Xindong 20) grain. However, confocal laser scanning microscopy with 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde and methanolic merbromin stains indicated that P amount influenced the microstructure of the starch granules. Starch granules from the 46 kg P ha−1 treatment released significantly more reducing sugars than those from the 0 and 92 kg P ha−1 treatments during digestion with alpha-amylase and amyloglucosidase digestion. Phosphorus application  significantly increased the relative expression of genes related to starch synthesis  and starch degradation . Phosphorus application also increased the transcript abundance of amylase genes at the periphery of the endosperm. We propose that P application, especially the 46 kg P ha−1 treatment, enhanced channels in wheat starch granules. These channels facilitated the transport of substances required for starch biosynthesis, thus increasing starch accumulation in wheat endosperm. These results provide insight into the potential mechanisms through which P influences the microstructure and biosynthesis of wheat starch.Phosphorus is an essential plant macronutrient which profoundly affects the yield and quality of wheat starch. In this study, scanning electron microscopy showed that P fertilizer amount (0, 46, and 92 kg P ha Phosphorus (P) is one of the main limiting factors for plant growth in natural ecosystems. The application of P fertilizer is often essential for crop production , adenosine diphosphate glucose pyrophosphorylase (AGPase), starch branching enzyme (SBE), starch debranching enzyme (DBE), and soluble starch synthase (SSS). The AGPase is activated by 3-phosphoglycerate and is inhibited by inorganic P in leaves. The activity of AGPase can be changed by altering the ratio of 3-phosphoglycerate and inorganic P, which in turn regulates starch synthesis  N (potassium chloride extraction), 15 mg kg−1 available P (Olsen), and 208 mg kg−1 available K (ammonium acetate extraction).The study was conducted at the Shihezi University Experimental Farm, Shihezi, China  from October 2014 to June 2015. The soil at the site is classified as gray desert soil by Chinese scientists and a −1 urea  was applied to the soil. The plots were drip irrigated at 10–12 d intervals three times before winter and six times after winter dormancy. Urea was applied via drip irrigation at elongation (45 kg ha−1), heading (75 kg ha−1), and flowering (120 kg ha−1).Seeds of the winter wheat cultivar “Xindong 20” were supplied by the Agriculture College of Shihezi University. On the day of sowing, 75 kg ha−1 ; 46 kg P ha−1 ; and 92 kg P ha−1 . The P fertilizer  was applied in a 10-cm-deep band between each ridge. The plots were 2.4 × 3 m. Each plot was separated by a 50-cm-wide bare strip.The experiment used a randomized block design with three replications. Three P treatments were applied to the plots 160 d after sowing, when about 5% plants of the plants had turned green after dormancy. The P treatments were as follows: 0 kg P hain situ hybridization. The remaining grains were dried at 70°C to constant weight and weighed. Starch granules were isolated from grain (not oven-dried) collected at 35 DPA.Grain samples were collected from the middle region of the wheat spikes at 7, 14, 21, 28, and 35 days post anthesis (DPA). The samples were collected from each plot and then pooled to form one sample per treatment. Three subsamples were removed from each composite sample. These subsamples were frozen in liquid N for 5 min and then stored at −80°C for RNA extraction. Another three subsamples were fixed in 4% paraformaldehyde (pH 7.2–7.6) and 0.1% DEPC for histochemical analysis and −1 Tris-HCl, pH = 6.8; 10 mmol L−1 EDTA; 4% w/v SDS), three times with deionized water, and finally three times with acetone. The starch samples were then air dried.Starch granules were isolated using a modified version of the method used by Peng et al. . The embThe starch granules were sprinkled onto double-sided conductive adhesive tape attached to aluminum stubs and then coated with gold-palladium (60:40) particles (20 nm particle size) using a sputter coater . The morphology of the starch granules was examined using a field emission scanning electron microscope  at an accelerating voltage of 5–10 kV.−1 NaOH) was added to the mixture before heating in a boiling water bath. Absorbance was measured at 540 nm using a spectrophotometer . A standard curve was prepared using malt sugar solutions with concentrations of 0, 0.1, 0.3, 0.5, 0.7, 0.9, and 1.0 mg mL−1.Total amylase activity was measured as described by Liu . BrieflyTo measure α-amylase activity, β-amylase in the crude enzyme extract was inactivated by heating at 70°C for 15 min. All of the other steps were the same as the assay of total amylase activity described above. Beta-amylase activity was calculated as the difference between total amylase and α-amylase activity.−1 sodium acetate (pH 7.5), 1 mmol L−1 calcium chloride, and 0.02% (w/v) sodium azide]. Next, Protease Type XIV  was added to the suspension before incubating at 4°C for 24 h on a shaking table. After centrifuging at 3,000 g for 20 min, Protease Type XIV-treated starch granules were washed with deionized water and ethanol and then air dried.The isolated starch granules (2 g) were suspended in 40 mL of proteolytic buffer [50 mmol LProtease (Type XIV)-treated starch granules were stained with a methanolic solution of merbromin and 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) according to Kim and Huber . MerbromAfter staining with merbromin and CBQCA, the starch samples were transferred to glass slides and then photographed using a Zeiss LSM 510 CLSM system . Excitation was achieved with an Argonlaser (488 nm) operating at 30% power. Emission was detected through an LP505 emission filter.−1 NaOH. The mixture was heated in a boiling water bath for 10 min. After centrifugation , 500 μl of the supernatant was diluted 200 times with distilled water, 1 mL of 1 mol L−1 acetic acid and 1 mL iodine reagents  were added, color development conditions were room temperature for 10 min, absorbance was measured at 620 nm with a spectrophotometer . A standard curve was prepared using soluble starch solutions at 0, 20, 40, 60, and 80% (w/v).Grain starch content was determined according to the method of Zhao , with th−1, pH 4.8) containing 360U α-amylase  or 50U amyloglucosidase . The samples were incubated on a shaking incubator . Next, 50 μL of 1 mol L−1 HCl was added to the samples. The reaction was stopped by adjusting the pH to 7 using 1 mol L−1 NaOH.Alpha-amylase and amyloglucosidase digestion was conducted according to Tang et al. . After i−1 NaOH, 18.5% w/v NaKC4H4O6·4H2O, 0.5% w/v phenol, 0.5% w/v NaHSO3) was added to 0.1 mL of supernatant. The mixture was heated in a boiling water bath for 5 min. The absorbance was measured at 540 nm with a spectrophotometer . A standard curve was prepared at using glucose at concentrations of 0, 100, 200, 300, 400, 500, and 600 μg mL−1.The extent of starch degradation was determined by measuring the concentrations of reducing sugar produced by starch hydrolysis. Reducing sugar concentrations were determined using a modification of the method described by Bernfield . After cAGP1, AGP2, SS1, SS2, SS3, SS4, GBSS1, GBSS2, SBE1, SBE2A, SBE2B, ISO1, AMY1, AMY2, AMY3, AMY4, BAM1, BAM2, BAM3, BAM4, BAM5, BAM6, and BAM7 were designed using Primer Premier 5.0 software according to sequences published in the National Center for Biotechnology Information (NCBI). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The reference control was wheat ACTIN gene. The specificity of the primers was tested and the PCR conditions were optimized using gradient PCR and agarose gel electrophoresis . The primer sequences are presented in Supplementary Table The primers of ACTIN gene.The RNA was extracted from de-embryonated wheat grain using RNAiso Plus  and Fruit-mate  kits according to the manufacturer's instructions. Total RNA quality was tested using agarose gel electrophoresis. First strand cDNA was synthesized using a reverse transcription kit . The cDNA quality was tested by amplifying the wheat The rt-qPCR reaction solution was prepared with a SYBR Premix Ex Taq Kit . The components are presented in Supplementary Table A mathematical model was used to determine the relative expression of target gene compared with the reference gene. The relative quantification was calculated with the following formulae:ACTIN gene was used as the reference. The gene is the favored reference for studying wheat genes because it is highly conserved in cell integrity, motility, and structure.The wheat AMY4, BAM1, and BAM5 gene fragments were amplified using PCR. The fragments were collected using an EasyPure Quick Gel Extraction Kit . The fragments were linked with pEASY-T3 cloning vectors, and then the vectors were transformed to pEASY-T1 competent cells using a pEASY-T3 cloning kit . After screening and culturing, the plasmids were isolated using a TIANprep Mini Plasmid Kit  and sequenced by Sangon Biotech (Shanghai) Co., Ltd. Based on the sequencing results, the plasmids were digested with a restriction enzyme, either Nco I  or Pst I . The linearized plasmids were used to synthesize antisense and sense probes via in vitro transcription. This was performed using a Dig RNA Labeling Kit .The AMY4 was as follows: 5′-UUGGUUUCCGAUGGUGUUGUCCAAGAACAGGCAGCUCGCAAUGGCGGGAUCAUUAAGAACGGGAGAGAAAUCCUAUUGCAGGCUUUUAAUUGGGAAUCCCAUAAACACAAUUGGUGGAGUAAUUUAGAGGGCAGAGUUGCCGACAUUGCUA-3′.The antisense probe sequence of BAM1 was as follows: 5′-ACUCAGGAAUGCAAGGCCUCAUGGCAUCAACAAGAGCGGCCCUCCUGAGCACAAGCUGUUUGGAUUCACCUACCUCCGGCUGUCGAAUCAGUUGGUGGAGGGACAAAACUAUGUCAAUUUCAAGACCUUUGUUGACAGAAUGCAUGCCAACCUGCCUCAUGACCCAU-3′.The antisense probe sequence of BAM5 was as follows: 5′-UGAACCGGAACCUGUUCGACGGCGACAACUGGCGACGGUUCGUCGCGUUCGUGAAGACCAUGGCCGACGGCGGCGCGAGGACGGCGCUGCCCAGGUGCGACACUGGGCACUCGGAUCUGUACGUGGGGUUCGUUGA-3′.The antisense probe sequence of The method for making a paraffin section was modified from Ausubel et al. . Wheat gin situ hybridization was performed with an Enhanced Sensitivity ISH Detection Kit I, POD . The paraffin sections were observed and photographed using a stereo microscope .The paraffin sections were dewaxed and rehydrated. Then, 2-KI (0.1%) for 5 min after dewaxing and rehydration. The sections were washed with deionized water, and then observed and photographed using a stereo microscope .Paraffin sections containing wheat grain were stained with IP < 0.05. Image processing was performed using Adobe Photoshop CS6.The data was analyzed by one-way ANOVA using Microsoft Excel and SPSS 13.0 software. Significance comparisons were made by Duncan's multiple range test at Grain weight increased across time and was significantly enhanced by P application Figure . SimilarThe morphological characteristics of the starch granules was observed using SEM Figures . The A-tTo study the effects of P fertilizer on the micro-structure of starch granules, protease (Type XIV) -treated granules were stained with methanolic merbromin and CBQCA and then visualized by CLSM. The results showed that P fertilizer caused substantial changes in the histochemical patterns of the starch granules. Fluorescence was clearly visible and strong in large areas of many starch granules in NP Figures . In contDrought-induced microstructural changes to starch granules may facilitate the transfer of chemicals  into the matrix of the starch granule and accelerate hydrolysis  and the highest GBSS2 expression (at 7 DPA).The expression pattern of SS1, SS2, and SS3 transcripts were greatest at 7 and 14 DPA and then decreased across time. In contrast, the SS4 transcripts remained steady between 7 and 21 DPA and then increased significantly at 28 DPA. In HP, the SS1, SS2, and SS3 transcripts were gradually increased from 7 to 14 DPA, whereas SS4 showed little expression on any sample date.In NP, the SBE1, SBE2A, and SBE2B), SBE1 transcripts were most abundant. In NP, SBE1 transcript abundance was greatest at 21 DPA, whereas the transcript abundances of SBE2A and SBE2B were both greatest at 7 DPA and then decreased. In HP, the transcripts of SBE1, SBE2A, and SBE2B were gradually increased from 7 to 14 DPA then decreased during the remaining time.Among the genes encoding starch branching enzyme  and starch degradation enzymes .BAM1 and BAM5 were the highest among the seven BAM genes. In addition, the relative expression of AMY4 increased sharply to high levels during late grain filling. For these reasons, AMY4, BAM1, and BAM5 mRNA were localized using in situ hybridization  was increased by P-induced increases in the number of pits and channels in wheat starch granules (Zu et al., AMY4, BAM1, and BAM5 transcript abundance at the periphery of the endosperm at 28 DPA. This indicates that during late grain filling in NP, the meristematic region at the periphery of the endosperm still maintained metabolic activities to support the relatively abundant transcripts of genes involved in starch synthesis and degradation. This explains the increased starch content in NP.Wheat endosperm cells are differentiated from the meristematic region at the periphery of the endosperm. Therefore, the youngest cells are found at the outer edge of the endosperm and the oldest at the center (Bradbury et al., The results of this study show that P fertilizer significantly altered microstructures in the starch granules. This is important because the channels may provide greater surface area for the transport of starch-synthesizing enzymes and substrates needed for starch synthesis. The study also indicated that P fertilizer significantly affected starch accumulation by influencing the expression of genes related to starch biosynthesis and degradation. Further study is necessary to understand the mechanism by which P influences starch morphology and biosynthesis. Such information may provide information helpful for increasing wheat yield and starch quality. The latter could have important implications for the food industry.RZ: Substantial contributions to the acquisition, analysis, and interpretation of data for the work; Drafting the work and revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. KF, CAL: Substantial contributions to the acquisition, analysis, and interpretation of data for the work; Revising the work critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. CEL, CYL: Substantial contributions to the conception and design of the work; Drafting the work and revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nowadays, there are many techniques to compensate bone atrophies of the posterior maxilla in order to obtain an implant-supported rehabilitation.This case series describes the Ebanist technique: a sinus lift procedure to be used in case of extremely resorbed bone crests (≤3 mm) allowing simultaneous implant placement.With a dedicated cylindrical trephine bur, it is possible to harvest a cylinder of bone from a fresh mineralized frozen homologous bone block graft and to simultaneously create a trapdoor on the recipient site. The trapdoor cortical bone is detached from the sinus membrane and removed. Dental implant is placed into the graft before the grafting procedure since the cylindrical block, once inserted in the recipient area, is not able to oppose sufficient resistance to the torque needed for implant placement.Second-stage surgery and following prosthetic rehabilitation were performed after 5 months. In all cases, implant stability was manually checked and no pathological symptoms or signs were recovered at any follow-up visit.This technique can be considered a valid procedure for implant therapy on atrophic posterior upper maxillae, when the residual bone crest is extremely resorbed. Nowadays, there are many techniques available to treat bone atrophies of the posterior maxilla in order to obtain a functional and esthetic rehabilitation with an implant-supported restoration -4. The sThe most commonly used and described sinus lift techniques are the lateral and the transalveolar approach. The lateral sinus lift is distinguished by an osteotomy of the lateral wall of the sinus , whereasth century. This technique is particularly useful in case of extremely resorbed bone crests (≤3 mm), because it allows a sinus lift with simultaneous implant placement in one single procedure.The goal of this paper is to describe a new procedure: the Ebanist Sinus Lift technique (ESL), so-called due to its similarity with the technicality of Italian cabinet-makers of the 16The following case series will describe this new technique.This case series was conducted in full accordance with ethical principles, including the Declaration of Helsinki, and each participant gave a written consent.11).In 2007, a 61 years old healthy woman (B.S.) was referred to a private dental practice for rehabilitation of her edentulous upper right posterior sextant, affected by severe bone atrophy .A fresh mineralized, non-irradiated, frozen, homologous bone graft was used, provided by the National Bank of Tissue - General Hospital of Treviso , certified by the National Health Ministry. The ELS technique is simplified by the use of special trephine burs . This system is a set of 4 burs with increasing diameter that allows the surgeon to harvest a cylinder of bone from the fresh block graft and to simultaneously create with the same diameter a trapdoor on the recipient site  and the host cortical bone was detached from the sinus membrane and gently pushed within the sinus .These trephines have a small cylindrical pivot at the center that takes engagement in a small 2 mm-diameter hole previously performed with a round bur, both on the recipient site and on the graft block. This device does not swing, granting an extremely precise cut and consequently a correct insertion of the graft into the planned recipient area. A trapdoor was created on the patient ridge with the exact diameter of the bone graft previously prepared  around the trapdoor, enough to expose bony anchorage areas and to set in the graft without any tensions.5a, b) because the cylindrical block, once inserted in the recipient area, could not be able to oppose sufficient resistance to the torque needed to place the implant.The implant  was placed into the graft before the grafting procedure . A slow resorbable membrane was used to completely cover and protect the graft, then the flaps closure was obtained with horizontal mattress sutures.Consequently, the shape of the cylindrical graft matched perfectly the size of the recipient site, simplifying the insertion of the allograft. After its placement, the cortical part of the block was gently thinned down until its removal, in order to allow its complete substitution during the healing processes .An implant-supported fixed prosthetic rehabilitation after sinus lift performed with the ESL technique was suggested to a 66 years old healthy woman (A.A.) with monolateral posterior edentulism of the upper left maxilla  and the host cortical bone was detached from the sinus membrane and removed .After local anesthesia, a full thickness crestal incision was performed on the upper right posterior edentulous ridge. Using the ESL dedicated burs, a circular trapdoor was created in the posterior area .A cylindrical bony graft was harvested from a block of fresh mineralized, non-irradiated, frozen, homologous bone, using the same diameter ESL bur used to create the trapdoor. A dental implant  was placed into the graft before its placement in the recipient site. Since the shape of the cylindrical graft matched perfectly the size of the recipient site, the combination of graft and implant was inserted into the recipient site . The left side was treated with traditional implant therapy in the premolar area, and with implant placement in association with the ESL technique in the molar area.A 67 years old healthy man (R.A.) was referred in a private dental practice asking for a fixed rehabilitation. The patient presented a bilateral edentulous posterior atrophic bone crest of the upper maxilla  was placed in it.12). A slow resorbable membrane was used to cover the graft. Flaps were finally sutured with horizontal mattress sutures.Taking advantage of the perfect match between graft and recipient site, the cylindrical block with the dental implant was placed and resulted clinically stable , it was chosen to perform implant therapy in association with the ESL technique.A 54 years old healthy woman (M.) asked for an implant-supported fixed rehabilitation of her upper posterior left sextant. Due to the extremely atrophic crest  before the grafting procedure. The graft perfectly matched the recipient site and appeared clinically stable . A slow resorbable membrane covered the graft and flaps were sutured.Cortical bone was removed from the sinus membrane and a block of fresh mineralized, non-irradiated, frozen, homologous bone was chosen for this case. From the graft, a cylindrical block was created, using a bur 1 mm bigger in diameter than the one used to crete the trapdoor. A dental implant  was placed in the graft  was recommended immediately after surgery together with 4 mg of betamethasone. A cold/soft diet and appropriate oral hygiene were recommended for 2 weeks. Patients were not allowed to wear removable dentures before implant uncovering. Patients were seen after 7 days for sutures removal and after other 14 days for wound healing control.In all cases, postoperative recovery was uneventful: soft tissues healed well and no signs of inflammation were present.Dental implants remained covered underneath the soft tissues and monthly follow-ups were planned for 5 months, when the second surgery was scheduled to uncover the implants.For each implant, stability was manually checked tightening the abutment screws with a 20 Ncm torque. No pathological symptoms or signs  were recovered at any follow-up visit  for each implant.16A) after implant placement and no peri-implant bone loss.Radiographic assessments of case #. 1 showed no pathological signs 6 years , 4 years  and 3 years  after implant therapy.Cases #. 2, #. 3 and #. 4 also didn’t show pathological signs nor bone loss respectively 3 years .Grafting the floor of the maxillary sinus has become the most common surgical intervention to increase insufficient alveolar bone height before the placement of dental implants in the posterior maxilla .The indication for a sinus lift procedure is clear when the residual bone shows 10 mm or less height in the posterior maxilla . The useOn the contrary, the use of the osteotome technique is able to gain about 3 to 9 mm of vertical bone height increase .The selection of the suitable sinus lift technique is therefore mainly founded on the height of pre-implant residual bone. When the residual bone is more than 5 mm of height, the transcrestal technique is the most indicated . On the Limitations for implant placement in the posterior maxilla are related for sure to residual bone height and width but also to the property of maxillary bone. Poor bone quality and quantity in posterior maxilla are considered the reasons for the decreased primary implant stability and, therefore, the increased failure rates .It has been shown that the implant survival rate decreases with the redurction of the pre-implant residual bone height . Most ofThe authors suggest the use of a fresh frozen cancellous bone graft (FFB) for the ESL technique. Due to its handling process, FFB undergoes a procedure of removal of the antigenic sites from the bone surface, showing angiogenesis induction and enhanced immune tolerance . MoreoveConcerning the clinical outcome of prosthetic rehabilitation, it has been shown  that impDifferent studies , 23 showConcerning the recipient site, great importance is given to the bucco-palatal bone width in its most coronal portion and to the presence of possible anchorage anatomical areas. For example, the presence of a bone septum or even the convergence of palatal and/or buccal bony walls of the sinus floor may be strategic areas useful to improve graft stability. The minimum ridge width required to perform the surgery with the cylindrical graft is 7 mm, because a graft with smaller dimensions could fracture during implant insertion.Contrary, the thickness of the residual bone in the recipient area is quite irrelevant. The reason is that both the trapdoor and the graft are designed with an equal width and diameter to that measured between the palatal and the buccal bony walls of the sinus.In order to perform a more precise surgery it is advisable to create a surgical template that allows the correct positioning of the implants, facilitates the development of anchorage areas and permits a precise placement of the graft. This same template will also help to easily locate the implants during the second stage surgery.All the modifications to the original sinus lift techniques presented in the literature -10, 24 hFollowing the positive results achieved in this case, it would be useful to prepare a randomized clinical trial to compare the ELS technique with other transalveolar sinus lift procedures.


In vivo assays cannot always be conducted because of ethical reasons, technical constraints or costs, but a better understanding of the digestive process, especially in infants, could be of great help in preventing food-related pathologies and in developing new formulas with health benefits. In this context, in vitro dynamic systems to simulate human digestion and, in particular, infant digestion could become increasingly valuable.To simulate the digestive process through the use of a dynamic model of the infant gastroenteric apparatus to study the digestibility of starch-based infant foods.Lactobacillus paracasei CBA L74. After the digestion the foods were analyzed for the starch concentration, the amount of D-glucose released and the percentage of hydrolyzed starch.Using M.I.D.A (Model of an Infant Digestive Apparatus), the oral, gastric and intestinal digestibility of two starch-based products were measured: 1) rice starch mixed with distilled water and treated using two different sterilization methods  and 2) a rice cream with (premium product) or without (basic product) an aliquot of rice flour fermented by An in vitro dynamic system, which was referred to as M.I.D.A., was obtained. Using this system, the starch digestion occurred only during the oral and intestinal phase, as expected. The D-glucose released during the intestinal phase was different between the classical and HTST methods . The same analysis was performed for the basic and premium products. In this case, the premium product had a significant difference in terms of the starch hydrolysis percentage during the entire process.The M.I.D.A. system was able to digest simple starches and a more complex food in the correct compartments. In this study, better digestibility of the premium product was revealed. With the current increase in the prevalence of food-related pathologies  –2, therein vitro digestive system based on infant physiology including the oral, gastric and small intestinal phases of digestion, to estimate the starch digestibility of two different categories of baby foods: rice starch-based foods and rice cream-based foods.The aim of the present study was to develop a dynamic Bacillus licheniformis  and was supplied at a concentration of 10000 U/ml on soluble starch at pH 6.5 and 40°C. Glucoamylase was obtained from Aspergillus niger  and had an activity of 1300 AGU/g. Pepsin  was obtained from porcine gastric mucosa and had an activity between 3200 and 4500 U/mg protein. Creon® 10000 U.Ph.Eur. , Italy), which is normally used in the treatment of pancreatic exocrine insufficiency, was available as brown/clear capsules. Each capsule contained lipase (10000 Ph.Eur. units), amylase (8000 Ph.Eur. units), and protease (600 Ph.Eur. units). CaCl2, KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6, (NH4)2CO3 and HCl were purchased from Sigma-Aldrich .All materials were standard analytical grade. α-amylase was obtained from Rice starch  was provided by Heinz Italia S.p.A, Latina, Italy. Water and rice starch were selected to outsource the fine tuning of the M.I.D.A. The volume selected was 45 ml, and the mass percentage of starch contained in it was 5.5% w/v, which corresponds to the average starch content reported on the nutritional label of different baby foods. This mixture of rice starch and water was then autoclaved in two different ways to simulate the main sterilization techniques for baby foods: the classical sterilization technique   and High-Temperature-Short-Time (HTST) .Both sterilization treatments were carried out in a lab autoclave , to reproduce the stabilization processes generally used for low acid products.Lactobacillus paracasei CBA L74, which was referred to as the “premium product”. The fermentation process has been previously described [Lactobacillus paracasei CBA L74 , International Depository Accession Number LMG P-24778. The fermentation was started in the presence of 106 bacteria, achieving 108 CFU/ml after a 20 h of incubation at 37°C. After heating at 85°C for 20 sec, to inactivate the live bacteria, the fermented cereal was spray dried (target outlet temperature: 85°C), and a 15% w/w aliquot was added to the rice cream to obtain the premium product. The final fermented cereal powders contained bacterial bodies and fermentation products.The rice cream-based foods were both made up of rice cream  and mineral water. In particular we used mineral water and rice cream (13.64% w/v), based on a recipe recommended for six-month-old infants, which corresponded to approximately 3 spoons of rice cream with 180 ml of water added, as suggested by the label of common commercial products, fermented or not fermented. For these analyses proteolytic enzymes were also added to the Simulated Gastric Fluids (SGF). The first food was a commercial rice cream, which was referred to as the “basic product”, and the second food was the same product with an 15% w/w addition of rice cream fermented by escribed . Briefly2 and the corresponding electrolyte stock solutions, whose composition is given in The Simulated Digestive Fluids were the following: Simulated Salivary Fluid (SSF), Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF). They were made up of distilled water, enzymes, CaClin vitro digestion procedure. The compositions of the simulated digestive fluids for the in vitro digestion of the rice starch-based foods and the rice cream-based foods are shown in 3 required to adjust the pH to the proper value in each digestive compartment was determined in a test prior to digestion and then added to each simulated digestive fluid. In particular, the gastric pH choosen was near 3, because of the gradual reduction observed from the first month  to the second year of life (pH 1–2) [As suggested by Minekus M et al , the Sim(pH 1–2) . In the in vitro dynamic digestive system, referred to as the M.I.D.A. was designed for this study reproducing the physiology of the gastrointestinal tract of a six-month-old infant. It consisted of four consecutive compartments simulating the esophagus, the stomach, the pyloric sphincter and the intestine as shown in Fig 1A.An These compartments (except for the pyloric sphincter) were composed of two concentric tubes. Food and the digestive juices flowed in the inner tube, while the annular space between the concentric tubes allowed the continuous circulation of water at 37°C, which was pumped from a thermostatic bath to maintain the normal human body temperature in each compartment.Fig 1B, was made entirely of Pyrex and had an inner diameter of 10 mm and a lenght of 90 mm. In the terminal area, the tube had a curvature and was connected to the next compartment by means of conically tapered ground glass joints. The gastric compartment was made up of two conical glass elements and two white concentric silicon rubber tubes, as illustrated in The esophageal compartment, shown in The inner tube of the stomach compartment had an inner diameter of 35 mm, a wall thickness of 1 mm and a length of 185 mm. The outer tube of the stomach compartment had an inner diameter of 40 mm, a wall thickness of 2 mm and a length of 210 mm. The global length of the stomach, including the conical glass elements, was 385 mm, while the overall volume of the gastric compartment was approximately 280 ml, as the capacity of stomach in infants varies between 90 and 500 ml . The pylin vitro digestion protocol was developed based on the consensus paper from INFOGEST “A standardised static in vitro digestion method suitable for food–an international consensus” [The experimental nsensus” .in vitro digestion, 45 ml of the food was split into nine sterile 10-ml Falcon tubes . Then, in turn, 5 ml of the SSF was added to each Falcon tube, and the food-SSF mixture was placed in an incubator at 37°C for 2 min simulating the oral compartment. After the oral phase, one of the nine boluses was immediately frozen at -20°C and stored for successive analysis. The other boluses went through gastric digestion.The only enzyme having physiological significance in the oral phase was α-amylase, and its activity in this work corresponded to the value suggested for adults by the consensus paper from INFOGEST because of the similar levels between salivary α-amylase in the adult and in 6-month-old-infants . The boly(t) represents the fraction of the chyme remaining in the stomach at time t; β is the coefficient describing the shape of the curve; T/2 represents the gastric emptying half time calculated using After incubation, every two minutes, 10 ml of bolus was inserted into the esophageal compartment together with 10 ml of SGF. Porcine pepsin was added to the SGF during gastric digestion of the rice cream-based foods only and was not required for rice starch-based foods because of the low protein content in isolated rice starch . The enzT/2 depends on V0, which is the volume of food going through digestion, and on K, which is the caloric density of the foods. The value of T/2 was 2.14 min for the rice-starch based foods and 3.17 min for the rice cream-based foods. The value of β was fixed to 1.5 according to Havenaar R, et al [R, et al . The gasR, et al  to allowDuring intestinal digestion, the volumes of chyme reported in Twenty-one samples were collected during each experiment. The starch content was quantified enzymatically  using the Megazyme Total Starch Assay Kit . Samples were filtered through Whatman No. 1 filter paper; then, 2 ml was taken and placed into 15 ml glass test tubes along with 8 ml of 95% v/v ethanol. These solutions were mixed vigorously on a vortex mixer and, after standing at room temperature for 30 min, were centrifuged at 1800 g for 10 min. The supernatant solutions were decanted, and starch was redissolved in 1 ml of water using a boiling water bath to aid redissolution. The volumes were adjusted to 3.9 ml with 100 mM acetate buffer at pH 5.0. Thereafter, 0.1 ml of AMG diluted 50-fold was added, and the solutions were incubated in a water bath at 50°C for 30 min. Aliquots of 0.1 ml of the diluted solutions were transferred to glass test tubes, and 3 ml of the GOPOD reagent was then added. After incubating the tubes at 50°C for 20 min, the absorbances for each sample were read against a blank at 510 nm.D-glucose was measured using the Megazyme D-glucose Assay Kit : 3 ml of GOPOD reagent was added to 0.1 ml of samples containing D-glucose and incubated at 40–50°C for 20 min. Absorbances were read at 510 nm against the blank. Digestion experiments were performed in triplicate for each food. The results are represented as the mean grams of starch and D-glucose in each sample as a function of the time of sampling.The degree of conversion of starch was calculated using t according to Eq (The D-glucose released (g) was calculated by subtracting the amount of D-glucose at t = 0 min  from the amount of D-glucose detected in each sample collected at time ng to Eq :D−glucot was calculated using the %SH is the percentage of starch hydrolysis at the sampling time t; Sh is the amount of starch hydrolized to D-glucose (g); 0.9 represents stoichiometric constant of D-glucose content conversion into starch; GR is the amount of released D-glucose calculated using Eq  evaluated differently according to the tested foods. For the starch-based foods, Si is the starch content measured before the sterilization treatments. For the rice cream-based foods, Si is the starch content measured after dissolving the rice cream into water.The amount of starch digested into D-glucose at each time of sampling using Eq ; and Si p value. Differences were considered statistically significant if there was a probability greater than or equal 95% to refuse the null hypothesis.Student t-test was used. Each test was conducted in triplicate. GraphPad Prism version 7.0  was used for the statistical analysis. Assuming no difference (the null hypothesis), the significance was determined using the in vitro digestion process is shown schematically in  It was comprised of three phases replicating the oral, gastric and intestinal phases of digestion in vivo.The Fig 2 shows an overall view of the digestion times (B), the starch level at T0 (C) and the separate steps of the digestion . The different starch content during the oral phase (from 0 min to 2 min) in the rice starch-based foods  is reproduced in in vitro digestion (from 122 min to 248 min) is described in The evolution of the starch content for both classical and HTST methods during the last stage of  the amount of D-glucose released both during the entire digestion (A), at T0 (B) and during each separate step  is shown. At the time 0 point , gastric (B) and intestinal steps (C). Starch hydrolysis increased during the oral , the starch level at T0 (B) and the separate digestion steps  are shown. Interestingly, basic and premium products had different starch contents at 0 min  suggesting that the fermentation by Lactobacillus paracasei CBA L74 was able to reduce the starch content.In at 0 min  before tThe different starch content during the oral phase (from 0 min to 2 min) in the rice cream-based foods (basic and premium) are reproduced in in vitro digestion (from 122 min to 248 min) is described in The evolution of the starch content both for basic and premium products during the last stage of  the D-glucose released during the entire digestion (A), the D-glucose present at T0 (B), and the D-glucose released during the separate steps  are shown. At time 0 , gastric (B) and intestinal steps (C) of digestion. The percentage of starch hydrolysis observed during the oral digestion for both of the rice cream-based foods is illustrated in D-glucose produced through the enzymatic hydrolysis of starch in both the rice cream-based foods, basic and premium, was calculated using t time 0 , the fert time 0  and intet time 0 , as expein vitro digestion of the rice cream-based foods is reported in The percentage of starch hydrolysis during the last stage of the ad hoc system for child digestion was developed by Havenaar et al. [in vitro digestive system that is able to simulate the main stages of digestion, from oral to intestinal, of a six-month-old infant. The dynamics of our M.I.D.A system was guaranteed by the continuity of the simulated gastrointestinal tract, which was composed of three compartments, that were not considered as simply separate flasks but as districts interacting with each other. In each of these compartments, we simulated the physiologic temperature, pH and enzymatic profiles. Our model is, to the best of our knowledge, the only one resembling an infant gastrointestinal tract and that has been used for the purpose of studying the digestibility of nutrients, such as starch. Starch is the primary nutrient involved in energy intake since it is a source of D-glucose, which is the primary substrate for cell metabolism. The digestibility and absorption of starch has significant nutritional and physiological implications both in children and in adults. The extent of starch digestibility is influenced by the nature of the starch, food processing and the physiological status [“Tell me what you eat and I will tell you who you are” well summarize the importance of studying nutrition and digestion, in particular in the pediatric age. For this purpose, in vivo studies represent the best route to obtain informations, but only a few studies have been done in vivo on infants, because of ethical reasons and costs. For this reason few digestive dynamic systems have been described in the literature –8, 29. Tr et al. , the TIMl status  and is gThe readout of starch digestion was the amount of residual starch, the D-glucose released and the percentage of starch hydrolysis in time. The first analysis was independent of the amount of starch loaded, whereas the other two were corrected for the initial amount loaded in the system. The M.I.D.A dynamic digestive system could digest starch no matter what sterilization method was used, in the correct compartment and allowed a punctual monitoring of the digestion process.Lactobacillus [The next step was to challenge the M.I.D.A. system with a more complex food, specifically fermented or unfermented commercial rice cream. The rational of this experiment was to test the digestibility of the same very common baby food before and after the fermentation process carried out with a particular strain of bacillus . Fermentbacillus , the enhbacillus  or, morebacillus .In particular understanding the mechanisms of infant food digestion, fermented or unfermented, in the infant gastrointestinal tract is a key step for developing new foods with health benefits for the infant. In our experiments, the fermentation process alone was sufficient to reduce the starch amount in the rice cream confirming that fermentation is a process that helps food digestibility. Interestingly, during the intestinal phase of digestion, the percentage of starch hydrolysis in the premium product was significantly greater than the basic product. The percentage of starch hydrolysis is related to the starch present at time 0, consequentely taking into account the initial different amounts of the starch in the two foods tested. The starch content and the D-glucose released, instead, do not take into account the initial different amounts of the starch; this is why no statistically significant differences were observed between these two products in term of starch content and D-glucose released.Increased starch digestibility in the premium food could be related to the amylolytic properties of the fermenting microflora that bring about the cleavage of amylase and amylopectin to maltose and D-glucose , 43. AddIn conclusion, the M.I.D.A. system was able to digest starch in the correct compartment and demonstrated that fermented baby food is better digested than the unfermented baby food.Altough human clinical trials undoubtedly remain the best approach to study food digestion , they ca


The Brazilian Journal of Cardiovascular Surgery (BJCVS) is grateful for the reviewers, listedbelow, which collaborated in this edition. Without their work would be impossible to keep thehigh scientific standard of our journal.Alfredo Inácio FiorelliDouglas BolzanGibran Roder FeguriGilberto BarbosaLindemberg SilveiraMagaly Arrais dos SantosMarcos Aurélio Barboza de OliveiraMelchior LimaMichel Silva ReisReinaldo BestettiRenata TrimerRicardo LimaRui M. S. AlmeidaSergio Francisco dos Santos Junior


Ipomoea batatas (L.) Lam.). Although numerous studies have analyzed the accumulation and properties of starch in sweet potato SRs, the transcriptomic variation associated with starch properties in SR has not been quantified. In this study, we measured the starch and sugar contents and analyzed the transcriptome profiles of SRs harvested from sweet potatoes with high, medium, and extremely low starch contents, at five developmental stages . We found that differences in both water content and starch accumulation in the dry matter affect the starch content of SRs in different sweet potato genotypes. Based on transcriptome sequencing data, we assembled 112336 unigenes, and identified several differentially expressed genes (DEGs) involved in starch and sucrose metabolism, and revealed the transcriptional regulatory network controlling starch and sucrose metabolism in sweet potato SRs. Correlation analysis between expression patterns and starch and sugar contents suggested that the sugar–starch conversion steps catalyzed by sucrose synthase (SuSy) and UDP-glucose pyrophosphorylase (UGPase) may be essential for starch accumulation in the dry matter of SRs, and IbβFRUCT2, a vacuolar acid invertase, might also be a key regulator of starch content in the SRs. Our results provide valuable resources for future investigations aimed at deciphering the molecular mechanisms determining the starch properties of sweet potato SRs.The starch properties of the storage root (SR) affect the quality of sweet potato ( Ipomoea batatas (L.) Lam.) is an important food crop that is widely grown throughout the world due to its stable yield, rich nutrient content, low input requirement, multiple uses, high yield potential, and adaptability under a range of environmental conditions . These results improve our understanding of the biochemical and molecular mechanisms underlying starch accumulation and starch–sugar interconversion during SR development in sweet potato.Three sweet potato varieties with different SR starch properties , Xushu 22 (XS22), and Shangqiu 52-7 (SQ52-7)) were selected for transcriptional profiling. Plants of the three varieties were cultivated at the experimental station of the Sweet Potato and Potato Research Institute, Southwest University, Chongqing, China, in 2013 and 2014. For each variety, fifty plants were produced from transplants and grown in unfertilized washed sand in the greenhouse, at temperatures of between 22 and 28°C. After transplanting into the field, roots from five individual plants were sampled at 50, 65, 80, 95, 110, 125, and 140 days after transplanting (DAP) and were divided in two samples; one sample was used to quantify starch properties (including starch and sugar content and composition) and the other one was immediately frozen in liquid nitrogen and used for RNA extraction. Samples were collected and assessed in triplicate each year. Fifteen samples, including the SRs sampled at 65, 80, 95, 110, and 125 DAP from YS33, XS22, and SQ52-7 in 2013 were used for transcriptome analysis.Total RNAs were extracted from the root tissues and residual DNA was digested using the RNAprep Pure Plant Kit with DNase I  according to the manufacturer's instructions. The total RNA samples were examined by agarose gel electrophoresis, and the concentration and quality of RNA were determined with a NanoDrop ND-2000 Spectrophotometer . RNA quality was verified using a 2100 Bioanalyzer RNA Nanochip  and all the samples had an RNA Integrity Number (RIN) of > 8.5. The RNA samples were used for transcriptional profiling and qRT-PCR analysis. A total of 20 μg of RNA from each sample was used for cDNA library preparation.The dry matter, starch, amylose, and sugar contents of the SRs were determined for each sample. Dry matter was measured as previously described . For each trait, a two-way analysis of variance (ANOVA) was used to determine the effect of the genotype and developmental stage as well as their interaction. A Tukey's multiple comparison test was then employed to determine the statistical significance between varieties at each of the five developmental stages. Statistical comparisons were considered significant at Q < 20 were removed. All the filtered reads from the 15 libraries were de novo assembled using Trinity  with paired-end method and default parameters as previous study on optimal assembly strategy  sequencing using the Hiseq2000 platform was performed at Beijing Yuanquanyike Biotech, Beijing, China, according to the manufacturer's instructions . To obtain clean reads, all of the raw reads were filtered with the following process. Firstly, reads that failed the built-in Failed Chastity Filter in the Illumina software according to the relation “failed-chastity ≤ 1,” using a chastity threshold of 0.6, on the first 25 cycles were excluded. Secondly, reads with adaptor contamination were discarded. Thirdly, low-quality reads were masked with ambiguous sequences “N.” Finally, reads with more than 10% RRID: SCR_005476). The unigene expression levels were quantified and normalized using the CLC Genomics Workbench version 3.7.1  in terms of reads per kilobase of exon model per million mapped reads (RPKM). Then, this commercially available software was used to test for statistically significant differences in unigene expression between two samples. DEGs were screened based on the following combined criteria: q-value [false discovery rate (FDR)] < 0.01 and absolute fold change of RPKM > 2.Genes differentially expressed between different genotypes at the same developmental stage, and between different developmental stages from the same genotype were screened. Firstly, we quantified the expression level of unigenes in the 15 samples. All the filtered reads were mapped to the unigenes in the newly assembled transcriptome using bowtie2-2.1.0  alignment  between unigenes and sequences derived from public databases, including the UniProtKB/Swiss-Prot Database (http://www.expasy.ch/sprot), TrEMBL Database (http://www.ebi.ac.uk/uniprot), the Conserved Domain Database  (http://www.ncbi.nlm.nih.gov/cdd), the Pfam Database (RRID: SCR_004726) (http://pfam.xfam.org/), NCBI Non-redundant Protein (Nr) Database (http://www.ncbi.nlm.nih.gov), and the Eukaryotic Orthologous Groups  Database (http://www.ncbi.nlm.nih.gov/COG), was performed, and the best alignments were used to determine the sequence direction of unigenes.BLASTx (E <1.0E-5) using Blast2GO . The unigenes were further classified using GO Slim (http://www.geneontology.org/GO.slims.shtml). To assign the detected unigenes to biological pathways, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation was conducted using the online KEGG Automatic Annotation Server . The DEGs were analyzed for GO category enrichment and KEGG pathway enrichment using AgriGO  and KAAS, respectively, using Fisher's Exact Test and FDR correction.Gene ontology (GO) annotation analysis of the unigenes was performed using the high-score BLAST matches in the Swiss-Prot and TrEMBL Proteins Database , according to the manufacturer's instructions. Gene-specific primers were designed with Geneious Pro 4.8.5 to be between 18 and 27 bp long, with a GC content of 40–60%, a melting temperature (Tm) of 57–63°C, and a PCR product range of 160–300 bp. For genes that have been sequenced previously, the full-length sequences were used to design primers. Primers used in this study are listed in Table RRID: SCR_002865) version 20.0, and tests of significance were two-sided.Pearson's correlation analysis of gene expression and trait values was performed with SPSS , the mean dry matters of the three genotypes over the 3 years (2011–2013) were 34.469 ± 2.385%, 27.957 ± 1.137%, and 13.725 ± 1.473%, respectively, while the corresponding average starch contents were 23.623 ± 2.073%, 17.961 ± 0.989%, and 5.588 ± 1.280%, respectively , indicating that YS33 SRs might accumulate higher levels of dry matter components in addition to starch than XS22 SRs.The starch content of the total dry matter in SQ52-7, XS22, and YS33 ranged from 55.472 to 74.885%, 74.436 to 88.458%, and 72.829 to 86.795%, respectively, and peaked at 95 DAP Figure . SQ52-7 The starch composition changed during SR development in the three tested varieties Figures . AlthougWe measured the content of various sugars, including sucrose, fructose, glucose, and maltose, in the dry matter of the 15 SRs using HPLC. There was no detectable sucrose accumulation in the SRs of YS33 at 65, 80, and 95 DAP Figure . In XS22http://www.ncbi.nlm.nih.gov/Traces/sra) under accession number SRP075405.A total of 8.35E + 09 high-quality reads were generated with an average trim read length of 98.47 bp. For each sample, from 5.27E + 07 to 6.14E + 07 raw reads were obtained, and the total number of bases ranged from 5.33E + 08 to 6.21E + 08. After trimming the adapter sequences and removing low quality sequences, approximately 7.91E + 09 sequences from all 15 samples were assembled into 241,386 transcripts and 112,336 unigenes (≥ 200 bp) using Trinity. The average length of the transcripts and unigenes was 973.89 and 662.39 bases, respectively Table . The uniE-value of ≤ 1.0E-5  with known gene sequences at a cut-off -5 Table , indicat0% with kE-value cutoff of < 1.0E-5. We mapped 11873 unigenes to 321 KEGG pathways, and the highly-represented pathways included Ribosome , Protein processing in endoplasmic reticulum , Plant-pathogen interaction , Plant hormone signal transduction , and Starch and sucrose metabolism .To assign the detected unigenes to biological pathways, all of the unigenes were compared against the KEGG pathways database using BLASTX with an r2) of transcriptomes among SRs ranged from 0.85 to 1  and a β-amylase (BMY) gene (comp94608_c0_seq1) were only expressed in XS22 SRs, and three transporters, including a glucose transporter encoding unigene (comp104813_c0_seq1) and two glucose-6-phosphate/phosphate translocator (G6PPT) encoding unigenes (comp32901_c0_seq1 and comp33012_c0_seq1), were only expressed in XS22 SRs. We found that the AGPase large subunit 4 encoding unigene IbAGPb3 was only expressed in YS33 SRs at 95 DAP.After excluding unigenes with undetected expression RPKM < 0.5), 19115 and 24456 unigenes specifically expressed in one developmental stage and one genotype were identified, respectively. Genotype-specific unigenes accounted for 35.5% of the total number of expressed unigenes, which is approaching the percentage of commonly expressed unigenes in the sweet potato genotype (48.4%), indicating that transcriptome variation was greater among genotypes than among developmental stages , the GBSS encoding gene IbGBSSI  showed higher expression in YS33 and XS22 than in SQ52-7 at most developmental stages. In addition, genes  encoding UDP-glucose pyrophosphorylase (UGPase), SBEI, and INVinh, respectively, and the G6PPT transporter encoding gene (comp83665_c1_seq1) also exhibited high expression in these SR samples. Among the low expression group, some unigenes were expressed at low levels throughout SR development in the three genotypes investigated, such as unigenes  encoding sucrose-phosphate synthase (SPS), two BMY encoding genes (comp48829_c0_seq1 and comp63470_c0_seq1), and a disproportionating enzyme (DPE) encoding gene (comp79218_c0_seq1).Cluster analysis assigned these DEGs into two groups Figure , based oWe also found that most DEGs involved in starch and sucrose metabolism were highly expressed at 95 DAP in the three genotypes, suggesting that 95 DAP might be a critical stage in sucrose metabolism or starch biosynthesis in sweet potato SRs.IbAGPb3 showed weak expression in YS33 at 95 DAP (RPKM > 0.5), and was not expressed in the other samples (RPKM < 0.5). QRT-PCR analysis indicated that this gene was expressed at higher levels in XS22 and YS33 at 95 DAP than in other samples, but its expression level was very low compared with that of the other AGPase encoding genes ] involved in starch granule formation Figure  showed ds Figure .IbISBEI, IbISBEII and two IbGBSS genes showed high expression throughout SR development in all three genotypes. Two SSS genes, which were not previously reported in sweet potato, also showed relatively high expression in the SRs of all three genotypes. Expression of a starch synthase III encoding gene (comp89307_c0_seq2) was higher than that of starch synthase I encoding gene (comp87190_c0_seq4) in the three genotypes, but both of these genes were expressed at higher levels in YS33 or XS22 than in SQ52-7 , implying that these two genes might be novel starch synthase encoding genes in sweet potato.Starch phosphorylase (SP) was reported to be involved in starch granule formation . DBE was involved in amylopectin synthesis  catalyzes amylose biosynthesis, and SSS and SBE are essential for amylopectin biosynthesis in higher plants  also participates in starch degradation and biosynthesis . Three of these genes exhibited high levels of expression in the 15 samples Figure . UnigeneP Figure .SuSy genes (comp87700_c1_seq4 and comp87700_c2_seq1) showed extremely high levels of expression during SR development in the three genotypes. Four SuSy genes  showed similar expression patterns; however, it remains to be determined whether these unigenes represent the same SuSy gene  and UGPase (comp83799_c0_seq1) expression were positively correlated with starch content in the dry matter  in the three genotypes, indicating their critical roles in SR starch accumulation in sweet potato.Sucrose synthase (SuSy) catalyzes the reversible conversion of UDP-glucose and fructose into sucrose, but mainly acts to decompose sucrose into UDP-glucose and fructose to provide substrate for starch synthesis Table . We detee Figure . UDP-glue Figure . Expresse Figure . CorrelaSucrose synthesis involves SPS, which converts UDP-glucose and fructose-6-phosphate into sucrose-6-phosphate; sucrose-phosphate phosphatase (SPP), which converts sucrose-6-phosphate into sucrose  (comp87686_c0_seq4) and UDP-glucuronate 4-epimerase (GAE) encoding genes (comp84725_c1_seq1) showed differential expression during development, but not among genotypes Figure . The twoIbβfruct2 (comp85641_c0_seq1), showed differential expression in the SRs of the three genotypes. Expression analysis demonstrated that this gene had higher expression in SQ52-7 than in XS22, and lowest expression in YS33 . Invertase activity is regulated by the invertase inhibitor (INVinh)  was also observed. Expression of the INVinh unigene comp70068_c0_seq1 was negatively correlated with that of Ibβfruct2 (r2 was -0.601 – −0.975), and the expression of another INVinh unigene (comp80373_c0_seq2) also showed a significant negative correlation with that of Ibβfruct2 . However, INVinh has been reported to mediate the post-translational regulation of invertase, and the activity of one invertase might be regulated by several INVinhs  and the other of which encodes a probable aquaporin. We detected DEGs encoding three groups of aquaporin: the plasma membrane intrinsic protein (PIP), tonoplast intrinsic protein (TIP), and Nodulin 26-like intrinsic membrane protein (NIP). Although none of the aquaporin unigenes showed differential expression among the SRs of the three genotypes examined, they did exhibit dynamic changes in gene expression during SR development  of the dry matter in SRs , but we found that starch accumulation in the dry matter of XS22 SRs was higher than that in YS33 SRs at most detected developmental stages. The starch and sugar contents in the dry matter of SRs in XS22 were both higher than those in YS33 at most developmental stages. Compared to YS33, the lower starch content per SR fresh weight detected in XS22 was mainly due to the lower dry matter content and higher water content in the SR at the harvest stage, and not due to starch accumulation in the dry matter. Thus, the dry matter or water content of SR flesh, combined with the starch accumulation in the dry matter, determine the starch content of SRs, and differences in water content and starch accumulation in the dry matter of SRs also contribute to differences in SR starch contents among genotypes.XS22 contained a lower SR starch content at the harvest stage (17.961 ± 0.989%) than did YS33 (23.623 ± 2.073%), and the dry matter content of XS22 SRs (27.957 ± 1.137%) was also significantly lower than that of YS33 SRs (34.469 ± 2.385%) (To decipher the molecular mechanism underlying starch accumulation in SRs, we detected DEGs encoding key enzymes involved in starch granule formation. Surprisingly, these genes were not always expressed at much lower levels in the SRs of SQ52-7, which have a low SR starch content and starch accumulation in the dry matter, than in those of YS33, which have a high starch content and starch accumulation in the dry matter. Furthermore, there was not a direct correlation between the expression of genes encoding any of the enzymes involved in starch degradation, including SP, DPE, and BMY, and starch content traits in the SRs of the three genotypes. These results indicate that the expression of genes encoding starch granule-synthesizing or degradation enzymes are not the decisive factors contributing to the SR starch content variation among sweet potato genotypes.IbAGPb1A, IbAGPb1B, and IbAGPa1, which encode the AGPase large subunit 1A and 1B and small subunit 1, respectively, were markedly expressed in developing SRs, and IbAGPb2, IbAGPb3, and IbAGPa2, which encode the AGPase large subunit 3 and 4, and small subunit 2, respectively, were expressed at low levels compared with those of the other three AGPase subunit encoding genes. Especially IbAGPb3, which was scarcely expressed in sweet potato SRs. This result is similar to the findings of a previous study that examined the expression of genes encoding AGPase subunits in sweet potato (Zhou et al., IbAGPb3 was only expressed in leaves and IbAGPb2 was expressed in sink tissues at low levels. However, our results also showed that IbAGPb3 was expressed at 95 DAP in the SRs of YS33 and XS22 (Figure AGPase catalyzes the formation of ADP-glucose, the substrate for starch synthesis, and thus constitutes the first, rate-limiting step in starch biosynthesis (Hannah and James, 2 Figure ; thus, tInterestingly, the expression of most DEGs encoding key enzymes involved in starch and sucrose metabolism, such as AGPase, GBSS, SSS, SP, and SuSy, peaked at 95 DAP. This result is in agreement with our finding that starch accumulation in the dry matter of SRs of the three genotypes examined all peaked at 95 DAP, indicating that 95 DAP is an important time point in starch biosynthesis. This developmental stage should be the focus of future studies of SR starch traits of sweet potato.The amylose content and amylose to amylopectin ratio are important factors affecting starch structure and properties. In sweet potato, amylose-free and high-amylose transgenic sweet potato plants have been produced by inhibiting the expression of genes encoding sweet potato GBSSI and SBEII through RNA interference, respectively (Shimada et al., It was reported that the two types of SBE, SBEI and SBEII, differ in substrate specificity and expression patterns (Zeeman et al., Starch accumulation is a dynamic process, which includes the synthesis, degradation, transport, and conversion of sucrose and starch (Zeeman et al., Genes that affect sucrose cleavage and metabolism may be central regulators of starch accumulation in storage organs. Two pathways mediate sucrose cleavage in the cytosol; in one, sucrose is converted to glucose and fructose by invertase, and in the other, sucrose is converted to UDP-glucose and fructose by SuSy (Li and Zhang, Ibβfruct2, which encodes a vacuolar acid invertase, was expressed at high levels in SQ52-7, which had a low starch content, and at low levels in YS33, which had a high starch content. This negative correlation between Ibβfruct2 expression and SR starch content indicates that the vacuolar acid invertase encoded by this gene regulates the starch content of sweet potato SRs. While there is little previous report of a vacuolar acid invertase regulating the starch properties of sweet potato SRs, Tanaka et al. (SRF1, which encodes a Dof zing finger transcription factor, regulates the SR starch content through negatively regulating Ibβfruct2 expression, but not by directly regulating the starch biosynthetic pathway. Previous studies also demonstrated that invertase might influence the starch content of storage organs (Tang et al., We found that a et al.  demonstrIbβfruct2 and the SR starch content. Moreover, INVinh might also regulate the starch content by inhibiting invertase activity, so the expression of the detected INVinh encoding genes, which is inversely correlated with that of Ibβfruct2 in the SR, might also be positively correlated with SR starch content.Here, we propose a model in which IbβFRUCT2 and its inhibitor regulate the starch content of sweet potato SRs Figure . FirstlyIn this analysis, we revealed the variation of total dry matter, starch accumulation, and sugar content in SRs derived from three genotypes during SR development, identified DEGs involved in starch and sucrose metabolism in sweet potato SRs, analyzed the expression pattern of these genes and the correlation between gene expression and variation in starch properties. Some of these genes had previously been reported in sweet potato, whereas others are novel genes that had not been detected. Our results reveal critical candidate loci involved in the biosynthesis and metabolism of starch and sugar during SR development, and the data produced in our study represent a useful resource for researchers aiming to decipher the physiological and molecular mechanisms underlying the starch and sugar properties of sweet potato SRs.KZ wrote the paper, designed and led much of the experimental work, and carried out the transcriptome analysis. ZW carried out much of the experimental work, performed trait measurements and qRT-PCR detection. DT assisted with field manipulation and plant management. HL, KLuo, YL and XW contributed to SR collection, trait measurement, and sample preparation. CL and JW provided sweet potato varieties for the study and helped with plant management. KLu and JD performed data analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Argüelles and Argüelles-Prieto ask an important question: are editors responsible for journal impact factor (IF) mania? We previously suggested that an obsession with publishing scientific work in a journal with the highest possible IF is primarily driven by scientists themselves, creating a tragedy of the commons regarding the allocation of scientific prestige . As publAs gatekeepers of the publication process, journal editors are unavoidably complicit in maintaining the IF economy. However, just because editors must work within this reward system does not make them responsible for IF mania. The primary responsibility of journal editors is to select high-quality work for their journals and to maintain the journals’ reputation. The preoccupation with journal IF, also referred to as “impactitis” , is geneFurthermore, we disagree with the statement that the “scientific community has universally accepted that the so-called ‘top journals’ only publish the most relevant papers, which are at the frontier of new knowledge.” The notion that quality can be measured by the publication venue has been emphatically rejected by the San Francisco Declaration of Research Assessment . In factFinally, we dispute the notion that editors should not be allowed to submit papers to the journals that they serve. Argüelles has made this argument before , and we


Randomized clinical trials (RCTs) represent the best study design for minimizing bias when investigating the effectiveness of a form of treatment. RCTs also are the building blocks of systematic reviews and meta-analyses, which allow more generalizable conclusions to be drawn about therapeutic efficacy , provideUnfortunately, meta-analyses can only include those data that the authors can find. Many prospective trials are started but never finished, and many more are completed but never published –5. The RSelective publication of this sort, sometimes called publication bias or positive-outcome bias, is harmful for at least two reasons. First, if two studies—one showing positive results and one showing no difference between interventions—have been performed, and the study with the positive outcome is more likely to be published and therefore available for meta-analysis, meta-analyses will tend to inflate the apparent benefits of a treatment and de-emphasize its harms , 8. ThisA solution to this problem has existed for some time. RCTs should be registered in one of the numerous publicly available, free, searchable, well-maintained repositories of clinical trials before the first patient is enrolled –11. In aThe Journal of Bone and Joint Surgery, The Bone & Joint Journal, and Clinical Orthopaedics and Related Research® have either required or encouraged trials to be registered but permitted it to be done retrospectively . We now all concur that this is not sufficient. Retrospective registration merely to allow a paper to be considered for publication by a journal does not allow the identification of no-difference findings, minimize data dredging, help patients find care, or support ethics panels in their important work. The International Committee of Medical Journal Editors (ICMJE) has required that its member journals insist on prospective registration for more than a decade now [For several years, cade now . It is tThe Journal of Bone and Joint Surgery, The Bone & Joint Journal, and CORR® require registration in a publicly searchable clinical trials registry prior to the publication of RCTs, and the registry number of the trial will be published in the electronic and print versions of these papers. Until the end of 2017, which we consider a transition period, this registration may take place either prospectively or retrospectively; but as of January 1, 2018, authors of all RCTs that began after the publication of this editorial must demonstrate proof of prospective registration to be considered for review by any of our three journals.With that in mind, as of now, We understand that there will be rare situations in which prospective registration is not possible or not appropriate, and we will be receptive to such claims. In these situations, the authors will be expected to explain in the limitations section of the paper the reasons for non-registration, and the editors will add a note to the title page of such a paper explaining why the exception was permitted. However, simple omission of prospective registration will not be considered a suitable excuse; the principles at stake here are too important.We encourage all orthopaedic journals to join us in setting and maintaining this important principle underlying the reporting of research in our specialty.


The purpose of this study was to evaluate the shear bond strength (SBS) of two different resin cements  and Variolink N ) to 112 zirconia specimens with airborne-particle abrasion and 20%, 30%, or 40% hydrofluoric acid (HF) for 1 or 2 h. A total of eight specimens were used to observe the phase transformation after surface treatments. Six specimens were treated only with HF etching and the average surface roughness (Ra) was analyzed. A one-way ANOVA test was applied for SBS and the effect of HF concentration on Ra. An independent t-test was performed for the comparison of Panavia F 2.0 and Variolink N, and the influence of the HF application time on Ra. A higher HF solution increased SBS and Ra. HF etching produced a lower rate of monoclinic phase transformation. Panavia F 2.0 showed a higher SBS than Variolink N. Due to its aesthetic quality and strength, metal ceramic restoration, which is supported by the metal structure, has been used for a long period of time in the areas requiring aesthetics . Metal cThere have been many studies on adhesives for zirconia. However, unlike traditional ceramics, zirconia has a high crystalline phase content, which makes the surface of zirconia unable to be etched by a low concentration of HF ,9,10,11.In recent years, studies have reported that the zirconia surface can be etched by corroding zirconia grains with high-concentration acids at room temperature ,21,22. HCommercial zirconia disc  was used in this in vitro study. By using an automatic cutting machine , 126 square-shaped specimens (15 × 15 × 1.5 mm) were prepared. One surface of each of the specimens was polished with up to 1200 grit size abrasive papers.n = 14) for SBS testing and debonded zirconia surface observation. The control group (C) was sintered in a furnace  according to the manufacturer's instruction  [A total of eight specimens were preserved for the analysis of the phase transformation following surface treatments. Thereafter, six specimens were also preserved for the analysis of average surface roughness (Ra) by only the HF etching procedure. As shown in ng time) . These sThen, 14 specimens were named as group NoHF. Twenty percent, 30%, and 40% HF solutions were experimentally prepared using 48% HF solution , distilled water, and an electronic scale. The abraded-sintered specimens and six preserved specimens were treated with 20%, 30%, or 40% HF solutions for 1 h or 2 h in a plastic box . The conComposite resin tubes (diameter: 2.379 mm) were fabricated using light-polymerized flowable resin  and mold  . After i2, using a light-curing unit   and specimens were placed on the stand. A 10-MDP-containing composite resin cement  and a conventional composite resin cement  were selected. After mixing the cement according to the manufacturer’s instructions, two composite tubes were pressed perpendicularly into the zirconia specimens under about 500 g . Residua3M ESPE) .All specimens were immersed in distilled water for 24 h. The specimens were divided into two subgroups (the non-thermocycled group and the thermocycled group). The latter group was thermocycled 5000 times between 5 °C and 55 °C in water baths with a dwelling time of 30 s, according to ISO 10477 ,26.The shear bond strength test was performed at a crosshead speed of 1 mm/min in a universal testing machine . The load was applied at the interface between the composite tube and the zirconia specimen until the composite tube was dislodged. The maximum load was recorded automatically.After the SBS test, randomly selected specimens were Pt-coated by an ion sputter . An observer blinded to the surface treatment examined the debonded surfaces with a field emission-scanning electron microscope (FE-SEM) .X-ray diffraction (XRD)  was used to detect phase transformation by the HF etching. The diffractograms were obtained using cu-kα radiation at 40 kV and 200 mA, from 20° to 70° at the scan speed of 3°/min and a 0.02° step size; the peak intensity ratio was obtained automatically.A scanning probe microscope (SPM)  was applied to evaluate the Ra of HF-etched specimens. Three areas of a representative specimen of each group, without the airborne-particle abrasion process, were selected. Subsequently, 5 μm × 5 μm images with 256 × 256 pixels were taken by a using non-contact mode, with a scan rate of 0.5 Hz.t-test was performed. To examine the effect of thermocycling, a paired t-test was applied.All statistical analyses were performed using SPSS 20.0 Statistics . The level of significance of α = 0.05 was assumed to mark statistical significance. All results were described as the means ± standard deviation. A one-way ANOVA test followed by the least significant difference test for post hoc comparisons was performed for SBS and the effect of HF concentration on the Ra. For the comparison of Panavia F 2.0 and Variolink N, as well as the influence of the HF application time on the Ra, an independent The SBS values of each group are presented in p < 0.05). Two hours of etching with 20% HF increased the Ra value more than 1 h of etching (p < 0.05). Two hours of etching with 30 or 40% HF did not increase the Ra value more than 1 h of etching. FE-SEM photographs of debonded surfaces are presented in Monoclinic peaks were noted in all etched groups and in group C . In partZirconia is widely applied in the field of dental prostheses due to its aesthetics, excellent biocompatibility, and strength ,21. For Several studies were carried out to increase the adhesion between zirconia surface and cement by using mechanical and chemical methods. Even though an ultra-short pulsed laser has been experimentally applied to treat the zirconia surface, alumina sandblasting is a typical mechanical method employed in clinics . Kulunk In recent years, there have been reports on HF-etched zirconia ,21,22,32In the present study, the SBS of the groups treated with HF solutions after 110-μm alumina airborne-particle abrasion was significantly higher than that of group C and group NoHF. Therefore, the null hypothesis was rejected. Lee et al.  reported(1)High-concentration HF etching and airborne-particle abrasion improved SBS.(2)Panavia F 2.0 showed higher SBS than Variolink N, regardless of thermocycling.(3)An increase in the HF concentration produced a higher Ra value.(4)HF etching resulted in a lower rate of monoclinic phase transformation than airborne-particle abrasion.Various bonding primers have been used to enhance the durability of resin cements-zirconia restorations in clinical practices. However, bonding primers were not applied in this study. Further research with bonding primers and HF etching would be necessary to improve clinical relevance and SBS. In addition, the physical properties of etched zirconia should also be investigated for long-term clinical success. Under the limitations of this study, the following conclusions can be made: