Ribonucleotide reductase small subunit B (RRM2B) is a stress response protein that protects normal human fibroblasts from oxidative stress. However, the underlying mechanism that governs this function is not entirely understood. To identify factors that interact with RRM2B and mediate anti-oxidation function, large-scale purification of human Flag-tagged RRM2B complexes was performed. Pyrroline-5-carboxylate reductase 1 and 2  were identified by mass spectrometry analysis as components of RRM2B complexes. Silencing of both PYCR1 and PYCR2 by expressing short hairpin RNAs induced defects in cell proliferation, partial fragmentation of the mitochondrial network, and hypersensitivity to oxidative stress in hTERT-immortalized human foreskin fibroblasts (HFF-hTERT). Moderate overexpression of RRM2B, comparable to stress-induced level, protected cells from oxidative stress. Silencing of both PYCR1 and PYCR2 completely abolished anti-oxidation activity of RRM2B, demonstrating a functional collaboration of these metabolic enzymes in response to oxidative stress. Hence, RR activity is crucial for maintaining cellular levels of dNTPs, which are used for DNA synthesis during DNA replication and DNA repair. Mammalian RR holoenzyme consists of two large subunits, RRM1, and two small subunits, RRM2 or RRM2B. In most cell types, expression of RRM1 is constant throughout all phases of cell cycle, whereas RRM2 expression is low in G1 phase12/M15561 to S phase, whereas RRM1/RRM2B complex regulates DNA repair89Ribonucleotide reductase (RR) catalyzes conversion of ribonucleoside diphosphate (NDP) to deoxyribonucleoside diphosphate (dNDP), a rate-limiting step in Rrm2b null mice show attenuated dNTP pools following oxidative stress and depletion of mitochondrial DNA contentAlthough it is well characterized that RRM2B is involved in the process of DNA repair, mutations of RRM2B have been identified in patients with mitochondrial DNA depletion syndrome, suggesting that RRM2B is an essential gene for the maintenance of mitochondrial DNA11121314in vitro biochemical assayWe previously reported that purified recombinant RRM2B protein alone has intrinsic catalase activity to convert hydrogen peroxide to water and oxygen in an In this study, we employed purification of RRM2B complexes followed by mass spectrometry analysis to identify novel RRM2B-associating factors, which might collaborate with RRM2B to antagonize overt oxidative stress. Using this approach, we successfully identified Pyrroline-5-carboxylate reductase 1 and 2  as RRM2B-interactors. Most importantly, we formally demonstrated functional cooperation, either directly or indirectly, between RRM2B, PYCR1 and PYCR2 in response to oxidative stress.To purify RRM2B complexes and identify associating factors, we first established several stable human 293 T-REx cell lines expressing RRM2B proteins, which were either untagged or tagged with Flag-, hemagglutinin (HA)- or OneStrep-epitope at the N- or C-terminus, upon induction by doxycycline see . RRM2B eSince anti-Flag-beads immunoprecipitated RRM2B complex most efficiently, compared to anti-HA and StrepTactin beads and N-terminal tagging preserved complex formation with RRM1, a large-scale purification of RRM2B complexes was performed from whole cell lysate of 293 T-REx-Flag-RRM2B cells. 293 T-REx cells expressing untagged RRM2B were used as negative control. Flag-RRM2B complexes were first immunoprecipitated by anti-Flag-beads and eluted with urea. An aliquot from each step was examined by Western blotting to validate the purification procedure C. Flag-Rbona fide RRM2B associating-factors. Other than RR subunits, we did not identify those RRM2B- or RR-associated proteins reported previously from our group or others using different approaches31920212223241-pyrroline-5-carboxylate (P5C) to proline. Given that PYCR1 and PYCR2 as well as proline are known to mediate anti-oxidation activity in vivo26A mixture of urea-eluted RRM2B complexes was subjected to protease digestion and analyzed by mass spectrometry. Data analysis identified 154 candidate proteins. The list of the candidate proteins was then sorted by the intensity ratio of the Flag-RRM2B sample and the negative control. Among 154 candidates, 96 proteins had an intensity ratio of more than 10. The top three hits were the bait, RRM2B, and known associating protein RRM1 and RRM2, thus demonstrating that our methodology was able to pull down in vivo  is the third member of PYCR protein family. PYCR1 and PYCR2 are closely related (85% identity), whereas PYCRL is the most distant member (it shares 45% homology with PYCR1 and PYCR2). Unlike PYCR1 and PYCR2 which are located in mitochondria, PYCRL is a cytosolic protein and utilizes cytosolic pool of P5C to produce prolineFinally, we performed indirect immunofluorescent staining followed by confocal microscopy to analyze cellular localization of both RRM2B and PYCR2 in HFF-hTERT cells. We observed distinct RRM2B-punctate staining in the cytoplasm and faint staining throughout the cells  in which the stress response pathway remains intact. Very low amount of RRM2B was detected in the cytosolic fraction in the unstressed cells and undetectable in the mitochondrial fraction. When overexpressed, small fraction of RRM2B was detected in the mitochondria . To inveBoth PYCR1 and PYCR2 are involved in proline synthesis. Hence, it is conceivable that deficiency of these enzymes can trigger stress response in normal human cells. To test the hypothesis, we expressed multiple shRNAs by pSUPER.retro.puro retroviral or pLKO.1 lentiviral vectors to silence expression of PYCR1 or PYCR2 in HFF-hTERT cells see . While sCIP1 and RRM2B were also induced, suggesting activation of p53 pathway  targeted against the 5′-untranslated region of the mRNA individually or in combination. Co-injection of two pycr MO caused profound defects during embryonic development demonstrating the essential functions of pycr isozymes in vivo . Hydrogen peroxide was purchased from Avantor Performance Materials, Inc. . Doxycycline was purchased from Sigma-Aldrich .Human telomerase reverse transcriptase (hTERT)-immortalized normal human foreskin fibroblasts (HFF-hTERT) , Flp-In™ T-REx™ 293 cells and 293T cells were cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and penicillin and streptomycin (Thermo Fisher Scientific Inc.). All cells were incubated at 37 °C and 5% COUntagged, Flag-, HA- and OneStrep-tagged human RRM2B cDNAs were generated by polymerase chain reaction (PCR) using oligonucleotides as primers listed in Flp-In™ T-REx™ 293 cells were co-transfected with pOG44 and pcDNA5/FRT/TO-untagged or -tagged RRM2B vectors. Transfected cells were selected with 100 μg/ml hygromycin for two weeks. Expression of RRM2B cDNA was induced by 1 μg/ml doxycycline for 24 hours.Retroviruses and lentiviruses were generated as previously described293 T-REx-RRM2B (negative control) and -Flag-RRM2B cells were incubated with 1 μg/ml doxycycline for 24 hours and harvested. Immunoprecipitation, elution and digestion of RRM2B protein complexes were carried out as described previouslyLiquid chromatography-mass spectrometry and data analyses of the digested immunoprecipitated samples were carried out as previously describedTwenty million cells were harvested for sub-cellular fractionation. Dounce homogenization (80 strokes for HFF-hTERT cells and 110 strokes for 293 T-REx-RRM2B cells) and differential centrifugation were performed to separate mitochondria from cytosol components by using Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific Inc.) and according to the instruction by the manufactor.Cells were rinsed with PBS and lysed with buffer A  supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors  for 30 min on ice. Whole cell lysate was cleared by centrifugation for 10 min. Protein concentration was quantified by BCA kit (Thermo Fisher Scientific Inc.). Equal amount of total proteins was incubated with Strep-Tactin resins , ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich), anti-HA agarose (Sigma-Aldrich), normal mouse IgG , normal rabbit IgG (Santa Cruz Biotechnology Inc.) or antibodies to PYCR1 , PYCR2 , RRM2B , for three hours to overnight at 4 °C with rocking. Protein A or protein G beads (Santa Cruz Biotechnology Inc.) were added to the antibody-antigen reaction for additional one hour. Beads were collected by centrifugation at 10,000x g for 15 seconds and then washed with ice-cold buffer A three times before boiling in SDS-PAGE sample buffer.CIP1 , TOM20 , TUBA , human p53  and β-ACTIN (Sigma-Aldrich).Procedures for immunoblotting were as describedHFF-hTERT cells transduced with retrovirus expressing Flag-RRM2B were seeded onto glass coverslips, fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 in PBS. Cells were stained for 1 h with affinity-purified rabbit anti-RRM2B antibody followed by a 30-min exposure to anti-rabbit IgG-AlexaFluor 647, mouse anti-PYCR2 antibody followed by a 30-min exposure to anti-mouse IgG-AlexaFluor 594 and DAPI before mounted in ProLong® Gold Antifade Mountant (Thermo Fisher Scientific Inc.). Fluorescence images of RRM2B and PYCR2 were captured with wide-field DeltaVision deconvolution microscope , equipped with 60x/1.42 N.A. oil-immersion objective lens. Both microscope and camera were controlled by SoftWorX application suite software. Stacks of optical section images, with image size of 512 × 512, were collected for all fluorochromes. All images were deconvolved by using SoftWorX software (Applied Precision Inc.), and later analyzed with VoloCITY software (PerkinElmer).HFF-hTERT cells expressing cox4-DsRed were transduced with pMSCVhyg vector expressing RRM2B or pLKO.1 lentiviral vectors expressing non-silencing shRNA and shRNAs directed to silence PYCR1, PYCR2 or both. Following hygromycin or puromycin selection, cells were seeded onto coverglasses in triplicate and fixed in 4% paraformaldehyde for 10 minutes at 37 °C. Fixed cells were washed with PBS and stained with DAPI before mounted in ProLong® Gold Antifade Mountant (Thermo Fisher Scientific Inc.). Mitochondrial morphology was visualized by Zeiss Upright LSM510 2-Photon microscope  and photographed. Mitochondrial morphology was categorized into three groups, mostly long tubular, intermediate phenotype and mostly fragmented. At least 100 cells were scored from each coverglass.Transduced HFF-hTERT cells following puromycin selection were seeded in 60 mm dishes at low density (200–500 cells per dish). Cell colonies were fixed and stained with 0.5% Crystal Violet in methanol:acetic acid  two to three weeks following seeding. For hydrogen peroxide sensitivity assays, cells were treated with hydrogen peroxide in serum free media for 10 minutes at 37 °C after overnight seeding. Treated cells were rinsed with complete media two times and incubated for 2-3 weeks before fixation and staining.5 per well) following puromycin selection. Cell numbers were counted two days and three days after seeding. For hydrogen peroxide sensitivity assays, cells were treated with hydrogen peroxide in serum free media for 10 minutes at 37 °C after seeding for overnight. Treated cells were rinsed with complete media for two times and incubated for additional two days before cell counting.Transduced HFF-hTERT cells were seeded in 6-well plates  sodium citrate and 0.1% (v/v) Triton X-100) and digested with RNAse A (2 μg/ml) before analysis by fluorescence-activated cell sorting.Danio rerio) were obtained from zebrafish core facility of Taipei Medical University and maintained at 28 °C on a 14 h light/10 h dark cycle. All animal procedures were approved by Taipei Medical University Institutional Animal Care and Utilization Committee (TMU-IACUC). The methods were carried out in accordance with the approved guidelines. Embryos were incubated at 28 °C and different developmental stages were determined according to the Zebrafish BookZebrafish . The pycr1 and 2 MOs were designed according to a previous reportpycr MOs were as follows: pycr1 MO 5′-CAGCTCCGATAAATCCCACACTCA T-3′, pycr2 MO 5′-CCG CTCCAATGAAGCCCACACTCAT-3′, and pycr3 MO 5′-CTGAAGCACCTGACA GACTCATGGT-3′. Each MO was dissolved in 1 Danieau solution containing 0.5% phenol red to a concentration of 5 and 7.5 ng. The pycr MOs were injected into at the 1- to 2-cell stage of wild-type zebrafish embryos using a microinjection system consisting of a SZX16 stereomicroscope  and an IM-300 Microinjector . The MO-mediated effects were evaluated at 24 h and 48 h (2 dpf) after injection. The phenotypes of fish morphants were categorized in three groups, normal, mild and severe. The embryos morphants were observed under an Olympus IX70-FLA inverted fluorescence microscope , photographed by the SPOT digital camera system , and assembled by ImageJ programThe antisense morpholino oligonucleotides (MOs), against zebrafish We performed Student’s t tests with two-tailed distribution and two-sample equal variance in Excel .How to cite this article: Kuo, M.-L. et al. PYCR1 and PYCR2 Interact and Collaborate with RRM2B to Protect Cells from Overt Oxidative Stress. Sci. Rep.6, 18846; doi: 10.1038/srep18846 (2016).


Starch-rich crops form the basis of our nutrition, but plants have still to yield all their secrets as to how they make this vital substance. Great progress has been made by studying both crop and model systems, and we approach the point of knowing the enzymatic machinery responsible for creating the massive, insoluble starch granules found in plant tissues. Here, we summarize our current understanding of these biosynthetic enzymes, highlighting recent progress in elucidating their specific functions. Yet, in many ways we have only scratched the surface: much uncertainty remains about how these components function together and are controlled. We flag-up recent observations suggesting a significant degree of flexibility during the synthesis of starch and that previously unsuspected non-enzymatic proteins may have a role. We conclude that starch research is not yet a mature subject and that novel experimental and theoretical approaches will be important to advance the field. Starch is an insoluble, non-structural carbohydrate composed of α-glucose polymers. It is synthesized by plants and algae to store energy in a dense, osmotically inert form. Starch has significant value for humans: it serves as the main carbohydrate source in an equilibrated diet and as a renewable raw material for industry. For instance, starch is extensively used as a thickener and texturizer in processed foods, as it gelatinizes to form pastes when heated in water. Starch pastes also have innumerable uses in the non-food sector, such as in the production of paper and board , 2, of bBased on its biological functions, starch is often categorized into two types: transitory starch and storage starch. The starch which is synthesized in the leaves directly from photosynthates during the day is typically defined as transitory starch, since it is degraded in the following night to sustain metabolism, energy production and biosynthesis in the absence of photosynthesis. If this night-time carbohydrate supply is reduced—for instance in mutants impaired in starch synthesis—plants grow more slowly and experience acute starvation . The staStarches from different botanical sources vary in terms of their functional properties  and thus in their end-uses. This variation stems from differences in the structure of starch, such as the size of starch granules, their composition, and molecular architecture of the constituent polymers . Still, Arabidopsis thaliana and the single-celled green alga Chlamydomonas reinhardtii. Although their starches have no direct industrial value, many aspects of starch biosynthesis appear to be widely conserved within the Viridiplantae clade. Thus, discoveries made in these systems are likely to have broad relevance. It is nevertheless always important to bear in mind the cellular and metabolic context in which starch is made. Variation in conditions between tissues and species can have a strong influence on the amount and structure of starch. Such differences may explain why, in some cases, different phenotypes result from similar genetic perturbations. In the long run it will be important to understand both the basic starch-biosynthetic process and tissue-specific factors that affect it.This review focuses on the mechanisms of starch biosynthesis and seeks to provide a broad overview of our current knowledge, while highlighting recent advances. Significant steps in our basic knowledge have been made through the analyses of model systems such as the plant 5 and a branching level of 4–5 %  )Starch-branching enzymes (E.C. 2.4.1.18) belong to the α-amylase superfamily of enzymes  and class II (family A) enzymes. Class I enzymes generally occur as a single isoform in planta, with the exception of Arabidopsis, which does not contain a class I BE . Class IProtein-sequence homology revealed a putative third class of BE. Genes belonging to this class III were found in Arabidopsis , rice, E. coli (EcGlgB), while transferring chains of similar length to plant BEIIs, differed in that it preferentially placed new branches only three glucose units away from an existing branch point [EcGlgB was expressed in an Arabidopsis line deficient in its endogenous BEs (be2/be3 double mutants) [EcGlgB restored the synthesis of insoluble glucans [The specific placement of branches by BEs is believed to be a major determinant of the cluster structure of starch . However, due to our limitations in assessing the actual distribution of branch points, investigations on the specificity of BE isoforms often focused more on the lengths of transferred chains rather than their placement, primarily by comparing the CLDs of glucans before and after the in vitro modification by a BE. Accordingly, class I BEs tend to transfer slightly longer chains than BEII and are more active towards amylose, whereas class II BEs typically prefer amylopectin –190. Furch point . This stmutants) . Neverth glucans , 193, alamylose-extender (ae) phenotype. This phenotype was observed in beIIb mutants of maize [beIIa mutants of durum wheat [ae phenotype is characterized by altered amylopectin structure with longer external and internal chains and, in some cases, increased levels of amylose [ae starch is of special interest both for industry and human health [Mutants of BEI have been analyzed in maize , 195, riof maize  and riceof maize , in beIIum wheat , in mutaum wheat , 203) anum wheat , 205. Th amylose , 206–208n health , 30. In n health , 209. UpIn potato tubers, repression of BEII led to amylopectin with longer chains , but a hbeIIa mutants have normal endosperm starch, but mostly replace leaf starch by a glucan of low molecular weight and a low degree of branching [Some mutations in BE affect leaf starch metabolism. Maize ranching , 214. Alranching . It was E. coli glycogen BE to restore some insoluble glucan synthesis [Collectively, the numerous studies of BEs show that, as expected, branching activity is absolutely required for amylopectin synthesis. They also show that BE specificities differ and that they significantly influence the final glucan structure. Furthermore, their activity relative to that of SSs is important, which is illustrated by the change in glucan structure when expressed to differing extents , 212. Neynthesis , 193. ItPlant DBEs hydrolyze α-1,6-linkages and release linear chains. They belong to the glycoside hydrolase family 13 (CAZy: ) and shastarch-excess (sex) phenotypes [ISA1 and ISA2 are involved in debranching during the synthesis of amylopectin and are thus in the focus here. They act together as a heteromultimeric enzyme, and ISA1 additionally forms active homomultimers in some species (described below)—hereafter we use “ISA” to refer to both homomeric and heteromeric activities. In contrast, ISA3 and LDA primarily debranch starch during its degradation and mutants of these enzymes often have enotypes –220. Nevenotypes , 221–223C. reinhardtii [C. reinhardtii and in japonica rice , do isa1 mutants completely fail to produce starch [ssII allele [Mutants lacking ISA partly replace starch by a water-soluble polysaccharide in the endosperms of maize , 225, rinhardtii . In termnhardtii . In moste starch , 229; inI allele .isa1/isa2/isa3/lda mutant lacking all DBEs [ssI/isa double mutants have longer A and B1 chains and produce significantly more starch than isa single mutants [A common model to explain the accumulation of phytoglycogen is the “trimming” model . Accordiall DBEs . This suall DBEs , 223, maall DBEs  and riceall DBEs , 222. Hoall DBEs . This reall DBEs , and sugall DBEs  and riceall DBEs  suggeste mutants  Fig. 5)isa1/isa2 mutants , 121.FigAtISA1–AtISA2 in E. coli [S. cerevisiae  impeded the accumulation of glycogen instead of yielding a more amylopectin-like glucan. Second, far fewer glucans were observed in the Arabidopsis ssII/ssIII double mutant compared with the ssII/ssIII/isa triple mutant [ssII/ssIII plants showed structural similarities to glycogen in that they had much shorter chains than wild-type amylopectin and were partly found in the water-soluble fraction [Interestingly, recent studies suggest that ISA itself can act in a degradative fashion under some circumstances. First, expression of  E. coli  and S. ce mutant . Notablyfraction . These disa mutants [A purely degradative action of ISA may also provide an explanation for the formation of numerous small granules/particles in  mutants , 236. In mutants . Howeverisa1, isa2 and isa1/isa2 mutants show identical phenotypes [AtISA1 and AtISA2 have a high native molecular mass, interact with each other and are stable only in the presence of their partner [AtISA1 and AtISA2 were present [E. coli [Depending on the species, ISA1 either acts exclusively in heteromultimeric complexes formed between ISA1 and ISA2, or both as heteromeric and homomeric complexes. The Arabidopsis enotypes . Moreove partner , 235. Ca present . These d present , 239. Si present , althoug[E. coli .C. reinhardtii [C. reinhardtii ISA1 had an elongated structure and paired with another ISA1 in a tail-to-tail fashion, although the existence of higher multimers could not be excluded [isa2 mutants, which have only the ISA1 homomer, had similar endosperm starch synthesis as wild-type plants, suggesting that the ISA1 homomers are sufficient [ssIII mutant background, loss of ISA2 resulted in the accumulation of phytoglycogen in maize kernels [In other species it is clear that ISA1 also acts as a homomultimer. One or several homomeric complexes of ISA1 were found in addition to at least one ISA1/ISA2 complex in rice and maize grains and in nhardtii –242. In fficient , 243. Ho kernels —an intri kernels . Thus, bFor the past decade there has been mounting evidence for the formation of complexes between starch-biosynthetic enzymes and for the phosphorylation of these enzymes. Recently, two additional complexes were identified which are distinct as they involve proteins without apparent catalytic functions  consistently appear less in the granule-bound fraction [In fraction , 244. Thfraction . Furtherfraction , 246. Sifraction . In bothfraction , 126. Thfraction , 246 andA potential biological function for complex formation could be to channel substrates from one enzyme to another—a newly created branch resulting from BE action could be directly elongated first by SSI and then by SSII, thereby increasing the overall efficiency of the process. It is also possible that the complex confers enzymatic specificity; for instance, steric limitations of the whole complex could define the length of a chain that is transferred by a BE or where exactly a new branch is placed. Tetlow and Emes  further 2+-dependent protein kinases in phosphorylation of BEIIb.Similarly, the role of phosphorylation of BEs and some SSs  remains It is clear that there will be much need for detailed biochemical and structural analyses of these protein complexes in the future. Particularly the creation of mutant proteins that are still active, but unable to interact with its enzymatic partners, would help in establishing the importance of the complexes.The reversible phosphorylation of starch is best characterized in Arabidopsis where it is pivotal for efficient degradation. It is believed that phosphorylation of glucosyl units at the C6 or C3 positions  dilsf2 mutants have increased levels of C3 phosphorylation at the end of the day, even though these mutants almost completely degrade their starch at night. This suggests that phosphate groups introduced during the day are normally also being removed by LSF2 [However, glucan phosphorylation is not confined to the dark period. Arabidopsis leaf starch made during a single photoperiod is C6- and C3-phosphorylated, showing that both GWD and PWD are active during the day. Furthermore,  by LSF2 . In addi by LSF2 . Incubat by LSF2 .gwd mutants, which are essentially phosphate free, have a normal internal structure but differ in their granular surface, which is irregular and displays shorter chains [gwd mutants also have only little net starch synthesis during the day. This could mean that lack of GWD inhibits starch synthesis generally [gwd mutants starve during the night, which can influence the utilization of photoassimilates the following day).The relevance of phosphorylation during synthesis is not clear yet. Starches from Arabidopsis r chains . These denerally . Howeverenerally . It, thun + glucose-1-phosphate ⟷ n+1 + orthophosphate. Higher plants have two classes of phosphorylase: a plastidial and a cytosolic one. The plastidial class, named Pho1, PhoL (because of its low affinity towards glycogen) or PHS1, carries a 78-amino acid insertion near the glucan-binding site and shows highest affinity towards linear glucans, especially malto-oligosaccharides. By contrast, the cytosolic class, named Pho2, PhoH or PHS2, prefers branched substrates such as glycogen [α-Glucan phosphorylase (EC 2.4.1.1) catalyzes the reversible reaction glycogen , 259.The function of Pho1 is ambiguous, to the extent that it is not even clear whether it acts in a synthetic or phosphorolytic  way in vivo. The plastidial concentration of orthophosphate typically exceeds that of glucose-1-phosphate manifold , which wbeII mutants of maize and barley [Nevertheless, the gene expression and/or enzyme activity of Pho1 correlates with that from known starch-biosynthetic genes in potato tubers  and the d barley , 126, PhC. reinhardtii—which differs from vascular plants in having two plastidial isoforms, PhoA and PhoB—loss of PhoB resulted in reduced starch content. The remaining starch furthermore had a higher relative amylose content and structural modifications [In ications . Still, ications .pho1 mutant of Arabidopsis [ssIV mutants and those impaired in maltose metabolism [There has also been speculation that PhoI may be involved in starch degradation. However, no significant influence on leaf starch content was noted upon repression of the leaf isoform of Pho1 in potato  or in thbidopsis –272. Altbidopsis , 273. Motabolism , 272.In recent years, several novel proteins have been identified that influence starch structure and/or amount. In some cases, the impact on starch may be an indirect one whereas in others, the proteins may be part of a hitherto undiscovered aspect of the starch-biosynthetic process. In either case, the analysis of the underlying mechanisms has the potential to provide new insights into facets of starch biosynthesis that go beyond the core enzyme machinery, such as the regulation of starch synthesis or the influence of the plastidial environment on granule formation and morphology.flo6 phenotype results from altered ISA1 function. This is an important hypothesis to test given the importance attributed to ISA in trimming nascent amylopectin to facilitate its crystallization.Peng and colleagues described the rice Floury Endosperm6 (FLO6), a plastidial protein with a CBM48 and the capacity to bind starch . Grains Another CBM48-containing plastidial protein, Protein Targeting To Starch (PTST), has been described in Arabidopsis , 104. Inlest mutants had a wild-type phenotype, overexpression lead to a low-starch phenotype, similar to that of est1 mutants [Another class of non-enzymatic proteins was very recently discovered in Arabidopsis. Designated Early Starvation1 (EST1), this plastidial starch-binding protein was identified because mutants lacking it display a premature depletion of transitory starch at night . This ap mutants .Changes in starch granule morphology in cereal endosperms have also been linked to perturbations of other cellular processes including ER stress , failureOsbZIP58 and Rice Starch Regulator1 (RSR1) were shown to regulate the expression of a series of starch-biosynthetic genes in rice grains, thereby influencing starch structure and granule packing [Finally, other studies have begun to uncover regulatory factors that appear to influence starch structure and/or content by controlling the expression of starch-biosynthetic genes. For example, the transcription factors  packing , 282. Si packing .This diverse set of newly identified proteins serves to illustrate that there is much still to understand about starch biosynthesis and the cellular context in which it occurs. It is worth noting that most of the proteins described above are evolutionarily conserved within higher plants, and cross-species analyses will be an essential aspect of further investigations.Here, we have presented recent research findings in the context of established knowledge and ideas about starch synthesis. Ongoing genetic studies both in model and non-model species continue to raise evidence for enzymatic interdependencies, but also take us closer to understanding the requirements for the synthesis of a crystallization-competent glucan. This is well illustrated by studies of the ISA DBE: its contribution to promoting crystalline starch is highly dependent on the prior action of synthetic enzymes to produce suitable substrates for it, and some aberrant substrates are prone to degradation by ISA, while others are capable of crystallization in its absence. The assembly of enzymes into complexes, emerging as a common theme in cereal storage starch biosynthesis, and the participation of non-enzymatic protein factors add potential new layers of complexity to this already intricate process.Saccharomyces cerevisiae with the vision that this could allow us and others to disentangle starch biosynthesis from concomitant degradation and characterize in detail the functional interplay between starch-biosynthetic enzymes. The demonstration that starch can be made in a heterologous system would also show that we know which enzymatic set(s) are sufficient for starch biosynthesis and stimulate other complementary research. Together with empirical data from in vitro studies, it would represent an excellent starting point for the creation of mathematical models capable of simulating the starch-biosynthetic process. Furthermore, a heterologous system could serve as a valuable tool to aid the targeted modification of our starch crops, given that we are still not able to produce the variety of starches required by industry in planta.Arguably, one major stumbling block is the lack of a simple heterologous system for starch granule synthesis. We are working towards the production of such a system in the yeast


Crowns were cemented with resin cement, submitted to artificial aging in a chewing simulator , and tested for fracture strength. Two remaining crowns referring to PM and GM groups were submitted to a chemical composition analysis to measure the level of yttrium after aging. One-way ANOVA and Tukey's test (P = .05) indicated that monolithic zirconia crowns presented similar fracture strength , which was higher than bilayer crowns (2060.4 N ± 810.6). There was no difference in the yttrium content among the three surfaces evaluated in the monolithic crowns. Thus, monolithic zirconia crowns present higher fracture strength than bilayer veneered zirconia after artificial aging and surface finishing does not affect their fracture strength.The purpose of this study was to evaluate the effect of design and surface finishing on fracture strength of yttria-tetragonal zirconia polycrystal (Y-TZP) crowns in monolithic (1.5 mm thickness) and bilayer (0.8 mm zirconia coping and 0.7 mm porcelain veneer) configuration after artificial aging. Bovine incisors received crown preparation and Y-TZP crowns were manufactured using CAD/CAM technique, according to the following groups ( The increase of esthetics' demand has led to the development of metal-free restorations without metallic components . Dental However, the strength of all-ceramic crowns relies upon the core as well as the veneer material, whereby a bilayer system with a strong and tough Y-TZP core veneered with translucent but brittle porcelain tends to fail prematurely. Moreover, these bilayer systems have several disadvantages including the multistep manufacturing process, low toughness of the veneer material, and weak bonding between veneer layer and coping . TherefoThe alternative to circumvent all the bilayer systems' disadvantages is to replace the veneer/core bilayer with a monolithic restorative system . Monolit2O3), or ceria (CeO2) may be added to zirconia to stabilize the tetragonal and stronger phase at room temperature [Fabricating zirconia monolithic restorations could improve the mechanical stability and increase the range of indications of those prostheses. However, its wear behavior and chemical stability have not yet been fully clarified. Zirconia presents three different crystal configurations depending on the temperature: monoclinic from room temperature to 1170°C; tetragonal from 1170°C to 2370°C; and cubic at temperatures above 2370°C. When cooling after sintering, this material undergoes volume expansion of 3% to 5%, which is related to the transition from tetragonal to monoclinic phase. Nonetheless, many oxides such as calcium (CaO), magnesium (MgO), yttrium  and yttrium (Y2O3) to form yttrium hydroxide (Y(OH)3), which steadily drains the stabilizer, allowing for local conversion to the monoclinic phase [The aging process may induce yttrium loss and compromise the stability of the tetragonal phase of zirconia-based restorations, leading to uncontrolled tetragonal-to-monoclinic transformation . It has ic phase , 28. Apaic phase . In spitHence, the purpose of this study is to compare the fracture strength and failure mode of two Y-TZP monolithic systems, either polished or glazed, and bilayer veneered Y-TZP crowns after prolonged artificial aging. The content of yttrium of the monolithic crowns after artificial aging was also investigated. The null hypothesis was that the crown design, monolithic or bilayer, had no effect on fracture strength of aged zirconia crowns.Thirty-two healthy bovine incisors were used in this study, and a standardized crown preparation was performed in a lathe machine  with the following dimensions: 4.2 mm diameter occlusal base, 6.0 mm diameter cervical base, and 7.0 mm axial height . The tapn = 10) according to the crown fabrication technique: PM group: monolithic zirconia polished crowns (1.5 mm thickness); GM group: monolithic zirconia glazed crowns (1.5 mm thickness); BL group: zirconia copings with hand-layered porcelain veneering (0.8 mm core and 0.7 mm porcelain thickness). Two additional crowns, referring to PM and GM groups, were submitted to an electron probe microanalysis (EPMA) to quantify the yttrium content after aging.Specimens were randomly distributed in three groups . All zirconia crowns and copings were designed by the same technician with Lava Scan Design System. Then, zirconia blocks  were milled by using the Lava CNC 500 milling machine (3M ESPE). After the milling procedure, all copings and crowns were sintered in a furnace (Lava Furnace 200) for approximately 11 hours. The fully sintered crowns referring to PM were finished and polished with diamond wheels and bristle brushes . The crowns referring to GM received glaze firing after the sinterization. A silicone impression was taken from one finalized specimen of PM in order to duplicate its 1.5 mm thickness to control the final thickness of the veneered crowns. Copings referring to BL were veneered with the powder build-up technique with Lava Ceram veneer ceramic (3M ESPE). The thickness of the veneered porcelain and the contour of the final crown were verified by measuring the crown at different locations with a digital caliper, and the firing cycle was controlled by an experienced dental technician to ensure standardized crowns.The crowns were cleaned for 10 min in an ultrasonic bath , and 10 specimens of each group were cemented on their respective prepared tooth with a self-adhesive phosphate-based luting resin . A static load of 5 kg , 30 was After luting, specimens were stored in distilled water at 37°C for 24 hours and submitted to an aging procedure: 2 500 000 cycles, 80 N, at 37°C under artificial saliva bath . LoadingAged specimens were loaded in a universal testing machine  under deionized water bath at room temperature, with a 5 mm diameter ball indenter (stainless steel) at a crosshead speed of 0.5 mm/min. The maximum fracture load was measured by applying compressive load to the occlusal surface until the crown failed. Catastrophic fracture failure was considered as either the presence of visible cracks or sudden load drops or even acoustic events of chipping or fracture.The crowns were optically examined after fracture testing, and failure modes were divided into total core fracture, chipping of the veneer, or fracture at core/veneer interface. One representative specimen from each group was mounted on stubs with carbon adhesive tape and colloidal silver paint. Then, specimens were gold-sputtered and observed under scanning electron microscopy (SEM).The two remaining specimens referring to PM and GM were used for quantification of yttrium content. The yttrium level was measured in 10 points starting from the worn occlusal surface  up to the most inner point of the coping of PM and GM and in a surface away from the occlusal load in PM undamaged. Compositional analyses were performed by using electron probe microanalysis (EPMA) on an electron microprobe (camera SX-50/51 DCI 1300 DLL) with 40-degree take-off angle and beam energy of 15 keV.Fracture strength and yttrium content were separately analyzed by using SPSS 19.0 for Windows (SPSS Inc.). One-way analysis of variance (ANOVA) and Tukey's test were used to compare mean and standard deviation (SD), with 95% confidence levels for both fracture strength and yttrium content.P = .002, P = .930) and significantly (P = .002) higher than the results for the bilayer crowns . 2.5 million mechanical cycles were selected to simulate 5-year aging in the oral environment, considering that an average adult would perform around 500 000 loading cycles/year , 34. HowAll crowns survived the artificial aging in the chewing simulator. This result indicates a stable performance of zirconia-based crowns under a constant load of 80 N during 5 years. Previous studies that evaluated the clinical performance of zirconia-based restorations demonstrated a survival rate of 79–100% after 5 years –15, withThe null hypothesis that the Y-TZP crown design, monolithic or bilayer, has no effect on fracture strength was rejected. Therefore, the present study showed higher fracture strength of monolithic zirconia crowns in comparison to the bilayer configuration.Previous studies analyzing the fracture strength of all-ceramic monolithic crowns indicate a superior performance for the monolithic design. Monolithic lithium disilicate restorations and hand-layer veneered Y-TZP core evidenced that the highest fatigue load-to-failure values were presented by monolithic crowns and the lowest for veneered Y-TZP crowns . Even thAnother in vitro study evaluated the load-bearing capacity of four different zirconia based crowns, including zirconia core with veneer layer produced either by powder build-up or CAD/CAM technique, glazed monolithic zirconia, and polished monolithic zirconia. The results showed that zirconia in bilayer configuration had significantly lower load-bearing capacity than the other crowns' design . NeverthIn the present study, the groups referring to monolithic crows, polished and glazed, showed a total core fracture pattern. This result was expected, since PM has only one material layer and GM has a thin glaze layer which leads to a bulk structural fracture. On the other hand, all the bilayer crowns showed fracture at core/veneer interface. Failure mode at the veneer layer has been reported for bilayers crowns, most commonly in powder build-up technique rather than in the sintering or pressed veneering technique , 17. Thi2O) interacts with yttrium (Y2O3) generating yttrium hydroxide (Y(OH)3), which totally compromises the stabilizer, leading to local yttrium deficiency that results in transformation of tetragonal to monoclinic phase. Another mechanism suggested is that water breaks the bond between Zr and O, resulting in localized stress growth as a result of −OH movement inside the crystal structure. This motion causes lattice fault that acts as nucleating agents for posterior crystalline changes. And the last theory is that O2– from water breakdown fills oxygen vacancies [There is still no consensus about the system triggering LTD, but three different rationales have been suggested in the literature. The first hypothesis is that water  after aging Vita In-Ceram YZ in boiling water for 7 days, confirming the first hypothesis for LTD's origin . HoweverIn the current study, there was no difference in the yttrium content among occlusal worn surfaces and undamaged surfaces. Thus, this result can support the hypothesis that the chemical composition of monolithic crowns was not affected by the occlusal loading.The results of this study demonstrated that monolithic zirconia-based crowns might have reliable fracture strength after 5 years of occlusal loading. Indeed, the fabrication of monolithic zirconia restorations might allow for extended clinical application, reducing a major drawback, which is fracture of veneering ceramic. However, future researches concerning whether temperature or ph variations can influence the fracture strength and chemical stability of monolithic zirconia crowns after artificial aging should be conducted. And in vivo studies should be performed to evaluate the clinical behavior of monolithic zirconia restorations.According to the results of this study, Y-TZP monolithic crowns (polished and glazed) present higher fracture strength than bilayered veneered Y-TZP crowns. There was no evidence of yttrium depletion after 2.5 million cycles in artificial aging.


One hundred years after the original formulation by Petrus J.W. Debije (aka Peter Debye), the Debye Scattering Equation (DSE) is still the most accurate expression to model the diffraction pattern from nanoparticle systems. A major limitation in the original form of the DSE is that it refers to a static domain, so that including thermal disorder usually requires rescaling the equation by a Debye-Waller thermal factor. The last is taken from the traditional diffraction theory developed in Reciprocal Space (RS), which is opposed to the atomistic paradigm of the DSE, usually referred to as Direct Space (DS) approach. Besides being a hybrid of DS and RS expressions, rescaling the DSE by the Debye-Waller factor is an approximation which completely misses the contribution of Temperature Diffuse Scattering (TDS). The present work proposes a solution to include thermal effects coherently with the atomistic approach of the DSE. A deeper insight into the vibrational dynamics of nanostructured materials can be obtained with few changes with respect to the standard formulation of the DSE, providing information on the correlated displacement of vibrating atoms. Based on the orientational average of the intensity distribution, in 1915 Debye derived his equation for scatteringQ = 4 π sin(Θ)/λ the wavevector transfer modulus , f the atomic scattering factor and rij the magnitude of the distance between any two of the N atoms composing an atomistic aggregate.being e.g. the pioneering electron diffraction study by Germer & White inN atoms, N2 terms, which is computationally demanding if particles exceed a few tens of nanometers; moreover, the sample size and shape distributions must be properly represented, therefore implying a further increment in the number of terms to be computed.Despite the apparent simplicity of Q, whereas the B-factor is defined as However, in recent years the ever-increasing power of computers has alleviated computational problems, especially considering that 456The effect of the DW factor is to depress the diffracted (aka “Bragg”) intensity for the dynamic disorder brought in by the thermal displacement. While it is generally correct to provide for such an effect, two points should be considered. First, (i) the theory underlying the traditional DW factor is based on several simplifying assumptions, among which ignoring correlations between atomic displacements; furthermore, (ii) intensity removed from Bragg peaks should appear as a TDS signal. So far (i) has been largely ignored in DSE applications, whereas (ii) was only treated by approximate models in RS approachIn general terms, the instantaneous scattering amplitude from a small crystal is a sum of phase terms, weighted on the atomic scattering factorsri + δi(t), includes a static component, ri, referred to an average position, plus a dynamic term, the time-dependent  displacement δi(t).where the instantaneous atomic position, I(Q) = F(Q)F ∗(Q), which has to be time-averaged to account for the interaction of photons  with matter in a given experiment. Using compact notation, rij = ri − rj and δij = δi − δj  crystal whereas the following terms introduce thermal effects with increasing accuracy.I(Q), is given by the  orientational average of θ and ϕ depicted in Fig. 11δij over Q, which can be expressed as,The intensity from an ideal powder of small crystals, The scattered intensity, assuming spherical scattering factors and considering for the sake of discussion terms up to the second order in the series expansion, is therefore given byThe solution of the integrals leads to the expressionrij direction, rij.The time-averaged terms represent the square of the pair displacement projection respectively along the i.e.i.e.kij,The time average of s) values associated to the s-th shell Ss, which can be calculated separately for different coordination shells. Denoting with the apex  with numbers below unity limiting the depressing effect of thermal vibrations on Bragg peaks as atoms tend to move coherently therefore preserving the crystal order. On the other hand, kij values between 1 and 2 would be expected for optical vibrations, as in aggregates composed of different atomic species.The or atoms . Correlain silico case of study, the powder diffraction pattern of a palladium sphere (diameter 7.40 nm), obtained by taking the average of the outputs of 1112kij parameters.It is interesting to validate the approach empowered by kij parameters corresponding to the first twenty-five shells and kij values asymptotically tend to unity. It should be noticed that each kij refers to atoms lying along equivalent crystallographic directions  analysis  on more As reported in The most recurrent particle is depicted in kij values refined by the StoRM code (red circle) are reported as a function of the shell number and interatomic distance. The same figure also shows MD values for the most representative truncated cube of the distribution in kij markedly below unity demonstrate the strong correlation in the thermal motion of neighbor atoms, which extends to several shells before gradually approaching the uncorrelated condition (kij = 1).Information on the correlated thermal displacement is depicted in D, which can be refined to give the best-match with the observed MSD data. For the discussed case of study, the best fit of D = 235 K, significantly lower than the literature value of 272(18)K for bulk palladium15B-factor, with respect to that obtained from the DW factor, by averaging The TDSE also provides a more accurate B-factor for the powder of palladium nanocubes is 0.62 Å2, higher than the literature reference for the bulk phase (B = 0.45(6) Å2 1311192021with the normalization resulting from kij values between experimental results and MD simulations reported in e.g. the capping agent protecting the particles), not considered in the simulation. Therefore, experimental values of the kij parameters, and particularly the deviation from the CD model, grant access to information on the atomic displacement correlation in nanostructured particles, related to the phonon dispersionDifferences in Q values have been employed to investigate correlated atomic displacements by means of the atomic Pair Distribution Function (PDF) approach2526272826Q data. Distinctive features of the microstructure, like those related to domain shape  peak in kij parameters) affect the entire powder pattern, involving both diffuse and Bragg intensity, not only peak broadening. This fact is illustrated by kij . Even more interestingly, the effect of temperature on vibrational properties can be evaluated and the analysis of their modification as a consequence of the interaction with a specific environment (e.g. during oxidation/reduction or in operando) can be performed, therefore shedding new light on in vivo structural and dynamical features of nanostructured particles.In conclusion, the presented extension to the Debye scattering equation (i) grants access to information on the correlated thermal displacement in nanostructured particles and (ii) concurs to enhance and strengthen the detail of information retrieved from scattering data, as demonstrated in How to cite this article: Scardi, P. and Gelisio, L. Vibrational Properties of Nanocrystals from the Debye Scattering Equation. Sci. Rep.6, 22221; doi: 10.1038/srep22221 (2016).


Simultaneous bilateral distal biceps rupture is rarely reported, but several issues has to be taken into account and the here presented case brings up some of these aspects.This case presents a simultaneous bilateral distal biceps ruptures, and due to delayed diagnosis, bilateral muscle retraction had occurred. Surgical reconstruction was challenged both by social circumstances and by previous harvesting of the Semitendinous tendon on one side. The surgery was performed as a staged approach, using a Semitendinosus graft at one side and later using a Quadriceps tendon graft on the opposite site. At follow-up 14 month postoperatively the Oxford Elbow Score was 92 percent for both elbows and the MRI’s bilaterally demostrates that the grafts are tight.The Quadriceps tendon is a previously non reported type of graft material for this type of surgery. Based on the experience from this case it is concluded that six weeks after trauma the degree of retraction of the distal biceps tendon can impair direct repair.When reconstruction of the ruptured distal biceps tendon is needed, subjective normal forces can be obtained using both the semitendosus graft as well as the quadriceps graft, however with a minor increased donor site morbidity with respect to the quadriceps graft. Simultaneous bilateral distal biceps rupture is rare. It is generally accepted that early surgical treatment is recommended for distal biceps rupture, since the outcome of conservative treatment often results in reduced strength and negative consequences for activities of daily living and especially more strenuous activities  and a concomitant incision more proximal to the fossa, the lacertus fibrosus was found ruptured and the biceps tendon was dissected and found retracted approximately 8 cm. After extensive mobilization by blunt finger and scissors dissection around the m. biceps brachii, a tendon gap of 3 cm to the tuber radii, in combination with a thin tendon sustained with a longitudinal rupture as well, prevented primary repair. From the ipsilateral lower limb a good quality semitendinosus tendon graft was harvested. The tuberosity radii foot print was debrided and a central placed 5 mm metal Corkscrew anchor loaded with double Fiberwire  sutures was inserted and the graft was sutured firmly to footprint and then merged into the biceps tendon and secured in the correct length, by evaluation of the tension in fully elbow extension and pronation, using Orthocord #2 suture.Similar exposure and technique was used as in the right arm and also the preoperatively findings were equal compared to the left arm. With no easy access to allograft tissue and with a lag of hamstring graft based on previous harvesting bilaterally, it was decided to use the ipsilateral quadriceps tendon. A 8 mm wide, 4 mm thick and 8 cm long full thickness quadriceps tendon graft was harvested and reinserted corresponding to the technique described for the right arm.Both elbows were subject to the same rehabilitation protocol. The elbow was unloaded by a 90 degrees solve universal arm sling for four weeks. Guided full range of movement with passive exercises and active extension and pronation exercises for the elbow and forearm was allowed from day one postoperatively and physiotherapy was started after one week. Active exercises for elbow flexion against gravity and forearm supination, was allowed after 4 weeks. A maximum of 2 kg load after 6 weeks and a maximum of 10 kg load were allowed after 10 weeks.At 4–5 month follow-up, normal range of movement had been achieved and muscle strength was subjectively recovered. The biceps muscle belly appeared displaced a few centimeters proximal bilaterally. See Figure From this case we have demonstrated that good results can be obtained by delayed reconstruction of distal biceps ruptures, both by using semitendinosus and by using quadriceps grafts. The patient presented achieved full range of movements and regained elbow and forearm strength. Regardless of whether a repair or reconstruction is warranted, you have to decide if to repair either both tendon ruptures at the same time, or to do it in a staged manner. Several factors must be considered such as the patients’ social and occupational situation, hand dominance, medical comorbidities and general health. In cases with simultaneous repair or reconstruction, the mobility of the arms are reduced based on the postoperative regime, that normally restricts active flexion and supination for a 2–6 weeks, according the surgeons preferences. With the staged approach the patient is allowed complete use of the non-operated arm for activities of daily living while convalescing from surgery of the opposite arm. There is currently no evidence to guide this, and the decision should be made in collaboration with the patient. From the literature it seem that comparable good results can be obtained for both acute tendon reinsertion and for later reconstruction of the tendon gap (Wiley et al. Complete ruptures of the distal biceps tendon are classified as acute or chronic, depending on the time period between injury and diagnosis. Ruptures occurring after 4 weeks are generally considered chronic (Bell et al. Based on the experience from this case we can conclude that six weeks after trauma the degree of retraction of the distal biceps tendon can impair direct repair. Subjective normal forces can be obtained using both the semitendinosus graft as well as the quadriceps graft, however the quadriceps graft gives a minor increased donor site morbidity the first year.Written informed consent was obtained from the patient for the publication of this report and any accompanying images.


International Journal of Medicine and Medical Sciences, vol. 6, no. 9, pp. 201–210, 2014.[27] P. R. da Costa, D. da Cunha Sá-Caputo, A. Arnóbio et al., “Whole-body vibration and benefits for people with osteoarthritis: a system review,”In our paper entitled “Whole-Body Vibration Exercise for Knee Osteoarthritis: A Systematic Review and Meta-Analysis” , there wThese corrections do not change any conclusion or findings of the paper.


Oxidative stress is one of the key mechanisms affecting the outcome throughout the course of organ transplantation. It is widely believed that the redox balance is dysregulated during ischemia and reperfusion (I/R) and causes subsequent oxidative injury, resulting from the formation of reactive oxygen species (ROS). Moreover, in order to alleviate organ shortage, increasing number of grafts is retrieved from fatty, older, and even non-heart-beating donors that are particularly vulnerable to the accumulation of ROS. To improve the viability of grafts and reduce the risk of posttransplant dysfunction, a large number of studies have been done focusing on the antioxidant treatments for the purpose of maintaining the redox balance and thereby protecting the grafts. This review provides an overview of these emerging antioxidant treatments, targeting donor, graft preservation, and recipient as well. Ischemia/reperfusion injury (IRI) is present in many medical situations, specifically in organ transplantation. This event can lead to immediate and long-term graft dysfunction, such as allograft rejection, delayed graft function (DGF), and even primary nonfunction (PNF) . It is dOxidative stress, known as an imbalance between the generation of ROS and antioxidant defense system, is the disease mechanism most commonly involved in IRI. It has been proven that ischemia initiates the noxious generation of ROS, while the reoxygenation process during reperfusion is responsible for most ROS production, activation of complement system, and inflammatory response . EvidencThe ROS-induced injurious effect on graft and recipient is related to various posttransplant complications. Besides, marginal grafts are significantly vulnerable to the oxidative stress and thus restrict graft pool, which aggravates organ shortage. To alleviate such adverse outcomes, a volume of preclinical and clinical studies against oxidative stress are under investigation, targeting consecutive process throughout the transplantation, including donor, graft preservation, and recipient as well. Despite some new discoveries in the mechanism against ROS, the clinical results remain controversial. Therefore, we summarize some advanced development of antioxidant treatments in organ transplantation and their corresponding mechanisms.2+, and preventing nucleus injury and DNA fragmentation [Local ischemic preconditioning (LIPC), a widely accepted antioxidant approach, is a brief period of ischemia/reperfusion that leads to tolerance of subsequent ischemia/reperfusion injury (IRI). In animal studies, LIPC has been proven to be an efficient tool to protect most organs , particularly via antioxidant pathway . Concernentation . From beentation . Howeverentation  nor has entation . These centation . To furtentation . MoreoveκB signaling pathway [Hydrogen is a reducing gas that displays antioxidant properties and exhibits protective effects against graft IRI and dysfunctions. Though application of mechanical ventilation (MV) in intensive care unit supports donor's life, it can also provoke oxidative stress and inflammatory response, causing ventilator-induced lung injury (VILI) and reducing graft viability . To prev pathway . Therefo pathway , though  pathway . Another pathway . Nowaday2O2 and superoxide, are highly potent and specific agents to the ROS-induced injury and not consumed in reaction with ROS. However, due to inability to cross cell membrane barriers and fast elimination, a significant hurdle in the clinical translation lies in the insufficient delivery of these enzymes to targeted sites, especially the vascular endothelium suffering oxidative injury [High levels of ROS play a pivotal role in transplant-associated IRI, especially in cadaveric donors. Antioxidant enzymes (AOEs), such as catalase and SOD that detoxify He injury . It is ae injury . To enhaApplication of vascular immune targeting AOEs to specific endothelial epitopes has been proven to be an effective donor preconditioning method in lung transplantation model. The nanosized conjugates, consisting of AOEs and specific antibodies, can be directed against the endothelial determinant accumulation in vascular endothelium after intravenous administration and eventually delivered into endothelial cell, thus alleviating oxidative stress. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is such an endothelial epitope and the specific nanosized particles  have been examined in a porcine lung transplantation model, in which the immune targeting treatment allows immediate reconstitution of pulmonary gas exchange and microcirculation, and improve both graft and recipient outcomes . MoreoveκB pathway, curcumin, to target delivery to renal tubular epithelial and antigen-presenting cells. In a renal IR model, liposomal curcumin provided cytoprotection effect through multiple antioxidant mechanisms following renal IR injury [Liposomes are artificial vesicles consisting of one or more phospholipid layers, with an aqueous core enclosed; lipid-soluble antioxidants can be incorporated into the lipid bilayer, while water-soluble antioxidants  can be encapsulated in the aqueous space. NAC is a well-known antioxidant which can function both as a ROS scavenger and as a precursor of reduced glutathione, thus modulating the redox status. To improve the bioavailability, NAC is encapsulated in the aqueous space of liposome, gaining a higher protective potency than the free drug. Liposomal NAC has been administered separately in rodents to protect lung  and liveR injury .Conclusively, immune targeting therapy and liposome formulation are promising approaches to facilitate the delivery of AOEs and nonenzymatic antioxidants before ischemia process. Though there is no associated animal transplantation study performed, we hold that the carriers can help the antioxidants administered to a donor quickly bind to endothelium and subsequently protect graft from IRI. However, these carriers must be carefully tested in terms of toxicity, activation of defense systems, inflammation or thrombosis, and aggregation in circulation and embolism of the microvasculature.Ex vivo graft preservation is necessary for allocating and transporting the graft to its recipient. It has been widely accepted that primary occurrence of ROS-induced injury arises from the cellular alteration during the ischemia process, especially in cold preservation period. Several approaches including machine perfusion and modified preservation solutions have been developed to reduce the injury.Machine perfusion (MP) is increasingly used as an alternative method to overcome the present shortage of donors by expanding the graft pool and prolong the storage time. It is a dynamic technique using a continuous flow of solutions to perfuse and maintain residual metabolism of the graft . In recePolyethylene glycol (PEG), synthesized as liner or branched polymers in different sizes, functions as an alternation of hydroxyethyl starch (HES) contained in UW solution due to its low viscosity. As an “immunocamouflage” agent, PEG binds covalently to various biological surfaces and forms complexes with cell membrane lipids, membrane proteins, or carbohydrates, preventing osmotic swelling as well as lipid peroxidation (LPO) in graft cold storage. PEG is also an effective free radical scavenger and can modulate oxidative stress during preservation. Owing to these protective effects, several PEG-based preservation solutions, including Polysol, IGL-1 solution, and SCOT, have been developed for organ preservation.Polysol solution is a colloid-based low-viscosity organ preservation solution containing vitamins, amino acid, and a variety of ROS scavengers , which possess strong antioxidant capacity. Polysol solution is applied on a steatotic rat liver perfusion model and significantly attenuates LPO to nearly one fourth of that in HTK control . In rat 2/s), higher sodium, and lower potassium compared with UW solution. The application of IGL-1 solution has been reported in the SCS of pancreas [Institute Georges Lopez-1 (IGL-1) solution is characterized by lower viscosity  has shown its protective potential in a pancreatic islet transplantation model, reducing IRI and ameliorating the long-term outcome of recipients' immune response . To examHydrogen-rich preservation solution has been proven to have high antioxidant potential and tested in liver, kidney, pancreas, bone marrow, lung, and intestinal cold storage . The antNO is a kind of free radical diatomic gas and gaseous signaling molecule. The protective potential of NO is associated with the reduction of superoxide anion-induced tissue toxicity and the inflammatory response. Furthermore, NO can modulate mitochondrial energy generation and thus decrease ROS formation during I/R period. Kageyama et al.  investigCarbon monoxide (CO) is also a gaseous signaling molecule and possesses a high affinity for heme prosthetic group. CO supplemented to preservation solution has been proven to improve the graft function in experimental studies , 47. A p2S) is considered as the third gaseous signaling molecule with properties to help relax vascular smooth muscle, inhibit apoptosis, modulate inflammatory response, and alleviate oxidative stress [2S) has been proven to possess better antioxidant potential in vitro when compared to that of L-arginine (a source of endogenous NO). The effective doses of both compounds in human body are believed to be 56 μM for H2S and 1.2 g/mL for L-arginine, respectively, which provides essential information for subsequent human study [Hydrogen sulfide  plays an essential role in cell death during IRI, induced by ROS and reversely aggravating the oxidative stress . To protAscorbic acid (AA) is a potent physiological extracellular scavenger of ROS. AA has been supplemented into HTK and Polysol solution to prevent ROS. Noticeably, high-concentrated AA has been reported to aggravate hepatic IRI owing to its excess reduction of iron . Alpha-tκBp65 and MDA expression, as well as ameliorating the destructive capacity of oxygen free radicals. From bench to bedside, induced by three cycles of brief 5 min repetitive ischemia and reperfusion via clamping the exposed external iliac artery, Wu et al. [Remote ischemic postconditioning (RIPoC) is induced by several cycles of I/R on a remote tissue (arm or leg) to produce systemic protection against IRI in distant organs, without direct access to the vessels of the organ of interest. RIPoC can increase antioxidant capacity of liver and kidney temporarily by reducing NF-u et al.  demonstru et al. , 58 perfu et al. . Althougu et al. . NeverthSimilar to LIPC, local ischemic postconditioning (LIPoC) is defined as rapid and intermittent interruptions of blood flow in the early phase of reperfusion after a prolonged period of ischemia. In a canine autotransplantation model, after flushing and static preservation of the kidney for 24 hours, LIPoC was performed with six cycles of 10 or 30 seconds or three cycles of 1-minute I/R before final reperfusion. The result indicates enhanced level of expression of SOD and decreased levels of MDA, implying that LIPoC may protect the graft via an antioxidant pathway , whereasOxidative stress is a common cause of PNF, DGF, and allograft rejection, especially in marginal donors. This review summarizes the innovative antioxidant treatments for the donor, graft preservation, or recipient designed to improve the graft viability and long-term outcome (Tables Although SCS has been effective for decades on optimal organs preservation, the preservation protocol is not adapted to the increasing marginal grafts which is able to extend the graft pool. Thus, the novel antioxidant preservation solution, as well as various supplements, requires more mechanism researches and pragmatic RCT. Combined utilization of antioxidant approaches may be more promising than attempts to reinforce the antioxidant capacity of organs by a single agent functioning as ROS scavenger. Even though the antioxidant potential of NMP is still unclear, this approach is worth more intensive researches. To this end, though a combination of antioxidant treatments seem to provide the best outcome, accurate models for preclinical studies and unified protocols for clinical trials are needed before the treatments can be translated into clinical practical. Moreover, the potential adverse effect of local and systemic antioxidant interventions to donors, grafts, and recipients, such as host defense hazard and prooxidant effects, should not be neglected. Antioxidant administration should also be controlled at a gradual, controlled rate, thus avoiding burst release and comprising effect.


The world population is aging and the number of old people is continuously increasing. Arterial structure and function change with age, progressively leading to arterial stiffening. Arterial stiffness is best characterized by measurement of pulse wave velocity (PWV), which is its surrogate marker. It has been shown that PWV could improve cardiovascular event prediction in models that included standard risk factors. Consequently, it might therefore enable better identification of populations at high-risk of cardiovascular morbidity and mortality. The present review is focused on a survey of different pharmacological therapeutic options for decreasing arterial stiffness. The influence of several groups of drugs is described: antihypertensive drugs , statins, peroral antidiabetics, advanced glycation end-products (AGE) cross-link breakers, anti-inflammatory drugs, endothelin-A receptor antagonists, and vasopeptidase inhibitors. All of these have shown some effect in decreasing arterial stiffness. Nevertheless, further studies are needed which should address the influence of arterial stiffness diminishment on major adverse cardiovascular and cerebrovascular events (MACCE). Although evidence of the importance of PWV is growing, there was no study reported in which a decrease of cardiovascular mortality due to reducing arterial stiffness by pharmacologic approaches had been observed. Nevertheless, we believe that there is sufficient proof of PWV being an important cardiovascular risk factor and that such a study is very much needed. Therefore, in what follows we review all known pharmacological approaches capable of decreasing arterial stiffness. Importantly, it should be noted that the effects of pharmacologic agents on stiffness are usually slight or modest, but not substantial. Thus, new therapeutic approaches to decrease arterial stiffness are highly desirable.The world population is aging so the number of old people is continuously increasing , 2. WithConductive arteries propel the pressure wave generated by the heart, that is, the ejection of blood from the left ventricle. This wave is reflected at the impedance mismatch points , from where it travels backwards to the heart. Consequently, the observed generated wave is the sum of the forward travelling wave (moving from the heart) and the reflected wave (travelling backwards towards the heart) . In younThe low blood pressure elastic modulus of the elastin component of arterial media dominates the mechanical behavior of the arterial wall, making it distensible . At high in vivo is not possible due to uncertainties arising from nonlinear behavior, viscoelasticity, anisotropy, active tone, residual stresses, and tethering [The arteries become stiffer with increasing age and disease . Increased stiffness results from structural changes, such as fragmentation of elastin, an increased amount of collagen, arterial calcification, glycation of both elastin and collagen, and cross-linking of collagen by advanced glycation end- products (AGE) –17. Thesethering . The metethering .Increased arterial stiffness leads to increased PWV and central arterial pressure, resulting in higher arterial pulsatility. The latter leads to damage of the microcirculation in several organs, especially the highly perfused ones, such as myocardium, the kidneys, and brain . Taking Endothelial function and arterial stiffness are two different aspects of arterial disease, which are interconnected as their pathophysiological background is similar. Nitric oxide (NO) has been shown to contribute importantly to arterial compliance or distensibility . ArteriaAccording to the literature, several drugs have been shown to influence arterial stiffness: antihypertensives, statins, peroral antidiabetics, AGE cross-link breakers, anti-inflammatory drugs, endothelin-A receptor antagonists, and vasopeptidase inhibitors. The majority of them act predominantly on the dynamic component of arterial stiffness and to a lesser extent on the structural component in arterial wall remodeling, whereas only AGE cross-link breakers act directly on the structural component. Influence of particular drugs or drug groups on arterial stiffness is summarized in  per se leads to a change in the properties of the vessel [Antihypertensive drugs have been implicated in arterial stiffness diminishment but vary in their degree of effect. The various antihypertensive drug classes have been more or less extensively evaluated in this regard. Unequivocally, the renin-angiotensin system inhibitors proved to be superior to all other antihypertensive drugs in reducing arterial stiffness. There are different reasons for and understanding of this phenomenon, but the most probable explanation lies in the profibrotic action of the renin-angiotensin system, as the turnover of the extracellular matrix in the arterial walle vessel . In whatThere had been some evidence that angiotensin-converting enzyme (ACE) inhibitors lead to arterial compliance improvement , but it Intuitively, it might be expected that angiotensin receptor blockers (ARBs or sartans) would produce the same effect as ACE inhibitors. In a trial with patients with resistant hypertension who were receiving three antihypertensive drugs in maximal dosages, including ACE inhibitors, the addition of valsartan for two weeks resulted in reduction of Aix . When va per se as the other mentioned drugs. The mechanism of action is through heart rate reduction, as this influences the viscoelastic properties of the arterial wall. Reduced heart rate also leads to increased wave reflections, a lower reduction in aortic than brachial systolic blood pressure, and reduced pulse pressure amplification. Peripheral vasoconstriction, achieved by, for example, atenolol, is an additional mechanism responsible for the negative effect on wave reflections [Beta-blockers without vasodilating properties have been shown to have a weaker effect on arterial stiffness and central pulsatile hemodynamics than vasodilating drugs of other groups. Nevertheless, they showed the same extent of reduction of arterial stiffnesslections –77. The lections . Differelections . Thus thlections . New, inlections , 74–77.Calcium channel blockers also lower PWV and reduce wave reflections, but to a lesser degree than renin-angiotensin inhibitors. The largest amount of evidence is for the dihydropyridine calcium channel blocker amlodipine , 49, 55.Diuretics seem to have no beneficial effect on pulsatile hemodynamics. Many agents were studied, including hydrochlorothiazide, which showed a neutral effect on reduction of central blood pressure and a neutral effect on pulse pressure amplification , 55. ConThe aldosterone antagonist spironolactone proved to reduce PWV and Aix when adjusted for blood pressure, compared to bendrofluazide . The benDirect renin inhibitor, that is, aliskiren, the only one available, was evaluated in diabetes mellitus type 1 and type 2 patients where it reduced PWV as well as Aix, thus showing a beneficial destiffening effect , 71, 72.Nitrates have also been studied in this regard. As vasodilating drugs, they influence the smooth muscle cells of large arteries, leading to possible arterial destiffening. Their effect was evaluated in hypertensive patients, where isosorbide mononitrate proved to amplify systolic and pulse pressure but substantially reduced Aix. On the other hand, they did not influence PWV, therefore leading to the conclusion that their effect on arterial stiffness was minimal .It seems that arterial stiffness reduction can be obtained with different antihypertensive drugs. The highest effects are exerted by inhibitors of the renin-angiotensin system. Stiffness reduction seems to be correlated with the dose of antihypertensive drug. Long-term treatment also seems to have a greater effect. Mechanisms behind this are the slow extracellular matrix turnover, the long-term constant of arterial remodeling, and the necessity that the target tissue systems influence changes in arterial stiffness more than blood pressure .Statins (HMG-CoA reductase inhibitors), besides their basic action in reducing low-density lipoprotein (LDL) cholesterol, also have several additional protective/beneficial effects (pleiotropic effects) on the cardiovascular system . There aγ) agonists, was shown not only to improve insulin resistance and glycemic control, but also to decrease arterial stiffness in patients with type 2 diabetes mellitus [Treatment with glitazones, peroxisome proliferator-activated receptor gamma  were shown to be effective in improving arterial stiffness in several studies, including patients with chronic inflammatory diseases [Inflammation states, such as inflammatory bowel disease , 122, rhdiseases –107. In diseases –110. Cordiseases . Conflicdiseases , 112.The serum concentration of endothelin-1 was shown to correlate with aortic elasticity parameters in 152 subjects with essential hypertension . TherefoVasopeptidase inhibitors are a new class of drugs that act on two key enzymes in the metabolism of vasoactive peptides: they inhibit ACE to reduce vasoconstriction and inhibit endopeptidase, which is involved in the degradation of several natriuretic peptides, to enhance vasodilation , 127. InNew, innovative therapeutic pharmacological options are emerging and show some promise . Short tArterial stiffness progressively increases with age and was found to be a risk factor of cardiovascular disease. Therefore, besides identifying solely classical cardiovascular risk factors, it appears to be more and more important to assess arterial stiffness, which could be easily measured noninvasively and expressed as PWV. This modified or advanced approach enables better cardiovascular risk stratification. We believe that such an approach should be introduced into the treatment and prevention of cardiovascular disease. At the present there are several pharmacological agents which could influence arterial stiffness. Consequently, the present review focused on a survey of different pharmacological therapeutic options for decreasing arterial stiffness. However, new and more effective treatment is highly desirable. Furthermore, future studies should address the influence of decrease in arterial stiffness on major adverse cardiovascular and cerebrovascular events (MACCE).


Library and Archives Canada (LAC) contains a number of resources relevant to Indigenous heritage, among them the School Files Series which contains government records created from 1879 to 1953 about residential and day schools operating in Canada and available on microfilm. In recent years, many of these microfilm images have been digitized and made available on the LAC and other websites.The school records contain information on admissions, discharges, day-to-day operation and financial reports. They also contain material on family background, health examinations and death registers. The material is organized using a series of filing systems and is accompanied by helpful guides and search tools. Much of the data are “open-coded” meaning the material is available for public use. Researchers may freely consult these records without restriction.The Tri-Council Policy Statement on Research Involving Humans states that research involving publicly available data is exempt from the requirement for ethical review. However, the statement also recommends that researchers interpreting the ethical framework in Indigenous contexts uphold the principles of respect for persons, concern for welfare and the advancement of justice.To that end, we have asked the authors of this paper to provide a brief commentary on the ethical issues arising from their work. We have also asked Ry Moran, Director of the National Centre for Truth and Reconciliation, to comment on the implications of this type of research for the development and implementation of processes surrounding data access and use.Tracey Galloway Deputy Editor International Journal of Circumpolar Health University of Toronto Mississauga Mississauga, ON, Canada Email: tracey.galloway@utoronto.caRhonda Johnson Editor-in-Chief International Journal of Circumpolar Health University of Alaska Anchorage Anchorage, AK, USA Email: rmjohnson2@uaa.alaska.edu


It also allowed the identification of rapid and slow phases in starch digestion. Upon gelatinisation, both k and C∞ increased with dramatic changes notably in C∞; however after starch samples had been stored for 24 h at room temperature, k was not affected but C∞ decreased. This suggests that retrograded starch is virtually inert to amylase action. Both k and C∞ were strongly related to the increase in degree of order of the α-glucan chains, monitored by FTIR-ATR spectroscopy, in retrograded starch.The rate and extent of digestibility of starch were analysed using the logarithm of the slope (LOS) method. Digestibility curves with α-amylase were obtained for starches in their native, gelatinised and 24 h retrograded form. A LOS plot of the digestibility curves was then constructed, which allowed the rate constant ( Amylose and amylopectin are the two major α-glucan polymers of starch, with amylopectin having a higher average molecular weight and a more branched structure than amylose. Both contain glucose units joined by α 1–4 glycosidic bonds, which can be hydrolysed by α-amylase to produce oligosaccharides, especially maltose. The branches in amylopectin are formed through α 1–6 glycosidic bonds, which are linkages that are resistant to α-amylase action . OligosaIt is now widely documented that starch-rich food products with similar starch contents can produce different postprandial blood glucose and insulin responses in human subjects. Therefore there is considerable interest in understanding the basis for the observed differences in rates of intestinal starch digestion following a starch-rich meal. These differences have prompted studies of factors, such as the chemical and physical structure and properties of starch at the molecular and granular levels, that affect the rate and extent of α-amylase action on numerous native and hydrothermally processed starches .in vitro starch digestion with pancreatic α-amylase to mimic in vivo amylolysis and predict the likely in vivo glycaemia resulting from α-amylase acting on starch-rich food materials. This allows foods to be classified on the basis of their potential glycaemic index. The in vitro approach to determining the rate of starch digestion has advantages over in vivo studies  in being considerably cheaper to perform and no ethical authorisation is required. In addition, the in vitro experiments are less time consuming , which in studies performed in vivo is known to escape digestion in the small intestine  is a surface analytical method that is used to examine the external surface of starch samples. FTIR-ATR therefore allowed determination of the relative proportions of ordered and disordered structures for dispersions of native, gelatinised and 24 h retrograded starch. The FTIR spectra were used to compare the organisation of starch granules with their susceptibility to amylolysis  and wild type pea starch were gifts from Prof. C. Hedley and Prof. T. Bogracheva . Purified potato starch was bought from the National Starch and Chemical Company . Maize, waxy maize and high amylose maize starch were gifts from Dr. C. Pelkman at Ingredion. Rice starch was obtained from Sigma–Aldrich Ltd. Durum wheat grains for durum wheat starch extraction were bought from Millbo, Italy and extracted by the method described in at 25 °C . The pur2.2Starch moisture was determined gravimetrically by weighing the starch sample onto pre-dried aluminium pans that were then heated overnight at approximately 103 °C. Upon removal, the dried weight was recorded and the moisture content was calculated by the difference between fresh weight and dry weight. The amylose/amylopectin content for the starches was analysed using the iodine dye binding method of 2.32CO3 stop solution in Eppendorf tubes. The ice-cold stop solution rapidly cools the reaction sample and raises the pH to a level where amylase is no longer active. The Eppendorfs were then centrifuged and the supernatant was collected and used for determination of reducing sugar concentration by a Prussian blue assay (2CO3 in distilled H2O) and solution B (1.18 mM K3Fe(CN)6 in distilled H2O) were added. Samples were then boiled for 15 min and allowed to cool at room temperature before 750 μL of solution C (3.11 mM NH4Fe(SO4)2, 0.1% (w/w) SDS, 0.2% (v/v) H2SO4 in distilled H2O) was added. The tubes were allowed to stand at room temperature for 2.5 h before the absorbance was recorded at 695 nm. Maltose standards ranging from 0 to 100 μM were treated similarly and used for quantification of the content of reducing sugar expressed as maltose equivalents. For studies of gelatinised material, starch was heated for 20 min at 90 °C and then cooled to 37 °C before α-amylase was added to produce a final concentration of 2.25 nM. Starch containing retrograded material was prepared by similar heat treatment followed by a storage period of 24 h at room temperature before α-amylase was added to give an enzyme concentration of 2.25 nM for digestibility measurements.A 5 mg/mL solution of native starch was prepared in PBS and incubated at 37 °C with 4.5 nM (approximately 0.33 IU) porcine pancreatic α-amylase on an inclined rotating table to allow constant end-over-end mixing. Samples were withdrawn at timed intervals up to 120 min and transferred to ice-cold 0.3 M Naue assay . Each sak, and the total digestible starch, C∞, are obtainable from plots of LOS against time.The slopes of digestibility curves were measured at various time points throughout the incubation period, converted to logarithmic form and then fitted to the first-order kinetic model  and allowed to cool to 37 °C, before FTIR spectra were taken. Retrograded starch was prepared by a similar heat treatment procedure but stored at room temperature for 24 h before a spectrum was taken. A spectrum for distilled H2O was also measured and subtracted from the final sample spectra before the data were normalised and compared  starch. The absence of discontinuities in LOS plots obtained for gelatinised and 24 h retrograded starches, suggests that retrogradation has not brought about the production of a new structural element that can be digested, albeit at a slower rate.Retrograded starch is primarily retrograded amylose because the relatively short and mostly linear amylose chains can re-associate within 48 h, whereas the bulky amylopectin can take several days to re-associate . Sievertper se is digested at a slower rate than gelatinised starch since k values for gelatinised and 24 h retrograded starches are similar. If retrograded starch is virtually inert towards α-amylase, the measured k value will only reflect the digestion of starch in the digestible/accessible form that remains within the preparation, i.e., material that has not become retrograded. Therefore, the decrease in C∞ values, coupled with unchanged k values, for the retrograded preparations relative to the gelatinised starch, seems to provide evidence that retrograded starch is inert to α-amylase action over the time scale of the experiment. Inhibition by product maltose of amylase activity seems an unlikely explanation for the decrease in C∞. The Ki for maltose at 9–14 mM is indicative of a weak inhibitor (In our preparation of retrograded starch, the bulk of the starch material present is still in the gelatinised, rapidly digested, form but the quantity of non-digestible starch is increased by retrogradation of amylose particularly . There inhibitor  and its 4In vitro digestibility curves for different botanical starches in their native, gelatinised and retrograded starch forms are well fitted by the first-order kinetic equation. This allows for the accurate determination of C∞ and k values from a LOS plot. All starches in their native or processed forms display different digestibility rates due to variations in the proportion of amorphous material, resulting in a distinctive rate constant for different starch fractions. The digestibility rate constants of the various gelatinised starches are virtually identical because they represent an intrinsic catalytic property of amylase ( amylase . Where dUpon 24 h retrogradation, the quantity of digestible starch is decreased compared with gelatinised starch due to changes in starch crystallinity/order. Evidence for changes in starch crystallinity was obtained from FTIR-ATR spectra, which indicated an increase in the degree of ordered structure.in vivo of starch in foods and the consequent glycaemia.This study illustrates that LOS plots can be applied to different botanical sources of starches and that the rate and extent of digestion can be accurately determined for native and processed starches. Values for the maximum extent of digestion are important for predicting the total digestibility