An increase in the pCO2 during the bloom suggests that most of the additionally fixed carbon was rapidly consumed.The North Pacific Subtropical Gyre (NPSG) is the largest ecosystem on Earth, and it plays a critical role in global ocean productivity and carbon cycling. Here, we report a rare and striking ~2000-km-long phytoplankton bloom that lasted over one month in the western part of the NPSG in summer 2003. The bloom resulted from the co-occurrence of a northward-shifted North Equatorial Current (NEC) supplying additional phosphate, and strong eddy activity that fueled productivity and spread chlorophyll mainly through horizontal stirring. The extensive one-month bloom had a maximum Chl concentration of six times the summer mean value and collectively fixed an additional five teragrams (5 × 10 The eddies are often observed as a long west-east band generated by shearing activity between the eastward-flowing Subtropical Countercurrent (STCC) and the westward-flowing North Equatorial Current (NEC)4. As a result of the beta planetary effect, the eddies move westward at a speed of approximately 0.1 m/s6. Seasonally, the eddies are more active in spring and summer than in other seasons7. The eddies were more active from 1996 to 1998 and from 2003 to 2008 on an interannual time scale3. In particular, the eddies in 2003 were the strongest among the years from 1998 to 2009 due to a strong STCC enhanced by a surface Ekman temperature gradient convergence within the STCC region3.The western part of the North Pacific Subtropical Gyre  is a hotspot of eddies8. The mixed layer depth is generally deeper than 100 m in winter and shallower than 30 m in summer9. Strong vertical stratification in summer inhibits the mixing of the surface water with the deep, nutrient-rich water. These conditions result in a chronically nutrient-limited surface layer, making the NPSG the least biologically productive region per unit area in the global ocean10.In the wNPSG, a vertical entrainment associated with a mixed-layer deepening balances the annually integrated surface thermal forcing13. The mechanisms driving these blooms include nitrogen fixation by diazotrophs15, eddy interactions16, internal waves17, and mixing at the subtropical front18. To date, there are no reports of large-scale summertime phytoplankton blooms in the wNPSG in the literature. Chlorophyll (Chl) concentrations observed from satellite ocean colour images in the wNPSG are usually lower than 0.06 mg/m3 in summer20. In July 2003, a huge bloom over ~2000 km long with Chl concentrations >0.2 mg/m3 occurred within the area bounded by 135°E to 165°E and 15°N to 28°N, as revealed by satellite images , altimetry-derived eddy kinetic energy (EKE), Argo floats, surface drifters, conductivity-temperature-depth (CTD), surface seawater nutrient and partial pressure of CO2 (pCO2) data. As the Chl blooms resemble rings of eddy-like patterns and the EKE was particularly strong in 2003, we first examined the co-variability of the Chl and eddies and the intensity of the eddies in 2003 compared to other years. From the Argo and CTD’s vertical temperature profiles, we demonstrate that there was a possible deepening of the vertical mixing when the bloom occurred in the wNPSG in 2003. We also analysed the drifter data and performed an eddy composite analysis to show that the stirring induced by the strong eddy currents was the main mechanism sustaining the summertime bloom.We utilized a combination of satellite and 3 in July 2003  was observed between the time series of the EKE and Chl  between the EKE and Chl of approximately 2 weeks  in the waters south of 17°N and north of 26.5°N24. The near-complete consumption of phosphate in the subtropical region is linked to active nitrogen fixation by a number of diazotrophs, particularly nanoplanktonic cyanobacteria24.The high Chl concentrations at the southern part of the eddies in Fig. The strong stirring by the eddy currents at the southern edge of the bloom likely encapsulated the high-phosphate waters from the northern front of the NEC to the bloom area. Indeed, the surface phosphate concentrations observed by the Japan Meteorological Agency (JMA) cruises within the bloom region were higher (>50 nM) in July 2003 than in other years from 2001 to 2005 for the same period Fig. . Hence, 3 to 0.30 mg/m3  and shipboard CTD (ID H9TO) within the bloom area Figs  and 9 sh00121 and/m3 Fig. . The 29 /m3 Fig. , roughly/m3 Fig. . This coThe monthly mean AOT, a proxy for atmospheric deposition, within the strongest bloom region (145–155°E and 17–25°N) shows that the atmospheric deposition during summer 2003 was rather weak compared to the summertime values in other years Fig. . In addipCO2 during the bloom period indicates that this additional input of carbon was rapidly consumed and respired as CO2.This study reports the largest summer phytoplankton bloom ever recorded in the wNPSG. The bloom emerged as eddy features in the space domain and varied with eddies in the time domain. Our findings show that the eddies were responsible for the bloom mainly via horizontal stirring with anomalous phosphorous supplied from the NEC. The extremely strong eddy currents in summer 2003 swiftly spread the high Chl waters to the east and west with a speed faster than that of the eddy westward motion. Our findings differ from the current understanding of eddy-induced blooms that are westward-propagating and driven by the westward eddy motion. In addition, coinciding with the northward extension of the high-phosphate NEC waters, strong eddy stirring could have encapsulated the high-phosphate waters within the bloom area, further sustaining the blooms. The absence of the bloom when the aerosol measurements were high indicates that atmospheric deposition did not contribute to macronutrient loading to the bloom area. The extraordinary 2003 summertime bloom not only resulted in an increase in Chl six times that of the mean values but also collectively fixed an additional five teragrams of carbon during the one-month bloom period. However, the concomitant increase in the http://www.globcolour.info), which provides a merged Chl product with good coverage from sensors such as SeaWiFS, MERIS, MODIS-Aqua, and VIIRS. Daily altimetry SSH-anomalies (SSHAs) at 0.25° resolution were obtained from the Archiving, Validation, and Interpretation of Satellite Oceanographic (AVISO) data . For data consistency, the daily SSHAs were averaged at 8-day intervals according to the dates of the Chl. The geostrophic velocity anomalies  were derived from the zonal and meridional gradients of the 8-day SSHAs2, and the EKE was estimated as follows: 0.5*[(u′)2 + (v′)2]. The anomalies of the Chl in this study refer to deviations of the Chl concentration from the long-term mean of monthly means. The net primary production (NPP) rates were calculated based on the Vertically Generalized Production Model (VGPM)28. The VGPM includes Chl, photosynthetic active radiation from GlobColour, and sea surface temperature (SST) from Optimum Interpolation SST29 as input parameters. The in situ CTD, Argo, and drifter observations were obtained from the NOAA’s Atlantic Oceanographic and Meteorological Laboratory (http://www.aoml.noaa.gov/). The IDs of the concomitant CTD and Argo data are H9TO and Q5900121, respectively. The drifter data were used to calculate the eddy-current speed. The in situ nutrient data measured by the Japan Meteorological Agency were obtained via NOAA’s World Ocean Database 201330 (https://www.nodc.noaa.gov/OC5/WOD/pr_wod.html).All satellite observations used in this study cover a common period from 1998–2014. Daily, eight-day, and monthly Chl and AOT at 1/4° resolution were obtained from the European Space Agency’s GlobColour project .Composite analysis was adopted to show mean distributions of the Chl concentration and NPP in cyclonic and anticyclonic eddies. The SSHA data within 135–165°N and 15–28°N were selected to track the eddy centres for the composite analysis. The eddy centre was defined by the maximum and minimum SSHAs for the anticyclonic and cyclonic eddies, respectively. For the cyclonic-eddy composite, a zonal distance between the maximum SSHAs at the west and east of each cyclonic eddy was obtained to normalize the x- and y-axes of the maps. We selected every cyclonic eddy with the SSHA difference above 0.08 m between the eddy-centre SSHA and the maximum SSHAs at its sides along the eddy-centre latitude. The 0.08 m value was selected to be twice as large as the SSH accuracy of approximately 0.04 m to avoid errors in satellite-altimetry measurementsThe original datasets used in this study can be obtained from different data centers noted in the “Methods” section. The post-processed datasets generated during and analysed during this study are available from the corresponding author on reasonable request.


To the Editor: We read with great interest the Sarajevo Declaration on Integrity and Visibility of Scholarly Publications published as the editorial in the Croatian Medical Journal ( Journal . We appr Journal . TherefoWe thank the authors for the Declaration and feel that, with our suggestion taken into account, we can enforce the ultimate goal of the Declaration, which is to improve scientific and publishing practice.


Arthritis Research & Therapy [Arthritis Research & Therapy are freely available to communities around the world.We aim to be a broad, online and independent journal with a global readership and community of authors Fig.  that insLike all journals, we strive for fair and fast peer review. We are unusual in having two Editors-in-Chief (in two different continents) and we have established a strong group of experts who handle submissions across our six sections: Immunology and Pathology, Pharmacology and Therapeutics, Epidemiology and Clinical Trials, Bone and Cartilage Biology, Imaging and Outcomes, Genetics and Epigenetics.We would like to highlight emerging and exploratory studies and in particular we would like to be the journal of choice for informative science linked to clinical trials. We don’t believe that basic science can only be successfully conducted in animal models. Animals model processes; humans display the pathology.We would like to be a journal that publishes across the wide spectrum of autoimmune rheumatic and musculoskeletal conditions. Clinical trials are changing and new devices and diagnostic tests are emerging which will be subjected not just to the current test of “equivalence” but which will now need to be tested in randomised clinical studies. We want to be at the forefront of publishing the science that accompanies these trials, as well as the new methodologies on which they are based.What should the aim, scope and ambition of a journal be? After two years as Editors-in-Chief of  Therapy , we haveSo in summary we hope that the following statement accurately summarizes these points and reflects the scope of our journal:Arthritis Research and Therapy is an international, open access, peer-reviewed journal, publishing original articles in the area of musculoskeletal research and therapy as well as reviews, commentaries and reports.“Established in 1999, A major focus of the journal is on the immunologic processes leading to inflammation, damage and repair as they relate to autoimmune rheumatic and musculoskeletal conditions, and which inform the translation of this knowledge into advances in clinical care.Original basic, translational and clinical research is considered for publication along with results of early and late phase therapeutic trials, especially as they pertain to the underpinning science that informs clinical observations in interventional studies.”


Starch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications. Its metabolism has been subject of extensive research over the past decades. Despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. Moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. This study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. Moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism.We identified 77 genomic loci encoding enzymes involved in starch metabolism. Novel isoforms of many enzymes were found. Their analysis will help to elucidate mechanisms of starch biosynthesis and degradation. Expression analysis of starch genes led to the identification of tissue-specific isoenzymes suggesting differences in the transcriptional regulation of starch metabolism between potato leaf and tuber tissues. Selection of genes predominantly expressed in developing potato tubers and exhibiting an expression pattern indicative for a role in starch biosynthesis enabled the identification of possible transcriptional regulators of tuber starch biosynthesis by co-expression analysis.This study provides the annotation of the complete set of starch metabolic genes in potato plants and their genomic localizations. Novel, so far undescribed, enzyme isoforms were revealed. Comparative transcriptome analysis enabled the identification of tuber- and leaf-specific isoforms of starch genes. This finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. Putative regulatory proteins of starch biosynthesis in potato tubers have been identified by co-expression and their expression was verified by quantitative RT-PCR.The online version of this article (doi:10.1186/s12864-016-3381-z) contains supplementary material, which is available to authorized users. Solanum tuberosum) is one of the world’s most important crop plants. Potato tubers are rich in starch and contain minerals and vitamins as well as essential amino acids. Due to its nutrient composition and the high starch content, it serves as staple food, animal feed and feedstock for many industrial purposes including bioethanol production and food thickener. Moreover, tuber starch is used in the paper and textile industry.Potato , namely the Myb-related CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) , S2 [RH Leaf], BV_P_S [DM Tubers ], S7 [RH Young Tuber], S8 [RH Mature Tuber] and S15 [RH Tuber Sprout]. For each starch gene FPKM values were extracted and further analyzed by calculating mean FPKM values for leaf and tuber tissue, respectively, and determining the ratio between the average values. Co-expression analysis was conducted in Excel by applying the function PEARSON on all genes using the same query genes as in the microarray analyses.


GWD1 in rice. Transgenic rice grain starch had 9-fold higher 6-phospho (6-P) monoesters and double amounts of 3-phospho (3-P) monoesters, respectively, compared to control grain. The shape and topography of the transgenic starch granules were moderately altered including surface pores and less well defined edges. The gelatinization temperatures of both rice flour and extracted starch were significantly lower than those of the control and hence negatively correlated with the starch phosphate content. The 6-P content was positively correlated with amylose content and relatively long amylopectin chains with DP25-36, and the 3-P content was positively correlated with short chains of DP6-12. The starch pasting temperature, peak viscosity and the breakdown were lower but the setback was higher for transgenic rice flour. The 6-P content was negatively correlated with texture adhesiveness but positively correlated with the cohesiveness of rice flour gels. Our data demonstrate a way forward to employ a starch bioengineering approach for clean modification of starch, opening up completely new applications for rice starch.Starch phosphorylation occurs naturally during starch metabolism in the plant and is catalysed by glucan water dikinases (GWD1) and phosphoglucan water dikinase/glucan water dikinase 3 (PWD/GWD3). We generated six stable individual transgenic lines by over-expressing the potato  Clean protocols for starch modification is therefore desired and starch bioengineering directly in the plant is an attractive approach2.Starch is the major carbohydrate of cereal grain and a primary dietary component of human energy intake. Grain starch also serves the major source for food and non-food applications such as in thickening, gelling, sizing, binding and adhesion. Owing to its non-optimal physicochemical properties, raw starch functionality does not meet industrial processing and product demands and therefore expensive and polluting post-harvest chemical modifications, e.g. phosphorylation producing so called “starch phosphate” to inhibit retrogradation, are frequently requiredin vivo covalent modification of starch. In the early 20th century, the presence of small amounts (0.2–0.4% w/w) of monoesterified phosphate groups was detected in potato (Solanum tuberosum) tuber starch3. Subsequently, it has been verified that phosphorylation takes place in virtually all plant species5. In potato tuber starch, a majority of the phosphate monoesters (70–80%) are bound at the C-6 position of the glucosyl unit, while C-3 phosphorylation makes up 20–30%6. Two types of glucan water dikinaseshave been demonstrated to catalyse starch phosphorylation: α-Glucan, water dikinase 1  and α-Glucan, water dikinase 3/phosphoglucan, water dikinase (PWD/GWD3). GWD1 specifically phosphorylates starch at the C-6 position, catalyzing the transfer of the β-phosphate from ATP to a glucosyl residue7 and then PWD recognizes the C-6 pre-phosphorylated glucan and subsequently phosphorylates C-3 hydroxyls9. Phosphorylation by GWD1 disrupts the crystalline structure of the starch surface and increases the hydrolytic action of plastidial β-amylases11. RNA interference-mediated down-regulation of GWD1 in the wheat endosperm resulted in decreased starch-bound phosphate, an increase in grain size and plant biomass but unaltered starch content12. Starch phosphorylation stimulates starch degradation in Arabidopsis mutant plants in which the activity of GWD1 or PWD/GWD3 is decreased. As a result, these plants exhibit significantly increased leaf starch contents with the phenotype of the GWD1-deficient plants being more severe13. However, the GWD1 enzyme level remains largely constant throughout the diurnal cycle and also starch biosynthesis seems to be affected by starch phosphorylation as demonstrated by both metabolic and structural effects in Arabidopsis14 and barley15. Expression of the potato GWD1 in barley also affects grain germination in barley16. In support for a role of starch phosphorylation in both biosynthesis and degradation, GWD1 catalyzed phosphorylation of crystalline starch induces physical repulsion between starch segments5 thereby solubilising crystalline structures17 providing accessibility for biosynthetic or hydrolytic enzymes. Hence, GWD1 and PWD/GWD3 play complex and dependent key roles in starch degradation and biosynthesis pathways.Phosphorylation is the only known 20. Hence, phopshorylated starch can improve starch functionality and can be possibility produced directly in the cereal grain, which shows tremendous potential economic and environmental advantage2. Engineering the expression of GWD1 or PWD/GWD3 is the obvious strategy to alter starch phosphate in the crop. The manipulation of GWD1 activity in the crop has been described in some patents including the overexpression in wheat21 and corn22 leading to increased viscosity of the starch paste. Overexpression of the potato GWD1 gene specifically in the developing barley endosperm resulted in grain starch with ten-fold increased phosphate content, and starch granules showing altered morphology and lower melting enthalpy23.From a technological point of view, mono-phosphorylated starch has increased hydration capacity, thereby influencing starch pasting properties, gel strength and clarity, stickiness and viscosity24. Rice endosperm starch contains low concentrations of starch bound phosphate esters (≈1 nmol/mg)8, which limits its usage in various industrial processes. Whether introduction of potato GWD1 gene into rice could produce starch with higher bound phosphate esters needs to be addressed, which may introduce starch phosphate into the smallest granule found among cereals. The objective of this study is to express the potato GWD1 gene in rice  endosperm amyloplasts, and to investigate the morphological and physicochemical properties of the resulting modified rice starch.Rice is a crucial cereal crop in developing countries and the milled grains are composed of approximately 80–90% starchStGWD1) coding for the potato starch phosphorylator GWD1. Stable transgenic rice lines were generated by Agrobacterium-mediated transformation using pUCED-Hord:TP-StGWD:NOS vector  and StGWD1 as selection markers , pasting temperature (PT), breakdown (BD) and setback (SB) were significantly lower for transgenic lines as compared to control Table . The Glc27, while amylose content has been reported to be positively related to gel hardness29, and the interplay between phosphate and amylose in paste and gel systems can be complex30. For the transgenic rice lines, the higher amylose content can explain the increased hardness of GWD1 and GWD2 flour.The hardness of gels prepared from rice flour of RGD3-6 was significantly higher than that of the control. However, gels prepared from RGD1 and RGW2 flour had lower gel hardness than control  , fb2 (DP 25-36) and fb3 (DP ≥ 37) populations32. The profiles of the control and transgenic lines were rather similar but specific changes could be detected. Generally, the average chain length of amylopectin from transgenic lines was higher, which is in agreement with the notion that phosphorylated chains in starch granule are typically longer, compared to non-phosphorylated amylopectin chains4. Compared to control, the short main peak DPs of transgenic lines were somewhat decreased (from DP15 to DP14) and the content of longer chains were increased in lines RGD1, RGD2 and RGD5. Hence, we found fewer chains in the fb1 (DP 13-24) population and more chains in the fb3 (DP ≥ 37) population as compared to control. Correlation analysis demonstrated that the Glc-6-P content was negatively correlated with the proportion of DP 13-14 (r = −0.863), but positively correlated with the proportion of DP 25-36 (r = 0.941)  endosperm, which lead to about 9-fold and double increase in Glc-6-P and Glc-3-P, respectively, consisting with the results of Carciofi et al.23. The degree of substitution for industrial, chemically modified monostarch phosphate is less than 0.5%  corresponding to approximately 50 nmol/mg. This level is in the same range as for highly phosphorylated potato starch5. For the chemically modified rice starch, the phosphate content was increased by 3-fold or 4-fold, which displayed significantly increased food freeze-thaw stability38. Although the phosphate content in the transgenic rice was not as high as highly phosphorylated potato, we suppose that the content of starch phosphate in these lines is in a concentration range sufficient for application where stabilized starch is required.Potato glucan water dikinase 1 (StGWD1) is a 155 kDa protein, and is known to be involved in starch metabolism by adding phosphate groups to amylopectin40. The same models show that a phosphate ester at the C-3 position imparts molecular strain in the double helical motif thereby preventing optimal crystalline packing40. Following studies indicated that starch phosphate esters can stimulate hydrolytic enzyme activity in vitro and have a stabilizing effect on the β-amylase-glucan ligand complex, supporting that starch phosphate esters can direct amylases to phosphorylated spots by GWD1 and PWD/GWD3 catalyzed on starch granules41. Hence, the presence of pores in starches with GWD1 over-expressed rice lines indicates that the presence of GWD1 seems to stimulate starch degradation in rice endosperm.The starch granules of transgenic lines showed only minor morphologically differences including the presence of surface pores and slightly distorted edges and irregular surfaces found in some granules. Molecular force-field models indicate that a phosphate ester can be linked at the C-6 position to amylopectin without disturbing its double helical structure17. Phosphorylation also influences the physicochemical properties of starch, i.e. decreases the melting enthalpy for starch as deduced from starch extracted from tubers of GWD1 antisense suppressed potato lines31. DSC data for the transgenic rice starch showed that the dissolution temperature parameters To, Tp and Tc and dissolution enthalpy (ΔH) of hyper-phosphorylated starch in GWD1 over-expression lines were significantly lower than control. Amylose is known to suppress ΔH31, so the suppression of ΔH can be a combined effect of increased amylose and starch phosphate. The low dissolution temperatures found can be an effect of phosphate-induced disturbance of the crystalline lattice of the granules even though this parameter was not affected in the potato system31. In the barley system To, Tp, Tc are not different for hyper-phosphorylated starch as compared to control but ΔH is decreased despite the constant amylose content23. Our correlation analysis for the rice system demonstrated that the degree of phosphorylation in GWD1 over-expressed rice lines was negative correlated with ΔH, but not at significant level. For potato tuber starch, a strong negative correlation has been found between melting enthalpy of the lintnerised starch and the phosphate content42. Hence, the effects of starch phosphate monoesters are apparently complex not the same for different starch granule systems. Factors including the amount and distribution of amylose, protein and lipids are also important.GWD1 efficiently catalyzes the phosphorylation of crystalline maltodextrins and thereby induces the solubilization of both the neutral and the phosphorylated glucans in these crystalline aggregatesP < 0.05, Supplementary Table 23, where amylose was not affected demonstrating different metabolic routes in these two plants. For the potato tuber system, decreased starch phosphate content, due to RNA interference suppression of the GWD1, resulted in increased amylose content19. Hence, the different effects on amylose content in the phosphorylation-modified rice, barley and potato models are not consistent and needs further investigation, e.g. quantification of the amylose synthesising enzyme such as granule bound starch synthase. Pasting profiles using RVA can reveal effects of specific molecular structures, including phosphate monoesters and amylose, on starch granule swelling and disintegration of starch granules during heating and shear43. Especially the degree of phosphorylation is known to enhance the pasting capability of starch19. It is reported that the pasting viscosity parameters are highly correlated with the AAC for rice starches44. Our data showed that the AAC was negatively correlated with PV and CPV, while both G-6-P and G-3-P had significant negative correlation with most RVA parameters, notably PV and BD, but positively affected the SB and the gel hardness  found in the rice system is supposedly directed mainly by the high amylose content and less by the increased phosphate.The AAC was higher in all the transgenic lines as compared to control Fig.  and was 46. If phosphorylated starch modification carried out directly in the crop by transgenic biotechnology, modern breeding or mutagenesis, starch functionality can be improved with little or no requirement for post-harvest processing, which has tremendous potential economic and environmental advantages2. We here produced hyper-phosphorylated starch in rice by endosperm-specific transgenic overexpression of potato StGWD1 resulting in an increase of starch phosphate content and the improvement of starch functionality. Our data demonstrate a way forward to employ a starch bioengineering approach for clean modification of starch.Phosphorylated starches are of tremendous value for technical applications, and this type of modification yields a significant hydration capacity producing clear and highly viscous starch pastesStGWD1 in rice . The transgenic lines had 9-fold and double higher Glc-6-P and Glc-3-P, respectively and increased amylose content. The starch granules displayed only minor morphological alterations, notably the presence of surface pores and moderately distorted edges and surfaces. The novel starch introduces unique combinations of functionality for rice starch, such as reduced gelatinization temperature, decreased pasting viscosity, increased gel formation capacity and increased gel hardness.Rice starch contains only low concentrations of starch bound phosphate monoesters, which limits its usage in various industrial processes. We produced six stable individual transgenic lines with hyper-phosphorylated starch by the overexpression of the japonica variety “Zhonghua11” (O. sativa L.) was used as the parent cultivar. The plants were grown in the greenhouse at the Zhejiang University, Huajiachi Campus, Hangzhou, China. The developing seeds were harvested 15 days after flowering (DAF), immediately frozen in liquid nitrogen and stored at −80 °C. Mature seeds were harvested 40 DAF for extraction of starch and analysis of physicochemical properties.Rice D-Hord:TP-StGWD:NOS was the same as that for the transformation of barley  methodTotal RNA was extracted using UNIQ-10 Column Total RNA Purification Kit (Sangon Biotech) and then reverse transcribed to cDNA using Frist-Strand Synthesis of cDNA kit (Promega) according to the manufacturer’s instructions. The specific primers used in the subsequent RT-PCR are listed in Supplementary Table 50, their concentration measured using NANODROP 2000 spectrophotometer  and sodiumdodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE)51. Separated proteins were blotted onto polyvinylidene fluoride fluoride (PVDF) membranes using a transblotter and GWD1 was detected using western blotting according to Crofts et al.52 using rabbit polyclonal antiserum directed against StGWD1 as probe20.Soluble proteins were extracted according to Chen and Bao28. Rice flour preparation was according to Bao et al.28 and starch was extracted according to Kong et al.26.The mature rice grains were sun-dried until the seed moisture was around 12%, and stored at room temperature for three monthsThe starch samples were attached to specimen stub and sputter coated with gold using Eiko IB5 before viewing with scanning electron microscope (TM-1000).53. AAC was measured using the iodine staining method28, calculated using a standard curve made from 4 rice samples with known amylose content.G-6-P and G-3-P contents were quantified using high-performanceanion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD)et al.28.The paste property of rice flour was determined byusing a Rapid Visco Analyzer . According to the method of AACC61-02, 3.0 g of rice flour was placed an aluminum canister, and 25.0 g ofdistilled water was added. The heating profile was: (1) constant 50 °C for 1 min; (2) linear ramp to 95 °C until 4.8 min; (3) constant 95 °C until 7.3 min; (4) linear ramp down to 50 °C at 11.1 min and (5) constant 50 °C until 12.5 min. Three primary parameters were calculated from the pasting curves: peak viscosity (PV), hot paste viscosity (HPV), and cool paste viscosity (CPV). Three secondary parameters were calculated from primary parameters: breakdown viscosity (BD = PV − HPV), setback viscosity (SB = CPV − PV) and consistency viscosity (CS = CPV − HPV). The viscosity parameters were measured in Rapid Visco Units (RVU). Pasting temperature (PT) was determined according to the method proposed by Bao The sample cans containing rice starch gels formed in RVA analysis were sealed by Parafilm and kept at 4 °C for 24 h. Textural properties were determined by a TA-XT2i Texture Analyzer  equipped with Texture Expert software (version 1.2) in a two-cycle programme. Hardness, adhesiveness, and cohesiveness were calculated using the Texture Expert software program (Version 5.16).o), Peak (Tp), Conclusion temperatures (Tc) and enthalpy (ΔHg) of gelatinization were calculated by a Universal Analysis Program, version 4.4 A .Thermal properties were monitored using a Differential Scanning Calorimeter model Q20 , and the parameters of Onset  and Tukey’s test to determine significant differences by SAS statistical software . Correlation analyses among the different parameters were calculated by the Proc Corr procedure.Supplementary Table 1 to 3, Fig. 1 & 2


I am pleased to write this guest editorial on the occasion of the tenth anniversary of the Journal of Applied Clinical Medical Physics and I wish to thank the present Editor‐in‐Chief, George Starkschall, for the invitation to do so.When we started ten years ago the idea of an online journal was quite new. Systems for handling an entirely electronic journal including submission, review, editing, and advertising had not been fully developed. Most journals that were online at that time were primarily print journals that were posted on the Internet. JACMP being a new journal had no print version. Our early partner was the American Institute of Physics Publishing who was, at that time, developing a system for online publishing. Their help and expertise were vital to the success of the journal and they went beyond the terms of their contract to help get the journal started. When the American College of Medical Physics (ACMP) asked me to undertake this task and to become the Editor‐in‐Chief, I knew nothing about electronic publishing and had more to learn than anyone else. But AIP Publishing proved a good mentor and Volume 1 Issue 1 went online just one‐and‐a‐half years after the College gave the initial go‐ahead.Several criteria for the journal had been established by the ACMP: it was to be entirely electronic, there would be no print version; it was to be a free journal supported by advertisers; it was to be a journal devoted to the clinical applications of medical physics; its targeted readers and contributors were medical physicists worldwide; it aimed to have a short turnaround time from submission to publication – although this, of course, would depend upon how quickly referees submitted their reports and authors returned their revised manuscripts, and how user‐friendly the system was. That most of these aims have been met is a tribute to the Editors‐in‐Chief, the editorial boards, the advertisers, the referees, the authors and publishing partners who have served the journal over the last ten years. Vital to those initial four years was the manuscript manager and I am indebted to Georgeanne Moore who learned the electronic publishing business with me and then handled all the manuscripts while I was Editor‐in‐Chief. I was also fortunate to have an excellent editorial board that put in a lot of time and deserve much of the credit in getting the journal started.The journal would not have been possible without the support and encouragement of the members of the American College of Medical Physics and, in particular, the vision of Michael Mills who campaigned early for a journal and who served as the third Editor‐in‐Chief. Getting the journal started was not inexpensive and the College made the commitment to underwrite the cost .As a reminder of those early years, I keep in my office printed copies of the first three volumes of the journal. In Volume 1 we averaged slightly less than five papers per issue. As I write this editorial, the last four issues have averaged nearly 15 papers  per issue. This represents a three‐fold increase in 10 years, and the trend continues upward. The journal is well and going strong.The mission statement of the journal was published in the first issue and states: “…to publish papers that will help clinical medical physicists perform their responsibilities more effectively and efficiently for the increased benefit of the patient.” And this statement still appears on every issue. A quick review of the papers published over these last ten years shows that it has lived up to this expectation. And it has truly become an international journal with papers being published from around the world (over 20 countries) and – it can be concluded – read worldwide.Each year our first and second year medical physics’ students are required to present a twenty‐minute summary of a published paper that they find interesting and believe would be of interest to their fellow students. Increasingly they are choosing articles published in JACMP.It appears that it is firmly established in the list of medical physics journals that should be read by those entering the field.Congratulations to the present Editor‐in‐Chief, my friend and colleague George Starkschall. I wish him and the journal continuing success in the coming years.Peter Almond, Ph.D


We report a case of spontaneous reattachment of an extensively dislocated graft after nDSAEK.Graft detachment is a complication of nonA 54-year-old male underwent penetrating keratoplasty (PKP) for keratoconus in his left eye in 2001. Following graft opacity due to rejection, a second PKP was implemented in May 2014. The graft was kept in good condition after the reoperation and yet, visual acuity (VA) declined due to cataract. PEA+IOL was then performed in May 2015. Because edema appeared in the graft 6 months after the PEA+IOL, nDSAEK was carried out in May 2016. Although the donor graft well attached immediately after the nDSAEK, the graft was almost completely dislocated 3 h later except a temporal part. Air was reinjected into the anterior chamber on the following day and the detachment was resolved. Despite of the treatment, about 1/5 of the graft remained detached and the detachment deteriorated to 3/4 of the graft 9 days later. Because the patient could not decide whether to undergo another operation immediately, we decided to follow him up first and found that the partially detached graft reattached spontaneously 1 month later during the follow-up. Although the cornea had a mild edema remaining in the superior temporal area, his BCVA improved to 1.0. Three months later, the graft remained in position and the cornea kept its transparency.Spontaneous reattachment was observed during the follow-up in a case that had shown a comparatively extensive graft dislocation after nDSAEK. Currently, Descemet stripping automated endothelial keratoplasty (DSAEK) is the most frequently performed surgical treatment for corneal stromal edema due to corneal endothelial dysfunction. In Japan, postoperative complications of cataract surgery or laser iridotomy are the major causes for corneal endothelial dysfunction along with a small number of cases of Fuchs dystrophy. After Kobayashi et al. reported non-Descemet’s stripping automated endothelial keratoplasty (nDSAEK) –3, nDSAEGraft dislocation is the most common postoperative complication either after DSAEK or nDSAEK . Graft dHere we report a case of spontaneous reattachment of dislocated graft after nDSAEK that had occurred during follow-up with good visual prognosis.The patient was a 54-year-old male without any systemic diseases. He underwent PKP in 2001 for keratoconus in his left eye, and received another PKP in May 2014 due to opacity caused by graft rejection. After the second PKP, the graft was in good condition but visual acuity declined because of progressed cataract. Subsequently, phacoemulcification with intraocular lens implantation (PEA + IOL) was carried out in May 2015. Six months later, graft edema appeared due to bullous keratopathy [Fig. Under local anesthesia, nDSAEK was performed in May 2016. We prepared a temporal scleral tunnel incision 5.0 mm wide and used a precut donor graft of 8.5 mm. After making four venting incisions, we performed inferior iridectomy with a vitreous cutter. The nDSAEK button was pulled into the AC using a double glide technique  with an No intraoperative complications were noted. However, the graft almost completely detached 3 h later except a temporal part. Air was reinjected on the following day and the attachment was improved although approximately 1/5 of the graft remained detached in the inferior nasal area. We determined that air reinjection would not further improve the condition and decided to follow him up, which was also the patient’s wish for not having any more operations immediately. Nine days after the nDSAEK, graft dislocation progressed further Fig. . The areUnexpectedly, spontaneous graft attachment was observed at 1 month after the nDSAEK and the corneal edema had almost subsided leaving only a mild edema in the superior temporal cornea. His vision improved subjectively and objectively, and the BCVA was 1.0 Fig. . Three mWe experienced a case of a large nDSAEK graft detachment that had spontaneously reattached 1 month after surgery. Our case demonstrated that a close observation without immediate additional operations could be an option for a large but partial graft detachment in the early postoperative period after nDSAEK.Fuchs corneal dystrophy is the leading cause for corneal endothelial dysfunction in Europe and North America and with Fuchs dystrophy, the recipient Descemet’s membrane and endothelium must be removed to eliminate the guttae that distort vision. In Japan, cataract surgery and argon laser iridotomy are the main factors for corneal endothelial dysfunction and there is no need to strip the Descemet’s membrane. Therefore, nDSAEK has been widely adopted for the treatment of corneal edema. Graft dislocation is a postoperative complication that occasionally occurs after DSAEK and nDSAEK. Reportedly, the decision to remove the Descemet’s membrane or not does not make a remarkable difference in the frequency of postoperative graft dislocation –3. When On the other hand, spontaneous graft reattachment has been reported. Hayes and colleagues reported 12 cases of graft dislocation after DSAEK that were followed up without resurgery . In theiIn the reported case, graft attachment did not appear to have any problem immediately after the nDSAEK, however, graft detachment started 3 h later. We considered 3 possible causes for the graft detachment: 1) The posterior surface of the cornea could have been flattened by the site of the corneal suture since the patient had previously received PKP. 2) The corneal graft of 8.5 mm in diameter might have been relatively too large. 3) Because the operation was performed under local anesthesia and the vitreous pressure was high, the air injection might have been insufficient and caused the fluid to remain between the host cornea and the donor graft.Likewise, graft detachment could occur in various cases. Although persistent graft detachment can cause poor visual outcome due to corneal fibrosis, the decision between follow-up and immediate reoperations should be made carefully on a case-by-case basis considering the additional burden on patient’s physical condition or possible complications such as iris damage, primary graft failure, and infections.In conclusion, our case has demonstrated that even in eyes with a large but partial graft detachment after nDSAEK, the graft detachment can be monitored up to a month for spontaneous reattachment when further intervention appears to be difficult. Although good visual outcomes were obtained in our case, further investigations on the mechanism of spontaneous reattachment, how often it occurs, and in what cases it is most likely to occur will be necessary.


Sino-Japanese Symposium on Coagulation, Fibrinolysis, and Platelet first held in Hanchow, China in 1990, and again in Taipei, Taiwan in 1992. Thanks to the persistence and hard work of the conference founders and the participation of colleagues from countries around the world, that small, oligo-country conference has evolved into a regional conference now held every two years in cities of the Asia-Pacific region. Since its launch in Taipei, Taiwan in 2000, the APSTH Congress has been, and is now recognized as a key event in our field of thrombosis and hemostasis research.It begins with the State of the Art 2016: Research and Review from the 9th Congress of the Asian-Pacific Society on Thrombosis and Hemostasis, the first-ever publication of our APSTH conference proceedings. Truly, another big milestone.Taiwan has the honor of hosting the congress for the second time in 16 years with a program designed to foster international exchange and friendship in the pursuit of the newest medical knowledge on thrombosis and hemostasis. The best proof of our persistence in this endeavor is now lying before you, Thrombosis Journal, Dr. Yukio Ozaki, and Rachael Sykes from the supplements team at BioMed Central, and the support of Toby Hung, Jeff Boice, and Shih-Yao Lin which were instrumental and invaluable in the preparation and publication of these proceedings. Thank you all.The contributions in this first edition of APSTH proceedings consist of a balanced mixture of basic and clinical research and review papers. It is our hope that this APSTH publication will provide insight and help to advance our understanding in the various fields of thrombosis and hemostasis. We are grateful to the contributing authors for their excellent work. We are indebted to the many international reviewers who rose to the challenge of publication time constraints to provide us their insights and opinions. We wish to acknowledge the guidance of the editorial office of We wish you happy reading.Supplement Editorial Committee, APSTH 2016Ming-Ching Shen, organizerShu-Wha Lin, co-editorChing-Ping Tseng, co-editorJiaan-Der Wang, co-editor


In addition, shear bond-strength (SBS) tests were conducted to determine the effect of 10-MDP concentration on the bonding effectiveness to zirconia. These SBS tests revealed a 10-MDP concentration-dependent SBS with a minimum of 1-ppb 10-MDP needed. 31P-NMR revealed that one P-OH non-deprotonated of the PO3H2 group from 10-MDP chemically bonded strongly to zirconia. 1H-31P HETCOR NMR indicated that the 10-MDP monomer can be adsorbed onto the zirconia particles by hydrogen bonding between the P=O and Zr-OH groups or via ionic interactions between partially positive Zr and deprotonated 10-MDP (P-O−). The combination of 1H NMR and 2D 1H-31P HETCOR NMR enabled to describe the different chemical states of the 10-MDP bonds with zirconia; they not only revealed ionic but also hydrogen bonding between 10-MDP and zirconia.Currently, the functional monomer 10-methacryloyloxy-decyl-dihydrogen-phosphate (10-MDP) was documented to chemically bond to zirconia ceramics. However, little research has been conducted to unravel the underlying mechanisms. This study aimed to assess the chemical interaction and to demonstrate the mechanisms of coordination between 10-MDP and zirconium oxide using  Zirconia ceramics possess several advantageous properties such as their pleasing esthetic color, high fracture toughness, and optimal biocompatibility. Recently, yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) ceramics are widely used for fixed dental prostheses and dental implants. Reliable bonding and perfect marginal integrity along the tooth-cement-zirconia interface are essential for the clinical durability of all-ceramic zirconia restorations7910et al.et al.17et al.et al.et al.et al.et al.To improve bond strength and durability of resin-based materials to zirconia, many surface treatments, such as silica tribochemical coating and silica organoprecursor deposition, were developed to facilitate chemical bonding, particularly by means of silane chemical interaction with the deposited silica81314151H and 31P magic angle spinning (MAS) nuclear magnetic resonance (NMR) and two-dimensional (2D) 1H → 31P heteronuclear correlation (HETCOR) NMR. Shear bond-strength (SBS) tests were conducted to determine the effect of 10-MDP concentration on the bonding effectiveness to zirconia. The hypothesis tested was that the chemical interaction of 10-MDP with zirconium oxide relies solely on ionic interaction of 10-MDP− with partially positive zirconium.There exists a definitive need to clearly understand the underlying mechanisms of chemical interaction on an atomic level. The primary chemical interaction of 10-MDP with zirconium oxide must be fully elucidated, so to design acidic functional monomers with improved bonding performance in light of the ultimate aim to prolong the clinical lifetime of zirconia restorations. The aim of this study was therefore to determine the type of chemical interactions that occur between 10-MDP and zirconium oxide using 1H MAS NMR spectra of ZrO2 and 10-MDP-coated zirconia (10-MDP_ZrO2). A broad resonance at 5.0 ppm was assigned to the Zr-OH groups and adsorbed water molecules from the surface hydrated layer around the zirconia particles2 specimens. The former sharp resonances were assigned to the methacryloyloxy groups of the 10-MDP monomer1H → 31P HETCOR NMR measurements of 10-MDP_ZrO2 (31P dimension showed characteristic 1H resonances assigned to P-OH bonds at 8–11 ppm2-O-P bonds of the methacryloyloxy groups at 4.0 ppm in δ(1H)− and partially positive Zr, respectively.In the MDP_ZrO2 , slices 1H → 31P HETCOR NMR spectrum of 10-MDP_ZrO2. Slices taken at 4.0, 6.8, and 11.0 ppm in the 1H dimension show a broad characteristic 31P resonance that should be assigned to the P atoms of the adsorbed 10-MDP. For comparison, the 31P NMR signal of the 10-MDP monomer in the acetone solution is also shown (31P) are associated with the monomer and dimer of 10-MDP, respectively31P feature can be deconvoluted into four peaks around −13.7, −7.7, −2.6, and 0.3 ppm in δ(31P). These peaks are derived from the 10-MDP dimer and 10-MDP monomer. The weak peak at 0.3 ppm (10.0%) can be assigned to physisorbed and hydrogen-bonded 10-MDP monomers that do not cause deprotonation of any P–OH groups, while the weak peak at −13.7 ppm (21.4%) was assigned to physisorbed 10-MDP dimers that did not cause deprotonation of any P–OH groups31P chemical shift implies that the 10-MDP monomer can interact with the surface hydrated layer of the zirconia particles, not only via hydrogen bonding but also via ionic interactions between P–O− anions and positive Zr4+ groups.so shown . The shaet al.et al.16This study investigated the mechanisms of chemical interaction between 10-MDP and zirconia in order to establish a surface-binding model of 10-MDP onto zirconia. The SBS data showed that higher concentrations of 10-MDP promote bond strength . This fi1H MAS NMR spectra of 10-MDP showed a predominant peak at 4.2 ppm. A slice taken at −2.8 ppm in the 1H dimension was related to P-OH at 11.0 ppm and CH2-O-P at 4.0 ppm. A slice taken at −7.1 ppm in the 1H dimension demonstrated detection of P-OH at 8–11 ppm, Zr-OH at 6.8 ppm, and CH2-O-P at 4.0 ppm. These findings indeed confirm the presence of phosphate groups originating from 10-MDP at the zirconia surface, even after washing with acetone, thereby indicating that the strong interaction of 10-MDP with zirconia resisted washing. Hydroxyl groups were found in the phosphate groups and at the zirconia surface; these hydroxyl groups interacted with each other. This is in agreement with results presented in a previous studyWe performed NMR analyses to gain a better understanding of the chemical state at the interface of 10-MDP with zirconia. 1H fast MAS NMR studies, the hydrogen bonding was found to have self-assembled monolayers. 1H and 31P MAS NMR were also useful to reveal the nature of the interaction of phosphoric acid with zirconia271H → 31P heteronuclear correlation (HETCOR) confirmed the spectral assignment, identifying heteronuclear through-space proximities with sufficient sensitivity for 31P detection at all the solid specimen surfaces. The 1H projection of the 2D 1H → 31P HETCOR NMR spectrum showed a small shoulder at 11.0 ppm due to the P-OH proton  (−7.7 ppm). The combination of the 1H NMR and 2D 1H → 31P HETCOR NMR spectroscopy analysis confirmed the different chemical states of 10-MDP bonding to zirconia. The current study revealed not only ionic bonding between 10-MDP and zirconia, but also hydrogen bonding. Therefore, the hypothesis of the study that the chemical interaction of 10-MDP relies solely on the ionic interaction of 10-MDP− with partially positive zirconium was rejected.It was reported that the pKahe right  indicateWe conclude from the results of this study that one P-OH group of the 10-MDP molecule interacts with zirconia, while the other OH group interacts with P=O of another neighboring 10-MDP molecule. In this study, when a large amount of 10-MDP was used, interaction among different 10-MDP molecules through intermolecular hydrogen bonding was observed; thus, the 10-MDP concentration should be optimized in future investigations.2O3 particles  to ensure that the cement bonded strongly to the sandblasted zirconia surface and would not fail during the SBS test. The sintered zirconia plates were solely polished with 15-μm diamond particles to minimize surface retention . A cement film thickness of about 25 μm was reached, as was measured using Feg-SEM . All specimens were stored in distilled water for 24 h at 37 °C.Sintered zirconia round bars  and sintered zirconia plates (10 × 10 × 3 mm) were prepared by Tosoh . The zirconia round bars were sandblasted  with Aletention  and shouThe specimens were mounted on a material-testing machine , and shear stress was applied at a cross-head speed of 0.5 mm/min. After SBS testing, all failed specimens were analyzed using a light microscope (40×)  to assess the fracture pattern, which was used to classify the samples as having failed ‘cohesively’, ‘adhesively’, or ‘mixed’ (involving both cohesive and adhesive failure regions). For statistical analysis, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used with α = 0.05.2/g; Sigma Aldrich, St. Louis, MO, USA) was mixed with a 50-ml solution of 25 wt% 10-MDP  in acetone for 20 min via ultrasonic agitation, prior to being left without agitation for 40 min. The 10-MDP coated particles were separated from the mixed solution by centrifugation followed by a thorough wash with acetone in order to remove excess unbound functional monomer. For comparison, zirconia powder without any treatment was also prepared.To analyze the chemical interaction of 10-MDP with zirconia using NMR, 2 g of zirconium (IV) oxide nanopowder stabilized with 3 mol% yttria  and recycle delays of 90 s. The signals of approximately 790 pulses were accumulated with NH4H2PO4 as an external reference (1.0 ppm vs. 0 ppm 85% H3PO4). 1H high-power decoupling was used during the 31P acquisition. 1H MAS-NMR spectra were taken at 499.8 MHz with a pulse length of 1.15 μs (pulse angle of π/4) and 120 s recycle delays; here, the signals from approximately four pulses were accumulated with adamantane (C10H16) as an external reference (1.91 ppm vs. 0 ppm TMS). For comparison, the 1H MAS NMR spectrum of zirconia powder without any treatment and the 31P NMR spectrum of the 10-MDP monomer in an acetone solution were also measured.The local structure around H and P in 10-MDP coated zirconia particles was examined by solid-state MAS NMR. The NMR experiments were carried out using an Agilent DD2 500 MHz NMR spectrometer . A zirconia rotor with a diameter of 3.2 mm was used for the 1H → 31P heteronuclear correlation (HETCOR) experiments were performed using cross-polarization with a contact time of 3 ms, a recycle delay of 5 s and 96 scans per t1 increments; 64 t1 slices were acquired, where the rotor spinning frequency was maintained at 15 kHz.Two-dimensional (2D) Study repeatability was guaranteed by having conducted each NMR analysis twice.How to cite this article: Nagaoka, N. et al. Chemical interaction mechanism of 10-MDP with zirconia. Sci. Rep.7, 45563; doi: 10.1038/srep45563 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Background. The color masking ability of a restoration plays a significant role in coveringa discolored substructure; however, this optical property of zirconia ceramics has not been clearly determined yet. The aim of this in vitro study was to evaluate the color masking ability of a zirconia ceramic on substrates with different values.Methods. Ten zirconia disk specimens,0.5 mm in thickness and 10 mm in diameter, were fabricated by a CAD/CAM system. Four substrates with different values were prepared, including: white (control), light grey, dark grey, and black. The disk specimens were placed over the substratesfor spectrophotometric measurements. A spectrophotometer measured the L*, a*, and b* color attributes of the specimens. Additionally, ΔE values were calculated to determine the color differences between each group and the control,and were then compared with the perceptional threshold of ΔE=2.6. Repeated-measures ANOVA, Bonferroni, and one-sample t-test were used to analyze data. All the tests were carried out at 0.05 level of significance.Results. The means and standard deviations of ΔE values for the three groups of light grey, dark grey and black were 9.94±2.11, 10.40±2.09, and 13.34±1.77 units, respectively.Significant differences were detected between the groups in the ΔE values (P<0.0001).The ΔE values in all the groups were more than the predetermined perceptional threshold(ΔE>2.6) (P<0.0001).Conclusion. Within the limitations of this study, it was concluded that the tested zirconia ceramic did not exhibit sufficient color masking ability to hide the grey and black substrates. The masking ability has been defined as the ability to hide a colored background.5Metal ceramic restorations have shown long-term success due to good mechanical properties.*, a*, and b* define lightness, red/green value, and yellow/blue value, respectively, which can be measured via spectrophotometry. The ΔE is calculated to determine color differences using this formula:A method to evaluate the masking ability of restorative materials is to measure a ΔE color difference in CIELab color system. In this color system the color attributes of L*ab=[(L*2-L*1)2+(a*2-a*1)2+(b*2-b*1)2]1/2ΔE5 This formula can detect even a small amount of color difference between natural dentition and restorations.10 An opaquematerial with ideal masking ability has a ΔE color difference close to zero when being placed over a black and white substrate.8 In this case the color of the material is not affected by the substrate’s color. As spectrophotometers and colorimeters can recognize even a small amount of color change which cannot be detected by human eyes, limits have been defined for the perceptional threshold and the acceptable clinical threshold based on the ΔE value.5 It has been considered that the acceptable clinical threshold is more than the perceptional threshold.11 If the ΔE color change is more than the threshold, a color match will be rejected. The perceptional threshold range is1‒5.5of ΔE in the literature.14which is the most commonly used formula forΔE.18Some investigations have assessed various factors affecting the final color of zirconia restorations, including substrates,22 cements,23 ceramic veneers and their thicknesses,26 glazing process,24and laboratory techniques.27Several studies have evaluated the optical properties of different ceramic materials.19concluded that the color of a substructure affected the color of posterior zirconia-based restorations on a metal post and core or a prefabricated post and acomposite resin core, ranging from 1.2 to 3.1 of ΔE. Choi & Razzoog20evaluated the masking ability of zirconia ceramics with and without porcelain veneer and concluded that the unveneered zirconia ceramic was rather capable of masking the different tested substrates. Oh & Kim22 concluded that abutment shade, ceramic thickness and coping type affected the color resulting from zirconia restorations.Pecho et al28 concluded that clinicians shouldpay attention tothe optical property differences betweenzirconiaceramics and human dentin to achieve optimal esthetics in restorative dentistry. Kurtulmus-Yilmaz and Ulusoy29showed that zirconia ceramics were less translucent than lithium disilicate glass ceramic, and had partial translucency with some translucency differences among the zirconia systems. Tuncel et al30reported that the translucency of zirconia was influenced by the coloring procedure and the grain size. The masking ability of a zirconia ceramic is related to its color coverage over underlying structures, including cement and dental substrates. Masking a dental substrate with cements may not be feasible because different shades do not exist for all the cements; in addition, availability of different cement shades allows minor esthetic corrections.21Therefore the color masking ability of a zirconia ceramic on different substrates may be essential to achieve proper clinical results. The L*attribute, which is related to the value or lightness of a ceramic or a substrate, is the most effective factor in the resultant color.20 Therefore, the aim of this in vitro study was to evaluate the color masking ability of a zirconia ceramic on the substrates with different values. The null hypothesis was that the examined zirconia ceramic would show sufficient color masking ability to hide the tested substrates.Suputtamongkol et alA sample of maximum 10 is needed in a repeated-measure design with 3 replicates and the effect size of 0.81 (variance within groups=4 and variance explained by the effect=2.6), with α=0.05 and β=0.1. A G power of 3.1.3 was implemented for sample size calculation.Therefore, 10 zirconia disk specimens were fabricated. The disk specimens were placed onto four substrates with different values, including: white (W), light grey (LG), dark grey (DG) and black (B). Spectrophotometric measurements were performed on the specimens.22All the zirconia disks were sintered at 1500°C for a 12-hour process in a sintering furnace . A micrometer  with an accuracy of 0.002 mm was employed to measure the thicknesses of the disks. The disks were adjusted to a thickness of 0.5±0.01 mm. An adjustment and polishing kit  was used to reduce the thicknesses according to the above-mentioned acceptable range. In case of lack of the acceptable thickness, the disk was excluded from the study. The zirconia disks were polished, cleaned in an ultrasonic bath  containing 98% ethanol for 15 minutes and finally dried.A CAD/CAMsystem  milled zirconia blocks  to prepare zirconia disks. The disks were 0.5 mm in thickness and 10 mm in diameter.22 White and black Teflon materials were milled to fabricate the white and black substrates according to the above-mentioned dimensions. In order to fabricate grey substrates, a putty silicone impression  was taken from the black substrate to prepare a mold. Half a gram and one gram of carbon graphite were separately added to the liquid of an auto-polymerized acrylic resin , to make the light and dark grey substrates, respectively.20 The liquid and powder of the acrylic resin were mixed and applied to the mold. After 5 minutes the polymerization of the acrylic resin was completed. The grey acrylic substrates were polished with 800-grit silicon carbide abrasive papers for 10 minutes. All the substrates were cleaned in the same ultrasonic bath containing 98% ethanol for 15 minutes Four cylindrical substrates with different values, including white (W), light grey (LG), dark grey (DG) and black (B), were prepared. The substrates were 10 mm in diameter and 10 mm in height. minutes . The CIE31 A putty silicone material  was adapted to the mouthpiece of the spectrophotometer to match the conditions of spectrophotometry for all the specimens and to prevent external light. The specimens were located at the center of this putty mold. Before each measurement the spectrophotometer was calibrated by the white and green calibration plates, respectively. The disks were placed on the substrates with a water drop in between to prevent refraction of light.5 Each disk specimen was placed on each of the four substrates and the color measurements were carried out. All the color measurements were conducted at the center of the specimens marked by a pen on the monitor screen of spectrophotometer, and the color attributes of L*, a*, and b* were recorded for each specimen. ΔE was calculated to determine the color differences of a disc on different substrates. To compare the three substrates of LG, DG and B with the substrate W, the ΔE values were measured in three situations, including W-LG, W-DG and W-B. The aforementioned formula was employed to calculate ΔE. The ΔE=2.6for perceptional threshold was hypothesized in this study.14A spectrophotometer  was employed for spectrometric measurements.*, a*, b* and ΔE in the four groups of W, LG, DG and B, repeated-measures ANOVA was employed. Pairwise comparisons of the groups were performed by Bonferroni test. STATA  was used to compare the ΔE values with the threshold of 2.6 using one-sample t-test. All the tests were carried out at 0.05 level of significance.A normal distribution of data was confirmed in all the groups by Kolmogorov-Smirnov test (P>0.05). SPSS 21  was used for the analysis of data. In order to compare the color attributes of L* values for the four groups of W, LG, DG, and B were 88.35±1.46, 78.46±1.71, 78.06±1.66, and 75.38±1.35units, respectively  was employed. The null hypothesis of µ≤2.6 was rejected for ΔEW-LG, ΔEW-DG and ΔEW-B (P<0.0001).The means and standard deviations of the ΔEectively   were more than the threshold of ΔE=2.6. This demonstrated that the color changes induced by the substrates were beyond the perceptional threshold. Tracing the L*, a*, b* and ΔE values . It was concluded that the color of the tested zirconia ceramic was affected by its substrate. A comparable consequence was gained from the present study, though the substrates and zirconia ceramics used were not similar.Oh & Kim20assessed the masking ability of a zirconia ceramic with and without porcelain veneer. The color differences induced by zirconia ceramic and porcelain veneer were compared with the substrates alone, and it was concluded that the unveneered zirconia ceramic was rather capable to mask the different tested substrates. However, in the present study the color differences induced by the substrates were compared with a white substrate and the results revealed that the tested zirconia ceramic could not hide the substrates. In other words, Choi &Razzoog20 calculated the ΔE between the substrate (as a control) and the zirconia ceramic with the substrate, while we calculated the ΔE between the zirconia ceramic with the white substrate (as a control) and the zirconia ceramic with the other substrates. The difference in the results might be attributed to the mentioned methodological approaches. It should be considered that incomplete masking ability of a ceramic on discolored background substructures might lead to unpleasant esthetic results. On the other hand, the highest color changes, reported by Choi & Razzoog,20 occurred in the L* and b* values. Similarly the highest amount of color differences were caused by the L* attribute in the current investigation.Choi & Razzoog29showed that zirconia-based all-ceramic systems had lower translucency than lithium disilicate-based glass ceramics, this lower level of translucency of zirconia is not sufficient to hide the substrate color according to the results of this study. Tuncel et al30showed that zirconia as a framework material had smaller grain size and more translucency compared to monolithic zirconia. In addition, colored zirconia framework material exhibited similar grain size and was less translucent than non-colored zirconia framework material.30 The results of the present study, which showed the effect of substrate color on the zirconia ceramic color, were consistent with those of a study by Tuncel et al,30 indicating the high translucency of non-colored zirconia framework.Although Kurtulmus-Yilmaz and UlusoyBased on the results of this study, the grey and black substrates can change the color of zirconia core beyond the perceptional threshold. Thus, zirconia-based restorations may be contraindicated on the black and grey discolored teeth, or the negative effects of these discolorations might be reduced by increasing the zirconia core thickness, applying sufficient porcelain veneers, and using suitable luting agents.32 The present study assessed the specific effects of three substrates with different values in this respect. In addition, the zirconia core veneering materials may affect the color of zirconia-based restorations, which were not evaluated in this study. Therefore consideration of this factor is suggestedin future studies. This research had some limitations such as testing a non-coloredzirconia ceramic without veneer. More investigations are suggested in this respect.Light transmission via a zirconia core structure can be predictable in zirconia-based restorations, and there forecements and dental substrates might affect the color of these restorations.Within the limitations of this study it was concluded that the tested zirconia ceramic did not exhibit sufficient color masking ability to hide the grey and black substrates.This paper has been entirely drawn up from a “M.D. Thesis” which was successfully completed by Dr. Mahdiye Javadi Sharif under the supervision of Dr. Farhad Tabatabaian.FT contributed to the concept and design of the work. The acquisition, analysis, and interpretation of data were accomplished by FT, MJS, and MN. FT,FM and MJSdrafted and revised it critically for intellectual content. All the authors read and approved the final manuscript.This project was supported and funded by the authors.The authors declare no competing interests with regards to the authorship and/or publication of this article.Not applicable.


To examine the role of pheromone signaling in vivo, we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro, no extracellular complementation occurred in vivo. This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo. In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum.Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency plasmid transfer  Enterococcus faecalis, an important nosocomial pathogen. Previous studies showing efficient conjugative plasmid transfer in the gastrointestinal (GI) tracts of experimental animals did not examine how the enterococcal sex pheromone response impacts the efficiency of transfer. Our study demonstrates for the first time pheromone-enhanced, high-frequency plasmid transfer of E. faecalis plasmid pCF10 in a mouse model in the absence of antibiotic or bacteriocin selection. Pheromone production by recipients dramatically increased plasmid transfer in germfree mice colonized initially with recipients, followed by donors. The presence of a coresident community of common gut microbes did not significantly reduce in vivo plasmid transfer between enterococcal donors and recipients. In mice colonized with enterococcal recipients, we detected plasmid transfer in the intestinal tract within 5 h of addition of donors, before transconjugants could be cultured from feces. Surprisingly, pCF10 carriage provided a competitive fitness advantage unrelated to antibiotic resistance or bacteriocin production.Horizontal gene transfer is a major factor in the biology of  Enterococcus faecalis  in liquid medium in vitro. In donors carrying pCF10, a low level of induction (in the absence of recipients) can also be triggered by host factors in serum, via a mechanism involving differential sequestration of donor-produced iCF and CF peptides  tracts of hamsters and pigs following antibiotic-induced suppression of the normal gut microbiota has been observed  was not restored in the intestine by coresident CF-producing recipients (CF+ recipients), suggesting that natural pheromone signaling occurred in microniches where mating cells were colocalized. The data also suggest that pCF10 may contribute to competitive fitness of its host cell in the gut by an unknown mechanism.Our goal was to answer several basic questions related to plasmid transfer of pCF10 An overview and summary of the experimental setup and time line are depicted in 10.1128/mBio.00037-18.4TABLE S1 TABLE S1, DOCX file, 0.01 MB.Numbers of recipients, donors, and transconjugants in feces. Fecal samples were obtained from 12 mice in a preliminary experiment. The donor strain was OG1Sp(pCF10), and the recipient strain was OG1RF. Download Copyright © 2018 Hirt et al.2018Hirt et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00037-18.5TABLE S2 TABLE S2, DOCX file, 0.01 MB.T/D ratios in two preliminary experiments with wild-type (CF+ [C+]) and cCF10-deficient mutant (CF− [C−]) as the established recipient in the germfree mouse model. In the CF− experiment, the mice are inoculated with the C+ recipient on day 4. The T/D ratios are averages ± standard deviations for four mice. The T/D ratios in the upper, middle, and lower sections of the intestine and in feces are shown. Download Copyright © 2018 Hirt et al.2018Hirt et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the −2 to 10−1 5 h after donor inoculation. These ratios remained at that level in all sections of the intestinal tract throughout the experiment, suggesting that even at the low densities found at the earliest time point, the donor populations had good access to recipients. A significantly higher T/D ratio was observed on day 4 in the upper intestinal tract , whereas the difference in the T/D ratio for the middle and lower sections of the intestinal tract was not significant.Experiment 1 used wild-type recipient and wild-type donors. Recipients, donors, and transconjugants established at the highest cell density in the lower intestinal tract, whereas the upper intestinal tract supported the lowest bacterial populations as expected  to C. Tr3 to 104 to one 5 h after donor inoculation. This ratio shifted down by the end of the experiment to approximately 80:1 recipient to donor  3 days before introduction of the donor  were inoculated with a defined community  with a peak T/D ratio (10−1) reached in the upper intestinal tract at day 4 and a slight decrease at day 7 . A comparison of T/D ratios from experiments with and without a competing community is shown in P = 0.0162), with no significant impact in the upper and middle intestinal sections . Modest interference of the competing flora with plasmid transfer efficiency was also observed in the feces (P = 0.02). A comparison of the overall cell numbers of recipients, donors, and transconjugants in both experiments shows that the total enterococcal population densities were barely affected (F. prausnitzii and R. faecis) did not establish in our system. At the end of the experiment, A. muciniphila comprised 30% of the total population, while B. ovatus, B. vulgatus, and E. faecalis each comprised about a fifth of the total population. C. sporogenes was present at approximately 3%. Over the course of the experiment, the number of C. sporogenes showed the least variation, suggesting a well-defined niche , acquired pCF10 at <1/10,000 the wild-type level. Transfer into the CF− recipient strain was substantially rescued during in vitro matings containing mixtures of CF+ and CF− recipients if the CF+ strain comprised at least 10% of the total recipient population. A partial rescue of the CF− phenotype was also obtained by the addition of 10% human serum to the mating mixture, increasing the T/D ratio to 10−4 . In two mice sacrificed 5 h after the addition of donors, the T/D ratio was 10−3 . Although the numbers of transconjugants increased over time as in experiment 1, the T/D ratio remained in the range of 10−5 to 10−4. Levels of plasmid transfer into the CF− recipient were substantially lower at all time points; for example, both total transconjugants and transconjugants/donor values were reduced by 2 to 3 orders of magnitude on day 4, which showed the highest overall frequencies with either recipient  in the total enterococcal population increased during the experiments reported here and in preliminary experiments (data not shown). This suggests that pCF10 provides one or more factors that confer a selective advantage to plasmid-carrying cells in the absence of antibiotic or bacteriocin selection. A candidate for this selective advantage is aggregation substance (AS), encoded by the t strain . The selt strain  providedt strain , 16. Thest cells , 18 and st cells  with subst cells , includiE. faecalis that has not been addressed is the role of the conjugation-inducing pheromones such as cCF10 in vivo. Licht et al. showed that cCF10 had biological activity in intestinal mucus in vitro , the results from experiment 3 demonstrated the requirement of cCF10 for high-frequency plasmid transfer in vivo. However, plasmid transfer to the CF− recipient still occurred with a T/D ratio of 10−4, which was considerably higher than that observed in vitro with the same donor and recipient  Fold increase/decrease of recipients (R) and donors (D) from day 1 to day 4. (B) Fold increase/decrease of transconjugants (TC) and the transconjugant/donor (T/D) ratio from day 1 to day 4. Download FIG S3, TIF file, 0.4 MB.Rate of increase of Copyright © 2018 Hirt et al.2018Hirt et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. faecalis cell numbers and plasmid transfer rate, except in the lower intestinal tract. The failure to complement the pheromone-negative strain with a wild-type recipient suggests the need for close contact between recipient and donor in vivo. These studies represent the initial investigation into how plasmid carriage and cell-cell signaling may impact the ecology of E. faecalis in the GI tract. The ability to introduce a variety of genetic constructs into this well-defined model and to vary the overall complexity of the community will ultimately enhance our understanding of important questions related to how antibiotics and other inducers of GI tract dysbiosis impact the transition of the enterococci from harmless commensal organisms to lethal nosocomial pathogens.In summary, we have shown that pCF10 plasmid transfer occurs within 5 h of donor introduction in a germfree mouse model in the intestinal tract. Early sampling and results from the day 4 time points suggest that the upper intestinal tract provides the most favorable environment for plasmid transfer. The introduction of a simple multispecies community did not substantially impact In vivo transfer experiments employed either a CF+ wild-type recipient OG1RF  . Stocks of E. faecalis were made and frozen at −80°C; isolates were grown from stocks before each experiment. A defined community of Bacteroides ovatus, Bacteroides vulgatus, Clostridium sporogenes, Akkermansia muciniphila ATCC BAA-835, Faecalibacterum prausnitzii ATCC 27755, and Roseburia faecis DSMZ 16840 was used as competing GI microflora. For these bacteria, stock solutions were made of each strain in prereduced medium inside an anaerobic chamber and frozen at −80°C.nt OG1RF  and OG1Ent OG1RF  or an isCF+ and CF− recipients were grown and enumerated on tryptic soy agar (TSA) plates with 50 µg/ml rifampin, OG1ES on TSA with 10 µg/ml erythromycin, and OG1Sp(pCF10) on TSA with 630 µg/ml spectinomycin plus 10 µg/ml tetracycline. Transconjugants were selected with 50 µg/ml rifampin plus 10 µg/ml tetracycline for OG1RF(pCF10) and 10 µg/ml erythromycin plus 10 µg/ml tetracycline for OG1ES(pCF10).All animal experiments were approved by the institutional IACUCs at both the Mayo Clinic, Rochester, MN , 26, and7 CFU of wild-type recipient OG1ES/mouse on day −3 and 2.9 × 107 CFU of mutant CF− recipient (OG1RF background)/mouse on day 4 (7 CFU of mutant CF− recipient (OG1RF background)/mouse on day −3 and 1.0 × 107 CFU of wild-type recipient OG1ES/mouse on day 4  and at 1 (n = 4), 4 (n = 4), and 7 (n = 4) days by selective plate quantitative culture. The upper GI section includes the duodenum plus the proximal jejunum, the middle GI section includes the distal jejunum and the ileum, and the lower GI section consists of the colon. Fecal samples were collected from individual mice and placed in 1 ml sterile saline, homogenized, quantitatively cultured, and reported as log10 CFU/milliliter. The GI tissue sections were aseptically dissected, and the fecal contents were removed, sliced open longitudinally, weighed, and placed into 1 ml sterile saline. They were vortexed for 30 s, sonicated for 5 min , and vortexed an additional 30 s. The sonicate fluid was quantitatively cultured, and log10 CFU/gram of tissue was calculated. The frequency of plasmid transfer is reported as transconjugant/donor (T/D) ratio.Fourteen mice were used for each of three experiments; donors, recipients, and transconjugants were enumerated from fecal samples on days 1, 4, and 7 and from GI tissue  at 5 h . Plasmid transfer was assessed after 4 h coculture of donor and recipients by enumeration of donors and transconjugants by plating on selective plates as described above.The defined microbial community used was characterized by partial 16S rRNA gene sequencing of the communities found on days 1, 4, and 7 in mouse feces and also in an aliquot of the original gavage input. Fecal DNA was isolated by previously described methods , 31. LibAnalysis was performed by the in-program suite of the Prism7 software package .


Microscale bacterial distribution would provide clues to understand bacterial lifestyle and nutrient utilization. However, the detailed bacterial localization around dietary factors in the intestine remains uninvestigated. Therefore, we explored microscale habitats in the murine intestine by using histology and fluorescent in situ hybridization, focusing on dietary factors. This approach successfully revealed several types of bacterial colonization. In particular, bifidobacterial colonization and adhesion on granular starch was frequently and commonly observed in the jejunum and distal colon. To identify the bacterial composition of areas around starch granules and areas without starch, laser microdissection and next-generation sequencing-based 16S rRNA microbial profiling was performed. It was found that Bifidobacteriaceae were significantly enriched by 4.7 fold in peri-starch areas compared to ex-starch areas. This family solely consisted of Bifidobacterium pseudolongum. In contrast, there was no significant enrichment among the other major families. This murine intestinal B. pseudolongum had starch-degrading activity, confirmed by isolation from the mouse feces and in vitro analysis. Collectively, our results demonstrate the significance of starch granules as a major habitat and potential nutritional niche for murine intestinal B. pseudolongum. Moreover, our results suggest that colonizing bifidobacteria effectively utilize starch from the closest location and maintain the location. This may be a bacterial strategy to monopolize solid dietary nutrients. We believe that our analytical approach could possibly be applied to other nutritional factors, and can be a powerful tool to investigate in vivo relationships between bacteria and environmental factors in the intestine.Each species of intestinal bacteria requires a nutritional source to maintain its population in the intestine. Dietary factors are considered to be major nutrients; however, evidence directly explaining the  The gut microbiota are known to modulate host health and diseases including metabolic syndrome, inflammatory bowel disease, and cancer , and modin vitro studies. However, studies have shown that nutrients identified in vitro sometimes fail to expand the target organisms or also expand non-targeted organisms when fed to humans or test animals [Indigenous bacteria are known to successfully maintain their numbers in the intestinal microbiota; hence, they are presumed to have established an ecological or nutritional niche that enables them to thrive. Information regarding what nutrients are directly utilized by which bacteria has accumulated through  animals . Thus, iTo elucidate the niche of organisms, it is important to understand their spatial distribution, which may reflect their required environmental factors. In particular, detailed information on bacterial lifestyle will be provided by analyses with an appropriate spatial resolution that corresponds to the activities of target organisms. In intestinal microbiology, several groups have analyzed and reported microscale spatial organizations of microbiota –7, and aAll animal experiments were performed according to the guidelines of the Yakult Central Institute and were approved by the Animal Experimental Committee of Yakult Central Institute.C57BL/6J Jcl mice (6 weeks old) were purchased from CLEA Japan  and maintained in a specific pathogen-free facility. All mice were fed CE-2 chow  by the breeder before purchase, and then fed MF chow  and water ad libitum after purchase. The mice were anesthetized with sodium pentobarbital and sacrificed after 5 weeks, on Day 35 or 36 (the day of purchase was considered to be Day 0), at 11 weeks of age. Animals were monitored daily and weighed twice a week. No individual animal became ill or died.Two batches of animals were used in this study. Batch #1 with 4 mice were subjected to microscopic observation and laser microdissection (LMD), and batch #2 with 6 mice was used for bacterial culture and fecal microbiota analysis. Batch #2 was also subjected to microscopic observation and LMD, except for two mice which were used for preparatory experiment for LMD. Consequently, the sections of 8 mice were subjected to LMD analyses, and sections from 6 mice were successfully analyzed (described later). According to the manufacturer, major starch sources of MF chow are corn and wheat (mainly bran), and standard composition (w/w) of MF chow is as follows; 7.9% moisture, 23.1% crude protein, 5.1% crude fat, 5.8% crude ash, 2.8% crude fiber, 55.3% Nitrogen-free extract.The whole gastrointestinal tract with digesta was fixed using methacarn solution (methanol/chloroform/acetic acid = 6:3:1) for 15–60 min or modified Carnoy’s solution (ethanol/chloroform/acetic acid = 6:1:6) [FISH was performed as previously described . The FISFor starch staining, after the FISH washing step, a drop of Lugol's solution  was placed onto sections. After 30–60 s, the slide was washed briefly with water and dried immediately. Purple-stained starch granules were confirmed before and after mounting. Once mounted, specimens were observed within minutes, before Lugol's staining disappeared. Solutions for Alcian blue-PAS staining were purchased from Muto Pure Chemicals. For co-staining, FISH was performed after Alcian blue-PAS staining.Fluorescence microscopy images were captured by assigning pseudocolors to acquired monochrome images at each wavelength.4 μm2 (approximately 25–50 regions from 2–3 sections) for each peri-starch sample, 1.0–2.0 x 105 μm2 for each ex-starch sample.A part of the distal colon with digesta was subjected to LMD using a PALM MicroBeam IV system . Colon sections of 8 mice were first prepared, and sections of single mouse was omitted, since they had no intestinal content. Ten-micrometer paraffin sections were stained with Lugol’s solution and briefly washed with water. Because sections were observed without FISH staining, bacterial accumulation and taxonomy were indistinguishable during sample collection. Purple-stained starch granules and the surrounding regions (40-μm diameter) were excised from sections and collected into an AdhesiveCap 500 polymerase chain reaction (PCR) tube  as peri-starch samples. All of the observed starch granules in each section were collected, except for those present as chunks within intact plant tissue. We judged these granules as inadequate for analysis because FISH analysis confirmed that there were no or few bacteria in intact plant tissues that contained chunks of starch. In parallel, areas without starch granules were randomly selected and collected as ex-starch samples. Similar amount was collected for both groups; 3.5–6.0 x 10http://blast.ncbi.nlm.nih.gov/) using database of The All-Species Living Tree Project [DNA was extracted from LMD samples by using the DNeasy Blood & Tissue Kit  according to the manufacturer’s instructions. As previously described –14, the  Project  to deter Project , and at http://rdp.cme.msu.edu/) was used to obtain reference 16S rRNA gene sequences of type strains of Bifidobacterium. The sequences were aligned, and sequence motifs specific for B. pseudolongum subsp. globosum and B. pseudolongum subsp. pseudolongum were selected. The designed Bpl190 probe  soluble starch is contained in the product). Plates were flooded with 1:1-diluted Lugol's solution for approximately 30 s. Subsequently, Lugol's solution was removed and starch degradation was evaluated by formation of a clear zone around the colonies.Isolates were cultured on modified GAM agar and harvested. Genomic DNA was extracted as previously described . The 16STo explore the bacterial microscale localization, the intestinal contents of conventionally raised mice were paraffin-embedded, and sections were analyzed using several staining methods. In these preparations, the overall structure of the digesta was maintained. Alcian blue-periodic acid-Schiff (PAS) staining revealed components of the intestinal digesta and their distribution . Plant cFISH analysis of the intestinal digesta revealed several types of bacterial localization. For example, rod-shaped bacteria accumulated onto granular structures , and manTo identify the bacteria-enriched granules, several staining methods were applied. Alcian blue-PAS and FISH co-staining indicated that the granules were PAS-positive but Alcian blue-negative . This suThe chow used in this study contained maize and wheat, which both mainly consist of starch. Starch is a neutral carbohydrate, with a diameter of 5 to 20 μm . Some stClostridium cluster XIVa, XIVb and Clostridium leptum subgroup in the colon , but not with probes for other dominant bacteria; lactobacilli in the jejunum and he colon . Colonizhe colon  and fecehe colon .Erysipelotricaceae and Lactobacillaceae showed some difference between two sample sets. The difference is probably due to the different DNA extraction methods employed for each sample set. The results support overall adequacy of LMD-NGS analysis and therefore we considered comparison of bacterial population between two LMD samples will not be disturbed , consistent with the preceding FISH analysis. In contrast, populations of other major families  did not show significant enrichment around starch .To quantitatively and comprehensively characterize the local bacterial composition around the starch granules, LMD and next-generation sequencing (NGS)-based 16S rRNA microbial profiling was performed. First, starch granules were visualized by Lugol’s staining, and then areas surrounding starch (peri-starch areas) and areas without starch (ex-starch areas) were randomly sampled . Major bisturbed . The popd starch , and we Bifodobacteriaceae in both peri-starch and ex-starch areas were B. pseudolongum . Regarding the NGS data, we double-checked at the species-level that the starch-surrounding bacteria were indeed B. pseudolongum by using a FISH probe  analysis . Therefore, this hypothesis should be further examined in future studies. It will be useful to phenotypically identify and analyze localization of potential competitors which are sharing the same environment with bifidobacteria.In FISH analysis, only bifidobacteria were detected as starch-colonizing bacteria, and other major bacterial groups showed no association with starch. This bifidobacterial species selectivity was also demonstrated by comparing the microbial composition of peri-starch areas and other areas using LMD and NGS. Furthermore, the adhesion was intense and observed consistently in the jejunum, distal colon, and feces. This suggests that the adhesion is maintained from the upper to the lower intestinal tract with strong affinity. These multiple pieces of evidence raise the possibility that this adhesion is a bacterial strategy for exclusive monopolization of favorable nutrients; the colonizing bifidobacteria effectively utilize the nutrients from the closest location, and restrict utilization by other bacteria by occupying the surface of the nutrient. This hypothesis is substantiated by amylolytic activity of a Bifidobacterium [in vitro [in vivo, at least by adhesion, and imply a degradation process independent of other bacteria.This idea might partly explain the observation of a bifidobacterial response on starch intake in previous reports . Preacterium , 33. Mulin vitro , 34–36. B. pseudolongum, which is a common species in the intestine of animals including rodents [Bifidobacterium, Bacteroides, and other species including Eubacterium rectale and Ruminococcus bromii are major human colonic bacteria with starch-utilizing ability [E. rectale or R. bromii were not detected in this study. Among these starch-utilizing bacteria, adhesion to starch granules has been evaluated among major bifidobacterial species [Bacteroides thetaiotaomicron [E. rectale [R. bromii [Bacteroidaceae, a family that contains many amylolytic species, was slightly larger in ex-starch areas compared to peri-starch areas. Similarly, the fractions of Rikenellaceae and an unidentified clade belonging to Clostridiales were significantly larger in ex-starch areas. Unfortunately, we were not able to identify the species in these families and for most of other OTUs, because of the low sequence identity. This is due to the lack of registered sequences for murine bacterial species in current reference database. More information on biochemical characteristics of murine intestinal bacterial species is necessary for detailed and precise discussion on how certain bacteria were excluded from peri-starch area. In general, bacteriocin-like substances or antibacterial metabolites can serve as mediators of such exclusion. Wang et al. reported that several species or clades of human colonic bacteria, in particular Bacteroides and Fusobacterium, can degrade only soluble or gelatinized starch but not granular starch, while Bifidobacterium can also degrade granular starch [This study identified only one bifidobacterial species,  rodents . However rodents , 39. The ability –36, 40,  species . Adhesivaomicron , E. rect rectale , and R. . bromii , althougr starch .in vivo report that describes species-specific localization of intestinal bacteria onto a single dietary factor and the relationship between bacterial localization and utilizing ability. Our analytical approach could possibly be applied to nutritional factors other than starch, and could be a powerful tool to investigate in vivo relationships between bacteria and environmental factors in the intestine. Further studies on the local microbial community around single dietary factors will be important, as metabolites or degradation products may be concentrated near the sites of colonization and affect the local community. Additionally, it remains unknown how adhesion onto nutrients affects the colonizer’s phenotype and influences the host physiology. These issues should be addressed in the future.To our knowledge, this is the first S1 FigA cross section of murine jejunum was stained by (a) Periodic acid-Schiff (PAS) and Alcian blue, and subsequently (b) stained by FISH .(PDF)Click here for additional data file.S2 FigA paraffin-embedded cross section of feces collected on Day 8 was stained by Bif153 (green), Lab158 (red), DAPI (blue), and Lugol’s solution. Bif153-positive bifidobacteria colonizing starch granules in feces, similar to that seen in the intestine. (a) Bright-field microscopy. (b) Fluorescent microscopy.(PDF)Click here for additional data file.S3 Fig2, and subjected to DNA extraction and following analyses.  Before LMD.  After LMD.Colon content in peri-starch area  and ex-starch area  in sections were collected by LMD. Arrowhead in (a) indicates a starch granule stained purple by Lugol’s solution. The area surrounding starch granules (a) and the area in the rectangle in (c) were separately collected. Approximately 25–50 areas surrounding starch were collected to obtain at least 35,000 μm(PDF)Click here for additional data file.S4 FigFecal bacterial composition of six mice (#2–1 to #2–6) were examined from the beginning of feeding (day 0) until day 35, and shown at family level. Four of them (#2–1 to #2–4) were analyzed by LMD and results were shown in (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableValidation of Bpl190 probe and reference strains used in this study. ++; strong, +; positive, −; negative, w; weak.(PDF)Click here for additional data file.