The isotopic composition of mother-calf pairs is correlated, and the estimates of the maximum mother-to-calf isotopic difference was ~1.4‰ for δ13C and between 1 and 1.5‰ for δ15N values. Change in δ15N values among epidermal layers in calves was associated with the transition from fetus to consumption of maternal milk. It is here proposed when lactation influences calf epidermis, δ15N values decrease consistently from the outermost to the innermost layer. However, if a calf was born only few days before collection, epidermis integrates more variable δ15N patterns because gestation still affects the isotopic composition of the layers. The possibility of calculating mother-to-calf nitrogen isotope fractionation, and the regularity of changes between calf layer δ15N values, allowed results of an isotopic clock model to predict the age of each calf when sampled with its mother. This model has the potential to be a straightforward method to estimate the beginning of lactation, therefore calf birth date when direct observations are not feasible. The non-lethal remote collection of epidermis appears to be an effective tool for the study of the physiology of reproduction of baleen whales. The parallel study of the three epidermal structural layers highlighted the importance of considering the unique mother-calf pair physiological status at the time of sampling time when stable isotope results are interpreted.The isotopic composition of baleen whales’ epidermis structural layers can give information about dietary change over time. This study investigated if epidermis layers integrated isotopic values that record physiological changes from gestation to lactation. Epidermis tissues (n = 43) were collected from free ranging lactating female gray whale and calves during the beginning of three breeding seasons. Modelling of δ Cetacean epidermis has three instead of the five layers normally found on terrestrial animals, which allows whales to better adapt to conditions found in marine environments including higher density, greater heat capacity and greater water pressure ,2. Cetac1/2)”, respectively, and are expected to be influenced by tissue growth, catabolic turnover and body mass [13C values can identify dietary carbon sources and potentially animal movements and δ15N values can indicate possible prey items and trophic linkages [Stable isotope analysis (SIA) of skin biopsies collected from free-ranging individuals has been widely used in the study of cetaceans . Resultsody mass ,8. Most ody mass –14. δ13Clinkages .13C and δ15N values across epidermal layers were interpreted to represent temporal change and were used to infer intra-specific shifts in foraging preferences and seasonal and regional movements. Particularly, the isotopic composition of SB reflects animals’ diet at the time of sampling, while that of the SC integrated the isotopic composition of past diet [15N values differences appear to follow clearer patterns than δ13C values. This was justified by the greater potential of nitrogen compared to carbon to integrate differences between isotopic compositions of prey from different areas. The precise time period represented by each epidermal layer, however, it is not well known and could vary according to factors such as species and environmental conditions [13C and δ15N values were compared in stranded striped dolphins , bycaught common dolphins (Delphinus delphis) [Eschrichtius robustus) [Recent results of SIA on epidermis strata subsamples of blue , and of nditions . In addidelphis)  and straobustus) , no sign13C and δ15N values across whales’ epidermal layers could be used to estimate how long ago an animal changed diet and if different layers record and integrate movement patterns or changes in metabolic and nutritional conditions. Nutritional and physiological status, such as gestation, lactation, and nutritional stress can affect the isotopic composition of mammals [Megaptera novaengliae) epidermis  to evaluate the effects of pregnancy [13C and δ15N values are expected to decline during pregnancy, because of lipid mobilization, increase of protein synthesis and decreases in nitrogen excretion [15N in the fetus relative to placental blood and to a balance between the tissues of the mother-fetus pair [13C and δ15N values were about 1.13‰ and 1.5‰ higher than those of the respective mothers between muscle tissues [13C values between mothers and fetuses, whereas the δ15N value of the mothers was on average 0.7‰ lower than the fetus [13C and δ15N values were reported to increase in fetuses and decrease in mothers with time during pregnancy. In grey seals, fetus red blood cells and serum δ13C values were lower than those of their mothers , whereas fetus δ15N values were higher  [13C values in pregnant relative to lactating fin whales, and no significant differences were found in δ15N values. Habran et al. [13C values in serum of lactating female northern elephant seals sampled 5 days after parturition (assumed to reflect gestation) compared to 22 days after birth (assumed to represent lactation), but no significant differences were reported for δ15N values. On the other hand, in lactating grey seal serum, δ13C appeared to decrease and δ15N values to increase significantly between beginning and end of lactation (about 15 days apart) [13C and δ15N values were found from fetal to calf epidermis in two bowhead whale specimens, although after weaning δ15N values of calf epidermis increased [15N values were reported to increase significantly between day 5 and 22 after birth, while serum δ15N values appeared to follow an opposite decreasing trend for the same time frame [13C values of whiskers in the offspring of southern elephant seals [15N increased significantly. Conversely, stable isotope patterns of both red blood cells and serum did not change due to lactation in grey seals pups [13C and δ15N values during lactation has only been investigated in pinnipeds [13C and δ15N values were reported to be generally higher in pups than in mothers at the final stage of lactation [13C values and hair δ13C and δ15N values were found to be significantly lower than those of their respective mothers’ tissues [13C and δ15N values during pregnancy and lactation can differ depending on species and tissue analyzed.Based on this information, it is unclear if changes in δ mammals ,19,20. I mammals –28 and o mammals ,29,30. Aregnancy , and anoregnancy ,25,31. Pregnancy . Pinniperegnancy ,26,27,33regnancy ,24,25,28xcretion . Simultahe fetus . In soutn et al.  found sime frame . The δ13innipeds –23,25,28innipeds ,25,28. Wactation ,25 and aThe eastern gray whale is a good model animal to study because, like other baleen whales, it is a capital breeder and its migration patterns and feeding habits are well documented ,35,36. G15N values [Here we investigated if the transition from gestation to lactation is recorded in the isotope composition of the three structural layers of gray whale lactating females and calves. Skin biopsies were collected from free-ranging and alive gray whale mother-calf pairs during the first two months spent in their main calving area, Ojo de Liebre lagoon. We determined epidermal inter-layer isotopic variability in mother and calf individuals, calculated the mother-to-fetus isotope fractionation directly from our sampled pairs and investigated if the expected dependency between mother and calf δThe institution that granted the collection of skin biopsies was SEMARNAT  in México. Codes: SGPA/DGVS/0937, SGPA/DGVS/011543/17, SGPA/DGVS/010876/18 and SGPA/DGVS/12644/19.We analyzed the epidermis of 43 free-ranging female eastern gray whales (n = 19) and calves (n = 24) collected in Ojo de Liebre lagoon , Baja California Sur, Mexico, in February 2011 and 2018 and in January 2019. In 2011, 6 females and 5 calves were chosen randomly from gray whale mother-calf pairs that were forming typical temporary pods in the study area. Attempts made to obtain samples from both mother and calf of a single pair were however unsuccessful. Photo-ID and behavioral data were not collected. In 2018 and 2019 we applied focal sampling methodology  to motheDuring February 2011 and 2018 we sampled all organisms with a 5 m long stainless steel pole equipped with a modified stainless steel biopsy point . We usedAll biopsies were taken when animals surfaced to breathe, in the emerged dorsal area between the dorsal and caudal fin. Once collected, tissue samples were wrapped in aluminum foil, placed inside sterile plastic bags, and stored on ice until we reached land . Thereafter, the biopsies were stored in liquid nitrogen and then frozen at -80°C. In between every biopsy event, punches were cleaned, sterilized, and stored in alcohol.For each biopsy, we separated the epidermis from dermis and hypodermis at the Centro de Investigaciones Biológicas del Noroeste, La Paz, Mexico. The tissue was cut while frozen with a sterile scalpel.Based on previous histological studies of other cetaceans’ epidermis ,3,39, SSStable isotope analyses were performed at the SOEST Stable Isotope Biogeochemical Facility of the University of Hawai‘i (UH) at Mānoa and at the U.C. Davis Stable Isotope Facility. Samples were powdered and weighed into tin cups , and their C and N isotopic composition was measured using a Costech elemental combustion system (Model 4010) coupled to a Thermo-Finnigan Delta plus XP isotope ratio mass spectrometer through a Conflo IV interface (Mānoa) and with an Elementar Vario Micro Cube elemental analyzer  interfaced to an Isoprime VisION isotope ratio mass spectrometer (Davis). Glycine and tuna muscle tissue were used as reference materials to normalize the samples and to correct for instrument drift at Mānoa and Alfalfa flower, bovine liver, enriched alanine, glutamic acid and nylon 6 at Davis.2 for nitrogen. Accuracy and precision at UH were < 0.2 ‰, as determined from multiple laboratory reference materials extensively calibrated using National Institute of Science and Technology reference materials and analyzed every 10 samples.All isotope values are expressed in delta (δ) notation relative to V-PDB for carbon and atmospheric NWe completed statistical analysis and graphical visualizations with the statistical software package R version 3.5.0 for Windows (R core Team 2017). Probability values < 0.05 were considered to be the critical statistical level of significance.15N values for full incorporation rate of blue whale epidermis [13C and δ15N values depend on those of the mother, because both the fetus and the calf feed directly on mother’s tissues. Previous observations [The investigation of marked subepidermal cells in two bottlenose dolphins  and in opidermis  was of 1pidermis . Based orvations ,36 suggelmer”) to test differences in δ13C and δ15N values in epidermal layers (lme4 package for R). The repeated measure design of individual’s epidermal layer isotopic values and the link between calf and mother isotopic values due to gestation and lactation were set as random effects. The values were allowed to vary within the boundaries of an individual ID and within the boundaries of mother-calf pairs IDs . Estimated marginal means  were computed and p-values adjusted for multiple comparison by Tukey. This post-hoc analysis was performed to estimate layer specific differences in mothers and calves.We used linear mixed effect models . Additi13C and δ15N values were analyzed with paired t-tests. Mother and calf epidermis mean δ13C and δ15N values were compared, then the isotopic composition of each epidermis layer. Data normality was checked using the Shapiro-Wilk normality test. If data were not normally distributed, we used the paired two-samples Wilcoxon test, specifically the Wilcoxon Rank Sum and Signed Rank tests (“exactRankTests” package for R).Mother-calf pair epidermal layer δt1/2), defined as the time required for a certain isotope to reach the mid-point between steady state with the old and new diets [Generally, after a change in the isotopic composition of a diet, the isotopic composition of a tissue changes with time. This change can be described by an exponential decay curve :δt=, old diet) is different from the δ15N values of calf epidermis layers in steady state with nutrition from lactation (δ(∞), new diet). Therefore, the timing of this dietary change can be interpreted as an estimate of the time of calf birth. Depending upon the timing of calf birth, dietary changes in δ15N values may visible in different epidermal layers. If, for example, a calf was born just prior to sampling, one could expect that only the newest epidermal layer (SB) would integrate nutritional inputs from lactation. On the other hand, if a calf were several weeks old at the time of sampling, the change to nutrition from lactation would be recorded in the SS and SC, or SS only. Here, we propose the use of 15N values of each calf-mother pair:t1/2 of 42 ± 16 days [In this study, we assumed that the δ 16 days .15N turnover [15N values of both mother and calf are considered to represent always full gestation (δ(0)), independently from calf birth. On the other hand, the second method relies on the trend in δ15N values measured here among organisms’ epidermal layers and it assumes that full gestation effects could be visible in any layer as a function of the time since calf birth. A decline in δ15N values is indeed expected in mothers from conception to parturition, and the speed of these changes appears to increase together with mother and fetus weight gain [15N values from calf SC δ15N values. In the second case, the highest δ15N value of mother’s epidermis is subtracted from the highest value of the corresponding calf, independent of the layers where they were found. Finally, for each method, we assume that the largest mother-to-fetus nitrogen isotope fractionation value measured approximates calf δ(0) as:δ(∞)was calculated by the rearrangement of t) could not be represented by any of the δ15N values recorded in the sampled calf epidermis, because we did not know t, or calf age, the main unknown variable of our model. Jenkins et al. [15N values recorded at a specific time after birth and consistent between different species. In 11 terrestrial mammals of different size and diet specializations approximately 15 days after birth, mother’s blood components  has been shown to continue to reflect δ(0) values, and calf blood incorporates δ15N values that are about equal to those of its mother δ(0) [15N turnover rates [15N epidermis values is an indication of the 15 days after calf birth. In other words, when mother-calf δ15N values are equal it indicates calf δ(t) of 15 days and we can apply Eqs Nitrogen isotope fractionation between mother and offspring is inconsistent between species , howeverturnover ,13, and ght gain ,48. If ts et al.  present her δ(0) . In thiser rates ,47 and wδ(0) and δ(∞)values were used to create the exponential decay curve representing the change from gestation to lactation inputs. In addition, the age of each epidermis layer was calculated independently using 15N values of calf epidermis at t1/2 were calculated using For each calf, the calculated 13C and δ15N values of all gray whale together ranged from -15‰ to -20.5‰ and from 10.6‰ to 16.6‰, respectively δ15NCalf—Mother value. For mother-calf pairs where δ15N values decreased from SC to SB  the two methods gave similar results, because SC always recorded the highest values. For the remaining mother-calf pairs, “method 1” generally gave higher mother-to-fetus nitrogen isotope differences than “method 2”. The highest calculated isotope difference was 1.5 ‰ (“method 1”) and 0.9 ‰ (“method 2”). Given that both of these values were from mother-calf pairs sampled in January 2019 ) ) , which wn (δ(0)) . To explCalves birth dates calculated using both the 1.5 ‰ and the ~1 ‰ mother-to-fetus isotopic differences agreed with known times for gray whale parturition. A timeline of gray whale reproduction, gestation and lactation periods expected for this species is shown in 13C and δ15N values highlighted intra- and inter-individual differences, due to epidermis layers, age class, and year of sampling. The isotopic composition of mother-calf gray whale pairs did not differ, thus suggesting direct nutrient transfer from maternal to offspring tissues. Changes in δ15N values among mother-calf epidermal layers corresponded closely to the transition from gestation to lactation, which is a dietary change from placental blood to maternal milk in calves. Previous works [15N values among epidermal layers reflects dietary information, and this study is the first to use this information to investigate a baleen whale species during breeding. Predicted gray whale mother-to-fetus epidermis nitrogen isotope fractionation was similar to that reported in the literature for different tissues and different mammal species [The potential of whale epidermal tissue isotopic composition to integrate physiological and nutritional effects was supported by the isotopic patterns found among gray whale lactating females and calves. The modelling of δus works  showed t species ,29,48. W species ,13, it w13C and δ15N values differed between organisms sampled during different years. On the other hand, δ13C and δ15N values changed among epidermal layers and age classes in similar ways and, consequently, year of sampling was not included in the best-fitted models as a fixed effect.By focusing on the collection of mother and calf individuals, we were able to determine a priori important characteristic that are difficult to detect during sampling campaigns of free-ranging animals, such as sex, age, and reproductive status. Data modelling highlighted the relevance of variables as epidermis layers , age class  and the interaction between the two for the interpretation of SIA results for both carbon and nitrogen. Mean δ13C values is generally complicated by the high lipid content of maternal milk [13C values during pregnancy, and results based on a study of fin whale muscle tissues [13C values during lactation. These changes were likely associated with lipid mobilization during gestation and lactation. During pregnancy, lipids are metabolized and used directly to provide energy during final gestation. On the contrary, during lactation, the production of lipid-rich maternal milk with low δ13C values would result in an enrichment of 13C in tissues proteins of females relative to gestation. Here, epidermis δ13C values both increased and decreased among layers of lactating gray whales , when animals were supposed to have just started to lactate, while stronger inter-individual variability was found in February mothers (2011 and 2018). The only evidence reported in the literature for calves is from two bowhead whale specimens [13C between fetus and calf, due to maternal milk consumption. In gray whale calves, only 9 out of 24 individuals had lower δ13C values in their SB (newest layers) compared to SC (oldest layer)  . The absC values . Model a15N values allowed a more refined interpretation of data. In lactating females, mean and inter-layer δ15N patterns appeared to vary among years of sampling  relative to layers representing early life outside the maternal womb (δ15N = 12.4 ‰). Conversely, in other capital breeding marine and terrestrial mammal offspring, SIA do not unambiguously support the decreasing trend in δ15N values found here. In northern elephant seals, for example, a decrease in δ15N values was observed in serum but not in red blood cells between the initial and the final stage of lactation [15N during lactation [15N values can both decrease or increase in cubs plasma during lactation. On the other hand, grizzly bear offspring δ15N values are reported to decrease in their plasma during 3 months of lactation [15N values found in the epidermis layers of most gray whale calves could be associated with lactation effects. A similar interpretation was given by Horstmann-Dehn et al. [15N values found in the new epidermis of the bowhead calf compared to those of its epidermal fetal patches. On the other hand, when more variable patterns are recorded, as for some 2019 individuals, we suggest that calf birth occurred so close to sampling time that δ15N values most likely did not yet reflect lactation effects; an assumption that needs confirmation. Assuming gray whale epidermal cell turnover is similar to that of bottlenose dolphin [15N values between 10 and 20 days after birth. Pregnant gray whales are expected to give birth in the breeding areas of Mexico from the end of December to the beginning of March [15N values represent different periods of time, with respect to birth and the onset of nursing.Calf δing 2019 . Top-seld calves . To the actation . Conversactation . In polaactation , SIA resactation . All then et al.  for the  dolphin  and belu dolphin , maternaof March , however13C and δ15N values differed between mothers and calves always in their SC, and then isotopic values tended to converge in their SS and in their SB, respectively  δ15N values of calf epidermis at birth, representative of life in the womb, can be calculated from the observed mother-to-calf nitrogen isotope fractionation; and 3) 15 days after calf birth, δ15N values of mother and calf epidermis are equal.Calf-specific changes in δck model ,45,46 asck model . Because13C and δ15N values could have already been influenced by prey inputs, thus no longer reflecting full fasting. During the first months of lactation, capital breeders produce milk that is composed of fatty acids derived largely from the metabolism of its blubber lipids [15N values ). In addition, no information is available for the mother-to-fetus isotope fractionation in cetacean epidermis. Previous results suggest mother-to-fetus nitrogen isotope difference of ~1‰ for blood, plasma, and hair in other mammals such as elephant seals and humans [15Ncalf-mother if sampled calves are born within few days of sampling. In that case, SB δ15N values would differ little from the other epidermal layers, and predictions could result in a discrepancy called “the edge-effect bias” [The collection of maternal milk from baleen whale is logistically challenging and no literature data is available to determine if milk isotopic composition varies during lactation stages . Availabr lipids . By sampd humans ,48, and d humans . Interesct bias” . This co15N values at steady state with maternal milk ) from direct sampling, because calf weaning occurrs in the polar feeding areas months after their stay in the breeding lagoon. For this reason, we estimated calf δ(∞) using information available in the literature for other species mother-calf pairs for which δ15N differences between mother and calf was known at a specific time after birth [15N values of plasma and red blood cells were similar 12 to 14 days after parturition in those mother-calf pairs. Nothing similar has been reported in marine mammals, however, we assumed these data could be relevant for two reasons. First, due to the peculiar histology of its structural layers, whale epidermis combines turnover rates that vary from a few days to months [15N values from SC to SB, while the nitrogen isotope composition of mothers’ epidermis was less variable. In all mother-calf pairs collected in 2018 and in 3 couples from January 2019, δ15N values were similar at a certain point  [15N values from them and their calves, from birth to weaning.Our model represents a potential method to estimate calf age based on mother-calf pairs epidermis δcidents) . A syste15N values during breeding have the potential to be useful for the study of other baleen whales during the same life-history phase. The western gray whale is listed as an endangered population by the IUCN. A precise estimate of gray whale calf birth dates among different years would allow the detection of possible alterations in the known patterns of the species’ reproduction and to immediately investigate the correlated causes. Additional studies are necessary to monitor changes with time in δ15N values of both mothers and calves. By collecting epidermis biopsies at the beginning and at the end of their stay in the study area, it may be possible to gain insights on the isotopic patterns of milk. Moreover, it may help to corroborate the higher expected turnover rate in calf epidermis, as well as the persistence of pregnancy effects on a mother’s δ15N value. Future investigation of feeding dynamics of pregnant and lactating baleen whale individuals emerges as critical. The success of their prolonged reproductive cycles varies according to the energy balance that exists between accumulation of nutrients and their use to supply specific physiological needs. In a time in which ethic statements strongly influence the management of conservation, the findings of this study could help to go beyond questionable and old-fashioned practices, such as scientific whaling. Non-lethal collection of epidermis biopsies and successive stable isotope analysis provide an effective tool for the study of reproduction, feeding, and population dynamics of migratory aquatic species.The eastern gray whale is a good model animal and variation among its epidermal layer δS1 File(XLSX)Click here for additional data file.


Here we report the pH induced self-assembly of equilibrium zwitterionically charged block co-polypeptide nanotubes into hierarchical nanotube fibers. Natural materials, such as collagen, can assemble with multiple levels of organization in solution. Achieving a similar degree of control over morphology, stability and hierarchical organization with equilibrium synthetic materials remains elusive. For the assembly of peptidic materials the process is controlled by a complex interplay between hydrophobic interactions, electrostatics and secondary structure formation. Consequently, fine tuning the thermodynamics and kinetics of assembly remains extremely challenging. Here, we synthesized a set of block co polypeptides with varying hydrophobicity and ability to form secondary structure. From this set we select a sequence with balanced interactions that results in the formation of high-aspect ratio thermodynamically favored nanotubes, stable between pH 2 and 12 and up to 80 °C. This stability permits their hierarchical assembly into bundled nanotube fibers by directing the pH and inducing complementary zwitterionic charge behavior. This block co-polypeptide design strategy, using defined sequences, provides a straightforward approach to creating complex hierarchical peptide-based assemblies with tunable interactions. Due to their biocompatibility, biodegradability and their versatility in chemistry,Synthetic hierarchical materials often form kinetically trapped structures, which can be very stable,Here we use BCPPs with a defined amino acid composition and sequence, inspired on previous observed assembly behavior.x[KGE]y, see Using solid-phase peptide synthesis (SPPS),–1. At both concentrations similar behavior was observed. For [ALV]5[KGE]2 this resulted in macroscopic phase separation. Assembly of [ALV]5[KGE]2 could be induced by a DMSO solvent switch procedure  sequence, or a decrease of the hydrophilic (KGE) sequence length, [ALV]3[KGE]5 was synthesized. CryoTEM showed identical nanotube assemblies compared to [ALV]3[KGE]4  soluble unimers of [ALV]5[KGE]2, to (2) nanotubes of both [ALV]3[KGE]4 and [ALV]3[KGE]5 that are in equilibrium with the solution, to (3) kinetically trapped structures with [ALV]4[KGE]3 and finally (4) phase separation of [ALV]5[KGE]2  indicates that both the morphology and the energetics of the formation pathways can be controlled by the relative block lengths of the [ALV]x[KGE]y system.CryoTEM further showed that the most hydrophobic peptide [ALV]9 ± 1 nm  and an ii.e. folding behavior) of these macromolecules was investigated using Fourier transform infrared (FTIR) spectroscopy, by analyzing the amide I vibrations  experiments monitoring structure formation with static light scattering (SLS) measurements. CryoTEM studies at room temperature before and after thermal annealing and for a reheated part of the sample to 58 °C (equipment limit) during vitrification were used to provide further insight into the system behavior.To investigate if the [ALV]10 R(q) scales linearly with the logarithm of the scattering vector log10 q, with a slope close to –1, corresponding to the presence of cylindrical shaped assemblies or nanotubes with a fractal dimension of 1.56During each heating step the total light scattering intensity at 90° was measured to determine the equilibration time. This showed that during every step full system equilibration occurred within 80 seconds after reaching the target temperature Fig. S7. At roomq values (<2 × 107 m–1), indicating a progressive increase in attractive interactions between individual cylinders with increasing temperature. This strongly implies a tendency of the cylinders to form bundles at high temperatures. This trend is observed with a single exception at 60 °C at a q of 1.1 × 107 m–1, likely due to the presence of intermediate aggregates. At 58 °C cryoTEM indeed showed the presence of large populations of bundles of relative short nanotubes. These bundles were present alongside a minor population of long (>2000 nm) curved nanotubes and some sheet like structures which possibly resulted from the unfolding of nanotubes . FTIR showed the formation of dominant β-sheet arrangements in all cases with amide I maxima between 1628–1624 cm–1  the introduction of secondary structure (beta sheets) in the hydrophobic block, that provides the nanotubes with the required stability under different self-assembly conditions, (2) the reversible introduction of a zwitterionic regime in the hydrophilic blocks that allowed to direct the inter-nanotube interactions through pH variation.Importantly, the thermodynamic stability of the nanotubes is a key factor in uniformity of the nanotube formation process, which, together with their high aspect ratios make this system an ideal candidate for further investigation as a peptide hydrogel system.This project received funding from the 4TU High-Tech Materials research program “New Horizons in designer materials”, the Marie Sklodowska-Curie Action project “LPEMM” and from the Netherlands Organization for Scientific Research .There are no conflicts to declare.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary informationClick here for additional data file.


We found that in addition to inhibiting PKA activity, both PKI (6–22) amide and full-length PKIα facilitated the activation of multiple isoforms of protein kinase C (PKC), albeit at much higher concentrations than necessary to inhibit PKA. Thus, our results call for appropriate interpretation of experimental results using PKI as a pharmaceutical agent. Furthermore, our study lays the foundation to explore the potential functions of PKI in regulating PKC activity and in coordinating PKC and PKA activities.G-protein-coupled-receptor (GPCR) signaling is exquisitely controlled to achieve spatial and temporal specificity. The endogenous protein kinase inhibitor peptide (PKI) confines the spatial and temporal spread of the activity of protein kinase A (PKA), which integrates inputs from three major types of GPCRs. Despite its wide usage as a pharmaceutical inhibitor of PKA, it was unclear whether PKI only inhibits PKA activity. Here, the effects of PKI on 55 mouse kinases were tested in  G-protein-coupled receptor (GPCR) signaling is highly regulated, and its exquisite spatial and temporal specificity has important implications on its function. Spatially, GPCRs and their signaling components are located in specific cell types, subcellular compartments, and microdomains, and their spatial localization specifies their functions . TemporaOne important intracellular integrator that lies downstream of multiple GPCRs is protein kinase A (PKA) . PKA is pkiα reduces neuronal excitability and eliminates hippocampal plasticity , the regulatory subunits of PKA, and the endogenously expressed PKA inhibitory peptide, PKI . PKI binasticity . TherefoIn addition to the important functions of endogenous PKI, shorter peptides of PKI, for example PKI (6–22) amide, are widely used as pharmaceutical agents to inhibit PKA activity . HoweverHere, we analyzed the effect of PKI on 55 kinases, selected based on their similarity to PKA and their expression patterns. We confirmed that both PKI (6–22) amide and full length PKIα inhibited PKA activity with sub-nanomolar IC50. In addition, we found that at high concentrations, PKI (6–22) amide inhibited calcium/calmodulin-dependent protein kinase I (CamK1). Surprisingly, at concentrations often used in pharmacology experiments, PKI (6–22) amide facilitated the activity of multiple PKC isoforms, rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), and p70S6 Kinase (p70S6K). Synthesized full length PKIα also facilitated the activity of ROCK1 and multiple PKC isoforms. These results are important not only for interpretation of experiments using PKI as a pharmacological agent, but also sheds light on potential biological functions of endogenous PKI.PKA inhibitor (PKI) fragment (6–22) amide was purchased from Tocris (Cat. No. 1904). Full length mouse PKIα was synthesized from L amino acids and then purified with high-performance liquid chromatography (HPLC) by the Koch Institute Biopolymers and Proteomics Facility. Mouse PKIα shares 74 out of the 76 amino acids with human PKIα, and the two differing amino acids are not in the 6–22 region.in vitro assays with purified kinases with the Thermo Fisher Scientific SelectScreen Kinase Profiling Service. CamK1, DAPK1, and NUAK1 (ARK5) were screened with the Adapta Universal Kinase Assay, and the rest of the kinases were screened with the Z′-LYTE Peptide Kinase Assay  amide was used to for the initial screen because this short peptide is a potent inhibitor of PKA and has been widely used to inhibit PKA activity in numerous studies. We first determined with a high concentration of PKI (6–22) amide (5 µM) the kinases whose activities are PKI sensitive at all and therefore merit further studies with dose response curves. As expected, 5 µM PKI (6–22) amide effectively inhibited PKA activity by 85%. PKI (6–22) amide had no effect on the majority of other kinases. However, at 5 μM, PKI (6–22) amide inhibited five other kinases by more than 10%. Surprisingly, PKI (6–22) amide (5 µM) enhanced the activity of 20 kinases by more than 10% (expressed in Since we were surprised by the facilitating effect of PKI (6–22) amide on the PKC family, we set out to disambiguate the potential effect of PKI (6–22) amide on PKC in the kinase reaction, on the protease reaction (development reaction), or on fluorescence itself. PKI (6–22) amide did not induce any significant fluorescence readout change when added in or after the development reaction, but did induce a large change when added in the kinase reaction for PKC epsilon . These cTo determine the concentration at which PKI (6–22) amide exerts effects, the dose-response curves of PKI (6–22) amide on activities of 11 selected kinases were measured. These kinases included 1) PKA, 2) all five other kinases whose activity is inhibited by more than 10% at 5 μM, and 3) five selected kinases whose activity is increased by more than 10% at 5 µM – three of these are PKC isoforms. The IC50 of PKI (6–22) amide for PKA was 0.61 nM, comparable to the value determined from previous studies  Figure . PKI 6–. HoweverFull length PKI may display different potency and specificity compared with the short peptide. Therefore, although the screen with PKI (6–22) amide above was useful in determining the specificity of PKI as pharmaceutical agents, it is important to perform dose-response curves with full length PKI to assess whether endogenous PKI may alter activities of kinases other than PKA. Full length mouse PKIα protein was synthesized and purified by High-Performance Liquid Chromatography (HPLC), and was subsequently used to determine the dose response curve of seven kinases .PKIα inhibited PKA activity with an IC50 of 0.11 nM, with higher potency than PKI (6–22) amide . PKIα diIn order to understand the specificity of PKI on kinases, we screened both PKI (6–22) amide and full length PKIα against a panel of 55 kinases. In addition to inhibiting PKA activity, PKI (6–22) amide also inhibited CaMK1, and facilitated the activities of p70S6K, ROCK1, and multiple PKC isoforms. Full length PKIα, at high concentrations, also facilitated the activity of ROCK1 and multiple PKC isoforms. These results inform the design and interpretation of experiments using PKI as a pharmacological agent, provide a starting point to explore novel functions of endogenous PKI, and raise the interesting possibility that PKI may act as a molecular switch between PKA and PKC activity.We found that in addition to inhibiting PKA activity, PKI also facilitated the kinase activity of multiple PKC isoforms. This was surprising because previous studies found little influence of PKI on PKC . In prinAGRTGRRQAIIAHDILVAA, whereas we used PKI (6–22) amide whose sequence is TYADFSGRTGRRNAIIA . Second, the forms of PKC used are different: we used purified human recombinant PKC isoforms expressed in insect cells, whereas previous studies used partially purified PKC without clear distinction of isoforms  amide and other short peptides of PKI are widely used in pharmacological experiments to inhibit PKA activity. They are often used at between 1 and 10 μM, either intracellularly through a whole-cell recording pipette, or applied in the bath in cell-permeant myristoylated forms. Here, we found that PKI (6–22) amide, at these concentrations, also inhibits CamK1, and facilitates the activity of multiple PKC isoforms, possibly acting as a positive allosteric modulator. Future directions will involve testing the effects of PKI (6–22) amide on kinase activity in a cell-based system. Importantly, for any users of PKI to perturb PKA functions, these additional targets of PKI need to be taken into account to design experiments and interpret results.Despite the additional targets of PKI identified in our study, PKI seems more specific than the other commonly used PKA inhibitors, H89 and KT 5720 . This maWe found that full length PKIα, one of the endogenously expressed isoforms, enhanced the activity of ROCK1 and multiple PKC isoforms at high concentrations. This raises the interesting possibility that PKI acts as a molecular switch to regulate the balance between PKA and PKC activity. However, the validity of this hypothesis depends on the endogenous concentration of PKIα, which is hard to determine, partially because the local concentrations of PKIα within a subcellular compartment may be higher than what can be determined with traditional biochemical methods. Our study therefore lays the foundation for future explorations on how endogenous PKI impacts cellular signaling and confines it in space and time through non-classical targets.


Non-evoked miniature release of neurotransmitters is increasingly recognized as playing an important role in neural function and is implicated in synaptic plasticity, metaplasticity, and homeostasis. Spontaneous miniature release events (minis) are usually measured electrophysiologically by recording the miniature postsynaptic currents (mEPSCs) that they evoke. However, this indirect technique can be confounded by changes within the postsynaptic neuron. Here, using the fluorescent probe SynaptopHluorin 2×, we have developed an optical method for the measurement of minis that enables direct assessment of release events. We use the technique to reveal that the frequency of minis following incubation of hippocampal neurons with Amyloid β oligomers (Aβo) is increased. Electrophysiological mEPSC recordings obtained under the same conditions report a decrease in frequency, with the discrepancy likely due to Aβo-induced changes in quantal size. Optical quantal analysis of minis may therefore have a role in the study of minis in both normal physiology and disease, as it can circumvent potential confounds caused by postsynaptic changes. Until quite recently presynaptic “miniature release events” (minis) were thought to be functionally unimportant, however, it is now clear that they serve distinct physiological roles. Miniature release mechanisms are, in part, separate from those of evoked release  at the soma of the postsynaptic neuron. This approach introduces some important confounds into the measurements. First, it is not possible to attribute events measured at the soma to specific synapses, precluding the study of a specific contribution of minis at a specific site, or those arising at clusters of synapses on specific axonal or dendritic branches. The latter may be particularly important as there is clear evidence that dendritic branches are an important spatial unit for synaptic regulation  with SypH 2× (gift of Dr. Y. Zhu) plasmids using Lipofectamine 2000 (Invitrogen). To investigate the effects of Aβo, cells were incubated in Aβo  for ove1–42 peptide (Abcam) and oligomers were synthesized according to a validated protocol . The peptide was incubated at room temperature for at least 1 h to establish monomerization and randomization of the structure. The HFIP was aliquoted and allowed to evaporate overnight, followed by 10 min in a Savant Speed Vac. The resulting peptide was stored as a film at −80°C. The film was dissolved in anhydrous dimethylsulfoxide  to 5 mM, diluted to approximately 100 μM with Ham’s F12  and briefly vortexed. The solution was incubated at 4°C for 22–24 h, and soluble oligomers obtained by centrifugation at 14,000 g for 10 min at 4°C.Experiments were conducted using a single batch of Aβl Klein, . This wil Klein, . Briefly4Cl unquenching. When required, APs were evoked by passing 20 V, 1 ms current pulses from a custom-made stimulation box via platinum electrodes. We have previously shown that each of these 20 V pulses has a greater than 97% probability of eliciting a single AP in stimulated neurons in our experimental setup  with APV (50 μM) and NBQX (10 M) to block recurrent activity. TTX (1 μM) was added for experiments investigating minis. NH4Cl unquenching was achieved by replacing Tyrodes solution with a Tyrodes solution containing 50 mM NH4Cl in substitution of 50 mM NaCl. For experiments examining minis, folimycin (10 nM) was added after 10 baseline images  during all non-data acquisition periods. Suitable fields of boutons were selected using the readily visualized resting fluorescence signal exhibited by SypH 2×, the additional criterion being that boutons show normal, healthy morphology with a regular outline, and relatively modest resting fluorescence signal that increases following NHe images . For exp2, 2 mM Na2-ATP, 0.2 mM Na-GTP, 10 mM HEPES). The internal solution was allowed to diffuse for 2 min before mEPSCs were recorded using an Axopatch 2A amplifier (Axon Instruments) and WinWCP software (Strathclyde Electrophysiology Software). Recordings were then analyzed using Mini Analysis from Synaptosoft, and mEPSCs were detected manually.mEPSCs were recorded from dissociated hippocampal cultures at DIV 14–18. These recordings were carried out in whole-cell voltage-clamp, holding cells to −70 mV, using patch electrodes (5–8 MΩ) filled with Cs internal solution  data, with the optimal number of Gaussians determined by the model resulting in the lowest Akaike Information Criterion (AIC). The Gaussian distributions were ranked according to their expected values, with the rank corresponding to the number of release events (q). Having identified the boundaries (defined as the intersections between Gaussian distributions), we assigned each bouton within those boundaries as having undergone the number of miniature release events to which that distribution corresponds. To compare different conditions with different numbers of boutons, the relative proportions of boutons in each Gaussian distribution rather than the absolute numbers were used.Boutons were selected for analysis using a 2 μm diameter ROI. Experiments imaging pHluorins were analyzed using a custom-written MATLAB script. For analysis of minis, background fluorescence was subtracted from data points, which were then normalized to the bouton’s NH4Cl signal. A frequency distribution was then generated in the same range as before, and two Gaussians fitted. Having found the intersection of the Gaussians, observations below the intersection were designated as not released, while those above were assigned as a release event. Probability of release (Pr) was then calculated for each bouton, discarding any bouton that did not release at all or had fewer than 15 usable measurements.For one AP experiment, data points were background adjusted as before, and for each simulation, a response was measured as a response (average two points after stim) - baseline (average of first five points). These were then normalized to the NH4Cl signal as before. The maximum response was calculated as the average of two points immediately following the end of stimulation minus the average of the first eight baseline time points.For 100 AP experiments, data points were background adjusted and normalized to the NHTo directly probe release, we used SypH 2×, a genetically encoded, presynaptically expressed reporter of vesicle fusion  to reveal the maximal SypH 2× response at each terminal (2R = 0.98), with the interpeak distance representing quantal size  at pH 7.4 to isolate spontaneous miniature release events. Images were taken at 1 Hz over 10 s to give a baseline signal before the addition of folimycin (10 nM). After waiting for 10 min a series of images was again acquired at 1 Hz for 30 s. Over the last 10 s, SypH 2× was unquenched by alkalization using a buffer containing NHterminal . All resterminal . Backgroterminal . The exp4Cl-normalized responses and fitted this with two Gaussian distributions (corresponding to either no release or one release event) using a custom-written objective curve fitting script. We again derived q as the interpeak distance (q estimate of comparable magnitude to that derived from the mini-experiment (q = 0.028 ΔF/F4ClNH). The proportion of events that showed successful release was 21%, corresponding to an average probability of release across all events of 0.21 , with q = 0.44 ΔF/F4ClNH, similar to that of control , well documented to cause robust increases in mini frequency  was assessed. The boundaries of each pair of Gaussians were calculated, and boutons that responded within each range were assigned as corresponding to that number of release events  form  with q = 0.037 ΔF/F4ClNH (Cultured neurons expressing SypH 2× were incubated in media containing Aβo (200 nM) for over an hour and miniature release events were then measured as described above with the Aβo concentration maintained in the experimental buffer. Once again, responses were plotted as a frequency distribution in the same range as the control, and four Gaussians were objectively fitted . The meaF/FNH4Cl , identicq = 0, q = 1, etc.) to investigate changes in mini frequency as before  amplifier and a cesium-containing internal solution to increase input resistance  and at mammalian central synapses that upon partial or complete blockage or loss of neurotransmitter receptors at the postsynaptic terminus  there is a swift increase in the probability of evoked presynaptic release as a result of mechanisms of homeostatic synaptic plasticity  might result from the induction of homeostatic plasticity in response to postsynaptic weakening caused by Aβo, as can occur rapidly when the postsynaptic terminus is blocked or pharmacologically suppressed  Act, 1986 (UK).Ethical review and approval was not required for the animal study because HT and AJ planned and designed the study. HT performed the experiments. RT assisted with coding and analysis of SypH 2× data. AJ and NE provided oversight for the work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


VPD increased recently, but does not yet exceed the maximum values reconstructed during the Medieval Warm Anomaly. The most humid conditions in the Siberian North were recorded in the Early Medieval Period and during the Little Ice Age. Increasing VPD under elevated air temperature affects the hydrology of these sensitive ecosystems by greater evapotranspiration rates. Further VPD increases will significantly affect Siberian forests most likely leading to drought and forest mortality even under additional access of thawed permafrost water. Adaptation strategies are needed for Siberian forest ecosystems to protect them in a warming world.Newly developed millennial δ Although precipitation is low in the Siberian North, due to low temperatures, water loss is not yet as large as observed in European forest ecosystems5. However, increasing surface air temperature7 over the period 1950–2008 driven by the anthropogenic CO2 increase, may also lead to drought stress accordingly12. By enhancing the atmospheric vapor pressure deficit (VPD)14, even in remote subarctic regions, increased evapotranspiration impacts in particular the tree’s water relation. Ongoing anthropogenic warming already has significant negative effects on Siberian forest ecosystems, leading to increased permafrost degradation and CO2 emissions15. Along with temperature increases, increasing VPD14 has been shown to result in low stomatal conductance3, thereby responding to water shortage in trees—even in regions affected by permafrost3. While water and nutrient supply for plants in the Siberian North predominantly depends on freeze–thaw processes in the active soil layer17, wildfire-induced changes can also significantly affect the active soil layer depth and seasonal dynamics with long-term consequences for carbon, nutrient and water balance of the ecosystem19.Precipitation and permafrost-thawed water during the summer are crucial for trees growing in regions with severe temperature limitations and predominantly continental climate conditions20, and (2) a strong temperature increase up to 6 °C with increased precipitation bringing more frequent flooding events21. Based on these projections by the climatic models it becomes clear that the recent rapid climatic warming propose a heterogeneous changes in precipitation patterns.Climate models project drastic changes in climate even under the optimistic RCP 2.6 scenario for the Siberian regions for the period 2081–2100 relative to 1986–2005: (1) a strong temperature increase and reduced moisture availability leading to forest die-back and northward shift of boreal ecosystems22. Tree-ring width, latewood density and wood anatomical parameters record mainly summer air temperature signals for the Eurasian subarctic25, while little is known about the past hydro-climatic regime 25. Additional paleoclimatic proxies like stable carbon isotopes in tree rings should therefore be considered, which can provide information not only about temperature but also about moisture changes in the temperature limited environment26. The carbon isotope ratio (13C/12C) is impacted by the same environmental variables as photosynthetic rate (A) and stomatal conductance (gs)27, which can be an ideal tracer of moisture changes. Low δ13C values usually indicate high air humidity (low VPD) and good water supply  in contrast to higher (less negative) δ13C values by indicating drought (low gs).To reduce such uncertainties in climatic models, knowledge about past climate variability is crucial and can be provided by annually resolved tree-ring records28, irradiance31, and hydroclimate changes34. A recent review by the Past Global Changes (PAGES) Hydro2K Consortium35 compared climatic models and different paleoclimatic archives36 to estimate hydroclimate variability and change over the Common Era. However, studies of stable isotope in tree-rings from remote Siberian regions are still rare and urgently needed, because the impact of anthropogenic activity is still relatively low in these regions, and started much later26 than in industrialized and highly populated areas20.Stable isotopes have been used to derive reconstructions for subarctic regions of millennial air temperatures13C) isotopes, to reconstruct a vapour pressure deficit (VPD) over the past ~ 1,500 years.To reveal the impact of recent climate changes on Siberian forests we used the imprinted isotope signal from living and dead larch trees stored in permafrost as a climate proxy to assess pre-industrial  and anthropogenic  changes in stable carbon (δ13C chronology was constructed based on larch (Larix cajanderi Mayr.) tree-ring cellulose samples with annual temporal resolution for the period from CE 516 to 2004  from 0.2 to 0.9 of mean carbon isotope values are observed for the periods CE 780–810 and CE 1060–1090 when compared to the climate norm (i.e. periods of 30 years before and 30 years after the shifts) Fig. .13C values were revealed mainly for the Medieval Climate Anomaly  period with a mean value of − 23.9‰ and a maximum value of − 21.8‰ and for the Recent Warming Period  with a mean value − 23.9‰ and a maximum value of − 22.3‰. Standard deviation of the MCA (SD = 0.64) was similar to the RP (SD = 0.66) (Table High δ6) Table .13C values were detected during the Early Medieval Period  and during the Little Ice Age  periods, where minimal values were − 26.05‰ (mean − 24.54‰) and − 27.49‰ (mean − 25.03‰), respectively  and verification (1982–2004) periods, and for the whole period of observations (1959–2004) showed significant (p < 0.01) and stable statistical relationships over the full length of the study period ] for the calibration period, as well as R2 and coefficient of efficiency (CE) values for the verification period  was found for 1538 . The highest simulated VPD value in the past 1,489 years was in 1,036  (≈ 11.2 mbar or + 3.5σ), while a greater number of negative anomalies over the past periods were recorded during the LIA (− 3.4σ)   were revealed for the period CE 1516–1616. For the periods, CE 1116–1616 and CE 1716–1916 anomalies were within the range of − 2.09 σ to − 2.88 σ with the SD from 0.5 to 0.9, respectively to both periods Table .Maximum values of the positive (dry) simulated VPD anomalies (+ 3.5σ) were revealed for the MCA  and the RP (CE 1901–2004) (+ 2.9σ) periods, highlighting extreme anomalies during the medieval period compared to the recent period. Less pronounced increasing July VPD trends were detected for the periods CE 616–716, 1116–1216, 1316–1416, and 1416–1616.38 showed a drastic VPD increase and a relative humidity decrease after the 2000s for other sites worldwide.Reconstructed July VPD is highly correlated with the CRU gridded July maximum air temperature Fig. a and Julp < 0.05) with our VPD reconstruction indicating drought conditions in the study region for the period with available data for the July evapotranspiration CE 1979–2004  trees.Reconstructed July VPD showed highest values during the Medieval Climate Anomaly period (800–1070) in the Siberian North compared to the period (1870–2004). This finding is similar to reconstructions of June–July air temperatures for this region using tree-ring widths from the same trees42. However, most likely wet conditions during the LIA were recorded in pollen records from the Canadian Arctic43. Our findings suggest a trend towards higher simulated VPD  across the entire period (516–2004 CE) compared to the global average of 1%14. The drastic thawing of permafrost in the subarctic forest and reduction of precipitation with increasing evaporative demand44 could increase the risk of forest decline and mortality at these sites in the (near) future.Dry conditions during the MCA are recorded in tree rings from Fennoscandia and Arctic Canada45 but also was dry in northeastern Yakutia and started earlier (since 800 CE) compared to the western part of the Eurasia subarctic31  and the recent periods, while humid conditions were recorded during early first millennia and the Little Ice Age. Helama et al.29 reported extremely dry MCA compared to LIA in the post-industrial period (1850–present)42.The reconstruction of July VPD showed that MCA was not only warm13C values are associated with negative VPD anomalies and probably result from less isotopic discrimination driven by high stomatal conductance values and moderate net photosynthesis. The RP is characterized by increasing temperature and development of drought conditions with increasing VPD of ca. 0.8 mbar (CE 1950–2004) compared to the past (CE 516–1949).Negative anomalies of δ27. Furthermore, trees most likely adapt their root system to changing environmental conditions or will ultimately die off due to water shortage. Further eco-hydrological studies are needed for this unique Siberian region to develop adaptation strategies for Siberian forest ecosystems to protect them in a warming world.Significantly correlated spatial distribution patterns of maximum July air temperature and July evapotranspiration with the reconstructed VPD chronology are further suggesting the development of drought conditions in the permafrost region under investigation. We thus conclude that increased VPD under raised air temperature and evapotranspiration may trigger stomatal closure to minimize water loss46. The seasonal thaw depth of permafrost is not more than 30–50 cm, reaching maximum thaw depth (up to 70 cm) in July3. Soil moisture in the upper layers decreases during the summer season41. Soil dryness in July and August depends on the amount of precipitation during these months. The soil temperature at a depth of 20 cm is still below freezing point at the end of June, but may increase up to + 4 °C in July according to the daily measurements by Fyodorov-Davydov et al.46.The study site is situated in northeastern Yakutia, Indigirka , Sakha Republic Fig. a, RussiaLarix cajanderi Mayr.) were collected from 200 to 350 m.a.s.l. near At-Khaya mountain   Fig. a. A typi E) Fig. b are rep E) Fig. c. Only w47.Larch stem discs and tree cores were selected from the available material, which was collected during several expeditions between 1997 and 2004 Table 45,47.47. All 48 selected subsamples needed for the construction of new stable carbon isotope chronology were re-measured and cross-dated using the standard dendrochronological Software 48.Subsamples were selected based on previously calendar dated tree-ring width series and later on were cross-dated using the reference tree-ring width chronology49 were excluded from the analyzed wood samples. Then, each annual ring of individual or pooled wood sample was enclosed into a filter bag and α-cellulose extraction was performed according to the method described by Loader et al.50 and Boettger et al.51. Samples were prepared for each individual year and each larch tree separately for the period from 1945 to 2004. However, small sample size resulted in the need to pool material, i.e. annual rings from four trees were milled together for all of the other periods. To check coherence and offset between pooled tree-ring cellulose samples we measured trees individually for every 10th year 52 between δ13C chronologies of four individual trees is 0.95 for raw and 0.90 for atmospheric corrected according δ13C atmospheric CO2 data for the period from 1948 to 2004.Cross-dated subsamples were split for each annual ring separately with the scalpel. Average age of trees used for the carbon isotope analysis was 308 years. First 50 years of the juvenile stage13C/12C using an isotope ratio mass spectrometer delta-S  linked to two elemental analyzers  via a variable open split interface . The 13C/12C was determined by combustion under excess oxygen at a reactor temperature of 1020 °C, operating in continuous flow mode.Stable carbon isotope analysis was performed within several research projects during the period from 2005 to 2017 at the stable isotope facility of the Paul Scherrer Institute, Villigen, Switzerland. Samples (0.2–0.3 mg) were weighed into tin capsules for the analysis of the 2 exclusion in helium53. This system was linked to a IRMS .Samples for the periods CE 550–910, 995–1250, 1280–1630, 1670–1795, and 1830–1895 were analyzed with a vario PYRO cube  via thermal decomposition at 1,450 °C and conversion to CO under O54.Both systems the EA-IRMS and the PYRO cube yielded very similar precisions (± 0.2‰) and the values from the two instruments were in high agreements (better than the measurement precisions)sample is the molar fraction of 13C/12C of the sample and Rstandard, of the Vienna Pee Dee Belemnite (VPDB) standard. The precision of measurements is σ ± 0.1‰.The isotopic values were expressed in the delta notation relative to the international standard in the Eq. :1\docume53 and further modified by Weigt et al.54, where δ13Ccorrected = 1.1142 δ13Craw + 1.4504. This correction equation is established by measuring a subset of samples via combustion with the elemental analyser. Additional quality control standards are used in each analysis run to test the stability and reproducibility of the system.As the pyrolysis method via PYRO cube involves a small contribution of carbon from the reactor filling to the measuring gas, this needs to be corrected as proposed by Woodley et al.13C data of cellulose for all years after 1,800 were corrected for the Suess effect (decline of the 13C/12C ratio of atmospheric CO2) using δ13C values of atmospheric CO2 obtained from the South Pole ice core and the Mauna Loa Observatory, Hawaii55 (https://www.cmdl.noaa.gov./info/ftpdata.html). This correction is necessary because the emission from fossil fuel combustion and biomass burning have resulted in decreasing δ13C values of atmospheric CO2. De-trending procedure was applied for the δ13C isotope chronology for comparison with climate data to check if decadal variability are still robustly explained by climate parameters.The δ13C chronology against temperature, precipitation and vapor pressure deficit data available from the Chokhurdach weather station  for the period 1959–2004. Gridded July temperature and evaporation data were used (https://climexp.knmi.nl/correlate.cgi) to correlate the reconstructed July VPD and present distribution of the signal along the spatial scale.Response function analysis was applied using Statistica 13.3 Software to evaluate the climate response of the Yakutia δ56 was computed (https://spei.csic.es/) for the period 1979–2004 to reveal and estimate drought conditions with a gridded net 1° × 1° spatial resolution and a monthly time resolution (available under the Open Database License https://opendatacommons.org/licenses/odbl/1.0/).The Standardised Precipitation-Evapotranspiration Index (SPEI)13C tree-ring cellulose chronology (C-IND) was regressed against VPD instrumental data (1959–2004). C-IND shows significant correlation with VPD data, with the highest correlation being observed with the month of July (r = 0.49) (Table s)57 were computed to test the robustness of the transfer function  Table . Split-pion Fig. . Calibra13C versus VPD.Trends were calculated as the slope of the linear regression from 1959 to 2016 CE for the δ13C in tree-ring cellulose and earlier obtained June-July air temperature reconstruction from tree-ring width for northeastern Yakutia45 were analyzed for climatic anomalies in comparison 20 1870–2004. Anomalies are shown relative to each analysed period from the mean and standard deviation over the 1489 years. Deviations ≥  ± 3σ from zero were considered as extreme anomalies, ≥  ± 2σ as anomalies.A new developed July VPD reconstruction derived from isotopic composition of δon Table . NormaliSupplementary Information.


The sentence describing manual contouring times should have read “Manual segmentation of the head and neck took a mean 86.75  min/scan for an expert reader and 73.25  min/scan for a radiation oncologist. The autogenerated contours represented a 93% reduction in time.” This article has been corrected.1In the Original Investigation titled “Evaluation of Deep Learning to Augment Image-Guided Radiotherapy for Head and Neck and Prostate Cancers,”


Ocimum basilicum L.) as one of the most popular herbs. In basil most research has focused on increasing secondary metabolites with light spectra. However, knowledge about the effect of light intensity  and spectra on growth and morphology is key for optimizing quality at harvest. The impact of PPFD and spectrum on plant growth and development is species dependent and currently few studies in basil are available. Understanding the response to End-Of-Production (EOP) light of growth and morphology is important for successful vertical farming. We performed a comprehensive series of experiments, where the effects of EOP PPFD, fraction of blue and their interaction on the growth and morphology were analyzed in two green and one purple basil cultivar. In addition, the impact of different EOP intensities and duration of far-red were investigated. We found that increasing the PPFD increased fresh mass, dry matter content and plant height in all three cultivars. The responses were linear or quadratic depending on the cultivar. A high fraction of blue (>90%) increased plant height and decreased the dry mass partitioning to the leaves. The only interaction found between the fraction of blue and overall PPFD was on plant height in the green cultivar whereas other growth parameters and morphology responded stronger to PPFD than to the fraction of blue light. Plant dry matter production was increased with the addition of far-red. Far-red EOP intensity treatments enhanced the fraction of dry mass partitioned to the leaves, whereas a prolonged far-red treatment enhanced partitioning to the stem. Both plant fresh mass and dry matter content were improved by applying high PPFD shortly before harvest. Light spectra were found to be of less importance than PPFD with respect to plant dry matter content. Light use efficiency (LUE) based on fresh mass decreased with increasing PPFD whereas LUE based on dry mass increased with increasing PPFD, when given as EOP treatments. The overall physiological mechanisms of the light intensity and spectral effects are discussed.Vertical farming is becoming increasingly popular for production of leafy vegetables and herbs, with basil ( Ocimum basilicum L.) that is used for its unique aroma. Besides aroma other important quality parameters include yield, plant morphology and fresh mass and dry matter content  ranging from 400–700 nm . IncreasSeveral studies have focused on increasing the secondary metabolites in basil, however, fewer studies have elucidated the effect of PPFD, light spectra and the interaction of the two on the growth and morphological features. The primary attribute of crops in a commercial production system is biomass, that is fresh and dry mass of leaves. Other relevant attributes include morphology such as short internodes and increased partitioning of carbon to the leaves. Knowledge of the response of basil to changes in light intensity and spectra will allow for a fully controlled plant production and a desired growth and morphology. To optimize production in vertical farming, it has been proposed to focus the lighting strategy during the first part of cultivation cycle on optimizing biomass increase, while the last period before harvest the lighting strategy should focus on optimizing product quality by End-Of-Production (EOP) treatments . We aimeO. basilicum L.) was grown in a climate chamber in a vertical farming set-up with twelve compartments of the size 0.8 × 1.3 m in table area and a plant density of 123 plants m–2. Two green cultivars (Emily and Dolly) and one purple cultivar (Rosie) Sweet basil, were used; all cultivars were derived from Enza Zaden, NL. Seeds were germinated under red-white LED light  varying between 150 and 200 μmol m–2 s–1  or Hanwell data loggers  with deviations within 10% and 1°C from the set points. To maintain air temperature around 25°C fans were installed in high light treatments above the lamps blowing out of the individual compartments. Plants were kept well-watered through an ebb and flood system based on plant needs and growth stage. For the first 3 weeks of the growth, plants were watered once every second day for 10 min and after that once every day for 10 min. High light and high blue treatments were given an extra round of watering when needed. The nutrient solution consisted of NO3– 8.5 mM, SO42– 1.5 mM, HPO42– 1.5 mM, NH4+ 1.5 mM, K+ 5.5 mM, Ca2+ 4.0 mM, Mg2+ 1.5 mM, Cl– 0.2 mM, Fe3+/Fe2+ 30 μM, Mn2+ 5 μM, Zn2+ 5 μM, H2BO3– 35 μM, Cu+/Cu2+ 1 μM, MoO42– 1 μM with pH 5.7 and EC 1.7 dS m–1 before transplant and with an EC of 2.3 dS m–1 after transplant.Basil  treatments of 9, 33, 65, and 100% out of the total PPFD of 300 μmol m–2 s–1. In three other treatments the plants were grown under red white light of 300 μmol m–2 s–1 for 20 days after which they were exposed for 5 days to different blue light treatments of 33, 65, and 100%. Day length was 16 h.In Experiment 2 the response of cultivar Dolly to different fractions of blue light applied either throughout the growth (25 days) or as EOP treatment during 5 days before harvest were investigated. The different fractions of blue light were created by using different ratios between pure blue  and red white LEDs. Seeds germinated for 15 days under 150 μmol m–2 s–1 red white LED light for 15 days. After transplant the plants were grown for another 15 days under red white light of 200 μmol m–2 s–1. Five days before harvest plants were exposed to treatments of 100 μmol m–2 s–1 PPFD with 9% blue, 100 μmol m–2 s–1 PPFD with 90% blue, 300 μmol m–2 s–1 PPFD with 9% blue and 300 μmol m–2 s–1 PPFD with 90% blue. The different fractions blue light were created by using different ratios between pure blue  and red white LEDs. Day length was 18 h.In Experiment 3 the response of cultivars Rosie and Dolly to EOP treatments with increased fractions of blue light and the interaction with PPFD during 5 days before harvest were investigated. Seeds of both cultivars germinated under 200 μmol m–2 s–1 red white LED light for 15 days. After transplant the PPFD was increased to 300 μmol m–2 s–1 of red white LED light for 15 days. EOP treatments were applied 5 days before harvest with 0, 50, or 100 μmol m–2 s–1 far-red  added to the 300 μmol m–2 s–1 of red white LED light. This resulted in treatments with a total photon flux density (PFD) of 303, 350, 400 μmol m–2 s–1 (400–800 nm). Day length was 16 h.In Experiment 4 the response of cultivar Emily to EOP treatments with increasing intensities of far-red in addition to the PPFD during 5 days before harvest were investigated. Seeds germinated under 150 μmol m–2 s–1 red white LED light for 10 days. After transplant the plants continued to grow under 150 μmol m–2 s–1 red white light for another 21 days. No far-red was applied or additional far-red  (180 μmol m–2 s–1) was applied during 1 week (as EOP treatment) or 3 weeks (throughout the growth). This resulted in treatments with a total PFD of 152, 330, 330 μmol m–2 s–1 (400–800 nm). Day length was 18 h.In Experiment 5 the response of cultivar Emily to different durations of far-red before harvest were investigated. Seeds germinated under 150 μmol m2 were counted as true leaves, leaf area was measured with a leaf area meter LI-3100C . Leaves and stem were separated and weighed for fresh mass and dry mass. Dry mass was measured after drying for 48 h at 80°C.Plant height was measured from the surface of the stone wool block to the height of the apex. Leaves of a minimum size of 1 cmnm400–700  m–2 d–1) was calculated as:Daily light integral (molnm400–800  m–2 d–1) was calculated as:Daily radiation integral (molnm400–700 –1) was calculated as:Light use efficiency (g molnm400–800 –1) was calculated as:Radiation use efficiency (g mol2 g–1) was calculated as:Specific leaf area (SLA) (cmDry matter content (%) was calculated as:The experiments were carried out as complete randomized block designs. Each experiment was repeated in time, which represented the blocks. In each experiment six small compartments were used for plant growth. Each light treatment was done in one compartment and repeated in time. For each repetition the position of the light treatments in the six compartments were randomized. Generally 5 or 6 representative plants from the light compartment were sampled for the analyses. For statistical analyses, the average values of each block were used as one replicate. Experiment 1 and 3 (cv. Dolly) were carried out three times, experiment 3 (cv. Rosie) 4 times and experiment 2, 4, and 5 and two times.post hoc LSD test. Treatment effects were tested at a probability level of 5%, unless an experiment had only two blocks in which case probability level of 10% was applied . Experiments on light intensity, blue light and far-red were analyzed with One-way Analysis of Variance (ANOVA), while the blue and blue light × light intensity experiment were analyzed with a two-way ANOVA, followed by  applied . Further–2 s–1 displayed very brittle leaves that easily broke at the petiole and broke easily when handled. For both cultivars light use efficiency (LUE) based on dry mass increased with increasing PPFD, but decreased when based on fresh mass  in cv. Dolly. Plants showed a fairly similar response to the fraction of blue light, both to the EOP and throughout the growth treatments. The largest difference was found between the 100% blue treatment and the other treatments which also included red and green light. Plant height increased quadratically while the fraction of dry mass partitioned into the leaves decreased quadratically with increasing blue light ; in fact–2 s–1) and height was higher at 90% compared to 9% blue light for both cultivars. These results were similar as in the experiments where either the PPFD or the fraction blue were studied separately (–2 s–1whereas no change in LUE was found for cv. Rosie (Photosynthetic photon flux density could play an important role in the response to blue light. Therefore the interaction of PPFD and fraction of blue light was studied in a purple (Rosie) and a green cultivar (Dolly) , 4. The parately , 2. For parately , 4B and parately , 4F. Morv. Rosie .–2 s–1 far-red to the PPFD of 300 μmol m–2 s–1 red-white light (–2 s–1) far-red were applied for 0, 1, and 3 weeks  (–2 s–1 (DLI 12.9 mol m–2 d–1) (–2 s–1 (DLI 28.8 mol m–2 d–1)  the optimal growth conditions. This consideration, is based on both LUE parameters and growth and morphological parameters .Optimal PPFD for basil growth  has been suggested to be 250 μmol mm–2 d–1) , 224 μmom–2 d–1)  and 500 m–2 d–1) . In our ultivars . FurtherBlue light and red:blue ratio have been intensely studied. Red light is the most efficient color driving photosynthesis but adding blue to a red background improves the overall photosynthesis . Yet, thBased on our findings a spectrum with 9% blue while the remaining part of the spectrum being 70% red and 19% green can be maintained throughout the growth of basil as an increased amount of blue did not improve the plant fresh mass nor the dry matter content. Furthermore, no differences were found between the EOP and throughout the growth blue light treatment.Amongst morphological parameters plant height is one that has been recorded in numerous studies. Blue light usually suppresses elongation  but in aShade avoidance syndrome like responses were also found when we grew plants under additional far-red light , 6A wher–2 s–1) (–2 s–1)   or after(–2 s–1) . Howeverincrease . The decincrease . This isincrease . Althougincrease  it decreWe showed that growth  and morphology (plant height and leaf area) were significantly affected by EOP increase in PPFD. Interestingly, LUE based on fresh mass decreased with increasing PPFD whereas LUE based on dry mass increased. The plant fresh mass did not respond to the fraction of blue light while plant dry matter content was reduced at the combination high fraction of blue and a high PPFD. When the spectrum consisted of either 90 or 100% blue, either applied as EOP treatments or throughout the growth shade avoidance syndrome was induced and plants grew taller resulting in more fresh and dry mass partitioned to the stem. Therefore, a high fraction of blue in the spectrum is not desirable for basil growth as the leaves are the consumed part.Addition of far-red for basil during growth is most beneficial when added as EOP treatment before harvest and only in a lower dosage at a high PPFD as it increases dry matter content of both leaves and stem.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.DL, EW, CN, and LM conceptualized the research plan. DL, EW, and LM designed the experiments. DL carried out the experiments, analyzed the data, and wrote the manuscript. EW and LM provided critical feedback on the manuscript. CN provided critical comments to the overall structure of the manuscript. All authors reviewed and approved the final manuscript.CS Nicole was employed by company Signify. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


D 570–98. These data illustrate the potential of the plastic quarry dust composite application in construction industry and model for regeneration of waste plastics for green building technologies.In this article data on impact strength, flammability and water absorption tests for innovative plastic-quarry dust composite is presented. The composites were prepared through moulding with virgin HDPE and PP plastics and quarry dust. The quarry dust was used at 0%, 5%, 20%, 40%, 60% and 80% weight percentages. The prepared samples were characterised for strength, fire resistance and hydrophobic properties using Charpy impact, flammability and water absorption tests respectively. For impact strength test was conducted according to ISO 179–1:2010 (E). The flammability test was conducted according to ASTMD 2863 while the water absorption test was carried out in accordance to ASTM  The data is useful to evaluate the application of the prepared samples in the construction industry and model for plastic waste recycling in the construction industry.•This data can benefit researchers of materials in construction industry, construction engineers and environmentalists in the quarry industries.•The data can further be developed to evaluate specific applications in the building sector such as in roofing tiles, walling materials, etc.•The data shows the influence of the appropriate choice of plastic-quarry dust proportions for optimal performance. It is an indicator of the need for future optimisation studies on the mixture constituents for enhanced performance of plastic-based composites as a construction material.122.1CU (kJ/m2), were computed for each sample using c = the corrected stored energy in Joules,Eh = the thickness of the test specimen (mm)b = the width of the tests specimen (mm)Charpy impact test was conducted according to ISO 179–1:2010 (E) using a Charpy impact tester . The samples were cut according to ISO 2818 into 100 mm × 10 mm × 4 mm sizes (sample shown in 2.23/s), Low Velocity (3.2 cm3/s) and High Velocity (4.8 cm3/s). The readings of the flow metre were then recorded and used to calculate the oxygen index according to the following equation.Flammability test was conducted according to ASTMD 2863 to accurately determine the relative flammability of the composites using the Dynisco Limiting Oxygen Index Chamber . The samples were burnt inside the chamber (as shown in 2.3D 570–98 Water absorption test was conducted according to ASTM