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Christian Mistegård Jørgensen,1 Nils Jensen Boe,1 Stine Munk Hald,1 Frederik Meyer-Kristensen,1 Mie Micheelsen Norlén,1 Christian Ovesen,1,2 Sören Möller,3,4 Birgit Bjerre Høyer,3 Jonas Asgaard Bojsen,5 Mohammad Talal Elhakim,5 Frederik Severin Gråe Harbo,5 Rustam Al-Shahi Salman,6 Larry B Goldstein,7 Jesper Hallas,8 Luis Alberto García Rodríguez,9 Magdy Selim,10 David Gaist1 1Research Unit for Neurology, Odense University Hospital, Odense, Denmark; University of Southern Denmark, Odense, Denmark; 2Department of Neurology, Bispebjerg Hospital, Copenhagen, Denmark; 3Open Patient Data Explorative Network (OPEN), Odense University Hospital, Odense, Denmark; 4Department of Clinical Research, University of Southern Denmark, Odense, Denmark; 5Department of Radiology, Odense University Hospital, Odense, Denmark; University of Southern Denmark, Odense, Denmark; 6Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK; 7Department of Neurology and Kentucky Neuroscience Institute, University of Kentucky, Lexington, KY, USA; 8Department of Clinical Pharmacology, Pharmacy and Environmental Medicine, University of Southern Denmark, Odense, Denmark; 9Centro Español Investigación Farmacoepidemiológica, Madrid, Spain; 10Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USACorrespondence: David Gaist, Research Unit for Neurology, Odense University Hospital, Odense, Denmark; University of Southern Denmark, Odense, Denmark, Email dgaist@health.sdu.dkPurpose: To estimate the strength of association between use of antithrombotics (AT) drugs with survival after spontaneous intracerebral hemorrhage (s-ICH) comparing oral anticoagulant (OAC) or platelet antiaggregants (PA) with no AT use and in active comparator analyses OAC vs PA, direct oral anticoagulant (DOAC) vs vitamin K antagonist (VKA), and clopidogrel vs aspirin.Patients and Methods: We identified patients ≥ 55 years with a first-ever s-ICH between 2015 and 2018 in Southern Denmark (population 1.2 million). From this population, patients who had used an AT at the time of ICH were identified and classified as OAC or PA vs no AT (reference group), and for active comparator analyses as OAC vs PA (reference group), DOAC vs VKA (reference group), or clopidogrel vs aspirin (reference group). We calculated adjusted relative risks (aRRs) and corresponding [95% confidence intervals] for 90-day all-cause mortality with adjustments for potential confounders.Results: Among 1043 patients who had s-ICH, 206 had used an OAC, 270 a PA, and 428 had no AT use. The adjusted 90-day mortality was higher in OAC- (aRR 1.68 [1.39-2.02]) and PA-users (aRR 1.21 [1.03-1.42]), compared with no AT. Mortality was higher in OAC- (aRR 1.19 [1.05-1.36]) vs PA-users. In analyses by antithrombotic drug type, 88 used a DOAC, 136 a VKA, 111 clopidogrel, and 177 aspirin. Mortality was lower among DOAC- vs VKA-users (aRR 0.82 [0.68-0.99]), but similar between clopidogrel vs aspirin users (aRR 1.04 [0.87-1.24]).Conclusion: In this unselected cohort from a geographically defined Danish population, 90-day mortality after s-ICH was higher in patients with prior use of an OAC compared with no AT use or patients using a PA. Mortality was slightly lower for patients using a DOAC than a VKA. Mortality was also higher in PA- vs no AT-users, but there were no differences in mortality between clopidogrel vs aspirin.Keywords: stroke, intracerebral hemorrhage, stroke prevention, oral anticoagulants, platelet antiaggregants

Purpose: Vitamin D may induce tolerance in the intestinal immune system and has been shown to regulate the phenotype of tolerogenic intestinal dendritic cells (DCs) in vitro. It is unknown whether vitamin D supplementation affects human intestinal DCs in vivo, and we aimed to investigate the tolerability and effect on intestinal CD103+DCs of high-dose vitamin D3 treatment in healthy subjects.Methods: Ten healthy subjects received a total of 480,000 IU oral vitamin D3 over 15 days and colonic biopsies were obtained before and after intervention by endoscopy. Lamina propria mononuclear cells (LPMCs) were isolated from the biopsies, stained with DC surface markers and analysed with flow cytometry. Snap-frozen biopsies were analysed with qPCR for DC and regulatory T cell-related genes.Results: No hypercalcemia or other adverse events occurred in the test subjects. Vitamin D decreased the number of CD103+ DCs among LPMCs (p = 0.006). Furthermore, vitamin D induced mRNA expression of TGF-β (p = 0.048), TNF-α (p = 0.006) and PD-L1 (p = 0.02) and tended to induce IL-10 expression (p = 0.06). Multivariate factor analysis discriminated between pre- and post-vitamin D supplementation with a combined increased qPCR expression of PD1, PD-L1, TGF-β, IL-10, CD80, CD86, FOXP3, NFATc2 and cathelicidin.Conclusion: High-dose vitamin D supplementation is well tolerated by healthy subjects and has a direct effect on the CD103+ DCs, local cytokine and surface marker mRNA expression in the colonic mucosa, suggestive of a shift towards a more tolerogenic milieu. [ABSTRACT FROM AUTHOR]

Mie Micheelsen Jensen,1 Stine Munk Hald,1,2 Line Marie Buch Kristensen,1 Nils Jensen Boe,1,2 Frederik Severin Gråe Harbo,3 David Gaist1,2,4 1Department of Neurology, Odense University Hospital, Odense, Denmark; 2Neurology Research Unit, Department of Clinical Research, University of Southern Denmark, Odense, Denmark; 3Department of Radiology, Odense University Hospital, Odense, Denmark; 4Open Patient Data Explorative Network (OPEN), Odense University Hospital, Odense, DenmarkCorrespondence: David GaistDepartment of Neurology, Odense University Hospital, J.B. Winsløwsvej 4, Odense C, 5000, DenmarkTel +45 65412485Fax +45 65413389Email dgaist@health.sdu.dkPurpose: Danish registries could be an attractive resource for studies of recurrent intracerebral hemorrhage (re-ICH). We developed and validated algorithms to identify re-ICH in the Danish Stroke Registry (DSR) and the Danish National Patient Registry (DNPR).Patients and Methods: Using multiple sources, we followed-up an inception cohort with verified first-ever spontaneous ICH (n = 2528) for their first re-ICH in 2009– 2018 (study period). We used verified cases of re-ICH (n = 124) as the gold standard to assess the performance of register-based algorithms for identifying re-ICH. For each cohort member, we traced events of re-ICH (ICD-10-code I61) in the study period according to DSR and DNPR, respectively. For each registry, we tested algorithms with a blanking period (BP) – ie, a period immediately following the index ICH during which outcome events were ignored – of varying length (7 days– 360 days). The algorithm with the shortest BP that returned a positive predictive value (PPV) of ≥ 80% was considered optimal. We also calculated negative predictive value (NPV), sensitivity, and specificity of each algorithm and [95% confidence intervals] for all proportions.Results: The optimal algorithm for DSR (BP 30 days) had a PPV of 89.5% [82.2– 94.0], NPV 98.8% [98.2– 99.1], sensitivity 75.8% [67.6– 82.5], and specificity 99.5% [99.2– 99.7]. The optimal algorithm for DNPR (BP 120 days) had a PPV of 80.6% [71.7– 87.2], NPV 98.1% [97.5– 98.6], sensitivity 63.7% [55.0– 71.6], and specificity 99.2% [98.8– 99.5].Conclusion: Simple algorithms accurately identified re-ICH in DSR and DNPR. Compared with DNPR, DSR achieved higher PPV and sensitivity with a shorter BP. The proposed algorithms could facilitate valid use of DSR and DNPR for studies of re-ICH.Keywords: stroke, recurrent stroke, intracerebral hemorrhage, epidemiology, validity, register-based research

Stine Munk Hald,1,2 Christine Kring Sloth,1 Mikkel Agger,1 Maria Therese Schelde-Olesen,1 Miriam Højholt,1 Mette Hasle,1 Helle Bogetofte,1 Ida Olesrud,1 Stefanie Binzer,3 Charlotte Madsen,1 Willy Krone,4 Luis Alberto García Rodríguez,5 Rustam Al-Shahi Salman,6 Jesper Hallas,7 David Gaist1,2,8 1Department of Neurology, Odense University Hospital, Odense, Denmark; 2Department of Clinical Research, Neurology Research Unit, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark; 3Department of Neurology, Lillebaelt Hospital, Kolding, Denmark; 4Department of Radiology, Odense University Hospital, Odense, Denmark; 5Centro Español Investigación Farmacoepidemiológica (CEIFE), Madrid, Spain; 6Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK; 7Clinical Pharmacology and Pharmacy, Department of Public Health, University of Southern Denmark, Odense, Denmark; 8Odense Patient Data Explorative Network (OPEN), Odense University Hospital, Odense, DenmarkCorrespondence: David GaistDepartment of Neurology, Odense University Hospital, J.B. Winsløwsvej 4, 5000 Odense C, Odense, DenmarkTel +45 65412485Fax +45 65413389Email dgaist@health.sdu.dkPurpose: To establish the validity of intracerebral hemorrhage (ICH) diagnoses in the Danish Stroke Registry (DSR) and the Danish National Patient Registry (DNPR).Patients and Methods: Based on discharge summaries and brain imaging reports, we estimated the positive predictive value (PPV) of a first-ever diagnosis code for ICH (ICD-10, code I61) for all patients in the Region of Southern Denmark (1.2 million) during 2009– 2017 according to either DNPR or DSR. We estimated PPVs for any non-traumatic ICH (a-ICH) and spontaneous ICH (s-ICH) alone (ie, without underlying structural cause). We also calculated the sensitivity of these diagnoses in each of the registers. Finally, we classified the location of verified s-ICH.Results: A total of 3,956 patients with ICH diagnosis codes were studied (DSR only: 87; DNPR only: 1,513; both registries: 2,356). In the DSR, the PPVs were 86.5% (95% CI=85.1– 87.8) for a-ICH and 81.8% (95% CI=80.2– 83.3) for s-ICH. The PPVs in DNPR (discharge code, primary diagnostic position) were 76.2% (95% CI=74.7– 77.6) for a-ICH and 70.2% (95% CI=68.6– 71.8) for s-ICH. Sensitivity for a-ICH and s-ICH was 76.4% (95% CI=74.8– 78.0) and 78.7% (95% CI=77.1– 80.2) in DSR, and 87.3% (95% CI=86.0– 88.5) and 87.7% (95% CI=86.3– 88.9) in DNPR. The location of verified s-ICH was lobar (39%), deep (33.6%), infratentorial (13.2%), large unclassifiable (11%), isolated intraventricular (1.9%), or unclassifiable due to insufficient information (1.3%).Conclusion: The validity of a-ICH diagnoses is high in both registries. For s-ICH, PPV was higher in DSR, while sensitivity was higher in DNPR. The location of s-ICH was similar to distributions seen in other populations.Keywords: stroke, intracerebral hemorrhage, epidemiology, validity, register-based research

Stine Munk Hald,1,2 Christine Kring Sloth,1,2 Sabine Morris Hey,1,2 Charlotte Madsen,1 Nina Nguyen,3 Luis Alberto García Rodríguez,4 Rustam Al-Shahi Salman,5 Sören Möller,2,6 Frantz Rom Poulsen,7 Anton Pottegård,8 David Gaist1,2,6 1Department of Neurology, Odense University Hospital, Odense, Denmark; 2Department of Clinical Research, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark; 3Department of Radiology, Odense University Hospital, Odense, Denmark; 4Spanish Centre for Pharmacoepidemiologic Research (CEIFE), Madrid, Spain; 5Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK; 6Odense Patient Data Explorative Network (OPEN), Odense University Hospital, Odense, Denmark; 7Department of Neurosurgery, Odense University Hospital, Odense, Denmark; 8Clinical Pharmacology and Pharmacy, Department of Public Health, University of Southern Denmark, Odense, Denmark Purpose: The purpose of this study is to establish the validity of intracerebral hemorrhage (ICH) diagnoses in the Danish Stroke Registry (DSR) and the Danish National Patient Registry (DNPR).Patients and methods: We estimated the positive predictive value (PPV) of ICH diagnoses for a sample of 500 patients from the DSR (patients recorded under ICH diagnosis) and DNPR (International Classification of Diseases, version 10, code I61) during 2010–2015, using discharge summaries and brain imaging reports (minimal data). We estimated PPVs for any ICH (a-ICH) and spontaneous ICH (s-ICH) alone. Furthermore, we assessed PPVs according to whether patients were recorded in both or only one of the registries. Finally, in a subsample with ICH diagnoses with access to full medical records and original imaging studies (extensive data, n=100), we compared s-ICH diagnosis and hemorrhage location after use of extensive vs minimal data.Results: In the DSR, the PPVs were 94% (95% CI, 91%–96%) for a-ICH and 85% (95% CI, 81%–88%) for s-ICH. In the DNPR, the PPVs were 88% (95% CI, 84%–91%) for a-ICH and 75% (95% CI, 70%–79%) for s-ICH. PPVs for s-ICH for patients recorded in both registries, DSR only, and DNPR only were 86% (95% CI, 82–99), 80% (95%CI, 71–87), and 49% (95%CI, 39–59), respectively. Evaluation of extensive vs minimal data verified s-ICH diagnosis in 98% and hemorrhage location in 94%.Conclusion: The validity of a-ICH diagnoses in DSR and DNPR is sufficiently high to support their use in epidemiologic studies. For s-ICH, validity was high in DSR. In DNPR, s-ICH validity was lower, markedly so for the small subgroup of patients only recorded in this registry. Minimal data including discharge summaries and brain imaging reports were feasible and valid for identifying ICH location. Keywords: stroke, epidemiology, register-based research, intracranial hemorrhage

Objective: To evaluate associations between oral anticoagulant (OAC) discontinuation and risk of ischaemic stroke (IS) among patients with atrial fibrillation (AF)., Methods: We undertook a population-based cohort study with nested case-control analysis using UK primary care electronic health records (IQVIA Medical Research Data-UK) and linked registries from the Region of Southern Denmark (RSD). Patients with AF (76 882 UK, 41 526 RSD) were followed to identify incident IS cases during 2016-2018. Incident IS cases were matched by age and sex to controls. Adjusted ORs for OAC discontinuation (vs current OAC use) were calculated using logistic regression., Results: We identified 616 incident IS cases in the UK and 643 in the RSD. ORs for IS with any OAC discontinuation were 2.99 (95% CI 2.31 to 3.86, UK) and 2.30 (95% CI 1.79 to 2.95, RSD), for vitamin K antagonist discontinuation they were 2.38 (95% CI 1.72 to 3.30, UK) and 1.83 (95% CI 1.34 to 2.49, RSD), and for non-vitamin K antagonist oral anticoagulant discontinuation they were 4.59 (95% CI 2.97 to 7.08, UK) and 3.37 (95% CI 2.35 to 4.85, RSD). ORs were unaffected by time since discontinuation and duration of use. Annually, up to 987 IS cases in the UK and 132 in Denmark could be preventable if OAC therapy is not discontinued., Conclusions: Our results suggest that patients with AF who discontinue OAC therapy have a significant twofold to threefold higher risk of IS compared with those who continue therapy. Addressing OAC discontinuation could potentially result in a significant reduction in AF-attributed IS., Competing Interests: Competing interests: LAGR works for CEIFE, which has received other research funding from Bayer AG. LAGR has also received honoraria for serving on advisory boards for Bayer AG. DG has received honoraria from AstraZeneca (Sweden) for participation as a co-investigator on a research project outside the submitted work, and receiving speaker honorarium from Bristol-Myers Squibb outside the submitted work. YB and PV are employees of Bayer AG. GB is an employee of Bayer AB. MS has served on the steering committees and led sub-studies from trials sponsored by Bayer and has served as a consultant and received speaker’s honoraria from Bayer. MS has also served as a consultant to Portola, Bristol Myers Squibb and Janssen. LCS, SMH and JH report no potential conflicts of interest., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

A major challenge in affluent societies is the increase in disorders related to gut and metabolic health. Chronic over nutrition by unhealthy foods high in energy, fat, and sugar, and low in dietary fibre is a key environmental factor responsible for this development, which may cause local and systemic inflammation. A low intake of dietary fibre is a limiting factor for maintaining a viable and diverse microbiota and production of short-chain fatty acids in the gut. A suppressed production of butyrate is crucial, as this short-chain fatty acid (SCFA) can play a key role not only in colonic health and function but also at the systemic level. At both sites, the mode of action is through mediation of signalling pathways involving nuclear NF-κB and inhibition of histone deacetylase. The intake and composition of dietary fibre modulate production of butyrate in the large intestine. While butyrate production is easily adjustable it is more variable how it influences gut barrier function and inflammatory markers in the gut and periphery. The effect of butyrate seems generally to be more consistent and positive on inflammatory markers related to the gut than on inflammatory markers in the peripheral tissue. This discrepancy may be explained by differences in butyrate concentrations in the gut compared with the much lower concentration at more remote sites.

Purpose: Low intake of dietary fibre is associated with the development of type 2 diabetes. Dyslipidaemia plays a key role in the pathogenesis of type 2 diabetes. Knowledge of the impact of dietary fibres on postprandial lipaemia is, however, sparse. This study aimed in subjects with metabolic syndrome to assess the impact on postprandial lipaemia and features of the metabolic syndrome of a healthy carbohydrate diet (HCD) rich in cereal fibre, arabinoxylan and resistant starch compared to a refined-carbohydrate western-style diet (WSD)., Methods: Nineteen subjects completed the randomised, crossover study with HCD and WCD for 4-week. Postprandial metabolism was evaluated by a meal-challenge test and insulin sensitivity was assessed by HOMA-IR and Matsuda index. Furthermore, fasting cholesterols, serum-fructosamine, circulating inflammatory markers, ambulatory blood pressure and intrahepatic lipid content were measured., Results: We found no diet effects on postprandial lipaemia. However, there was a significant diet × statin interaction on total cholesterol (P = 0.02) and LDL cholesterol (P = 0.002). HCD decreased total cholesterol (-0.72 mmol/l, 95% CI (-1.29; -0.14) P = 0.03) and LDL cholesterol (-0.61 mmol/l, 95% CI (-0.86; -0.36) P = 0.002) compared with WSD in subjects on but not without statin treatment. We detected no other significant diet effects., Conclusions: In subjects with metabolic syndrome on statins a 4-week diet rich in arabinoxylan and resistant starch improved fasting LDL and total cholesterol compared to subjects not being on statins. However, we observed no diet related impact on postprandial lipaemia or features of the metabolic syndrome. The dietary fibre x statin interaction deserves further elucidation.

Recently, the intestinal microbiota has been emphasised as an important contributor to the development of metabolic syndrome. Dietary fibre may exert beneficial effects through modulation of the intestinal microbiota and metabolic end products. We investigated the effects of a diet enriched with two different dietary fibres, arabinoxylan and resistant starch type 2, on the gut microbiome and faecal short-chain fatty acids. Nineteen adults with metabolic syndrome completed this randomised crossover study with two 4-week interventions of a diet enriched with arabinoxylan and resistant starch and a low-fibre Western-style diet. Faecal samples were collected before and at the end of the interventions for fermentative end-product analysis and 16S ribosomal RNA bacterial gene amplification for identification of bacterial taxa. Faecal carbohydrate residues were used to verify compliance. The diet enriched with arabinoxylan and resistant starch resulted in significant reductions in the total species diversity of the faecal-associated intestinal microbiota but also increased the heterogeneity of bacterial communities both between and within subjects. The proportion of Bifidobacterium was increased by arabinoxylan and resistant starch consumption (P<0.001), whereas the proportions of certain bacterial genera associated with dysbiotic intestinal communities were reduced. Furthermore, the total short-chain fatty acids (P<0.01), acetate (P<0.01) and butyrate concentrations (P<0.01) were higher by the end of the diet enriched with arabinoxylan and resistant starch compared with those resulting from the Western-style diet. The concentrations of isobutyrate (P = 0.05) and isovalerate (P = 0.03) decreased in response to the arabinoxylan and resistant starch enriched diet, indicating reduced protein fermentation. In conclusion, arabinoxylan and resistant starch intake changes the microbiome and short-chain fatty acid compositions, with potential beneficial effects on colonic health and metabolic syndrome., Trial Registration: ClinicalTrials.gov NCT01618526.

A malignant tumor is not merely an accumulation of neoplastic cells, but constitutes a microenvironment containing endothelial cells, fibroblasts, structural components, and infiltrating immune cells that impact tumor development, invasion, metastasis, and outcome. Hence, the evolution of cancers reflects intricate cellular and molecular interactions between tumor cells and constituents of the tumor microenvironment. Recent studies have shed new light on this complex interaction between tumor and host immune cells and the resulting immune response. The composition of the immune microenvironment differs across patients as well as in cancers of the same type, including various populations of T cells, B cells, dendritic cells, natural killer cells, myeloid-derived suppressor cells, neutrophils, and macrophages. The type, density, location, and organization of immune cells within solid tumors define the immune contexture, which has proved to be a major determinant of tumor characteristics and patient outcome. Lung cancer consists mostly of non-small cell lung cancer (85%); it is our most deadly malignant disease, with the 5-year survival rate being merely 15%. This review focuses on the immune contexture; the tumor-suppressing roles of tumor-infiltrating lymphocytes; and the relevance of this immune contexture for cancer diagnostics, prognostication, and treatment allocation, with an emphasis on non-small cell lung cancer., (Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Photosynthetic electron transport can involve either a linear flow from water to NADP, via Photosystems (PS) II and I or a cyclic flow just involving PSI. Little is known about factors regulating the relative flow through each of these pathways. We have examined photosynthetic electron transport through each system in plants of Arabidopsis thaliana in which either the PSI-D1 or PSI-E1 subunits of PSI have been knocked out. In both cases, this results in an imbalance in the turnover of PSI and PSII, such that PSII electron transport is limited by PSI turnover. Phosphorylation of light-harvesting complex II (LHCII) and its migration to PSI is enhanced but only partially reversible and not sufficient to balance photosystem turnover. In spite of this, cyclic electron flow is able to compete efficiently with PSI across a range of conditions. In dark-adapted leaves, the efficiency of cyclic relative to linear flow induced by far-red light is increased, implying that the limiting step of cyclic flow lies in the re-injection of electrons into the electron transport chain. Illumination of leaves with white light resulted in transient induction of a significant non-photochemical quenching in knockout plants which is probably high energy state quenching induced by cyclic electron flow. At high light and at low CO(2), non-photochemical quenching was greater in the knockout plants than in the wildtype. Comparison of PSI and PSII turnover under such conditions suggested that this is generated by cyclic electron flow around PSI. We conclude that, when the concentration of PSI is limiting, cyclic electron flow is still able to compete effectively with linear flow to maintain a high DeltapH to regulate photosynthesis.

When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions (e.g. heat, chilling or drought), and can result in the production of reactive oxygen species (ROS). Here, we present evidence for a novel protective process - feedback redox regulation via the redox poise of the NADP(H) pool. Photosynthetic electron transport was studied in two transgenic tobacco (Nicotiana tabacum) lines - one having reduced levels of ferredoxin NADP+-reductase (FNR), the enzyme responsible for reducing NADP+, and the other reduced levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the principal consumer of NADPH. Both had a similar degree of inhibition of carbon fixation and impaired electron transport. However, whilst FNR antisense plants were obviously stressed, with extensive bleaching of leaves, GAPDH antisense plants showed no visible signs of stress, beyond having a slowed growth rate. Examination of electron transport in these plants indicated that this difference is due to feedback regulation occurring in the GAPDH but not the FNR antisense plants. We propose that this reflects the occurrence of a previously undescribed regulatory pathway responding to the redox poise of the NADP(H) pool.

Potato (Solanum tuberosum) is the fourth largest crop worldwide in yield, and cv. Kuras is the major starch potato of northern Europe. Storage starch is packed densely in tuber amyloplasts, which become starch granules. Amyloplasts of soil-grown mini-tubers and agar-grown micro-tubers of cv. Kuras were purified. The mini-tuber amyloplast preparation was enriched 10-20-fold and the micro-tuber amyloplast approximately fivefold over comparative total protein extracts. Proteins separated by SDS-PAGE were digested with trypsin, analysed by mass spectrometry and identified by mascot software searches against an in-house potato protein database and the NCBI non-redundant plant database. The differential growth conditions for mini- and micro-tubers gave rise to rather different protein profiles, but the major starch granule-bound proteins were identical for both and dominated by granule-bound starch synthase I, starch synthase II and alpha-glucan water dikinase. Soluble proteins were dominated by starch phosphorylase L-1, other large proteins of the classes 'starch and sucrose metabolism', 'pentose phosphate pathway', 'glycolysis', 'amino acid metabolism', and other proteins such as plastid chaperonins. The majority of the identified proteins had a predicted plastid transit peptide, supporting their presence in the amyloplast. However, several highly expressed proteins had no transit peptide, such as starch phosphorylase H, or had a predicted mitochondrial location. Intriguingly, all polyphenol oxidases, a family of enolases, one transketolase, sulfite reductase, deoxynucleoside kinase-like and dihydroxy-acid dehydrase had twin-arginine translocation motifs, and a homologue to dihydrolipoamide dehydrogenase had a Sec (secretory) motif; these motifs usually target thylakoid-like structures.

The pgr5 mutant of Arabidopsis thaliana has been described as being deficient in cyclic electron flow around photosystem I, however, the precise role of the PGR5 protein remains unknown. To address this issue, photosynthetic electron transport was examined in intact leaves of pgr5 and wild type A. thaliana. Based on measurements of the kinetics of P700 oxidation in far red light and re-reduction following oxidation in the presence of DCMU, we conclude that this mutant is able to perform cyclic electron flow at a rate similar to the wild type. The PGR5 protein is therefore not essential for cyclic flow. However, cyclic flow is affected by the pgr5 mutation under conditions where this process is normally enhanced in wild type leaves, i.e. high light or low CO(2) concentrations resulted in enhancement of cyclic electron flow. This suggests a different capacity to regulate cyclic flow in response to environmental stimuli in the mutant. We also show that the pgr5 mutant is affected in the redox poising of the chloroplast, with the electron transport chain being substantially reduced under most conditions. This may result in defective feedback regulation of photosynthetic electron transport under some conditions, thus providing a rationale for the reduced efficiency of cyclic electron flow.