The extent to which ADRβ2 polymorphisms and in particular their haplotypes contribute to severe asthma is unknown.ADRβ2 polymorphisms and haplotypes with asthma severity.To determine the association of ADRβ2 polymorphisms. The cohort  included 2296 non-asthmatics, 386 mild asthmatics, 172 moderate asthmatics and 125 severe asthmatics. Haplotype frequency and haplotype pair for each subject was determined using the PHASE algorithm.Caucasians (n = 2979) were genotyped for 11 per se. Haplotype pair 2/4 was associated with asthma severity  but not with asthma per se. Prevalence of haplotype pair 2/2 appeared to decrease with asthma severity . Two new haplotypes were identified, occurring exclusively in asthmatics at a frequency of ≥ 1%. In addition, a positive association between carriage of ADRβ2 +523*C and increased risk of atopy was discovered.The three asthmatic cohorts were comparable in age and gender but were distinguishable from each other in terms of symptoms, spirometry, medication use and health care utilisation (p <0.001). None of the polymorphisms showed a genotypic or allelic association with asthma diagnosis or severity. Nine haplotypes were identified and no association was found with asthma diagnosis or severity ADRβ2 haplotype pair 2/4 is associated with severe asthma and is consistent with findings of poor bronchodilator response in mild asthmatics who are also haplotype 2/4. Asthma is a common inflammatory airways disease affecting 12% of adults in the Australian population. Approximately 10% of these have severe persistent disease and remains poorly controlled despite maximal therapy. Response to medications varies significantly between individuals. Although medication adherence is a significant issue, it is estimated that up to 70% of variability in therapeutic responses to pharmacotherapy is genetically based.ADRβ2) agonists are the most commonly prescribed and used inhaled asthma therapy. Their efficacy is partially dependent upon their molecular conformation and properties and the density, structure and conformation of the ADRβ2 on the cell surface. The expression/regulation of ADRβ2 is altered by several single nucleotide polymorphisms (SNP) within the promoter, coding and the 3′UTR domains. ADRβ2+46*G (Gly16) results in increased agonist-stimulated receptor down-regulation while ADRβ2+79*G (Glu27) is highly resistant.ADRβ2+491*T (Ile164) alters receptor binding affinity with reduced activation in vitroin vivo responses to terbutaline in healthy and cardiac failure subjects. β2 adrenergic receptor  in mild asthmatics homozygous for ADRβ2+46*A is associated with adverse outcomes.Clinical studies in mild asthmatics have shown differential clinical response attributable to genetic variation. ADRβ2 polymorphisms and asthma have almost exclusively involved those with mild or moderate disease while the significance of genetic variation in severe asthma remains unclear.ADRβ2 polymorphisms and severe asthma produced contradictory results.ADRβ2+79*G (odds ratio 1.91) 1 and tachyphylaxis with formoterol in ADRβ2+46*G homozygotes ADRβ2+46 or +79 genotype in fatal or near fatal asthma.ADRβ2+46 and +79) per se but rather requires analysis of rarer variants and haplotypes.Larger pharmacogenetic studies on ADRβ2 gene dictates that alleles at different locations of the ADRβ2 gene with opposing in-vitro effects are commonly co-inherited as a haplotype and the outcome of their interaction is hard to predict. Receptors with ADRβ2+79*G polymorphism are highly resistant to agonist-induced downregulation in vitro yet co-inheritance of ADRβ2+46*G favours ADRβ2+46*G induced down-regulation.ADRβ2 haplotypes are assessed in addition to individual genotypes.ADRβ2 gene derived from the maternal and paternal copy of chromosome 5. This in effect means that ADRβ2 gene is inherited, not simply as genotypes or single haplotype, but as a haplotype pair  Asthma Genetic Cohort. Non-asthmatic controls (n = 200) were recruited through contacting individuals randomly selected from the metropolitan telephone directory. Mild, moderate and severe asthmatic cohorts were similarly recruited but enriched by recruiting from specialty asthma clinics and hospital databases.1 % predicted, use of oral steroids in the last 12 months, daily inhaled corticosteroid dose, weekly frequency of use of rescue medication, weekly frequency of daytime symptoms, weekly frequency of nocturnal awakening due to asthma, unplanned visits to doctor in the last 12 months and hospital admissions for asthma in the last 12 months.1 greater than 80% predicted), frequency of daytime or night-time symptoms and use of rescue medication use less than twice per week with no unplanned medical visits or hospitalisations in the previous 12 months while treated with low dose inhaled corticosteroid therapy. Severe refractory asthma patients had to meet the American Thoracic Society (ATS) Criteria Asthma (n = 683) was defined as having a respiratory physician-diagnosis of asthma based upon clinical history, spirometry, salbutamol reversibility and/or positive methacholine challenge tests. Mild and moderate asthma were defined as published previously using a number of criteria including lung function  underwent spirometry in accordance with the ATS guidelines as well as allergen skin prick testing to 5 common aeroallergens: cat, dog, house dust mite, mould mix and grass pollen mix. Atopy was defined as positive skin reaction  to at least one allergen. The study was approved by the Human Research Ethics Committees at Sir Charles Gairdner and Royal Perth Hospitals. All subjects provided written consent to all study procedures including allergy and genetic testing.To improve the power to detect a true genetic association, we expanded the non-asthmatic LIWA cohort to include 2096 unrelated Caucasians ascertained by QIMR and included in a recent GWAS of asthma.ADRβ2 gene . A total of 11 SNPs of the Rβ2 gene  were genADRβ2 haplotype and haplotype pairs for each individual patient were inferred from the genotype data using the PHASE program. (v2.0). The PHASE prediction algorithm was run several times to ensure that the result was reproducible. The number and characteristics of haplotypes with frequency > 1% remained consistent. For each individual, only the most likely HP (predicted with over 80% accuracy) was considered in the final analysis.Patient characteristics are expressed as mean with standard deviation (SD), unless otherwise stated. Demographic data and clinical parameters were compared between all groups using ANOVA and Tukey's test or where pooled data was used, Student's t-test for continuous data or with Chi square or Fishers' exact test (where appropriate) for categorical data.Genotypic and allelic frequencies were calculated for each patient group stratified by asthma severity or atopy. Univariate analysis was then performed using chi-square tests and logistic regression analysis using SPSS v18.0 . Power analysis showed that a difference of 10% in allelic and genotypic frequency could be detected, with 80% power and Bonferroni-corrected p-value of 0.05.Frequency of the 5 most common HPs were calculated for mild, moderate and severe asthma group. The differences were tested using the odds ratio method where only a more stringent p value of 0.01 was considered statistically significant (Bonferroni-corrected). The prevalence of the HPs in the three asthma cohorts were also assessed using a logistic regression trend test performed on SAS v9.2  to further support the association with asthma severity.The overall demographic and clinical characteristics of the study cohort are summarised in The severe, moderate and mild asthmatics were distinguishable in all clinical parameters including frequency of symptoms, lung function, medication use and health care utilisation  included 1248 non-asthmatics with mean age 31  and 848 individuals  with no available asthma information that were considered as unselected controls to improve power.ADRβ2+46*A and ADRβ2+79*C in the entire study cohort was 0.36 and 0.56, respectively. ADRβ2+491*T was only seen in the heterozygous form in 13 asthmatics  and 37 non-asthmatics. ADRβ2 SNPs frequencies were comparable for all study groups and were within Hardy-Weinberg equilibrium  for 2′ and one 5′UTR SNP (ADRβ2-1023) for 4′. These two new haplotypes were found exclusively in asthmatics , followed by 2/2 (19.3%), 2/6 (14.8%), 4/4 (12.2%), 4/6 (10.9%) and 6/6 (3.0%). There was no association between any HPs and asthma diagnosis per se . Howeverper se . The preper se . In compper se . There wThe association with atopy was also present but not significantly strengthened in the relevant haplotype analysis inclusive of the +523CC genotype. Prevalence of atopy was also similar amongst the 6 most common haplotype pairs. .ADRβ2 haplotypic features. Given the heterogeneity in asthma pathogenesis and clinical manifestations, not unexpectedly, ADRβ2 polymorphisms were not associated with asthma diagnosis or severity when individual SNPs or haplotypes were examined in isolation. However the frequency of the most common haplotype pair 2/4 was significantly higher in severe asthmatics with a raised odds ratio compared with mild asthmatics. The potential role for this ADRβ2 haplotype pair in the pathogenesis of severe asthma was further supported by a significant, positive trend in the prevalence of haplotype pair 2/4 with increasing asthma severity. In addition, carriage of ADRβ2+523*C was associated with increased risk of atopy.This is the largest cohort of asthmatics and healthy controls to be studied in detail with respect to their ADRβ2 SNPs, successful genotyping for the entire cohort at each SNP locus and by performing the algorithm multiple times. In the vast majority of our cohort (99%), the final haplotype pair for each individual was predicted with over 98% specificity. Each of Drysdale's haplotypes are distinct and specifically defined by the subset of SNPs genotyped in this study.The haplotypes in this study were statistically inferred via the PHASE algorithm which is a well-recognised, time-efficient method for haplotype reconstruction using unphased genotype data derived from population samples such as ours. The accuracy and predictive power of PHASE algorithm was enhanced given our large sample size, the tight linkage disequilibrium between ADRβ2 polymorphisms similar to those used by Drysdale et alUsing a subset of 11 ADRβ2 haplotypes and mild asthma ADRβ2 haplotypes and mild asthma. Five HPs  accounted for 80% of our subjects which is similar to that reported in mild or moderate asthma. ADRβ2 SNPs.While previous studies have investigated the relationship between in vivo. Whether the coinheritance of haplotype 2 and 4 affects receptor dimerisation and therefore function of the combined receptor contributing to asthma pathogenesis needs to be considered and further explored.The association between HP2/4 with asthma severity was unexpected but significantly substantiated by the findings from formal trend tests across the 3 asthma severity groups. It is worth noting that haplotype 2 and haplotype 4 are heterogeneous at the main functional locus as well as many others and it is therefore rather difficult to know how or which functional effects would be specifically driven when inherited as a haplotype pair. ADRβ2 preferentially exists as dimerised pair ADRβ2 haplotypes and any individual clinical parameter used to define asthma severity. This is not surprising given that asthma is heterogeneous and classification of disease severity can be difficult when the various parameters used in defining severity are inter-related in many ways. In this study, there were no association between the inheritance of ADRβ2 haplotypes and diagnosis of mild or moderate asthma. Inflammation plays a critical role in the pathogenesis of asthma but there is increasing evidence for considerable variability in the pattern of inflammation between individuals which most likely contributes to phenotypic differences in disease severity and treatment responses. Detailed examination of the role of ADRβ2 haplotypes in the pathogenesis of asthma is beyond the scope of this study. However, it is unlikely any one gene can fully account for the pathogenesis of severe asthma but ADRβ2 haplotypes may explain some of the therapeutic resistance seen in severe asthma. Mild asthmatics homozygous for ADRβ2+46*A bronchodilate to SABA1 and increased risk of exacerbation if SABA or LABA are used regularly.ADRβ2 haplotypes defined by Drysdale and colleagues.There was no correlation between et alet alADRβ2 agonist. The response for HP2/4 was intermediate while HP4/4 showed a much weaker response. apriori selection of patients with a positive BDR has led to selection bias is unclear. However, the implication that BDR can alter depending on prior treatment and HPs takes on much greater importance in severe asthma particularly with the current finding of a higher frequency of the less favourable HP2/4 and lower frequency of the more responsive HP2/2 and 2/6. ADRβ2 agonist and therefore similar to population studies by Drysdale el al. It is possible that the loss of protective, more responsive haplotypes 2 and 6 in severe asthmatics leads to a dynamic increase in the frequency of the most common haplotype pair in any given population, thus explaining the apparent association between HP2/4 and severe asthma.Data on the effect of HPs on acute bronchodilator response (BDR) is limited and controversial. Taylor ADRβ2 haplotypes with respect to in vitro receptor characteristics and response to different ADRβ2 agonists needs addressing. Panebra et al reported variable baseline ADRβ2 expression and agonist-induced downregulation between ADRβ2 haplotypes. ADRβ2 agonists. Whether such observation translates to clinically important differences in acute bronchodilator response and tachyphylaxis respectively requires further investigation. Furthermore, the relative effect of each haplotype when coinherited as haplotype pair 2/4 is unknown. This would be best performed in a prospective pharmacogenetic study complementing in vitro studies to characterise the effect of haplotype pairs on receptor expression, function and regulation as well as pharmacological response. Such studies would provide important insights into whether individuals with severe asthma might benefit from alternative treatment strategies that minimise excessive use ADRβ2 agonists.To further enhance the current study data the functional significance of et al demonstrated significantly higher total IgE levels and prevalence of atopy in individuals homozygous with the ADRβ2 haplotype -47T/-20T/+46A/+79C/+252G. The strong correlation between ADRβ2+523CC genotype and atopy is a novel finding. The precise mechanism for this is unclear and is beyond the scope of the current study. It is most likely that ADRβ2 +523C acts as a tag or proxy for the actual genetic variation associated with atopy. This would not be surprising given that the ADRβ2 gene is located on chromosome 5q, a region where multiple candidate gene polymorphisms has been associated with atopy and/or IgE production. These genes include interleukin 4 (IL4) ADRβ2+523C>A is a synonymous polymorphism in which both alleles produce the same protein without any changes in amino acid sequence. It was long assumed that synonymous SNPs are “silent” and inconsequential, as the primary sequence of the protein is retained. However, it has been shown in other disease states that synonymous polymorphisms can predict disease susceptibility if it is in linkage with another polymorphism of the same or different gene that contributes to disease pathogenesis. One such example is the association between the immune-related GTPase family M(IRGM) gene and Crohn's inflammatory bowel disease. ADRβ2 agonists in the treatment of asthma, there is persistent background concern about their safety and efficacy. Functional polymorphisms of ADRβ2 potentially generate differential treatment response and suboptimal outcomes for asthmatics treated with regular SABA or LABA. Most studies have investigated only a limited number of ADRβ2 polymorphisms in mild or moderate asthmatics. However, it is the overall haplotype that best predicts receptor characteristics and treatment response. This study identified 13 new ADRβ2 haplotypes and demonstrated, for the first time, that the ADRβ2 haplotype pair 2/4 is associated with asthma severity. Further functional and clinical studies are needed to determine the role of ADRβ2 polymorphisms/haplotypes both in the development of severe asthma and in influencing therapeutic responses to ADRβ2 agonists. The positive association between ADRβ2+523CC genotype and atopy is novel. This most likely reflects its linkage with another candidate gene or causal polymorphism.Despite widespread use of


Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D2Q15K2) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross β-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the β-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm−1. CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm−1) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm−1) signals than in surrounding tissues at the resonance for the cross β-sheet polyQ in vitro.Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates  Caenorhabditis elegans nematodes in vivo and the strong signal at 2845 cm−1 assigned to the C-H vibrational stretch. Other regions of the spectrum can give information about other important molecules; for example, imaging at 1090 cm−1 of phosphate groups of chromosomes within cells undergoing mitosis −1 to 950 cm−1 to make a comparison between un-differentiated and differentiating murine embryonic stem cells Coherent anti-Stokes Raman scattering (CARS) microscopy can be applied in living cells and provides vibrational contrast at a speed that is orders of magnitude higher than conventional Raman microscopy −1 to ∼1800 cm−1 contains bands that can be considered as ‘fingerprints’ for the structure of proteins, given a level of knowledge/reference data it is possible to gain an understanding of the structure of protein molecules directly from the Raman spectrum of the ‘pure’ protein −1), β-sheet  and disordered protein structures (1670 and 1220 cm−1). Like Raman spectroscopy, CARS methods have also been used to evaluate protein structure in vitro. Perhaps the most important study in this area is the polarization sensitive CARS spectroscopy of the amide I band of various purified proteins in vitro, a result of a collaboration between the groups of Chikishev and Greve −1.Since the Raman spectral region from ∼1000 cmi.e.in vivo. The reason for this is that the CARS voxel sizes are not sufficiently small to allow detection of anything other than a mixture of biomolecules.So far CARS images obtained within this spectral region have not been recorded with a view to identifying particular molecules. Even though images have been obtained by tuning the excitation lasers for molecular vibration modes assigned to amide I, II or II of peptide bonds, in vivo aggregates are on the micron scale and most importantly, a significant volume of published work demonstrates that EGFP/other-tagged polyQ peptides within living mammalian cells and in C. elegans follow an aggregation process In CARS microscopy voxels of the order of a few hundred nanometres are interrogated, thus protein aggregates, with a size on the micron scale, are ideal candidates for evaluation using CARS methods. In the studies described in this paper we interrogate polyglutamine (polyQ) aggregates, that are implicated in Huntington’s disease in vivo, the various steps required are illustrated in in vivo by CARS microscopy relies upon effective characterisation of similar polyQ aggregates in vitro. The benchmark Raman spectral information for polyQ aggregates in vitro allows for the identification of protein structure in vivo, if common spectral signatures are observed.This is a novel approach for protein structure evaluation in vitro from a number of different polyQ peptide sequences as well as huntingtin exon 1 (htt exon 1) has previously been reported; the fibres have a particular morphology, often showing short, wide filaments 2Q15K2 give patterns with characteristic cross-β reflections 2Q15K2) as evaluated by Perutz et al. 2Q15K2 was incubated in water and the resulting short, stocky fibres detected and imaged by electron microscopy  show vepatterns . The cal2Q15K2 (with the solvent background subtracted) is shown in −1 is from an artefact produced by the spectrometer’s CCD camera. In the amide I and III regions, from 1600–1700 and 1200–1340 cm−1 respectively −1 originates mainly from C-C and C-N stretching of residues in β-sheets, while the amide I band at 1659 cm−1 originates from the carbonyl stretching mode. Raman marker bands for β-sheet in the amide I region are typically reported at ∼1690 cm−1 and a small shoulder at this position is also observed here. This is the first report of a strong β-sheet Raman marker band at 1659 cm−1 that we are aware of, however Sharma et al.2Q15K2 is particularly homogenous and rigid.It is unfortunate that purified proteins or peptides, already characterised by NMR or crystallography methods, have been rarely assessed by Raman spectroscopy methods. The same sample of fibres as used for the electron microscopy and X-ray diffraction studies, was then examined by Raman spectroscopy and the resulting Raman spectrum of D2Q15K2 our attention turned towards the potential for the analysis of the protein structure in vivo using CARS microscopy. The spontaneous Raman cross-section is typically 10−30et al. −1, remains strong but is only slightly shifted to lower wavenumber in the CARS spectrum (50% non-resonant background contribution); see −1 in the Raman spectrum is less easy to observe in the predicted CARS spectra with a 50% non-resonant background contribution (−1) is both (i) shifted to lower wavenumber and (ii) weaker as a function of non-resonant contributions in the predicted CARS spectra  a signature band for the protein structure and (ii) where non-resonant contributions have limited effect on the peak intensity and shift; it is for this reason that the amide I peak (seen in the Raman spectrum at 1659 cm−1) is that chosen for the following studies. It should be noted that the concentration of polyQ within an aggregate is anticipated to be higher than almost any other protein or molecule in the surrounding tissue (‘the background’), thus if a β-sheet structure of polyQ, as seen in vitro, is also present in vivo, then a CARS image obtained at ∼1657 cm−1 is anticipated to be optimal. Recent progress in stimulated Raman scattering, With a knowledge of the potential CARS spectrum, it is possible to establish the optimal vibrational frequency for imaging using our single frequency CARS microscopy method where the aim is to evaluate the structure of the aggregates of polyglutamine C.elegans strains expressing Q40-YFP fusion proteins, as previously reported for other studies et al., C. elegans strains expressing Q40-YFP are shown to contain both polymorphic distribution of the chimeric protein Q40-YFP structures identified as diffuse and focused fluorescence patterns. And as already elegantly demonstrated, with aging there is an accumulation of aggregates into foci from the diffuse structures and the timescale for formation of the polyQ aggregate foci correlates with the toxicity for the C. elegans strains expressing Q40-YFP. Transgenic et al. −1 with a rather weak band at 1662 cm−1. Thus by selecting the strong signature bands identified in the Raman (and predicted CARS) spectra that of D2Q15K2, at 1690 cm−1 and ∼1655–1659 cm−1, images to establish the presence of the β-sheet structure of polyglutamine in vivo are obtained. The spectral resolution of the CARS microscope is sufficiently high (3 cm−1)  have been studied by Tonge C. elegans strains expressing Q40-YFP were imaged by CARS microscopy from day 4 (day 1 adult) and older, at wavenumbers spanning the fingerprint region (1220–1750 cm−1) −1 to detect lipids. et al., with aging there is an accumulation of aggregates from diffuse structures at approximately day 4 for C. elegans strains expressing Q40-YFP and this correlates with toxicity. −1 showing the lateral view of the pharyngeal region of the nematode, C.elegans at day 4  and sur−1 is the most intense. The aggregate, labelled as ‘a’ in −1 are co-located (See −1 see −1 (or at 1690 cm−1 (−1 is far weaker in the centre of the aggregate ‘d’ than in the surrounding regions (Shown in 690 cm−1 ). In fac regions .−1, is not present in aggregate ‘d’, and present only in the diffusely distributed regions ‘c’ and the centre of aggregates labelled as ‘a’ and ‘b’ in −1 is observed in the centre of the aggregates in −1 (or 1690 cm−1 (C.elegans (Q40-YFP) imaged at 5 days old or older, under similar conditions, show no evidence for a CARS signal at 1650–1700 cm−1 in the single densely aggregated Q40-YFP regions, identified by fluorescence; the diffusely located Q40-YFP is not observed at this time , which 690 cm−1 . C.elegahis time . It is tet al., the influence of aging of the worms is correlated with the aggregation into foci of the chimeric fusion protein Qn-YFP and its toxicity, which is characterised by motility in vivo remains little understood in vivo assemble into cross β-sheet structures as found in fibres of the peptide, D2Q15K2 and htt exon 1 obtained in vitro. These structures migrate into foci, following which the polyQ cross β-sheet structure undergoes a metamorphosis into a different form. It is possible that this is a result of proteins, such as heat shock proteins, which co-assemble into the foci and disrupt the prevailing polyQ structure.As outlined previously by Morimoto C. elegans strains expressing Q40-YFP showed that the fluorescence of diffuse Q40-YFP structures recovered within 3 seconds and focal aggregates did not recover within 30 seconds, thus it had already been invoked by others that the diffuse and foci Q40-YFP structures exist as different biochemical states. C. elegans is different and not currently possible to identify.Previous fluororescence recovery after photobleaching (FRAP) studies on 4 day old in vivo. It is unfortunate that the protein structure of purified proteins, peptides and assemblies that have already been characterised in vitro by NMR or crystallography methods, have rarely been assessed by vibrational spectroscopy methods, such as Raman or infrared spectroscopy. The generation of libraries of Raman spectra of characterised proteins have the potential to enable CARS microscopy to become an invaluable tool for the identification of protein structure in vivo, particularly for misfolded proteins in protein aggregates that are a hallmark for age associated diseases.This is the first time that CARS microscopy has been used for the evaluation of protein secondary structure 2Q15K2 was purchased from Pepceuticals Ltd, Nottingham, UK as a lyophilised powder at >95% purity. The peptide was dissolved in 2 micron filtered distilled water and allowed to assemble over three days at 5 mg/ml at room temperature.The peptide D4 µl of the peptide solution (assembled) was placed on 400 mesh, formvar/carbon coated copper grids (AGAR Scientific) and blotted using Whatman filter paper. The grid was then washed twice with filtered distilled water before staining using 2% w/v uranyl acetate in water and blotting. The grid was examined using a Hitachi7100 electron microscope operated at 80 kV and pictures were taken using a Gatan CCD camera.10 µl droplet of peptide solution (assembled) was placed between two waxed filled capillaries and allowed to air dry in a sealed environment. The partially aligned fibre sample was placed on a goniometer and X-ray fibre diffraction data was collected using a Rigaku rotating anode (CuKα) and RAxis IV++ detector. Exposure times were 15–20 minutes and the specimen to detector distance was set to 160 mm. Diffraction patterns were examined using CLEARER. 2Q15K2, assembled) (5 mg/mL), as used for the electron microscopy and X-ray fibre diffraction studies, was pipetted into a quartz anti-reflection coated microfluorescence cell  prior to Raman spectroscopy measurements. A ChiralRAMAN spectrometer  operating in the backscattering mode, utilising a Nd:VO4 laser exciting at 532 nm (700 mW) and with a spectral resolution of 7 cm−1 was used to collect the Raman data. Using methods as previously reported for peptides and proteins 3 and a total data collection time of 1 minute. Raman spectra were repeated over a 24 hour period and found to be consistent.The peptide solution (DC. elegans strain expressing the chimeric fusion of polyglutamine (Q40) to yellow fluorescent protein (YFP) as previously reported The −1 (5 ps pulses at 800 nm). All images are 512×512 pixels and obtained in 2 seconds.The CARS setup is based on a Coherent Paladin Nd:YVO4 laser and an APE Levante Emerald optical parametric oscillator (OPO). The fundamental (1064 nm) of the laser is used as Stokes, whereas the signal from the OPO (tunable between 700–1000 nm) is used as the pump and probe. The beams are scanned over the sample by galvano mirrors  and focused by a 20×0.5NA objective lens into the sample. Both beams have a power of several tens of milliwatts at the sample. The forward generated CARS signal is filtered from the pump and Stokes light and detected by a photomultiplier tube Figure S12Q15K2 that were used for the X-ray diffraction and Raman spectral studies.Transmission electron microscopy image of fibrils of D(TIF)Click here for additional data file.Figure S22Q15K2 (5 mg/ml) and predicted M-CARS spectra with various contributions of non-resonant background .Raman spectrum of the fibrils of D (Data relates to (TIF)Click here for additional data file.Figure S32Q15K2 (5 mg/ml) in water.Raman and CARS spectra of fibril of D (a) CARS spectrum of fibril of D2Q15K2. (1200–3000 cm−1) and Raman spectrum (1200–1800 cm−1) (with the solvent background subtracted) (b) Expanded view of CARS spectrum of fibril of D2Q15K2. (1200–1800 cm−1). Samples were prepared for CARS spectra using 100 µl of the fibril containing aqueous solution  which was added to a glass slide, dried slightly in air and covered with a cover slip and sealed with varnish. The spectral resolution is 7 cm−1 with 50 mW maximum average power on the sample, with an 80 MHz repetition rate and ∼5 ps pulses. The objective was a 1.2NA water immersion objective, the beam was set to scan at 25 us per pixel, and at the magnification used each pixel was a little more than one focal spot wide. The rest time between irradiation events at a single focal volume was ∼1 second, and imaging was never carried out in one sample region (∼100×100 microns) for more than 3 minutes continuously. All other CARS parameters are as detailed in the Materials and Methods section and reference 29.(PDF)Click here for additional data file.Figure S4Schematic showing the pharyngeal region of the nematode, C.elegans in relation to images shown in . (i) Image as for (TIF)Click here for additional data file.Figure S5Image of the lateral view of the nematode, C. elegans (Q40-YFP) by CARS (1690 cm1−) and fluorescence microscopy. This is a similar view as for the data shown in −1). , fluorescence as green contours. There is a magnified image of aggregate ‘a’. A slight shift of the viewing regions between experiments means that the image acquired at 1690 cm−1 had to be shifted by x = 12 pixels = 2.2 µm and y = 2 pixels = 0.37 µm, therefore the region of the worm shown here is approximately (but not exactly) the same as in −1. Frames labelled ‘a’, ‘b’ and ‘c’ are those evaluated in (TIF)Click here for additional data file.Figure S6Image of the lateral view of the nematode, C. elegans (Q40-YFP) obtained by CARS microscopy (2850 cm1−). This is a similar view as for the data shown in −1).(TIF)Click here for additional data file.Figure S7Evaluation of the CARS (1657 cm1−) image of the nematode, C. elegans (Q40-YFP) labelled as aggregate ‘a’ (shown inand 4(iii)). (i) CARS (1657 cm−1) intensity profile along muscle (with plot (iii) of intensity as a function of distance) (ii) CARS (1657 cm−1) intensity profile across the muscle (with plot (iv) of intensity as a function of distance). Figure (iii) and (iv) are intensity plot of CARS (1657 cm−1) signal as a function of distance (black), the intensity profile (averaged over 3 µm) is fitted with a Gaussian curve (red).(TIF)Click here for additional data file.Figure S8Evaluation of the CARS (1657 cm1−) and fluorescence image of the nematode, C. elegans (Q40-YFP) labelled as aggregate ‘a’ in. Figure (i) shows the fluorescence (green contour plot) and the CARS (1657 cm−1) signals (black/grey). The red line follows the tissue structure in the muscle cells. In figure (ii) is a plot is the CARS signal intensity (a.u.) along the red line and the Gaussian fit.(TIF)Click here for additional data file.Figure S9Evaluation of the CARS and fluorescence image (1690 cm1−) of the nematode, C. elegans (Q40-YFP) labelled as aggregate ‘a’ in  (i) CARS (1690 cm−1)  and fluorescence images (green contour) for aggregate ‘a’ and adjacent muscle. Red line follows tissue structure through centre of aggregate. (See −1). Figure (ii) CARS intensity (a.u.) as a function of distance along the red line of upper figure (black line) and fit of the data (red line).(TIF)Click here for additional data file.Figure S10Evaluation of the CARS and fluorescence image (1690 cm1−) of the nematode, C. elegans (Q40-YFP) for region ‘b’ in  (i) CARS (1690 cm−1)  and fluorescence images (green contour) for region ‘b’. Figure (ii) shows the plot of CARS intensity (a.u.) as a function of distance (pixels) corresponding to the pixels followed by the blue and pink lines in figure (i).(TIF)Click here for additional data file.Figure S11Evaluation of the CARS (1657cm1−) and fluorescence image of the nematode, C. elegans (Q40-YFP) inas ‘c’ and ‘d’. The figure (i) shows the fluorescence (green contour plot) and the CARS (1657 cm−1) signals (black/grey). The data evaluated in the lower plot is obtained from the maximal fluorescence indicated by the pink/red line in figure (i). The figure (ii) shows a plot of the intensity of the fluorescence (green) and the CARS signal (black) as a function of distance along maximal fluorescence (in pixels). A fit of the data is shown in red. The fluorescence signal intensities correlate until a point ∼1× the width of the aggregate on the RHS. The CARS signal at 1657 cm−1 is negative as compared to the adjacent LHS region.(TIF)Click here for additional data file.Figure S12Evaluation of the CARS (1657cm1−) image of the nematode, C. elegans (Q40-YFP) shown inas ‘c’ and ‘d’. (i) The upper figure shows the fluorescence (green contour plot) and the CARS (1657 cm−1) signals (black/grey). The CARS data evaluated in the lower plot is obtained along the pink/red line in (i). The lower plot (ii) shows the intensity of the CARS signal (red) where the fluorescence is maximal (−1 within the aggregate (d in (TIF)Click here for additional data file.Figure S13Evaluation of the intensity of the CARS (1690 cm1−) signals in the image of the nematode, C. elegans (Q40-YFP) in the region defined in  Figure (i) shows the fluorescence (green contour plot) and the CARS (1690 cm−1) signals (black/grey) for region ‘c’ in −1 is negative as compared to the adjacent LHS region within the aggregate region.(TIF)Click here for additional data file.Figure S14Images of the lateral view of the pharyngeal region of the nematode, C. elegans (Q40-YFP) at 2 day old adult (day 5) obtained by CARS and fluorescence microscopy (wavenumber identified in each image). The fluorescence is identified in green. There is no evidence for a CARS signal in any of the images at the wavenumber identified).(TIF)Click here for additional data file.


To examine the independent and combined association of physical activity (PA) and sedentary behavior (SB) on both systolic (SBP) and diastolic blood pressure (DBP) in adolescents from two observational studies.Participants from two cross-sectional studies, one conducted in Europe  and the other in Brazil , were selected by complex sampling. Systolic and diastolic blood pressure (outcomes), PA and SB, both independently and combined, and potential confounders were analyzed. Associations were examined by multilevel linear regression.Performing the recommended amount of PA (≥60 min/d) attenuated the effect of SB on DBP in BRACAH study girls and in boys from both studies. In contrast, PA did not attenuate the effects of SB on the SBP of girls in the HELENA study. The combination of less than recommended levels of PA with 2–4 h/d of sedentary behavior was found to be associated with increased SBP in boys from both studies.Meeting current PA recommendations could mediate the association between SB and DBP in both sexes. In boys, the joint effect of low levels of PA and excessive sedentary activity increases SBP levels. Longitudinal studies are required to confirm these findings. Chronic non-communicable diseases (NCDs) are the main source of disease burden worldwide and are thus a major public health problem Among the factors that may influence blood pressure levels , patterns of physical activity (PA) and sedentary behavior (SB) have been shown to have inverse Although the effects of PA and SB on BP have mainly been examined in isolation, there are studies suggesting that these behaviors have an aggregate effect on adolescents Reproducing the same results in different population groups with different characteristics would increase their biological plausibility and provide a higher level of scientific evidence. For this reason, we tested the hypothesis, separately, in two cross-sectional studies conducted with adolescents: Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) and Brazilian Cardiovascular Adolescent Health (BRACAH).Thus, we hypothesized that higher levels of PA would attenuate the adverse effect of high SB levels on BP and that the combined effect of low PA and high SB levels may contribute to increased BP levels and that these effects may be different depending on where adolescents live.The HELENA study was based on data from a random sample of European adolescents who were tested on a wide range of nutrition and health-related parameters. The data were collected in 2006 and 2007 in ten cities from nine European countries. A detailed description of the HELENA sampling and recruitment methodology, harmonization processes, data collection, analysis strategies and quality control activities has been published elsewhere Data from the BRACAH study were collected in 2007 in the city of Maringá, PR, Brazil, population approximately 330,000 . The adolescents were selected by random sample and evaluated on a broad range of cardiovascular risk factors and various health behavior parameters. The complete sample size methodology of this study has been described previously For the current study, we selected adolescents from HELENA and BRACAH with complete data regarding gender, age, systolic BP (SBP), diastolic BP (DBP) (outcomes), PA levels, SB, socioeconomic status, parental education, regular tobacco consumption, body mass index and waist circumference. These variables are described in detail below.A total of 3,308 adolescents from the HELENA study (12.5–17.5 years old) and 991 adolescents from the BRACAH study (14.0–17.5 years old) met all the inclusion criteria and were included in the analyses.In both studies, BP measurements were performed following the recommendations for adolescent populations Systolic and Diastolic BP were measured by the arm blood pressure oscillometric monitor device OMRON® M3 (HEM 742) in the BRACAH study and the OMRON® M6 (HEM 70001) in the HELENA study. The OMRON® M3 (HEM 742) has been clinically and epidemiologically validated for adolescents by the Brazilian Research GroupThe PA and SB levels were considered independent variables and measured by means of questionnaires in both studies. The questionnaire model used for PA measurements in both studies was developed to assess PA levels (moderate-to-vigorous levels) in adolescents In the HELENA study, PA was also measured with accelerometers  for seven consecutive days, with a minimum of 8 hours recording/day for at least 3 days During weekdays, how many hours do you usually spend watching television?"-"During weekdays, how many hours do you usually spend on computers?-"During weekdays, how many hours do you usually spend playing video games?" Sedentary behavior was totaled and classified into the following categories: 0–2 h/d; >2–4 h/d; ≥4 h/d according to Dunstan et al. Sedentary behavior levels were assessed with a structured questionnaire, including questions on time habitually spent in front of the television, the computer and/or playing video games. In both studies, the questionnaire used questions such as "We also established six clusters of PA according to PA recommendations Questionnaire:<60 min/d of PA +>4 h/d of SB;<60 min/d of PA +2–4 h/d of SB;<60 min/d of PA +<2 h/d of SB;≥60 min/d of PA +>4 h/d of SB;≥60 min/d of PA +2–4 h/d of SB;≥60 min/d of PA +<2 h/d of SB;Accelerometer (using PA recommendations and tertiles of sedentary time):rd tertile of SB;<60 min/d of PA of PA +3nd tertile of SB;<60 min/d of PA +2st tertile of SB;<60 min/d of PA +1rd tertile of SB;≥60 min/d of PA +3nd tertile of SB;≥60 min/d of PA +2st tertile of SB;≥60 min/d of PA +1The potential confounders for this study were:Country (HELENA only):Age (years):Socioeconomic status: based on the family's household goods. In the HELENA study, the same definitions were used in previous HELENA studies Parental education: determined with a self-reported questionnaire and classified into four levels: elementary education, lower secondary education, upper secondary education and university degree.Regular tobacco smoking: defined as the regular consumption of at least one cigarette per day for a minimum of one month 2). BMI was used as a continuous variable in the analysis. Wearing light clothes and no shoes, the adolescents' height was measured to the nearest 0.1 cm with a wood stadiometer and their body mass to the nearest 0.1 kg with a calibrated portable digital scale.Body mass index (BMI): calculated as weight (kg)/height, percentages  and 95% confidence intervals (CI95%). Multilevel linear regression models using fixed effects intercept were fitted to analyze the relationship between each BP level and independent variables st) to test the associations between BP and two separate measures of individual behavior; 2nd) to test the extent to which country-specific characteristics and contextual variables mediate the associations between SBP and DBP levels and PA and SB.The multilevel analyses were performed with two objectives: 1p<0.001).Stata 12  was used for all statistical calculations. All analyses were adjusted for the clustered nature of the sample using the "svy" set of commands and stratified by gender, since interactions between sex and the studied variables were observed  we conducted a comparative analysis between the PA and SB levels found with the questionnaires and the PA measures found with and without the use of accelerometers. No significant differences were found for either sex (p = 0.406 for boys and p = 0.714 for girls).Subject characteristics, sociodemographic/socioeconomic variables, BP levels and PA and SB levels (independent and combined) are shown in a supplementary file. rd tertile of SB and systolic BP ; ≥60 min/d+>4 h/d β = −3.02 (95%CI = −5.14–−0.90); and ≥60 min/d+2–4 h/d β = −6.05 (95%CI = −10.16–−1.94) remained significantly and negatively associated with DBP in the girls from the BRACAH study.β = −2.31 (95%CI = −4.23–−0.39); and ≥60 min/d+<2 h/d β = −1.21 (95%CI = −3.11–−0.71). While in the BRACAH study, a significant association was observed in this category: ≥60 min/d+2–4 h/d β = −4.33 (95%CI = −7.90–−0.76). Moreover, the “<60 min/d +2–4 h/d” cluster was directly associated with systolic BP in both studies, with the largest effect presented by HELENA study adolescents  Differences between studies might be explained by 1) associations between behaviors analysed and determinants of BP  and 2) susceptibility of individuals to different social environments and cultures Our results differ from those of Ekelund et al. (2006), who found no differences in BP levels in the PA and SB cluster measured by accelerometers. On the other hand, our results corroborate studies that have evaluated the effects of PA and SB separately on BP levels We observed that boys with low PA and high SB levels showed higher levels of systolic BP. This finding agrees with results from others studies in which adolescents with low PA and high SB presented low levels of cardiorespiratory fitness Furthermore, in our European female sample, we found that adequate PA levels do not attenuate the effect of high levels of SB on systolic blood pressure, probably because: 1) females have reduced PA levels and 2) estrogens have a more powerful influence on BP levels than PA during adolescence Accelerometers allow the study of activity patterns, and can establish the dose-response relationship between activity and health outcomes. On the other hand, self-reported PA data may not accurately reflect activity patterns due to recall bias and/or social desirability bias Our results are of importance since the behavioral patterns under consideration during adolescence tend to continue into adulthood Since there were some methodological differences between the HELENA and the BRACAH studies  data from both studies were analyzed separately, but we used the multilevel analysis in order to control the influence of contextual (country-specific) variables, since several studies have shown that they influence PA and SB patterns A limitation of this study is its cross-sectional design; consequently, causality cannot be established. Moreover, it was not possible to adjust the analysis for other potentially BP-associated factors in either of the two samples, such as genetics or intrauterine development, but we developed an adjusted analysis for large potential confounders. On the other hand, the diverse geographic origin of the samples, the use of objective measures to assess PA and SB and multilevel adjusted analysis are some of the main strengths of our study.According to our results, meeting current PA recommendations could mediate the association between SB and DBP in both sexes. In boys, the joint effect of low levels of PA and excessive sedentary activity increases SBP levels. These results suggest that regular PA should be promoted and SB discouraged in adolescent populations to prevent elevated blood pressure and its consequences in adulthood.Table S1Characteristics of the samples from the HELENA study and the BRACAH study.(DOC)Click here for additional data file.


Echinochloaoryzicola(syn.E. phyllopogon) is an exotic weed of California rice paddies that has evolved resistance to multiple herbicides. Elimination of seedlingsthroughcertain weed control methods can limit the spread of this weed, but is contingent on accurate predictions of germination and emergence timing, which are influenced by seed dormancy levels.In summer annuals, dormancy can often be relieved through stratification, a period of prolonged exposure to cold and moist conditions.We used population-based threshold models to quantify the effects of stratification on seed germination of four E. Oryzicola populations at a range of water potential (Ψ) and oxygen levels. We also determined how stratification temperatures, moisture levels and durations contributed to dormancy release. Stratification released dormancy by decreasing base Ψ and hydrotimerequired for germination and by eliminating any germination sensitivity to oxygen. Stratification also increased average germination rates (GR), which were used as a proxy for relative dormancy levels. Alternating temperatures nearly doubled GR in all populations, indicating that seeds could be partially dormant despite achieving high final germination percentages. Stratification at Ψ = 0 MPa increased GR compared to stratification at lower water potentials, demonstrating that Ψ contributed to regulating dormancy release. Maximum GR occurred after 2-4 weeks of stratification at 0 MPa; GR were often more rapid for herbicide-resistant than for herbicide-susceptible seeds, implying greater dormancy in the latter. Manipulation of field conditions to promote dormancy alleviation of E. oryzicola seeds might improve the rate and uniformity of germination for seed bank depletion through seedling weed control. Our results suggest field soil saturation in winter would contribute towards E. oryzicola dormancy release and decrease the time to seedling emergence. EchinochloaoryzicolaVasing. (syn. E.phyllopogonStapf ex Kossenko) is a morphological mimic of rice (Oryza sativa) that can germinate and initiate shoot growth under hypoxia in flooded paddies E. oryzicola control E. oryzicola now hinges on maximizing weed seedling recruitment in order to eliminate such seedlings prior to planting the crop The temperate region summer annual weed E. oryzicola seed, the PBTM approach predicted with useful accuracy the germination responses of seeds to shifting temperature and water availability and their subsequent emergence from field soils Poaceae seeds typically possess non-deep physiological dormancy (NDPD), which indicates that seed dormancy release and increases in germination rates (speed)vary along a continuum of time and environmental conditions E. oryzicola germinated similarly E. oryzicola populations trace their origin to a single introduced biotype dispersed throughout California rice fields Population-based threshold models (PBTM) have been developed to describe germination responses to temperature, water potential E. oryzicola seed populations that emerge in spring E. oryzicola seed dormancy release and that dormancy levels could perhaps be manipulated using wintertime irrigation to increase the rate of springtime germination and weed seedling recruitment E. oryzicola seeds to transition from aerobic respiration to anaerobic alcoholic fermentation As in many summer annual species with NDPD E. oryzicola seed dormancy alleviation, we sought here to: 1) quantify stratification effects upon germinationof seeds of R and S populations of E. Oryzicola across a range of moisture and oxygen levels; and 2) ascertain the relative contributions of alternating temperatures and of stratification temperature, water potential and duration towards dormancy release in R and S E. oryzicola populations. This knowledge will contribute towards the accuracy of germination-based predictions of seedling emergence as affected by the dormancy status of the seed and thus improve the timing and efficacy of weed control programs.To understand the environmental requirements for E. oryzicola seeds (spikelets) of four populations  representing the range of phenotypic variability previously reported in California −2 s−1 of photosynthetic photon flux density (PPFD) from metal halide and high pressure sodium lamps to maintain a 16-h day length; soluble fertilizer  was applied through irrigation as needed. Seeds were harvested from panicles at the time of seed shattering in early fall, stored at 20°C for 3 weeks to approximate typical early autumn temperatures and thereafter stored at 3°C, approximating mid-winter temperatures. Water content of seeds kept in dry storage was 7 to 9% (dry weight basis).Ψ) or oxygen concentration (Ox) in seeds that had been stratified (wet-chilled) for dormancy removal or not stratified (dry-chilled). Seeds of each population were subjected during 90 days to either stratification by 10 cm immersion in 500 ml water and storage at 3°C to simulate wintertime stratification Ψ and Ox experiments that followed were conducted in a growth chamber at a constant 25°C, as this falls within the range of optimal temperatures for germination in these populations [unpublished data], and 12 h day length under 200 µmol m−2 s−1 PPFD  to satisfy any light requirements for germination. Germinated seeds were counted and removed daily for the first 5 days of the experiment and every 2–3 days thereafter until day 24. Seeds were counted as germinated whenever coleoptile growth reached ≥ 1 mm. At the end of each experiment non-germinating seeds were tested for viability using tetrazolium The role of dormancy in determining the range of environmental conditions that allow germination was assessed in spring 2008 in two concurrent experiments by comparing germination responses to gradients of water potential (Ψ levels of 0 (pure DI water), −0.2, −0.4 and −0.7 MPa. These Ψ levels were verified with a dewpoint water potential meter  at the beginning and end of the experiment. Experimental units were designed to prevent PEG or moisture loss through evaporation, and there was no change in Ψ levels over the period of germination. The experimental unit was a 14×14×5 cm transparent plastic container placed inside a 3.8 L airtight re-sealable clear plastic bag −1and bubbledinto containers Ψ levels were main plots and populations × chilling treatment were randomized sub-plots.Because of this species’ association with flooded environments et al. Ox) of 21, 10, 1, 0.01 and 0.001% created using pressurized 21% O2 and premixed O2/N2 ratios ,and supplied to containers holding 300 mL of DI water. Upon being established in each container through an initial 5-minute flush of gas at a rate of 3000 mL min−1, Ox treatments were continuously distributed into containers as described above. Immediately before inflow into the containers, O2 concentrations in gas mixtures were verified with a headspace trace oxygen analyzer . Gas entered the containers via an airtight grommet-lined opening and emerged through an aquarium bubbler fixed to the bottom center of the container. Inflow of gas created a positive pressure inside the sealed bag that permanently pushed air outwards through a syringe needle Ox levels were main plots and populations × chilling treatment were subplots randomized within.Using the same experimental arrangement described in the preceding section and following Al-Ani Final germination was expressed as a percentage of the total number of seeds in a treatment.Hθ) for germination that is defined as:The hydrotime population-based threshold model proposed by Gummerson Ψ is experimental water potential, bΨ(g) is the base water potential below which germination is prevented for a given fraction g of the seed population, and gt is time to germination of g. The model also assumes bΨ follows a normal distribution among seeds and is thus responsible for within-population variation in germination timing. Probit analysis was used to estimate the parameters of bΨ(50) is median bΨ and σΨb is the standard deviation in bΨ among seeds in a population. We combined germination data from the range of experimental Ψ treatments and used where bOx) across the seed population and a constant oxygen time (Oxθ) to germination for all seeds. Germination responses are described as a function of the logarithm of the oxygen concentration:Ox is daily average oxygen percentage in the micro-environment surrounding the seed, bOx(g) is the base or minimum level of oxygen just allowing germination of a given fraction g of seeds, and gt is time to germination for that same fraction. Parameter values for Stratification effects on the germination response to decreasing oxygen were analyzed using an oxygen-time threshold model analogous to the hydrotyme model −2 s−1 PPFD, and 80% RH. Germinated seeds were counted and removed daily over two weeks.Three experiments evaluated the roles of temperature fluctuations and stratification conditions  in releasing dormancy. Populations AM (S) and RD (R) Sets of dry-stored non-chilled seeds, not exposed to any dormancy-releasing treatment, were placed in Petri dishes and simultaneously germinated at either constant 20°C or at alternating 14/26°C night/day using growth chambers set as described above. Four replicate sets of seeds per population were randomly distributed within each temperature regime.Seeds of each population were placed in Parafilm-sealed Petri dishes containing 50 ml of DI water, wrapped in aluminum foil to control for light effects on dormancy release, and randomly arranged in covered 6.1 L plastic storage boxes in dark rooms set for constant 2.5, 5, and 7.5°C temperature regimes. These temperatures were selected because 5°C is the optimum chilling temperature for cold stratification in many species Ψ spanned the range from no water stress to stress below −1.5 MPa, which is the permanent wilting point of many herbaceous species Ψ levels were established and verified as described earlier, andwereverified every three weeks. Upon completion of stratification treatments, seeds were germinated as described earlier. Treatments were arranged in a split-plot factorial where population x Ψ combinations were in main plots and stratification durations were in sub-plots; there were two replicate sets of seeds in a completely randomized design.Seeds of each populationwere placed in Parafilm-sealed Petri dishes containing 50 mL PEG solutions of either 0 (DI water), −0.4, −0.8 or −1.6 MPa and kept under a constant 5°C for either 0, 3, 4, 7, 10, 14, 17, 28 , 35, 57 or 92 days. The drc add-on package G is cumulative germination, a is the upper asymptote (the lower asymptote is assumed to be 0), x is days, c is time to 50% germination and b is the slope. The inverse of the thus calculated c parameter equals the GR Ψ, whereas median times to germination vary non-linearly with respect to these factors; while germination tests can be done at warmer temperatures, germination time course at all suboptimal temperatures can be predicted based on the GR and base temperatures Dormancy-releasing effects of alternating temperatures and of stratification temperature, moisture stress and stratification duration were assessed by comparing germination rates (GR). The GR for a given treatment was calculated as the inverse time to median germination , which was estimated for each blocked replicate by fitting cumulative germination data with a three-parameter log-logistic equation using the R statistical program with the Ψ when stratified  compared to nonstratified seeds (Ψb) for the CR population. The hydrotime model fitted better the germination of stratified seeds, as indicated by the lower RMSE values (All populations germinated to high percentages (85–90%) across ratified . Howevernditions  by reduced seeds . StratifE values . Model pE values . The twoOx dropped to 1%, andfinal germination of nonstratified CR seeds was notably low and insensitive to oxygen availability . Germination time courses of stratified E. oryzicola seeds in all populations were almost insensitive to hypoxia and sensitivity was even limited in nonstratified seeds (bOx(50) of 0.04 ppm (not shown). Given this general lack of responses, an oxygen threshold model could only be fit to nonstratified seeds of three of the populations germinating under Ox of 1 to 21%, but model parameters still reflected only minor sensitivity to Ox and were not statistically different across populations (not shown). This minor sensitivity disappeared under stratification and germination rates were faster than for non-stratified seeds under all Ox levels  in all populations . AverageE. oryzicola populations within the range of stratification temperatures studied , and maximum GR values declined with Ψ<0 MPa and the enhancement of GR due to stratification varied (p <0.05) with the duration of wet-chilling and moisture stress during that period . Thus GRh Ψ<0 MPa. Lengthenegative . Averageulations .Hθ and bΨ(50)indicated dormancy removal and broadened the range of water potentials at which germination could occur following stratification E. oryzicolaΨ fell to −0.7 MPa (bΨ(50) that reflected the inability of nonstratified CR seeds to germinate(bΨ(50) due to stratification is consistent with similar reductions from after-ripening in red rice PolygonumReductions in both −0.7 MPa , and in germinate. The oveE.oryzicolato increased ability to germinate under hypoxia through anaerobic fermentation Ox levels in three nonstratified populations  (Oxlevels for the nonstratified seeds of these three populations was narrow and the estimated bOx(50) values (∼ 0.04 ppm) fell well below those of most other species E. oryzicola exhibited limited sensitivity to hypoxia consistent with the known ability of this weed to germinate under flooded conditions Oxlevels  seedsacross Ox levels (bΨ(50) value  . NeverthOxlevels  could sux levels  could be0) value  rather tΨ and Oxlevels  for E. oryzicolaDormancy release by stratification was expressed primarily through increases in germination rates.Studies with other species have documented enhancement of stratification effects as stratification temperatures were reduced below Tb for E. oryzicola germination Ψ , suggesting lower dormancy levels. Greater basal ethylene levels, which can play a causal role in the metabolic response to submergence Winter flooding is often practiced by California rice growers to facilitate stubble decomposition  of time . Herbici of time  and whenE. oryzicola seed dormancy was manifested primarily by reduced GR rather than by lower final germination percentages, and was released by alternating temperatures and by stratification. Stratification led to dormancy release characterized by hastened germination rates of unstressed seeds, enhanced ability to accrue hydrotime and germinate under drier conditions, and a minor increment of the ability to germinate under hypoxia. There was, otherwise, little germination response to oxygen availability in the E. oryzicola seeds tested, consistent with the wetland habit of this weed. The effects of stratification on GR were generally more sensitive to changes in Ψ than to temperature. Dormancy levels may have been lower among R populations compared to S populations in these experiments,but R seeds may also be more prone to secondary dormancy induction as stratification duration increases. A weed seed bank depletion program based on favoring weed emergence for subsequent control, such as with the stale seedbed technique, would benefit from optimizing environmentalconditions for weed seed germination. Based on our results, and depending on the degree of dormancy of the population, E. oryzicola dormancy release to enhance weed seed germination would benefit from field soil saturation in winter. Thiswill decrease time to seedling emergence,allowing for early-season weed control and a shortened crop planting delay.In summary,


The molecular network sustained by different types of interactions among proteins is widely manifested as the fundamental driving force of cellular operations. Many biological functions are determined by the crosstalk between proteins rather than by the characteristics of their individual components. Thus, the searches for protein partners in global networks are imperative when attempting to address the principles of biology.We have developed a web-based tool “Sequence-based Protein Partners Search” (SPPS) to explore interacting partners of proteins, by searching over a large repertoire of proteins across many species. SPPS provides a database containing more than 60,000 protein sequences with annotations and a protein-partner search engine in two modes (Single Query and Multiple Query). Two interacting proteins of human FBXO6 protein have been found using the service in the study. In addition, users can refine potential protein partner hits by using annotations and possible interactive network in the SPPS web server.http://mdl.shsmu.edu.cn/SPPS/.SPPS provides a new type of tool to facilitate the identification of direct or indirect protein partners which may guide scientists on the investigation of new signaling pathways. The SPPS server is available to the public at  The flowchart representation of the SPPS web server is shown in http://www.ncbi.nlm.nih.gov), UniProt (http://www.uniprot.org/), and PIR (http://pir.georgetown.edu).The input format of query protein sequences for SPPS server is the standard FASTA. The sequences of proteins can be either derived from in-house experiments, or directly taken from the databases embodying protein sequences, such as NCBI . In addition, a download link is available for all known interactions of the query protein if “Uniprot Entry Name” of the query protein is submitted, which were collected from several PPI databases .As the result of ‘Single Query’, the output in SPPS is split into three main sections, namely, “Protein Partner List”, “Protein Partner Network” and “Result annotation”. “Protein Partner List” includes all predicted protein partners along with their overall confidence scores in probability. The confidence score is provided for each potential protein-protein interaction as described in the Contrary to “Single Query”, the output of “Multiple Query” mainly divides into “Direct Interaction Prediction” and “Indirect Interaction Prediction” sections. The probability score is always provided for direct PPI predication. One or two intermediate proteins linking two query proteins are also estimated if “Consider indirect interaction” option in “Multiple Query” mode is checked. All probabilities of prediction in the output are defined the same way as “Single Query”.In addition to the “Search” option, SPPS also offers all training data, models and standalone software under its “Download” page, which facilitate users in their local machines if they have a great number of data to predict.To test the reliability of the SPPS server, we searched for the interacting protein partners for human F-box protein 6 (FBXO6) using “Single Query” and predicted a few direct interactions not included in the training models from the latest literature among several species using “Multiple Query”.FBXO6 protein is involved in the endoplasmic reticulum-associated degradation pathway by mediating the ubiquitination of glycoproteins. FBXO6 interacts with the innermost chitobiose in N-glycans of glycoprotein substrates by a small hydrophobic pocket in FBA domain and the introduction of point mutation into the residues in that pocket (FBXO6 null) impaired the binding activity toward its glycoprotein substrates Besides“Single Query”mode, SPPS server also provides “Multiple Query” to quickly evaluate whether two proteins are directly and indirectly interactive based on the score of probability, which may help biologists to choose the most probable protein-protein interaction pairs for further experimental validation. Many directly interacting proteins from the latest literatures in 2011 SPPS server has been running since 2007 and is updated annually. Competitive prediction of our algorithm has been evaluated in 2010 among sequence-based PPI prediction methods By bringing together a protein-partner search engine and protein databases in a single program, the SPPS server is a convenient tool for the identification of potential protein partners of query proteins such as kinases, regulatory factors, and other components of much more complex protein machineries. This web server can also be used in constructing the signal transduction network for a known protein or a novel protein whose function is unknown. In general, one protein may interact with at least several partners including upstream and downstream regulators. As illustrated by the example of FBXO6, SPPS provides a good predictive ability for potential protein partner hits. These are useful guidelines for further experimental validation of signaling network around any given protein.However, SPPS still has certain limitations, one of them being that the number of the models is not enough for covering all the species. The second one is that SPPS server has not considered interspecific interactions, such as the interactions between viral and human proteins, which may be vital in exploring targets responsible for infectious diseases. The running time for “Single Query” job is still slow due to large database and limited CPUs. To overcome these shortages, we are currently (i) collecting original interaction data produced by using the yeast two-hybrid based methods, mass spectrometry, protein chips and hybrid approaches to construct training models for more species, (ii) developing new kernel of SVM to adapt crossover interactions between different species, (iii) planning to provide more CPUs to accelerate the running process.http://mdl.shsmu.edu.cn/SPPS/.Discovering protein partners in large-scale network has been unprecedentedly appreciated due to the requirement to address the complicated process of biological systems by means of integrated technology. SPPS provides a new type of tool to facilitate the identification of direct or indirect protein partners and guides scientists to design new experimental directions. The SPPS server is available at a public web service http://www.uniprot.org/), which is carried out by a Python script “Updater” at a 6-month interval. The database currently consists of more than 60,000 non-redundant protein sequences, with species covering “Homo sapiens”, “Mus musculus”, “Caenorhabditis elegans”, “Drosophila melanogaster”, and “Saccharomyces cerevisiae”, as shown in SPPS requires a sufficient number of known protein sequences covering a diverse range of species. The protein sequences in our database were retrieved from UniProt f(x of observation ixi)g(x to derive probabilities by Eq. (1)A and B are estimated by minimizing the negative log-likelihood of the training data, Eq. (2),For the SPPS server, we further enhanced the algorithm by probability. Putative protein partners are ranked by the value of probabilities. Platt's approach was used to derive posterior probabilities for the estimated class membership 293T cells transfected with either Flag-FBXO6 WT or Flag-FBXO6 Null were lysed in 6 ml of lysis buffer  for 20 min with gentle rocking at 4°C. Lysates were cleared using centrifugation , the supernatant was subjected to immunoprecipitation (IP) with 50 µl of anti-FLAG M2 affinity resin (Sigma) overnight at 4°C with gentle inversion. Resin containing immune complexes was washed with 1 ml ice cold lysis buffer 4 times followed by three 1 ml Tris Buffered Saline (TBS) washes. Proteins were eluted with two 50 µl 150 µg/ml 3×Flag-peptide (Sigma) in TBS for 30 minutes, and the elutions were pooled for a final volume of 100 µl. Proteins in each elution were precipitated with cold acetone and the resulting pellet washed 2 times with cold acetone. Proteins were separated by 10% SDS-PAGE and transferred to NC membrane . After blocking with 5% nonfat milk in PBS, membranes were immunoblotted with indicated antibodies, followed by HRP-linked secondary antibodies . The signals were detected by SuperSignal West Pico Chemiluminescent Substrate kit  according to manufacturer's instructions.Figure S1WT or 293T FBXO6Null were immunoblotted with the indicated antibodies.Immunocomplexes from either 293T FBXO6 Both FBXO6WT and FBXO6Null interacted with Cullin1, only FBXO6WT interacted with the DDOST and RPN1.(TIF)Click here for additional data file.


Implementing a recall paradigm without hypnosis, we use functional MRI (fMRI) to explore and compare nociceptive and centrally-driven pain experiences. We posit that a trace of a recent nociceptive event can be used to create sensory-re-experiencing of pain that can be qualified in terms of intensity and vividness. Fifteen healthy volunteers received three levels of thermal stimuli  and subsequently were asked to recall and then rate this experience. Neuroimaging results reveal that recalling a previous sensory experience activates an extensive network of classical pain processing structures except the contralateral posterior insular cortex. Nociceptive-specific activation of this structure and the rated intensity difference between physical and recalled pain events allow us to investigate the link between the quality of the original nociceptive stimulus and the mental trace, as well as the differences between the accompanying neural responses. Additionally, by incorporating the behavioural ratings, we explored which brain regions were separately responsible for generating either an accurate or vivid recall of the physical experience. Together, these observations further our understanding of centrally-mediated pain experiences and pain memory as well as the potential relevance of these factors in the maintenance of chronic pain. A growing body of neuroimaging literature has focused on and compared the neural correlates of simulated action and perception. These studies demonstrate that brain regions critically involved in motor, sensory and emotional processing can be activated without peripheral inputs Some groups have employed hypnosis to induce imagined or suggested sensations of pain in the absence of nociceptive input, and results suggest some overlap with neural activity during physical pain As an alternative approach, our paradigm uses a pain recall task, a form of mental imagery, that avoids the need for hypnosis or confounds of empathy-related processing. Mental imagery techniques have previously been shown to be useful in eliciting analgesic responses, with these methods focusing on the powerful effect of suggestion Beyond this, we wanted to determine whether one could simulate pain as a near sensory experience if the recalled event occurred temporally close to the original painful stimulus. Several studies have suggested that a memory “trace” of a painful experience may exist, but in a limited form such that sensory re-experiencing is not available after a painful event 15 healthy volunteers  were recruited after screening for absence of any prior history of pain, neurological or psychiatric disorders, and not meeting any of the exclusion criteria for MR experimentation. Written informed consent was obtained in accordance with the Declaration of Helsinki and the study was approved in full by the Central Oxfordshire Research Ethics Committee (C02.086).The imaging session consisted of two blocks of 15, six-second thermal stimuli to the dorsum of their left hand. Subjects were presented with three types of thermal stimuli  that had previously been defined by subjective thresholding in the scanner. Warm was defined as 0/10, low pain was defined as 2/10 and high pain was defined as 6–7/10 . Overall, there were 10 repeats for each stimulus type, pseudo-randomized over the two imaging blocks. Each repeat consisted of a visual stimulus reading either “Feeling warm” or “Feeling pain,” during which the individual would receive either a warm or noxious thermal stimulus. An eleven second gap (accompanied by a fixation cross) followed. The length of this “time-to-test” interval was chosen based upon data suggesting it would allow accurate pain memory recall intensity of the recalled event and the vividness of the evoked sensation, measuring how close to reality it felt. At three points during the study, subjects were asked to rate the stimulus intensity of the real thermal event using a VAS. The three thermal conditions were pseudo-randomised both within the experiment and across subjects.During the recalled event, subjects were instructed to visualise the location of the previous thermal stimulus, changes in intensity, and the context surrounding the thermal experience while attempting to simulate the sensation. Using a visual analogue scale (VAS), subjects were then instructed to use a slider to rate both the A thermal resistor developed in-house and controlled by in-house written software was used to increase skin temperature. The device's ramp rate is such that the desired stimulating temperature is reached within one second of the triggered event and sustained evenly for the remaining five seconds of the stimulus. The device records the varying temperature at the site of stimulation demonstrating a return to baseline within six seconds. This device has been reliably used within several previous studies Visual stimuli included a fixation cross and four six-second prompts: “Feeling warm,” “Feeling pain,” “Imagine feeling warm” and “Imagine feeling pain”. Visual analogue scales (VAS) were presented to obtain online ratings for intensity of recalled pain and vividness. Each scale was presented for six seconds. The “Imagined intensity” scale was anchored with no pain at the minimum and extremely painful at the maximum. Similarly, vividness was anchored with not vivid and extremely vivid. All visual stimuli were projected onto a screen visible to the subject via prism glasses. Visual stimulation was continuous throughout the experiment.3. Scans were acquired continuously throughout the experiment. High resolution, T1-weighted, structural scans (64 slices at 1×1×1 mm3 voxel size) were obtained for each individual for anatomical overlay of brain activation.Functional imaging was conducted using a 3 Tesla Siemens/Varian MRI system with a bird-cage radio frequency coil and four channel phased-array receiver coil. A gradient echo-planar imaging (EPI) sequence was used with a TR  = 3 s; matrix  = 64×64; TE  = 30 ms; 41×3 mm axial oblique slices; volumes  = 294; FOV = 192×192; voxel size  = 3×3×3 mmOnline ratings for intensity of recalled pain and vividness of recalled pain were grouped according to the preceding physical stimulus given  and the individual means and standard deviations calculated. One-way ANOVAs were used to test for statistical difference between the average ratings for all three conditions, for both vividness and intensity across individuals. Individual ratings across conditions of vividness and intensity ratings were correlated using Pearson correlation coefficient. Temperatures of the thermode at the skin surface were recorded once every 500 ms. The mean and standard deviation of these temperatures across epochs prior to and during the recalled events were calculated per individual and then across subjects.www.fmrib.ox.ac.uk/fsl). Pre-statistic processing included: motion correction using MCFLIRT  Version 5.63, part of FSL  = 10.98, p<0.01; Intensity: recalled warm mean ± SE  = 1.08±0.43, recalled low pain mean ± SE  = 2.52±0.42; recalled high pain mean ± SE  = 4.49±0.6).In post-scan debriefing sessions, all of the subjects reported the ability to recall the thermal events during the cued “imagined” conditions. The ability of healthy volunteers to recreate a similarly intense experience was demonstrated by the fact that ratings for intensity of recalled pain were related to the actual stimulus applied . Ratings for intensity of recalled sensory events showed a significant positive linear trend across intensities one-way ANOVA: F = 2.32, p = 0.11 Vividness: warm mean ± SE  = 4.33±0.53, low pain mean ± SE  = 4.27±0.47; high pain mean ± SE  = 5.66±0.46).Furthermore, the significant difference between imagined low and imagined high pain ratings, despite the lack of a cue for this difference, confirms that subjects accurately recreated and matched the recalled event to the prior sensory experience. Despite all three conditions being rated as significantly different for intensity, vividness ratings reveal that subjects were equally able to imagine warm and low pain stimuli, whereas “high pain” stimuli were rated as significantly more vivid (one-way ANOVA: F(14) = −2.364, p<0.05) and physical high pain vs. recalled high pain (t(14) = 2.844, p<0.05). No significant difference was observed between physical low pain vs. imagined low pain (t(14) = −0.603, p = 0.556). Individual averages were taken of the recorded temperatures of the stimulating thermode prior to and during the recalled events to ensure that nociceptive fibres were not stimulated (mean ± SD temperature at onset across subjects and across imagined conditions  = 37.8±1.2°C).To explore the perceptual differences between physical and recalled pain events, subjective ratings of the thermal stimuli were acquired at three time points during the study . A comparison with rated intensity of imagined events revealed a significant difference between physical warm vs. recalled warm  pain should differ from recalling pain. This difference was investigated using a mixed effects analysis group contrast of physical high pain versus imagined high pain which revealed nociceptive-specific activity in only one brain region: contralateral (right) posterior insula , Table 3(14) = 3.0527, p<0.01), the anterior insula shows two separable but comparable BOLD responses to the physical and recalled pain events  = 1.7, p>0.05 see Consistent with our hypothesis that the recalled task draws upon a trace of the original nociceptive event, a conjunction analysis of recalled and physical pain conditions revealed significant activation in most pain processing structures including bilateral anterior insula, ACC, thalamus and SI , suggestOur trace hypothesis however, suggests a link between these two events. As such, we posited that a greater response during the original stimulus should predict greater activation during recall. By extracting PEs from the posterior insula  exploring the difference between physical and imagined pain  revealed isolated activity in only one brain region: contralateral posterior insula , Table 3Supporting the mixed effects group contrast data , when commonalities between recalled imagined and physical pain conditions were further explored using a conjunction analysis, it revealed activation of several of the classical pain processing structures , with moA contrast identifying neural activation specific to recalling a painful event (imagined high pain > imagined warm) revealed activity in the thalamus, premotor cortex, SI, PAG, and the cerebellum . This ovBeyond activation of pain processing structures, in both imagined pain conditions (imagined low pain > baseline contrast and imagined high pain > baseline contrast – Confirming a report by Morley Previous studies have suggested that a memory trace of a painful experience may exist but in a limited form such that sensory-re-experiencing is not available long after a painful event In our study we demonstrate that, without hypnosis, a recall task was sufficient to create a quantifiable sensory re-experiencing of pain that activated all of the classical pain structures except the contralateral posterior insula. Identifying one structure unique to processing nociceptive pain may aid in further characterising the differences between peripherally and centrally mediated pain experiences. We suggest that the imagined pain events were created by using a recent and retrievable memory trace, with structures in the working memory network enabling this retrieval. The concept of a pain trace may be helpful in better understanding the efficacy of therapies including hypnosis and mental imagery tasks