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Additional file 3: Table S2. Number of RNA sequencing reads obtained for each sample. Figure S1. HPLC chromatograms of skin extracts. Figure S2. Neutral lipid analysis by thin-layer chromatography (TLC).

The nonribosomal enterotoxin tilivalline was the first naturally occurring pyrrolobenzodiazepine to be linked to disease in the human intestine. Since the producing organism Klebsiella oxytoca is part of the intestinal microbiota and the pyrrolobenzodiazepine causes the pathogenesis of colitis it is important to understand the biosynthesis and regulation of tilivalline activity. Here we report the biosynthesis of tilivalline and show that this nonribosomal peptide assembly pathway initially generates tilimycin, a simple pyrrolobenzodiazepine with cytotoxic properties. Tilivalline results from the non‐enzymatic spontaneous reaction of tilimycin with biogenetically generated indole. Through a chemical total synthesis of tilimycin we could corroborate the predictions made about the biosynthesis. Production of two cytotoxic pyrrolobenzodiazepines with distinct functionalities by human gut resident Klebsiella oxytoca has important implications for intestinal disease.

Arsenic-containing lipids (arsenolipids) are novel natural products recently shown to be widespread in marine animals and algae. Research interest in these arsenic compounds lies in their possible role in the membrane chemistry of organisms and, because they occur in many popular seafoods, their human metabolism and toxicology. Progress has been restricted, however, by the lack of standard arsenolipids and of a quantitative method for their analysis. We report that the certified reference material CRM 7405-a (Hijiki) is a rich source of arsenolipids, and we describe a method based on HPLC-ICPMS/ESMS to quantitatively measure seven of the major arsenolipids present. Sample preparation involved extraction with DCM/methanol, a cleanup step with silica, and conversion of the (oxo)arsenolipids originally present to thio analogues by brief treatment with H2S. Compared to their oxo analogues, the thioarsenolipids showed much sharper peaks on reversed-phase HPLC, which facilitated their resolution and quantification. The compounds were determined by HPLC-ICPMS and HPLC-ESMS, which provided both arsenic-selective detection and high resolution molecular mass detection of the arsenolipids. In this way, the concentrations of two arsenic-containing hydrocarbons and five arsenosugar phospholipids are reported in the CRM Hijiki. This material may serve as a convenient source of characterized arsenolipids to delineate the presence of these compounds in seafoods and to facilitate research in a new era of arsenic biochemistry.

Rationale. The uptake of arsenate by algae from oceanic waters and its transformation to arsenosugars and arsenolipids is well established, but the biosynthetic pathways remain largely unknown. Methodology. We investigated these pathways by using time-series experiments over 48 h to follow the formation of organoarsenic species from arsenate-enriched medium (15 mu g As L-1) by the unicellular alga Dunaliella tertiolecta cultured under batch and continuous culture conditions. We used complementary mass spectrometry methods for the determination and quantification of 14 arsenic species; an additional three species could be quantified but remained unidentified. Results. The alga rapidly methylated the arsenate to dimethylarsinate (DMA), which then served as the precursor to arsenosugars and arsenolipids; the concentrations of these complex organoarsenicals increased throughout the experiments accompanied by a concomitant reduction in DMA concentrations. The pattern of compounds formed by the alga was similar for both batch and continuous cultures, but the concentrations were 2-3-fold higher in the continuous culture samples and the increases with time were much clearer. Discussion. The data suggest that the arsenosugars and the arsenolipids were mostly formed simultaneously from DMA, although there was an indication that the arsenic phospholipids were at least partly also being formed from the arsenosugars. Overall, the data are consistent with a direct biosynthesis of DMA from arsenate by D. tertioleta, and thereafter a non-specific incorporation of DMA into commonly available alga metabolites encompassing various sugars and lipids. Refereed/Peer-reviewed

To investigate the release and degradation of arsenolipids present in the marine brown macroalga Ecklonia radiata, tissues were collected in various stages of decomposition from intertidal environments, while tissues were also decomposed in laboratory-based microcosms prepared using combinations of autoclaved and natural (non-autoclaved) seawater and sand. Field collected macroalgae samples contained 20–120 μg g−1 total As of which 1–10% were arsenolipids comprising mainly an arsenic hydrocarbon (AsHC; 3–13% of total arsenolipids) and four di-acyl arsenic phospholipids (AsPLs; 86–95%). Additionally, a mono-acyl AsPL was found in all water-column decomposing samples. Arsenolipid concentrations in live tissues were similar to those in tissues decomposing in the water-column (1.3–2.9 μg g−1 dry mass), which were both up to four times higher than those in decomposing tissues collected from intertidal environments (0.7–1.3 μg g−1 dry mass). In the microcosm experiments, the arsenolipid content of E. radiata decreased substantially as decomposition proceeded. In the majority of microcosms, more than 75% of the arsenolipids present initially disappeared within 5 days with only the AsHC persisting until day 60 (the length of the experiment). This study demonstrates that the habitat in which decomposition occurs influences the release and degradation of arsenolipids with the greatest losses occurring when tissues decompose in intertidal environments. Microbial diversity, biomass, and overall activity are thus likely to play important roles in the persistence of arsenolipids in decomposing algae. Refereed/Peer-reviewed

The influence of arsenate and phosphate levels in water on the formation of arsenic-containing lipids (arsenolipids) and water-soluble arsenicals by a unicellular marine alga was investigated by exposing Dunaliella tertiolecta to five regimes of arsenic and phosphate, and determining the biosynthesized organoarsenicals with HPLC/mass spectrometry. Under all conditions, the major arsenolipid produced by D. tertiolecta was the novel phytyl 5-dimethylarsinoyl-2-O-methyl-ribofuranoside (AsSugPhytol546) representing ca. 35-65% of total arsenolipids. The new compound contains a phytol aglycone and a methoxy group replacing a sugar hydroxyl-two structural features not previously observed for arsenolipids. Minor arsenolipids were several previously reported arsenosugar phospholipids (AsSugPLs, in particular AsSugPL958 and the previously unknown AsSugPL978), the relative quantities of which increased with increasing phosphate exposure, and an arsenic-containing hydrocarbon (AsHC360), which remained unaffected by the different treatments. The relative amount of total arsenolipids produced by D. tertiolecta remained remarkably constant (ca. 45% of total As) and independent of the culture conditions. In contrast, with rising As-concentrations we observed an increase of hydrophilic arsenicals, which were dominated by arsenate and arsenosugars. The results highlight a possible major difference in arsenic biochemistry between macroalgae and unicellular algae with potential implications for how various algae handle their natural arsenic exposure in the world's oceans. Refereed/Peer-reviewed

Objective: Growing evidence indicates that exposure to metals are risk factors for cardiovascular disease (CVD). We hypothesized that higher urinary levels of metals with prior evidence of an association with CVD, including non-essential (cadmium , tungsten, and uranium) and essential (cobalt, copper, and zinc) metals are associated with baseline and rate of change of coronary artery calcium (CAC) progression, a subclinical marker of atherosclerotic CVD., Methods: We analyzed data from 6,418 participants in the Multi-Ethnic Study of Atherosclerosis (MESA) with spot urinary metal levels at baseline (2000-2002) and 1-4 repeated measures of spatially weighted coronary calcium score (SWCS) over a ten-year period. SWCS is a unitless measure of CAC highly correlated to the Agatston score but with numerical values assigned to individuals with Agatston score=0. We used linear mixed effect models to assess the association of baseline urinary metal levels with baseline SWCS, annual change in SWCS, and SWCS over ten years of follow-up. Urinary metals (adjusted to μg/g creatinine) and SWCS were log transformed. Models were progressively adjusted for baseline sociodemographic factors, estimated glomerular filtration rate, lifestyle factors, and clinical factors., Results: At baseline, the median and interquartile range (25 th , 75 th ) of SWCS was 6.3 (0.7, 58.2). For urinary cadmium, the fully adjusted geometric mean ratio (GMR) (95%Cl) of SWCS comparing the highest to the lowest quartile was 1.51 (1.32, 1.74) at baseline and 1.75 (1.47, 2.07) at ten years of follow-up. For urinary tungsten, uranium, and cobalt the corresponding GMRs at ten years of follow-up were 1.45 (1.23, 1.71), 1.39 (1.17, 1.64), and 1.47 (1.25, 1.74), respectively. For copper and zinc, the association was attenuated with adjustment for clinical risk factors; GMRs at ten years of follow-up before and after adjustment for clinical risk factors were 1.55 (1.30, 1.84) and 1.33 (1.12, 1.58), respectively, for copper and 1.85 (1.56, 2.19) and 1.57 (1.33, 1.85) for zinc., Conclusion: Higher levels of cadmium, tungsten, uranium, cobalt, copper, and zinc, as measured in urine, were associated with subclinical CVD at baseline and at follow-up. These findings support the hypothesis that metals are pro-atherogenic factors., Competing Interests: DISCLOSURES The authors have no conflict of interest to disclose. The views expressed in this manuscript are those of the authors and do not necessarily represent the views of the National Heart, Lung, and Blood Institute; the National Institutes of Health; or the U.S. Department of Health and Human Services.