Several protein-targeted RNA aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. We examined the structural accommodation of an RNA aptamer that binds bacterial r-protein S8. The core of the primary binding site for S8 on helix 21 of 16S rRNA contains a pair of conserved base triples that mold the sugar-phosphate backbone to S8. The aptamer, which does not contain the conserved sequence motif, is specific for the rRNA binding site of S8. The protein-free RNA aptamer adopts a helical structure with multiple non-canonical base pairs. Surprisingly, binding of S8 leads to a dramatic change in the RNA conformation that restores the signature S8 recognition fold through a novel combination of nucleobase interactions. Nucleotides within the non-canonical core rearrange to create a G-(G-C) triple and a U-(A-U)-U quartet. Although native-like S8-RNA interactions are present in the aptamer-S8 complex, the topology of the aptamer RNA differs from that of the helix 21-S8 complex. This is the first example of an RNA aptamer that adopts substantially different secondary structures in the free and protein-bound states and highlights the remarkable plasticity of RNA secondary structure. Over the past several years, high-resolution structure studies of ribonucleoprotein complexes have revealed a wealth of detailed information on structural motifs that contribute to protein–RNA specificity . RNA molrplE gene at the beginning of the spc operon  and 16S rRNA is a well-studied interaction that is specified by an internal loop. The binding of r-protein S8 to 16S rRNA has been extensively characterized using a variety of techniques including chemical modification and protection assays , filter c operon . The trahis site .spc mRNA binding sites. Notably, these protein–RNA interactions are maintained within the 30S ribosomal RNA subunit. These complexes reveal that the S8-RNA binding involves electrostatic and hydrogen-bond interactions and shape complementarity.In addition to structural conservation of the primary RNA binding sites for S8, the overall fold of S8 r-proteins is conserved. The S8 protein has two domains, N- and C-terminal , and thexponential enrichment (SELEX) study suggested the presence of a base triple (G597-C643)-U641 located in the internal loop of helix 21 (642 was present in the selected aptamers (641 participates in a bifurcated hydrogen bond with the G597-C643 base pair and the A642 base (595-(A596-U644)  . These e96-U644) ,24. A co triplet .The nucleotide sequence and secondary structure of the primary binding site for r-protein S8 on helix 21 are highly conserved Figure . These cBacillus anthracis and E. coli S8 r-proteins were expressed as N-terminal 6X-His tagged fusion proteins. The rpsH genes were polymerase-chain-reaction-amplified from genomic DNA, cloned into the pET28b vector (Novagen) using the Nhe1-Xho1 restriction sites, and the sequences confirmed. The proteins were expressed in BL21(DE3) cells, isolated in the form of inclusion bodies, and dissolved with 7 M urea as described  column that was equilibrated with buffer A . The column was washed with buffer A plus 20 mM imidazole and 500 mM NaCl and the protein was eluted using 200 mM imidazole in buffer A. Fractions were collected and those containing S8 (>95% purity) were combined and the protein renaturated over 3 days via serial dialysis in buffer B  containing decreasing molar concentrations of urea: 7.0, 4.0, 2.0, 1.0, 0.5 and 2× 0.0 M. The refolded S8 proteins were concentrated (Amicon) and quantified using the Bradford method.The escribed . The B. X)CUUCGG(NY)GGGAAGCCUC-3′. X and Y designate the number of randomized nucleotides  so that the sum of X and Y was fixed at 16. The aptamer sequence chosen for study was identified after 10 rounds of selection and forms a secondary structure with a symmetric internal loop, in contrast to the asymmetric internal found in natural S8 binding sites. Additional details of the selection are given in Supplementary Information.The RNA aptamer selection was performed as described  using 5′in vitro using T7 RNA polymerase and a synthetic DNA template. Unlabeled and isotopically labeled RNA molecules were prepared as described . For fluorescence anisotropy experiments, 5′-fluorescein-labeled RNA hairpins were prepared by ligation of a 5′-fluoroscein-labeled RNA heptamer (Dharmacon) with in vitro transcribed RNA sequences corresponding to the aptamer or the r-protein S8 binding site on helix 21. The labeled hairpins were PAGE purified, dialysed against 150 mM KCl and 10 mM sodium cacodylate, pH 6.6, and stored at −80°C.The aptamer molecule for X-ray crystallography Figure  was purcescribed . The pol1H- probe) and 600 MHz and 800 MHz  spectrometers and NMR spectra were processed and analyzed using Felix 2007 .Spectra were acquired on Varian Inova 500 MHz  experiments and 2D HCN experiments were used to identify intra-residue base-ribose correlations. Pyrimidine C2 and C4 resonances were assigned from H6-C2 and H5-C4 correlations using 2D H(CN)C and 2D CCH-COSY experiments and a 2D H(N)CO experiment for uridine NH- resonances  and 3D 13C-edited NOESY spectra (tm = 180 and 360 ms). Assignments and distance constraints for the exchangeable resonances were derived at 12°C from 2D NOESY spectra (tm = 160 and 360 ms) acquired in 90% 1H2O. 3JH-H, 3JP-H and 3JC-P coupling constants were estimated using DQF-COSY, 31P-1H HetCor and CECT-HCP  sonances . SequentCECT-HCP  experime−1 Å−2 to 30 kcal mol−1 Å−2 for the NOE and from 2 kcal mol−1 rad−2 to 30 kcal mol−1 rad−2 for the dihedral angle constraints. After minimization, NOESY spectra were re-examined for predicted NOEs absent from the constraint list. The calculations were repeated using revised constraint lists and eight structures were selected for the final refinement using criteria that included lowest energies, fewest constraint violations and fewest predicted unobserved NOEs. A second round of rMD was performed on these structures using a starting temperature of 300 K followed by cooling to 25 K over 28 ps of rMD. The eight refined structures were analyzed using Xplor-NIH and Chimera. The data and structure statistics are reported in Supplementary Table S1.Structure refinement was carried out with simulated annealing and restrained molecular dynamics (rMD) calculations using Xplor-NIH v2.19 . The aptB. anthracis S8 were obtained by sparse matrix screening of S8 at 10 mg/ml at 4ºC. Preliminary results were followed by optimization of the successful condition manually using the sitting drop vapor diffusion method. The best-quality crystals were grown in 48–51% Tacsimate at 20ºC. No cryoprotectants were required for cryopreservation in liquid nitrogen.Crystals of 2, 75 mM KCl, 2 mM DDT, 20 mM MOPS pH 7.0) and S8 protein  in a 1:1 mole ratio to a final concentration of 150 μM followed by incubation for 1 h on ice. The final crystallization condition was 0.3 M di-ammonium hydrogen citrate, 100 mM sodium chloride, 16% PEG 3350 and 10 mM spermidine at 10°C.S8-aptamer complexes were formed by mixing RNA aptamer  was 99.9%.Crystals of S8-RNA were passed briefly through cryoprotectant solutions consisting of 0.3 M sodium citrate pH 7.0, 100 mM sodium chloride, 10 mM spermidine supplemented with 5, 10, 15, or 20% (v/v) glycerol. Diffraction data for the S8-aptamer complex was collected to 2.6 Å resolution using a NOIR-1 Molecular Biology Consortium (MBC) detector system at the beamline 4.2.2 at the Advanced Light Source synchrotron . The crystal belonged to space group P212121 with unit cell parameters g D*TREK  with RmeB.anthracis S8 was solved by the standard method of single anomalous dispersion (SAD). Heavy atom sites from the metabolically incorporated selenomethionines were found by the online application SHARP (R factor of 22.3% and Rfree of 23.3%.The structure of on SHARP . SAD eleon SHARP  and map on SHARP . Moleculon SHARP . The modB. anthracis S8 r-protein (solved in-house) as a search model. The initial solution suggested one monomer per asymmetric unit consistent with the Matthew's coefficient of 3.16 (65% of solvent). The molecular replacement was further confirmed by the initial (2Fo-Fc) map generated using Coot (R of 18.9% and Rfree 27.0% (Supplementary Table S2). Ramachandran plots and root-mean-square deviations (rmsd) from ideality for bond angles and lengths for S8/RNA were determined using a structure validation program, MolProbity .A Beacon 2000 fluorescence polarimeter (PanVera Corp.) was used for the fluorescence anisotropy experiments. 5′-fluorescein-labeled RNA hairpin samples were extensively dialyzed against a buffer of 25 mM Tris-Acetate (pH 7.6) and 150 mM potassium acetate. RNA samples were heated to 90°C for 60 s, snap cooled on ice and dialyzed against 25 mM Tris-Acetate (pH 7.6), 150 mM potassium acetate and 10 mM magnesium acetate. The concentration of RNA was kept constant at 1.0 nM and the concentration of S8 protein ranged 1.0–500 nM. Samples were mixed by addition of protein solution to RNA and incubated at 4°C for 30 min. Four measurements were averaged for each S8 concentration. Experiments were performed in triplicate. The apparent E. coli and Bacillus 16S rRNA in addition to non-natural binding sites with symmetric internal loops. Electrophoretic mobility shift assays (EMSAs) were used to qualitatively assess the S8 binding of non native-like aptamers and the sequence . The Bacillus S8 protein binds the RNA aptamer with a Kd of 110 ± 30 nM and the helix 21 site with a Kd of 180 ± 60 nM. The affinity of E. coli r-protein S8 for the RNA molecules are 8–10-fold tighter, Kd = 19 ± 4 nM and Kd = 28 ± 7 nM for the aptamer RNA and helix 21 RNA, respectively. The affinity of E. coli r-protein S8 for the helix 21 sequence element is consistent with filter-binding measurements  for an RNA construct containing the three-way junction formed by Helices-20–21–22 , which are indicative of base pairing. The inter-nucleotide phosphate 31P resonances are clustered between −3.32 and −5.05 ppm except the U27pC2831P resonance that has a chemical shift of −2.40 ppm. A complete list of resonance assignments is given in Supplementary Table S2.Sequential assignments for the non-exchangeable resonances were made using 2D NOESY and 3D 10-G13 and A26-A29 and is flanked on one side by a distorted A14-U25 base pair. The internal loop is characterized by two non-standard base pairs, a sheared A-G and a U-U, and a Watson–Crick G-C base pair. The bases of A10 and A29 form an inter-strand stack with each other. The spectral data support the presence of these base–base interactions, but the arrangement of nucleotides is not as tightly ordered as observed in other structures. The H1′ resonance of U27 has a chemical shift of 5.03 ppm and is consistent with a partially sheared base pair configuration between G13 and A26 . As with the neighboring G13-A26 pair, the gap between the uridine bases is relatively wide and the imino protons are accessible for solvent exchange. Residue C28 pairs with G11 as indicated by the NH2 and C2 resonances of C23, but the G NH resonance exchanges with solvent and is not observed. The A10 and A29 bases each extend across the helix axis with A24 stacked on the G11-C28 base pair. This conformation is supported by unusually strong cross-strand H2-H1′ NOE cross peaks. The A10 base is laterally displaced toward the minor groove and is positioned slightly below the plane of the C9-G30 base pair. In the converged structures, the A10 NH2 consistently forms a hydrogen bond with the C9 O2. The moderately downfield-shifted A10 N6 resonance (82.5 ppm) is consistent with this hydrogen bond.The global fold the aptamer is a hairpin capped by a canonical UUCG tetraloop and the stem interrupted by an eight-nucleotide internal loop Figure . The int and A26 . The U1213 ribose has a C2′-endo ring pucker conformation and the uniformly small (<5 Hz) P–C2′ coupling constants for the loop residues place the ϵ torsion angles in the trans conformation characteristic of A-form RNA. Although torsion angles α and ζ were left unconstrained, the ζ angle between U27 and C28 is trans-like rather than −gauche in all structures. This configuration is consistent with the relative downfield 31P shift of the involved phosphate. α and ζ at other positions are consistently −gauche or exhibit −trans/gauche variability between converged structures.The sugar-phosphate backbone conformations of the aptamer nucleotides within the internal loop are surprisingly uniform Figure . Only thBacillus ribosomal protein S8 was solved by molecular replacement using the structure of unliganded B. anthracis S8 and refined against a 2.6 Å data set. The refined model contains all 38 nucleotides of the aptamer and residues 4–132 of the S8 protein.The crystal structure of the aptamer RNA-2 Figure  in complThe S8 protein has two closely packed domains that are composed of the N- and C-terminal halves of the molecule Figure . The N-tsyn configuration about the glycosidic bond. The canonical A-form helical stem of the aptamer is interrupted by an internal loop that includes nucleotides A10-A14 on the 5′ strand and U25-A29 on the 3′ strand. This central core of the aptamer has several non-standard structure features and is characterized by a complex network hydrogen bonds among the bases. A10 and A29 at one end of the loop adopt a cis Watson–Crick/Watson–Crick base pair via an A10 N1-A29 NH2 hydrogen bond and weak A10 H2-A29 N1 hydrogen bond . Stacked against the A10-A29 pair is a G11-(G13-C28) base triple. The base of G11 is coplanar with the Watson–Crick G13-C28 base pair and is joined to the pair through hydrogen bond G13 O6-G11 NH2. The G11 base is further locked into position by a hydrogen bond between G11 O6 and U25 2′-OH. This base triple stacks on a base quartet composed of residues U12, A14, U25 and U27  and A14 (U25 O2 to A14 NH2) and is coplanar with A14 and U25  and the A642 N3 atom. A26 functionally replaces A642 in the S8-aptamer complex . The R124 side chain NH2 interacts with the G597 N3 and U598 O4′. In the aptamer-S8 complex, an interaction analogous to Y85-G597 involving the G11-(G13-C28) base triple is not observed. Many of the other interactions present in the S8-RNA structures correlate with earlier biochemical analyses (640 2′-OH present in native complexes. Substitution with deoxy-A at 640 does not affect protein binding (24 (which is isomorphic with A640).Despite the obvious sequence and structural differences between the native RNA sites and the aptamer, the structure of the aptamer is dramatically remodeled in the S8 complex to produce a conformation with remarkable similarities to native S8-RNA complexes . Alignme coli S8 , M. jannschii S8  and A. alicus S8  result ilicus S8 . Also, manalyses ,47,48. O642 and the T106-S107 (E. coli S8) peptide bond is recapitulated. This interaction is facilitated in 16S rRNA and spc mRNA by the odd number of nucleotides in the internal loop. In the aptamer, the stacking of the A26 base is made possible when the G13 and A26 bases shift above and below the plane of the base quartet, respectively. In the natural RNA targets, A642 participates in an i to i+1 base pair with residue 641  and in T. thermophilus, G595-(C596-G644). In the spc mRNA binding site, the corresponding base triple is A+80-(A+81-U+11). Although the base triples are isosteric, the non-Watson–Crick residue of the base triple in the aptamer RNA, G11-(G13-C28), is not sequential with either nucleotide of the base pair. This nucleotide topology difference for the aptamer base triple is reflected in the local geometry of the backbone on the face opposite the bound S8 protein. The backbone geometry at the G11-U12 step is characteristic of the loop E motif . Nodes of electropositive density also are centered at residues C17 and U25, but an electronegative patch in this primary binding site contours to the minor groove edge of A26. The electropositive surface charge that lines the secondary RNA binding site on S8 is more extensive than that on the primary face, but RNA binding in this region is weaker and non-specific. Given the potential for multiple charge–charge interactions, it is somewhat surprising that the secondary binding site was not identified as a preferred target during the selection. However, a site that accommodates multiple types of interactions  might be favored since electrostatic contributions toward binding diminish due to shielding effects caused by increasing salt concentrations used during the selection.Two sites on r-protein S8 interact with 16S rRNA, one site involves helix 21 and a second site involves helix 25. Therefore, S8 presents at least two surfaces for an RNA aptamer. The helix 21 binding site on S8 is lined with a strip of electropositive charge along which the phosphate backbone of the aptamer traverses from residues AProtein surfaces present specific structured sites, or epitopes, that are recognized by aptamers and often the same protein epitope can bind aptamers of different sequence and potentially different structure . The chathree-nucleotide loop, yet forms many of the same interactions with capsid protein as the natural RNA ligand. In the case of the NF-κB p50 homodimer, the RNA aptamer forms a hairpin with a seven-nucleotide internal loop capped by a GNRA tetraloop (Three complexes that offer a basis for comparison of free and bound aptamers as well as comparison of binding modes of aptamer and natural targets involve the MS2 capsid protein, NF-κB p50 homodimer and nucleolin. Aptamers against the MS2 capsid protein have the same basic secondary structure as the natural RNA binding site, an RNA hairpin capped by a four-nucleotide loop, and form many of the same protein–RNA interactions . One claetraloop . In the etraloop ,57. Thusetraloop . Nucleotetraloop ,58.As with the NF-κB and MS2 capsid protein complexes, the interactions between the aptamer and S8 recapitulate those of the native complexes. However, only the S8 aptamer has significant structural dissimilarity between free and protein-bound forms Figure ,23,24. RCoordinates have been deposited in the Protein Data Bank under accession numbers PDB ID: 2LUN, solution structure of the RNA aptamer, and 4PDB, crystal structure of the S8-aptamer complex. Chemical shifts have been deposited in the Biomolecular Magnetic Resonance Bank under accession numbers BMRB ID: 18532.Supplementary Data are available at NAR Online.


The previous decade has seen long non-coding RNAs (lncRNAs) rise from obscurity to being defined as a category of genetic elements, leaving its mark on the field of cancer biology. With the current number of curated lncRNAs increasing by 10,000 in the last five years, the field is moving from annotation of lncRNA expression in various tumours to understanding their importance in the key cancer signalling networks and characteristic behaviours. Here, we summarize the previously identified as well as recently discovered mechanisms of lncRNA function and their roles in the hallmarks of cancer. Furthermore, we identify novel technologies for investigation of lncRNA properties and their function in carcinogenesis, which will be important for their translation to the clinic as novel biomarkers and therapeutic targets.The online version of this article (doi:10.1186/s12943-016-0530-6) contains supplementary material, which is available to authorized users. Our understanding of cancer biology was drastically changed by the genomic revolution of the last decade, marked by the conclusion of the human genome project and the development of novel DNA sequencing technologies , 2. The Large-scale cDNA sequencing projects, together with technological advancements such as tiling arrays and the next generation RNA sequencing provided an unprecedented view of the transcriptome complexity –15. SurplncRNAs remained elusive even in the genomics era due to their low expression levels and their presence in specific cell types, tissues or narrow time frames –25. TheylncRNAs have been observed to regulate complex cellular behaviours such as growth, differentiation and establishment of cell identity that are commonly deregulated in cancer –36. Somede novo assembly of all available public datasets [lncRNAs were initially observed in carcinogenesis due to their differential expression compared to normal tissues. High expression in tumour tissues of some of the first identified lncRNAs such as h19, MALAT1 and PCA3 was recognised before the availability of next-generation sequencing technologies –39. RNA datasets , 40, mardatasets .Fig. 1InThere are two main drivers for altered expression of lncRNAs in tumours. First, abundance of some lncRNAs can be altered due to the cancer-induced change of copy number of their genomic loci. This phenomenon has been observed for FAL1 in ovarian and PRAL in hepatocellular carcinoma , 43. SecPresence of lncRNAs in specific tumours can also be observed based on their overlap with the cancer risk loci identified through genome-wide association studies (GWAS). For example, association of ANRIL with glioma and basal cell carcinoma as well as PTCSC3 with thyroid cancer were discovered based on the known risk loci established through genotyping of cancer patients , 50 ReceAlthough lncRNAs are scarcely functionally annotated , their mcis-acting activity. PCE3, as the first lncRNA involved in cancer was only recently found to have the same mode of action, with the discovery of its antisense protein oncogene PRUNE2 [Chromatin remodelling was one of the first identified functions of lncRNAs. Epigenetic remodelling is frequently achieved through interaction of a lncRNAs with PRC2 (reviewed in ), a prote PRUNE2 . Howevere PRUNE2 . Besidese PRUNE2 –67. Othee PRUNE2 . lncRNAse PRUNE2 –71.A large number of lncRNAs exert their oncogenic function through direct interaction with proteins or protein complexes as scaffolds or allosteric activators/inhibitors. CTBP1-AS, CCTA2 and ZBTB7A interact with transcription factors and modify their activity –74. SomeSome lncRNAs have recently been found to act as competing endogenous RNAs (ceRNAs), by binding miRNAs (“sponging”) and reducing their inhibitory effect on their natural targets (reviewed in ). There cis for Khps1, or in trans for MEG3 [In addition to the previously described mechanisms, lncRNAs have recently been observed to employ the following set of diverse strategies. lncRNAs can modify the phosphorylation state of proteins by masking phosphorylation motifs, like LINK-a and HIF1a . Furtherfor MEG3 , 90. Othfor MEG3 .In summary, lncRNAs act through an increasingly wide range of mechanisms that compete with proteins in terms of their diversity and regulatory potential. In the next section we discuss how these mechanisms impact upon cell transformation to cancer phenotype.In 2000, Hanahan and Weinberg described six properties required for cell transformation, termed hallmarks of cancer. These included self-sustained growth signalling, insensitivity to growth inhibition, apoptosis avoidance, uncontrolled proliferation, angiogenesis and metastasis , 93. lnclncRNAs promote self-sufficiency in growth signals mostly by acting on the activation on the first step of the signal transduction, the signal receptors. Multiple lncRNAs have been observed to specifically bind nuclear receptors either alone or in a ribonucleoprotein complexes (reviewed in ). SRA1 sGrowth inhibition or its evasion can also be regulated by lncRNAs, and mostly involves influence on tumour suppressors that regulate cell cycle such as cyclins, CDKs, CDK inhibitors and p53 (reviewed in ). This cApoptosis or controlled cell death is one of the key pathways for control of carcinogenesis (reviewed in ). Some lMaintenance of telomeres as nucleoprotein structures that stabilize ends of chromosomes is a key requirement for limitless replicative potential of cancer cells. Telomeres shorten in dividing cells, so a ribonucleoprotein complex telomerase is required to elongate telomeric repeats through reverse transcription of an internal template RNA. Shortening of telomeres induces production of a lncRNAs TERRA, that is transcribed from the subtelomeric regions . Under nMultiple lncRNAs have been implicated in regulation of nutrient supply to tumours, mostly by regulating vascular endothelial growth factor (VEGF) that is essential for formation of blood vesicles. Transcription of VEGF was recently reported to be regulated by lncRNAs HOTAIR  and MIATMultiple lncRNAs increase invasiveness of cancer cells and facilitate metastasis. Examples of these include h19 , MALAT1 lncRNAs can be involved in a number of other processes related to cancer. Some lncRNAs promote a metabolic switch to glycolysis and lactic acid fermentation termed the Warburg effect . lincRNAThe number of annotated transcribed genomic elements has increased by 100 % in the last decade, the majority of which are in the non-coding space and have a defined function in less than 1 % of cases . Such a The first challenge is presented by the lack of an exhaustive definition of the full cancer transcriptome, regardless of the cell or tissue type. Currently, a major obstacle to analysis of cancer transcriptome is the alignment of sequence reads to the consensus human genome. Ideally, all the reads would be aligned to a genome sequenced by single-molecule DNA sequencing, but the cost and the quality of this technology are still keeping it away from mainstream research. The next issue is the limited dynamic range of transcript detection for RNA sequencing. This can already be solved by applying the recently developed CaptureSeq method for targeted enrichment of transcripts from specific regions of interest . FurtherAfter defining the non-coding elements of the transcriptome, the second challenge is the systematic identification of lncRNAs properties that could lead to identifying their cellular function. This can be achieved by investigating their location in the cellular compartments, structural properties as well as possible interactors.Quantified localization of lncRNAs through microscopy techniques can provide important information about their properties. RNA-Fish as an established technique for RNA localisation has recently been used to identify subcellular location of multiple lncRNAs, in addition to their expression across a population of cells, spatio-temporal behaviour and coexpression with proximal mRNAs .Structure of biological molecules is vital to their function, and several techniques have been developed to investigate secondary and tertiary structures of lncRNAs. Techniques such as Parallel Analysis of RNA Structure (PARS)  and FragAssessing the function of lncRNAs by identifying their binding partners can be performed depending on the type of interaction. Binding of RNA to DNA or proteins can be assessed with ChIRP-seq or ChIRP-MS respectively (Chromatin Isolation by RNA purification followed by sequencing or mass spectrometry) , 136. ThThe final challenge in defining lncRNA functions is developing loss- and gain-of-function lncRNA studies. The RNA interference technology is being supplemented by the powerful CRISPR/Cas-9 system, a newly developed genome-editing technology that allows easier manipulation of lncRNAs behaviour . CRISPR Long non-coding RNAs are fine-tuners and regulators of key biological processes. Though we have only started to annotate their function in various aspects of cell transformation and metastasis, they are already filling in the major gaps of our understanding of cancer biology. It will be exciting to see the next decade migrate from the perception of lncRNAs as a side act in biological regulation to the center of new biological concepts, paradigms and drug therapies. Watch this space.ceRNA, competing endogenous RNAs; CHART, capture hybridization of analysis of RNA targets; ChIRP-seq or ChIRP-MS, chromatin isolation by RNA purification followed by sequencing or mass spectrometry; CLIP-seq, cross-linking immunoprecipitation sequencing; GWAS, genome-wide association studies; hMeRIP, hydroxymethylcytosine sites in RNA; lncRNA, long noncoding RNA; MeRIP, methylation RIP-seq; PARS, parallel analysis of RNA structure; RAP, RNA antisense purification; RIP, RNA immunoprecipitation; RNA-MaP, RNA on a massively parallel array RNA-MITOMI, RNA-mechanically induced trapping of molecular interactions; SHAPE, selective 2′-hydroxyl acylation and primer extension; SNP, single nucleotide polymorphism


Evidence of some recovery was found at Bramston Reef with living coral genera similar to what was described in c.1890 present in 2012. In contrast, very little sign of coral re-establishment was found at Stone Island suggesting delayed recovery. These results provide a valuable reference point for managers to continue monitoring the recovery (or lack thereof) of coral communities at these reefs.Long-term data with high-precision chronology are essential to elucidate past ecological changes on coral reefs beyond the period of modern-day monitoring programs. In 2012 we revisited two inshore reefs within the central Great Barrier Reef, where a series of historical photographs document a loss of hard coral cover between c.1890–1994 AD. Here we use an integrated approach that includes high-precision U-Th dating specifically tailored for determining the age of extremely young corals to provide a robust, objective characterisation of ecological transition. The timing of mortality for most of the dead  For instance, using anecdotal comparisons of a collection of historical photographs taken from c.1890 onwards of reef flat and reef crest communities from 14 locations, WachenfeldIn this study we revisit Bramston Reef and Stone Island; two of the historical photograph locations in Wachenfeldin situ faviids photographed in 1994. The first photograph was taken looking south-east over Bramston Reef with Gloucester Island and Cape Gloucester as the landmark feature in the background ]. Living coral genera included Acropora, Goniastrea, Montipora, Goniopora, Lobophyllia, Favites, Turbinaria, Pocillopora and Favia. Despite a high abundance of dead faviid colonies, mean dead coral cover was low (7.4 ± 6.4%) as most surfaces were covered by algae, accounting for 45 ± 9% of the benthic composition To assess the recovery, or lack thereof, of coral communities, the same sites at Bramston Reef and Stone Island photographed in 1994 were located using the bearings and geographic features described by Wachenfeldckground , while tckground . In bothmainland . The deamainland , howeverAcropora and sub-massive colonies believed to have died in situ. Here, mean live coral cover was extremely low (0.09 ± 0.12%) with only Acropora captured in the analysis  able to grow to levels slightly higher than MLWS height21in situ live/dead coral colonies relative to MLWS height.The uppermost limit of open-water coral growth on the GBR approximates the mean low water spring (MLWS) tide level plus or minus 25 cm due to inter-annual variation in tidal behavior, exposure to wave action, and the varying tolerance of different coral species to exposure, with some faviid corals  with an average elevation of 0.26 ± 0.09 mLAT. Towards the edge of the reef , benthic composition closely resembled what can be seen in the historical photographs with numerous large dead faviids creating an undulating topography with substrate height being on average 0.08 ± 0.21 (1sd) mLAT (N = 6). The elevation of six massive dead faviids from this zone was reported to be within error of the MLWS level, ranging in height between 0.57 to 0.66 mLAT [average 0.60 ± 0.03 (1sd)]  at the same location as the 1994 photographs, ranging between −0.31 to 0.49 mLAT and progressing through three distinct zones . From th3 (1sd)] . Large l.70 mLAT .N = 3)] where large fossil microatolls up to 2.6 m in diameter characterized the benthos. The reef flat then abruptly stepped down to 0.15 ± 0.22 mLAT (N = 9) between ~20–240 m and was largely still submerged at low tide. Here, living tabular Acropora sp. and Porites sp. colonies were occasionally found. Towards the reef crest and at the location of the historical photographs, substrate height increased to 0.23 ± 0.11 mLAT (N = 2) at a distance of 240–290 m from shore  that predominately feature in the 1994 photographs of Bramston Reef, revealed the timing of mortality to have mostly occurred between 1984.6 ± 4.7 and 1993 ± 10, analytically almost indistinguishable from a weighted mean of 1990.4 ± 1.3 (2-sigma) (234U/238U activity ratios (ranging between 1.143–1.150) similar to that of modern corals reported elsewhere on the GBR2324232Th, with samples containing on average 1.34 ppb (range 0.30 to 4.57 ppb) and resulted in uncorrected 230Th age errors of between 0.6 and 1.8 years (High-precision U-Th dates of 17 randomly collected dead 2-sigma) . Only th.8 years .in situ colonies sampled at the reef crest of Stone Island were identified as Acropora sp. and Porites lobata and were found to be more than 6,000 years old (SIS1T2.2 and SIS1T2.4). The 230Th age data is in agreement with 14C ages obtained from fossil microatolls close to the reef edge at Stone Island in an earlier study230Th age data from the remaining seven samples  cluster around two more recent time periods in the mid-20th [between 1964.1 ± 6.7 and 1977.5 ± 5.6 (N = 3)] and mid-21st [between 2006.4 ± 3.1 and 2007.3 ± 2.9 (N = 4)] Century and overlap in age with those from Bramston Reef . When both the Stone Island and Bramston Reef 230Th age data are considered together, the ages return a weighted mean of 1970.4 ± 9.6 (N = 4) and 2007.2 ± 1.4 (N = 5) for each of the two periods. Detrital 232Th was slightly higher in the Stone Island samples with average concentrations of 4.73 ppb (range 0.99 to 23.4 ppb), which consequently resulted in larger uncorrected 230Th age errors of between 0.6 and 29 years , Bramston Reef was still characterized by many large faviid colonies, dead and overgrown by algae and sediment, as well as a large number of small living faviid colonies. Yet there was evidence of some small increase in coral cover, primarily driven by tabular r genera . In addir genera . At Stonin situ faviid colonies sampled from Bramston Reef revealed the timing of mortality to have mostly occurred around 1990.4 ± 1.3 AD, precisely bracketing the time period during which two major events are likely to have caused significant mortality in coral communities. The first event occurred on the 11 December 1989 where an extremely low tide of −0.1 mLAT was reported at the Bowen tidal gauge station; the lowest on record for the period spanning 1986–2013  since a disturbance event of similar severity affected this reef.Although outside the time period that brackets the mortality event, anomalous SSTs preceding the extreme low tide and flood in 1987 that reportedly caused coral bleaching in the central GBR36230Th age errors), its coincidence with major flooding in 1970 is noteworthy230Th age data and the timing of these events suggests that factors associated with major flooding may be primarily responsible for mortality in coral communities in this region. The lowered salinity from the flood plume could also have been responsible for mortality on the reef slope at both Bramston and Stone Island, resulting in the low level of coral cover observed in 2012 , a similarly ‘luxuriant crop of seaweeds…mixed with coral growths, (was) conspicuous…[p. 15  precisely dating to 2007.2 ± 1.4 AD suggests that living corals were present until very recently, although coral cover is unlikely to have been high because the time period between 1994 [where no Acropora and very few massive coral colonies were foundin situ dead corals available for sampling in 2012. Indeed, photographs and descriptions of the damage at Poole Island [less than 8 km south-east of Stone Island and Bramston Reef  either had low levels of mortality and/or obtained high coral cover as a result of the regeneration of surviving tissue e.g.4143For Bramston Reef, there has been some increase in coral cover following the 1991 ± 1.3 AD disturbance event over a ~21 year time period. For Stone Island, the limited evidence of coral growth since the early 19th Century suggests that recovery is severely lagging. Even if a ‘healthy’ community did exist in the 1960–70s, it still took 40–50 years to recover following the 1918 disturbance event. These timescales are similar to (or in the case of Stone Island exceed) documented recovery rates of reefs that have undergone almost 100% mortality, have experienced changes to the reef structure or reflect a chronically disturbed (e.g. eutrophic) environment .14C) ages obtained from fossil microatolls sampled across the reef flat at Stone Island revealed much of the development to have taken place between 6,870 ± 280 and 6,090 ± 190 yBP  together with quantitative geochemical and ecological data is a unique and valuable method to reef managers to fill the knowledge gap that exists prior to monitoring programs. A similar approach can be applied to other reef locations where an accurate chronology for the timing of substantial declines in coral cover, shifts in species composition and associated drivers are required.Bramston Reef  and Stone Island reef  are a nearshore and fringing reef, respectively, that are located within Edgecumbe Bay, central GBR . NeighboAcropora spp., massive faviid and Porites corals .Using the geographical features in the background of the photos taken in 1994 as a reference For the Bowen region, mean sea level (MSL) and MLWS height is 1.76 mLAT and 0.67 mLAT, respectivelyin situ coral samples were collected from Bramston Reef and Stone Island in order to constrain the timing of coral loss observed in the historical photographs. To determine the time of death of the massive in situ faviids present in the 1994 photographs of Bramston Reef, a subsample of carbonate material was collected from the top of 17 randomly selected colonies using a hammer and chisel along transects 1, 2 and 4 , as well as along the elevation transect. Care was taken to avoid sampling bias with both small and large colony sizes sampled (range 22–71 cm). Given that the colonies were covered in mud and algae at the time of collection, this also prevented any bias towards preservation state. Only nine dead corals believed to be in situ  were found along transects 1 and 2 at the reef crest of Stone Island.Dead et al.24Samples were identified to species level in the laboratory and high-purity  aragonite material from each of the dead faviid colonies was carefully vetted under a light microscope then prepared for U-Th age dating on a Nu Plasma Multi-Collector Inductively Coupled Plasma Mass Spectrometer (MC-ICP-MS) in the Radiogenic Isotope Facility at the University of Queensland following methods and procedures described in Clark 230Th contributions in order to obtain precise and accurate 230Th ages for such young coral samples. The detrital 230Th end-member for corals sampled from Bramston Reef was determined from a 230Th/232Th versus 234U/238U isochron by analysing three samples of coeval material from each of the following colonies: BRS1-R2  and BRS1T2.3 . U-Th data for each sample was plotted in 230Th/232Th versus 234U/238U space and the 230Th/232Th0 ratios calculated from the y-intercept (with 2σ). The detrital component for the site was determined to be 0.81 ± 0.01 (1σ), based on the average of the 230Th/232Th0 ratios. This value, together with a GBR weighted mean 230Th/232Th0 value of 1.066 ± 20% determined by Clark et al.230Th present in the dead coral samples and allow for accurate 230Th age estimatesAll U-Th ages were corrected for initial/detrital 230Th age (years)230Th age was calculated by dividing the distance from the surface of the colony to the sampling location (cm) by the average linear extension rate (cm yr-1) for that coral species. Average linear extension rates for P. verweyi colonies and G. aspera colonies were determined to be 0.88 ± 0.23 (± 1sd) cm yr−1 and 0.73 ± 0.05 cm yr−1, respectively, based on dead coral colonies that were slabbed into a 7 mm thick section down the main growth axis and X-rayed  to be able to measure the length of each annual density banding couplet  in an attempt to identify the primary driver for mortality.The mortality age (or surface age) of each sampled colony was determined by subtracting the number of annual growth bands above the sampling location  from the corrected  couplet . The hig couplet . Kernel How to cite this article: Clark, T. R. et al. Historical photographs revisited: A case study for dating and characterizing recent loss of coral cover on the inshore Great Barrier Reef. Sci. Rep.6, 19285; doi: 10.1038/srep19285 (2016).


Schizosaccharomyces pombe ribosomal (r)RNAs and generated the first complete PTM maps of eukaryotic rRNAs at single-nucleotide resolution. There were 122 modified sites, most of which appear to locate at the interface of ribosomal subunits where translation takes place. We also identified PTMs at specific locations in rRNAs that were altered in response to growth conditions of yeast cells, suggesting that the cells coordinately regulate the modification levels of RNA.We present a liquid chromatography–mass spectrometry (LC-MS)-based method for comprehensive quantitative identification of post-transcriptional modifications (PTMs) of RNA. We incorporated an in vitro-transcribed, heavy isotope-labeled reference RNA into a sample RNA solution, digested the mixture with a number of RNases and detected the post-transcriptionally modified oligonucleotides quantitatively based on shifts in retention time and the MS signal in subsequent LC-MS. This allowed the determination and quantitation of all PTMs in  To date, >100 different types of PTMs have been found in various types of cellular RNAs, including transfer (t)RNA, rRNA, small nuclear RNA, small nucleolar (sno)RNA and messenger (m)RNA ,3, and tet al.  and RNase A were obtained from Sigma-Aldrich. Sodium cytidine-13C9 5′-triphosphate and sodium uridine-13C9 5′-triphosphate were purchased from Santa Cruz Biotechnology, Inc. RNase T1 was purchased from Worthington and further purified by reversed-phase LC. Triethylammonium acetate buffer (pH 7.0) was purchased from Glen Research. Chemically synthesized oligonucleotides that mimic S. pombe 5S rRNA (positions 90–115) with or without modifications of ribose or base molecules were obtained from JBioS. The chemical structures of these synthetic oligonucleotides are given in Supplementary Table S1.Standard laboratory chemicals were obtained from Wako Pure Chemical Industries. Sodium guanosine-2 at 37°C, and the medium was changed every 2 or 3 days.HeLa cells (American Type Culture Collection) were cultured in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Hana-Nesco Bio) and 100 U/ml penicillin-streptomycin (Invitrogen) in a humidified atmosphere containing 5% COSchizosaccharomyces pombe (American Type Culture Collection) was used throughout this study. The cells were grown in YE medium  at 30°C and harvested during logarithmic growth phase (∼1.0 × 107/ml) as determined by counting cells in culture.Wild-type SP6 Total RNAs were prepared from HeLa cells or yeast cells using the TRIzol reagent (Invitrogen Life Technologies). To obtain 5S, 5.8S or 18S rRNA, total RNAs were applied to a reversed-phase LC system  equipped with a capillary PLRP-S 4000 column , eluted with a 60-min linear gradient from 11.6% to 14% acetonitrile in 100 mM triethylammonium acetate (pH 7.0) including 0.1 mM ammonium phosphate dibasic at a flow rate of 50 μl/min at 60°C and fractionated by monitoring ultraviolet absorption at 260 nm . Only rRin vitro transcription of our internal standard RNAs, the DNA of human 5.8S rRNA and S. pombe 5S, 5.8S, 18S and 25S rRNA was amplified by polymerase chain reaction (PCR) from genomic DNA. The primers used for the PCR are given in Supplementary Table S1. The amplified DNA of each rRNA was inserted into the XhoI/EcoRI, XhoI/HindIII, KpnI/HindIII or HindIII/NotI sites of plasmid pBluescript II KS . Before in vitro transcription, the plasmid was linearized with SpeI to terminate the product at the end of the rRNA. To synthesize RNA, 2 μg of template DNA was incubated and transcribed using a Megascript T3 kit (Invitrogen). When RNA was synthesized, guanosine-13C10 5′-triphosphate, cytidine-13C9 5′-triphosphate or uridine-13C9 5′-triphosphate solution was used instead of the respective 5′-triphosphate reagent that contained carbons with a natural isotope distribution. The RNA was precipitated in ethanol, solubilized in nuclease-free water and then purified further by reversed-phase LC as described above.To construct the plasmids for 2 and 1 mM DTT. Before RNase H digestion, the sample RNA was denatured at 65°C for 10 min. The RNase H digests were directly loaded onto reversed-phase LC columns described above to purify the resulting RNA fragments.Sequence-specific RNase H cleavage was performed using a guide DNA to target the RNA site . DNA molThe LC system used was as described ,35. The S. pombe genome database (http://www.pombase.org/downloads/datasets). We used the following search parameters: the maximum number of missed cleavages was set at 1; the variable modification parameters included two methylations per RNA fragment for any residue; RNA mass tolerance of 20 ppm and MS/MS tolerance of 750 ppm were allowed. When Ariadne identified a methylated oligonucleotide, the MS/MS spectrum was manually inspected to distinguish the methyl group attached to the base or sugar by the presence or absence of methylated base loss from the parent ion. Where necessary, the position of methyl group on the nucleotide base was predicted based on the structure of Saccharomycescerevisiae rRNA (http://people.biochem.umass.edu/fournierlab/snornadb/main.php) (We used the software Ariadne  for dataain.php) .et al.  with various acrylonitrile amounts and ethanol percentages and for different incubation times. Under the optimized conditions determined , about 30% of the pseudouridine and 5% of the uridine molecules were cyanoethylated (data not shown).RNA samples were digested before the derivatization reaction. The digests were cyanoethylated by the method developed by Mengel-Jorgensen et al. , with miDenaturing polyacrylamide gel electrophoresis and in-solution RNase T1 and RNase A digestion of RNA were performed as described .in vitro-transcribed RNA does not carry PTMs and (ii) RNA PTMs generally cause a shift in the retention time (Rt) and MS signal in LC-MS. Our strategy is depicted and described in Figure R2 > 0.99) with an average standard deviation of <5% . SILNAS can also be used to determine the site of pseudouridylation, a ‘mass-silent’ modification found abundantly in many types of RNA, by the shift of retention time in LC or by coupling the cyanoethylation with acrylonitrile to discriminate pseudouridine (Ψ) from uridine .SILNAS is based on the fact that (i) an 71AAUUGmCA78Gp was 92% methylated . The SILNAS-based quantitative analysis actually provided the levels of modification at all of the PTM sites in S. pombe rRNAs (described below). Figure S. pombe 5.8S and 18S rRNA, respectively.To validate SILNAS, we analyzed PTMs in human 5.8S rRNA, a 157-nucleotide RNA containing two methylated nucleotides and two Ψs   and wereSchizosaccharomyces pombe; i.e. 5S, 5.8S, 18S and 25S rRNA consisting of 119, 160, 1842 and 3498 nucleotides, respectively. We easily ascertained the complete chemical structures of the 5S and 5.8S rRNAs and found no PTMs in 5S rRNA and only a single Ψ at position 76 in the 5.8S rRNA  and 75 in large subunit (LSU) rRNA (one in 5.8S and 74 in 25S rRNA). SSU rRNA contained 23 sites of pseudouridylation, 19 sites of 2′-O-methylation, 2 sites of base acetylation, 2 sites of base dimethylation, a single site of base monomethylation and a single site of 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridylation , and LSU rRNA contained 32 sites of pseudouridylation, 37 sites of 2′-O-methylation and 6 sites of base monomethylation . Our analysis also assigned two acetylcytidines at positions 1297 and 1815 in 18S rRNA. For more than three decades, it has been reported that 18S rRNA from a broad range of eukaryotic cells, from budding yeast to mammals, has two acetylcytidines , and we could easily map all the modified nucleotides on S. cerevisiae rRNAs. By surveying the resulting 3D modification map, we readily recognized that most  modified nucleotides were located in functionally important interior regions of the ribosome, including the peptidyl transferase center (PTC); the A, P and E sites of tRNA and mRNA binding; the polypeptide exit tunnel and the interacting surfaces of both the SSU and LSU .S. pombe rRNAs were almost fully modified (>85%), whereas the extent of modification at several sites was ∼10–60% (Supplementary Table S3). Of particular interest is that the extent of modification of specific nucleotides was significantly affected by the growth conditions of the yeast cells from which the RNA was extracted. We actually estimated the quantitative levels of ∼40 modification sites in 5.8S and 18S rRNAs of S. pombe cultivated under different growth temperatures, including Ψ, Am, Cm, Um, Gm, ac4C, m7G, m62A and m1acp3Ψ in 5.8S and 18S rRNA. Among those, we found that the modification levels of several Ψs varied with growth temperature to the significant extent, while the levels of most others were nearly 100% regardless of growth temperature (Supplementary Table S3). Namely, the pseudouridylation at U76 in 5.8S rRNA increased from 44% to 90% in response to raising the growth temperature from 17°C to 35°C. Likewise, the baseline pseudouridylation at U1231 in 18S rRNA was 22% at 17°C and increased to 68% at 35°C (Figure The SILNAS-based quantitative analysis of the stoichiometry of PTM at each modification site showed that the extent of PTM is site specific. Namely, most of the 122 PTM sites in the C Figure –21.In this study, we described an MS-based method, SILNAS, for comprehensive PTM analysis of RNA and presented its application to construct the first complete PTM map of eukaryotic rRNAs. We expect that it will serve as a useful resource for studies of the structure, function and biogenesis of the ribosome. SILNAS compares with previous methods ,29 that Supplementary Data are available at NAR Online.


Agroup of twenty-three individuals consumed 300 g/d of cooked red meat without (HRM diet)or with 40 g/d of HAMSB (HRM+HAMSB diet) over 4-week periods separated by a 4-week washoutin a randomised cross-over design. Stool and rectal biopsy samples were collected forbiochemical, microbial and immunohistochemical analyses at baseline and at the end of each4-week intervention period. The HRM diet increased rectal O6MeG adductsrelative to its baseline by 21 % (P< 0·01), whereas the additionof HAMSB to the HRM diet prevented this increase. Epithelial proliferation increased withboth the HRM (P< 0·001) and HRM+HAMSB(P< 0·05) diets when compared with their respective baselinelevels, but was lower following the HRM+HAMSB diet compared with the HRM diet(P< 0·05). Relative to its baseline, the HRM+HAMSB diet increasedthe excretion of SCFA by over 20 % (P< 0·05) and increased theabsolute abundances of the Clostridium coccoides group(P< 0·05), the Clostridiumleptum group (P< 0·05),Lactobacillus spp. (P< 0·01),Parabacteroides distasonis (P< 0·001) andRuminococcus bromii (P< 0·05), but loweredRuminococcus torques (P< 0·05) and theproportions of Ruminococcus gnavus, Ruminococcus torquesand Escherichia coli (P< 0·01). HRM consumptioncould increase the risk of CRC through increased formation of colorectal epithelialO6MeG adducts. HAMSB consumption prevented red meat-induced adduct formation,which may be associated with increased stool SCFA levels and/or changes in the microbiotacomposition.Epidemiological studies have identified increased colorectal cancer (CRC) risk with highred meat (HRM) intakes, whereas dietary fibre intake appears to be protective. In thepresent study, we examined whether a HRM diet increased rectalO Although there is a genetic component in CRC development, diet and otherlifestyle factors are estimated to explain as much as 30–50 % of the global incidence of thedisease. According to the recent report from the World Cancer Research Fund and AmericanInstitute for Cancer Research (WCRF/AICR), there is convincing evidence that intake of red and processed meat increases therisk of CRC, whereas intake of dietary fibre is protective.Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies worldwide,accounting for 10 % of all cancers and for approximately 20 % of all cancer-related deaths indeveloped countries. For red meat, in particular, it has been suggested that its high content of haemFe is a substantial contributor. Red meat undergoes fermentation in the colon that might alter the microbiotacomposition and result in the production of potentially genotoxic products that could play arole in oncogenesis. These agents include N-nitroso compounds (NOC), a complex mixture ofnitrite-derived products formed either in processed meat itself or endogenously in the humangut via bacterial metabolism. NOC are alkylating agents that generate DNA adducts in humancolonocytes after high red meat consumption. We have recently shown that the level of the pro-mutagenic adductO6-methyl-2-deoxyguanosine (O6MeG) is increased in murine colonocytesafter consuming a diet high in red meat. O6MeG is a known toxic and mutagenic base modification that, ifunrepaired, can induce GC → AT transition mutations  and also recombination events or mutations in the form of sister chromatidexchanges. More recently, it has been suggested that high red meat consumption can increasethe expression of certain oncogenic microRNA.A variety of mechanisms have been proposed to link red and processed meat consumption and therisk of CRC. Of the major SCFA, butyrate is of particular interest as it appears to be thepreferred metabolic substrate for colonocytes, and butyrate also promotes a normal cellularphenotype. In vitro studies with CRC cell lines have shown that butyrateinduces apoptosis, reduces cell proliferation and promotes differentiation. Animal experiments have shown that butyrate may reduce colorectal carcinogenesisby enhancing the apoptotic response to methylating carcinogens.Dietary fibre is a heterogeneous group of compounds, principally indigestible carbohydratesof plant origin that include NSP, starches that escape digestion in the small intestine and oligosaccharides. One possible mechanism for the reduction in therisk of CRC by dietary fibre is the production of SCFA via fermentation by the large-bowelmicrobiota. This protective effect correlated most closely with large-bowel butyrate levels,supporting a role for fermentation in risk modification. Acylated starches , in which the acyl group is linked to the starch framework by an esterbond, can deliver specific SCFA to the large bowel where bacterial esterases release the SCFA.Ingestion of butyrylated high-amylose maize starch increases colonic butyrate levels inanimals and humans. Accordingly, the present study was carried out in healthy individuals todetermine whether high red meat consumption generated O6MeG adducts in rectalepithelial cells, and whether concurrent consumption of high red meat and butyrylatedhigh-amylose maize starch opposed this effect (primary aim). We also investigated the effectsof these diets on other indices of colonic health including rectal proliferation, colonicfermentation products and microbiota composition, as these might participate in the generationof adducts.Increasing large-bowel butyrate supply has the potential to improve colonic function andlower disease risk. RS is thought to be particularly effective in this regard as itsfermentation generally favours butyrate production. Red meat and fibre (including RS) aregenerally consumed together as components of foods. Our animal studies have shown that dietaryRS is able to oppose colonocyte DNA strand breaks, telomere shortening and pro-mutagenic DNAadduct formation in rodents fed red meat. Participants on the HRM arm of the study were asked to consume 250 ml ofreduced-fat milk or orange juice per d to match the HRM+HAMSB intervention. During theintervention periods, participants reduced their intake of their habitual diet toaccommodate the extra 300 g red meat. Participants were instructed to maintain their usualdiet during the study but to avoid consuming high levels of protein or fibre, or probioticsupplements, except those prescribed for the study. Participants were also asked to avoidconsuming, or record the use of, any medication that could interfere with bowel function(including antibiotics). Participants were monitored by a trial nurse (weekly) anddietitian (at the end of each 4-week period) to ensure that diet and interventionguidelines were followed, and weight was kept stable. Details of medical history andmedications, weight, bowel health and symptoms, and adverse events were collected by thetrial nurse throughout the study. Composition of the participants' diets and compliancewith the interventions was assessed using weighed food diaries that were completed by theparticipants at the end of each 4-week dietary period, 3 d before each clinic visit. Fooddiaries were entered into FoodWorks Professional 7 Nutrition Calculation software (XyrisSoftware) by a dietitian, to calculate energy and macronutrient intake based on Australianfood composition tables and food manufacturers' data. The present study was approved bythe Flinders Clinical Research Ethics Committee , and all volunteers gave written informed consent.The present trial was registered in the Australian New Zealand Clinical Trials Registry asACTRN12609000306213 (http://www.anzctr.org.au).The present study was conducted as a double-blind, randomised cross-over trial consistingof two intervention periods of 4 weeks each, preceded by a 4-week run-in (baseline) andseparated by a 4-week (washout) period . AgroupStool and rectal pinch biopsy samples were obtained at the completion of the 4-week entryperiod (baseline) and at the end of each intervention period. A complete faecal collectionwas made by all participants for the last 48 h of each dietary period, and the sampleswere stored in portable freezers ( − 20°C). At each visit to the Flinders Medical Centreclinic, an experienced gastroenterologist collected four rectal mucosal biopsies usingalligator forceps through a 25 cm rigid sigmoidoscope; this procedure was performedwithout bowel preparation or prior dietary restriction. Biopsies were formalin-fixed anddehydrated through gradient alcohol and xylene before being embedded in paraffin wax.. Total SCFA concentration was calculated as the sum of acetic, propionic,butyric, isobutyric, caproic, isovaleric and valeric acid concentrations. Totalbranched-chain fatty acids concentration was calculated as the sum of isobutyric andisovaleric acid concentrations. Phenol and p-cresols were measured in thefaeces by using vacuum microdistillation and HPLC. Faecal NH3 concentration was measured by using the indophenolblue method. Aqueous extracts of the faeces were prepared by diluting 1 g faeces with4 ml of distilled water, homogenised and centrifuged , and total apparent NOC were measured by chemical denitrosation with HBr andchemiluminescence detection of the released nitric oxide using a thermal energy analyser(TEA). Concentrations were calculated by comparing the TEA response of a faecalwater sample with the response of an N-nitrosodipropylamine standard(16·6 μg/ml), and values were expressed as total apparent NOC (ng/ml).Faecal samples were thawed at 4°C, pooled, homogenised, and then subsampled for analysis.For the determination of SCFA, weighed portions were diluted at 1:3 (w/w) with deionisedwater containing 1·68 mmol heptanoic acid/l as an internal standard ,and processed for SCFA analysis using GC as described previously6MeG adduct load was performed using animmunohistochemical detection method. The immunohistochemical measurement of O6MeG adducts has beenpreviously used for many years mainly in different animal species; however, this is the first time it has been applied to human colonic crypts.The specificity of the monoclonal antibody has been validated by RIA. In brief, rectal biopsies were embedded in paraffin and sectioned at 4 μm,and their O6MeG adduct load was quantified using an anti-O6MeGantibody (Squarix Biotechnology); this antibody is listed as being specific for humantissue. Antigen retrieval (10 mm-citrate buffer) was performed, followed by RNasetreatment (20 μl RNase A (10 mg/ml), Thermo Fisher Scientific; 5 μl RNase T (10 units/ml),Thermo Fisher; 100 μl PBS (pH 7·4) and stopped with a 5 min treatment with NaCl solution(140 mm)). DNA unwinding was achieved using alkali treatment before applying Special Block A(Covance Laboratories) for 30 min. The O6MeG antibody (1:1000) was applied tothe slides overnight at room temperature, followed by Special Block B (CovanceLaboratories), before applying poly-horseradish peroxidase (HRP) anti-mouse IgG. Sectionswere counterstained with haematoxylin, and chromogen 3,3′-diaminobenzidinetetrahydrochloride (DAB) was used to visualise positive O6MeG staining. Allslides were independently and randomly coded before quantification of nuclear staining forO6MeG with a computer image analysis protocol. Overall, twenty appropriate crypts were visualised using an OlympusMicropublisher 3.3 RTV camera and Olysia Bio-report software (Olympus). Camera andmicroscope settings were calibrated before each image to ensure analytical consistency. Toidentify a linear path through a single row of nuclei along the crypt axis for all imagestaken, image analysis software developed by the CSIRO Mathematics Informatics andStatistics division, ‘Imview’ and ‘R for Windows’ 2.1.0, was used. Raw colour , luminescence (L), normalised colour values and colourratio (RoB = ¼ r/b) data points were calculated for eachpixel along the length of the linear path. The number of cells within each half crypt wascounted, and the calculated RoB ratio was then averaged for each nucleus within individualcrypts. Total O6MeG values/crypt were achieved by summation of the ratio valuefor each nucleus along the crypt axis. Representative sections of one individual from eachtreatment group showing the immunohistochemical staining are shown in . Slides were visualised by brown nuclear staining, and assessed as the numberof Ki-67-positive cells/crypt.The quantification of the O. In brief, DNA was extracted using a repeat bead beating and column clean-upmethod, and qPCR assays amplified the 16S ribosomal RNA (rRNA) gene (or in the case ofsulphate-reducing bacteria the adenosine-5′-phosphosulfate reductase(aps) gene) using primers that targeted bacterial species or groups ofinterest. The primer pairs and their amplification conditions are listed in onlineSupplementary Table S5. Data are expressed as absolute abundances and as a proportion oftotal bacteria. Bacterial targets were chosen for their relevance to gut health. In otherwords, we selected key species (e.g. F. prausnitzii) or groups ofbacteria (e.g. C.coccoides group) that were responsible for the production of butyratefollowing fermentation of complex carbohydrates, particularly RS, by bacteria suchRuminococcus bromii. P.distasonis was chosen because of its potential role in the cleavage ofbutyrate from the butyrylated RS used in the study. We also examined changes in somepotentially enteropathogenic species (e.g. E.coli), as well as in general groups such asLactobacillus and Bifidobacterium that are regarded asmarkers of bowel health by many. A range of other bacteria such as those implicated in gutmucus barrier turnover and inflammatory bowel disease, such as A.muciniphila, were also targeted. Sulphate-reducing bacteria were included todetermine whether the production of toxic hydrogen sulphide could contribute tolarge-bowel DNA adduct formation in response to red meat treatment.Extraction of DNA from stool samples and subsequent quantitative real-time PCR (qPCR) wasperformed and analysed according to the method used by Christophersen and colleagues. Analyses were carried out using a linear mixed-effects model, with subjectsas the random effect, on either base-10 logarithm-transformed data, where necessary, or onuntransformed data for each of the variables.All statistical analyses were performed with scripts in R, version 3.0.1, using the Rstatistical package6MeG, epithelial proliferation, certain bacterial species but not SCFA . This was indicated by asignificant difference between the baseline level and the washout level of the responsevariable or a significant interaction between the week of diet consumption and theresponse variable. Therefore, the data analyses used in the present study were only thoseof the first period of the study (i.e. only measurement weeks 0 and 4). As a result, theanalysis reported herein was carried out using only the baseline and the first-perioddata. The comparison between the groups in the first period of the trial was carried outusing a linear mixed-effects model, testing for changes from the baseline and a differencebetween the treatments. For P value for the difference between the two treatments at week4, and these were tested using either the original data or the log 10-transformed data asappropriate.Initial analyses were carried out using the two periods of the trial, including thebaseline and washout periods. However, data analyses of the initial study showed that someof the response variables had carry-over effects, including the primary end-pointO+ version 1.06 (PRIMER-E). A Pvalue < 0·05 was considered significant.The effects on the overall composition of the gut microbiota were analysed by combiningall qPCR assays and performing a permutational-based multivariate analysis. Data werelog-transformed before producing a resemblance matrix using Euclidean distance.Differences between the interventions were tested on first-period cross-sectionalcomparison only using Permanova6MeG, epithelial proliferation, certain bacterial species but not SCFA . There was a significant increasein the rectal crypt O6MeG adduct load when the participants consumed the HRMdiet first compared with all the other intervention stages); however, when theparticipants consumed the HRM+HAMSB diet as the first intervention, there was no change inthe O6MeG adduct load with the subsequent consumption of HRM ). There was a significant effect of treatment and treatment orderon PCNA-positive cells/crypt  and (B)). For theparticipants on the HRM or HRM+HAMSB diet as their first intervention, the PCNA-positivecells significantly increased (P< 0·001). For those on the HRM orHRM+HAMSB diet (received as their first intervention), the positive cells significantlydecreased after consuming their final treatment compared with the first treatment.Participants who consumed the HRM diet as the first treatment had significantly higherPCNA-positive cells/crypt compared with those who consumed the HRM diet as the secondtreatment (P< 0·001). We also observed that numbers of somebacteria in the washout phase were significantly different from those during the entryperiod or the dietary interventions (see online Supplementary Tables S3 and S4). Theanalysis revealed a diet-order effect on the microbiota composition. When the volunteersconsumed the HRM+HAMSB diet as the first intervention, their microbiota compositionsignificantly differed from that of the entry levels (P= 0·02), as wellas from that of the subsequent HRM intervention (P= 0·02) and the washoutlevels (P= 0·005) in the same volunteers. Furthermore, the microbiotacomposition of these volunteers consuming the HRM+HAMSB diet first was also significantlydifferent from that of those consuming HRM first (P= 0·01). However, whenthe volunteers consumed the HRM diet first, the microbial composition during thesubsequent consumption of the HRM+HAMSB diet was only different from that of the washoutlevels (P= 0·02).Initial analyses were carried out using the two periods of the trial, including baselineand washout periods. However, data analyses of the initial study showed that some of theresponse variables had carry-over effects, including the primary end-pointO6MeG,epithelial proliferation and certain bacterial species. The study was still adequatelypowered based on the primary end-point ‘O6MeG’. Calculations using a two-tailedt test with a power of 80 % with means of 60·8 and 77·4 and a standarddeviation of 15 showed that group sizes of five were adequate to detect a differencebetween the baseline and the HRM intervention.The results arising from the respective baseline periods and the first arm of the dietaryintervention (i.e. at week 4 of the intervention) are described in detail below, as theresults of the second arm (cross-over) showed carry-over effects for Osem 1·8) years and mean body weight 79·8 (sem 5·6) kg) completed theHRM intervention as the first diet period, while thirteen  years and body weight 82·4 (sem 3·5) kg) completedthe HRM+HAMSB intervention first.Recruitment commenced in July 2009, with each participant followed up for the 4-monthduration of the interventions. Data collection was completed by September 2010. A total oftwenty-five participants were assigned randomly, with twelve allocated to the HRM dietaryintervention first and thirteen allocated to the HRM+HAMSB dietary intervention first.However, two participants withdrew before the commencement of the intervention diets; onedue to unrelated medical problems and the other due to intolerance of the first rectalbiopsy. Approximately one-third of the participants on the trial diets reported increasedflatulence. Of the volunteers, ten  and 82·8 (sem 3·3) kg after the HRM and HRM+HAMSB interventions,respectively.Participants maintained consistent body weight, with mean weights of 77·1 (P< 0·05) and HRM+HAMSB (P< 0·01)interventions. Fibre intake was decreased in the HRM group at week 4 compared with itsbaseline level (P< 0·01). Fibre intake was significantly lower inthe HRM group than in the HRM+HAMSB group after 4 weeks (P< 0·01).Fe intake from meat was significantly higher for the HRM and HRM+HAMSB interventions atweek 4 compared with their respective baseline levels(P< 0·001).There was no difference between the diets for reported intake of energy, total andsaturated fat, total carbohydrates and sugar, starch, alcohol or total Fe intake . Compare6MeG adduct load was increased at week 4 in the HRM group compared withits baseline level (P< 0·01); however, the addition of HAMSB tothe HRM diet prevented this increase (P< 0·001) andHRM+HAMSB (P< 0·05) groups; however, the number was lowerfollowing the HRM+HAMSB intervention than the HRM intervention as was the stool concentrations of acetate, propionateand total SCFA (P< 0·05). Faecal p-cresolconcentration was lower in the HRM+HAMSB group at 4 weeks compared with its baseline level(P< 0·01) and the HRM group (P< 0·05).Branched-chain fatty acids, phenols, NH3 and NOC were unaffected by thetreatment.The results of the stool analyses are presented in Parabacteroides distasonis relative to its baseline level(P< 0·0001) and the HRM group (P< 0·001).Compared with its baseline level, HRM+HAMSB consumption increased the numbers ofLactobacillus spp. (P< 0·01), theClostridium coccoides group (P< 0·05), theClostridium leptum group (P< 0·05) andRuminococcus bromii (P< 0·05), but lowered thenumbers of Ruminococcus torques (P< 0·05). Whenbacterial numbers were expressed as a percentage of total bacteria, the proportion ofP. distasonis was increased by the consumption of the HRM+HAMSB diet at4 weeks compared with its baseline level and the HRM group at 4 weeks (bothP< 0·0001). Lower proportions of Ruminococcusgnavus (P< 0·01), R. torques(P< 0·01) and E. coli(P< 0·01) were evident in the HRM+HAMSB group at 4 weeks comparedwith its baseline level. The HRM+HAMSB group also had lower proportions of R.torques (P< 0·05) and E. coli(P< 0·05) than the HRM group at 4 weeks. When the results of allqPCR assays were combined and then analysed statistically to gain an indication of theimpacts of the treatments on microbial diversity, it was found that the microbialdiversity during the HRM+HAMSB intervention was different from that at baseline(P< 0·05) and during the HRM intervention(P< 0·01); however, the composition at baseline and the HRMintervention did not differ.Significant changes in stool bacteria in response to the diet are presented in 6MeG) in the colon, whereasco-consumption of a fermentable carbohydrate can reduce this effect. We have now shown that when free-living healthy human subjects consumed theirnormal habitual diet containing at least an additional 300 g red meat over a 4-week period,there was increased formation of the O6MeG adduct in the rectal epithelium. Thisincrease in adduct formation might account, in part, for the increased risk of CRCassociated with consuming high levels of red meat.Previously, we reported that feeding a diet rich in red meat to rodents can increase thelevel of the pro-mutagenic DNA adduct  all point towards SCFA, particularly butyrate, to be the key mediators inpreventing meat-induced DNA adducts and DNA strand breaks in colonic mucosa. Butyrate is apreferred metabolic substrate for colonocytes, and this SCFA has strong anti-tumorigenicproperties in vivo and in vitro. In the present study, ingestion of HAMSB in conjunction with HRM was also ableto favourably influence the colonic luminal environment, as evidenced by increased levels ofSCFA and a reduction in the potentially toxic protein fermentation productp-cresol. This elevation of faecal butyrate with HAMSB confirms previousstudies in human subjects, and has the potential to improve colonic health and offer protection againstCRC. Although consumption of a blend of types 2 and 3 RS in a recent human trial ofhereditary CRC failed to reduce tumour incidence, the relatively low daily intake of RS used in that study may have beeninsufficient to increase SCFA levels in stool (which were not measured). At least 20 g ofRS/d may be needed to increase stool levels of SCFA.The present study also confirms that dietary fermentable carbohydrate in the form of HAMSBcan protect against red meat-induced colorectal DNA lesions in humans, and is consistentwith epidemiological evidence that dietary fibre consumption reduces the risk of CRC. Thepresent study and our previous work in rodents6MeG adducts with the HRMintervention is dietary haem. Haem is abundant in red meat, the majority of which derivedfrom the diet passes into the large bowel. We recently identified dietary haem as an agent that can increaseO6MeG adducts in the colon of mice. Haem Fe-rich meat has also been shown to increase alkylated DNA adducts in anin vitro digestion system. Dietary haem may also increase the production of reactive oxygen species,causing cellular toxicity and pro-mutagenic lesions. Other factors, such as bile acids, could also contribute to adduct formation and DNA damage more broadly. Haemmay also be responsible for the increased rectal cell proliferation in response to HRM andHRM+HAMSB consumption, as evidenced by more PCNA-labelled cells/crypt. Haem is associatedwith increased epithelial proliferation in the colon of rodents, and can injure the colonicsurface epithelium by generating cytotoxic and oxidative stress.A reasonable explanation for the increase in O6MeGadduct load. High dietary haem and red meat have previously been associated with increasedluminal NOC in humans. A dose–response relationship has been described between red meat intake andfaecal NOC: low faecal NOC (374 μg/kg) at low red meat intake (60 g/d) and a 4- or 5-foldincrease in faecal NOC with increased red meat intake of 240 and 420 g/d, respectively. Lewin et al. also observed an increase in faecal NOC in volunteers fed 420 g/d of red meatin comparison with a vegetarian diet, and suggested that NOC are important genotoxinsinvolved in the generation of alkyl adducts. Although we observed an increase in the O6MeG adduct load with HRMintake (300 g/d), we are unable to completely explain the lack of the effect of HRM onfaecal NOC. One possible explanation is that the other studies had a very high level of control with the meals being consumed in anexperimental facility. In the present study, we only controlled for the amount of meat inthe diet so that other factors in the diet may account for the discrepancy.In the present study, we anticipated that NOC would increase in the stool of theparticipants consuming the HRM diet, and that this would explain a higher OC. leptum group and R. bromii, a member of theC. leptum group, in response to the consumption of RS as HAMSB isconsistent with the effects previously observed in human subjects and with the central rolethat this bacterium appears to have in RS degradation(–). This increase provides further evidence  that HAMSB was being consumed by the participants and was reaching thelarge bowel where it was available for fermentation. The numbers of P.distasonis were also increased by the HAMSB treatment, which is also consistentwith changes in humans following consumption of butyrylated RS. HAMSB is a chemically modified RS, and the forms of RS (classified as RS4)have been shown to be more likely to stimulate the growth of P. distasonis. In line with the HAMSB treatment, stool excretion of butyrate increased,primarily from the release of the bound butyrate; however, the higher number of bacteria inthe C. coccoides group could also have contributed as a number of butyrateproducers are classified in that group.There is growing recognition of both the importance of the large-bowel microbiota in humanhealth and the strong role of diet in modulating its composition and metabolic activities.Using a suite of qPCR assays that targeted a range of bacteria important to gut health, wedemonstrated significant shifts in the composition of the gut microbiota in response to theHRM+HAMSB, but not HRM, intervention. Our observation of an increase in the stool numbers ofthe E. coli , R. gnavus and R.torques , supporting the potential of HAMSB to promote gut health. We did not observeany clear indication of the effects of HRM on the composition of the gut microbiota.However, the range of bacteria that we targeted is limited, and a more detailed analysis ofpopulations may reveal changes. This may give an insight into the mechanisms of HRM-inducedadduct formation and reasons for the associated increased risk of CRC. Furthermore,in vitro experiments have demonstrated that the formation of alkylatedDNA adducts appear to depend on the microbial composition. The changes that we have observed support the idea that the increases in stoolSCFA levels and the associated protection against dietary HRM-mediated colorectal tissuedamage that have occurred in response to dietary RS treatment are at least partly mediatedby the gut microbiota, through both cleavage of the esterified butyrate and fermentation ofthe RS substrate. Part of the protective effect may also be attributable to reducing numbersand activities of bacteria with potential for harm.When the numbers of bacteria were expressed as a proportion of total bacteria, the additionof HAMSB to the diet also lowered the numbers of 6MeG adducts’. Participants allocatedthe HRM+HAMSB intervention in the first period did not have a HRM-induced increase inO6MeG adducts during the subsequent period. This contrasted with the increasein adduct formation that occurred when HRM was consumed first. This suggests that theconsumption of HAMSB is able to protect against the damage caused by the HRM diet. We havepresented in detail the analyses of the data from the first period . Presentation ofthe data from the first arm reduces the statistical power of the study compared with that ofthe full cross-over. However, the primary end-point ‘O6MeG’ was still adequatelypowered, and the effect of HAMSB on SCFA  is magnified. A furtherlimitation of the study was that the right-sided colonic mucosa could not be evaluated, asbiopsies were only taken from the rectum. This limits the application of the results toproximal colorectal carcinogenesis, particularly as genotypic differences between proximaland distal cancers exist. More invasive studies are warranted to investigate whether the same effectsobserved in the rectum occur in other regions of the large bowel.A limitation of the present study was that the randomised cross-over design resulted in aperiod effect for the primary end-point ‘O6MeG adducts. These findings might explain the increased risk for CRCassociated with HRM consumption, and could point to a beneficial effect ofbutyrate-generating RS.In summary, our findings show that high dietary red meat intake has detrimental effects onthe colorectum by increasing pro-mutagenic DNA adducts and epithelial cell proliferation.Conversely, increasing luminal butyrate levels with HAMSB prevented the accumulation ofOFor supplementary material accompanying this paper visit http://dx.doi.org/10.1017/S0007114515001750.click here to view supplementary material


Acyrthosiphon pisum, genome we provide an updated gene annotation and expression profile of putative sugar transporters. Finally, gut expressed sugar transporters are functionally expressed in yeast and screened for glucose and fructose transport activity.Phloem feeding insects, such as aphids, feed almost continuously on plant phloem sap, a liquid diet that contains high concentrations of sucrose (a disaccharide comprising of glucose and fructose). To access the available carbon, aphids hydrolyze sucrose in the gut lumen and transport its constituent monosaccharides, glucose and fructose. Although sugar transport plays a critical role in aphid nutrition, the molecular basis of sugar transport in aphids, and more generally across all insects, remains poorly characterized. Here, using the latest release of the pea aphid, de novo approach, we identified 19 sugar porter (SP) family transporters in the A. pisum genome. Gene expression analysis, based on 214, 834 A. pisum expressed sequence tags, supports 17 sugar porter family transporters being actively expressed in adult female aphids. Further analysis, using quantitative PCR identifies 4 transporters, A. pisum sugar transporter 1, 3, 4 and 9  as highly expressed and/or enriched in gut tissue. When expressed in a Saccharomyces cerevisiae hexose transporter deletion mutant (strain EBY.VW4000), only ApST3 (previously characterized) and ApST4 (reported here) transport glucose and fructose resulting in functional rescue of the yeast mutant. Here we characterize ApST4, a 491 amino acid protein, with 12 predicted transmembrane regions, as a facilitative glucose/fructose transporter. Finally, phylogenetic reconstruction reveals that ApST4, and related, as yet uncharacterized insect transporters are phylogenetically closely related to human GLUT (SLC2A) class I facilitative glucose/fructose transporters.In this study, using a ApST4, and the transport specificity of its product is consistent with ApST4 functioning as a gut glucose/fructose transporter. Here, we hypothesize that both ApST3 (reported previously) and ApST4 (reported here) function at the gut interface to import glucose and fructose from the gut lumen.The gut enhanced expression of The online version of this article (doi:10.1186/1471-2164-15-647) contains supplementary material, which is available to authorized users. Acyrthosiphon pisum, catabolizes 15 - 30% of its ingested sucrose in oxidative pathways. A larger proportion, close to 50% of the ingested sucrose, is incorporated into aphid tissues . Ap. ApN. lude novo search to identify 19 sugar porter family transporters in the latest version of the A. pisum genome (assembly Acyr_2.0). Compared to our previous study, which identified 29 putative sugar transporters  also transport sugars. As with the sugar porter family, previously described, sugar transporters within the solute:sodium symporter family are also most extensively characterized in humans (Reviewed in ). Human sporters , 11, 26.A. pisum encodes a large family of sugar porter family transporters. Based on gene expression patters, only 4 transporters  are predicted to play a functional role at the gut interface. Here we characterize A. pisum sugar transporter 4, ApST4, as a facilitative glucose/fructose transporter. Our gene expression and functional transport analyses are consistent with ApST4 playing a role in aphid sugar transport, functioning at the gut interface to transport glucose and fructose. Here, we demonstrate that ApST4 is, structurally, functionally and phylogenetically related to mammalian GLUT class I facilitative transporters. By placing transporters in a phylogenetic framework this work will facilitate prediction and testing of as yet uncharacterized sugar transporters across related taxa.In summary, this study demonstrates that Acyrthosiphon pisum (Harris), lineage UY2 -glucose (Perkin-Elmer) or D-[U-14C]-fructose (Perkin-Elmer), depending on the experiment. At given intervals 2 OD of cells were collected by rapid filtration onto nitrocellulose membranes (0.2 μm pore size) under vacuum and washed with an excess of ice-cold distilled water. Transport of 14C-glucose or 14C-fructose into yeast cells was determined by liquid scintillation counting. Glucose and fructose transport into yeast cells expressing ApST4 increased linearly for at least 30 min. Thus, all uptake assays were stopped after 10 min in the linear phase of uptake. Sugar selectivity experiments were performed by adding a 2-fold molar excess of unlabeled sugar  to the standard 14C-labeled fructose uptake assay. pH dependence was analyzed by performing uptake assays in either 50 mM citrate buffer (pH 4 – 6), or 50 mM phosphate buffer (pH 7 – 9), as indicated. The influence of pH gradient was determined by preincubating cells for 5 min with either 1 μM 2,4-dinitrophenol (DNP) or 1 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Sodium ion (Na+) dependence was analyzed by performing standard uptake assays with the addition of 50 mM NaCl to the transport buffer, control experiments were run in parallel, where NaCl was replaced with an equimolar amount of choline chloride. Kinetic parameters Km and Vmax were estimated by fitting uptake rates at various substrate concentrations (as indicated) to the Michaelis–Menten equation using non-linear regression . All uptake assays were performed in triplicate, using an independent yeast clone for each replicate and uptake data were corrected for background uptake into cells transformed with an empty pDR195 vector.Uptake assays in whole yeast cells were based on methods described by . Brieflyhttp://www.cbs.dtu.dk/services/TMHMM/)  in insect genomes. Table S3. Summary of annotated A. pisum solute:sodium symporter (SSS) family transporters. Table S4. Quantitative PCR primers for A. pisum sugar transporters and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Table S5.A. pisum sugar transporter coding sequence primers for Saccharomyces cerevisiae expression constructs. (DOCX 2 MB)Additional file 1: Figure S1: Western blot analysis of recombinant A. pisum sugar transporters expressed in yeast.