Croatian Medical Journal (CMJ) pays a lot of attention to its visual identity. This visual identity includes not only fonts, colors, page organization, logo, but all the published items. The cover page, as the most noticeable part of the CMJ’s visual identity, is considered to be a record of the journal’s activities, history, and progress. Every issue of the CMJ has a specially designed cover page that corresponds to the main topic of the issue. In this vein, the title page of the current issue is related to the 9th International Society for Applied Biological Sciences (ISABS) Conference to be held in June 2015 at the Croatian island of Brač.The CMJ was considered as an object of biomedical analysis and in this way was transformed from a scientific journal to a virtual patient. The humanized CMJ as a subject of forensic study, in order to reveal its genetic  identity was expected to provide its DNA. As the journal does not abruptly change from issue to issue, one can agree that its hereditary information is carried through the time and its generations. This hereditary information in the real word does not correspond to any biological molecule, but it is in the domain of the CMJ’s cultural history. Still, for this cover page and this particular topic, it was assumed that the DNA was a hereditary molecule determining the CMJ.The topic of the current issue is Forensic and Anthropologic Genetics and Individualized Medicine. In order to design the cover page, our designer (Maša Vukmanović) identified key words – forensics, genetics, and individual, and tried to find the visual analogues for their combination. The CMJ’s DNA, the cover page designer decided to look for CMJ’s genes represented in the published articles. The structure of the articles was analyzed and every article was transformed into a ladder of signals obtained by electrophoresis (CMJ, and at the same time corresponds to its identity, enabling its potential forensic identification.To assess the phoresis . The artCMJ, we strongly believe that the unity of science and arts can offer substantial benefits not only to our journal, but also to science and human culture in general.The analogies of science and arts are frequently evoked . Art can


By introducing the coloring liquids, it is claimed that it is possible to make the color of frameworks fabricated from zirconium oxide extremely close to the natural tooth color. The purpose of this research was to evaluate the effect of two staining techniques on value changing in zirconia crowns. Three groups A, B, and C, each containing ten zirconia crowns, were used. The zirconium cores samples were fabricated by a CAD/CAM device. Group A was left uncolored, Groups B was submerged for two minutes in A2 coloring liquid and Group C was stained with brush. Then all cores were sintered and the porcelain was applied by using the layering technique. Ultimately, the crowns color was determined using a spectrophotometer. Their color changing (∆E) and value changing (∆L) in relation to A2 color were also assessed. The data were analyzed with one-sample t-test, post-hoc Tukey, and one-way ANOVA tests with significant level set at 0.05.p= 0.001). The highest mean value was 78.31±1.22 belonging to group C (staining with brush) and the lowest mean value was 76.99±0.65 belonging to group B (submerging). The results of post-hoc Tukey regarding both ∆E and ∆L variables showed a significant difference between groups A (uncolored) and C (staining with brush) with P∆E=0.006 and P∆L=0.039, respectively. A significant difference between group B (submerging technique) and C (staining with brush) were shown when these two variables were compared . The mean value in all groups was higher than the value obtained from A2 color samples , it is recommended to use the submerging technique for staining zirconia cores. In recent years, esthetic dentistry has boomed to a great extent as patients’ demands have increased greatly and developments in approaches and new materials have improved clinicians abilities in providing esthetic treatments.The color compatibility has always been the first and foremost step in restorative and prosthetic dentistry.Color is a phenomenon resultant of visual perception which depends on the light reflected from or passed through an object.Visual color matching through making comparison between a patient’s tooth and the standard sample is the most common approach used in clinical dentistry.Visual evaluation is generally accepted to be unreliable and to have variable outcomes, while instrumental measurements have concrete and quantitative results.Selecting restorative materials compatible with the natural tooth color has always been one of the most complex issues in restorative dentistry.Porcelain shade selection guides entirely differ from the restorations that are used in restoring these teeth. People, as well, have different abilities in selecting colors. In addition, changes in the lighting conditions of environment can cause changes in color perception. Besides, porcelain thickness can produce a remarkable effect on the final color.In order to achieve maximum esthetics, it is essential for the restoration to restore the translucency as well as the color. Translucency of restorative material produces a natural and real appearance in the restorations. Natural teeth demonstrate variable colors and translucencies all over their structures. Therefore, translucency has the same importance as color in the process of color adjustment in achieving restoration esthetics.Although a majority of restorations are still fabricated from PFM, metal-free restorations have become much more popular. Unique cUsing liners has been suggested to compensate for the negative effect of the white color of zirconia on the restoration final color. It has been claimed that these liners decrease the strength of zirconia-veneer bond. As a substitute for these liners, newer zirconia coloring liquids which are used before sintering or pre-colored zirconia are recommended, although the advantages of these materials are under discussion.et al.1.2.1.2.The results of this research demonstrated that the value increased in all A, B, and C groups which was a predictable outcome. Porcelain shade selection guides completely differ from the restorations which are applied in restoring these teeth. Normally, porcelain shade- selection guides are in the shape of tabs, which have a high thickness (4mm). Most shade selection guides do not have metal or zirconia basis and are fabricated from ceramic materials and differ from the final restoration, while thet al.[et al.,[Johnston et al. in 1989,The material will be more opaque if the majority of light passing through the ceramic spreads out extremely and be reflected diffusely. In contrast, when there is only part of the light which is dispersed and most of it is transmitted diffusely, the material is more translucent.This issuet al.,[According to Aboushelib et al., coating The number of scientific reports on the color of zirconia core restorations is very limited; therefore, this subject needs to be investigated extensively in future studies. The results of the present research demonstrated that the type of zirconia cores staining technique has an influence on all ceramic crowns value. Therefore, it is recommended to consider staining technique as one of the influential factors on the final color of zirconia crowns.It is recommended to use the submerging technique of staining Zirconia cores in order to reproduce closer value of natural teeth.


Corneal perforation in severe Stevens-Johnson syndrome (SJS) presenting great therapeutic difficulties, the imperative corneal transplantation always result in graft failure and repeated recurrence of perforation. The aim of this study was to evaluate the effectiveness of a modified small tectonic keratoplasty (MSTK) with minimal corneal graft in the management of refractory corneal perforation in severe SJS.Refractory corneal perforations in ten patients (10 eyes) with severe SJS were mended with a minimal corneal patch graft, under the guidance of anterior chamber optical coherence tomography, combined with conjunctival flap covering. The outcome measures included healing of the corneal perforation, survival of the corneal graft and conjunctival flap, relevant complications, and improvement in visual acuity.Corneal perforation healed, and global integrity was achieved in all eyes. No immune rejection or graft melting was detected. Retraction of conjunctival flap occurred in one eye, which was treated with additional procedure. Visual acuity improved in six eyes (60%), unchanged in three eyes (30%) and declined in one eye (10%).The MSTK combined with conjunctival flap covering seems to be effective for refractory corneal perforation in severe SJS. Severe Stevens-Johnson syndrome (SJS) usually affects the eyes with extensive area of necrosis of the ocular surface, which could result in corneal ulcer or even perforation-3. BecauOrthotopic corneal transplantation is commonly used in treating corneal perforation. But in eyes with severe ocular surface disorders, persistent epithelial defects can lead to graft melting, repeated recurrence of perforation or even loss of vision. Even thSmall corneal graft has been successfully used in treating peripheral corneal perforations as it faces less risks of graft failure-14. PortThe study was approved by the Institutional Review Board of Shandong Eye Hospital and was conducted in accordance with the principles of the Declaration of Helsinki. Possible benefits and risks were explained to all patients, and informed consent was obtained.The medical records of ten patients (10 eyes), with refractory corneal perforations resulting from severe SJS, who were treated with modified small tectonic keratoplasty (MSTK) combined with conjunctival flap covering were retrospectively reviewed. All the perforations were conducted at Shandong Eye Hospital of Shandong Eye Institute between July 2008 and June 2011. The diagnosis of SJS was mainly based on a history of severe inflammation of the oral, dermal, and tracheal mucous membranes after taking medicine or having an infection. CornealAll the patients were immediately administered with ofloxacin eyedrops  twice per hour at the first presentation. Combined MSTK and conjunctival flap covering was performed within 24 hours. The resected tissues were subjected for etiological examination.The trephine size was determined as 1.0 mm larger than that of the corneal perforation according to the surgeon’s experience for the first two patients. From the third patient, HD-OCT was used to minimize the size of corneal trephine just as illustrated in Figure All surgery was performed under retrobulbar anesthesia by the same surgeon. Necrotic tissues were cleared from the surface of the corneal ulcer. Viscoelastic materials were then injected from the perforation  to maintain the anterior chamber at a safe depth. Meanwhile, the iris incarcerated in the perforation was restored Figure A and A’.A full-thickness corneal button was obtained from a cryopreserved donor cornea using a corneal trephine with the same diameter as (when the recipient bed was ≤3 mm) or 0.25 mm larger than (when the recipient bed was >3 mm) the recipient bed Table . The donThe bulbar conjunctiva near corneal lesions with good elasticity and rich blood vessels was selected as the donor site of conjunctival flap. After subconjunctival injection of 0.5 to 1 mL of 2% lidocaine hydrochloride, a double pedicle conjunctival flap, 1 mm wider than the corneal lesion, was obtained 2 mm away from the limbus  daily for 3 days. TobraDex eyedrops  and 1% cyclosporine A  were administered four times daily for the first month and tapered thereafter. Preservative-free artificial tears were administered 6 ~ 8 times daily according to the ocular surface conditions.The patients were followed up weekly for 4 weeks and monthly or bimonthly thereafter. Visual acuity, slit lamp appearance, and intraocular pressure (IOP) were evaluated. All complications were recorded and disposed. Surgical success was defined as healing of the ulcer and perforation, and survival of the conjunctival and corneal grafts. Failure was defined as corneal graft melting, recurrence of an ulcer or perforation in the original position.SPSS 16.0 was used for statistical analysis. The change in BCVA was analyzed using the Wilcoxon signed-rank test. P values of less than 0.05 were considered statistically significant.The patients were 4 females (40%) and 6 males (60%), with an average age of 48.8 years . The maximum size of each corneal perforation ranged from 2.0 mm to 4.0 mm. There was an irregular corneal ulceration with the outer diameter of 4.0 mm to 7.5 mm around all the perforations Figure A. All thThe mean follow-up period was 21.1 ± 11.8 (SD) months . Corneal perforations were healed, and the integrity of the globes was restored in all patients  during the follow-up period. A progressive regression of conjunctival flap was observed at 1 month after surgery, and the conjunctival flap became semi-transparent when healing of the corneal lesions was completed Figure B. In oneThe corneal grafts presented mild edema within the first month and became semi-transparent ever since Figure B. No immVisual acuity was improved in 6 eyes (60%), unchanged in 3 eyes (30%), and declined in one eye (10%) at the final follow-up. There was a significant difference between the preoperative and postoperative visual acuities (P = 0.043). The IOP of all the patients was within the normal range throughout the study period.Because of prolonged ocular surface morbidities, corneal perforations in severe SJS still face great challenge of recurrence, and the final visual function is always pessimistic. In this series of 10 patients, the corneal perforations were successfully repaired with MSTK and conjunctival flap in 9 patients. Tarsorrhaphy was required for remedial reason in one patient with conjunctival flap failure to adhere to the corneal lesion. All corneal grafts remained stable during the follow-up of no less than 1 year, and improved vision was acquired in more than 50% of the cases.Efficacy of AMT in treating small corneal perforation has been well established but unable to promote corneal stability in patients with severe corneal thinning. Also, bTraditional orthotopic PK, using a large-diameter graft, could achieve comparable anatomical and functional success in treating corneal perforation. But in patients with severe SJS, the corneal grafts are facing high risk of immune rejection, persistent epithelial defection, infection, graft melting, and resultant recurrence of corneal perforation. Tugal-TDue to the fact that the corneal graft was much smaller than the ulcer, a ‘ditch’ was left around the graft Figure C*. The eIn this case series, MSTK combined with conjunctival flap achieved favorable effects. The success of this combined procedure may be attributed to the following aspects. First, the corneal graft was quite small, which resulted in a lower risk of immune rejection and melting. Second, the corneal graft restored the integrity of the eye and added a rigid barrier between the aqueous humor and the conjunctival flap, which facilitated adherence of conjunctival flap to the corneal lesions. Third, the conjunctival flap helped to prevent secondary infection by covering the space around the small-diameter corneal graft. Fourth, the flap aided in the absorption of necrotic tissues and promoted vascularization of the wounded cornea, thus prDue to the complexity of corneal perforations in SJS, most patients may have poor visual acuity after treatment, and additional treatment for improving eyesight can be performed. Moreover, conjunctival flaps could not be obtained for protective necessities in patients who do not have enough healthy conjunctivas. Hence, permanent tarsorrhaphy would eventually be resorted to.In summary, combined MSTK and conjunctival flap covering is effective in treating corneal perforations in SJS. The eye globe could achieve tectonic integrity with a minimal corneal allograft under the protection of conjunctival flap, and the vision could be partly restored. This combined procedure might also be considered for the treatment of refractory corneal perforations associated with other severe systemic immune diseases.The authors declare that they have no competing interests.WS designed the study, performed all the operations, participated in interpretation of the data and revised the manuscript critically for important intellectual content. FW, SL and HG assisted in surgeries and overall management of the patients, participated in the data acquisition and analysis. FW also drafted the manuscript and revised it. TW participated in the data acquisition and analysis, contributed to manuscript revision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2415/14/97/prepub


Preoperative quantitative assessment of coronary angiography (CAG) and real-time graft size and graft flow assessment using epigraftic ultrasonography in the operative field have been performed to ascertain the patency in the right coronary artery bypass grafting (CABG).This study aims to evaluate the efficacy of the quantitative coronary artery and graft assessment to increase the patency of the right CABG.CABG was performed in 200 patients from January 2010 to December 2014. Ninety-three patients underwent all of preoperative CAG, real-time graft assessment and postoperative CAG with a total of 93 grafts anastomosed to the right coronary artery. Severity of coronary artery stenosis was evaluated by CAG. The size of target right coronary artery was measured by ultrasonography in the operative field. Parameters about graft flow were obtained from flow velocity curve in all the graft. The grafts were divided into two groups: patent grafts  and failing grafts . All factors were compared in these two groups and evaluated by logistic analysis and receiver operating characteristic (ROC) curve analysis.The overall patency as measured by postoperative CAG was 94.6% (88/93). There were 7 failing grafts including 5 occlusion and 2 competitive slow flow. Logistic regression analysis revealed that the percentage of graft size measured by ultrasonography divided by the size of the right coronary artery (graft-RCA size mismatch) and the pulsatility index (PI) were independent predictors of early graft failure . ROC curve analysis revealed that graft-RCA size mismatch > 2.08 and PI > 4.55 were predictors of graft failure.In this series, epigraftic ultrasonography depicted graft flow clearly. Combination of preoperative quantitative coronary artery assessment and real time graft assessment was essential to predict graft failure during the CABG. This technique may increase the patency of the right coronary artery bypass grafting.


These particles were significantly more efficacious inducers of heme oxygenase-1 (HO-1) than n-6 PUFA corn or saturated FA-rich palm CRLPs. Mechanistically, HO-1 induction by all CRLPs requires NADPH oxidase 4, with PUFA-containing particles additionally dependent upon mitochondrial reactive oxygen species. Activation of both p38 MAPK and PPARβ/δ culminates in increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression/nuclear translocation and HO-1 induction. These studies define new molecular pathways coupling endothelial cell activation by model CMRs with adaptive regulation of Nrf2-dependent HO-1 expression and may represent key mechanisms through which dietary FAs differentially impact progression of endothelial dysfunction.Circulating levels of chylomicron remnants (CMRs) increase postprandially and their composition directly reflects dietary lipid intake. These TG-rich lipoproteins likely contribute to the development of endothelial dysfunction, albeit via unknown mechanisms. Here, we investigated how the FA composition of CMRs influences their actions on human aortic endothelial cells (HAECs) by comparing the effects of model CMRs—artificial TG-rich CMR-like particles (A-CRLPs)—containing TGs extracted from fish, DHA-rich algal, corn, or palm oils. HAECs responded with distinct transcriptional programs according to A-CRLP TG content and oxidation status, with genes involved in antioxidant defense and cytoprotection most prominently affected by  Atherosclerosis and CVD are driven by endothelial dysfunction, a condition associated with low-grade systemic inflammation and characterized by inappropriate inflammatory activation of vascular endothelial cells (ECs). This disruption of endothelial homeostasis is coupled with impaired anti-inflammatory and antioxidant defense capacity, resulting in reduced resistance to oxidative and inflammatory insult, excessive reactive oxygen species (ROS) production, and development of oxidative stress . Thus, iECs are continuously challenged by the complex molecular milieu of circulating nutrients, cells, and blood constituents, including TG-rich lipoproteins (TRLs) . TRLs increase postprandially, but while endogenous lipoprotein involvement in CVD development is well-documented, how exogenous TRLs carrying dietary fats influence endothelial homeostasis is far less well understood, despite strong evidence implicating CMRs in the initiation of endothelial dysfunction –6. This n-3 PUFAs, particularly DHA, reduce plasma indices of oxidative stress when ingested as part of a high-fat meal  to test the overarching hypothesis that model artificial CMRs carrying n-3 PUFAs exert greater cytoprotective effects than those rich in n-6 PUFAs or SFAs. This comparative analysis showed that A-CRLPs of varying FA content switch EC gene expression toward a program of antioxidant defense, dominated by induction of the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent gene, heme oxygenase-1 (HO-1), with n-3 PUFA-containing particles exhibiting the greatest efficacy. Our studies identify a new redox-dependent pathway through which model postprandial TRLs regulate EC gene expression and this may provide a molecular explanation for the relative beneficial effects of n-3 PUFAs in the postprandial phase.Because ex vivo TRL fractions contain a complex mixture of both exogenous and endogenous lipoproteins, distinguishing the actions of VLDLs and their remnants from those of CMRs is challenging. Here, we have used our validated tractable model of ApoE-containing CMRs, “artificial TG-rich CMR-like particles” (A-CRLPs) , to estaChemicals and reagents were from Sigma-Aldrich  unless otherwise stated.® , corn oil, and unrefined palm oil  using SupelcleanTM LC-Diol SPE tubes. Oils were dissolved 1:10 in hexane, applied to preconditioned columns, and the flow-through discarded. TGs were eluted in 9:1 hexane:dichloromethane, assessed for purity by thin-layer chromatography, and stored under argon at −80°C  as per the manufacturer’s instructions, and their oxidation state assessed by measuring malondialdehyde using the thiobarbituric acid reactive substance (TBARS) assay, as described previously  was prepared by ultracentrifugation to remove chylomicrons, CMRs, VLDLs, and IDLs and the remnant-free plasma stored at −20°C. A-CRLPs containing human ApoE were prepared with TGs from fish, DHASCOeviously , using 7eviously   and used for experiments at passages 6–8. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured in M199 supplemented with 20% FCS and ECGS as previously described , 23, andhttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=alateuokffyxtgp&acc=GSE80067).Experimental details of a preliminary microarray performed in HAECs exposed to A-CRLPs of differing TG composition, together with its statistical analysis, are given in the supplementary file. The dataset has been placed in the GEO repository  for 20 min before exposure to A-CRLPs. For longer incubations, ECs were challenged with A-CRLPs prior to addition of 10 μM DHR or 10 μM 2′7′-dichlorofluorescein-diacetate (DCF-DA) for 20 min, or 5 μM MitoSOX (ThermoFisher) for 10 min, as indicated. After washing in PBS, fluorescence was measured in a plate reader  using excitation/emission wavelengths of 205/230 nm for DHR and DCF-DA, and 510/580 nm for MitoSOX.MAPKα, PPARβ/δ, Akt1, or Akt2 (20 nM) complexed to ESCORT III in OptiMEM (see supplementary Table 1). Transfection media were replaced with EGM-2 and experimental incubations performed 48–72 h posttransfection.HAECs in 6-well plates were cultured in EGM-2 until 50% confluent and washed with OptiMEM . Cells were transfected for 4 h with scrambled negative control siRNA or siRNAs targeting NADPH oxidase (NOX)4, p38® Green JumpStart™ Taq ReadyMix™ with MgCl2 was performed in a Chromo4 thermocycler  using primers listed in supplementary Table 2. Data were processed using Opticon Monitor 3 software and normalized to the validated reference gene, GAPDH.RNA was extracted using the GenElute™ mammalian total RNA miniprep kit according to the manufacturer’s instructions. Reverse transcription of 0.5 μg RNA per sample with 1 μM oligo dT primer (MWG Eurofins) and 1 U/ml RNaseOUT  was performed with the OmniscriptRT kit  in a total reaction volume of 20 μl for 1 h (37°C). Real-time PCR using SYBR3VO4 and then lysed in 76.5 mM Tris-HCl containing 10% (v/v) glycerol, 2% (w/v) SDS, 1 mM Na3VO4, and 10 μl/ml protease inhibitor cocktail on ice for 10 min. Proteins (30 μg) were resolved by SDS-PAGE (10%), transferred to polyvinylidene difluoride membranes, and immunoprobing was performed as described previously .HUVECs on 1% gelatin-coated glass coverslips were treated as indicated, washed with PBS, fixed in paraformaldehyde (4%), permeabilized with 0.1% Triton X-100, and then blocked in 3% BSA/1% goat serum in PBS for 30 min. Coverslips were washed in PBS/0.1% Tween (three times for 5 min) and incubated with anti-Nrf2 antibody  diluted in 3% BSA/1% goat serum for 1 h. Following further washing, coverslips were incubated for 1 h with anti-rabbit Alexafluor 594 secondary antibody . Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and images collected using an SP5 confocal microscope and Leica Application Suite advanced fluorescence software version 2.6  with a 40× HCX PL FLUOTAR PH2  objective lens. Mean Nrf2 intensity per DAPI-defined nucleus was quantified using VolocityP < 0.05 was considered statistically significant.Data were collated in Microsoft Excel  with appropriate statistical analyses performed using GraphPad Prism  as detailed in the figure legends. Data are expressed as mean ± SEM and n-3 PUFA-containing particles prepared using TGs from fish and DHASCO® oils were more oxidized than those containing corn or palm oil TGs, and assessment of EC viability showed no deleterious effects of any of the A-CRLPs over a range of TG concentrations . In normal healthy humans, the TG content of the postprandial CMR fraction at 5 h, corresponding to peak lipemia, approximates 300 μM   and thats 300 μM ; hence, n-3 PUFA-containing A-CRLPs generally promoting greater fold induction than those enriched in n-6 PUFAs or SFAs. HO-1, for example, was upregulated by 6.84-fold (P = 0.004), 5.48-fold (P = 0.054), 3.78-fold (P = 0.0074), and 1.86-fold (P = 0.11) by fish, DHASCO®, corn, and palm A-CRLPs, respectively. Subsequent extensive and independent quantitative (q)PCR validation of selected genes identified by the array confirmed that fish and DHASCO® A-CRLPs significantly increased expression of HO-1, SRX, and TXR mRNAs in HAECs after 4 h, with particularly robust effects on HO-1 expression (® A-CRLPs)  and SFA-containing A-CRLPs without effect . Transcripts showing increased expression (>1.5-fold) 4 h after exposure to A-CRLPs included those encoding antioxidant proteins and protective molecules such as metallothioneins. Nrf2 target genes involved in antioxidant defense, such as HO-1, sulfiredoxin (SRX), and thioredoxin reductase (TXR), were particularly affected, with pression –32. CornA-CRLPs) , and alsA-CRLPs) . Of the A-CRLPs)  and promA-CRLPs) . Additiot effect . Incorpot effect . Given tt effect . As antit effect . Togethen-3 PUFA fish and DHASCO® A-CRLPs showed significantly greater ability to induce HO-1 and SXR at 16 h than n-6 PUFA- or SFA-enriched A-CRLPs, with the latter showing a consistent trend toward HO-1 and SRX induction . Similarly, all particles reduced TXNIP mRNA after 16 h, but fish and DHASCO® A-CRLPs did so to a significantly greater extent than corn and palm A-CRLPs , indicating divergent effects of sustained exposure to n-3 PUFA or SFA A-CRLPs on NOX4 expression.Our data show that A-CRLPs induce antioxidant response gene expression in HAECs and increase ROS production, with probucol-containing particles corroborating the importance of oxidative state/ROS production for A-CRLP-driven gene expression. Because of the high level of HO-1 induction observed compared with that of the other validated genes, and the divergent effects of articles , 2, we fbit NOX2   preincubated cells, DHASCO® A-CRLPs induced a significant increase in both DCF and MitoSOX fluorescence, but did not significantly increase either signal in cells preincubated with MitoTEMPO . These studies showed that NOX4 siRNA, but not noncoding scrambled siRNA, significantly reduced the ability of A-CRLPs to increase HO-1 expression at the mRNA level  with a sitoTEMPO . Taken t® A-CRLPs. In addition, while Nrf2 mRNA was unaffected by A-CRLPs (not shown), Nrf2 protein expression was increased in cells exposed to n-3 PUFA-containing particles, and attenuated by Nrf2 siRNA  in HUVECs  and PPARβ/δ (GSK0660) were then used to examine involvement in A-CRLP-induced HO-1 induction. Neither SB202190 [which effectively reduced p38MAPK phosphorylation ] nor GSK0660 influenced basal HO-1 expression, but both inhibitors reduced A-CRLP-induced HO-1 induction at the protein  or PPARβ/δ significantly reduced the increased HO-1 mRNA evident in cells exposed to A-CRLPs .Nrf2 is a transcription factor that controls the adaptive response to endogenous and exogenous oxidative stress. Here, we identified several Nrf2-dependent genes modified by A-CRLPs, including antioxidant and cytoprotective molecules, demonstrating that modulation of Nrf2-regulated targets is one key EC response to lipoprotein challenge. HO-1 is a multifunctional enzyme, generating products with protective, anti-apoptotic, and anti-inflammatory actions , 32 and n-3 PUFA-containing A-CRLPs, but we detected no change in Nrf2 mRNA, as reported for oxidant-induced Nrf2 . Nonesterified DHA has been reported to inhibit the modest increases in EC VCAM-1 expression elicited by “atherogenic” mixtures of TRLs isolated from hyperlipidemic individuals , yet this did not correlate directly with their relative effects on HO-1 expression or their oxidative state indicated by TBARS content (n-3 PUFA A-CRLPs > n-6 PUFA A-CRLPs). Oxidation of the relatively n-6 PUFA-rich corn A-CRLPs may occur more slowly than the n-3 PUFA-containing particles, potentially explaining the difference in their TBARS contents. In addition, all A-CRLPs, including the SFA-rich particles, affected TXR and NADPH quinone oxidoreductase-1 expression to an equivalent extent after 16 h, despite the lower ability of SFA CRLPs to induce ROS compared with the other CRLPs. In keeping with their action to increase expression of cytoprotective genes, A-CRLPs had no detrimental effects on HAEC viability, so the ROS generated in response to these particles likely act as physiological signaling molecules rather than reflecting deleterious “oxidative stress” or lipid oxidation within ECs, cells which rely primarily on glycolysis and not on FA oxidation for energy  and of products of spontaneous oxidation in ECs, the activities of lipid-metabolizing enzymes, such as cyclooxygenases, lipoxygenases, and epoxygenases, operative in ECs, generate a plethora of oxidized FA metabolites that may contribute to the altered cell signaling and downstream gene expression described here. The specific oxidized lipid species contributing to the adaptive antioxidant/protective response triggered in ECs by rent DHA , suggest® A-CRLPs and increased by palm CRLPs, in keeping with previous studies demonstrating that nonesterified DHA decreases, whereas palmitate increases, NOX4 (n-3 PUFA-rich particles, with no effect on the response to palm A-CRLPs. Thus, our data suggest that NOX4 activity and mitochondrial ROS regulate HO-1 induction by A-CRLPs, with SFA-rich palm A-CRLPs inducing HO-1 primarily via NOX4 and n-3 PUFA-rich A-CRLPs using both mechanisms to upregulate endothelial HO-1. While it is known that inappropriate expression of NOX4 can lead to redox imbalance and pathology (A combination of pharmacological and siRNA approaches strongly implicated NOX4, highly expressed by ECs , 54, as es, NOX4 , 56 and athology , the NOXathology  and thatathology .MAPK, implicating these kinases in the control of ECs by dietary FAs. Given the known pro-survival actions of the Akt and MEK-ERK signaling pathways, we hypothesized that these might be key mediators of the antioxidant defense responses triggered by A-CRLPs. However, although Akt1 appears to regulate basal HO-1 expression, we found that p38MAPK is the dominant pathway coupled to downstream regulation of A-CRLP-driven Nrf2-dependent HO-1 expression, consistent with a growing body of evidence linking p38MAPK activation to cell survival and oxidative stress resistance (We showed that HAECs exposed to A-CRLPs exhibited enhanced phosphorylation of Akt, ERK1/2, and p38sistance , 60–62. PPARβ/δ is a lipid ligand-inducible transcription factor with established roles in the regulation of lipid metabolism and endothelial function , 63. AltThe modest stimulation of HO-1 induction by SFA-rich palm CRLPs, together with their lack of effect on cell viability, suggest that these particles are not overtly deleterious to ECs, contrasting with reports that nonesterified palmitate alone drives lipotoxicity and EC dysfunction in vitro . HoweverMAPK and parallel activation of the lipid sensor, PPARβ/δ (see Collectively, our data are consistent with a mechanism in which A-CRLP treatment results in activation of NOX4 leading to redox-dependent activation of p38


Background: Cereal crops, particularly wheat, are a major dietary source of starch, and the bioaccessibility of starch has implications for postprandial glycemia. The structure and properties of plant foods have been identified as critical factors in influencing nutrient bioaccessibility; however, the physical and biochemical disassembly of cereal food during digestion has not been widely studied.Objectives: The aims of this study were to compare the effects of 2 porridge meals prepared from wheat endosperm with different degrees of starch bioaccessibility on postprandial metabolism  and to gain insight into the structural and biochemical breakdown of the test meals during gastroileal transit.Design: A randomized crossover trial in 9 healthy ileostomy participants was designed to compare the effects of 55 g starch, provided as coarse (2-mm particles) or smooth (<0.2-mm particles) wheat porridge, on postprandial changes in blood glucose, insulin, C-peptide, lipids, and gut hormones and on the resistant starch (RS) content of ileal effluent. Undigested food in the ileal output was examined microscopically to identify cell walls and encapsulated starch.Results: Blood glucose, insulin, C-peptide, and glucose-dependent insulinotropic polypeptide concentrations were significantly lower  after consumption of the coarse porridge than after the smooth porridge (P < 0.01). In vitro, starch digestion was slower in the coarse porridge than in the smooth porridge . In vivo, the structural integrity of coarse particles (∼2 mm) of wheat endosperm was retained during gastroileal transit. Microscopic examination revealed a progressive loss of starch from the periphery toward the particle core. The structure of the test meal had no effect on the amount or pattern of RS output.Conclusion: The structural integrity of wheat endosperm is largely retained during gastroileal digestion and has a primary role in influencing the rate of starch amylolysis and, consequently, postprandial metabolism. This trial was registered at isrctn.org as ISRCTN40517475. Diets containing a high proportion of foods with a low glycemic index (GI)Compelling evidence reveals that food structure plays a critical role in influencing the bioaccessibility of starch and other macronutrients and the subsequent metabolic responses –11. CurrA common misconception is that any food containing whole grains, even in flour form, is slowly digested. However, the high GI of white and whole-meal wheat bread is linked to structural characteristics of the constituent flour, which contains mostly ruptured cells with high-bioaccessibility starch , 19. PreThe aim of this postprandial ileostomy study was to provide insight into the structural and biochemical degradation of wheat endosperm  during gastroileal digestion and its effects on metabolic responses . We hypothesized that porridge prepared from coarsely milled wheat endosperm (containing intact cells) would be digested more slowly and therefore elicit a lower glycemic response than a nutritionally matched porridge prepared from finely milled endosperm (lacking intact cells). This unique study in healthy participants with an ileostomy has provided new insight into both the mechanistic aspects of starch digestion and its metabolic consequences.Healthy participants aged 20–76 with ileostomies were recruited between September 2012 and July 2013 via the Ileostomy Association in London. All participants had undergone a proctocolectomy for ulcerative colitis, pure colonic Crohn disease, or lower bowel cancer at least 1 y before taking part in the study. The study protocol was registered as ISRCTN40517475 and approved by the relevant ethics committee in the United Kingdom (REC 12/LO/1016). All volunteers gave their written informed consent after being provided with oral and written information about the aims and protocol of the study.2) <20 or >35, fasting plasma glucose >7 mmol/L, serum cholesterol >7.8 mmol/L, or serum triacylglycerols >3 mmol/L. Fasting plasma glucose concentration, BMI, blood pressure, liver function, and blood cell counts were confirmed to be within prescribed limits before enrollment in the study. All participants were otherwise healthy, with no history of diabetes or signs of diagnosed gastrointestinal conditions, as confirmed by means of a full medical history and examination. The usual dietary habits of individuals were assessed from a 3-d diet diary, and the nutrient intake was analyzed with the use of NetWisp v.3.0 (Tinuviel Software).All participants had healthy small bowels with well-functioning ileostomies with <10 cm of small intestine resected. Exclusion criteria included the following: BMI (in kg/mThis study was one part of a 2-branch investigation (study 1 was on lipid bioaccessibility and study 2 on starch bioaccessibility) with common participant recruitment and screening processes for both branches. The participant characteristics, study details, and results presented in this publication are for the starch study only.A randomized crossover study design was used in which each participant received 2 experimental meals (in random sequence) separated by at least 1 wk. The random allocation of the treatment sequence was based on a computer-generated random number sequence. The study investigators generated the random allocation sequence, enrolled participants, and assigned participants to interventions. Because of the different physical appearance and texture of the test meals, study participants were not blinded; however, all samples were coded so that those investigators assessing outcomes  were blinded. All screening and study visits took place at the Clinical Research Facilities at St. Thomas’ Hospital, London.On the day preceding the study, participants were provided with a standardized low-fiber evening meal and advised to drink plenty of water. Participants were also instructed to avoid food and drink high in sugar, caffeine, or alcohol and to refrain from strenuous exercise both on the day preceding the study and throughout the study period.An overview of the study day protocol is provided in Meals low in resistant starch (RS) were provided after blood and effluent collections at 4 h, and again at 8 h (dinner with dessert), to minimize interference with ileal RS measurements. Participants were required to eat an identical menu for both study visits. Foods for all meals were purchased from Sainsbury’s, London. Lunch  consisted of a leafy salad with cheese and either tofu, chicken breast, beef, or tuna, served with yogurt and juice (from concentrate), and contained <4 g starch. Dinner consisted of mashed potato with fish, chicken, lamb, or beef and cooked carrots, and was followed by a high-calorie  dessert. Dinner with dessert contained up to 50 g starch in total, but the majority (at least 85%) of the starch in this meal originated from mashed potato, which contained starch that previously was found to be readily digested and absorbed  of the specific test meals . This caTest meals consisted of 2 freshly prepared porridges made from 77 g durum wheat endosperm, cooked in water and flavored with 17 g low-sugar black currant jam (Sainsbury’s Reduced Sugar Blackcurrant Jam) and 61 g jelly . Proximate analysis of the durum wheat  was performed by Premier Analytic Services, and additional direct measurements of available carbohydrate (starch and sugar) contents were performed with the use of a modified Megazyme kit  method . On the Supplemental Figure 1) confirmed that the coarsely milled particles contained a high proportion of intact cells, whereas particles <0.21 mm consisted almost entirely of ruptured cells. Durum wheat (Triticum durum L. “Svevo”), donated by Millbo, was selected for this study because its fracture properties make it particularly well-suited to the milling techniques used.The durum wheat endosperm component of the meal was provided in the form of finely milled flour (<0.2 mm) to create a smooth porridge or coarsely milled macroparticles (∼2 mm) to create a coarse porridge. The smooth and coarse porridges were predicted to be high- and low-bioaccessibility starch test meals, respectively. The endosperm material was prepared from de-branned durum wheat grains with the use of a Satake TH050 roller-mill (Satake Europe), as described previously . MicrostThe porridges were cooked under standardized heating conditions by simmering smooth or coarse wheat endosperm in water (while stirring vigorously) to achieve total and uniform gelatinization of the starch. Next, the porridge was allowed to cool for 5 min, during which time the jam and jelly flavoring was stirred in. The liquid component of the meal was standardized at 449 mL by providing the required volume of drinking water to make up for evaporative losses. The meal was served immediately thereafter to prevent further starch retrogradation, which is known to decrease the susceptibility of starch to amylolysis . Both teC∞). Because the Prussian blue assay quantifies products as maltose (not glucose) and either underestimates or fails to account for other minor starch digestion products , it slightly underestimates the extent of overall starch digestion. For purposes of comparison, therefore, all digestibility data were expressed as a percentage of total hydrolyzable starch based on maltose equivalents, with the use of extracted durum wheat starch, which had been hydrothermally processed to fully gelatinize the starch  during duodenal digestion is an important determinant of the duration and magnitude of the glycemic response , 22, 23.e starch , as a ree starch .2 EDTA-coated tubes preloaded with either 100 μL aprotinin  for GIP and PYY analyses or 40 μL dipeptidyl-peptidase-IV inhibitor (Merck Millipore) for GLP-1 and cholecystokinin analyses, and kept on ice for 15 min followed by centrifugation and plasma storage. All blood samples were centrifuged at 1500 × g for 15 min at 4°C, and aliquots of the supernatant were collected and stored at either −40°C  or −80°C (for gut hormones) until analyzed.Venous blood samples for glucose, insulin, C-peptide, triacylglycerols, and NEFA analyses were collected and handled as described previously . Blood sIleal effluent was collected every 2 h for up to 10 h and overnight at the convenience of participants. Effluent samples were obtained from the participants, who emptied the contents of their ileostomy pouches into Whirl-Pak (Nasco) specimen bags at predetermined time points. Immediately after each effluent collection, the sample was weighed. A small subsample was taken directly into the fixative , and the remaining effluent was then homogenized . For starch and sugar analysis, ∼2 g of the homogenized effluent was weighed into 4 × 15 mL BD Falcon tubes (BD Biosciences) preloaded with 95% (vol:vol) ethanol to inactivate any residual enzyme activity. These samples were stored overnight at 4°C before starch and sugar analysis. Subsamples of the homogenized effluent were also taken for determination of dry matter, and these were frozen immediately (−80°C) to prevent further degradation. Overnight samples were collected into specimen bags by the participants ad libitum and subsequently placed in absorbent padding between eutectic freezer blocks  in a polystyrene box to freeze the samples at the time of collection. These frozen samples were stored for future analysis.Plasma glucose, serum triacylglycerol, and serum NEFA concentrations were determined on an iLab 650 auto-analyzer (Instrumentation Laboratories) with the use of Glucose Oxidase and Triglyceride IL test kits (Instrumentation Laboratories) and a Randox NEFA kit (Randox Laboratories). Serum insulin and C-peptide and plasma GLP-1, GIP, and total PYY (1–36 and 3–36 forms) concentrations were determined by standard methods as described previously . Cholecyg at 22°C, and the resulting supernatant was analyzed for total reducing sugar, while the pellet was analyzed for total starch content. Total reducing sugar was determined by 3,5-dinitrosalicylic acid assay in which 15 μL of the supernatant (diluted in water if necessary) was transferred into a 96-well plate  to which was added 5 μL of 3M NaOH and 20 μL 3,5-dinitrosalicylic acid reagent consisting of 1.0% (wt:vol) 3,5- dinitrosalicylic acid, 1.6% (wt:vol) NaOH and 30% (wt:vol) sodium potassium tartrate, prepared in deioinized water. Standard samples containing known concentrations of maltose were also included. The plate with samples was then sealed and incubated in a water bath at 90°C for 5 min, after which 200 μL deionized water was added. The absorbance of standards and samples were then measured at 544 nm with the use of a FluoStar Optima plate reader (BMG Labtech), thereby enabling total reducing sugar concentrations to be calculated as maltose equivalents. Starch determinations were carried out with the use of a scaled-down version  present in the ileal effluent, because the sugars are likely to be products of starch degradation , as described in previous studies , 29. To  version  of the M1, in which t is the time interval between ingestion and ileal recovery of RS for samples 0–N.Mean transit time (MTT) was calculated  as shown2 with 5% wt:vol potassium iodide] and viewed on a Zeiss Axioskop 2 mot plus light microscope. Images were captured with a Zeiss AxioCam HRc and AxioVision v3.1 microscope software (Carl-Zeiss).For examination by light microscopy, samples of ileal effluent were immersed in Karnovsky’s fixative , and left to fix at room temperature for at least 24 h. The samples were subsequently rinsed in sodium cacodylate buffer (0.1M), dehydrated through increasing concentrations of ethanol, and then infiltrated with freshly prepared LR white resin mixture (Sigma-Aldrich) prepared in 1.98% (wt:wt) benzoyl peroxide with the use of absolute ethanol as the transition solvent. Finally, resin-embedded samples were sealed in BEEM 00 capsules and polymerized at 60 ± 2°C for 24 h. The cured samples were trimmed and sectioned (0.5–1.0 μm) on an Ultracut E Reichert-Jung microtome mounted with a glass knife. Sections were stained with Lugol’s iodine [2.5% (wt:vol) It tests were used to identify time points with significantly different values between the 2 meals. The Bonferroni method was used to correct for multiple comparisons. All statistical analyses were performed on data obtained between 0 and 240 min, and additional subanalysis was performed for glucose, insulin, C-peptide, and GIP data obtained from AUC values calculated for the 0–120 min period, because this is the most dynamic part of the blood response curves to a carbohydrate-rich meal and is a common indicator of glycemia . Graphs and incremental AUCs were obtained with SigmaPlot 12.5 (Systat Software). Statistical analysis was performed with IBM SPSS Statistics 20.0. Data were checked for normality with the use of a Shapiro-Wilks normality test before analysis. A repeated-measures ANOVA was performed with time and meal as “within” factors to evaluate the effect of the test meals on postprandial responses. When significant meal × time effects were detected, Student’s paired glycemia . StatistThe cooked particles of wheat endosperm used to make the smooth and coarse porridge meals were found to have contrasting starch amylolysis characteristics  and one further participant withdrew during the second intervention because of illness . Because we could not rule out the possibility that the abdominal discomfort experienced by this participant was related to the test meal, no further interventions were attempted with this individual. A total of 9 participants (7 women and 2 men) completed the study and participant characteristics are shown in A consort flow-chart of participants through the study is shown in P = 0.038) and over 240 min for insulin (P = 0.046), C-peptide (P = 0.018), and GIP (P = 0.024), but not for glucose (P = 0.229). Also, significant meal × time interactions over 240 min were observed for glucose (P < 0.001), insulin (P = 0.035), C-peptide (P < 0.001), and GIP (P < 0.001). After 120 min, the incremental AUC for glucose, insulin, C-peptide and GIP were 33%, 43%, 40%, and 50% lower (P < 0.01), respectively, after consumption of coarse porridge than after smooth porridge. The smooth porridge, which consisted of highly bioaccessible starch, elicited a rapid and large glycemic response within the first 60 min, followed by a reduction in glucose with an overshoot to nearly 1 mmol/L below the fasting value. In contrast, the coarse porridge elicited a smaller rise in blood glucose and insulin concentrations, with a much slower rate of decline after 90 min; indeed, the glucose concentrations remained above fasting even after 4 h. The peak concentrations of glucose, insulin, C-peptide, and GIP were 12%, 32%, 37%, and 60% higher (P < 0. 01), respectively, after smooth than after coarse porridge and were reached within 90 min of ingesting the test meals.Plasma glucose (0–120 min), serum insulin, C-peptide, and plasma GIP concentrations were significantly lower after the coarse porridge  than after the smooth porridge containing high-bioaccessibility starch , significant meal × time effects were observed over 240 min for GLP-1 (P = 0.009) and PYY (P = 0.053). Plasma GLP-1 and PYY concentrations increased rapidly after test meal consumption and returned to baseline concentrations within 2 h of test meal ingestion. Peak concentrations after smooth and coarse porridge consumption were not significantly different . Cholecystokinin concentrations fluctuated throughout the 4 h period, and no clear pattern was evident (data not shown).Although there was no main effect of the meal on GLP-1 and PYY . Statistical analysis showed that the test meals had no effect on the pattern of the response curves ; however, there was a tendency toward a lower triacylglycerol response after the coarse porridge  are shown in porridge .Supplemental Figure 4). A total of ∼3 g of RS  was recovered at the terminal ileum during the 10 h after ingestion of both smooth and coarse porridge meals . For both test meals, the amount of RS recovered over the entire 24-h collection period, estimated from cumulative data, was higher than the 10-h values . However, when considering the microstructural observations (see the next section) it seems likely that the starch measured in the overnight samples may have originated from sources other than the test meals . Thus, the RS recovered during the first 10 h may be more representative of the RS content of the porridge test meals than the 24-h samples.The smooth and coarse porridges did not have any significant effects on the total amount of ileal output or meal transit, or on the amount of RS in ileal effluent  were freshly prepared, differing only in particle size (2 mm compared with <0.2 mm), and were swallowed with minimal mastication. This design ensured that any observed metabolic effects could be attributed to the structural properties of the test meals during gastroileal digestive transit.Our in vitro digestion experiments clearly demonstrated that starch entrapped within the cells of the coarse wheat endosperm particles was hydrolyzed (by α-amylase) at a considerably slower rate than the exposed starch in the fine flour particles. Hence, the structural integrity of dietary fiber, as intact cell walls, can strongly influence the rate at which entrapped starch is made bioaccessible and thereby affect the time course of glucose absorption into the portal blood.On the basis of the in vitro data showing marked differences in the rate of amylolysis between coarse endosperm and flour, we hypothesized that the same materials would also elicit differences in the in vivo glycemic responses. Indeed, the coarse porridge evoked significantly lower glycemic, insulinemic, and C-peptide responses compared with the smooth porridge. The magnitude of the reductions in glycemia and insulinemia achieved in the present study were similar to, if not greater than, the glycemic reductions achieved in some previous studies with the addition of soluble fiber or use of whole grains , 34. OurMicroscopic examination of ileal effluent revealed that coarse (2-mm) particles of wheat endosperm retained structural integrity during the first 10 h of gastroileal transit but that the majority of encapsulated starch was eventually digested from seemingly intact cells. To our knowledge, this is the first time that such microstructural observations have been reported for wheat endosperm digested in the upper gastrointestinal tract of humans. After 10 h, the majority of the test meal had reached the terminal ileum, and only negligible amounts of RS remained , irrespective of particle size. This is consistent with previous ileostomy studies of white wheat bread and cornflakes  and suggIt has been suggested that limiting the bioaccessibility of nutrients may reduce the magnitude of gut hormone responses , 38. TheAlthough the test meals were rich in starch and contained little fat (1.6 g/portion), postprandial reductions in triacylglycerol and NEFA concentrations were observed. These reductions probably reflect the net effect of the insulin-mediated uptake of fatty acids and the inhibition of lipolysis in adipose tissue , 43. No Triticum aestivum (bread wheat), are affected by processing and digestion, but the results of the current work on durum wheat, which is used in bread- and pasta-making, demonstrate proof of principle.Preserving the natural structural integrity (intact cells) of wheat endosperm, and indeed other edible plant tissues, during processing could provide a means of limiting the rate and/or extent of energy uptake from food postingestion and may have significant and important applications in the prevention and management of cardiometabolic diseases. Although wheat breads containing 50–80% whole grains have previously been shown to elicit lower glycemic responses than white bread , granaryThere is now convincing evidence that the tissue structures of some plant foods  can be retained to a greater or lesser extent during mastication  and gastIn conclusion, these results provide the first evidence that the structural integrity of wheat endosperm is a major factor in influencing starch bioaccessibility and that manipulating the structural integrity of the endosperm has the potential to greatly influence postprandial metabolism.


Uncooked corn-starch (UCCS) has been the mainstay of therapy for the hepatic glycogen storage diseases (GSD) but is not always effective. A new starch (WMHMS) has demonstrated a more favourable short-term metabolic profile.To determine efficacy and safety of a new uncooked starch (WMHMS) compared to UCCS over 16 weeks treatment with each.A double-blind cross-over study of 10 adults  with GSD Ia and Ib. After an individualised fast, subjects were randomised to take a 50 g starch-load of either WMHMS or UCCS. Starch-loads terminated when blood glucose was < 3.0 mmol/L or the subject felt subjectively hypoglycaemic. Anonymous biochemical profiles were assessed by 2 investigators and a starch administration schedule recommended. Each starch was delivered in coded sachets and intake was monitored for the following 16 weeks. After a washout period, the protocol was repeated with the alternative product.4 subjects failed to establish therapy on the cross-over limb. Data from 7 paired starch load showed: longer median fasting duration with WMHMS , slower decrease in the glucose curve (0.357 versus 0.632 mmol/hr p = 0.028) and less area under insulin curves for the first 4 hours (p = 0.03). Two of six subjects took 50% or less WMHMS compared to UCCS and one took more. Plasma triglycerides, cholesterol and uric acid were unchanged after each study phase.WMHMS leads to significant reduction in insulin release and reduced starch use in some GSD patients. The glycogen storage diseases (GSDs) comprise a group of rare inherited disorders of glycogen metabolism. GSD I (OMIM 232200) is caused by reduced activity of glucose-6-phosphatase ; GSD Ia by deficiency of the hydrolytic enzyme; and GSD Ib by deficiency of the endoplasmic reticulum transmembrane glucose 6-phosphate transport protein, G6P translocase. The major metabolic consequence of ineffective function of G6Pase is hypoglycaemia, provoked by relatively short fasts. Secondary metabolic disturbances include hyperlactataemia, hyperuricaemia and hyperlipidaemia [Maintaining blood glucose concentration in the normal range improves secondary biochemical features as well as clinical parameters such as growth. The introduction of continuous nasogastric glucose polymer feeds showed this clearly ,3. The sApart from sustained normoglycaemia without excessive insulin rise, the features of an ideal starch for treatment of patients with the hepatic GSDs include suppression of secondary biochemical abnormalities, palatability, convenience, few side-effects and maintenance of normal appetite , , prepared in an emulsified form (WMHMS)  has shown improvement in the duration of normoglycaemia and glucose profile in particular of patients with glycogen storage disease type Ia and Ib, ,12, but The study protocol was approved by the ethics committee nominated by the UK Central Office for Research Ethics Committees (COREC). All adults that attended the Charles Dent Metabolic Unit clinic, London UK with a enzymatic and/or molecular proven diagnosis of GSD Ia or Ib, that were taking uncooked corn-starch as part of their dietary regimen were invited to participate. All recruited subjects gave written informed consent to participate.The study had a randomised double-blind cross over design. Subjects were allocated an anonymous reference number and were randomly allocated to receive either UCCS or WMHMS. Each starch was manufactured using food-grade techniques and packaged in identical sachets bearing a reference code. The patient reference numbers and sachet reference code were paired by Vitaflo Ltd and the supervising physicians  were blinded to this pairing. The supervising physicians devised a safe personalised fasting period for each patient, prior to each starch load based on previous corn-starch loads and medical history.Patients attended in the morning after their individualised fast. An intravenous cannula was placed in the patient’s arm and baseline blood samples were collected. When the participant’s blood glucose was below 4.5 mmol/L, 50 g of the nominated starch mixed in cold water was taken orally. Blood samples were performed at 30 minute intervals for the next 120 minutes and hourly thereafter. No further intake, apart from drinking water, was allowed. The starch load test ended when the patient had fasted for 10 hours, the plasma glucose was ≤3.0 mmol/L on the bedside glucose analyser  or the patient wished to end the test. When the blood glucose was ≤ 3.5 mmol/L, blood tests were performed at 30 minutes intervals until the test end.Plasma was extracted from whole blood samples immediately at the bedside. Plasma glucose and lactate were analysed immediately . Serum to measure insulin was frozen within 30 minutes of collection and subsequently thawed and analysed by a solid-phase, two-site chemiluminescent immunometric assay . At baseline, “fasting” cholesterol, triglycerides and uric acid were taken.Data from the first starch load was used to guide management using the particular starch for the subsequent 16 weeks. After a further washout period of 2 – 4 weeks on normal treatment, participants re-attended for a further starch load with the alternative starch.The glucose and lactate data from the load test were interpreted by two investigators who have experience in interpreting these results (PJL and MPC). Neither of these investigators had any other subject-related role in the study. They were blinded to the identity of both the participant and the starch. They were given some background information of normal starch dosing and the last available corn-starch load data for that participant. On the basis of this information a safe frequency of daytime dosing for the given starch was discussed, agreed and prescribed. The participants were asked to take the nominated starch for 16 weeks. If the volunteer took an overnight infusion of glucose polymer, this was not altered.The starch was delivered in boxes with individual sachets bearing a reference code identifying the type of starch only to representatives from Vitaflo Ltd. The participants were asked to take the nominated starch as recommended by the blinded prescribers. However, it is recognised that some patients take less or more starch doses in the home environment – the participant was asked to note what they actually took and this was noted by a telephone call made by one member of the research team on a weekly basis.For the 16 week study period, participants were asked to keep a record of subjective episodes of hypoglycaemia. Whilst measurement of blood glucose was not recommended, participants were also asked to keep a record of any blood glucose measurement that was taken. One member of the research team contacted the participant to note these data as well as general information about symptoms and appetite over the preceding week. If the participant had an inter-current illness such as diarrhoea or vomiting necessitating additional glucose polymer use or was admitted to hospital for intravenous dextrose, the entire week’s data for this period was omitted from analysis. A maximum of 2 consecutive weeks and 4 total omitted weeks were allowed, from each 16 week period, to remain included in the trial. The participant was asked to complete a diet diary after they had taken the nominated starch for at least 6 weeks. They were asked to write down everything they ate for a 3 day period and the nutritional content of the diet including the starch taken were analysed by an experienced state registered dietician (MFL) using specialised software At the end of the 16 week study period, the research volunteer was asked to attend an outpatient clinic. They were weighed and examined and “fasting” plasma triglycerides, cholesterol and uric acid were taken. This was followed by a washout period of 2 – 4 weeks and the patient re-attended for a starch load with the alternative starch. Pre-starch-load preparation including fasting period and assessments were identical. The subject was asked to take the same meals at the same times the day before the second starch-load as they had for the first one.t-test for each individual. Total energy intake and energy intake derived from both treatment and dietary carbohydrate were obtained from analyses of dietary diaries and compared by paired non-parametric analyses  All analyses were two-tailed. Test durations were compared both paired and unpaired by Wilcoxon’s sign rank test and the unpaired data plotted on a Kaplan-Meier curve as shown in Figure s Figure , as was s Figure . Data we10 volunteers were recruited to the study. Patient details are shown in Table Whilst on UCCS, 4 of 8 subjects were admitted into hospital – all for reasons not deemed to be trial related – one for dysphagia related to a substance ingested by mistake, a second one for haematuria and abdominal pain probably related to a renal stone (this subject withdraw from the second limb of the study on WMHMS) and the remaining two for vomiting associated with hypoglycaemia during an inter-current viral illness. Whilst on WMHMS, 1 subject with GSD Ib was admitted twice to hospital, firstly with post streptococcal glomerulonephritis and secondly with a viral lower respiratory tract infection (this subject subsequently withdrew without taking UCCS), and the complications not deemed to be trial related.8 starch loads were performed using UCCS with a median duration of 5 hours and 9 starch loads with WMHMS with a median duration of 7.5 hours , and (980 vs 2100 g), p < 0.001 for both. One subject took more WMHMS than UCCS (1020 g vs 690 g p < 0.001), but this was related to better compliance with prescribed therapy after she was notified in the washout period about deteriorating long-term renal function. This was substantiated by an increase of 4.3 Kg weight over the washout period on normal therapy prior to starting WMHMS. The reduction of starch use in two subjects whilst taking WMHMS was not matched by broadening of dietary intake. As a consequence their total energy intake reduced from 2410 and 3044 Kcal/day to 1876 and 2454 respectively. There was no difference in median weight change between the 2 populations: + 0.25 Kg for WMHMS and – 0.05 Kg for UCCS. Patient specific data is indicated in Table Mean weekly starch use by the 6 patients that completed the study is shown in Figure  980 vs 200 g, p <The dietary regimen for the treatment of type I glycogen storage disease has previously been called the “intensive regimen”, with hypoglycaemia a predominant feature after even quite short fasting intervals ,14. For Waxy maize starch differs from typical corn-starch in that it has a high proportion of amylopectin. It has been subject to research interest due to its low glycaemic index . ChemicaOnly two individuals took significantly less WMHMS than UCCS over the course of this study. Part of the reason for this was because of the safety aspect of the study design. Three individuals that took overnight pump feeds of glucose did not have this aspect of their treatment altered. Consequently, there was a reduced period of 14 or 15 hours in which any dose of studied carbohydrate could be taken. The only real options in that time period were to prescribe either two doses at 7 hour intervals or three doses at 4 – 5 hour intervals; for each of these patients, the more conservative 4 hourly regimen was selected for both starches, even if the data suggested someone could fast for six hours comfortably. These subjects, who place naso-gastric tubes themselves every night, were particularly compliant, only ever taking more than the prescribed dose, but never less. Therefore, no difference is seen for these 3 individuals. By contrast, the 3 subjects who take corn-starch both day and night had more flexibility with prescriptions and also varied the amount they took more. Two of these three subsequently took significantly less WMHMS and the other subject complied more with the prescribed dose due to adverse long-term deterioration in renal function.13CO2 with WMHMS. This may have been confounded by the age range of subjects and the variable duration of tests. Sustained incretin release after UCCS ingestion has been shown when compared to other carbohydrates. This may also account for increased insulin release when UCCS is compared to waxy maize starch [The mechanism of potential extended duration of WMHMS needs more investigations. Our previous study , demonste starch . There ae starch . It is pTM) with UCCS (Argo TM) and dextrose at rest, followed by an hour of exercise and a subsequent 2 hour recovery period [WMHMS has now been studied in healthy volunteers allowing its role to be investigated under different physiological stresses to patients with GSD. Roberts et al studied 9 male competitive cyclists during and after intense exercise . They coy period . In the y period .The significant lifelong carbohydrate intake that these patients take put them at particular risk of insulin resistance and non-insulin dependent diabetes mellitus, although there are few reports of these complications . Data frThis study with a small number of patients with GSD Ia and Ib, demonstrated that WMHMS can be safely incorporated into the dietary regimen of these patients. Our experience shows that these patients are a difficult group to study. From a metabolic perspective, they are brittle with a tendency for hypoglycaemia after very short fasts with marked fluctuation of related variables, insulin and lactate, as well as longitudinal indices of metabolic control such as triglycerides and uric acid. These study difficulties have been commented on specifically on a meta-analysis of GSD Ia dietary therapy . Co-morbThis study has demonstrated an improved glucose and insulin profile after 50 g of WMHMS was given when compared to UCCS. The study provides preliminary data that the starch can be safely introduced into the dietary regimen of patients without untoward effects. Further studies determining mechanism of action, role in other disorders, optimal dosing and long-term effects should be performed.


HHP-treated potato starch (PS) exposed to α-amylase  showed a significantly greater degree of hydrolysis and amount of reducing sugar released compared to α-amylase at a concentration of 0.04% (w/v) or 0.02% (w/v). Native PS (NPS) granules have a spherical and elliptical form with a smooth surface, whereas the hydrolyzed NPS (hNPS) and hydrolyzed HHP-treated PS granules showed irregular and ruptured forms with several cracks and holes on the surface. Hydrolysis of HHP-treated PS by α-amylase could decrease the average granule size significantly (p <0.05) from 29.43 to 20.03 μm. Swelling power decreased and solubility increased with increasing enzyme concentration and increasing pressure from 200–600 MPa, with the exception of the solubility of HHP-treated PS at 600 MPa (HHP600 PS). Fourier transform infrared spectroscopy (FTIR) showed extensive degradation of the starch in both the ordered and the amorphous structure, especially in hydrolyzed HHP600 PS. The B-type of hydrolyzed HHP600 PS with α-amylase at a concentration 0.06% (w/v) changed to a B+V type with an additional peak at 2θ = 19.36°. The HHP600 starch with 0.06% (w/v) α-amylase displayed the lowest value of To (onset temperature), Tc (conclusion temperature) and ΔHgel . These results indicate the pre-HHP treatment of NPS leads to increased susceptibility of the granules to enzymatic degradation and eventually changes of both the amorphous and the crystalline structures.The effect of high hydrostatic pressure (HHP) on the susceptibility of potato starch  suspended in water to degradation by exposure to bacterial α-amylase  for 40 min at 25°C was investigated. Significant differences ( Solanum tuberosum) is the third most important food crop in the world after rice (Oryza sativa) and wheat (Triticum aestivum) in terms of human consumption [Potato  on the susceptibility of PS to α-amylase hydrolysis and to determine the morphology, thermal properties and crystal properties of native, HHP-treated and hydrolyzed HHP-treated PS. This knowledge will provide a better understanding of modified PS as a functional agent in industry.–1) was purchased from Sigma Chemical Co., Ltd. . All other chemicals used in this study were of analytical grade.A Chinese potato cultivar (Qingshu no. 9) frequently used for industrial starch production was selected for this study. Qingshu no. 9 was grown in North Eastern China. Bacterial α-amylase  sodium bisulfite (1 kg of tuber in 1 L of solution) for 10 min and then blended in a domestic juice extractor for 3–4 min. The resulting slurry was passed through a fine muslin cloth to separate the cell debris as described by Peshin . The sus–1 and was kept at 200, 400 or 600 MPa for 40 min, which were optimized by single-factor experiment (data not shown). After each treatment, the pressure was decreased to atmospheric pressure at ~100 MPa min–1, the starch was decanted, freeze-dried and stored in a desiccator. Control starch (NPS) was treated at atmospheric pressure (0.1 MPa) at room temperature (~ 25°C).Treatment with HHP was done in a discontinuous HHP machine  at a pressure of 600 MPa in a steel vessel of 0.6 L capacity (60 mm × 210 mm). A mixture of starch  and water in a polymer pouch was placed into the vessel containing water as the pressure medium. The temperature inside the vessel was controlled at 25°C by a circulating thermostatically controlled water bath. In each experiment, pressure was increased at a rate of ~100 MPa ming for 15 min. The hydrolysis residue was washed with water and ethanol. The hydrolyzed starch obtained was air-dried in a forced-air oven at 50°C for 24 h. The percentage (dry weight basis) of hydrolysis was calculated as follows [W0 is the weight (dry basis) of untreated starch and W1 is the weight (dry basis) of hydrolyzed starch.The starch samples were hydrolyzed with bacterial α-amylase, in duplicate, essentially as described by Franco and Ciacco  but with follows :HydrolyThe starch granules were observed using a scanning electron microscope . The starch samples were sprinkled onto double-sided tape mounted on an SEM stub, coated with gold and placed into the SEM chamber. Photomicrographs were taken using an SEM apparatus at an accelerating voltage of 15 kV.D50).Particle size distribution of the starch granules was measured with a laser particle size analyzer . The mean granule size of each starch sample was calculated as the mean volume diameter (–1 over a temperature range of 30°C–120°C. Peak temperature (Tp); onset gelatinization temperature (To); conclusion temperature (Tc) and enthalpy of gelatinization (ΔHgel) were recorded. The gelatinization temperature range (R) was calculated as 2(Tp−To) [Thermal properties of the starches were analyzed using a differential scanning calorimeter  essentially as described by Kaur et al.  but withTp−To) The FTIR spectra of the native and hydrolyzed HHP-treated starches were obtained with an IR spectrophotometer . Starch samples were blended with KBr powder and pressed into thin films before the measurement.–1 were recorded.The XRD patterns of native and hydrolyzed HHP-treated starches were collected with X’pert PRO  according to the modified method described by Chang et al. . The difg for 15 min. The supernatant was decanted into an evaporating dish and dried at 105°C for 5 h. The dried supernatant and the residue were weighed. The solubility  and swelling power  were calculated as follows:Swelling power and solubility of the starches were determined essentially as described Crosbie  but withp < 0.05. All data were expressed as mean ± standard deviation.All experiments were done in triplicate and statistical analysis was done with SAS version 9.2  to determine difference of means using Duncan’s multiple range test functions. Differences were considered to be statistically significant at –1 and 16.53%, respectively. Hydrolyzed starch with HHP600 by 0.06% (w/v) α-amylase yielded the greatest amount of reducing sugar and the highest percentage of hydrolysis, 29.64 mg mL–1 and 49.36%, respectively. Using an increased enzyme concentration with hNPS and HHP hydrolyzed starches increased the yield of reducing sugar and the percentage hydrolysis. This observation was based on an increased amount of glucose released as a result of the cleavage of α-(1→4) glycosidic bonds. In addition, owing to the effect of high pressure on the starch granule, some hydrogen bonds in the amorphous area were broken and the amorphous growth ring regions were hydrated; therefore, amorphous lamellae were formed in the crystalline domain [During hydrolysis with α-amylase, the α-(1→4) linkages of the interior of polymeric starch chains were cleaved and produced simple sugars as further hydrolysis products of maltose, maltotriose and α-dextrins. The varying level of HHP influenced the action of the enzyme at different concentrations on granular starch . The rede domain  and hydrWe found a significant difference between hNPS and HHP-treated hydrolyzed starch at all three concentrations of enzyme . The outD50) (29.43±0.1 μm) and diameter range (1.45–130.37 μm). These findings could be attributed to the partial gelatinization of starch granules. PS, which has a B-type pattern and a greater resistance to pressure compared to other starches [D50 of the starch granules decreased with increase in enzyme concentration. This could be attributed to the degradation of starch granules after enzyme digestion. For better understanding and monitoring the extent of hydrolysis, SEM photographs of native, HHP-treated, native hydrolyzed and hydrolyzed HHP600 potato starch are shown in The shape and granule size of various modified starches are given in starches , was notTo) ranged between 52.42°C (for hydrolyzed HHP600 PS with 0.06% (w/v) α-amylase) and 58.37°C (for hNPS with 0.06% (w/v) α-amylase). The peak temperature (Tp) ranged between 57.59°C (for PS HHP-treated at 400 MPa) and 63.19°C (for hydrolyzed HHP600 PS with 0.06% (w/v) α-amylase). Gelatinization enthalpies (ΔHgel) ranged between 4.41 J g–1 (for hydrolyzed HHP600 PS with 0.06% (w/v) α-amylase) to 10.92 J g–1 (for hNPS with 0.06% (w/v) α-amylase). Compared with NPS, a reduction was found in gelatinization temperatures and enthalpy with increasing pressure treatment, which was accordance with the results of lotus seed starch treated with HHP [To and Tp) and the enthalpies of gelatinization (ΔHgel) shifted to higher values with increasing enzyme concentration. Two events occur during endothermic transition First, gelatinization, primarily water molecules diffuse freely into the amorphous region of starch. Second, they penetrate the crystalline region [Zea mays var. ceratina), maize (Zea mays var. indentata), rice (Oryza sativa), potato (Solanum tuberosum) starch and wheat (Triticum aestivum) starch [The gelatinization parameters for native, HHP-treated, native hydrolyzed and hydrolyzed HHP-treated PS are given in with HHP . Starch with HHP . For hNPe region . Enzymat) starch .R-value between the samples suggested a transformation in the crystalline regions of the starch granules [R-value for the various starch types might be owing to the differences in the strength of heterogeneous crystals.The difference in granules . Gunaratgranules  suggesteOverall, the decrease in transition temperatures and enthalpies of gelatinization of the hydrolyzed HHP-treated samples demonstrated a reduction in the amorphous area of the granules, which was accompanied by a change in the crystalline regions following a cooperative process in the case of HHP, but led to more crystalline structure in the hNPS starches.600 potato starch are presented in –1) represent complex vibrational stretches associated with intermolecular hydroxyl groups [600 PS with 0.02% and 0.04% (w/v) α-amylase showed the same broad bands from 3100–3700 cm–1 (600 PS with 0.06% (w/v) α-amylase showed a greater intensity  glycosidic linkage (C-O-C). The peaks at 1081, 1156, and 1020 cm–1 were characteristic of the anhydrous glucose ring C-O stretch [–1 was sensitive to changes of crystallinity [FTIR spectra of native, native hydrolyzed and hydrolyzed HHPl groups  The nati700 cm–1 , while hntensity . The shaibration , as shown starch , of whic stretch . The banallinity . Compare600 PS are presented in 600 starches, however, displayed different behavior. The intensity of the peaks was changed slightly owing to the loss of crystallinity during treatment with HHP [600 potato starch with 0.06% (w/v) α-amylase was in the transformation from B type to B+V type with a majority of B-type crystallite. It might be owing to an increased proportion of short amylose chains, which tend to form double helices [The XRD pattern of NPS, native hydrolyzed and hydrolyzed HHPwith HHP . The sma helices . In addi600 sample hydrolyzed with different concentrations of enzyme showed decreased solubility compared to hydrolyzed HHP-treated (200 and 400 MPa) starches at different enzyme concentrations. It is likely high pressure induced formation of the amylose–lipid complex and inhibited amylose leaching, which led to decreased solubility [The swelling power and solubility of NPS, hydrolyzed NPS, HHP-treated and hydrolyzed HHP-treated PS are presented in lubility .D50 value, thermal properties and crystalline structure pattern. Swelling power and solubility decreased with increasing level of HHP, owing, at least in part, to change in the intramolecular structure of starch. The hydrolyzed HHP-treated starch, however, showed decreased swelling power but increased solubility with increasing HHP level and enzyme concentration. By contrast, hydrolyzed starches HHP-treated at 200 and 400 MPa showed little or no difference in structural, morphological or functional properties compared to the native form, which showed that potato starch with B-type pattern are resistant to high pressure, especially below 600 MPa. The information obtained from this study suggests the possibility of modifying the physicochemical, functional and morphological properties of PS without heat treatment, obviating any undesirable side effects. This modification process could be practical in the food and related industries.This study demonstrated the effect of HHP and enzymatic hydrolysis with α-amylase at different levels of pressure and concentration of enzyme on various physicochemical properties of potato starch. Pre-HHP treatment of starches at 600 MPa increased susceptibility to enzymatic hydrolysis with an increased amount of reducing sugar produced. Further, there was significant change of the


Solanum tuberosum L.) a small fraction of starch is converted into the reducing sugars glucose and fructose. Reducing sugars accumulate in response to cold temperatures. Even small quantities of reducing sugars affect negatively the quality of processed products such as chips and French fries. Tuber starch and sugar content are inversely correlated complex traits that are controlled by multiple genetic and environmental factors. Based on in silico annotation of the potato genome sequence, 123 loci are involved in starch-sugar interconversion, approximately half of which have been previously cloned and characterized. By means of candidate gene association mapping, we identified single-nucleotide polymorphisms (SNPs) in eight genes known to have key functions in starch-sugar interconversion, which were diagnostic for increased tuber starch and/or decreased sugar content and vice versa. Most positive or negative effects of SNPs on tuber-reducing sugar content were reproducible in two different collections of potato cultivars. The diagnostic SNP markers are useful for breeding applications. An allele of the plastidic starch phosphorylase PHO1a associated with increased tuber starch content was cloned as full-length cDNA and characterized. The APHO1a-H allele has several amino acid changes, one of which is unique among all known starch/glycogen phosphorylases. This mutation might cause reduced enzyme activity due to impaired formation of the active dimers, thereby limiting starch breakdown.Starch accumulation and breakdown are vital processes in plant storage organs such as seeds, roots, and tubers. In tubers of potato ( Solanum lycopersicum) and potato (Solanum tuberosum) are closely related species with highly syntenic genomes and high gene sequence similarity  with amylose content of rice grains , starch branching enzyme , ADP-glucose pyrophosphorylase (large subunit) , alpha-amylase (AMY), and beta-amylase (BMY) associated with starch properties of maize kernels (INV), L-type starch phosphorylases (PHO1), soluble starch synthase (Sss), and AGPaseS were associated with chip or processing quality (corresponding to reducing sugar content) and tuber starch content, in some cases also with tuber yield and starch yield  probed genes with key functions in starch-sugar interconversion for association of SNPs with tuber starch and sugar content, chip quality, tuber yield, and starch yield; (ii) cloned and functionally characterized a starch phosphorylase cDNA allele diagnostic for increased tuber starch content and better processing quality; (iii) identified SNP alleles and haplotypes corresponding to known SSCP markers diagnostic for tuber quality traits; (iv) assessed the diagnostic value of markers associated with processing quality in new germplasm; and (v) provide an overview on the genomic organization of the genes functional in starch-sugar interconversion based on the potato genome sequence , yield, starch content, tuber shape, and plant maturity  were part of a second, independent population of 205 tetraploid genotypes (the ‘BRUISE’ population)  in response to cold storage  using the following cycling conditions: Initial denaturation 4 min at 94°, 34 cycles denaturation at 94° for 45 sec, annealing for 30 sec at the temperature specified in InvDE141 gene was amplified as described . In the case of triallelic SNPs additional classes such as AAAC or AABC were assigned. The SNP allele dosage in heterozygous individuals  was estimated from the height ratio of the overlapping base calling peaks manually and using the Data Acquisition and Analysis Software DAx . For statistical analysis, genotypes were converted in numerical values from 0 to 4  or 0 to N .A total of 50 ng of genomic DNA template in 25 μL of 1x ammonium buffer , 2.5 mM MgClStp23-8b, StpL-3b, StpL-e3, Pain1-8c, and InvGE-6f were performed as described previously , 0.2 mM of each dNTP, 1 μM each forward and reverse primers  including 0.2 mM each dNTP, 1 μM each forward and reverse primers, and 1.5 U of Taq-Polymerase  or FastStart High Fidelity PCR System (Roche) or AccuPrime Pfx DNA Polymerase (Invitrogen Life Technologies). Cycling conditions were as follows: initial denaturation for 2 min at 94°, 20 cycles of denaturation (30 sec at 94°), annealing (30 sec at Ta according to Escherichia coli plasmid vector TOPO XL (Invitrogen Life Technologies) and transformed into competent cells of strains DH5α or One Shot ccdB Survival (Invitrogen Life Technologies) according to the manufacturer’s instructions. Plasmid DNA was isolated using the Plasmid Mini Kit . Plasmid insertions were custom Sanger sequenced at the Max-Planck-Genome-Center Cologne on Abi Prism 377, 3100 and 3730 sequencers (Applied Biosystems) using BigDye-terminator v3.1 chemistry. Sequences were aligned and examined for SNPs. An SNP was considered factual when it was detected in at least three clones originated from two independent amplifications.Total RNA was extracted from tubers and leaves of the tetraploid varieties Diana, Theresa, Saturna, and Satina. Leaf RNA was isolated using the ToTALLY RNA Total RNA Isolation Kit . Tuber RNA was isolated either with the PureLink Plant RNA Reagent  or using the protocol described in  primers . PHO1a cQubit Fluorometer (Invitrogen) and stored at −20°. A total of 50 µg of total native protein were loaded onto NativePAGE Novex 3–12% Bis-Tris gels (Life Technologies) and electrophoresed at 200 V and 4° in a XCell SureLock Mini-Cell (Invitrogen), following the manufacturer’s instructions. Glucan-forming activity of phosphorylase was tested as described . The homogenate was centrifuged at 14,800 g for 15 min at 4°. The supernatant was removed, and total protein was quantified with a escribed . After ehttp://www.ncbi.nlm.nih.gov/) and SPUD DB Potato Genomics Resource (http://solanaceae.plantbiology.msu.edu/pgsc_download.shtml) by text searches. Annotated potato loci (PGSC0003DMG40*******) corresponding to the genes were obtained using the BLAST sequence alignments tool in (http://potato.plantbiology.msu.edu/integrated_searches.shtml). Gene number, genomic position (pseudomolecules v4.03), and expression profiles were retrieved from the potato genome browser S. tuberosum group Phureja DM1-3 (http://potato.plantbiology.msu.edu/cgi-bin/gbrowse/potato/). Exon-intron structure was deduced from aligning cDNA with genomic sequence using Spidey (http://www.ncbi.nlm.nih.gov/spidey/) or from RNAseq data available in the potato genome browser. cDNA sequences were assembled and analyzed using the software tools BioEdit . SNPs with a MFA <1% (MFA was present in simplex in less than eight genotypes of the CHIPS-ALL population) were not considered. Markers associated with chip quality in the CHIPS-ALL population were tested by analysis of variance for reproducibility of the MFA effect in the SUGAR40 panel. Three measurements of tuber reducing sugar content (mg/100 mg dry weight) in the SUGAR40 panel .Gene specific primers suitable for amplicon sequencing were designed for eight genes with important functions in starch-sugar interconversion according to the literature . PositioGWD; File S2). The representative minor frequency SNP allele 3259GWD-G had a positive effect on TSY. The rare (frequency 2–3%) SNP allele 3452GWD-G increased TSC and TSY . The rare haplotype 11112A11140PWD-A was associated with lighter chip color, particularly after cold storage, whereas another low-frequency allele, 10758PWD-A, showed the opposite effect.Phosphoglucan water dikinase ciations . The mosBMY-8/2 is one of nine potato genes annotated as beta-amylase . BMY-8/2 is 3.6 kbp long, has four exons, and is highly expressed in most tissues. Eight of 14 SNPs scored in exon four showed trait associations  (SssI-4b that was associated with lighter chip color and greater TSC and TSY in the CHIPS-ALL population (Soluble starch synthase I (SssI) . None ofPGM-3 analyzed in this study (AJ240053) is not annotated in the current version (4.03) of the potato genome. Aligning the full-length cDNA with the genome sequence revealed that PGM-3 is located in a 16.2-kbp region on chromosome III (Table S1). The gene has at least 21 exons and is expressed in all tissues. The sequencing of an amplicon spanning exons one to three revealed 15 SNPs including two triallelic SNPs. Three SNPs showed associations, in particular the common allele 468PGM-3-C, which was associated with increased TSC. The triallelic SNP marker 441 ACPGM-3-T was the only one in the whole set, which showed above threshold significant association with tuber yield. The direction of the effect, however, was inconsistent between genotypic groups (Besides two genes annotated as plastidic phosphoglucomutase (c groups .AGPaseS) is one of three expressed genes encoding the large (L) subunit of glucose-1-phosphate adenylyltransferase (Table S1). The 5-kbp gene has 14 exons and is ubiquitously expressed. Twenty-five SNPs were scored in an amplicon located in exons three to six. Nine SNPs in five LD groups were associated with chip color, TSC, and TSY. The low-frequency SNP haplotypes 1259C1612AGPaseS-C and 1284T1411C1457AGPaseS-C corresponded to the diagnostic SSCP markers AGPsS-10a and AGPsS-9a (1286AGPaseS-G and 1348 C1592AGPaseS-G) or positive (1392AGPaseS-C) trait associations (ADP-glucose pyrophosphorylase S (AGPsS-9a . The thrciations .Table S1), five of which have been previously cloned and characterized, the soluble acid invertase Pain-1 (InvGE and InvGF (InvCD111 and InvCD141 (Pain-1 with the same exon-intron structure is the locus PGSC0003DMG400004790 (INV-8/2) on chromosome VIII. This putative invertase gene, which is orthologous to the equally uncharacterized tomato gene LIN9 (http://www.ncbi.nlm.nih.gov/gene/778304), has not been functionally characterized so far. It has a length of 4.1 kbp, seven exons, and is expressed mainly in flowers. Expression in tubers is very low . With one exception (PGM-3-snp408 and KT-InvInh-snp395/396 on chromosome III), SNPs associated with tuber quality traits showed very low or no LD among each other. This indicated that most of the observed associations were independent from each other.LD between all SNPs scored in the CHIPS-ALL population was strongest between SNPs within the same locus. Strong LD blocks included SNPs with physical distances up to approximately 700 bp. Weaker LD was observed between physically linked loci such as PHO1a (Stp23 in (Table S1). The characterized cDNA sequence (D00520) matches to four small scaffolds containing different parts of the gene , which are not anchored to the current physical map. The gene comprises 15 kbp, has 15 exons, and is highly expressed in all tissues. In total 161 full-length cDNAs (2901 bp) were cloned from leaf and tuber tissue of cultivars Diana, Theresa, Saturna, and Satina. Satina served as negative control because this cultivar lacked the SSCP markers Stp23-8b and Stp23-8a associated with increased tuber starch content and better chip quality in the CHIPS-ALL population . The sequence of cDNA 1 was obtained from all four cultivars and was identical to GenBank accessions D00520 and X52385  on potad X52385 . We refe 7, 8, 9 .Stp23-8b and Stp23-8a, which are both diagnostic for increased chip quality, TSC, and TSY . In contrast, the distributions of the haplotype 22A322G824A2776PHO1a-T and the Stp23-8b marker in the 34 varieties were, with one exception, identical, indicating that cDNA 5, subsequently referred to as APHO1a-H, corresponded to Stp23-8b (Table S2). Eleven standard varieties were simplex for the APHO1a-H allele. Only cv Saturna contained APHO1a-H in duplex dosage. None of the cDNAs corresponded to SSCP marker Stp23-8a. Genotyping the BRUISE85 panel with the allele specific marker Stp23-8b   showed that the amino acid substitutions at positions 8, 275, 647 and 932 were not specific for PHO1a-HA, while Thr108 and Asn926 were unique for PHO1a-HA. Sequence alignments of more than 200 alpha-glucan phosphorylases from plants, animals, fungi and bacteria (not shown) confirmed that the nonconservative substitution of alanine108 by threonine was exclusively present in PHO1a-HA. Crystal structures of plant PHO1a proteins are not available. In silico simulation of PHO1a structure based on the crystal structure of yeast glycogen phosphorylase indicated that the Ala108Thr substitution is located in the α2 helix, a conserved region involved in the interaction between the dimers of the bacterial ortholog maltodextrin phosphorylase MALP -3b, StpL(PHO1b)-3e, Pain1-8c, InvGE-6f) or by amplicon sequencing . Significant effects on reducing sugar content (P ≤ 0.05) were observed for 23 marker alleles in all genes tested. Except for BMY-3-snp2533, the direction of the MFA effect was the same as in the CHIPS-ALL population . Approximately half of these genes have been cloned and characterized before in potato and/or tomato (Table S1). Seven of the previously characterized genes (5.7%) were not annotated in the current potato genome draft sequence. Including the results of this study, 25 loci functional in starch-sugar interconversion have been analyzed for association of DNA polymorphisms with tuber quality traits (Table S1) (The annotated potato genome sequence (able S1) .INV-8/2, the candidate genes probed for association in this study were selected according to their known function in plastidial starch biosynthesis and degradation (SssI tagged two (GWD) to nine alleles (PHO1b) per locus, which were associated with either increased or decreased tuber starch content, starch yield, and/or chip quality in the CHIPS-ALL population. Effects on tuber yield were negligible. The high number of SNP-trait associations , particularly associations with TSC suggest that allelic DNA variation in at least some of the tested starch metabolizing genes contributes directly to the natural variation of tuber starch content, starch yield and, due to the inverse correlation between tuber starch and sugar content, chip quality. We cannot exclude, however, that the observed SNP-trait associations are indirect, resulting from LD with DNA variants in physically linked genes that are causal for the observed trait variation. At present, there is no genome-wide LD physical map available in potato, which would allow an estimate of the physical distances flanking the analyzed functional candidate loci, which should be considered for containing the causal gene(s). The recombination frequency in distal chromosomal regions is greater compared with pericentromeric regions  are two further candidate genes annotated, which are both expressed in tubers, a fructokinase  as well as heterodimers (PHO1a-PHO1b) -3b; PHO1 markers evaluated in both populations. However, due to the correlation between tuber starch content and susceptibility to bruising, for a positive allele effect on tuber starch content there is a penalty by higher bruising susceptibility and vice versa. As this correlation is not absolute and additional candidate gene markers for starch corrected resistance to bruising have been identified  and 1103InvGE-A . Even if some of these 123 genes are annotation artifacts, pseudogenes or genes with a different in vivo function than suggested by in silico annotation, this number is much larger than the number of loci that have been actually evaluated for DNA polymorphisms and their associations with tuber quality traits. Furthermore, the metabolic scheme in 2 fixation and carbon partitioning between source and sink tissues (Searching the potato genome for genes functional in starch-sugar interconversion  based on tissues . Except Genetic dissection of tuber quality traits by genome-wide QTL linkage and association mapping based on restriction fragment length polymorphism and amplified fragment length polymorphism markers identified in the order of 15 to 25 QTL for tuber starch content and processing quality . Neverth