ZT of 2.5 at 923 K by the cumulative integration of several performance-enhancing concepts in a single material system. Using non-equilibrium processing we show that hole-doped samples of PbTe can be heavily alloyed with SrTe well beyond its thermodynamic solubility limit of <1 mol%. The much higher levels of Sr alloyed into the PbTe matrix widen the bandgap and create convergence of the two valence bands of PbTe, greatly boosting the power factors with maximal values over 30 μW cm−1 K−2. Exceeding the 5 mol% solubility limit leads to endotaxial SrTe nanostructures which produce extremely low lattice thermal conductivity of 0.5 W m−1 K−1 but preserve high hole mobilities because of the matrix/precipitate valence band alignment. The best composition is hole-doped PbTe–8%SrTe.The broad-based implementation of thermoelectric materials in converting heat to electricity hinges on the achievement of high conversion efficiency. Here we demonstrate a thermoelectric figure of merit  Alloying lead telluride has proven a good strategy to improve its thermoelectric properties. Here, the authors use a rapid temperature quenching to prepare compounds alloyed above the thermodynamic solubility limit, which results in band convergence and high thermoelectric figures of merit. ZT=S2σT/κ=S2σT/(κel+κlat), where S is the Seebeck coefficient, σ is the electrical conductivity, T is the temperature and κ is the thermal conductivity that is comprised of an electronic part (κel) and a lattice part (κlat). PbTe-based materials are the top-performing thermoelectrics in the temperature range of 500–900 K (refs EL−Σ) of 0.15–0.20 eV at 300 K between them121415EL−Σ to within a few kBT (kB is the Boltzmann constant). This decrease is referred to as effective band convergence, which makes the contribution of the Σ band to the charge transport very significant, especially when temperature is higher than 500 K, and produces much higher Seebeck coefficients9Thermoelectric materials technology can play a role in energy-saving alternatives for sustainable power generation, by providing a direct means for heat to electricity conversion123450 K refs , 11, thaZT of PbTe by strongly reducing the thermal conductivity. For instance, previous studies showed only a small equilibrium solid state solubility (less than1 mol%) of SrTe in bulk PbTe , early studies of PbTe–SrTe thin films187ZT of ∼2.5 at 923 K coupled with a new milestone of average ZT of 1.67 between 300 and 900 K in Na-doped PbTe–8%SrTe. This new approach is quite different from previous studies on PbTe (for example PbTe0.7S0.3 and (PbTe)0.65(PbS)0.25(PbSe)0.1  in comparison to Pb2+ (1.19 Å) and confirms the successful substitution of Sr for Pb in PbTe. We also note that water quenching produces a behaviour of the lattice parameter  is nearly temperature independent. The case is very different for p-type lead or tin chalcogenides featuring two or more valence bands1417232425EL−Σ and carrier redistribution between the two sub-bands1417RH which peaks at a temperature when the contribution from the two bands to hole transport is comparable2326TH) corresponding to the peak of RH is a measure of ΔEL−Σ and is greatly affected when the band structure is altered for example by alloying1723RH always peaks at ∼425 K  electronic structure calculations. Since Na serves only as a dopant in PbTe adjusting the carrier concentration, as has been demonstrated by numerical experiment studies and theoretical calculations2829nd ΔEL−Σ . When th−1 for x=0 and goes up to greater than 90 μV K−1 for x>6 at 300 K  and hole mobilities (μH) for all samples are given in m*) of each sample from the measured S and Np considering a single parabolic bandm* increases gradually from 0.82 me to 1.42 me (me is the free electron mass) with increasing SrTe content from 0 to 6 mol%. We have proposed above that the alloying of SrTe causes convergence between the two valence bands (L and Σ) of PbTe. The degeneracy (Nv) of the L band of PbTe is 4 while that of the Σ band is much larger, Nv=12 (refs Nv becomes 12–16 rather than 4 for the L band or 12 for the Σ band9m* is related to Nv through the relation m*=(Nv)2/3mb* (mb* is the band effective mass)9Nv) significantly increases m* by employing more degenerate valleys in the hole transport without seriously deteriorating μH  of the Pb0.98Na0.02Te–x%SrTe samples in this study are remarkably enhanced, with the maximum values exceeding 30 μW cm−1 K−2 around 500 K . We also compared the temperature-dependent power factors of Na-doped PbTe alloyed with 3 mol% MgTe, MnTe and SrTe in this study  and Sr2+ (1.32 Å)  makes the scattering of carriers from crystal defects less significant. As an evidence of this conjecture, we list the room temperature carrier mobilities for the three samples as follows: 69, 65 and 78 cm2 V−1 s−1 for 3% MgTe . The regTe ref. , MnTe . μH) as a function of temperature for x=6, 8 and 10 samples  with similar hole concentrations of 1.6–1.7 × 1020 cm−3. Clearly the changes of μH with increasing SrTe amounts are negligible. This is attributed to the favourable energy alignment of the valence bands of PbTe and SrTe as reported earlierT−1 from 300 to 400 K and T−4 from 400 to 700 K. The fast degradation of mobilities at elevated temperature in p-type PbTe-based compounds arises from the contribution of the heavy holes in the Σ valence band.Both the total κ,  and lattκlat should vary as 1/T before any bipolar diffusion becomes significant252632κlat starts to deviate from such a relationship when the contribution from bipolar diffusion (κbi) is significant , suggesting that alloy scattering plays a large role in the reduction of lattice thermal conductivity. An apparent departure of the experimental data from the Klemens model is observed for samples with higher SrTe content (x>6). This deviation is attributed to the strong scattering of phonons from the ubiquitous SrTe nanostructures78114344Using the Debye-Callaway model6 K ref. , average −1 ref. , and an  −1 ref. . The mod0.98Na0.02Te–8%SrTe, with grain sizes ranging from 1 to 3 μm on average, as indicated in Microstructural and compositional information was obtained using scanning electron microscopy (SEM), (scanning) transmission electron microscopy ((S)TEM) equipped with energy dispersive spectroscopy and secondary ion mass spectrometry (SIMS). EV) between PbTe and SrTe is only 0.06 eV with the valence band maximum (VBM) of PbTe lying slightly higher than that of SrTe  between SrTe precipitates and Pbx1−SrxTe matrix, 7846Our valence band alignment calculations indicate that the valence band energy offset  and 15% higher than the sample by Biswas et al. (ZT=2.2)  .ZT enhancement in this study with respect to the previous work is especially noticeable at T<800 K while at elevated temperature (800–923 K) this enhancement becomes less significant. In PbTe the change of the energy levels of the L and Σ valence bands with increasing temperature is an unusual phenomenon which causes band convergence. According to recent studies48T>800 K) the thermoelectric performance of PbTe–SrTe system would not benefit from the temperature-driven band convergence more than PbTe itself does. However, the enlarged bandgap (Eg) of PbTe by alloying with SrTe could play a significant role in improving the thermoelectric performance at elevated temperature (T>800 K), due to the suppression of bipolar diffusion. For example, the Seebeck coefficient of PbTe–SrTe shows no sign of downturn up to 900 K (T<600 K), the SrTe alloying-induced band convergence (decrease of ΔEL−Σ) makes the contribution from the heavy Σ valence band to the Seebeck coefficient become noteworthy. As a result, one can observe a large enhancement of Seebeck coefficient of PbTe–SrTe as compared to pure PbTe, see Pisarenko relationship between Seebeck coefficients and carrier concentrations , which is important for device applications, in the range of 300–900 K, is 1.67 for the high performing sample in the present study. In comparison, ZTave is 1.05 for PbTe and 1.37 for the lightly alloyed samples of Biswas et al.0.98Na0.02Te–8%SrTe sample showed negligible changes of thermoelectric properties during the multiple heating–cooling measurement cycles and after a 15 days of vacuum annealing at 823 K, suggesting an excellent thermal stability . Desired amounts of Pb, Sr, Na and Te were weighed and loaded into 13 mm diameter carbon coating silica tubes under an N2-filled glove box. The tubes were then evacuated to a residual pressure of ∼10−4 torr, flame-sealed, slowly heated up to 1,423 K in 11 h, soaked at this temperature for 12 h and subsequently ice water quenched to room temperature. The tubes containing the molten samples were periodically shaken to ensure the homogeneity of the compositions. The quenched ingots were further vacuum annealed at 873 K for 3 days. To probe into the bandgap variation of PbTe as a function of SrTe content using infrared spectroscopy (IR), a series of PbTe–x%SrTe ingots without Na doping  were also synthesized using the same route. All samples synthesized in this study are all single phase compounds within the detection limit of powder X-ray detection limit , Sr chunk , Na chunk  and Te shot  were used as the starting materials to synthesize 15 g of Pbon limit . For a t0.98Na0.02Te–x%SrTe were ground into fine powders using agate mortar under an N2-filled glove box, put inside a 12.7 mm diameter graphite die and densified by SPS  at 823 K for 5 min under an axial compressive stress of 60 MPa in vacuum. Highly dense disk-shaped pellets with dimensions of 12.7 mm diameter and ∼10 mm thickness were obtained  of 10 × 10 μm. The powder diffraction patterns were obtained with Cu Kα (λ=1.5418 Å) radiation in a reflection geometry on an Inel diffractometer operating at 40 kV and 20 mA and equipped with a position-sensitive detector. The lattice parameter was obtained using a Rietveld refinement method ((Scanning) transmission electron microscopy (S/TEM) and STEM energy dispersive spectroscopy experiments investigations were carried out using a JEOL 2100F microscope operated at 200 kV. The thin TEM specimens were prepared by conventional methods, including cutting, grinding, dimpling, tripod, with minimal duration of Ar-ion milling with a liquid Nt method .3 for simultaneous measurement of the Seebeck coefficient and electrical conductivity using an Ulvac Riko ZEM-3 instrument under a low-pressure helium atmosphere from room temperature to 923 K. Samples were spray coated with boron nitride spray to minimize outgassing except where needed for electrical contact with the thermocouples, heater and voltage probes. The uncertainty of the Seebeck coefficient and electrical conductivity measurements is 5%, which is widely accepted for Ulvac instrumentsThe obtained SPS-processed pellets were cut into bars with dimensions 12 × 3 × 3 mm3 for thermal diffusivity measurements. The samples were coated with a thin layer of graphite to minimize errors from the emissivity of the material. The total thermal conductivity was calculated from κtot=D·Cp·d, where the thermal diffusivity coefficient (D) was measured using the laser flash diffusivity method in a Netzsch LFA457, and the density (d) was determined using the dimensions and mass of the sample. Heat capacity (Cp) is estimated from the relation Cp/kB per atom atom=3.07+(4.7 × 10−4 × (T−300))15−3 or are above 97% of the theoretical densities  was calculated by subtracting the electrical thermal conductivity (κel) from κtot using a Wiedemann-Franz relationship κel=L·σ·T, where L is Lorenz number which can be obtained by fitting the Seebeck coefficient to the reduced chemical potential32Cp, D and L values for all the samples in this study can be found in ZT is <15%. Unless otherwise noted, all the thermoelectric properties were measured perpendicular to the sintering pressure direction, although no directional anisotropy effects (<3%) were observed in the charge transport properties.Highly dense SPS-processed pellets were cut and polished into a square shape of 6 × 6 × 2 mmensities . The uncensities , consideNp) was estimated using the relationship Np=1/eRH, where e is the elemental charge, and RH is the Hall coefficient. The Hall mobility (μH) was calculated using the relationship μH=σRH with σ being the electrical conductivity obtained from the ZEM-3 instrument. It should be mentioned here that the use of two different instruments to deduce carrier mobility data may cause some uncertainties.The high-temperature Hall measurement was performed on a homemade apparatus (University of Michigan) in an argon atmosphere. The Hall resistance was monitored with a Linear Research AC Resistance Bridge (LR-700), with constant magnetic fields of ±1 T applied by using an Oxford Superconducting magnet. The effective carrier concentration (−1) using a Nicolet 6700 FT-IR spectrometer. The reflectance versus wavelength data generated, were used to estimate the bandgap by converting reflectance to absorption data according to Kubelka–Munk equations: α/S=(1−R)2/(2R), where R is the reflectance, α and S are the absorption and scattering coefficients, respectively.Room temperature optical diffuse reflectance measurements were performed on finely ground powders to probe optical energy gap of the series. The spectra were collected in the mid-IR range −1. The calculated lattice constant of PbTe is 6.43 Å, which is very close to experimental result 6.463 Å. All structures are fully relaxed with respect to cell vectors and cell-internal positions. For the Pb and Sr species, the 5d electrons and 4p 5s states are respectively treated as valence states. The total energies were numerically converged to ∼3 meV per cation using a basis set energy cutoff of 400 eV and dense k-meshes corresponding to 4,000 k-points per reciprocal atom in the Brillouin zone.DFT calculations of pristine, stoichiometric PbTe, and Sr-doped PbTe were carried out. The calculations were performed using the generalized gradient approximation with PBE27Te27. For the isovalent doping of Sr, we consider a single Sr impurity  with Sr substituting for Pb. The symmetry of the original primitive cell was changed by the substitution defects in PbTe, thus, for the purposes of a more direct comparison with PbTe we transformed the eigenstates for defect structures into a so-called effective band structure in the primitive Brillouin zone of the parent compound PbTe using a spectral decomposition method55To investigate the movements of the conduction band and valence bands (L and Σ bands) with added Sr ions, we considered 3 × 3 × 3 supercells of NaCl-type PbHq  by placing H in the host material with a 128 atom supercell, calculating the total energy of this structure, and subtracting the energy of the corresponding pure host material, hydrogen chemical potential, and electron chemical potential:To assess the relative band alignments of the second phase SrTe and MgTe with host phase PbTe we utilize the findings of Van de Walle and Neugebauer, who demonstrated a universal alignment of the electronic transition level of hydrogen in a wide range of materials including semiconductors, insulators and even aqueous solutionsEV and EF are VBM and Fermi level (relative to the VBM). To select the most favourable interstitial H binding sites in host materials, multiple binding configurations are calculated. The electrostatic potential correction term ΔE is calculated by inspecting the potential in the supercell far from the impurity and aligning it with the electrostatic potential in bulkwhere The authors declare that the data supporting the findings of this study are available within the article and its How to cite this article: Tan, G. et al. Non-equilibrium processing leads to record high thermoelectric figure of merit in PbTe–SrTe. Nat. Commun. 7:12167 doi: 10.1038/ncomms12167 (2016).Supplementary Figures 1-21, Supplementary Tables 1-3, Supplementary Methods and Supplementary References.


O-linked β-N-acetylglucosamine (O-GlcNAc) in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT), and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA). Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology.Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with  Tau protein is a neuron-specific microtubule-associated protein (MAPs) that stabilizes microtubule and stimulates microtubules assembly . Tau hasO-GlcNAc (O-linked β-N-acetylglucosamin) in a healthy neuron. The level of O-GlcNAcylation is reduced under pathological condition. Then, tau phosphorylation sites are freely exposed to kinases resulting in the generation of hyperphosphorylated tau. Gong’s group firstly found that O-GlcNAcylation level in AD brains was 22% lower than that in normal brain, and hyperphosphorylated tau contained four-fold less O-GlcNAc than non-hyperphosphorylated tau . B. B12]. Bin vitro without post translational modification on tau  antibody. Then, the purified tau was labeled with an anti-O-GlcNAc antibody   d. Our reO-GlcNAcylation was increased by 5.5-fold . The intensities of tau-BiFC fluorescence were analyzed using Harmony 3.1 analysis software (PerkinElmer™). Error bars indicate standard deviation from two independent experiments. Each experiment was performed in triplicate.HEK293 tau-BiFC cells were plated in a black transparent 384-well plate under the same culture conditions described above. The next day, tau-BiFC cells were treated with Thiamet G , BZX2 or Forskolin (Sigma) at various concentrations. After the incubation, the fluorescence response in tau-BiFC cells was automatically imaged by using OperettaO-GlcNAcylation level in tau-BiFC cells, immunoblot assay was performed 24 h after chemical treatment. Total cell lysates were prepared using CelLytic M (Sigma) containing protease and phosphatase inhibitor cocktail (Sigma). Ten micrograms of cell lysates were separated on 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane . The blot was blocked with TBS-T  containing 4% bovine serum albumin (BSA) and then incubated with primary antibody solution; anti-phospho-tau Ser199 , anti-phospho-tau Ser396 , anti-β-tubulin , monoclonal antibody O-GlcNAc  and anti-Tau Ser262  at 4 °C overnight. The blots were developed with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody and enhanced chemiluminescence HRP substrate . Both bands (Tau-VN173 and Tau-VC155) intensities of the immunoblot signals from three independent experiments were quantified by ImageJ densitometry software . The average value was normalized with that of non-phosphorylated tau (TauSer262). Data are presented as normalized mean values ± standard deviation. The significance of the experiments was determined with paired t-test; *p < 0.05, **p < 0.01, ***p < 0.001.To quantify phosphorylation and g for 2 min at 4 °C and washed three times with 1 mL of PBS (pH 7.4). For the immunoblot analysis, immunoprecipitates were dissolved in 100 µL of Laemmli SDS sample buffer and heated for 5 min at 95 °C. Equal volume (20 µL) from all immunoprecipitated samples was loaded on 10% SDS-polyacrylamide gel.Anti-tau (TauSer262) antibody was used to immunoprecipitate tau proteins from tau-BiFC cell lysates. The anti-tau (TauSer262) antibody (2 µg) was pre-incubated with 50 µL (25 µL agarose/bed volume) of protein A-sepharose beads  for 1 hour with constant agitation at RT. The pre-incubated mixtures were gently centrifuged for 2 min and washed twice with PBS (pH 7.4). The tau-BiFC cell lysates (1 mg) were added to the pre-incubated mixtures and incubated overnight with constant agitation at 4 °C. The immunoprecipitated complexes were collected by centrifugation at 3000× O-GlcNAc in tau pathology and emphasize the importance of O-GlcNAcylation in controlling tau phosphorylation. For many years, tau phosphorylation has been considered the key mechanism initiating tau pathology. Here, we suggest the modification of the old paradigm: that tau phosphorylation is a secondary event caused by O-GlcNAc modification.In conclusion, our results indicate the protective role of


Microcebus murinus) from Madagascar present an excellent model for studies of torpor regulation in a primate species. In the present study, we analyzed the response of the insulin signaling pathway as well as controls on carbohydrate sparing in six different tissues of torpid versus aroused gray mouse lemurs. We found that the relative level of phospho-insulin receptor substrate (IRS-1) was significantly increased in muscle, whereas the level of phospho-insulin receptor (IR) was decreased in white adipose tissue (WAT) of torpid animals, both suggesting an inhibition of insulin/insulin-like growth factor-1 (IGF-1) signaling during torpor in these tissues. By contrast, the level of phospho-IR was increased in the liver. Interestingly, muscle, WAT, and liver occupy central roles in whole body homeostasis and each displays regulatory controls operating at the plasma membrane. Changes in other tissues included an increase in phospho-glycogen synthase kinase 3α (GSK3α) and decrease in phospho-ribosomal protein S6 (RPS6) in the heart, and a decrease in phospho-mammalian target of rapamycin (mTOR) in the kidney. Pyruvate dehydrogenase (PDH) that gates carbohydrate entry into mitochondria is inhibited via phosphorylation by pyruvate dehydrogenase kinase . In the skeletal muscle, the protein expression of PDK4 and phosphorylated PDH at Ser 300 was increased, suggesting inhibition during torpor. In contrast, there were no changes in levels of PDH expression and phosphorylation in other tissues comparing torpid and aroused animals. Information gained from these studies highlight the molecular controls that help to regulate metabolic rate depression and balance energetics during primate torpor.Gray mouse lemurs ( Cheirogaleus) and mouse lemurs (Microcebus), represent the only family among the primates with members capable of entering torpor/hibernation bouts Tb of ∼27 °C, whereas hibernation bouts can last up to 4 weeks with minimum Tb of 11.5 °C TbSeasonal hibernation and daily torpor are fascinating phenomena, whereby animals enter a state of hypometabolism during which most body functions are strongly suppressed One common theme in the field of hibernation is the involvement of signal transduction pathways, which restructure cellular metabolism to promote energy conservation and survival. The Akt pathway regulates events including apoptosis, protein synthesis, cell proliferation, and energy metabolism, and thereby lies at a junction between metabolism and cellular survival Zapus hudosnius), the percentage of PDH in the active form dropped from 15% in the heart and 29% in the kidney of euthermic animals to just 1% in animals that had been hibernating for 5–8 days PDK4 increased in skeletal muscle and white adipose tissue (WAT), together with increased amounts of PDK4 protein in the heart, skeletal muscle, and WAT of hibernating animals In anticipation of the winter season, seasonal mammalian hibernators enter a phase of hyperphagia resulting in weight gains of up to 40% as they fatten Although the behavioral and physiological adaptations supporting torpor in gray mouse lemurs have been well studied, the molecular mechanisms supporting daily torpor have yet to be fully elucidated. Since the insulin/IGF-1 pathway and PDH/PDK4 are central regulators of metabolism and survival, we investigated the phosphorylation status of key factors involved in these processes. Our results demonstrate that insulin/Akt signaling networks and PDH control are integral components of the hypometabolic state in lemur tissues.The insulin/Akt signaling pathway is regulated by posttranslational modifications such as protein phosphorylation at multiple distinct sites, which are indicative of the activity state of the target protein. As a result, antibodies that recognize these phosphorylation sites were used to monitor changes in the activity state of components of the insulin/Akt signaling pathway comparing control (aroused) and torpid conditions in gray mouse lemurs. To evaluate the response of Akt/mTOR pathway to daily torpor, the relative changes in levels of phosphorylated proteins were assessed in different tissues, including the skeletal muscle, heart, liver, kidney, brown adipose tissue (BAT), and white adipose tissue (WAT), using commercially-available multiplex panels. The phosphoproteins examined included IGF-1R (Tyr1135/Tyr1136), IR (Tyr1162/Tyr1163), IRS1 (Ser312), PTEN (Ser380), Akt (Ser473), GSK3α (Ser21), GSK3β (Ser9), TSC2 (Ser939), mTOR (Ser2448), p70S6K (Thr412), and RPS6 (Ser235/Ser236).P < 0.01), whereas phosphorylation levels of all the other targets remained unchanged (P < 0.05) (P < 0.05), whereas relative phosphorylation levels were comparable for the other proteins (P < 0.05)  was significantly higher during torpor, which is 4.89 ± 0.82-fold higher than in control aroused animals (nchanged . Among t < 0.05) . In the proteins . In the  < 0.05) .P < 0.05)  , which iP < 0.05) changed only in the skeletal muscle, but comparable in the other five tissues examined  was almost doubled in the liver during torpor , suggest2-terminal kinase (JNK), which has been shown to be involved in phosphorylation of IRS1 on Ser312 In muscle, the inactivation of IRS1 in torpid gray mouse lemurs should lead to decreased glucose uptake, glycogen synthesis, and/or rates of glycolysis. Evidence for reduced glycolytic flux and glycogen synthesis exists in mammalian hibernators TbInsulin signaling was also inhibited in WAT of gray mouse lemurs as indicated by a reduction in the phosphorylation state of the insulin receptor. Hibernators accumulate large reserves of triglycerides in WAT to fuel winter survival and also increase the proportion of polyunsaturated fatty acids to keep lipids fluid at low C. elegans and Drosophila melanogaster have single IGF-1/insulin-like receptors (DAF-2 in C. elegans and INR in D. melanogaster), and several homologs  are present in mammals daf-2 mutant showed extended lifespan, indicating that insulin/IGF-1 antagonizes longevity Interestingly, regulatory controls when present at the receptor level occur on IR rather than IGF-1R. While an array of genetic and environmental factors are thought to play roles in lifespan extension, crucial genes that are involved in modulating lifespan have been identified, including insulin receptors. Both Despite upstream changes, the relative phosphorylation levels of the downstream targets measured remained unaltered in muscle, WAT, and liver during torpor. This suggests that IR/IRS1 signaling during torpor may not be mediated by Akt-dependent signal propagation. While insulin/IR and IGF-/IGF-1R are strong activators of PI3K-mTOR, these signals also regulate Ras-ERK mitogen-activated protein kinase (MAPK) signal transduction , albeit One well-known role of Akt is the phosphorylation-dependent regulation of GSK3 The mTOR pathway has a major role in regulating cap-dependent translation In summary, while tissue-specific responses were observed, insulin/IGF1-dependent signal transduction was inhibited in muscle and WAT through regulatory events occurring on IRS1 and IR, respectively. As discussed above, inhibition of insulin signaling in muscle and WAT may act to balance the energy production-consumption equilibrium during the hypometabolic state. In contrast, activation of insulin-dependent signal transduction was indicated in the liver. However, the outcome may be Akt-independent in liver, since specific nodes of the Akt pathway failed to exhibit signs of regulation. In both the heart and the kidney, daily torpor led to inhibition of the mTOR pathway, suggesting repression of protein synthesis. In addition, elevation of GSK3α phosphorylation in the heart may contribute to the coordinated suppression of mitochondrial respiration during primate torpor. Finally, the protein expression of PDK4 increased significantly and this was matched with a significant increase in the phosphorylation of PDH at Ser300, suggesting the conversion of pyruvate to acetyl-CoA is significantly reduced in muscle during torpor. In conclusion, the present study provides insights into the regulation of PI3K/Akt signaling and fuel utilization in six tissues of the gray mouse lemur during torpor. Further studies may include carrying out deeper transcriptomic and proteomic analysis.−3 J/day versus 144 × 10−3 J/day) to enhance the depth of their torpor bouts. Control animals were capable of entering torpor and were fed ad libitum. Tb and locomotion were monitored and used to determine the state of torpor, which was assessed as a continuous reduction in Tb . Control animals were euthanized after arousal from a daily torpor bout (after spontaneous rewarming to 35–36 °C), whereas torpid lemurs were euthanized during a torpor bout . Samples of frozen tissues were packed in dry ice and air freighted to Carleton University where they were stored continuously at −80 °C until use.Standard procedures for holding, experimentation and sampling of gray mouse lemurs were used and all animal experiments were conducted by Dr. Martine Perret and the MECADEV team  as described by Giroud et al. 3VO4 and 10 mM ß-glycerophosphate) and protease  inhibitors. Samples were then incubated on ice for 30 min with occasional vortexing. Homogenates were centrifuged at 12,000 × g for 20 min at 4 °C and the supernatants were collected as total soluble protein lysates. Protein concentration of the lysates was determined using the Bradford assay  and then tissue extracts were standardized to 5 μg/μl and stored at −80 °C until further use.Protein extracts of tissue samples were prepared as per manufacturer’s instructions . Briefly, ∼50 mg aliquots of frozen tissue were weighed and homogenized 1:4 (w/v) with ice-cold lysis buffer  in a Dounce homogenizer with the further addition of phosphatase  and PDH  in the muscle, heart, liver, kidney, BAT, and WAT comparing control (aroused) and torpid mouse lemurs. The assay for the insulin/Akt Luminex® panel was performed following manufacturer’s instructions and all multiplex panels are subject to rigorous quality control and validation studies. Aliquots of protein homogenates (5 μg/μl) were combined with Milliplex MAP Assay Buffer 2 , with 17.5 μg total protein added per well. HeLa cell lysates treated with the dual specificity lambda phosphatase  were used as a negative control, while positive controls included insulin-stimulated HepG2 cells  and MCF7 cells stimulated with IGF-1 . The same protocol was followed for the PDH panel except for the use of Assay Buffer 1  with 10.5 μg protein added per well. For the PDH kit, HepG2 cell lysates catalog No. 47-234) were used as a positive control, whereas HepG2 cell lysates treated with dichloroacetate (DCA) , a PDK inhibitor, served as a negative control. Positive and negative controls were prepared as per manufacturer’s instructions.For the Akt panel, premixed phosphoprotein beads  for all the protein targets were provided as a 20 × stock, which were sonicated for 15 s, vortexed for 30 s, diluted to 1 ×, and vortexed once more for 15 s. After calibration with Assay Buffer, the bead mixture was gently mixed with a pipette and sonicated for 10 s and then 25 μl 1 × phosphoprotein beads was added to each well. Following the addition of the phosphoprotein beads, equal amounts of diluted cell lysate was added to each sample well, whereas HeLa, HepG2, and MCF7 cell lysates were added to control wells. The sample and control wells were incubated overnight at 4 °C on a plate shaker (600–800 rpm) protected from light. After removal of the lysate by vacuum filtration, all wells were washed twice with Assay Buffer 2 . Afterward, 25 μl 1 × biotin-labeled detection antibodies was added to each well and the filter plate was incubated on a plate shaker for 1 h at room temperature. Following removal of the antibody solution by vacuum filtration, the filter plate was washed twice (as above). Streptavidin–phycoerythrin  was then diluted in Assay Buffer and aliquoted into wells (25 μl). Following incubation on a plate shaker for 15 min, 25 μl of Amplification Buffer  was added to each well and incubated for 15 min. The SAPE/Amplification Buffer was then removed by vacuum filtration. The beads were resuspended in 150 μl Assay Buffer 2 and data acquisition was performed on a Luminex 100 instrument  with Milliplex Analyst software . Equipment settings were as follows: 50 events per bead, sample size of 100 μl, and gate settings of 8000–15,000.The PDH kit used magnetic beads , which were provided as a 1 × stock. Beads were sonicated for 30 s and vortexed, and 25 μl was added to each well following the addition of 25 μl each of Assay Buffer 1, lysate controls, or samples. The plate was incubated on a plate shaker (600–800 rpm) protected from light for 2 h at room temperature. The well contents were removed and the plate was washed 3 times using a magnetic plate washer. Afterward, 50 μl of detection antibody was added and incubated on a plate shaker at room temperature for 1 h before the washing step. Next, 50 μl SAPE was added to each well, incubated on a plate shaker at room temperature for 30 min. Finally, the beads were resuspended in 100 μl Sheath Fluid after wash and data acquisition was performed as described above.The relative protein expression of PDK4 was determined using a BlueGene Elisa kit  purchased from Life Sciences Advanced Technologies  following the manufacturer’s instructions. Aliquots of 50 μl of sample were added to wells of a microtiter plate that were pre-coated with antibodies, whereas negative control wells received 50 μl of phosphate-buffered saline (pH 7.2). Next, 5 μl of the supplied balance solution was added to each sample, followed by 100 μl of supplied antibody conjugate. Samples were covered and incubated for 1 h at 37 °C before the wells were washed with the supplied 1 × wash buffer (5 times). Next, 50 μl each of substrate A and substrate B were added to each well, covered, and incubated for 15 min at room temperature in the dark. Finally, 50 μl of stop solution was added to each well and the optical density at 450 nm was determined using a Thermoscan microplate reader.n = 4) normalized to control (aroused) values. Statistical analysis was performed using SigmaPlot statistical package (v.12) software. The two-tailed Student’s t-test was employed to assess differences between samples from aroused and torpid animals and difference was considered significant with P < 0.05 or P < 0.01.Bead- and ELISA-based assays used the median fluorescence intensity (MFI) and mean absorbance, respectively, to determine the relative protein levels. All numerical data are expressed as mean ± SEM (All authors contributed to the conception and design of the project and to the editing of the manuscript. MP and FP carried out the animal experiments; SNT, JZ, KKB, and CWW conducted biochemical assays. Data analysis and assembly of the draft manuscript were carried out by SNT, JZ, and KBS. All authors read and approved the final manuscript.The authors have declared no competing interests.


Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides  were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC50: 0.044 μmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit  and β-subunit . The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and β-subunits. In addition, the LRY sequence was found in the β-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and β-subunits of PE , and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities . Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins.We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse,  Suetsuna et al. also reported the antihypertensive effect of U. pinnatifida peptide on blood pressure in spontaneously hypertensive rat (SHR) [et al. screened ACE inhibitory activities of methanol and aqueous extracts prepared from twenty-six red algae at the coast of Jeju Iland in Korea [Lomentaria catenata showed the strongest ACE inhibitory activity and Lithophyllum okamurae recorded the second highest activity. Remarkable activities in the methanol extracts at 70 °C were observed in Grateloupia filicina, G. lanceolata and Sinkoraena lancifolia, however no significant activities were found in their aqueous extracts. He et al. also measured ACE inhibitory activities of the 48 hydrolysates from marine protein materials [Polysiphonia urceolata was relatively high. However, they did not study in detail, such as the structure of ACE inhibitory peptides and the original protein of inhibitory peptides.Angiotensin I converting enzyme ACE: EC 3.4.15.1) is physiologically important in the regulation of blood pressure catalyzing the production of angiotensin II and the destruction of bradykinin . Therefo.4.15.1 iin Japan . They isat (SHR) . They isin Korea . Among aaterials , and thePalmaria palmata) is a red alga distributed mainly in high-latitude coastal areas, and is popular in Ireland and Atlantic Canada as a food and a source of minerals. Recently, it was reported the dulse protein hydrolysates show inhibitory effects for renin [Laminaria sp.) farming areas in Hokkaido Prefecture, because it slows the growth of Kombu , phycocyanin (PC) and allophycocyanin (APC) that are divided on their spectral properties  ,15,16. AThen, we measured ACE inhibitory activity of dulse proteins and its hydrolysates. The dulse proteins indicated slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis a. The AC50: 0.044 μmol) has remarkably high activity, followed by VYRT (IC50: 0.14 μmol)  [The dulse protein hydrolysates were separated by reversed-phase high-performance liquid chromatography (HPLC) a. The el14 μmol) . Althougide (IW) , it is eide (IW)  which isAs described in the preceding section, nine ACE inhibitory peptides were isolated from the dulse protein hydrolysates. Next, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides.As shown in Escherichia coli Rosetta 2 (DE3)  in  2 (DE3) a, and thDulse was harvested off Usujiri, Hokkaido, Japan in February, and the samples were stored at −20 °C until use.l-histidyl-l-leucine (Hip-His-Leu), thermolysin (EC 3.4.24.27) from Bacillus thermoproteolyticus, pepsin (EC 3.4.23.1) from porcine stomach, trypsin (EC 3.4.21.4) from bovine pancreas and chymotrypsin (EC 3.4.21.1) from bovine pancreas were purchased from Wako Pure Chemical . Synthetic peptides (purity: 99.4%–99.8%) were purchased from Medical Biological Laboratories Co. . All other reagents were purchased from Wako Pure Chemical Industries .ACE from rabbit lung was purchased from Sigma Chemical Co. . Hyppuryl-v/w of distilled water at 4 °C for 7 h. The extract was centrifuged  at 4 °C, 15,000 g for 10 min, and then the supernatant was used as dulse proteins. The dulse proteins were hydrolyzed by 1.0 wt % of thermolysin at 70 °C for 3 h, and the reaction was ended by heat treatment at 100 °C for 5 min. Subsequently, the solution was centrifuged at 4 °C, 15,000 g for 10 min. The supernatant was dried by lyophilisation into dulse hydrolysates.The frozen samples were lyophilized and ground into a fine powder by Wonder Blender WB-1 . Proteins were extracted from the powder by adding 20 g for 10 min. The supernatants were dried by lyophilisation.The dulse hydrolysates were dissolved in distilled water and the solution was adjusted to pH 2.0. The hydrolysates were digested by 1.0 wt % of pepsin at 37 °C for 3 h (pepsin digests). After the reaction, the pepsin digests were adjusted to pH 8.0. Then, 1.0 wt % of trypsin was added to the pepsin digest and the mixture was incubated at 37 °C for 3 h (pepsin-trypsin digests). Subsequently, the pepsin-trypsin digests were treated by 1.0 wt % of chymotrypsin at 37 °C for 3 h (pepsin-trypsin-chymotrypsin digests). The digests were boiled for 5 min to inactivate the enzymes, and then centrifuged at 4 °C, 15,000 The dulse hydrolysates were dissolved in ultra pure water containing 0.1% trifluoroacetic acid (TFA) and applied to sequential filtration by Millex-GV (pore size: 0.22 μm) and Millex-LG (pore size: 0.20 μm). Peptides in the filtrate were isolated by reversed-phase HPLC with a Mightysil RP-18GP column (4.6 × 150 mm)  using a linear gradient of acetonitrile (1%–20%) containing 0.1% TFA at a flow rate of 1.0 mL/min.50 as an absolute quantity of peptide to inhibit 50% of 1.0 U ACE.ACE inhibitory assay was carried out according to the method of Cheng and Cushman  with somThe visible ray absorption spectrum of dulse protein extracts analyzed by a spectrophotometer .Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using a 0.1% SDS-13.75% polyacrylamide slab-gel by the method of Laemmli (1970) . The gelThe amino acid sequences of dulse ACE inhibitory peptides were analyzed by Edman degradation method using a protein sequencer  and Matrix Assisted Laser Desorption/Ionization Time Of Flight Tandem Mass Spectrometry (MALDI-TOF/MS/MS) method using a 4700 Proteomics Analyzer with Denovo Explorer software .Nco I site which includes an ATG sequence being applicable to a translational start codon was introduced to 5′-terminus of each cDNA, while a BamH I site was introduced to 3′-terminal. The modified cDNAs encoding α- and β-subunits of dulse PE were digested with Nco I and BamH I and were inserted into the expression plasmid pET-16b . The recombinant subunits (rPEα and rPEβ) were expressed in Escherichia coli Rosetta 2 (DE3)  by induction with isopropyl-β-D(−)-thiogalactopyranoside . The rPEα and rPEβ produced as inclusion bodies were dissociated with 8 M urea and 10 mM 2-mercaptoethanol. The dissolved solutions were centrifuged at 4 °C, 10,000 g for 10 min and the supernatants were renatured by dialyzing against 10 mM Tris-HCl buffer (pH 8.0) into dulse rPEα and rPEβ (Bacterial expression of recombinant α- and β-subunits of dulse PE was carried out according to the method of previous paper  with somand rPEβ a.


The experimentally identified sulfation, nitration and phosphorylation sites were collected and integrated with reliable predictions to perform large-scale analyses of in situ crosstalks. From the results, we observed that the in situ crosstalk between sulfation and nitration is significantly under-represented, whereas both sulfation and nitration prefer to co-occupy with phosphorylation at same tyrosines. Further analyses suggested that sulfation and nitration preferentially co-occur with phosphorylation at specific positions in proteins, and participate in distinct biological processes and functions. More interestingly, the long-term evolutionary analysis indicated that multi-PTM targeting tyrosines didn't show any higher conservation than singly modified ones. Also, the analysis of human genetic variations demonstrated that there is no additional functional constraint on inherited disease, cancer or rare mutations of multiply modified tyrosines. Taken together, our systematic analyses provided a better understanding of the in situ crosstalk among PTMs.Recent studies have indicated that different post-translational modifications (PTMs) synergistically orchestrate specific biological processes by crosstalks. However, the preference of the crosstalk among different PTMs and the evolutionary constraint on the PTM crosstalk need further dissections. In this study, the  The first question is whether PTMs recognizing the same type of amino acid residues are significantly co-occurred. For this issue, Wang et al. totally characterized 141 O-GlcNAcylation and >350 phosphorylation sites from the mitotic spindle and midbody samples of human HeLa cells, and observed that both PTMs extensively co-occupy at same serine/threonine (S/T) residues or adjacent regionset al. systematically identified ~1750 O-GlcNAcylation and ~16,500 phosphorylation sites from mouse synaptosomes, and revealed that only 8% (135) of O-GlcNAcylation sites are also phosphorylatedet al. detected that only ~22% of succinylation sites were also acetylated at the same lysines from mouse embryonic fibroblasts (MEFs)et al. demonstrated that succinylation and acetylation extensively co-occupy at same residues from prokaryotes and eukaryotes, whereas 66% of E. coli, 56% of S. cerevisiae, and 57% of mouse liver succinylation sites are overlapped with acetylationin situ crosstalk may not only be dependent on the PTM types, but also exhibit a tissue-specific manner. The second and more important question is whether amino acids targeted by multiple PTMs undergo additional evolutionary pressure against singly modified sites. Because the co-occurrence of O-GlcNAcylation and phosphorylation are similar to be random, Trinidad et al. suggested that there is little or no evolutionary pressure for the in situ crosstalk between two PTMsin situ crosstalk of protein lysine modifications (PLMs), although the long-term phylogenetic analysis among multi-species revealed multi-PTM targeting sites are only slightly more conserved than singly modified lysines, the short-term evolutionary results demonstrated that multiply modified lysines dramatically enriched more human disease-associated and rare variationset al. concluded that mutations of lysines with multi-PTMs undergo much greater purifying selection against singly modified lysine residuesRecently, the in situ crosstalk truly occurs among the three PTMs. For example, both phosphorylation and nitration can co-occur at Y125, Y133 and Y136 of α-synuclein (UniProt ID: P37840)30v-Src at Y87None of two questions mentioned above have been fully addressed, while different viewpoints were raised for both problems. To further clarify the controversial issues, more efforts should be taken. Recent progresses in the identification of substrates and sites for tyrosine modifications, such as sulfation242527http://tsp.biocuckoo.org), which exhibited superior performance than other existed tools by comparison. Together with a nitration sites predictor of GPS-YNO2vice versa. Our results demonstrated the in situ crosstalk between sulfation and nitration was significantly under-represented, and two PTMs prefer to regulate different functions. Moreover, by predicting potential sulfation and nitration sites in known phosphorylated substrates, we detected that a considerable proportion of tyrosine phosphorylation sites (24.5%) might be modified by either sulfation or nitration, and both sulfation and nitration preferentially target phosphorylated tyrosines rather than non-phosphorylated sites by 1.71- and 1.45-fold, respectively. Again, statistical results suggested sulfation and nitration prefer to crosstalk with phosphorylation in regulating potentially distinct biological processes and functions. Further analyses of the sequence and structure preferences revealed that different types of tyrosine modifications prefer to co-occur at distinct structural positions in proteins. Interestingly, whether PTMs prefer to in situ crosstalk or not has no correlation with the evolutionary constraint on multiply modified tyrosines. In our results, although three types of tyrosine modifications have distinct preferences for the in situ crosstalk, the long-term evolutionary analysis across eight vertebrate species revealed that multiply modified tyrosines are not more conserved than unmodified ones. In addition, by mapping all known or predicted tyrosine modification sites to human genetic variations, we observed that multiply modified tyrosines didn't significantly enrich inherited disease, cancer or rare mutations. Taken together, our results suggested that there is no functional constraint on multiply modified tyrosines, and the in situ crosstalk of tyrosine modification does not need additional natural selection.From the scientific literature, public databases and our previous studiesFor systematically analyzing the tyrosine sulfation and its crosstalk with other tyrosine modifications, a prerequisite is to establish a comprehensive and reliable dataset. However, the experimental identification of sulfation substrates is still labor-intensive and time-consuming, while only 273 known sulfation sites were collected. Thus, computational prediction of sulfation sites from protein primary sequences can serve as an alternative solution. Before developing the tool, we first analyzed the sequence profile of 202 non-redundant sulfation sites by WebLogoSp values of 85%, 90% and 95%, respectively , 0.9527 (4-fold), 0.9595 (6-fold), 0.9547 (8-fold) and 0.9563 (10-fold), respectively . Since tectively .Sn values for GPS-TSP, Sulfinator and SulfoSite at the equal level of Sp values  was much greater than SulfoSite (69.73%)  . In thisth, 2012) association files from the GOA database at the EBI (http://www.ebi.ac.uk/goa)Besides sulfation, protein tyrosine nitration (PTN) is also an important PTM, and predominantly implicated in a variety of fundamental processes such as RNA splicing, mRNA processing and translation272425p-value < 10−11) and nitrated  proteins. For the sulfated substrates, the top five most enriched biological processes are chemokine-mediated signaling pathway (GO:0070098), chemotaxis (GO:0006935), inflammatory response (GO:0006954), elevation of cytosolic calcium ion concentration (GO:0007204), and cell adhesion (GO:0007155), which are consistent with previously experimental observations2425With the hypergeometric distribution2) testp-value < 10−7). Indeed, sulfation was found to be preferentially involved in modifying membrane and extracellular proteins, while nitration prefers to attack substrates in cytosol  of 1.71 and 1.45, respectively  or nitration  at the same residues, separately. We only considered the predicted sulfated and nitrated proteins, whose sites were predicted from known phosphorylation sites. Clearly, sulfation prefers to in situ crosstalk with phosphorylation in a variety of biological processes, such as peptidyl-tyrosine phosphorylation (GO:0018108), positive regulation of phosphatidylinositol 3-kinase activity (GO:0043552), and transmembrane receptor protein tyrosine kinase signaling pathway (GO:0007169) , peptidyl-tyrosine phosphorylation (GO:0018108), and response to unfolded protein (GO:0006986)  pathwaysin situ crosstalks. The results suggested that the sulfation-phosphorylation crosstalk is significantly enriched in pathways of leukocyte transendothelial migration (hsa04670), tight junction (hsa04530), and adherens junction (hsa04520)  . In cont0006986) . By compsa04520) , whereassa04520) . Taken tin situ crosstalks. The experimentally identified PTM tyrosines including 273 sulfation, 1050 nitration and 24,242 phosphorylation sites were employed for the analysis. Furthermore, 17,015 and 25,306 predicted sulfation and nitration sites in phosphorylated substrates were also analyzed, while 2913 and 3689 phosphorylation sites were predicted to be sulfation- and nitration-phosphorylation crosstalk sites. With these datasets, we first analyzed the position distributions of modified tyrosines. In the result, known sulfation sites preferentially occur at N-terminal or C-terminal but not middle of proteins, whereas predicted sulfation sites slightly prefer to locate at C-terminal, as well as other types of tyrosine modifications and their crosstalks , and the ratio of modified/unmodified site (E-ratio) was only 1.04 , and the result was also consistent with a previous reportp-value < 0.01, E-ratio = 0.73). In this regard, the conservation of distinct tyrosine modifications is quite different. Adding more or less species for the evolutionary analysis didn't influence the results, as well as changing the YRCS threshold from 5/8 to 1.In our results, phosphorylation sites are slightly more conserved than unmodified tyrosines . Although nitration-phosphorylation crosstalk sites look like to be significantly more conserved than phosphorylation sites, the E-ratio of crosstalk/phosphorylation sites was only 1.02 . In this regard, the evolutionary pressure on tyrosines with nitration and phosphorylation is extremely weak against phosphorylation sites, and no signature of additional natural selection was observed for the sulfation-phosphorylation crosstalk. This might be due to tyrosine residues undergo stronger purifying selection than other types of amino acids, and both phosphorylated or non-phosphorylated tyrosines slowly evolve with a similar rate40YRCS threshold didn't influence the results (Data not shown).Furthermore, we analyzed the conservation of sulfation-phosphorylation and nitration-phosphorylation crosstalk sites against known phosphorylation sites . Unexpecin situ crosstalks of tyrosine modifications for the specific sites, but no additionally functional constraint was observed for the crosstalks.As previously describedAccumulative studies have exhibited the ubiquity and importance of PTM crosstalks5677510111219303132vice versa. The statistical results demonstrated that in situ crosstalk between sulfation and nitration was significantly under-represented . The result seems conflicting with known evidence that sulfation exclusively occurs in secreted pathways because two tyrosylprotein sulfotransferases, TPST-1 and TPST-2, locate in the trans-Golgi network, and almost all of sulfated proteins were identified in extracellular or membrane-related regions2425p-value > 0.05).By computationally predicting sulfation and nitration sites in known tyrosine phosphorylated substrates, we observed that up to 24.5% of known phosphorylation sites might also be modified by either sulfation or nitration, while statistical results suggested that both sulfation and nitration prefer to yrosines . Interesyrosines . Notably2) is ~46 Da, while the phosphate and sulfate groups are 79.9663 and 79.9568 Da, respectivelyin situ crosstalk between sulfation and phosphorylation. Since currently systematical profiling of sulfation sites is yet to be carried out, the predictions based on the limited small-scale experimental data could serve as a start for the studies of crosstalks between sulfation and phosphorylation. In addition, because the large-scale data set of tissue-specific sulfation or nitrations sites is still not available, whether the preference of in situ crosstalks of tyrosine modifications will be changed in different tissues or organs remains to be dissected. However, our current results still clearly demonstrated that different types of tyrosine modifications can have distinct preferences for the in situ crosstalk.Undoubtedly, our results will be helpful for further experimental consideration. For example, the molecular masses of a nitro group  for unmodified, singly, doubly and triply modified lysines are 88.8%, 91.1%, 93.8% and 100%, respectivelyFor the short-term evolutionary analysis, Gray in situ crosstalk of O-GlcNAcylation and phosphorylation because of nearly random co-occurrencein situ crosstalk or not, little or no additional evolutionary pressure exists on residues with multiple PTMs against singly modified sites.More interestingly, although no evolutionary pressure was detected for the http://tsp.biocuckoo.org/database.php). The protein sequences were retrieved from the UniProt databasehttp://weizhong-lab.ucsd.edu/cd-hit/) to cluster the protein sequenceshttp://tsp.biocuckoo.org/down.php.By searching the PubMed with multiple keywords such as “tyrosine sulfation”, “sulfation” and “sulfated”, we collected 273 experimentally identified tyrosine sulfation sites in 171 proteins, and integrated the dataset into the DOSS database , sensitivity (Sn), specificity (Sp), and Mathew Correlation Coefficient (MCC) were adopted and defined as below27To evaluate the performance of GPS-TSP for the prediction of tyrosine sulfation sites, four measurements of accuracy (The leave-one-out (LOO) validation and 4-, 6-, 8- and 10-fold cross-validations were carried out. The Receiver Operating Characteristic (ROC) curves and AROC (area under ROC) values were also calculated.http://www.cbs.dtu.dk/services/NetSurfP/), which calculates a probability score for each of three major types of secondary structures including α-Helix, β-Strand and Coil, respectivelyhttps://www.strubi.ox.ac.uk/RONN)To assess the position distribution of modified tyrosine residues in protein sequences, we equally separated a protein into three fragments, namely, N-terminal, Middle and C-terminal, and directly counted the number of residues in each part. The secondary structures were predicted NetSurfP server ver. 1.1 , G. gallus (CHICK), R. norvegicus (RAT), M. Musculus (MOUSE), C. familiaris (CANFA), B. Taurus (BOVIN), and S. scrofa (PIG) from the UniProt database http://www.clustal.org/omega/)YRCS of can be calculated as below: To analyzed the conservation of modified and unmodified tyrosines in N is the number of sequences with the maximum branch length (MBL), which is the maximum branch distance between any two species that contain a conserved tyrosine. The YN is the number of tyrosines appears in the column. The tyrosine residues with RCSY = 1 were regarded as conserved tyrosines. The change of the RCSY threshold (0.625 ~ 1) didn't influence the final results. The species tree of the eight organisms were taken from the Interactive Tree Of Life The th, 2012) in the NCBI ftp server, including 78,827 records with 15,641 non-redundant missense mutations. Moreover, we downloaded 73,270 cancer-associated variants from the CanProVar database st 2013, phase I release v3)We downloaded 153,839 human disease-associated single nucleotide polymorphisms (SNPs) from the GWASdbY.X. designed and supervised experiments. Z.P. and Z.L. performed experiments and data analysis. H.C., Y.W., T.G., S.U. and J.R. contributed to data analysis. Y.X., Z.P. and Z.L. wrote the manuscript with contributions of all authors.Supplemental experimental procedures and supplemental tables


Panthera onca) are species of conservation concern because they are suffering population declines and are keystone species in their ecosystems. Their large area requirements imply that unprotected and ever-increasing agricultural regions can be important habitats as they allow connectivity and dispersal among core protected areas. Yet information on jaguar densities across unprotected landscapes it is still scarce and crucially needed to assist management and range-wide conservation strategies. Our study provides the first jaguar density estimates of Colombia in agricultural regions which included cattle ranching, the main land use in the country, and oil palm cultivation, an increasing land use across the Neotropics. We used camera trapping across two agricultural landscapes located in the Magdalena River valley and in the Colombian llanos  and classic and spatially explicit capture-recapture models with the sex of individuals as a covariate. Density estimates were 2.52±0.46–3.15±1.08 adults/100 km2 in the Magdalena valley, whereas 1.12±0.13–2.19±0.99 adults/100 km2 in the Colombian llanos, depending on analysis used. We suggest that jaguars are able to live across unprotected human-use areas and co-exist with agricultural landscapes including oil-palm plantations if natural areas and riparian habitats persist in the landscape and hunting of both jaguar and prey is limited. In the face of an expanding agriculture across the tropics we recommend land-use planning, adequate incentives, regulations, and good agricultural practices for range-wide jaguar connectivity and survival.Large carnivores such as jaguars ( Due to their charisma and functional role in maintaining ecosystem integrity and services ,2 large Canis lupus), pumas (Puma concolor), leopards (Panthera pardus), and jaguars are able to live in human use landscapes [Abundance, density, and distribution estimates are key information for conservation and management strategies, and when they refer to modified areas they can provide valuable information on species tolerance limits . Becausendscapes ,19–21.The jaguar is the only living representative of the genus Panthera found in the New World and it is the largest cat existing in the Americas . It rangElaeis guineensis) landscapes. The latter are particularly of concern as a driver of impoverished habitat with unknown survival value for jaguars [As for the other large carnivores, protected areas are too few in number for long-term jaguar conservation, which requires a landscape approach with both protected and unprotected lands ,24. Howe jaguars , 27,28.Colombia is extremely important for range-wide jaguar conservation and connectivity due to its position between Central and South America . In ColoThe study was conducted across different private properties in the Departments of Santander and Casanare, Colombia. It did not involve manipulation or handling of any living organism as we used non-invasive methods for data collection. Following regulations from the Colombian Government, a study of such nature does not require permits or approval from an Institutional Animal Care and Use Committee or equivalent committee. Each landowner was consulted and personally granted us permission to access and collect data on his or her property.We conducted the study at two sites in Colombia . Site-I Site-II is located in the Orinoco River basin in the llanos region and in the Department of Casanare (5.9552203N -71.4833672E to 6.0812814N -71.2976157E). This area is naturally characterised by seasonally flooded tropical savannahs bisected by riparian forests, and the dominant land use is extensive cattle ranching with introduced grasses . Mean an2 per year [Jaguar prey species have been historically hunted at both sites and hunting still occurs for subsistence and commercial reasons . Killingper year . Widesprper year ,39.Camera trapping surveys were done between April and August 2014 at Site-I and in April-May 2014 at Site-II. We employed a camera design which is recommended for jaguar studies ,40,41 an2 (minimum convex polygon), while a continuous design and 53 station across 151.3 km2 at Site-II , where g0 is the probability of capture when the trap is located exactly at the centre of the home range, and sigma (σ) is a spatial parameter related to home range size [The most commonly used function and the one we also used is the half-normal detection function, which describes the probability of capture (P) of an individual i at a trap j as a function of distance (d) from the activity centre of the individual to the trap as follow: Pij = g0 exp , and more than 30 min apart for all other species [We calculated capture rates for jaguar prey species at the two sites using the total number of independent capture events of each species divided by the number of trap-nights and expressed as records per 100 trap nights ,58. Inde species ,59–61. I species ,62 henceWe recorded seven females (49 events) and three males (39 events) at Site-I and two females (8 events), three males (57 events), and one adult individual of unknown sex at Site-II . Four of2) at Site-I while N = 6.00 ± 0.00 (6.00–6.00) and density = 1.12 ± 0.13 (95% CI: 0.86–1.38) (N/100 km2) at Site-II.The best CR model for Site-I was Mh (AIC = 130.2), but M0 (AIC = 130.6) was also strongly supported (ΔAIC<2); whereas for Site-II the best CR model was Msex (AIC = 75.4) followed by Mh (AIC = 76.6), which also had strong support (ΔAIC<2) . Both suThe best SECR model (AIC = 924.2) for Site-I allowed g0 to vary with sex but had a fixed σ (SECR.sex.g0), while for Site-II the best model (AIC = 612.5) allowed both parameters to vary with sex (SECR.sex). However, SECR.sex and SECR.sex.g0 also had strong support (ΔAIC<2) for Site-I and Site-II respectively .2 and 102.1 km2 at Site-I and Site-II respectively, and to male home range estimates of 52.8 km2 at Site-I and 38.3 km2 at Site-IITherefore we report density estimates and parameters for both SECR models at both sites . Under tDasyprocta punctuata) and black agouti (Dasyprocta fuliginosa) being the most frequently captured species at Site-I and 2 respectively , peccaries, giant anteaters (Mymecophaga tridactyla), and capybaras on which jaguar depend in other regions [Caiman crocodilus) and turtles (Podocnemis and Trachemys sp) as found elsewhere [Carnivore densities are highly dependent on the prey base available ,70 and l regions –61 were  regions . It is tlsewhere .Site-II is part of the Llanos-Amazon JCU , indicatLower jaguar numbers in the llanos could also be due to retaliatory killing following livestock predation. Incidents of jaguar predation on livestock do occur ,73 howevUsually more males than females are recorded in camera trap studies because males tend to move more and have larger home ranges . This is2) and low detection probabilities at home range center (g0 = 0.01) the density bias for polygons like ours, ca. 150km2, is less than 10% [Our survey effort (47–53 camera stations) was more comprehensive than most jaguar studies, as only 15% of jaguar studies reviewed by Tobler and Powell  used > 4than 10% .2), whereas for males it was the opposite. Female home ranges are usually smaller than those of males’ [Jaguar home ranges in wetter habitats vary greatly: some studies –81 estimf males’ ,80,83. WBecause of sex-specific detection probabilities and home range sizes, including sex as a covariate reduces the bias in density estimates and produced better SECR models at both sites. However the best CR models at Site-1 did not include sex as a covariate and it could be because CR models do not include spatial behaviour, hence reducing differences between the sexes. Ultimately, with small sample sizes, partitioning the data into sex specific group is a trade-off between bias and precision. We also recommend larger camera polygons than ours to increase the number of individuals captured and achieve more accurate density estimates.We concur with other authors ,44,53, aIn the case of wide-ranging species such as large carnivores, human-use areas are important habitats for connectivity and dispersal between core protected areas as well as for resident and breeding populations ,19,89. TAs agriculture and oil palm cultivation continue to expand across the tropics they need to be integrated into range-wide jaguar conservation strategies. For long-term jaguar conservation it is key to engage landowners, implement land-use plans in both regions to maintain natural habitats in the landscape, and establishing further oil palm plantations in already disturbed areas, as identified by García-Ulloa et al. . Across S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S4 Appendix(DOCX)Click here for additional data file.S5 Appendix(DOCX)Click here for additional data file.


Spike-timing-dependent plasticity (STDP) has been well established between excitatory neurons and several computational functions have been proposed in various neural systems. Despite some recent efforts, however, there is a significant lack of functional understanding of inhibitory STDP (iSTDP) and its interplay with excitatory STDP (eSTDP). Here, we demonstrate by analytical and numerical methods that iSTDP contributes crucially to the balance of excitatory and inhibitory weights for the selection of a specific signaling pathway among other pathways in a feedforward circuit. This pathway selection is based on the high sensitivity of STDP to correlations in spike times, which complements a recent proposal for the role of iSTDP in firing-rate based selection. Our model predicts that asymmetric anti-Hebbian iSTDP exceeds asymmetric Hebbian iSTDP for supporting pathway-specific balance, which we show is useful for propagating transient neuronal responses. Furthermore, we demonstrate how STDPs at excitatory–excitatory, excitatory–inhibitory, and inhibitory–excitatory synapses cooperate to improve the pathway selection. We propose that iSTDP is crucial for shaping the network structure that achieves efficient processing of synchronous spikes. Activity-dependent plasticity of synaptic connections between neurons is crucial for cortical circuit development and memory  that consists of a single postsynaptic Poisson neuron excited by excitatory and inhibitory spike trains Figure . Followin Figure . All Synwe is modified by a quantity that depends on the current value of we and the spike-time difference Δ t = tpre − tpost:Excitatory weights are modified by a temporally Hebbian eSTDP rule , the difference LTP—LTD is set equal. Additionally, τipre = τipost for all iSTDP.The right and left sides of the iSTDP window can be either LTP or LTD depending on the sign of To stabilize iSTDP, each inhibitory weight is decreased by a small amount α for every presynaptic spike .In summary, the simultaneous strengthening of correlated excitatory and inhibitory inputs  should occur when iSTDP has an anti-Hebbian LTP component in this simple circuit (anti-Hebbian and symmetric iSTDP), and when inhibitory input spikes arrive after postsynaptic spikes with a sufficiently large Next, we verify our theoretical predictions for the SFC with simulations using a LIF neuron . In contrast to Figure detailed balance .A typical example of synaptic weight evolution with anti-Hebbian iSTDP is shown in Figure d , together with the shape of the iSTDP window. Note that we set α such that weights from background inputs remain weak.As in our analytical approach, we compared the effect of different iSTDP windows on the inhibitory weights of an inhibitory–excitatory pathway with correlated spike-times. The comparison of the Hebbian, anti-Hebbian, and symmetric iSTDP windows agrees with the theoretical predictions of expected drift in weights in Figure in , but not significantly on the delay d. The matching is not linear and depends on the learning window.The potentiation of excitatory and inhibitory weights with both anti-Hebbian and symmetric iSTDP exhibits a balance between correlated excitation and inhibition, as illustrated in Figure In summary, we find that simulations with the LIF neuron confirm the theoretical results with the Poisson neuron. Detailed balance in the weights from the correlated pathway can arise if the iSTDP window is anti-Hebbian or symmetric, but not if it is Hebbian.While detailed balance between excitatory and inhibitory weights can arise through anti-Hebbian or symmetric iSTDP Figure , anti-Hein governs the temporal width of input spike volleys, we evaluate τout for the postsynaptic response  is compared to two control conditions:The PSTH obtained from the simulations with specific inhibition the inhibitory weights are swapped to obtain unspecific inhibition (leading to the inhibitory inputs are omitted (black curve).in = 2.12 in the SFC with anti-Hebbian iSTDP, rout = 2.21 sp/s for specific inhibition, 2.23 sp/s for unspecific inhibition, and 38.94 sp/s for excitation only.The three conditions are characterized by different mean firing rates. For τout between specific inhibition and the unspecific inhibition control represents the particular contribution of detailed balance on output response sharpening. Likewise, removing the inhibitory inputs from the circuit and taking the difference with the unspecific inhibition control should reveal the response sharpening due to the general presence of inhibition . To evaluate the relative change of spiking probability induced by the input stimuli, we normalize the PSTH with respect to the mean postsynaptic firing rate . This gives a signal/noise ratio (SNR) for the output spikes following an event in this detection task. As can be seen in Figure d as in Figure in = 0.71–5.66 ms, in the range of the delay d, as illustrated in Figure d prevent a proper weight structure from developing with anti-Hebbian iSTDP, thus the sharpening of the response fails  component of iSTDP is crucial to achieve a balance between excitatory and inhibitory weights given correlated inputs, and assuming an inhibitory delay in the order of a few milliseconds. Moreover, interneurons can be recruited by Hebbian eSTDP in a self-organized fashion to develop inhibition through iSTDP onto output neurons. By selecting adequate delays in this disynaptic inhibition scheme, iSTDP sharpens the output firing response, enhancing the propagation of spike volleys.We investigated how the interplay between eSTDP and iSTDP shapes the excitatory and inhibitory weight distributions. In our model, correlations in inhibition follow correlations in excitation by a delay of up to 10 ms Figures , and 4, There is, to our knowledge, currently no experimental evidence of this kind of anti-Hebbian iSTDP. Some studies show evidence of anti-Hebbian STDP in excitatory synapses in the electric fish , the excitation–inhibition structure in synaptic weights arises from the recruitment of interneurons: specialization due to eSTDP, followed by the strengthening of inhibition onto output neurons induced by iSTDP. Hebbian eSTDP provides a sufficient degree of temporal correlation between the selected excitatory and inhibitory pathways onto the output neuron. This correlation is essential for anti-Hebbian iSTDP to select weights from adequate interneurons, whose firing is correlated with the output neuron.anti-correlated. We expect that Hebbian iSTDP for the inhibitory synapses from the interneurons would be an interesting choice in this case, to further reinforce the anticorrelation between excitation and inhibition onto the output neuron.One could also imagine other combinations of eSTDP-iSTDP for the interneurons in the FFC model. For example, if the eSTDP onto the interneurons is anti-Hebbian, excitation and inhibition onto the output neuron become in): for temporally broader spike volleys, larger delays are recruited . For the inputs, we considered sharp correlations at the scale of a few milliseconds, in line with the timescale of input correlations for which neurons in a balanced state are most sensitive, shown both experimentally t et al. . Propagat et al. . Our rest et al.  enforcedt et al. . In addit et al. . The pret et al. . We showt et al. . Quick bin, the well-timed inhibition may hyperpolarize the neuron so strongly that the responses exhibit a rebound, after inhibition vanishes  = ∑s δ(t − tks); likewise, Sim is the mth inhibitory spike train. Though ρpost may take on negative values in theory, we assume it is positive on average, and do not consider the case of no postsynaptic spiking. The EPSPs and IPSPs are summed together to obtain ρpost; * denotes the convolution of functions. For each EPSP at synapse k, the time course of the postsynaptic response for a single spike is described by the normalized kernel functions ϵe rescaled by the weight wek. For IPSP at synapse m, the same holds with ϵi and wim. In Figure e = 3 ms; likewise τi = 5 ms for all inhibitory synapses.The In order to evaluate the expected weight change, we calculate the pre-post spike-time correlations for excitatory and inhibitory inputs. We consider the situation when pre-post correlations are dominated by the effect of input correlations. Actually, we use spike-time covariances defined as . For the configuration described in Figure d. Denoting by C0(Δ t) the homogeneous covariance corresponding to Figure The angular brackets 〈 … 〉 denote the ensemble average over the randomness from the stochastic process. Considering spike trains with constant average firing rates and fixed pair-wise correlations, we can omit the dependence on kth excitatory input, the covariance Covek,post is given by the input covariance on which the postsynaptic response (EPSPs-IPSPs) operates:All covariances are defined in a similar manner to Equation (5). For the We(u) with the pre-post covariance Cek,post(−u), which yields:The subsequent STDP weight update is given by the integral value of the learning window mth inhibitory weight is given by:Similarly, the pre-post covariance and the expected change for the These formulas are used to generate Figure In Figures r, underlies a group of inhomogeneous Poisson processes which have rates that fluctuate around r. Final spike trains are derived from these inhomogeneous Poisson processes, and will appear to be homogeneous, correlated spike trains with stationary rate r. These correlated spike trains do not have Poisson statistics, because their autocovariance is modulated by their correlation. In order to have exponential cross-correlation functions (CCF) between these spike trains, the function employs the Ornstein–Uhlenbeck process. The time-constant of the exponential CCF is a parameter called τc in Brette  model. Its membrane potential ge and gi, that decay exponentially with conductance trace parameters τe and τi:With synaptic conductances k, ge is increased by wek, and for every inhibitory spike from synapse m, gi by wim. Intrinsic time constants of the neuron are not considered. All simulations are run with BRIAN, a python-based neural simulator .In Figure ipre = τipost = 20 ms), excitatory and inhibitory learning are slowed down , the eSTDP equilibrium value is higher (w0 = 0.08), and the value of the start-up weights is changed . Total simulation time is 1000 s.All synapses are plastic. The excitatory synapses onto both the output neuron and the inhibitory interneurons are subject to the same Hebbian eSTDP leaning window. The list of parameters that vary from the SFC is shown in Table To test the robustness against noise, we modify the FFC by adding 400 excitatory random inputs onto the interneurons and output neuron  are detected by binning the spike times of the 100 correlated inputs in the SFC in bins with width 0.5 ms, and counting the spikes in a sliding window of duration τTo evaluate the temporal acuity of the spikes fired in response to such events, we count the postsynaptic spikes in bins of 0.5 ms. This yields a peri-stimulus time histogram (PSTHs) around the time of the events, as shown in Figure F0. The number of spikes in each bin of the PSTH is then divided by F0, yielding the “normalized” PSTH as a deviation from average activity. This deviation is also the signal to noise ratio .Not only the latencies of spikes following the event, but also the excess of spikes compared to the baseline output firing rate contributes to the temporal acuity of the response. We obtain the average firing rate during the entire 300 s simulation, out, we compare the outcome of simulations to two controls:To study how the emerged inhibitory weight structure affects τ“specific inhibition”: the weights are as in the numerical simulation of the SFC;“unspecific inhibition control”: the inhibitory weights are swapped between the two input groups, leading to equivalent total inhibition, but abolishing the relation between the temporal structure of the spike trains and the strength of the weights that depended on them through iSTDP;“excitation only control”: the inhibitory inputs are removed completely, leaving only the excitatory inputs.In the unspecific inhibition control for the SFC, we aim to destroy the detailed weight structure that emerges, but preserve strong feedforward inhibition. After swapping the inhibitory weights between the correlated and random inputs, the weight strengths are adjusted down to obtain a postsynaptic firing rate similar to the specific inhibition configuration. Weight corrections are not performed for trials with mean weight smaller than 1. Excitatory weights are unchanged in all three conditions. In the FFC, the same three scenarios are applied. For specific inhibition, all weights are as obtained from the simulation. To obtain the unspecific inhibition condition, excitatory weights onto the interneurons are swapped between the winner and loser input pathways. The result of this manipulation is that an interneuron receiving strong inputs from the dark blue group and weak inputs from the light blue group, changes to receiving weak inputs from the dark blue group and strong inputs from the light blue group. This procedure leads to qualitatively the same control as in the SFC. Inhibitory weights are not adjusted further as in the SFC. For the excitation only control, the interneurons are omitted.In this study, Matthieu Gilson and Florence I. Kleberg designed the experiments, Florence I. Kleberg performed the experiments and analyzed the data, and Florence I. Kleberg, Matthieu Gilson, and Tomoki Fukai wrote the paper.The research was funded by RIKEN.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.


Background: Diverse flower communities are more stable in floral resource production along the flowering season, but the question about how the diversity and stability of resources affect pollinator reproduction remains open. High plant diversity could favor short foraging trips, which in turn would enhance bee fitness. In addition to plant diversity, greater temporal stability of floral resources in diverse communities could favor pollinator fitness because such communities are likely to occupy the phenological space more broadly, increasing floral availability for pollinators throughout the season. In addition, this potential effect of flower diversity on bee reproduction could be stronger for generalist pollinators because they can use a broader floral spectrum. Based on above arguments we predicted that pollinator reproduction would be positively correlated to flower diversity, and to temporal stability in flower production, and that this relationship would be stronger for the most generalized pollinator species.Materials and Methods: Using structural equation models, we evaluated the effect of these variables and other ecological factors on three estimates of bee reproduction , and whether such effects were modulated by bee generalization on floral resources.Results: Contrary to our expectations, flower diversity had no effect on bee reproduction, stability in flower production had a weakly negative effect on one of the bee reproductive variables, and the strength of the fitness-diversity relationship was unrelated to bee generalization. In contrast, elevation had a negative effect on bee reproduction, despite the narrow elevation range encompassed by our sites.Discussion: Flower diversity did not affect the reproduction of the solitary bees studied here. This result could stem from the context dependence of the diversity-stability relationship, given that elevation had a positive effect on flower diversity but a negative effect on bee reproduction. Although high temporal stability in flower production is expected to enhance pollinator reproduction, in our study it had a weakly negative—instead of positive—effect on the average number of brood cells per nest. Other environmental factors that vary with elevation could influence bee reproduction. Our study focused on a small group of closely-related bee species, which cautions against generalization of our findings to other groups of pollinators. More studies are clearly needed to assess the extent to which pollinator demography is influenced by the diversity of floral resources. There is a consensus that diversity enhances ecosystem functioning . SpeciesIt is well known that ecosystem productivity is positively associated to species diversity . We propClarkia, diverse communities provide more resources along the flowering season, sustaining a higher number of pollinator individuals per plant , Zuccagnia punctata (Fabaceae), Prosopis flexuosa (Fabaceae), Condalia microphylla (Rhamnaceae), Acantholippia seriphioides (Verbenaceae), and Opuntia sulphurea (Cactaceae). We selected sites with different flower abundance, composition and diversity. The region suffers from recurrent fires, which are mostly human-caused and are in fact the most common human disturbance ; the time elapsed since the last fire varied substantially among our study sites . We worked in fourteen 100 × 200 m rectangular study sites . These sites lie at 1,100–1,500 m above sea level, at the ecotone between the Monte desert and the Prepuna biomes . The plady sites .Trichothurgus laticeps Friese, lacks brood cells, as females lay eggs bare amidst a pollen mass; thus, for this species we used the length of the trap cavity occupied by pollen as an estimate of the number of brood cells. For the analysis, we used only the seven bee species that occupied at least 30 trap nests, as we judged smaller sample sizes unreliable for statistical analyses.We placed trap nests in six points per plot as shown in Floral resource availability was studied using flower density, as flowers represent the resource packages encountered by pollinators as they forage see also . Flower To evaluate the effects of flower diversity and temporal stability of floral resources on bee fitness and to assess the influence of other ecological factors on this relationship, we used structural equation models (hereafter SEM). We built a general initial model to explore the effects of flower richness, flower abundance, time elapsed since the last fire, elevation, and temporal stability in flower production on bee reproductive parameters. We estimated bee productivity at the community level using two proxies: total number of brood cells per site, and total number of nests per site. To evaluate reproduction at the species level we used three proxies: average number of brood cells per nest, total number of brood cells per site, and total number of nests per site. To estimate the average number of brood cells per nest we used only data of sites where species were present; for the total number of brood cells per site and the total number of nests per site we used data of all sites, as the absence of a species in a site represented zero abundance. Flower richness was used as a proxy of flower diversity; it was rarefied to remove the effect of flower abundance. Flower abundance was estimated as flower density per site. Time elapsed since the last fire was provided by park rangers . Temporal stability in flower production along the season was calculated as the inverse of its coefficient of variation see . For eacip, between two pairs of variables that are not directly connected with an arrow in the causal model, and then using those probabilities to calculate Fisher’s C statistic, which follows a chi-square distribution, k pairs of independent variables with their corresponding conditional variables constitutes the basis set /(1 − r)] (w = 1/var(r) = N − 3  . The groasis set . Indepen the set . We used(1 − r)]  to make  = N − 3 . We used = N − 3 , with a  − 3 . In this simplified model, the negative effect of temporal stability in flower production on the average number of cells per nest was weaker than in the complete model , flower abundance (estimated using flower density) and temporal stability of flower production along the flowering season (estimated as the inverse of coefficient of variation of the weekly flower abundance mean), fire (estimated as time elapsed since last fire), and elevation (estimated as meters above the sea). Y axis represents the path coefficients that rank from 1 to −1, and the x axis represents the effect of above mentioned ecological variables on three bee reproductive variables: A is the average brood cell number per nest per site, B is the total number of brood cells per site, and C is the total number of nests per site. In blue color are the ninety-five percent confidence limits of path coefficients obtained from bootstrap sampling of the distribution of path coefficients.Click here for additional data file.10.7717/peerj.2250/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.2250/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.2250/supp-8Supplemental Information 8Column names: “codigo.i” refers to bee species, “sitio” refers to site names, “mean.no.celd” is the main number of brood cells per nest per site, “celd.tot” is the total number of cells, “nidos.tot” is the total number of nests.Click here for additional data file.10.7717/peerj.2250/supp-9Supplemental Information 9Column names: “sitio” refers to site name, “abund.flores” is the flower abundance, “raref” is the flower richness rarefied, “altitud” is the altitude, “anio.q” is the time elapsed since last fire. The other columns were not used in this study.Click here for additional data file.10.7717/peerj.2250/supp-10Supplemental Information 10Column names: “id.trampa” is a identification number for bee individuals, “codigo.i” is the bee species, “site” is name of the site, “codigo.p” is the plant species consumed, “sum.sp” are the number of pollen grains observed, “sum.tot” is the total pollen grains observed for that bee individual , “prop” is the proportion of use of a given plant by a bee individual.Click here for additional data file.


N-terminal kinases (JNK) are members of the mitogen-activated protein kinase (MAPK) family that have important roles in signal transduction. The small molecule SP600125 is widely used in biochemical studies as a JNK inhibitor. However, recent studies indicate that SP600125 may also act independent of JNK. Here, we report that SP600125 can induce Src, type I insulin-like growth factor receptor (IGF-IR), Akt and Erk1/2 phosphorylation. Notably, these effects are independent of its inhibition of JNK. Inhibition of Src abrogates the stimulation of IGF-IR, Akt and Erk1/2 phosphorylation. IGF-IR knockdown blunts the induction of both Akt and Erk1/2 phosphorylation by SP600125. Moreover, combination of SP600125 and the Src inhibitor saracatinib synergistically inhibits cell proliferation. We conclude that SP600125 can activate Src-IGF-IR-Akt/Erk1/2 signaling pathways independent of JNK.c-Jun  N-terminal kinases (JNKs) belong to the mitogen-activated protein kinase (MAPK) family, and consist of more than ten isoforms encoded by three genes, namely JNK1, JNK2 and JNK3 [c-Jun SP600125 is an anthrapyrazolone compound that is widely used in biochemical studies as a JNK inhibitor. In addition, analogs of SP600125 have a variety of biological activities. For example, anthrapyrazolones themselves have been investigated as anticancer agents for their DNA-binding activity ,4. MoreoDespite SP600125 has been considered as a JNK inhibitor in a number of studies, recent studies demonstrate that it may also act in JNK-independent manner. For example, the ability of SP600125 to up-regulate REDD1 expression and inhibit voltage-dependent potassium ion channel is JNK-independent ,12. The To investigate the effects of SP600125 on Akt or Erk1/2 phosphorylation, HepG2 hepatoma cells were treated with different doses of SP600125 for 48 h, followed by western blot analysis of the levels of Akt and Erk1/2 phosphorylation. Treatment with SP600125 inhibited JNK phosphorylation and induced both Akt and Erk1/2 phosphorylaion in a dose-dependent manner A,B. MoreBoth Akt and Erk1/2 are classical effectors downstream growth factor receptors including IGF-IR. We found that treatment with SP600125 also increased IGF-1R phosphorylation in dose-dependent and time-dependent manner in various cancer cell lines . In addiBecause plenty of studies show that IGF-1R plays a pivotal role in regulating PI3K/Akt and Ras/MAPK signaling pathways , we wondAs we know, SP600125 is widely used as JNK inhibitor. To determine whether JNK is involved in SP600125-induced IGF-1R signaling, HepG2 cells and SMMC-7721 cells were treated with SP600125 or JNK inhibitor VIII, another specific JNK inhibitor. SP600125 induced IGF-1R, Akt and Erk1/2 phosphorylation in both HepG2 and SMMC-7721 cells A,B. JNK Previous studies showed that IGF-1R can be phosphorylated and then activated by Src  ThereforGiven that SP600125 induces Src phosphorylation, we tested if inhibition of Src can potentiate the anti-cancer activity of SP600125. We utilized EdU assays to detect the effect of combination of saracatinib and SP600125 on cell proliferation. While both saracatinib and SP600125 slightly inhibited HepG2 cell proliferation, combination of saracatinib and SP600125 significantly inhibited cell proliferation . These dPrevious studies demonstrate that SP600125 may potently inhibit voltage-dependent potassium ion channel and up-regulate REDD1 in JNK-independent manner ,12. G ivIn summary, this study uncovers that SP600125 activates Src-IGF-1R-Akt/Erk pathways independent of inhibition of JNK. While JNK appears to be an attractive target for treating cancer, especially hepatocellular carcinoma , activatSP600125 was purchased from Beyotime Institute of Biotechnology and Sigma. JNK inhibitor VIII was purchased from Merck . Saracatinib was purchased from LC Laboratories. Antibodies for p-IGF-1R (Y1135) (#2182-1) were purchased from Epitomics . Antibodies for IGF-1R (#3027), p-Akt (S473) (#4060), Akt (#3063), p-Erk1/2 (T202/Y204) (#4370S), Erk1/2 (#4695), p-Src (Y416) (#6943P), Src (#2123), p-JNK (T183/Y185) (#9251), JNK (#9258P) were purchased from Cell Signaling Technology . Antibody for β-actin (#KM9001) was purchased from Tianjin Sungene Biotech Co., Ltd., Tianjin, China.2.Hepatoma cell lines HepG2, SMMC-7721, Bel-7402 and cervical cancer cell line Hela were all obtained from Cell Lines Bank, Chinese Academy of Science . The cells were maintained in Dulbecco Minimal Essential Medium (DMEM) containing 10% fetal bovine serum and 50 units/mL of penicillin and 50 µg/mL streptomycin sulfate, and incubated at 37 °C in a humidified atmosphere of 5% COAll siRNAs were custom-synthesized products of Ribobio Co., Ltd. . The target sequence of siIGF-1R is CAATGAGTACAACTACCGC. The target sequence of siJNK1/2 is GAAAGAATGTCCTACCTTC. The negative siRNA (siCtr) was provided by Ribobio Co., Ltd. . The double-stranded siRNA was dissolved in DEPC-treated water. Subconfluent proliferating cells were transfected with Lipofectamine 2000  according to the instructions of manufacturer. The final concentration of siRNAs was 50 nM.3, 1 mM NaF). Protein concentrations were quantified by BCA protein assay . Samples containing 30 μg of protein were resolved by SDS-PAGE and transferred to PVDF membrane . Membranes were incubated with primary antibodies overnight at 4 °C and appropriate HRP-secondary antibodies for 1 h at room temperature. Detection was performed with chemiluminescent agents . Images were gathered by Alpha Innotech’s FluorChem imaging system . Densitometric analysis was performed with Adobe Photoshop .Total protein extracts were harvested by lysis with RIPA buffer   medium for 2 h. Then the cells were treated with 4% Polyoxymethylene for 30 min, 2 mg/mL glycine for 10 min, and incubated with 0.5% TritonX-100 for 10 min. The cells were incubated in Apollop < 0.05.All statistical analysis was performed with one-way ANOVA. All statistical tests were two-tailed, and difference to be considered to be statistically significant when The current study demonstrates that SP600125 activates Src-IGF-IR-Akt/Erk1/2 pathways in a JNK-independent manner.