Saccharomyces cerevisiae purged of its endogenous glycogen-metabolic enzymes. Systematic variation of the set of biosynthetic enzymes illustrated how each affects glucan structure and solubility. Expression of the complete set resulted in dense, insoluble granules with a starch-like semi-crystalline organization, demonstrating that this system indeed simulates starch biosynthesis. Thus, the yeast system has the potential to accelerate starch research and help create a holistic understanding of starch granule biosynthesis, providing a basis for the targeted biotechnological improvement of crops.Starch, as the major nutritional component of our staple crops and a feedstock for industry, is a vital plant product. It is composed of glucose polymers that form massive semi-crystalline granules. Its precise structure and composition determine its functionality and thus applications; however, there is no versatile model system allowing the relationships between the biosynthetic apparatus, glucan structure and properties to be explored. Here, we expressed the core Arabidopsis starch-biosynthesis pathway in DOI:http://dx.doi.org/10.7554/eLife.15552.001 Most plants and algae produce a carbohydrate called starch, which provides the plant with a dense store of energy. Starch is also the main carbohydrate in our diet and its unusual physical properties mean that it has many industrial uses. It is made of two different sugar-based molecules known as glucans and forms large, partially crystalline granules inside plant cells. Several enzymes are known to be involved in making starch, yet it is not clear exactly how the process works.Animals and fungi cannot make starch but they do make another type of carbohydrate called glycogen, which is also a glucan. Yeast is a single-celled fungus that is often used in research because it is easy to genetically engineer and quick to grow. To study the plant enzymes that make starch in more detail, Pfister et al. aimed to genetically engineer yeast to make their own starch.Arabidopsis thaliana were inserted into mutant yeast cells that were unable to make glycogen. The experiments show that all the plant enzymes are active in yeast and retain the roles that they perform in plants. Some of the enzyme combinations yielded glucan granules that occupied a large part of the yeast cell. These granules had many of the physical characteristics of plant starch, showing that yeast can be used as a system to better understand how starch is made.For the experiments, different combinations of enzymes involved in starch production in a plant called Important next steps will be to insert more plant proteins into the yeast and to fine-tune the production of these proteins. This should help researchers to design starches with desired properties in yeast and ultimately engineer crop plants to produce them.DOI:http://dx.doi.org/10.7554/eLife.15552.002 Starch is an agricultural raw material coveted for its nutritional value, but also for its functional properties, which have extensive applications in the food industry, in paper manufacturing, and in the production of biodegradable materials, amongst others . FurtherBoth industrial use and nutritional benefits depend on distinct biophysical characteristics and therefore on the underlying structure of starch. Starch is comprised of two α-polyglucan components (here named glucans): large moderately-branched amylopectin molecules and interspersing small, near-linear amylose molecules . Both arAmylopectin biosynthesis involves multiple enzyme activities, often composed of several isoforms with distinct specificities: four classes of starch synthases (SSs) elongate glucan chains using ADPglucose as glucosyl donor, creating α-1,4 glucose linkages; two classes of branching enzymes (BEs) introduce branches in the form of α-1,6 linked chains; and at least one isoamylase-type debranching enzyme hydrolyzes some branches again, probably tailoring the glucan for crystallization . In contin planta has been empirical and slow, and the full potential of this important renewable resource has not been realized.Starches from different plants vary in terms of amylose content, amylopectin structure and glucan modifications (e.g. level of phosphorylation) . HoweverHere we created a model system in yeast that allows the expression of multiple starch-biosynthetic enzyme combinations in a targeted, controlled and fast manner. Using a suite of molecular and biochemical analyses, we show that the enzymes are functional and, in the right combinations, capable of producing semi-crystalline starch-like granules. This shows that our yeast system is a highly tractable experimental system in which starch biosynthesis can be simulated and the capacities of different interdependent enzymatic combinations can be evaluated, bringing us much closer to a holistic view of this critical process.Saccharomyces cerevisiae CEN.PK113-11C, which had been developed for stable heterologous gene expression , SS2 (At3g01180), SS3 (At1g11720) and SS4 (At4g18240), the branching enzymes BE2 (At5g03650) and BE3 (At2g36390) and the isoamylase encoded by ISA1 (At2g39930) and ISA2 (At1g03310) (see BE1 (At3g20440), a third Arabidopsis gene annotated as branching enzyme . This yielded a second fraction of water-soluble but methanol-precipitable glucans  and a third fraction of methanol-soluble glucans .To quantify glucan accumulation, we cultured yeasts in shake flasks using galactose as the sugar source in the main cultures. The cells were harvested after 6 hr, as growth of yeast strains producing glucans was reduced afterwards (not shown). After homogenization of the cells and centrifugation at 6000 Most lines without ISA synthesized high amounts of soluble glucans, which reached around 10% of the wet weight . Light mMost lines expressing ISA produced water-insoluble glucans as well as soluble glucans . These lInterestingly, the lines which expressed ISA but neither SS3 nor SS4  had very low glucan levels when grown in liquid culture  revealed that SS4 consistently promoted the synthesis of insoluble glucans, both in absolute and relative terms . Reciprocally, the increase in the proportion of insoluble glucans caused by SS4 was strongest in the lines without SS2 .To monitor the effect of ISA and SSs on the presence of insoluble glucans, we re-calculated these as percentages of total glucans . As mentve terms . In contvs. 5). In contrast, when together with SS4 and ISA, having BE3 alone reduced both the total glucan content and the percentage that was insoluble. In all instances, however, having BE3 alone rather than both BEs, was accompanied by a shift towards longer wavelengths of maximum absorption [λmax] after iodine staining.During the creation of the library of strains described above, we observed lines that produced insoluble glucans despite lacking ISA: Strain I and strain M, containing BE3 as sole BE and either SS2 or SS3, respectively, contained up to 19% of their glucans in an insoluble form . The equTogether, these data show that each enzyme activity influences insoluble glucan formation in a distinct manner. They also highlight the complex functional interplay between both enzyme classes and isoenzymes of each class that are collectively responsible for starch synthesis.Studies of plant mutants and of individual enzymes in in-vitro have suggested that SS classes have distinct preferences in terms of glucan chain elon gation . To invess2 mutants from Arabidopsis and other species  at the expense of intermediate-length ones (DP = 11–25) . This re species . The mag species .SS3 and SS4 did not influence the lengths of short and intermediate chains in a distinct manner (not shown), which is in-line with reports from Arabidopsis mutants . HoweverThe presence of water-insoluble glucans prompted us to test whether these possessed the characteristics of genuine starch granules. We therefore performed a series of microscopic and biophysical analyses, focusing on the insoluble glucans from line 29 .The insoluble glucans were laid down as distinct spherical particles that were several micrometers in diameter, as determined by scanning electron microscopy . The sizn = 6] from line 29 = 0.44 ± 0.01 e−Å−3, corresponding to a mass density of 1.36 ± 0.04 g mL−1; Arabidopsis amylopectin density = 0.441 ± 0.004 e- Å−3, corresponding to 1.36 ± 0.01 g mL−1; see Materials and methods for details)  .gbss mutant plants , from that of soluble glucans from the corresponding line without ISA  and from that of linear glucans such as potato amylose (λmax634 nm). The similarity in absorption spectra indicates that the insoluble glucans from line 29 form comparable secondary structures to those in plant amylopectin, i.e. double helices between adjacent chains. Notably, also the iodine absorption spectra of insoluble glucans from yeast strains expressing only subsets of SSs  were dissimilar to that from line 29, re-affirming the influence of SSs on glucan structure  of purified and dried glucans resulted in a diffraction pattern typical of many starches, where glucan chains form double helices that align with an inter-helical spacing of ~1.6 nm, indicative of a B-type allomorph . Small-aCollectively, these analyses show that the insoluble glucans from line 29 form granular particles which display the typical internal organization of semi-crystalline plant starch.To our knowledge, this study represents the first example of where insoluble, starch-like granules have been produced in a non-plant system. It provides direct evidence that SSs, BEs and ISA are all key enzymatic components for starch granule formation. We furthermore show that the heterologously expressed enzymes retain their specificities in yeast and propose that this system can be used as an advanced tool for studying the overall process of starch biosynthesis in unprecedented speed and depth.isa1isa2  and limit-dextrinase (At5g04360), at least two active β-amylases (BAM1 [At3g23920] and BAM3 [At4g17090]), the α-amylase AMY3 (At1g69830), the disproportionating enzyme DPE1 (At5g64860) and the α-glucan phosphorylase PHS1 (At3g29320) . In geneisa1isa2 , the be2  cases, insoluble glucans were only obtained when ISA was present . The proisa mutant background restored granule formation in the mesophyll cells  which synthesizes only little glycogen overall but accumulates it in single colonies, presumably due to stochastic initiation events  even restored some granule formation  were overrepresented in the yeast glucans, in particular when ISA was present , indicating that at least part of it may actually be insoluble.Another difference between the glucans made in yeast and in plant cells concerns the presence of soluble polysaccharides: while virtually all plant glucans are normally laid down as insoluble starch granules, every yeast strain accumulated also substantial amounts of soluble glucans. The percentages of insoluble glucans from total glucans varied depending on the complement of enzymes present (as discussed above). Still, it did not exceed ~70% in the best cases . Even with advanced genome editing and transgenesis methods, the creation of a set of homozygous plant lines may take substantially longer than recreating and analyzing the pathway in detail in yeast, particularly in the light of the emerging systems for combinatorial gene assembly . The yeaUnless otherwise noted, chemicals were purchased from Sigma-Aldrich and restriction enzymes from Fermentas (Thermo Fisher Scientific).3 × 6 H2O (10 µg L−1) by NH4Fe(SO4)2 × 10 H2O (16.5 µg L−1) and the addition of histidine and uracil (20 mg L−1each). The medium was supplemented with either 2% (w/v) glucose (named MIN-N-Glu) or 2% (w/v) galactose . YPD plates were prepared as for liquid medium, but included 2% (w/v) bacto agar (BD). Plates containing synthetic complete (SC) or complex medium with glycerol (YPG) were prepared as described (−1). Uracil was omitted in SC-ura plates. Plates with synthetic medium and galactose , used for iodine staining of yeast colonies were prepared like SC plates but with replacement of glucose by 2% (w/v) galactose. Plates with 5-fluoroorotic acid (FOA plates) are SC plates supplemented with 0.1% (w/v) 5-fluoroorotic acid (Fermentas).Complex medium consisted of 1% (w/v) Bacto yeast extract (BD) and 2% (w/v) Bacto peptone (BD), supplemented with either 2% (w/v) glucose (medium named YPD) or 2% (w/v) galactose . Sugars were added after autoclaving; 20% (w/v) glucose stock was separately autoclaved and 20% (w/v) galactose stock was filter-sterilized through a 0.22 µm filter unit. Minimal medium lacking nitrogen (MIN-N medium) was prepared as described , except escribed , except Arabidopsis thaliana mutants are listed in gbss ptst mutants  was kindly provided by Prof. Barbara A. Halkier, Department of Plant and Environmental Sciences, University of Copenhagen, Denmark. Yeast strains were generated by sequential integration of constructs either at loci of the yeast expression platform (S. cerevisiae strains used in the present study are presented in CEN.PK113-11C (platform  (designeS. cerevisiae (BIOMATIK) was used for SS2 as it was not expressed otherwise. The E. coli APGase glgC-TM carries three amino-acid changes  that render it insensitive to allosteric regulation  or FLAG tags (for SS4 and BE1), respectively. A C-terminal HA tag was also added to glgC-TM. All CDS were fused to a ca. 450 bp PGAL1promoter, except for ISA1 and ISA2 that were fused to the bidirectional PGAL10-PGAL1promoter (Coding sequences (CDS) of Arabidopsis genes less the chloroplast transit peptides as predicted by ChloroP  were clogulation . SS2, SSpromoter . These ppromoter  or modifpromoter .GSY1 gene, was PCR-amplified from chromosomal DNA of CEN.PK113-11C using primers with 5' SbfI and 3' AscI and BclI restriction sites  and BstAPI and ligated into the pX-3_GSY1_down vector digested with the same restriction enzymes. The resulting vector was named pGSY1. Vectors targeting GSY2, GLC3, GLG1, GLG2, GPH1, GDB1 were generated by replacing the recombination sites of pGSY1 by the sequences homologous to the beginnings and ends of the respective genes using the 5' AvrII and 3' SacII restriction sites (for the recombination sites 'up') or the 5' SbfI and 3' AscI sites (for the recombination sites 'down'), respectively. All restriction sites were added via primers during PCR amplification on CEN.PK113-11C chromosomal DNA. The resulting vectors were either used directly for disruption of genes  or modified to contain a starch-biosynthetic gene as described above . Plasmids were verified by sequencing. Details on plasmids and primers for their generation are provided in To target constructs to other loci, the two flanking recombination sites (5' site named 'up' and 3' site named 'down') were replaced by other CEN.PK113-11C sequences of between 300 and 800 bp in length. First, a 'GSY1 down' construct, targeting the 3’end of the on sites . The proURA3 marker for subsequent transformation, yeasts were selected on plates containing 0.1% 5-fluoroorotic acid (FOA plates), positive colonies streak purified on YPD and purified colonies tested for petiteness on YPG plates and loss of marker on SC-ura plates. Marker loss was further confirmed by PCR . After incubation of the main culture for 6 hr, single-cell colonies were obtained by plating cells on an YPD plate. No loss of construct loss was detected among all 42 single-cell colonies tested by stringent PCR spanning promoter and coding sequences of the expression constructs (depicted in the last eight rows of 600) of 0.3 or YPD  to a starting OD600 of 0.1. After growing for 6 hr, cells were pelleted, washed twice with water and the cell pellet flash frozen in liquid nitrogen. The wet weight is the weight of the cell pellet after carefully removing the supernatant prior to freezing. Samples were stored at −80°C. Replicate cultures arise from independent pre-cultures of a single yeast line. Yeasts were grouped according to replicate number (instead of line/genotype) in a non-random way during growth and processing.Yeasts were grown at 30°C with 260 rpm shaking. Unless otherwise specified, all yeasts were grown in complex medium. Yeast lines from YPD plates were first inoculated in YPD and grown overnight. Cells from these pre-cultures were inoculated in flask main cultures with either YPGal  to a starting optical density at 600 nm . Therefore, the cells were pelleted, once washed with the respective MIN-N medium and then resuspended in the MIN-N medium to reach the same volume as in YPgal/YPD. After additional shaking for 3 hr, cells were harvested as described for cultures in complex medium. Replicate cultures arise from independent main cultures.For growth of yeast lines that express BE3 or ISA1/ISA2 only , a speci−1]. The cells were then homogenized by vigorous vortexing for 45 to 50 min, and complete disruption of cells was confirmed at random using a light microscope. Samples were subject to centrifugation for 5 min at 6000 g, thereby fractionating the soluble fraction  and the insoluble fraction .Unless otherwise noted, all steps were performed at 4°C. Yeast cell pellets from shake-flask experiments were resuspended in 3 volumes 1.12 M perchloric acid and 4 volumes glass beads . To calculate the volume of the cell pellets, the wet weight [mg] was multiplied with the factor 0.9 ). To ensure complete cell lysis, the samples were subject to centrifugation for 5 min at 6000 g, the supernatant discarded, the pellet frozen in liquid nitrogen, thawed and the digestion repeated. Complete lysis was confirmed by light microscopy. In addition, at least one negative control (yeast without insoluble glucans) was included in each preparation. Samples were then subjected to centrifugation at 6000 g for 5 min, the pellets (containing insoluble glucans and cell debris) once washed with water and then resuspended in glucan buffer . To separate insoluble glucans from cell debris, the suspension was sequentially laid onto two 100% Percoll cushions  and the insoluble glucans pelleted by centrifugation for 5 min at 2500 g in a swing-out rotor. The glucan pellet was then treated with proteinase K for 16 hr at 37°C  in glucan buffer supplemented with 3.2 mM CaCl2 and purified on a third 100% Percoll cushion as before. The pellet containing the highly pure insoluble glucans was washed three times with glucan buffer to remove proteinase K, then extensively in water to remove SDS, then cooled to 4°C until use.Yeast cell pellets from liquid cultures with complex medium  were lysed for 3 hr with gentle shaking in 100 mM potassium phosphate buffer, pH 7.5, supplemented with g. The pellet was first once washed with 0.5% (v/v) SDS, then extensively in water, then cooled to 4°C until use.For isolation of plant starch granules, rosettes from Arabidopsis wild-type plants (WS ecotype) were ground in liquid nitrogen. After resuspending the powder in starch buffer  and sequential filtering through 100 µm, 30 µm and 15 µm nylon nets, the suspension was laid onto a single 95%  Percoll cushion and starch granules pelleted by centrifugation for 20 min at 2500 For visualization, the glucans were coated with platinum  and imaged with a Leo 1530 Gemini SEM  using a 7-kV electron beam.g at 25°C, resuspended in 100 mM MES buffer, pH 6.5, then kept on ice until use (2 to 3 hr). Before injecting cells into the tips of glass micro-capillaries, glycerol was added to the cell suspension as cryo-protectant to a final concentration of ~17% (v/v). Purified, dry, amylose-free Arabidopsis starch granules (gbss ptst mutant starch) were resuspended in water 12 hr before injection into the capillary tips. Filled capillary tips were flash frozen in liquid ethane and stored briefly in liquid nitrogen before mounting for tomography measurements.Yeasts were grown in complex medium with galactose (inducing condition) as described above. After 6 hr incubation of the main culture, cells were harvested by centrifugation for 3 min at 500 8 photons s−1. The specimens were mounted on a 3D piezoelectric positioning stage on top of a rotation stage 3.7 mm downstream of the focus, where the beam had a diameter of about 8.2 μm. Coherent diffraction patterns from the specimen were recorded with an Eiger 500 k detector with 75 μm pixel size , corresponding to 131 points per scan. At each scanning position, diffraction patterns were recorded with an exposure time of 0.1 s. Ptychographic scans were repeated at 192 rotation angles of the specimen with respect to the beam ranging from 0 to 179.0625° with a step size of 0.9375°. During the entire acquisition, a cryo jet was blowing nitrogen gas at 110 K onto the glass capillary, ensuring that the frozen specimen remained in the vitreous phase for the measurements. Ptychographic reconstructions were performed using 200 iterations of a difference map algorithm (6 Gy.Cryo X-ray ptychographic tomography measurements  were perxel size  placed 7xel size  with an lgorithm  followedlgorithm . Using 5lgorithm . The dos3 to 303 voxels were randomly selected within a single tomogram among regions of high density (presumably constituting the insoluble glucans) from individual Arabidopsis starch granules or individual cells of line 29, respectively. For each selected region, the electron density value at the maximum of the histogram  was determined. The mean and standard deviation were calculated from values of the six cubic regions. Average electron densities were converted to mass densities (6H10O5) with 27% (w/w) hydration, as suggested for B-type starch (−Å−3 (corresponding to a mass density of 1.364 ± 0.004 g ml−1) for Arabidopsis starch and 0.44 ± 0.01 e−Å−3 (corresponding to a mass density of 1.36 ± 0.03 g ml−1) for insoluble glucans from yeast line 29. We additionally estimate a systematic error of ~0.003 e−Å−3 (corresponding to ~0.01 g ml−1) in the absolute electron density measurement caused by noise in the tomographic reconstructions. The error given in the results section is the sum of this systematic error and the S.D.For measurements of electron densities of insoluble glucans, 6 cubic regions with 20ensities  using ane starch . Note th+ microfocused beam  with the λCuKα = 0.15418 nm radiation in order to obtain direct information on the scattering patterns. The scattering intensities were collected by a Fujifilm BAS-MS 2025 imaging plate system  and a 2D Triton-200 X-ray detector . An effective scattering vector range of 0.05 nm−1 < q < 25 nm−1 was obtained, where q is the scattering wave vector defined as q = 4π sin θ / λCuKα with a scattering angle of 2θ.Insoluble glucans were purified as described for SEM, but additionally washed in 80% (v/v) ethanol and desiccated under low pressure for two days. The powder was used directly for WAXS measurement. For SAXS measurements, a ~10% [w/w] glucan suspension in water was injected into capillaries, let stand until the glucans had settled, and diffraction patterns were acquired of glucan-enriched regions. SAXS and WAXS experiments were performed using a Rigaku MicroMax-002Klebsiella planticola pullulanase , 0.04 U of Pseudomonas isoamylase (Megazyme) and 10 mM sodium acetate, pH 4.8. The reaction was stopped by heating to 99°C for 5 min. The samples were clarified by centrifugation for 1 min at 16,000 g and the supernatant applied to sequential 1.5 mL columns of Dowex 50WX4  and Dowex 1 × 8 . The glucans were eluted in 4 mL water, lyophilized and re-dissolved in water by heating to 99°C for 5 min. In addition, non-debranched controls of soluble and insoluble glucans from several yeast lines were prepared by omitting the debranching enzymes. Malto-oligosaccharides were treated in the same way as these non-debranched controls. Chains were separated on a HPAEC-PAD system  using a CarboPack PA-200 column, a flow rate of 0.5 mL min−1 and the following program: 0 to 12.5 min, a linear gradient from 95% eluent A (100 mM NaOH) and 5% B  to 60% A and 40% B; 12.5 to 50 min, a linear gradient to 15% A and 85% B; 50 to 52 min, a linear gradient to 10% A and 90% B; 52 to 60 min, 10% A and 90% B (column wash); 60 to 80 min, 95% A and 5% B (column equilibration) . Peaks were identified using standard linear glucan chains with degrees of polymerization (DP) from 2 to 7 . Peak areas were determined with Chromeleon software and summed within the depicted range . Arabidopsis leaf starch from wild-type (WS ecotype) and mutants was extracted using perchloric acid as described . Soluble−1 and 2.8 g L−1 in water for insoluble and soluble glucans, respectively) were heated to 99°C for 10 min and then clarified by centrifugation at 16,000 g for 1 min. One volume of supernatant was mixed with 7 volumes of iodine/potassium iodide solution , and the absorption in steps of 1 or 2 nm immediately measured. The spectrum from a water blank with iodine solution was subtracted from the glucan absorption spectrum. The absorptions in the wavelength range provided in the Figures (420 to 780 nm in Iodine absorption spectra from soluble and insoluble glucans from yeast (from perchloric-acid extraction), Arabidopsis and potato amylopectin and potato amylose  were obtained using an adaptation of a described method . Glucan Cells from YPD plates were resuspended in water and dropped onto an SC-gal plate, which was then incubated at 30°C for 24 hr. Subsequently, native suspensions of amylose-free Arabidopsis and potato starch and pre-boiled potato amylose (type III) in water were dropped onto the plate. Once dry, the plate was exposed to vapor of Lugol's solution and immediately photographed.Cells from liquid cultures in complex medium were mixed with one volume Lugol's solution and immediately imaged using an Axio Imager.Z2 light microscope equipped with a 100X oil-immersion objective .2, 1 mM CaCl2, 2% [w/v] glutaraldehyde; final concentrations) for 5 min, pelleted by centrifugation for 3 min with 1000 g and resuspended in glutaraldehyde fixative . After incubation for 16 hr at 4°C, cells were pelleted and washed four times with water, then once with 100 mM sodium cacodylate buffer, pH 6.8. Cells were post-fixed at 4°C with increasing concentrations of osmium tetroxide in 100 mM sodium cacodylate, pH 6.8, starting from 0.2% (w/v) osmium tetroxide for 5 hr, followed by 0.5% (w/v) for 16 hr and finally 1% (w/v) for 2 hr. Cells were pelleted by gentle centrifugation and washed three times with 100 mM sodium cacodylate, pH 6.8, at 4°C, then once with water at 25°C. Cell dehydration was performed by sequential washes in increasing ethanol concentrations , six washes in 100% ethanol and two washes in 100% acetone whereat each step had a total duration of 5–8 min including centrifugation. Finally, cells were infiltrated with epon , by gradually increasing its concentration in acetone, beginning with 25% (v/v) for 2 hr, then 50% (v/v) for another 2 hr, followed by 75% (v/v) for 16 hr and five times 100% for a total period of 7 hr. Finally, cells were transferred to gelatin capsules filled with freshly prepared 100% epon and incubated at 60°C for 48 hr for the polymerization of the embedding medium.Cells grown in liquid cultures in complex medium  were pre-fixed with glutaraldehyde  sections were cut with a diamond knife, placed on formvar/carbon-coated copper grids, stained with 2% (w/v) uranyl acetate and Reynold’s lead citrate. Images were acquired using a FEI Morgagni 268 TEM.g at 4°C), frozen in liquid nitrogen and stored at −80°C until use. Protein amounts were determined using a Bradford-based protein assay (Bio-Rad) with bovine serum albumin (BSA) as standard. Equal loading was confirmed in SDS-PAGE.For isolation of soluble proteins, yeast cell pellets from liquid cultures with complex medium were resuspended in 3.3 volumes of native extraction buffer  and 4.3 volumes of glass beads  and homogenized by vortexing for 6 min in total at 4°C with cooling in between. Rosette material from two pooled four-week-old Arabidopsis plants (harvested in the middle of a 12 hr day) was homogenized in the same extraction buffer as above in an all-glass homogenizer. Soluble proteins were separated from cell debris by two sequential centrifugation steps  protein assay (Pierce) with BSA as standard.For total protein extracts, yeasts were grown and homogenized as above, but with non-native extraction buffer . The extracts were heated to 99°C for 12 min, then clarified by centrifugation for 5 min at 16,000 −1for 3.5 hr at 4°C. After washing for 30 min at 4°C in 50 mM Hepes-NaOH, pH 7.0, and 10% (v/v) glycerol, the gel was incubated overnight at 25°C with gentle shaking in 50 mM Hepes-NaOH, 10% (v/v) glycerol, 2.5 mM AMP, 50 mM glucose 1-phosphate and 28 U (per gel) phosphorylase a (from rabbit muscle).To monitor branching enzyme and isoamylase activity, 15 µg of native soluble proteins were loaded on a 7.5% (w/v) native polyacrylamide gel containing 0.015% (w/v) oyster glycogen and run at 10 V cm−1for 3.5 hr at 4°C. The gels were incubated for 16 hr at 25°C with gentle shaking in 100 mM HEPES-NaOH, pH 7.5, 2 mM DTT, 10% (v/v) glycerol, 0.5 mM EDTA, 0.5 M trisodium citrate (tribasic) and 0.8 mM ADPglucose. Both types of gels were stained with ~1:3 diluted Lugol's solution to visualize glucan-modifying enzyme activities.For starch synthase activity, 22.5 µg of proteins were separated on a 7.5% (w/v) native polyacrylamide gel containing 0.3% (w/v) oyster glycogen using 10 V cmAB_439706)  and goat anti-mouse IgG (H + L)-HRP secondary antibody , respectively. Chemiluminescence was visualized using the WesternBright ECL HRP substrate kit (Advansta). The immunoblot shown in For the immunoblots shown in _439706)  or anti-t tests or ANOVA . Multiple comparisons of untransformed percentages of insoluble glucans .The Arabidopsis Genome Initiative gene codes for the Arabidopsis genes used in this study are the following: platform  are as f In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your paper has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor.Thank you for submitting "Recreating the Synthesis of Starch Granules in Yeast" for consideration by The reviewers have discussed their comments with one another, and, based on the individual reviews and the reviewers' discussion, the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Starch is the most important storage carbohydrate in plants, with key roles in plants and serving as the major dietary source of carbohydrates for humans. It l also has many non-food applications. Differences in starch structure between and within species are of great biological, nutritional and technical importance. Starch is also immensely complex, consisting of an unbranched glucan (amylose) and a branched glucan (amylopectin), which form a semi-crystalline starch granule. This physicochemical complexity sets starch apart from all other storage glucans, including the widely distributed soluble glycogen. Starch structure and the role of different enzymes in generating this structure have been intensively studied in several plant species including the referenced system Arabidopsis and also many crops including maize and potato. This has occurred out of curiosity, but also frequently driven by possible applications. The approach has mainly involved using forward or reverse genetics, followed by chemical and physical analysis of the resulting changes in starch structure. These studies have defined a set of enzymes that are involved in starch synthesis – or more precisely, whose modification / deletion alters the levels or especially the structure of starch. These genes are being used to try to modify starch structure in Arabidopsis and several crop plants.However, although very successful, this previous research does not provide a full understanding of how starch is synthesized, and the structure of starch and starch granules. This requires the reconstitution of the entire system from defined parts, i.e. synthetic biology.This is the potentially game-changing advance that is reported by Zeeman and colleagues.Starting with a yeast strain in which all glycogen-metabolizing genes are deleted, the individual components can be added all together, or in combinations or alone to test how a starch-like structure can be created. This paper convincingly shows that each of the individual enzyme is active, and that when all are present they form a starch-like structure which is analyzed chemically and physically to show the properties are much more similar to starch from Arabidopsis than to glycogen.A. thaliana. Numerous other strains produced α-glucans of distinct primary structure and morphology.Specifically, using a combinatorial approach, the authors examined nearly 30 recombinant yeast strains containing plant starch synthases (SS), branching enzymes (BE), and a debranching isoamylase heterodimer (ISA). A particular strain ("line 29") expressing four SS, two BE, and the ISA heterodimer, along with an ADP-glucose pyrophosphorylase, was able to recapitulate the abundant production of insoluble starch granules with a structure highly similar to those from wild-type The authors also convincingly argue why this is best done is yeast, being faster and more generalizable. Studies of starch structure in mutants are unavoidably complicated by the simultaneous presence of starch degrading enzymes that may – and almost certainly do – act on incorrectly formed starch. The use of a completely heterologous system allows to rigorously dissect how starch and starch grains are formed.As such, this work demonstrates and validates a powerful new model system to dissect the complex, crucially topical process of starch biosynthesis across plant species. This system also has the potential to explore the effects of protein mutants/variants, post-translational modifications, and cooperativity with additional proteins and enzymes. The study appears to be technically sound and performed with a high degree of rigor.In short, this research is a tour-de-force and opens new perspectives for understanding how starch is synthesized and using this knowledge to modify starch structure.Essential revisions:1) As mentioned at the end of the Discussion, starch grains contain amylopectin and amylose and the latter is formed by GBSS. In this study, GBSS was omitted, and the focus was on formation of an amylopectin-like macromolecule. This does need to be justified. It may be important to compare the amylopectin-like structure formed in yeast with the structure of amylopectin in gbss1 mutants . This can probably be done very easily, probably even using information available in the literature.2) Consideration should be given to why the repeat structure of the insoluble glucan in strain 29 is not the same as for Arabidopsis starch. The fact that the enzymic reconstruction omitted one branching enzyme (BE1) should be discussed. Although the authors have published that knock out of this gene has no discernible phenotype in Arabidopsis, it remains possible that in the yeast system this could make an important contribution to starch structure.3) It would be helpful to have experimental data for results in yeast that were not predicted by the work from plants. For example, there are examples within the yeast system where insoluble glucan is produced in significant amounts  line I w4) The analysis of Arabidopsis mutants has suggested that the role of SS4 in starch biosynthesis is due to its ability to prime granule formation. However, the yeast results suggest that SS3 and possibly SS2 might also be able to prime insoluble glucan accumulation. Some discussion of these results needs be offered as well as comparison of the 'starch structure' between lines 5, 7 and 29 to compare the arrangements of glucan formed by limited complements of enzymes compared to the full complement in line 29.5) Looking at 6) In the first sentence of the Discussion, 'genuine' starch granules may be an overstatement, especially as GBSS and presumably amylose are missing. Further on in the same paragraph, 'the highest degree of similarity' can perhaps be rephrased – what the authors mean is probably that the glucan was most similar to those from Arabidopsis when all enzymes were included, but this sentence could also be read otherwise.7) Some of the discussion /comparison of the results in yeast and plants that is at the moment in the Results should be moved to the Discussion. Splitting it between the Results and Discussion does not help to bring over the main messages. It also leaves the Discussion a little short on new insights and a little long on what might be done in the future. Also, more discussion of the roles on glycogenines in yeast, and the ability of the cocktail of enzymes to create a starch-like structure without a seeding polysaccharide or glycoprotein might be added.8) The authors have put much emphasis on the results from strain 29, which carries genes encoding the entire complement of starch biosynthetic enzymes defined in Arabidopsis (except GBSS and SBE1). However, the presentation of their analysis might be improved by comparing and contrasting insoluble glucan made in Arabidopsis and yeast, with additional data on the structure of the insoluble glucans produced in yeast in strains where such production was unexpected. An analysis of what did not work as expected rather that only what did work, or a systematic discussion of the contribution of each component in isolation and in combination could provide a better interpretation of the data already generated as well as establishing how robust the yeast model is for starch synthesis in plants.9) For a general reader, a brief account of the colors obtained with iodine with different glucans (including glycogen) would be very helpful.10) Similarly the method used to separate soluble and insoluble glycans should be briefly explained in the text.11) The authors should include a precise description of the glycosidic linkages of amylose and amylopectin in the second paragraph of the Introduction. […]Essential revisions:1) As mentioned at the end of the Discussion, starch grains contain amylopectin and amylose and the latter is formed by GBSS. In this study, GBSS was omitted, and the focus was on formation of an amylopectin-like macromolecule. This does need to be justified. It may be important to compare the amylopectin-like structure formed in yeast with the structure of amylopectin in gbss1 mutants . This can probably be done very easily, probably even using information available in the literature.max) of 550 nm after complexion with iodine , very close to that of gbss ptst amylopectin (548 nm). These points are now mentioned in the manuscript. We used starch from the Arabidopsis gbss ptst mutant  in addition to wild-type starch for structural analyses  where comparisons had not previously been made. We are confident that the absence of GBSS (and PTST) does not alter amylopectin structure in Arabidopsis mutants.The amylopectin chain-length distribution is not altered upon loss of GBSS in Arabidopsis . Likewise, the amylopectin from wild-type Arabidopsis starch (separated from amylose by size-exclusion chromatography) has a wavelength of maximal absorption (λ2) Consideration should be given to why the repeat structure of the insoluble glucan in strain 29 is not the same as for Arabidopsis starch. The fact that the enzymic reconstruction omitted one branching enzyme (BE1) should be discussed. Although the authors have published that knock out of this gene has no discernible phenotype in Arabidopsis, it remains possible that in the yeast system this could make an important contribution to starch structure.be1 mutant of Arabidopsis was first reported to have a wild-type phenotype , while the be2be3 double mutant was reported to have no starch synthesis (i.e. a BE null mutant). Later, the be1 mutant was reported to be embryo lethal . We nevertheless agreed with the reviewers that it was interesting to test the function of BE1 in the yeast system. We therefore expressed it in two different backgrounds; 1) together with SS3 and the AGPase . This strain only contained amylose-like glucans like its parental line with just SS3 and the AGPase, indicating that BE1 alone has little or no branching activity. Second, we included BE1 in strain 29 (i.e. in addition to the other amylopectin-synthesizing enzymes). Here again, BE1 did not alter glucan production or structure . Thus, the absence of BE1 cannot explain the atypical repeat structure in the insoluble glucans from strain 29. Rather we believe that the repeat size stems from an enzymatic imbalance; more specifically excessive synthase activity relative to branching activity – a hypothesis we state clearly in the Discussion of the revised manuscript (section “Current limitations and future applications of the yeast system”) and which we will test in future research.Most plant species contain two classes of branching enzymes (class I and II). In Arabidopsis, however, both BE2 and BE3 fall into class II, and there is no class I branching enzyme . The gene annotated as BE1 belongs to the putative class III of branching enzymes . However, no branching enzyme activity or role in starch metabolism has been reported for this or any other enzyme of the putative class. The 3) It would be helpful to have experimental data for results in yeast that were not predicted by the work from plants. For example, there are examples within the yeast system where insoluble glucan is produced in significant amounts  line I wmax values (wavelength of maximum absorption) when only BE3 is present. This indicates that more chains are able to form secondary structures, which presumably renders part of the glucans insoluble, despite the absence of ISA. We now discuss this finding and the role of isoamylase during the formation of insoluble glucans more extensively in the Discussion section “Role of ISA, SS isoforms and BEs during the formation of insoluble glucans”.The reviewers raise an important point here: the production of insoluble glucans by SS2 in the absence of ISA is interesting. In the revised manuscript, we have investigated this aspect systematically for all SSs. For this we created an additional set of yeast strains expressing only BE3 (like in line I) instead of both BE2 and BE3 revised . These d4) The analysis of Arabidopsis mutants has suggested that the role of SS4 in starch biosynthesis is due to its ability to prime granule formation. However, the yeast results suggest that SS3 and possibly SS2 might also be able to prime insoluble glucan accumulation. Some discussion of these results needs be offered as well as comparison of the 'starch structure' between lines 5, 7 and 29 to compare the arrangements of glucan formed by limited complements of enzymes compared to the full complement in line 29.In the revised manuscript we now distinguish more clearly the priming of granule formation (i.e. whether a given SS can efficiently use available primers or generate its own) and whether the glucan product made by the concerted action of a given SS, BE and ISA combination can crystallize.We therefore investigated the capability of each SS isoform to initiate glucans in the absence and presence of the yeast’s endogenous glycogenins , as described in review point 3.5) Looking at We believe that the overrepresentation of DP > 18 goes along with the altered width of the lamellar repeat and is due to an imbalance in enzyme activity . The overrepresentation of DP>18 is mentioned in the Results section “Yeast glucans have semi-crystalline properties of starch” and in the Discussion section “Current limitations and future applications of the yeast system”. There is also references given in that section to a paper where plant BE mutants were shown to have a longer lamellar repeat than normal. We argue that this can be explained by our imbalance theory.6) In the first sentence of the Discussion, 'genuine' starch granules may be an overstatement, especially as GBSS and presumably amylose are missing. Further on in the same paragraph, 'the highest degree of similarity' can perhaps be rephrased – what the authors mean is probably that the glucan was most similar to those from Arabidopsis when all enzymes were included, but this sentence could also be read otherwise.We have changed “genuine” starch granules to “insoluble, starch-like granules” and rephrased the paragraph accordingly.7) Some of the discussion /comparison of the results in yeast and plants that is at the moment in the Results should be moved to the Discussion. Splitting it between the Results and Discussion does not help to bring over the main messages. It also leaves the Discussion a little short on new insights and a little long on what might be done in the future. Also, more discussion of the roles on glycogenines in yeast, and the ability of the cocktail of enzymes to create a starch-like structure without a seeding polysaccharide or glycoprotein might be added.We have searched the manuscript for inappropriate discussion in the results and moved these to the Discussion.We have also included the analysis of additional yeast strains still expressing the glycogenins in the manuscript new , which s8) The authors have put much emphasis on the results from strain 29, which carries genes encoding the entire complement of starch biosynthetic enzymes defined in Arabidopsis (except GBSS and SBE1). However, the presentation of their analysis might be improved by comparing and contrasting insoluble glucan made in Arabidopsis and yeast, with additional data on the structure of the insoluble glucans produced in yeast in strains where such production was unexpected. An analysis of what did not work as expected rather that only what did work, or a systematic discussion of the contribution of each component in isolation and in combination could provide a better interpretation of the data already generated as well as establishing how robust the yeast model is for starch synthesis in plants.As described above (point 3), we have added a systematic analysis of the “unexpected” production of insoluble glucans, i.e. in the absence of isoamylase revised , togethe9) For a general reader, a brief account of the colors obtained with iodine with different glucans (including glycogen) would be very helpful.We have added an explanation to the Results section (“Yeast strains produce high amounts of glucans”).10) Similarly the method used to separate soluble and insoluble glycans should be briefly explained in the text.We have added an explanation to the Results section (“Yeast strains produce high amounts of glucans”).11) The authors should include a precise description of the glycosidic linkages of amylose and amylopectin in the second paragraph of the Introduction.We have added a precise description in the second paragraph of the Introduction.


Eight methylation sites encompassing five genes, LPP, CPT1A, APOA5, SREBF1, and ABCG1, were significantly associated with PPL response at an epigenome-wide level (P < 1.1 × 10−7), but no methylation site reached epigenome-wide significance after adjusting for baseline TG levels. Higher methylation at LPP, APOA5, SREBF1, and ABCG1, and lower methylation at CPT1A methylation were correlated with an increased TG-PPL response. These PPL-associated methylation sites, also correlated with fasting TG, account for a substantially greater amount of phenotypic variance (14.9%) in PPL and fasting TG (16.3%) when compared with the genetic contribution of loci identified by our previous genome-wide association study (4.5%). In summary, the epigenome is a large contributor to the variation in PPL, and this has the potential to be used to modulate PPL and reduce CVD.Postprandial lipemia (PPL), the increased plasma TG concentration after consuming a high-fat meal, is an independent risk factor for CVD. Individual responses to a meal high in fat vary greatly, depending on genetic and lifestyle factors. However, only a few loci have been associated with TG-PPL response. Heritable epigenomic changes may be significant contributors to the unexplained inter-individual PPL variability. We conducted an epigenome-wide association study on 979 subjects with DNA methylation measured from CD4 Postprandial lipemia (PPL) refers to the changes in plasma lipoproteins following food consumption. PPL is highly correlated with fasting plasma TG concentrations, yet some evidence suggests that PPL may be an independent risk factor for CVD –4 in wayEnvironmental factors exert significant effects on gene expression, in some instances through epigenetic mechanisms . Thus, eWe hypothesize that individuals respond to environmental exposures by modulation of the epigenome, which elicits changes in the PPL response that could alter CVD risk. Of different forms of epigenetic modification, DNA methylation is the most extensively studied for its technical feasibility at the epigenome-wide scale, cost-effectiveness, well-established standard analysis platform, and its apparent relation to nutrition . The obj1) a high-fat meal test; and 2) a 3 week treatment of fenofibrate (160 mg). Participants were requested to stop the use of lipid-lowering medication for at least 4 weeks and to refrain from alcohol for 24 h prior to their study visit. Diet history questionnaires were used to collect demographic, lifestyle, and dietary data  from families of European descent at two field centers: Minneapolis, MN and Salt Lake City, UT. GOLDN was designed as an intervention study to identify genetic factors that determine lipid responses to two interventions: ary data . The stuPostprandial TG responses were calculated based on the growth curve models of TG as the function of times, as described . Briefly+ T cells, which represent the most common lymphocytes in whole blood , CD4+ T cells were isolated from frozen buffy coat samples that were collected at visit 2 (baseline) before fenofibrate intervention. DNA was extracted from these cells using DNeasy kits   detection P > 0.01 and 1.5% of samples have missing data; or 2) >10% of samples have no adequate intensity  based on the β scores of all autosomal CpG sites that passed quality control were calculated using the prcomp function in R (v12.12.1). Four PCs were used in the EWAS.Different cell types in whole blood may have contrasting methylation patterns. Thus, to minimize the confounding effect of cell type differences and increase the consistency of methylation measures across samples, we restricted DNA methylome analysis to CD4le blood , 22. Usierlands) . Genome-erlands)  using Ilntensity . For adjntensity , 25. At P > 10−6.The detailed procedure of genome-wide genotyping in GOLDN has been described , 26. In t-test.As the epigenome is known as the fingerprint of individuals in response to lifetime exposures up to the time point when the samples were collected, an individual epigenotype depends on local environments. Therefore, individuals with the same genotype may have different epigenotypes under different environments. For such reasons, the best discovery and replication in EWASs should be done within the same population. We randomly split the whole population (n = 979) into two-thirds as a discovery sample (n = 653) and one-third as a replicate sample (n = 326) using Proc Surveyselect in SAS v9.4  while holding the distributions of baseline TG, BMI, and sex similar between the two samples. To examine the differences in clinical characteristics between the sexes among the discovery and replication, we performed a 2, age3, study site, and the first four PCs for T cell impurity as fixed effects, and kinship as a random effect. The kinship matrix was generated based on family pedigree  and repeated the analysis using the same models (with or without adjusting for the baseline TG) as in the discovery and replication stages. Because PPL-TG response traits are strongly correlated with fasting TG, we also conducted an EWAS for fasting TG with the entire population using the same method and model (without adjusting for baseline TG).In the discovery stage, we modeled the association between methylation β score at each CpG site and PPL response measures using a linear mixed model , adjustipedigree . The anagree  that reached epigenome-wide significance at P ≤ 1.1 × 10−7 (P < 0.05/4 = 0.0125). After adjustment for baseline TG, only two methylation sites (cg00574958 and cg17058475 at CPT1A) were replicated in the replication sample .We first conducted epigenome-wide association tests for each of four PPL-TG traits , with P values ranging from 3.39 × 10−4 to 1.05 × 10−7. At CPT1A, four CpG sites were highly correlated with each other, with correlation coefficients ranging from 0.643 to 0.843  and similar associations with the AUC. Thus, these four CpG sites, located at the promotor region of CPT1A, likely represent one methylation region at CPT1A. Interestingly, three other methylation sites at LPP, SREBF1, and ABCG1 are also correlated with these four CPT1A methylation sites . In contrast, cg12556569 at APOA5 was independent of all seven methylation sites, but when adjusting for baseline TG, no methylation site reached epigenome-wide significance. Lastly, there was no significant association between methylation sites and the other three PPL-TG measures of uptake, clearance, and AUI.Applying the concept of meta-analysis, we next conducted a third epigenome-wide association analysis by combining the discovery and replication samples and using the same models . We then conducted an EWAS for fasting TG with the entire population (n = 979) using the same method, and found six of eight AUC-associated methylation sites were significantly associated with fasting TG at the epigenome-wide significance , and two other sites (cg12556569 and cg11024682) at APOA5 and SREBF1 almost reached the epigenome-wide significance .In this population, we found that AUC was strongly correlated with fasting TG , we estimated the combined variance contribution of all eight CpG sites together. This yielded a value of 14.9% for AUC variance. As AUC is strongly correlated with fasting TG, we also estimated the combined variance contribution of the eight sites to the phenotypic variance of fasting TG and determined this to be 16.3%. In contrast, the genetic variance contribution of the two previously identified genetic loci (rs964184 and rs10243693)  with the genotype of SNP rs964184 . However, methylation levels at any of the identified CpG sites were not associated with the rs10243693 genotype. In addition, we also examined all SNPs within 50 kb upstream or downstream of each of the eight methylation sites for association with AUC. For cg12556569 at APOA5, there were 20 SNPs . For the other seven methylation sites, none of the SNPs evaluated reached significant association after correction for multiple testing.We examined the correlation between the eight identified methylation sites and two genetic variants (rs964184 and rs10243693) that we previously found in GOLDN to be associated with AUC   and the duration of these changes after consumption of a high-fat meal . In termCHIANTI) , lendingr = 0.853; supplemental Table S6). Hence, it is not unexpected that identified epigenetic variants that are associated with AUC are also highly correlated with fasting TG, thus contributing to the risk of CVD.Individuals respond diversely to a high-fat meal, with TG reaching the highest peak 3.5–4 h after the meal and returning to baseline at 10 to 12 h after consumption , and decreased methylation of the same four sites has been associated with increased fasting plasma TG : rs964184 (r = −0.689), rs662799 , and rs3135506 . The correlation between cg12556569 and rs964184 was also observed in other populations of European descent  and a high-carbohydrate low-fat diet was reported for plasma TG in Han Chinese women . The other three PPL traits are weakly (r = 0.15 for clearance slope), negatively (r = −0.10 for uptake slope), or not (r = 0.036 for AUI) correlated with baseline TG. On the other hand, as the PPL was limited to 6 h with only 3 measures , AUC may not reflect the length of time needed to capture the catabolic side of the PPL, and the data points were not dense enough to precisely calculate the absorption and synthesis of TRLs (and their catabolism). Still, AUC may provide a more comprehensive view of the overall PPL response than the other three phenotypes. Additionally, the other three PPL traits  may not be properly captured with methylation measures in the CD4+ T cells. Therefore, the fact that identified epigenetic loci are associated with AUC only, not the other three measures of PPL, could reflect the particular characteristics of epigenetic loci that are induced by the environment.All eight identified epigenetic loci in this population are associated with the AUC-PPL phenotype, but there is no significant epigenetic locus that is associated with the other three PPL phenotypes: uptake, clearance, and AUI. We have examined the correlation between all four PPL phenotypes and baseline TG . Only AUC is highly correlated with baseline TG , while other loci were marginally significant with AUC at P < 5.5 × 10−6. In essence, our findings imply that the identified epigenetic loci at CPT1A, SREBF1, and ABCG1 can link obesity to hyperlipidemia (high TG) and elevated postprandial TG response, and then to the risk of CVD. This observation potentially defines a mechanism by which obese subjects show increased PPL response to a high-fat meal, with a subsequent increased risk for CVD.It is well-established that obese individuals exhibit elevated postprandial TG in response to a high-fat meal compared with nonobese individuals , 58, butrt Study . To examMany GWASs have identified genetic loci for a given trait, but often these loci account for only a small fraction of genetic variation and phenotypic variation, raising the issue of missing heritability . Within APOA5, can lead to reduced expression, and then slow clearance of TG and elevated PPL (CPT1A and SREBF1)  . However SREBF1) . DNA met SREBF1) . As such


Submission to journals takes a lot of time and format related submission requirements vary greatly from one journal to another. Lack of time and motivation in academia reduces scientific outputs and demotivates researchers. Author-based journal selection system (ABJSS) is a platform for pooling manuscripts conceived to minimize the time spent for manuscript submission and to increase scientific output. The system will provide two types of account: “Author” and “Journal Administrator”. Each account type will have its own abilities and permissions.The ABJJS system is an ongoing project that will be designed in cooperation with IT experts and academicians and it will be presented to the scientific world as soon as it secures sufficient support. Original works, reviews, case reports and letters are obviously important for any evident-based academic activity. In addition to the labor-intensive and challenging nature of writing, authors also face several problems during the publication period. Submission to journals takes a lot of time and format related submission requirements vary greatly from one journal to another. The submission process may take several days, and the evaluation process by the journal between several months and years while; overall the time between admission and publication can take a few months or even 1-2 years. This challenging and time-consuming process may lead to frustration and low performance of authors most of whom even give up the submission process altogether.Lack of time and motivation in academia reduces scientific outputs and demotivates researchers. Unfortunately, this problem has not been addressed in the scientific literature because the authors have not tried to solve this problem enough. Even so, even when we send this letter, we could not find a suitable reference.Author-based journal selection system (ABJSS) is a platform for pooling manuscripts conceived to minimize the time spent for manuscript submission and to increase scientific output. The aim is to help volunteer member journals view submitted manuscripts and inform authors about required revisions and estimated time for publication. Authors will be able to upload their abstracts and keywords to the ABJSS. The full text will be available only upon a direct demand by a journal and an authorization by the author. Just as authors send journals an undersigned declaration of conflict of interests, accompanied to their submitted journals, journals will guarantee that they will never use the manuscript data and otherwise they will have temporary publication ban due to ethical violation.Authors may receive requests for revision or delay by several journal, but they can accept only one. If an author accepts a proposal from a journal, s/he cannot receive proposals from any other journal until s/he withdraws his/her article as the system does not allow him/her to do it. This will solve authors' problem of lack of time and motivation while preventing journals from becoming victims.Sample images of the designed project are shown in this write-up. In the The system will provide two types of account: “Author” and “Journal Administrator”. Each account type will have its own abilities and permissions.Author will be able to upload abstracts and evaluate full-text view requests from Journal Administrators. Once an Author accepts a request from a Journal Administrator, he/she will not be able to contact with other Journal Administrators through the system related to that specific scientific paper.Journal Administrator will be able to apply advanced search for abstracts such that the system will provide description type and topic selection from a wide variety of options. In addition, the system will let the Journal Administrator to generate complex search filters by involving AND and OR operations between keywords.System security will be handled in such a way that the paper abstracts and account information  will be kept in system by encrypting the texts with 128bits AES encryption methodology.Redundancy will be provided through periodically generated database images through the SVN version control tool. These database images will be kept in an offline location which will be independent from the system server.The ABJJS system is an ongoing project that will be designed in cooperation with IT experts and academicians and it will be presented to the scientific world as soon as it secures sufficient support. It is planned to be used initially for the medical literature; but its integration with other fields will be also highly useful.Onur Ozturk: Conceptualized and designed the project.Fatih Ileri: Drafted the initial manuscript, critically reviewed the manuscript, and approved the final manuscript as submitted.


Hordeum vulgare L.) grain by measuring temporal changes in (i) grain amylose and amylopectin content, (ii) starch synthase activity, and (iii) the relative expressions of key starch-related genes.Starch consists of two types of molecules: amylose and amylopectin. The objective of this study was increase understanding about mechanisms related to starch accumulation in hulless barley , granule bound starch synthase (GBSS), and starch branching enzyme (SBE) increased steadily during grain filling, reaching their maximums 20–25 days after anthesis. The activities of SSS and SBE were greater in Ganken 5 than in either Beiqing 6 or Kunlun 12. The expression of GBSSI may control starch synthesis at the post transcriptional level. GBSSI is mainly responsible for amylose synthesis whereas SSSI and SBEII are mainly responsible for amylopectin synthesis in amyloplasts.The results suggest that SBE and SSS may control starch synthesis at the transcriptional level, whereas The online version of this article (doi:10.1186/s40529-017-0184-8) contains supplementary material, which is available to authorized users. Starch is the main end product of carbon fixation during photosynthesis. Starch consists of two major components, amylose and amylopectin. Amylopectin is composed of short α-1,4-linked chains of glucose. About 5% of these chains are linked together by α-1,6 linkages Manners . AmyloseSeveral enzymes are involved in starch biosynthesis. ADP-glucose pyrophosphorylase (AGPP) catalyzes the first reaction in starch synthesis, producing the activated glucosyl donor ADP-glucose  and SBE . Several multiple isoform enzymes are plant species-specific. Among the species examined so far, each isoform of SSS and SBE plays a distinct role in amylopectin biosynthesis  of starch. In fact, the process of grain filling in hulless barley is the process of starch accumulation. Hulless barley includes both waxy and non-waxy cultivars. The starch in waxy cultivars is composed of amylopectin (98.9%), whereas the starch in non-waxy cultivars is composed of both amylose (16.2–23.3%) and amylopectin (74.1–78.4%). The objectives of this experiment were (i) to compare the physiological and biochemical characteristics of starch biosynthesis during endosperm development in waxy and non-waxy cultivars of hulless barley and (ii) to study the differential expressions of AGPP, GBSS, SSS, and SBE genes at different grain filling stages.The field experiment was conducted in 2015 at the Agriculture Experiment Station of Shihezi University, China. Three hulless barley cultivars were used in this study: Beiqing 6 (non-waxy), Kunlun 12 (non-waxy), and Ganken 5 (waxy).2. The row spacing was 20 cm. There was 2 cm between plants within each row. The fertilizer and irrigation practices were the same as those used by farmers in the regions.The barley was sown in April. Each cultivar was grown in three replicate plots. The area of each plot was 5 mPlants were tagged as they flowered. Forty spikes with the same flowering date were collected from each plot 5, 10, 15, 20, 25, 30, and 35 days after anthesis (DAA)  were determined according to the “double-wave-length” method of He (−1) and amylopectin . The absorbance of the amylose standards was measured at 554 nm (Aλ1) and 490 nm (Aλ2)  on the y-axis. The linear regression equation of the standard curve was Y = 0.0079x − 0.0139 (R2 = 0.99). The standard curve for amylopectin was prepared in the same way only absorbance was measured at 542 nm (Aλ1) and 713 nm (Aλ2). The ΔA was calculated by subtracting Aλ2 from Aλ1. The linear regression equation of the standard curve was Y = 0.0059x + 0.0191 (R2 = 0.99). Total starch contents were determined by summing the amylose content and amylopectin content.Starch, amylose, and amylopectin contents  were put into 100 mL beakers. Small amounts of anhydrous ethanol were added to the beakers followed by 10 mL of 1 mol L−1 HCl. A 0.5 mL aliquot of iodine reagent (0.1% I2 + 1% KI) was added to the samples and then distilled water was added to bring the final volume to 50 mL. The samples were mixed and allowed to stand at room temperature for 20 min. Standard solutions were prepared of amylose (0–30 μg mL−1) and amylopectin (40–120 μg mL−1).A 3-mL aliquot of each solution was mixed with 20–30 mL distilled water. The pH of the solutions was adjusted to 3.5 with 0.1 mol L−1) was calculated by multiplying the starch content (%) by the seed weight (mg grain−1).The absorbance the main and particular wavelengths for amylose were 554 and 490 nm, respectively. The main and particular wavelengths for amylopectin were 542 and 713 nm, respectively , where W is starch accumulation, t is days after anthesis, K is the maximum accumulation amount, and a and b are constants. The equation can be rewritten as: y = k/(1 + eA + BT) where A = ln (a) and B = −b. The following characteristics were calculated for starch, amylopectin and amylose accumulation using CurveExpert 1.4 software: (i) time when the starch accumulation rate was at its maximum (Tmax) = −A/B; (ii) starch accumulation at 90% of its final amount (D) = [ln (1/9) − A]/B; (iii) the maximum starch accumulation rate (Vmax) = −KB/4; (iv) the mean starch accumulation rate (Vmean) = K/D; and (v) the initial growth power (C0) = K/(1 + eA) . The samples were homogenized by grinding in an ice bath and then centrifuged at 10,000 rpm for 30 min at 4 °C. The supernatant, referred to as enzyme preparation 1, was used to determine the activities of AGPP, SSS, and SBE activity. The sediment was resuspended with 5 mL of buffer solution and then centrifuged at 1000 rpm for 3 min at 4 °C. The supernatant, referred to as enzyme preparation two, was used to determine GBSS activity.Enzyme extraction was done using the method of Cheng and Jiang . Twenty −1 Hepes–NaOH (pH 7.4), 1.2 mmol L−1 ADPG, 3 mmol L−1 PPi, 5 mmol L−1 MgCl2 and 4 mmol L−1 DTT. The total reaction volume was 130 μL. The solutions were kept at 30 °C for 20 min and then the reactions were terminated by placing the samples in boiling water for 1 min. The solutions were centrifuged at 10,000 rpm for 10 min. A 100-μL aliquot of the supernatant was collected and mixed with 5.2 μL of colorizing agent consisting of 5.76 mmol/L NADP, 0.08 unit P-gucomutase, and 0.07 unit G6P-dehydrogenase. The absorption of the samples was measured at 340 nm. One unit of AGPP activity was defined as the amount of enzyme causing a one unit per min increase in absorbance.The AGPP activity was measured by mixing enzyme preparation 1 (20 μL) into a reaction mixture(110 μL) consisting of 100 mmol L−1 Hepes–NaOH (pH 7.4), 1.6 mmol L−1 ADPG, 0.7 mg amylopectin, and 15 mmol L−1 DTT. The total reaction volume was 56 μL. The solutions were kept at 30 °C for 20 min. The reactions were terminated by placing the reaction mixtures in boiling water for 1 min followed by cooling in a water bath. Next, the samples were mixed with 20 μL of a reaction solution consisting of 50 mmol L−1 Hepes–NaOH (pH 7.4), 4 mmol L−1 PEP, 200 mmol L−1 KCl, 10 mmol L−1 MgCl2, and 1.2 units of pyruvate kinase. The solutions were kept at 30 °C for 20 min. The reactions were terminated by placing the mixture in boiling water for 1 min. The samples was centrifuged at 10,000 rpm for 10 min. A 60-μL aliquot of the supernatant was collected and then mixed with 43 μL of color liquid including 50 mmol L−1 Hepes–NaOH (pH 7.4), 10 mmol L−1 glucose, 20 mM MgCl2, 2 mmol/L NADP, 1.4 units hexokinase, and 0.35 units G-6-P-dehydrogenase. The solutions were kept at room temperature for 10 min. The enzyme activity was assayed spectrophotometrically at 340 nm. One unit of GBSS activity was defined as the amount of enzyme causing a one unit per min increase in absorbance  into a reaction mixture (36 μL) consisting of 50 mmol L−1 HCl. The solutions were then mixed with 500 μL dimethylsulfoxide and 700 μL of iodine reagent (0.1% I2 + 1% KI). The enzyme activity was assayed spectrophotometrically at 540 nm. One unit of SBE activity was defined as the amount of enzyme causing a one unit per min increase in absorbance.The SBE activity was measured by mixing 50 μL of enzyme preparation 1 into a reaction mixture containing 50 mM Hepes–NaOH (pH 7.4), 5 mM G1P, 1.25 Mm AMP, and phosphorylase a (54 unit). The total reaction volume was 1.45 mL. The solutions were kept at 30 °C for 20 min and then the reactions were terminated by placing the samples in boiling water for 1 min. The reaction was terminated by adding 50 μL of 1 mol LThe procedure for measuring SSS activity used enzyme preparation 2 rather than enzyme preparation 1. In all other aspects, the procedure to determine SSS activity was the same as that used to determine GBSS activity. One unit of GBSS activity was defined as the amount of enzyme causing a one unit per min increase in absorbance at 340 nm.AGPP . Total RNA from endosperm was isolated using Fruit-mate™ for RNA purification (TaKaRa) and RNAiso Plus (TaKaRa Code: 9108). The RNA was reverse transcribed with the Script cDNA synthesis kit (TaKaRa). The synthesized cDNA was subjected to PCR for 45 cycles. The conserved regions of the gene sequences of The RT-PCR efficiencies (E) were calculated from the slopes in Light Cycler software using the equation E = 10[−1/slope]. Calculations for the test precision and test variability are based on the crossing-point (CP) variation from the CP mean value. A mathematical model was used to determine the relative expression ratio (R) of an unknown sample to the control, and expressed in comparison to a reference gene  and days after flowering (x).Starch, amylose, and amylopectin contents increased across time in all three cultivars Fig. a, c, e. −1).Grain starch contents are affected by the starch accumulation rate during grain filling. There were temporal differences in the accumulation rates of starch, amylose, and amylopectin among the three hulless barley cultivars Fig. b, d, f. The data in Fig. The amylopectin/amylose ratio increased to a maximum and then decreased in both Beiqing 6 and Kunlun 12 Fig. . This sh−1 d−1 at 25 DAA, whereas Kunlun 12 reached 21.86 nmol grain−1 d−1 and Ganken 5 reached 21.10 nmol grain−1 d−1 at 20 DAA.The AGPP activity increased to a maximum and then decreased in all three cultivars Fig. a. There −1 d−1) followed by Kunlun 12 (20.39 nmol grain−1 d−1) and then Beiqing 6 (18.00 nmol grain−1 d−1). This is because amylopectin is the major component of starch in Ganken 5. Overall, SSS activity was greater in Ganken 5 than in either Kunlun 12 or Beiqing 6 during the entire grain filling process.The SSS activity in all three cultivars increased to a maximum about 20 DAA and then decreased Fig. b. There −1 d−1) and 25 DAA in Kunlun 12 (19.87 nmol grain−1 d−1). The amylose content of Kunlun 12 was also greater than that of Beiqing 6. Overall, these results indicated that GBSS regulates amylose synthesis in barley and determines the amylose content of the grain. The role of GBSS is more important during late grain filling than during early grain filling.No GBSS activity was detected in Ganken 5 during grain filling Fig. c. There The SBE directly affects grain amylopectin content. The SBE can cut α-1, 6-glycosidic bonds, thereby forming branching sugar chains. The SBE activity in all three cultivars increased to a maximum about 20 DAA and then declined Fig. d. GankenPrevious studies indicated that starch accumulation rates were more consistently related to the activity of AGPP than to the activities of SSS, GBSS, and SBE . AGPP is comprised of two large subunits and two small subunits, each of which is encoded by distinct genes. Our study indicated no significant differences in GBSS I and GBSS II. The expression of GBSSI appears to be mostly confined to storage tissues. GBSSII is encoded by a separate gene and is generally expressed more in leaves and other non-storage tissues that accumulate transient starch. The results of the present study indicated that the relative expression of GBSSI increased to a maximum at 20 DAA and then declined in the non-waxy cultivars Beiqing 6 and Kunlun 12 . The SSS I expression increased to peak at 10 DAA, but then declined. The SSS I expression of Ganken 5 was significantly less than that of Beiqing 6 and Kunlun 12 between 25 and 35 DAA. The SSS genes are exclusively involved in amylopectin biosynthesis and SSS I is the major SSS form in cereal endosperm. This may be one reason for the decline in the amylopectin/amylose ratio in Beiqing 6 and Kunlun 12.The SSS genes are exclusively involved in amylopectin biosynthesis. The distribution of the SSS genes between the stroma and starch granules within plastids varies among plant species, tissue types, and plant development stages. In our study, the relative expression of SBEI and SBEII) differ in the length of glucan chains transferred in vitro and in their substrate specificities. In cereals, there are two closely related forms of SBEII . These two forms differ in chain-length specificity in vitro, with SBEIIb transferring shorter chains than SBEIIa during extended incubation. In the present study, temporal changes in SBEI, SBEIIa, and SBEIIb were similar in all three cultivars . The enzyme is known to be largely extraplastidial  in cereal endosperm, but plastidial in other cereal tissues and in all tissues of non-cereal plants  result in amylose-free starch in endosperm and pollen grains. The waxy locus in cereals is encoded by GBSSI, which catalyzes the elongation of amylose. In addition to its role in amylose biosynthesis, GBSSI is also responsible for the extension of long glucans within the amylopectin fraction  differ in the length of glucan chains transferred in vitro and in their substrate specificities. SBEII, compared with SBEI, transfers shorter chains, exhibits greater affinity towards amylopectin, and exhibits greater rates of branching with amylose  lay a foundation for identifying genes involved in the regulation of starch metabolism and (ii) provide valuable insight into the mechanism of metabolic regulation of starch biosynthesis under various physiological conditions.GBSS I expression was later than that of SSSI, SSSII, SBEIIa, and SBEIIb. This may explain the decline in the ratio of amylopectin to amylose during grain filling in Beiqing 6 and Kunlun 12 (Additional file GBSS I expression was less in Ganken 5 than in Beiqing 6 and Kunlun 12. Overall, we conclude that GBSS I is mainly responsible for amylose synthesis whereas SSS I and SBE II are mainly responsible for amylopectin synthesis in amyloplasts.The amylopectin/amylose ratio gradually declined in both Beiqing 6 and Kunlun 12 Fig. . Further


Journal clubs are important mechanisms for teaching how to approach the scientific literature critically and for disseminating findings. Papers from high-impact journals often dominate journal club selections, a practice that reinforces the unscientific emphasis of placing high value on publishing venue rather than scientific content and critical analysis of the publications. We suggest improving journal clubs by including preprints rather than focusing completely on published papers. This change in practice might benefit the scientific enterprise in numerous ways, including by providing direct criticisms to preprint authors before publication, deemphasizing publishing venue, teaching students the art of reviewing papers, and making journal clubs more current by discussing unpublished data. A journal club is a meeting to discuss a scientific paper. The practice of journal clubs originated in medicine and provided a mechanism for physicians to discuss new findings in the field . JournalCell, Nature, or Science. When these journals and their offshoots published by Cell Press and Nature Publishing Group  were added, the total was 59 (53%). Journals published by scientific societies, such as those published by the American Society for Microbiology (such as mBio), the National Academy of Sciences , and the American Association of Immunologists , were a minority, accounting for less than 30% of journal selections.In a typical journal club, the presenter provides a detailed description of the paper. The goal is to show trainees how to read the literature critically and to discuss new information. A nonscientific survey of immunology and microbiology journal club publication choices at four universities found in open Internet websites provided some insight into journal choices. Of 112 journal club publication choices, 30 (27%) were from In our opinion, the quality of journal clubs has been degraded in recent years as another deleterious consequence of the impact factor mania that has gripped the biological sciences . Specifihttp://asapbio.org/preprint-info/biology-preprints-over-time).In recent years, the biological sciences have experienced the phenomenon of the preprint movement –5. Altho–There are numerous reasons why using preprints for journal clubs instead of published articles might improve that experience while also benefiting the scientific enterprise. (i) Preprints are free and available to all with an Internet connection. This might be a major advantage in resource-poor settings where the selected article is behind a pay wall and not accessible to all. Although such costs are often minimized by distributing the article to journal club members, that practice can run afoul of restrictions by certain publishers. (ii) Preprints are often the most current form of scientific information since they are deposited before the article is published. Given that publication of a manuscript can be associated with significant delay as a result of peer review and publication procedures, such as the generation and correction of article proofs, using preprints in journal clubs will heighten the immediacy of the information. (iii) Presenting preprints will give the journal club members the ability to participate in peer review, since comments and criticisms generated during the discussion can be posted on preprint servers. For preprints deposited in the Biorxiv server, there is a built-in commenting service that allows the comments to be directly linked to the manuscript. Having more eyes look at each manuscript might improve the literature by reducing the frequency of inappropriately reproduced images, which plagues up to 4% of all published articles . Hence, http://asapbio.org/preprint-journal-clubs). In this editorial, we strongly endorse those suggestions. Institutions that adapt this practice will further empower the preprint movement.In summary, we see the introduction of preprints into journal club choices as a positive step that might improve the scientific enterprise at many levels, from providing additional means for prepublication criticism to refocusing the discussion to the scientific issue of the work. The use of preprints in journal clubs was previously suggested by others, who argued for some of the same benefits noted above. A Web platform to facilitate peer review of preprints is already available (


Recent engineering technologies have developed powerful techniques to characterize the mechanical properties of cell and matrix at nanoscale levels. Extracellular matrix (ECM) influences mechanical tension and activation of pathogenic signaling during the development of chronic fibrotic diseases. In this short review, we will focus on the present knowledge of the mechanisms of how ECM stiffness is regulated during the development of liver fibrosis and the molecules involved in ECM stiffness as a potential therapeutic target for liver fibrosis.Tissue and matrix stiffness affect cell properties during morphogenesis, cell growth, differentiation, and migration and are altered in the tissue remodeling following injury and the pathological progression. However, detailed molecular mechanisms underlying alterations of stiffness Each tissue/organ has an optimum stiffness level. The tissue/organ stiffness changes in response to biochemical and physical stimuli during the development or due to pathological conditions such as chronic fibrotic disease and cancer progression , 2.Atomic force microscopy (AFM) allows surface topography of tissues to be imaged with a nanometer spatial resolution using a sharp tip attached to a cantilever. In addition to surface imaging, AFM enables the measurement of mechanical data from tip/sample interaction . AFM is Many studies suggest that ECM stiffness affects biological properties of cells and tissues. For example, naive mesenchymal stem cells cultured on soft matrices that mimic brain exhibit neurogenic phenotype, whereas those cultured on stiffer matrices that mimic muscle exhibit myogenic phenotype, suggesting that matrix elasticity governs lineage and phenotypes of stem cells . NIH3T3 β/type V collagen-mediated , c-myb, and MEF-2) and acquiring contractile, proinflammatory, and fibrogenic properties . S. S107].  effects , suggestn damage . It remain vitro –111 and  in vivo , 113, rafibrosis . The monfibrosis . These fAs described in this review, each of the recent studies suggests that AFM could be a powerful characterization tool to understand mechanistic changes associated with liver disease. Thus, interest in such nanoscale measurements of liver matrix stiffness during the development and progression of liver fibrosis are likely to increase in future work, particularly with the development of new imaging modalities and AFM hardware that improve the quality of data obtained for biological tissues.


Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health (WestJEM). We continue to grow and thrive, extending our scope and reach throughout the nation and the world.I want to take this opportunity to update our readers, reviewers, and supporters regarding the growth and stature of the WestJEM is indexed in all the world’s sources, including MEDLINE/Index Medicus, PubMed, PubMed Central, Europe PubMed Central, Embase, EBSCO/CINAHL, SCOPUS, HINARI  and Clarivate (formerly Thomson-Reuters) Emerging Sources Index. As an open access journal, we are members of the Directory of Open Access Journals, which indexes our abstracts.One of the measures of a successful journal is its ability to make its published research available to other researchers, policy makers and thought leaders through robust indexing. st of 76 journal titles in emergency medicine. This is equivalent to the Clarivate (former Thomson-Reuters) impact factor that is commonly used to gauge a journal’s influence. This ranking can be found at: http://www.scimagojr.com/journalrank.php?category=2711&area=2700&order=cpd&ord=desc&page=1&total_size=76 and lists WestJEM as the 3rd ranked (of 12), fully open-access journal in the specialty.The journal’s two-year impact factor from Scimago Journal and Country Rank (SJR) is 1.136 for 2016. This ranks us 21http://www.scimagojr.com/journalsearch.php?q=19900193277&tip=sid&clean=0 (Image 1).The trend for impact factor can be found at: th of 75 journal titles in emergency medicine, and rising.Our three-year impact factor from Scopus 2016 CiteScore journal metrics is 0.95 (Image 2). This ranks us 25https://www.scopus.com/sourceid/19900193277?origin=sbrowse#tabs=1Comparison to other journals in the specialty can be found here: WestJEM score can be found at: https://www.scopus.com/sourceid/19900193277?origin=sbrowse#tabs=0And specific WestJEM is one of a few fully open-access emergency medicine journals in the world, the full published paper is available to anyone with an internet connection without charge. Furthermore, authors published in WestJEM retain their own copyright, and are not asked to sign over rights to their intellectual property as a condition of publication, like subscription-based journals. WestJEM’s article processing fee upon acceptance is the lowest among open-access journals. There is never a fee to submit a paper.Why does this matter? The more robustly indexed a journal, the more likely a researcher interested in your work is to find, and cite, your article. Given that WestJEM is distributed electronically to more than 23,000 readers, researchers and clinicians, and mailed to more than 4,000. Content is also distributed through Medscape, with some 250,000 hits per year (http://www.medscape.com/viewpublication/21595). There were 1.8 million article views and downloads of WestJEM papers in 2016. Clearly, distribution is wide and vast, offering the greatest chance of your work being cited.Each issue of WestJEM papers is tracked through Altmetrics, with our highest impact papers achieving more than 400 notices by news outlets, tweets, Facebook reposts, and academic reader inclusions. https://www.altmetric.com/details/9119550Immediate impact on social media of WestJEM is mainstream. The scientific quality of papers continues to increase, as the journal contributes to the greater good of the specialty. With the population health and public policy niche of the journal, this ultimately benefits our community and society.Publishing in Thank you for your faith in our efforts.