BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling:  Hordeum vulgare L.) is the fourth most important cereal crop both in terms of cultivated area and tonnage harvested; global production being mostly used as animal feed and for the malting industry (http://faostat.fao.org). Only 5% of the global production of barley is used as ingredients in food preparation, but nevertheless barley grains are a valuable functional food for the high content of soluble dietary fiber Barley  as well as mutants containing up to 70% amylose in the endosperm have been described Starches with different amylose/amylopectin ratios have different properties that influence their possible use for either nutritional purposes or industrial transformations Starch granule size distribution is another important parameter that may affect technological properties and end-use of each particular type of starch BMY1 (beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Seven mutant lines present starches with potentially interesting features for nutritional uses and/or industrial applications, including an altered amylose/amylopectin ratio or an unusually high percentage of A-granules or A-granules that are larger than in wild type starch.In a previous work using a TILLING strategy, novel allelic variants in genes involved in starch metabolism in barley seeds were identified 3) mutagenized barley population including 4,906 M3∶4 families Beta-amylase 1 (BMY1), Granule-Bound Starch Synthase I (GBSSI), Limit dextrinase 1 (LDA1), Starch Synthase I (SSI) and Starch Synthase IIa (SSIIa) (4 lines (three generations of selfing after mutation induction), which have been verified to be homozygous for the mutation (not shown). Mutant lines and cv. Morex were grown in open field following standard agronomic practice in 0.5-m long one-row plots (approx. 12 plants per plot) using a randomized design with two replicates. For each line, grains harvested  from two replicates were bulked. The same experiment was carried out in two years. Grains from separated years constituted the biological replicates.Details on the TILLING-based molecular screening for the five starch metabolism enzymes were reported in  (SSIIa) . For eacSSIIa for which 2.5 g of seeds were used). Starch grains were purified as described in Starch was extracted by grinding the grains to a fine powder in a pepper mill. About 5 g of seeds, corresponding to about 100 seeds, were used for each line and for each biological replicate . The relative content of amylose was determined using both the Amylose/Amylopectin assay kit  following the manufacturer instructions, and by an iodometric assay as reported in Sestili et al. The morphology of starch samples was analyzed using a scanning electron microscopy (SEM) Hitachi S-4000. The samples were glued by a carbon type on an aluminum stub and gold coated (2 nm thick layer) before observations. For each sample two sets of 10 pictures at two magnifications (1000x and 2500x) were randomly collected. These two magnifications allowed a good estimation of the size of large and small granules. The granule size was estimated using the software ImageJ for image processing and analysis. The two main axes for each granule were recorded and 200–500 grains were measured for each sample. Percentage of granules with major axis lower than 8 µm (B-granules) was recorded in 10 pairs of pictures (at different magnification) for each genotype. Statistically significant differences between mutants and wild type mean values were detected by Student's t-test (P<0.01). The frequency analysis was carried out tacking classes of 2 µm.The X-ray powder diffraction patterns were recorded using a Philips X'Celerator diffractometer with Cu Ka radiation (l = 1.5418 Å) and equipped with a Ni filter. The samples were scanned for 2θ angles between 5° and 30°, with a resolution of 0.02°. The degree of crystallinity of samples was quantitatively estimated following the method of Nara and Komiya In the diffraction patterns only peaks associable to A-type and V-type crystallinities were detected. To estimate the relative amount of A-type and V-type crystallinities in the starch samples from the diffraction patterns, only well isolated diffraction peaks were considered. The one at 15.1° is diagnostic of the A-type crystallinity and the one at 19.7° is diagnostic of the V-type one. These diffraction intensities has been normalized on the sum of their intensities.3BMY1 GBSSI, LDA1, SSI and SSIIa) were phenotypically characterized in this study (SSIIa (Starch Synthase IIa) that showed shrunken kernels with an empty cavity inside , and mutant 1517-SSIIa, carrying a nonsense mutation in Starch Synthase IIa, contained much more amylose than the wild type , SSI and GBSSI SSIIa. Notably this mutant appeared to lack also SBEII and SSI isoforms. Moreover, although mutant 1284-SSI has no premature stop codon in the SSI gene, a drastic reduction of the SSI band was observed in starch granules . With the exception of 1517-y shaped . A quanty shaped , with a  spheres . B-granuBMY1, GBSSI and SSIIa (SSI (57%) and 5850-SSI (62%), but relatively more abundant in mutant 1284-SSI of the same gene (85%) and in mutant 905-LDA1 of limit dextrinase I (85%) (The percentage of B-granules (<8 µm) in wild-type purified starch was 73% (SD). A similar value was found in mutants of nd SSIIa . In the  I (85%) . All the I (85%) .SSI and 1039-SSIIa, both missense mutations), A-type granules were significantly smaller (−25% major axis) than wt ones (17.1 µm) (SSI mutant were significantly larger (+15%) than wt ones. The average diameter of B-particles varied between 2.3 and 3.5 µm in all samples, with no significant differences between wt and mutants  ; P<0.01. mutants . Interes in size .i.e. 78–83% A-type and 17–22% V-type. Interestingly, however, in the low-amylose 1090-GBSSI mutant, crystallinity was almost exclusively of the A-type (92%) whereas in the high amylose 1517-SSIIa mutant crystallinity was prevalently of the V-type (76%)  .Starch structure and chemical composition are genetically determined by a large set of genes GBSSI) waxy locus and barley cultivars with altered GBSSI activity contain altered levels of amylose in grains, ranging between 0 and 41% of total starch waxy mutants Granule-bound starch synthase I (GBSSI with a G493E point mutation. Grain starch of this mutant (1090-GBSSI) contains less than 10% amylose (vs. 30% of wild-type) and is thus defined low-amylose or near-waxy. Crystallinity of 1090-GBSSI starch was found to be largely A-type, with a minor contribution of the V-type pattern. A similar profile has been previously reported in low amylose barley Here we report a new allele of Plant starch synthases (both granule bound and soluble isoforms) are proteins of about 60–120 kDa that belong to the glycosyltransferase family GT-5 GBSSI gene that results in strongly diminished expression of the enzyme GBSSI seems to modulate, rather than inactivate, enzyme activity as demonstrated by the residual content of amylose (10%) detected in its starch. Moreover, this effect is obtained without altering protein expression, as suggested by the SDS-PAGE pattern identical to the wild-type. Indeed, mutation G493E may prove useful for the understanding the little known catalytic mechanism of GBSSI.Several near-waxy cultivars are known in barley, all carrying a large deletion in the promoter region of the LDA1) and is apparently involved both in starch biosynthesis and degradation In vivo, the activity of LDA1 is regulated by a proteinaceous inhibitor LDI i.e. inhibition of granule initiation) and lower amylose/amylopectin ratio LDA1 (905-LDA1) with a higher percentage of small B-granules that further supports the role of this enzyme in starch granule initiation.Together with isoamylases (ISAs), limit dextrinases  constitute the set of starch debranching enzymes. The role of ISAs in removing excess branches of amylopectin formed by branching enzymes is well known LDA1 has been solved LDA1, that in mutant 905-LDA1 is substituted by a more bulky isoleucine, belongs to the carbohydrate binding module CBM48 and is located close to the interface with the catalytic domain  and the role of LDA1 in controlling granule nucleation is further supported.The tridimensional structure of barley c domain . It is pSSI to SSIV), and some of them are represented by more than one isoform SSIV is probably involved in starch granule initiation SSI preferentially synthesize short glucan chains using short amylopectin chains as substrate SSII and SSIIIin vivoIn plants, soluble starch synthases are divided into four classes with different specificity , while the third mutant had more B-type granules (1284-SSI). Morever, the A-granules of mutant 5850-SSI were more abundant but smaller in size and, symmetrically, the A-granules of mutant 1284-SSI were larger but less frequent. Only in mutant 1132-SSI the higher percentage of A-granules was not compensated by a reduction in size. The unexpected phenotypic difference between the three mutant lines may be due to currently unknown additional background mutations present in the genome of TILLMore mutant lines In cereals SSISSI is localized at the base of a loop involved in the formation of a high affinity binding site for maltooligosaccharides. This site is 30 Å away from the putative catalytic site, but is believed essential for colocalizing branched glucans and SSI, thereby favoring catalysis. Moreover, the starch phenotype associated to the G576D mutation  may suggest a role of this site in controlling the final size of large starch granules. The other two point mutations here described , and a SSIIa null mutant (1517-SSIIa) characterized by small/shrunken seeds and containing less starch with more amylose (48% of grain starch is made of amylose in this mutant). The SDS-PAGE analysis of starch extracted from 1517-SSIIa confirmed the absence of the protein SSIIa, together with SBEII and SSI isoforms. The simultaneous absence of SSIIa, SBEII and SSI in the starch granule was already observed in SSIIa mutants of barley, bread and durum wheat SSIIa was largely characterized by a V-type diffraction pattern suggesting the formation of lipid-amylose complexes, similarly to those observed in the sex6 mutant SSIIa, in spite of the smaller size of A-granules, no other starch parameters including crystallinity were significantly affected. In barley endosperm, SSIIa was shown to extend short amylopectin glucan chains of 3–8 glucose units to chains of up to 35 units SSIIa negatively affects amylopectin synthesis, more than amylose synthesis SSIIa is fully consistent with these results.Starch crystallinity of 1517-GBSSI) in controlling amylose biosynthesis and, conversely, the role played by soluble starch synthase IIa (SSIIa) in controlling amylopectin synthesis. Starch granule initiation appeared to be controlled by limit dextrinase (LDA1), and size of A-granules by starch synthases IIa. Thanks to their physical-chemical properties, these new alleles deserve further attention in order to investigate their possible interest in nutritional uses or industrial applications.TILLING of five genes encoding enzymes involved in starch metabolism enabled us to identify seven new alleles that are associated with new starch phenotypes in terms of amylose/amylopectin ratio, or crystal packing, or distribution of A- and B-granules, or size of A-granules . Our resFile S1BMY1 (B), 2682-BMY1 (C), 1090-GBSSI (D), 905-LDA1 (E), 1132-SSI (F), 1284-SSI (G), 5850-SSI (H), 1039-SSIIa (I), 1517-SSIIa (L). Scale bars: 10, 20, and 30 µm. Table S1, Water content in whole flours of TILLING mutant lines. Dry weight was obtained after incubation of samples at 80°C for 24 h. Data are means ±SD (n = 4).Figure S1, SDS–PAGE separation of starch protein extract from cv. Morex and barley mutants. Figure S2, SEM analysis of starch granules from cv. Morex (A) and barely mutants 2253-(PDF)Click here for additional data file.


Furthermore, a web application that will potentially be implemented in the IEDB was created to allow users the capacity to generate customized epitope sets.Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell vaccines and immunotherapies. In this context, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of T and B cells. However, the ever-growing information contained in the Immune Epitope Database (IEDB) and the differences in quality and subjects studied between epitope assays make this task complicated. In this study, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the IEDB. From the sets generated, three epitope sets  were experimentally validated by detection of T cell reactivity  Adaptive immunity is based on the recognition of specific molecular structures, named epitopes, by either antibodies/B cell receptors or T cell receptors. Antibodies and B cell receptors bind a wide variety of structures, including proteins and carbohydrates. In the case of protein ligands, antibodies can recognize either a series of contiguous residues (linear epitopes) or a set of residues encoded in disparate regions of the protein sequence and brought together in the three dimensional structure of the protein ligand (discontinuous epitopes).T cells recognize a complex between MHC molecules (named HLA in humans and H-2 in mouse) and, in most cases, a peptidic epitope of 8–16 residues in length . In the Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell-based vaccines and immunotherapies –8. AccorAs immune investigations proceed over time, many different epitopes from various organisms have been identified. Alternatively, large-scale epitope identification can reveal hundreds of potential epitopes –12. The This large amount of information might, in some cases, pose a challenge for the identification and selection of appropriate sets of epitopes for use in specific contexts. Thus, it is clearly desirable, given the ever-growing body of information contained in the IEDB, to develop tools to enable the efficient generation of sets of validated reference epitopes for any antigenic source of interest. in vivo assays or recognized by IgE in the case of allergens are considered more biologically relevant than linear epitopes recognized in ELISA assays and elicited by peptide immunization. In the case of T cell reactivity, again by way of example, multimer/tetramer staining assays or readouts based on ICS or ELISPOT assays, are preferred over older assay platforms such as Thymidine incorporation following multiple in vitro restimulations with peptides. These high quality assays have been selected based on our experience and judgment; however, in the web tool developed, the user will be capable of customizing it by his/her needs, that is, select only epitopes derived from neutralization or ELISA assays or any other desired selection.While an epitope, according to classic definitions, is any structure capable of interacting with T and B cell receptors, in practice the consensus in the scientific community is that certain types of assays identify the most relevant and validated epitopes. In the context of antibody reactivity, by way of example, epitopes identified on the basis of X-ray structures of Ag/antibody complexes, biological activity, and ex vivo, without manipulation. This is because manipulations, such as in vitro expansion, are known to profoundly change the phenotype and characteristic of the T cells [ ex vivo detection of human T cell responses is often difficult, largely because the immune response in any individual may target many different epitopes, and different individuals typically recognize unique epitope repertoires. Simultaneous use of many different epitopes as a pool might represent a powerful approach to detecting T cell responses, because even if the frequency of T cells recognizing each individual epitope may be below the limit of detection, a pool of a large number of epitopes  might pass the limit of detection.Indeed, for many applications, it is desirable to study T cells T cells –17, thusAn important consideration in the definition of reference sets of epitopes is how to factor the number of individual donors or experiments in which a given structure is reported to elicit a positive response, and particularly if this validation is provided in multiple independent studies. For example, different studies often report on essentially the same epitope but utilize different nested, truncated, or frame-shifted version of the same sequence, leaving uncertainty on how to combine the data or which particular version of the epitope to select for testing. Clarification of a general approach for combining data from such disparate studies would greatly facilitate the generation of nonredundant sets of epitopes.In the present study, we have attempted the definition of an automated process to generate reference sets of high quality epitopes for various disease indications. The resulting tool, made available to the scientific community, provides a standardized and reproducible platform to automatically extract and process relevant data from the IEDB without the need of complex analysis and judgment calls from the user. At the same time, the tool also offers flexibility to enable the end user to design sets meeting specific user-defined criteria. We have also analyzed the data currently available in the IEDB, to determine how many sets of pathogen or autoantigen specific epitopes could be identified on the basis of the data available to date.Epitope data was derived from the IEDB database as of October 2014. MySQL was used to run queries and directly work with the database itself. The web page application is written in PHP/HTML code with a MySQL connection that allows communication between the database and the user interface.Two independent scoring systems were developed to allow ranking and sorting of the epitopes. The first was based on the type of assays used to characterize the epitopes and the second on the frequency by which each epitope was recognized. ex vivo or in vitro configuration. To provide that ex vivo assays are always ranked higher, we assigned an ex vivo configuration a value of 4, and an in vitro configuration a value of 1, and calculated a final assay score by multiplying the assay type and configuration values. In the case of B cell epitopes, X-ray structure, biological activity, and in vivo assays were assigned a score of 1, as well as epitopes that present IgE; for these epitopes, the effector origin was not taken in consideration. Since each epitope can be associated with multiple records, each describing different assays and thus with different assay scores, the highest assay score, reflecting the highest level of validation reached for that epitope, was selected and carried forward.Regarding the assay type scoring system for MHC class I or class II epitopes, in our selection we included epitopes defined by multimer/tetramer staining, ELISPOT, and ICS assays. We arbitrarily associate a numerical parameter value of 3, 2, and 1 to these assay types, respectively. Each of these assay types can be used in either anN is the total of subjects tested and R is the number of positive responses. The square root is a correction factor, approximating one standard deviation for the number of responding donors. This gives a higher score to epitopes studied with larger sample sizes. In case there is no information of subjects tested, the epitope will be assigned R = 1, N = 1 if the assay outcome is positive and R = 0, N = 1 if it was negative. The RF score (RFS) also takes into account all data for a given epitope across all publications. We decided to use an older and simpler RF definition, as compared to the one used in [P) = 1 and total subjects (T) = 1; the new definition gives a RFS = 1 (0.04–1.00) whereas the older version gives an RFS = 0. In contrast with epitope 2 with P = 8 and T = 11, the new version gives RFS = 0.73 (0.43 : 0.92) and the older RFS = 0.47. In order for the RFS to reflect the difference between epitopes 1 and 2 in terms of the total number of subjects, the latter should be assigned a higher score to be used for ranking and filtering the data, which at the end benefits the user giving simpler and useful results.In terms of scoring each epitope on the basis of the frequency by which it was recognized, we utilized a previously described Response Frequency (RF) score . The RF  used in , as it iBoth the assay score and RF score are calculated for each epitope and provided in the results. This allows further ranking or selecting epitopes based on different thresholds for these criteria.For MHC class I epitopes, it is generally observed that a length of about 8–11 residues is optimal for T cell recognition and use in assays. Because of the structure of the class I binding groove, distinct class I sequences typically represent unique epitopes, even if they are nested within a longer sequence that is also recognized by T cells. Accordingly, for the present study, we have not subjected class I epitopes of nested or overlapping character to further processing.For MHC class II epitopes, however, optimal epitopes are usually longer than the minimal T cell recognized 9-mer core. In general, class II epitopes are optimally of 13–20 residues in length [In the case of donors with latent tuberculosis infection (LTBI), leukapheresis or whole unit blood samples from 10 adults were obtained from the University of California, San Diego, Antiviral Research Center (AVRC) clinic. Donors were classified as LTBI based on positive QuantiFERON-TB Gold In-Tube (Cellestis), as well as a physical exam and/or chest X-ray that was not consistent with active tuberculosis. Because Dengue virus (DENV) prevalence is low in the San Diego area, most LTBIs are DENV naïve.To obtain DENV seropositive samples, anonymous blood donations from healthy adults were obtained by the National Blood Center, Ministry of Health, in the area of Colombo, Sri Lanka. Plasma of the associated donation was tested for serology using the flow-based U937+DC-SIGN neutralization assay  as previously described , 20. BecAll Samples were collected and used following guidelines from the Institutional Review Boards (IRB) of LJI and the Medical Faculty, University of Colombo .15-mer peptides were synthesized as crude material on a small (1 mg) scale by Mimotopes  and/or A and A (San Diego). PBMCs were purified by density gradient centrifugation  from 100 mL of leukapheresis sample or 450 mL of whole blood, according to manufacturer's instructions. Cells were cryopreserved in liquid nitrogen suspended in fetal bovine serum (Gemini Bio-products) containing 10% dimethyl sulfoxide.6 cells/well) were incubated with peptide pools (1 μg/mL) for 2 hrs. Brefeldin A (1 μg/mL) (BD Bioscience) was added to the mixture and incubated for another 4 hours . Cells were then washed and stained for cell surface markers using anti-CD3-Alexa Fluor 700 (UCHT1), anti-CD8-V500 (RPA-T8) (both from BD Biosciences), anti-CD4-APC EFluor780 (RPA-T4), anti-CD45RA-EFluor450 (HI100), and anti-CCR7(CD197)-PerCPCy5.5 (G043H7)  for 30 mins on ice. Cells were then washed, fixed with 4% paraformaldehyde, blocked with human sera, and stained for intracellular IFNγ using anti-IFNγ-FITC . Samples were acquired on a BD LSR II flow cytometer. The frequency of cells responding to the TB/DENV-specific peptides was quantified by determining the total number of gated subset+ and cytokine+ cells and background values subtracted  using FlowJo software (Tree Star).PBMCs . For this purpose we adapted the categorization adopted by Seymour et al. . The relIn ex vivo or in vitro), utilized to derive the Assay Score, are also given. All epitope tables from the categories shown in http://dx.doi.org/10.1155/2015/763461.As a result of the processes described above, we generated sets of epitopes for the various categories. As an example, Having established the conceptual framework for selection of epitope sets, we next expanded our applicability. Autoimmune epitopes are identified by the fact that both source antigen and host organism are the same . As listed in We considered expanding the scope of the study to also select epitopes recognized by species other than humans. In this case, the second most frequently represented host species is mouse. Accordingly, an option was created in the web application (next section) to allow selection of murine epitopes. The number of murine epitopes identified is listed as a separate column in  in vivo assays as most biologically relevant. In addition, we included induction of IgE subclass responses, as this type is most relevant in the context of allergic diseases. The number of B cell epitopes identified accordingly is also listed as a separate column in Finally, we also expanded our analysis to allow selection of B cell/antibody epitopes. In this case, we set a 5 to 20 residue size window and initially selected X-ray structure, biological activity, andFinally, we developed a tool, which will be hosted by the IEDB as an additional link in the search results page and will be part of the next IEDB update release in fall 2015. This tool allows generation of specific epitope sets following the default criteria described above but also allows users to customize the generation of novel sets.A sample screen shot of the main interface is shown in An “advanced options” webpage can be accessed from the main page, and a sample screen shot of this option is shown in  ex vivo T cells assays require relatively substantial amounts of each epitope (in the 1–0.1 microgram/mL range), compounded by the fact that peptide solubility in solvents most widely applicable to preparing peptide stock solutions, such as DMSO, is usually limited to about 20–40 mg/mL. Since solvents like DMSO are toxic in cellular assays at concentrations above 0.5%, this would seem to effectively limit the number of peptides that can be safely included in a pool to about 20.To experimentally validate the usefulness of the tool we decided to synthesize some of the actual peptide sets identified by the tool and experimentally test them for recognition by human T cell responses. One of the main challenges for testing large pools for T cell recognition is thatHowever, in many cases the solubility of one peptide is not drastically influenced by the presence of other peptides . For this reason, we predicted that it might be possible to make pools of peptides already dissolved in a solvent like DMSO, mix the solutions, and relyophilize the pool of pools. Indeed, we routinely find these “sequentially lyophilized” pools, once resuspended, to be much more soluble than the individual components. Mycobacterium tuberculosis, based on an RF score > = 0.1 and being recognized ex vivo .Accordingly, we synthetized a set of 207 EBV human CD8/class I epitopes, identified by the default setting described above . In addition, we also synthetized a set of 92 CD8/class I epitopes derived from DENV virus, obtained by selecting only peptides with RF > = 0.01 , and a set of 86 epitopes CD4/class II epitopes derived fromPeptides corresponding to these three sets of epitopes were pooled and tested with human PBMC as a source of T cells. For these experiments we selected PBMC from 5 individuals infected with DENV virus and likely uninfected with TB (see methods for details) and PBMC from 5 LTBI individuals and likely uninfected with DENV. Because of the high incidence of EBV infection worldwide , we assu ex vivo stimulation, the IFNγ response was measured by ICS  and DENV donors. The reactivity was lower in the DENV donors than in the LTBI donors (P = 0.03). This lower, but detectable, reactivity is explained by the fact that the DENV seropositive individuals are vaccinated against MTB with M. bovis BCG and are likely exposed to nontuberculous mycobacteria [ ex vivo reactivity in 3 out of 5 donors is in line with what was detected in previous studies [P = 0.004 for LTBI and P = 0.05 for DENV).PBMC were stimulated with the DENV CD8 pool, MTB CD4 pool, and EBV CD8 pool. After Figures . The gatBI donor  and a DEBI donor . The IFNbacteria . In the  studies . As expeWe devised a strategy that allows automatically filtering datasets to select epitopes of appropriate size, defined restriction, and assay type for use in characterizing responses to specific indications. While querying the IEDB database can also generate these sets, a certain degree of complexity in the queries and the setting of multiple parameters would be necessary. In our application, the epitope sets are automatically generated, while the user is still enabled to change the default settings to generate validated epitope sets matching specific criteria.We further identified which epitope categories are supported by current IEDB data, and found that reference epitope sets could be produced for 43 categories with data currently available in the IEDB. The number of such categories, broadly based on previous epitope classification work , is undoWhile these actual epitope sets are provided as tables within the paper, we implemented a web application to automatically generate epitope sets, based on the fact that the IEDB content is rapidly growing and new epitopes are added to the IEDB in each of its biweekly updates. We plan to continuously gather feedback on this web application from the scientific community, and to implement changes and modifications through the main IEDB website . ex vivo. As such, these sets of epitopes represent potentially valuable resources for diagnostic purposes, as well as for further detailed characterization of the immune response to specific targets of immunological interest.Finally, to illustrate applicability in an actual experimental setting, we selected and synthesized peptide sets corresponding to EBV, DENV, and MTB epitopes. These epitope sets were used to measure immune reactivity in human cells. The experimental testing of these epitope sets demonstrated the applicability of these sets as a valuable resource to allow detection of T cell responsesMycobacterium tuberculosis sets. The compressed file “Epitope Tables” contains the epitope tables for mouse (H-2) and human (HLA) of T cell class I (H-2 I and HLA I) and class II (H-2 II and HLA II). In the case of B cell epitopes, they are divided into discontinuous (Mouse B cell Discont and Human B cell Discont) and linear (Mouse B cell Linear and Human B cell Linear). For each of these directories, the epitope tables of each category (Table 2) are found inside.”Supplemental tables 1A, 1B and 1C contain the epitope sets used in the experimental validation of the EBV, DENV and


Transit-Time Flow Measurement (TTFM) is a valid method of intraoperative graft verification. TTFM shows a low diagnostic accuracy due to high number of false positive.High-Resolution Epicardial Ultrasonography (HR-ECUS) is a well-recognized procedure for coronary anastomosis and stenosis evaluation with high sensitivity and specificity.The aim of the study is to verify if the combined use of TTFM and HR-ECUS in intraoperative graft verification process can increase the diagnostic accuracy of procedure itself.From November 2009 to December 2014, 741 patients underwent isolated CABG. A total number of 1733 grafts were performed; all the grafts were intraoperatively verified by means of both TTFM and HR-ECUS.Among all grafts considered functioning at TTFM, 7 (0.4%) were failing at HR-ECUS and promptly redone. These were confirmed as true positive at graft revision due to technical error. HR-ECUS confirmed the good functioning of the remaining grafts already demonstrated by TTFM; among them, 8 showed high troponin I release , whereas the remaining had no high TnI release . In 2 of 39 grafts malfunctioning at TTFM, HR-ECUS confirmed the graft failure. Finally, in 35 cases, HR-ECUS did not confirm TTFM diagnosis demonstrating a full patency of the anastomosis; these grafts had an uneventful clinical course (true negative). The main result of this study is the increase of PPV from 10% with TTFM to almost 100% of TTFM + HR-ECUS, avoiding many unnecessary graft revisions.In intraoperative graft verification procedure the combined use of TTFM and HR-ECUS increase diagnostic accuracy of the verification process close to 100%. Both methods should be used intraoperatively in order to achieve the best outcome for the patient.


P < 0.05) in the crude digestibility level, thermal properties, and the water separation (syneresis) of starch gels (7.0% db) during cold and frozen storage were observed due to the modification. Hydroxypropylation increased the gel stability, water solubility, digestibility, and storage stability of the native starches in the cold storage to a significant level. Lowered gelatinization and retrogradation enthalpies as well as gelatinization temperature were observed for derivatized starches compared to the native starch. Low levels of pasting stability with increased levels of breakdown and reduced cold paste viscosity were observed in the hydroxypropylated starch samples except for the Malaysian cultivar (S5). Chemically modified starch gels stored under cold storage did not show a syneresis for two weeks in the cycle and the frozen storage showed much improved stability in the starch gels within the four-week cycle. Chemical modification of sweet potato starch with hydroxyl propyl substitution can enhance the functional characteristics of the native starch which will improve its potential application in the food industry.Starches obtained from different cultivars of sweet potatoes commonly consumed in Sri Lanka, were chemically modified with hydroxypropyl substitution, to analyze the changes in the physicochemical properties. Significant changes ( Starches have a wide spectrum of applications both in food and other industries such as textile and cosmetics. Native starches have restricted usage in food processing operations, distribution, and storage conditions due to the unfavourable characteristics prevailing. Native starches which have been modified either by physical or chemical treatments show much improved functional attributes that will have a broader area of usage in food processing operations . ChemicaRheological, morphological, and physicochemical characteristics can be improved through the chemical modification of the native starch by cross-linking, substitution, or reacting with acids or alkali. The amount of the chemical reagent required to achieve the functional properties needed in the starch may vary depending on the starch source, reagent type required for the substitution, the degree of substitution of the chemical reagent on the starch source, and the required range of properties in the final modified starch product , 6. The 3 in sweet potato starch mostly took place within the central regions of the starch granules and substitution taking place throughout the granules was observed by [Hydroxypropylation (HP) is commonly used in the chemical modification of the native starch due to much improved thermal characteristics upon gelatinization. Hydroxypropyl groups are being substituted to the native starch in the presence of alkali catalysts and the monosubstitution group in HP of the food grade starch should not exceed the 10% level . It was erved by . Substiterved by , 13.The main source of the modified starch used in the food industrial applications in Sri Lanka is the corn starch and the country has to incur more expenditure in importing the product. Therefore it is important to utilize the alternative products which are feasible to manufacture locally with little or no modifications to improve the economic status of the country. Previous studies done by us using these commonly consumed sweet potato cultivars in Sri Lanka showed much favourable functional characteristics both in the native form as well as the physically modified form due to hydrothermal treatment . CurrentMatured roots of sweet potatoes after about 2-3 days of harvesting were randomly selected from three different locations in Dhambulla, Gokarella, and Horana areas in Sri Lanka. Extracted starches from S1 (Wariyapola red), S3 (Wariyapola white), S4 , S5 , and S7 (CARI 273) cultivars were analysed for functional characteristics.µm sieve, sealed, and packed for analysis.Starch separation was carried out according to the method described by . Fresh r2SO4 was mixed in a centrifuge tube. The pH was adjusted to 11.3 with 1 M NaOH and 4.5 mL of 3.0% propylene oxide was added for the substitution since previous studies have shown more effective substitution levels at this level [HP groups were substituted to the native starch according to the method described by . A starcis level . Capped Ws). The supernatant was dried until a constant weight (W1) was obtained in a drying oven at 100°C. The SP and WSI were calculated as follows:W1/0.1 × 100%.Swelling power and the WSI of the native and the chemically modified starches were determined by the method described by . WeighedµL distilled water on the aluminum DSC pan and kept at ambient temperature for 1 h to equilibrate the mixture. The DSC pan was scanned from 30 to 120°C at a rate of 10°C/min. An empty pan was kept as a reference and all the measurements were performed in triplicate. The corresponding enthalpy (J/g) was expressed on the dry weight basis of the starch.The thermal characteristics of starch gelatinization and retrogradation were determined by using differential scanning calorimeter (DSC) . Approximately 3 mg of starch was mixed with 9 HR (J/g) was measured for triplicated samples by keeping an empty pan as a reference.A starch gel was prepared by using a ratio of 1 : 1 starch to water in the DSC pan and equilibrated for 1 h. After the equilibration the DSC pan was heated using a convection oven at 120°C for 10 mins to gelatinize the starch. The DSC pan with the gelatinized starch was stored at 4°C for 24 h to initiate the process of nucleation. Prior to scanning by the DSC, the sample was kept at 40°C for 7 mins. The DSC pan was heated from 30 to 120°C at a rate of 10°C/min. Change in ΔPasting properties of the native and the substituted starches were determined by using a rapid visco analyzer . A starch sample with approximate weight of 2.0 g/db was mixed with 25.5 g of distilled water in the RVA canister to a total sample weight of 27.5 g (8.1% starch concentration). The total pasting cycle was set for 22 mins; the homogenized slurry was heated at 50°C for 1 min and then at 95°C for 12.5 mins. Then the sample was cooled to 50°C within 7.5 mins and held at 50°C for 1 min to complete the cycle. Triplicate measurements were taken for all the samples.In vitro starch digestibility of native and HP starches was measured by using the method described by [2, 30 mg gelatin, and 30 mg pancreatin were added . The capped tubes were kept at 37°C in a shaking water bath for 12 h and the reaction was terminated by adding 5 mL of 1% H2SO4. Digested suspension was centrifuged at 20,000 ×g for 10 mins and the supernatant was decanted. The remaining residue pellet was dispersed with 15 mL of 80% ethanol and recentrifuged for 5 mins. The resulting pellet was dried at 50°C for 6 h and then at 80°C to a constant weight. Sample was weighed at the ambient temperature and the percentage weight loss after the in vitro digestion was considered as the crude digestibility level of the starch. A sample blank without pancreatin was kept as a control to adjust the results.ribed by . A starcSyneresis of starch gels of native and modified starches during cold storage and frozen storage was determined with slight modifications to the methods described by , 18. A sStarch pastes were stored in the freezer (−18°C) for 1 week and then thawed at 40°C for 2 h to determine the freeze-thaw stability. The thawed samples were centrifuged at 3000 ×g for 10 mins and the residue was weighed after discarding the supernatant. The gels were refrozen following the measurement of expelled water to repeat the cycle. All starch samples were subjected to four freeze-thaw cycles. Cumulative values for expelled water were obtained after calculating each percentage of expelled water from the remaining gel. Total syneresis was calculated by the addition of mean values of the expelled water from the triplicated samples, after each refrigeration or freeze-thaw treatment.P < 0.05).All numerical results were averages of three independent replicates. Data were analysed by one-way analysis of variance (ANOVA) using Minitab Ver 14.0. The statistical significance was determined by using Tukey's test (P ≤ 0.05) of swelling power and water soluble index compared to their native forms in all the cultivars studied. No significant difference in SP and WSI were observed in the chemically modified S1, S3, and S5 starch types  was observed in the digestibility of native and hydroxypropylated S7 starch. This may be due to the lower degree of substitution of HP groups into the granular matrix than the other chemically modified starch types and the resulting crystallite structure may have not influenced the enzyme attack in the S7 starch type. Initial X-ray diffraction study prior to the treatment and after together with analysis of the degree of substitution would give more evidence in this. S3, S4, and S5 starches showed nearly twofold increases in digestibility due to derivatization  has changed from 15.5–17.1°C in the native starches to 13.9–16.8°C when substituted. Each starch type showed a reduced level of gelatinization temperature range than its native form due to chemical modification (To) and conclusion temperature (Tc) may due to the change in heterogeneity of the starch molecular structure due to the substitution. Significant reduction (P < 0.05) in the peak temperature (Tp) of gelatinization was observed in all the chemically modified starches. Reduced values for the transition temperatures , and enthalpy (ΔH) of gelatinization for HP starches and further reduction of the values with increased levels of molecular substitution were reported by [The gelatinization temperature range (fication  and the orted by .Hg) in the HP starches . Melting enthalpy of retrogradated amylopectin ranged from 3.5 to 5.8 J/g and from 1.3 to 2.5 J/g in native starches and the HP starches, respectively  in the cold paste viscosity (CPV) of the substituted and the native S5 starch.Pasting can be defined as series of processes that take place following the gelatinization, the dissolution of starch due to granular swelling, release of granular components gradually, and total disruption of the granules. When gelatinized starch paste is subjected to cooling the rapid reassociation of linear amylose through forming a gel matrix governing the extent of viscosity increases in starch pastes. Representing of RVA curve for our analysis is given in High level of paste stability was observed in chemically modified S5 starch compared to other substituted starch types. Significant increase in the peak viscosity was observed in the HP substituted S7 starch . ReducedP < 0.05) was shown in the native starch gels of S4 and S7 (P < 0.05) of water release from the gel matrixes of the derivatized starches during the fourth week may have been caused by the retardation of the retrogradation process due to the high affinity to water by the HP groups. Inconsistent pattern of water loss shown by the native starches during the four-week refrigeration cycle may be due to the comparably free formation of starch nuclei for crystallization caused by the noninterruption by the HP groups.HP has greatly influenced the reduction of syneresis in the native starch during the four-week refrigeration cycle. More than tenfold increase in the water loss (%) was observed in the native starch during this period and a significantly high level of water loss  in the syneresis (%) of S1, S4, S5, and S7 and S3 showed a slightly high level of expelled water compared to the other types. As observed in the cold storage, native forms of the S7 showed a high level of syneresis and a low level of water loss in the S3 cultivar  the in vitro digestibility level, swelling power, water solubility, and starch gel stability during the frozen and the refrigerated storage. Chemically modified starches showed lower gelatinization temperatures and no significant effect on the pasting characteristics was observed except for the S5 starch. Chemical modification has significantly reduced the retrogradation levels in S3 and S7 types while preventing the syneresis in S4 type during the cold storage. Significant level of increase in the percentage digestibility, SP, WSI, paste stability due to high SB, CPV, and low BD and also the increased level of stability during the refrigerated and frozen storage were observed in S5 cultivar due to HP substitution. With much improved functional characteristics due to chemical modification with substituted HP groups, tested starches showed a high potential in being used as a food ingredient in the industry and the characterization of these starches with different levels of HP should be tested to achieve the desired level of pasting properties.Hydroxypropyl substitution to native starches significantly increased (


Punctured extinct cave bear femora were misidentified in southeastern Europe (Hungary/Slovenia) as ‘Palaeolithic bone flutes’ and the ‘oldest Neanderthal instruments’. These are not instruments, nor human made, but products of the most important cave bear scavengers of Europe, hyenas. Late Middle to Late Pleistocene (Mousterian to Gravettian) Ice Age spotted hyenas of Europe occupied mainly cave entrances as dens , but went deeper for scavenging into cave bear dens, or used in a few cases branches/diagonal shafts (i.e. prey storage den type). In most of those dens, about 20% of adult to 80% of bear cub remains have large carnivore damage. Hyenas left bones in repeating similar tooth mark and crush damage stages, demonstrating a butchering/bone cracking strategy. The femora of subadult cave bears are intermediate in damage patterns, compared to the adult ones, which were fully crushed to pieces. Hyenas produced round–oval puncture marks in cub femora only by the bone-crushing premolar teeth of both upper and lower jaw. The punctures/tooth impact marks are often present on both sides of the shaft of cave bear cub femora and are simply a result of non-breakage of the slightly calcified shaft compacta. All stages of femur puncturing to crushing are demonstrated herein, especially on a large cave bear population from a German cave bear den. Potok Cave), Slovenia ) and LatCrocuta crocuta spelaea) carnivore den which overlaps with another Aurignacian camp site, but again, it has no Middle Palaeolithic Neanderthal occupation signs (cf. [Other cave bear cub femora with holes (‘bone flutes’) were then reported from the Istállóskö Cave, Hungaria (cf. ). This wgns (cf. ).Brodar  reportedAll aforementioned femora and other cave bear bones with ‘holes’  were compiled and studied by the ethnologist/musician Omerzel-Telep , without2.2et al. [° rotation of which is corrected herein (a), (c) the general bone taphonomy of cave bear bones, and (d) carnivore jaw functions, especially hyenas, correctly presented herein , who miet al. , (b) theet al. ), the 18d herein a, (c) thd herein .2.3et al. [Crocuta crocuta spelaea, will be discussed as the oval hole producer herein (Panthera leo spelaea), leopards (Panthera pardus spelaea), Ice Age spotted hyenas (Crocuta crocuta spelaea) and Ice Age wolves (Canis lupus spelaeus)—which are known now to be cave bear killers, and main consumers in mountain regions, where mammoth steppe megafauna were absent [In this contribution, not only sole carnivore damage can be demonstrated on all previously published ‘pseudo-bone flutes’, which were already revised in some cases ,16 figu. Herein,et al. ; see figr herein , based or herein . The Icer herein . All fore absent ,18–21. Te absent   and large cave bears (Ursus ingressus) and large amount of material in different destruction stages from the Weiße Kuhle Cave, but also some relevant bones from the den sites Keppler Cave, Zoolithen Cave, Sophie's Cave, Große Teufels Cave and the Czech Sloup Cave.First, from the literature available, ‘bone flutes’ were compiled and reinterpreted herein with new drawings . From thGeneral cave bear bone damage by large carnivores  is present in all of those large cave bear dens. On average 80% of the cub, and 20% of the adult cave bear bones have large predator damage. The comparison focused on the presence/absence and positions of round–oval puncture marks. Herein the bone damage stages 1–3  are presented in detail for cave bear femora of cubs, subadult and adult cave bears. This material is composed and compared from the aforementioned publications to end the long discussion about ‘Middle Palaeolithic Neanderthal bone flutes and oldest instruments’. Tooth mark types, shapes and especially their positions on both sides of the shaft ends, or the middle part, were identified as the antagonistic upper and lower jaw tooth marks of hyenas.U. ingressus) from the latest Late Pleistocene  of Hermann's Cave (cf. details in [U. s. eremus) from Sophie's Cave, but those were well identified to have resulted from porcupine (rodent) incisive teeth . Other tails in ).Finally, the position of the bite mark holes and their orientation in oval pits are calculated at similar more or less cylindrical in the middle of the shaft formed longbones: radius, femur and tibia.The figured ‘bone flutes’ of Divje Babe Cave 1, Mokriška Jama Cave and all other Slovenian ‘pseudo-bone flutes’ are housed in the National Museum in Ljubljana, Slovenia and Hungary (NMLS). Oase Cave material was studied in the Institute Emil Racovita, Romania (IR). Weiße Kuhle Cave (Germany) bones are in the PaleoLogic Research Institute, Czech Republic (PAL), Perick Cave material is kept in the collection of the Perick Cave club house in Hemer, Germany (PCH). Hermann's Cave material was analysed in the collection of the Rübeland show caves, Germany (RC), Keppler Cave material is in the Statdmuseum Menden, Germany (SMM), and Große Teufels Cave bones are kept in the show cave of the village of Pottenstein (PO).4.4.13 and antagonistic lower jaw P3–4, sometimes parts of M1, which attributes it only to the crushing teeth triangle of hyenas , and in a second step (damage stage 2) the other joint was cut off using the scissor-dentition on very small-sized femora –7. The lenas cf.  and 3. Ienas cf. , whereasenas cf. , 7 and 8enas cf. , do not 4.2The amount of bone material is still not enough to present clear statistics. However, with the herein used Weiße Kuhle material being very representative for a large cave bear den, puncture holes are found in the cave bear cub humerus (4×), ulna (0×), radius (2×), femur (13×) and tibia (5×). The position of the holes is mostly on the herein studied 19 cub femora, on the ventral side, and not on the dorsal convex side cf. –7. The f5.5.1All ‘cave bear cub femora bone flute’ sites failed to date into the ‘Neanderthal times’ because all are not of Neanderthal  human, but are instead from modern human Aurignacian occupations in ‘archaeological layers’ at entrances of cave bear dens, cf. ,7,9, or 5.2Ursus spelaeus subsp.  do not show such holes in femora  and the large U. ingressus, and from mountainous regions, where Ice Age spotted hyenas were around all over Europe  and are from smaller early cave bear forms of r et al. ); intere regions ). As is ngressus ,23,58,59ngressus ,23,57. T. map in ).5.3Drilled holes were produced experimentally for a reconstruction of a ‘cave bear cub bone flute’ (cf. ) and are5.4Small parallel rectangular scratches on the pseudo-bone flute of Divje Babe Cave 1 were misinterpreted as cut marks (cf. ). Also o5.5et al. [U. s. eremus and full herbivorous U. ingressus  are never used to crack longbones (e.g. ). The deet al. , becauseet al. . The tomet al. ) restudyet al. ). Indeedet al. ,61,67; fus (e.g. ) cave beus (e.g. ). Some a canines . Microsc canines ) declare canines ,70,71).  canines ,19–21,72 [et al. , those sk et al.  was inco [et al. , this in [et al. –22 (figu5.6It is no wonder then that further incorrectness about cave bear bone taphonomy at Divje Babe Cave 1 was published (cf. ), becaus6.Ursus spelaeus subsp. eremus  and spelaeus  and U. ingressus  used caves all over Europe for cub raising and hibernation. All the large carnivore punctured cave bear cub femora (and other punctured bones) appear always in small to large cave bear den cave/cave entrance contexts. This sometimes overlaps with hyena dens and human camp sites at cave entrances only, where cave bear den, carnivore den and human remains are even mixed up (partly separated in layers), all over Europe due to competition for and seasonal use of cave entrances/rock shelters. The cave bear bones with round–oval, larger puncture marks can be well attributed solely to the main cave bear scavenger of Europe—the Ice Age spotted hyena Crocuta crocuta spelaea. This main Late Pleistocene bone destructor in Europe is known recently with more than 150 den sites (95% are cave sites) all over Europe. At cave bear dens hyenas left, by periodic scavenging, up to 20% of damaged bones, whereas also lions (cave bear killers), leopards and wolves played a larger role in the cave bear hunting/scavenging, even deep in caves. Those indeed also left, in some cases, round–oval, larger punctures in cave bear bones, but with their canines only in soft spongiosa , and never in any bone shaft compacta. Neither carnivores nor cave bears (herbivorous) used their canine teeth to crush longbones, or any other bones. Therefore, all other top predators—except hyenas—can be excluded, at least for the round–oval punctures in cave bear longbone shafts. Only hyenas have developed a carcass destruction and butchery strategy, also for cave bears. This strategy is demonstrated, herein in detail, on cave bear femora destruction , which is presented in three stages and for different aged individuals—cubs (less than 1 year), subadults (1–2 years) and adults. Cub bones are ‘soft’ and thin-walled in the bone shaft compacta, which increases in subadults to adults. This explains why puncture marks are found only in cub (less 1 year) femora, and partly in subadults, whereas they are absent completely in adults, because hyenas cracked those bones into pieces with the premolar triangle teeth (i.e. bone crushing teeth) for access to the bone marrow and easier swallowing of those pieces for the bone collagen use. Hyenas left, therefore, ‘pseudo-bone flutes’ during the Late Middle to Late Pleistocene all over Europe in cave bear dens, and on different cave bear species/subspecies. This is known due to lack of breakage on most of the cave bear cub femora, which generally show additional diagonal zigzag margins (from chewing joints by scissor teeth of hyenas) or have triangular or smaller scratch tooth marks. This even allows reconstruction, in some cases in detail, the tooth mark of the upper and lower jaw teeth of hyenas—the last tooth mark of the premolar bone crushing triangle of the powerful jaws of the last hyenas of Europe. Finally, some flakes and refitted cub femora, both with tooth mark holes, prove the bone cracking activities at cave sites.The ‘cave bear cub femora with holes’ are, in all cases, neither instruments nor human made at all. All cave bear pseudo-bone flutes are not dated to Neanderthal Middle Palaeolithic Mousterian layers, but instead, if possible to date, to Late Palaeolithic, Aurignacian/Gravettian layers. There, where they are dated absolutely (Divje Babe Cave 1) are without archaeological context at all, and simply of cave bear den use during the MIS 3–5d. At these times, different cave bear subspecies


Regarding the paper ‘HIV Vaccine Awareness Day: sustaining the momentum’ by Kathleen M MacQueen, Mitchell Warren.Journal of the International AIDS Society, Citation: MacQueen KM and Warren M. Journal of the International AIDS Society 2016, 19:21202. http://www.jiasociety.org/index.php/jias/article/view/21202 ∣ http://dx.doi.org/10.7448/IAS.19.1.21202Published in Reference 6 is missing in the reference list, and should be cited in the text according to the following: “This is no different than the requirements for advancing research on a cure for HIV [6], on new methods …”.The correct reference list is displayed below.


Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis  The biosynthesis of starch in plant chloroplasts depends on a novel protein that targets starch synthase to the growing starch granules; this represents a potential target for the biotechnological modification of starch. Read the Synopsis. Arabidopsis thaliana fail to make any amylose in starch. This is because the GBSS protein, which normally binds to starch, cannot bind in the absence of PTST. This discovery sheds new light on a previously unknown protein targeting process by which enzymes are delivered to the starch. Furthermore, our discovery highlights PTST an ideal target gene for biotechnology.Starch plays a vital role in our everyday lives. It is not only a major dietary carbohydrate, but is also used to manufacture household products such as pharmaceuticals, paper, and textiles. Plants produce starch as a means of storing energy; it is composed of two glucose polymers—amylopectin and amylose. While amylose is present in a smaller quantity than amylopectin, it has a major impact on starch processing. Being able to control the amount of amylose is therefore a goal for biotechnology. Amylose is made by the enzyme GRANULE BOUND STARCH SYNTHASE (GBSS), which was for decades believed to be the only protein required for amylose production. We now report here that a second protein, PROTEIN TARGETING TO STARCH (PTST), is involved in the process. Mutants lacking the PTST protein in the model plant  Starch is a vitally important plant-derived material that is widely used as food and in manufacturing of non-food products. Many plants accumulate starch during photosynthesis within leaf chloroplasts, where it acts as a major storage carbohydrate. The degradation of starch at night provides carbohydrates to fuel respiration and growth when photosynthesis is not possible . Starch In its native form, starch exists as semi-crystalline, insoluble granules that are typically between 1 to 100 μm in diameter, depending on botanical source . It is cDetailed information on the biochemistry of starch synthesis can be found in recent reviews ,6. Briefin planta  Triton X-100) using a Waring blender. Insoluble material was resuspended in the extraction medium and sequentially filtered through 30 μm and 15 μm nylon nets. Starch granules were separated from the filtrate at 2,500g for 15 min over a 95% (v/v) Percoll cushion (Sigma-Aldrich). The starch pellet was then washed in 0.5% (w/v) SDS. Excess SDS was removed by extensively washing the pellet in water. The pellet was then washed once in 80% (v/v) ethanol, and dried under vacuum overnight.Entire rosettes of 4-week-old plants were homogenised in starch extraction medium  as described previously .For scanning electron microscopy (SEM), purified granules were visualised in a Quanta 250 FEG microscope (FEI).v/v) glutaraldehyde; 0.05 M sodium cacodylate (pH 7.4), for 7 h at 22°C. The segments were washed three times with 0.1 M sodium cacodylate (pH 7.4), and then incubated at 4°C overnight in 1% (w/v) osmium tetroxide in 0.1 M sodium cacodylate (pH 7.4). Samples were then washed three times in cold 0.1 M sodium cacodylate (pH 7.4), and once with water. The samples were dehydrated in a series of aqueous ethanol solutions from 50% (v/v) to 100% ethanol, then finally with 100% acetone. The segments were embedded in Epon epoxy resin (Fluka). Sections were cut with a diamond knife and stained with a 1% (w/v) toluidine blue prior to microscopy. Images were acquired on an AxioImager Z2 microscope fitted with a 100× oil-immersion lens with 1.4 numerical aperture, and AxioCam monochrome camera (Carl Zeiss).For light microscopy analysis of starch granules in chloroplasts, leaf segments from 3-week-old plants were fixed in 2% . Insoluble material was pelleted at 20,000g, and soluble proteins were collected in the supernatant. Protein content was determined using the Protein Assay kit (Bio-rad), and equal amounts of protein per sample were loaded onto SDS-PAGE gels.For the extraction of leaf soluble proteins: young leaves were homogenised with a pestle and mortar in protein extraction medium . The suspension was then heated at 95°C for 5 min and insoluble material was removed by centrifugation. Volumes of soluble protein and insoluble protein extracts corresponding to equal leaf area were loaded onto SDS-PAGE gels.For the fractionation of soluble and insoluble proteins: 7 mm leaf discs were harvested from young leaves at the end of day and snap frozen in liquid Ng for 10 min. Proteins in the supernatant were separated on SDS-PAGE gels. The gels were stained with the Silver Stain Plus kit (Bio-rad).For the extraction of starch granule-bound proteins: dried purified starch granules were resuspended at a concentration of 33 mg/ml in SDS-PAGE loading medium (see above) and heated at 95°C for 10 min. Gelatinised starch was then pelleted at 20,0006 recombinant proteins, respectively.For immunoblotting, proteins were transferred onto a PVDF membrane following SDS-PAGE. GBSS was detected using polyclonal antibodies raised either against the purified pea GBSS protein  or againFor building the phylogenetic tree, sequences were retrieved on the NCBI and Phytozome databases using pBLAST. The alignment was constructed using both ClustalW on Bioedit  and MAFFThe predictions of coiled coils were carried out on the COILS/PCOILS server . ResultsE. coli, a PTST-cTP::pGEX-4T-2 IPTG-inducible expression vector was cloned as follows: A full length cDNA clone encoding the PTST protein was obtained from the RIKEN Bioresource Center . The region encoding the PTST protein  was amplified with BamHI and NotI restriction sites using the following primers, 5´-ATGGATCCGCTTCTACTCGAAAACATTAC-3´ and 5´-TAGCGGCCGCCTATTCCACCACTAAAACATTG-3´, and cloned into the pGEX-4T-2 vector .For GST-tagged PTST recombinant protein expression in E. coli, a GBSS-cTP::pET24a+ IPTG-inducible expression vector was constructed. Arabidopsis cDNA was prepared from wild-type Columbia plants using the RNeasy Plant total RNA extraction kit (Qiagen) and Wizard Reverse transcription kit (Promega). The cDNA region encoding the GBSS protein  was amplified from the cDNA preparation with NheI and XhoI restriction sites using the following primers: 5´-ATGCTAGCTGTGAGAAAGGAATGTCTGTG-3´ and 5´-ATCTCGAGCGGCGTCGCTACGTTCTC-3´. The amplicon was cloned into the pET24a+ vector (Novagen/Merck) in frame with the C-terminal His-tag.For GBSS recombinant protein expression in AtSSI.All point mutations were generated using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s instructions. For the generation of the chimeric GBSS protein, the full length cDNA of Arabidopsis GBSS was synthesised by Biomatik, with the coding sequence of the coiled coil helix (amino acids 440–454) replaced with the Arabidopsis SSI cDNA sequence encoding amino acids 497–511 of E. coli BL21 (DE3) CodonPlus cells (Agilent Technologies) harbouring the expression vector plasmids were cultured in LB medium at 37°C. Protein expression was induced by adding 1 mM IPTG, and grown overnight at 20°C or 18.5°C for GST-PTST and GBSS, respectively. For GST-PTST purification, cells were resuspended in lysis medium  and lysed with a microfluidiser (Microfluidics). The lysate was subject to centrifugation at 20,000g for 10 min, and the supernatant was incubated with Glutathione Sepharose 4B resin . The resin was washed five times in triton wash medium , five times in wash medium , and subsequently eluted in elution medium . Free GST was also purified as described for GST-PTST from cells harbouring the empty pGEX-4T-2 vector plasmid. For GBSS purification, the recombinant protein was purified from the lysate using Ni2+-NTA agarose (Qiagen) using a previously described protocol  BSA, 0.01% [v/v] Triton X-100) for 30 min at 20°C with maize starch . The reactions were mixed end-over-end on a spinning wheel during the incubation. The substrates were pelleted at 5,000g for 30 s, and unbound proteins were collected in the supernatant. The substrates were then washed three times in binding medium, and bound proteins were eluted in elution medium . Proteins were detected by SDS-PAGE followed by immunoblotting (see above).Recombinant GST-PTST (1 μg) was incubated in binding medium . The PTST and GBSS constructs were recombined into the Gateway destination vectors, pB7YWG2.0  and pEaAgrobacterium tumefaciens (strain GV3101), and cultures were grown at 28°C in LB medium supplemented with the appropriate antibiotics for 22–24 h. Cells were pelleted at 5,000g for 15 min and resuspended in water to an OD600 of 1. The cell suspension was then infiltrated into the intercellular spaces between abaxial epidermal cells of intact Nicotiana benthamiana leaves using a 1 ml plastic syringe. Infiltration of 0.2 ml into each spot yielded approx. 5 cm2 coverage of leaf area. After infiltration, plants were placed away from strong light for 48 h.Expression constructs were transformed into http://rsbweb.nih.gov/ij/).The plants were placed under illumination for 12 h prior to microscopy. Images were acquired on a Zeiss LSM 780 confocal microscope (Carl Zeiss), with a 40× water-immersion lens with a 1.1 numerical aperture. YFP signals were monitored using an argon laser at 514 nm wavelength for excitation, and emitted light was captured between a wavelength window of 518 to 557 nm. CFP signals were monitored using an argon laser at 458 nm for excitation, and emitted light was captured between 462 to 500 nm. Autofluorescence of chlorophyll was monitored using an argon laser at 514 nm for excitation, and emitted light was captured between 662 to 721 nm. Images were processed using ImageJ software (ptst-1 plants as described previously [The full-length coding sequence of the Arabidopsis GBSS protein was amplified from an Arabidopsis cDNA preparation (described above), with the following primers: 5´-ATGGCAACTGTGACTGCTTCTTCTAA-3´ and 5´-CGGCGTCGCTACGTTCTC-3´. The amplicon was cloned into the Gateway-compatible entry vector, pCR8 using the pCR8/GW/TOPO TA kit (Invitrogen). The construct was recombined into the Gateway-compatible pEarleyGate103 vector  in frameeviously . Screeniw/v) amylopectin and 0.1% (w/v) β-limit dextrin were conducted as described previously [w/v) amylopectin, and the gels were incubated overnight in the starch synthase activity medium (described above for “In-gel GBSS assay for recombinant proteins”). Activity was visualised by staining the gel in Lugol’s solution, followed by extensive destaining in water.Zymograms on native PAGE gels containing 0.1% (eviously . Starch S1 Data(TXT)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.S6 Data(DOCX)Click here for additional data file.S1 Figp < 0.05 were observed at any time point. Numerical data used to generate the plot are provided in Plants were grown under a 12-h light (0–12 h) and 12-h dark (12–24 h) regime. Entire Arabidopsis rosettes were harvested every 4 h and starch was quantified. Values are the mean ± standard error of the mean (SEM) from 4–5 plants. Note that values at the 0-h time point are replotted from the 24-h time point. No significant differences between mutants and wild type (Col) at (TIF)Click here for additional data file.S2 Figptst-2 starch granules. (B) Wild-type granules (Col) versus sex4, ptst sex4, and gbss sex4 starch granules. Numerical data used to generate the plots are provided in Purified granules were dissolved in 0.5 M NaOH and separated over a Sepharose CL-2B column. Fractions were mixed with an iodine solution, and absorbance was recorded at 595 nm. (A) Wild-type granules (Ws) versus (TIF)Click here for additional data file.S3 Figptst starch granules, and a dilution series of proteins from wild-type starch, were separated by SDS-PAGE. GBSS was detected by immunoblotting using a GBSS-specific antiserum. The equivalent mass of starch loaded is indicated above each lane, and the corresponding band intensity calculated using ImageJ densitometry software is indicated below. Numerical data used to generate the plot (band intensity) are provided in Granule-bound proteins extracted from purified Arabidopsis (TIF)Click here for additional data file.S4 FigA. thaliana GBSS and SS1 proteins, together with barley SS1 (HvSS1) and rice GBSS1 (OsGBSS1) proteins. Structural features from the solved HvSS1 and OsGBSS1 structures are depicted below the respective sequences, where helices (h) are represented in blue and sheets (s) are represented in orange. The bottom panel shows the sequence of the chimeric GBSS protein, where the helix containing the coiled coil from AtGBSS has been swapped with the homologous helix on AtSS1, together with an indication of COILS prediction scores. The swapped regions are highlighted in yellow. Full alignments used to generate these figures are provided in All alignments were generated using ClustalW, and the region surrounding the predicted coiled coil of GBSS is shown. (A). Sequence alignment of GBSS sequences from representative species of Viridiplantae. The COILS prediction scores for each individual sequence are indicated below the sequence. Amino acid positions containing Lys441, Glu449, and Glu452 are highlighted in yellow. (B) Generation of the chimeric protein between GBSS and SS1, which does not have a predicted coiled coil. The top panel shows the sequence alignment for (TIF)Click here for additional data file.S5 Fig2+-NTA resin. Unbound proteins were removed in the supernatant (Flowthrough), and the resin was washed five times. Bound proteins were eluted twice with high imidazole. Proteins were detected by immunoblotting with anti-His for GBSS-His6, and anti-GST for GST-PTST, and free GST.Purified recombinant proteins  were incubated together with a Ni(TIF)Click here for additional data file.S6 Figi and Wf) by immunoblotting with anti-GST. (B) The indicated amounts of recombinant GST-PTST protein were incubated with wild-type (WT) or waxy (wx) maize starch. Starch binding assay was carried out as described for (A). Unbound proteins were detected in the supernatant (Soluble), while bound proteins were detected in the pellet fraction (Pellet). The amount of bound proteins increased as the amount of protein used in the assay increased. Starch was therefore not limiting for the amounts of protein tested.(A) Purified GST-PTST, the W217A/W255A variant, and free GST (1 μg each) was incubated with wild-type (WT) maize starch, or Sephadex G-10 as a non-glucan control. After centrifugation, unbound proteins were collected in the soluble fraction. After three washes, bound proteins were eluted from the starch pellet. Protein was detected in the soluble (S) and pellet (P) fractions, as well as the initial and final washes (W(TIF)Click here for additional data file.S7 Figp < 0.05 between wild-type and ptst mutant at any DP. Numerical data used to generate the plot are provided in ptst mutants produce starch with normal granule morphology. Starch granules were purified from Arabidopsis rosettes and examined under a scanning electron microscope (SEM). Bar = 5 μm. (C) The number of starch granules per chloroplast is not affected by the loss of PTST. Resin-embedded leaf tissue was sectioned and stained with toluidine blue prior to light microscopy. Bar = 10 μm. (D) Zymogram analysis of soluble enzymatic activities in the ptst and gbss mutants. Soluble protein extracts of Arabidopsis leaves were separated on native PAGE gels containing 0.1% (w/v) amylopectin for visualising hydrolytic activities, 0.1% (w/v) β-limit dextrin for visualising debranching enzyme activities, and 0.15% (w/v) amylopectin for starch synthase activities. Equal amounts of protein were loaded per lane (30 μg). Following electrophoresis, gels were incubated in activity medium, and bands were observed by staining with iodine solution. The starch synthase activity gel was extensively destained in water. No differences were observed in the banding pattern between mutants and wild type (Col).(A) Amylopectin chain length distribution is not affected by the loss of PTST. Starch was debranched enzymatically and prior to analysis with high performance anion exchange chromatography (HPAEC-PAD). The area of individual peaks corresponding to chains with a given degree of polymerisation (DP) was expressed as a percentage relative to the summed peak area for DP 2–50. Values represent mean ± standard error of four biological replicates. No significant differences were detected at (TIF)Click here for additional data file.S8 FigData was obtained from the Genevestigator database, consisting of curated Rice Genome 51K array experiments. As expected, GBSS1 was mainly expressed in the endosperm, while GBSS2 was mainly expressed in leaf tissue. PTST was expressed in both leaf and endosperm tissues. Numerical data used to generate the plot are provided in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Tg′), as it was hypothesized that such temperatures might cause different effects on retrogradation. The Tg′ value of fully gelatinized waxy rice starch gel with 50% water content and the enthalpy of melting retrograded amylopectin in the gels were investigated using differential scanning calorimetry. Starch gels were frozen to −30°C and stored at 4, 0, −3, −5, and −8°C for 5 days. The results indicated that the Tg′ value of gelatinized starch gel annealed at −7°C for 15 min was −3.5°C. Waxy rice starch gels retrograded significantly when stored at 4°C with a decrease in the enthalpy of melting retrograded starch in samples stored for 5 days at −3, −5, and −8°C, respectively, perhaps due to the more rigid glass matrix and less molecular mobility facilitating starch chain recrystallization at temperatures below Tg′. This suggests that retardation of retrogradation of waxy rice starch gel can be achieved at temperature below Tg′.The retrogradation rate of waxy rice starch gel was investigated during storage at temperatures in the vicinity of the glass transition temperature of a maximally concentrated system ( The retrogradation of starch has been defined as a process which occurs when the molecules comprising gelatinized starch begin to reassociate in an ordered structure . RetrogrTg′, has been defined as the glass transition of a maximally freeze-concentrated system [Tg′ plays a key role in the quality and storage stability of frozen products because the rate of deteriorative changes in frozen food is closely related to Tg′ [Tg′, where the food matrix is in a glassy state, molecular mobility decreases and consequently reduces the rate of deteriorative changes involving molecular mobility. Due to the fact that retrogradation of gelatinized starch involves the movement and rearrangement of starch chains to form a junction zone [Tg′ should be different. Although there have been some studies on the influence of storage temperatures on the extent of retrogradation, these studies used ambient temperature, refrigeration (4 or 5°C), and frozen storage (−20°C) [Tg′. Moreover, all of these experiments used samples which were gelatinized in differential scanning calorimeter (DSC) pans. This static gelatinization might not mimic the real heating process of starch suspension which usually includes stirring the suspension during application of heat. Heating with shear would completely paste the starch suspension while heating without shear might cause incomplete pasting. Moreover previous studies [ Glass transition is a second-order phase transition that occurs over the temperature range at which amorphous solid materials  are transformed to a metastable leathery state . A specid system . Tg′ pl to Tg′ , 15. Belion zone , we hypo (−20°C) , 17 but  studies  have shoWaxy rice starch was made from Thai waxy rice grains (RD 6), grown in the Kalasin province area and aged at least six months.μm with mean diameter of 3.8 ± 0.7 μm. The protein, fat, and ash contents of waxy rice starch were 0.20 ± 0.00, 0.05 ± 0.00, and 0.03 ± 0.00 g/100 g dried solid by the AACC Method 46-12, 30-25, and 08-01, respectively [Rice kernels were soaked in water for 4 h and then ground with water using a double-disc stone mill. The slurry was centrifuged for 15 min and dried at 45°C for 15 h. The flour was ground in a hammer mill and passed through a 100-mesh sieve; then it was stored at room temperature in sealed plastic bags. For the isolation of waxy rice starch from waxy rice flour, the method of Hogan  was usedectively . The moiectively . The amyA starch suspension (10% w/w) was prepared by mixing the waxy rice starch in distilled water and stirring continuously at 250 rpm for 1 h followed by 200 rpm at 95°C for 1 h. The gelatinized starch sample was then put in stainless steel DSC pans. Water in each sample was allowed to evaporate at room temperature until the final water content was 50% as determined by weighing. The final sample weight in each pan was 20–30 mg. The pans were hermetically sealed to prevent moisture loss. The sealed pans were separated into two sets with one set being used for glass transition determination and the other set being used in the retrogradation study.Tg′ of a nonannealed sample and held at this temperature for 15 min, and cooled back to −60°C at 10°C/min and reheated to 25°C at 5°C/min to locate the Tg′. The Tg′ was indicated by an onset temperature of the heat capacity change, which was determined using the computer software program associated with the Perkin Elmer instrument. All measurements were run in triplicates.A Pyris-1 DSC  equipped with an intracooler subambient accessory was used. Nitrogen gas was used as the purge gas at a flow rate of 20 mL/min during calibration and measurements. The instrument was calibrated using indium and ice. An empty pan was used as a reference sample. Each sample in a sealed DSC pan was cooled to −60°C and then heated to 25°C at 5°C/min to determine the glass transition temperature of a nonannealed state. For the isothermal annealing treatment, the samples were cooled to −60°C and held for 15 min, heated to three different annealing temperatures  in the vicinity of the The sealed DSC pans with gelatinized starch were frozen to −30°C in a cooling bath and held for 1 h and then immediately stored at −8, −5, −3, 0, and 4°C for 5 days. Storage at −8, −5, and −3°C was done in a cooling bath while the samples at 0 and 4°C were kept in a low temperature incubator . After storage, the pans were left to stand for 30 min at room temperature to equilibrate and then heated from 25 to 120°C in the DSC at 10°C/min to observe the melting peak of the retrograded starch gels. All measurements were performed in triplicate.Tg′ and is independent of the initial solids fraction (before freezing) [Tg′ values of the gelatinized waxy rice starch isothermally annealed at different temperatures are presented in Tg′ value of the gelatinized waxy rice starch was about −5°C in the nonannealed state. At the three different annealing temperatures of −4, −7, and −10° C, the Tg′ value of gelatinized waxy rice starch was −4, −3.5, and −4.2°C, respectively. The annealing temperature of −7°C resulted in the highest Tg′ value and the most clearly detectable among the three annealing temperatures. This might have occurred because this temperature, which was slightly below the Tg′ of the gelatinized waxy rice starch (−5°C), was high enough to have sufficient molecular mobility for ice formation and yet also low enough to maintain the amorphous glass matrix as discussed by Lim et al. [Tg′ result of −3.5°C agreed relatively well with Slade and Levine [Tg′ values of gelatinized wheat starch, gelatinized waxy corn starch, and cooked rice stick noodles were −5, −6, and −4°C, respectively.In a system that is allowed to form the maximum amount of ice, the glass transition of this maximally freeze-concentrated matrix occurs at reezing) . Howeverm et al. . Our Tgd Levine , Roos and Levine , and Isrd Levine  who founTg′ for 5 days showed differences in enthalpy of melting of the retrograded starch gels . This indicated that at temperature below the glass transition temperature, the movement of starch chains to form junction zone of retrogradation was hindered. The results also agreed with Baik et al. [In this study, waxy rice starch was selected because it showed a well-defined melting peak of retrograded amylopectin at temperature range of 40–75°C. The method of gel preparation used in this study included (1) shearing during heating, which provided a fully gelatinized starch sample, and 2) control of the water content at 50% to obtain the maximum retrogradation extent. Gelatinized waxy rice starch samples stored at various temperatures in the vicinity of the  control  Figures . The peak et al.  who repoTg′ of the gelatinized waxy rice starch gel annealed at −7°C for 15 min was –3.5°C. The results showed that rice starch gels retrograded substantially when stored at 4°C for 5 days. Decreases in the enthalpy of melting of retrograded starch gel were observed in samples stored at –3, −5, and −8°C. This was due to the more rigid glass matrix and less molecular mobility at temperatures below Tg′. These results suggested that the retardation of retrogradation of waxy rice starch gel can be manipulated by a temperature below Tg′ and acceleration could be carried out at temperature above Tg′.The onset of


Starch makes up more than half of the calories in the human diet and is also a valuable bulk commodity that is used across the food, brewing and distilling, medicines and renewable materials sectors. Despite its importance, our understanding of how plants make starch, and what controls the deposition of this insoluble, polymeric, liquid crystalline material, remains rather limited. Advances are hampered by the challenges inherent in analyzing enzymes that operate across the solid–liquid interface. Glyconanotechnology, in the form of glucan-coated sensor chips and metal nanoparticles, present novel opportunities to address this problem. Herein, we review recent developments aimed at the bottom-up generation and self-assembly of starch-like materials, in order to better understand which enzymes are required for starch granule biogenesis and metabolism. Here, we have highlighted the potential of bottom-up synthetic biology approaches to produce starch-like surfaces in a format suitable for both structural analysis and real-time kinetic assessment of enzyme action thereon. These glyconanotechnology methods will help to pave the way to a more complete understanding of natural starch metabolism and may be used to inform understanding and exploitation of the natural processes, including the recapitulation of starch production in non-starch producing organisms.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.