Journal of Applied Clinical Medical Physics, we conclude our twelfth volume. Looking ahead to Volume 13, we will find several changes occurring with the Journal. The first of these changes is in the sponsorship of this periodical. As many of our readers may be aware, beginning on January 1, 2012, the American College of Medical Physics (ACMP), the sponsor of this Journal, will cease to exist, and many of the activities of the ACMP will be taken over by the American Association of Physicists in Medicine (AAPM). Beginning in 2012, the AAPM will sponsor two journals, a scientific journal, Medical Physics, and a clinical journal, the JACMP. Instead of reporting to the ACMP Board of Chancellors, the Journal's editorial board will report to the AAPM Board of Directors. However, you, as readers, will see no change in the nature of the manuscripts published in the JACMP, nor any change in the editorial policy, editorial procedures, or mission of the Journal.With the publication of this issue of the A change that you will see, however, is that, beginning with Volume 13, we will be increasing our publication frequency to six issues per year, rather than four. Increasing our frequency of publication is a move that we have been considering for several years, and is a consequence of the increase in number of manuscripts submitted, and subsequently accepted, for publication. We believe the increase in publication frequency will have several advantages. First of all, the turnaround for accepted manuscripts will be faster. In order to meet our publisher's requirements, the deadline date for acceptance of a manuscript for publication in Volume 13 Number 1, scheduled for publication around February 15, is October 15, 2011. Any manuscripts accepted after that date would be published in Volume 13 Number 2. With our quarterly publication schedule, the publication date would be around May 15; hence, with the increased frequency of publication, the publication would occur one month earlier. A second benefit goes to our advertisers, whose message now can reach our readers six times a year, rather than four times a year. We hope that the decrease in turnaround time, coupled with the increased frequency of the Journal, will prove to be advantageous to both our readers and our advertisers.JACMP. Names of Associate Editors can be found below, and names of reviewers can be found in a Supplementary File.Finally, I would like to acknowledge the difficult and time‐consuming work of our Associate Editors and reviewers in maintaining the high quality of manuscripts published in the Associate Editors (including Guest Associate Editors) for Volume 12 include the following: Nzhde Agazaryan, Salahuddin Ahmad, John Antolak, Peter Balter, Pat Cadman, Marco Carlone, Nathan Childress, Geoff Clarke, Laurence Court, Luca Cozzi, Larry DeWerd, Bill Erwin, Kent Gifford, Rebecca Howell, Jennifer Johnson, Tommy Knöös, Stephen Kry, Harish Malhotra, Osama Mawlawi, Charles Mayo, Moyed Miften, Mike Mills, Eduardo Moros, Firas Mourtada, Ben Nelms, Jennifer O'Daniel, Tinsu Pan, Niko Papanikolaou, Donald Peck, Matt Podgorsak, Joann Prisciandaro, Jim Rodgers, John Rong, Yi Rong, Isaac Rosen, Surendra Rustgi, Bill Salter, Mehrdad Sarfaraz, Jeff Shepard, Chengyu Shi, Alf Siochi, Eric Slessinger, Jason Stafford, Lu Wang, Chuck Willis, Kamil Yenice, Al Zacarias, and Ron Zhu.Thank you to our Associate Editors and our reviewers, and especially to our authors, without whose contributions we would have no Journal.George Starkschall, PhDEditor‐in‐ChiefNovember 15, 2011


Despite literature documenting limited and asymmetrical benefits along with ethical issues, short-term international volunteering is increasingly popular among North American university students as a perceived advantage when applying to professional healthcare schools or the job market. Academic institutions are also encouraging students to pursue international experiences in order to cultivate values as global citizens. These experiences are most typically limited to economically privileged students. Furthermore, international activities in developing countries often lack a pedagogy of social justice and may confirm a simplistic understanding of development. Brazilian educator Paulo Freire’s “liberation pedagogy” provides a framework for understanding the limitations of international volunteering, whereby the presence of privileged volunteers implementing Western models of development may hinder aspects of local movements. Regardless, university students face intense competition in accessing opportunities, such as medical school, and pay large sums to participate in volunteering to strengthen their academic credentials. We propose that these students form “the voluntariat.” They simultaneously play two roles by, first, contributing to the conditions that oppress the very communities in which they volunteer and, second, by playing a role as objects of oppression by the liberal institutions of learning and employment to which they are attempting to gain access. She was doing HIV awareness… and she’s teaching people how to use condoms…you’re a seventeen-year-old girl straight out of high school in the UK and you’re going to teach adults [how to use a condom] …We actually just tolerate these things that you do ‘cause we like need your money and you have that colonial relationship…but nobody buys the bullshit. 1Having spent many late nights scouring online premedical forums in search of the elusive factor that guarantees admission to medical school, international volunteering seemed to be the hot ticket in town. Of those who responded to the question in the 2015 post-Medical Colleges Admission Test (MCAT) questionnaire n = 26,459), 27% reported having volunteered internationally.59, 27% rIn my interviews for scholarships and jobs, the background experience that I believe gives my applications some advantage over others is my past research experience on eye cancer in Kenya and clean cook stove technology in India. Although I make an effort to realistically convey my limited contributions to these international projects and distinguish them from service work, I perceive the individual interviewing me for the coveted position is already focused on my global experience. This fascination with an otherwise 2-in-1 combination of tourism and altruism extends beyond admission committees and employers to higher-level academic institutions seeking to increase the employability of their graduates.Despite the increasing number of students engaging in international experiences, emerging literature in global health and international development has become increasingly critical of international volunteering. International activities are typically short-term 1–8 weeks) weeks8 I8Crane’s allusion to the 1884 Berlin Conference, where Africa was divided amongst European nations, is an eerie reminder of Africa’s colonial past and implies that this exploitive relationship may not be over.President Gertler’s report recognizes that participation in international experiences seems to be limited to select students – those who can afford the high costs and do not face cultural norms that may prevent them from engaging in international travel.The disparities among those interacting in these situations can give rise to deleterious consequences. Tausch et al. have shown that positive contact of disadvantaged groups with advantaged groups can decrease motivation for collective action by leading to decreased in-group identification and positive attitudes towards the advantaged group.12What are future physicians learning from these experiences? International work can widen perspective on different ways of living around the world and expose young doctors to new pathologies.In order to make sure their funding is not jeopardized and that the governments of the countries they work in will allow them to function, NGOs have to present their work in shallow framework, more or less shorn of a political or historical context.Many international volunteer programs are not designed to imbue development principles and rely on the sensationalist imagery of thankful smiles in the midst of extreme poverty to provide a cultural experience.16Volunteers typically perceive themselves as belonging to a separate category from those they encounter in foreign, usually, poorer countries. This separation allows multiple dichotomies to flourish: “us/them,” “local/global,” “developed/developing.” by the oppressed rather than for them, criticizing teachers who unknowingly perpetuate domination when working with the oppressed.13Brazilian educator Paulo Freire’s “liberation pedagogy”st century: “voluntariat.” Where the “proletariat” forms a class of workers who do not own capital and must sell their labour, the voluntariat is its counterpart. The voluntariat is driven by i) “the white man’s burden” – the responsibility that the Western world feels to improve the conditions of the developing world and ii) the need to gain an advantage in an employment market where higher education admission to professional schools is increasingly competitive, despite higher education not guaranteeing a liveable income in his/her field of education. Produced by the intersection of globalization and neoliberalism, the voluntariat constitutes a pool of young, unskilled, middle-class labourers from the global North, available internationally for short terms for free or, more typically, by paying a fee. In a global capitalist economy, their existence seems fantastical. And yet, the exploitation of the worker has been so intensified that individuals are willing to pay to gain access to the employment market. In traditional volunteering, the volunteer does not receive monetary compensation. In its current manifestation, the voluntariat furnishes her/his own reproduction of labour by paying for the experience. Simultaneously, while attempting to provide goods and services for the maintenance of the labour force in the community in which s/he serves, the voluntariat promotes the integration of these communities into the capitalist system by creating health workers who become objects for exploitation, opening new niche markets in the process. In this way, the voluntariat not only contributes to the oppression of the community in which they operate, but is simultaneously the object of oppression by liberal institutions, in this case the employment market and graduate schools.I propose a new term to account for the particular position that international volunteers occupy in relation to development and economic conditions in the 21The U of T’s President’s Report suggests that international experiences for university students could accompany the quest for recruiting graduate students from “key markets” abroad.In order to navigate the forces that shape the lives of patients, internationally-based education for medical students must demonstrate awareness of these factors to form physicians who are also active global citizens. The aim of this paper is not to discourage all international experiences but to understand that they must be accompanied by a pedagogy based on social justice. Indeed, my own undergraduate experience benefitted greatly from the international research work I was able to pursue by allowing me to apply theories from the classroom, work more intimately with my professors, and interact with professionals from a variety of backgrounds. Volunteers must be encouraged and trained to identify the ways they, themselves, might be complicit in the suffering of the populations with which they are working rather than seeing themselves as saviours. Instead of relying on sensational imagery, medical education must prioritize reflection on the systematic causes of health inequities using a social justice lens.


A combination of conventional genetic analyses and the latest mapping method, MutMapPlus, revealed that both of these lines harbour novel independent mutant alleles of IIbstarch branching enzyme  (BEIIb) gene. In age1, amino acid substitution of Met‐723 to Lys completely abolished BEIIb enzyme activity without significant reduction in its protein level. A transposon insertion in an intron of BEIIb gene reduced BEIIb protein level and activity in age2. Production of a series of the mutant lines by combining age alleles and indica‐type IIastarch synthase  allele established stepwise alteration of the physicochemical properties of starch including apparent amylose content, thermal property, digestibility by α‐amylase and branched structures of amylopectin. Consistent with the alteration of starch properties, the results of a sensory evaluation test demonstrated that warm cooked rice of the mutants showed a variety of textures without marked reduction in overall palatability. These results suggest that a series of the mutant lines are capable of manipulation of cooked rice textures.Physicochemical properties of storage starch largely determine rice grain quality and food characteristics. Therefore, modification of starch property is effective to fine‐tune cooked rice textures. To obtain new resources with modified starch property as breeding materials, we screened a mutant population of a  Starch constitutes most of the weight of cereal grains and is the most important carbohydrate glucose polymer for humans as a dietary source of energy. Starch consists of linear or slightly branched amylose and highly branched amylopectin. The amylose content and chain length distribution (CLD) of amylopectin branches are the predominant determinant of the physicochemical properties of starch and the cooking properties of grains.et al., et al., Starch is synthesized by the orchestrated functional interactions of four classes of enzymes, ADP‐glucose pyrophosphorylase, starch synthase (SS), starch branching enzyme (BE) and starch debranching enzyme (DBE) , which produce active and inactive SSIIa, respectively  are enriched, and short chains (DP < 11) are depleted in indica varieties compared to japonica varieties  6 and 7), short (DP 6–10) and long (DP > 33) amylopectin chains, respectively , and BEIIa has a more extensive transfer preference (DP 6–15), while BEI transfers intermediate chains (DP 26–39) in addition to short chains (DP 6–12) , exhibit severe effects on grain appearance and starch properties  compared to the original cultivar Kinmaze  solved the bottleneck by greatly reducing the number of times of the phenotyping process and accelerated identification of the causal mutation governing the agronomic trait. MutMap is one of the procedures and is based on a crossing of the mutant to the original parental line used for mutagenesis, followed by resequencing of the genome DNA of a bulk of F2 segregants with mutant phenotypes obtained by self‐pollination of F1  by a combination of the conventional mapping and MutMapPlus procedures. A point mutation of BEIIb gene in age1 allele caused BEIIb‐deficient phenotype, which is permissive for cooked rice even though there was a complete disappearance of its enzyme activity. In addition, a transposon insertion into the same gene yielded another age allele, named age2, resulting in milder modification of the activity. Both age alleles exhibited moderate effects on cooked rice textures as well as the physicochemical properties of their starch. Our work would not only provide breeding materials for fine‐tuning cooked rice textures and confirm the general versatility of MutMapPlus but also shed novel insights into the regulation of BEIIb functions in starch synthesis.In this study, to provide novel breeding resources with linked DNA markers for fine‐tuning cooked rice textures, we screened the mutant lines exhibiting altered grain starch properties and identified two novel mutant alleles of m urea solution, in which Nipponbare starch is gelatinized. Among the 3300 mutant lines examined, we found two recessive mutant lines whose starches were hardly gelatinized in the urea solution , its enzyme activity was completely abolished  and its enzyme activity were lower than that of Nipponbare. An additional smaller molecular weight protein was detected in age2 , immunoblot (IB) and activity staining analyses. and Aoki   array analysis indicated that the mutated locus in both age1 and age2 was mapped within the region between 11 and 21 Mb on chromosome 2  was also peaked at the same location (the expected Δ(SNP index) is 0.67)  to Lys (M723K)  Figure . Within age1 phenotype, constructs consisting of a genomic fragment of WT or M723K mutant‐type BEIIb gene including the 2.4 kb upstream region from the translation start site were introduced into age1 . Furthermore, whole‐genome resequencing data confirmed that no SNPs introducing amino acid substitutions were found within the region from 10 Mb to the end of chromosome 2 in age2  were designed with an additional mismatch introduced in the third nucleotide from the 3′ end, while a reverse primer is common  harbouring SSIIaindica allele  , 1.5%, agew Figure .Tp in age1 and age2 lines was 10 °C and 6 °C higher than that of Nipponbare, respectively , suggesting that a combination of SSIIaindica and age2 alleles provided an additive or even transcending effect on the elevation of Tp.The thermal properties of purified starch were analysed by differential scanning calorimetry. age1 and age2 showed similar CLD patterns, in which the proportion of short chains (DP 6–17) was significantly reduced, and that of intermediate (DP 19–33) and long chains (DP > 41) was increased. Consistent with BEIIb activity  and intermediate chains (DP 13–25) were decreased and increased, respectively, as reported previously  Figure . Amylopeage1, age2 and SSIIa‐NIL were digested slower than those of Nipponbare   showed a significant reduction of stickiness in SSIIa/r Figure . Howeverr Figure . These page mutant lines with moderate changes of grain starch properties and identified them as novel mutants of BEIIb by a combination of conventional mapping and MutMapPlus. Before application of MutMapPlus, we tried to identify the causal mutation of age1 by MutMap. However, the Nipponbare mutant population, from which we found age mutants, was generated two decades ago, and the original parental plant subjected to the mutagenesis was not available. Furthermore, as the Nipponbare individuals, which were used for the crossing to establish the F2 progenies were not preserved, we sequenced different individuals to build the reference sequence for the mapping analysis. Under these situations, a trial of MutMap analysis gave multiple candidate regions, where SNP indexes approached 1, throughout the genome due to background SNPs among Nipponbare individuals used for mutagenesis, crossing and reference sequencing, and failed to pick the age1 causal mutation  was almost the same, whereas those to short chains (DP 6–12) were different. A similar trend was observed in the effect of SSIIaindica allele. The effect of age1 and SSIIaindica on starch properties was severely attenuated in SSIIaindica/age1 compared to that of age2 and SSIIaindica in SSIIaindica/age2  analysis of soluble protein (SP) and proteins loosely (LBP) or tightly (TBP) bound to starch granule in developing seeds. Protein fractions were prepared as reported by Asai et al.  plots of rice chromosomes generated by MutMapPlus analysis of age1. SNP index and Δ(SNP index) plots were generated by MutMapPlus analysis of bulked F2 mutant progeny obtained by a crossing between age1 and Nipponbare. (a) SNP index of the mutant (M) bulk. (b) SNP index of the wild‐type (WT) bulk. (c) Δ(SNP index) calculated by subtraction of the index of WT bulk from that of M bulk. Red lines indicate average value of SNP index or Δ(SNP index) obtained by the sliding window analysis of 4 Mb intervals with 50 kb increment. Candidate region confined by the Fisher's P value of <0.05 is indicated with a red box.Click here for additional data file.Figure S3 Investigation of urea concentration for gelatinization of starch of vector control lines of Nipponbare and age1. Halved rice grains were incubated with various concentrations of urea solution overnight and gelatinization of starch was evaluated by iodine staining. Transgenic plants were grown in a greenhouse. Temperatures after flowering were set at 27 °C/22 °C during 15 h light/9 h dark periods, respectively.Click here for additional data file.Figure S4 Investigation of urea concentration for gelatinization of endosperm starch of F1 seed in allelism test. Endosperm powder of F1 seeds (2.5 mg) was incubated with various concentrations of urea solution (150 μL) and gelatinization of starch was evaluated by the volumes of sediments. Grains used for this experiment were ripened in a warm and closed room in summer  The distribution of the length of amylopectin side chains was examined by HPAEC‐PAD, and then the difference in the profiles between EM10 and Kinmaze or age1 and Nipponbare was calculated by subtracting the ratio of a chain of given length of the sample with that of the mean value of Kinmaze or Nipponbare, respectively. Values are the means of three biological replicates. (b) Grain appearance of age1 and EM10. Images by reflected light (left panel) and transmitted light (right panel) are shown. Grains used in this experiment were ripened in a plant incubator.Click here for additional data file.Figure S8 Comparison of chain length distribution between single and double allele lines. The difference in the chain length profiles of amylopectin between the double allele lines and their parental single allele lines was shown. The values were calculated by subtracting the ratio of a chain of given length of the double allele line with that of the single allele line shown in Figure Click here for additional data file.Table S1 SNPs identified in age2 by BWA mapping and Coval extraction between 10 Mb and 3′ end of chromosome 2.Click here for additional data file.Table S2 Eating quality of the cooked rice of the mutant lines as measured by a sensory evaluation test.Click here for additional data file.Table S3 Primers used in this study.Click here for additional data file.Table S4 Peptide sequences of antigens for raising polyclonal antibodies.Click here for additional data file.Appendix S1 Experimental procedures.Click here for additional data file.


Next-generation sequencing (NGS) approaches are commonly used to identify key regulatory networks that drive transcriptional programs. Although these technologies are frequently used in biological studies, NGS data analysis remains a challenging, time-consuming, and often irreproducible process. Therefore, there is a need for a comprehensive and flexible workflow platform that can accelerate data processing and analysis so more time can be spent on functional studies.We have developed an integrative, stand-alone workflow platform, named CIPHER, for the systematic analysis of several commonly used NGS datasets including ChIP-seq, RNA-seq, MNase-seq, DNase-seq, GRO-seq, and ATAC-seq data. CIPHER implements various open source software packages, in-house scripts, and Docker containers to analyze and process single-ended and pair-ended datasets. CIPHER’s pipelines conduct extensive quality and contamination control checks, as well as comprehensive downstream analysis. A typical CIPHER workflow includes: (1) raw sequence evaluation, (2) read trimming and adapter removal, (3) read mapping and quality filtering, (4) visualization track generation, and (5) extensive quality control assessment. Furthermore, CIPHER conducts downstream analysis such as: narrow and broad peak calling, peak annotation, and motif identification for ChIP-seq, differential gene expression analysis for RNA-seq, nucleosome positioning for MNase-seq, DNase hypersensitive site mapping, site annotation and motif identification for DNase-seq, analysis of nascent transcription from Global-Run On (GRO-seq) data, and characterization of chromatin accessibility from ATAC-seq datasets. In addition, CIPHER contains an “analysis” mode that completes complex bioinformatics tasks such as enhancer discovery and provides functions to integrate various datasets together.Using public and simulated data, we demonstrate that CIPHER is an efficient and comprehensive workflow platform that can analyze several NGS datasets commonly used in genome biology studies. Additionally, CIPHER’s integrative “analysis” mode allows researchers to elicit important biological information from the combined dataset analysis.The online version of this article (doi:10.1186/s12859-017-1770-1) contains supplementary material, which is available to authorized users. Understanding the precise regulation of transcriptional programs in human health and disease requires the accurate identification and characterization of genomic regulatory networks. Next-generation sequencing (NGS) technologies are powerful, and widely applied tools to map the in vivo genome-wide location of transcription factors (TFs), histone modifications, nascent transcription, nucleosome positioning, and chromatin accessibility features that make up these regulatory networks. Although NGS technologies can be used in diverse ways to investigate numerous aspects of genome biology, reaching sound biological conclusions requires the exhaustive analysis of these datasets to recognize and account for many potential biases  includinProperly implemented pipelines are essential to genome and chromatin biology studies, but often fail to implement the features required to overcome five major challenges: (1) quickly processing large batches of data with minimal user input, (2) remaining highly customizable for different experimental requirements, (3) conducting comprehensive quality control assessments of sequencing datasets to identify potential areas of bias, (4) reducing the issues associated with building, maintaining, and installing multiple pipelines and bioinformatics software, and (5) increasing reproducibility among researchers.Despite the many computational approaches that already exist to analyze NGS datasets, there are no currently available tools designed to tackle all five challenges simultaneously. ChiLin, HiChIP, bcbio-nextgen, and MAP-RSeq offer powerful command-line data analysis pipelines, but are limited to chromatin immunoprecipitation (ChIP) coupled with sequencing (ChIP-seq) and whole transcriptome sequencing (RNA-seq) studies. Galaxy, an open, web-based platform for data analysis , offers To overcome these previous obstacles, we devised CIPHER, an integrated workflow platform that automates the processing and analysis of several commonly used NGS datasets including ChIP-seq, RNA-seq, Global Run On sequencing (GRO-seq) , microcoCIPHER manages to overcome the five previously mentioned obstacles by: (1) parallelizing all the steps in a typical pipeline therefore taking full advantage of a desktop’s or cluster’s available RAM and CPUs, (2) providing command line flags to alter the majority of parameters at each step, (3) incorporating extensive quality control software and providing detailed QC reports specific to each pipeline, (4) combining pipelines for several of the most commonly used NGS techniques into a single, standalone tool, and (5) using a lightweight Docker containers to package all the required software dependencies to run CIPHER into a standardized environment.In this report, we demonstrate that CIPHER is a fast, reproducible, and flexible tool that accurately processes and integrates NGS datasets by recreating the results of two published studies, and comparing CIPHER’s speed and ease of use to two other ChIP-seq and RNA-seq pipelines. We further validate CIPHER’s built-in random-forest based enhancer prediction model by identifying potentially functional enhancers in various human cell lines.Many previously described NGS workflows are developed using scripting languages such as Python or Perl as a ‘glue’ to parse datasets, and automate the series of commands that make up a processing pipeline. In contrast to these approaches, CIPHER was designed using Nextflow, a specialized, and new workflow language that is built around the Unix pipe concept . By usinCIPHER can be run with default settings by specifying the “--mode”, “--config”, “--readLen”, “--lib”, “--fasta”, and “--gtf” flags. The “--mode” flag indicates the type of NGS pipeline you wish to run from the currently available workflows , while the “--config”, “--readLen” and “--lib” flags provide information regarding file locations, read length and type of sequencing (e.g. single-ended or pair-ended), respectively, so that the pipeline runs the appropriate processes. Finally, the “--fasta” and “--gtf” flags indicate reference annotation information that is required for mapping and downstream exploration such as differential gene expression (DGE) analysis. In the case that the user is not familiar with reference FASTA and GTF files or where to acquire them, providing the “--download_data” flag will automatically download the appropriate Ensembl/Gencode reference files for a specified organism, if it exists (e.g. “--download_data hg19” will download Gencode fasta and gtf files for the hg19 human genome).cipher.readthedocs.io). By default, CIPHER will output processed files into a “./report” directory (which can be changed by specifying the “--outdir” flag). The output includes various files and is largely dependent on the pipeline mode specified, but in general provides quality control reports in pdf or html format, gzipped fastq files of raw sequences after trimming and adapter removal, sorted BAM files of mapped and unmapped alignments along with various files that contain detailed statistics regarding the number of unique, multimapped, and low-quality reads, as well as normalized track files in bigWig format for visualization. Further pipeline dependent downstream analysis such as narrow and broad peak calls for ChIP-seq, differential gene expression lists for RNA-seq, nucleosome positions and DNase hypersensitive sites (DHS) for MNase-seq and DNase-seq respectively, unannotated transcription units for GRO-seq, and chromatin accessible sites for ATAC-seq are also output. Notably, unlike other published pipelines  and splice-unaware mappers , and supports both single-ended and pair-ended sequencing datasets. Thus, to appeal a broader audience, CIPHER allows the user to choose from five different aligners  to fit any experimental condition and dataset.By default, CIPHER will map reads to a reference genome using BBMap, a fast short-read aligner for both DNA and RNA-seq datasets, that is capable of mapping very large genomes containing millions of scaffolds with very high-sensitivity and error tolerance. Because read alignment often requires a large amount of flexibility for specific datasets (e.g. removing the first 5 nucleotides from the 5′ end of a read), CIPHER enables the user to set all of an aligner’s parameters at the top level.To ensure properly sound biological conclusions, it is crucial that the user accurately and thoroughly evaluates the quality of their sequencing datasets. To this end, CIPHER incorporates a number of quality control tools to identify potential biases, contaminations, and errors in NGS datasets.N, multimappers), estimated fragment length by calculating cross-coverage score, and the percentage of reads that overlap called peaks (known as FRIP) when possible.All pipelines integrate FastQC , an openFingerprint plots that predict enrichment of ChIP-seq datasets are generated to judge how well a ChIP experiment worked using deeptool’s  “plotFinMultiQC  is used For our purposes, peak calling refers to the identification of TF and histone binding domains, nucleosome positions, DHS, and chromatin accessible sites Fig. . There aBy default, CIPHER will estimate the fragment length for each sample using the SPP R package , and bypcipher.readthedocs.io.Nucleosome positions are determined using the DANPOS2 software suite  Fig. 2b2b. DANPODHS characterize chromatin accessible regions in the genome where TFs can bind Fig. . While sA similar approach to the identification of chromatin accessible sites from ATAC-seq data is used. CIPHER takes advantage of MACS2 flexible algorithm to call peaks in a similar manner to DHS. However, “--extsize” of 73 and “--shift” of −37 is used since the DNA wrapped around a nucleosome is about 147-bp in length.To visualize binding site, gene expression, chromatin accessibility, and gene annotation information, various visualization tracks (e.g. bedGraphs and bigWigs) are produced. Deeptools  is used DGE analysis generally refers to the up- or down-regulation of transcripts produced by a cell in response to or because of an aggravation (e.g. knock-out of a gene/genomic domain or knock-down of a certain factor). CIPHER’s DGE pipeline is straightforward and includes basic mapping, quantification, and DGE analysis steps. As previously described, mapping is completed by the user’s choice of aligner, while quantification is accomplished by the featureCounts module  of the SrandomForest package) was constructed using the classical concept of binary classification trees, with each feature being the average coverage signal of a marker within a set distance along a genomic element. CIPHER takes RPM-normalized bedgraph files of DNase-seq and ChIP-seq as input to build the RFT model.Enhancers are short DNA sequences that act as TF binding hubs, and function in a spatio-independent manner to fine-tune promoter activity at distances ranging hundreds of bases to megabases. To predict enhancers, we developed and applied a random-forest tree (RFT) machine-learning model that combines chromatin accessibility (DNase-seq) and chromatin signature datasets obtained from ChIP-seq  Fig. . The RFTcaret package in R. Notably, CIPHER’s enhancer prediction-model accuracy achieved slightly above 93%, which means that the majority of ‘true’ enhancers were identified during our ‘testing’ stage, indicating reliably efficient enhancer identification functionality.RFT model construction underwent two stages: training and testing. In the ‘training’ stage, a forest is constructed using two classes of genomic elements (one class containing a previously determined set of enhancer elements from the Encyclopedia of DNA Elements (ENCODE) project  and a seThe provided reference genome is split into 200-bp bins, and the enhancer prediction model categorizes each window into “enhancer” or “non-enhancer” bins Fig. . Bins thCIPHER’s “analysis” mode was created to take advantage of CIPHER’s broad NGS data processing workflows. In “analysis” mode, CIPHER can run several functions that integrate various input files and combines them to answer a more specific or typically more complex biological question. Currently, CIPHER contains two main analysis functions. We have already touched on CIPHER’s enhancer prediction functionality, but “analysis” mode also contains a “geneExpressionNearPeaks” function that calculates fragments per kilobase per million mapped reads (FPKM) and transcripts per kilobase per million mapped reads (TPM) normalized expression values of genes near an input list (e.g. list of peaks or enhancers). This is accomplished by taking the Stringtie  output fTo validate CIPHER’s potential in NGS data analysis, we used data from the Gene Expression Omnibus repository (GEO) to re-create two previously published studies: a ChIP-seq study from McNamara et al.  and a GRTo determine if CIPHER’s workflows are appropriate for typical NGS studies, we downloaded the raw data from two studies , 43 and The first study by McNamara et al. consisted of several ChIP-seq datasets, and provided evidence that KAP1, also known as TRIM28, acts as a scaffold to recruit the 7SK snRNP complex to gene promoters to facilitate productive transcription elongation in response to stimulation. Their bioinformatics analysis showed that 70% of all genes in the human genome containing a form of RNA polymerase II (Pol II) that is paused at promoter-proximal regions (defined as −250/+1000 from the known TSS), also contained the KAP1-7SK snRNP complex as revealed by co-occupancy of KAP1 and three subunits of the 7SK snRNP complex . The same authors also published a thorough methods paper that provided a detailed experimental description and analysis of ChIP-seq datasets , in whicTo determine whether we could reproduce McNamara et al.’s results using CIPHER, we processed six of their ChIP-seq datasets . Given that CIPHER does not include a Bowtie aligner, we used the more recent Bowtie2 aligner  but all other settings were left as default. CIPHER processed all ChIP-seq datasets (~30 million reads per dataset) in 7 h and 23 min. We then identified ~26,000 promoter-proximal regions ) and conducted co-occupancy analysis of called peaks. Our analysis revealed 14,397 KAP1-7SK snRNP target genes as opposed to the original manuscript’s 14,203  on the activation of the Pol II paused form in a process called ‘Pol II pause release’ . The stuWe decided to reproduce one important section of this previous study by processing six GRO-seq datasets: 2 non-target (NT) replicates, and 4 Brd4 knockdown (KD) replicates. As previously done, we left all settings at default except for altering the “--aligner” flag to use Bowtie2. CIPHER processed all six GRO-seq datasets (~50 million reads per dataset) in approximately 10 h. DGE analysis of NT versus BRD4 KD resulted in 2528 differentially expressed genes at an FDR < 0.001 .We first downloaded and installed both HiChIP and MAP-RSeq standalone versions. While both pipelines provided virtual machines (VM) that already came packaged with all the necessary software and dependencies, we found that these VMs were clunky, slow and only really meant to demonstrate the pipeline for testing purposes. While both pipelines provided detailed instructions on how to manually install all the software and dependencies that were required, users who are unfamiliar with bash or Unix commands would have significant trouble installing them. Furthermore, both pipelines provided a version of their pipelines that could only be run exclusively on a SGE cluster, greatly limiting their use.cipher.readthedocs.io). Importantly, CIPHER can be easily run on several cluster services including SGE, SLURM, LSF, PBS/Torque, NQSII, HTCondor, DRMAA, DNAnexus, Ignite, and Kubernetes without altering the original script, thus vastly increasing the flexibility and usage of our workflow platform.In comparison, CIPHER only requires the manual installation of Nextflow and Docker, greatly reducing the number of obstacles a new user may encounter during their setup. By default, CIPHER will automatically fetch Docker containers that hold all the required software and dependencies that are needed to run the pipeline, without the slow-down that comes with a typical VM. In cases where the user does not or cannot use/install Docker, we have provided detailed instructions on how to download all the software required to run CIPHER using the Anaconda package manager in our documentation  to fastq(s), control ID, and marker ID for each sample vastly reducing the time and complexity of the initial startup.Finally, we ran each of the pipelines on single and multiple in-house datasets to test their speed. For ChIP-seq we first ran a single sample and its associated input (~30 million reads each) and then conducted another run that included five samples and their associated input (~30 million reads each). We found that for the single sample dataset, HiChIP took approximately 2X longer than CIPHER . However, the difference in run time became vastly more noticeable when the pipelines were run on multiple datasets (6 samples), in which HiChIP took approximately 4X longer to finish than CIPHER .Fairly similar results were obtained with the MAP-RSeq pipeline, where processing a single RNA-seq sample  took approximately 1.5X as long using MAP-RSeq than CIPHER (~8 h versus ~6 h), while processing 18 samples  took approximately 15X as long using the MAP-RSeq pipeline . These speed differences are likely the result of CIPHER’s innate ability to process a large number of datasets in parallel, while both HiChIP and MAP-RSeq have to process datasets serially (e.g. one at a time). Together this demonstrates the ease-of-use and speed of CIPHER compared with other pipelines.We downloaded ChIP-seq (H3K4me1) from the ENCODE project in human colonic cancer cells (HCT116) to obtain real quality distributions. We then down-sampled the original fastq files into three different datasets containing 1 M, 5 M and 10 M reads using BBMap’s “reformat.sh” script. Using a dataset of 25 Ilumina TruSeq adapters we randomly added adapters to the reads using the “addadapters.sh” script from the BBMap suite with “qout = 33” and “right” flags set, to ensure that adapters will be 3′-type adapters. This ensures that adapters will be added at a random location from 0 to 149, and possibly run off the 3′ end of the read, but the read length always stays at 150. If the adapter finishes before the end of the read, random bases are used to fill in the rest. Using this approach, about 50% of all reads get adapters. Once the adapter is added, each of the adapter nucleotides is possibly changed into a new nucleotide, with a probability from the read’s quality score for that nucleotide to simulate sequencing error.Speed tests were conducted using the “time” Unix command for 1 M, 5 M and 10 M reads and averaging the completion times over 3 runs. Accuracy was estimated by replacing each read’s original name with a synthetic name indicating the read’s original length and length after trimming. For example, “@0_150_15” means that the read was originally 150 bp long and 15 bp after trimming because an adapter was added at position 15 (0-based). This allows BBMap’s “addadapters.sh” script (with the “grade” flag set) to quantify both the number of bases correctly and incorrectly removed, as well as the percentage of true-positive adapter sequences remaining and non-adapters removed.Performance tests showed that BBDuk outperforms the speed category by a large margin, with Trimmomatic not far behind, and Cutadapt being extremely slow Fig.  while acTo compare mappers against each other, we generated a dataset using Teaser , which iResults showed that BBMap and BWA-MEM correctly mapped more simulated reads  than Bowtie2 (56.545%) Fig. . All thrBBMap, BWA-MEM and Bowtie2 appear to perform on par in terms of accuracy, performance tests for memory usage and speed indicated that BBMap was slower and used larger amounts of RAM than either of the other two programs Fig. . HoweverEnhancers are short DNA-sequences that can regulate basal gene transcription over distances ranging from a few kilobases to megabases. Enhancers are characterized by the presence of various genomic features including: (1) an accessible chromatin landscape, (2) distinct chromatin signatures, (3) TF binding, and (4) bi-directional transcriptional activity as revealed by the presence of enhancer-derived non-coding RNAs (eRNAs) based on GRO-seq data , 48.Previous studies have shown that it is possible to accurately predict enhancer elements using machine-learning models by combining these various regulatory features –53. HoweTo validate CIPHER’s enhancer prediction functionality, we identified enhancer elements in two cell lines (HCT116 and HeLa). Using ChIP-seq and DNase-seq datasets from the ENCODE project, we generated average coverage profiles for H3K4me1, H3K27ac, H3K4me3, and DNase-seq. These coverages profiles were fed into CIPHER’s “analysis” mode.Enhancer activity can be inferred from the presence or absence of histone markers. Enhancers are typically marked with high levels of H3K4me1, in contrast to promoters that are marked with higher levels of H3K4me3. More recently, H3K27ac and high eRNA content have been found to distinguish functionally active from primed or latent enhancers , 54 Fig. Thus, pChromatin state profiles were evaluated by constructing heatmaps for active and primed enhancers ranked by decreasing levels of chromatin accessibility Fig.  and c. TFurthermore, while both active and primed enhancers contained comparable levels of H3K4me1, active enhancers contained larger H3K27ac levels (average coverage: 0.72 versus 0.099), and stronger eRNA sense (6 versus 1) and anti-sense (5 versus 1) read coverage compared with primed enhancers, consistent with increased enhancer activity Fig. . MoreoveUsing CIPHER in combination with our previous stringent cut-off, we also predicted enhancers in other cell lines: 38,045 active and 10,600 primed enhancers in HeLa Fig. , and 38,CIPHER is a robust, and comprehensive NGS data analysis workflow suite with numerous functions and quality control metrics. It integrates pipelines for several of the most commonly generated datasets used in current genome biology studies and features an “analysis” mode that conducts complex bioinformatics challenges such as enhancer identification and integrative dataset analysis functions. CIPHER is extremely easy to run and makes use of Docker containers so there are no dependency issues. Entire datasets can be reproduced among researchers starting from raw data in a single command. Here we re-created the results of two published studies, briefly compared CIPHER’s ease of use and speed to two other automated pipelines and provided performance metrics for several adapter decontamination and mapping tools. We further validate CIPHER’s enhancer-prediction model in various human cell lines.Although CIPHER has combined several comprehensive and thorough pipelines for commonly used NGS approaches, there are still quite a few challenges that remain to be addressed. CIPHER’s current RNA-seq pipeline is largely optimized for typical two-type experimental designs (e.g. WT versus KO) and must be rewritten to ensure multi-design experiment DEG analysis and time-series analysis. We also plan to include pipelines for genome-wide association studies (GWAS) and de novo transcriptome assembly in the near future. Additionally, CIPHER currently only runs entire workflows, but we are aware that individuals may prefer to use only a subset of tools to complete certain tasks, thus it will be beneficial to allow this type of modular tool selection in the near future. As new or improved methods/software become available, the modular design of CIPHER will enable their smooth integration into our existing pipelines.


European Journal of Psychotraumatology, the European Society for Traumatic Stress Studies (ESTSS) and the team at Taylor and Francis.Happy New Year to our readers around the world – some of them it just started, for others it will soon begin – from the here), a popular Swedish author, historian and expert in economic globalization, our progress has been unprecedented. On almost any index things are markedly better now than they have ever been. We are safer, healthier, there is more prosperity and less inequality.According to Johan Norberg . Relatively few asylum seekers find their way to Western Europe, by the way. Countries like Turkey, Pakistan, Lebanon, and Iran host most of them   to see what else we published in 2016 and before. With the e-alerts function one can easily receive each newly published paper when it comes out in your mailbox.Please check out our new journal website  as ESTSS owns the journal. By the way, all around the world are welcome to join the society.The fees will still remain relatively low, especially for members of the Some things did not change, I am very pleased to continue with almost the same team of associate editors . We have an active editorial board and are very grateful to all board members and external reviewers for their reviewing of papers for the journal.The neurobiology of PTSD (Ruth Lanius)Bayesian statistics (Rens van de Schoot)PTSD Symptomics: Analyzing individual PTSD symptoms and their network configurations in traumatized populations (Cherie Armour & Eiko Fried)ISTSS Highlights 2016 (Paul Frewen & Christian Schmahl),Treatment for refugees (Christine Knaevelsrud) – call soon to come out.Child Maltreatment Across the Lifespan- highlights of the ESTSS meetingTraumatic Grief (Paul Boelen & Geert Smid)e-Health for trauma related symptomsWe will have several guest editors throughout the year for special issues. Ongoing & Planned special issues for 2017 include:European Society for Traumatic Stress Studies meeting in Odense, Denmark . Save the date! So we watch the year to come with great interest. I look forward to reading and sharing with you the exciting things that psychotrauma professionals are doing in 2017.Finally, we are planning our next Editorial Board Meeting in Denmark at the With best wishes for a happy New Year 2017.


The latest achievements in 2-dimensional (2D) material research have shown the perspective use of sandwich structures in nanodevices. We demonstrate the following generation of bilayer materials for electronics and optoelectronics. The atomic structures, the stability and electronic properties of Moiré graphene (G)/h-BN bilayers with folded nanoholes have been investigated theoretically by ab-initio DFT method. These perforated bilayers with folded hole edges may present electronic properties different from the properties of both graphene and monolayer nanomesh structures. The closing of the edges is realized by C-B(N) bonds that form after folding the borders of the holes. Stable ≪round≫ and ≪triangle≫ holes organization are studied and compared with similar hole forms in single layer graphene. The electronic band structures of the considered G/BN nanomeshes reveal semiconducting or metallic characteristics depending on the sizes and edge terminations of the created holes. This investigation of the new types of G/BN nanostructures with folded edges might provide a directional guide for the future of this emerging area. Two-dimensional structures are attractive materials for application use, because they have many unique properties. For example, graphene – a plane of hexagonally arranged carbons - has such properties as high strength and thermal conductivity, as well as a high mobility of charge carriers. However, it has a significant disadvantage, i.e. the lack of energy gap, which makes its use in electronics extremely limited. The band structure varies depending on the number of layers, their mutual orientation23et al.et al.It is well known that when bilayer graphene or graphite is cut by electron beam, their edges are connected5h-BN) is another monolayer material which is a dielectric analogue of graphene. It has some advantageous properties: higher thermal and chemical stabilities than graphene, mechanical robustness, resistance to oxidation and good optical propertiesh-BN nanomeshes were grown on crystal surfaces using different techniques14Hexagonal boron nitride  centers are considered here. Naturally, the asymmetric shape of the holes should result in a severe disruption of symmetry and corresponding changes in the electronic properties of graphene as in the case of single-layered nanomeshes67C146 consists of 280 atoms 80 atoms  and has 80 atoms . Holes c80 atoms  similar 0 atoms 5: three wNq = 6(2q − 1) + Nq−1 atoms from each layer . The G/BN Moiré meshes with such ≪round≫ holes will be denoted as Mθ{AAoq}.A ≪round≫ hole is formed by recessing the hexagonal pieces from the AA-area center of the corresponding Moiré structure ,c. The io1} structure with the smallest holes  with the same ≪round≫ hole structures and the same supercells  correspond to the indexes of two unit supercell vectors in the unit cell vectors of graphene. According to the rules in the paper [26] our GNMs are metallic with the view very similar to graphene band structure. However the Fermi level is a little lower than in graphene, because of electron localization near the edges. Hydrogen atoms on the GNM hole edges do not break the symmetry of graphene nanomesh sublattices.It is interesting to compare these electronic spectra with the band structures of corresponding monolayer graphene nanomeshes -atoms are equal to δC,B(N) ≈ 1.96, 1.18 (1.21) Å and δC,B(N) ≈ 2.32, 0.99 (1.03) Å, respectively, in the regions outside of the inner torus parts (the Z-axis is perpendicular to the layers). Thus, the structure corrugation of the graphene part of the considered G/BN meshes can explain the Dirac point splitting in contrast to the electron bond spectra of monolayer GNMs  + Nq−1 in AB area can be created in Moiré structures with smaller angles 0° ≤θ < 6°.The bigger holes with radius F that shows strong localization of electrons on all holes  retain the metallic properties.We also consider three holes in the supercell of the mesh by the adding of two holes in AB-stacking areas of the nanomesh M6 with AA-≪round≫ holes . After fll holes . The foradius R1  and the  0.16 eV . A minibtual gap  which is, AB∇Δ1} . Corresp, AB∇Δ1} . The shio2, AB∇Δ1} has a 0.32 eV second band gap in the Γ point above the conductivity zone, whereas we obtain the smaller value of gap (0.25 eV) over EF in the corresponding GNMo2,∇Δ1.It is remarkable that if one compares 27C55 consists of 110 atoms. In such a small unit cell, only one ≪symmetric≫ hole from three possible ones can be formed in one of the AA, ABB or ABN centers. Two types of AB areas are situated between AA-regions: ABB and ABN with B- atom and N- atom in their centers as it has been shown earlier.Further, we continue to study the formation of closed holes in the Moiré pattern θ = 10.9° whose supercell (BN)o1} similar to the strained mesh M6{AAo1} and opening a gap in its band structure  in terms of [26] has metallic property  holes.In this work only the simplest meshes Mθ{AA(AB)2 smooth folded hole with radius Finally, we have modeled a new type of ≪round≫ holes in the AB-regions of BN/G bilayers which can be organized also by knocking the atoms with an electron focusing beam with nm point on the AB- area of a bilayer (see for example [8]). A ≪round≫ hole can be formed by knocking 8 atoms . The clo∇1} with six B atoms on each hole edges has metallic character. Electron doping of the BN part opens the gap on the Dirac point of carbon branches situated at 0.3 eV up Fermi level – Δ1}  unit cell with triangle holes is metallic with a narrow metallic band near the Fermi level .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Dementia & Neuropsychologia has recently been indexed in the Literatureon the Health Sciences in Latin America and the Caribbean (LILACS) database, makinginformation contained in the papers we publish more visible to the regional scientificcommunity. Our goal is to be indexed in other international databases over theforthcoming months. Together with the free full text availability provided by our sitedemneuropsychol.com.br, we are confident we are going inthe right direction toward making our journal a better vehicle for publication ofmanuscripts from Latin America and elsewhere.Dementia & Neuropsychologia carries on its mission of publishingresearch on aspects of dementia and neuropsychology that are pertinent to developingcountries, particularly issues relevant to populations with low or heterogeneouseducational background. In this issue, we publish two papers reporting the influence ofeducational level on performance in two well-known tests: the Mini-Mental StateExamination and the Mini-Cog.Dementia & Neuropsychologia has published abstracts ofseveral symposia held in Brazil, but we aim to publish abstracts of events from othercountries in Latin American and abroad. Carrying these presentations is a significantpart of our mission, because abstracts of meeting presentations are hard to find whennot published as special supplements or part of a journal.Since its launch, In this issue, we are publishing the abstracts of the IPA 2009 International Meeting heldin Rio de Janeiro on 04-07 May, and sponsored by the International PsychogeriatricAssociation in cooperation with the Brazilian Association of Geriatric Neuropsychiatry(ABNPG). Hosting this International Meeting in Brazil is a significant achievement forthe ABNPG in its effort toward raising the quality of scientific research in the fieldand providing Brazilian and Latin American researchers with an opportunity toparticipate as speakers in international congresses and symposia.Dementia & Neuropsychologia.Publishing the abstracts of the IPA 2009 International Meeting represents a significantmilestone, enabling platform and poster presentations to reach a broader scientificcommunity. We regard abstracts as the forerunners of potential full manuscripts forfuture publication in Ricardo NitriniEditor-in-Chief


BioMed Research International has retracted the article titled “Exogenous IL-4-Expressing Bone Marrow Mesenchymal Stem Cells for the Treatment of Autoimmune Sensorineural Hearing Loss in a Guinea Pig Model” . Figures


Here we show that leaves of gwd, sex4, bam4, bam1/bam3 and amy3/isa3/lda starch breakdown mutants accumulate higher levels of starch than wild type (WT) leaves when cultured under continuous light (CL) conditions. We also show that leaves of CL grown dpe1 plants impaired in the plastidic disproportionating enzyme accumulate higher levels of maltotriose than WT leaves, the overall data providing evidence for the occurrence of extensive starch degradation in illuminated leaves. Moreover, we show that leaves of CL grown mex1/pglct plants impaired in the chloroplastic maltose and glucose transporters display a severe dwarf phenotype and accumulate high levels of maltose, strongly indicating that the MEX1 and pGlcT transporters are involved in the export of starch breakdown products to the cytosol to support growth during illumination. To investigate whether starch breakdown products can be recycled back to starch during illumination through a mechanism involving ADP-glucose pyrophosphorylase (AGP) we conducted kinetic analyses of the stable isotope carbon composition (δ13C) in starch of leaves of 13CO2 pulsed-chased WT and AGP lacking aps1 plants. Notably, the rate of increase of δ13C in starch of aps1 leaves during the pulse was exceedingly higher than that of WT leaves. Furthermore, δ13C decline in starch of aps1 leaves during the chase was much faster than that of WT leaves, which provides strong evidence for the occurrence of AGP-mediated cycling of starch breakdown products in illuminated Arabidopsis leaves.Although there is a great wealth of data supporting the occurrence of simultaneous synthesis and breakdown of storage carbohydrate in many organisms, previous  A substrate or “futile” cycle is a metabolic cycle of simultaneous synthesis and breakdown of a compound for which the net balance consists solely on the dissipation of energy ,2. In caStarch is the main storage carbohydrate in plants. Synthesized by starch synthases (SS) using ADP-glucose (ADPG) as the sugar donor molecule, and branching enzymes, this polyglucan accumulates in photosynthetic and non-photosynthetic tissues of plants. In mesophyll cells of leaves, up to 50% of the photosynthate is retained within the chloroplasts during the day in the form of starch , which iS1A Fig). This enzyme catalyzes the conversion of fructose-6-phosphate (F6P) from the CBC into glucose-6-phosphate (G6P), which is then converted into ADPG linked to starch biosynthesis by the stepwise reactions of plastidic phosphoglucomutase (pPGM) and ADPG pyrophosphorylase (AGP). This interpretation of starch biosynthesis implies that AGP is the sole source of ADPG, and functions as the major regulatory step in the starch biosynthetic process [Arabidopsis, genetic evidence showing that transitory starch biosynthesis occurs solely by the CBC-pPGI-pPGM-AGP-SS pathway has been obtained from the characterization of mutants impaired in pPGI, pPGM and AGP. Leaves of mutants totally lacking either pPGM and/or AGP accumulate ca. 1–3% of the wild type (WT) starch, whereas leaves impaired in pPGI accumulate ca. 10% of the WT starch [It is widely accepted that, in mesophyll cells of illuminated leaves, the whole starch biosynthetic process resides exclusively in the chloroplast . Accordi process ,30. In AT starch –39.S1B Fig) [gwd, sex4, bam3, bam1/bam3, bam4, mex1, mex1/pglct-1, dpe1 and amy3/isa3/lda. Leaves of these plants cultured under short day (SD) (12 h light/12 h dark) conditions accumulate high levels of starch, a phenotype that has been ascribed to reduced starch degradation during the dark period [The pathway of nocturnal starch breakdown in mesophyll cells of leaves is relatively complex and requires the coordinated actions of a suit of enzymes  . The iniS1B Fig) . RemovalS1B Fig) . A set oS1B Fig) ,40. MaltS1B Fig) . BAM4, aS1B Fig) . GlucoseS1B Fig) . G1P canS1B Fig) ,41. In Ak period .Evidence has been provided that, in addition to the CBC-pPGI-pPGM-AGP-SS pathway, plants possess additional/alternative pathway(s) of transitory starch biosynthesis wherein (i) hexose-phosphates and/or ADPG occurring in the cytosol enter the chloroplast for subsequent conversion into starch, (ii) CBC and the pPGM-AGP-SS starch biosynthetic pathway are not connected by pPGI, and (iii) starch synthesis and breakdown simultaneously occur during illumination thus allowing the formation of a substrate (starch) cycle wherein pPGM and AGP (but not pPGI) play an important role in recycling back to starch its own breakdown products ,39,42,43gwd leaves accumulate higher levels of starch than WT leaves when plants are cultured under continuous light (CL) conditions. Moreover, recent metabolic flux analyses carried out using illuminated Arabidopsis plants cultured in 13CO2-enriched environment revealed labeling of the starch breakdown product maltose [14CO2 pulse-chase and starch-preloading experiments using isolated chloroplasts [14CO2 pulse-chase experiments on illuminated leaves showing no significant loss of 14C from starch during the chase period [Supporting the occurrence of starch breakdown during illumination in leaves, several independent studies have shown that most of the enzymes involved in starch breakdown are redox-activated at physiologically relevant potentials occurring in the illuminated chloroplast –48. Also maltose . Additioroplasts  or culturoplasts  in whiche period ,54, whice period ,50,53–5514CO2 pulse-chase method employed to investigate the possible occurrence of starch breakdown in illuminated leaves is based on the enzymatic digestion of ethanol precipitates from crude extracts using amylolytic enzymes (e.g. amyloglucosidase and α-amylase) and measurement of the label in the supernatant [13CO2 pulse-chase experiments using WT and AGP null aps1 plants, and analyzed changes in stable carbon isotope ratios (δ13C) in starch by using high-performance liquid chromatography (HPLC) combined with isotope ratio mass spectrometry (IRMS). Contrary to the widely accepted paradigm that starch breakdown solely occurs during the night in Arabidopsis leaves, results presented in this work provide strong evidence supporting the occurrence of extensive starch degradation, and AGP-mediated cycling of starch breakdown products in illuminated Arabidopsis leaves.The ernatant ,54. Howeernatant . To invegwd leaves accumulate higher levels of starch than WT leaves when plants were cultured under CL conditions. This phenomenon can be ascribed to either one of the following reasons: (i) starch degradation operates in WT illuminated leaves (but not in gwd leaves), or (ii) starch phosphorylation by GWD exerts a negative effect on starch biosynthesis during illumination. To differentiate between these possibilities we measured the starch content in leaves of gwd, sex4, bam1/bam3, bam4 and amy3/isa3/lda starch breakdown mutants germinated and permanently cultured under CL conditions. The rationale behind this experimental approach was that, if starch breakdown solely occurs during the dark period, leaves of starch breakdown mutants other than gwd should accumulate WT levels of starch when cultured in the absence of a dark period. Conversely, if starch degradation also occurs during the day, leaves of the above starch breakdown mutants should accumulate higher levels of starch than WT leaves. As shown in Fig 1, both iodine staining and quantitative starch content measurement analyses revealed that leaves of all the above starch breakdown mutants accumulate higher levels of starch than those of WT plants cultured under CL conditions. The overall data thus provide strong evidence for the occurrence of starch breakdown in Arabidopsis leaves during illumination.Caspar et al.  reported2 fixation rates (An). As shown in S2A Fig, An values of the above mutants were comparable to those of WT plants. We also measured the maximum catalytic activities of AGP and carried out non-reducing western blot analyses of the small, catalytically active AGP subunit (APS1), which in leaves is present as a mixture of ca. 50 kDa active (reduced) monomers and ca. 100 kDa inactive (oxidized) dimers formed by intermolecular links involving Cys bridges [S2B Fig) and in the levels of ca. 50 kDa APS1 monomers in leaves of WT and starch breakdown mutants . We also measured the maximum catalytic activities of SS and carried western blot analyses of SS4, an important determinant of starch content [S2B Fig) and in the levels of SS4 could be found between WT and starch breakdown mutants . Therefore, high levels of starch in leaves of CL grown starch breakdown mutants cannot be ascribed to enhanced photosynthesis or to high SS and/or AGP activities, but more logically to reduced starch breakdown.To examine for the possible occurrence of pleiotropic effects that could explain the high starch content phenotype in all the above starch breakdown mutants we measured their net photosynthetic CO bridges . These a content ,59. No dmex1 mutant impaired in the chloroplastic MEX1 maltose transporter accumulate high levels of maltose when cultured under SD conditions [mex1 plants causes growth retardation since nonphotosynthetic metabolism is prevented during the night and maltose over-accumulation causes chloroplast dysfunction [mex1/pglct double mutants impaired in both the MEX1 and the chloroplastic pGlcT glucose transporter [mex1 and mex1/pglct plants germinated and permanently cultured under CL conditions. We also measured the fresh weight (FW) and chlorophyll contents of these plants. Assuming that amylolytic starch breakdown is the sole source of maltose in Arabidopsis leaves, we reasoned that if starch breakdown solely occurs during the dark period, leaves of WT, mex1 and mex1/pglct plants cultured under CL conditions should not accumulate any maltose. Furthermore, mex1 and mex1/pglct plants should accumulate WT levels of chlorophyll and display WT growth phenotypes. Conversely, if starch degradation also occurs during the day, leaves of WT plants should accumulate maltose when cultured under CL conditions. Furthermore, maltose content in mex1 and mex1/pglct leaves should be higher than that of WT leaves. Moreover mex1 plants should have reduced chlorophyll content and growth. As shown in Fig 2A, leaves of CL grown WT plants accumulated maltose. Notably, levels of this disaccharide in leaves of CL grown mex1 and mex1/pglct plants were exceedingly higher than those of WT leaves . Furthermore, mex1 plants accumulated low levels of chlorophyll in their leaves , showed reduced photosynthetic capacities  and were small  when compared with WT leaves. These phenotypes were even more severe in the mex1/pglct mutant .Maltose is the major starch breakdown product exported from the chloroplast to the cytosol at night as strongly supported by the fact that leaves of the nditions . Lack offunction ,60. Thisnsporter , indicatmex1/pglct plants we cultured mutant plants on MS medium with or without sucrose supplementation. As shown in Fig 2E, the defective growth of mex1/pglct plants was found to be mostly rescued by the external supply of sucrose. It should be emphasized that, irrespective of the presence of sucrose in the culture medium, leaves of CL grown mex1/pglct plants accumulated high levels of maltose . Thus, the defective growth of the mex1/pglct mutant cultured in sucrose-free medium is neither ascribed to pleiotropic effects nor to maltose over-accumulation, but essentially caused by a shortage of carbohydrates as a consequence of limited photosynthesis and restricted export of photoassimilates from the chloroplast to the cytosol. The overall data thus provide further evidence for the occurrence of starch breakdown in leaves during illumination.To examine for the possible occurrence of pleiotropic effects that could explain the severe dwarf phenotype of the CL grown dpe1 mutant accumulate high levels of maltotriose  when plants are cultured under SD conditions [dpe1 plants germinated and permanently cultured under CL conditions. The rationale behind this experimental approach was that if starch degradation occurs during the day, dpe1 leaves should accumulate higher levels of maltotriose than WT leaves. In support of this presumption, we found that maltotriose levels in leaves of dpe1 plants cultured under CL conditions (120.2 ± 17.1 nmol/g FW) were exceedingly higher than those of WT leaves (9.3 ± 1.1 nmol/g FW).DPE1 plays an important role in starch degradation as strongly supported by the fact that leaves of the nditions . To furt14CO2 pulse-chase method employed to investigate the possible occurrence of starch breakdown and cycling in illuminated leaves is based on the measurement of the 14C-label in amyloglucosidase and α-amylase digests of ethanol precipitates obtained from crude extracts of plants [The chloroplast is equipped with a set of enzymes  that are capable of recycling the starch breakdown products into starch . We nextf plants ,54. Howef plants .aps1 plants. As shown in Fig 3, these analyses revealed that TOC contents are higher than starch carbon contents in both WT and aps1 amyloglucosidase digests, which shows that amyloglucosidase is capable of releasing carbon compounds other than starch glucose molecules from Arabidopsis leaf ethanol precipitates. Therefore, starch cycling studies in illuminated leaves should be based on methods capable of discriminating between carbon from starch compounds (glucose) and non-starch compounds in amyloglucosidase digests.As a first step to investigate the possible occurrence of AGP-mediated mechanisms of starch cycling in illuminated Arabidopsis leaves we explored whether amyloglucosidase digestion of Arabidopsis leaf ethanol precipitates is capable of releasing carbon compounds other than starch-based glucose molecules. Towards this end, we compared the starch carbon and total carbon (TOC) contents in amyloglucosidase digests of leaves of WT and 13C/12C) ratios, isotopic enrichments and turnover of a variety of carbohydrates [13CO2 pulse-chase experiments using WT and aps1 plants cultured under long day (LD) conditions in 13CO2-enriched environment, and carried out time-course HPLC/IRMS analyses of δ13C in the glucose molecules of amyloglucosidase digests obtained as illustrated in S3 Fig. We also used the very low starch pgi1-2 mutant as control. We reasoned that if AGP-mediated starch cycling occurs during illumination, the rate of increase of δ13C in starch of aps1 leaves during the pulse should be higher than that of WT leaves and pgi1-2 leaves, since only glucose molecules coming from newly fixed 13C enriched CO2 will be incorporated into starch in aps1 leaves. In contrast, glucose molecules derived from both newly fixed 13C enriched CO2 and 13C non-enriched starch breakdown products will be incorporated into starch in WT and pgi1-2 leaves. Moreover, reduction of δ13C in starch of aps1 leaves during the chase should be faster than that of WT and pgi1-2 leaves. This would be the case since glucose molecules derived from the breakdown of 13C enriched starch will not be recycled back to starch in aps1 leaves, and only glucose molecules coming from newly fixed 13C non-enriched CO2 will be incorporated into starch. In contrast, glucose molecules from both newly fixed 13C non-enriched CO2 and 13C enriched starch breakdown products will be incorporated into starch in WT and pgi1-2 leaves. Conversely, if starch cycling does not operate during illumination, δ13C kinetics in starch during the 13CO2 pulse and chase in aps1 leaves should be comparable to that of WT and pgi1-2 leaves.Because of its precision, sensitivity and accuracy, HPLC/IRMS is now considered the most suitable technique for measuring stable carbon isotope . As expected, δ13C values in starch of both WT and aps1 leaves increased during the pulse and then dropped during the subsequent chase . Notably, in strong support of the idea that AGP-mediated starch cycling occurs in illuminated leaves, 13C enrichment in starch of aps1 leaves during the pulse was faster than that of WT leaves . Furthermore δ13C decline in starch of aps1 leaves during the chase was faster than that of WT leaves . This is especially meaningful considering that δ13C kinetics in starch of pgi1-2 leaves during the 13CO2 pulse and chase was comparable to that of WT leaves , indicating that pPGI is not involved in starch cycling.As shown in 13C kinetics in starch of aps1 and WT plants could be the consequence, at least partly, of the exchange of glucose moieties at the granule surface with free sugars or sugar phosphates by SP or DPE1, which in the very low starch aps1 mutant may be accentuated since the surface area to volume ratio of granules is very much greater than in WT plants. However, this scenario is highly unlikely since the δ13C kinetics in starch of WT and pgi1-2 leaves were very similar .A possibility cannot be ruled out that the differences observed in the δ13C enrichment in different metabolites of primary metabolism in Arabidopsis plants cultured in 13CO2-enriched environment showed that labeling kinetics in CBC intermediates are much faster than that of sucrose [S1A Fig), these results implied that starch would label faster than sucrose. However, kinetic analyses of 13C enrichment in starch were not performed [Previous studies of  sucrose . Provideerformed .13C in the sucrose of 13CO2 pulsed-chased WT leaves and compared them with that of starch. We also analyzed the δ13C kinetics of glucose and fructose. We reasoned that if starch cycling does not operate during illumination, δ13C kinetics in starch during the 13CO2 pulse and chase should be faster than that of sucrose, especially under conditions of very active starch accumulation. Conversely, if starch cycling occurs, δ13C kinetics in starch during the 13CO2 pulse and chase should be comparable or even slower than that of sucrose. As shown in Fig 5, sucrose labeled faster than glucose and fructose, which is consistent with [13C in sucrose during the pulse was exceedingly higher than that of starch, even under conditions of most active starch accumulation . Furthermore δ13C decline in sucrose during the chase was faster than that of starch . Overall, these data are consistent with the idea that starch is subject to continuous cycling during illumination.To further investigate the possible occurrence of starch cycling in illuminated leaves we carried out kinetic analyses of δent with . Notablyaps1, pgm and aps1/pgm leaves [The operation of starch cycling through a mechanism involving AGP, and the occurrence of WT levels of ADPG in m leaves ,39 are cpgm, aps1, aps1/pgm, mex1/pgm and mex1/aps1 plants cultured under LD and CL conditions, and compared them with that of WT leaves. The rationale behind this experimental approach was that if pgm, aps1 and aps1/pgm leaves do not actively synthesize starch, they should not accumulate any maltose. Conversely, if pgm, aps1 and aps1/pgm leaves synthesize starch, they should accumulate substantial levels of maltose. Furthermore, mex1/pgm and mex1/aps1 leaves should accumulate higher levels of maltose than pgm and aps1 leaves.To investigate the possible occurrence of simultaneous synthesis and breakdown of starch in mutants impaired in pPGM and AGP during illumination we conducted high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and gas chromatography-mass spectrometry (GC-MS) analyses of the maltose contents in leaves of pgm leaves presented contrasting results. Thus, whereas [pgm leaves accumulate WT levels of maltose during illumination, [pgm leaves do not accumulate any maltose. Therefore, to correctly identify maltose by HPAEC-PAD we added known amounts of maltose to extracts of leaves that had been previously digested with maltase, an enzyme that catalyzes the hydrolytic breakdown of maltose. Further confirmed by GC-MS, these analyses revealed that leaves of pgm, aps1 and aps1/pgm plants accumulate ca. 40–50% of the WT maltose content under both LD and CL conditions . Moreover, mex1/aps1 and mex1/pgm leaves accumulated exceedingly higher levels of maltose than aps1 and pgm leaves, and ca. 3-fold more maltose than WT leaves in the two photoregimes .Previous HPAEC-PAD analyses of maltose content in  whereas  reportedination,  reportedpgm/gwd and aps1/gwd plants, and compared them with those of pgm and aps1 leaves. We reasoned that if starch breakdown occurs in leaves of CL grown pgm and aps1 plants, pgm/gwd and aps1/gwd leaves should accumulate higher levels of starch than pgm and aps1 leaves. In line with this presumption, we found that pgm/gwd and aps1/gwd leaves accumulate ca. 5-fold more starch than pgm and aps1 leaves , which corresponds to 10–15% of the WT starch . Similar results were obtained using plants cultured under LD conditions (not shown). To examine for the possible occurrence of pleiotropic effects that could explain the enhanced levels of starch by gwd incorporation we measured An under varying Ci in WT, gwd, aps1 and aps1/gwd plants. Consistent with [An values in aps1 leaves are lower than those of WT at any Ci , which could explain to some extent the low starch content of this mutant. Moreover, An values in aps1/gwd leaves were still lower than those of aps1 leaves, indicating that incorporation of the gwd mutation exerts a negative effect on the photosynthetic capacity of aps1 plants. Therefore, enhanced levels of starch in leaves of CL grown aps1/gwd plants can logically be ascribed to reduced starch breakdown.We also measured the starch contents in leaves of CL grown ent with ,66, we faps1 and pgm mutants.The overall data thus provide strong evidence for the occurrence of active starch synthesis and breakdown in leaves of the very low starch mex1 and mex1/pglct mutants accumulate high levels of maltose in leaves and display a dwarf phenotype under both SD and CL conditions  strongly indicates that MEX1 and pGlcT not only participate in the export of starch breakdown products during the night but, together with the triose-P translocator (TPT), co-participate in the export of photoassimilates from the chloroplast to the cytosol for their subsequent conversion into sucrose to support growth. Thus, according to this interpretation of starch metabolism, photosynthate export from the chloroplast to the cytosol during illumination would involve the synthesis and subsequent export of starch breakdown products as schematically illustrated in Fig 8. This view is consistent with previous reports showing that mesophyll cells of mutants impaired in TPT can export starch breakdown products to the cytosol during illumination [Results presented in this communication provide strong evidence for the occurrence of extensive starch breakdown in Arabidopsis leaves during illumination. The fact that ditions . Based on the results presented in Fig 3, we propose that the persistence of constant radioactivity levels in amylolytic extracts of plants during the chase in 14CO2 pulse-chase experiments [13C kinetics in WT and aps1 plants cultured in 13CO2-enriched environment  provide evidence for the occurrence of a mechanism of AGP-mediated starch cycling in illuminated Arabidopsis leaves as schematically illustrated in Fig 8. The occurrence of starch cycling is consistent with the interpretation on the starch granule formation according to which debranching enzymes and DPE1 would play important roles not only in starch breakdown, but also in the synthesis and maturation of the starch granule [The e period ,54. Assue period ,54. Howeeriments ,54 could granule ,27. Inhe13C starch conducted in this work do not provide a quantitative estimation of the magnitude of metabolic fluxes involved in synthesis and breakdown of starch, and recycling of breakdown products to starch during illumination. The fact that aps1/gwd leaves accumulate 10–15% of the WT starch would indicate that a maximum of 10–15% of the starch accumulated in the leaf is subject to degradation during illumination. This inferred value, however, is an underestimation of the true flux through starch breakdown, since the low starch content of aps1/gwd leaves could be partly the consequence of the extremely reduced photosynthetic capacity of the mutant .Labeling kinetics analyses alone do not provide information about the magnitude of metabolic fluxes . Such anMost observations on storage carbohydrate cycling are consistent with a role for the cycle as a switch mechanism, allowing the cell to rapidly utilize storage carbohydrates in adapting to sudden environmental changes. Thus, we hypothesize that starch cycling may entail important advantages such as rapid metabolic channeling toward various pathways, especially in response to physiological and biochemical needs imposed by the environment. Maltose molecules derived from starch breakdown during illumination could play an important role in protecting proteins, membranes and the photosynthetic electron transport in response to temperature stress  or in suArabidopsis thaliana WT (ecotypes Col-0 and Wasilewskija Ws-2) and the gwd (SALK_077211), sex4 (SALK_126784), bam4 (SALK_037355), mex1 (SALK_201638), pglct (SALK_078684), bam1/bam3, dpe1 (SALK_207318), amy3/isa3/lda, ss4 [aps1 (SALK_040155), pgm (GABI_094D07), aps1/pgm [pgi1-2 [mex1 with either aps1, pgm and pglct, self-pollinating the resulting heterozygous mutants, and PCR screening for homozygous progeny using the oligonucleotide primers listed in S1 Table we produced the mex1/aps1, mex1/pgm and mex1/pglct double mutants, respectively. Unless otherwise indicated, plants were germinated and permanently cultured on soil in growth chambers under either LD (16 h light (100 μmol photons sec-1 m-2)/8 h dark photoperiod, 22°C during the light period and 18°C during the dark period) or CL conditions . At the indicated times, leaves were harvested, immediately freeze-clamped, ground to a fine powder in liquid nitrogen with a pestle and mortar, and stored at -80°C.The study was carried out using lda, ss4 , aps1 . A 50 mL syringe  and needle  were filled with 99.9 ‰ 13CO2 and placed on a syringe pump, which delivered 13CO2 to the growth cabinet. This system allowed homogenous labeling of the CO2 in the growth cabinet throughout the pulse phase. During the chase plants were transferred to a growth cabinet lacking a 13CO2 enriched atmosphere. At the indicated times, leaves were harvested and stored at -80°C as described above.For One g of the frozen powder (see above) was resuspended at 4°C in 3 mL of 100 mM HEPES (pH 7.5), 2 mM EDTA and 2 mM dithiothreitol, 1 mM PMSF and 10 mL/L protease inhibitor cocktail (Sigma P9599), and centrifuged at 14,000 x g for 20 min. The supernatant was desalted by ultrafiltration on Vivaspin 500 centrifugal concentrator (Sartorius) and the protein extract thus obtained was assayed for enzymatic activities. AGP activity was measured following the two-step assay method described in . SS actiFor immunoblot analyses of SS4, protein samples (30 μg protein/lane) were separated on 10% SDS-PAGE, transferred to nitrocellulose filters, and immunodetected by using antisera raised against SS4 as primary antibody , and a g2 1% (w/v)) for 30 min, rinsed briefly in deionized water and photographed.Plants harvested at the 18 days after sowing (DAS) stage were fixed by immersion into 3.7% formaldehyde in phosphate buffer. Leaf pigments were then removed in 96% ethanol. Re-hydrated samples were stained in iodine solution (KI 2% (w/v) and IS3 Fig). The ethanol-insoluble material was pelleted by centrifugation, washed three times with 1 mL 80% ethanol, resuspended in 300 μL of 0.2 M KOH and heated for 1 h at 95°C as described in [Aspergillus niger . Control samples contained heat-denatured amyloglucosidase. The digests and controls were adjusted to 75% (v/v) methanol and 1% (w/v) KCl, and incubated for 1 h at 4°C. After centrifugation at 15,000 x g for 20 min, the methanol/KCl-insoluble material was removed. The supernatant was dried at 60°C in a drying oven, and water (100 μL) was added . The resulting sample (designated as “amyloglucosidase digest”) was used for subsequent measurements of TOC and starch contents. For measurement of TOC content, aliquots of the amyloglucosidase digests and controls were molecularly filtered through thoroughly rinsed VIVASPIN  membranes, loaded in tin containers (5 x 9 mm) and dried in a drying oven at 50°C for 15 hours. TOC content in the residue was then determined using a NC 2500 elemental analyzer  as indicated in http://www.ru.nl/science/gi/facilities/elemental-analysis/cn-elemental/. Starch carbon content was calculated considering that 1 mol of glucose contains 72 g of carbon.A 0.15 g aliquot of the frozen powder of plants (see above) was resuspended in 1 mL of 80% (v/v) ethanol  obtained from leaves of 13CO2 pulse-chased plants and controls were conducted essentially as described in [3PO4) and oxidant reagents (0.3 M Na2S2O8) were each 50 μL/min.HPLC/IRMS analyses of δribed in . High-pe13C value of -27.21 ± 0.02%. Stable carbon isotope ratios were reported in the delta-notation:sample and RVPDB are the 13C/12C ratio in the sample and international standard (Vienna Pee Dee Belemnite), respectively. Peak identification of glucose was based on retention times in comparison with external standards. Glucose concentration measurements were based on peak areas of the separated compounds and calibrated against external standards.Isotopic ratio measurements were carried out on a Delta V Advantage isotope ratio mass spectrometer (Thermo Electron). The reference gas was regularly calibrated against phthalic acid  with a δ13CO2 pulse-chased plants (see above) was resuspended in 1 mL of 80% (v/v) ethanol . After centrifugation at 15,000 x g for 20 min, the supernatant was dried at 60°C in a drying oven, and water (100 μL) was added . Sucrose, glucose and fructose molecules were then subject to HPLC/IRMS analyses of δ13C.A 0.15 g aliquot of the frozen powder of leaves of S2 Fig). Maltose and maltotriose from supernatants were then determined by HPAEC-PAD on a DX-500 Dionex system by gradient separation with a CarboPac PA20 column according to the application method suggested by the supplier. We checked the reliability of the method of maltose detection and measurement by adding known amounts of maltose to supernatants previously digested with maltase. Maltose was also measured by GC-MS essentially as described in [For measurement of maltose and maltotriose contents, 0.15 g of the frozen powder (see above) was resuspended in 1 mL of 80% ethanol, left at 70°C for 90 min and centrifuged at 13,000 x g for 10 min  and by HPAEC-PAD. Chlorophyll content was quantified according to .Gas exchange determinations were conducted as described in  using a Presented data are the means (± SE) of three independent experiments, with 3 replicates for each experiment. The significance of differences was statistically evaluated with Student´s t-test using the SPSS software. Differences were considered significant if p<0.05.S1 Fig(A) During the day, photosynthetically fixed carbon is either retained within the chloroplast to fuel the synthesis of transitory starch, or exported to the cytosol as triose phosphates by means of TPT to be subsequently converted into sucrose. (B) During the night, starch is remobilized thereby providing maltose and glucose molecules that are exported to the cytosol and metabolized to support sucrose synthesis and growth. The enzyme activities involved are numbered as follows: 1, pPGI; 2, pPGM; 3, AGP; 4, SS; 5, β-amylase; 6, AMY; 7, debranching enzymes; 8, DPE1; 9, SP. In “B”, maltose and glucose are transported from plastid to the cytosol via the MEX1 and pGlcT transporters, respectively.(EPS)Click here for additional data file.S2 Fig2 fixation rates, (B) total AGP and SS activities, (C) non-reducing western blot of AGP and (D) western blot of SS4. In “A”, gas exchange determinations were conducted at 25°C with a photosynthetic photon flux density of 350 μmol m-2 s-1 and with a CO2 concentration of 450 μmol mol-1.(A) Net photosynthetic CO(EPS)Click here for additional data file.S3 Fig(EPS)Click here for additional data file.S4 Fig0 and aps1 plants, and (B) Ws-2 and pgi1-2 plants cultured under LD conditions.Time-course of starch content in leaves of (A) Col- In “A” and “B”, the experiment was conducted using plants at the 26 DAS and 22 DAS growth stages, respectively. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment.(EPS)Click here for additional data file.S5 Fig2 assimilation rate (An) versus intercellular CO2 concentration (Ci) in 20 DAS WT (Col-0), aps1, gwd and aps1/gwd leaves.The graphic represents the curves of net CO(EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.