Grafting with rootstocks is essential for the culture of many perennial fruit crops and is increasing being used in the production of annual fruits and vegetables. Our previous work based on microarrays showed that transcripts encoding enzymes of both primary and secondary metabolism were differentially expressed during graft union formation in both homo-grafts (a genotype grafted with itself) and hetero-grafts (two different genotypes grafted together). The aim of this study was to profile primary and secondary metabolites, and quantify the activity of phenylalanine ammonia lyase (PAL) and neutral invertase (NI) in the scion and rootstock tissues and the graft interface of homo and hetero-grafts of grapevine 1 month after grafting. Table-top grafting was done on over-wintering stems (canes) of grapevine and the graft interface tissues  were compared to the surrounding rootstock and scion tissues. The objective was to identify compounds involved in graft union formation and hetero-grafting responses.A total of 54 compounds from primary and secondary metabolism  and the activity of two enzymes were measured. The graft interface was associated with an increase in the accumulation of the branched-chain amino acids, basic amino acids, certain stilbene compounds and higher PAL and NI activity in comparison to the surrounding woody stem tissues. Some amino acids and stilbenes were identified as being accumulated differently between the graft interfaces of the scion/rootstock combinations in a manner which was unrelated to their concentrations in the surrounding woody stem tissues.This study revealed the modification of primary metabolism to support callus cell formation and the stimulation of stilbene synthesis at the graft interface, and how these processes are modified by hetero-grafting. Knowledge of the metabolites and/or enzymes required for successful graft union formation offer us the potential to identify markers that could be used by nurseries and researchers for selection and breeding purposes. Grafting is widely used in horticulture because rootstocks can provide resistance to soil borne pathogens and abiotic stresses as well as to influence scion growth and performance . GraftinVitaceae. Stilbenes are accumulated in grapevine tissues in response to a number of abiotic and biotic stress treatments, and are thought to protect against oxidative stress in addition to being phytoalexins [PHENYLALANINE AMMONIA LYASE (PAL), first and committed step in the phenyl propanoid pathway, has been associated with incompatibility responses in Prunus spp. [Because of its agronomic importance, most studies of graft union formation focus on incompatibility responses, related to either poor graft-take soon after grafting or delayed incompatibility responses occurring months to years after grafting. Accumulation of certain secondary metabolites at the graft interface has been observed in incompatibility responses in fruit trees , 11, olioalexins , 17. In nus spp. , 19 and nus spp. , yet theWe have previously characterized the changes in transcript abundance at the graft interface of compatible homo-grafts  and heteV. vinifera cv. Cabernet Sauvignon (CS/CS) 28 d after grafting showed a distinct primary metabolite profile in comparison with the scion and rootstock tissues. This graft interface tissue  had higher water, protein and Glc contents, and a lower concentration of starch compared to the surrounding tissues  in the graft interface tissue while in the scion and rootstock, the protein content was approximatively 1.7 mg g− 1 FW. Glucose concentration was higher in the graft interface (3 nmol g− 1 FW) in comparison with the scion and rootstock tissues (2 nmol g− 1 FW). Fructose accumulated slightly at the interface, but the difference was not significant. On the contrary, there is almost two times less starch in graft interface compared with the scion and rootstock tissues. The total concentration of amino acids did not differ between the three tissues studied, but the concentration of some individual amino acids was different between the graft interface and surrounding tissues. In particular, Gln and GABA contents were significantly higher, while Arg, Lys, His, Phe and Tyr contents were lower in the interface samples of CS/CS compared to the woody tissues of scion and rootstock.The proliferation of callus tissues at the graft interface of the homograft V. berlandieri x V. rupestris cv. 1103 Paulsen (1103P) and V. riparia cv. Gloire de Montpellier (RG)  and as oligomers  . Our anatrans-ε-viniferin , one tetramer (isohopeaphenol) and two monomers (trans- and cis-astringin) were highly increased at the graft interface.The concentration of stilbenes at the graft interface of CS/CS and CS/RG was considerably higher than that of CS/1103P  accounted for 49% of the total variance, with 31% explained by PC1 and 18% by PC2  in oilseed rape [WIND1 [WIND1 in grapevine was not differentially expressed between the rootstock and graft interface tissues [Some changes in amino acid concentrations between the scion and rootstock stem and graft interface tissues of CS/CS grafts overlap with the metabolome reprogramming of wound induced callus in tomato  and indueed rape . The APEeed rape . Tomato eed rape , the trae [WIND1 , and the tissues  and some tissues , whereas tissues , but theThe comparison of the profile of primary metabolites between the different genotypes studied showed that CS had considerably higher levels of starch and Arg (and some other minor amino acids) than the two American rootstock species RG and 1103P. This could be due to differences in the formation of reserves in the autumn (both storage amino acids and their quantity) and/or differences in the mobilization of reserves from dormancy until 28 d after grafting. The higher starch and Arg content of the graft interface of CS/CS could be related to the higher concentration of starch and Arg in the rootstock (and scion) wood of CS than the other two genotypes, however, the lower water content suggests that these samples contained less callus tissue.V. riparia are known to be early developing genotypes when grown un-grafted [Some amino acids were specifically accumulated at the graft interface of the hetero-grafts in a manner which was unrelated to their rootstock tissue concentrations such as the high accumulation of branched chain amino acids , Asn and Gln at the graft interface of CS/RG. Branched chain amino acids have an essential role in protein synthesis and the enzymes encoding their synthesis are generally highly expressed in young tissues . Similar-grafted .The concentration of Pro was higher in all tissues of the homo-graft, CS/CS, than the two hetero-grafted combinations, CS/RG and CS/1103P, suggesting that hetero-grafting reduced Pro accumulation both locally at the graft interface and at a short distance away in the scion. Proline is known to accumulate in plant tissues in response to abiotic stress, it has numerous functions in growth and development , and impGenerally, the concentration of flavanols (particularly epicatechin) decreases at the graft interface compared to the surrounding woody tissues, presumably the wood has a high concentration of flavanols, which gets diluted as the callus cells develop. Flavanol concentration, and particularly epicatechin, has been measured in a number of studies of graft incompatibility , 37, 38,trans-ε-viniferin (the major stilbene found in CS wood), and 3 trimers: trans- and cis-miyabenol C, and α-viniferin. Stilbenes have antioxidant capacities [The increase in the abundance of transcripts encoding stilbene synthases at the graft interface of CS/CS 28 d after grafting was associated with an increase in the total concentration of stilbenes, which was primarily due to an increase in the concentration of the dimer pacities  as such pacities , potentipacities , a hormopacities .Generally, the coupling of an increase in stilbene concentration and a decrease in flavonoid concentration is frequently observed in grapevine responses to stress including wounding .V. vinifera  [Trans-ε-viniferin is the main stilbene reported in canes for all cultivars [− 1, which is consistent with our results.The three genotypes of grapevine studied, CS, RG and 1103P, had different stilbene profiles in the rootstock wood tissues, genotypic variation in stilbene concentration has been previously reported in different cultivars of inifera  ,44 and iinifera  . Trans-εultivars , 43, 44,Vitis spp. show variation in the induction of stilbene synthesis in response to pathogens [The accumulation of stilbenes at the graft interface of the different scion/rootstock combinations is generally related to the concentration of stilbenes in the surrounding woody tissues; however, some stilbenes are specifically accumulated at the graft interface. Different genotypes of athogens  and ultrathogens , suggestathogens .Grafting is a considerable stress to plants, resulting in the stimulation of wound responses and healing processes . Metabolite profiling of primary metabolites at the graft interface of grapevine has revealed that the metabolome of the graft interface is reprogrammed to support callus cell proliferation, by the accumulation of Gln and GABA, and the reduction of Phe, Arg, Tyr and Lys concentrations. Globally, the concentration of flavanols decreased and the concentration of stilbenes increased at the graft interface in comparison to the surrounding tissues for all scion/rootstock combinations. There were genotypes specific differences in the profile of flavanols and stilbenes in the rootstock tissues, and although the concentration of these metabolites at the graft interface often reflected these genotype-specific differences, some specific metabolite accumulation patterns were observed. A small number of stilbenes showed scion/rootstock specific accumulation patterns; this could suggest that hetero-grafting two different genotypes together triggers specific changes in secondary metabolism and plant defense responses at the graft interface.Vitis vinifera cv. ‘Cabernet-Sauvignon N’ , V. riparia cv. ‘Riparia Gloire de Montpellier’  and the V. berlandieri x V. rupestris hybrid cv. ‘1103 Paulsen’  hardwood was collected from a vineyard in France in January  and stored as one meter long stems in a cold chamber (4 °C) until grafting in March. The identification of the plant material was done by the Centre de Ressources Biologiques de la Vigne, collection ampélographique de Vassal, Montpellier, France, by simple sequence repeats markers. No permissions were required to obtain this plant material. The scion CS was grafted onto RG (CS/RG) and 1103P (CS/1103P) as well as homo-grafted onto CS rootstocks (CS/CS); all these scion/rootstock combinations are highly compatible with grafting success rates of 87–95%. Dormant, stored stems (of approximately 0.8 to 1.2 cm in diameter) were taken out of the cold chamber 2 days before grafting and soaked in water at room temperature in order to rehydrate. One-bud cuttings were made for scions and two-node de-budded cuttings were made for rootstocks shortly before grafting. Bench mechanical omega grafting was performed on scion/rootstock pairs of approximately the same diameter. Grafts were briefly dipped into melted wax and placed into humid sawdust filled boxes for callusing at 28 °C for 28 d. Three pools of 15 graft interface zones (approximately 5 mm in length including both scion and rootstock tissues) were harvested and immediately snap frozen in liquid nitrogen. Two independent grafting experiments were done in the spring 2011 and 2012; some of this material was also used for previously published transcriptomic studies [ studies  The procedure of ethanolic extraction was followed as described by . Glucose− 1  according to the gradient described by [Free amino acids were measured after derivation with 6-aminoquinolyl-N-hydroxy-succinimidyl-carbamate  as described by . Amino aribed by . ChromatFor the measurement of NI and PAL activities, 500 mg of frozen tissue were extracted in 4 mL of 100 mM Tris-HCl buffer pH 8.8 containing 3 mM EDTA (ethylenediaminetetraacetic acid), 0.05% spermidine (w/v), 4 mM β-mercaptoethanol, 2 mM dithiothreitol, 0.1% triton X-100, a mixture of protease inhibitors , 1% (w/v) bovine serum albumin and 5% (w/v) polyvinylpolypyrrolidone. After centrifugation , the protein extract was desalted using a PD-10 column  equilibrated with 25 mL of 100 mM Tris-HCl pH 8.8, 3 mM EDTA.The NI activity was assayed using a protocol from . For the− 1; 0 min: 18% B; 3.5 min, 33% B; 6.5 min, 33% B; 12 min, 40% B; 13 min, 95% B; 16 min, 95% B; 16.5 min, 18% B. A calibration curve with catechin (Sigma), epicatechin (Sigma) and B2 (extrasynthese) in the range 0.04–50 mg L− 1 in 50% methanol was prepared. Detection was performed with a Triple Quadripole  operating under the following conditions: gas temperature: 350 °C, gas flow: 11 L min− 1, nebulizer 15 psi, voltage: 3000 V. Quantifier ions for each molecule were adapted from the literature [− 1; 0 min: 10% B; 3 min, 20% B; 10 min, 33% B; 13 min, 33% B; 14 min, 50% B; 17 min, 95% B; 22 min, 95% B. A calibration curve with resveratrol, piceid, piceatannol, astringin, pallidol, parthenocissin, ε-viniferin, ω-viniferin, δ-viniferin, miyabenol C, hopeaphenol, isohopeaphenol, vitisin A and vitisinol C in the range 0.011–50 mg L− 1 was prepared. 1 μL was injected for each sample and standard. Detection was performed with a Triple Quadripole  operating under the following conditions: gas temperature: 350 °C, gas flow: 11 L min− 1, nebulizer 15 psi, voltage: 3000 V; mode of detection: MRM (transitions adapted) [One hundred mg of plant material were extracted in 1 mL of methanol in an ultrasound bath for 10 min. After centrifugation, the supernatants were filtered and diluted in milli Q water (1:1) for the analysis of flavanols and stilbenes. Flavanols were separated by HPLC on an Agilent SB-C18 RRHD column  at 40 °C. The gradient was as follows: solvent A: 0.1% formic acid in milliQ water; solvent B: 0.1% formic acid in actonitrile (LC-MS grade); flow 0.4 mL minterature . Dimers adapted) .All data analysis and data presentation was done using the R environment  with theAdditional file 1:Table S1. A comparison of water content (% H2O), phenylalanine ammonia lyase (PAL) activity, neutral invertase (NI) activity and the concentration of some metabolites in the scion, rootstock and graft interface of Vitis vinifera cv. Cabernet Sauvignon homo-grafts 28 d after grafting. When the conditions of an ANOVA were met (Shapiro and Barlett tests), means and p values are given, when conditions of an ANOVA were not met, median (indicated by stars) and p values of Kruskal-Wallis test are given. P values adjusted with Benjamini-Hochberg (BH) test. Letters indicate results of post hoc Tukey tests.Additional file 2:Table S2. A comparison of water content (% H2O), phenylalanine ammonia lyase (PAL) activity, neutral invertase (NI) activity and the concentration of some metabolites in the rootstock wood of Vitis vinifera cv. Cabernet Sauvignon (CS) grafted with itself (CS/CS) and grafted with the rootstocks V. berlandieri x V. rupestris cv. 1103 Paulsen (CS/1103P) and V. riparia cv. Gloire de Montpellier (CS/RG) 28 d after grafting. When the conditions of an ANOVA were met (Shapiro and Barlett tests), means and p values are given, when conditions of an ANOVA were not met, median (indicated by stars) and p values of Kruskal-Wallis test are given. P values adjusted with Benjamini-Hochberg (BH) test. Letters indicate results of post hoc Tukey tests.Additional file 3:Table S3. A comparison of water content (% H2O), phenylalanine ammonia lyase (PAL) activity, neutral invertase (NI) activity and the concentration of some metabolites at the graft interface of Vitis vinifera cv. Cabernet Sauvignon (CS) grafted with itself (CS/CS) and grafted with the rootstocks V. berlandieri x V. rupestris cv. 1103 Paulsen (CS/1103P) and V. riparia cv. Gloire de Montpellier (CS/RG) 28 d after grafting. When the conditions of an ANOVA were met (Shapiro and Barlett tests), means and p values are given, when conditions of an ANOVA were not met, median (indicated by stars) and p values of Kruskal-Wallis test are given. P values adjusted with Benjamini-Hochberg (BH) test. Letters indicate results of post hoc Tukey tests.Additional file 4:Table S4. A comparison of the concentration of flavanols in the scion, rootstock and graft interface of Vitis vinifera cv. Cabernet Sauvignon homo-grafts 28 d after grafting. When the conditions of an ANOVA were met (Shapiro and Barlett tests), means and p values are given, when conditions of an ANOVA were not met, median (indicated by stars) and p values of Kruskal-Wallis test are given. P values adjusted with Benjamini-Hochberg (BH) test. Letters indicate results of post hoc Tukey tests.Additional file 5:Table S5. A comparison of the concentration of flavanols in the rootstock wood of Vitis vinifera cv. Cabernet Sauvignon (CS) grafted with itself (CS/CS) and grafted with the rootstocks V. berlandieri x V. rupestris cv. 1103 Paulsen (CS/1103P) and V. riparia cv. Gloire de Montpellier (CS/RG) 28 d after grafting. When the conditions of an ANOVA were met (Shapiro and Barlett tests), means and p values are given, when conditions of an ANOVA were not met, median (indicated by stars) and p values of Kruskal-Wallis test are given. P values adjusted with Benjamini-Hochberg (BH) test. Letters indicate results of post hoc Tukey tests.Additional file 6:Table S6. A comparison of the concentration of flavanols at the graft interface of Vitis vinifera cv. Cabernet Sauvignon (CS) grafted with itself (CS/CS) and grafted with the rootstocks V. berlandieri x V. rupestris cv. 1103 Paulsen (CS/1103P) and V. riparia cv. Gloire de Montpellier (CS/RG) 28 d after grafting. When the conditions of an ANOVA were met (Shapiro and Barlett tests), means and p values are given, when conditions of an ANOVA were not met, median (indicated by stars) and p values of Kruskal-Wallis test are given. P values adjusted with Benjamini-Hochberg (BH) test. Letters indicate results of post hoc Tukey tests.Additional file 7:Table S7. A comparison of the concentration of stilbenes in the scion, rootstock and graft interface of Vitis vinifera cv. Cabernet Sauvignon homo-grafts 28 d after grafting. When the conditions of an ANOVA were met (Shapiro and Barlett tests), means and p values are given, when conditions of an ANOVA were not met, median (indicated by stars) and p values of Kruskal-Wallis test are given. P values adjusted with Benjamini-Hochberg (BH) test. Letters indicate results of post hoc Tukey tests.Additional file 8:Table S8. A comparison of the concentration of stilbenes in the rootstock wood of Vitis vinifera cv. Cabernet Sauvignon (CS) grafted with itself (CS/CS) and grafted with the rootstocks V. berlandieri x V. rupestris cv. 1103 Paulsen (CS/1103P) and V. riparia cv. Gloire de Montpellier (CS/RG) 28 d after grafting. When the conditions of an ANOVA were met (Shapiro and Barlett tests), means and p values are given, when conditions of an ANOVA were not met, median (indicated by stars) and p values of Kruskal-Wallis test are given. P values adjusted with Benjamini-Hochberg (BH) test. Letters indicate results of post hoc Tukey tests.Additional file 9:Table S9. A comparison of the concentration of stilbenes at the graft interface of Vitis vinifera cv. Cabernet Sauvignon (CS) grafted with itself (CS/CS) and grafted with the rootstocks V. berlandieri x V. rupestris cv. 1103 Paulsen (CS/1103P) and V. riparia cv. Gloire de Montpellier (CS/RG) 28 d after grafting. When the conditions of an ANOVA were met (Shapiro and Barlett tests), means and p values are given, when conditions of an ANOVA were not met, median (indicated by stars) and p values of Kruskal-Wallis test are given. P values adjusted with Benjamini-Hochberg (BH) test. Letters indicate results of post hoc Tukey tests.


Manihot esculenta Crantz) is a root crop used as a foodstuff and as a starch source in industry. Starch functional properties are influenced by many structural features including the relative amounts of the two glucan polymers amylopectin and amylose, the branched structure of amylopectin, starch granule size and the presence of covalent modifications. Starch phosphorylation, where phosphates are linked either to the C3 or C6 carbon atoms of amylopectin glucosyl residues, is a naturally occurring modification known to be important for starch remobilization. The degree of phosphorylation has been altered in several crops using biotechnological approaches to change expression of the starch-phosphorylating enzyme GLUCAN WATER DIKINASE (GWD). Interestingly, this frequently alters other structural features of starch beside its phosphate content. Here, we aimed to alter starch phosphorylation in cassava storage roots either by manipulating the expression of the starch phosphorylating or dephosphorylating enzymes. Therefore, we generated transgenic plants in which either the wild-type potato GWD (StGWD) or a redox-insensitive version of it were overexpressed. Further plants were created in which we used RNAi to silence each of the endogenous phosphoglucan phosphatase genes STARCH EXCESS 4 (MeSEX4) and LIKE SEX4 2 (MeLSF), previously discovered by analyzing leaf starch metabolism in the model species Arabidopsis thaliana. Overexpressing the potato GWD gene (StGWD), which specifically phosphorylates the C6 position, increased the total starch-bound phosphate content at both the C6 and the C3 positions. Silencing endogenous LSF2 gene (MeLSF2), which specifically dephosphorylates the C3 position, increased the ratio of C3:C6 phosphorylation, showing that its function is conserved in storage tissues. In both cases, other structural features of starch  were unaltered. This allowed us to directly relate the physicochemical properties of the starch to its phosphate content or phosphorylation pattern. Starch swelling power and paste clarity were specifically influenced by total phosphate content. However, phosphate position did not significantly influence starch functional properties. In conclusion, biotechnological manipulation of starch phosphorylation can specifically alter certain cassava storage root starch properties, potentially increasing its value in food and non-food industries.Cassava ( Starch is one of the most important plant products. It is the major nutritive component of our major staple crops, including cereals such as maize, wheat and rice, as well as root crops like potato and cassava. In addition to its nutritional value, starch has a myriad of uses in the food and non-food industries . In order to fulfill these different functional uses, starch and starch derivatives need to have distinct physicochemical properties . Functional diversity is obtained partly by using starches from different botanical sources and partly through chemical, physical and enzymatic treatments, performed on starch after its extraction/gelatinisation to modify the constituent polymers .Manihot esculenta Crantz) is one of the worlds major starch crops. It is a perennial shrub in the Euphorbiaceae family and is commercially cultivated in tropical and subtropical regions  and Phosphoglucan Water Dikinase (PWD)  and Like-SEX Four 2 (LSF2), mainly through work on transitory leaf starch in Arabidopsis. SEX4 releases phosphate from both the C6 and C3 positions of amylopectin, with the preference for C6. In contrast, LSF2 is specific for C3-bound phosphate  . GWD andse (PWD) , 2006. Fse (PWD) . Thus, shosphate .starch-excess phenotypes in leaves and retardation in vegetative and/or reproductive growth, which is particularly severe in the case of gwd mutants , barley (Hordeum vulgare), maize (Zea mays), wheat (Triticum aestivum), and rice (Oryza sativa)  ,b and wat traits . Such biStGWD; both the wild-type protein and a redox-insensitive version of it), or silenced each of the endogenous phosphoglucan phosphatase genes (MeSEX4 and MeLSF2). Both approaches altered starch-phosphorylation in the predicted way resulting in starches with distinct properties.To alter starch phosphorylation in the storage root starch of cassava, transgenic plants were generated in which we either overexpressed the potato GWD gene (Manihot esculenta cv 60444) was used as the wild type. For overexpression of GWD, two constructs harboring coding sequence of either the wild-type potato GWD (pCAMBIA2300::StGWD) or a redox-insensitive version of it (pCAMBIA2300::StGWDm) were obtained from Mikkel Glaring . The redox-insensitive version contains a nucleotide modification leading to an amino acid substitution of cysteine 1084 to a serine (C1084S) at the peptide level. For RNA interference lines (RNAi), hairpin constructs targeting either the MeSEX4 or the MeLSF2 genes were designed. The protein sequences of AtSEX4 and AtLSF2 were used as a query to identify orthologous proteins in cassava . Hairpin constructs containing an antisense fragment, a loop, and a sense fragment designed for MeSEX4 or MeLSF2, were subcloned into a modified pCAMBIA1301 vector containing the S. tuberosum patatin B33 promoter. Transgenic cassava lines were obtained via Agrobacterium-mediated friable embryonic callus (FEC) transformation as described in  were vortexed with 600 μL RNA extraction buffer . Ethanol absolute (150 μL), potassium acetate  and chloroform: isoamyl alcohol  were then sequentially added to the mix, vortexed and separated by centrifugation at 13,000 g for 3 min. The supernatant (600 μL) was taken and further cleaned by vortexing with 600 μL phenol:chloroform:isoamyl alcohol (25:24:1). Ethanol was added to the supernatant and the mixture was incubated at −80°C for 30 min to precipitate nucleotides. The precipitate was washed with 80% (v/v) ethanol, dissolved in 75 μL DEPC water, and RNA precipitated by addition of 25 μL 8 M LiCl in DEPC water for 16 h at −20°C. The RNA pellet was washed with 80% (v/v) ethanol, dissolved in 30 μL DEPC water and DNase I treated. Complementary DNA was synthesized using the SuperScript III kit (Invitrogen) and oligo(dT) primers. Real-time quantitative PCR was carried out using the SYBR Green Supermix (Eurogentec) with Applied Biosystem 7500/7500 Fast. PP2A was used as a reference gene. Gene-specific transcripts were normalized to PP2A and quantified by the ΔCt method (Ct of gene of interest - Ct of PP2A gene). Primer sequences (5′–3′) were as follows: MeLSF2, TGAGGAACCCATATGAGTACCA and GCTGCAAATTTAGAATGTAGGCC (150-bp amplicon); MeSEX4, ATTCAGCATCTACGTGCAGA and CTCTTCCTAGCCCAGCAGTG (145-bp amplicon); MePP2A, TGCAAGGCTCACACTTTCATC and CTGAGCGTAAAGCAGGGAAG (150-bp amplicon).Cassava leaves or roots were powdered in liquid Ng) was homogenized in 1 mL ice-cold extraction medium  using a pre-chilled glass homogenizer. After centrifugation , protein in the supernatant was determined using the Bradford method , or against the Arabidopsis thaliana SEX4  were harvested at the end of the day or end of the night from each plant, cleared in 80% (v/v) ethanol, rinsed in water and iodine-stained with Lugol’s solution (Sigma-Aldrich).g, 10 min, 4°C) the insoluble material was washed four times in 80% (v/v) ethanol, resuspended in water and used for starch quantification. Starch in the insoluble fraction was gelatinized at 95°C for 15 min and digested to glucose at 37°C using α-amylase and amyloglucosidase (Roche). Starch content  was determined by quantifying the released glucose with a hexokinase/glucose-6-phosphate dehydrogenase-based spectrophotometric assay.The method of 2 and ground to powder using a Geno Grinder . Cassava root powder (10 mL) was homogenized in a Waring Blender in 100 mL of 50 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 0.5% (v/v) Triton X-100. Insoluble material was collected by centrifugation , resuspended in the same medium and sequentially filtered through 100 and 60 μm nylon meshes. Starch granules were purified by sedimenting through a 95% (v/v) Percoll cushion at 2,500 g for 15 min. The starch pellet was washed at least three times in 0.5% (w/v) SDS, and the SDS then washed away by at least three water washes. Purified starch was then dried under vacuum for 48 h. Subsequent analyses required more starch than was yielded by each plant. Therefore, equal quantities of starch from each line was pooled.Cassava storage roots were harvested from the wild type and from each of the independent transgenic lines. Roots from 3 or more plants per line were chopped, immediately frozen in liquid N2HPO4.Purified starch granules (5 mg) were suspended in water and acid-hydrolyzed in 50 μL 2 M HCl for 2 h at 95°C. The solution was neutralized with 1 M NaOH. Fifty μL of supernatant was then treated with 15 units of Antarctic Phosphatase . Released phosphate was determined using the malachite green assay in which is a complex formed between free phosphate, malachite green reagent and molybdate. Color formation was monitored spectrophotometrically at a wavelength of 660 nm. A standard curve correlating phosphate content and absorbance was generated using a dilution series of 0–10 nmol K31P NMR analysis were prepared according to 2, pH 6, and digested with α-amylase and amyloglucosidase (Roche). After adjusting the sample pH to 6 and adding 5% (v/v) D2O, 31P NMR spectra were measured on an Avance III 500 MHz spectrometer equipped with a CPQCI CryoProbe with cryo-cooled 31P preamplifier and a Z axis-pulsed field gradient unit (Bruker) at 303K. 31P spectra of cassava starch were obtained for 5–10 k transients . All spectra were processed, analyzed and plotted with Topspin 3.2 (Bruker). 31P spectra were calibrated indirectly with the DSS signal as external reference with Ξ= 0.404807356 . Samples were visualized using a Hitachi SU5000 microscope at an acceleration voltage of 5.0 kV and a working distance of 6–7 mm.g). The supernatant was applied to sequential 1.5 mL columns of DOWEX50 and DOWEX1 to remove the charged compounds. Neutral compounds were eluted in 4 mL water and freeze-dried. After re-dissolving in water by heating  linear chains were separated and detected by HPAEC-PAD  using a CarboPack PA-200 column as described previously (Purified starch (0.2 mg) was suspended in 450 μL water. Samples were heated to 100°C for 15 min and cooled to 20°C. Sodium acetate  was added together with 1 unit pullulanase and 0.04 units of isoamylase (Megazyme) to debranch the samples . The reaction was stopped by heating  and clarified by centrifugation . The starch granule suspension was further diluted in water to attain an obscuration level of about 10% and continuously stirred to prevent sedimentation.−1) was used for determining amylose content essentially according to y) = 0.0105 amylose content (x) – 0.2887 (R2 for the standard curve = 0.997).Purified starch in water . The paste clarity was determined according to 2 atmosphere at a heating rate of 5°C/min from 30 to 100°C. To evaluate paste viscosity, purified starch was analyzed using a RVA4500 Rapid Viscoanalyzer (Perten Instruments). Gelling properties were recorded by placing a 16% (w/v) starch/water slurry in the instrument and stirring for 10 s at 960 rpm and then at 160 rpm for the remaining time. The temperature profile was as follows: 50°C for 1 min, followed by a linear increase to 95°C over 3 min 42 s, hold at 95°C for 2 min 30 s, cool to 50°C over 3 min 48 s and hold at 50°C for 2 min. Viscosity was recorded continuously.Swelling power was measured using the method from t-tests, used for determining significant difference between modified starches and control samples in C3 phosphate percentages and in swelling power, were conducted with the Prism software, as were Dunnett’s multiple comparison tests for amylose content and starch granule size. The least significant values were calculated at 5% probability. Relationships between starch characters and starch physico-chemical properties for GWD-expressing lines were analyzed by the means of Pearson correlation and displayed as heat map with the relevant P-values indicated.Two-tailed unpaired StGWD, XM_006357557.2 and PGSC0003DMT400019845; MeLSF2, XM_021769917.1 and Manes.10G005000.1; MeSEX4 XM_021770800.1 and Manes.10G053500.1; MePP2A, XM_021767530.1 and Manes.09G039900.1: S.tuberosum B33 class I patatin promoter GQ352473.1; CaMV 35S promoters: X04879.1.The gene NCBI and/or Phytozome accession numbers, respectively, for the following genes/promoters are: StGWD protein or a mutant version of it (StGWDm) that is insensitive to redox regulation . The StGWD protein levels varied among the transgenic lines , G18, G2 (high expression), G16 (lower expression), and G21 (transgenic control) were selected for further analysis. From the 35S::StGWDm lines (hereafter referred to as ‘R’ lines), R9, R41, R46 (high expression), and R49 (transgenic control) were selected. In storage roots, the expression level was in some cases similar to that in leaves, but not in all of them. Of the G-lines, only G18 had detectable levels of StGWD protein while among the R-lines, both R9 and R46 had detectable levels of protein in storage roots  displayed severe growth phenotypes  grew well, like the GWD-expressing plants  that contained different amounts of phosphate in water at 55°C. The weight of wet sediment formed during heating was determined and the swelling power calculated based on the increased weight. The line with the highest phosphate, R9, had a significantly higher swelling power (19.4 g/g) compared to the wild type , the starch was stored at 4°C and the clarity re-measured over the subsequent 4 days. Light transmittance gradually decreased due to retrogradation, reaching a stable level after 48 h. While this pattern was observed in all our cassava lines Figure , the pas−1  was used to determine starch thermal properties during gelatinization. No significant correlation was observed between starch phosphate content and gelatinization temperature or enthalpy  and paste clarity , but not gelatinization temperature, granule size or amylose content  and compositional characteristics  of starch strongly suggest that the phosphate content of cassava starch influences swelling power  led us to overexpress the s barley  and rices barley , where tStGWD itself contribute to the high levels. Such other factors could include the concentration of ATP, which may be higher in potato amyloplasts than in cassava root amyloplasts. Alternatively, differences in the structure of amylopectin between potato and cassava may affect the degree to which StGWD acts. It was shown previously that GWD has much higher activity on branched substrates with longer rather than shorter chains  or for targeted genome editing , to create similarly improved, transgene-free cassava lines (Our data demonstrate that it is feasible to alter the phosphate content of cassava root starch and thereby its functional properties by over expressing redox insensitive va lines . FurtherStGWD, MeSEX4, and MeLSF2 cassava transgenic lines, which was done by CH, the 31P NMR analysis, which was done by FD, and the RVA analysis, which was conducted by JL and JK. WW and SZ wrote the manuscript with input from all co-authors.WW, CH, and SZ conceived the project. WW performed most of the experiments, except for the generation of 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.


Aim: The aim of this study is to investigate the clinical features of graft dysfunction following living kidney transplantation and to assess its causes.Methods: We retrospectively analyzed a series of 366 living kidney transplantation indication biopsies with a clear etiology and diagnosis from July 2003 to June 2016 at our center. The classifications and diagnoses were performed based on clinical and pathological characteristics. All biopsies were evaluated according to the Banff 2007 schema.Results: Acute rejection (AR) occurred in 85 cases (22.0%), chronic rejection (CR) in 62 cases (16.1%), borderline rejection (BR) in 12 cases (3.1%), calcineurin inhibitor (CNI) toxicity damage in 41 cases (10.6%), BK virus-associated nephropathy (BKVAN) in 43 cases (11.1%), de novo or recurrent renal diseases in 134 cases (34.7%), and other causes in nine cases (2.3%); additionally, 20 cases had two simultaneous causes. The 80 cases with IgA nephropathy (IgAN) had the highest incidence (59.7%) of de novo or recurrent renal diseases. After a mean ± SD follow up of 3.7 ± 2.3 years, the 5-year graft cumulative survival rates of AR, CR, CNI toxicity, BKVAN, and de novo or recurrent renal diseases were 60.1%, 31.2%, 66.6%, 66.9%, and 67.1%, respectively.Conclusions: A biopsy is helpful for the diagnosis of graft dysfunction. De novo or recurrent renal disease, represented by IgAN, is a major cause of graft dysfunction following living kidney transplantation. Many pathological morphological studies have focused on graft dysfunction based on an indication biopsy, but few etiological analyses have been conducted in a large case series.Biopsy is the gold standard for the diagnosis of graft dysfunction. Biopsy results change the clinical diagnosis in 36% and the therapy for 59% of patients . An indiA total of 366 patients who underwent an indication biopsy following living kidney transplantation from July 2003 to June 2016 at our center were included . The patThe indications for the biopsy with reference to 2009 KDIGO Clinical Practice Guideline [A needle biopsy guided by ultrasonography was performed with an 18-gauge needle (Bard). Each sample had at least six glomeruli under light microscopy, electron microscopy, and immunofluorescence analyses  were performed on all biopsies and were observed by two senior pathologists in an independent and blinded fashion. The diagnosis and classification were determined by senior pathologist, some were consulted with doctor in charge, according to the Banff 2007 schema combined with clinical examinations, including therapeutic drug monitoring and BK virus testing in the urine and sera.We analyzed the incidence and the 5-year graft cumulative survival rate of the various causes of graft dysfunction. Due to a great discrepancy in diseased time of different causes, the survival time after transplantation may not reflect the clinical features of graft dysfunction, we defined the time of follow-up began from the day of diagnosis by biopsy instead of the day of transplantation.U test or chi-square test, p values lower than .05 were considered statistically significant, the multiple comparisons were performed with Kruskal–Wallis test and the results were corrected with Bonferroni correction.Data were analyzed with IBM SPSS Statistics software version 22.0 . The comparisons of categorical variables were performed with the Mann–Whitney The 366 cases  includedChronic rejection (CR) occurred in 62 cases (16.1%), and the occurrence was similar in each year, as well as 41 cases (10.6%) of CNI toxicity damage. We adjusted the dosage according to the blood concentrations or converted immunosuppression from CNI to rapamycin (RAPA). With early control of the blood pressure and hyperlipidemia, the 5-year graft cumulative survival rates were 31.2% and 66.6%, respectively.Forty-three cases (11.1%) had BK virus-associated nephropathy (BKVAN), most of which occurred within 1–2 years after transplantation. None of the patients were diagnosed with BKVAN after more than 7 years. After the treatment of immunosuppression reduction and replacement of FK506 with CsA, the 5-year graft cumulative survival rate was 74.3%.De novo or recurrent renal diseases occurred in 134 cases, representing the highest incidence (34.7%) . These dde novo or recurrent renal disease and CNI toxicity damage in five cases, de novo or recurrent renal disease and CR in five cases, and de novo or recurrent renal disease and AR in six cases.Graft dysfunction with other causes occurred in nine cases (2.3%), including acute pyelonephritis, hypertension, and chronic obstruction nephropathy. Twenty cases had two causes simultaneously; these causes were AR and CR in four cases, de novo or recurrent renal diseases. However, the reduction in the incidence was not associated with a better prognosis, and the rejection episodes were more severe than previously reported [With the wide use of effective immunosuppression, the incidence of AR has been reduced markedly in recent years . The overeported . Due to AR has a high incidence in the first year but can occur at any time after transplantation . This paAlthough most AR cases can be cured, many studies have indicated that AR is still the main reason cause of graft dysfunction ,10 and ade novo or recurrent renal diseases occupied the first position of the various causes of graft dysfunction in our results.Unlike in deceased donors, in living transplantation, early steroid withdrawal has been shown to be a safe intervention in the management of low immunological risk ,15. Howede novo or recurrent renal diseases are the third leading cause of graft dysfunction, following CR and death with functional graft [All forms of original nephropathy may recur after transplantation, including IgAN, FSGS, MN, and MPGN. However, great discrepancies exist in their clinical features and prognoses. Data have shown that al graft .IgAN is the most common recurrent nephropathy and recurs in 9–61% of patients . Moreovede novo cases is difficult. Thus, we conjecture that these two periods may represent a peak incidence of recurrent and de novo FSGS, respectively. To test the hypothesis, more biopsy samples should be collected preoperatively. Recurrent FSGS is prone to occur in males and children [Recurrent FSGS develops in approximately 30% of patients after the first transplantation . We founchildren , but moschildren  reportedBKVAN resulted in graft failure in 7% of the cases and emerged as one of the most important infectious diseases after transplantation . SignifiSince the pathological and immunological mechanisms remain unknown, no reliable treatment strategy has been proven to reverse the progression of some diseases, such as CR, recurrent IgAN, and FSGS. However, biopsy promotes the detection rate of the causes of graft dysfunction. Early and aggressive treatment could be provided to improve the prognosis.de novo or recurrent renal disease, represented by IgAN, is more frequent than rejection as a major cause of graft dysfunction following living kidney transplantation. When choosing to maintain immunosuppressive treatments for living transplantation, especially in patients with a high risk of recurrence, steroid withdrawal should be handled with caution, and an early biopsy should be performed.A biopsy is helpful for the diagnosis of graft dysfunction. To this end, we conclude that Despite the incidence and 5-year graft cumulative survival rates analyzed in our study, we did not perform a comparison of the significance between groups for the prognosis of graft dysfunction, influenced by the lack of information about the primary diseases of the recipients and other donor factors. In addition, a few patients did not visit and have biopsy in time when having an initial indication. So we could not compare the outcomes of the different causes in this study. To achieve this end, a complete database and survival analysis based on Cox regression is needed in a subsequent study.


Traditional compensation methods based on temperature-related parameters are not effective for complex total reflection prism laser gyro (TRPLG) bias variation. Because the high frequency oscillator voltage (UHFO) fundamentally affects the TRPLG bias, and the UHFO has a stronger correlation with the TRPLG bias when compared with the temperature, an introduction of UHFO into the TRPLG bias compensation can be evaluated. In consideration of the limitations of least squares (LS) regression and multivariate stepwise regression, we proposed a compensation method for TRPLG bias based on iterative re-weighted least squares support vector machine (IR-LSSVM) and compared with LS regression, stepwise regression, and LSSVM algorithm in large temperature cycling experiments. When temperature, slope of temperature variation, and UHFO were selected as inputs, the IR-LSSVM based on myriad weight function improved the TRPLG bias stability by 61.19% to reach the maximum and eliminated TRPLG bias drift. In addition, the UHFO proved to be the most important parameter in the process of TRPLG bias compensation; accordingly, it can alleviate the shortcomings of traditional compensation based on temperature-related parameters and can greatly improve the TRPLG bias stability. The total reflection prism laser gyros (TRPLGs) are applied in several systems . For exaThere are three major errors in conventional two-mode active laser gyros: mode locking, scale factor error, and null shift ,4. Mode At present, the traditional temperature compensation method of RLG bias is LS regression , which iThe TRPLG differs from the traditional RLG in the structure and control principle of the resonant cavity ,34. TherBasic block diagram of TRPLG is shown in The main factors that affect TRPLG bias are temperature, slope of temperature variation, and UHFO. The temperature variation causes a change in the refraction index of the prism, which causes a change in the optical path and its length, eventually altering the TRPLG bias. The temperature acquisition circuit is shown in The hardware structure of TRPLG bias compensation is shown in In the original weight space, the model is calculated as follows becomes :(2)minω,After using Lagrangian and optimal conditions, the following double problems are obtained:Core skills have been applied here With standard SVM as above in the solution of the QP problem, the number of training samples has a significant influence on the matrix scale, resulting in an excessive calculation amount and low calculation speed. Therefore, the extended algorithm LSSVM is studied. The equality constraints are used instead of the inequality constraints. Therefore, this optimization problem described in Equation (2) becomes as follows:This is just a ridge regression cost function formulated in the feature space. However, it should be noted that, when One constructs the Lagrangian as:After elimination of the variables The core skills of applying here are as follows:The resulting LSSVM model for function estimation becomes then (10)y(x)In accordance with Mercer’s condition, many kinds of kernel function To obtain a robust regression based on the LSSVM solution, one can weight the error variables  problem :(12)minωThe Lagrangian becomes:The unknown variables for this weighted LSSVM problem are denoted by the The choice of the weights Huber weight function isHampel weight function isLogistic weight function isMyriad weight function isThe IR-LSSVM requires to obtain the distribution information of the error based on the training of the LSSVM, and then the weight ed again .IR-LSSVM Algorithm is as follows:Given training data For the optimal Computer Determine the weights Solve the weighted LSSVM (14), giving the model The gyro used in the temperature experiment was the type 70 TRPLG produced by Xi’an North Jierui Optoelectronics Technology Ltd. , and is shown in The large temperature cycle was set up as follows: (1) the TRPLG was fixed with bolts in the temperature chamber, and tested for 2 h at 25 °C, (2) the temperature was then decreased at the rate of 1 °C/min until −40 °C and tested for 6 h, (3) increased at the rate of 1 °C/min to 70 °C and tested for 10 h, (4) decreased at the rate of 1 °C/min until 25 °C and tested for 4 h. The entire cycle was performed twice, once for training and once for compensation.The test software provided the data of temperature, slope of temperature variation, UHFO and TRPLG output, the slope of temperature variation based on the temperature difference per unit time, at intervals of 100 s. The relationship between the TRPLG output and different parameters in the large temperature cycling experiments are shown in For further comparison, the traditional LS model of TRPLG bias is given by (21)B=k0The TRPLG bias can be obtained by subtracting the projection of the earth’s rotation angle rate at this latitude from the TRPLG output. The compensation results of the TRPLG bias using different parameters based LS model are shown in To improve the regression effect, we introduced two multivariate stepwise regression models. Model 1 is a traditional second-order model based on temperature, slope of temperature variation, and its cross terms. The expression isThis study used interactive stepwise regression analysis to find the optimal regression equation. First, the first three items included in the initial model were specified as The compensation result of Model 1 is shown in As seen in The variables in Model 2 were introduced and removed using the interactive stepwise regression analysis method in Model 1. Ultimately, the The compensation result of Model 2 is shown in The IR-LSSVM algorithm is shown in The IR-LSSVM model of TRPLG bias compensation using single parameter cannot completely eliminate TRPLG bias drift. To complete high precision compensation, a bias composite compensation method based on IR-LSSVM model was used. We selected different composite parameters as inputs and TRPLG bias as outputs; the regression and compensation results are shown in To clearly illustrate the significance of the UHFO, we compared In summary, the UHFO is the most important parameter in the process of TRPLG bias compensation. It can make up for the shortcomings of traditional temperature compensation and can greatly improve the TRPLG bias stability.We compared the compensation results of IR-LSSVM model and LSSVM model. The LSSVM compensation model is expressed in Equation (10). To obtain an LSSVM model with the RBF kernel, two extra tuning parameters are needed: the regularization parameter The comparison of compensation results between the IR-LSSVM model and the LSSVM model is shown in To eliminate the complex TRPLG bias variation and improve TRPLG bias stability, we theoretically analyzed the factors affecting the TRPLG bias. The UHFO was introduced into TRPLG bias compensation based on temperature and slope of temperature variation; comparative analysis was performed in LS regression, stepwise regression, and IR-LSSVM. Large temperature cycling experiments can draw the following conclusions.First, in comparison with LS regression, multivariate stepwise regression can reduce partial TRPLG bias drift.Second, IR-LSSVM based on composite parameters can overcome the shortcomings of poor nonlinear fitting ability of the LS regression and stepwise regression.Third, the UHFO proved to be the most important parameter for TRPLG bias compensation; it can make up for the shortcoming of traditional compensation based on temperature-related parameters and can greatly improve the TRPLG bias stability.Fourth, when temperature, slope of temperature variation, and UHFO are used as inputs, the IR-LSSVM based on myriad weight function can completely eliminated TRPLG bias drift to achieve high precision compensation.In conclusion, the IR-LSSVM model proves to be accurate, reliable, and has a significant practical value in engineering.


Stent placement in the cephalic arch is being used with increasing frequency. Late complications of bare metal and stent grafts in dialysis access, in particular stent migration, are often under-reported and can lead to compromise of future dialysis circuits.A 52-year-old man developed acute arm swelling 2 days after creation of a left arm brachio-basilic arteriovenous graft. The axillary vein was found to be jailed by a previously deployed cephalic arch stent graft which had migrated into the subclavian vein. There was failure to cross through the fabric of the stent graft using conventional chronic total occlusion wires and techniques. A TruePath device was used successfully to cross through the fabric of migrated cephalic arch stent graft and recanalise the short subclavian-axillary vein occlusion.The adapted use of a drilling chronic total occlusion device to drill through the fabric of migrated stent graft was performed successfully to allow complete recanalisation of the occluded axillary vein. Stent-graft (SG) placement for cephalic arch stenosis in dysfunctional dialysis access is an option for patients, and a recent systematic review suggests that it may be more durable in the short term compared to angioplasty alone  initially created were abandoned. He underwent hemodialysis via a right brachiocephalic arteriovenous graft (AVG) but required multiple interventions to maintain patency. During a thrombolysis procedure, venoplasty of a cephalic arch occlusion resulted in a focal rupture for which conservative management with balloon tamponade was unsuccessful. An 8 × 60 mm Fluency SG  was deployed in the cephalic arch to manage the rupture. Following deployment, the SG showed no protrusion into the subclavian vein Fig. . On a suA new left loop brachiobasilic AVG was created inadvertently and the patient developed left arm swelling 2 days post-surgery. A graftogram showed complete occlusion of the axillary-subclavian vein from the migrated cephalic arch SG. Attempts to cross the axillary vein occlusion using 0.035″ standard guide wires, 0.018″ glide wires , a 0.014″ Winn 200 T  and 0.018″ Victory 25 g  CTO wires were all unsuccessful due to the inability to traverse the fabric of the Fluency SG , the peripheral end of the occluded SG was accessed Fig. . Using aLeft arm swelling improved significantly over the 3 days. Unfortunately, late infection of the AVG developed and was explanted 28 days post intervention. New long-term AVG dialysis access was created in the left lower limb. Six months after left subclavian-axillary vein stenting, an opportune catheter angiogram via his left groin AVG demonstrated continued patency, and without recoil, of the stented segment Fig. .Cephalic arch stenosis affects up to 34% of dysfunctional brachiocephalic AVFs (Bennett et al. Conventional techniques of crossing the occlusion were unsuccessful due to the SG fabric preventing wire passage. Due to vascular anatomy and potential inadvertent subclavian artery injury, sharp recanalisation employing a straight needle was deemed high risk. Bench-testing using a demonstration unit of the TruePath device and Fluency SG demonstrated controlled and easy traversal of the drilling tip of the device through the SG fabric. TruePath is guidewire-mounted mechanical recanalization device with a rotating distal diamond tip designed for true-lumen crossing of peripheral arterial occlusions. We adapted the previously reported balloon puncture technique, for placement of dialysis access catheters in occluded internal jugular veins (Too et al. The case report demonstrates the successful novel use of the TruePath device to drill through the fabric of a migrated cephalic arch stent graft which had jailed the axillary vein outflow causing acute arm swelling due to compromise of the axillary vein outflow. Migration of cephalic arch stent grafts are under-reported and can compromise future dialysis access options. The use of the TruePath device facilitated recanalization of the occluded axillary vein. The device and techniques used in this case can be utilised in similar situations, where conventional techniques would pose high risk of arterial injury or likely be futile.


Micromachines from March 2016. The journal was founded in 2009 by Prof. Miko Elwenspoek from the University of Twente, and published its first issue in 2010. Twenty years ago, I spent 6 months as a visiting PhD student in Prof. Elwenspoek’s research group of Transducers Science and Technology, working on thermal flow sensors. Taking over the responsibility from Prof. Elwenspoek is my personal honour that represents a true generation change in the leadership of our editorial board.It is my pleasure to assume the role of the Editor-in-Chief of Under Prof. Elwenspoek’s leadership, the journal has grown in both quantity and quality over the past five years. The number of submissions has almost doubled on a year-to-year basis. In 2015, even with a rejection rate of 52%, the number of published papers increased by 50.5% as compared to 2014. The journal has been indexed in all major databases and reached an impact factor of 1.269 (2014). As of March 2016, the journal has published 386 papers with 895 citations (778 citations without self-citations) and an H-index of 13. This performance is truly remarkable for our relatively young journal. The accomplishment was not possible without the leadership of the former Editor-in-Chief and the dedication of the highly efficient team in the editorial office. I would like to take this opportunity to thank our editorial office team for their wonderful support and dedication.Micromachines is serving the research community with an international, peer-reviewed and open-access forum for studies on micro- and nanoscale machines. Thus, traditionally the journal covers a range of topics related to Micro Electro Mechanical Systems (MEMS), Nano Electro Mechanical Systems (NEMS), Lab on a Chip (LOC), and other microdevices as well as their applications. Most of these systems and devices come from the legacy of silicon-based microtechnologies established over the past fifty years. To further improve the impact of the journal and to reach a broader audience, my vision for the journal is to broaden the subject areas beyond silicon-based microsystems and microdevices. Examples for this broader approach are bio-related applications, bio-inspired design of micromachines, micro and nanomachining of non-silicon materials, bottom–up construction of micro- and nanomachines. In fact, papers published in the past year already evidently supported this trend.The aim of An excellent example for biomedical applications of micromachines is the Special Issue “Biomedical Microdevices” that consists of 14 high-quality papers , micropuThe design of micromachines can find inspiration from nature, as natural systems are optimised machines perfected by millions of years of evolution. The bionic mimosa robot  and the Micromachines. I’m honoured to work with this outstanding editorial team as well as the dedicated team of the editorial office. Our collaborative effort will surely bring the journal to the next level of quality and quantity in the years to come. The year 2016 will not disappoint readers of Micromachines. We have a line-up of various exciting Special Issues reporting emerging topics such as three-dimensional (3D) printing, nanomaterials for MEMS, bio-inspired systems, bottom–up self-assembly, polymeric technology and microfluidic bioreactors. I am looking forward to the upcoming exciting journey with contributors and readers of Micromachines.An enthusiastic editorial board of renowned researchers representing the broad topics and interests of the journal currently supports


We report our surgical approach to SAEF consisting in removal of the infected graft and in situ reconstruction using an autologous venous graft from 2008 to 2017 were reviewed. Six of seven patients (86%) survived 30-day. In one case a graft thrombosis and acute lower limb ischemia occurred requiring re-operations. All patients received injective antibiotic therapy for 20 days, followed by oral therapy for 3 months. There were no major complications at long-term follow-up.Seven consecutive patients with SAEF treated with graft removal and Our results suggest that superficial femoral vein reconstruction of the abdominal aorta for SAEF is effective with an acceptable in-hospital mortality and low rate of major complications. We stress the importance of the deep femoral veins to create the graft because the large saphenous vein is often affected by significant intimal hyperplasia that can cause steno-occlusive complications. •In situ reconstruction for secondary aortoenteric fistulas is effective.•We stress the importance of the deep femoral veins to create the graft.•One of the largest series of secondary aortoenteric fistulas treated with venous graft.•Morbidity and mortality are acceptable at long-term follow-up. Seconin situ reconstruction using a novel autologous venous graft.As the treatment of choice for SAEF is still under debate, we describe our surgical approach to SAEF consisting in removal of the infected graft and 22.1in situ reconstruction using an autologous venous graft were reviewed, and the demographic and clinical data were analyzed. All procedures were performed by the same experienced general and vascular surgeon.The medical records of seven consecutive patients with SAEF treated at our academic university hospital, from 2008 through 2017, with removal of the infected graft and An institutional board  approved this retrospective case series and informed consent was obtained from all patients prior to each procedure. This study is compliant with the PROCESS 2018 guidelines  and has 2.2A midline xifo-pubic laparotomy and adhesiolysis were performed in order to access the retroperitoneal space. The bowel segment involved in the fistula was identified and a blunt dissection was carried out to find a correct cleavage plane between the intestinal wall and the prosthesis.The type, shape and dimensions of the required autologous venous graft were assessed before proceeding with the removal of the infected prosthesis. A 15- to 20-cm tract of the superficial femoral vein was isolated, reaching the confluence with the common femoral vein and preserving the deep femoral branches; the superficial femoral vein was sectioned, only after ligation and section of its confluents . The venThe next step was the isolation of the bowel from the infected prosthesis. After accurate debridement, the intestine was closed with a running monofilament suture. Aorta and iliac/femoral clamping were performed with previous heparinization, and the infected prosthesis was excised. The next step was removal of any residual infected or necrotic tissue, including the aortic margins and the retroperitoneal inflammatory tissue. The excised tissue was sent for microbiological examination in all cases. Subsequently, a proximal end-to-end anastomosis was performed, and after anastomotic leakage testing the remaining sutures were completed . Once an3All the seven patients identified were men with a mean age of 69 (range 62–73) years, who had undergone surgery for a sub-renal aortic aneurysm. The previous surgery was aorto-aortic replacement in four (57%), aorto-bifemoral in two (29%), and aorto-bisiliac in one case (14%). Repair of the aortic aneurysm was performed with a dacron prosthesis in all cases. Six patients were affected by hypertension, one had pancreatic cancer, one other had chronic obstructive pulmonary disease (COPD) and previous surgery for bowel obstruction; finally, one patient suffered from rheumatoid arthritis. There was hematemesis in 6 cases (86%), melena in 5 (71%), and abdominal pain in 3 (43%). The mean preoperative hemoglobin value was 11.8 (range 7.3–15.0) g/dL.A computed tomography angiography (angio-CT) was performed in all cases, as well as a doppler ultrasound (US) to evaluate the superficial femoral veins. A gastroduodenoscopy (EGDS) was carried out in 6 patients (86%). One patient underwent traditional angiographic examination.The mean time lapse between the prosthesis placement and repair with autologous graft was 78 (range 23–192) months. All patients were sent to the intensive care unit (ICU) postoperatively for a mean stay of 10 (range 3–25) days. Six patients (86%) received blood and plasma transfusions. Postoperative heparin prophylaxis was given in all cases until full mobilization or hospital discharge. The 30-day survival was 86% (6/7). In one case (the patient with the largest number of comorbidities) a graft thrombosis and acute lower limb ischemia occurred requiring re-operations, but the patient died in the 25th postoperative day. All patients received injective antibiotic therapy for 20 days based on the results of antimicrobial susceptibility testing, followed by prophylactic oral therapy for three months.All patients were observed 6 months after the operation and then at 1-year intervals, and they were evaluated with vascular physical examination and doppler US. Further examinations  were performed as needed. The mean postoperative follow-up time was 63 (range 9–125) months. There were no major complications; a mild bilateral lower limbs edema occurred in one case and was treated with elastic stockings.4Brock  first dein situ reconstruction techniques have been developed using new silver implants, rifampicin and arterial or venous homografts.The goals of SAEF surgical treatment are control of bleeding (if present), removal of the prosthesis and any peri-prosthetic tissue, and restoration of bowel and vascular continuity. The most used vascular reconstruction method is the extra-anatomic bypass , which an situ reconstruction with deep and superficial veins in 20 patients, the majority of them suffered from aortic prosthetic infection but there was aorta-enteric erosion and aorta-enteric fistula in one case each. The authors found a significantly higher failure rate after saphenous vein reconstruction compared to deep vein reconstruction. Nevelsteel et al. [Results on the replacement of an infected graft with arterial or venous autografts in 24 patients have been first reported by Ehrenfeld et al.  in 1979.l et al.  reportedin situ reconstruction with autologous venous graft was used for the treatment of aortic prosthetic infection without fistula in the majority of patients reported by Clagett [in situ reconstruction technique with deep venous graft for treatment of SAEF is effective. To our knowledge, this is one of the largest single-center series of SAEF treated with venous homograft and observed for a very long follow-up. The in-hospital mortality (14%) was acceptable, only mild postoperative complications occurred, and no re-infections, occlusions or failures of the homograft have been documented at long-term follow-up. We preferred the use of the superficial femoral vein to create the graft because the large saphenous vein is often affected by significant intimal hyperplasia that can cause steno-occlusive complications [The technique of  Clagett  and Neve Clagett . In our ortality % was accin situ repair, preferably with a venous graft, may achieve the best result. More recently, Heinola et al. [in situ femoral veins replacement. Eighteen patients had SAEF, and the Authors suggest treating this condition with an aortic endograft as bridging technique only. They concluded that in situ reconstruction with femoral veins presents acceptable morbidity and mortality, and remains the treatment of choice for aortic graft infections at their hospital. A very recent meta-analysis on 402 patients (92 with SAEF) treated for aortic stent-graft infection after endovascular repair concluded that surgical treatment is a better option compared with conservative management [Only recently, the use of endovascular techniques showed excellent short-term results for SAEF treatment, representing an alternative to open surgery or a “bridge” towards open surgery, after patient stabilization in cases of bleeding ,15. Unfoa et al.  reviewednagement . TherefoIn conclusion, the optimal management of SAEF is still unclear. Our results suggest that superficial femoral vein reconstruction of the abdominal aorta for SAEF is effective with an acceptable in-hospital mortality and low rate of major complications. We stress the importance of the deep femoral veins to create the graft because the large saphenous vein is often affected by significant intimal hyperplasia that can cause steno-occlusive complications.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Not commissioned externally peer reviewed.An institutional board approved the study and informed consent was obtained from patients prior to each procedure.University of Sassari).This study was supported by institutional funding (Feo CF wrote the paper.Feo CF, and Ginesu GC designed the study.Pinna A and Galotti F performed the research and acquired the data.Paliogiannis P, Fancellu A and Porcu A analyzed the data and contributed to the revision of the manuscript.Researchregistry5210.Claudio F Feo.Panagiotis Paliogiannis.The authors declare that they have no conflict of interest.


Foxtail millet is considered a ‘smart food’ because of nutrient richness and resilience to environments. A diversity panel of 92 foxtail millet landraces preserved by Taiwan indigenous peoples containing amylose content (AC) in the range of 0.7% to 16.9% exhibited diverse physiochemical properties revealed by a rapid viscosity analyzer (RVA). AC was significantly correlated with 5 RVA parameters, and some RVA parameters were also highly correlated with one another. In comparison to rice, foxtail millet contained less starch (65.9–73.1%) and no significant difference in totals of resistant starch (RS), slowly digestible starch (SDS), hydrolysis index (HI), and expected glycemic index (eGI) according to in vitro digestibility assays of raw flour with similar AC. RS was significantly positively correlated with AC and four RVA parameters, cold paste viscosity (CPV), setback viscosity (SBV), peak time (PeT), and pasting temperature (PaT), implying that suitable food processing to alter physicochemical properties of foxtail millet might mitigate hyperglycemia. This investigation of pasting properties and digestibility of diverse foxtail millet germplasm revealed much variation and showed potential for multi-dimensional utilizations in daily staple food and food industries. Setaria italica) is considered one of the crops of the future because of its relatively high nutrient contents and great adaptability to environments, which provide resilience to climate change. Ranking second in the world production among millets, foxtail millet was domesticated around 6,000 BC and mainly cultivated as a staple food in southern Europe and temperate, subtropical, and tropical Asia [Foxtail millet , SBV (R2 = 0.82), PeT (R2 = 0.59) and PaT (R2 = 0.57), but a significant negative correlation with HPV (R2 = −0.7)  . The riculations ,31, and ns [time . A signins [time   and BDV (R2 = 0.94), but negative correlations with CPV (R2 = −0.28) and SBV (R2 = −0.34) (R2 = −0.45), SBV (R2 = −0.59), PeT (R2 = −0.55), and PaT (R2 = −0.29). However, CPV showed significant positive correlations with SBV (R2 = 0.93), PeT (R2 = 0.43), and PaT (R2 = 0.56). SBV showed a significant positive correlation with PeT (R2 = 0.31) and PaT (R2 = 0.56), while PeT and PaT were also significantly positively correlated with each other (R2 = 0.46). The correlations among these parameters indicated that the pasting properties of foxtail millet starch provided reference information for food applications.PKV had significant positive correlations with HPV (= −0.34) . HPV shoThe distribution patterns of these RVA parameters were various among waxy, low-AC, and non-waxy types . VariatiSix RVA parameters showed a significant difference, except for PKV . The nonSpecific applications of particular foxtail millet genotypes in diets and/or food processing might be suggested according to the evaluation of physicochemical properties, revealed by scatter plots of 92 accessions along 2 correlated RVA parameters . In geneTo mimic the starch digestibility in human digestive systems, the in vitro starch hydrolysis assay was performed on 9 foxtail millet accessions and 3 rice varieties with various AC. In rice, the AC of IR8, TN11, and Hung-No were 20.7%, 12.2%, and 1.4%, respectively. For foxtail millet, three non-waxy accessions with 14.4–16.9% AC, two low-AC accessions with 6.0–7.7% AC, and 4 waxy accessions with 1.3–1.9% AC were selected. The rice flour in this study was made of polished white rice, while the foxtail millet flour was made of dehusked but unpolished grains because polishing is not a required process for utilization of foxtail millet. The starch content of foxtail millet ranged from 65.9% to 73.1%, while those of three rice cultivars ranged from 88.9 to 91.1% . Starch indica rice with the highest AC, IR8, had the slowest hydrolysis rate, indicating the least digestible starch. The hydrolysis rate of glutinous rice, Hung-No, was higher than japonica rice, TN11. In foxtail millet, the hydrolysis rate from highest to lowest was waxy, low-AC, and non-waxy. Also, the hydrolysis rates of all examined foxtail millets were higher than the 3 rice varieties, indicating that foxtail millet starch was relatively easy to digest.The starch digestibility of grain flours was assessed by in vitro enzymatic hydrolysis lasting 120 min . In geneStarch was classified into RDS, SDS, and RS according to the time of digestion . In the In foxtail millet, high eGI were observed in waxy accessions, while no significant difference was detected between non-waxy and low-AC accessions. In rice, the eGI of Hung-No was also significantly higher than TN11 followed by IR8, while the latter two varieties showed no significant difference. Raw foxtail millet starch possessed higher HI and eGI than rice in all AC levels, indicating that foxtail millet starch was easier to digest than rice though they contained less starch . The chap < 0.001) was observed in the postprandial glucose level of patients who consumed a millet-based dosa when compared to those who consumed a rice-based dosa [The starch property of foxtail millet-based food was significantly different under boiling, steaming, and extrusion preparations; for example, high eGI can be found in millet porridge, followed by millet pancake, and cooked millet . The stased dosa . Millet-sed dosa ; moreoveR2 = 0.59) but not significantly correlated with RDS and SDS. From the perspective of the physicochemical property of amylose, long-linear chains characteristic in starch granules interact with amylopectin and lipid, and hence amylose is resistant to enzyme digestion [The digestibility of starch is diverse and influenced by particle size, composition, physicochemical properties, food processing conditions, and plant types . The corigestion . RS contigestion ,47. As aindica rice, IR8, exhibited the highest RS and lowest HI and eGI because of high AC (>20%)  , and manobserved . Physiocobserved ; thus, aOryza sativa) varieties, IR8 (subspecies indica), TN11 (japonica), and Hung-no (indica glutinous cultivar), were included for comparison. IR8, developed by the International Rice Research Institute (IRRI), is a semi-dwarf and high-yielding rice variety widely considered to have played an important role in the rice Green Revolution; TN11 is a leading variety in Taiwan. Rice varieties were grown in TARI Chiayi branch in a RCBD with two replicates in 2015.A diversity panel of 92 foxtail millet accessions was evaluated . Table . All accFoxtail millet grain samples were dried at 37 °C for a week. Dehusked grains were transversely cut and fixed on the specimen holder by carbon conductive double-faced adhesive tape. The grain surfaces were gold coated in an ion sputter E-1010  for 60 seconds at 2.4 kV, and images of grain surfaces were taken at 1500, 3000, and 6000× magnification by using FEI Quanta 200 .2-KI) assay with minor modification [2O, 0.5 mL of 1 M acetic acid and 0.5 mL of iodine solution (2 % KI and 0.2 % I2). The AC of each sample was calculated according to the standard curve based on the absorption at O.D. 620 nm .AC was determined by the iodine-staining  to obtain RVA profiles. Dehusked foxtail millet grains were ground into fine flour and sieved through a 0.43 mm mesh (40 Mesh). Approximately 3 g of foxtail millet flour was mixed with 25 mL of water in an RVA sample canister. The sequential temperature curve for 23 min was as follows: (1) holding at 50 °C for 10 sec at 960 rpm and then incubating at 50 °C for 1 min; (2) heating to 95 °C and holding for 3.7 min; (3) cooling to 50 °C and holding at 50 °C for 3 min . Seven Rd-Glucose Assay Kit  and then converted to starch content.The total starch content of foxtail millet was determined by using Megazyme starch assay kit  according to AOAC Method 996.11 (AACC Method 76-13.01). The total glucose content was determined by the 2O in the tube, shaking for 10 min, and then centrifuging at 1500 g for 10 min; 15.0 mL of pancreatin supernatant was saved, subsequently adding 0.75 mL of amyloglucosidase (1200 U mL−1) and 1 mL of invertase (3000 U mL−1). Starch digestion was initiated by adding 5 mL of the enzyme mixture and then incubating at 37 °C for 3 h under horizontal shaking at 200 rpm. During the incubation, 0.5 mL of each sample was added into 10 mL of absolute ethanol and mixed well every 20 min. These digested samples were collected at 7 time points, specifically 20 (G20), 40, 60, 80, 100, 120 (G120), and 180 min (TG), and the supernatant was saved after centrifugation at 1500× g for 5 min. The glucose portion in the supernatant was measured according to the glucose oxidase-peroxidase method by using a GOD-POD diagnostic kit . The content of three starch fractions, RDS, SDS, and RS was calculated on a dry basis with the following equations and expressed as percentage of total starch [The flour of two to four foxtail millet accessions from each AC level was analyzed by using in vitro starch hydrolysis based on Englyst et al. (1999) with some modifications. In rice, IR8, TN11, and Hung-No, representing high AC, intermediate AC, and waxy type were included for comparison. Approximately 0.5 g flour was dispersed in 10.0 mL of freshly prepared pepsin solution (50 mg of pepsin and 50 of guar gum in 10 mL of 0.05 M HCl) and incubated at 37 °C for 30 min at 200 rpm. Subsequently, 10.0 mL of 0.1 M acetate buffer  was added to form a buffer at pH 5.2. The enzyme mixture was prepared by dispersing 3.0 g of pancreatin in 20.0 mL of Ht correspond to the percentage of starch hydrolyzed at t time. C∞ is the equilibrium concentration and k is the kinetic constant in t time. Area under curve (AUC) was obtained each sample by using the equation:tf and t0 are the final (180 min) and initiate (0 min) hydrolysis time, respectively.The estimated glycemic index (eGI) was calculated based on glucose measurements at 7 time points mentioned above during starch hydrolysis. The kinetics of in vitro starch digestion was calculated according to the first order equation :(4)C=C∞(japonica rice TN11. The expected (eGI) was converted by following equation:H100 is the glucose content measured at 100 min.The hydrolysis index (HI) was calculated by dividing the area under curve (AUC) value of each sample with the AUC of the reference food, p < 0.05.AC and the starch hydrolysis parameters were estimated for 3 samples and expressed as mean ± SD. For AC, RVA, and starch hydrolysis parameters, one-way ANOVA was performed to investigate statistical significance of differences between parameters, followed by Fisher’s LSD. Pearson correlation coefficients between variables were calculated with a significance threshold of


Z.J. Zhang, M.L. Zheng, Y. Nie and Z.Q. NiuDepartment of Anesthesiology, the Cangzhou Central Hospital, Cangzhou, Hebei, ChinaRetraction for: Braz J Med Biol Res | doi: 10.1590/1414-431X20176825 | PMID: 29267506Click here to view Specialist message]The Brazilian Journal of Medical and Biological Research was contacted by one specialist questioning the validity of this study. [The authors were contacted on May 7 and 22, 2018 to answer the questions one-by-one. As of June 29, 2018, we did not receive an answer from the authors.10.1590/1414-431X20176825> PMID: 29267506.The Editors decided to Retract the article: “Comparison of Arndt-endobronchial blocker plus laryngeal mask airway with left-sided double-lumen endobronchial tube in one-lung ventilation in thoracic surgery in the morbidly obese” that was published in volume 51 no. 2 (2018)  in the Brazilian Journal of Medical and Biological Research <The Brazilian Journal of Medical and Biological Research informed all authors before the publication of this Retraction.


We review the scope, content, and focus of the peer-reviewed journal, Research on Aging (SAGE), publishing its 41st volume this year. We will discuss how scholarship produced from researchers around the globe has changed over the years. Data on submissions, acceptance rates, and the important role of an international editorial board will be presented. The review process will be described, along with suggestions on how to increase chances of success when submitting original research. Although Research on Aging is sometimes considered to focus primarily on social gerontology, the scope in recent years has widened considerably, with manuscripts in aging studies published from such fields as economics, psychology, demography, public health, and public policy, as well as from sociology, and social work, among others. One of several special issues forthcoming in the journal will be described to demonstrate the possibilities for international impact.


NFATc1) and c-Fos were measured, and protein levels of NFATc1 and c-Fos were investigated. The zirconia specimens had rhomboid-like shapes with smooth surfaces and exhibited no difference in surface roughness compared to the titanium specimens. Morphologies of differentiated osteoclasts on both materials were similar. TRACP activity on the zirconia showed comparable results to that on the titanium. The mRNA value of NFATc1 on the zirconia was higher than that on the titanium at day four. The protein level of c-Fos was expressed thicker on the zirconia when compared to the titanium at day two. The results of this study suggest that zirconia material provides adequate osteoclastogenesis behaviors for dental implant use.Zirconia is worth studying as an alternative to dental titanium implants to overcome the disadvantages of titanium. This study investigated the surface characteristics of the zirconia implant material and osteoclastogenesis responses on the surface compared with titanium. Yttrium oxide-stabilized 5% tetragonal zirconia polycrystalline specimens were manufactured, and osteoclast-precursor cells were cultured and differentiated into osteoclasts on the specimens. Surface shape, roughness, and chemical composition were evaluated. After culturing, cell morphologies and differentiation capacity were analyzed using tartrate-resistant acid phosphatase activity (TRACP). mRNA of two critical transcription factors, nuclear factor of activated T-cells 1 ( Titanium (Ti) has been studied for years not only for dental implants but also for medical fields including orthopedic implants because of its great biocompatibility and high bone-tissue affinity ,2,3. In 2O3)-stabilized 5% tetragonal zirconia polycrystals (Y-TZP) and is used in dental restorations [The negative effects of Ti have driven many researchers to investigate the potential of ceramic implants for use in dental applications. Plecko et al.  comparedorations . Zirconiorations ,20. Zircorations . Osteoblorations . In perspective of bone physiology, cooperative interactions occur between osteoblasts and osteoclasts, which contribute to bone formation and resorption. And both cells are required for bone remodeling after implant insertion . Bone neWe hypothesized that zirconia would be an acceptable material for osteoclast differentiation and have conducted experimental osteoclastogenesis on the surface of zirconia and Ti. The current study investigated the characteristics of zirconia compared to Ti surfaces assessing visual and quantitative properties of osteoclasts on zirconia and Ti disks. Two indicating factors for differentiation of osteoclasts in mRNA and protein level were examined to analyze gene expression.2, 5.1 mol% Y2O2, 0.25 mol% Al2O3) . Each disk had uniform dimensions with a 15-mm diameter and 1-mm thickness. Thirty machine-surfaced Ti disks (15 mm in diameter and 1 mm in thickness) were manufactured from commercially pure Ti grade IV  as a control. All disks were washed in distilled water and ultrasonically cleaned with alcohol. Disks were then dried and autoclaved at 120 °C for 30 min. The disks surfaces were coated with a platinum film and viewed with a field emission scanning electron microscope (FE-SEM)  using an acceleration voltage of 15 kV at three magnifications . 3D images and surface roughness were obtained by confocal laser scanning microscopy (CLSM) . Energy dispersive X-ray spectroscopy (EDS)  was conducted to assess the chemical composition of the disks. Each evaluation was conducted with three disks for each group.As the experimental group, thirty zirconia disks were fabricated via cold isostatic pressing at 200 MPa then sintered for 2 h at 1650 °C in air. Zirconia specimens were composed of yttria-stabilized tetragonal zirconia polycrystals  containing 10% fetal bovine serum , 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were suspended in 250 µL α–MEM, and were directly seeded on top of the disk surface at a density of 2 × 104 cells/disk, not on the residual space of the well. After adhering cells to the disk for 6 h, the medium was changed to cover the entire well [Murine RAW264.7 macrophage/monocyte cells  were cultured on the surfaces of zirconia and Ti disks . Disks wire well . One hunAfter three days of culture on the disks, cells were fixed with 2% glutaraldehyde in phosphate buffered saline (PBS) for 10 min at room temperature . Cells wTartrate-resistant acid phosphatase (TRACP) enzyme activity was measured to quantitatively evaluate osteoclast differentiation activity. Cells were treated with RANKL for three and five days, and TRACP activity was measured using the TRACP assay kit  according to the manufacturer’s protocol. The TRACP activities of the cultured cells were examined by a colorimetric assay utilizing the conversion of colorless p-nitrophenol phosphate to colored p-nitrophenol. The concentration of p-nitrophenol was measured spectrophotometrically by taking sample absorbance readings at 405 nm . Replicate measurements were performed with 4 disks for each group.glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and two target genes, nuclear factor of activated T-cells 1 (NFATc1) and c-Fos. Total ribonucleic acid (RNA) was isolated from cell samples after two and four days of treatment with RANKL. Cells were lysed by adding the TRIzol® reagent  according to the manufacturer’s instructions. The homogenized samples were incubated for 5 min at room temperature to permit the complete dissociation of nucleoprotein complexes. Then, 0.2 mL of chloroform was added to each sample, and the samples were mixed vigorously and then incubated at room temperature for 3 min. The samples were centrifuged at 12,000× g for 15 min at 4 °C. Following centrifugation, the mixture was separated into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA was precipitated from the aqueous phase by mixing with 0.4 mL of isopropyl alcohol. The samples were incubated at room temperature for 10 min and centrifuged for 10 min at 4 °C. The RNA precipitate formed a gel-like pellet on the side and bottom of the tube. After removing the supernatant, the RNA pellet was washed with 1 mL of 75% ethanol and centrifuged at 12,000× g for 5 min at 4 °C. The RNA pellet was dried for 5 min. RNA was dissolved in RNase-free water and incubated at 55 °C for 10 min. Total RNA was quantified using a Nanodrop Spectrophotometer ; 1 μg of total RNA was reverse transcribed to cDNA at 42 °C for 50 min using the Superscript II (SSII) reverse transcriptase (Invitrogen) that contains oligo (dT), 2.5 mM dNTP, 5xFS buffer, 0.1M DTT, SSII enzyme, and RNase out.Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted to investigate mRNA levels in osteoclast cells for one reference gene, c-Fos and NFAcT1 were normalized to GAPDH.Real-time PCR was performed in an iCycler (Bio-Rad) using SYBR green (Invitrogen) detection. Each reaction contained 5 μL of cDNA, 0.4 μL of the forward and reverse specific primers , and 0.4c-Fos, and NFATc1, were assessed via Western blot analysis [g for 15 min at 4 °C. Harvested proteins were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane . The membrane was probed with specific antibodies  with a concentration of 2 μg/mL and the reactivity of the immune complexes were detected by using enhanced chemiluminescence  reagents.The protein expression levels of 2 genes, analysis . RAW264.t-test analysis . p-values less than 0.05 were considered statistically significant.Statistical significance for all data comparisons was determined by student In SEM views, the zirconia specimens had rhomboid-like shapes with smooth surfaces, and the Ti specimens exhibited a flat surface with striped patterns. .3D image of the microstructures on the surface of each disk is shown in EDS spectra revealed Zi peaks for zirconia disks and Ti peaks for Ti disks, confirming that both disks were pure and free from any contamination for further cell experiments . Differentiated osteoclasts morphologies are shown in At day three, absorbance value was 0.301 ± 0.016 for Ti and 0.286 ± 0.024 for zirconia disks a. At day3.4. mRNA Expression of c-Fos and NFATc1c-Fos mRNA was 1.205 ± 0.278 for zirconia and 0.921 ± 0.093 for Ti. At day four, the relative expression of c-Fos was 2.124 ± 0.474 for zirconia and 1.514 ± 0.124 for Ti. These results did not show any significant differences  was significantly higher than that on Ti (1.722 ± 0.158) (p = 0.02) (mRNA levels assessed by RT-PCR on RNA harvested from cells that were treated with RANKL for two and four days are shown in ferences a. At day = 0.02) b.c-Fos and NFATc1. We observed that c-Fos protein levels were slightly thicker for zirconia disks when compared to Ti disks at day two whereas protein levels on zirconia and Ti disks were similar at day three. Protein levels of NFATc1 were similar across all treatment groups.Osseointegration describes the structural and functional complex that occurs between an implant surface and the inner bone  which isThe present study may be the first study to compare osteoclast cell responses on zirconia and Ti materials directly. Given that osteoclast differentiation is likely to play an important role in peri-implant bone remodeling  we selecTo induce osteoclast differentiation, we allowed them to proliferate for 6 h and then treated them with RANKL. The effects of zirconia surface itself on osteoclast cellular responses were compared with those on Ti surface. In this study, a surface roughness of the zirconia disk did not show any difference with that of the Ti disk. Considering that the surface topography of materials contributed to differences in osteoclast behavior in previous reports ,38,39, tPotent TRACP activity is found in osteoclasts and is a suitable biochemical probe for osteoclast function ,41. TRACc-Fos and NFATc1 are known as major transcription factors required for the osteoclast differentiation of terminal stage and are strongly induced by RANKL stimulation [NFATc1 and c-Fos. Protein levels were assessed at one, two and, three days after treatment with RANKL. At day two, cells grown on zirconia showed higher levels of c-Fos protein relative to those grown on Ti. However, on day three, the abundance of c-Fos protein on both disks did not vary each other. Even though c-Fos is an important indicating factor for osteoclastogenesis, NFATc1 could induce osteoclast differentiation in the absence of c-Fos [NFATc1 are similar on both disks, it could induce comparable osteoclast differentiation even when there is no c-Fos protein. In the case of NFATc1, there were no significant differences in protein levels between cells grown on zirconia and Ti disks for any experimental time point. These observations indicate that zirconia induces similar osteoclast cell responses relative to Ti. mulation . Westernof c-Fos . In otheNFATc1 and c-fos after treatment with RANKL for two and four days. At day two, there were no differences in mRNA levels of either transcription factors for cells grown on either Ti or zirconia disks. There was not a significant difference in c-Fos mRNA level between zirconia and Ti disks at day four. mRNA level of NFATc1 of zirconia for day four was significantly higher than that of Ti. Annette et al. [We conducted real-time RT-PCR to assess mRNA level of e et al.  reportede et al. . Taken tNonmetallic materials with high mechanical properties are required . Low elaThis study investigated surface characteristics of zirconia implants and osteoclast behaviors in gene level on the surface. Visual evaluation of differentiated osteoclasts confirmed that osteoclastogenesis for both zirconia and Ti implant disks were comparable. Given that two transcriptional factors appearing similarly on both disks, it is possible that zirconia implants could function like Ti implants and induce favorable osseointegration.