After accounting for mycorrhizal association, sample size, and climatic range, foliar δ15N in P. balsamifera was more weakly related to mean annual precipitation and foliar N concentration than when measured across species, yet exhibited a stronger negative effect of mean annual temperature. Similarly, the effect of precipitation and elevation on Δ13C were stronger in a global data base of foliar Δ13C samples than observed in P. balsamifera. These results suggest that processes influencing foliar δ15N and Δ13C in P. balsamifera are partially normalized across its climatic range by the habitat it occupies or by the physiology of the species itself.Variation across climate gradients in the isotopic composition of nitrogen (N) and carbon (C) in foliar tissues has the potential to reveal ecological processes related to N and water availability. However, it has been a challenge to separate spatial patterns related to direct effects of climate from effects that manifest indirectly through species turnover across climate gradients. Here we compare variation along environmental gradients in foliar N isotope (δ Global gradients in foliar chemical traits, therefore, have the potential to reveal ecological controls on plant functioning. However, both intra and inter-specific variation contribute to relationships between foliar chemical traits and climate gradients3, and these relationships are not necessarily congruent4. Analysis of how individual species respond to climate gradients could provide greater insight into ecological controls on plant functioning, possibly aiding the understanding of plant response to global change.Spatial variation in foliar chemical traits represent phenotypic responses to environmental and genotypic factors. Because foliar chemicals regulate key biological functions, such as photosynthesis, spatial variation in foliar chemicals can also be used to understand the functioning of landscapes, including productivity and water relations of plants15N, which serves as an indicator of terrestrial N cycling6, varies systematically between species9, mycorrhizal associations11 and along gradients in climate12, and, when measured over time, can be used to infer ecosystem response to disturbance and climate change16. Local to regional-scale anthropogenic impacts on N cycling can also be important influences on foliar δ15N20. Mechanistically, processes that fractionate the stable isotopes of nitrogen occur to a greater extent when N availability  is high21. Thus, over time, processes associated primarily with gaseous N loss typically increase the δ15N value of the remaining inorganic N pool, causing 15N-enrichment in foliar tissues that draw from that pool. Conversely, systems experiencing low N availability have a more conservative N cycle and do not lose as much N with low δ15N values. Furthermore, when N availability is low, plants depend more strongly on N fixation, atmospheric deposition, and N from mycorrhizal fungi6. While the δ15N of N passed to plants through these processes differs, it is generally depleted in 15N relative to the enriched pool remaining following leaching and denitrification losses22. Therefore, continental- to global-scale patterns in foliar δ15N related to climate reflect climatic control on processes that influence N availability23, but these patterns must account for mycorrhizal association, and nevertheless, leave considerable small-grain variability related to local processes24.Of particular interest to understanding plant-environment relationships are the isotopic composition of nitrogen and carbon in leaves. Foliar δ13C) represents an integration of the balance of CO2 and water fluxes in plants, and variation in Δ13C is controlled by both environmental and genetic factors25. For example, there is a well-documented positive relationship between plant-moisture status and Δ13C across a variety of C3 plants e.g. refs 13C are both influenced by Ci/Ca, the ratio of intercellular to ambient [CO2]29. Dry conditions lead to reduced stomatal conductance  and a proportionately greater decrease in transpiration than photosynthesis31. This relative decline in transpiration causes increased water-use efficiency and an increase in the fixation of 13C relative to 12C. Δ13C decreases with increasing elevation28, likely related to a combination of atmospheric and edaphic factors32, but the mechanisms are unclear and investigation continues33.Carbon isotope discrimination )22 and mycorrhizal association between species influences foliar δ15N10. Similarly, C3 plants vary in foliar Δ13C due to variation in carboxylation efficiency and stomatal response to environmental conditions22. However, it is an outstanding question if these sources of variability among species strengthen or weaken the global to continental scale variability in foliar δ15N and Δ13C imposed by climate. The assembly of species adapted to environmental conditions at any given site, might be expected to reduce the effect of climate on foliar chemical traits. Alternatively, gradients in plant lifeform along climate or elevational gradients, such as from deciduous angiosperms to evergreen gymnosperms that exhibit divergent mean foliar Δ13C, could strengthen effects of precipitation and elevation28. To advance our understanding of the relative effects of climate and plant traits (which vary by species) on foliar isotopic composition, widely distributed observations of foliar δ15N and Δ13C in a single species are required. Yet, there remains a paucity of examples where foliar δ15N and Δ13C were measured in more than a small number of specimens  of the same plant species in its natural environment e.g.,28. Furthermore, because past work has analyzed foliar δ15N and Δ13C separately, the effect of N availability on Δ13C  remains understudied34.The existence of broad relationships between foliar isotopic composition and environmental factors suggest that these foliar chemical traits index the availability of nitrogen and water to plantsPopulus balsamifera for foliar [N], δ15N and Δ13C. Using these data, our objective was to explore the extent to which large gradients in climate-associated variation in foliar δ15N and Δ13C observed globally e.g., refs 4. While global interspecific variation in foliar traits is widely recognized as being valuable for understanding plant-nutrient relationships35, few studies have sampled a sufficient number of individuals to conduct the same analysis using intraspecific variation. Because interspecific variation in plant traits is generally stronger than intraspecific variation, we hypothesize that the global variation in foliar [N], δ15N and Δ13C of P. balsamifera will exhibit a weaker response to climate than observed in global samples collected from many species. To evaluate the extent to which patterns in P. balsamifera chemical traits correspond to patterns in plant functioning, we subsequently used structural equation modeling to compare the effects of climate variables and foliar [N] on Δ13C.Here we measure 755 specimens of the widely-distributed tree species 15N in P. balsamifera ranged from −20.6‰ to 8.8‰. Climates for sampled P. balsamifera trees spanned ~12 °C of mean annual temperature (MAT) (−1.9 °C to 10.2 °C) and ~2100 mm of mean annual precipitation (MAP) (390–2566 mm). In a model of foliar δ15N with MAT, log(MAP), and log[N] as model effects, the addition of P. balsamifera observations  exhibited little influence on model estimates compared with using the original global data set (n = 9828)  foliar δ15N increased with increasing MAT (particularly for MAT >0 °C), (2) foliar δ15N decreased with increasing log-transformed MAP, and (3) foliar δ15N increased with increasing log-transformed [N] . P. balsamifera foliar δ15N showed no relationship with log-transformed MAP (P = 0.6). The explanatory ability of mean climate factors and foliar [N] was low (R2 = 0.05), leaving considerable small-grain variability remaining in the P. balsamifera δ15N observations.Across the range of sites sampled Fig. , δ15N in15N and climate for P. balsamifera were also present when subsetting the global data to the same climate envelope as P. balsamifera. Repeated random samples of foliar δ15N, N, MAT and MAP from the global database within the climate envelope defined by the P. balsamifera samples resulted in model estimates for each effect on δ15N that were different than the model estimates using P. balsamifera alone  7.8%, 20.3%, and 100% of the time, respectively. Therefore, relative to the global data set, the effect of MAT on the P. balsamifera samples was more negative (P < 0.001), the effect of logMAP was removed , and the effect of log[N] was reduced, although still significant (P < 0.001).The unique patterns in relationships between foliar δone Fig. . The P. 13C in P. balsamifera ranged from 18.2‰ to 25.7‰. Elevation for sampled P. balsamifera trees ranged from sea level to 2092 m, which was 60% of the elevation range spanned by the global data set (sea level to 3500 m). After accounting for elevational differences between samples, residual Δ13C was plotted against logMAP . Further, after accounting for variation with MAP, high elevation P. balsamifera sites exhibited elevated residual Δ13C relative to the global samples at the same elevation. As a result, models using P. balsamifera samples attribute a smaller fraction of model variance to logMAP and sqrt(elevation)  and a negative direct effect of log(foliar [N]) on Δ13C . A positive direct effect of log(MAP)  and a negative direct effect of MAT  on log(foliar [N]) represent indirect effects of climate on Δ13C through foliar [N]. The direct effect of MAT on Δ13C was not significant, therefore, the only effect of MAT on foliar Δ13C is indirectly through foliar [N].To examine how climate and N availability jointly influence Δdel Fig. . Results15N between a global data set, including hundreds of different plant species, and a single widely distributed species, P. balsamifera, we found that broad scale patterns in the isotopic composition of foliar tissues observed globally is largely weakened when observed in a single species. Across global gradients in climate, foliar δ15N measured in a wide variety of species has been shown to correlate with MAT, MAP, and [N], and mycorrhizal fungi association10. Demonstrating the same for a single species is often difficult due to the limited climatic variability encompassed by the range of most species. For the widely distributed P. balsamifera, we found weak, but significant effects of MAT and foliar [N], but not MAP  exhibited significant effects of MAT, MAP and [N] on δ15N 7.8%, 20.3%, and 100% of the time . Comparing global and P. balsamifera samples, the largest difference is the effect of MAT, which had a significant negative effect on foliar δ15N in P. balsamifera. These patterns afford some speculation as to the mechanisms behind global foliar δ15N patterns. For example, increasing foliar δ15N with increasing MAT between −0.5 and 30 °C is thought to represent a gradient in the dominant pathway responsible for the loss of N from ecosystems, from DON leaching (a low fractionation pathway) at low MAT to denitrification (a high fractionation pathway) at high MAT10. For P. balsamifera, occupying a MAT range from −1.9 to 10.2 °C, the enrichment of 15N decreased with increasing MAT, which is counter to the broad global pattern, but consistent with the 7.8% of models from the global database that were significant across this temperature range. For P. balsamifera, therefore, one possible interpretation is that N availability decreases with increasing temperature. However, it is also possible that P. balsamifera becomes more reliant on NO3- as opposed to NH4+ with increasing temperature or is more likely to occupy sites with lower nutrient availability, leading to a small decline in foliar δ15N with increasing temperature22.Random samples from the global database within the climate envelope defined by the range of 15N was weaker for P. balsamifera than for the global samples. The global pattern of decreasing foliar δ15N with increasing MAP has been explained as a response to higher gaseous N loss in more xeric environments36 and low N availability  in wet environments37. For the relatively mesic environments studied here, there was no effect of MAP, suggesting that precipitation amount is potentially not a driver of denitrification for the sites inhabited by P. balsamifera across this gradient. This might be a consequence of the habitat preferences of P. balsamifera, which is for wet locations such as riparian zones and the edges of wetlands, which might act to normalize the effect of precipitation on δ15N across its range.After accounting for MAT, the effect of MAP on foliar δ15N was smaller for P. balsamifera than it was for the global samples. After accounting for MAT and MAP, for every 1% increase in foliar [N] δ15N increased 5‰. Considering a foliar δ15N range of ~30‰, N availability appears to be highly variable in P. balsamifera, possibly associated with variation in productivity across its range. However, it is also clear that there is considerable site-to-site variability in N availability unexplained by the mean climate factors studied here. This variability is likely due to any number of factors, such as site soil moisture conditions, variable anthropogenic inputs, and site disturbance history19.The effect of foliar [N] on foliar δP. balsamifera samples to the global database reduced the effect of MAP and elevation on foliar Δ13C. The effect of elevation on Δ13C has been discussed as related to temperature, the partial pressure of atmospheric CO2 or O2, irradiance, or edaphic factors28, but is still a poorly understood process33. There is also a trend towards greater dominance of evergreen gymnosperms at higher elevations, which have lower Δ13C values than angiosperms. Diefendorf et al.28 calculated that at the site level, evergreen gymnosperms on average exhibited 2.7‰ lower Δ13C than deciduous angiosperms. Therefore, not accounting for plant functional type in models will likely exaggerate the effect of elevation on Δ13C, but this is just one possible influence of elevation on Δ13C. By considering only P. balsamifera samples we explicitly account for interspecific variation, effectively removing most of the effect of elevation on Δ13C  at the same stomatal conductance34. For a single tree species with presumably similar nitrogen and water acquisition strategies across its range, the effects of increased N availability and reduced stomatal conductance at low precipitation sites have the effect of reducing Δ13C. We see these direct and indirect effects on Δ13C in our structural equation model , global variation across many species is exactly what is needed. By studying a single species, we control for one source of variability on foliar isotopes, providing insight into mechanisms that are unique to that species or the habitat it occupies. Specific to δ15N, measurements of P. balsamifera exhibit a negative effect of MAT that was not measured across a similar climate range in globally distributed foliar samples. Among the possible interpretations is that N availability to these trees is reduced along the southern range edge of this species. Likewise, for foliar Δ13C, the effect of elevation was reduced in P. balsamifera, which suggests that the habitat’s P. balsamifera occupies somehow normalize for the effect of elevation on foliar Δ13C. It is difficult to use these observations of P. balsamifera to generalize to other species or mechanisms driving geographic variability. However, it seems clear that global variation in foliar δ15N and Δ13C is driven by variation between species, each with its own unique relationships to climate and elevation. The importance of this finding will only be realized as further research attempts rectify intraspecific and interspecific isotopic patterns in a variety of species, ideally leading to stronger generalities of how climate affects resource availability.In summary, we generally found different responses to climate in plant foliar chemical traits measured in a single widely distributed species than the same measured in many species distributed globally. Foliar chemical traits in Populus balsamifera is a dominant deciduous tree across most of the boreal forest of North America   of the sample with a locational uncertainty of <30 m. Sites spanned nearly 23 degrees of latitude (39.87°N to 62.74°N) and 90 degrees of longitude (60°W to 150°W)  interfaced with a ThermoFinnigan Delta V + isotope ratio mass spectrometer . A MgClO4 trap was used to remove water vapor prior to the transfer of sample gases to the IRMS. The δ13C and δ15N data were normalized to the VPDB and AIR scales, respectively, using a two-point normalization curve with internal standards calibrated against USGS40 and USGS41. The long-term analytical precision (1σ) of an internal leaf standard analyzed alongside samples was 0.28‰ for δ13C and 0.24‰ for δ15N. Carbon isotope discrimination against 13C (Δ13C) was calculated according to Farquhar et al.29 as:13Cair value used was the δ13C measured in the atmosphere at Mauna Loa, Hawaii in June 2015 (δ13Cair = −8.6‰). Elevated Δ13C signifies greater increases in intercellular CO2 concentrations (Ci) than atmospheric CO2 concentrations (Ca), which is caused by increased isotopic fractionation due to either greater stomatal conductance and/or lower photosynthetic assimilation rates in C3 plants45. Foliar [N] was log transformed to achieve a normal data distribution for comparison with δ15N and climate parameters.We sampled 755 specimens of 47. Daily mean temperatures were calculated as Tmean = (Tmin + Tmax)/2. From the daily means, mean annual temperature (MAT) was calculated by first calculating annual means 1980–2015, which were then averaged. Similarly, mean annual precipitation (MAP) was calculated as the mean annual sum of daily precipitation, 1980–2015. MAP was log10 transformed to achieve a normal data distribution. The square root of elevation was calculated to achieve a normal data distribution.We attained gridded climate data from DAYMET 1980–2015, which is an interpolated product providing minimum and maximum temperature and precipitation totals at daily intervals15N (n = 9828) was acquired from Craine et al.10. Therefore, the number of measurements of δ15N of P. balsamifera was 7.7% of the global data set. The global data set included data on mean annual temperature (MAT), mean annual precipitation (MAP) both extracted from48, geographic location, and species. The global data set represented foliar samples from across the full spectrum of global climates, whereas the P. balsamifera data were constrained to climate conditions in northern North America. A global data set of foliar Δ13C in trees (n = 570) was acquired from Diefendorf et al.28. These data represent species mean Δ13C at each site , rather than individual observations, thus removing within-species variability at each site. This data set included site MAP and elevation.A global data set of foliar [N] and δet al.10 and Diefendorf et al.28 to build models relating δ15N and Δ13C to climate factors. We combined the global and P. balsamifera datasets and built ordinary least squares models of foliar δ15N and Δ13C with a selection of MAT, logMAP, log[N] (for δ15N), and logMAP and sqrt(elevation) (for Δ13C) as model effects. Models using the (1) global data sets, (2) the global data sets combined with the new P. balsamifera observations, and (3) the P. balsamifera observations alone were compared to investigate the impact of the addition of the P. balsamifera samples on the global relationships. To facilitate visualization, for each of the models using the combined data sets, we iteratively built models that left one predictor out and then plotted the model residuals against the remaining predictor. For δ15N, the global data set contained a sufficient number of observations within the climate range exhibited by the P. balsamifera data (n = 1937) to estimate the effects of MAT and MAP on foliar δ15N across samples spanning a similar range in these predictors. Therefore, we repeatedly (x1000) sampled 755 random observations from the global database constrained to have the same minimum and maximum climate values as exhibited by the P. balsamifera samples. To account for variation in mycorrhizal association between the many species represented in the global foliar δ15N database, we used normalization factors published by Craine et al.10 and applied an offset to each measurement so that mean foliar δ15N was the same for ericoid (+2.7‰), ectomycorrhizal (0‰), arbuscular (−1.2‰), and non-mycorrhizal (−3.2‰) plants. Using the 755 selected observations, we ran a model with MAT, logMAP, and log[N] as model effects and recorded the estimates for each predictor. We then repeated the model on each of the 1000 random resampled data sets and calculated the mean and standard deviation of the resulting model estimates for comparison with estimates of the same predictors on P. balsamifera foliar δ15N alone. There were insufficient samples in the global database to complete the same random resampling procedure for Δ13C.After completing a general survey of the data and evaluating the geographic and climatic range represented by the data, we followed procedures described by Craine 13C) is an outstanding question, particularly among widely distributed plant species that exist across a wide range of climate conditions. However, such relationships have rarely been evaluated for a single, widely distributed species. Therefore, we explored the importance of direct and indirect effects of MAT, MAP, and log[N] on Δ13C using a structural equation model (SEM). The model we used contained no latent variables, but did include all possible pathways between climate variables and the foliar chemical variables. This model was implemented in the R programing language using the Lavaan package version 0.5–2249.The impact of global patterns of N availability and resulting foliar N on water relations in plants (and foliar Δ


During fresh fruit consumption, sensory texture is one factor that affects the organoleptic qualities. Chemical components of plant cell walls, including pectin, cellulose, hemicellulose and lignin, play central roles in determining the textural qualities. To explore the genes and regulatory pathways involved in fresh citrus’ perceived sensory texture, we performed mRNA-seq analyses of the segment membranes of two citrus cultivars, Shiranui and Kiyomi, with different organoleptic textures.GAUT4 in pectin synthesis, CESA1, 3 and 6, and SUS4 in cellulose synthesis, CSLC5, XXT1 and XXT2 in hemicellulose synthesis, and CSE in lignin synthesis. Low levels, or no expression, of genes involved in cellulose and hemicellulose, such as CESA4, CESA7, CESA8, IRX9 and IRX14, confirmed that secondary cell walls were negligible or absent in citrus segment membranes. A chemical component analysis of the segment membranes from mature fruit revealed that the pectin, cellulose and lignin contents, and the segment membrane’s weight (% of segment) were greater in ‘Kiyomi’.Segment membranes were sampled at two developmental stages of citrus fruit, the beginning and end of the expansion period. More than 3000 differentially expressed genes were identified. The gene ontology analysis revealed that more categories were significantly enriched in ‘Shiranui’ than in ‘Kiyomi’ at both developmental stages. In total, 108 significantly enriched pathways were obtained, with most belonging to metabolism. A detailed transcriptomic analysis revealed potential critical genes involved in the metabolism of cell wall structures, for example, Organoleptic quality of citrus is easily overlooked. It is mainly determined by sensory texture perceived in citrus segment membrane properties. We performed mRNA-seq analyses of citrus segment membranes to explore the genes and regulatory pathways involved in fresh citrus’ perceived sensory texture. Transcriptomic data showed high repeatability between two independent biological replicates. The expression levels of genes involved in cell wall structure metabolism, including pectin, cellulose, hemicellulose and lignin, were investigated. Meanwhile, chemical component contents of the segment membranes from mature fruit were analyzed. This study provided detailed transcriptional regulatory profiles of different organoleptic citrus qualities and integrated insights into the mechanisms affecting citrus’ sensory texture.The online version of this article (10.1186/s12864-018-4669-y) contains supplementary material, which is available to authorized users. Fruit quality is determined by many attributes. The organoleptic properties are the vital quality attributes that influence consumer appeal . Among tPlant cell walls play a central role in determining the textural quality of plant-based foods . Fruit tCitrus fruit are hesperidia. The fruit are divided internally into 9–14 juice vesicle-containing segments by segment membranes . Fresh cCitrus unshiu Marcov × Citrus sinensis Osbeck (Kiyomi tangor) and Citrus reticulata Blanco ‘Shiranui’ are two citrus hybrid cultivars that are rapidly increasing in popularity. ‘Kiyomi’ is an orange-mandarin cross, while ‘Shiranui’ is a three-way hybrid between ‘Kiyomi’ and C. reticulata Blanco ‘Ponkan’ [In recent years, citrus hybrid cultivar production has rapidly expanded in China with the focus on consumer-desired properties, such as juiciness, sweet and seedless flesh, and easy peeling. ‘Ponkan’ . The twoHere, to explore the molecular mechanisms of segment membrane’ development, we performed an RNA-seq analysis. Segment membranes were sampled at two developmental stages. The genes in the biosynthesis and regulatory pathways involved in determining the composition of cell walls and the middle lamella were emphasized. To clarify the putative association between cell wall composition and the differences in the two cultivars’ organoleptic properties, the major components in the mature fruit’s segment membranes were analyzed. The transcriptome analysis of segment membrane development combined with cell wall composition analyses should enhance our understanding of the effects of segment membranes on citrus organoleptic qualities.Five-year-old ‘Kiyomi’ tangor and ‘Shiranui’ trees cultivated in a citrus research orchard located in Suining, Sichuan Province, China, were used. Fruit samples were harvested at two developmental stages, 150 and 210 days after flowering, assigned as first and second stages, respectively. At the first stage, fruit development occurred during the early expansion period, while at the second stage, fruit expansion had finished, and fruit reached their maximum size and began to mature. For each cultivar, fruit samples were collected from three different trees. Thus, each tree was an independent biological replicate. For RNA-seq, two independent randomly chosen biological replicates were used. Ten representative fruit from each tree were collected at each sampling stage. Segment membranes separated from fruit segments were immediately frozen in liquid nitrogen and kept at − 80 °C until use. Total RNA was extracted using an RNAprep Pure Plant Kit , which was specifically designed for materials rich in polysaccharides and polyphenolics. Extracted RNA was treated with DNase  to remove genomic DNA. RNA quality was surveyed by separation in 1.0% agarose gels, stained with ethidium bromide and observed by UV light.Total RNA samples from two trees of each cultivar at two developmental stages, for a total of eight samples, were used for RNA-seq library construction and sequencing. Sequencing was performed on an Illumina HiSeq 2000 platform. RNA quantity and integrity were determined using a Nanodrop 2000  and a Bioanalyzer 2100 . RNA-seq libraries were constructed using the Illumina TruSeq RNA Library Prep Kit v2  following standard protocols. Poly(A)-containing transcripts were enriched from the total RNA using oligo(dT)-coated magnetic beads. For each sample, adapters with unique barcodes were ligated to the end-polished cDNA fragments. The libraries were amplified by PCR and quantitated by Qubit 2.0 (Thermo Scientific).r > 0.97), and differentially expressed genes (DEGs) were identified from each comparison using edgeR [Kallisto  was usedng edgeR . DEGs weng edgeR . Gene Onng edgeR , and KOBng edgeR  was applng edgeR  enrichme18 primer with the Transcriptor First-Strand cDNA Synthesis Kit with gDNA Eraser . The Applied Biosystems StepOnePlus™ Real-Time PCR System equipped with StepOne™ Software V2.1  was used. The β-actin expression level was used for normalization. Water was the negative control. Each reaction was replicated three times. To confirm the specific amplification of desired PCR products, melting curves were analyzed. The qRT-PCR data were analyzed using the comparative ΔΔCT method [First-strand cDNA was synthesized from total RNA using an anchored-oligo (dT)The weight ratios of segment membranes to whole segments of mature fruits were determined. For each cultivar, the three trees sampled for RNA-seq and qRT-PCR were used. The data from each tree were regarded as a replicate. Ten fruits were sampled from each tree. Fruit segments were separated and weighed. After carefully removing juice pulp from inside the segment, the segment membranes were weighed. The segment membrane’ weight ratio was given as the value (%) of the segment membrane to fruit segment.The segment membranes used for weight-ratio determination were dried and ground to a 60-mesh powder. The powder was used for pectin content and pectin methylation analyses, and the following experiments, which included determinations of the lignin, cellulose and hemicellulose contents. The data from each tree were regarded as a replicate. Pectin content was determined by quantifying GalA using the carbazole colorimetric method . The extThe lignin content was the sum of the Klason insoluble and soluble lignin. Klason insoluble lignin was analyzed according to the Tappi T 222 om-88 as described by Bura et al. [Total RNAs from eight samples  were prepared and sequenced on an Illumina HiSeq 2000 platform. The output was an average 10 M reads (Paired-end 100-bp model) for each library. High repeatability was shown between two independent biological replicates. From a principal component analysis  < 0.005 and an absolute log2 to be 0.5413 (‘Kiyomi’ first-stage vs. ‘Shiranui’ first-stage), 0.7469 (‘Kiyomi’ second-stage vs. ‘Shiranui’ second-stage), 0.5313 (‘Kiyomi’ first-stage vs. ‘Kiyomi’ second-stage), and 0.7893 (‘Shiranui’ first-stage vs. ‘Shiranui’ second-stage).Eight DEGs were used in a qRT-PCR assay to further validate the transcriptome sequencing dataset. Primers used in quantitative RT-PCR experiments were listed in Additional file The GO enrichment analysis identified 76 GO categories as significantly enriched at the first stage, including 67 categories in ‘Shiranui’ and 33 in ‘Kiyomi’, with 24 categories that overlapped. At the second stage, in agreement with the biological processes majority in ‘Shiranui’ at the first stage, 72 categories were identified to be significantly enriched in ‘Shiranui’ and 42 in ‘Kiyomi’, with 28 categories overlapped, and totally 86 GO categories were significantly enriched at the second stage. In comparisons between the different developmental stages of each cultivar, the GO-enrichment analysis identified 131 biological processes as significantly enriched in ‘Kiyomi’, with 93 processes at both the first and second stages, and 55 overlapping processes. In ‘Shiranui’, 112 processes were enriched, 77 at the first stage and 80 at the second stage, with 45 overlapping processes.http://www.genome.jp/kegg/pathway.html). Most pathway groups belonged to the metabolism category, including “carbohydrate metabolism”, “energy metabolism”, “lipid metabolism”, “amino acid metabolism”, “metabolism of cofactors and vitamins” and “metabolism of terpenoids and polyketides”  in the second stage than in the first stage in both cultivars. GAUT1 and GAUT7 showed similar and relatively low expression levels , the homopolymer of GalA  linked in an α-1,4 configuration, is the most abundant pectic polysaccharide. HG biosynthesis is catalyzed by α-1,4-D-galacturonosyltransferase (EC 2.4.1.43), designated GAUT, and some GAUT-like (GATL) proteins . Twelve e lethal . Here, c complex . GAUT2 wof GAUT1 . SeveralPME isoform genes were detected in this research. Several PMEs showed higher expression levels than the HG biosynthesis-related GAUT and GATL genes. For instance, the average PME31 expression level from ‘Kiyomi’ and ‘Shiranui’ at the two developmental stages was 276.333, which was significantly higher than that of GAUT8 (17.261) or GATL3 (16.460). PME transcripts displaying expression levels greater than 10 were listed in Additional file PMEs showed higher expression levels in ‘Shiranui’ than in ‘Kiyomi’  study, in which gene expression was visually analyzed on ethidium bromide-stained gels [There is a group of pectinases that broadly control pectin degradation. Pectin methylesterase  is an enzyme that catalyzes the specific hydrolysis of the methyl ester bond at C-6 in GalA residues in the linear HG domain of pectin, which is secreted in a highly (70%–80%) methylesterified form. In total, 23 integral GAUT8 17.1 or GATLned gels .Fig. 3PMPMEI contigs that had expression levels greater than 10  family proteins, which restrain PMEs through a post-translational control mechanism, were also detected in this research. There were nine Seven genes encoding pectin acetylesterase E.C. 3.1.1.6), which is responsible for removing the acetyl residues of pectin, were found in our research. The pectin acetylesterase 8, 2 and 6 genes had expression levels greater than 10 and were listed in Additional file .1.6, whiArabidopsis protein databases. Twelve contigs, with expression levels greater than 10 in any sample, were shown in Additional file The deesterified pectin is subsequently degraded by pectinases that break the α-1,4-linkages. Pectinases function by hydrolysis, such as polygalacturonase (E.C. 3.2.1.15), or through transelimination mechanisms, such as pectate lyase (E.C. 4.2.2.2) and pectin lyase (E.C. 4.2.2.10). Here, 32 independent contigs belonging to the pectinase superfamily were detected, although the gene names of these contigs were not annotated by querying using a BLAST algorithm-based search against CESA genes, CESA1, 3, 4, 6, 7, 8 and 9, that were expressed in citrus fruit segment membranes  complexes in symmetrical rosette forms . We dete10 CESAs . Arabidoynthesis –34. The ynthesis –37. In oedundant . The absy absent . From thSUS3 and 4 showed significantly high expression levels comparing with those of SUS2 and 6 , which may be associated with cellulose synthesis [SUS4 displayed an expression pattern similar to those of CESA1, 3 and 6, which are associated with cellulose synthesis in primary walls. We hypothesized that SUS4 might be the main protein responsible for cellulose synthesis in citrus primary cell walls.Sucrose synthase  is another important enzyme involved in cellulose synthesis. Four SUS-encoding genes were detected in our research. ui’ Fig. . It was ynthesis . Here, cynthesis . In our CESA and SUS, the expression levels of the CELs were low , including endo-β-1,4-glucanase. Compared with ofibrils .Hemicellulose is a diverse group of polysaccharides. It constitutes roughly one-third of the wall biomass. In dicots, the hemicelluloses are primarily xyloglucans (XyGs) and xylans (Xyls). The former is most abundant in the primary cell wall, and the latter in the secondary cell wall .CSLC genes, CSLC4, 5 and 12, were detected in citrus segment membranes. CSLC4 and 5 showed similar but lower expression levels than CSLC12 , CSLC 7 (identity 73%) and CSLC 9 (identity 74%), which are expressed during different stages of ovule development [XyGs have a β-1,4-glucan backbone that is linked with different mono-, di- or trisaccharide side-chain xylosyl residues. XyG biosynthesis requires glucan synthase to form the glucan backbone and glycosyl transferases to produce the side chains. At least five types of enzymatic activities are involved in XyG biosynthesis: cellulose synthase-like C proteins (CSLCs), XyG xylosyltransferases (XXTs), galactosyltransferase, fucosyltransferase and acetyltransferase. All of the genes encoding these enzymes were expressed in this study , hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT) and caffeoyl shikimate esterase (CSE). In our data, PAL1, C4H2, 4CL2, C3H3, HCT (single gene) and CSE (single gene) were detected at considerable levels. After CSE, C4H2 was the highest expressed gene. Its average expression level in all eight samples was 61.683, which was greater than PAL1 (18.372), 4CL2 (38.071), HCT (9.259) and C3H3 (21.952)  and heterotrimeric (C4H1–C4H2–C3H3) membrane protein complexes that catalyze three converts, from cinnamic acid to p-coumaric acid, from p-coumaric acid to caffeic acid, and from p-coumaroyl shikimic acid to caffeoyl shikimic acid [C4H1 and C4H2, were confirmed in citrus. C4H2 was constitutively expressed at a significantly higher level than C4H1, which is expressed in response to wound induction, even in wounded tissues [C4H2 was extremely greater than the 0.111 C4H1 level, which implied that the putative heterodimeric complex of C4H2–C3H3 was preferred, rather than the complexes containing C4H1. In addition, the high level of C4H2 indicated that C4H2 might function in both the C4H2–C3H3 complex and in C4H2 alone. The CSE activity, combined with that of 4CL, produces caffeoyl-CoA from caffeoyl shikimate, bypassing the HCT reaction that occurs in the direct conversion [CSEs were highly expressed in our data, with an average value of 245.820. The high expression indicated that the CSE pathway was more effective than that of HCT, which had been suggested in Arabidopsis [CSE compared with other genes, it is important to try and identify additional CSE functions.The synthesis of caffeoyl-CoA from phenylalanine represents a subset of the upstream steps of the lignin biosynthetic pathway, particularly coniferyl alcohol (G lignin) and sinapyl alcohol (S lignin) . Six enzmic acid . Two C4H tissues . From ounversion . CSEs webidopsis . HoweverArabidopsis CCRs were cloned [AtCCR1 is preferentially expressed in tissues undergoing lignification, while, in contrast, AtCCR2 is involved in defense responses. In our research, CCR1 showed a negligibly low expression level, with an average of 0.107, which suggested the faint lignification of citrus segment membranes during the two developmental stages. Five CADs, CAD1, 4 and 7–9, were detected in the citrus segment membranes, and CAD4 (64.898 level) was more highly expressed than the others. Because the knockdown of CAD4 in Arabidopsis resulted in plants with relatively normal lignin contents [CAD4 expression level would lead directly to a higher lignin content.Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) are responsible for the final two steps of the lignin monomer synthesis pathway. Two e cloned , 68. AtCcontents , we did HCT, CSE and F5H (ferulate 5-hydroxylase), other genes involved in monolignol synthesis were detected to be members of multi-gene families. The highest expressing member of each gene family was compared among citrus samples  and COMT1 (caffeic acid O-methyltransferase), had higher expression levels at the second stage compared with the first stage in both ‘Kiyomi’ and ‘Shiranui’. The comparisons of gene expression levels in the two citrus cultivars at the same developmental stage showed that, PAL1, C4H2, C3H3, CCOAOMT1 and CSE were expressed higher in ‘Kiyomi’ at both stages, while 4CL2, HCT, CCR2, CAD4 and COMT1 were expressed higher in ‘Shiranui’. The series of enzymes participating in the lignin synthesis pathway represent multiple isoforms encoded by different genes. These genes vary in kinetic properties, distributions, expression patterns, and responses to antisense suppression by the different enzyme isoforms [In addition to les Fig. . Most geisoforms . Therefop < 0.001). Among the main components of the cell wall, the pectin, cellulose and lignin contents were all greater in ‘Kiyomi’, especially the pectin and lignin contents, which showed significantly higher levels than in ‘Shiranui’. The hemicellulose, unlike the other components, was more abundant in ‘Shiranui’ at 116.6 ± 1.43 mg g− 1 than in ‘Kiyomi’ at 103.2 ± 6.15 mg g− 1 (p < 0.05). The degree of pectin methylesterification was expressed as μl CH3OH g− 1. Based on the results  of segment membranes per segment, and the main components contents of segment membrane’ cell walls, including pectin, cellulose, hemicellulos and lignin, and the degree of pectin methylesterification were illustrated in Fig. lts Fig. , ‘ShiranSegment membranes are not targeted in breeding but are edible portions of citrus. The popular consumer-driven sensory properties of citrus segment membranes are thin, soft and brittle. The property of thinness increases the enjoyment of eating the juice sacs, which are the contents of the segments, rather than the segment membrane. Desirable brittleness in citrus segment membranes allows them to be easily crumbled while chewing the juice sacs. The softness of segment membranes is preferred to a harsh texture.The segment membrane’ weight of ‘Shiranui’ was suspected to determine its thinness, making ‘Shiranui’ more pulpous. Pectin in plant cell walls is part of the polysaccharide network, which is involved in the control of cell wall porosity and cell expansion , and it Lignin imparts strength and rigidity to plant walls, which are highly resistant to both mechanical disruption and enzymatic degradation , 73. In GAUT4 in pectin synthesis, CESA1, 3 and 6, and SUS4 in cellulose synthesis, CSLC5, XXT1 and XXT2 in hemicellulose synthesis, and CSE in lignin synthesis. Low levels, or no expression, of genes involved in cellulose and hemicellulose, such as CESA4, CESA7, CESA8, IRX9 and IRX14, confirmed that secondary cell walls were negligible or absent in citrus segment membranes. A chemical component analysis in mature fruit segment membranes showed that higher pectin, cellulose and lignin contents might result in the undesired organoleptic properties of ‘Kiyomi’, which resulted in the fruit being perceived as thick, glutinous and chewy.Our study provided a comprehensive transcriptomic insight into the citrus segment membrane’s cell wall structures and metabolism, and indicated the possible contribution of segment membrane cell-wall composition to fresh citrus’ organoleptic properties. The high repeatability of transcriptomic data and the detection of DEGs in ‘Kiyomi’ and ‘Shiranui’ indicated distinct differences during segment membrane development between the two citrus cultivars. A detailed transcriptomic analysis revealed potential critical genes involved in the metabolism of cell wall structures, for example, Additional file 1:Figure S1. Heatmap of differentially expressing genes. Figure S2. Verification of RNA-seq by qRT-PCT. Figure S3. Top 30 significantly enriched pathways based on up-expressed genes in ‘Kiyomi’ compared with in ‘Shiranui’ at the first-stage. Figure S4. Top 30 significantly enriched pathways based on up-expressed genes in ‘Shiranui’ compared with in ‘Kiyomi’ at the first-stage. Figure S5. Top 30 significantly enriched pathways based on up-expressed genes in ‘Kiyomi’ compared with in ‘Shiranui’ at the second-stage. Figure S6. Top 30 significantly enriched pathways based on up-expressed genes in ‘Shiranui’ compared with in ‘Kiyomi’ at the second-stage. Table S1. Primers used in quantitative qRT-PCR experiments. Table S2. Top 30 significantly enriched pathways based on up-regulated genes. Table S3. Genes involved in pectin metabolism. Table S4. Genes involved in cellulose metabolism. Table S5. Genes involved in hemicellulose (xyloglucans and xylan) metabolism. (PDF 808 kb)


Equisetum species. The biochemical details of the MXE reaction were incompletely understood — details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-β-d-glucan (MLG), with radioactive oligosaccharides of xyloglucan as the acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and gel-permeation chromatography (GPC). Equisetum HTG consistently cleaved the MLG at the third consecutive β-(1→4)-bond following (towards the reducing terminus) a β-(1→3)-bond. It then formed a β-(1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its xyloglucan endotransglucosylase/hydrolase subfamily membership. Using size-homogeneous barley MLG as the donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative to the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.Mixed-linkage glucan∶xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-β-glucanase (HTG), which among land plants is known only from  The cell wall, which surrounds virtually all plant cells, has a skeleton of cellulosic microfibrils tethered  by a hydrated extraprotoplasmic matrix composed mainly of carbohydrates. Cell expansion is regulated by cell-wall extensibility, which itself is controlled in part by modification of cell-wall polymers by apoplastic enzymes. In addition, the cell wall confers strength to mature tissues, pest and pathogen defence , cell siThe primary cell wall is typically construed as being composed largely of three structurally and functionally distinct polysaccharide fractions: cellulose, hemicelluloses and pectins. The occurrence, prevalence and structure of different cell-wall components often exhibit phylogenic, cell-type and stimulus-dependent variation, the result of the different evolutionary pressures and the differing characteristics conferred on cell walls by each component.d-glucopyranosyl backbone with various substitution patterns. Typically, the first two or three of every four consecutive glucose residues are α-d-xylosylated at position 6 -linked β-GG, XXXG ; see FryGG, XXXG  for an uGG, XXXG . The phyGG, XXXG , possiblGG, XXXG . XylogluGG, XXXG –12, suggd-glucan (MLG) — another hemicellulose — is an unbranched β-d-glucopyranosyl homopolysaccharide in which cello-oligosaccharide regions  are connected by single (1→3) bonds; these (1→3) bonds are never found consecutively , cellulase , cellulase (from A. niger), barley MLG (medium viscosity) and Iceland moss MLG (lichenan) were from Megazyme, Inc., Bray, Ireland. Merck (0.2 mm) silica-gel 60 TLC plates were from VWR . Tamarind xyloglucan was a generous gift from Dr K. Yamatoya . Equisetum arvense MLG was purified as described previously [3H]XGO-ols) were synthesised in-house, essentially as described by Hetherington and Fry [3H]XXXGol and [3H]XXXGXXXGol were ∼100 and 1.6 MBq mol–1, respectively. Dialysis tubing (12–14-kDa cut-off) was from Medicell International, Ltd, London, U.K. Miracloth was from Calbiochem (http://www.emdbiosciences.com). Solvents were from Fisher Scientific, U.K. Other chemicals came from Sigma–Aldrich, U.K.Microbial XEH  was a geeviously . Reducti and Fry , with XXKav [elution volume on GPC relative to totally excluded polymers (Kav = 0.0) and totally included low-Mr material ] 0.2–0.3 were pooled. The pool was rerun through the same column, and again the Kav 0.2–0.3 region was pooled as size-homogeneous MLG; the final yield was 7.7% of the total starting MLG.Three 10-ml aliquots of barley MLG (6.25 mg/ml) were separately fractionated by GPC on Sepharose CL-6B  and the fractions with E. fluviatile was grown outdoors in the King's Buildings pond in Edinburgh.For XEH digestion, 0.001% (w/v) microbial XEH in buffer  was incubated with the sample at room temperature for 1 h.−1 in the same buffer. One volume of lichenase solution was added to 1 volume of substrate (MLG) solution and incubated at room temperature for 4 h.For lichenase digests, lichenase was dissolved at 2 U mld-glucosidase (0.125 U ml–1 in the same buffer) at 20°C for various times.For glucosidase digestion, the sample was incubated with β-All enzyme reactions were stopped by the addition of 0.5 volumes of 50% (v/v) formic acid.E. fluviatile extracts, prepared in buffer as before  and MLG (donor substrate). Routinely, the reactions were stopped by the addition of 0.5 volumes of 50% (v/v) formic acid. MXE products (MLG–[3H]XGO-ol) were freed from unreacted oligosaccharides by precipitation with ethanol . After bench-centrifugation, the pellet was redissolved in water at 100°C and then reprecipitated with 75% ethanol. This was repeated until the ethanolic supernatant contained no detectable radioactivity . Details of the MXE reaction conditions for specific experiments are given in the following three paragraphs.3H]XXXGXXXGol), 0.75% (w/v) barley MLG and 50% (v/v) E. fluviatile extract was incubated at 25°C for 72 h, after which 46% of radioactivity had been incorporated into 75% (v/v) ethanol-insoluble material (MLG–[3H]XGO).For determination of the nature of the bond formed between MLG and xyloglucan (reported in 3H]XGO) were formed by incubation of reaction mixtures (20 µl) containing 15, 10 and 5 kBq [3H]XXXGol (84 MBq µmol−1) with 0.3% (w/v) E. arvense, barley and Iceland moss MLG, respectively, and 50% (v/v) E. fluviatile enzyme extract, for 16 h at 25°C. Ethanol was then added to 75% (v/v), which precipitated the MLG–[3H]XGO.For identification of the site of MXE cleavage of various MLGs  and a 40%-saturated (NH4)2SO4-precipitated enzyme preparation from E. fluviatile at 20°C. After 0, 2, 4 or 8 h, a 62.5-µl aliquot was stopped with formic acid, and the products were size-fractionated by GPC on Sepharose CL-6B.For exploration of the preferred regions of MLG cleavage during the MXE reaction , with a2SO4 XXXGol as the acceptor substrate and barley MLG as the donor substrate, we digested it with lichenase. This yielded a single 3H-oligosaccharide (assumed to be Glcn·XXXGol), which migrated slower than [3H]XXXGol on TLC and was purified by preparative TLC. Graded β-d-glucosidase treatment , we suggest that the two β-d-Glc residues were attached to each other, as (Glc2)·XXXGol. In contrast, [3H]XXXGol with two separate Glc monosaccharide substituents attached at different positions would have given two different intermediary octasaccharides, for which there is no evidence in Supplementary Figure S1. The linkage within the (Glc2) disaccharide was at this point still unknown. Furthermore, the site of attachment of the (Glc2) moiety to the XXXGol, indicated by the dot in ‘(Glc2)·XXXGol’, was also still unknown.Since β-(1→3) bonds act as ‘hinges’ between the relatively rigid cello-oligosaccharide repeat units of MLG, the bond attacked by HTG, like that attacked by other MLG-cleaving enzymes , which 2)·[3H]XXXGol with three hydrolases: a cellobiohydrolase and two cellulases [= endo-β-(1→4)-d-glucanases — a Trichoderma cellulase capable of digesting xyloglucan and an Aspergillus cellulase incapable of digesting xyloglucan] (Trichoderma cellulase were capable of digesting (Glc2)·[3H]XXXGol to [3H]XXXGol in the allotted time. Graded digestion of (Glc2)·[3H]XXXGol with Trichoderma cellulase showed that breakdown to [3H]XXXGol occurred without intermediate , confirming that the two Glc residues are removed, and thus linked, together. Aspergillus cellulase did not digest the (Glc2)·[3H]XXXGol.To investigate the unknown bonds, we digested the (Glcoglucan] . Both th2) disaccharide moiety, we purified (Glc2)·XXXGol by GPC and HPLC, then digested it with Trichoderma cellulase [expected to yield a non-radioactive (Glc2) disaccharide + [3H]XXXGol]. Aliquots of the digest were ‘spiked’ with authentic disaccharides and analysed by HPLC (2)·XXXGol had been hydrolysed to XXXGol and cellobiose. This showed that (Glc2)·[3H]XXXGol is GG·XXXGol, where GG is cellobiose. The bond between the GG and the XXXGol moiety is also likely to be a β-(1→4)-bond because it can be cleaved by cellobiohydrolase. Such a bond identity would also be consistent with HTG's GH16 family membership.To test the nature of the bond within the  was digested with lichenase and the single radioactive product, GG·[3H]XXXGXXXGol (the dot indicating that we do not yet specify to which position on the [3H]XXXGXXXGol the cellobiose unit is attached), was purified by GPC (Bio-Gel P-2) and then digested with microbial XEH, which is expected to cleave only the single GX linkage of the [3H]XXXGXXXGol moiety [3H]XXXGXXXGol and the initial acceptor substrate ([3H]XXXGXXXGol) yielded a single radioactive oligosaccharide, which comigrated with [3H]XXXGol (As a test of whether the bond created during the MXE reaction is a β-(1→4) bond to the non-reducing terminal Glc residue of the XGO, we exploited the fact that, regardless of the length of an XGO, the only free Glc 4-hydroxy group is at the non-reducing terminus. In contrast, other free hydroxy groups also occur throughout the oligosaccharide. Therefore, if HTG attaches MLG to a Glc 4-OH, the new bond will always be to the non-reducing terminus of the XGO, whereas if it attaches it to any other hydroxy group, the new bond could be elsewhere. To test this, we used the tetradecasaccharide [l moiety . MicrobiH]XXXGol . This inH]XXXGol . Since tH]XXXGol .Figure Equisetum MLG, barley MLG and Iceland moss MLG (known as lichenan), which have mainly cellotetraose units, mainly cellotriose units and almost only cellotriose units, respectively [3H]GGXXXGol, chromatographically indistinguishable from that obtained when barley MLG was the donor  bond in HTG's negative subsites during the MXE reaction. The fact that we see only a single product (GGXXXGol) means that the (1→3) bond in the negative subsites is always in the same place when the enzyme is acting on barley MLG. We reasoned that the structure of MLG substrates might affect the site of attack preferences relative to (1→3) bonds. Therefore, to further probe this apparent preference for cleavage relative to a (1→3) bond, we performed MXE–lichenase treatments on three structurally distinct MLGs: ectively . When eahe donor . This coEquisetum > barley > Iceland moss, since these MLGs have high, medium and low cellotetraose contents, respectively. Conversely, if the target is cellotriose, the order should be reversed. Assessed from the Vmax values of MXE activity on the three tested MLGs , the target is the cellotetraose unit. Given this knowledge, the deduced structure of the MXE/lichenase product, GGXXXGol ((1→3) bond ((1→4) bond in the MLG, which would agree with the action of all other known XTH family members.If the target site of HTG is a cellotetraose unit, then the effectiveness of the three tested donor substrates should be GGXXXGol , and the→3) bond . This suThis indicates that it is a requirement for MXE activity that a (1→3) bond links the β-Glc residues occupying subsites −4 and −3, and that (1→4) bonds interlink the β-Glc residues occupying subsites −3, −2, −1 and +1.The above experiments identify the precise molecular site (‘′’ in the sequence…G4G3G4G4G′4G3G4G…) targeted when HTG uses MLG as the donor substrate. As well as exhibiting specificity at this level, it is also possible that HTG might exhibit a preference for a general region of the MLG chain, e.g. near the non-reducing terminus, near the middle or near the reducing terminus; alternatively, the selection of G4G3G4G4G′4G3G4G sites for cleavage may be purely random with respect to the termini. Defining such preferences may help to define the biological role of HTG.) and a radioactive oligosaccharide as the acceptor , the preferred regions of cleavage can be distinguished by the size of the radioactive product —To test for any such preference, we adapted the method of Steele et al. . With a cleavage near the non-reducing terminus:cleavage near the middle:cleavage near the reducing terminus:3H. Each sample yielded a single peak of hexose residues indistinguishable from that of the initial donor substrate, indicating that only a very small proportion of the polysaccharide molecules was cleaved between 0 and 8 h — either by the MXE reaction itself or by any contaminating hydrolases  possess exactly one [3H]glucitol moiety. To allow direct comparison between the profiles of these two types of reading, we converted the mass profiles of the MLG . Since the donor molecules are far larger than the acceptor molecules, random cleavage should yield radioactive transglycosylation products averaging about half the Mr of the donor. Median sizes  preference similar to that of most XTHs [Hexose readings reveal the profiles  since ea the MLG  into mol the MLG  by refereviously . As expee et al. ) were esost XTHs . Our obsEquisetum enzyme from its GH16b subfamily co-member [one glycosidic bond towards the reducing terminus from a (1→3) bond. Another difference is that lichenase requires the (1→3) bond [Our demonstration that HTG cleaves an MLG chain three glycosidic bonds towards the reducing terminus from a (1→3) bond distinguishes this unique o-member  lichenas→3) bond , whereas→3) bond , which l→3) bond .not needed by HTG when xyloglucan or cellulose serves as the donor substrate  bonds in the positive subsites. Since HTG cleaves a (1→4) bond located three glycosidic bonds towards the reducing terminus from a (1→3) bond, and since MLGs contain very few cellopentaose or larger cello-oligosaccharide units, it would seem highly likely that a β-(1→3) bond usually exists between the glucose residues in subsites +1 and +2. We cannot say whether a β-(1→3) bond in this position is required for the MXE activity of HTG. To address this point, we would require polysaccharide substrates rich in cellopentaose or larger cello-oligosaccharide units. Nevertheless, the fact that no MLGOs, whether having a (1→3) or a (1→4) bond at the non-reducing end, are capable of acting as acceptor substrates — while XGOs do  — suggeectively ) since tEquisetum plants, whereas XET activity (due to numerous XTH isozymes resembling those of flowering plants) peaks in young, rapidly growing tissue [Equisetum stems — perhaps by linking the MLG (which extends into [) during the interpolymeric transglycosylation reaction between MLG  and xyloglucan , thus:Extractable MXE activity (due to HTG) peaks in late-season g tissue ,32. It mnds into , and is nds into ,70, the  units near the reducing or non-reducing ends or near the middle of the xyloglucan chain. It thus appears that the biological function of MXE is adequately fulfilled by randomly attacking any exposed lengths of the MLG chain.However, in contrast with this suggestion, we found that HTG attacks MLG stochastically, selecting its target cellotetraose unit randomly along the polysaccharide chain. In this respect, HTG's attack on MLG resembles XTHs' attack on xyloglucan where there is no noticeable discrimination between Equisetum MLG, which has a higher cellotetraose:cellotriose ratio than poalean and lichen MLG. It is unclear whether MLG or HTG appeared first during the evolution of the Equisetales; however, the enzyme is well adapted to its natural substrate. Nevertheless, an appreciable proportion of cellotetraose units do also occur in poalean MLG; thus, MXE activity might realistically be introduced into grasses or cereals by genetic manipulation. Any consequent effects on poalean wall mechanics could then provide new information on the role of HTG and potentially have the benefit of increasing wall strength in ageing stems in cereal crops. Furthermore, our data show that HTG exhibits no appreciable discrimination between cellotetraose units that are located near the reducing or non-reducing end, or middle, of MLG donor substrate chains.In conclusion, we have defined for the first time the precise site at which HTG selects and cleaves bonds when using MLG as the donor substrate. It selects cellotetraose units, cleaving the (1→4) bond located three glycosidic bonds towards the reducing terminus from a (1→3) bond. These sites are most abundant in


Melaleuca alternifolia oil. In situ exposure of polyterpenol to vapors of electron-accepting dopant reduced the optical band gap to 1.5 eV and increased the conductivity from 5.05 × 10−8 S/cm to 1.20 × 10−6 S/cm. The increased conductivity may, in part, be attributed to the formation of charge-transfer complexes between the polymer chain and halogen, which act as a cation and anion, respectively. Higher levels of doping notably increased the refractive index, from 1.54 to 1.70 (at 500 nm), and significantly reduced the transparency of films.Owing to their amorphous, highly cross-liked nature, most plasma polymers display dielectric properties. This study investigates iodine doping as the means to tune optoelectronic properties of plasma polymer derived from a low-cost, renewable resource, i.e.,  The employment of plasma polymerization techniques for synthesis of high-performance organic thin film materials has been investigated by many authors because these techniques allow the polymerization of a range of organic compounds, including those that may not necessarily polymerize through conventional thermo-chemical pathways ,2,3. PolMelaleuca alternifolia oil, by way of radio frequency polymerization. With a refractive index slightly above that of glass, transparency to the visible region, very smooth surface, and high chemical inertness and thermal stability, polyterpenol films have prospective applications in organic optoelectronics as encapsulating layers for the organic circuitry. Polyterpenol has also been demonstrated as an appropriate insulating material to improve the output characteristics of organic field effect transistors (OFET) and is a prospective candidate for full flexible organic electronic circuits [In this paper we report on the effect that in situ iodine doping has on optoelectronic and chemical properties of the plasma-polymerized terpinen-4-ol thin films, herein called polyterpenol. Polyterpenol is fabricated from non-synthetic mono terpene alcohol, a major constituent of circuits ,13,14.Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to analyze the changes in chemical structure of polyterpenol films as a consequence of doping. XPS survey spectra collected for pristine and iodine-doped polyterpenol samples deposited under the same conditions are depicted in −1) and methylene/methyl (1450 cm−1) bands indicates chain branching and suggests that, similar to pristine polyterpenol, iodine-doped film is comprised mostly of short polymer chains rather than long linear backbone structures [−1 region, which correspond to aliphatic C–I stretch, and may suggest incorporation of iodine atoms into the backbone of the polymer, similar to that observed in co-polymers of pyrrole and ethylenglycol doped with iodine during plasma polymerization [−1 region. These peaks are not inherent to the polymer, and arise as a result of inadequate correction for the background CO2 and H2O present in the ambient air. Similar artefacts have been observed previously [In the FTIR spectra of the monomer and pristine polymer sample  the prevructures . FTIR sprization . All FTI/methyl 150 cm−1 bThe shifts observed in C–H frequencies and skeletal C–C vibration bands can be attributed to high electronegativity of iodine atoms that can have marked effect on the spectrum of neighboring group frequencies. Integration of iodine into the polyterpenol molecular structure, most likely by abstraction of H, is further supported by the presence of the group frequency associated with conjugated C=C bond. Considering the complex chemical structure of polyterpenol thin film and presence of a large number of residual radicals commonly observed in plasma polymers, several different reactions with iodine are likely to occur. Formation of iodine oxidants during polymerization can lead to oxidation of alcohol moieties to ketones. Furthermore, light-induced homolytic dissociation of iodine may lead to formation of additional iodine radicals that can, in turn, initiate hydrogen abstraction. Therefore, in situ iodine doping results in a change in structure of plasma polymerized terpinen-4-ol thin film samples.Ψ, Δ) which are then parameterized as a function of wavelength in order to obtain thin film parameters, such as film thickness, surface roughness, refractive index, and extinction coefficient spectral dependencies, dielectric functions, and others [Rp and Rs Fresnel coefficients (for the p- and s-polarizations respectively) and the complex reflection coefficient  was found to be between 2.34 and 2.78 for pristine and between 2.52 and 2.97 for iodine-doped polyterpenol samples. This is similar to the values obtained for plasma polymerized linalyl acetate thin films [εionic) and orientational  phenomena in the bulk of the film accounting for the remainder [εionic and εorientational [For the dielectric function, the real and imaginary terms can be expressed as follows:position . From elin films . At freqemainder . Owing ttational . Indeed,tational ,24,25. Ftational . α is the absorption coefficient, hν the photon energy, B a constant dependent on the length of localized state tails, and n is determined by the nature of the electronic structure of the material, and gE the optical band gap [n can take values of ½ for direct, and 2 for indirect, transition; the n value of 2 is frequently chosen for amorphous materials and indicates a parabolic function for the density of states distribution in the band tails [The optical band gap of the polymer thin films were determined from absorption data obtained using UV-VIS spectroscopy and ellipsometry, and also by fitting a Tauc-Lorenz oscillator model to the ellipsometric data collected for the film samples. In amorphous semiconductors, the photon absorption follows the Tauc relation :(4)αhν=band gap . Parametnd tails ,30. Due nd tails ,32.n, k) was used to estimate the optical band gap values for pristine and iodine doped polyterpenol. As shown in αhν)2 and (αhν)1/2 as a function of photon energy (hν) and extrapolating the linear section of the curve to the abscissa, with the intercept denoting the values for the direct and indirect transition energy gaps. UV-VIS absorbance data was treated in a similar fashion to confirm the results (data not shown here). The results showed consistency between estimation methods and are outlined in Absorption coefficient data which resulted from the modelling of optical constants J=J0essed as :(6)βPF=entiated . The folJ–V characteristic of pristine polyterpenol can be described by a power law relation, where JnV, and n = 0.94. Such value of n indicates an approximately ohmic conduction mechanism [n is 2.05 and 4.68 for 2.5–10 V and 10–20 V applied voltage, respectively, indicating non-ohmic charge transport for these regions. Although n > 2 suggests a possibility of space charge limited conduction, fitting the J–V data, the relationship is more linear in lnJ–V0.5 plot, suggesting Schottky or Poole-Frenkel conduction  [−12–10−11 Ω−1·m−1 [−4–10−8 Ω−1·m−1 and 10−6–10−8 Ω−1·m−1, respectively [The conductivity of the samples was determined as a reciprocal of resistivity −1·cm−1) . In gene Ω−1·m−1 ,42. Thisectively ,45. In pectively .−9 Ω−1·m−1 and 1.20 × 10−8 Ω−1·m−1 for increasing doping levels . For comparison, plasma polymers deposited from iodothiophene have been reported to have conductivity of 1.1 × 10−6 Ω−1·m−1 [−5–10−6 Ω−1·m−1 [2, the electric conductivity ranged from 10−4 to 10−11 Ω−1·cm−1 [Doping increases the conductivity polyterpenol to 7.74 × 10 Ω−1·m−1 . Similar Ω−1·m−1 . When poΩ−1·cm−1 .Atomic force microscopy and nanoindentation analyses were executed to highlight the changes in physical and surface properties of the polymer films while contact angle measurements are conducted to evaluate wettability of the polymer samples. maxR), surface skewness (skwR), and coefficient of kurtosis (kurR) increased from 5.4 nm, 0.22, and 1.65 for pristine to 6.8 nm, 0.76, 2.51 for iodine-doped polyterpenol, respectively. Such values of skwR and kurR indicate a disproportionate number of peaks and a well spread out height distribution for the surface. The average roughness (aR) and RMS (qR) parameters of iodine doped films were found to be similar to the values for pristine polyterpenol, 0.56 nm and 0.42 nm for aR and qR respectively. Increasing the applied power or deposition time did not significantly increase the average surface roughness of iodine-doped films when examined at nanoscale (1 µm × 1 µm).Examination of the surface topography of the pristine and iodine doped polyterpenol thin films showed that the surfaces were smooth, defect free and uniform , Table 3Surface wettability studies were performed using water, diiodomethane and ethylene glycol. The average contact angle reduced from 68.7° to 61.6° for water, 71.63° to 47.1° for glycerol, and from 38.1° to 28.4° for diiodomethane, for pristine and iodine doped films, respectively. Increased hydrophilicity of doped samples was attributed to a significantly larger number of polar functional groups exposed to the top of the polymer surface. Contact angle measurements were also conducted on a number of iodine-doped samples stored for 60 days in an ambient environment, with an approximately 10% increase in respective contact angles for water and glycerol compared to those pertaining to freshly-deposited doped surfaces. Plasma polymerized thin films are frequently characterized by a significant number of radicals trapped within the bulk of the polymer. After the deposition, these radicals continue to react with one another, the polymer molecules, and the ambient environment. Rapid oxidation of polymer upon exposure to the air is common for plasma polymers, and results in the formation of oxygen-containing groups on the surface of the films, rendering the film more hydrophilic. Overtime, however, the polar moieties have a propensity to migrate into, or re-orientate themselves toward, the bulk of the film in an attempt to minimize the interfacial tension at the polymer/air interface. Such change in surface configuration can account for the change in the contact angle behavior of doped polymer films with time. Under similar exposure conditions, the surface configuration of pristine polyterpenol remains relatively constant, due to initially lower surface tension and consequent lower interfacial tension between the surface of polyterpenol and ambient air.The average contact angle for each liquid was used to calculate the surface free energy and its components, based on the Lewis acid/base method , and thePristine and iodine doped polyterpenol thin films were fabricated from non-synthetic terpinen-4-ol monomer  using the experimental setup described in . RF enerKα, hν = 1486.6 eV) operating at 150 W. An atomic force microscope (AFM)  operated in semi-contact mode was used to obtain images of the surface topography and to quantify the surface roughness. Surface wettability studies were performed using a contact angle system  employing the sessile drop method. An average of ten measurements per sample was obtained using sterilized nanopure H2O (18.2 M Ω·cm−1), diiodomethane  and ethylene glycol (Sigma Aldrich). The average contact angle for each liquid was used to calculate the surface free energy and its components, based on the Lewis acid/base method [J–V measurements, metal−insulator−metal (MIM) structures were prepared by depositing aluminium electrodes via thermal evaporation using a custom-made shadow mask placed beneath the samples.Thickness, roughness, and optical properties of the doped and pristine samples deposited on glass substrates were examined through variable angle spectroscopic ellipsometry  and UV-VIS spectroscopy . FTIR spectra of samples deposited onto KBr pallets were recorded at room temperature using a single beam FTIR spectrometer . X-ray photoelectron spectroscopy (XPS) survey spectra were collected using an Axis Ultra spectrometer , equipped with a monochromatic X-ray source (Al e method . For J–V−8 S/cm to 7.74 × 10−7 S/cm and 1.20 × 10−6 S/cm (at 20 V) for increasing doping levels. When subjected to annealing to 100 °C for 5 h, the band gap of doped polyterpenol increased slightly, the increase was attributed to desorption of loosely bonded iodine atoms from the surface of the film, and remained stable thereafter. Doping was found not to affect the surface morphology of the film. All films were smooth, defect-free, and uniform. Increased hydrophilicity of doped samples was attributed to a significantly larger number of polar functional groups exposed to the top of the polymer surface. Iodine doped samples were found to be monopolar in nature, largely manifesting electron-donor properties, although the electron-acceptor fraction of the surface tension for iodine doped samples was significantly higher than that of pristine polyterpenol, due to the presence of iodine atoms.Polyterpenol thin films were manufactured using RF plasma polymerisation. Iodine doping was carried out in situ, which led to significant changes in the chemical structure of the film. FTIR studies revealed that iodine was being incorporated into the backbone of the polymer, and that such incorporation resulted in the notable reduction in the methyl and methylene functional groups and appearance of C=C bond. Reduction in oxygen and increase in carbon atomic fractions as determined by XPS studies further indicated that abstraction of H, most probably by elimination of water, was one of the likely reaction pathways to take place during co-polymerisation. Higher levels of doping notably increased the refractive index of the thin films, from 1.54 to 1.70 at 500 nm wavelength, and significantly reduced the transparency of films, owing to the strong absorption of light characteristic of iodine species. Introduction of iodine impurity reduced the band gap of polyterpenol, by extending the density of states more into the visible region of the electromagnetic spectrum and an increase in C=C bonds in the iodine doped film layers compared to pristine polyterpenol. Doping increased the conductivity from 5.05 × 10


Cancer immunotherapy has primarily been focused on attacking tumor cells. However, given the close interaction between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME), CAF-targeted strategies could also contribute to an integrated cancer immunotherapy. Fibroblast activation protein α (FAP α) is not detectible in normal tissues, but is overexpressed by CAFs and is the predominant component of the stroma in most types of cancer. FAP α has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a post-proline bond. When all FAP α-expressing cells  are destroyed, tumors rapidly die. Furthermore, a FAP α antibody, FAP α vaccine, and modified vaccine all inhibit tumor growth and prolong survival in mouse models, suggesting FAP α is an adaptive tumor-associated antigen. This review highlights the role of FAP α in tumor development, explores the relationship between FAP α and immune suppression in the TME, and discusses FAP α as a potential immunotherapeutic target. In the past, cancer immunotherapy mainly focused on attacking tumor cells. Cancer immunotherapy is a promising treatment strategy against solid tumors; however, it cannot completely eradicate them. The biological complexity of the tumor microenvironment (TME) seems to be an obstacle for cancer immunotherapy, suggesting that using a strategy in which only tumor cells are targeted is inadequate to overwhelm the aggressively growing tumor. Therefore, a synergistic TME-targeted strategy is required for the development of more potent cancer immunotherapy . Modern In order to determine the role of FAP α-expressing stromal cells in immune suppression within the TME, a transgenic mouse model has been created with established Lewis lung carcinomas. Only 2% of the tumor cells in this model express FAP α; however, when all FAP α expressing cells  are destroyed, the tumors begin to die rapidly. Therefore, FAP α expressing cells are a non-redundant, immunosuppressive component of the TME . FurtherExtracellular matrix (ECM) breakdown, detachment of neoplastic cells from the primary site, and their subsequent invasion into the lymphatic vessels, capillaries, and the surrounding normal tissues, is the result of a complex interplay of numerous proteolytic enzymes, including serine proteases. Overexpression of serine proteases in carcinomas is correlated with enhanced tumorigenicity and adverse prognosis -12. TherFAP α was originally identified by a group in pursuit of a selective marker for activated fibroblasts. A monoclonal antibody named F19 was produced to define the FAP α-positive cell, which strongly labeled cultured fibroblasts, fibroblasts in fetal mesenchymal tissues, the reactive stromal fibroblasts of epithelial tumors, and tumor cells of sarcomas , 14. TheFAP α is a type II integral membrane serine protease that belongs to the dipeptidyl peptidase (DPP) subfamily, which has the ability to cleave the bond between proline and any other amino acids. This enzymatic activity has been shown to have an impact on a wide variety of bioactive signaling molecules . There iFAP α has both dipeptidyl peptidase and collagenase activity, and can degrade gelatin and type I collagen. However, the biological significance of FAP α cleavage of gelatin and type I collagen is still unknown. Nonetheless, FAP α is required for the generation of biologically active fragments of denatured collagen . RecentlIn vivo studies have demonstrated that increased FAP α expression is associated with increased tumor growth rate and promotes neovascularization [While different kinds of tumors have general features in common, they also have their own characteristics related to tumor location, size, stage, degree of cell malignancy, involvement of lymph nodes, and metastasis. In this section, we will examine the role of FAP α in different carcinomas. FAP α is expressed by more than 90% of human epithelial tumors, and there has been much interest in exploring FAP α as a therapeutic target in breast cancer. rization . Anotherrization . These tFAP α is overexpressed by CAFs in 85-90% of primary and metastatic colorectal cancers . High leA series of findings about the expression and role of FAP α in pancreatic carcinoma has suggested that FAP α-targeted immunotherapy may be a new treatment for pancreatic cancer patients. FAP α-induced reorganization of the ECM in TME promotes the invasiveness of pancreatic cancer cells . There i3 in size, tumor progression is critically dependent on the supporting TME [The complex interactions between the stroma and tumor, along with the regulatory signaling molecules in the TME, contribute to oncogenesis and tumor progression. The process of tumor invasion and metastasis is accompanied by angiogenesis and ECM degradation . In mostting TME . Previouting TME -43.in vitro have the ability to activate the host immune system. Even the use of tumor-specific antibodies and activation of antitumor immune cells does not alter the overall capabilities of these agents [However, tumor immune tolerance is a major impediment in cancer immunotherapy. For example, tumor vaccines proven to have therapeutic effects e agents . Therefoe agents -48, all e agents .In contrast to normal tissues, the vast majority of immune cells in the TME have lost their function. Furthermore, in cancer patients the composition of immune cells undergoes a change wherein the inhibitory subgroups, such as regulatory immune cells, myeloid-derived suppressor cells (MDSCs), and M2 macrophages are the dominate components . In addiFAP α is an immune-suppressive component in the TME. The first experiments that assessed the immunologic effects of perturbing the FAP α+ stromal cells used a direct approach of conditionally depleting this cell type from a mouse bearing immunogenic Lewis lung carcinoma cells expressing ovalbumin (LL2/OVA). These studies demonstrated that administering diphtheria toxin to these mice depletes approximately 80% of the tumoral FAP α+ cells, which comprised only approximately 2% of all tumoral cells, and caused rapid, adaptive immune-dependent reduction in tumor volume. Furthermore, when FAP α expression was inhibited, tumor shrinkage was seen accompanied by increased expression of IFN-γ and TNF-α, indicating the existence of immune suppression that was restricted to the FAP α+ cells . Using bTRP53 gene. The residual tumor is composed only of premalignant epithelial cells and inflammatory cells. Thus, a single protein, CXCL12, secreted from a single stromal cell type, the FAP α+ CAFs, explains the overriding immunosuppression by the FAP α+ cell in a model of human pancreatic ductal adenocarcinoma. CXCL12 is the reason for immune suppression, while abrogation of FAP α positive cells permits immune inhibition of tumor growth and enhances the efficacy of constructed immunotherapeutic antibodies [In subsequent studies, immune suppression by the FAP α+ CAFs was mediated by CXCL12, the chemokine that binds to cancer cells and excludes T cells by a mechanism that depends on signaling by the CXCL12 receptor, CXCR4 . T cellstibodies . Furthertibodies . TherefoAs previously mentioned, FAP α is a transmembrane serine protease that is highly expressed on CAFs present in >90% of human epithelial tumors, and plays a significant role in tumor progression and metastasis . Targetiin vivo that matters [As reviewed above, FAP α belongs to the serine proteinase family, has both collagenase and dipeptidase activities which can degrade gelatin, collagen, and other substrates of dipeptidase, and promotes tumor growth, migration, invasion, metastasis and ECM degradation. Therefore, it was hypothesized that selectively blocking the enzymatic activity of FAP α may be a method of targeting it in tumor development. Val-boroPro, also called Talabostat, was the first inhibitor of the proteolytic activity of FAP α used in phase II clinical trials successively performed in patients with metastatic colon cancer, non-small cell lung cancer, and melanoma. Val-boroPro was administrated alone or combined with other non-specific anti-tumor drugs, but minimal clinical response was observed with the addition of Val-boroPro -64. Therin vivo diagnostic and tumor-targeting studies [The disappointing clinical outcome of Val-boroPro does not exclude the potential role of FAP α's proteolytic activity in tumor invasion and metastasis, or that an inhibitor antibody may be a potent therapeutic target. An inhibitory scFv antibody, named E3 was identified, which competitively inhibits FAP α function . This sc studies . Combina studies , 69. AdvEarly work on FAP α-targeting monoclonal antibodies investigated the toxicity, imaging, and bio-distribution of a 131-labeled monoclonal antibody (131I-mAbF19) against FAP α in patients with hepatic metastases from colorectal carcinoma. The 131I-mAbF19 was administered by intravenous injection and no toxicity was observed . Furtherin vitro were restricted to target cells overexpressing the FAP α antigen, consistent with other studies [in vitro screening method was used to determine whether dendritic cells transfected with mRNA encoding products of FAP α are capable of stimulating cytotoxic CD8+ (CTL) responses from human peripheral blood mononuclear cells. It was demonstrated that CTL responses could be consistently generated against FAP α. To enhance the immunogenicity of the mRNA-translated FAP α product, a lysosomal targeting signal derived from lysosome-associated membrane protein-1 (LAMP-1) was fused to the COOH terminus of FAP α to redirect the translated product into the class II presentation pathway. Dendritic cells transfected with mRNA encoding the FAP α-LAMP fusion product stimulated enhanced CD4+ and CD8+ T-cell responses [Vaccines targeting FAP α provide another therapeutic strategy that takes advantage of the restricted distribution of FAP α in tumor sites. Scientists have constructed a DNA vaccine directed against FAP α , and imm studies , 60. Moresponses .in vitro results confirmed that 1-MT could be dissociated from the FAP α τ-MT vaccine and inhibit intracellular IDO activity [Furthermore, in our research, we have developed a new tumor vaccine, FAP α τ-MT, which was produced by conjugating 1-MT to a FAP α. The activity . In a FAactivity . These rCancer cells are embedded in stroma, the connective tissue framework of solid tumors. Stromal cells and cancer cells depend on each other for mutual paracrine stimulation, and stromal fibroblasts are probably required for cancer cells to survive and grow , 79. RecFAP α is a tumor-associated antigen which is a serine protease involved in extracellular matrix remodeling and highly expressed on reactive stromal fibroblasts in >90% of human epithelial carcinomas, but is not detectable in normal adult human tissues , 84. StrIn the context of immunotherapy involving T cells targeting cancer cells, an agent targeting FAP α-expressing cells might increase therapeutic efficacy against both solid tumors and metastatic cells -89. Elim


The relationship between diabetes and oxidative stress has been previously reported. Exercise represents a useful non-pharmacological strategy for the treatment in type 2 diabetic (T2DM) patients, but high intensity exercise can induce a transient inflammatory state and increase oxidative stress. Nutritional strategies that may contribute to the reduction of oxidative stress induced by acute exercise are necessary. The aim of this study was to examine if n-3 PUFA supplementation intervention can attenuate the inflammatory response and oxidative stress associated with high intensity exercise in this population. As a primary outcome, lipoperoxidation measurements (TBARS and F2-isoprostanes) were selected.Thirty T2DM patients, without chronic complications, were randomly allocated into two groups: placebo (gelatin capsules) or n-3 PUFA (capsules containing 180 mg of eicosapentaenoic acid and 120 mg of docosahexaenoic acid). Blood samples were collected fasting before and after 8 weeks supplementation. In the beginning and at the end of protocol, an acute exercise was performed (treadmill), and new blood samples were collected before and immediately after the exercise for measurements of oxidative stress and high-sensitivity C-reactive protein (hs-CRP).p = 0.005). Supplementation also significantly reduced TRAP levels after exercise  for − 33,884 , p = 0.004, Cohen’s d effect size = 1.12), but no significant difference was observed in n-3 PUFA supplementation group in lipoperoxidation parameters as TBARS (mean difference and 95% CI to − 3.8 (− 10–2.4) for − 2.9 (− 1.6–7.4) or F2-isoprostanes (mean difference and 95% CI -0.05 (− 0.19–0.10) for − 0.02 (− 0.19–0.16), p > 0.05 for both.After the supplementation period, a decrease in triglycerides levels was observed only in n-3 PUFA supplementation group (mean difference and 95% CI of 0.002 (0.000–0.004), PUFA n-3 supplementation reduced triglycerides as well as TRAP levels after exercise, without a significant effect on inflammatory and oxidative stress markers.This study is registered at ClinicalTrials.gov with the registration number of NCT03182712. The redox imbalance resulting from the increase of reactive oxygen and nitrogen species, and the concomitant lowering of antioxidant defenses have been associated to micro- and macrovascular diabetic complications . A low concentration of vitamin E was used in the formulation to prevent n-3 PUFA oxidation, as previously recommended . Subject1C) was measured by an immunoassay; urinary albumin was measured by immunoturbidimetry.Venous blood samples were taken after fasting from the antecubital vein in EDTA coated (for plasma) or plain (for serum) tubes using standard aseptic techniques. Urine samples for measurement of albuminuria also were collected only before and at the end of the supplementation period. Total cholesterol, HDL-cholesterol, triglycerides and glucose were measured by enzymatic colorimetric assays; Friedewald’s formula was used to calculate for LDL-cholesterol fraction . GlycateBefore and after each submaximal test, blood samples were immediately centrifuged at 4 °C, 4000 rpm, for 5 min, and plasma or serum were separated and stored at − 75 °C for further analysis. We analyzed hs-CRP after each testing using nephelometry. Lipid Peroxidation was assessed by two distinct techniques: 1) Plasma F2-isoprostanes analysis, using a commercial kit (Cayman®), and 2) TBARS analysis, based in the thiobarbituric acid reactivity with malondialdehyde, a lipid oxidation end-product . Non-enzt-test for independent samples or the Mann-Whitney test were performed. A generalized estimating equation (GEE) followed by Bonferroni’s post hoc test was used to verify supplementation and exercise effects adjusted for age, gender, duration of diabetes and use of drugs. The GEE model for each outcome was based on the goodness of fit. The normality of the residuals was verified by normal Q-Q plot. Some cardiometabolic variables were transformed due to the high variability among individuals, using a) logarithmic transformation for hs-CRP and F2-isoprostanes, b) square root transformation for albuminuria, uric acid and SOD, and c) inverse transformation for triglycerides and HbA1C. Cohen’s d was used to verify the effect size of the means. The accepted significance level was p < 0.05. Data were reported as mean ± standard deviation, and difference mean and 95% confidence interval (95% CI).Data were analyzed on SPSS 25.0 for Windows . Normality was assessed by the Shapiro-Wilk test, and Levene’s test was used for homoscedasticity. For baseline value comparison between groups, Student’s p = 0.03).Figure p > 0.05 for both). Table P = 0.005), compared with baseline values, and by 26% when compared with the placebo group (P = 0.05). No other interaction condition vs time was observed in the experimental groups.After the supplementation period, individuals did not change your body weight . Table d effect size was consider high for this mean difference (d ≥ 0.8). Although no statistically significant interaction was found for SOD levels (p = 0.095), the n-3 PUFA supplementation group presented an increase after supplementation , with a tendency in their concentrations after exercise. There was no significant difference for hs-CRP between the groups.Regarding submaximal exercise tests, there were no differences in time duration in both groups before (14 ± 5.02 min and 15 ± 6.1 min) and after supplementation (15 ± 4.7 min and 13 ± 5.8 min) for n-3 PUFA supplementation and placebo groups, respectively  were not attenuated by n-3 PUFA supplementation.2 (load dependent) caused by exercise can lead to increased blood and tissue levels of reactive oxygen species [2peak) in type 1 diabetic subjects. In our study, exercise performed with intensity equivalent to 70–80% of VO2peak was not able to increase the production of lipoperoxides as measured by TBARS and F2-Isoprostanes (primary outcomes). If exercise intensity was to be an issue, our data are corroborated by the findings of Davison et al. [As previously mentioned, physical exercise is considered a powerful tool to prevent and to treat diabetes and the associated comorbidities. Nowadays, due to the lack of time, many people are using high intensity exercise sessions (with a lower volume), as an alternative, to improve their health, metabolic function and body composition. However, high intensity exercise can induce a transient inflammatory state and increase oxidative stress , 11 that species . Laakson species  and Atal species  showed in et al. , that diP = 0,002). After treating healthy volunteers with 3.6 g/day of n-3 PUFA, Nalsen et al. [P = 0.015) reduction in lipoperoxidation as measured by F2-isoprostanes concentration. The difference in results among studies may be related to different exercise protocols, duration of the tests, and different methods employed when measuring the oxidative stress markers.We hypothesized that including n-3 PUFA to our subjects would induce benefits when they exercise at high intensity, however, with except of changes in TRAP, our intervention fail to change levels of lipoperoxidation, SOD and UA. Accinni and colleagues  also foun et al.  did not Regarding SOD and UA, our secondary outcomes, there were no significant changes after the exercise, similarly to the results described by Laaksonen et al.  and AtalSubjects who develop T2DM have high levels of inflammatory markers such as hs-CRP when compared to healthy subjects , 38. SupPlasma glucose and HbA1c did not change significantly after the period of supplementation. Some studies reported increased levels of glucose, HbA1c and low insulin activity, in type 2 diabetes, after the consumption of large amounts of fish oil (around 10 g/day) . HoweverTo the best of our knowledge, this is the first study to examine the interactions between n-3 PUFA supplementation and high-intensity exercise on oxidative stress and inflammation in subjects with T2DM. However, we recognize that the study has several limitations. First, the sample size was small, although the effect size on the variables with statistical significance was high. So, at this point, should be interpreted with caution before being extrapolated to the general population. Second, we have only studied acute effect of high intensity exercise; therefore, it is not possible to state that the effects would be similar after other intensities of exercise. Finally, we did not analyze the supplement offered to the participants, so we cannot be 100% sure if the product provides the amount of n-3 PUFA. We believe in the suitability of the company, but we suggest that future studies test the product before the beginning of the trial. Despite some limitations, the strength of this study is that we analyzed a large number of variables related to oxidative stress, including two measurements of lipoperoxidation (TBARS and F2-siprostanes) and measurements of enzymatic (SOD) and non-enzymatic antioxidants (uric acid and TRAP). Thus, this study contributes to narrow the gap in the literature on the effects of antioxidants nutrients in oxidative stress parameters after acute exercise. Further clarification is needed regarding the clinical relevance of the n-3 PUFA supplementation for T2DM in different intensities of exercise.P values. On the other hand, n-3 PUFA cause reduction of triglyceride levels, at rest, without altering membrane oxidative damage or inflammation (as assessed by hs-CRP). Studies including other inflammation markers such as IL-8, MCP-1, eHSP70, or others, are required to better assess this response. Likewise, longer periods of observation and/or and other intensities of exercise may be necessary to induce significant increments in oxidative stress parameters in diabetic patients.Eight weeks of supplementation with n-3 PUFA did not attenuate oxidative stress parameters after a high intensity exercise in T2DM subjects, with exception to TRA