Recent studies have shown that their mechanisms of action result in decrease cell migration, cell invasion, cell proliferation, and metastasis of PCa cells.Prostate cancer (PCa) is the most frequently diagnosed cancer and the third highest cause of cancer-related deaths in men in the U.S. The development of chemotherapeutic agents that can bind PCa tumor cells with high specificity is critical in order to increase treatment effectiveness. Integrin receptors and their corresponding ligands have different expression patterns in PCa cells. They have been identified as promising targets to inhibit pathways involved in PCa progression. Currently, several compounds have proven to target specific integrins and their subunits in PCa cells. In this article, we review the role of integrins inhibitors in PCa and their potential as therapeutic targets for PCa treatments. We have discussed the following: natural compounds, monoclonal antibodies, statins, campothecins analog, aptamers,  Integrins are transmembrane protein receptors that attach cells to the extracellular matrix (ECM) or that bind ligands secreted by other cells ,2. They The Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute have estimated there will be 161,360 new cases of PCa in 2017 . CurrentThe α2β1 integrin is a well-known receptor for collagen, which is highly expressed in PCa and its activation/phosphorylation has been implicated in PCa progression. This integrin is the dominant cell-surface protein that has the most relevant role in the invasion of PC3 PCa cells when compared to other integrins [Gleditsia sinensis (GS) is a natural herb that has been used in oriental medicine to treat many diseases, such as carbuncles, suppuration, obesity, and swelling [G. sinensis against different types of cancer, such as gastric cancer, lung cancer, colon cancer, and PCa [G. sinensis extract has shown to affect α2β1 integrin expression, and to have regulatory effects in cell migration and adhesion in PCa cells. Recent studies have shown that the administration of non-toxic levels of water extracted from G. sinensis (50 μg/mL) significantly inhibited collagen-mediated cell migration and cell adhesion in PC3 cells through the inactivation of the expression of the α2β1 integrin [G. sinensis extract specifically inhibits the expression of the α2 subunit (not the β1 subunit) [swelling . Its the and PCa . G. sinesinensis 0 μg/mL ssubunit) ,7,8,9. Isubunit) ,5,6,9. CG. sinensis-mediated inhibition of cell migration and adhesion was shown to be more effective in controlling the progression of late stages in metastatic prostate tumors compared to early stage PCa. Furthermore, a study using an in vivo xenograft tumor model showed that mice treated with a 200 μg/mL oral dose of G. sinensis extract developed significantly smaller tumors compared to non-treated, nude mice [G. sinensis in vivo when they observed that treatment with high concentrations (>50 μg/mL) resulted in DNA fragmentation and induction of programmed cell death [G. sinensis extract inhibits collagen, which is a mediator of cell migration and cell adhesion in PC3 PCa cell line via the inactivation of the α2β1 integrin.d-pinitol is a phytochemical that was identified as the active and main ingredient in soy-based foods and legumes [d-pinitol. This phytochemical functions as an osmolyte in plants by improving tolerance to drought or heat stress [ legumes ,10. Matut stress . In termt stress ,12. Recet stress .d-pinitol has been shown to inhibit cell migration and invasion at non-cytotoxic concentrations (0 μM to 30 μM) in the PC3 and DU145 androgen-independent PCa cell lines [d-pinitol reduces αvβ3 integrin mRNA expression, resulting in the inhibition of metastasis [d-pinitol downregulates αvβ3 integrin expression through two important pathways. First, this compound inhibits the FAK/c-Src kinase phosphorylation pathway, which plays an important role in cell motility and invasion in a dose-dependent manner [d-pinitol decreases p65 phosphorylation in the NF-κB signal transduction pathway [d-pinitol in PCa cells is mediated through the modulation of αvβ3 integrin cell surface expression.ll lines . Also, itastasis . d-pinitt manner ,13. Seco pathway . This pa pathway . TogetheIntegrins that contain the αv subunit contribute too many cellular functions that have been shown to promote malignancies, such as melanoma, renal cancer, colorectal cancer, and PCa . InhibitOn the other hand, β1 integrins are highly expressed in the basal cell layer and are localized in the basal cell/stromal interface, where integrins interact with the ECM . The actMonoclonal antibodies (mAbs) represent the largest class of therapeutic agents that specifically recognize cell surface antigens ,14,15. TThe 33B6 mAb was developed as an inhibitor of the activated conformation of β1 integrin . Lee et The αvβ3 integrin is highly expressed in invasive and metastatic PCa cells and can bind ligands such as fibrinogen . Recent Studies are currently validating the use of statins for PCa treatment ,17. InteThe ability of statins to control tumor cells and their microenvironment could be useful in improving the efficacy of the currently available chemotherapeutic drugs when using a combination chemotherapeutic approach ,18. SimvScientists are striving to develop new anticancer compounds partly because conventional cancer drugs have limited specificity and bioavailability . The devKong et al. investigated the antitumor activity of 10-HCPT in 22RV1 androgen-dependent PCa cells, which have high αvβ3 integrin expression, and in MCF-7 breast cancer cells, which express low levels of αvβ3 integrin . The mecThe α6β4 integrin belongs to the group of laminin-332 binding integrins that are present in endothelial, epithelial, and Schwann cells, as well as in keratinocytes ,24. The Aptamers, are short single stranded oligonucleotides with high affinity and specificity for target molecules, including proteins, peptides, metal ions, small molecules, and cancer cells, due to their unique three-dimensional folding ,25,27. T50 of 149 nmol/L [The selection of an integrin α6β4-specific DNA aptamer (IDA) to block the α6β4 integrin/laminin-332 interaction was recently reported by Berg et al. . IDA inh9 nmol/L . The bin9 nmol/L . In addi9 nmol/L . Also, t9 nmol/L ,25,26.The endothelial cell α5β1 integrin fibronectin receptor can recognize single ligands, and has been shown to play a role in metastatic invasion during extravasation and angiogenesis ,32,33,34Peptide pharmaceuticals have been identified as important therapeutic compounds for cancer because of their low toxicity and high specificity . The PHSN-acetylation and C-amidation makes PhScN resistant to proteolytic degradation [Peptide pharmaceuticals with simple structures, such as PhScN, have several advantages compared to other therapeutic compounds, including high potency and specificity. Their small size allows them to penetrate tissues that may not be reached by larger molecules. However, a major disadvantage of these agents is their rapid degradation by proteolytic enzymes, such as aminopeptidases and carboxypeptidases . Neverthradation ,31,35.The expression of the αvβ1, αvβ5, αvβ3, and α2β3 integrins correlates with the ability of cells to adhere and migrate to ECM proteins in vitro, as well as the aggressiveness of the cancer cell lines in vivo ,37,38. TContortrostatin (CN) is a non-toxic component in the venom of the southern Copperhead snake that functions as a platelet integrin antagonist, thereby inhibiting their aggregation ,37,38,39CN has been shown to inhibit PC3 cell growth, detachment, and migration . These rThe expression of αvβ3 and αvβ5 integrins in advanced PCa is upregulated compared to normal prostate cells because they are functional integrins with metastasis initiating properties . The mos50 value of 2.0 nM in PC3 PCa cells [GLPG0187 is a non-peptide RGD integrin receptor antagonist . GLPG018Ca cells ,43. In vCa cells ,44. InteCa cells . In addiCa cells . These rCa cells .N-telopeptide of type 1 collagen (uNTx) [MK-0429 is a non-peptide small molecule inhibitor of the αvβ3 integrin that has demonstrated potent inhibition of osteoclast formation and osteoclastic bone resorption in both preclinical and clinical studies . A studyn (uNTx) . Resultsn (uNTx) . In othen (uNTx) .Cilengitide is a cyclic pentapeptide and RGD mimetic that selectively and competitively antagonizes ligand binding to αvβ3 and αvβ5 integrins in vitro . In vitrIn summary, the study of integrin inhibitors have helped to characterize integrin specificity  and haveThis review discusses the use of integrin inhibitors as anti-PCa treatments. The studies reviewed here have shown that targeting integrins and their subunits can affect diverse signaling pathways that are responsible for PCa progression. The development and discovery of new molecules, natural compounds, drugs, formulations, non-peptide, and peptide inhibitors that can target specific protein-ligand binding sites of interest in PCa remains a challenge. Collectively, the data provided supports the important pharmacological effects of each integrin inhibitor as anticancer agents. We classified integrin inhibitors as new active agents for PCa treatment and emphasized various aspects of their inhibitory properties, such as high specificity for their target and low toxicity. Nevertheless, it is important to mention that there is a need for additional research to investigate the biological role of integrins in bone microenvironments, because this is another potential target in the progression of metastatic PCa cells. This research will significantly contribute to a better understanding of the cellular behavior of integrins and their ECM ligands in different stages of PCa. On the other hand, it is important to know how to control integrins by targeting other biological pathways, such as cell metabolic pathways. A better understanding of how to inhibit integrins will drive this area of research forward and will enrich what is currently known about how to treat PCa cancers that are resistant to chemotherapy, will help in the development of new biomarkers that can be used for personalized therapy, and will improve actual chemotherapeutic strategies and help in the developments of new ones. In sum, targeting the biomolecular interactions between integrins and their inhibitors has great potential in PCa therapeutics.


Artemisia capillaris→ Heteropappus altaicus→ A. sacrorum. The diversity pattern was one of low–high–low, with diversity peaking in the 10–20 year phase, thus corresponding to a hump-backed model in which maximum diversity occurring at the intermediate stages. A spatially aggregated pattern prevailed throughout the entire period of grassland recovery; this was likely linked to the dispersal properties of herbaceous plants and to high habitat heterogeneity. We conclude that natural succession was conducive to the successful recovery of native vegetation. From a management perspective, native pioneer tree species should be introduced about 20 years after abandoning croplands to accelerate the natural succession of grassland vegetation.Analyzing the dynamic patterns of species diversity and spatial heterogeneity of vegetation in grasslands during secondary succession could help with the maintenance and management of these ecosystems. Here, we evaluated the influence of secondary succession on grassland plant diversity and spatial heterogeneity of abandoned croplands on the Loess Plateau (China) during four phases of recovery: 1–5, 5–10, 10–20, and 20–30 years. The species composition and dominance of the grassland vegetation changed markedly during secondary succession and formed a clear successional series, with the species assemblage dominated by  Ansai CWe used the approach of substituting space for time—i.e., using plots of different ages in the same study area—to investigate grassland vegetation dynamics in the recovery phases during secondary succession. Data were collected in September 2011 from a total of 36 sampling plots  at nine locations and from four recovery phases after cropland abandonment: 1–5, 5–10, 10–20, and 20–30 y. Three 1 m × 1 m quadrats  were chosen along the diagonals of each plot for detailed inventories of the grassland vegetation. All trees, shrubs, and herbs were identified and measured for their base diameter, layer coverage, total height, and growth condition. Local villagers and land documents verified the time of abandonment of each plot. Farming practices had been similar across all nine locations for >40 y before their abandonment. These abandoned croplands were allowed to undergo natural vegetation succession with no influence from human activity  or to species replacement between sites (turnover) , βsne is the nestedness component of the Sørensen dissimilarity, a is the number of species shared between two cells, b is the number of unique species at the poorest site, and c is the number of unique species at the richest site.where βq = 0), the exponential of the Shannon entropy , and the inverse Simpson concentration . These proposed estimators are accurate for both rarefaction and short-range extrapolation.Hill numbers—i.e., the effective numbers of species—are often used to characterize the taxonomic, phylogenetic, or functional diversity of a species assemblage  was estimated as the mean of 200 bootstrap replications, with 95% confidence intervals drawn , each of which was divided into n (n ≥ 2) small equal quadrats (the “S-quadrats”) /n if the occurrence of species i is also random. The relationship can be extended for the analysis of a community with s species as follows:Shiyomi et al.  proposedyi = lg(vi/n2) and xi = lg[pi(1-pi)/n], the relationship between yi and xi is expressed by the simpler regression:where α′ and β are constants intrinsic to the plant community, and ζ is a random variable. For the logarithms of both variances, denoted by i = lgζi is the residual term for the difference of species i from the regression line, which indicates the community tendency. Species on the y = x line have random patterns, species above the line have aggregated patterns, and species below the line have regular patterns. The tendency of the spatial patterns in the community is random when α = 0 and β = 1; however, the distribution of individual species may not be random, depending on the value of εi.where α = lgα′and β are constants estimated from the samples, and εy = x over the range of x, the spatial patterns of a community tend to be more heterogeneous than those found in a random distribution. The degree of spatial heterogeneity of species i is expressed by δi as:For regression lines above i may thus be similar to that of εi if β is near 1 and α is near 0. The heterogeneity of the entire community (δc) can be expressed by the weighted average heterogeneity:The distribution of δAll statistical analyses used the R version 3.3.2  Keng had the highest IVs and were the dominant species in the 1–5y recovery phase; H. altaicus (Willd.) Novopokr., B. ischaemum, Setaria viridis (Linn.) Beauv., and Leymus secalinus (Georgi) Tzvel. were the dominant species at 5–10 y; A. sacrorum, A. leucophylla (Turcz. ex Bess.) C. B. Clarke, S. bungeana, and H. altaicus were the dominant species at 10–20 y; and A. sacrorum, A. leucophylla, Lespedeza bicolor Turcz., and S. bungeana were the dominant species at 20–30 y. The grassland at the site underwent a clear successional series, with species assemblages dominated by A. capillaris→ H. altaicus→ A. sacrorum.The species SOR) and turnover dissimilarity (βSIM) showed similar trends among the four phases: being highest for 10–20 y, but with similar values at 5–10 y and 20–30 y, but lowest for 5–10 y. The nestedness dissimilarity (βSNE) clearly differed among the four recovery phases in this order: 10–20 > 20–30 > 5–10 > 1–5 y.The variation in species composition among the four recovery phases was estimated by the beta-diversity analysis Figure . Total dq = 0, 1, and 2 compared the plant diversities among the four recovery phases , Shannon diversity (q = 1), and Simpson diversity (q = 2) showed consistent trends: increasing gradually from 1–5 y to 10–20 y, yet remaining stable with diversity peaking at 10–20 y, then decreasing in the final phase (20–30 y). Nonetheless, the confidence intervals for species richness overlapped.Coverage- and individual-based rarefaction and extrapolation curves for the Hill numbers s Figure . The refq = 0, 1, and 2 for compared the plant diversities among the four recovery phases , Shannon diversity (q = 1), and Simpson diversity (q = 2) had patterns consistent with those for species diversity based on the abundance data .Coverage- and sample-based rarefaction and extrapolation curves for Hill numbers s Figure . All fouIn sum, a comparison of species diversity based on both abundance data and incidence data found that the diversity pattern of grassland vegetation followed a trend of “low–high–low,” with species diversity highest during 10–20 y of succession.R2 values (coefficients of determination) for all phases indicated that the power law adequately explained the mapping data and successfully identified high spatial heterogeneity. All of the regression lines were mostly above the y = x line over the observed ranges of x. The spatial patterns of the four phases were judged to be more heterogeneous than would be expected from a random pattern, as they displayed an overall aggregated pattern according to the power-law graph.Figure p, and spatial heterogeneity, δ, of the four recovery phases. Here, we used the 15 most dominant species with their maximum abundances , L. bicolor (H9), Potentilla bifurca Linn. (H13), and Bidens pilosa Linn. (H4) were highly heterogeneous. By contrast, L. daurica (Laxm.) Schindl. (H7), S. viridis (H2), A. argyi Levl. et Vant. (H15), and A. leucophylla (H8) were less heterogeneous and had intermediate δ values. The species A. capillaris Thunb. (H3) had an approximately random pattern. In the next phase, of 5–10 y, S. oleraceus (H10), B. pilosa (H4), L. secalinus (H11), and L. bicolor (H9) were all highly heterogeneous, whereas S. bungeana (H5), P. bifurca (H13), D. moldavica (H12), and S. viridis (H2) were less heterogeneous and had intermediate δ values. The distribution of species H. altaicus (H6) followed an approximately random pattern. In the third, of 10–20 y, S. oleraceus (H10), B. pilosa (H4), L. secalinus (H11), and S. viridis (H2) were all highly heterogeneous. Conversely, L. bicolor (H9), S. bungeana (H5), A. argyi (H15), and D. moldavica (H12) were less heterogeneous and had intermediate δ values. The species A. sacrorum (H1) had an approximately random pattern. In the final phase assessed, that of 20–30 y, D. moldavica (H12), A. argyi (H15), and L. daurica (H7) were highly heterogeneous. P. bifurca (H13), A. leucophylla (H8), S. bungeana (H5), and H. altaicus (H6) were less heterogeneous and had intermediate δ values, while A. sacrorum (H1) showed a more or less random pattern.In the first 1–5 y of succession, c) increased gradually for the recovery phases of 1–5 y (δc = 0.077), 5–10 y (δc = 0.071), and 10–20 y (δc = 0.142), was largest in the 10–20 y phase, but then abruptly decreased to a low level in the phase of 20–30 y (δc = 0.078).The spatial heterogeneity for the entire grassland community , was often later succeeded by the perennial species A. sacrorum during grassland succession. The perennial species H. altaicus has always been an associated species, and though it continues to survive and grow over the long term it cannot ultimately form a climax grassland community. The importance value of A. sacrorum increased across the recovery phases; it was the dominant species in the 10–20 y phase, but obviously more so at 20–30 y. We therefore conclude that the grassland vegetation at our study site is adaptable to the environment: it was able to form a stable community after 20 years of natural recovery of abandoned cropland.After cropland abandonment, the grassland species composition and dominance changed markedly during the four phases of 1–30 y of secondary succession. A clear successional series emerged, whereby the species assemblage was dominated by A. capillaris, S. oleraceus, and B. pilosa) and the late-successional species  coexisted in the 10–20 y phase. Chen et al. . We therefore recommend that some native pioneer tree species, such as Pinus tabuliformis Carr., Betula platyphylla Suk., and P. davidiana Dode., be considered for introduction to the grassland 20 years after abandonment, to accelerate the process of succession and to promote community stability.Natural succession assists in the recovery of vegetation. However, natural succession over a long period (>20 years) may be needed to establish stable vegetation cover, especially in grazed areas or where erosion is still a large problem. Our results indicated that successional age was a key factor in controlling species composition and diversity, as it further affected the recovery and development of the vegetation. Furthermore, the succession of grassland vegetation may be accelerated by introducing pioneer tree species. The succession of vegetation on the Plateau is as follows: annual herbs→ perennial herbs→ shrubs→ pioneer trees→ climax trees. Our study found that the grassland communities which formed after 20 years of natural recovery on abandoned cropland were dominated by some shrub and semi-shrub species, namely The species composition, diversity patterns, and spatial heterogeneity changed markedly during the secondary succession (1–30 y) of grassland vegetation on the Loess Plateau. The pattern of plant diversity was of low, then high, and then low again, with the highest diversity observed in the 10–20 y phase. An aggregated spatial pattern was pronounced throughout the entire period of grassland recovery. Natural succession assisted the recovery of vegetation to abandoned croplands. However, the results suggest that 20 years after abandonment occurs, native pioneer tree species should be introduced to accelerate the natural succession of grassland vegetation.CS, SX, and GL initiated and designed the research. SX and GL obtained funding for this study. CS, SX, and GL collected the materials and performed the experiments. CS, ZC, and SX analyzed the data and wrote the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


APOE) status interacts with vascular risk factors (VRFs) to predict the progression of white matter hyperintensities (WMHs) on brain MRI scans over a specific period of life in older age when the risk of dementia increases. At age 73 years, baseline VRFs were assessed via self-reported history of diabetes, hypertension, smoking, and hypercholesterolemia, and via objective measures of blood HbA1c, body mass index, diastolic and systolic blood pressure, and blood high-density lipoprotein to total cholesterol (HDL) ratio. APOE e4 allele was coded as either present or absent. WMH progression was measured on MRI over 3 years in 434 older adults, in a same-year-of-birth cohort. APOE e4 carriers with either a self-reported diagnosis of diabetes  or higher glycated hemoglobin levels  exhibited greater WMH progression, and the former survived correction for multiple testing. All other APOE-VRF interactions were nonsignificant . The results suggest that carrying the APOE “risk” e4 allele increases the risk of greater age-related WMH progression over the early part of the eighth decade of life, when combined with poorer glycemic control. The interaction effect was robust to co-occurring VRFs, suggesting a possible target for mitigating brain and cognitive aging at this age.We examined whether apolipoprotein E ( T1-, T2-, T2*-, and FLAIR-weighted sequences were coregistered at a resolution of 1 × 1 × 2 mm. A semi-automated multispectral fusion method  combined2.5APOE status  as predictors, alongside an interaction term (APOE e4 status × VRF), for each VRF. We used full information maximum likelihood estimation to reduce bias due to missingness, under the assumption of “missing at random”  are reported throughout. Variance inflation factors (VIFs) among VRFs was ascertained using “vif” in the “usdm” package for R. In a supplementary, a posteriori analysis, we further explored interactions between APOE status and both HbA1c and diabetes. We split APOE e4 and e2/3 groups by diabetes diagnosis or high/low HbA1c level (median split at 5.59 DCCT), visualized, and tested (t-tests) group differences in WMH progression.WMH volumes at age 73 and 76 years were expressed as a proportion of intracranial and corrected for age in days on the date of image acquisition. Residuals of the regression between log WMH volume at Waves 2 (IV) and 3 (DV) were derived to index WMH volume change (ΔWMH) over a mean of 3.71 (SD = 0.27) years. Using the “sem” function in the “lavaan” package (v.0.5–22) in R v3.2.2, we used ΔWMH as the dependent variable in models with VRFs and  random” , includi random” : age 11 3t (865.34) = 3.685, p < 0.001) in the total sample. Associations among the VRFs were modest  or 2  factors of general vascular risk. A total of 136 participants were APOE e4 carriers, who were not significantly different to non-e4 carriers in terms of age, cross-sectional WMH volume at either wave, male:female ratio, MMSE score at Wave 2, or VRF status. APOE e4 carriers did, however, show a significantly lower MMSE score at Wave 3 , but these did not survive FDR correction.Effects of VRFs on WMH volume change—without reference to s sample . The maiβ = 0.160, p = 0.002) or higher HbA1c  showed greater WMH progression, but only for APOE e4 carriers. Only the interaction between APOE e4 and diabetes survived FDR correction. Correcting these relationships for other, co-occuring VRFs  and HbA1c . Further group-wise analysis showed that participants with diabetes, or high HbA1c, who carried the APOE e4 allele exhibited significantly greater change in WMH volume progression over 3.7 years than all other groups  point of assessment, the current data suggest that carrying the e4 allele continues to influence the degree of WMH progression at this specific age. In isolation, diabetes was not associated with WMH progression  with HbA1c might ostensibly corroborate the biological plausibility of this result. Having only 2 waves of data makes our results more susceptible to artifacts such as regression to the mean  are unknown, and may affect these findings. We also relied on self-report for information on a variety of clinical diagnoses. Nevertheless, the use of blood biomarkers such as HDL ratio and HbA1c allow objective measures, independent of self-report for some VRFs. More importantly, although the current total sample is relatively large, the proportion of individuals with both diabetes and the e4 allele was small, thus the specific diabetes-mean see  and otheAPOE e4 carriers. This work requires replication in other larger prospective imaging studies, and also at different ages to examine whether, and which, interactions contribute to life-course WMH volume changes. Furthermore, APOE is only one of the many potential genetic contributors to WMH burden in older age. Its consistent association with dementia and cognitive decline make it an obvious candidate for interaction studies, but WMH risk is likely to be polygenic (The reduction of average blood glucose levels is identified here as a potentially meaningful predictor of WMH progression in the mid-1970s among olygenic , and futThe authors declare no potential conflicts of interest.


High-performance oscillators, atomic clocks for instance, are important in modern industries, finance and scientific research. In this paper, the authors study the estimation and prediction of long-term stability based on convex optimization techniques and compressive sensing. To take frequency drift into account, its influence on Allan and modified Allan variances is formulated. Meanwhile, expressions for the expectation and variance of discrete-time Hadamard variance are derived. Methods that reduce the computational complexity of these expressions are also introduced. Tests against GPS precise clock data show that the method can correctly predict one-week frequency stability from 14-day measured data. Timing technology is important in modern finance , industrThe difference between an oscillator’s output signal from its nominal value can be divided into deterministic and random parts. The oscillator’s random behavior is well-documented by a class of noise processes called power-law noise (PLN) . While tOn the other hand, distinctions between deterministic and random behavior are blurry . It is oThis paper studies the estimation of oscillator stability under the influence of frequency drift. In f (Fourier) frequency and It is well documented that high performance oscillators are influenced by power-law noises (PLN). PLN processes are conventionally defined by their power spectral densities (PSD):M, RWFM) . HoweverHowever, PSD measured from an oscillator signal is not used solely in practice. Since the PSD estimates are “noisy” , time dok is used as a generic form of different variances. The majority of measured data nowadays are digital. e is Euler’s number, A time-domain variance fined in :(6)Φk(αits PSD:σk2(τ)=∫0le Σk(τ) . The disIf we denote:Equation . Insteadfunction . For exawing way :(11)Sx(The autocorrelation function of PLN processes has the following asymptotic form when t>>τ :(12)RRx(quations –(14), WaRx(quatioσ^k2(τ):Varσ^y2:ΦAVAR:ΦAVAR:Varσ^y2 of:chniques , and we h subject to:The basic idea of stochastic ONA is:Equation , ε>0,10),(34)3τ)−6Rxt+τ,τ+6Rx=uations (Eσ^z2(τ)=t . Hence, we replace the symmetric two-time autocorrelation function in Equation t≥τ0, them fixed, are normally distributed. It is easy to see that Equation Φ, B(ε) aquations . The unfEquation  will be Equation .In GPS MCS clock prediction, only PLN of m, the complexity of Equations (Although for Equations  and 38)38), Waltquations , 16), N-dimensional column vector i-th component of ℓ such that:In order to estimate the values of Equations , 16), ,  and 38)38), we dquations , (16), (quations  and (38)quations , (16), (quations  and (38)if if To calculate the values of By using the auxiliary vector To test the modified stochastic ONA method, a strong presentation of deterministic frequency drift should be seen. In the real data test of , frequenThe oscillator should be somehow well-modeled. If the dominant variation of the frequency standard has not been model and it has major influence on frequency stability estimates, stochastic ONA will not function properly. For instance, stochastic ONA fails to predict the long-term AVAR and HVAR from MJD.52437.0-52451.0 GPS SVN.36 (PRN.06) precise clock data. AVAR and HVAR estimated from the data imply strong periodic behaviors.To test the method proposed in this article, we predict The prediction of GPS SVN.45 onboard clock long-term stability shows how stochastic ONA behaves when it cannot distinguish RWFM from frequency drift. As shown in h that holds for Equation  formulated the influence of frequency drift on Allan (AVAR) and modified Allan (MVAR) variances; (ii) derived expressions for discrete Hadamard variance (HVAR); (iii) simplified the formulations for the case of GPS MCS (master control station) clock prediction; and (iv) introduce a method that reduces the computational complexity of Walter’s characterization of AVAR and MVAR.For the SVN.45 satellite clock, stochastic ONA cannot find a set of noise intensity coefficients for which Equation  holds foFor the SVN.41 onboard clock, stochastic ONA does find noise intensity coefficient values that hold for the probabilistic constraints . In thisTo test stochastic ONA and the model, we predict In summary, the method introduced in this paper can predict long-term stability superimposed with influences of frequency drift. Criterion  takes thriterion  assumes


S-gene mutations in the post-immunization era in China. From 2005 to 2013, 1077 hepatitis B cases under 15 years of age reported through Chinese National Notifiable Disease Reporting System (NNDRS) were successfully sequenced of S-gene in Shandong province, China. A total of 97 (9.01%) cases had amino acid (aa) substitution in the “α” determinant of HBsAg. The yearly prevalence from 2005 to 2013 maintained at a relatively stable level, and showed no significant change (P > 0.05). Multivariate logistic regression analysis demonstrated that the prevalence of “α” mutations was independently associated with the maternal HBsAg status (P < 0.05), and not with surveillance year and hepatitis B vaccination (P > 0.05). The hottest mutation position was aa126 , and aa 145 . Mutated residue 126 tended to occur less frequent, while that of residue 145 was more frequent with increasing year. Our data showed that there was no increase in the frequency of HBV “α” mutations over time during the post-immunization period. However, long-term vaccination might enhance the change of HBV mutational pattern, and G145 mutation was becoming dominant.Limited information is available about the temporal trend in the prevalence and evolution of hepatitis B virus (HBV)  An estimated 350 million population were chronically HBV infected, with 600,000 deaths annually related to HBV worldwide2. In most HBV endemic areas, maternal-infant vertical transmission is a major route for the HBV chronic infection (approaching 90%)4. Interruption of early HBV transmission through effective immunization is the most cost-effective strategy for the prevention of primary infection.Hepatitis B virus (HBV) infection and HBV-related complications remains a major global public health problem5. It is well documented that neutralizing antibodies produced during natural infection, or following active or passive immunization against HBV are targeted to the conformational epitopes of the “α” determinant6. Hence, single or multiple mutations occurring within this region can lead to conformational changes with altered antigenicity and can allow replication of mutated HBV in vaccinated population, posing a potential threat to the long-term success of massive vaccination7. Such mutations are generally behaved as vaccine escape mutants (VEMs). Early investigations have revealed that VEMs were mainly occurred in the “α” determinant8. G145R mutation was the first identified VEMs to be described in 19909, and later several other notable VEMs  at different positions associated with HBV breakthrough infections have also been repeatedly documented worldwide11. Apart from causing immune failure, VEMs are found in association with HBV occult infection13, and also have been proven to reduce the binding of antibodies raised against wild-type small envelop protein under experimental conditions15.The surface (S) region of HBV surface antigen (HBsAg), utilized in current recombinant hepatitis B vaccine (HepB), contains a highly conserved antibody-neutralizing epitope cluster which spans amino acids (aa) 124–147 within the major hydrophilic region (MHR) of HBsAg, and is referred to as “α” determinant. The “α” determinant is considered to be in the form of two major loops with cysteine-disulfide bonds16. With the successful control of HBV achieved by massive hepatitis B vaccination, one of the remaining concerns might focus on the issue of VEMs, which may potentially challenge the global prevention program and the eventual eradication of HBV infection17.In China, HepB was recommended to infants since 1992 and was introduced into the national children immunization program in 2002, with the prevalence of HBsAg among the children under 5 years of age decreasing from 9.67% in 1992 to 0.96% in 20062, which is inhabited by a population of 95.79 million (2010 census data). With the implementation of national immunization program, HBsAg prevalence among children aged 1–14 years has dramatically decreased from 8% in 1992 to 1.36% in 200618. Since 2005, surveillance of hepatitis B case under 15 years of age has been implemented in the province. We used the blood samples collected in this surveillance system during 2005–2013 to analyze the trend of the prevalence and pattern of HBV S-gene mutations in the era of massive hepatitis B vaccination, evaluating the influence of the current national universal vaccination immunization program on HBV surface mutations.Shandong province is located in the eastern part of China and covers an area of 156,700 km19. After being reported through Chinese National Notifiable Disease Reporting System (NNDRS), all hepatitis B cases aged 0–14 years, including acute and chronic cases, were investigated using a standard questionnaire through interviewing with their parents by the staffs of the Center for Disease Control and Prevention (CDC) at county level. All investigators were trained at provincial CDC before the investigation. The cases’ demographic information , hepatitis B vaccination history, clinical characteristics and epidemiological risk factors  were collected. Hepatitis B vaccination history was obtained according to immunization certificate, if not, by parents’ description. Blood samples were collected for each case in the hospitals and then transferred to the provincial CDC and stored at −70 °C until use.The surveillance on hepatitis B cases was initiated in all 140 counties of Shandong province since 2005. The definition of hepatitis B case was based on clinical and serological criteria in the hospitalThe ethical approval was given by Ethics Review Committee of the Shandong CDC, and the study was conducted in accordance with the ethical standards of the Declaration of Helsinki. Written informed consents for the use of their clinical samples were obtained from the children’s legal guardians.TM and evaluated under UV light. Samples that were found to be negative in the one-step PCR were amplified by two-step PCR using HBVSF2  and HBVSR2  inner primers in second round. The amplification conditions were the same as in the one-step PCR. All necessary precautions to prevent cross-contamination were strictly followed, and negative controls were included in each step of the molecular assays.HBV DNA was extracted from 200 ul serum with QIAamp DNA Blood Mini Kit  according to manufacturer’s instructions. DNA was amplified by two primers, HBVSF1  and HBVSR1  in a 50-UL reaction. Amplification conditions were as follows: 94 °C for 5 min, then 35 cycles at 94 °C for 35 s, 58 °C for 30 s, 72 °C for 35 s, and finally 72 °C for 10 min. Amplified products were visualized by 1.5% agarose gel electrophoresis stained with GelRed20, including 28 HBV sequences of different genotypes (A-H) obtained from GenBank. The reliability of the phylogenetic tree was tested using the bootstrap test with 1,000 replicates. The HBV serotype was deduced from the amino acid (aa) at positions 122, 127, 134, 159, and 16021. The envelope gene aa sequence of each sample was determined by translation of the nucleotide sequence according to the S open reading frame (ORF). Amino acid mutations were identified by comparison of the target sequence with HBV reference sequences of the same genotype. The sequences obtained were submitted to GenBank with the assessions KX067076-KX067778.The positive PCR products were purified and sequenced directly with a BigDye Terminator v3.1 Cycle Sequencing Kit . Sequences were analyzed by an ABI Prism 3130X genetic analyzer (Applied Biosystems). The HBV genotype was identified by phylogenetic analysis using neighbor-joining method after estimation of genetic distance using the Kimura two-parameter method χ2 test or Fisher’s exact test, as appropriate. The χ2 test for trend was used to analyze the trend of proportions with year. A logistic regression model was used to examine associations between the prevalence of “α” mutations and putative risk factors. All statistical tests were two sided. P value < 0.05 was considered statistically significant. All statistical analysis was performed by SPSS 13.0 for Windows .Comparison of categorical variables among different groups was analyzed using From 2005 to 2013, a total of 3120 cases of hepatitis B under15 years of age  were reported by NNDRS in Shandong province, China. The case number decreased with year during this period. Blood samples were collected from 2194 (70.32%) of them. Due to an insufficient volume of serum, HBV-DNA extraction was completed only for 1248  cases, with S gene successfully sequenced in 1077 cases   based on Lys122, Arg160, Val177 and Pro178; four strains (0.37%) were specified as Adrq−, based on Lys122, Arg160 and Ala177; ninety-six strains (8.91%) belonged to adw2, based on Lys122, Lys160 and Pro127; three strains (0.29%) were specified as adw3, based on Lys 122, Lys160 and Thr127; thirteen strains (1.21%) belonged to ayr, based on Arg122 and Arg 160; six strains (0.56%) belonged to ayw1, based on Arg122, Lys160, Pro127, Phe134 and/or Ala159.Phylogenetic analysis of S gene indicated that three HBV genotypes were circulating in Shandong province. Genotype C was predominant, accounting for 93.59% (1008/1077); followed by genotype B for 6.13% (66/1077) and genotype D for 0.28% (3/1077). No other genotypes were detected. The majority of HBV strains belonged to HBsAg subtype In 1077 acquired sequences, a total of 97  strains had aa substitution in the “α” determinant region of HBsAg. The yearly prevalence of “α” mutations during 2005–2013 was 6.11% (8/131), 9.90% (10/101), 9.73% (11/113), 6.62% (9/136), 9.22% (19/206), 9.33% (7/75), 12.75% (13/102), 10.26% (8/78), and 8.89% (12/135), respectively, showing no statistically significant difference.To further investigate the profiles of “α” determinant mutations, 403 cases with missing information on hepatitis B vaccination and/or maternal HBsAg status were excluded. Table 8 IU/ml. The mutations cases’ characteristics were summarized in Supplement-1.Fifty-seven out of 674 cases with complete information had “α” mutations. Documented anti-HBs titer and HBV DNA levels were available in 42 cases among them. Of these, five cases  had a protective level of anti-HBs . Quantification of HBV DNA revealed levels ranging from <500 to 3.84 × 1027. Notably, two T131N/M133T (a novel N-linked glycosylation site) double mutations associated with I126S were observed in our study15. In addition, the other six mutations types  were not yet identified as VEMs, and D144N was a newly identified mutation  had demonstrated low ability to bind antibodies and were identified as VEMs previously27. Seventy-one  out of 107 mutations were located in the 1st loop (aa 124–137) and 37  in the 2nd loop (aa 139–147) of this antigenic domain.The most prevalent mutation position was aa 126 , and aa145 , all of which were recognized as VEMsχ2trend = 7.74, P = 0.005). By contrast, the percentage of aa 145 was 8.82%, 26.32% and 44.44% in the corresponding period, showing a significantly upward trend   increased from 60.8% for children born in 1992–1997 to 93.2% in 2002–200534, which was reported to be a major driving force for the selection of VEMs7.The present study found a much higher prevalence of “α” mutations among children with HBsAg-positive mother compared with those whose mother was HBsAg-negative. The reason might be the fact that most children born to HBsAg-positive mother were given additional hepatitis B immunoglobulin (HBIG) besides three doses of hepatitis B vaccine to interrupt HBV vertical transmission35. Mutations at aa 126 had been found frequently in HBV isolates from patients with chronic HBV infection with or without immunization, and most of these mutations were considered to be natural occurrence10. Residue 145 is located in the 2nd loop structure, known to be the best-described escape mutant position. G145 mutation is highly associated with vaccination selection, and neutralizing antibody induced by vaccination is no longer recognizes the mutated epitope27. The well-known G145R/A mutations were often found in vaccination failure settings10. Again, an in vitro model revealed that HBV with a G145R mutation shared the replication efficacy with equal to wild-type virus36.Based on an alarming number of sequences obtained in the present study, several important findings were observed concerning the mutation patterns. Of the 11 positions where mutations occurred in “α” determinant region, the most frequent mutation position was aa 126 , and 145 , all of which were known to act as VEMs, accounting for more than half of the total mutation strains wither the “α” determinant. So, aa 126 and 145 might be the hottest mutation positions among HBV-infected children. Residue 126 is located in the 1st loop structure of the “α” determinant, and aa changes in this region may lead to HBV adaptive evolution, resulting in the changes of antigenicity and virulence increasedOur study found mutated residue 126 tended to occur less frequent, while that of aa 145 was more frequent with increasing year. It was largely attributable to an increasing massive vaccination and favoring selective pressure on HBV, and accordingly indicating that the substitutions in the aa positions 145 were more associated with anti-HBs immune pressure than those in other epitope sites of “α” determinant region. Our results strongly suggested although the prevalence of “α” determinant mutations showed no significant change in the post-immunization era, the patterns of these mutations had been remarkably changed under the pressure of vaccination. As a result, G145 mutation was becoming more and more dominant with the vaccination selection and needed to be incorporated into future vaccine design.26. Additionally, a small amount of P127T, A128V, G130E, T131P/N, M133T/I, F134L and D144N mutations was observed in our study, including the less frequent T131N/M133T double mutations. T131N/M133T can create a novel N-linked glycosylation site, and play a role in some HBV immune escape mutants15. Further biological functional analyses of these mutations are needed to determine whether they are able to escape existing vaccines. To the best our knowledge, D144N is a new identified mutation in the present study.Four other types of VEMs , which were known to be associated with virus secretion and lower reactivity in HBsAg assays, were also detected37. However, Hsu et al. confirmed that G145R virus could circulate as a stable strain for 8 years of follow-up38. Such mutations could also contribute to HBV transmission in children born to HBsAg-positive mothers and vaccinated individuals40.So far, it is still uncertain when these mutations emerged, or their duration in circulation. A previous study proved that more than half of HBV-infected children could spontaneously lose the predominance of circulatory mutant virus by reversion to wild-type12. The point mutations of “α” determinant were detected by direct sequencing instead of cloning and sequencing assay and quasispecies mutations might be ignored in this study, which led to the underestimation of actual prevalence of “α” determinant mutations.There were still several limitations in our study. The data related to the administration of HBIG backgrounds in the studied children was not been collected. No children with HBV occult infection were involved in our study, who were reported to harbor even more mutationsIn conclusion, the present study revealed that the prevalence of “α” determinant mutations remained stable in post-immunization period. However, long-term hepatitis B vaccination enhances the change of epidemic types in “α” determinant mutations. G145 mutation was becoming dominant. Given the facts that hepatitis B vaccine, HBIG and antiviral drug would more and more widely used in the future and possibility of the transmission of such “α” determinant mutation has not be complete ruled out, an ongoing surveillance with longer years is desperately needed.Supplementary Information


Fat tissue is well known for its capacity to store energy and its detrimental role in obesity and metaflammation. However, humans possess different types of fat that have different functions in physiology and metabolic diseases. Apart from white adipose tissue (WAT), the body’s main energy storage, there is also brown adipose tissue (BAT) that dissipates energy as a defense against cold and maintains energy balance for the whole body. BAT is present not only in newborns but also in adult humans and its mass correlates with leanness. Moreover, “brown-like” adipocytes have been detected in human WAT. These “brown-in-white” (brite) or beige cells can be induced by cold and a broad spectrum of pharmacological substances and, therefore, they are also known as “inducible brown adipocytes.” Activation of brown and/or brite adipocytes reduces metabolic diseases, at least in murine models of obesity. Thus, brown/brite adipocytes represent the “brite” side of fat and are potential targets for novel therapeutic approaches for treatment of obesity and obesity-associated diseases. Obesity and overweight have been conclusively shown to increase the risk of type 2 diabetes, hypertension, hypercholesterolemia, cardiovascular disease, and certain types of cancer . ObesityAn important pathophysiological basis of obesity and obesity-associated disorders is the increase in adipocyte size due to the uptake/storage of excess energy in the form of lipids, which causes cellular stress and an inflammatory response that spreads throughout the whole body (metaflammation) .In addition to the “bad” white fat that represents, for many people, the “dark side” of fat tissue, there is also a bright side: brown fat takes up glucose and lipids and burns energy to generate heat. Moreover, cold exposure and different pharmacological stimuli bring out “brown-like” cells in white fat depots. These cells function similar as brown adipocytes and are also termed brite or beige cells.White adipose tissue (WAT) is the major adipose organ in adults and is the main storage site of energy in the form of triacylglycerols. Whenever fuel is required, fatty acids are released from WAT by lipolysis. This process is initiated by norepinephrine which binds to the beta-adrenergic receptors on white adipocytes leading to the generation of cAMP. The second messenger cAMP activates protein kinase A and, in turn, stimulates the hormone-sensitive lipase releasing free fatty acids from triacylglycerol in the lipid droplets. WAT composes as much as 20 % of the body weight of healthy adult humans. Although it is widely distributed throughout the whole body, separate/specific depots can be distinguished: visceral white adipose tissue (vWAT) mainly surrounds internal organs, whereas superficial or inguinal white adipose tissue (igWAT) is located beneath the skin.Morphologically, a white adipocyte is a unilocular cell and contains a single large lipid droplet that pushes the nucleus close to the plasma membrane. Mitochondria are located mainly in the thicker portion of the cytoplasmic rim near the nucleus. Beyond simple fat storage, WAT is also a secretory and endocrine organ that secretes hormones , interleukin 6 (IL-6), metallothionein, resistin, and etc.) and has an important role in metabolic homeostasis, inflammatory processes, and vascular homeostasis . AlthougDevelopment of WAT is initiated in the mesoderm during the embryonic period . Murine The transcriptional regulation of WAT adipogenesis involves the activation of several transcriptional factors including PPARγ and C/EBPs . PPARγ iUCP1, several markers for brown adipocytes have been described including, peroxisome-proliferator-activated receptor γ-coactivator 1α (PGC-1α), cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea), Zic1, Lhx8, Eva1, and Epsti1 [Brown adipose tissue (BAT) is a special type of adipose organ found in almost all mammals including mice, rats, rabbits, sheep, bears, and humans . Pigs ard Epsti1 .BAT develops earlier than WAT during embryogenesis (as early as day 9.5) , 22, 55.N-methyltransferase 1 (EHMT1), C-terminal-binding proteins (CtBPs), and early B cell factor-2 (EBF2) [During BAT development, some positive regulators have been identified so far, including protein PR domain containing 16 (PRDM16), PPARα, bone morphogenetic protein 7 (BMP7) and Orexin. Although PRDM16 was shown to be dispensable for brown adipocyte development , it is r2 (EBF2) , 41, 42.2 (EBF2) . BMP7 an2 (EBF2)  as well 2 (EBF2) .UCP1, Cidea, PGC-1α, PRDM16, and CCAAT/enhancer-binding protein β (C/EBPβ). In mice, Zic1 and Hoxc9 have been identified as the most specific markers for classical BAT and brite fat, respectively [Cd137, Tbx1, Tmem26, Cited1, and Shox2 have been suggested [Brite/beige fat, which is also known as inducible brown adipose tissue, functions as an extra or reserve brown adipose tissue that can be induced by cold exposure to dissipate energy , 32. Briectively . In addiuggested .Fig. 1WhThere is no consensus concerning the mechanism of “browning” and the embryonic origin of brite adipocytes. There is evidence that brite adipocytes arise from pre-existing white adipocytes , 52. On PRDM16 and PPARα/γ play critical roles in brite cell development. Their positive regulatory effect has been shown to be related to an induction in PGC-1α expression  and a stSince white fat is often viewed as “bad” or as the “dark side” of adipose tissue, one might be inclined to overcome or ease obesity via inhibition of adipose tissue expansion , 26. It An alternative might be to further the “brite side” of fat by increasing the number of brown and/or of brite cells. According to the evidence of numerous animal models –33, 53,


P = 0.007) and mild asthmatics . Also, the GCRα expression level had significant inverse correlation with age in SRA . Glucocorticoid receptors are related to, but not directly responsible for GC resistance. Since the GCRα/GCRβ expression ratio decreased in SRA, studies are needed to assess its value in diagnosing GC resistance.The response to glucocorticoids (GCs) therapy classifies severe refractory asthma (SRA) and mild asthma, so the glucocorticoid receptors (GCRs) gene expression may be involved in SRA pathogenesis. Thus, it is aimed to compare the expression levels of two GCR isoforms (GCRα and GCRβ) in SRA, mild asthmatics, and healthy controls. Total RNA was isolated from the peripheral blood mononuclear lymphocytes of 13 SRA patients, 14 mild asthma patients and 30 healthy volunteers. The expression levels of GCR isoforms were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). The expression level of GCR isoforms did not show any significant difference between the cases/control groups. However, the relative expression analysis between asthma/control, SRA/control and SRA/asthma groups was in the order of 0.933, 0.768 and 0.823 for GCRα and 0.697, 1.014 and 1.454 for GCRβ, respectively. Also, the expression fold change of GCRα/GCRβ in asthma, SRA and control groups was 786.88, 445.72 and 588.13, respectively. The GCRα and GCRβ isoforms did not show any correlation in SRA; but they had significant correlation in both healthy volunteers (r = 0.490,  Nearly 250,000 premature deaths occur annually due to asthma. Asthma continues to be a major cause of disability and poor quality of life. It is also a major cause of health resource utilization and a public health concern worldwide  and compliance with treatment has been checked, but still have poor asthma control or frequent (≥2) severe exacerbations per year despite the prescription of high-intensity treatment or can only maintain adequate control when taking systemic corticosteroids and are thereby at risk of serious adverse effects of treatment” .Glucocorticoids (GCs) are among the mainstays of treatment in asthmatics . Classifi.e.NR3C1 gene), altered expression level of the GCR gene, GCR modification, effects of other transcription factors and defective histone acetylation (Glucocorticoids exert their effects mainly via binding to their intracellular receptors (GCRs). So, among the proposed mechanisms of GCs resistance, the possible role of GCRs in SRA development is prominent -9. The rtylation -12.NR3C1) gene on chromosome 5q31.3. The GCRα is one of the four known GCR isoforms generated by alternative splicing. It has been detected in all tissues and binds to GCs. Then, the GCRα-GCs complex translocate to the nucleus and implements its role by two mechanisms. First, as a transcription factor, it binds to promoter regions and down regulates many inflammatory genes  or up regulates anti-inflammatory genes . Second, the complex may interact with other transcription factors like AP-1, NF-AT, NF-κB or STAT family members  are less understood  in PBMLs of SRA patients, in order to investigate differences among asthmatics and healthy volunteers.Accordingly, as the difference in GCR expression levels is a proposed hypothetical mechanisms for occurrence of GC resistance, this study was performed to evaluate the expression level of GCRα and GCRβ  of Tehran and the National Research Institute of Tuberculosis and Lung Diseases (NRITLD) from 2012 through 2014.Case Selection and SamplingThirteen SRA patients were selected sequentially from asthma clinic of NRITLD, according to the criteria mentioned in the 2011 international consensus for definition of severe asthma . The casThe 30 volunteer participants in the control group had no clinical or paraclinical history of disorders at the time of the study. Demographic and clinical data gathering, together with blood sampling, were performed by a trained expert clinician. The blood samples were immediately incubated at 4 °C for up to two h prior to lab procedures.All of the participants voluntarily agreed to enroll in the study and signed written informed consent forms. Their demographic and clinical data were kept confidentially and there was no intervention to be applied throughout the clinical management of the cases. The ethical committee of the NRITLD approved all stages of the study under the code sbmu1.REC.1391.1 dated May 14, 2012.RNA Extraction and cDNA Synthesis®-H  solution based on its relevant protocol. Using RNXPlus solution , total RNA was isolated according to the manufacturer’s instructions and kept at -80 °C until molecular analysis. The quality and quantity of the extracted RNA were checked by agarose gel electrophoresis and spectrophotometry, respectively.After drawing 5 milliliters of venous blood, PBMLs were separated by LympholyteGAPDH) gene and the qualified ones were kept at -80 °C to be studied by qRT-PCR.Using Oligo-dT, random hexamer and reverse transcriptase enzyme , cDNAs were synthesized from 3 micrograms of isolated RNAs. The prepared cDNAs were checked by simple PCR on glyceraldehyde 3-phosphate dehydrogenase  for each sample  and Power SYBR Green I PCR Master Mix , qPCR was done according to the manufacturer’s protocol. The qRT-PCR was started by a preliminary denaturing stage at 95 °C for 15 min followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 60 °C for 20 sec and extension at 72 °C for 20 sec.Statistical Analysist-test and one-way ANOVA with a Tukey’s HSD post-test were used to seek statistical differences between the mean ΔCts of the asthma, SRA, and control groups. Also, considering the optimal efficacy of the designed primers (97.2% for GCRα and 94.3% for GCRβ), the 2-ΔΔCt method , which was used for further analysis. Independent sample t method  was usedParticipant Characteristicsi.e. ANOVA (Tukey’s HSD) test showed that the mean age of the control group was significantly different from that of asthma and SRA groups  and males participated more in the control group. The body mass index (BMI), which may represent a general index for nutritional condition and physical health of participants, had no significant differences among the three groups. The ethnicity of participants was nearly the same in the three groups .Demographics of subjects are summarized in Comparison of Gene Expressions-ΔΔCt values (i.e. expression ratio) of asthma/control, SRA/control and SRA/asthma groups for GCRα were in order of 0.933, 0.768, and 0.823. Similarly, these values for GCRβ were calculated to be 0.697, 1.014, and 1.454, respectively. These findings indicated that GCRα expression in SRA was lower than controls and asthma cases (ratio < 1) and vice versa for GCRβ , respectively. In general, GCRα and GCRβ expressions were not significantly different between genders. As indicated in for GCRβ . On the Correlation with Gene ExpressionsP = 0.17) and GCRβ . Additionally, P = 0.007) and mild asthmatics . However, there were no significant correlation between the expression of GCRα and GCRβ isoforms in SRA. The dot plot diagrams are presented in Supplementary file.There was no significant correlation (Pearson’s test) between age of the participants and the ΔCts of GCRα  may account for GC resistance , 12.The detailed differences in structure, function, and localization of the GCRα and GCRβ explain why GCRβ, in contrast to GCRα, does not bind to ligand (GC) and cannot affect gene expression by itself. Instead, as a dominant negative, GCRβ may represses the transcriptional activity of GCRα . While sTotally for all participants, the GCRα and GCRβ expressions (ΔCts) were neither associated with age nor significantly different in sex groups. So, it seems that the significant difference of age and gender between the control and case groups (asthma and SRA) did not affect the interpretation of the results.Also, the lack of correlation between BMI and other variables  shows thetc.); hence, there was the possibility of confounding in the results. Thus, these factors were not directly included in the analysis. Furthermore, to prevent bias, it was defined that the cases had to have the sampling criteria (according to GINA for asthma and IMI for SRA) for more than two years. Also, the cases were clinically controlled by the least corticosteroid regimens for at least two months. On the other hand, it seems that prescription of Inhaled Corticosteroids (ICS) had negligible systemic effects  may interfere in the mechanism of GCR expression in mild and severe asthmatics.Our results showed that age and GCRα ΔCts were negatively correlated in asthma cases (more in SRA), while they were not correlated in controls. This means that direct correlation between age and GCRα expression, seen in asthmatics, increased with disease severity. Also, correlation between GCRα ΔCts and GCRβ ΔCts in asthmatics was higher than in controls; but this correlation was not seen in SRA. Thus, some affecting factors (-ΔΔCt method indicated the followings.The preliminary review of our findings showed that there were no significant differences in GCRα and GCRβ expressions (mean ΔCts) among the cases and controls. Meanwhile, more detailed analysis by 2The comparison of GCRα fold changes (expression ratios) between the ratios of asthma/control (0.933), SRA/control (0.768), and SRA/asthma (0.823) showed that GCRα mRNAs are produced in decreasing order in controls , asthma, and SRA. Also, these figures for GCRβ were 0.697, 1.013, and 1.453, respectively. It means the decreasing order of GCRβ expression is SRA, control, and asthma . The latet al., (2012) who tried to introduce a marker for GC resistance in asthma. Our results were comparable with their findings that in PBMLs of SRA cases, the GCRβ expression increased while the GCRα/GCRβ expression ratio decreased  explain the decrease of GCRα/GCRβ expression ratio; the findings that have been showed by IHC study previously . But forecreased . in-vitro study showed that corticosteroids down regulate GCRα expression in respiratory cell lines dose-dependently  are involved. It worth to be noted that we studied clinical expression where many factors can interfere. For example, the stability of GCR mRNA may be influenced by estrogens and iron. Also, some cytokines (i.e. IL-2 and IL-4) can decrease GCRα expression without affecting GCRβ  can significantly increase GCRβ expression  to obtain pulmonary samples. Furthermore, the restricted inclusion criteria made our sample size limited and it may be partly the reason of non-significant results. Furthermore, asthma and its severe form are complex multifactorial disorders, which have no unique definition. Thus, various sampling criteria and bioenvironmental factors may yield in different results of various studies. It seems that well-defined longitudinal comprehensive studies of clinical, molecular, and bioenvironmental factors with reasonable sample size will provide more valid and reliable results.It should be noted that this study just assessed the GCR activity at the transcriptional level of SRA cases. While some studies at the proteomic level have supported our findings , 44, manFinally, we would like to re-emphasize that there are other isoforms of GCRs, which may play roles, especially when GCRα and GCR-γ expressions are inevitably evaluated altogether.in-vivo quantitative study indicates that GCRs are related to, but not directly responsible for SRA pathogenesis and their expression evaluation is not valuable for diagnosis of GC resistance. The lack of correlation and wide gap between GCRs’ expression in SRA show that GCRβ does not inhibit GCRα in a competitive manner. On the other hand, it seems that the reduction in GCRα/GCRβ expression ratio is a major notable finding, but not a causative factor, in GC resistance and further studies on its role in diagnosis or treatment of GC resistance cases may be useful. However, other causative and/or confounding factors and mechanisms  most probably interfere with the complex pathogenesis of GC resistance in severe asthmatics.The present


Ag-Mg antisite defects significantly increase the thermoelectric performance of α-MgAgSb by increasing the number of band valleys near the Fermi level. For Li-doped α-MgAgSb, under more Mg-rich conditions, Li will substitute on Ag sites rather than on Mg sites and may achieve high thermoelectric performance. A secondary valence band is revealed in α-MgAgSb with 14 conducting carrier pockets.Engineering atomic-scale native point defects has become an attractive strategy to improve the performance of thermoelectric materials. Here, we theoretically predict that Ag-Mg antisite defects as shallow acceptors can be more stable than other intrinsic defects under Mg-poor‒Ag/Sb-rich conditions. Under more Mg-rich conditions, Ag vacancy dominates the intrinsic defects. The  S is the Seebeck coefficient, σ is the electrical conductivity, T is the absolute temperature, and κ is the total thermal conductivity, which consists of both electronic (κe) and lattice (κl) components3. A high ZT value indicates good thermoelectric properties, and one therefore should try to increase the power factor (κ = κe+κl). A large power factor can be achieved by (a) increasing the density of states near the Fermi level , and (b) by increasing the energy dependence of the carrier mobility using energy filtering18. Meanwhile, forming a solid solution21 and creating strong lattice anharmonicity28 can achieve low lattice thermal conductivity. A recent study has proposed that engineering atomic-scale native point defects can simultaneously optimize the thermal and electrical performances of thermoelectric materials30, which is becoming an attractive strategy to improve ZT values. Native point defects play important roles in conduction in semiconductors, and they can change the band structure29.Thermoelectric materials can perform direct conversion between electrical and thermal energy. Thermoelectric performance is quantified by the figure of merit, 31 shows superior thermoelectric properties in the low temperature range44. Great efforts have been devoted to understanding and enhancing the unique thermoelectric properties of α-MgAgSb. The carrier concentration of α-MgAgSb-based materials is relatively low at room temperature, which leads to its high electrical resistivity. To overcome this limitation, extrinsic doping, including Na doping35, Cu doping36. In doping38, and changing the Sb content41 have been used to increase the carrier concentration of α-MgAg0.97Sb0.99 or α-MgAgSb, although the electrical resistivity (1‒4.5 × 10−5 Ω·m) is still larger than those of good thermoelectric materials, such as CoSb3 (0.3‒1 × 10−5 Ω·m)45 and Bi2Te3 (1‒1.5 × 10−5 Ω·m)46. Liu et al. used Li doping to increase the carrier concentration of MgAg0.97Sb0.99, and a high average ZT39 of 1.1 from 300 K to 548 K was achieved.The α phase of MgAgSbet al. reported that Ag vacancy could increase the ZT for α-MgAgSb30. Moreover, the Ag vacancy concentration can be tuned by the hot pressing temperature, which they denoted as the recovery effect. Therefore, it is necessary to explore the conditions for forming intrinsic defects and their influence on the electronic structure.Intrinsic defects represent another effective way to tune the carrier concentration to enhance the thermoelectric performance. Moreover, extrinsic point defects strongly influence the native point defects. Recently, Liu p-type conduction. Moreover, the AgMg point defect in α-MgAgSb may have higher ZT than the Ag vacancy. For Li-doped α-MgAgSb, the doping formation energies strongly depend on the chemical potentials. Under more Mg-rich conditions, Li will substitute on Ag sites (LiAg) rather than on Mg sites (LiMg), and a larger ZT can be achieved by LiAg doping than by LiMg doping. By reasonably controlling the chemical potential, both the antisite defect AgMg and Li substitution on Ag sites of α-MgAgSb can be obtained, and the products may be promising thermoelectric materials for low temperature power generation.In this work, the chemical potentials and defect formation energies of native point defects and Li doping in α-MgAgSb at all possible charge states are studied by using density functional theory. We found that the defect formation energies strongly depend on the chemical potentials. Ag vacancies and Ag-Mg antisites (Ag on Mg sites) are the dominant defects that act as shallow acceptors, which determine the Engineering intrinsic defects may be an effective way to improve the thermoelectric performance of α-MgAgSb. Due to the complex phase transitions and the appearance of secondary phases, previous experimental works have shown that it is difficult to synthesize pure phase α-MgAgSb. Different types of native point defects may easily appear in α-MgAgSb. Thus, it is necessary to first explore the conditions for forming intrinsic point defects in α-MgAgSb.V is the average difference between the local potentials far from the defect in the defective supercell and the corresponding ones in the perfect supercell47.The defect formation energy We calculated the accessible range of chemical potentials for the equilibrium growth conditions of α-MgAgSb. Under equilibrium conditions for the crystal growth, the steady production of the host material, α-MgAgSb, should satisfy the following equations:3Sb2, and Ag3Sb), the chemical potential To maintain the stability of MgAgSb during growth and avoid any competing phases  can  can 8) cmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimMg, VAg, SbI, and AgMg should be acceptor-like defects, whereas VSb, MgI, AgI, and MgAg should be donor-like defects. Under Mg-poor‒Ag/Sb-rich conditions  (GGA-PBE) exchange-correlation functional in the Vienna ab-initio simulation package (VASP), and predicted that α-MgAgSb is a semimetal37. Using the local density approximation (LDA) exchange-correlation potential as implemented in the VASP, Ying et al. found that α-MgAgSb is a semiconductor with an indirect band gap of 0.1 eV38. Because the GGA and LDA exchange correlation potentials always underestimate the band gap of crystals, we calculated the band structure of α-MgAgSb as implemented in WIEN2k with the Tran-Blaha modified Becke-Johnson (TB-mBJ) exchange correlation potential, as is shown in Fig. et al. also calculated the band structure by using the mBJ functional and the Heyd-Scuseria-Ernzerhof (HSE) approach as implemented in VASP42. They did not consider the k-path of Z-A, however, so they thought that the VBM was at X. As shown in Fig. k is the wave vector, EF is the Fermi energy, and ħ is the reduced Planck’s constant. According to Eq. :Previous experimental work showed that Li doping can significantly increase the Mg, LiAg, LiSb, and LiI, have been included in our calculation. Because of the large formation energy for LiSb, we only show the LiSb with zero charges.Based on the representative chemical potential points, we can calculate the chemical potential of Li, and the values of the chemical potentials at points A and B are −0.8857 eV and −0.8163 eV for I is a shallow donor. At point A, substitutional Li on Mg sites has the lowest formation energy. At point B, substitutional Li on Ag sites has much lower formation energy than on Mg sites. Thus, at point A, Li doping can lead to good p-type conductivity, while at point B, Li doping cannot change the conductivity type of α-MgAgSb because of LiAg with zero charges. The formation energy for Li substitution on Mg sites at point A is smaller than for substituting Li atoms on Ag sites or Sb sites, implying that the Li atoms prefer to occupy the Mg sites rather than the Ag or Sb sites at point A. On the other hand, at point B (more Mg-rich conditions), Li substitution on Ag sites of α-MgAgSb is the most stable structure. Therefore, we can substitute Li on Mg sites or Ag sites in α-MgAgSb by controlling the chemical potential of Li under different conditions.The calculated impurity formation energies of the doping systems are plotted in Fig. 47LiAg48Sb48 and Mg48Ag47LiSb48 are shown in Fig. 49. Therefore, increasing the band mass should be detrimental to the carrier mobility. Multiple degenerate valleys may produce a large 6. As can be seen in Fig. We also investigated the electronic structure and the transport properties for α-MgAgSb with Li doping on Mg sites and Ag sites at a doping level of 0.02. The calculated band structures of Mgp orbitals. The large band degeneracy NV and heavy band effective mass can jointly contribute to the high Seebeck coefficient. Moreover, for p-type α-MgAgSb, the carrier concentration largely depends on the number of band valleys near the Fermi level, which is mainly due to the fact that more carriers can be activated across the band gap. High carrier concentration may help to increase the electrical conductivity. Therefore, Li doping may play an important role in achieving a higher Seebeck coefficient and electrical conductivity, which will lead to a large ZT for Mg47LiAg48Sb48. Recently, Liu et al. used Li doping to increase the carrier concentration of MgAg0.97Sb0.99, thus decreasing the electrical resistivity, and a high average ZT39 of 1.1 from 300 K to 548 K was achieved.To explain the reason why the Γ point becomes higher and moves toward the Fermi level, we calculated the partial charge densities near the Fermi level at the Γ point using VASP, as shown in Fig. 48Ag47LiSb48 is shown in Fig. The calculated band structure of Mget al. found that the appearance of three-centered Mg-Ag-Sb bonds in α-MgAgSb results in low intrinsic lattice thermal conductivity50. To investigate the elastic properties of α-MgAgSb with intrinsic defects and Li doping, the stress-strain method was used to calculate the elastic constants and other elastic properties51. A small finite strain is applied on the optimized structure, and then the atomic positions are optimized. The elastic constants are obtained from the stress of the strained structure. The calculated elastic constants of MgAgSb and Mg0.98Li0.02AgSb are listed in Table 52, indicating that they are elastically stable. On the other hand, the positive eigenvalues of the elastic constant matrix for each compound further prove that they are elastically stable. From the calculated elastic constants Cij, the polycrystalline bulk modulus B and shear modulus G were estimated using the Voigt-Reuss-Hill approximation53. A high (low) B/G ratio of a material indicates that it is ductile (brittle), and the critical value is about 1.7554. The calculated B/G ratios for MgAgSb and Mg0.98Li0.02AgSb are larger than the critical value (1.75), indicating that they are all ductile materials.Ying T decay can only continue, however, until the minimum lattice thermal conductivity (κmin) is reached, as defined by Cahill56. At high temperature (T > ΘD), κmin can be approximated by the following formula:V is the average volume per atom, and vs and vl are the shear and longitudinal sound velocities, respectively. As a fundamental parameter, the Debye temperature is connected with many physical properties of solids, such as the specific heat, melting point, and elastic constant. At low temperatures, the vibrational excitations arise solely from acoustic vibrations. One of the methods used to calculate the Debye temperature is based on the elastic constant data. The Debye temperature is given by:kB, h, NA, ρ, M, and n are the Boltzmann constant, Planck’s constant, Avogadro’s number, density, molecular weight of the solid, and number of atoms in the molecule, respectively. The average wave velocity vm in polycrystalline materials is approximately given by57Thermal conductivity of a material includes both electronic and lattice thermal conductivity. The electronic contribution to the thermal conductivity is described by the Wiedemann-Franz relation, vs and vl can be obtained using the polycrystalline shear modulus G and the bulk modulus B from Navier’s equation as follows58:B and G can estimate using the Voigt-Reuss-Hill approximation from the calculated elastic constant data, which were obtained by the stress-strain method53. The calculated elastic constants and the minimum lattice thermal conductivity are listed in Table Ag (Mg48Ag47Sb48), AgMg (Mg47Ag49Sb48), LiMg (Mg48LiAg48Sb48), and LiAg (Mg48Ag47LiSb48), the calculated κmin values are 0.55 W/mK, 0.53 W/mK, 0.54 W/mK, 0.53 W/mK, and 0.54 W/mK, respectively. As shown in Eq. Ag and LiMg defects induce an obviously decreasing shear modulus in α-MgAgSb, which indicates that VAg and LiMg defects weaken the resistance against shear deformation of α-MgAgSb. Thus, the shear sound velocity decreases due to Ag vacancy and LiMg doping. Consequently, the minimum lattice thermal conductivity values are reduced due to Ag vacancy and LiMg doping. For AgMg and LiAg defects, the decrease in the shear modulus is not so large compared with VAg and LiMg defects. Thus, the change in the minimum lattice thermal conductivity due to AgMg and LiAg defects is smaller than that due to VAg and LiMg defects.For α-MgAgSb, VZT needs to have a large S (found in low carrier concentration semiconductors or insulators) and a large σ . The carrier concentration dependence of the Seebeck coefficient and the electrical conductivity are shown in Eqs ZT needs S, the carrier concentration, n, the electrical conductivity relative to relaxation, σ/τ, the thermopower relative to relaxation, and the figure of merit, S2σ/τ, as a function of temperature, as shown in Fig. Equation  suggestsAg (Mg48Ag47Sb48), AgMg (Mg47Ag49Sb48), LiMg (Mg48LiAg48Sb48), and LiAg (Mg48Ag47LiSb48) are all positive over the entire studied temperature range, indicating p-type transport for the four types of defects. The n and σ/τ, of AgMg are lower than those of VAg, while AgMg has the larger S2σ/τ owing to its large S. Liu et al. reported that Ag vacancy can be rationally engineered by controlling the hot pressing temperature, and a high peak ZT of ~1.4 and an average ZT of ~1.1 can be achieved30. α-MgAgSb containing AgMg point defects may have higher ZT than with Ag vacancy because α-MgAgSb with AgMg has a larger S of LiAg-doped α-MgAgSb is larger than for LiMg-doped α-MgAgSb. Although LiAg-doped α-MgAgSb has the lowest n and σ/τ, the S2σ/τ of LiAg-doped α-MgAgSb is larger than that with LiMg defects, due to the large S, as shown in Fig. et al. found that the average ZT can reach as high as 1.1 from 300 K to 548 K when there is Li doping on Mg sites of MgAg0.97Sb0.9939. Thus, under more Mg-rich conditions, LiAg doping may lead to a larger ZT than for Li substitution on Mg sites. Thus, good thermoelectric performance as a result of the antisite defect AgMg and as a result of Li substitution on Ag sites in α-MgAgSb can be predicted.As can be seen in Fig. p-type conduction of experimentally synthesized α-MgAgSb. Moreover, for α-MgAgSb, the AgMg antisite defect may induce a higher ZT than Ag vacancy, due to the more numerous band valleys near the Fermi level than with AgMg in α-MgAgSb. α-MgAgSb has a secondary valence band with 14 carrier pockets, which indicates that heavily p-type doping may lead to a high thermoelectric performance in α-MgAgSb. For Li-doped α-MgAgSb, Li doping on Ag sites has a lower formation energy than on Mg sites under more Mg-rich conditions, and LiAg may lead to a larger ZT than for Li doping on Mg sites. Thus, engineering atomic scale defects is an effective strategy for enhancing the thermoelectric properties of α-MgAgSb, and the achieved high ZT demonstrates that AgMg antisite defects and the substitution of Li on Ag sites in α-MgAgSb could lead to materials with good potential for future application in the thermoelectric area.In this work, we investigated the defect formation energies, the electronic structure, and the thermoelectric performance of the host α-MgAgSb and the effects of substitutional Li doping of α-MgAgSb, by using density functional theory combined with semiclassical Boltzmann theory. We found that the formation energies strongly depend on the chemical potentials. Ag vacancy and Ag-Mg antisite defects are the dominant defects, acting as the shallow acceptors that determine the 59, as implemented in WIEN2k62. The Tran and Blaha modified semi-local Becke–Johnson exchange correlation potential (TB-mBJ)63 was used, which is known to give much more accurate band gaps than the standard Engel–Vosko generalized-gradient approximation (EV-GGA)64. The muffin-tin radii were chosen to be 2.5 a.u. for Mg, Ag, and Sb. The cut-off parameter Rmt × Kmax = 9 (where Kmax is the magnitude of the largest k vector) was used, and the self-consistent calculations were performed with 2000 k-points in the irreducible Brillouin zone; the total energy was made to converge to within 1 mRy. The electrical transport properties were then calculated by using semiclassical Boltzmann theory66 within the constant scattering time approximation, as implemented in the Boltzmann Transport Properties (Boltz-TraP) code67. This approximation has been used to calculate the transport coefficients of some known thermoelectric materials and very good agreement with experimental results was achieved69.The electronic structure of α-MgAgSb was investigated using the full-potential linearized augmented plane wave methodAg (Ag vacancy), VMg (Mg vacancy), VSb (Sb vacancy), MgI , AgI , SbI , AgMg (Ag on Mg site), and MgAg (Mg on Ag site). In the case of Li doping, we simulated interstitial doping (LiI) and substitutional doping, including LiMg (Li doping on Mg site), LiAg (Li doping on Ag site), and LiSb (Li doping on Sb site).We simulated various defects in α-MgAgSb, along with Li doping, using a supercell that contained 144 atoms. We considered three intrinsic point defects, vacancy, interstitial, and antisite. Because of their large formation energies, cation/anion antisites, such as Mg or Ag on the Sb site and Sb on the Mg or Ag sites, are not discussed in this study. The intrinsic defects considered in this study include Vc axis, with half of the Mg-Sb pseudocubes filled with Ag, although the pseudocubes where silver atoms are located are quite different from those in half-Heusler compounds70. Such a complex lattice structure may lead to a relatively small thermal conductivity.As shown in Fig. Ag, AgMg, LiMg, and LiAg using the supercell (144 atoms in MgAgSb supercell), corresponding to a doping level of 2% for α-MgAgSb. We also fixed the lattice constants, only optimizing the internal coordinates. The electronic structures of Mg48Ag47Sb48, Mg47Ag49Sb48, Mg47LiAg48Sb48, and Mg48Ag47LiSb48 were calculated with WIEN2k. Other parameters were in accordance with the calculations for α-MgAgSb.We also studied the electronic structure and thermoelectric properties of V


Actinidia arguta cultivars: “HB” (“Hongbaoshixing”) and “YF” (“Yongfengyihao”) at two different fruit developmental stages: 70d (days after full bloom) and 100d (days after full bloom). Metabolite and transcript profiling was obtained by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer and high-throughput RNA sequencing, respectively. The identification and quantification results of metabolites showed that a total of 28,837 metabolites had been obtained, of which 13,715 were annotated. In comparison of HB100 vs. HB70, 41 metabolites were identified as being flavonoids, 7 of which, with significant difference, were identified as bracteatin, luteolin, dihydromyricetin, cyanidin, pelargonidin, delphinidin and (−)-epigallocatechin. Association analysis between metabolome and transcriptome revealed that there were two metabolic pathways presenting significant differences during fruit development, one of which was flavonoid biosynthesis, in which 14 structural genes were selected to conduct expression analysis, as well as 5 transcription factor genes obtained by transcriptome analysis. RT-qPCR results and cluster analysis revealed that AaF3H, AaLDOX, AaUFGT, AaMYB, AabHLH, and AaHB2 showed the best possibility of being candidate genes. A regulatory network of flavonoid biosynthesis was established to illustrate differentially expressed candidate genes involved in accumulation of metabolites with significant differences, inducing red coloring during fruit development. Such a regulatory network linking genes and flavonoids revealed a system involved in the pigmentation of all-red-fleshed and all-green-fleshed A. arguta, suggesting this conjunct analysis approach is not only useful in understanding the relationship between genotype and phenotype, but is also a powerful tool for providing more valuable information for breeding.To assess the interrelation between the change of metabolites and the change of fruit color, we performed a combined metabolome and transcriptome analysis of the flesh in two different  Actinidiaceae, genus Actinidia), a kind of perennial and deciduous plant, is one of the fruit trees that have been domesticated and cultivated successfully in the last century, and in recent years has been grown commercially worldwide [Actinidia comprises a total of 76 species and about 125 known taxa worldwide [Actinidia chinensis and A. deliciosa—have been cultivated commercially [Kiwifruit  and “Yongfengyihao” (“YF”)—were selected to perform metabolome and transcriptome analysis. All metabolites that could be detected by instrument were identified and then used for differential analysis. Finally, 41 metabolites related to flavonoid biosynthesis were obtained, 7 of which presented significant difference in the comparison between HB100 and HB70. Through combined metobolome and transcriptome analysis, 2 pathways—flavonoid biosynthesis and galactose metabolism—were significantly different in terms of change during fruit development. In flavonoid biosyntheis, 8 differentially expressed structural genes were obtained. In addition, 7 transcription factor genes related to flavonoid biosynthesis were obtained by transcriptome analysis. RT-qPCR results and cluster analysis revealed the genes AaF3H, AaLDOX, AaUFGT, AaMYB, AabHLH and AaHB2 could be determined as candidate genes on which to conduct further study. Through integrated analysis of identified metabolites with significant difference and related genes involved in flavonoid biosynthesis, the association between metabolites and genes was explained so as to provide valuable information for revealing the red coloring of A. arguta.To investigate the metabolite changing rule associated with color change during A. arguta fruit development ” and “c18002_g1 (named AaMYB)”, “c102250_g1” and “c86342_g1 (named AabHLH)”, and “c127253_g1 (named AaHB1)” and “c129627_g1 (named AaHB2)” belonged to “MYB”, ”bHLH“ and “HB” families, respectively , the comprehensive analysis of metabolome and transcriptome was performed using Pearson’s Correlation Coefficient ,29. The AaMYBC1, AaMYB, AabHLH, AaHB1 and AaHB2  were clustered into one class -epigallocatechin. It is generally known that transcription factor plays a role by binding the promoter of structural genes. Therefore, we speculate the hypothesis that the three transcription factors AaMYB, AabHLH, and AaHB2 activate expression of AaF3H, AaLDOX and AaUFGT by binding the promoter of these three structural genes, inducing the accumulation of the 7 metabolites described above in flavonoid biosynthesis. Specifically, AaMYB, AabHLH, AaHB2, AaF3H, AaLDOX and AaUFGT were the candidate genes obtained from this study and could be used for further study, revealing the red mechanism of A. arguta.In order to better understand the relationship between metabolites and genes in flavonoid biosynthesis, all results of metabolites and genes were combined to establish a network, aiming to show the relationship between gene expression and metabolite accumulation more intuitively . Among tFagopyrum esculentum [Camellia sinensis [Ficus carica L. [A. arguta cultivars “HB” and “YF” at two developmental stages, a total of 28,837 metabolites were obtained, of which 13,715 had been annotated , “Hongbaoshixing”  and “Yongfengyihao” , were selected as experimental materials. Two different sampling stages of fruit development, defined in days after full bloom (DAFB), were 70 DAFB  and 100 DAFB . ‘YF’ fruit selected as a control case were green throughout the whole development process and were also sampled at 70d to 100d . Briefly, 20 μL of sample was extracted with 120 μL of precooled 50% methanol, vortexed for 1 min, and incubated at room temperature for 10 min; the extraction mixture was then stored overnight at −20 °C. After centrifugation at 4000× 4H2O) and solvent B (IPA:CAN = 9:1 + 0.1% formic acid). Gradient elution conditions were set as follows: 0~0.5 min, 95% B; 0.5~9.5 min, 95% to 65% B; 9.5~10.5 min, 65~40% B; 10.5~12 min, 40% B; 12~12.2 min, 40~95% B; 12.2~15 min, 95% B. The injection volume for each sample was 4 µL.All chromatographic separations were performed using an ultra-performance liquid chromatography (UPLC) system . An ACQUITY UPLC BEH Amide column  was used for the reversed phase separation. The column oven was maintained at 35 °C. The flow rate was 0.4 mL/min and the mobile phase consisted of solvent A  was used to detect metabolites eluted form the column. The Q-TOF was operated in both positive and negative ion modes. XCMS software 3.2.0  was used to control the chromatograph and mass spectrometer.MSConvert was used to transform LC-MS raw data into the mzXML format, which was then processed by the XCMS, CAMERA and metaX toolbox, implemented in the R software ,51,52. Thttp://www.plantcyc.org/), KEGG (http://www.kegg.jp/) and in-house (http://spldatabase.saskatoonlibrary.ca/), HMDB (http://www.hmdb.ca/) databases were used to perform level-one and level-two identification and annotation. Screening and quantitative analysis for differential metabolites were conducted using metaX software (http://metax.genomics.cn/).In order to explain the physical and chemical properties and biological functions of metabolites, the online PLANTCYC , Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer  and RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system , respectively. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina®  following manufacturer’s recommendations and were sequenced on an Illumina Hiseq platform. According to the manufacturer’s instructions, NEBNext® Ultra™ RNA Library Prep Kit for Illumina®  was used to generate sequencing libraries, which were then sequenced on an Illumina Hiseq platform.RNA isolation, purification and monitoring, and cDNA library construction and sequencing were performed as previously described . Brieflyp-value < 0.05 found by DESeq.Transcription factors were predicted using iTAK software . The ide® 480 real-time PCR system with a 96-well plate was used to conduct an amplified reaction consisting of 95 °C for 5 min, followed by 45 cycles of 10 s at 95 °C, 20 s at 60 °C, and 20 s at 72 °C in a volume of 10 μL. At the end of each experiment, a melt-curve analysis was carried out using the default parameters (5 s at 95 °C and 1 min at 65 °C). The Actinidia β-actin was used for normalization [Total RNA was extracted using a modified CTAB (cetyltrimethyl ammonium bromide) method . The teslization . All anaCt−ΔΔ method [The relative expressions were calculated using the 2t method , and Gra