Metabolic changes have been frequently associated with Huntington’s disease (HD). At the same time peripheral blood represents a minimally invasive sampling avenue with little distress to Huntington’s disease patients especially when brain or other tissue samples are difficult to collect.We investigated the levels of 163 metabolites in HD patient and control serum samples in order to identify disease related changes. Additionally, we integrated the metabolomics data with our previously published next generation sequencing-based gene expression data from the same patients in order to interconnect the metabolomics changes with transcriptional alterations.This analysis was performed using targeted metabolomics and flow injection electrospray ionization tandem mass spectrometry in 133 serum samples from 97 Huntington’s disease patients (29 pre-symptomatic and 68 symptomatic) and 36 controls.ALDH1B1, MBOAT1, MTRR and PLB1) that showed significant association with the changes in metabolite concentrations.By comparing HD mutation carriers with controls we identified 3 metabolites significantly changed in HD (serine and threonine and one phosphatidylcholine—PC ae C36:0) and an additional 8 phosphatidylcholines  that exhibited a significant association with disease severity. Using workflow based exploitation of pathway databases and by integrating our metabolomics data with our gene expression data from the same patients we identified 4 deregulated phosphatidylcholine metabolism related genes  contains supplementary material, which is available to authorized users. HTT gene, resulting in a mutant huntingtin protein (The Huntington’s Disease Collaborative Research Group Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder that presents itself through motor dysfunction, psychiatric disturbances and cognitive decline. The pathology is caused by an expanded CAG repeat in the Deregulation of energy and metabolic pathways have been repeatedly implicated in HD  and flow injection electrospray ionization tandem mass spectrometry (FIA-ESI–MS/MS). A total of 163 different metabolites were quantified simultaneously by the platform in 10 µL serum. The metabolite panel consists of 14 amino acids, Hexose (H1), free carnitine (C0), 40 acylcarnitines (Cx:y), hydroxylacylcarnitines (C(OH)x:y), and dicarboxylacylcarnitines, 15 sphingomyelins (SMx:y) and N-hydroxyacyloylsphingosylphosphocoline (SM(OH)x:y), 77 phosphatidylcholines  and 15 lyso-phosphatidylcholines. Lipid side chains are denoted as Cx:y, where x represents the number of carbons in the side chain and y the number of double bonds. The assay procedures of the p150 kit as well as the metabolite nomenclature have been described in detail previously  and an Ultravap nitrogen evaporator , beside standard laboratory equipment. Mass spectrometric (MS) analyses were done on an API 4000 LC–MS/MS System  equipped with a 1200 Series HPLC  and a HTC PAL auto sampler  controlled by the software Analyst 1.5. Data evaluation for quantification of metabolite concentrations and quality assessment was performed at the Genome Analysis Center of the Helmholtz Zentrum München using the MetIDQ™ software package, which is an integral part of the AbsoluteIDQ™ kit. Internal standards served as reference for the calculation of metabolite concentrations in µM.Metabolite concentrations were determined using the targeted metabolomics kit AbsolutePeripheral blood was collected from 29 presymptomatic, 68 symptomatic and 36 control, non-fasting individuals with institutional review board approval and after informed consent. For serum sample isolation, blood was collected in BD vacutainer Z tubes (no additives) and was allowed to clot for 1 h at room temperature. Tubes were spun at 1300 g for 10 min at room temperature, were aliquoted and stored at −80 °C. Detailed information about the UHDRS clinical scores and CAG repeat lengths of all the patients and controls as well as gender age and BMI information can be found in Supplementary File 1.To ensure the robustness of downstream statistical analyses, all data provided from the MetIQ software package were subjected to three quality control steps. For the first step the coefficient of variance was calculated for each experimental plate. To achieve this five aliquots of a reference plasma pool were measured on each plate together with the cohort samples. The coefficient of variance was calculated as the standard deviation to mean ratio for all five reference samples per metabolite and per experimental plate. All metabolites with a mean coefficient of variance of all plates, higher than 25 % were excluded from further analysis. All metabolites with a missing value rate larger than 5 % were also excluded. In the second step any outlying data points with a value greater than mean ± 5 SD of all measurements for this metabolite were excluded. Additionally, two Huntington disease samples were excluded due to high BMI values (outliers). For samples with less than, or equal to, three independent outlying points only the independent data points themselves were excluded. After these quality control steps 114 out of 163 metabolites and 133/138 samples remained. After the above steps, when missing values were detected these were imputed using the R package “mice”. Finally, all metabolite concentrations were transformed using natural logarithm and before applying the experimental linear modeling analysis.http://www.r-project.org/) was used. After the metabolite concentrations were log-transformed, linear modelling statistical tests were applied. In specific, in the first model (disease status) the HD mutation carriers’ and control individuals’ groups were coded as the main covariate and tested in a linear model using gender  age and BMI as additional (continuous) covariates. In the second model (disease severity) a four group categorical variable vector was used as the main covariate. The following groups were defined: Group 1—(n = 36) control, Group 2—(n = 29) pre-symptomatic (TMS score ≤5), Group 3—(n = 31) symptomatic  and Group 4—(n = 37) advanced symptomatic individuals . For both models the final P value that was used to judge the validity of our findings was extracted using the ANOVA function on the two nested linear models; the reduced linear model containing only the covariates of gender, age and BMI and the full model additionally containing the main disease group categorical covariate. Since many identified metabolites showed a high degree of correlation (see Supplementary File 2), the Bonferroni method was judged too strict for multiple testing correction. Therefore, the experiment-wide significance threshold that was used was 1.34E−03. This value was calculated using the matrix spectral decomposition method and the eigenvalues of the metabolites correlation matrix (matSpDlite) (Li and Ji Platform for RIKEN Metabolomics (PRIMe) tool for Microsoft excel   and lower limit of quantification (LLOD). In order to analyze the group structure of the metabolomic dataset across controls, presymptomatic and symptomatic HD patients, we performed a partial least square discriminant analysis (PLS-DA). The symptomatics group  exhibited a clear shift from the control (group 1) and the pre-symptomatic group 2) samples. The scores plot for the symptomatics, pre-symptomatics and control groups and for the first two principal components can be seen in Fig.  samples.Previous studies have shown that calculating the ratios of individual metabolite concentrations can reduce dataset variation. Furthermore, such metabolite ratio changes have been connected to altered enzymatic reactions and pathways, can be used as an approximation of the associated enzymatic activity , ALDH1B1 , KMT2A (lysine specific methyltransferase 2A), MBOAT1 (membrane bound O-acyltransferase DC1), MTRR (methionine synthase reductase), PISD (phosphatidylserine decarboxylase), PLB1 (phospholipase B1) and HADH (hydroxyacyl-CoA dehydrogenase). The correlation values of each of the 8 genes with the 8 significant metabolites from our 4 group linear modeling analysis are represented in Fig. MBOAT1, PLB1, ALDH1B1 and MTRR the correlations with the majority of the 8 metabolites were high (r > 0.6) while for the other 4 genes the correlations were average or poor. The highest associations were observed between the genes ALDH1B1, MTRR and PLB1 with phosphatidylcholines PC ae C.38:0, PC aa C36:5 and PC aa C38:6 , PGLYRP1 (peptidoglycan recognition protein 1) and TKTL1 (transketolase-like 1). Finally, for the serine and threonine metabolites we could not identify any unique common genes. It should be noted however that the above phosphatidylcholine related gene ALDH1B1 was also present in our threonine KEGG reaction list and similarly PISD and PLB1 were also present in our serine KEGG reaction list.To further explore potential molecular connections of the identified metabolites with HD relevant or novel disease state/progression mechanisms we combined our targeted metabolomics dataset with our previously published next-generation sequencing gene expression data from the same patient and control cohort ) and phase II conjugation (containing gene MTRR and serine and threonine), supporting the results from our first pathway analysis. All the overlapping pathways reported for the Wikipathways analysis can be seen in Supplementary File 6.To expand on the above findings, we performed a second analysis using the WikiPathways Web Service and all 10 significant metabolites (8 phosphatidylcholines and 2 amino acids) from both of the above metabolomics linear models, in order to investigate further connections between potential metabolomics and transcriptomic pathways. The metabolite-gene pathways with the highest overlap of genes and metabolites we identified were glycerophospholipid biosynthesis  that changed gradually with disease severity. These consisted of changes in inter acyl-alkyl-phosphatidylcholines ratios but also changes in the sphingomyelins to phosphatidylcholines ratios. Sphingomyelins are a group of sphingolipids found in mammalian cell membranes and especially membranes that surround nerve cell axons Slotte . A decreMTRR, PLB1 and ALDH1B1) exhibited especially high correlation with specific diacyl and acyl-alkyl phosphatidylcholines that were downregulated in HD in the metabolomics dataset. More specifically, MTRR is involved in the proper function of methionine synthase and folate metabolism Supplementary material 2 (PDF 240 kb)Supplementary material 3 (PDF 2398 kb)Supplementary material 4 (PDF 392 kb)Supplementary material 5 (DOCX 18 kb)Supplementary material 6 (DOCX 13 kb)Supplementary material 7 (DOCX 12 kb)Below is the link to the electronic supplementary material.


Alteromonas spp. are heterotrophic gammaproteobacteria commonly found in marine environments. We present here the draft genome sequence of Alteromonas macleodii MIT1002, which was isolated from an enrichment culture of the marine cyanobacterium Prochlorococcus NATL2A. This genome contains a mixture of features previously seen only within either the “surface” or “deep” Alteromonas ecotype. Alteromonas spp. are copiotrophic bacteria widely distributed throughout the marine environment, where they can play a notable role in the processing of dissolved organic carbon pools  waters in the Mediterranean. Genomic analyses have shown that A. macleodii strains cluster into two phylogenetic groups, termed the “surface” and “deep” ecotypes, correlated with where they were found in the water column   (We isolated 49°44′W) . The enr49°44′W)  at 21°C Genomic DNA libraries were prepared as previously described  and sequN50 of 79,162. The total assembly length and G+C content (44.6%) are consistent with those of other sequenced A. macleodii isolates  strain, including a set of hydrogenases and metal resistance genes.The draft genome contains 125 contigs with a total length of 4,667,157 bp and an isolates . PhylogeProchlorococcus NATL2A strain with which it was coisolated, as NATL2A is relatively abundant during mixing events and also occupies an intermediate position between high and low-light-adapted ecotypes of Prochlorococcus (–This mixture of traits normally associated with either the surface or the deep-water environment raises the possibility that MIT1002 may contain adaptations allowing it to withstand vertical mixing. This provides an intriguing parallel with the distribution and physiology of the rococcus –15.JXRW00000000. The version described in this paper is version JXRW01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Here, we study large areaMoS2-graphene-heterostructures formed by direct transfer ofchemical-vapor deposited MoS2 layer onto epitaxial graphene/SiC. We showthat via a direct transfer, which minimizes interface contamination, we can obtainhigh quality and homogeneous van der Waals heterostructures. Angle-resolvedphotoemission spectroscopy (ARPES) measurements combined with Density FunctionalTheory (DFT) calculations show that the transition from indirect to direct bandgapin monolayer MoS2 is maintained in these heterostructures due to the weakvan der Waals interaction with epitaxial graphene. A downshift of the Raman 2D bandof the graphene, an up shift of the A1g peak of MoS2 and asignificant photoluminescence quenching are observed for both monolayer and bilayerMoS2 as a result of charge transfer from MoS2 to epitaxialgraphene under illumination. Our work provides a possible route to modify the thinfilm TDMCs photoluminescence properties via substrate engineering for future devicedesign.Two-dimensional layered transition metal dichalcogenides (TMDCs) show great potentialfor optoelectronic devices due to their electronic and optical properties. Ametal-semiconductor interface, as epitaxial graphene - molybdenum disulfide(MoS Recently Han et al.developed a novel seeded CVD patterned growth method to obtain highly crystallineMoS2 flakes on oxidized silicon substrate2 grown by this approach has high crystallinity, with large flakes(between 20–100 μm) and electrical and opticalproperties comparable to exfoliated material. These CVD-grown flakes are suitable fortransfer onto epitaxial graphene/SiC substrate in order to obtain large area ofMoS2/graphene heterostructures. Using a transfer process  measurementswere used to study the electronic structure of the MoS2/epitaxial grapheneheterostructure. Due to a weak interlayer coupling the electronic structure of grapheneand MoS2 are well retained in their respective layers. However the bandalignment in the MoS2/graphene heterostructure allows under illumination acharge transfer process from MoS2 to graphene. This is revealed by adownshift of the graphene 2D Raman bands, an upshift of the A1g Raman band ofMoS2, and a strong quenching of photoluminescence (PL) ofMoS2/graphene heterostructure. Complementary we performed photocurrentmeasurements to elucidate how the presence of the semiconductor affects thephotoconductive properties of graphene. In that respect, this work may open a new way ingraphene optoelectronics by modulating the graphene photoelectric response through 2Dmaterials interfacing.One well-established method to produce high quality wafer-scale monolayer graphene is theepitaxial approach based on the graphitization of the Si face of SiC132 flakes grown bychemical vapor deposition on oxidized silicon substrates that were then transferredonto epitaxial graphene grown on SiC(0001) , we were ableto obtain several flakes with various stacking order and orientation in a singletransfer step .For the following experiments, the monolayer, bilayer and multilayer coverage wasestimated from optical analysis to be around 83%, 15% and 2% respectively.TMDCs/graphene heterostructures were made from MoSiC(0001) . ThegraiC(0001) . The CVDsolution . Afterwaubstrate . We finafer step . The MoS2/graphene/SiC heterostructure, we measured its band structure byangle-resolved photoemission spectroscopy (ARPES) at the Cassiopéebeamline of Synchrotron Soleil. The small x-ray spot size(50 × 50 μm2)allowed the measurement of the band structure of a single flake (monolayer orbilayer) forming the MoS2/graphene heterostructure. The photoelectronintensity is presented in 2 valence band. A close inspection of the K point of grapheneBrillouin zone is shown in 2 flake andgraphene underlayer is critical. In order to obtain a perfect alignment of theBrillouin zone of the two materials we need a mismatch angle of zero degree. As wecan see in 232 are not visibleindicating a non-zero mismatch angle for this flake. However we clearly see thegraphene band structure, in particular the π bands of graphene preservetheir linearity characteristic of a massless Dirac fermions signature, indicating ahigh structural quality of the MoS2/graphene heterostructure. Moreover,similar to pristine monolayer graphene, the Dirac point (ED) is locatedat 0.3 eV below the Fermi level (FL). From a linear fit, using therelation E = ħvFk, we obtain the value of the FermivelocityvF ~ 1.1 × 106 m/s,which matches the expected value for monolayer graphene on SiC. As the lineardispersion and Fermi velocity of the pristine graphene is preserved in theMoS2/graphene heterostructure, we can infer that the MoS2transfer does not affect the electronic structure of monolayer graphene in theheterostructure formation. In addition, this feature is expected theoretically sincein van der Waals heterostructures, the superposition of each layer electronicstructure constitutes a good approximation of the electronic structure of themultilayer. Differently from previous work of Diaz et al. no signature ofinterlayer hybridization2flake and the graphene underlayer. However identifying the dependence of this effecton the mismatch angle would clearly require a further work and go much beyond themain objective of this paper. 2 on epitaxial graphene,along the K-Γ-K direction in the hexagonal Brillouin zone, therespective second derivative are shown in 2 presents only one band atthe Γ point (maximum at Binding Energy (BE)~ −1.68 eV ± 0.05 eV),and this structure evolves into two branches in the case of bilayer MoS2(maximum at BE~ −1.25 eV ± 0.05 eV).This evolution is representative of the splitting of the bands due to the weak vander Waals interaction between the two MoS2 layers. The relative positionof the top of the valence bands at the Γ point in the bilayer film iscloser to the Fermi level than the one obtained from the monolayer  and Γ points. However, when considering the monolayerband structure, the top of the valence band becomes very flat near the Γpoint, leading to a direct gap at the K (K′) point. The evolution of thevalence band at the Γ point provides a straightforward method toidentify the thickness of ultrathin MoS2 films, and also proves the highquality of MoS2 transferred on epitaxial graphene. The epitaxial grapheneunderlayer does not affect the MoS2 band structure, as expected for a vander Waals heterostructure. However, we do not exclude the presence of the universalbuckled form of 2D crystal in our MoS2 layerIn order to better understand the electronic properties of theMoSonolayer . This in2/graphene heterostructure, micro photoluminescence (micro-PL) andmicro-Raman measurements are performed at room temperature (see method).To further investigate the electronic properties of mono and bilayerMoS2 todirect gap semiconductor in monolayer MoS2. The PL signal is stronglyenhanced when a direct band gap is present. The same behavior was also observed forthe as grown MoS2 on SiO2 , Γ−1 is between8–14, and presents a huge decrease as the thickness of graphene layer isreduced (~2 in the case of 5 nm FLG). In our case, we haveonly one graphene layer, meaning that as for SiO2 we can supposeΓ−1 ~ 1.Therefore, in both cases we can neglect the effect of optical interferences, anddirectly compare the PL raw data of MoS2/SiO2 andMoS2/graphene heterostructures as shown in 2/SiO2 heterostructure strain canexist due to the different thermal expansion coefficients of MoS2 and theSiO2 substrate during the MoS2 flakes growing2/graphene heterostructure38392 substrate). This phenomenonwas explored also as a function of the laser power. In 2 PL intensity evolveslinearly with increasing laser excitation412 and graphene. But in the case of graphene underlayer the PLsignal is quenched for each laser power.In the case of vertical vdW heterostructures, possible PL intensity variations couldarise from the interference effects due to different optical constants and thicknessof the different layers forming the heterostructure2 to thegraphene which hinders the recombination of electron−hole pairs createdby the photoexcitation322 and graphene , but rather to astandard hopping from an electron in MoS2 conduction band to anunoccupied state at the same energy in graphene43This phenomenon is the signature of electron transfer from MoS2/graphene heterostructure and pristine graphene layer−1. Besides thetypical second-order Raman bands that originate from the SiC substrate, the threemain structures typical of graphene are present on the pristine graphene andMoS2/graphene spectra: i) the D band (defect induced mode), ii) the Gband (in-plane vibration mode) and iii) the 2D band (two-phonon mode)2/graphene heterostructure within the wavenumberrange between 350–450 cm−1 twonew peaks are present. These two characteristic features correspond to the in-planevibration (1g) of Moand S atoms in the MoS2 film341g and 2 layers. The averageRaman spectra obtained from each layer are shown in −1,~21 cm−1 and24 cm−1 corresponding to monolayer,bilayer and multilayer (~three layers) MoS2,respectively342 on graphene and onSiO2 substrates result of a Lorentzian fitting, are narrower than those on SiO2. As in the case of the PL spectra, the upshift of the in plane Raman mode2, and confirms that the photoelectrons generated bythe Raman laser are transferred from the MoS2 to graphene. At the sametime the G peak presents an upshift. If we focus our attention on monolayerMoS2/graphene we have an upshift of~3 cm−1 ± 1cm−1.From this shift we can obtain a quantitative analysis of the level of electrondoping under illumination5357582/graphene as −1implies a change in the graphene Fermi level of ~70 meV.From these values we can calculate the variation in the n-doping of graphene usingthe relation vF = 1.1 × 106 m/s),we obtained an electron density for pristine graphene|N| ~ 7 × 1012 cm−2which increases to|N| ~ 1013 cm−2for 1ML MoS2/graphene under illumination.A detailed analysis of the Raman spectra of the monolayer and bilayerMoS2 decorated graphene) at roomtemperature while illuminating the sample with light energy(λ = 405 nm(hν ≈ 3 eV)) above theMoS2 band gap because of its thickness and limited coverage. The photocurrent generation in TMDCheterostructure generally suffers from two main limitations from an applied point ofview, which are their slow response time610.8. Such dependence isweaker than for large gain system like graphene–PbS quantum dot hybridsystem2 decorated graphenesystem over a larger range of light flux. While modulating the incident laserintensity thanks to a signal generator we can extract the frequency dependence ofthe graphene-MoS2 systemSince the spot diameter of the laser is 1 mm2 on epitaxial graphene layer. From the PL, ARPES and micro-Ramandata presented in 2/graphene heterostructurepresents a good long-range order at large scale. Our ARPES measurements on theheterostructure showed that graphene and MoS2 largely retained theiroriginal electronic structure indicating weak van der Waals interactions between thetwo crystals. The PL quenching in the MoS2-graphene heterostructure andthe upshift of the A1g Raman mode of MoS2 and the downshift ofthe 2D Raman mode of graphene confirm the charge transfer between theMoS2 and graphene layer. Our work suggests that the opticalproperties of MoS2 are strongly affected by the underlayer graphene. Thefact that an interaction is visible between MoS2 and graphene is a clearsignature of the quality of the wet transfer process and of the absence ofinterfacial contaminations. Moreover, this charge transfer can be influenced by theunderlayer graphene doping, varying the band alignment in the heterostructure, whichshould be considered in device design and fabrication. Furthermore, efficientphotoresponse was observed on large 2D MoS2/epitaxial graphene devices,opening a new way in graphene optoelectronics.In summary, we have studied the electronic properties of the wet transfer of largearea MoS2) at1550 °C to produce well-ordered atomic terraces of SiC.Subsequently, the SiC sample is heated to 1000 °C at apressure of about 10−5 mbar and thenfurther heated to 1550 °C in an Ar atmosphere. Thisgraphitization process results in the growth of an electrically active graphenelayer on top of the buffer layer, covalently bound to the substrateex-situ to perform different measurements.Monolayer graphene studied in this paper is produced via a two-step processbeginning with a starting substrate of 4H-SiC(0001)2/SiO2 samples were grown via CVD in a 1″quartz tube furnace. Microliter droplets of saturated ammonia heptamolybdatesolution were dried onto the corners of a Si/SiO2 growth substratethat had previously been coated with a layer of sodium cholate (1% solution spincoated 4000 rpm for 60 sec). Sodium cholate is a known growthpromoter, acting to increase diffusion of the molybdenum source by increasingthe surface adhesive energy relative to the adatom cohesive energy2) was used to bring sulfur vapor into the furnace fora 30 min growth period. The sample was then rapidly cooled bycracking open the furnace and sliding it downstream with respect to the quartztube.MoSThe PL measurements were carried out using a confocal commercial Renishawmicro-Raman microscope with a 100× objective and a Si detector(detection range up to ~2.2 eV). The Raman spectrameasurements were performed on the same microscope using a 532 nmlaser in an ambient environment at room temperature. To ensure thereproducibility of the data, we followed a careful alignment and optimizationprotocol. In addition, the excitation laser was focused onto the samples withspot diameter of ~1 μm and incident power of~5 mW. The integration time was optimized to obtain asatisfactory signal-to-noise ratio. We obtained Raman spatial maps by rasterscanning with 0.3 μm step size using a precision 2Dmapping stage.° and 16 meV. The meandiameter of the incident photon beam was smaller than50 μm. All ARPES experiments were done atroom temperature.The ARPES measurements were conducted at the CASSIOPEE beamline of SynchrotronSOLEIL . We used linearly polarized photons of50 eV and 90 eV and a hemispherical electron analyzerwith vertical slits to allow band mapping. The total angle and energyresolutions were 0.25For electrical measurements the samples are electrically characterized in air atroom temperature. Temporal and power dependence of the current are obtainedwhile biasing the sample and measuring the current with a Keithley 2634B assourcemeter. Illumination is ensured by a 405 nm blue laser diodewith tunable light intensity. The frequency dependence of the photocurrent ismeasured while the sample is biased using a Keithley 2634B. The output signal isamplified in a Keithley 427 current amplifier and the signal acquired on HPoscilloscope.3d5for S and Mo were used with cutoff radii (in atomic units)s = 4.3, p = 4.7,d = 5.5 (S) and s = 5.0,p = 5.6, d = 4.8 (Mo). In thisstudy we have considered standard unit cells of 3, 6 and 9 atoms to describerespectively a mono-bi- and trilayer of MoS2. Each configuration hasbeen relaxed using a sample of 32 k-points in the Brillouin zone. In case ofmultilayer MoS2, we have considered the most stable AB stacking, andthe equilibrium distance has been determined using theLCAO-S2 + vdW formalism712 are providedin First-principles calculations have been performed using a very efficient DFTlocalized orbital molecular dynamic technique (FIREBALL)676869How to cite this article: Pierucci, D. et al. Large area molybdenumdisulphide - epitaxial graphene vertical Van der Waals heterostructures. Sci.Rep.6, 26656; doi: 10.1038/srep26656 (2016).


Our results demonstrate that optical properties suitable for photovoltaic applications, in addition to spatial electron-hole separation, are possible but efficient charge transport may be a limiting factor.The increasing efficiency of the inorganic-organic hybrid halides has revolutionised photovoltaic research. Despite this rapid progress, the significant issues of poor stability and toxicity have yet to be suitably overcome. In this article, we use Density Functional Theory to examine (Pb Both exhibit the same distorted [Pb2I6]2− unit, hypothesised to be due to the relatively strong dispersion interactions between the iodine atoms and the π bonding system on the organic linkers21π system. Of particular interest, however, was the observation of a photocurrent enabled by charge transfer between the organic and inorganic networks, allowed by the relative energies offset between the valence band of [Pb2I6]2− and the methylviologen LUMO and confirmed by experiment and Density Functional Theory (DFT) calculations272I6) · (H2DPNDI) · (H2O) · (NMP) crystal observed to absorb across the entire visible spectrumThe lowering of dimensionality when compared to the methylammonium lead halides, and corresponding structure, is also consistent with a related hybrid lead iodide, MVPbab initio Simulation Package (VASP)2930313738404143−1. For the pseudopotential treatment of Pb, 6s, 6p and 5d orbitals were considered valence.All calculations were performed using periodic DFT using the Vienna 2DPNDI]2+ and [Pb2I6]2− frameworks together, the solvent molecules in the crystal are bound solely by intramolecular dispersion forces and hydrogen bonding, which most GGA functionals are unable to correctly describe. To account for this, the D3 van der Waals correction method produced by Grimme et al.In addition, while electrostatic forces hold the [Het al.2I6]2− network (lead s states and iodine p), while the conduction band is wholly situated on the organic ligands - predominantly carbon, oxygen and nitrogen p states. This suggests that this compound exhibits spatial charge separation on photoirraditation: in a direct transition, electrons will transfer onto the organic network, while the holes remain on the lead iodide nanowires. This observation is supported by plotting the partial charge density maps for the valence band maximum (VBM) and conduction band minimum (CBM) seen in et al.p states, is small. The VdW corrected calculations showed very similar DOS plots to the corresponding uncorrected calculation in 0 and 1.96 m0 for the conduction and valence band respectively. These values are much higher than the equivalents for methylammonium lead iodide (MAPI) obtained using DFT (0.23 m0 and 0.29 m0 respectively)2I6]2− compared to the lead iodide perovskite, which is enforced by the larger organic cation hindering the orbital overlap. However, the charge separation from the lead and iodide-based valence band may strongly reduce potential recombination losses expected from low conductivity4041To further assess this compound’s capability as an absorber, the electronic band structure was calculated with each of the PBEsol-based methods, and in addition using hybrid functionals PBE0 and HSE06, using the relaxed PBEsol structure. The resulting diagrams are shown in α against energy is shown in 4 cm−1) just above the fundamental band gap, which, in comparison to the HSE06 result, would mean absorption within the optimal 1.0–1.5 eV range. This supports the experimental absorption measurements by Liu et al. that demonstrated that the compound does absorb within the visible electromagnetic rangeTo further investigate the optical behaviour of this system, the optical absorption was calculated; however, due to the large size of the system, such a calculation was only possible at the PBEsol level. The resulting plot of the attenuation coefficient et al.et al.3NH3I and PbI2, the commonly observed byproducts of MAPI degradation. As such, we have probed the thermodynamics of the analogous reaction for this compound:In addition, the thermodynamic stability of the compound was assessed in comparison to MAPI’s decomposition pathway. As noted above, theoretical study by Zhang rH = +2.03 eV, indicating that this compound does not have a similar intrinsic instability, and should be more resistant to decomposition than MAPI.Utilising structures from the literature21512I6, that further exploration of photoactive organic cations in hybrid photovoltaic materials could lead to very useful device properties like efficient charge separation and suitable electronic band gaps, together with improved stability.The structure-property relationships of similar dense hybrid inorganic-organic materials are still relatively unexplored, but it is evident from the study of this compound, and those on MVPb2I6) · (H2DPNDI) · (H2O) · (NMP) have been examined using a variety of DFT methods, with a focus on assessing its capability as a photovoltaic material. The compound was shown to have an indirect band gap of 1.26 eV with the hybrid HSE06 functional, and spatial valence band and conduction band separation due to the inclusion of a photoactive organic cation, which may lead to the reduction of recombination losses. This comes at the cost of the large size of the cation leading to lower [Pb2I6]2− connectivity and corresponding high effective carrier masses. It is clear that photoactive organic cations, like DPNDI, can allow suitable band gaps for photovoltaic devices, hence further effort into producing such hybrid materials without sacrificing connectivity, and resultant mobility, is highly recommended.The crystal and electronic structures of the hybrid inorganic-organic lead iodide .


Apis dorsata) accounts for the evolution of a series of defence strategies to protect the colonies from predation. In particular, the concerted action of shimmering behaviour is known to effectively confuse and repel predators. In shimmering, bees on the nest surface flip their abdomens in a highly coordinated manner to generate Mexican wave-like patterns. The paper documents a further-going capacity of this kind of collective defence: the visual patterns of shimmering waves align regarding their directional characteristics with the projected flight manoeuvres of the wasps when preying in front of the bees’ nest. The honeybees take here advantage of a threefold asymmetry intrinsic to the prey–predator interaction: (a) the visual patterns of shimmering turn faster than the wasps on their flight path, (b) they “follow” the wasps more persistently (up to 100 ms) than the wasps “follow” the shimmering patterns (up to 40 ms) and (c) the shimmering patterns align with the wasps’ flight in all directions at the same strength, whereas the wasps have some preference for horizontal correspondence. The findings give evidence that shimmering honeybees utilize directional alignment to enforce their repelling power against preying wasps. This phenomenon can be identified as predator driving which is generally associated with mobbing behaviour (particularly known in selfish herds of vertebrate species), which is, until now, not reported in insects.The open nesting behaviour of giant honeybees (The online version of this article (doi:10.1007/s00114-014-1220-0) contains supplementary material, which is available to authorized users. Giant honeybees have evolved shimmering behaviour for collective defence  from 20 km away to the close vicinity of the experimental giant honeybee colony. After its relocation, the paper nest was encased with a curtain built of white and black linen. Utilizing the intrinsic positive phototaxis of forager wasps, we guided them through a tunnel of linen towards the experimental giant honeybee colony . The video films were formatted as jpg-sequences and processed by image analysis software  to detect wasp movements and shimmering activities of the honeybees. In total, 6,891 frames with wasps flying in front of the bees’ nest were collected  in pairs of successive frames  referred to the focus frame at the time ti  represented the “motionless” state; and in the second class, changes in luminance values of Δ lumpx > 5 signalled “movement”. This movement coding was used to calculate the coordinates of the horizontal position (xsh [fi]) and of the vertical position (ysh [fi]) of the momentary gravity point of the shimmering wave at the time ti as explained in Eqs. px = 1,2,..,npx gives the number of segmented pixel areas at the focus frame fi, and am gives the pixel size of the spot mpx.Motion patterns of shimmering (sh) were detected by the differences in pixel luminance (lumsh) of the direction of the shimmering wave was calculated , the positions of head and abdominal tip were interactively determined by mouse clicks  as their topological projections even under weak contrast. The momentary projected direction of the flight path (θw) at the time point t [fi] was calculated by determining the displacement of the thorax point of the wasp per frame interval (Eq. The wasps (w) typically hovered and scanned at an average distance of 50 cm from the honeybee nest  and those of the shimmering waves .w and sh) as a function of a time lag applied to one or the other of them. This method considers both counterparts at every time point in two roles: (a) as the driving signaller, which provokes the other party’s response, and (b) as the driven responder, which reacts to the signaller after a specific time lag. In the continuous sequence of interacting signals, the individual traits of both participants were separated by their roles as stimulating (driving) cue and as driven response to this cue by cross-correlating both time series  using positive and negative time-lag filters. Therefore, cross-correlation of the two time series  delivers a measure for the level of interaction between initiating cue and driven response (abbreviated as cue → response), whereby the sign of the time lag determined the state as signaller or responder. Consequently, two aspects of interaction can be considered: (a) the course of the gravity centre of shimmering as response to the flight course of the threatening wasp, abbreviated further on as w → sh and (b) the wasp’s flight course as response to the propagation direction of the shimmering wave (sh → w).The interaction between wasps and shimmering bees was categorized by the similarity between the time series of the counterparts  was classified as ipsi-directional (I), contra-directional (C) or intermediate (im). Proper ipsi-directional coincidence is given for any time point ti by equity of the angles  and proper contra-directional coincidence is given by opposite angles (θsh = θw + 180°). For statistical purposes, we expanded this narrowly limited paradigm of coincidence by the range of Δ θA = 45°  with θsh = θw ± Δ θA and contra-directionality (DIRC) with θsh = θw + 180° ± Δ θA . For simplification, we categorized the alignment conditions  = cat θ [sh]–cat θ [w] and for the aspect of sh → w this difference is Δ cat θ (sh → w) = cat θ [w]–cat θ [sh]  per inter-frame interval. Two modes of alignment can be distinguished: (a) ipsi-directionality (abbreviated as xI or yI), if the movement components of both counterparts  changed within a single inter-frame interval with the same sign . (b) Contra-directionality (abbreviated as xC or yC), if the movement components differed in their signs . Each step of interaction represents one pair of alignment defined by the movement steps of both counterparts . This allows distinguishing ipsi-directional , two intermediate alignment conditions  and contra-directional .Per definition, two parties are randomly aligned if both movements equal to a Einstein , the matvalue  and sign (=qsign) of each of the directional components for every step of movement of both parties in interaction. A path of movement can be described by its heading factor h which refers to the statistical difference of the probabilities (P) between positive directions  and negative directions  over time according to Eqs. In this paper, we introduce a mathematical movement heading model which uses this split-component method on the basis of a doubly randomized algorithm (with 0.00 ≤ rnd (q) ≤ 1.00) to separately assign 0 and y0) at the time t0 and the heading factors for both directional components, the value of the subsequent movement step was defined by Δx = rnd [Δxvalue] and Δy = rnd [Δyvalue]. Its sign was determined by initializing functions  and a threshold algorithm regarding the time interval (Δt = ti−t0) which defines the sign of both directional components (Δc as Δx or Δy) as positive for rnd [Δcsign] < hc and negative for rnd [Δcsign] > hc. In the model, the theoretical position of both parties at the time point ti was calculated by integration of the x- and y-components according to Eq. Given the position of both parties  was positive in 75% of the cases (represented here by the time steps Δt) and it was negative in the complement of 25% of cases.For explanation, the heading factors hwalking parties can be described by the momentary values of their headings. Random alignment is given if the headings of each of them are zero (hx[w] = hx[sh] = hy[w] = hy[sh] = 0). In terms of the normalized distributions of alignment, these random walk conditions result in uniform rates of the alignment components (r [DIR1-4] = 0.25). Variation of the heading factors of the two parties, reflected in the probability histograms in Fig. S3B/Online Resource Directional alignment of two w and sh) display moving patterns which happen, topologically seen, on roughly parallel vertical planes  and only in four from eight angular cat θsh sectors .When wasps prey in front of a giant honeybee nest, they provoke shimmering waves  and, nonetheless, independent from the wasp’s movement direction . The wasp produced turning velocities of ωw and sh) can be particularly demonstrated if their alignment rates are viewed synchronously  were significantly higher than those of contra-directional alignment . Similar results are achieved for the broader comparison, with the expansion of ipsi- and contra-directionality by combinations of angular sectors  which was traced in singular sessions  and proved for the entirety of sessions investigated ; it refers to synchronous assessment (lag = 0) of all sessions (nss = 50) observed.Coincidence in the movement directions of both parties  by utilizing the principle that any reaction happens after any stimulus but with delay. Fast flyers, such as wasps and bees, display latency periods not shorter than 20 ms. Therefore, a time window of up to five frames (≡100 ms) will be sufficiently long to trace any responsiveness of each of the parties expressed by their motions. For the graphical summarization in Fig. sh → w) are displayed by positive time shifts (lag [w after sh] > 0), and the responses of the bees (w → sh) are displayed by negative time shifts (lag [w after sh] < 0). The results document ipsi-directional dominance (Did: RI > RC) in both aspects of interactions under all time-shift conditions investigated  whereas Did decreased slightly but significantly with increasing absolute lags.The streams of behaviours as documented in the Movies id [│lag│ > 0] with those under synchronous assessment as reference (ref D = Did [lag = 0]), separately for every session (nss = 50) for wasps and shimmering bees. The differences of the Did values between both lag conditions (ΔDid = Did [│lag│ > 0]–ref D) decreased with the absolute lag value , the ΔDid values differed slightly from the reference, whereas for wasps as responders (sh → w) the ΔDid values decreased much stronger with increasing time shifts  differed more strongly than with wasps as responders.We compared the values of Don nss = 0 for wasfts Fig. . Consequid values of each session to classify two categories of ipsi-directional dominance  displaying the supra-reference status of ipsi-directional dominance (supra-Did), whereas for ΔDid < 0 the alignment response was classified as sub-reference status (sub-Did). The rates of supra-Did traits  and the magnitudes of supra-Did values at bigger latencies . For that, four positional conditions were distinguished . The alignment rates were sorted into four directional (DIR1–4) and four positional (POS1-4) classes while for each experimental session (nss = 50) the total rate of inter-frame intervals was normalized as 1.0. The findings display preference of the wasps for specific positions at the nest, which probably results from site properties of the honeybee nest (and which would certainly differ with other nests). For the shimmering bees as responders , in which the wasp as stimulus was top-right (tr) to the gravity centre of the shimmering wave. Cross-referencing with the other aspect of interaction .The histograms Figs.  and 7b edefence hypothesis of shimmering has been proved for the aspect that shimmering waves have the potential to repel threatening wasps away from the nest  were distinguished into four DIR1–4 classes  whereby the rates decreased with increasing latency periods. The bees as responders (w → sh) aligned at a lower level . This gives evidence that the bees responded to all flight directions of the wasp with the same strength of alignment, conforming to the predictions of the defence hypothesis of shimmering. Conversely, the wasps as responders (sh → w) were more prone to horizontal than to vertical alignment with shimmering. This is displayed by the DIR xI yC graph in Fig. ipsi-directionality (DIR xI yI) and x-based contra-directionality .Using the split-component method see , the attes Figs.  and 7e. vel Fig.  under pa1-4], see Figs. χ2) criterion. In the simplest assumption that both parties (w and sh) equal in the headings  the best match will result at a heading of h = 0.34  were permutated stepwise. Figure S3D/Online Resource x[w] = 0.59, hy[w] = 0.67, hx[sh] = 0.62), for which the missing vertical heading component was determined at the maximum of the respective regression polynomial P[χ2] = f (hy[sh]) with hy[sh] = +0.154. Otherwise, the best-match permutation for the equity conditions of all partial headings  can be assessed according to the look-up table of Fig.S3/Online Resource x [w] = 0.18 and hy [w] = 0.34 and of the associated shimmering waves with hx [sh] = 0.24 and hy [sh] = 0.16.For the best match with the empirical data, the two pairs of headings  in alignment under synchronous assessment conditions . Such wasps usually hover or scan in front of the bees’ nest, preying on the stationary curtain bees or ambushing homing or departing forager bees in flight  are compared in a stimulus–response paradigm by considering latencies, also asymmetries between the alignment responses of shimmering bees and wasps were traced  is hidden behind the linen, HD-camera , two cameras for stereoscopic imaging , laser vibrometer , and infrared camera ; the cyan arrow (n2) marks a small second queen-less colony. (D) Close-up of the experimental Apis dorsata nest from the front; the wasp paper nest (E) was relocated behind it. (JPEG 19.2 MB)Fig. S1 Experimental site and setup.  Back view of the hotel in Sauraha  with the honeybee nest at the second floor; the cover of white linen protected the experimental site from sunlight, and guided the wasps from and to the relocated paper nest (positioned behind the yellow circles) but also to pass them along the experimental honeybee nest. (C) The experimental Online Resource 2w) and shimmering (sh). (A) Angular deviation between the direction of flight of the wasp  and the direction of the shimmering wave  assessed in two subsequent frames . Equity in directions of w- and sh-movements is given by θw = θsh. In the noted example, the direction of the shimmering wave was cat θsh = [8] and that of the wasp flight path was cat θw= [7]. For the aspect of interaction w → sh , both directions result in a deviation of the shimmering wave from the wasp’s flight direction (as reference: θref = θw) with Δ cat θsh-w = cat θsh - cat θw = [+1]; for the reverse aspect (sh → w), the deviation was Δ cat θw-sh = cat θw - cat θsh= [−1]. (B) Ipsi-directional alignment is displayed by the white sectors [+1],[0],[−1], contra-directional alignment by the black sectors [+3],[4],[−3],, the deviations Δ cat θref = [+2] or [−2] represent here intermediate alignment. These definitions are applied for both aspects  whereby ipsi-directionality is given by [cat θref -1] ≤ Δ cat θw,sh ≥ [cat θref +1] and contra-directionality by [cat θref +4 -1] ≤ Δ cat θw,sh ≥ [cat θref +4 +1]. (JPEG 2.52 MB)Fig. S2 Definitions of angular relations between the movements of wasp  to a straight walk with hx = hy = 0.50, where the movement components Δx, Δy were positive (directed to the right and upwards) in 75% of all cases (≡time steps), and negative (directed to the left and downwards) in the compliment of 25% of cases. (B) The rate histograms concerning the four categories of directional alignment  of both model parties . The best match with the empirical data of Figs. 1–4 patterns from the headings in the model. (D) Determination of the best match between the empirical data as displayed in the Figs. x[w] = +0.18, hy[w] = +0.34, hx[sh] = +0.24) while hy[sh] was varied (abscissa). The best match was here achieved at the maximum of the regression function (P[χ2] = f (hy[sh]); R2 = 0.9828) at the ordinate value of P[χ2] = 0. 968 corresponding with the abscissa value of hy[sh] = 0.17. (E) Best-match conditions (max P[χ2]) between the empirical data  Typical paths under headings ranging from High Resolution Image (TIFF 3.04 MB)Online Resource 4ipsi-directional dominance (Did) under time-shift conditions. (A) Graphical definitions under time-shift conditions with the difference between the rates of ipsi- directional and contra-directional alignment under synchrony conditions as the session-specific reference (ref D= ∆RI-C [lag=0]): supra Did ≡ ∆RI-C [│lag│>0] > ref D; sub Did ≡ ∆RI-C [│lag│>0] < ref D. (B) Rates of supra-Did values (R1 [supra Did]) which refer to the normalization with R1 [supra Did] + R1 [sub Did] = 1.0 per lag condition; e.g. the value R1 [supra Did] = 0.4 means that 40% of the sessions (nss = 50) show supra Did under the respective lag, and 60% show the complimentary relation of sub-reference Did . (C) Session-specific rates, given as means (full circles) and SEMs , regarding R2 [supra Did] and R2 [sub Did]; for explanation, the ordinate value R2 [supra Did] = +0.08 means that the rates of inter-frame intervals ; negative R2 [supra Did] values refer to the relative number of sub Did cases. The results show that the R2 [supra Did] values of w→ sh were larger  than that of sh → w at time shifts of more than 40 ms (│lag│= 3-4). (JPEG 2.59 MB)Fig. S4 Supra-reference and sub-reference ns nss = 0 show suOnline Resource 5Apis dorsata) nest . In the film, the wasp approaches the nest from the left side. A curtain of white linen was placed behind the bees’ nest  to urge the wasp to pass the bees’ nest when homing to or departing from the paper nest. The film documents 179 ff (≡3.58 s) showing the nest in a quiescent state before the wasp comes into the image at frame 52 . The incoming wasp provokes a wave propagating in up-right direction, which makes the wasp fly downwards and turn around. This initiates a second wave which propagates in up-left direction alike the wasp, which lastly disappears behind the linen curtain again at the left side of the image. (AVI 1.82 MB)Movie 1 A free-flying wasp provokes shimmering waves at the surface of a giant honeybee Online Resource 7Movie 3 The same episode as in Movie 2/ Online Resource 6 but only the moving objects (wasp and the shimmering bees) differential images are visualized (see Methods). The motion information is here segmented, so that differences in the original luminance between subsequent images (fps=50 Hz) with ∆ lum > 5 resulted in a white spot. The yellow line gives the contour of the nest. (AVI 821 kb)Online Resource 8blue as low values and red as the largest differences. (AVI 1.26 MB)Movie 4 The same episode as in Movie 3/ Online Resource 7, but without the tracking of the wasp’s flight, and the difference values of the original luminance between two subsequent images were not segmented by a threshold luminance value but coded by a rainbow scale with Online Resource 9Movie 5 The same episode as in Movie 4/ Online Resource 8, but with the tracking of the wasp’s flight. (AVI 1.35 MB)Online Resource 10Movie 6 The same episode as combined in the movies 1 & 4/ Online Resource 5–Online Resource 11Movie 7 The same episode as in Movie 6/ Online Resource 10, but with the tracking of the wasp’s flight. (AVI 2.28 MB)Online Resource 12Movie 8 The same episode as in movie 2/ Online Resource 5 but with slow motion . (AVI 8.91 MB)Online Resource 13Movie 9 The same episode as in movie 5/ Online Resource 9 but with slow motion . (AVI 8.80 MB)Online Resource 14Movie 10 The same episode as in movie 7/ Online Resource 11 but with slow motion . (AVI 7.83 MB)Below is the link to the electronic supplementary material.


Currently there is no mandatory labelling of allergens for non-pre-packed foods. Therefore, when buying products sold loose, consumers with food allergy rely on voluntary information provided orally by the staff or in a written form in the salesroom. The aim of this study was to evaluate whether the staff in bakeries is able to give advice regarding a safe product choice for food allergic consumers and to what extent bakery products, which are sold as “cow’s milk-free” by the staff, contain cow’s milk protein.®FAST MilkRIDASCREEN (RBiopharm)).Staff of 50 bakeries in four different neighbourhoods in Berlin was interviewed regarding selling non-pre-packed foods to customers with food allergy. Bakery products being recommended as “cow’s milk-free” by the staff were bought and cow’s milk protein levels were measured with ELISA ((In 30/50 bakeries (60%) the staff reported that they serve customers with food allergy at least once a month, in 12/50 bakeries (24%) at least once a week. Most of the staff  stated that they are able to advise food allergic consumers regarding a safe product choice. Altogether 73 “cow’s milk-free” products were sold in 44/50 bakeries to the study personal. Cow’s milk protein could be detected in almost half of the bakery products (43%), every fifth product (19.2%) contained > 3 mg cow’s milk protein.Most of the staff in Berlin bakeries felt confident in advising costumers with food allergy. However cow’s milk was detectable in a great number of bakery products which was sold by the staff as “cow’s milk-free”. In some products the quantity of cow’s milk protein exceeded an amount where approximately 10% of cow’s milk allergic children show clinical relevant symptoms.


PPARβ−/− mice have reduced metabolic activity and are glucose intolerant. We examined changes in bone and muscle in PPARβ−/− mice and investigated both the mechanism behind those changes with age as well as their response to exercise. Compared with their wild type, PPARβ−/− mice had an accelerated and parallel decline in both muscle and bone strength with age. These changes were accompanied by increased myostatin expression, low bone formation, and increased resorption. In addition, mesenchymal cells from PPARβ−/− had a reduced proliferation capacity and appeared to differentiate into more of an adipogenic phenotype. Concomitantly we observed an increased expression of PPARγ, characteristic of adipocytes. The anabolic responses of muscle and bone to exercise were also diminished in PPARβ−/− mice. The periosteal bone formation response to direct bone compression was, however, maintained, indicating that PPARβ controls periosteal bone formation through muscle contraction and/or metabolism. Taken together, these data indicate that PPARβ deficiency leads to glucose intolerance, decreased muscle function, and reduced bone strength. On a molecular level, PPARβ appears to regulate myostatin and PPARγ expression in muscle and bone, thereby providing potential new targets to reverse bone fragility in patients with metabolic disturbances.Fragility fractures are recognized complication of diabetes, but yet the underlying mechanisms remain poorly understood. This is particularly pronounced in type 2 diabetes in which the propensity to fall is increased but bone mass is not necessarily low. Thus, whether factors implicated in the development of insulin resistance and diabetes directly impact on the musculoskeletal system remains to be investigated.  Several cohort studies and meta-analyses have demonstrated that people suffering from type 2 diabetes mellitus (T2DM) have a higher risk of fracture despite their higher-than-average bone mineral density (BMD) –3. The s–PPARβ−/− mice are metabolically less active and develop glucose intolerance (–Transcription factors of the peroxisome proliferator-activated receptor (PPAR) family  are known to play an important role in energy balance. PPARγ and more recently PPARβ have been shown to be implicated in metabolic dysregulation. More precisely, a decrease in PPARβ expression results in decreased glycolysis and lipogenesis in the liver while increasing fat infiltration in skeletal muscle –13. Thus–olerance . Conversolerance –17. Henclerance –.PPARβ−/− mice have recently been reported to have increased bone turnover and decreased bone mass  and have been previously described  at baseline and at 15, 30, 60, 90, and 120 minutes after an ip injection of glucose at 2 g/kg body weight. Blood was obtained after an incision at the base of the tail.In vivo hindlimb grip strength was measured using an automated grip strength  as previously described . Each moPPARβ−/− and PPARβ+/+ mice. The mice were trained 5 days per week for 6 weeks. The exercise protocol was determined and adapted to the maximal capacity of PPARβ−/− mice to perform an exercise on a treadmill: at a speed of 16 m/min, for 30 minutes, with an inclination of 5%. During the first acclimatization week, the treadmill speed and the duration of each running session were gradually increased from 8 m/min for 10 minutes to 16 m/min for 30 minutes. For the last 5 weeks, the running sessions consisted of 16 m/min for 30 minutes with a treadmill inclination of 8°, corresponding to moderate exercise at this specific age  were measured in vivo by dual-energy X-ray absorptiometry  at 12 and 18 weeks of age in the exercise experiment; at 13 and 16 weeks of age in the axial compression experiment; and at 1, 4, 12, and 18 months for the aging experiment. Lean limb mass was evaluated by positioning the region of interest perpendicular to the vertebral column and with the corner of the region of interest aligned to the anterior margin of the hip.−1), trabecular connectivity density (millimeters−3), and structural model index. For the cortical bone at the femoral and tibial midshaft, we measured the cortical tissue volume , cortical bone volume , the marrow volume (cubic millimeters), and the average cortical width (micrometers). To evaluate bone marrow adiposity tibias, we processed protocol with osmium staining as described  was used to assess the trabecular microarchitecture in the excised fifth lumber spine body and distal femur and cortical bone geometry at the midshaft femoral diaphysis as previously described . Trabecuescribed . After lThe night before the mechanical testing, bones were thawed slowly at 7°C and then maintained at room temperature. A three-point bending test was performed on the femur as follows: bones were placed on the material testing machine on two supports separated by a distance of 9.9 mm, and the load was applied to the midpoint of the shaft. The mechanical resistance to failure was tested using a servocontrolled electromechanical system  with the actuator displaced at 2 mm/min. Outcomes measured were ultimate force, stiffness, and energy as described previously .After 48 hours of fixation, bones were dehydrated in absolute acetone and embedded in methyl-methacrylate (Merck), and 8-μm-thick transversal sections of the midshaft were cut with a Leica Polycut E microtome (Leica Corp Microsystems AG) and mounted unstained for the evaluation of fluorescence. Five-micrometer-thick sagittal sections were stained with modified Goldner's trichrome, and histomorphometric measurements were performed on the secondary spongiosa of the proximal tibia metaphysis and on the endocortical and periosteal bone surfaces in the middle of the tibia using a Leica Q image analyzer (Leica Corp) at ×40 magnification. All parameters were calculated and expressed according to standard formulas and nomenclatures : mineralSupplemental Material). Relative quantities were calculated with the formula, relative quantity = E − cycle threshold using an efficiency (E) of 2 by default. For each gene the mean quantity was calculated from triplicates for each sample, and this quantity was normalized to the similarly measured mean quantity of the glyceraldehyde-3-phosphate dehydrogenase normalization gene. Finally, normalized quantities were averaged for three to four animals.The whole tibia was excised, and both tibial extremities were cut to remove the bone marrow from the diaphysis by flushing with cold PBS. Muscle gastrocnemius was frozen by dipping them on liquid nitrogen. Muscle and tibial diaphysis plus extremities were immediately pulverized to a fine powder and homogenized in peqGold Trifast (peQLab Biotechnologie GmbH) using FastPrep system apparatus (QBiogene). Total RNA was extracted and then purified on minicolumns  in combination with a deoxyribonuclease treatment  to avoid DNA contamination. Single-stranded cDNA templates for quantitative real-time PCR analyses were carried out using SuperScript III reverse transcriptase (Invitrogen AG) following the manufacturer's instructions. Quantitative RT-PCR was performed using predesigned TaqMan gene expression assays  digestions of calvaria from 2- to 3-day-old PPARβ+/+ or PPARβ−/− mice, half issued from male and half from female pups. Cells from the third to fifth digestions were pooled and cultured in αMEM (Gibco), supplemented with 10% fetal calf serum (Amimed), antibiotics , glutamine , amino acids (Amimed), and amphotericin B . Parameters measured included cell proliferation, differentiation, and relative gene expression of osteoblast and osteocytic markers .Primary osteoblast cultures were isolated from PPARβ−/− and PPARβ+/+ adult mice. For this purpose, after removing soft tissue and epiphysis, bone marrow was flushed with DMEM medium. Marrow was then submitted to a 15-min enzymatic digestion. Collagenase activity was neutralized and marrow was then resuspended in αMEM (Gibco) supplemented with 10% fetal calf serum (Amimed), antibiotics , glutamine , amino acids (Amimed), and amphotericin B . The cells were plated at 106/cm3 cells in a T75 flask and incubated at 37°C with 5% CO2, and the media were changed every 3 days until the cells reached 80% confluence. Outcomes were the quantification of MSC PPARβ−/− and PPARβ+/+ differentiation into adipocyte and osteoblast populations. For adipogenesis, when MSCs reached 100% of confluence, adipogenesic media  were added. After 3 days of the adipogenic medium, cells were returned to a medium with DMEM, l0% fetal calf serum, l μm dexamethasone, and 5 pg/mL insulin that was refreshed twice per week. After 14 days in culture, adipocytes were visualized by oil red staining and quantified at 540 nm on a spectrophotometer. For osteoblastogenesis, when the MSCs reached 100% of confluence, osteogenic media  were added and replaced twice per week. After 14 days in culture, calcium depositions were visualized by alizarin red S staining and quantified at 405 nm on a spectrophotometer.Mesenchymal stem cells (MSCs) were isolated from the femurs of Blood from all mice was obtained by a submandibular collection at 1 and 18 months of age. After centrifugation, serum was removed and stored at −80°C until analysis. Serum carboxy-terminal collagen cross-links and osteocalcin were measured according to the manufacturer's instructions . Myostatin was measured in serum by an ELISA with a kit from United States Biological Corp) following the manufacturer's instructions.PPARβ−/− and PPARβ+/+) by paired or unpaired t tests. In the mechanical loading experiments, we compared stimulated and nonstimulated tibia in the same animal using a paired t test. For the exercise investigation, we compared sedentary vs trained animals using unpaired t tests. To compare the effect of genotype and the response to loading , we used a two-way ANOVA. As appropriate, post hoc testing was performed using Fisher's protected least squares difference. The P value of interaction between the genotype and loading  was mentioned only when it was found to be significant. We then tested the effects of repeated measures within groups (for the effects of aging) by a one-way repeated-measures ANOVA with the genotype used as a factor. Differences were considered significant at P ≤ .05. Data are presented as mean ± SEM.We first tested the effects of exercise or loading within groups . We found the same trend at the age of 12 months but the difference was not statistically significant.First, we confirmed that PPARβ−/− and PPARβ+/+ mice, indicating that PPARβ deficiency did not affect embryonic or early postnatal development. Between 1 and 12 months of age, total and limb lean mass gains were lower in PPARβ−/− vs PPARβ+/+ . Maximal running distance was significantly reduced in old PPARβ−/− vs PPARβ+/+ mice, whereas the maximal speed was unchanged . IGF-1 expression was not significantly different between PPARβ−/− and PPARβ+/+ mice. BMD gain was unaffected by PPARβ deficiency until 4 months of age. However, by 12 months of age, total body and femur BMDs were significantly lower in PPARβ−/− vs PPARβ+/+  , whereas the bone-forming indices were lower, particularly at the periosteal surfaces at a latter age , indicating that bone turnover and coupling is altered in the absence of PPARβ. In addition, we found higher circulating levels of myostatin in PPARβ−/− mice compared with PPARβ+/+ mice.Age-related changes in trabecular microarchitecture followed a similar pattern in the distal femur and caudal vertebrae, although in the femur the loss of BV/TV occurred earlier and was more dramatic A. At theP < .05) C. In the+/+ mice , D and E+/+ mice F. These tter age , G–J. CoPPARβ−/− mice compared with PPARβ+/+ mice at 4, 12, and 18 months . Expression levels of genes associated with osteoblast and osteocyte differentiation, as well as Foxo1 and Hmox, were significantly altered in PPARβ−/− mice , and Tnsf11b (Opg) gene expression was decreased  and an increased propensity to differentiate into adipocytes  and to a lesser extent in PPARβ−/− mice .Exercise increased body weight, limb lean mass, and force in −/− mice , A–C. Ex−/− mice , H and IPPARβ−/− was secondary to the muscle dysfunction and/or could be explained by an intrinsic failure of the bone mechanotransduction. To this aim, mice were subjected to direct axial compression of the tibia in vivo for 2 weeks. Contrasting with exercise, axial compression of the tibia significantly increased BMD and CtBV in both PPARβ+/+ and PPARβ−/− mice . Interestingly, mechanical deterioration occurs before cortical structure changes are observed, consistent with the notion of early alterations in the material properties of the bones in these mice. Second, exercise was not able to improve muscle and bone parameters in PPARβ−/− mice are metabolically less active and glucose intolerant from young adulthood to older age . Thus, increased myostatin expression in PPARβ-deficient mice may at least partly explain their poor muscle strength.First, PPARβ deficiency not only alters the proportion of type II/type I fibers and muscle oxidative function  but also31–Opg expression and osteoclastogenesis through the β-catenin signaling pathway , and/or other myokines, known to be involved in the musculoskeletal system in response to exercise (Igf-1 is not significantly affected; however, Bmps and Mstn are expressed, respectively, less and more in PPARβ−/− mice compared with their wild-type littermates. Thus, further studies aimed at investigating whether the use of a myostatin inhibitor or BMP treatment can restore the bone response to exercise of PPARβ−/− mice are warranted.Third, exercise was unable to restore muscle and/or bone mass and strength in diabetes , 45 and diabetes , 47. In pression , 49, wittructure . Alternaexercise , 52. In PPARβ−/− mice, two major hypotheses are suggested. First, the absence of PPARβ may alter the structure of the bone matrix, particularly at the endocortical surface, and therefore impair its mechanotransduction properties, as we previously reported for the periosteal surfaces in periostin-deficient mice (Runx2 and increased PPARγ expression, respectively (To potentially explain the absence of bone formation at the endocortical surfaces in loaded ent mice . Second,ent mice , 14. Theectively , two traPPARγ levels in muscle and bone, thereby providing potential new mechanisms to explain bone fragility related to metabolic syndromes and potentially identifying new molecular targets for the treatment of muscle and bone fragility in diabetes.In conclusion, the absence of PPARβ leads to three major systemic changes, namely glucose intolerance, decreased muscle strength, and exercise capacity, together with low bone formation and response to exercise. At the molecular level, PPARβ appears to regulate myostatin and


N1), dimensionless axial magnetic force strength parameter (N2), dimensionless tangential magnetic force strength parameter (N3), and magnetic Reynolds number (Rem) are illustrated graphically and discussed in detail. Applications of the study include automotive magneto-rheological shock absorbers, novel aircraft landing gear systems and biological prosthetics.In the current article, a combination of the differential transform method (DTM) and Padé approximation method are implemented to solve a system of nonlinear differential equations modelling the flow of a Newtonian magnetic lubricant squeeze film with magnetic induction effects incorporated. Solutions for the transformed radial and tangential momentum as well as solutions for the radial and tangential induced magnetic field conservation equations are determined. The DTM-Padé combined method is observed to demonstrate excellent convergence, stability and versatility in simulating the magnetic squeeze film problem. The effects of involved parameters, i.e. squeeze Reynolds number ( Understanding magneto-hydrodynamics (MHD) is strongly related to the comprehension of physical effects which take place in MHD. When a conductor moves into a magnetic field, electric current is induced in the conductor and creates its own magnetic field (Lenz’s law). Since the induced magnetic field tends to eliminate the original and external supported field, the magnetic field lines will be excluded from the conductor. Conversely, when the magnetic field influences the conductor to move it out of the field, the induced field amplifies the applied field. The net result of this process is that the lines of force appear to be dragged accompanied by the conductor. In this paper the conductor is the fluid with complex motions. To understand the second key effect which is dynamical we should know that when currents are induced by a motion of a conducting fluid through a magnetic field, a Lorentz force acts on the fluid and modifies its motion. In MHD, the motion modifies the field and vice versa. This makes the theory highly non-linear , 2.et al.  left unchanged, the following normalization condition is imposedThe Páde approximant is a rational fraction and the notation for such a Padé approximant is:(x)QM(x),where PL + 1 independent numerator coefficients and M independent denominator coefficients, which make L + M + 1 unknown coefficients in all. This number suggests that normally the [L/M] ought to fit the power series Eq , g (k), m (k) and n (k) are the differential transforms of f (η), g (η), m (η) and n (η) are displayed by:α, β, γ, κ and ω are constants. By substituting Eq (f (η), g (η), m (η) and n(η).MATHEMATICA software. The Padé approximant is employed to extend the convergence radius of the truncated series solution. As it is illustrated in Figs α, β, γ, κ and ω.Taking differential transform of Eqs –11), on, on11), 0,n(1)=ω,where α,r+2)))=0,(k+1)(k+r+1)))=0,where f 1)(k+2)k+(k+4)f=1+γ3ω))η5+⋯,where thN1), magnetic Reynolds number (Rem = N1Bt), dimensionless axial magnetic force parameter (N2), dimensionless tangential magnetic force parameter (N3) on the velocity and induced magnetic field components in the gap between the disks and also on the torques at the lower and upper disk. Computations are performed for the evolution of velocity components  and induced magnetic field components  with dimensionless axial coordinate (η). Representative values are used to simulate physically realistic flows. dg(0)/dη) and upper disk (dg(1)/dη) for various values of squeeze Reynolds number (N1) and axial magnetic force number (N2).The nonlinear ordinary differential equations subject to the boundary conditions are solved via DTM-Padé method for some values of the four key parameters i.e. squeeze Reynolds number (Rem) on the axial and tangential velocity distributions and induced magnetic field distributions . The magnetic Reynolds number defines the ratio of the fluid flux to the magnetic diffusivity. This parameter therefore is instrumental in determining the diffusion of magnetic field along streamlines and is analogous to the classical Reynolds number in viscous hydrodynamics, the latter controlling the vorticity diffusion along the streamlines. When N1 is large this implies fast vertical velocity of the upper disk and vice versa for small values of this squeezing parameter. The variation in Rem has almost little effect on the axial velocity distribution. As it is obvious from g arises at the lower disk (η = 0) for all cases. In other word, the azimuthal velocity of the fluid decreases as we move from the lower disk towards the upper disk. In addition, the tangential velocity distribution is a decreasing function of magnetic Reynolds number. With an increase in Rem from 1, 2 through to the maximum value of 10, there is a strong decrease in the axial induced magnetic field component (m) and tangential induced magnetic field component (n). For all cases the maximum values of m and n arise at the upper disk (η = 1). It is to certify that for higher values of Rem the squeeze film must possess much higher electrical conductivities.Figs N2) on the axial and tangential velocity distributions and induced magnetic field distributions  are illustrated in Figs N2 is large this means fast vertical velocity of the upper disk and vice versa for small N2. It can be seen that an increase in N2 from 0 to 5 induces a significant decrease in the axial velocity between the disks. The tangential velocity distribution (g) is also directly affected by an increase in squeeze Reynolds number. As the axial component of the magnetic force N2 increases, the azimuthal velocity g increases. Hence, the axial magnetic force N2 can be used to increase the azimuthal velocity of the fluid. The increase of g with N2 agrees with the results obtained by Elshekh and Elhady [N2 will be dominant Ω1. As the results present, an increase in the dimensionless axial magnetic force parameter causes to decrease the axial induced magnetic field distribution (m) and increase in the tangential induced magnetic field distribution (n). The tangential velocity profile and tangential induced magnetic field also become significantly more curved with higher N2 values. The current results are an expected result since the normal component of the induced magnetic field between the two disks must increase with the increase of the normal component of the external applied magnetic field.The effects of dimensionless axial magnetic force parameter (d Elhady  and Hamzd Elhady . The effN3) on the tangential velocity distribution (g) and tangential induced magnetic field distribution (n), respectively. The tangential velocity distribution slightly increases with the increases in the dimensionless tangential magnetic force parameter from 0.5 to 5. Increasing the dimensionless tangential magnetic force parameter causes to decreases the tangential induced magnetic field distribution.Figs The present study has displayed novel DTM-Padé solution, the combination of differential transform method and Padé approximation, for two nonlinear magneto-hydrodynamic squeeze film problems. Applications of the study include in automotive magneto-rheological shock absorbers, novel aircraft landing gear systems and biological prosthetics. The transformed dimensionless equations have been formulated and solved with robust boundary conditions. Exceptional stability and convergence characteristics have been demonstrated with the DTM. The physical key parameters emerging have been investigated graphically in detail including dimensionless axial magnetic force strength parameter, dimensionless tangential magnetic force strength parameter and magnetic Reynolds number. The results illustrated that the tangential velocity distribution is a decreasing function of magnetic Reynolds number. Further, the tangential velocity distribution is directly affected by an increase in squeeze Reynolds number. In addition, an increase in the dimensionless axial magnetic force parameter causes to decrease the axial induced magnetic field distribution and increase in the tangential induced magnetic field distribution. Moreover, increasing the dimensionless tangential magnetic force parameter causes to decreases the tangential induced magnetic field distribution.


Influenza virus contains two highly variable envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Here we show that, while HA evolution is much more complex than NA evolution, it still shows abrupt punctuation changes linked to punctuation changes of NA. HA exhibits proteinquakes, which resemble earthquakes and are related to hydropathic shifting of sialic acid binding regions. HA proteinquakes based on shifting sialic acid interactions are required for optimal balance between the receptor-binding and receptor-destroying activities of HA and NA for efficient virus replication. Our comprehensive results present a historical (1945–2011) panorama of HA evolution over thousands of strains and are consistent with many studies of HA and NA interactions based on a few mutations of a few strains. A previous paper  showed tHow large must a protein be to be “sufficiently large” for long-range forces to dominate its functionality? Methods based on short-range interactions  within Euclidean structures have emphasized chemical-protein epitope interfaces that are usually limited to 15–20 amino acids, although recently these have been expanded in yeast-based deep sequencing studies to 50 amino acids [N + 1  (4 < N < 17) in the bioinformatic survey of 5526 PDB segments, as expected from SOC and Darwinian evolution [Short-range packing interactions die out for amino acid sequences longer than 9, leaving mainly water-protein roughening interactions at longer range. The packing interactions are so strong that the energies associated with them change little with tertiary conformations. The long-range dominance of hydropathic interactions is the reason that the evolution of solvent accessible surface areas follows power laws for each centered amino acid in segments of length 2N + 1   < N < 17volution .W = 111. On an historic scale, going back to the 1918 pandemic, these HA1 changes are large and dramatic, as shown in As noted in , most ofW = 111; this choice of W will be discussed below. The various evolutionary punctuations of companion NA sequences [The 565 amino acids of compacted HA exhibit a rich Euclidean structure composed of two chains, HA1 and HA2 . The twoequences .Uniprot lists nine glycosylation sites in A3DRP0, Memphis/10/1996 HA1  and only one on HA2 (498). Glycan microarray analysis has revealed glycosylation differences between either remote H1N1 strains  at a few sites . The mulBefore we leave the 1976 New Jersey outbreak, we can use it to make a simple connection between HA and NA mutations. As noted in the abstract of [There are about 30 HA 1934 Puerto Rico sequences in Genbank, with an overall 99% identity. If we use BLAST to track subsequent evolution of these sequences , we find a few intermediate sequences from Melbourne  and then 1947 New Jersey (90%) and 1976 New Jersey (85%). Thus (as expected) there has been much more HA drift than NA drift. However, much of the HA drift is due to frequent aa mutations, so that HA positives (as defined by BLAST) are still 91% between 1934 Puerto Rico and 1976 New Jersey.RKD(Wmax) and RMZ(Wmax) with Wmax = 17 of the water packaging film with two scales, KD and MZ. These roughnesses  showed well-defined plateaus connected by punctuated decreases (vaccination programs) or increases (migration).  for NA for the two scales is around 90%.) The superior NA resolution of the MZ scale has led us to report HA results here only for the MZ scale. The sliding window width used for calculating the NA roughnesses was set at Wmax = 17, although 21 (closer to the transmembrane thickness) would have been equally good. This width was also used in displaying chain hydroprofiles 〈ψ(j)W〉. Note that W functions as a modular length, which appears naturally in the model by optimizing its resolution.The methods used here are almost the same as these used in  for NA bW = 17 does not occur for HA. Instead, at each punctuation of NA, the HA chain hydroprofile 〈ψ(j)W〉 exhibits hydrophobic stabilization over selected blocks whose edges tend to coincide with one or both of the edges of the sialic acid binding site 130–230. Because this site is so wide, we have chosen to display hydroprofiles 〈ψ(j)W〉 with W = 111. To test this choice, we selected several HA sequences from Hawaii 2007. As was shown in [RMZ(17) are large  [RMZ(1) differences are only 1.5% of RMZ(1), which is only 2∑, where ∑ is the sum of the σ's of each subset [RMZ(1) differences. Their 〈ψ(j)111〉 chain profiles exhibit a striking sign difference reversal near 180, presumed to be associated with a change in sialic acid binding . This shows that W = 111 is an excellent choice for HA1 sliding window width, as expected from its similarity to the length of the sialic acid binding site. An interesting point is that there is excellent correlation (87%) of R(111) for HA1 for the MZ and KD scales for the period 1918–2001, but this correlation breaks down with the advent of swine flu.The historical 1945-) directed evolution of roughnesses towards smaller values found for NA with 945- dire binding . A smallW, as well as the dominance of overall interactions with sialic acid on the length scale W ~ 111, explains why the details of glycosylation interactions on the length scale of glycosylation spacing  are important only for large-scale strain differences. A survey of 50 immunodominant epitopes  spanning HA Calif 04/2009 revealed a distinct subset that had nearly equal autoantibody interactions almost twice as strong as the remaining epitopes [−7. Note that the strong interactions were equally strong for all four cases. This is what one expects, as the two kinds of extrema are weighted equally in calculating the variance, which does not depend on the sign of the deviation from average. However, the equal weighting is surprising in Euclidean terms, as the hydrophobic/hydrophilic epitopes are on the inside/outside of the globular cluster, and inside/outside are not obviously equivalent. Finally, one of the sites identified as significant by glycan microarray analysis [The large value of epitopes . There aanalysis  is 190, ψMZ averages = 〈ψMZ〉 nearer 155  than HA. This is shown in σ from Hawaii 2011. Here for HA we find systematic parallel motion of 〈ψMZ〉 and 〈ψKD〉 with peaks at Fort Dix (New Jersey 1976) and swine flu (2009), as shown in ψMZ〉 and 〈ψKD〉 in a year where there was little activity (we used 1996) and using strains widely dispersed geographically.Perhaps the simplest difference between HA and NA profiles is the fact that NA profiles have overall σ with the Fort Dix outbreak hydrophobic peak and successive outbreaks of the swine flu pandemic , we find that the 〈ψ〉 peaks are ασ above background, with α ~ 4 for the MZ scale and ~3.5 for the KD scale. This is certainly an impressive resolution, but can we not do better? Can we not connect these average changes to the chemistry of HA?In We can. We return to ψ(j)111〉 HA1 chain profiles for the 1976 migration-vaccination punctuation are shown in RMZ(17) roughness and an even larger increase for RKD(17) [The expanded 〈RMZ(17) occurred in connection with the vaccination program of the American army began in 1944, and its benefits were largest in the Netherlands 1954 sequence [The first punctuation (A-B) of NA RMZ7 occurreWhat does this precursive pattern mean? It is mysteriously similar to an earthquake with a precursor and an aftershock, but that is not so mysterious after all . If protThe 1989–2003 NA smoothing gain ended with the advent of the “swine flu” strains, which appeared in successive outbreaks, first in Hong Kong 1999 and next in New York in 2003 and in Berlin in 2005. The characteristic HA feature of these strains was a large hydrophobic increase in the N terminal sub-130 block, which was almost identical for New York and Berlin . These hAs shown in With a larger data base, we can study the response to the 2007 human swine flu vaccination in one locality, which was done for North Carolina and for Norway. The results for North Carolina see  show a lThese large jumps in HA block hydropathicity are mediated by strings of single mutations, which were not seen in NA. Also NA contracted slightly and stiffened in response to the swine flu vaccination program, while HA expands and softens, another example of NA-HA “balance” . One is W = 111 block hydropathic roughnesses have a secondary effect on NA at W = 111? This question is addressed in W = 17 NA peak or precede the 2007 swine flu peak. In other words, HA evolution may be coupled to NA evolution but not in a simple way. This point is brought out in (b), which shows only one peak, placed between the two HA peaks. There appears to be some measure of complementarity of HA and NA evolution on the sialidase W = 111 scale. Overall we recognize that H1N1 HA and NA evolution is dominated by at most two length scales associated with N-glycans and sialic acid. Because our analysis is hierarchical (it distinguishes between increasing and decreasing roughness) and includes detailed quantitative aspects of sequence evolution on large length scales not accessible to similarity analysis, it could be useful in refining binding data [Could HA ing data , 21. Ouring data .Both here and in our previous paper on NA , we haveW ~ 20 as a configuration coordinate revealed opposing punctuations due to migration (increasing roughness) and vaccination programs (increasing smoothness) [W ~ 100. The HA1 profiles have a characteristic broad and deep (V-shaped or butterfly) hydrophilic minimum in the sialic acid binding region 130–230 and are strongly hydrophilic. The overall evolution of HA1 shows primarily shifts in hydropathicity of three chain blocks, 50–130, 130–230, and 230–300. These punctuated block shifts closely resemble earthquakes [The analysis of NA using hydropathic roughness with othness) . Here wethquakes –17 in onthquakes  of shortThe central limitation of prior studies of viral kinetics has been their low resolution, limited to the large N-glycan spacing length scale , 11 or tπ-like sliding water package conformational dynamics, not σ-like backbone compression. Finally, the HA analysis leads to a block model of the HA water packaging structure that includes HA fractures  . Mechanioprotein . It is poprotein , 31.Readers who wish to connect the methods used here to those of modern mathematics could consult , especia