Triplicate microcosms of fecal slurries were enriched with lactate and/or starch. Metabolic products  were measured and microbial community compositions determined using Illumina 16S rRNA sequencing over 12-hour intervals. We report that patterns of change in short chain fatty acid levels and pH in our in vitro system are similar to those seen in in vivo laminitis induction models. Community differences between microcosms with disparate abilities to clear excess lactate suggest profiles conferring resistance of starch-induction conditions. Where lactate levels recover following starch induction conditions, propionate and acetate levels rise correspondingly and taxa related to Megasphaeraelsdenii reach levels exceeding 70% relative abundance. In lactate and control cultures, taxa related to Veillonellamontpellierensis are enriched as lactate levels fall. Understanding these community differences and factors promoting the growth of specific lactate utilizing taxa may be useful to prevent acidosis under starch-induction conditions.Laminitis is a chronic, crippling disease triggered by the sudden influx of dietary starch. Starch reaches the hindgut resulting in enrichment of lactic acid bacteria, lactate accumulation, and acidification of the gut contents. Bacterial products enter the bloodstream and precipitate systemic inflammation. Hindgut lactate levels are normally low because specific bacterial groups convert lactate to short chain fatty acids. Why this mechanism fails when lactate levels rapidly rise, and why some hindgut communities can recover is unknown. Fecal samples from three adult horses eating identical diets provided bacterial communities for this  Horses are hindgut fermenters, adapted to grazing continually on marginal forages that change seasonally, thus slowly . The hinLaminitis is a chronic, crippling disease, accounting for 15% of all lameness in horses in the United States, with over 27% unable to return to normal work, and 4.7% mortality . It is cFirmicutes, with Bacteroidetes, Proteobacteria, and Verrucomicrobia as other major phyla. Studies have detected a greater proportion of fibrolytic bacteria than starch and lactate utilizing bacteria in the cecum than in the colon [Surveys of equine hindgut bacteria using culture based methods  and 16S he colon ,21, reflin vivo models of laminitis induction using starch gruel or oligosaccharide [Streptococcus bovis/ equinus group , have bincrease ,26,27. Bincrease ,28. In oincrease .Lactate levels in the hindgut are normally low due to the activity of lactate utilizing bacteria. It is unclear why this mechanism fails during conditions of starch induction. While studies of lactate producers have pointed to specific taxa that proliferate during the developmental stage of laminitis ,26,27, lin vitro model system to track bacterial metabolites and community shifts over time in response to enrichment with starch and/or lactate. Patterns of changes in lactate concentration and pH were similar to those reported in published in vivo studies [In this study we used fecal samples collected from 3 healthy, adult horses eating an identical pasture based diet in an  studies ,23. IlluThis study was carried out in accordance with the guidelines set forth by the Morris Animal Foundation and applied by the Institutional Animal Care and Use Committee at the University of Massachusetts, Amherst. We thank Dr. Carlos Gradil  for generously lending his time and expertise in sampling, and the Hadley Farm, University of Massachusetts, Amherst, MA for providing three horses for this study.Fecal samples were manually collected from the midrectum of three Morgan geldings cohoused at the University of Massachusetts, Hadley Farm and fed identical hay based diets. None of the animals had received antibiotics, anthelmenthics, or other medications for at least three months prior to sampling. Samples were protected from oxygen exposure during collection by inverting the glove around each as it was removed, and placing each immediately in a container evacuated of oxygen. Samples were kept as close to 39°C as possible in a heated, insulated container during transport and the following steps of dilution and inoculation. In an anaerobic chamber, samples were diluted to make a 10% slurry in anaerobic dilution media prepared as described by Bryant . This slp-phenylenediamine and 187.5 µl 10 mM iron (III) chloride were added, stoppered immediately, and incubated for 20 minutes at room temperature. The resulting suspension was spun down and the absorbance of the supernatant was measured at 670 nm in comparison to standard solutions . pH was determined using EMD colorphast pH strips .Short chain fatty acids  were measured for samples taken over the time course using high performance liquid chromatography (HPLC)  with an Aminex HPX-87H column . Headspace gases were analyzed using a gas chromatograph  fitted with a HayeSep DB column 100/120 . Hydrogen sulfide levels were measured using the methylene blue assay as in Cline  modifiedDNA was extracted from each sample as described elsewhere ,34 with GTGCCAGCMGCCGCGGTAA-3’) and universal reverse 806R (5’- Illumina adapter- Golay bar code- Reverse primer pad- Reverse primer linker- GGACTACHVGGGTWTCTAAT-3’). Each PCR reaction mixture contained 10 ng of genomic DNA, 2.5 µl 10X buffer, 2.0 µl MgCl2 (25 mM), 2.0 µl dNTP (2.5 mM each), 5.0 mM (each) forward and reverse primers, 1.25 µl (25 µg) BSA , 0.25 µl (1.25 U) Ex Taq , and molecular grade water to reach a volume of 25 µl. PCR was performed with 3 min of initial denaturation at 94°C followed by 30 cycles of the following program  followed by a final extension at 72 °C for 7 min. PCR products were cleaned using Qiagen MinElute kit  as directed and quantified using the Quant-iT PicoGreen assay . Equal concentrations were pooled and sequenced using the Illumina MiSeq platform at the Dana Farber Cancer Institute, Molecular Biology Core Facilities .Amplification of the V4 region of the 16S rRNA gene and attachment of Illumina adaptors and bar codes for multiplexing samples (reverse read only) was done in triplicate as described elsewhere . BrieflySequences were demultiplexed and trimmed of bar codes and primer sequences, then filtered for quality and reverse and forward reads were assembled into contigs using FastQC . SequencDespite variation in the pH response between the three horses, in all of the starch and starch/lactate enrichments, the pH dropped to below 6 by hour 12, and reached levels between 4 and 5 by hour 18 . These vThe three horse cultures differed dramatically in the ability to attenuate accumulated lactate. The lactate was reduced to below detectable limits in the control and lactate enriched treatment groups of all three horse cultures, however the peak was higher for horse 3 cultures and took longer to drop. In the starch and starch/lactate enrichments the differences were more dramatic. Lactate persisted at maximum levels in horse 3 cultures over the full time course while dropping for horse 1 and 2 cultures by hour 36.Headspace gases (hydrogen and methane) and hydrogen sulfide levels measured at 12 hour intervals did not show consistent differences between treatment and control conditions due to variation between horses especially for the starch and starch/lactate cultures .As shown in Rarefaction curves of the number of sequences per sample by observed species  indicateFirmicutes were the most abundant group by time 48 in all cultures regardless of enrichment conditions, with a corresponding decline in all other major groups, namely the Verrucomicrobia, Spirochaetes, Proteobacteria, and Bacteroidetes, however at this taxonomic level, clear differences between horse and treatment groups were not apparent.Taxonomic assignments at the phylum level  showed tFirmicutes. The Veillonellaceae, a family with known lactate utilizing species [Veillonellaceae dropped to less than 5% in horse 3 cultures, while in horse 1 and 2 cultures this group was the highest in abundance, making up more than 70% of the total sequences  under control and lactate conditions for horse 3 was also found in horse 1 and 2 cultures, it reached a lower abundance in both. While horse 3 control and lactate enriched cultures were able to attenuate lactate, it persisted in high concentrations in starch and starch/lactate enrichments in which no member of the Veillonellaceae was highly abundant.Identification of specific taxa that were most abundant by hour 48  suggesteMegasphaeraelsdenii (100% identity) was the most abundant OTU in horse 1 and 2 cultures, especially in the starch and starch and lactate enrichments, reaching relative abundances of over 70%. While this OTU was present in single numbers at Time 0 in all three horse samples, and persisted in horse 3 in low numbers under all culture conditions  and represented 97.7% of the Lactobacillaceae sequences across the dataset. This OTU was present in all three horse cultures, reaching abundances of 20% and greater in horse 1 in starch and starch and lactate enrichments at time 36, but then falling to 10% or less by time 48  together accounting for less than 50% of the sequences in this group , followed by an increase in the Lactobacillaceae as the abundance of Streptococcaceae falls.No study thus far has tracked microbial changes over the time course of starch induction in horses using 16S rRNA deep sequencing as described here, however, the changes that we observed in community composition are similar to what has been reported in equine and ruminant culture and probe based studies ,14,43,44Despite a sample size of three horses, we were able to observe between-horse differences in the ability to attenuate accumulated lactate as has been described in previous studies ,23.in vitro model may provide insight into microbial mechanisms of resistance and/or recovery.Certainly the microbial dynamics reported here may not reflect the actual community compositions of the caecum and large intestine of horses who recover or resist conditions of lactic acidosis, however the elucidation of endogenous lactate utilizers that thrive under conditions of low pH following starch enrichment in our Streptococcus species during the first 24 hours of starch induction followed by an increase in Lactobacilli species noted in other studies [Streptococcuslutetiensis group  of the16S gene is inherently limited [Streptococcuslutetiensis group may be especially difficult to distinguish due to their close phylogenetic relationships [While we observed the pattern of increase in  studies ,23 we exus bovis ) to be m studies ,24,29. O limited ,46 for a limited , and susionships ,48. SequStreptococcuslutetiensis and Lactobacillus sp as major lactic acid producers [Megasphaera, Veillonella, Selenomonas, Propionibacterium, and Anaerovibrio as key lactate utilizers. In fact, strains of Megasphaeraelsdenii have been shown to be effective in preventing lactic acidosis in cattle and are under development as probiotic therapies [While studies have pointed to roducers ,28,29, rroducers ,44,49 haherapies –52.in vitro model of starch induction, we report here changes in the abundance of specific microbes associated with the reduction of lactic acid. As we observed at time 0 in this study, 16S rRNA surveys of the equine gut microbiome have shown that under normal conditions, the Veillonellaceae (known lactate utilizers) comprise 1% or less of the total bacterial abundance [in vivo study did not see a difference in the abundance of Veillonellaceae in response to dietary change despite an increase in lactate levels [Veillonellaceae in that study did not increase in abundance as lactate accumulated, or which specific taxa were present in those horses.Using deep sequencing of the 16S rRNA gene of fecal communities challenged with starch, lactate, or both in an bundance –19. One e levels . It is uMegasphaeraelsdenii was highly abundant in all starch and starch/lactate cultures in which lactate accumulated and was attenuated. This taxa was present in very low abundance in cultures in which lactate persisted. It is unclear why this taxa fails to proliferate in any of the horse 3 cultures while reaching such high abundances under all conditions in horse 1 and 2 cultures. A second OTU related to Veillonellamontpellierensis was highly abundant in control and lactate enrichments specifically in horse 2 and 3 cultures, but did not thrive in the starch or starch/lactate enrichments, suggesting that other factors such as pH or competitive interactions exert selective pressure under starch and starch/lactate enrichment conditions.In our study we observed that one particular taxa closely related to Megasphaeraelsdenii OTU and the reduction of lactate, conclusive evidence that this taxa is responsible for reducing lactate levels will require further study. It is possible that other community members with lactate utilizing capabilities are playing active roles as well.While our data indicates a relationship between the presence of the Understanding the factors stimulating or preventing the proliferation of lactate utilizers in the horse gut microbiome could provide valuable information about why some horses are more sensitive to starch induction, and the microbial basis behind mechanisms of resistance.in vitro model for starch induced laminitis in horses as described here could provide a convenient and cost effective means to understand the microbial dynamics underlying colic and laminitis, and test hypotheses for ways to prevent or interrupt the progress of these equine diseases. Specific taxa in the family Veillonellaceae were highly abundant in starch enriched cultures that were able to attenuate lactate. These could provide useful insights into mechanisms of recovery or resistance, and could be valuable, individually or in consortia, as probiotics to prevent starch induced colic and laminitis.A robust Figure S1Hydrogen sulfide concentrations over time by horse.Concentration (mM) of hydrogen sulfide measured by the Cline (methylene blue) assay from each horse and culture condition at 12 hour intervals.(TIF)Click here for additional data file.Figure S2Headspace gas concentrations over time by horse.Concentration (mM) of hydrogen and methane gases measured by gas chromatography from each horse and culture condition at times 9, 20, 32, and 45 hours.(TIF)Click here for additional data file.Figure S3Rarefaction curves by horse.Observed species by number of sequences per sample for each horse dataset generated using a sampling depth of 9685 (the minimum number of sequences per sample and default parameters in QIIME.)(TIF)Click here for additional data file.Table S1Alpha diversity estimates for each sample.Chao1, observed species, Shannon Index and Good’s coverage for each horse dataset generated using default parameters in QIIME.(XLSX)Click here for additional data file.


Analysis of ancient DNA has provided invaluable information on past ecologies, ancient populations, and extinct species. We used a short snippet of highly variable mitochondrial control region sequence from New Zealand’s moa to characterise a large number of bones previously intractable to DNA analysis as well as bone fragments from swamps to gain information about the haplotype diversity and phylogeography that existed in five moa species.Pachyornis geranoides, Dinornis novaezealandiae, and Dinornis robustus were highly structured and some appear to have occupied the same geographic location for hundreds of thousands of years. In contrast, populations of the moa Anomalopteryx didiformis and Euryapteryx curtus were widespread, with specific populations of the latter occupying both the North and South Islands of New Zealand. We further show that for a specific area, in this case a North Island swamp, complete haplotype diversity and even sex can be recovered from collections of small, often discarded, bone fragments.By targeting such ‘snippets’, we show that moa populations differed substantially in geographic structure that is likely to be related to population mobility and history. We show that populations of Short highly variable mitochondrial ‘snippets’ allow successful typing of environmentally damaged and fragmented skeletal material, and can provide useful information about ancient population diversity and structure without the need to sample valuable, whole bones often held by museums. Dinornis were the giants of this ratite family, exceeding 200 kg in weight and 200 cm in height, while the coastal moa Euryapteryx curtus was only the size of a large turkey and weighed little more than 9 kg New Zealand’s flightless moa ratite (Order: Struthioniformes) represented one of the most extensive animal radiations known with at least 9 species in 6 genera Of all the ancient animals, moa are one of the most studied, with successful DNA extractions from moa bones, coprolites, eggs, and feathers. To date, genetic data from over 300 moa specimens are available, with a number of phylogenies having been constructed in attempts to clarify the number of moa species that existed Most DNA analyses of moa, and animals in general, have been restricted to selected regions of the mitochondrial genome, with the most studied area being the highly mutagenic hyper-variable-region I (HVRI). The variation inherent in this ∼300 bp region is capable of discriminating not only all moa species, but also harbours a strong phylogeographic signal. Using this region, a large number of moa samples have been successfully genotyped to give a comprehensive understanding of the various moa mitochondrial haplotypes that existed in New Zealand, most during the Holocene period. However, a significant number of gaps in moa phylogeography remain due to difficulty in the amplification of DNA extracted from rare older and/or more degraded bones. A number of these samples derive from regions in New Zealand where moa have been uncharacterised.DNA damage is driven by moisture, light, and temperature Use of relatively short highly informative DNA sequences has proved invaluable for typing a number of species, for example, equids Phylogenetic analyses of approximately 683 bp of the control region of 107 moa representing all known species identified a highly variable 30 bp region capable of not only identifying species, but also populations . This reAnomalopteryx didiformis, Euryapteryx curtus, and Dinornis novaezealandiae , 4 (A. didiformis), 17 (D. robustus), 28 , and 18 (E. curtus). As most locations are represented by only one or two samples, an approximate indication of phylogeographic genetic diversity within each of these species can be obtained by simply dividing the number of haplotypes by the number of locations from which samples were sourced. Relatively high ratios, suggesting high phylogeographic/genetic diversity, were obtained for D. novaezealandiae (1.1), P. geranoides (0.58), D. robustus (0.5), and E. curtus (0.72) while a very low ratio was obtained for the moa A. didiformis (0.13). As most moa bones date to the Holocene All bones sampled represented five of the nine currently recognised moa species, and consisted of P. geranoides and D. novaezealandiae, can be divided into predominantly eastern and western variants. In addition, moa bones sampled from middens in the Coromandel area recovered three previously uncharacterized haplotypes for D. novaezealandiae: D13, D23, and D24  joining the North and South Island. These bridges would have inevitably provided opportunities for population mixing A. didiformis, few haplotypes were found, with a single haplotype covering all samples from the South Island and only three being detected in the North Island. This may be the result of a bottleneck in this species, followed by rapid intra-island expansion.The spanning network of equences  fails toWe also analysed 11 moa samples from middens in the Taupo and Coromandel region in the North Island. Three haplotypes  from the Coromandel area were not found anywhere else and may be indicative of this general region. Our small sample size and the absence of moa samples from natural sites in these areas did not allow us to determine whether early moa hunters may have transported moa from neighbouring locations Thirty degraded moa bone fragments were analysed from Tiniroto Swamp, near Gisborne on New Zealand’s North Island. Twenty-four of these bone fragments were succesful for the amplification of the ‘snippets’, with a surprisingly large number (11) also allowing amplification of sex-specific nuclear DNA markers We show here that small DNA fragments recovered from small, highly degraded and/or old bones may provide useful information on species and populations. The ability to characterize animal populations using very short miochondrial ‘snippets’, from often discarded bone samples may in addition prove useful for the study of species where the destructive sampling of valuable whole bones is undesirable, such as those used by museums for display purposes or morphological analysis.Moa bone samples were kindly supplied by the following: Canterbury Museum (CM), Auckland Institute and Museum (AIM), University of Auckland (AU), University of Auckland Geology Dept (AUG), Otago Museum (OM), Whanganui Regional Museum (W), the Museum of New Zealand Tongarewa (MNZ), and the private collection of Derek Mickelson, Taihape (DM).All bone samples were treated using criteria set for the analysis of DNA from ancient material Approximately 50 mg of bone scrapings were incubated with rotation overnight in 0.8 ml of 0.5 M EDTA/0.001% Triton X100, then centrifuged, the EDTA/Triton X100 was decanted, and 0.4 ml of SET buffer  was added with 40 ul of 10% SDS, approximately 0.5 mg of dry dithiothreitol (DTT), and 0.5 mg of dry proteinase K. The mix was incubated with rotation at 56°C overnight, then extracted with phenol/chloroform followed by chloroform, before 200 ul of the extract was purified using a Qiagen DNeasy® Blood and Tissue Kit. The DNA was finally eluted from the silica column with 40 ul of 0.001% Triton X100.Ancient DNA precautions: All DNA extractions were carried out in a physically separate, dedicated ancient DNA laboratory. This facility is separated by 500 metres from the main laboratory in another building where amplifications were performed. A number of sequences were obtained in both directions from separate amplifications and in some cases from multiple extractions. Sequences from several samples were verified by LH at Massey University’s Ancient DNA facility in Auckland, New Zealand.4)2SO4, 2.5 mM MgCl2, 1 mg/ml BSA, 200 uM each of dGTP, dUTP, dCTP, and dATP, 0.5 uM of each primer mcrshFF and mcrshRR Approximately 2 ul of DNA was amplified by polymerase chain reaction (PCR) in 10 ul volumes containing 50 mM Tris-Cl pH 8.8, 20 mM  and moaWR (5′ATGTTAAGCAATGCTCTATGACA). Second, negative samples were tested for the presence of amplifiable nuclear DNA by targeting a 72 bp autosomal PCR product using the moa-specific autosomal primers msox9F (5′CTGCTCCTTGAATCTGATGA) and msox9R (5′CTTAGGCCAACGATACGAAA). Third, samples that amplified using the autosomal primers, were retested using the W-specific primers.Phylogenetic rates  for ∼683D. robustus; NC_002672) in TCS 1.21 Minimum spanning networks were constructed for each species using a highly variable control region segment from bases 554–580 Click here for additional data file.Figure S2Sample locations. The location of samples analysed is shown. Locations separated by less than ∼25 km are grouped as a single location.(TIFF)Click here for additional data file.


Herpesviridae family and SV40, BKV, JCV, MCV, WUV, KIV, LPV, HPyV6, HPyV7, and TSV from the Polyomaviridae family in 54 fresh frozen breast tumour specimens. Relevant clinical data and basic lifestyle data were available for all patients. The tissue samples were DNA extracted and real-time PCR assays were used for viral detection.Several different viruses have been proposed to play a role in breast carcinogenesis. The aim of this study was to investigate the prevalence of a subset of viruses in breast cancer tissue. We investigated the prevalence of 12 DNA viruses: EBV and CMV from the The highest prevalence, 10% (5/54), was found for EBV. MCV, HPyV6, and HPyV7 were detected in single patient samples (2% each), while WUV, KIV, JCV, BKV, LPV, SV40, TSV and CMV were not detected in the 54 breast cancer specimens analysed here. Further investigations are needed to elucidate the potential role of viruses, and particularly EBV, in breast carcinogenesis. Breast cancer is the most common type of cancer in women worldwide Polyomaviridae family which has traditionally contained only two true human-host specific viruses (JCV and BKV). In the last four years, there has been a flurry of new human polyomaviruses discovered, however their role in various human cancers has not been thoroughly evaluated. In 2007, WU and KI polyomaviruses  were nearly simultaneously discovered by separate groups in the respiratory tracts of children presenting with respiratory disease SV40 is a non-human member of the Herpesviridae family (EBV and CMV) together with ten members of the Polyomaviridae family  in breast tumour specimen.In this study we investigated the prevalence of two members of the We analysed 54 breast cancer tissue samples that were removed as a part of treatment from patients undergoing surgery at the Wesley Hospital, Brisbane, Queensland, Australia between 2003 and 2007. These samples were used in a previously published study into the role of human papillomaviruses in breast cancer Written informed consent was obtained from all patients, and the taking of fresh frozen breast cancer tissue samples for this project was approved by the Princess Alexandra Hospital Human Research Ethics Committee (PAH 2007/057).Tumour tissue samples were snap frozen and stored at −80°C. Prior to extraction the samples were grinded in 1.5 ml tubes on dry ice. The tissue samples were then homogenized in Trizol , and DNA extraction was carried out following the protocol provided by the manufacturer. The DNA extracted from the breast tissue specimens was stored at −20°C until analysed.Viral detection was achieved with both published and specifically designed real-time PCR (rtPCR) assays. Samples were screened in duplex for JCV (JL1) and BKV (V3a) using previously published methods Samples positive by the HPyV6-VP2 and HPyV7-LT screening assays were confirmed with the use of secondary rtPCR assays HPyV6-LT and HPyV7-VP2 , respectA common reaction mix was used for all rtPCR assays. Briefly, the rtPCR consisting of 12.5 µl Quantitect Probe PCR Mix , 10 pmol of each primer, 4 pmol of each probe and 2 µl of template in a 25 µl final reaction was performed in a Rotorgene 3000  under the following conditions: 15 minutes incubation at 95°C, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute.For positive controls, the MCV, WUV/KIV, JCV/BKV, CMV, and EBV screening assays used known clinical positive samples, for the LPV and TSV screening assays whole-genome plasmids were used, for the HPyV6 and HPyV7 screening assays synthetic oligo controls were used, and for the SV40 screening assay the SV40-integrated COS-1 cell line was used.Only samples which were both positive for the screening and confirmatory assays were considered “positive”. Other sample targets which were well characterised, such as JCV, BKV, EBV, CMV, WUV, KIV, or did not produce any positives  did not need confirmation.A PCR assay targeting the human L1 sequence was used to ensure that our sample contained human DNA and that no PCR inhibiting agents were present, as described in our previous study For comparisons, we used the t-test for normally distributed continuous variables and chi-square or Fisher's test  for categorical variables. All analyses were conducted in SAS (version 9.2) and all significance tests were two sided at α = 0.05.Fifty-four Australian breast cancer tumours were analysed for the presence of ten different viruses from the herpes and polyomavirus families.The mean age of the 54 women studied was 57 years and the mean size of the study tumours was 24 mm (range: 8–120 mm), with a majority of patients diagnosed with invasive ductal carcinoma (87%). Other relevant clinical features for the 54 patients studied can be found in in situ hybridisation or immunohistochemistry on the EBV positive samples, and thus, could not determine the cellular origin of the detected virus.The highest prevalence of the viruses investigated here in the breast tumour samples was 10% (5/54) which was found for EBV. None of the ten adjacent healthy tissues were EBV positive, including one originating from an EBV positive patient. Four of five EBV positive samples were grade 3 tumours, with the remaining one being grade 2. All five EBV positive women were postmenopausal, and were on average 10 years older than the EBV negative women , however this age difference was not statistically significant (p = 0.09). In a recent paper, the EBV positive cells in breast tumours were found to be infiltrating lymphocytes, while no virus was detected in the malignant breast cells MCV, HPyV6, and HPyV7 were detected in single cases (2% each) of the breast tumour samples. The MCV positive patient was a 58 year old woman with a grade 1 tumour of mixed type  breast cancer. The patient with the HPyV6 positive tumour was 52 years and had a grade 2 ductal breast tumour, while the HPyV7 positive patient was 78 years and had a grade 2 lobular breast tumour. The MCV, HPyV6 and HPyV7 positive samples produced high cycle threshold values of 31.9, 38.5, and 40.2, respectively, which is suggestive of low viral genome copy numbers. Given the evident low viral loads, and that MCV, HPyV6 and HPyV7 are polyomavirus with skin tropism, it is unclear whether these findings represent true infection or merely sample contamination originating from the skin during surgical excision or from skin shedding during sample processing.WUV, KIV, JCV, BKV, LPV, SV40, TSV, and CMV were not detected in the 54 breast cancer specimens analysed in this study.et al performed on Tunisian breast tumours did not find any BKV DNA positive tumours, but 23% of the specimens were found to be JCV positive. The JCV positive tumours in this paper were all invasive ductal type carcinomas. The majority of our samples (87%) were also of invasive ductal type, but we did not identify JCV in our population. Real-time PCR is generally accepted to be at least one log more sensitive than conventional PCR, and increasing PCR cycle numbers tends to increase the final yield and sensitivity of the assay. Considering Hachana et al used 35 cycles on their conventional PCR for the detection of JCV, while we used 45 cycles in a real-time PCR assay, we believe that assay sensitivity cannot explain the divergent JCV detections. In view of the Tunisian samples and our samples being similar breast tumour type , and the previous study's apparent lower JCV assay sensitivity, it is difficult to explain the vast difference in JCV detections between our sample sets. Furthermore, Hachana et al found a significant correlation between multiple viral infection  and “triple negative” phenotype  JCV and BKV were discovered 40 years ago and findings of these two viruses have been reported in various types of tissues, but infections have mainly been associated with urinary tract diseases and mild upper respiratory tract infections et al.None of our specimens were positive for the monkey origin polyomavirus SV40. A study by Hachana CMV is an interesting virus in regard to breast cancer, even though there is no solid evidence of an involvement in breast carcinogenesis. Thirty to 40% of CMV infections are acquired during the first year of life and the route of transmission is mainly through breast milk from the mother To conclude, we found very low or no prevalence to the viruses investigated in this study apart from EBV, although due to the histological limitations of the study, we could not draw robust conclusions on EBV's role in breast cancer. The study data suggests the lack of these DNA viruses' involvement in breast cancer, however further investigations and larger studies are needed to elucidate if EBV plays a role in breast carcinogenesis.


However, little is known about the molecular mechanisms underlying these interactions. We investigated whether plants respond differently when roots or shoots are induced. We mimicked herbivore attack by applying JA to the roots or shoots of Brassica oleracea and analyzed molecular and chemical responses in both organs. In shoots, an immediate and massive change in primary and secondary metabolism was observed. In roots, the JA-induced response was less extensive and qualitatively different from that in the shoots. Strikingly, in both roots and shoots we also observed differential responses in primary metabolism, development as well as defense specific traits depending on whether the JA induction had been below- or aboveground. We conclude that the JA response is not only tissue-specific but also dependent on the organ that was induced. Already very early in the JA signaling pathway the differential response was observed. This indicates that both organs have a different JA signaling cascade, and that the signal eliciting systemic responses contains information about the site of induction, thus providing plants with a mechanism to tailor their responses specifically to the organ that is damaged.Plants respond to herbivore attack by rapidly inducing defenses that are mainly regulated by jasmonic acid (JA). Due to the systemic nature of induced defenses, attack by root herbivores can also result in a shoot response and  In their natural environment, plants are subject to attacks by a wide variety of root and shoot herbivores. Plants respond to above- or belowground herbivore feeding by increasing the production of defense compounds vice versa, thereby affecting the performance of herbivores and natural enemies in the above- and belowground compartment Local JA production or ectopical JA application also induces systemic responses in undamaged or untreated plant parts. Systemic JA induction of defense responses also occurs from roots to shoots and Brassica plants Brassica oleracea and analyzed the transcriptome profiles in both organs with the genome-wide Arabidopsis thaliana 70 bp oligo chips. The Brassica genus is closely related to A. thaliana, which is reflected by an average 87% sequence homology in the coding regions of homologous genes (http://ukcrop.net/brassica.html). Moreover, the suitability of these long oligo A. thaliana arrays to analyze gene expression in Brassica species has been demonstrated before Compared to what is known about JA-induced responses in shoots, we know relatively little about the role of the JA signaling pathway in local and systemic root-induced responses Brassica oleracea were germinated on glass beads and water for one week, and the seedlings were transferred to individual 1.3 L pots containing sterilized plain river sand. The pots were maintained in a greenhouse at 21°C (day) and 16°C (night), room humidity 60%. Natural daylight was supplemented with sodium lamps to maintain the minimum PAR at 225 µmol.m−2.s−1 with a photoperiod of 16:8 (L:D). Twice, and later three times per week, the plants were provided with sufficient half-strength Hoagland solution with a doubled P-content to maintain the water percentage in the pots at 14% w/w Seeds from a wild accession (The Netherlands) of http://ag.arizona.edu/microarray/). Spot intensities were determined using ImaGene® 7.0 software , and transcript abundance was estimated as the natural log of the spot mean minus the mean of the local background. Transcript levels were normalized by centering to the median value of all genes on the slide. We analyzed the data separately for each combination of organ and time point (thus splitting the experiment into six subexperiments) and all data checks and analyses described below were carried out separately for each of the six subsets of slides. For each slide we set a threshold for spot detection at the 95th percentile of the distribution of negative controls. The analyses only included those probes for which at least two of the three replicates were above the detection threshold in each of the SJA, RJA and CON treatments. We further excluded all probes from the analysis whose intensity scores in all treatments and in all replicates were within the lowest quartile of the distribution of intensity scores of all genes on the slide. These low-expression probes could include A. thaliana genes that are either absent in Brassica or that are too dissimilar between both species to permit effective hybridization. One slide  was discarded as it showed overall low gene expression values, and visual inspection of residuals indicated that the statistical model did not fit well to data of this slide. Of the 29109 Arabidopsis gene probes present on the slide we included 24007 in our analysis.Total RNA was isolated with Trizol® Reagent  and further purified on RNeasy Mini Spin Columns . To allow all possible comparisons between treatment groups, a single color hybridization was performed on the 29,000 element Arabidopsis 70-mer Oligonucleotide Microarrays based on the Qiagen-Operon Arabidopsis Genome Array Ready Oligo Set (AROS) Version 3.0. Hybridization and scanning of the microarrays was performed by the Microarray Hybridization and Analysis Services at the University of Arizona, USA, following their standard procedures  resulted in a gene list that was more difficult to interpret biologically.ANOVA models were fitted for each probe to test effects of treatments on transcript levels using SAS 9.1 software . Residuals were tested for normality (Shapiro-Wilk test) and were examined visually for homogeneity of variances, confirming overall good conformation to standard ANOVA model assumptions. We interpreted Arabidopsis TAIR 9 January 2010 version, with some modifications based on more recent publications. Heat map construction and clustering of genes was done with the MultiExperiment Viewer software package from the TM4 microarray software suite http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi).All gene annotations were done according to the http://www.ncbi.nlm.nih.gov/geo/) and received the series record number GSE38784.To fulfill the MIAME requirements, all microarray data were deposited in the Gene Expression Omnibus (GEO) database  according to the manufacturer’s instructions. Subsequently, all samples were diluted five-fold with water. The qPCR amplification mix consisted of: 5 µl diluted 1st strand cDNA, 0.2 µl forward primer (10 µM), 0.2 µl reverse primer (10 µM), 12.5 µl qPCR SYBR green mix , and 7.1 µl H2O. The qPCR was performed on the Rotor-Gene 3000  according to the following protocol: an initial denaturation for 15 min at 95°C, followed by 45 cycles of 30 s at 95°C, 30 s at 58°C, 30 s at 72°C. The relative expression levels of the target genes were calculated by normalization with the expression of the two reference genes GAPC2 and PP2ATo verify the gene expression profiles obtained by microarray analysis, an RT-qPCR analysis was performed on separate batch of total RNA from the same pool of biological samples as described above. For primer design, orthologous sequences of the respective A separate batch of leaf discs taken as above was used to analyze glucosinolate levels at 6, 18 and 30 h after induction. The discs were freeze-dried, ground, extracted and analyzed on reversed phase HPLC as described in JA was applied either to the shoots  or to the roots , whereas control (CON) plants received a mock treatment. Systemic (untreated) leaves and the whole root system were harvested 6, 18 and 30 h after JA induction. To validate the gene expression patterns obtained by microarray analysis, we performed an RT-qPCR analysis for five genes involved in defense signaling and secondary metabolite production and compared the results to those of the microarray . Even th2 of the fold change in gene expression between RJA or SJA and the control treatment was calculated, resulting in twelve different transcriptome profiles. To analyze differences and similarities between all transcriptome profiles, correlation coefficients between the fold changes for every gene were calculated  also showed considerable differences depending on the tissue as well as the site of JA induction . Already2 of the fold changes of SJA over RJA were considered in addition to the fold changes of RJA or SJA over CON. Analysis of the SJA/CON and RJA/CON fold changes showed that shoots overall responded more extensively to JA treatment than roots  and amino acid degradation bins were specifically more affected by SJA, while genes in the cell wall protein and the glucosinolate bins responded more strongly to RJA . In rootGiven the contrasting responses after RJA and SJA of genes involved in primary metabolism, we measured the amino acid and sugar content in roots and shoots harvested 1, 3, 7 and 14 days after JA treatment using HPLC. As for the gene expression patterns , the effSUC1, SUC5, SUC7) was observed, indicating an increased sucrose loading into the phloem, which was not seen after SJA treatment. Cytosolic Invertase 1 (CINV1), encoding a key enzyme in sucrose degradation, was highly up regulated in RJA treated roots and to a lesser extent in SJA treated roots. In the shoots, Isoamylase 3, encoding a starch degrading enzyme, showed an almost 4 fold higher repression after RJA than SJA in at 18 h , a histone methyltransferase that epigenetically controls several processes related to flower development SPA1, which is involved in the regulation of circadian clock and photoperiodism VSP2, encoding a vegetative storage protein, showed a much higher expression after RJA than SJA in the shoots at 18 and 30 h  were also more strongly induced in shoot tissues after RJA than after SJA at all three time points  involved in terpenoids synthesis were differentially regulated by RJA and SJA , the sesquiterpene synthesis gene TPS21, and the homoterpene synthesis gene TPS04. After RJA, the monoterpene synthesis genes TPS10 and one of the TPS-CIN were induced in the shoots, as well as the sesquiterpene genes TPS13 and TPS21. In contrast, the CYP82G1 gene, involved in homoterpene synthesis, was significantly repressed in the shoots after RJA treatment, whereas TPS04 did not respond. These differential responses after RJA and SJA match previous studies showing that plants treated with JA to their shoots increased monoterpene, sesquiterpene and homoterpene emissions, whereas only monoterpene emissions increased when JA was applied to the roots BrassicaceaeIn the secondary metabolism bin, four genes ( and SJA . A detaid to SJA . No signOPR3 and AOC2) encoding enzymes involved in JA synthesis showed a large differential response to RJA and SJA  gene expression differed depending on the site of JA application. In the roots, RJA significantly induced AOC3 at 6 h and all 3 AOC genes at 18 h, while SJA only strongly induced AOC2 at 18 h. AOC is involved in the synthesis of 12-oxophytodienoic acid (OPDA), and OPR3 is responsible for the reduction of OPDA. The differential expression of both enzymes might have large effects on the concentration of OPDA itself, and probably also on the concentration of JA. Even though OPDA is a precursor in the synthesis of JA, the compound itself is also known to trigger a COI1 independent defense response A. thaliana roots causes a JA and OPDA accumulation in the shoots at respectively 30 min and 6 h, while in roots JA and OPDA does not increase in concentration at these time points In the hormone bin, two genes  and some genes involved in regulation of plant development. Moreover, a root JA induction mainly induced aliphatic glucosinolate synthesis, while a leaf JA application resulted in an indole glucosinolate synthesis. Also the volatile synthesis was differentially influenced by a root or shoot JA application. Genes encoding enzymes involved in the synthesis of mono-, sesqui- and homoterpenes were induced after a shoot JA application, while only monoterpene biosynthesis genes were induced after a root JA induction. This indicates that plants not only adapt their growth and development, but also their defense response specifically to the organ that is induced. This raises questions about the nature of the systemic signal, which is not yet known. Artificial wounding of Figure S1To verify the microarray expression data, for five defense related genes a comparison was made between the gene expression levels measured by microarray analysis (right column) and by RT-qPCR (left column). Although there were some small differences in the level of induction measured by RT-qPCR and microarray hybridization, the overall measured expression profiles were very similar. Expression is shown as fold changes compared to mock treatment at 6 h, 18 h and 30 h. Error bars represent the standard error of the mean.(TIF)Click here for additional data file.Figure S2Amino acid concentrations were measured by HPLC in the roots after RJA, SJA and CON at day 1, 3, 7 and 14. RJA resulted in a significant reduction in the concentration of glutamine at day 1 and (iso)leucine at day 1 and 3, whereas SJA caused a significant decrease of the concentration of (iso)leucine at day 1 and threonine at day 3. Concentrations are expressed in nmol/mg dry plant material after RJA , SJA  or control treatment . Error bars represent the standard error. Treatments that resulted in a statistically significant different concentration compared to control are marked with an asterisk .(TIF)Click here for additional data file.Figure S3Sugar concentrations were measured by HPLC in the roots after RJA, SJA and CON at day 1, 3, 7 and 14 after JA application. RJA and SJA caused a decline in sucrose concentration compared to CON at day 1 and 3. Concentrations are expressed in nmol/mg dry plant material. RJA, dotted line and open circles; SJA, dashed line and squares; CON; solid line and triangles. Error bars represent the standard error. Samples with a significantly different concentration compared to control are marked with an asterisk .(TIF)Click here for additional data file.Figure S4Gene expression in the Major CHO Metabolism bin in roots and shoots. (a) In all treatments, sucrose synthesis was repressed. In roots, a strong induction of the genes for sucrose export across the plasma membrane was only observed after RJA. A clear induction of Cytoplasmic Invertase 1 (CINV1) in roots after RJA, and to a lesser extent after SJA, indicates an increased degradation of sucrose into glucose and fructose. (b) In the shoots, a transient induction of genes involved in amylose synthesis and starch degradation at 6 h after RJA and SJA was observed, followed by strong repression of the latter thereafter. Beta-amylase-5 (Bam5), involved in starch degradation into maltose, was strongly induced in the shoots at all time points after RJA and SJA, in the roots after SJA, and to a much lesser extent in the roots after RJA. For genes that were significantly differentially expressed in at least one of the treatment groups, a heat map is shown representing the log2 fold changes in expression compared to control treatment. RRJA, Root tissue RJA treatment; RSJA Root tissue SJA treatment, SRJA, Shoot tissue RJA treatment, SSJA, Shoot tissue SJA treatment.(TIF)Click here for additional data file.Figure S5The differential response to RJA versus SJA is not due to a JA concentration effect. Glucosinolate concentrations  in leaves of Brassica rapa (Yellow Sarson) plants treated with increasing amounts of JA on the roots (RJA) or shoots (SJA). Glucosinolates were measured by HPLC on samples harvested seven days after treatment and grouped by biosynthetic origin: indole (black bars) and aliphatic glucosinolates (white bars). Letters over the bars indicate significant differences between treatment groups for indole  and aliphatic  glucosinolate levels. MANOVA analysis revealed an overall significant treatment effect , whereas JA concentration was not significant . The treatment×JA concentration effect was not significant either . Separate analysis of indole and aliphatic glucosinolates by ANOVA revealed similar patterns for each group. Combined with the results of the Tukey HSD analyses, this indicates that the JA response for both indole and aliphatic glucosinolates is saturated at 500 ug per plant. Moreover, the lack of response of the indole glucosinolates in RJA plants could not be alleviated by adding more JA to these plants.(TIF)Click here for additional data file.Table S1Primer sequences used for RT-qPCR.(DOCX)Click here for additional data file.Table S2Expression of genes showing at least a 3-fold change after RJA versus SJA.(XLSX)Click here for additional data file.


Growing teratoma syndrome is a rare occurrence with an ovarian tumor. Anesthesia has been reported to be difficult in cases of growing teratoma syndrome of the cystic type due to the pressure exerted by the tumor. However, there have been no similar reports with the solid mass type. Here, we report our experience of anesthesia in a case of growing teratoma syndrome of the solid type.The patient was a 30-year-old Japanese woman who had been diagnosed with an ovarian immature teratoma at age 12 and had undergone surgery and chemotherapy. However, she dropped out of treatment. She presented to our hospital with a 40cm giant solid mass and severe respiratory failure, and was scheduled for an operation. We determined that we could not obtain a sufficient tidal volume without spontaneous respiration. Therefore, we chose to perform awake intubation and not to use a muscle relaxant before the operation. At the start of the operation, when muscle relaxant was first administered, we could not obtain a sufficient tidal volume. An abdominal midline incision was performed immediately and her tidal volume recovered. Her resected tumor weighed 10.5kg. After removal of her tumor, her tidal volume was maintained at a level consistent with that under spontaneous respiration to avoid occurrence of re-expansion pulmonary edema.We performed successful anesthetic management of a case of growing teratoma syndrome with a giant abdominal tumor. Respiratory management was achieved by avoiding use of a muscle relaxant before the operation to maintain spontaneous respiration and by maintaining a relatively low tidal volume, similar to that during spontaneous respiration preoperatively, after removal of the tumor to prevent re-expansion pulmonary edema. The histological diagnosis was an immature teratoma with metastasis to her lymph nodes. She underwent chemotherapy for a short time after the surgery, but then dropped out of treatment and did not receive further therapy. She gradually developed a recurrent abdominal mass and remained under observation. At age 30, she presented to our hospital requiring an operation, and tumor extirpation was scheduled.2) 88 to 90%. Laboratory tests revealed an increased fibrin degradation product of 15.3μg/mL and D-dimer of 2.6μg/mL. Pulmonary function tests showed a low vital capacity (VC) based on the predicted value (%VC 37.7%) and arterial blood gas analysis revealed hypoxia .A physical examination revealed a huge abdominal mass and a chest X-ray revealed bilateral diaphragmatic elevation. She could not adopt a supine position because of the tumor causing respiratory distress Figure . A compu2, and cerebral regional oxygen saturation  were continuously monitored throughout the surgery. After the percutaneous sheaths were inserted, lidocaine 4% was sprayed on her upper airway, while midazolam 1mg and fentanyl 50μg were given intravenously and awake fiberoptic intubation was performed in the Semi-Fowler’s position. We did not use muscle relaxant before the operation because we were concerned about ventilation if spontaneous respiration was lost. During intubation, her tidal volume (TV) decreased slightly because of weakness of spontaneous respiration, but SpO2 was maintained and intubation was performed uneventfully. After intubation, midazolam 4mg was administered and anesthesia was maintained with 1.5% sevoflurane. Her TV with spontaneous respiration controlled by pressure support ventilation and positive end-expiratory pressure (PEEP) of 5cmH2O was kept between 250 and 350mL.Prior to induction of general anesthesia, an arterial catheter  was inserted into her radial artery and a central venous catheter  was inserted in her left internal jugular vein. Furthermore, percutaneous sheaths  were inserted in her femoral artery and vein to use for percutaneous cardiopulmonary support (PCPS) as necessary. Radial arterial pressure, electrocardiogram, capnography, SpO2O airway pressure under pressure controlled ventilation or manual ventilation. However, an abdominal midline incision was performed by the surgeon and her TV gradually increased. Her TV further increased and SpO2 recovered after median sternotomy. An inspiratory pressure of 20cmH2O and PEEP of 8cmH2O resulted in a TV of 300 to 350mL. Arterial blood gas analysis at a fraction of inspired oxygen of 0.6 gave results of PaCO2 41.8mmHg and PaO2 197.5mmHg.After surgeons completed preparation for the operation and the surgical site was disinfected, a muscle relaxant (rocuronium 50mg), fentanyl 200μg and 0.3μg/kg/minute remifentanil were administered. Spontaneous respiration was lost and we were unable to obtain a sufficient TV with less than the 20cmH2O and PEEP of 8cmH2O, which resulted in a TV of 350mL. After her spontaneous respiration recovered, she was ventilated with pressure support and PEEP to maintain a TV of 300 to 350mL. She was extubated on postoperative day (POD) 2 and discharged from the ICU without any complication on POD 5.Resection of her tumor caused massive blood loss that required blood transfusion. During the operation, 3 to 5μg/kg/minute dopamine was given and her mean arterial pressure was kept above 60mmHg. Finally, her tumor was removed completely and had a weight of 10.5kg. On histological examination, the tumor was diagnosed as a mature teratoma without malignancy. Her total blood loss was 9790mL and 22 units of red cell concentrate, 19 units of fresh frozen plasma, and 20 units of platelet concentrate were transfused. After removal of her tumor, the same TV was maintained with lower inspiratory pressures and PEEP to prevent re-expansion pulmonary edema (RPE). On completion of the operation, she was transferred to the intensive care unit (ICU) and ventilated with an inspiratory pressure of 18cmH20 even if muscle relaxants are used [20 and to prepare for PCPS in a situation of worsening hypoxia, while seeking to prevent lung injury from high airway pressures. As expected, spontaneous respiration disappeared after administration of the muscle relaxant and we were unable to obtain a sufficient TV with positive pressure ventilation. An abdominal midline incision was performed and TV improved immediately. Overall, our strategy was to maintain spontaneous respiration for as long as possible and we recommend this approach in patients with a giant abdominal tumor that cannot be reduced in mass preoperatively.Cases of GTS may present with a giant abdominal tumor ,3 and geare used ,7. In thCases with GTS involve a giant mass that is sometimes very close to great vessels, but complete surgical resection is required to prevent recurrence ,9. ThereFinally, there was a risk of RPE after removal of the tumor. RPE develops upon rapid expansion of chronically collapsed lungs through a mechanism of increased pulmonary vascular permeability ,11, withIn summary, we have described a procedure for successful anesthetic management in a case of GTS with a giant abdominal tumor. Induction and maintenance of anesthesia had several associated risks and circulatory management required extensive transfusion. Respiratory management was achieved by avoiding use of a muscle relaxant before the operation to maintain spontaneous respiration and by maintaining a relatively low TV, similar to that during spontaneous respiration preoperatively, after removal of the tumor to prevent RPE.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.GTS: Growing teratoma syndrome; ICU: Intensive care unit; PaCO2: Partial pressure of carbon dioxide; PaO2: Partial pressure of oxygen; PCPS: Percutaneous cardiopulmonary support; PEEP: Positive end-expiratory pressure; POD: Postoperative day; RPE: Re-expansion pulmonary edema; SpO2: Pulse oximeter oxygen saturation; TV: Tidal volume; VC: Vital capacity.The authors declare that they have no competing interests.NO was the major contributor in writing the manuscript. HIm and TT supervised the treatment of our patient and collected case data. HIs and HB revised the manuscript. All authors approved the final version of the manuscript.


In future scenarios of ocean acidification, calcifying organisms are expected to be less competitive: among these two key elements of the rocky subtidal food web, coralline algae and sea urchins. In order to highlight how the effects of increased pCO2 on individual calcifying species will be exacerbated by interactions with other trophic levels, we performed an experiment simultaneously testing ocean acidification effects on primary producers  and their grazers (sea urchins). Artificial communities, composed by juveniles of the sea urchin Paracentrotus lividus and calcifying  and non-calcifying  macroalgae, were subjected to pCO2 levels of 390, 550, 750 and 1000 µatm in the laboratory. Our study highlighted a direct pCO2 effect on coralline algae and on sea urchin defense from predation (test robustness). There was no direct effect on the non-calcifying macroalgae. More interestingly, we highlighted diet-mediated effects on test robustness and on the Aristotle's lantern size. In a future scenario of ocean acidification a decrease of sea urchins' density is expected, due to lower defense from predation, as a direct consequence of pH decrease, and to a reduced availability of calcifying macroalgae, important component of urchins' diet. The effects of ocean acidification may therefore be contrasting on well vegetated macroalgal assemblages and barren grounds: in the absence of other human impacts, a decrease of biodiversity can be predicted in vegetated macroalgal assemblages, whereas a lower density of sea urchin could help the recovery of shallow subtidal rocky areas affected by overfishing from barren grounds to assemblages dominated by fleshy macroalgae.Temperate marine rocky habitats may be alternatively characterized by well vegetated macroalgal assemblages or barren grounds, as a consequence of direct and indirect human impacts ( Mean surface ocean pH has decreased by approximately 0.1 unit between pre-industrial time and the 1990s 32−, used by many species to build calcareous shells and skeletons. Temperate rocky reef communities, as coral reefs, are particularly threatened by ocean acidification, since most of their organisms use carbonates to build calcareous structures. These organisms are well known to be particularly sensitive to acidified conditions  or high-magnesium calcite  pCO2 on photosynthesis and/or calcification of calcifying algae show complex and species-specific responses, with variable results depending on the pathway of carbonate deposition and on its relative amount The partial pressure of COi.e. fucoids) in favour of turfs e.g. urbanization of the coastline and overfishing (that favour proliferation of sea urchins).Along temperate Mediterranean coasts, shallow subtidal areas are dominated by macroalgae that provide a range of ecosystem services such as food source, shelter from predation and disturbance, proper settlement substrate and nursery for marine vertebrates and invertebrates. Macroalgal communities are particularly sensitive to anthropogenic disturbances and are, accordingly, used for ecological status assessment under the European Water Framework Directive 2000/60/EC 2pCO2 negatively affects their recruitment Macroalgae also exhibit different sensitivities to increased sea temperature and ocean acidification e.g. overfishing, Sea urchins are generally the most effective benthic herbivores in shallow subtidal areas in situ records in naturally acidified areas Sea urchins also show sensitivity to decreased pH: their carbonate structures (skeleton and grazing apparatus) are made up of the very soluble high-magnesium calcite, both in adult, juvenile and larval stages in situ observations in naturally acidified areas Macroalgae and sea urchins therefore strongly interact to shape the ecological state of rocky ecosystems. Studies on their response to ocean acidification have been so far performed separately through laboratory experiments on isolated species pCO2 on species interactions are still poorly known and the extent of consequences at the ecosystem level hard to assess. Most of the laboratory studies on ocean acidification have been focused on fitness and physiological processes of individual species pCO2 effects on upper trophic levels may be modulated (ameliorated or exacerbated) by indirect effects intrinsic to interactions with other trophic levels The effects of elevate Corallina elongata), and non-calcifying  macroalgae, and sea urchins (Paracentrotus lividus), under 4 different pCO2 conditions, relevant to present conditions and future scenarios In this context, we performed one of the first, to our knowledge, laboratory experiments on ocean acidification taking into account species interactions. We used calcifying  is faster and purportedly more sensitive to pH decrease e.g. of jaw and test growth) under short-term experimental conditions may better be assessed.The experimental design enabled testing the macroalgal responses to increasing of Paracentrotus lividus, about 4 months old, were provided by a sea urchin hatchery in Camogli , where they had been reared after in vitro fertilization. A total of 144 juveniles, with a test diameter of 5 to 6 mm, had been randomly selected and moved to the Laboratoire d'Océanographie de Villefranche, where the experiment was performed, one month before the start of the experiment. Algal specimens were collected in the Bay of Villefranche  at a depth of less than 5 m, and transported to Villefranche laboratory. No specific permits were required for collecting specimens in the present location: the Bay of Villefranche is not subject to particular protection restrictions. The location is not privately-owned or protected in any way. For each of the 3 selected algal species, Corallina elongata, Cystoseira amentacea and Dictyota dichotoma, 32 samples of around 5 g fresh weight (FW) were collected, cleaned of epiphytes, fastened with a rubber band and placed in experimental aquaria. The field collection did not involve endangered or protected species. All algal specimens were acclimated in a thermostated room at 22°C, for at least one week before the start of the experiment: the relatively short acclimation period was due to the decision of not adding nutrients for the macroalgal culture. We decided not to add any culture medium to the tanks in order to avoid possible biases due to fact that nutrient enrichment and pH interplay in different directions on growth of non-calcifying and coralline algae Juveniles pCO2 levels, chosen according to best practices pCO2  =  390 µatm (control), (2) optimistic scenario, pCO2  =  550 µatm, (3) realistic scenario (close to what expected for 2100), pCO2  =  750 µatm and (4) pessimistic scenario, pCO2  =  1000 µatm.Four pCO2 levels in the experimental tanks were obtained by bubbling pure-CO2 using a continuous pH-stat system . In the control tank, pCO2 was maintained at 390 µatm by bubbling CO2-free air produced by stripping CO2 from ambient using soda lime, and adjusting to the appropriate level through the IKS system. pH values corresponding to each pCO2 level were estimated based on desired pCO2, total alkalinity (AT), temperature and salinity using the R package seacarb Unfiltered seawater, pumped from a depth of 10 m in the Bay of Villefranche, was continuously supplied to four 200 l header tanks. The chosen pCO2 level  with only algae and two aquaria  with algae and sea urchins were set up. The design enabled us to tease apart weight loss due to sea urchin grazing and direct pCO2 effects on algal species. One of the three different algal species was allocated to each of the three experimental units in each aquarium , 4 aquaria for each O2 level . Three saquarium ; two 5 g−2s−1, corresponding to the irradiance at ca. 5 m depth in the Bay of Villefranche in June-July Irradiance values in the aquarium were maintained at about 215 µmol photons mAT) measurements were collected twice a week, filtered on GF/F membranes and immediately analyzed. AT was determined potentiometrically using a Metrohm titrator (Titrando 80) and a glass electrode  calibrated using first NBS buffers  and then using TRIS buffer solutions. Triplicate titrations were performed on 50 ml sub-samples at 25°C and AT was calculated as described by Dickson et al. (2007). Titrations of standard seawater provided by A.G. Dickson (batch 106) yielded AT values within 2.4 µmol kg−1 of the nominal value (standard deviation  =  4.6 µmol kg−1). All parameters of the carbonate chemistry were determined from pHT, AT, temperature and salinity using the R package seacarb Seawater samples for total alkalinity (Algae and urchins wet weights were measured at the beginning and at the end of the experiment (precision: 0.001 g). At the end of the experiment, all specimens were air dried and stored for following analyses. Macroalgal weight loss under experimental conditions was calculated as the difference between algal wet weight at the beginning and at the end of the experiment.A subset of 24 sea urchins  was dissected and immersed in 1% (w:v) sodium hypochlorite for 30 min in order to remove organic matter and facilitate the separation of structural elements. The length of all portions of Aristotle's lantern and the diameter of the test were measured under a stereomicroscope and the ratio between the length of the jaw pyramids of the Aristotle's lantern and the diameter of the test was calculated (hereafter referred as jaw/test ratio).Test robustness was measured on another sub-set of 24 sea urchin specimens using a custom-made device designed to measure the static force required to crush sea urchin tests  images of different parts of sea urchins  were taken using an Environmental Scanning Electron Microscope  at the University of Genoa.pCO2 on calcifying (C. elongata) and non-calcifying  algae in presence or absence of P. lividus on the percent algal weight loss (arcsin transformed) was tested using a 3-way crossed ANOVA, after test for normality and homogeneity of variance: factor 1 “algal species”, 3 levels ; factor 2 “pCO2”, 4 levels ; factor 3 “urchins”, 2 levels (absent/present). Replicate values (n = 2) were the mean weight losses of the two samples of algae in each experimental unit. Student–Newman–Keuls (SNK) tests were performed for the a posteriori comparisons of means to check for differences among factor levels when the ANOVA detected significant effects.The effect of C. elongata, C. amentacea, D. dichotoma); factor 2 “pCO2”, 4 levels . Replicate values (n = 2) were the mean values derived from urchins investigated in each independent experimental unit. A 2-way ANOVA, testing the same factors, and SNK tests were performed also on untransformed data of the normalised weight required to crush the test, in order to detect potential differences in test robustness among urchins fed with different algae and kept at different pCO2 levels.Then, 2-way crossed ANOVA and SNK tests was performed on untransformed data to assess potential changes in the jaw/test ratio of the sea urchins fed with the three macroalgae and exposed to the four different pH levels. Considered factor were: factor 1 “algal species”, 3 levels (T was maintained at an average (±SD) of (1) 8.09 ± 0.04, (2) 7.98 ± 0.06, (3) 7.84 ± 0.04 and (4) 7.70 ± 0.03, in the four treatments, respectively. AT levels remained stable across treatments and during the whole experiment.Parameters of the carbonate chemistry are reported in C. amentacea and D. dichotoma showed larger weight loss than C. elongata, irrespective of the pCO2 level: across all pH treatments C. amentacea lost around 90–100% of its weight when grazed by urchins and 55–65% when not grazed  and absence of sea urchins (pCO2” interaction (p<0.05) and of the factor “urchins” (p<0.001) on all algal species. The SNK test on the factor “urchins” confirmed the expected larger weight loss when urchins were present at all pCO2 levels . For the interaction between factors “algal spp.” and “pCO2”, SNK test revealed a significantly larger weight loss of C. elongata at elevated pCO2 treatments than at the control conditions (pCO2 (550 = 750 = 1000) > 390 µatm; p<0.01; Although irradiance and temperature in the experimental aquaria were chosen in order to mimic natural conditions, no growth of algal thalli was observed during the experiment in any of the treatments. Accordingly, all algal specimens showed a general decrease in weight in all treatments, including in the experimental units without sea urchins. As expected, weight loss was much larger in the units where urchins were present and represented between 65 and 100% of the amount of algae placed in the aquaria at the beginning of the experiment. In general, t grazed . D. diche absent . C. elonreatment . Yet, we urchins . The stapCO2 level, occurred during the first week . The remaining ones were in very good conditions after one month, and showed positive increase in size. The jaw/test ratio showed a weak significant difference only between control pCO2 and 750 µatm  and no significant interaction between factor “algal species” and “pCO2” was observed  and the SNK test highlighted significant higher values of the ratio in urchins fed with C. elongata, compared to C. amentacea and D. dichotoma (C. elongata > (C. amentacea  =  D. dichotoma); p<0.01). In Some mortality of sea urchins, unrelated to the d ANOVA; . SignifipCO2 and algal species on test robustness  > 1000 µatm; p<0.05) and changed according to the diet, across all pH treatments: tests of urchins fed with C. elongata were significantly stronger compared to urchins fed with the two non-calcifying species (C. elongata > (C. amentacea  =  D. dichotoma)). Scanning Electron Microscope images performed on the apical disc, spines and, Aristotle's lantern of sea urchins showed that only the Aristotle's lantern was affected by the experimental treatments, while the other portions investigated did not reveal clearly detectable differences. The surface of latero-radial sides of the Aristotle's lantern, usually characterized by fine and dense tridimensional mesh of calcite trabeculae pCO2 . The density and preservation of the trabecular structure of the Aristotle's lantern of urchins that grazed on non-calcifying macroalgae exhibited a larger damage at elevated pCO2 , suggesting a significant impact in a few decades.The weight loss of the non-calcifying species, pCO2 compared to experimental units where urchins were absent. More interestingly, the presence of urchins increased algal weight loss at any elevate pCO2 compared to controls. The apparently smaller weight loss of C. elongata in the grazed condition . An increase in growth of non-calcifying species at elevated pCO2 was anticipated based on previous studies 2 world, except when other constituents such as nitrogen, phosphorus, and iron are limiting e.g. light). Additionally, in the Mediterranean Sea, the macroalgal biomass is highest in the spring; growth is much lower in June-July, the time at which the experiment was performed.All algal species showed a weight loss during the experiment. It did not prevent testing the effects of the main factors  is reported to increase mortality, decrease growth and cause a thinner and more easily breakable test in one year old urchins Few studies have focused on the juvenile stage, which is potentially one of the most susceptible to acidified conditions, given their higher growth rate compared to adults. In fact, even a small change in pCO2 effects on test robustness: sea urchins maintained at the lower pH (pHT 7.7) exhibited a less robust test than the ones maintained at higher pH. Yet, lower test robustness, per se, did not affect mortality during the short experimental time. In addition to direct pCO2 effects, our study points out that the effect of ocean acidification on juveniles of Paracentrotus lividus is mediated by their diet, both concerning test robustness and jaw size, potentially affecting grazing capacity. The food source caused differences in the robustness of the test: urchins fed with C. elongata displayed a significantly stronger test than the ones fed with the two non-calcifying species.The present study shows significant T 7.7 in sea urchin sensitivity and the role of coralline in their diet for strengthening the test) are in agreement with the findings of Hall-Spencer et al. (2008) in naturally acidified areas: a threshold value of mean pHT 7.8 is reported below which sea urchins are absent; accordingly, in these pHT conditions coralline algae are missing. Combining the results of these two studies suggests that a pHT value of 7.7–7.8 and the lack of corallines in urchin diet does not increase directly the sea urchin mortality but, rather, makes them more susceptible to predation, because of the less robust tests.These findings  , may be due to the different toughness of the algal tissues. It is hypothesized that sea urchins need a larger grazing apparatus (relative to the body size) when fed with more calcified algal structures: in Corallina-fed urchins the ratio decreases sharply from the controls to the first intermediate pCO2 level, showing a fast response to C. elongata decalcification, that makes the alga more easily grazable Additional diet related differences were observed in the jaw/test ratio, a parameter notoriously variable according to the trophic condition of the individual 00 µatm) . No diffmentacea . The difpCO2. While the Aristotle's lantern of urchins fed with C. amentacea and D. dichotoma looked generally less tough in all treatments, Corallina-fed urchins displayed denser and smoother structures in the controls compared to controls fed with the other algae, with a clear reduction in thickness and increase in corrosion as a function of increasing pCO2.Scanning Electron Microscope images on latero-radial sides of Aristotle's lantern highlighted, for all sea urchins exposed to experimental conditions, a progressive weakening of the calcite trabecular structure as a function of increasing C. elongata, even at the higher pCO2, makes the test of the juveniles much stronger and their jaws larger compared with specimens fed with non-calcifying algae. These findings, that show a direct pCO2 effect on sea urchin defense from predation (test robustness) but also strong indirect effects mediated by the diet on test robustness and on jaw size, were achieved thanks to the experimental setup of an artificial community, designed for assessing interactions among primary producers and their grazers.Taken together, these results suggest that the uptake of carbonate from the diet is fundamental in modulating sea urchin response to ocean acidification. The carbonate content of e.g. predation, grazing, bioturbation), could have consequences at the ecosystem level pCO2 and diet, sea urchins showed different morpho-functional features, more as a consequence of diet than of pCO2. Notwithstanding these differences, the grazing pressure of the urchins was similar across all the treatments. The observed combined effects of ocean acidification and macroalgal diet on test robustness and on jaw/test ratio may have severe consequences at the ecosystem level. The decrease in test robustness due to the diet and pCO2 is anticipated to make juveniles of P. lividus more susceptible to predation by fish, e.g. Diplodus spp. Ocean acidification, whether causing the loss of keystone/critical species or the reduction in their activity .The consequent reduced sea urchin density is expected to cause a reduction of their grazing pressure on macroalgal assemblages, with different effects according to the state of the habitat. In barren ground habitats, the reduced abundance of sea urchins would favor the recolonisation of non-calcifying macroalgae e.g. Cystoseira), in absence of coralline macroalgae, will be potentially prevented by the installation of turfs, able to inhibit their recruitment and known to be promoted in the framework of cumulated human impacts (see The recovery of foundation species (acts see .pCO2 conditions and cumulated human impacts.These findings stress the need to move from experiments on individual species to species interactions in order to better understand the both direct and indirect effects driven by top-down and bottom-up processes, in order to build more reliable predictions of future scenarios under the interaction of high


Socioeconomic inequalities in diet-related health outcomes are well-recognised, but are not fully explained by observational studies of consumption. We provide a novel analysis to identify purchasing patterns more precisely, based on data for take-home food and beverage purchases from 25,674 British households in 2010. To examine socioeconomic differences (measured by occupation), we conducted regression analyses on the proportion of energy purchased from (a) each of 43 food or beverage categories and (b) major nutrients. Results showed numerous small category-level socioeconomic differences. Aggregation of the categories showed lower SES groups generally purchased a greater proportion of energy from less healthy foods and beverages than those in higher SES groups , while higher SES groups purchased a greater proportion of energy from healthier food and beverages (28% vs. 24%). At the nutrient-level, socioeconomic differences were less marked, although higher SES was associated with purchasing greater proportions of fibre, protein and total sugars, and smaller proportions of sodium. The observed pattern of purchasing across SES groups contributes to the explanation of observed health differences between groups and highlights targets for interventions to reduce health inequalities. •We give a novel, detailed account of purchasing by SES in 25,000 British households.•Lower SES groups purchased a greater proportion of less healthy food and drink.•Higher SES groups purchased proportionally more high-fat dairy and alcohol.•Overall, SES differences were less marked for nutrients than for food groups.•We highlight food groups to target for interventions to reduce health inequalities. Recent focus in the UK and elsewhere on the social determinants of health inequalities  has raisFood and drink purchasing are determinants of consumption, yet their role in the aetiology of health disparities is underexplored. While it is often argued that there are social class-based patterns in dietary behaviours, beyond social disparities in the consumption of fruits and vegetables , relativ1.How do patterns of purchasing of (a) food and drink categories, and (b) the nutritional content of food and drink, vary by SES?2.Are any observed purchasing patterns consistent with the extant empirical evidence for differences in health outcomes by SES?The analysis reported in this paper contributes to this objective by addressing two core questions:KWP's commercial panel comprises over 25,000 British households, recruited via stratified sampling, with targets set for region, household size, age of main shopper and occupational group. KWP offer vouchers from high street retailers as compensation for participation. Households provide demographic information when joining the panel, followed by annual updates. Households record all purchases  brought back into the home using barcode scanners (with barcodes provided to record non-barcoded products like fruit). KWP match scanned records to their nutritional data.n = 25,674; see To be included in KWP's final datasets, households must meet quality control criteria (meeting thresholds for data recording and purchasing volume/spend (based on household size) every four weeks). Panellists also upload digital images of checkout receipts, which KWP use to verify the accuracy of scanner data. We obtained KWP data on take-home purchasing of food and drink for the 52 weeks ending 26th December 2010, and analysed all households that reported at least 12 weeks' data , comprising three groups: Higher Managerial and Professional (A&B); White Collar and Skilled Manual (C1&C2); and Semi-skilled and Unskilled Manual (D&E). Notwithstanding its well-known limitations , this clWe classified all scanned food and beverage products into one of 43 categories  or ‘less healthy’  were conducted for each category and nutrient separately, with dummy variables for SES group, and controlling for age, gender, ethnicity , number of adults in the household, number of children in the household, and region. Reported significance levels were adjusted for multiple testing using Bonferroni's correction.Lower SES groups bought greater proportions of their total energy from 9/21 less healthy categories , accounting for 34.8% of group D&E's total energy purchased (6.5 percentage points more than group A&B). In contrast, higher SES groups bought greater proportions of energy from 5/21 less healthy categories , accounting for 15.3% of group A&B's total energy purchased (2.7 percentage points more than group D&E).A greater proportion of energy was bought from 4/7 neutral categories by higher SES groups , whereas no neutral categories were purchased proportionally more by lower SES groups.For healthier categories, there was a difference by SES group for 10/15 categories, with all these due to a higher proportion of energy being bought by higher SES groups. For example, in the juice category, on average the percentage of energy purchased from juice was 81% greater in the A&B group than in C1&C2 group, while percentage of energy from juice purchased by group D&E was 44% less than group C1&C2. Together these accounted for 21.8% of group A&B's total energy purchased, an extra 4.2 percentage points compared to group D&E.p < 0.001).Household purchasing differed by SES group for 28 of our 43 categories. The extent of the differences identified here are more widespread than suggested by previous work, with the proportions purchased from dairy products, pasta and rice, bread products, processed potatoes, sweet snacks and puddings, and wine all showing significant differences by SES. In general, purchasing by lower SES groups was characterised by proportionally less energy from healthier food categories (4 percentage points difference between groups A&B and D&E) and proportionally more energy from less healthy food categories (5 percentage points difference between groups A&B and D&E). While the SES differences were mainly small in absolute terms, at the population-level and over time, these differences are likely to have cumulative effects on health risks.Given that health considerations played a role in determining the categories used in this study, category-level differences would be expected to be reflected at the nutrient-level. Differences were indeed observed, with proportionally more purchasing of fibre, protein and total sugars by the higher SES groups, and proportionally less sodium purchased by the highest SES group. Both sodium and fibre have been linked to health outcomes, with excess sodium consumption being associated with high blood pressure and elevated risk of stroke  and highHowever, the other differences in nutrient purchasing are less relevant to health outcomes. Health effects of total protein intake are equivocal , and theThe current study has a number of strengths. It has extended previous work on socioeconomic differences in food purchasing, using a large dataset based on observed data and narrower health-based categories than in previous studies, to reveal small socioeconomic differences in purchasing across a wider range of categories. It used a large sample that may inform the design, tailoring and targeting of population interventions  that aimIt also has a few limitations: as the data do not include out-of-home purchasing, the socioeconomic differences reported need to be treated with caution as they represent only part of the diet. Higher SES groups generally consume more out-of-home than lower SES groups , and theThe UK government's ‘Healthy Lives, Healthy People’ White Paper highlights the importance of ‘improving the health of the poorest, fastest’ , 4. The


The family food environment is an important influence in the development of children’s dietary habits. Research suggests that influences of current dietary behaviour and behaviour change may differ. The aims of this paper were to: (1) investigate the association between the food environment at baseline and change in children’s saturated fat intake; and (2) to explore whether a change in the food environment was associated with a change in children’s saturated fat intake.Secondary analysis of a 12 week cluster randomised controlled trial in 133 4-13 year old children. Families were randomly allocated to parental education regarding changing to reduced-fat dairy foods or a comparison non-dietary behaviour. The interventions were family focused. Parents received education from a dietitian in 3x30minute sessions to facilitate behaviour change. Parents completed a comprehensive questionnaire capturing three domains of the food environment – Parent knowledge and attitudes; shaping practices; and behaviours and role modelling. Children’s dietary intake was assessed via multiple 24-hour recalls at baseline and week 12. Changes in the family food environment and primary outcome (saturated fat) were calculated. Hierarchical linear regression models were performed to explore the association between baseline and change in food environment constructs and change in saturated fat intake. Standardised Beta are presented (p<0.05).After adjustments for child and family demographics, higher levels of perceived food availability (β=-0.2) at baseline was associated with greater reduction in saturated fat intake, where as higher perceived responsibility (β=0.2), restriction (β=0.3) and pressure to eat (β=0.3) were associated with lesser change in saturated fat. An increase in nutrition knowledge (β=-0.2), perceived responsibility (β=-0.3) and restriction (β=-0.3) from baseline to week 12 were associated with greater reduction in saturated fat intake.The present study was one of the first to quantify changes in the family food environment, and identify a number of factors which were associated with a positive dietary change. Because interventions focus on behaviour change, the findings may provide specific targets for intervention strategies in the future.Australia New Zealand Clinical Trials Registry ACTRN12609000453280. Children’s diet quality is poor with implications for health in childhood-3 and laThe family food environment is a complex domain, and is thought to include parental factors such as nutrition knowledge, their parenting styles and feeding practices, role modelling, food availability and accessibility, as well as children’s own individual characteristics and behaviours. Over thAlthough the current literature suggests the family food environment is important, the majority of this evidence has come from cross-sectional research. Longitudinal research and interventions can provide stronger evidence of cause and effect. Interventions change behaviour by changing mediating variables, and interventions are more likely to be effective if the variables are strongly related to the behaviour. Given tA small number of longitudinal studies have examined the family environment in general but fewer have focused on the food environment. One Australian study of adolescents found mother’s modelling of healthy eating, and support from the family and best friends were associated with positive changes in fruit and vegetable intakes. A seconThis paper utilises data from a longitudinal randomised controlled trial examining the effects of parental behavioural nutrition education to encourage a change in children’s dairy food choices from regular to reduced fat varieties. The intervention was based on the family food environment model. ParentsThe study was approved by the Commonwealth Scientific Industrial Research Organisation (CSIRO) Food and Nutritional Sciences Human Ethics committee, was registered in the Australia New Zealand Clinical Trials Registry (ACTRN12609000453280), and the method is described in detail elsewhere. In brieThe comparison intervention was also family based; however, it focused on reducing children’s screen time behaviours (with the aim of maintaining current diet). Families also received a standard intervention booklet and equal contact with the dietitian.Children’s dietary intake was measured at baseline and end of intervention (week 12) via three 24-hour diet recalls. The recalls covered two weekdays and one weekend day, and one was conducted face-to-face, the other two via telephone. The three pass recall is a well established method and is described in detail elsewhere. ParentsThe primary outcome was change in percentage of energy from saturated fat. Grams of saturated fat intake was converted to kilojoules (multiplied by 37 kilojoules/gram), and the percent contribution to total daily energy intake calculated. The average percentage of energy from saturated fat over the three recalled days was calculated.At baseline and at the end of the intervention parents completed a questionnaire encompassing a broad range of aspects of the family food environment.Nutrition knowledge was measured using the General Nutrition Knowledge Questionnaire, validatDietary Guidelines for Children and Adolescents suggest children should consume how many serves of dairy foods each day?”, “Reduced fat milk contains less fat than low fat milk.”. One point was assigned for each correct answer (out of a possible 3).Three additional questions specific to dairy were also asked: “What type dairy foods do experts say children over the age of 2 years should eat?”, “The Five factors from the validated, and commonly used, Child Feeding Questionnaire (CFQ) were meaThe family food environment was captured by combining questions from three different questionnaires. Questions from the Family Food Environment (FFE) questionnaire were useThis was the first time questions about the family food environment from these different sources have been combined, therefore exploratory factor analysis was used to establish the validity of the family food environment factors in this sample. The data was assessed and supported the use of factor analysis. Principal axis factor analysis revealed seven factors with Eigen values exceeding 1, but an inspection of the Screeplot revealed a six factor solution. Varimax rotation was performed to aid the interpretation, and the rotated solution is discussed. For all questionnaires, negatively worded questions loaded negatively, and were reverse coded in the creation of factor scores. Factor scores were created by calculating the mean score for the items loading onto each factor (in most cases out of a possible 5). The six factors of the family food environment were perceived responsibility, perceived fresh food availability, perceived adequacy of child’s diet, meal preparation views, food involvement in meal preparation, role modelling of eating behaviours, and TV interruptions to meals. The factors structures were generally similar to the original questionnaires,8. Facto2). Parent/caregiver weight status was classified using the World Health Organization definition, with BMI ≥25 kg/m2 overweight and ≥30 kg/m2 obese[Height and weight of children and parents/caregivers was measured. Participants were weighed  and height measured with a stadiometer  while lightly clothed, without shoes, using a protocol consistent with international standards. Height m2 obese. For chim2 obese. Given tm2 obese. Childrem2 obese.Family demographic information was collected at baseline. Details were sought about parents’ gender, highest level of education, employment status, estimated annual household income. Child sex and date of birth were also reported.Differences in the baseline family food environment factors between groups was assessed using Independent Samples T-tests (significance p<0.05). Change scores were calculated for the family food environment variables and percent energy as saturated fat (change=Week 12 follow-up minus baseline). The effect size estimate of the change in family environment and dietary outcomes was calculated (mean change/change in SD). Values of <0.1 were considered small, 0.3 medium, and >0.5 large were used for interpretation of effect size. The depa priori based on known associations with both predictor and outcome (i.e. family food environment constructs and dietary intake). These included child gender, age, BMI z score, household income, caregiver education and BMI. The relationships between each of household income (4 categories) and education  and saturated fat were explored and both showed linearity. Therefore, for regression analysis these were treated as continuous variables to reduce the size of the model. All variables were considered normally distributed.The independent predictor variables were baseline family food environment factors for Model 2-4a, and the change in family food environment factors for Models 2-4b. A positive score reflected an increase in factor scores between baseline and follow-up. Socio-demographic covariables were selected Hierarchical linear regression models were performed to explore the association between socio-demographic variables, baseline and change in family food environment constructs and change in saturated fat intake. As the aim of the analysis was to consider the associations between family food environment and change in children’s percentage of energy from saturated fat, regardless of the mode of change, data from the intervention and control groups were pooled, and treatment was not included in the analysis.18.0 .Due to sample size limitations, three domains of the family food environment were explored in separate models. Inclusion of factors into these models was theoretically derived,37. ModeData analysed were based on 133 children from 86 families, representing 91% of the study sample. Primary caregivers were majority female (87%), 48% were within the normal weight range, and 29% were overweight. Fifty-six percent were tertiary educated, 44% worked part time and 54% reported a household income between A$52,400-114,399. The average child age was 8.9 (SD=2.9) years, 59% were male and 68% of the sample were categorised within the normal weight status category . A negative value represents a decrease in saturated fat intake from baseline to follow up  were associated with change in saturated fat. Parent reported perceived responsibility for children’s food intake  at baseline was positively associated with change in saturated fat and perceived fresh food availability  was negatively associated. Higher perceived responsibility at baseline was associated with a lesser change in saturated fat. Conversely, higher perceived fresh food availability was associated with a greater change in saturated fat. The baseline knowledge and attitudes model accounted for 12% of the variance in change in saturated fat (p=0.006)  and pressure to eat  at baseline were positively associated with change in saturated fat intake Model 3a). Higher restriction and pressure to eat were associated with a lesser change in saturated fat intake over the course of the intervention. The baseline parent shaping practices model (Model 3a) accounted for 16% of the variance in change in saturated fat intake (p<0.001) . The RCT was funded by Dairy Australia. The study was conducted and this manuscript prepared without input from Dairy Australia (the funding body). Dairy Australia approved this manuscript for publication. All authors declare no conflicts of interest.GH and RG were responsible for the design and completion of the RCT. GH and RG conceived this study. KL provided statistical expertise, assisted with the data analysis and interpretation. RG conducted the analysis. GH drafted the manuscript. GS assisted with the data analysis and draft of the manuscript. All authors read and approved the final manuscript.