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Oral and gut Bacteroidetes produce unique classes of serine-glycine lipodipeptides and glycine aminolipids that signal through host Toll-like receptor 2. These glycine lipids have also been detected in human arteries, but their effects on atherosclerosis are unknown. Here, we sought to investigate the bioactivity of bacterial glycine lipids in mouse models of atherosclerosis. Lipid 654 (L654), a serine-glycine lipodipeptide species, was first tested in a high-fat diet (HFD)-fed Ldlr -/- model of atherosclerosis. Intraperitoneal administration of L654 over 7 weeks to HFD-fed Ldlr -/- mice resulted in hypocholesterolemic effects and significantly attenuated the progression of atherosclerosis. We found that L654 also reduced liver inflammatory and extracellular matrix gene expression, which may be related to inhibition of macrophage activation as demonstrated in vivo by lower major histocompatibility complex class II gene expression and confirmed in cell experiments. In addition, L654 and other bacterial glycine lipids in feces, liver, and serum were markedly reduced alongside changes in Bacteroidetes relative abundance in HFD-fed mice. Finally, we tested the bioactivities of L654 and related lipid 567 in chow-fed Apoe -/- mice, which displayed much higher fecal glycine lipids relative to HFD-fed Ldlr -/- mice. Administration of L654 or lipid 567 for 7 weeks to these mice reduced the liver injury marker alanine aminotransferase, but other effects seen in Ldlr -/- were not observed. Therefore, we conclude that conditions in which gut microbiome-derived glycine lipids are lost, such as HFD, may exacerbate the development of atherosclerosis and liver injury, whereas correction of such depletion may protect from these disorders., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Coffee brewed from capsules contain estrogenic chemicals (ECs) that may harm the reproductive system. However, there are no studies investigating whether consuming capsule coffee causes these ECs to present in urine., Objective: Compare the effects of consuming capsule coffee vs. a plastic-free (French press) method on the appearance of ECs in urine., Methods: Participants (n = 30) were randomized to consume 540 mL of capsule or French press coffee once, then switched and consumed the other coffee after washout. Urine samples were collected prior to consumption, at 6 h and 24 h. Coffee and urine samples were analyzed for nine ECs using ultra-performance liquid chromatography with tandem mass spectrometry: bisphenol A (BPA), bisphenol F (BPF), bisphenol S, di(2-ethylhexyl) phthalate (DEHP), benzophenone, 4-nonylphenol (4-NP), dibutyl phthalate, caprolactam and dimethyl terephthalate., Results: In coffee samples, BPF (French press: 13.9 ng/mL, capsule: 16.1 ng/mL) and DEHP (capsule: 1.12 ng/mL) were present. In 6 h urine samples, the detection frequency for DEHP was 6.7% in capsule and 13.3% in French press coffee. BPF was detected in only one urine sample post-consumption., Conclusion: Consuming capsule coffee did not increase urinary EC exposure compared to consuming French press coffee., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Toll-like receptor 2 (TLR2) activation has been implicated in the pathogenesis of periodontal disease but the identity of the TLR2 agonists has been an evolving story. The serine/glycine lipids produced by Porphyromonas gingivalis are reported to engage human TLR2 and will promote the production of potent pro-inflammatory cytokines. This investigation compared the recovery of serine/glycine lipids in periodontal organisms, teeth, subgingival calculus, subgingival plaque, and gingival tissues, either from healthy sites or periodontally diseased sites. Lipids were extracted using the phospholipid extraction procedure of Bligh and Dyer and were analyzed using liquid chromatography/mass spectrometry for all serine/glycine lipid classes identified to date in P. gingivalis . Two serine/glycine lipid classes, Lipid 567 and Lipid 1256, were the dominant serine/glycine lipids recovered from oral Bacteroidetes bacteria and from subgingival calculus samples or diseased teeth. Lipid 1256 was the most abundant serine/glycine lipid class in lipid extracts from P. gingivalis, Tannerella forsythia , and Prevotella intermedia whereas Lipid 567 was the most abundant serine/glycine lipid class recovered in Capnocytophaga species and Porphyromonas endodontalis . Serine/glycine lipids were not detected in lipid extracts from Treponema denticola, Aggregatibacter actinomycetemcomitans , or Fusobacterium nucleatum . Lipid 1256 was detected more frequently and at a significantly higher mean level in periodontitis tissue samples compared with healthy/gingivitis tissue samples. By contrast, Lipid 567 levels were essentially identical. This report shows that members of the Bacteroidetes phylum common to periodontal disease sites produce Lipid 567 and Lipid 1256, and these lipids are prevalent in lipid extracts from subgingival calculus and from periodontally diseased teeth and diseased gingival tissues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nichols, Bhuse, Clark, Provatas, Carrington, Wang, Zhu, Davey and Dewhirst.)

In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA's c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that 'ectopic' production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete., Competing Interests: The authors have declared that no competing interests exist.

Coffee brewed from capsule machines may contain estrogenic chemicals migrated from plastic, but the estrogenic activity of capsule coffee has not been evaluated. This study evaluated the estrogenic activity of capsule coffee using the VM7Luc4E2 estrogen receptor transcriptional activation assay. Estrogenic potentials of six capsule coffee samples were calculated using relative maximum amplitude response of E2 (>15%RME2 indicative of estrogenic activity) and estradiol equivalent factor (EEF). Estrogenic chemical content was determined using ultra-performance liquid chromatography with tandem mass spectrometry. All capsule coffee samples possessed estrogenic activity (48-56%RME2). EEFs were 6-7 orders of magnitude lower than that of E2, (1.2 × 10 -7 -1.7 × 10 -6 ), indicating substantially weaker estrogenic potencies. Bisphenol A, bisphenol F, benzophenone, 4-nonylphenol, dibutyl phthalate, and dimethyl terephthalate were detected in capsule coffee. Capsule coffee exhibited estrogenic activity in vitro, and its estrogenic chemical content is likely driving its estrogenicity, warranting further investigations to fully understand the degree to which they are related and to predict the estrogenic potential based on the concentration of estrogenic chemicals., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Algal lipids are important molecules to store energy in algae and transfer energy in the marine food chain, and are potential materials for high value nutraceuticals (e.g., omega-3 fatty acids) or biofuel production. However, how lipid biosynthesis is regulated is not well understood in many species including Eutreptiella from the phylum of Euglenozoa. Here, we characterized the fatty acid (FA) profile of an Eutreptiella species isolated from Long Island Sound, USA, using gas chromatography-tandem mass spectrometry (GC/MS/MS) and investigated their biosynthesis pathways by transcriptome sequencing. We discovered 24 types of FAs including a relatively high proportion of long-chain unsaturated FAs. The abundances of C16, C18, and saturated FAs decreased when phosphate in the culture medium was depleted. Among the 24 FAs, docosahexaenoic acid (C22:6 ∆4,7,10,13,16,19 ) was most abundant, suggesting that Eutreptiella sp. preferentially invests in the synthesis of long-chain polyunsaturated fatty acids (LC-PFAs). Further transcriptomic analysis revealed that Eutreptiella sp. likely synthesizes LC-PFAs via ∆8 pathway and uses type I and II fatty acid synthases. Using RT-qPCR, we found that some of the lipid synthesis genes, such as β-ketoacyl-ACP reductase, fatty acid desaturase, acetyl-CoA carboxylase, acyl carrier protein, ∆8 desaturase, and Acyl-ACP thioesterase, were more actively expressed during light period, and two carbon fixation genes were up-regulated in the high-lipid illuminated cultures, suggesting a linkage between photosynthesis and lipid production. The lipid profile renders Eutreptiella sp. a nutritional prey and valuable source for nutraceuticals, and the biosynthesis pathway documented here will be useful for future research and applications., (© 2020 Phycological Society of America.)

Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue-destructive processes in human periodontitis. Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require the engagement of Toll-like receptor 2 (TLR2). Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2, but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6. The novel lipid class, termed Lipid 1256, consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide previously termed Lipid 654. Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes. Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms. Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Nichols et al.)

Necrotizing enterocolitis (NEC) is a devastating intestinal inflammatory disease of premature infants associated with gut bacterial dysbiosis. Using 16S rRNA-based methods, our laboratory identified an unclassified Enterobacteriaceae sequence (NEC_unk_OTU) with high abundance in NEC fecal samples. We aimed to identify this bacterium and determine its potential role in the disease. NCBI database searches for the 16S sequence, selective culture systems, biotyping and polymerase chain reaction were employed to refine classification of NEC_unk_OTU and identify toxin-encoding genes from the index NEC case. Bacterial cytotoxin production was confirmed by mass spectrometry and apoptosis assays. Additional fecal samples from 9 NEC and 5 non-NEC controls were analyzed using similar methods and multi-locus sequence typing (MLST) was performed to investigate clonal relationships and define sequence types of the isolates. NEC_unk_OTU was identified as Klebsiella oxytoca , a pathobiont known to cause antibiotic-associated hemorrhagic colitis, but not previously linked to NEC. Including the index case, cytotoxin-producing strains of K. oxytoca were isolated from 6 of 10 subjects with NEC; in these, the K. oxytoca 16S sequence predominated the fecal microbiota. Cytotoxin-producing strains of K. oxytoca also were isolated from 4 of 5 controls; in these, however, the abundance of the corresponding 16S sequence was very low. MLST analysis of the toxin-positive isolates demonstrated no clonal relationships and similar genetic clustering between cases and controls. These results suggest cytotoxin-producing strains of K. oxytoca colonize a substantial proportion of premature infants. Some, perhaps many, cases of NEC may be precipitated by outgrowth of this opportunistic pathogen.

The objective of this study was to examine exposure to estrogenic chemicals (ECs) via capsule coffee. Twenty-two brands of capsule coffee and 15 brands of French press coffee for comparison were brewed, and their contents of ECs were identified and quantified using ultra-performance liquid chromatography with tandem mass spectrometry. Exposure to ECs in coffee were compared to tolerable daily intake guidelines to assess potential hazard to health. Benzophenone was the most frequently detected EC in capsule coffee (mean concentration ± SD: 20.37 ± 47.07 ng/mL, n = 6), followed by bisphenol A (BPA, 0.31 ± 0.71, n = 4), dibutyl phthalate (1.41 ± 3.58, n = 3), 4-nonylphenol (0.67 ± 1.82, n = 3) and bisphenol F (BPF, 0.49 ± 1.54, n = 2). BPA and BPF were each detected in 3 French press coffee samples (0.29 ± 0.58 and 0.85 ± 1.75 ng/mL, respectively). Two French press coffee brands purchased as ground coffee rather than whole bean were positive for ECs (BPA in one and BPF in both). Hazard indexes were below 1.0 for each EC for both coffee types. These results indicate that there is EC contamination in capsule and French press coffee, but the quantities of ECs are low relative to established safety guidelines., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors. Published by Elsevier B.V.)

The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids., (Copyright © 2020 American Society for Microbiology.)

Furocoumarins are a class of photoactive compounds found in several plant species and may be responsible for the observed association between consumption of citrus products and the risk of skin cancer. Furocoumarin contents of several foods have been reported previously, but no comprehensive database of furocoumarin content of foods is currently available. Therefore, this study aimed to determine the distribution of furocoumarins in popularly consumed foods in the U.S. Samples of three varieties of each of 29 foods known or suspected to contain furocoumarins were purchased, prepared for analysis using a solid phase extraction method, and analyzed using UPLC-MS/MS for the presence of seven major furocoumarins. Most foods measured contained more than one furocoumarin, and some contained all seven of the furocoumarins examined. Total furocoumarin concentration was greatest in fresh parsley (23215 ng/g), grapefruits (21858 ng/g), lime juice (14580 ng/g), grapefruit juice (95341 ng/g), and limes (9151 ng/g). Bergamottin was found in the greatest proportion of foods sampled (23 of 29), followed by bergapten (19 of 29) and 6'7'-dihydroxybergamottin (16 of 29). These measurements will enable more accurate estimation of dietary furocoumarin exposure and will strengthen future epidemiological work investigating the relationships between furocoumarin intake and health outcomes.

Furocoumarins are a class of organic compounds found in a variety of vegetables and fruits. Relatively little is known about the absorption and excretion of these compounds following ingestion. The objective of this study was to identify furocoumarins in grapefruit and grapefruit juice and observe their kinetics in blood and urine. The furocoumarins detected in grapefruit using UPLC-MS/MS were bergamottin, 6',7'-dihydroxybergamottin (6',7'-DHB), epoxybergamottin, and bergaptol. Bergamottin, 6',7'-DHB, bergaptol, and bergapten were detected in grapefruit juice. In this study of 6 males and 3 females, only bergamottin and 6',7'-DHB were detected in plasma, whereas in urine, four distinct furocoumarin metabolites as well as bergaptol, 6',7'-DHB, 8-methoxypsoralen (8-MOP), bergamottin, and psoralen were identified. Following grapefruit ingestion, furocoumarins were detectable in plasma as early as 15 min and in urine within 1 h. They remained in plasma for up to 3 or more hours and in urine as late as 24 h.

A streamlined method has been developed for the isolation and analysis of polycyclic aromatic hydrocarbons in avian blood cells and plasma utilizing quick, easy, cheap, effective, rugged, and safe extraction in combination with novel phospholipid cleanup technology. A variety of traditional extraction and cleanup techniques have been employed in the preparation and analysis of polycyclic aromatic hydrocarbonsin a variety of matrices; liquid-liquid partitioning, solid-phase extractions, gel permeation chromatography, and column chromatography are all effective techniques, however they are laborious and time consuming processes that require large amounts of solvent. Using quick, easy, cheap, effective, rugged, and safe extraction coupled with phospholipid cleanup, samples can be quickly screened while maintaining high throughput and sensitivity. With a liquid chromatography approach, analysis times may be kept short at 16 min while maintaining high analyte recovery. Recoveries in quality control samples ranged from 70 to 109%, with average surrogate recoveries of 80.6 ± 1.10%. The result of using a quick, easy, cheap, effective, rugged, and safe extraction approach in conjunction with phospholipid cleanup is a methodology that significantly reduces sample preparation time and solvent use while maintaining high sensitivity and reproducibility., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Endocrine disrupting substances (EDSs) have the potential to disturb sensitive hormone pathways, particularly those involved in development and reproduction. Both fresh and estuarine water bodies receive inputs of EDSs from a variety of sources, including sewage effluent, industrial effluent and agricultural runoff. Based on current literature, freshwater species appear to respond to lower levels of EDSs than estuarine or marine species. Therefore, effects elicited by EDSs in freshwater teleosts may not be an accurate representation of how EDSs affect teleosts in estuarine and marine environments. To address this potential difference, a short-term reproductive bioassay was conducted under conditions of low and high salinity using mummichog (Fundulus heteroclitus), a euryhaline species that is native to the east coast of North America. The goals of this study were to determine the response of mummichog when exposed to an androgenic EDS and whether salinity affected the response. A model androgen, 5α-dihydrotestosterone (DHT), was selected for this experiment. Impacts on reproduction were evaluated at multiple biological levels, including physiological (sex steroid levels), organismal (gonad size and gonad morphology), and functional (egg production) endpoints. Under conditions of high salinity, egg production was significantly reduced at all exposure concentrations. Under conditions of low salinity, there were no significant differences based on DHT treatment; however, egg production in all treatment groups including the control were significantly reduced relative to the high salinity control group. Other reproductive endpoints, such as sex steroid production, showed stronger correlation to fecundity in females than males. This study demonstrates that mummichog fecundity is sensitive to androgenic endocrine disruption while also underscoring the importance of how changes in salinity, an environmental variable, can impact reproduction., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A new protecting group for the alcohol functionality was devised and shown to be removed photochemically under ultraviolet light in the presence of a radical scavenger in high yields.