Terpenes, a large family of natural products with important applications, are commonly associated with plants and fungi. The diterpenoids dehydroabietic and abietic acids are defense metabolites abundant in resin, and are used as biomarkers for conifer plants. We report here for the first time that the two diterpenoid acids are produced by members of several genera of cyanobacteria. Dehydroabietic acid was isolated from two cyanobacterial strains and its identity was confirmed spectroscopically. One or both of the diterpenoids were detected in the cells of phylogenetically diverse cyanobacteria belonging to four cyanobacterial ‘botanical orders’, from marine, estuarine and inland environments. Dehydroabietic acid was additionally found in culture supernatants. We investigated the natural role of the two resin acids in cyanobacteria using ecologically-relevant bioassays and found that the compounds inhibited the growth of a small coccoid cyanobacterium. The unexpected discovery of dehydroabietic and abietic acids in a wide range of cyanobacteria has implications for their use as plant biomarkers. Two norabietane diterpenoids have been reported from Microcoleus lacustris171820Despite that the overwhelming majority of described terpene molecules are found in plants and fungi, a small number of terpenoids have been known for decades to be produced by bacteria. These include geosmin and 2-methylisoborneol 1 in two marine strains and the LC-HRESIMS (Liquid Chromatography coupled to High Resolution Electrospray Ionization Mass Spectrometry) detection of 1 and 2 in an extended group of cyanobacteria, indicating that the diterpenoids are widely distributed among this group of organisms.As part of our ongoing studies on cyanobacterial secondary metabolite diversity, we have been exploring diverse strains of cyanobacteria from our in-house collection (LEGE Culture Collection) for their small-molecule constituents. We report herein the NMR (Nuclear Magnetic Resonance)-guided isolation of 2Cl2/MeOH (2:1), and fractionated using normal-phase vacuum liquid chromatography (VLC). Upon 1H NMR inspection of the resulting fractions, a set of aromatic signals (n-hexane/EtOAc (3:2). These resonances were followed by 1H NMR analyses of fractions resulting from successive rounds of normal-phase chromatography, until compound 1 was obtained with sufficient purity for detailed structural analysis.A crude organic extract (1.18 g) was obtained from repeated extraction of biomass from the colonial cyanobacterium strain LEGE 10388 with CH signals  was dete1 was carried out using a combination of HRESIMS as well as 1D and 2D NMR data . The identity of the isolated natural product was confirmed by comparison of 1H NMR and HRMS data to a commercially available standard  described above for the Synechococcales cyanobacterium unidentified strain LEGE 10388 and Plectonema cf. radiosum LEGE 06105. 1H NMR signals that could indicate the presence of 1 were found for fractions originating from eight other strains, of marine, estuarine and inland origins, namely Nostoc sp. LEGE 06077, Nostoc sp. LEGE 07365, Chroococcidiopsis sp. LEGE 06174, Synechocystis sp. LEGE 06079, Synechocystis salina LEGE 06099, Leptolyngbya ectocarpi LEGE 11425, Leptolyngbya sp. LEGE 07084 and Nodosilinea sp. LEGE 13457  that, in all likelihood, corresponds to abietic acid  for 1 and <1–7 μg/g (d.w.) for 2  for 2 . No 1H Nscreened .1, a phylogenetic analysis was carried out with the objective of providing an overview of the phylum Cyanobacteria as well as the placement of the screened strains within it. Dehydroabietic acid-producers were found to be distributed throughout the cyanobacterial evolutionary tree, as expected from their morphological diversity (Nodosilinea strains and a red-pigmented Leptolyngbya ectocarpi LEGE 11425), and also within the Synechocystis lineage and the order Pleurocapsales clade  and Oscillatoriales lineages.To better understand the diversity of the strains in our culture collection for which we detected the production of iversity . In partes clade . Strains1 for activity towards organisms that may interact with cyanobacteria in the natural setting. In particular, we evaluated the compound for antialgal and anticyanobacterial activity as well as for toxicity towards small crustaceans, which are known to feed on cyanobacteriaS. salina LEGE 06099, the green microalga Chlorella vulgaris and the crustacean Artemia salina were exposed to concentrations of 1 up to 50 μg mL−1 (167 μM), no inhibitory or stimulatory effects on growth were observed (data not shown). However, the marine picocyanobacterium Synechococcus cf. nidulans LEGE 07171 was inhibited by 1 . To clarify whether the anticyanobacterial interaction observed could have a relevant ecological meaning, we investigated whether the two terpenoids were released to the culture medium by actively growing cultures of cyanobacteria. We analyzed supernatants from two of the producing strains, Nostoc sp. LEGE 06077 and Nodosilinea sp. LEGE 13457, using LC-HRESIMS and detected only 1, which was also the most abundant compound inside the cells (1 in the supernatants indicates that it is two orders of magnitude lower than the minimum inhibitory concentration (MIC) observed in the S. nidulans LEGE 07171 assays (We tested the most abundant ted by 1 . Given the cells . Estimat1 assays .1–and most likely 2 (which we detected in very low amounts in our samples by LC-HRESIMS)–are widespread in cyanobacteria. This is, to our knowledge, the first report of any of these compounds among bacteria. Apart from vascular plants, 1 has been reported from a epiphytic lichen and its mycobiontArmillaria mellea, which is parasitic on several conifers1 and 2 as biomarkers for plants needs to be reassessed. The two diterpenoid acids are used extensively as higher plant biomarkers in paleobotanical and geochemical studies251 and 2 as markers for conifers is not called into question by the present discovery, mainly because other biomarkers originating from different biosynthetic routes are commonly used in conjunction with the resin acids1 and 2 of plant origin in studied materials is likely to be much higher than that of cyanobacterial origin, as cyanobacteria do not often reach high cell densities in the environment  were tentatively identified by GC-MS analysisS. salina LEGE 06099 and Synechocystis sp. LEGE 06079 are closely related to Synechocystis sp. PCC 6803  and have been shown to be inherited vertically31From our phylogenetic analysis, it is clear that the distribution of resin acid-producing cyanobacteria is transversal in the cyanobacterial phylum. We were unable to detect any of the the two resin acids in some strains, but it is possible that these were present at levels below the limit of detection. The small number of strains studied here restricts our ability to infer whether the present distribution is associated with horizontal gene transfer (HGT) events or if the production of 1 and 2 take place in plastidsSynechocystis sp. PCC 6803) are available in public databases, we searched these for homologs of a conifer abietadiene synthase, but could not find any strong or widespread homologies, although hypothetical proteins with weak homology were identified  in antibacterial assays e.g.39401 was also found to inhibit methanogenesis in different bacteriaC. elegans2 has shown mild cytotoxicity against cancer cells1 and 2 were genotoxic to marine organisms48S. nidulans LEGE 07171 and the fact that the compounds are released to the medium by actively growing cyanobacteria suggests that 1 and 2 may act as allelochemicals. Still, MICs observed in the S. nidulans assay were much higher than the estimated levels of 1 in the culture media -labda-11,13-diene-8α,15-diol) as a response to wounding in tobacco plantsThe defensive roles of re media  and, whi1, we have established that this compound is produced by cyanobacteria. Our subsequent investigation on the production of 1 and 2 in cyanobacteria brings up important questions regarding diterpene biosynthesis evolution at the bacterial and plant level. Future research should clarify whether the two metabolites are also frequent in other plants and algae. Access to the genes involved in the biosynthesis of the two resin acids in cyanobacteria will facilitate the study of their actual distribution. Also relevant will be to determine whether other biosynthetically-related resin acids  are found in cyanobacteria. Therefore, studies employing 1 and 2 as biomarkers need to not only take into account that these are not exclusive from the plant world but also consider the possibility that other diterpenes that are commonly associated with plants may have a (cyano) bacterial origin.To conclude, by isolating metabolites from two cyanobacteria and elucidating their structures to reveal their identity as 1D and 2D NMR data were acquired in either a 400 MHz Bruker Avance III or a 600 MHz Bruker Avance II HD equipped with a 5 mm cryoprobe. The samples were dissolved in deuterated chloroform .All reagents were ACS grade quality or higher, unless stated otherwise. All solvents were ACS grade quality except for those used for HPLC sample preparation and separations, which were HPLC gradient grade.1 and 2 is depicted in −2 s−1):dark cycle (14:10h) at 25 °C. Freshwater and estuarine strains were cultured in Z8 medium−1 NaCl , MN medium−1 of synthetic sea salts (Tropic Marin) (strain LEGE 11425). The culture media used for the growth of marine strains were additionally supplemented with 20 μg L−1 of vitamin B12.A list of the cyanobacterial strains used in this study for the prospection of Synechocystis salina LEGE 06099 and Synechococcus cf. nidulans LEGE 07171) were maintained in Z8 medium supplemented with 25 g L−1 NaCl. The microalga Chlorella vulgaris LEGE Z-001, also used as a target organism, was cultured in Z8 medium. Light and temperature conditions for the biological assay target strains were the same as those detailed above for the other cyanobacterial strains.Stock cultures of cyanobacterial strains used as target organisms in biological assays (2Cl2/MeOH (2:1). The resulting crude organic extract (1.18 g) was subjected to vacuum liquid chromatography (VLC) using silica gel 60  as stationary phase and a step-wise mobile phase gradient from 100% n-hexane to 100% EtOAc and then to 100% MeOH, yielding ten fractions. One of these  was found by 1H NMR to contain a set of downfield signals of interest and was further fractionated using a silica gel SPE cartridge , using a step-wise gradient from 10% EtOAc in hexane to 100% EtOAc. Subfractions eluting with 20% EtOAc in hexane  contained the 1H NMR peaks of interest, together with those corresponding to an aliphatic contaminant; these subfractions were pooled and subjected to another round of normal phase chromatography using an SPE cartridge , to yield 1  with enough purity for detailed 1D and 2D NMR analyses. The contaminant was still present in this sample, but to a much lesser extent than in the previous subfraction and did not interfere with the structure elucidation process using 1D and 2D NMR data, due to minimal superimposition with signals from 1.Lyophilized biomass (13.9 g) from multiple cultures of the cyanobacterium was repeatedly extracted by percolation with a warm (<40 °C) mixture of CHPlectonema cf. radiosum LEGE 06105 was extracted repeatedly with a 2:1 mixture of CH2Cl2/MeOH and the resulting crude extract (0.70 g) fractionated using VLC, as detailed for the colonial Synechococcales strain LEGE 10388. Upon inspection of the VLC fractions by 1H NMR, signals consistent with the aromatic moiety of a dehydroabietane were present in fraction E , which was further separated on a SPE cartridge  as detailed above. The fraction containing the 1H NMR signals  was subjected to two rounds of analytical-scale HPLC purification using a Synergi Fusion-RP column . The initial separation was carried out under isocratic conditions at 99% MeCN (aq) with a flow of 1.2 mL min−1 and the peaks eluting within tR = 1–7 min were re-injected into the HPLC system and separated isocratically using 75% MeCN (aq), to yield pure 1 .The lyophilized biomass (9.5 g) obtained from 1H NMR spectra of fractions from our library originating from the strains depicted in Plectonema cf. radiosum LEGE 06105 from which 1 had already been isolated) were carefully inspected for the presence of the characteristic set of signals that originate from 1–a doublet (J ~ 8 Hz), a doublet of doublets  and another doublet (J ~ 2 Hz) around δ7.16, δ7.00, and δ6.88 ppm, respectively. The fractions that contained such signals, as well as fractions of similar polarity  or crude extracts from the strains whose fractions did not contain the afore-mentioned aromatic signals were then submitted to LC-HRESIMS analysis. A portion of the material was dissolved in MeOH to a final concentration of 0.5 mg mL−1 (fractions) and 1 mg mL−1 (crude extracts) and injected (20 μL) into an HPLC system composed of an Accela PDA detector, Acella Autosampler and Acella 600 Pump (Thermo Fischer Scientific) to perform the chromatographic separation. For HRESIMS, the system was hyphenated with a LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fischer Scientific) controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1.0. A linear gradient from 40 to 100% MeCN (aq) over 30 minutes with a flow rate of 0.8 mL min−1 was used in conjunction with a Gemini C18 column . The capillary voltage of the electrospray ionization source (ESI) was set to 3.0 kV. The capillary temperature was 300 °C. The sheath gas and auxiliary gas flow rate were at 40 and 10 (arbitrary unit as provided by the software settings). The capillary voltage was −48 V and the tube lens voltage −247.79 V. To estimate the concentrations of 1 and 2 in the samples, calibration curves were carried out in the above system and conditions, using serial dilutions of commercial standards of 1  and 2  in MeOH. Extracted Ion Chromatograms (299.20 ± 0.01 m/z for 1 and 301.21 ± 0.01 m/z for 2) were used for the integration of the standard chromatographic peaks. The purity of the standard material was not taken into account for the estimations which, in the particular case of 2, may have led to an overestimation of up to 33% of the levels of the metabolite in the samples. An unknown difference in LC-HRESIMS conditions between: i) injections of commercial standard 1 and of the samples featured in 2), led to a delay of ~0.8 min in retention times. For clear interpretation of 2 was applied.Cyanobacterial biomass extraction and VLC fractionation, performed as described above, is routinely carried out in our laboratory as part of bioactive secondary metabolite isolation efforts e.g.Nodosilinea sp. LEGE 06104, Synechococcales cyanobacterium LEGE 10388, Nostoc sp. LEGE 07365, Leptolyngbya ectocarpi LEGE 11425 and Nodosilinea sp. LEGE 13457, using a commercial kit , according to the instructions provided for Gram-negative bacteria. The purified gDNA was inspected for integrity in an agarose gel stained with GelRed (Biotium) and was then used as a template for the amplification of a major portion of the 16S rRNA gene. PCR amplification was carried out as previously detailed57KT951669-KT951672, KU569325). The 16S rRNA gene sequences for the other cyanobacteria strains used in this study  was isolated from fresh biomass of is study  had been1 and 2 in this study, as well as publicly available sequences from selected  unicellular, colonial, filamentous non-heterocystous and heterocystous cyanobacteria were used to construct a phylogenetic tree. An alignment of the 16S rRNA gene sequences from the 10 strains producing 1, the 5 strains that were tested but for which 1 was not detected, the 56 selected cyanobacteria and from Chloroflexus aurantiacus strain J-10-fl (outgroup) was carried out using MAFFT through the Geneious v8.0 software package. The alignment was truncated to a core region (754bp). jModelTest2The 16S rRNA gene sequences from all the cyanobacteria strains screened for the presence of 1 and 2 up to 5 mg mL−1 were prepared in DMSO  and tested at 1% (v/v) in anticyanobacterial, antialgal and also in toxicity assays with Artemia salina (only for compound 1), which were carried out as detailed previouslySynechocystis salina LEGE 06155, Synechococcus cf. nidulans LEGE 07171 and the green microalga Chlorella vulgaris LEGE Z-001 were diluted in fresh culture medium to an OD 750 nm (200 μL) of ~0.1 (measurement with the microplate lid on) and added (198 μL) to microplate wells containing two microliters (2 μL) of the stock solution of 1 or 2. Optical densities (750 nm) were measured in a microplate reader  right after inoculation and following a 7-day exposure period. For the A. salina assay, aliquots (198 μL) of 15–20 freshly hatched nauplii (from dried cysts) in artificial seawater were added to the microplate wells that contained two microliters (2 μL) of the stock solution of 1. Mortality rates were determined after 48h. An antibiotic cocktail , was used as positive control for cyanobacteria, and potassium dichromate (4 μg mL−1) was used as a positive control for the green microalga and A. salina, while DMSO  was used as a negative control for all target organisms. When growth inhibition caused by 1 or 2 was observed, dose-response curves were produced and IC50 values calculated  using Prism 6 (GraphPad Software).Stock solutions of Nodosilinea sp. LEGE 13457 culture growing at 19 °C in Z8 medium for 120 days and a Nostoc sp. LEGE 06077 growing at 25 °C in Z8 medium for 21 days were filtered through 0.22 μm polyethersulfone syringe filters. The spent media were colorless and transparent, and there was no macroscopic evidence of cell senescence or lysis in any of the cultures. One-hundred milliliters (100 mL) of each of the supernatants were then liquid-liquid extracted with 200 mL of EtOAc. For each sample, the solvent in the combined organic layers was removed under reduced pressure and the residue was weighed and dissolved in a known volume of MeOH (<2 mL). Each solution was filtered through a 0.22 μm syringe filter  to remove insoluble material, injected (20 μL) and analyzed in the LC-HRESIMS system described above under the same chromatographic and detection conditions described for the intracellular fractions. Estimation of the levels of 1 was also carried out using a calibration curve as detailed for VLC fractions.A How to cite this article: Costa, M. S. et al. The conifer biomarkers dehydroabietic and abietic acids are widespread in Cyanobacteria. Sci. Rep.6, 23436; doi: 10.1038/srep23436 (2016).


Objective: To compare the maternal and umbilical cord serum selenium concentrations in Low and normal birth weight neonates. Materials and methods: A case-control study was carried out in Vali-Asr and Akbarabadi Hospitals (Jan. to Dec. 2013). Two groups; case group; 91 mothers who delivered a low birth weight (LBW) neonate and control group; 86 subjects who delivered a normal birth weight neonate were selected. Immediately after birth, 5 ml of maternal blood and umbilical cord blood were collected, and sent to laboratory to assay Se concentrations. To compare both groups' blood Se concentration, data were analyzed in SPSS 16.0.Results: Eighty six (48.6%) mothers with normal birth weight neonates and 91 (51.4%) mothers with low birth weight infants entered the study. Mean maternal mothers' age and mean maternal blood Se were 28.55 + 5.90 years and 79.3756+26.46915. A significant association was seen between maternal blood and cord blood Se level in control and case group  and . On the other hand no differences were seen between 2 groups' maternal blood Se level . Umbilical Cord blood Se concentration was not also different between case and control group .Conclusion: We found that maternal and umbilical cord blood Se concentrations were not different in low and adequate birth weight infants, however; umbilical cord Se concentrations were positively correlated with maternal blood Se concentrations. Selenium (Se) is a trace element that plays an important role in many humans' biochemical functions . SeleniuThe recommended daily intake of selenium is 50–200 µg. A significant correlation is seen between selenium dietary intake and its concentration in whole blood . Se and Many studies have demonstrated selenium deficiency in several obstetric and reproductive complications like preterm deliveries and premature infants , 2. BogdThe status of selenium is important in the neonates and trace nutrient deficiency may play an important role in the development of some problems . There iA case-control study was carried out in Vali-Asr and Akbarabadi Hospitals during January to December 2013. Two groups of mothers were selected for the study: case group; 91 mothers who delivered a low birth weight neonate (birth weight < 2500 gram) and control group; 86 subjects who delivered a normal birth weight neonate. The exclusion criteria were a past history of high risk pregnancy or systemic disease, prenatal Se supplements intake, twin or triplet delivery or any congenital malformations. 0C and sent to laboratory to assay mother's and neonate's blood Se level. Immediately after birth, 5 ml of maternal blood and 5 ml of umbilical cord blood  were collected, labeled, stored at -20 All maternal and neonatal demographic data including mother's age, parity, blood Se concentration, occupation status, neonatal sex, weight, UC blood Se level, were recorded in questionnaires. Participants gave informed consent before entering the study.To compare both groups' blood Se concentration, data were analyzed in SPSS 16.0. Independent T test, chi square and regression test were applied where applicable. The value of p < 0.05 was considered as level of significance.No intervention was performed in our study and patients’ data were considered confidential. Ethics approval for the study was obtained from the institutional review board of Tehran University of Medical Sciences .Eighty six (48.6%) mothers with normal birth weight neonates and 91 (51.4%) mothers with low birth weight infant sentered the study. 149 (84.7%) mothers were unemployed and 87 mothers (49.2%) had diploma and higher education, Mean maternal mothers' age and mean maternal blood Se were 28.5+ 5.90 years and 79.3+ 26.46, respectively. Of 177 babies, 89 of them (50.3%) were male. Demographic data is shown in A significant association was seen between maternal blood and cord blood Se level in control group . This relation was also found in case group . Our results showed that paired maternal-neonatal blood selenium concentrations were strongly correlated.On the other hand no differences were seen between two groups' maternal blood Se level . UC blood Se concentration in two groups were not significantly different . In case group, mothers' or cord blood Se concentration were not significantly correlated with parity, gestational age and birth weight. Details are shown in We could not find a significant correlation between maternal age and blood Se concentration, too .± 24.3798, 77.32 ± 26.12 µg/ml that means in our country umbilical cord selenium is in normal range. These ranges can also be used as reference data besides other reports  (In present study we compared the umbilical cord and maternal blood selenium concentrations in low and normal birth weight neonates. Based on our results, Mean umbilical cord Se levels in normal and low birth weight neonates' were 73.899  reports .In recenWe also found that Se levels in cord blood of both groups were lower than in their maternal blood, suggesting partially mother-fetus Se transfer .Our study demonstrated a significant positive relationship between umbilical cord and maternal blood selenium concentrations. Our finding was consistent to Dobrzynski et al. study that has been shown strong correlations between Se concentrations in the blood components of the umbilical cord and maternal blood . Sun et We found no significant difference between maternal and umbilical Se concentrations in two groups with different birth weights. It is possible that other influencing factors mask or overpower the role of Se on neonatal birth weight. The relationship of birth weight with the mother and newborn's Se concentrations at birth was previously studied and confirmed no differences between appropriate and small-for-gestational-age newborn infants . Also DoThere are some limitations to our study. Low selenium in early pregnancy may have a predictive value for fetal birth weight. Maternal Se serum concentrations were not measured in various gestational age-based groups that could provide valuable data. In addition, Se in the UC may be influenced by other factors like maternal weight or underlying disease that we did not consider in our study. We suggest more studies in different groups in different areas of Iran.We found that maternal and umbilical cord serum Se concentrations were not different in low and adequate birth weight infants. Additionally, maternal serum Se concentrations were positively correlated with umbilical cord Se concentrations.


Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq. The AG8 genome assembly used novel methods to produce a haploid representation of its heterokaryotic state. The whole-genomes of AG8, the rice pathogen AG1-IA and the potato pathogen AG3 were observed to be syntenic and co-linear. Genes and functions putatively relevant to pathogenicity were highlighted by comparing AG8 to known pathogenicity genes, orthology databases spanning 197 phytopathogenic taxa and AG1-IA. We also observed SNP-level “hypermutation” of CpG dinucleotides to TpG between AG8 nuclei, with similarities to repeat-induced point mutation (RIP). Interestingly, gene-coding regions were widely affected along with repetitive DNA, which has not been previously observed for RIP in mononuclear fungi of the Pezizomycotina. The rate of heterozygous SNP mutations within this single isolate of AG8 was observed to be higher than SNP mutation rates observed across populations of most fungal species compared. Comparative analyses were combined to predict biological processes relevant to AG8 and 308 proteins with effector-like characteristics, forming a valuable resource for further study of this pathosystem. Predicted effector-like proteins had elevated levels of non-synonymous point mutations relative to synonymous mutations (dN/dS), suggesting that they may be under diversifying selection pressures. In addition, the distant relationship to sequenced necrotrophs of the Ascomycota suggests the R. solani genome sequence may prove to be a useful resource in future comparative analysis of plant pathogens. Rhizoctonia solani is divided into several sub-species which cause disease in a range of plant species that includes most major agriculture, forestry and bioenergy species. This study focuses on sub-species AG8 which causes disease of cereals, canola and legumes, and compares its genome to other R. solani sub-species and a wide range of fungal and non-fungal species. R. solani is unusual in that it can possess more than one nucleus per cell. The multiple nuclei and sequence mutations between them made assembly of its genome challenging, and required novel techniques. We observed signs that DNA sequences originating from multiple nuclei in AG8 exhibit a high frequency of single nucleotide polymorphisms (SNPs) and more SNP diversity than most fungal populations. These SNP mutations also have similarities to repeat-induced point mutations (RIP). Moreover in AG8, RIP-like SNPs are not restricted to intergenic regions but are also widely observed in gene-coding regions. This is novel as RIP has previously only been reported in repetitive DNA of distantly-related fungi that have only a single nucleus per cell. We generated a list of 308 genes with similar properties to known plant-disease proteins, in which we found higher rates of non-synonymous mutations than normal.The fungus  Rhizoctonia solani  is a globally-distributed, soil-borne fungal phytopathogen employing a necrotrophic lifestyle. Collectively, the host-range of the R. solani species spans numerous plant species vital to the agriculture, forestry and bioenergy industries, including but not limited to: wheat, rice, barley, canola, soybean, corn, potato and sugar beet R. solani has been observed to increase in incidence and severity with increased adoption of conservation (no-till) farming techniques R. solani as a significant threat to global food security and other agro-forestry industries.R. solani species complex is comprised of fourteen anastomosis groups (AGs), most of which are reproductively incompatible with each other and are numbered AG-1 through AG-13. The ‘bridging isolate’ AG-BI is the exception, being compatible with multiple AGs The R. solani AG8, causative agent of bare patch of wheat, barley and legume species R. solani on wheat and barley production is estimated upwards of $77 million per annum and bare patch also remains a major problem for the production of wheat and other crops in the US Lupinus spp. (lupin) Medicago truncatula (barrel medic) ArabidopsisR. solani with genome sequences available are the biotrophic smuts Moniliophthora spp. R. solani is expected to be of importance in filling gaps in our knowledge of plant pathogen biology, which apart from rusts and smuts, is skewed towards the ascomycetes.Our study presents a comprehensive genome assembly and functional analysis of R. solani have also recently become publicly available, formerly being limited to EST libraries of AG1-IA R. solani AGs presents new opportunities for comparative genomics between R. solani anastomosis groups. The most comprehensive whole-genome study to date has been that of the rice pathogen AG1-IA http://www.rsolani.org with kind permission from Cubeta et al.), however a nuclear gene dataset and genome survey have not yet been published Significant genomic resources for other AGs of R. solani AG8 exists as a multi-nuclear heterokaryon in which individual R. solani cells may carry multiple nuclei and copy number can vary between cells. An average of 8 nuclei per cell has previously been observed in AG8, but numbers commonly ranged from 6 to 15 R. solanide novo genome assembly [available from GenBank: AVOZ00000000] and we also describe novel bioinformatic approaches used to overcome these challenges. This study also compares whole-genome synteny between R. solani anastomosis groups  and uses comparative genomics techniques to highlight genes and functions unique to AG8 and AG1-1A. Predicted properties of AG8 proteins have been leveraged to generate a list of 308 ‘effector-like’ genes that may be related to plant-pathogenicity. These collective resources will be important for further experimentation in this pathosystem.R. solani genome posed considerable challenges for genome assembly. To overcome these challenges we developed a novel genome assembly pipeline  and was abundant with LAGLIDADG and GIY-YIG intronic endonucleases. This is consistent with recent reports for the mitochondrial genomes of AG3 and AG1-IB, which are of similarly large sizes (235.8 kbp and 162.8 kbp respectively) and possess high abundances of endonucleases A single scaffold (Scaffold_77) of ∼140 kbp in length was predicted to represent the mitochondrial genome. The ends of the mitochondrial scaffold sequence were confirmed to be physically joined in a circular configuration by PCR  or Medicago truncatula seedlings separated by a permeable nitrocellulose membrane. This enabled collection of fungal tissue whilst reducing plant tissue contamination to negligible amounts. Alignment of RNA-seq data and proteins from related fungal species and pathogenicity gene databases were combined with in silico gene predictions to automatically predict gene structure annotations, which were then manually curated.To enable discovery and accurate annotation of protein-coding genes present in the AG8 assembly, particularly those expressed in the presence of plant tissues, three high-coverage Illumina RNA-seq libraries were aligned to the genome to delineate gene exon boundaries. To obtain transcript data for as many genes as possible, the libraries included one library of AG8 undergoing vegetative growth in culture and two “infection-mimicking” libraries. These libraries were derived from AG8 grown on water agar containing wheat  specifically conserved within the Basidiomycota , P. graminis f. sp. tritici  and Ustilago maydis (595). However AG8 was within a similar range to the average predicted secretome count across all fungi , which was predominantly comprised of necrotrophs.The density of gene-coding regions was relatively even throughout the assembled genomic scaffolds ii, withR. solani AG8 in the infection process, we predicted the functions of its 13,964 genes by comparison to the CAZy (Carbohydrate-Active enZyme) and Pfam (Protein family) databases. In total, we assigned CAZy annotations to 1,137 genes   PD40 beta-propeller [Pfam: PF07676]; Ankyrin [Pfam: PF13606] and leucine-rich repeats [Pfam: PF00560]. The most abundant peptidase domain was the CHAT (Caspsase HetF-Associated with TPRs) domain [Pfam: PF12770] which may be involved in programmed cell death. In summary, R. solani AG8 possesses a number of gene families whose members have a broad range of potential biological roles, for example those encoding caspases or protein-binding functions. Further study would be required to determine their relevance to plant pathogenicity or other lifestyle characteristics. These findings do however indicate that R. solani AG8 possesses a large number of carbohydrate-binding lectins of unknown function as well as a battery of CAZymes suitable for consumption of carbohydrates commonly found in cereal hosts, but also is geared towards the degradation of pectin.Pfam domains in R. solani AG8 and AG1-IA primarily infect two different hosts - wheat and rice respectively. Differences between them in their relative abundances of functionally-annotated genes may reveal important differences in their infection strategies. Overall, fewer Pfam domains were found to be significantly higher in AG1-IA than in AG8. In AG1-IA  and the bacteria Dickeya dadantii  Interestingly, AG8 had more members of two Pfams similar to ricinB lectins R. solani AG8 proteome  had a positive fold-change and 17 had a significantly higher relative expression in-planta   of the 308 predicted ‘effector-like’ genes were randomly selected and their mRNA expression relative to a set of 7 constitutively expressed genes was compared between Repeat-induced point mutations (RIP) are fungal-specific SNP mutations previously reported to be restricted to the filamentous Ascomycota (Pezizomycotina) R. solani to exhibit a bias towards CpG but not TpCpG. However, it should also be noted that hypermutations of CpG may also be caused by widely conserved processes involving the methylation of cytosine to 5-methylcytosine (5mC) and subsequent deamination which converts 5mC to thymine The small number of studies looking for RIP-like mutations in the Basidiomycota do not exhibit the characteristic CpA mutation bias observed in the Pezizomycotina Analysis of nucleotides immediately flanking heterozygous C↔T SNP sites in AG8 exhibited a CpG dinucleotide bias consistent with previous observations of ‘RIP-like’ cytosine hypermutations in the Basidiomycota Regardless of whether the underlying process is similar to RIP or not, CpG-biased hypermutation is likely to play an important role in the evolution of the AG8 genome. RIP has been recently proposed to have the potential to randomly introduce nonsense mutations, converting longer secreted proteins into small, secreted proteins thus making them gradually more effector-like The density of heterozygous SNP mutations within AG8 was compared to SNP densities between the genome assemblies of AG8 and AG1-IA, AG1-IB and AG3 . SNP denR. solani AG8 into a wider context. Similar to AG8, the Basidiomycete stripe rust fungus Puccinia striformis is heterokaryotic but exhibits a lower SNP density within its genome assembly of 5.98 SNPs/kbp P. striformis is binucleate and therefore only possesses 2 nuclei per cell as opposed to the 6–15 nuclei that have been observed within cells of R. solani AG8 Lentinula edodes) was reported to be 4.6 SNPs/kbp  Blumeria graminis), the SNP rate observed between pairs of isolates was lower at 1 SNP/kbp Rhizophagus irregularisLeptographium longiclavatumCoccidioides immitis and C. posadasii reported a rate of 23.7 SNPs/kbp relative to the C. immitus RS reference genome assembly  R. solani AG8  and much higher than that observed within a population of a multinucleate non-pathogen (R. irregularis). We speculate that the combination of multinuclearity and selection pressures relating to pathogenicity may be driving the accumulation of widespread heterozygous SNP diversity in R. solani AG8.Comparisons between individual genomes and fungal population genetics studies were also used to place the SNP diversity within lani AG8 . In concRhizoctonia solani AG8 . Previous observations of RIP in haploid fungal genomes have only reported its activity upon repetitive sequences R. solani AG8 isolate WAC10335 was isolated from lupin and provided by the Department of Agriculture and Food of Western Australia (DAFWA). Anastomosis group was confirmed by ribosomal ITS sequences and host-range was confirmed by inoculation assays on wheat, lupin and Medicago truncatulaR. solani does not readily produce sexual or asexual spores thus single spore isolation was not possible, therefore a single rapidly growing hyphal tip was excised from a colony growing on PDA and transferred to water agar containing 250 µg/ml cefotaxime. Pathogenicity of the resulting culture was confirmed as equivalent to the original. A pure in vitro culture of R. solani was produced by incubation in PDB at 25°C with gentle shaking for 7 days. Hyphae were filtered from the culture through sterile Miracloth and rinsed with sterile water. DNA was purified by grinding hyphal tissue in liquid nitrogen and suspension in DNA extraction buffer (2% (w/v) CTAB, 1.4 M NaCl, 0.2% (v/v) β-mercaptoethanol, 20 mM EDTA and 100 mM Tris-HCl) and mixing at 60°C. Following two rounds of chloroform/isoamylalcohol extraction, the aqueous supernatant was treated with RNase I (Invitrogen) at 20 µg/ml. The DNA was purified through an additional two rounds of chloroform/isoamylalcohol extraction and precipitated by adding 0.1 volumes of 3M NaOAc (pH 5.2) and 0.6 volumes isopropanol. The resulting DNA pellet was resuspended in 10 mM Tris-HCl (pH 8.0) buffer and quantitated by Qubit (Invitrogen) and BioAnalyser prior to sequencing.Two Illumina paired-end libraries of genomic DNA were sequenced, with 75 and 100 bp read lengths and median insert lengths of 250 bp and 300 bp respectively. Three Illumina genomic mate-pair libraries with insert lengths of 2 kbp, 5 kbp and 10 kbp were also obtained. Paired-end libraries were combined and trimmed for sequencing adapter/primer sequences, low-quality (<Q30), and low-complexity sequences via CutAdapt v1.1 Medicago or wheat infection-mimicking conditions. Under infection-mimicking conditions, AG8 was cultured on a film of nitrocellulose overlaid on water agar containing young sterilised Medicago truncatula or wheat seedlings. After seven days incubation at 25°C the film and hyphae were removed, ensuring negligible plant contamination in subsequent RNA extractions with TRIzol . Two sequencing libraries were generated per mRNA library, with 200 bp and 500 bp insert sizes. Transcript libraries were trimmed for contaminant sequences via Cutadapt v1.1 as per genomic reads.For the purpose of gene annotation, Illumina paired-end libraries of 100 bp read lengths were obtained from 3 mRNA libraries derived from AG8 grown under: vegetative conditions (7 days at 25°C in PDB with gentle shaking) (non-infection) and; de novo assembly methods. To this end, a novel pipeline was developed for AG8  Exon splice-junctions were determined by aligning six RNA-seq libraries to the AG8 assembly via TopHat 2.0.4  in silico gene prediction via GeneMark-ES v3.2.3 −3, top 20 hits) and the taxonomic distribution of their corresponding lowest-common ancestor taxa was summarised with MEGAN5  The library-specific and the combined transcriptome assemblies were used to determine exon structure using PASA r2012-06-25 . The output of PASA was passed to the EvidenceModeller r2012-06-25 auto-annotation pipeline R. solani AG8 and other sequenced fungi from JGI IMG v4 05, alg.-conn. = 0.1, coverage = 0.5, percent_identity = 25, adaptive_similarity = 0.95, inc_pairs = 1, inc_singles = 1, selfblast = 1, unambiguous = 0) R. solani AG8 and 86 other fungi ; predicted molecular weight ≤30 kDa; and ≥4 cysteine amino acids. Molecular weights and amino-acid compositions were predicted with Bio::Tools::SeqStats (BioPerl) Heterozygous SNP mutations derived from genomic read alignment to the final genome assembly, as described above, within all genes and predicted ‘effector-like’ genes were tested for: 1) stop-codon bias; 2) gene structure location bias with SNPeff in vitro grown PDB culture. Wheat samples were inoculated with millet seeds pre-infect with WAC10335 and grown in pots of vermiculite for 7 days at 24°C. Wheat seeds were surface-sterilised and germinated on moist filter paper at 4°C for 4 days, then planted into pre-infected vermiculite pots and covered by a layer of fresh fine vermiculite. The pots were transferred to a growth room at 16°C and 12 hours light/day (150 µmol.m−2.s−1) for 7 days. Plants were harvested and root and above ground tissues separated. RNA was extracted from root tissue using Trizol reagent (Sigma) according to the manufacturer's instructions and cDNA produced using Superscript III (Invitrogen) following the manufacturer's instructions. Quantitative PCRs used SsoFast EvaGreen Supermix (BioRad).Gene expression of selected genes .A total of 29 out of 308 predicted ‘effector-like’ pathogenicity genes were selected for testing based on their assigned functional annotations. Seven control genes were also selected based on stable expression, averaging ≥70 FPKM and ≤0.1× fold change between libraries, across the three RNA-seq libraries discussed in this study and/or for putative functions suggesting stable expression patterns (e.g. actin and tubulin). Primer pairs were designed from coding-exon sequences (CDS) using primer3 Figure S1R. solani AG8 proteins by BLASTP to NCBI Protein. Higher level taxa contain protein counts both for widely-conserved R. solani AG8 proteins for which that taxon has been assigned as its lowest-common-ancestor, as well as cumulative counts for all lower-level taxa contained within.Summary of “lowest-common-ancestor” taxa assigned to 8,449 (TIF)Click here for additional data file.Figure S2R. solani AG8 compared to species from JGI Integrated Microbial Genomes.Pfams abundant in (TIF)Click here for additional data file.Table S1R. solani anastomosis group host-ranges (A) and supporting publications (B).Summary of known (XLSX)Click here for additional data file.Table S2R. solani AG8 genome assembly.Heterozygous SNP (A) and dinucleotide (B) mutations in the (XLS)Click here for additional data file.Table S3R. solani AG8, AG1-IA and AG3 depicted in Whole-genome alignments between assemblies of (XLSX)Click here for additional data file.Table S4R. solani AG8. (A) Summary tables of repeat proportions within the R. solani AG8 genome assembly. (B) Curated classifications of repeat types. (C) Genomic coordinates of repetitive DNA regions mapped by RepeatMasker. (D) Repeat predictions made via TransposonPSI. (E) Consensus sequences of de novo predicted AG8 repeat families in FASTA format.Repetitive DNA content of (XLS)Click here for additional data file.Table S5R. solani AG8 genome assembly. (A) ncRNA predictions made via Infernal and their genomic coordinates. (B) Summary of Infernal predictions. (C) tRNA predictions made via tRNAScan.Summary of non-coding RNA regions predicted within the (XLSX)Click here for additional data file.Table S6R. solani AG8 genes to NCBI Protein database by BLASTP.Best matches of manually curated (XLSX)Click here for additional data file.Table S7R. solani AG8 and 86 fungal species.Comparison of SignalP 4.1 predictions between (XLSX)Click here for additional data file.Table S8R. solani AG8 genome. (A) Annotations made via the CAZymes Analysis Toolkit. (B) Manual curation of data presented in (A). (C) Summary counts of CAZyme content of R. solani AG8.Carbohydrate-active enzymes (CAZymes) present in the (XLSX)Click here for additional data file.Table S9R. solani AG8 and species of the JGI Integrated Microbial Genomes database. Data is summarised for Pfams abundant in AG8 in (A) and presented in full in (B).Pfams comparisons between (XLS)Click here for additional data file.Table S10R. solani AG8 and AG1-IA. (A) Counts of genes with Pfam annotations for both AGs. (B) Pfams significantly enriched in AG1-IA relative to AG8. (C) Pfams significantly enriched in AG8 relative to AG1-IA. (D) Full Pfam annotations for AG1-IA. (E) Full Pfam annotations for AG8.Comparison of Pfam annotations between (XLSX)Click here for additional data file.Table S11R. solani AG8 and 197 species. Compared species include fungal, oomycete, insect and prokaryotic species exhibiting a wide range of pathogenicity phenotypes.Summary of orthology relationships between (XLSX)Click here for additional data file.Table S12R. solani AG8 and data supporting their prediction.Predicted ‘effector-like’ plant pathogenicity genes of (XLSX)Click here for additional data file.Table S13R. solani AG8 proteins to known plant pathogenicity motifs.Summary of matches in (XLSX)Click here for additional data file.Table S14in vitro culture. (A) Raw CT values for all genes tested. (B) Genes with significantly up-regulated mRNA expression in planta.Summary of relative mRNA expression of selected predicted ‘effector-like’ genes. Selected genes were isolated from 7 dpi infected wheat versus 7 day old (XLSX)Click here for additional data file.Table S15R. solani AG8.Multiple alignments of the most abundant CAZyme families in (XLSX)Click here for additional data file.Text S1R. solani AG8.Methods used to assemble a haploid representation of the multinucleate genome of (DOCX)Click here for additional data file.Text S2R. solani AG8. Confirmation of circularity and correct scaffolding across internal gap of mitochondrial Scaffold_77 by PCR and its predicted mitochondrial genes and non-coding RNA regions.The mitochondrial genome of (DOCX)Click here for additional data file.Text S3De novo MEME search for novel motifs among the 308 R. solani AG8 effector candidates.(TXT)Click here for additional data file.


Amorphous perfluoroalkenyl vinyl ether polymer devices can store a remarkably powerful electric charge because their surface contains nanometre-sized cavities that are sensitive to the so-called quantum-size effect. With a work function of approximately 10 eV, the devices show a near-vertical line in the Nyquist diagram and a horizontal line near the −90° phase angle in the Bode diagram. Moreover, they have an integrated effect on the surface area for constant current discharging. This effect can be explained by the distributed constant electric circuit with a parallel assembly of nanometre-sized capacitors on a highly insulating polymer. The device can illuminate a red LED light for 3 ms after charging it with 1 mA at 10 V. Further gains might be attained by integrating polymer sheets with a micro-electro mechanical system. Storage systems for electrical energy have been investigated extensively over the past three decades1234343). A common requirement for electric storage is a surface with nanometre-sized cavities and high electrical resistance. We assumed a surface structure consisting of a distributed constant-equivalent circuit of resistance and capacitance, and one that is analogous to the active carbons in EDLCs.To develop solid supercapacitors without liquid solvents, we studied the capacitance of nanocrystalline de-alloyed Si-Al alloy ribbons68TM  with nanometre clusters containing the organic siloxanes. The fluorine atom is well known as the most electronegative element in the contact electrification seriesWe report here about the superior electric storage on a polymer with high electrical resistance. We selected amorphous perfluoroalkenyl vinyl ether polymer (APVEP) film, CYTOP2, lit the LED for 3 ms  at 1 mHz  + 4.48(ФCPD). This means that they can store electric charges; the same is true for the TiO2–x layerWe investigated the surface characteristics to understand the microscopic origin of the APP’s higher electric storage. IV drop at long distance from electrodes.We now turn to an explanation of the superior electric storage on the APP. The APP structure consists of PVDF main chains containing C = O and N-H radicles18We demonstrated the storage potential for the APP given its nanometre-sized cavities on an uneven surface. The APP has a high work function, measured at 10.13 eV, and it succeeded in illuminating an LED. We inferred that the potential for electric storage is related to the quantum-size effect derived from the TF theory. The integration of the film with a micro-electro mechanical system is likely to provide even higher levels of charge storage for portable electric application.The APVEP film with nanometre-cluster was prepared by doping a 3-aminopropyl (triethoxy) silane-coupling reagent into the APP. Sheet specimens with a thickness of 15 μm were fabricated on the Si substrate by spin-coating. The specific density, glass-transition temperature, and water-adsorption rate was 2.03, 471 K, and 0.01%, respectively−4 W was used to verify the electric storage.Scanning Kelvin probe-force microscopy  based on the measurement of electrostatic force gradient was applied to measure the absolute electrical potential between the Pt-coated cantilever tip at 0 and 20 V and the APP surface as the work-function difference. The DC charging/discharging behaviour was analysed at 10 V, 1 pA~1 mA for ~300 s at room temperature, with a complex impedance between 1 mHz and 1 M Hz and 10 mV, using the galvanostatic charge/discharge on a potentiostat/galvanostat . A red LED lump with standard power of 2 × 10How to cite this article: Fukuhara, M. et al. Superior electric storage on an amorphous perfluorinated polymer surface. Sci. Rep.6, 22012; doi: 10.1038/srep22012 (2016).


They used the Normalized Difference Vegetation Index (NDVI), a satellite-derived measure, to determine greenness within 100 m of each mother’s home during pregnancy.The authors reported that quartile increases in greenness were associated with higher birth weight for babies born at term (37 weeks or later) and decreased likelihood of having a moderately or very preterm birth, or a small-for-gestational-age baby. Because air and noise pollution can affect birth outcomes, they used detailed models to adjust for these environmental exposures. But in this study living in more “walkable” neighborhoods was associated with lower birth weight and increased risk of preterm birth.“We thought [noise and air pollution] were the causal pathways of how greenness influenced birth outcome,” says lead author Perry Hystad, an assistant professor of environmental and occupational health at Oregon State University. “We didn’t find that at all … which really suggests other pathways, particularly psychosocial or psychological factors, may be important.” Of the walkability findings, he says, “This really underscores the complexity of how the built environment can influence health and why more research is needed that examines multiple exposures together, rather than each in isolation.”“The study was well designed and well conducted,” says Payam Dadvand, an assistant research professor at the Centre for Research in Environmental Epidemiology in Barcelona, who was not involved with the study. “Although the study faced limitations in accessing data on some important covariates like indicators of individual socioeconomic status, a wide range of sensitivity analyses conducted by the authors can provide confidence in the findings.”“This paper did a thorough job of controlling for confounding by the built environment, which strengthens the overall evidence that greenness can positively influence birth outcomes,” says Geoffrey Donovan, a research forester with the Portland Forestry Sciences Laboratory of the U.S. Department of Agriculture Forest Service, who also was not involved with the study.The present study used an index of greenness ranging from –1 to 1, finding a threshold level of 0.15 above which greenness was associated with improved birth outcomes. No associations were observed under 0.15, which corresponded to dense urban areas, along major roadways, and the downtown core.“I was intrigued by this finding,” says Donovan. “As far as I know it is the only paper to have done this. My only mild concern is that at the extreme ends of a data set, statistical relationships often have a lot of variability, so it’s best to be very cautious when making inferences. … I’d like to see it replicated before we start talking about policy implications.”Hystad agrees. “This is where replicating the study in other cities with different NDVI levels—both very low and very high—will be important,” he says.“Another interesting observation of this study is the suggestion that ethnic minorities might be less likely to benefit from greenness,” says Dadvand. “This is consistent with our study in Bradford, United Kingdom,Howard Frumkin, dean of the University of Washington School of Public Health, says greenness is a highly innovative “exposure” in the environmental health sciences. “It’s innovative in that it relates to health promotion, not health threat, and it is a different kind of exposure—not a chemical, but a hard-to-quantify concept of greenness,” he says. “It’s also very actionable; it may be easier to promulgate tree-planting policies in cities than to bring chemical exposures down to very low levels. This is really a game-changing development in the field.”


We aimed to evaluate the association of birth weight SDS with insulin resistance, blood pressure, and auxology in children and adolescents born 23–42 weeks of gestation.We studied 143 singleton children and adolescents aged 9.3 ± 3.3 years (range 2.0–17.9 years). Clinical assessments included insulin resistance measured by HOMA2-IR, auxology, and blood pressure from sphygmomanometer measurements. Continuous associations were examined, and stratified analyses carried out. For the latter, participants were divided into those of below-average birth weight  and above-average birth weight .Irrespective of gestational age, lower birth weight SDS was associated with progressively greater HOMA2-IR (p<0.0001) and higher fasting insulin concentrations (p<0.0001). Decreasing birth weight SDS was associated with higher systolic (p = 0.011) and diastolic (p = 0.006) blood pressure. Lower birth weight SDS was also associated with decreasing stature (p<0.010). The BABW group was ~40% more insulin resistant than AABW participants (p = 0.004), with the former also displaying fasting insulin concentrations 37% higher (p = 0.004). BABW participants were 0.54 SDS shorter than those of higher birth weight (p = 0.002). On average, BABW participants had not met their genetic potential, tending to be shorter than their parents (p = 0.065). As a result, when corrected for parents' heights, BABW participants were 0.62 SDS shorter than those born of higher birth weight (p = 0.001). Sub-group analyses on participants born appropriate-for-gestational-age (n = 128) showed that associations of birth weight SDS with both insulin resistance and stature remained .Decreasing birth weight SDS  is associated with adverse metabolic profile and lower stature in children and adolescents. Numerous studies have shown that low birth weight is associated with short- and long-term adverse health outcomes –4. Childth centile, while the same weight at 41 weeks would place him in the 5th centile.However, most studies have examined associations with birth weight expressed in kilograms or grams. These are less accurate measures of health at birth as they do not take into account the gestational age of the individual. For example, among those born at term, a 3.0 kg boy born at 37 weeks of gestation has a birth weight in the 50Thus, studies using standard deviation score (SDS) rather than kilograms provide a more accurate assessment of birth weight effects, as it accounts for gestational age. As a result, we aimed to assess the association of birth weight SDS with insulin resistance, blood pressure, and auxology in children and adolescents born across the gestational age range.Ethics approval was provided by the Northern Ethics Committee . Written informed consent was obtained from parents or guardians. Verbal and written informed consent was also obtained from participants, except for the youngest children unable to write who provided verbal consent in the presence of parents or guardians. This study was performed in accordance with all appropriate institutional and international guidelines and regulations for medical research, in line with the principles of the Declaration of Helsinki.Children and adolescents were recruited for a study examining the effects of prematurity on endocrine regulation of growth . All parClinical assessments were carried out during a single visit to the Maurice and Agnes Paykel Clinical Research Unit , after an overnight fast. Heights and weights were measured on parents and participants, with body mass index (BMI) subsequently calculated. Each child's birth weight, height, and BMI were transformed into SDS ,14. Mid-Venous blood samples were collected and frozen at -80°C for later analysis. Plasma glucose was measured using an automated random-access analyser  with inter-assay coefficient of variation 1.2%. Insulin levels were determined by enzyme immunoassay  with inter-assay coefficient of variation <5%.www.dtu.ox.ac.uk/homacalculator/). Blood pressure was measured at the clinic by the same researcher using a standard mercury sphygmomanometer, with an appropriately-sized cuff on the non-dominant arm. Measurements were recorded on each child while seated and after a 5-minute rest. All subjects had pubertal development assessed by the same experienced paediatric endocrinologist. Participants were defined as pubertal by having Tanner stage 2 breast development in girls and testicular volume >3 ml in boys or evidence of adrenarche.Homeostasis model assessment of insulin resistance (HOMA2-IR) was obtained from fasting insulin and glucose values using the HOMA2 calculator v2.2.3  and BMI SDS. Stratified analyses were also carried out comparing participants of below-average birth weight  versus those of above-average birth weight  using linear mixed regression models as described above. Baseline comparisons between the two groups were carried out using one-way ANOVA and Fisher's exact tests. Note that the interaction between birth weight SDS and sex was assessed for all outcomes. In addition, sub-group analyses were carried out separately for prepubertal and pubertal participants, as well as only on those participants who were born appropriate-for-gestational-age (birth weight >-2 and <2 SDS).Univariate analyses were carried out in Minitab v.16 , while multivariate analyses were performed in SAS v.9.3 . All statistical tests were two-tailed and maintained at a 5% significance level. Demographic data are presented as means ± standard deviation (SD). Outcome data from stratified analyses are presented as model-adjusted means , with associated 95% confidence intervals.We studied 143 children and adolescents aged 9.3 ± 3.3 years (range 2.0–17.9 years), including 69 males (48%). Participants had a mean birth weight of-0.11 ± 1.17 SDS (range-3.39–2.90 SDS) spread over a Gaussian distribution . Sixty , fasting insulin concentrations , and diastolic blood pressure . Lower birth weight SDS was also correlated with lower height SDS  and lower height SDS–MPHSDS .Multivariate models corroborated the above findings. Lower birth weight SDS was associated with progressively greater HOMA2-IR (p<0.0001) and higher fasting insulin concentrations (p<0.0001) . DecreasSub-group analyses on the 93 prepubertal children showed that decreasing birth weight SDS was strongly associated with higher HOMA2-IR  and fasting insulin concentrations , and tended to be associated with higher systolic  and diastolic  blood pressures. Among the 50 pubertal participants, lower birth weight was associated with decreasing height SDS , decreasing height SDS–MPHSDS , increasing diastolic blood pressure , and increasing pulse rate .BABW and AABW participants were of similar age and sex ratio, as well as having a similar proportion of participants in puberty or born preterm . The BABBABW participants were 0.54 SDS shorter than those of higher birth weight . However, pubertal BABW participants had pulse rate that was significantly higher than the AABW group .Sub-group analyses examining only participants born appropriate-for-gestational-age showed that associations of birth weight SDS with insulin resistance and height remained, although attenuated . There wIndependently of birth weight, gestational age impacted on growth as previously reported . ShorterWe showed that decreasing birth weight SDS was associated with an adverse metabolic profile  as well as shorter stature in childhood and adolescence. While these findings were independent of gestational age, pubertal status, current adiposity, or parents' heights, decreasing gestational age was associated with progressively worse growth. Thus, the shortest children were those born both preterm and with low birth weight SDS. Importantly, subgroup analyses showed associations between birth weight SDS within the normal range and both insulin resistance and growth.Individuals born of low birth weight or SGA have been shown to have reduced insulin sensitivity ,7,16 andLow birth weight is associated with higher blood pressure in adolescence  and incrOne study has examined the final heights of individuals born of extremely low birth weight (500–999 g), and found that, although shorter, their stature was consistent with parents' heights . In contWe did not observe any association between birth weight and BMI SDS or total body fat percentage. These findings are not particularly surprising, as the increased risk of overweight and obesity in low-birth-weight subjects occurs in later life and rarely before early adulthood. Increasing obesity has been reported in adults who were born of lower birth weight SDS  or preteAlthough the data are conflicting, studies have shown an association between preterm birth and insulin sensitivity throughout the life course . In thisThe main limitation of our study was the wide age range of study subjects, including participants who were both pubertal and prepubertal. As puberty is known to be associated with increased insulin resistance  and chanIn conclusion, decreasing birth weight SDS across the gestational age range is associated with adverse metabolic profile and shorter stature in children and adolescents. In particular, there was a progressive reduction in insulin sensitivity with decreasing birth weight SDS across the normal range.


These findings suggest that compared to a traditional Cantonese diet high in cereals, eggs, and Cantonese soups, a diet high in fruits, nuts, and Cantonese desserts might be associated with a higher birth weight, while a varied diet might be associated with a greater birth weight and also a decreased risk of having a SGA baby.There was limited evidence revealing the association of Chinese maternal dietary patterns with fetal growth. We aimed to examine the relationship of maternal dietary patterns during pregnancy to neonatal birth weight and birth weight for gestational age in a Chinese population. A total of 6954 mother-child pairs were included from the Born in Guangzhou Cohort Study. Maternal diet during pregnancy was assessed using a self-administered food frequency questionnaire. Cluster analysis was used to identify dietary patterns. The following six dietary patterns were identified: “Cereals, eggs, and Cantonese soups” , “Dairy” , “Fruits, nuts, and Cantonese desserts” , “Meats” , “Vegetables” , and “Varied” . The mean neonatal birth weight Z scores of women in the above patterns were 0.02, 0.07, 0.20, 0.01, 0.06, and 0.14, respectively. Women in the “Fruits, nuts, and Cantonese desserts” and “Varied” groups had significantly heavier infants compared with those in the “Cereals, eggs, and Cantonese soups” group. Compared with women in the “Cereals, eggs, and Cantonese soups” group, those in the “Varied” group had marginally significantly lower odds of having a small-for-gestational age (SGA) infant after adjustment for other confounders (OR 0.77, 95% CI 0.57, 1.04,  Fetal growth is an important determinant of health and disease in a human’s entire lifetime. Studies suggest that restricted fetal growth is associated with an increased risk of childhood morbidity and chronic diseases in adulthood ,2,3,4, wAdequate maternal nutrition during pregnancy is vital for fetal growth . As a moThus far, there are several studies that have investigated the relationships between maternal dietary patterns and fetal growth ,24,25,26Hence, this study aims to examine the effect of different maternal dietary patterns during pregnancy on fetal growth in a Chinese population.The present study was based on the Born in Guangzhou Cohort Study (BIGCS), an ongoing population-based prospective cohort study from fetal life onwards that has been described in detail previously . BrieflyFor the present study, pregnant women, who were recruited between February 2012 and April 2015, and their infants were included as study subjects. A flowchart of the selection process of the study population is shown in Information about dietary intake was obtained with a self-administered food frequency questionnaire (FFQ) at the Q2 interview. We evaluated the validity and reproducibility of this FFQ elsewhere, and the FFQ has been shown to be a valid and reliable instrument to assess dietary intake . This FFInformation on birth weight and gestational age was obtained from the medical charts. Birth weight Z score was calculated on the basis of the birth weight reference in Guangzhou . Small-f2) was calculated by dividing weight in kilograms by height in meters squared. According to the Guidelines for Prevention and Control of Overweight and Obesity in Chinese Adults [2 (underweight), BMI 18.5–23.9 kg/m2 , BMI ≥ 24 kg/m2 (overweight or obese). The diagnosis of gestational diabetes mellitus (GDM) was made based on the outcomes of GDM screening [Several socio-demographic, health, and medical history characteristics were considered as possible confounders. Information regarding maternal age, education level , monthly income , parity , passive smoking during pregnancy , alcohol drinking during pregnancy , folic acid supplement use , pre-pregnancy maternal body measurements  was obtained from the Q1 questionnaires. Pre-pregnancy body mass index (BMI) . The K-means method was applied to classify participants into a predetermined number of mutually exclusive groups by comparing Euclidean distances between each participant and each cluster center in an interactive process until no further changes occur [Percentages of weekly intake of the above thirty food groups were taken into account to construct dietary patterns. Cluster analysis was performed using the k-means procedure in the R version 3.2.3  were reported for all socio-demographic factors , health characteristics , and medical history characteristics (GDM). These variables were cross- tabulated by dietary patterns, and significant differences were assessed by ANOVA for continuous variables or chi-square tests for categorical variables.Descriptive statistics .Six dietary patterns were identified . We labep < 0.05 for “Fruits, nuts, and Cantonese desserts”, β 0.06, 95% CI 0.05, 0.20, p < 0.05 for “Varied”, respectively) in the crude model. The results were consistent with those in the adjusted models. None of the other dietary patterns were significant predictors of birth weight Z score neither in the crude nor adjusted models.p < 0.05). The association was marginally significant after adjustment for socio-demographic and other confounders . No significant difference in the risk of having a LGA infant was observed among subjects in these patterns neither in the crude nor adjusted models.In the present large prospective study, we investigated the association of maternal dietary patterns during pregnancy with neonatal birth weight Z score as well as the risk of having a SGA or LGA baby in a Chinese population. The dietary patterns examined emerged from the foods that this population normally consume, and cultural Cantonese cuisine was also captured. We found that, compared with women in the “Cereals, eggs, and Cantonese soups” group, those in the both “Fruits, nuts, and Cantonese desserts” and “Varied” groups had infants with higher birth weight Z scores. Women in the “Varied” group had a lower risk of having a SGA infant than those in the “Cereals, eggs, and Cantonese soups” group.p < 0.05). Women in “Varied” not only had infants with higher birth weights  but also had a marginally significant risk reduction of having a SGA infant  in comparison to those in the “Cereals, eggs, and Cantonese soups” group. “Varied” was characterized by relatively high intakes of mixed food with high nutritional value, representing a more balanced and varied diet. Consumption of a variety of foods such as fruits, vegetables, and proteins is promoted in many dietary guidelines [In our study, 14.8% of the women followed the “Cereals, eggs, and Cantonese soups” dietary pattern, characterized by a high intake of foods that are part of local eating habits, and representational of a typical traditional Cantonese diet. Women in the “Cereals, eggs, and Cantonese soups” group might be subjected to nutritional inadequacy, and a high intake of carbohydrate and low protein intake was associated with increased risk of low birth weight . These fidelines ,35,36. Iidelines ,38. Previdelines ,40, whicMaternal diet is critical to fetal growth . Most ofBirth weight is an important health indicator and indirectly reflects the living conditions of populations. In our study, mothers in the “Cereals, eggs, and Cantonese soups” group were younger and had lower educational levels, while women in the “Fruits, nuts, and Cantonese desserts” and “Varied” groups had better socioeconomic status. In line with previous studies, our study also showed that better socioeconomic situations of mothers were commonly associated with healthy food consumption ,31, as wTo our knowledge this is the first prospective study to examine the relationship between maternal dietary patterns and fetal growth in a Chinese population with a relatively large sample size. Compared with an analysis based on individual nutrients or food items, dietary pattern analysis is able to capture synergistic effects among various nutrients and types of food, and the findings from dietary pattern analysis might be more comprehensive and informative for clinical practice and public health. Rather than factor analysis, cluster analysis can provide a clear description of exactly what is being consumed, and focus more on groups of people with a good or poor nutritional status, which is more valuable for a nutrition intervention design to target pregnant women in need . The present study had some limitations. Firstly, we did not collect data on portion sizes of food items, and were unable to calculate the amount of food consumption. As previous studies reported ,44, freqIn conclusion, the present prospective study in Chinese pregnant women showed that compared to a traditional Cantonese diet high in cereals, eggs, and Cantonese soups, a diet high in fruits, nuts, and Cantonese desserts might be associated with a higher birth weight, while a varied diet might be associated with a greater birth weight and also a decreased risk of having a SGA baby. These findings provide scientific evidence to support the advice to have a balanced and varied diet for pregnant women in the Chinese population. Pregnant women are recommended to consume different kinds of food, and increase the proportion of vegetables and fruits in their diet, especially for those who are younger and have low socio-economic status. Further studies are needed to clarify the associations between dietary patterns and fetal growth in other Asian populations.


Post-natal follow-up until age 1 year was performed with clinical assessments at 3, 6, and 12 months. Genome-wide DNA methylation was analysed on umbilical tissue in 19 AGA and 27 LGA infants. At birth, LGA infants had greater weight (p < 0.0001), length (p < 0.0001), ponderal index (p = 0.020), as well as greater head (p < 0.0001), chest (p = 0.044), and abdominal (p = 0.007) circumferences than AGA newborns. LGA infants were still larger at the age of 3 months, but by age 6 months there were no more differences between groups, due to higher length and weight increments in AGA infants between 0 and 6 months . Genome-wide analysis showed no epigenetic differences between LGA and AGA infants. Overall, LGA infants had slower growth in early infancy, being anthropometrically similar to AGA infants by 6 months of age. In addition, differences between AGA and LGA newborns were not associated with epigenetic changes.We evaluated the growth patterns of infants born large-for-gestational-age (LGA) from birth to age 1 year compared to those born appropriate-for-gestational-age (AGA). In addition, we investigated possible epigenetic changes associated with being born LGA. Seventy-one newborns were classified by birth weight as AGA (10 A systematic review and meta-analysis showed that high birth weight is independently associated with increased overweight risk in childhood and adulthood23There is no universal definition of oversize at birth. Nonetheless, babies born large-for-gestational-age (LGA) are usually defined by weight, determined as >90et al. reported rapid downwards shift in length increase during the first 3 months of life in LGA infants, as well as a slower than average weight gain in the first 6 monthset al. confirmed a ‘catch-down’ growth in both weight and length in LGA infants, mainly occurring during the first 3 months of life, leading to a substantial realignment on all growth parameters compared to infants born appropriate-for-gestational-age (AGA)789More than three decades ago, Davies FGFR2) has been identifiedSustained changes in growth and metabolism following an adverse fetal or early neonatal environment have been associated with mechanisms involving environmental regulation of gene expression12131618Thus, in the present study we aimed to evaluate the growth patterns of infants born LGA from birth to age 1 year in comparison to those born AGA. In addition, we aimed to assess whether there were epigenetic changes at birth associated with being born LGA.Ethics approval for this study was provided by the Northern Y Regional Ethics Committee . Written informed consent was obtained from parents or guardians. This study was performed in accordance with all appropriate institutional and international guidelines and regulations for medical research, in line with the principles of the Declaration of Helsinki.in vitro fertilisation, or born to mothers with type 1 diabetes or gestational diabetes, preeclampsia, gestational or pre-existing hypertension, chronic illnesses, or following maternal use of recreational drugs, tobacco, or alcohol during pregnancy. Other exclusion criteria were chromosomal or single gene defects, syndromal diagnosis, as well as having a first-degree relative or grandparent with diabetes or the metabolic syndrome.This study involved a prospective cohort of healthy infants recruited at birth from the Newborn Services, Auckland City Hospital , between March and September 2011. All infants were born at term (37–41 weeks of gestation) from singleton and uneventful pregnancies. Infants were excluded if conceived by th and 90th percentiles) or LGA (birth weight greater than the 90th percentile)All neonatal auxological measures were obtained by a single study investigator within 48 h of birth. These included weight, crown-heel length, as well as head, chest and abdominal circumferences. Birth weight was measured to the nearest 10 g using electronic infant scales, and birth weight data were transformed into standard deviations scores (SDS)2), with SDS corrected for sex onlyGestational age was determined by hierarchical integration of the following variables: date of last menstrual period, menstrual cycle length, ultrasound primarily at 16–20 weeks, and clinical assessment of gestational age at birth24Maternal obstetric history was recorded to clarify age at time of delivery, mode of delivery , parity, and the birth order of each infant. Maternal and paternal weights and heights, maternal pre-pregnancy weight and BMI, and weight and BMI at the end of pregnancy were obtained. Mean parental BMI was calculated as the average of maternal and paternal BMI. Mid-parental height SDS was calculated using standard formulaeUmbilical cord tissue was collected at birth, and frozen at −80 °C for later genome-wide DNA methylation analysis.All participants were re-evaluated by the same study investigator at 3, 6, and 12 months of age. Anthropometric measures, including weight, length, as well as head, chest, and abdominal circumferences were measured.Genomic DNA was isolated from umbilical tissue samples using the KingFisher Cell and Tissue DNA kit  according to manufacturer’s instructions. One μg of DNA was bisulfite treated using the EZ DNA Methylation™ Kit , and 500 ng of bisulfite-treated DNA was analysed using the Illumina Infinium 450 K methylation array platforms. To assess DNA methylation profile, we used the standard Illumina protocols. The bisulfide-converted samples were whole-genome amplified, and the amplified products were fragmented by an endpoint enzymatic process. The fragmented DNA was purified and hybridized to the Infinium Human Methylation 450 K BeadChips . During hybridization, the amplified and fragmented DNA samples anneal to locus-specific DNA oligomers residing on the bead chips. Single base extension reaction, washing, and staining were carried out using a TECAN Te-Flow chamber. The stained arrays were assessed for fluorescence intensities at the methylated and unmethylated bead sites using Illumina BeadArray Reader (Illumina Inc.). Quality control was performed using built-in controls. All samples passed quality control criteria . DNA methylation signals  from the scanned arrays were extracted using the methylation module of GenomeStudio® software version 2011.1 (Illumina Inc.). The β-value was used to estimate the methylation level of the CpG locus by calculating the ratio of intensities between methylated and unmethylated alleles . R package LumiDemographic characteristics between groups were compared with one-way ANOVA and Fisher’s exact tests. Anthropometric differences between groups were analysed using general linear regression models. Models accounted for important confounding factors . Models examining differences between LGA and AGA infants at 3, 6, and 12 months included age as a covariate. Binary logistic regressions were carried out to examine the factors affecting the likelihood of being born LGA or undergoing a caesarean section. Statistical analyses were carried out in SAS version 9.3  and Minitab v.16 . All statistical tests were two-tailed and maintained at a 5% significance level. Demographic data are presented as means ± standard deviations. Outcome data are presented as model-adjusted means , with associated 95% confidence intervals.For statistical analysis on differential methylation levels, probes with detection p-value > 0.05 were excluded. CpG sites with missing data for >10% of samples were also excluded from analysis. All CpGs residing on X and Y chromosomes were dropped from the analysis to eliminate systematic sex differences. We performed a multivariate linear mixed regression analysis that modelled the β-values as the dependent variables, with the variable group (AGA or LGA) as the primary independent variable, and including covariates for sex, birth weight, and ethnicity to adjust for potential effects of other covariate-dependent methylation. To assess significance while accounting for multiple testing, we used the Benjamini-Hochberg false discovery rate (FDR) procedureEligible pregnant women in early labour were recruited from the Delivery Suite at Auckland City Hospital. We aimed to recruit at a ratio of approximately two controls for each LGA participant. Of 57 LGA and 108 AGA babies eligible at birth, seventy-one infants (43%) attended all follow-up assessments  and were included in this study: 42 born AGA (18 boys) and 29 born LGA (18 boys) . The pri2 difference in mean parental BMI (p = 0.004). Prior to pregnancy, LGA mothers had BMI that was 2.7 kg/m2 greater than mothers of the AGA group (p = 0.004). Not surprisingly, every 1 kg/m2 increase in maternal pre-pregnancy BMI was associated with an 11% increase in the odds of a LGA infant being born (p = 0.014). In addition, increasing birth order was associated with an increased likelihood of LGA birth   . The rat3 greater (p = 0.020) than AGA infants. Further, LGA infants had greater circumference of the head , chest , and abdomen .At birth, LGA were larger than AGA infants based on all parameters assessed . LGA inf2 greater (p = 0.030), and had greater head  and abdominal  circumferences. However, there were no significant differences in ponderal index or chest circumference.By 3 months of age, there were persisting differences between LGA and AGA infants. LGA subjects were still 1.5 cm longer (p = 0.006), 713 g heavier (p < 0.0001), and of BMI 1 kg/mIn the first 6 months of life, LGA infants grew significantly slower than those born AGA: length increment 15.1 vs 18.3 cm, respectively (p < 0.0001) and weight increment 4.13 vs 4.87 kg, respectively (p = 0.002) . These dThe genome-wide methylation analysis was carried out on samples from 46 infants, including 19 AGA and 27 LGA infants. For this analysis, more than 485,000 DNA methylation sites covering 99% of human NCBI Reference Sequence (RefSeq) genes were examined at birth. 449,691 probes (92.6%) out of 485,577 passed the probe filtering criteria. The differential methylation analysis of CpGs sites showed no significant differences between LGA and AGA infants at birth  .Our results indicate that, despite being born oversized, LGA infants displayed slower length and weight velocity, so that by the age of 6 months LGA infants were anthropometrically similar to AGA infants. This suggests that LGA infants experience a slowing in growth in early infancy. In addition, no epigenetic differences in genome-wide methylation were found in LGA infants at birth.Alterations in the intrauterine environment can induce fetal developmental adaptations that might have long-lasting detrimental effects on the offspring5As mentioned previously, there is a paucity of data on growth outcomes of LGA infants. Our data corroborate previous studies showing that LGA newborns generally present greater adiposity besides being born oversized39ex utero in those born LGA, a putative role has been attributed to maternal influences to account for their increased growth in utero. For instance, fetal overgrowth in oversized infants of non-diabetic mothers has been linked to an energy-rich fetal environment associated with mild maternal hyperglycemia , maternal obesity, or excessive gestational weight gain42Nevertheless, we observed a subsequent slower growth in LGA infants, culminating in similar anthropometry in LGA and AGA groups by 6 months of age, which remained so at 12 months. These findings are in line with previous data showing a slowdown in weight and length gain from birth to age 6 months in most LGA infants7et al. reported a unique pattern of adiposity  at birth and still at age 12 months in LGA infants of diabetic mothers, in comparison to LGA of healthy mothers and AGA infants of diabetic mothers, supporting the additional detrimental effects of maternal diabetes on offspring growthHowever, the data on LGA infants are inconsistent and our results are in contrast with some studies reporting greater central adiposity at age 12 months in those born LGA compared to AGA infants9IGF2 and H19 are known to affect the fetal developmentIGF2 expression responsible for pre- and post-natal growth restriction (roughly 30% of Silver Russell syndrome cases), while hypermethylation induces IGF2 overexpression leading to overgrowth (Beckwith-Wiedemann syndrome)There is a large body of evidence showing that changes in the early life environment are associated with increased cardio-metabolic risk later in life16171820in utero associated with the LGA phenotype, which could possibly lead to heavier birth weight and altered body composition and metabolism, have only recently been examinedFGFR2 gene were identified as being associated with high birth weightPotential epigenetic changes in utero.In conclusion, our study showed that although LGA babies were larger and had greater adiposity at birth, a slowing in growth (length and weight) occurs in these infants in early infancy, leading to a similar anthropometry to AGA infants by 6 months of age. Of note, the differences observed in early life between LGA and AGA infants were not associated with epigenetic changes. Further research is needed to clarify the growth pattern of those born LGA in early infancy and childhood, and to elucidate possible mechanisms associated with overgrowth How to cite this article: Chiavaroli, V. et al. Infants born large-for-gestational-age display slower growth in early infancy, but no epigenetic changes at birth. Sci. Rep.5, 14540; doi: 10.1038/srep14540 (2015).