, unlike those of rodents . Howeverof labor , 11. Howof labor , 42. RU4of labor , 43. In of labor . All of practice . The expometrium , 45–47. in vitro . These fα2 receptor antagonists OBE022 and OBE002 are currently being used in a phase 2 clinical trial for pregnant women with preterm labor (α2 receptor have a complementary relationship in the process of parturition. Our results suggest that administration of an oxytocin receptor antagonist in combination with a PGFα2 receptor antagonist would be more effective for spontaneous preterm labor.Preterm birth is the main cause of neonatal morbidity and mortality: it causes 25% to 50% of long-term neurologic impairment in children and ∼70% of neonatal deaths and 36% of infant deaths . It is rrm labor . Perinatrm labor . We showα2 receptor signaling are major components for successful parturition. Our results thus indicate the transcriptional and functional hierarchy of uterotonics required for successful parturition.In conclusion, the oxytocin receptor is an upstream regulator of COX-2 and connexin 43 expression in the uterus during parturition, and both oxytocin/oxytocin receptor signaling and PGF


However, it is not clear what their carbon sink capacity will be under extreme climatic conditions. In this study, we used long-term (1998–2013) data from a C3 grassland Free Air CO2 Enrichment (FACE) experiment in Germany to study the combined effects of elevated [CO2] and extreme climatic events (ECEs) on aboveground biomass production. CO2 fertilization effect (CFE), which represents the promoted plant photosynthesis and water use efficiency under higher [CO2], was quantiffied by calculating the relative differences in biomass between the plots with [CO2] enrichment and the plots with ambient [CO2]. Down-regulated CFEs were found when ECEs occurred during the growing season, and the CFE decreases were statistically significant with p well below 0.05 (t-test). Of all the observed ECEs, the strongest CFE decreases were associated with intensive and prolonged heat waves. These findings suggest that more frequent ECEs in the future are likely to restrict the mitigatory effects of C3 grassland ecosystems, leading to an accelerated warming trend. To reduce the uncertainties of future projections, the atmosphere-vegetation interactions, especially the ECEs effects, are emphasized and need to be better accounted.Terrestrial ecosystems are considered as carbon sinks that may mitigate the impacts of increased atmospheric CO The rising atmospheric CO2 concentration contributes largely to global warming, and also stimulates ecosystem productivity5. It has been estimated that terrestrial ecosystems have sequestered about 25% of the anthropogenic carbon emissions over the past half-century6. Therefore, most ecosystems potentially act as carbon (C) sinks and mitigate the effects of increased [CO2]8. Concerns regarding the future capacity of ecosystems as C sinks have been raised due to the negative effects of extreme climatic events (ECEs)9. Extreme events such as prolonged heat waves, droughts, and frosts can significantly reduce ecosystem carbon uptake and productivity, thereby influencing the regional carbon cycle13 and gradually shifting the ecosystems from a C sink towards a C source10. Since ECEs have been projected to increase in both frequency and intensity15, studying the combined effects of ECEs and elevated atmospheric [CO2] ([eCO2]) on the carbon cycle of ecosystems is important for both understanding mechanisms16 and reducing predictive uncertainty17.Atmospheric carbon dioxide concentration [CO2] stimulates ecosystem productivity  directly through (i) enhanced photosynthesis18, or indirectly through (ii) reduced stomatal conductance22 and (iii) reduced respiration23. Accordingly, one may expect stronger CFEs at higher temperatures or in drier conditions25, and the negative effects of ECEs may be ameliorated through improving water use efficiency (WUE)26, increasing the plant carbon uptake27, and enhancing recovery after ECEs28. However, different studies have demonstrated that these theories are not applicable to all ecosystems30. In some experiments, [eCO2] was found to have no alleviating effect against ECEs32. On the contrary, [eCO2] may increase the risk of exposure to ECEs by extending the growing season length33. Meanwhile, ECEs can prevent plants from benefiting from [eCO2]34. The CFEs may be strongest under intermediate environmental conditions and vanish under more extreme weather conditions35. The inconsistencies in these results indicate an important role for ECEs in altering CFEs, and emphasize the necessity for more detailed studies, which have thus far been prevented due to the lack of suitable long-term continuous and high quality data36.Elevated [CO2] and ECEs on aboveground biomass production. The Gi-FACE experiment was carried out on a permanent grassland in the German federal state of Hesse, near Giessen  at 172 m a.s.l. . They were arranged in a randomized block design (three blocks). The CO2 fumigation began in May 1998 with an enrichment level of +20% [CO2] above the ambient level during daylight hours  experiments  in the world and studied the combined effects of  treatment. As a result, when considering total biomass, the treatment effect was only statistically significant for H2 when the second time section (2007–2013) was considered. This is reasonable as large initial biases of the forbs (incl. legumes) existed in the first few years of the experiment. As discussed in38, since 1997 before the start of the FACE up to the years before 2006/2007, there were more forbs and legumes harvested in the aCO2 plots than in the eCO2 plots. Benefited from the higher [CO2] in eCO2 plots, an increasing trend of ES was observed from 2001 to 2008 , but the positive effects of elevated [CO2] were still covered especially for the first time section (1998–2005/2006). Therefore, the ES of forbs (incl. legumes) is not a good indicator for the CFEs. In the following analysis, we will mainly focus on the reactions of the aboveground total biomass, as well as grass biomass to the emergence of ECEs. The results regarding forbs (incl. legumes) will be shown in the Supplementary Materials  from 1999–2011 into two groups according to the occurrence of ECEs, the ES changes were well separated , different ECEs were identified. For the spring growing periods, we identified extreme cold events in 2003, 2005, and 2013; spring hard frost events in 2005, 2010, and 2013; as well as extreme dry events in 2007ems Fig. . In fact012 Fig. , indicatass Fig. . If we rted Fig. . For theTmax higher than 30 °C, respectively , we found also high values in 2010 and 2013   Fig. . Only in10) Fig. . After r10) Fig. . Conversely Fig. 0, we fou10) Fig. . For theet al.35, who quantified CFEs as the slopes of productivity versus actual measured [CO2] to cope with the varying CO2 concentrations in real-world experiments. Fig. 2] were found  and elevated [CO2] enrichment, or PFG competitions. For instance, the decreased ES in both harvest of 2012 were most probably related to the extremely low [CO2] enrichment  where the decreased ES were not related to ECEs. Accordingly, the occurrence of ECEs is only a sufficient condition for the decrease of ES, not a prerequisite. The changes of ES can be influenced by other factors such as anomalous low . Properly quantified indexes that present the effects of ECEs may be important for future projections. In our work, we calculated the killing degree days (KDDs) as one measure of the extreme events. Actually, it can also serve as an useful indicator of the negative effects of high temperatures in summer. Before showing the relations between KDDs and ES in H2, it is worth noting that the enrichment level of [CO2] was set as 20% above the ambient level and in most years, the actual measured enrichment level was indeed around 20%  in 2012, and extremely high (48.53%) in 2013  the ECEs can down-regulate the CFEs, and (ii) a properly quantified index (e.g. KDDs) may help explain the changes in CFEs. As extreme climatic events such as drought, heat waves, etc., are projected to increase in both frequency and intensity−1 yr−1 up to 1995. Afterwards nitrogen fertilization was reduced to 40 kg N ha−1 yr−1. The harvested biomass of this species rich grassland is dominated by grass, with small amounts of forb and legume included. The FACE experiment started in May 1998 and the mean [CO2] enrichment is +20 % above ambient during daylight hours. There are three circular plots (rings) subjected to elevated [CO2] (eCO2), while another three circular plots (rings) served as controls with ambient [CO2] (aCO2)  at 172 m a.s.l. The local annual mean precipitation over the research period was 558 ± 92 mm and the annual mean 2m-air temperature was 9.4 ± 0.1 °C. The research area has been managed as a meadow. It was mowed twice a year and not ploughed for at least 100 years. The old, non-grazed grassland has been fertilized with 50–80 kg N hasee Fig. . They we2] plots and biomass averaged over the three ambient [CO2] plots were used to quantitatively show the CO2 fertilization effects. Besides biomass, daily soil moisture , semi-hourly 2m-air temperature, semi-hourly precipitation, as well as the hourly mean of CO2 concentration measured in the center of each ring, were used in this study. The soil moisture (averaged over the six rings) and precipitation are used for the determination of extreme dry events, while the 2m-air temperature records are used for the determination of extreme cold/hot events (as well as hard frost events and heat wave events), and also the calculation of Killing Degree Days (KDDs). With the measured CO2 concentration in each ring, we investigated the true [CO2] enrichment  and summer (H2) were used in this study. The spring harvest date was around the end of May (beginning of June) each year, while the summer harvest date was around the beginning of September  of aboveground biomass, which is defined as:Bio(eCO2) stands for the dry biomass matter obtained from eCO2 plots, while Bio(aCO2) represents the dry biomass matter obtained from the aCO2 plots. It is worth to note that there are three blocks in the experiment, and each block consist of two plots (one eCO2 plots and one aCO2 plots). Although the plots within each block are closely located  measured from the six plots (three blocks) were analyzed, respectively. Factors including time, CO2 treatment, block, as well as the interactions time × CO2, time × block were considered in the model. For each harvest (H1 and H2), the rmANOVA model was run for the full time period of 1998–2013. Meanwhile, as suggeted by the break point analysis in38, two half time sections, 1998–2005 and 2006–2013, were also analyzed by rmANOVA. If the p-values for the CO2 treatment is smaller than 0.05, we consider there were significant treatment effect and ES can be used to represent the CO2 fertilization effect.To check whether the effect size (ES) was indeed induced by the elevated [CO2 rings and in aCO2 rings, the product of RC in eCO2 rings and RC in aCO2 rings were calculated for each year  compared with the previous year, while negative products depict different changing directions (increase and decrease). By definition, increased grass percentage in eCO2 rings and decreased grass percentage in a CO2 rings may contribute to an increasing ES of grass biomass , while a decreased grass percentage in eCO2 rings associated with an increased grass percentage in aCO2 rings lead to a decreasing ES of grass biomass .To quantify the competition of grass with other plant functional groups (forb + legume), the Relative Changes (RCs) of grass percentage compared with that in the previous year were calculated Table , S2. To ear Fig. . PositivWe have defined different ECEs, including extreme cold and hot events, extreme dry events, hard frost in spring, as well as heat wave events. Their definitions are shown below. It is worth to note that (i) two times standard deviation (2 SD) was widely used for the determination of ECEs, but other threshold (e.g. 1.5 SD) has also been checked, which gives robust results; (ii) when studying the period-averaged (accumulated) temperature (precipitation), linear trends over 1997–2013 were removed before the analysis.Tmean < 0 °C) was exceptionally high ; or (iii) the consecutive days with Tmean < 0 °C was exceptionally long , we defined this growing period had experienced an extreme cold event  the minimum temperature averaged over this period was exceptionally low ; or (ii) the number of days with below-zero daily air mean temperature ; or (iii) the consecutive days with Tmax > 30 °C was longer than 5 days, we considered this growing period had experienced an extreme hot event  the maximum temperature averaged over this period was exceptionally high ; or (ii) the number of days with ent Figs , S5. EspExtreme dry events: For a given growing period, if (i) the precipitation accumulated over this period was exceptionally low ; or (ii) the soil moisture averaged over this period was exceptionally low ; or (iii) the consecutive rain free days were long enough to exceed 2 SD, we say this growing period had experienced an extreme dry event Figs , S7.36.Hard frost in spring: If in spring  after the first day of the growing season, the daily minimum temperature dropped below −10 °C, we defined it as a hard frost event in spring Fig. , which iTi, i = 1,2, …, n represent the daily maximum temperatures from the beginning to the end of the growing period. KDDs is one indicator that represent the negative impacts of high temperature. Different from40, where the threshold was 29 °C, here in this study we use 30 °C, as this is the threshold we used to determine heat wave events. Fig. KDDs is the sum of maximum temperatures in excess of 30 °C, as shown below,Supplementary Material


On applying different approximations, the enhancement of semiconducting gap follows the trend as GGA < mBJ < GGA + U. The maximum forbidden gaps observed by GGA + U method are Eg = 1.80 eV for Fe2TaAl and 1.30 eV for Fe2TaGa. The elastic parameters are simulated to determine the strength and ductile nature of these materials. The phonon calculations determine the dynamical stability of all these materials because of the absence of any negative frequencies. Basic understandings of structural, elastic, mechanical and phonon properties of these alloys are studied first time in this report.The structural and mechanical stability of Fe The technological applications exploiting these properties have been achieved successfully. Spintronic and thermoelectric applications are the offshoots of half-metallic ferromagnetism  and spin gapless attributes to the Seebeck effect useful for thermoelectric devices13. Within these dimensions, the materials with compatible lattice structure, high spin polarization and high Curie temperature are anticipated in practical spintronic applications. Magnetoelectronic devices mostly depend on the disproportionate number of majority and minority spin carriers, as exhibited ideally by half-metallic materials i.e. 100% spin polarization at the Fermi level. Such materials display the concoction properties of semiconductor and metal. Additional motive to delegate Heusler alloys in these applications is that these systems have the same crystallographic structure with different functional characteristics and some of them are even very close in electronic structure and composition15. Since the discovery of the NiMnSb Heusler alloy in 198316, a sequence of experimental as well as theoretical efforts (first principles simulations) were attempted to predict novel semiconductor or half-metallic systems. Among such compounds, transition metal based Heuslers have been widely investigated by material scientists worldwide. Predominantly, the Fe based Heusler structures constitute a vast family with semiconducting or half-metallic band profiles. For example, Fe2YSi  alloys were experimentally synthesized and predicted to be half-metallic alloys17. Fe2TiAl was reported to have thermoelectric applications18. Other materials like, Fe2TiSi, Fe2TiGe and Fe2ZrSi19, FeMnSi20, FeVRuSi21 and many more to report here have been investigated for their magnetic, semiconducting or mechanical properties. Using first-principle calculations, Fe2YZ  Heusler compounds with room temperature power factors 4 to 5 times larger than classical thermoelectrics were reported recently by Bilc et al.22. However, a little information is available on the electronic structure, mechanical stability, phonon dynamics and bonding characteristics of Fe2TaAl and Fe2TaGa alloys. In addition, the untouched lattice dynamical parameters and phonon properties are necessary to understand the intriguing physical properties and hence in this work, we tried to investigate their structural and mechanical stability, electronic and lattice dynamical properties in detail.Significant momentum in the study of intermetallic Heusler alloys has increased over the last decade as these systems exhibit numerous extraordinary capabilities in exposing the desired properties, extending from robust spin-polarization, half-metallic magnetism, magnetoresistance, shape memory effect, spin gapless semiconductor to giant magnetocaloric effect, phase transitions and thermoelectric effect2TaAl and Fe2TaGa type have been found to crystallize in a cubic structure with space group (Fm-3m) as depicted in Fig. 122. The corresponding atomic locations are Fe , Ta  and Z . The ground state structure is determined by geometry optimization via the total energy per unit cell (see Supporting Information) and thereby the equilibrium lattice constants, derivative of bulk modulus, total energy and equilibrium volume are obtained as shown in Table Full-Heusler ternary alloys of Fe23. These constants are determined by the linear response of a crystal towards the external forces, and are also associated with structural stability, equation of state (EOS), interatomic potential and phonon spectra24. Beyond this, the thermal properties including specific heat, thermal expansion, Debye temperature and Gruneisen parameter are exclusively linked to elastic constants. Consequently, the determination of elastic constants is indispensable to characterize a solid crystal. In this work, we have calculated the elastic constants of Fe2TaAl and Fe2TaGa systems using the generalized gradient approximation (GGA) and are listed in Table ij) are reduced to only three independent constants viz. C11, C12, and C44. From Table 12 < B < C11, (C11 − C12) > 0, (C11 + 2C12) > 0 and C44 > 0 is rigorously followed by the observed lattice constants and hence their mechanical stability is confirmed25. Further, to get the information about the different elastic moduli and other derivables, Viogt-Reuss-Hill method is used via the Eqns  stressesetc.) are summed up in Table 29. Concurrently, the ductile and brittle character of materials is directly linked to the critical value (1.75) of B/G ratio. If B/G is less than the critical value, then the material is ductile otherwise it is said to be brittle30. The covalent materials (e.g. Diamond) in principle are relatively hard and obviously brittle with a smaller Pugh ratio. The strong covalent bonds in such materials certainly produce a significant resistance resulting in a quite high hardness. Conversely, ductile materials with a high Pugh’s ratio are characterized by metallic bonding and low hardness. Consequently, the observed values of B, G and B/G clearly determine the Fe2TaAl alloy to be much harder and ductile from Fe2TaGa compound. This claim is also supported by the (C” = C12–C44) values because its positive value defines the ductile nature of the present alloys and if its value is negative, then the system is said to be brittle in nature31. Positive Cauchy pressure also specify the presence of metallic bonding in a material while as the negative value demonstrates the directional  and angular bonds. In the present set of calculations, Cauchy pressure predicted for both the Heusler alloys are positive which reflects their metallic character. Poisson’s ratio and its critical value; 0 < υ < 0.5 simply defines the plasticity of a crystal. Its small value reflects the maximum plastic character and conversely, the material is elastic in nature32. So, the elastic nature of these alloys can be observed from the υ values mentioned in Table 2TaAl alloy is purely isotropic but the Fe2TaGa alloy is anisotropic in nature31.Calculated results for the values of elastic parameters . Using the empirical equation  in their equilibrium structures, therefore, Slater-Pauling rule34; (Mt = Zt − 24) comprises the zero-spin magnetic moment for both these materials. Their non-magnetic character has earlier been reported in ref.22. The groundwork of structural optimization yields the equilibrium lattice constant, and the same is utilized to calculate the electronic band profile of Fe2TaAl and Fe2TaGa Heusler materials. Three different schemes; generalized gradient approximation (GGA), onsite Hubbard approximation (GGA + U) and modified Beckhe Johnson (mBJ) schemes have been employed and the spin polarized band profiles are displayed in Figs 2TaAl is observed to be a direct band gap material with Eg = 1.80 eV, while as Fe2TaGa is observed to be indirect band gap semiconductor with the corresponding energy gap of 1.30 eV. In the latter case, the valence band maximum (VBM) occurs at the Γ symmetry point and the conduction band minimum (CBM) occurs at the X symmetry point in its Brillouin zone. However, GGA and mBJ methods reveal both these materials to be the p-type indirect band gap semiconductors.Since both these alloys have 24 valence electrons  depicted in Fig. 37. Figure 2TaAl and Fe2TaGa system in its unit cell give rise to three acoustical and nine optical modes constituting a total of 12 phonon branches. But, in different directions, this number is reduced due to degeneracy. The optical modes obtained for Fe2TaAl are at around 510.19 cm−1 and 680.22 cm−1 and for Fe2TaGa are around 470.29 cm−1, 590.63 cm−1 at zone center Г and W points.Phonon dispersions are interesting phenomenon to understand the dynamical stability of a crystal system above and beyond knowing their thermal behavior, superconductivity, Raman and thermal spectroscopyωLO − ωTO) is called a reststrahlen band, which estimates the number of reflected electromagnetic waves. In our calculation It can also be seen from Fig. 2TaAl and −64.3 MeV for Fe2TaGa as the energy above the hull which is equal to the energy of formation of the compounds of interest from the phases that would be stable if the compound did not exist22. Therefore, our phonon results strengthen the stability criteria of these alloys. Moreover, we have calculated the cohesive energies (Ecoh) of these compounds using the following equation:2TaAl and Fe2TaGa per formula unit. The measurement of strength of the binding force between the constituent atoms in a solid structure is confirmed from cohesive energy its positive value indicates the stability of the material. The calculated values of the cohesive energy are 22.71 eV for Fe2TaAl compound and 20.67 eV for Fe2TaGa alloy. These observed values are significant and comparable to Co2TaSi (21.76 eV), Co2TaGe (19.18 eV)38 and Hf2Val (21.56 eV)39; and this specifies the large value of chemical bond energy. Therefore, the stability of the present materials is confirmed from the mechanical stability criteria, cohesive energy and phonon dispersion results.Since, the stability of both these materials has been confirmed via Convex Hull analysis with −303.3 MeV for Fe2TaAl and Fe2TaGa alloys have been examined using the density functional theory. Calculations of the electronic structure shows the indirect band gaps along Γ-L symmetry for all these materials. These compounds directly fall under the Slater Pauling rule that depicts their semiconducting behavior. Thus, we have confirmed from our studies that these full-Heusler alloys are narrow band gap semiconductors with 0.80 eV for Fe2TaAl and 0.61 eV for Fe2TaGa. In relation to electronic structure, the mechanical properties have been calculated. A theoretical study predicts the hardness and ductile nature of these materials and their ductility increases from Al > Ga and are potentially the possible hard semiconductor materials. In this study, the electronic, phonon and mechanical properties of cubic Fe2TaAl and Fe2TaGa alloys have been examined using the density functional theory. The calculation of lattice vibrations from DFPT gives the phonon dispersion. The coupling of optical and acoustic mode is an interesting phenomenon that we have observed in this study with zero LO-TO splitting at Г and W points of symmetry. The zero value of reststrahlen band is responsible for higher value of phonon scattering and reduced the lattice thermal conductivity.In this study, the electronic and mechanical properties of cubic Fe2TaAl and Fe2TaGa compounds, first-principles calculations using the full potential linearized augmented plane-wave method (FPLAPW)40 as executed in the WIEN2k Package41. This quantum mechanical code can precisely simulate the ground state structure, band gaps, dielectric properties, magnetic properties, and so on. The generalized gradient approximation (GGA)42, mBJ43 and onsite Hubbard correction (GGA + U)44, were adopted for the exchange-correlation potentials. In GGA + U calculations, we have used the U-J = 4.0 eV for Fe-d electrons with J = 0 according to the Dudarav’s method45. A dense k-point mesh of 10 × 10 × 10 was used in the Brillouin zone integrations. The basis functions are expanded up to RMTKmax = 7, where RMT is the smallest atomic radius in the unit cell and Kmax refers to the magnitude of the largest k vector in the plane wave expansion. The total energy convergence tolerance for the calculations was selected within 1 × 10−6 eV. Furthermore, the elastic properties were calculated by cubic elastic code46 and from these elastic constants other mechanical parameters were determined to discuss the mechanical response of these materials. DFT has proven to be one of the successful methods to predict the stability as well as ground state properties of the materials theoretically48. The lattice dynamical properties were analyzed in term of phonon frequencies as a second-order derivative of the total energy with respect to atomic displacements within the frame work of DFPT49 by using the Quantum Espresso package50.The investigation of structural, elastic, electronic and phonon properties of the FeLattice dynamics, mechanical stability and electronic structure of Fe-based Heusler semiconductors


Each model is capable of reproducing experimentally-observed data to some extent, but based on relative theoretical energies and reasonable reproduction of all variables considered, a microfibril based on 5 layers in a 34443 arrangement is predicted to be the most probable. A habit based on a 234432 arrangement is slightly less favored, and a 6 × 3 arrangement is considered improbable.Determining the shape of plant cellulose microfibrils is critical for understanding plant cell wall molecular architecture and conversion of cellulose into biofuels. Only recently has it been determined that these cellulose microfibrils are composed of 18 cellulose chains rather than 36 polymers arranged in a diamond-shaped pattern. This study uses density functional theory calculations to model three possible habits for the 18-chain microfibril and compares the calculated energies, structures,  Cellulose is one of the most important materials on Earth. As the foundation of plant cell walls, it has a critical role in the biosphere. Because it has evolved to provide strength and resist biodegradation, cellulose is commonly used in paper, clothing, and myriad materials. Efforts to convert cellulose into fermentable compounds have become commercialized recently in order to produce ethanol from recyclable feedstocks that minimize impact on the food supply.3 utilized X-ray and neutron diffraction to determine the crystalline structure of cellulose. This structure was used as the basis for density functional theory (DFT) calculations that subsequently reproduced the infrared, Raman, NMR and sum-frequency generation spectra of cellulose5. However, much of the research on cellulose has been performed on bacterial and/or plant-extracted cellulose neither of which represents the state of plant cellulose in muro7. Consequently, recent studies on non-extracted cellulose have been critical in helping to define the native state of cellulose within plant cell walls10.Landmark papers by Nishiyama and coworkers11. Furthermore, a diamond-shaped habit based upon cellulose polymer layers arranged in a 1 × 2 × 3 × 4 × 5 × 6 × 5 × 4 × 3 × 2 × 1 pattern was assumed to be the habit of this 36-polymer CMF . This view of the CMF was challenged by papers based on wide-angle X-ray scattering (WAXS)13. Based on diameter arguments, the 36-polymer CMF was judged to be too large, so the CMF would have to be smaller. This conclusion has been justified recently by the work of Nixon et al.14, Hill et al.15 and Venu et al.16 who have demonstrated that the CSC rosette is most consistent with a hexamer of trimers. Assuming only one cellulose polymer can be extruded from each of the CesA enzymes in the CSC19, the CMF should be made of 18 cellulose polymer chains rather than 36.Although the atomic structure of cellulose has been characterized, an outstanding question is the shape of cellulose microfibrils in plant cell walls. For decades, the cellulose microfibril (CMF) was thought to be comprised of 36 chains of cellulose polymers  based on the observation of “rosettes” formed by the cellulose synthase complex (CSC)The difference between 18 and 36 polymers in the fundamental plant CMF will be reflected in the habit of the CMF as well as the size. The shape of the CMF is far from purely academic because the surfaces displayed by the CMF will depend upon this arrangement of cellulose chains. In turn, the interactions of other plant cell wall components  will depend upon the nature of the exposed CMF surfaces. Biodegradation mechanisms of the cellulose will also depend heavily upon the nature of the CMF surfaces present. Hence, this study sought to determine the most probably arrangement of cellulose chains within the CMF in order to use this crystal habit as a basis for future studies of plant cell wall architecture, mechanics and biochemistry.21, we chose to use a similar methodology4 to predict the structure, energetics and spectroscopic properties of finite CMFs with various arrangements of 18 cellulose chains. The periodic DFT calculations in VASP were carried out in order to obtain atomic structures that could be used for comparison against observed X-ray and neutron diffraction studies2. Furthermore, a key prediction is the relative energies of each CMF habit. Although one cannot expect CMFs formed in plant cell walls to necessarily be at thermodynamic equilibrium, Iβ cellulose is known to be thermodynamically stable and only slightly higher in Gibbs free energy than the extracted cellulose II and III forms22. Our assumption is that it does not make biochemical sense for plants to produce a less thermodynamically stable form both in terms of assembly kinetics and in regards to ultimate strength and resistance to biodegradation.Based upon success in modeling cellulose atomic structure with density functional theory (DFT) calculations13C NMR chemical shifts (δ13C) of the model CMFs. The WAXS diffractograms were simulated using the CMF models taken from the structures as energy-minimized in VASP using the program WAXSiS24. δ13C values were calculated with Gaussian 0925 using the same model structures terminated after 4 glucan monomers along the c-axis with methyl groups to avoid unsatisfied valence at the terminal O atoms8 and following methods used in previous studies26.To supplement the atomic structure and thermodynamic criteria, this study also calculates the WAXS diffractograms and the 13. The first two have been suggested previously as shapes of the CMF (see ref.13), and the third is based upon a structure created for coarse-grained simulations of CMFs by Vin Crespi (The Pennsylvania State University).Three arrangements of the 18 cellulose polymers were created based upon the criteria that the CMF should attempt to minimize surface area, be reasonably symmetric and maximize the van der Waals forces between layers and H-bonding within layers. These three crystal habits are shown in Fig. et al.2 reveals that all three CMFs accurately reproduce observed interatomic distances and torsion angles , Ψ (C1-O4-C4-C5), χ (O5-C5-C6-O6) and χ′ (C4-C5-C6-O6) torsion angles calculated are in reasonable agreement with the experimental values (Table 2). There is no model that performs significantly better with respect to reproducing structural parameters, so we conclude that crystal habit has a minimal effect on these structural parameters.Comparison of the model atomic structures with the parameters reported for Iβ cellulose in Nishiyama s Tables  and 2. Ss Tables , but we 27, so although the D3 dispersion correction of Grimme et al.28 was employed, this empirically-derived estimate is subject to a significant level of uncertainty. In order to test how robust the DFT-predicted energies might be, single-point energy calculations were also performed on the tetramer CMFs in Gaussian 0925 using the Perdew-Wang29, M0530, and B3LYP32 exchange-correlation functions in both vacuum and aqueous solution conditions (IEF-PCM)33. Table Our second criterion for evaluating the probability of forming each CMF habit is the potential energy calculated for each. The periodic DFT calculations should better represent actual CMFs in that they are continuous rather than truncated after four glucan monomers. However, DFT has notable issues with estimating van der Waals forces13C NMR spectra of cellulose in vivo have been reported recently8 that can be used as a basis for evaluating the proposed CMF habits. Table 13C NMR spectra of cellulose have observed C4, C5 and C6 peak doublets that have been ascribed to the changes in chemical environment and/or structure that occur on the surface of cellulose compared to the interior . This interior to exterior or ordered to disordered shift is especially pronounced for C4 which changes from 88.5 to 91.5 ppm on the interior (iC4) to 84 to 86 ppm on the surface (sC4). A similar shift may occur for C6 from iC6 of 66 ppm to sC6 of 62 ppm, but the sC6 peak is harder to resolve due to overlap with other plant cell wall polymers . Our results  can match both the “iC4” and “sC4” peaks in Harris et al.9, and the surficial δ13C4 values are slightly higher than the interior δ13C4 values (83.9 ± 0.4–85.8 ± 0.6 ppm). Hence, the shift from 88.5 to 91.5 ppm to 84 to 86 ppm may not indicate interior versus exterior hydroxymethyl groups34. Furthermore, the shift is not necessarily the result of tg to gt rotations on the surface because none of the hydroxymethyl groups in our models are in a gt configuration. Instead, the difference may be caused by H-bonding or the lack thereof as well as other structural changes35.High-resolution et al.36 have found that some interior glucan chains in CMFs are more than one chain away from the nearest surface chains. Hence, the CMF of primary cell wall must have two types of interior cellulose chains: a core fraction not directly contacting with the surface and a bound fraction directly contacting with the surface. As shown in Supplementary Fig. et al. found using solid-state NMR techniques. Therefore, based on the core domain and s:i ratio, the 34443 model is preferred.Wang −1 and 16 nm−1 similar to the experimental data for Arabidopsis and Poplar  and Gaussian 0925 codes. Initial atomic structures were based upon the experimental model of Nishiyama et al.1 as supplied by Yoshi Nishiyama. The unit cell of cellulose was doubled to include 4 glucan units along the c-dimension, and 18 polymer CMFs were constructed in Materials Studio 2016. The simulation cell was expanded in the x- and y-dimensions to allow for space between the images of the CMFs in order to minimize any self-interactions. The model structures were converted into VASP input files (POSCAR) via a perl script written by Andrei V. Bandura . All atomic positions were relaxed during energy minimizations using the VASP code. The simulation cell lattice parameters were also relaxed until the c-dimension converged to a stable state in order to the cellulose chains to stretch or contract. After convergence of the chain lengths, the lattice parameters were then held constant in order to prevent artificial aggregation of the CMFs with their periodic images Density functional theory calculations in this study were performed with the Vienna Ab-initio Simulation Package (VASP41 with tetramethylsilane (TMS) and methanol as the primary and secondary standards43 in order to increase accuracy in reproducing experimental values. The modified Perdue–Wang exchange–correlation functional mPW1PW9144 along with the 6–31 G(d) basis set45 were used in the calculation of isotropic chemical shieldings for all nuclei. Only the added terminal methyl groups were relaxed in Gaussian 09 while all other atoms were fixed in the positions determined in the periodic energy minimizations performed with VASP. The 18-chain CMF structures (each chain contains 4 glucose units) were solvated in a box of TIP3P water46 followed the protocol detailed out in previous work35 using the Impact module of Maestro47, followed by 1000 steps of conjugate gradient minimization performed before 10 ps molecular dynamics simulations at 298 K with a 1 fs time step in the NVT ensemble using the OPLS_2005 force field48 keeping the atomic positions of cellulose molecules fixed. Only H2O molecules within 3 Å of the cellulose clusters were included for NMR calculations as it was found that H2O molecules further than 3 Å from the clusters had negligible effects on the calculated δ13C. An empirical correction of 49.5 ppm43 was used for the difference between the δ13C in methanol and TMS commonly used as an experimental 13C NMR standard41. This gives an isotropic chemical shielding of 193.0 ppm. To compute the δ13C for any C nucleus i in cellulose, we used: δ13Ci = 193.0 ppm - δ13Ci.NMR calculations used the multi-standard method of24.Small- and Wide-Angle X-ray Scattering curves of CMFs in solution were calculated based on explicit-solvent all-atom molecular dynamics (MD) simulations using WAXSiS49 and the Glycam06 carbohydrate force field50 at 300 K, at 1 bar, in the NPT ensemble, with 2 fs time step, 10 Å non-bonded interaction cutoff, and SHAKE-constrained hydrogen bonds. The positions of CMF were constrained using a force constant of 250 kcal/mol/Å2. Simulation frames are written every 0.5 ps so that the solvent configurations are uncorrelated23. From the MD trajectories, the theoretical scattering curves were then calculated using WAXSiS webserver. Two buffer subtraction methods have been used to calculate the scattering intensity: (i) total buffer intensity is subtracted, I(q) = Isample(q) − Ibuffer(q); (ii) the buffer intensity is reduced by the volume fraction v of the solute, I(q) = Isample(q) − (1 − v) Ibuffer(q)23. The two subtraction methods only slightly differ at small angles. As shown in Supplementary Fig. −1, and buffer scattering reduced by solute volume resulted in the 200 peaks at 16.5 nm−1, very close to the widely accepted value for 200 of cellulose I (around 16 nm−1)53. But they highly differ at wide angles around the water scattering peak (20 nm−1)23. As shown in Supplementary Fig. −1. However, both predicted the peak around 4 nm−1. The CRYSOL54 program was also used for calculation of theoretical diffraction patterns of the three CMF habits in water (with solvent density of 0.334 e/Å3) and in vacuum based on the atomic coordinate of the CMF structures obtained from the periodic energy minimizations performed with VASP.The 18-chain CMF structures were obtained from the periodic energy minimizations performed with VASP. Each chain contained 19 glucose residues Each CMF structure was solvated in a box of TIP3P water so that the CMF was surrounded in all directions by at least 20 Å. Short (50 ps) MD simulations were performed using AMBERPoplar tension wood sample was obtained as described in Sawada et al.55. The bottom 1 cm section of a seven week old stem from Arabidopsis thaliana colD was dried at 80 °C overnight before measurement. The XRD profiles were recorded as described in Sawada et al.55.The Supplementary Information


Due to limited instrumental measurements and historical pH records in the world’s oceans, seawater pH variability at the decadal and centennial scale remains largely unknown and requires documentation. Here we present evidence of striking secular trends of decreasing pH since the late nineteenth century with pronounced interannual to decadal–interdecadal pH variability in the South Pacific Ocean from 1689 to 2011 CE. High-amplitude oceanic pH changes, likely related to atmospheric CO2 uptake and seawater dissolved inorganic carbon fluctuations, reveal a coupled relationship to sea surface temperature variations and highlight the marked influence of El Niño/Southern Oscillation and Interdecadal Pacific Oscillation. We suggest changing surface winds strength and zonal advection processes as the main drivers responsible for regional pH variability up to 1881 CE, followed by the prominent role of anthropogenic CO2 in accelerating the process of ocean acidification.Increasing atmospheric CO Ocean acidification due to the industrial era is a major marine environmental concern, yet little is known on the historical ocean pH changes prior to human influence. Here, Wu et al. show that tropical South Pacific seawater pH is linked to ENSO pacing and has recently been decreasing rapidly. At present, the oceans take up more than 41% of anthropogenic CO2 emissions2, inducing the acidification of the ocean surface with major implications in marine carbonate chemistry and biological ecosystems3. Many modelling and experimental studies have indeed shown clear trends of shallow water acidification in lockstep with increasing atmospheric CO24. One of the major consequences is the decrease of carbonate saturation state that is crucial for calcifying organisms such as scleractinian corals to precipitate their aragonite skeletons5. These critical threats of ocean acidification (OA) on such marine organisms and ecosystems have been documented on the Great Barrier Reef (GBR) in Australia that witnessed a 14% decrease in coral calcification since 19906. Future projections of coral calcification rates suggest a possible additional decrease by up to 30% in the twenty-first century7. Moreover, end-of-century emission scenarios predicted by model simulations indicate unprecedented decrease by up to 0.4 pH units8 compared to the changes of only 0.2 pH units over glacial-interglacial cycles9.The concentration of atmospheric CO10 and limited station-based instrumental time series available over the past few decades11. The pH records that span only the most recent decades are far too short to precisely monitor the longer-term evolution of OA and to quantify its impacts on tropical ecosystems. To study the natural variability in oceanic CO2 uptake and carbonate chemistry changes, proxy records in modern marine archives such as corals provide a unique opportunity to go further back in time at a high resolution on the centennial scale. Additionally, it is crucial to investigate pH changes with links to ocean CO2 uptake as well as the relationship to sea surface temperature and forcing mechanisms at various timescales.Presently, accurate measurements of pH in the oceans are near non-existent aside from selected ship track measurementsδ11B signature in scleractinian coral skeleton. Coral-based reconstructions of ocean pH suggest notable recent acidification13 and decadal variability14. However, due to spatiotemporal constrains, the currently available coral-based pH reconstructions predominantly focus on the GBR and the marginal South China Sea16, whereas longer timescale pH changes of the open surface ocean from the Pacific Ocean remain poorly documented17.A method to document sub-seasonal to annual pH changes is based on the 2 inventory is found in the region between 14°S and 50°S18 and about 60% of the total oceanic anthropogenic CO2 is incorporated in the Southern Hemisphere oceans4  extending from the western Pacific warm pool  fluctuations. In addition, the timing of reconstructed pH variability in the South Pacific is coupled to prominent sea surface temperature variations modulated by the phases of ENSO and IPO. We suggest changing surface wind strengths and zonal advection processes across the Pacific Ocean as the main drivers responsible for South Pacific pH variability that is superimposed by the prominent role of anthropogenic CO2 increase in accelerating the process of OA.Here we present 323 years of annually resolved D. heliopora coral was collected in March 2015 at the Fausse Passe de Uitoé in New Caledonia  during the solar Maunder Minimum, the onset of industrial era from the late 1700s, and through the modern climate regime of today.The studied δ11B, δ13C and δ18O values for a total of 323 continuous years over the Common Era 26. Such value is directly related to sea surface salinity (SSS) responding to evaporation and freshwater input. Previous studies from the Pacific have demonstrated that coral δ18O variations over multiple timescales represent changes of SST, SSS, precipitation, or a mixture of those parameters depending on the environmental setting28.We generated annually resolved records of coral D. heliopora coral δ18O ratio to SST from New Caledonia was completed using annual gridded SST from the National Oceanic and Atmospheric Administration (NOAA), National Centers for Environmental Information (NCEI) and Optimum Interpolation SST version 2 (OI-SST)29. A 4-grid averaged OI-SST . Even though coral δ18O record is a combination of both SST and the δ18Osw, the coral δ18O-SST sensitivity of −0.24‰ C−1 is in agreement to the typically described relationship across the Pacific even with the use of annual resolution sampling  to the NOAA NCEI Extended Reconstructed SST version 4 (ERSST)33. A 4-grid regional annual average SST for ERSST (Supplementary Table δ18O-SST and ERSST yielded significant (p < 0.01) and high correlation coefficients between the most recent period of the record with the most accurate instrumental SST period  observations in the gridded SST timeseries was low. The observed lack of significant coherence over the mid-twentieth century in our analysis and verification may be reflecting the inadequacies of the gridded instrumental SST product. Nonetheless, the result over the verification period provides confidence in our chronology and the potential of the coral δ18O-SST proxy although it is likely that the contribution of δ18Osw is of significance. Thus, our δ18O record from New Caledonia suggests a strong secular warming and or freshening trend consistent with many coral records from the western Pacific with similar timing for the onset of warming in the mid-nineteenth century19  as well as their isotopic composition are pH dependent34 with a constant fractionation factor between the two aqueous boron species35. On the assumption that marine carbonates reflect the isotopic composition of seawater borate36, the δ11B signature of coral aragonite skeleton has been shown to record seawater pH37. Possible intra-colony differences of δ11B signature of coral aragonite skeleton were taken into account as studies of sub-seasonally resolved D. heliopora coral δ18O and Sr/Ca ratios have shown offsets between skeletal materials  and different coral polyps32. However, at the annual resolution, our test across multiple coral polyps revealed that skeletal material differences are inconsequential for δ11B ratios  compared to traditional δ18O and δ13C analyses makes it statistically likely that the sample comprises all different skeletal structures  and is thus representative of the whole coral skeleton. Our method demonstrates the reliability and reproducibility of annual δ11B ratio results from a new south Pacific coral species (D. heliopora) with larger skeletal structure than the commonly used Porites coral genus.To reconstruct seawater carbonate chemistry changes and potential modern ocean acidification from anthropogenic COδ11B signature to coral internal pH as studies indicated differences between empirically and theoretically determined fractionation factors for isotope exchange between boric acid and borate in seawater35. Furthermore, results based on a variety of coral species across temperate and tropical regions indicate consistent difference between coral internal calcification pH by approximately one-half of ambient seawater pH39. However, recent results also indicated possible modifications of coral internal pH due to external factors and species-specific processes41. Thus, we estimated the changes in seawater pH at New Caledonia using a moderate general calibration conversion39  to the one used in this study (D. heliopora) was chosen allowing for the establishment of biological and seawater pH linkage to climate-driven patterns. We focused our discussion on the relative pH changes instead of the absolute pH values because rigorous palaeo-pH reconstructions from coral δ11B signature require species-specific calibrations with robust quantification of physiological processes . Nevertheless, independently from a possible offset in our reconstructed pH using this calibration conversion, our observed trend and interannual to decadal variability from the measurements along the core will remain.Careful considerations were taken to convert our coral n39 Fig. . To be cδ11B, δ13C and δ18O, significant relationships were found. The significant correlation between δ11B and δ13C . Obvious perturbations outside of the regression line were observed and distinguished the most recent samples related to anthropogenic CO2 emissions in the atmosphere from those related to the older preindustrial era period  from the burning of fossil fuel into the ocean has caused considerable decrease in the δ13C of seawater DIC, known as the 13C Suess effect. The δ13C ratio depletion trend in atmospheric CO2 at five Pacific stations42, the Law Dome ice core record43, and archived in numerous Pacific coral records  throughout the interval of this study. For this particular coral genus, similar vertical extension rates have been reported throughout the Pacific31.As the ocean is one of the major global sinks of anthropogenic COδ13C values is neither related to tropical Pacific solar radiative forcing45 nor tropical volcanic forcing46  that are even greater than the 0.4 pH units predicted for the end-of-the-century , 37% of the variability in δ11B can be explained by δ13C from correlation analysis  coincided with changes in temperature most likely linked to the termination of the LIA, a period that was documented to be 1.4 °C cooler at New Caledonia51. The end of this period was however characterised by a maximum δ11B pH that is contrary to the recorded maximum δ18O enrichment  and decadal-interdecadal (11–41 years) timescales  events, the δ11B signature decreases from the preceding year translating into a surface seawater decrease in pH, and the opposite occurs during La Niña (LN) events with an increase in δ11B signature from the preceding year  displayed more pronounced depletion of δ11B than severe EN events and the very severe EN of 1997–98. Severe LN events are consistent with enrichment of δ11B indicating an increase in pH  are recorded in our coral as a series of major depletions indicating decreases in pH in New Caledonia. The fluctuations in our coral-based δ11B pH thus reflect the highly dependent nature of ocean CO2 uptake across the air–sea surface interface following the pacing of ENSO.The most recent ENSO events indicate possible changes of up to ±0.35‰ in coral 11B Fig. , which c pH Fig. . In addi56, more than 6000 km away  changes and zonal advection of surface heat in the Pacific Ocean as the primary cause and mechanism of surface ocean carbonate chemistry mixing and ultimately pH variability.A remarkable atmospheric teleconnection is witnessed by the compelling coherence and in-phase relationship of our New Caledonia reconstructed pH from the South Pacific to the most ENSO representative region, the Niño 3.4 region SST anomaly recordδ18Osw of the South Pacific . This relationship then shifted to a more prominent higher-frequency variability of 7 years (ENSO-related) to 16 years  to the ENSO frequency (4–8 years) in the last 100 years. Such observations are consistent with Central Pacific and Australian coral-based ENSO studies, where ENSO variance is significantly higher in the twentieth century than in any other time since the late Holocene59. We conclude that the non-stationary behaviour of long-term pH and SST variability observed in New Caledonia, which shifted to more pronounced interannual fluctuations after ~1850 cannot be due to simple local ocean circulation changes. This prominent shift rather suggests a likely anthropogenic influence with strong CO2 uptake by the ocean because the significant trend changes and emergence timing coincides with these frequency evolution in the post-industrial era.Cross-wavelets coherence analysis detecting frequency and relative phase relationship between our proxy records indicates significant non-stationary relationships between pH and SST/fic Fig. . Dominanδ13C that is related to the atmospheric 13C Suess Effect caused by the uptake of anthropogenic CO2 by the ocean. We argue that the onset of this modern acidification in the mid-1800s and the temporal coherence of δ11B pH and δ13C or marine carbonate chemistry changes are coupled to SST variability and have been consistently occurring for the last 300 years. The temporal coherence of the proxy records also has significantly changed in frequency since the emergence of the modern acidification. We implicate the changing strength of the westerlies and trade winds as important drivers forcing the pH and SST changes across the greater Pacific under possible decadal-enhanced ENSO variability (11–12 years). The changing surface ocean condition is a distinct process that modified the carbonate chemistry of the Pacific over the last three centuries with recent changes likely dominated by anthropogenic influence. Our results provide a new detailed perspective on the changes in modern OA that was likely influenced by the inseparable combination of the changing surface winds strength (trade winds and westerlies) and ENSO-related SST conditions driving the uptake of anthropogenic CO2 in the South Pacific.In summary, our reconstructed pH record from the South Pacific highlights that marine carbonate chemistry in the South Pacific is highly variable with pronounced high-amplitude pH oscillations since 1689 CE. We observed a significant emergence of secular decreasing pH trend since 1886 together with the dramatic depletion of oceanic D. heliopora coral was cored in 7–8 m water depth at the Fausse Passe de Uitoé, New Caledonia in the southwestern Pacific  and SEM analyses at the IRD, Laboratoire d’Océanographie et du Climat (LOCEAN) on the Analytical Platform ALYSES (IRD-UPMC) at Bondy, France. Approximately 500 mg of crushed coral powder (<20 µm) for all subsamples were analysed on a PANalytical X’Pert Powder X-ray Diffractometer that revealed 100% aragonite. Verification of the coral’s suitability for palaeoclimate research by representative SEM analysis was conducted on a Zeiss EVO LS15 SEM. The samples were carbon coated and analytical results demonstrated good preservation of primary porosity, clear dissepiments and well-defined centres of calcification  were omitted because of the presence of organic tissue layer remnants (~9–10 mm thick), which would have biased the geochemical results. Similarly, the coral section older than 1689 CE was avoided because the coral growth departed from the central vertical axis.Based on the X-radiograph positives of the coral slab with well-defined annual skeletal density banding, the chronology and age model of the coral colony was developed based on band counting and then verified by 31. Milling was completed with a Proxxon mill for coral powder collection at predetermined annual sampling intervals based on the X-radiographs  on the Analytical Platform PANOPLY (LSCE-GEOPS), at the Laboratoire des Sciences du Climat et de l’Environnement (LSCE), Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France. Details of the procedure  can be found in ref. 60. The initial (234U/238U) activity ratios of samples are in agreement with the initial (234U/238U) activity ratio of modern seawater . The ages presented herein , at the LSCE, CNRS, Gif-sur-Yvette, France. The isotope ratios are reported in ‰ deviation relative to the Vienna Pee Dee Belemnite (V-PDB). Long-term analytical precision based on repeated measurements of an in-house marble carbonate standard were ±0.04‰  for δ18O and ±0.02‰  for δ13C verified against NBS 19. Fifty samples or ~15% of the entire time series were analysed in duplicate and triplicate with robust average reproducibility better than ±0.05‰ (±1σ SD) for δ18O and ±0.02‰ (±1σ SD) for δ13C, which verified the homogeneity of our measured samples . Each coral powder solution as well as the international coral standard JCp-163, North Atlantic Seawater Standard VI (NASS VI), and the boric acid reference standard (NBS SRM 951) were purified with 60–80 mg of the anion exchange resin Amberlite IRA 743 following the Batch Method of ref. 62. The amount of resin added to each solution was sufficient for 100% extraction of the boron. Briefly, each individual batch of tubes  were slowly neutralised to a pH of 7–9 with 1N NH4OH over 4 h on a benchtop shaker with constant pH checks every 30 min. Once the pH remained stable between 8 and 9 pH units, the resin-added solutions were allowed to absorb the boron overnight. After overnight absorption, the individual tubes were rinsed five times with 18.2 MΩ milli-Q water on the benchtop shaker prior to boron extraction from the resin. The concentrated boron was eluted from the resin using four successive volumes of 1.25 ml 0.1N HNO3 (Optima-grade Ultrapure) for a total extracted volume of 5 ml. Each individual extraction steps (elution of 1.25 ml 0.1N HNO3) lasted at least 4 h to maximise boron extraction yield. Finally, the boron concentrations were adjusted to 150–200 ppb in 0.1N HNO3 (Optima-grade Ultrapure) for δ11B ratio analysis on the MC-ICP-MS.The pre-treated coral powder samples were then dissolved in 1.25 ml of 1N HNOδ11B) was determined using a double-focusing sector-field Multi-Collector Inductively Coupled Plasma Mass Spectrometer (Thermo Neptune Plus MC-ICP-MS) on the Analytical Platform PANOPLY (LSCE-GEOPS), at LSCE, Gif-sur-Yvette, France following established methods62. The δ11B ratio MC-ICP-MS analysis results are reported in ‰ deviation relative to the NBS SRM 951 standard (boric acid isotopic standard). The standards from each individual batch  were first measured using the ‘classic’ method in the following sequence per standard: (a) rinse solution (0.1N HNO3 and 0.04N HF), (b) blank solution : typical blank contribution is <~0.5%, and (c) the standard (JCp-1 or NBS SRM 951 or NASS VI). This process sequence is repeated until all the standards from each individual batch or batches are analysed. Upon the completion of this ‘classic’ method, a ‘rapid’ method sequence is initiated following the common standard-sample bracketing.Boron isotope composition  for sub-seasonally resolved isotope-based climate reconstructions32. Consequently, three sections of 20-year periods  were removed from the same coral core slabs for replication and test of reproducibility. The powder samples were collected on the same coral slabs from a different coral polyp on the same growth banding, adjacent but not overlapping to the original sampling transect  were calculated from coral skeletal δ11B ratios following the established equation, Eq.  constant for boron in seawater. For this study, we used the constant value, 8.58, because of the limited SST range  after correction of pKB16. In the above equation, δ11BSW (39.61‰67) and δ11BC represent the δ11B in seawater and coral, respectively. The α[B3-B4] is the fractionation factor for isotope exchange between boric acid [[B3-B4]) of 1.027235 to facilitate comparison to previously published studies of δ11B pH reconstructions17 and is also consistent with observations. The estimated seawater pH (pHsw)  Fig.  value waδ11B, δ13C and δ18O) of D. heliopora 71 for probability distribution.We used the Pearson’s product-moment correlation analysis to test the relationship between the different proxies , each coral proxy time series were time averaged over 8–10-year windows prior to running the change point analysis algorithm.Change point analysis was performed on the individual https://doi.pangaea.de/10.1594/PANGAEA.886966).The data reported in this paper have been deposited and fully available without restrictions at the information system PANGAEA (Data Publisher for Earth and Environmental Science; Supplementary InformationPeer Review File


Regulating the emergence of leaders is a central aspect of collective cell migration, but the underlying mechanisms remain ambiguous. Here we show that the selective emergence of leader cells at the epithelial wound-margin depends on the dynamics of the follower cells and is spatially limited by the length-scale of collective force transduction. Owing to the dynamic heterogeneity of the monolayer, cells behind the prospective leaders manifest locally increased traction and monolayer stresses much before these leaders display any phenotypic traits. Followers, in turn, pull on the future leaders to elect them to their fate. Once formed, the territory of a leader can extend only to the length up-to which forces are correlated, which is similar to the length up-to which leader cells can transmit forces. These findings provide mechanobiological insight into the hierarchy in cell collectives during epithelial wound healing. During collective cell migration, how leader cells emerge is poorly understood. Here, the authors find that small groups of mechanically-interacting follower cells pull on the future leaders to stochastically elect them to their fate. Effective collective migration, in many cases, requires formation of leader cells at the tissue boundary8. As an illustration, in the well-studied collective migration of a monolayer of epithelial cells mimicking wound healing, leader cells display a large lamellipodial structure at the front edge, move ahead of other non-leader or follower cells, and provide the guidance for the migrating group10. Beyond wound healing, leader cell formation has also critical influences on branching morphogenesis11 and metastatic invasion12 both in vivo and in vitro. The dynamics that dictate leader-cell selection is a central problem7, yet remains largely elusive, even for epithelial monolayers that show clear leader-follower organizations. It is not understood, for example, at what level, interface or bulk monolayer, the signals for formation of leader cells originate, and why only a fraction of cells at the front becomes leader cells. Many recent studies revealed diverse molecular and biomechanical traits of the leader cells14 and many discovered interesting biophysical aspects of epithelial mechanics19. While most of these studies describing formation and regulation of leader cells essentially attribute them to the interfacial properties20, some also indicate the possibility that the dynamics of leader cells could eventually be regulated by the dynamics of the bulk18. However, the nature of the contribution from bulk in the selection of leader cells remains mostly unknown as the events occurring at the onset of or preceding the leader cell formation remain obscure. Recent evidences showing long-lived traction patterns extending to several cell diameters21 and velocity patterns14, even in a confluent monolayer22, indicate the relevance of group dynamics in epithelial monolayers, but if and how the group dynamics might control the selection and regulation of leader cells at the margin remains unclear.Collective cell migration drives many critical biological processes including wound healing, organogenesis, and cancer developmentTo understand how leader cells emerge, here we have studied mechano-biological aspects of epithelial wound healing resolved in time and space. Using traction force and monolayer stress microscopy, we find that the leader cells at the wound-margin are effectively selected by the mechanical interactions of the follower cells located behind the leading edge. We demonstrate that the followers mechanically pull on the future leader, aiding in their polarization and protrusion. Combining experimental data with theoretical modeling, we are able to show that the territory of a leader extends only to the length up to which forces are correlated in the monolayer, which is similar to the length up to which leader cells can transmit forces. This finding, therefore, provides a mechanism for the formation of leader cells during collective cell migration, wherein we place mechanical interactions between the cells as the central player that determines when and where a leader cell would emerge.23. Initially for ~30–45 min, cells at the wound-margin did not exhibit any noticeable lamellipodial protrusion, which we name Phase 0  epithelial cells within confined areas and then lifted off the confinement to prompt two-dimensional sheet migration Fig. . This ine 0 Fig. . After te 0 Fig. . As the e 0 Fig. . This phe 0 Fig. . We alsoe 0 Fig. , we obsee 0 Fig. . These re 0 Fig. . Interese 0 Fig. . We alsoe 0 Fig. , pointin26. Moreover, as elucidated in normal and asthmatic bronchial epithelia, an unjammed monolayer displays considerably higher cell-matrix traction than a jammed one25. Further, a non-dimensional shape index (q) measuring the ratio of the perimeter to square root of the projected cross-sectional area (q = P/√A) of the individual cells can capture the specific state of the cells. Jammed cells were characterized with q < 3.81 and unjammed cells with q > 3.8125. In order to probe if the increase in traction stresses are associated with the possibility of local unjamming within a pre-migratory monolayer, we computed the shape index (q) for each individual cell. Indeed, follower cells behind the prospective leader cells displayed higher shape index than other cells within the same depth, at both temporal phases  and unjammed (fluid-like) as explained by dynamic heterogeneity of the monolayerses Fig. . Taken t23. Incidentally, on depleting the expression level of merlin with a specific small interfering RNA (siRNA), the resultant cell phenotype appeared intrinsically more unjammed and less correlated with their neighbors than the control cells as characterized by particle image velocimetry  even in an apparently quiescent and packed epithelial monolayer25. Since the emergence of the leader cells was preceded by the appearance of high stress regions behind them  separation distance showed excellent correspondence with the distribution of force correlation length (FCL)  in both MDCK and HaCaT cells : 162.4 ± 30.2 μm; 75 μm pattern: 143.3 ± 22.3 μm; 300 μm pattern: 166.2 ± 31.8 μm; mean ± s.d.; Fig. With these results in view, we next examined to what extent the contribution from the mechanobiology of bulk monolayer can prevail when the monolayer is presented with a perturbation at the interface. To this end, we generated monolayers with highly curved beak-shaped regions, using a soft-lithography based patterning technique Fig. . These hSubsequently, to test our hypothesis that force transduction determine the distance between the leader cells, we modulated the length-scale of force transduction by both chemical and physical methods. For chemical modification, we used the widely used pharmacological means of controlling the actomyosin contractility by treating the cells with a non-muscle myosin-II inhibitor, blebbistatin (5 μM), or a myosin-light-chain phosphatase inhibitor, calyculin A (1 nM). Blebbistatin reduced the contractile forces, while calyculin increased it. Then, as expected, blebbistatin treatment enhanced the ruggedness of the stress landscape and lowered the force correlation while calyculin treatment regularized the stress landscape and increased the correlation length Fig. , both in22, demonstrating the role of leader cell as a local guide that also exert high traction force8. Therefore, we presumed that the transition from phase-1 to phase-2 would happen when the number of followers per leader exceed the extent up-to which the leader cell can transmit forces. Subsequently, we computed the followers per leader by calculating the Euclidean distance between the velocities of followers, v and velocity of the leader cell, vL . By using default values as measured previously34 for cell and substrate parameters  as calculated theoretically  and cell elastic modulus (EC), showing LP as a function of EC. . Together, theoretical modeling and simulation results provide reasoning for the experimentally observed distance between leaders, dLL in Phase 1 and for the need of an outgrowth to transit to Phase 2 by placing the force localization length, LP as a central player.In order to explain why the distance between leader cells in phase-1 and the spatial extent of followers in phase-2 is limited to ent Fig. . Here, t LP Fig. . This in UL Fig. . Interes LP Fig. . This in35, we attempted to extend and demonstrate this hypothesis in the collective cell migration perspective. To this end, we designed a cell-pulling experiment wherein we cultured cells under confined conditions on an elastic silicone substrate, containing a micrometer wide trench. As described previously23, upon uniaxial stretching of this substrate, the cells residing on one side of the trench would exert pulling force on their neighbors on the other side , as described previously23 , beyond which new leaders are formed (Phase 2). Owing to its non-interfacial and mechano-biological nature, the mechanism of leader cell formation that we describe here is fundamentally distinct from the canonical perspective of interfacial regulation of leader selection. This does not exclude the possibility that feedback between interfacial curvature and motility will further stabilize leader cell identity18, as also supported by recent studies on the curvature sensing ability of epithelial cells36. Our results, however, support a view in which such effects are downstream of the mechanical selection of leader cells by their future followers . Once the leader cell is formed, its role as a puller plays an important role (Phase 1) and therefore, formation of another leader is prevented in a territory that is defined by how far the force from the leader is transmitted in the monolayer , 10 U ml−1 penicillin, 10 μg ml−1 streptomycin , and 5% fetal bovine serum . Human keratinocytes line  were cultured in high glucose dulbecco’s modified eagle medium  supplemented with GlutaMaxTM, 10% FBS, 10 U ml−1 penicillin and 10 µg ml−1 streptomycin.Madin-Darby canine kidney cells  were cultured in minimal essential medium  supplemented with 2 mM 37. Briefly, desired shapes of monolayers were designed in a QCAD program and transferred on transparencies using a high-resolution printer (JD Phototools). In a clean room facility, SU-8/25 negative photoresist  was spin-coated on a 2″ silicon wafer to a final thickness of about 50 μm. The wafer was then baked on a hot plate for 5 min at 65 °C followed by a second baking for 15 min at 95 °C. The transparencies containing the “photographic negative” of the pattern to be transferred were used as masks to illuminate the wafer with UV light for 12 s in Mask Aligner MBJ4 . To remove the unexposed photoresist, wafers were immersed in SU-8 Developer mr-Dev600 . The prepared wafers containing the desired geometric pattern were then treated with 1 H,1 H,2 H,2H-Perfluorooctyl-trichlorosilane to reduce surface adhesiveness. A sandwich consisting of the patterned wafer, 0.5 ml of uncured PDMS, a piece of parafilm, a piece of paper and a glass slide was put into a custom made molding press to obtain uniform pressure distribution. PDMS was pressed against the wafer in order to create thin PDMS membrane containing holes of desired shapes. The assembly was put into a compartment dryer at 65 °C for 100 min to allow PDMS polymerization. To prevent cell adhesion, prepared stencil masks were incubated in a solution of Pluronic F-127  for 30 min prior to use.Polydimethylsiloxane (PDMS) stencil masks with holes of defined shapes were fabricated in an adapted soft lithography process−1 fibronectin unless otherwise specified. Cells were seeded into the dish and incubated in a cell-culture incubator for 1 h until they adhere onto the fibronectin-coated glass accessible through the holes of the microstencils. The unattached cells were then removed by replacing the media. The set up was incubated again overnight or until a confluent cell monolayer of around 3000 cells/mm2 is obtained after which, the PDMS stencil was removed to trigger collective migration. Migration experiments were carried out at 37 °C and in 5% CO2 environment either inside a stand-alone cell culture incubator, or within an incubator staged over the microscope.For performing collective cell migration in defined patterns, PDMS microstencils with patterned holes or ibidi cell culture inserts (80209) were allowed to stick onto the customized glass bottom dishes (5 cm diameter) coated with 10 μg ml38. Briefly, glutaraldehyde activated glass bottom dishes (MatTek) were used to cast thin polyacrylamide (PAA) gel substrates (Young’s modulus of about 11 kPa) containing 0.5 μm fluorescent carboxylated polystyrene beads. These gel surfaces were then functionalized with sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate  and covalently coated with 0.5 mg ml−1 fibronectin (Sigma) to ensure cell attachment. A horizontal confinement was created on the functionalized PAA gels using thin PDMS blocks. Cells were seeded in the confined areas and grown until a confluent monolayer is obtained. Subsequently, confinement was released by lifting off the PDMS block and images for cells and beads were acquired as the cells migrated. After experiment, cells were trypsinized and resulting bead positions in relaxed state were obtained . The displaced images were aligned to correct for drift and compared to the reference image using particle image velocimetry to create a regular field of displacement vectors with a grid spacing of 5.44 μm. Displacement vectors were interpolated using cubic splines. From these vectors, traction stresses were reconstructed using regularized Fourier Transform Traction Cytometry39 with a regularization parameter chosen by Generalized Cross Validation40.Traction force microscopy was performed as described previously23. Force correlation length was computed by characteristic length scale of the spatial autocorrelation function of the average normal stresses as formulated elsewhere. Briefly, the extent to which the force propagates across the monolayer was obtained by the characteristic length-scale of the spatial autocorrelation function, C(r), of the average normal stress, which is known as the force correlation length30. C(r), was calculated as:Stresses within the monolayer were then calculated from the cell-substrate tractions using a force balance algorithm written in MATLAB (MathWorks) as described in our previous studyri from its spatial mean Merlin siRNA was purchased from Qiagen (5′ CAAAGAGAGGGAGACAGCCTTGGAT -3′) and was transfected by reverse transfection using Lipofectamine 2000 (Invitrogen), as instructed by the manufacturer. The scrambled siRNA was purchased from Qiagen.2 humidified incubator. During migration, Opti-MEM was replaced by MEM containing 5% FBS, 2mM l-glutamine and the respective inhibitor. For actin stainings, cells were fixed and permeabilized before adding Alexa fluor-488 labeled phalloidin (Thermo Fisher Scientific) for visualization of the actin cytoskeleton.Blebbistatin, an inhibitor of myosin II, and calyculin A, a phosphatase inhibitor, were obtained from Sigma. Powders of these drugs were dissolved in DMSO (Sigma) to make the stock. Before removing the confinement, cells were treated with 5 μM blebbistatin and 1 nM calyculin A respectively in Opti-MEM reduced serum medium for 1 h at 37 °C in a 5% CO23.The PDMS chamber for cell culture, was made by casting PDMS in a Plexiglas mold at an elastomer to crosslinker ratio of 10:1. The PDMS was cured for 2 h at 65 °C. The chamber was then peeled, sonicated in 70% ethanol and oxidized in an oxygen plasma environment for 1 min. This process coated a thin solid film of silica on top of the flexible PDMS membrane. A narrow trench was generated on the surface by scratching the silica-coated membrane with a micro-needle (tip size 20 μm) under an inverted table top microscope (Olympus CKX53). The PDMS chamber was then coated with 10 μg/ml fibronectin for 1 h at 37°C. Cells were then cultured until confluency on this fibronectin-coated membrane under confined conditions such that the trench is between 100 and 200 μm away from the cells  and relative displacement of UC (UC/UL) were plotted as exponential functions of distance. To compute the spatial extent of followers, velocity fields were derived by means of optical flow and Euclidean distance was calculated by comparing the velocity of leader cells from follower cells.The localization length, t-test for data following normal distribution and by Mann–Whitney test for the non-normal data. All values were given as mean ± s.d. (standard deviation) or s.e.m. or shown as boxplots. P-values greater than 0.05 were considered to be statistically not significant.Statistical analysis was carried out in Prism. All the data underwent normality test in Prism. Statistical significance was calculated by Student’s Relevant data and codes are available from authors upon request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Supplementary Movie 11


In the article titled “Cerium Oxide Nanoparticles Induced Toxicity in Human Lung Cells: Role of ROS Mediated DNA Damage and Apoptosis” , there wSupplementary MaterialsThe original uncropped and unadjusted Western Blots for all the panels in Figure 9a, 9b, and 9c.Click here for additional data file.


However, the notion of “FGF21 resistance” has been controversial partly due to difficulty in delineating the mechanisms underlying the physiological versus pharmacological effects of FGF21.  Here, key aspects of the term “FGF21 resistance” are discussed including; the origin and experimental context surrounding the term “FGF21 resistance”, new criteria for evaluating FGF21 sensitivity Paradoxical to these beneficial metabolic effects, circulating FGF21 is increased during obesity potentially suggesting a state of “FGF21 resistance”3. This hypothesis, however, has generated controversy within the field. In light of this and in response to a recent call for a unified definition of “FGF21 resistance”4, I would like to discuss the controversy surrounding “FGF21 resistance” during obesity, highlight unresolved questions and outline additional criteria for its definition.Fibroblast Growth Factor 21 (FGF21) has pleiotropic metabolic effects including increasing insulin sensitivity and energy expenditure, while decreasing body weight and sugar intake3. Shortly thereafter, an independent group also reported decreased FGF21 co-receptor expression in white adipose tissue and increased plasma FGF21 levels in obese mice5. However, based on dose response studies, these investigators concluded that circulating FGF21 is increased during obesity to maintain insulin sensitivity, and not due to “FGF21 resistance”5. Hence, the existence of “FGF21 resistance” during obesity has remained controversial with the prevailing question: how can “FGF21 resistance” exist if pharmacological dosing is still efficacious1? Potentially, FGF21 sensitivity during obesity may be akin to insulin resistance whereby the biological effect of endogenous FGF21 is lacking yet pharmacological dosing elicits an effect. Therefore, although effects of exogenous FGF21 should be evaluated in testing FGF21 sensitivity4, consideration of the dose, functional readout, and time-course of FGF21 action should be taken.“FGF21 resistance” was first used to describe decreased expression of the FGF21 receptor complex in epididymal white adipose tissue, increased plasma FGF21, blunted ERK phosphorylation, and attenuated reduction in plasma glucose following low dose administration of FGF21 that occurred in obese mice4, it is difficult to interpret tissue-specific decreases in receptor and signaling activation without understanding how that specific tissue mediates FGF21’s effects. For example, what is the relevance of decreased co-receptor expression in white adipose tissue? Adipose tissue is necessary for FGF21’s acute insulin sensitizing effect6 yet, different results have been reportedin vivo when either overexpressing or maintaining physiological levels of β-klotho during obesity8. Furthermore, brown adipocytes mediate the acute insulin sensitizing action of FGF216; meaning, that although decreased white adipose co-receptor expression has been used as a marker of “FGF21 resistance” we still do not understand the pathological relevance of this event.Although plasma FGF21 levels, FGF21 co-receptor expression and downstream signaling should be evaluated in defining “FGF21 resistance”6. To test chronic FGF21 sensitivity, weight loss and energy expenditure should be evaluated, although FGF21 signaling is difficult to assess since the specific tissue(s) mediating these effects of chronic FGF21 treatment remain undetermined.How then, should “FGF21 resistance” during obesity be defined? Different experimental designs are required when evaluating the acute insulin sensitizing action of physiological levels of FGF21 versus the chronic effects on body composition of pharmacological doses of FGF21. Acute FGF21 sensitivity should be determined via insulin tolerance test following co-injection of insulin and FGF21 in addition to assessing tissue specific glucose uptake and activation of the FGF21 signaling cascade in brown adipose tissues9, human FGF21 is proteolytically cleavedin vivo10. Therefore, the bioactivity of increased circulating FGF21 in humans remains unknown. It is possible that increased circulating FGF21 during obesity could serve a yet uncharacterized role. FGF21 has been shown to have central effects13, however whether or not central FGF21 co-receptor expression and signaling are altered during obesity remains unreported. Finally, caution is warranted in translating rodent studies to man. Although plasma FGF21 is elevated in obese and diabetic humansThere is still much to discover regarding FGF21 action but consideration of the points outlined here can help avoid ambiguity in defining “FGF21 resistance” during obesity. Undoubtedly, the definition of “FGF21 resistance” will continue to evolve as new physiological and pharmacological studies help unravel the mechanisms underlying FGF21’s metabolic actions.All data underlying the results are available as part of the article and no additional source data are required In the context of a discussion of FGF21 resistance and proteolytic cleavage, it is important to discuss recent developments as they pertain to the various circulating forms of FGF21. Specifically, it would be warranted to mention that it has been recently demonstrated that the ratio of ‘active’ FGF21 to total FGF21 can be modulated in humans  and that in certain disease states, expression of the protease FAP are altered.It would be helpful to mention the composition of the FGF21 receptor complex early in the manuscript, prior to discussion of tissue specific effects later in the article. Indeed, it is possible that there is local FGF21 resistance in specific tissues, as measured by pERK vs. traditional systemic hormonal resistance.When discussing translation of FGF21 results to man, it is important to consider that many of the proposed clinical candidates in this area have the site of FAP cleavage mutated, thus negating C terminal truncation (likely the most dramatic inactivation by endogenous proteases). While N terminal cleavage may still occur, it is likely that truncation would not impair action, unless the treatment was with wild type human FGF21.Inclusion of discussion of more current findings from numerous groups on tissue specific ablation/overexpression of FGF21 receptor components would add significantly to the manuscript, specifically, detailed discussion of central vs. peripheral action might be relevant to the topic of ‘FGF21 Resistance’.Another potential explanation for increased FGF21 in states such as obesity is that it may be a sustained homeostatic response to chronic insult. Indeed, FGF21 appears is elevated by a number of stressors (both acute and chronic), including oxidative stress, lipopolysaccharide (LPS), ethanol/alcohol and dietary stress (Fructose etc). Interestingly, when these stressors are removed, FGF21 levels typically normalize relatively rapidly.The opinion presented by Dr. Markan discusses an important topic in the FGF21 field, which has persisted as an unresolved issue for quite some time. While the report presents a balanced view, I would suggest the following potential additions/revisions:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Markan KR discusses the controversial “FGF21 resistance” in obesity. It is an important topic for discussion given the crucial role of FGF21 signaling in health and disease. However, I feel that the discussion should also incorporate more recent findings, especially the results of tissue-specific CKO studies which have shed light on the sites of FGF21 action (instead of just stating “the specific tissue(s) mediating these effects of chronic FGF21 treatment remain undetermined”). Also, highlighting examples of local FGF21 resistance  vs. systemic FGF21 resistance  would be necessary since the receptors for FGF21 are located in various cell types.     When the notion of “FGF21 resistance” was first introduced, it was an attractive idea to explain the “paradoxical” elevation in plasma FGF21 in obesity by analogy to other more established examples of hormone resistance where hormone insensitivity is associated with a feedback hormone production . Around that time, the site of receptor expression important for the metabolic action of FGF21 was poorly understood, but the receptor complex expressed in adipocytes was thought to be responsible for many of the chronic metabolic effects. A decrease in Beta-Klotho protein expression in white adipose tissues in obese mice was thus considered to be a potential mechanism for “FGF21 resistance”. More recently, mouse genetic studies have uncovered that the induction of weight loss, glucose lowering, stimulation of energy expenditure, and increased water consumption, are mediated by the receptor complex expressed in the nervous system, rather than adipocytes . This notion is completely overlooked by Markan KR, but in my view, is important when discussing “FGF21 resistance”.Beta-Klotho was introduced in the 3rd paragraph without an explanation. For readers who are not familiar with FGF21 signaling, it should be explicitly noted that FGF21 acts by activating membrane-bound FGFR/Beta-Klotho receptor complex.In the second paragraph, “blunted ERK phosphorylation” – which tissues were examined? Other comments:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. In this article, Dr. Markan expresses her opinion on the ongoing controversy surrounding FGF21 resistance in obesity. This issue has not been explicitly addressed in the past publications so the commentary piece should be a refreshing read for the researchers in FGF signaling field in particular and obesity in general. Having said so, I recommend revising the text along the points below:  1) In the Abstract, I suggest changing the verb “used” to “introduced”.  2) First paragraph: I am wondering if the word “paradoxical” is the appropriate term to use here. Surely one could argue that the observed increases in FGF21 serum level in obese mice/human subjects represent a natural feedback mechanism in response to the progressive worsening of insulin resistance.  Are there any published literature which correlate the severity of obesity with serum levels of FGF21? If there are, they should be discussed. 3) Clearly articulate/elaborate the difference between the two studies under scrutiny by putting particular emphasis on the different interpretations of dose response curves of FGF21 in these two studies. Also, please discuss how differences in experimental approach might possibly have influenced the disparate conclusions reached by the authors of these two studies. Explain whether only b-klotho co-receptor or both b-klotho and FGFR1c show reduced expression in obesity. Clearly identify tissues that lose expression of b-Klotho and/or FGFR1c and provide information on the extent of these expression losses if known. 4) For the sake of general readership, please introduce b-Klotho co-receptor and FGFR1c, the cognate receptor of FGF21, early on in the text perhaps even in the abstract. Please state that unlike classical paracrine FGFs, FGF21 operates through a dual receptor system.  5) The point brought up on the inactivating proteolytic cleavage of FGF21 is interesting and worthy of further discussion. Please identify whether the reported increases in FGF21 levels measure the levels of full length bioactive form. I.e. are the assays used capable to differentiating between full length “active” and cleaved “inactive” forms.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.