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Additional occurrence data are needed to better understand human exposure to per- and poly-fluoroalkyl substances (PFAS) from commercially available foods in the United States. The Food and Drug Administration’s (FDA) Total Diet Study (TDS) collects foods that are both nationally and regionally distributed. In 2018, 172 processed foods were collected from grocery stores around Lenexa, KS, as part of the TDS national collection. A previously developed method for the analysis of PFAS in foods as part of the TDS regional collection was modified and optimized for these samples. This method was single lab validated using 5 different matrices and method detection limits were calculated. During the analysis of these samples, challenges arose with method blanks and further investigation into statistical methods to distinguish between blank and sample concentrations were done. The confirmation of two short chain PFAS, perfluorobutanoic acid (PFBA) and perfluoropentanoic acid (PFPeA), was not possible using triple quadrupole mass spectrometry and a confirmation method was developed using high-resolution mass spectrometry. This technique was also used to investigate potential detections and interferents that fell within the retention time criteria for positive detections. In the national collection, positive detections of perfluorooctanesulfonic acid (PFOS) and perfluorononanoic acid (PFNA) were found in frozen fish sticks/patties, PFOS and perfluorodecanoic acid (PFDA) in canned tuna, and PFOS in protein powder. Concentrations were all below 150 ppt, and no other detects were confirmed above the method detection limits in any other foods.

Developing effective treatments for colorectal cancers through combinations of small-molecule approaches and immunotherapies present intriguing possibilities for managing these otherwise intractable cancers. During a broad-based, screening effort against multiple colorectal cancer cell lines, we identified indole-substituted quinolines (ISQ), such as N7,N7-dimethyl-3-(1-methyl-1H-indol-3-yl)quinoline-2,7-diamine (ISQ-1), as potent in vitro inhibitors of several cancer cell lines. We found that ISQ-1 inhibited Wnt signaling, a main driver in the pathway governing colorectal cancer development, and ISQ-1 also activated adenosine monophosphate kinase (AMPK), a cellular energy–homeostasis master regulator. We explored the effect of ISQs on cell metabolism. Seahorse assays measuring oxygen consumption rate (OCR) indicated that ISQ-1 inhibited complex I (i.e., NADH ubiquinone oxidoreductase) in the mitochondrial, electron transport chain (ETC). In addition, ISQ-1 treatment showed remarkable synergistic depletion of oncogenic c-Myc protein level in vitro and induced strong tumor remission in vivo when administered together with BI2536, a polo-like kinase-1 (Plk1) inhibitor. These studies point toward the potential value of dual drug therapies targeting the ETC and Plk-1 for the treatment of c-Myc–driven cancers.

Leishmaniasis, a disease caused by protozoa of the Leishmania species, afflicts roughly 12 million individuals worldwide. Most existing drugs for leishmaniasis are toxic, expensive, difficult to administer, and subject to drug resistance. We report a new class of antileishmanial leads, the 3-arylquinolines, that potently block proliferation of the intramacrophage amastigote form of Leishmania parasites with good selectivity relative to the host macrophages. Early lead 34 was rapidly acting and possessed good potency against L. mexicana (EC50 = 120 nM), 30-fold selectivity for the parasite relative to the macrophage (EC50 = 3.7 μM), and also blocked proliferation of Leishmania donovani parasites resistant to antimonial drugs. Finally, another early lead, 27, which exhibited reasonable in vivo tolerability, impaired disease progression during the dosing period in a murine model of cutaneous leishmaniasis. These results suggest that the arylquinolines provide a fruitful departure point for the development of new antileishmanial drugs.

Aminofutalosine synthase (MqnE) is a radical SAM enzyme that catalyzes the conversion of 3-((1-carboxyvinyl)oxy)benzoic acid to aminofutalosine during the futalosine-dependent menaquinone biosynthesis. In this Communication, we report the trapping of a radical intermediate in the MqnE-catalyzed reaction using sodium dithionite, molecular oxygen, or 5,5-dimethyl-1-pyrroline-N-oxide. These radical trapping strategies are potentially of general utility in the study of other radical SAM enzymes.

Sulfur modifications have been discovered on both DNA and RNA. Sulfur substitution of oxygen atoms at nucleobase or backbone locations in the nucleic acid framework led to a wide variety of sulfur-modified nucleosides and nucleotides. While the discovery, regulation and functions of DNA phosphorothioate (PS) modification, where one of the non-bridging oxygen atoms is replaced by sulfur on the DNA backbone, are important topics, this review focuses on the sulfur modification in natural cellular RNAs and therapeutic nucleic acids. The sulfur modifications on RNAs exhibit diversity in terms of modification location and cellular function, but the various sulfur modifications share common biosynthetic strategies across RNA species, cell types and domains of life. The first section reviews the post-transcriptional sulfur modifications on nucleobases with an emphasis on thiouridine on tRNA and phosphorothioate modification on RNA backbones, as well as the functions of the sulfur modifications on different species of cellular RNAs. The second section reviews the biosynthesis of different types of sulfur modifications and summarizes the general strategy for the biosynthesis of sulfur-containing RNA residues. One of the main goals of investigating sulfur modifications is to aid the genomic drug development pipeline and enhance our understandings of the rapidly growing nucleic acid-based gene therapies. The last section of the review focuses on the current drug development strategies employing sulfur substitution of oxygen atoms in therapeutic RNAs., In this review, we highlight the importance of sulfur modifications in natural cellular RNAs and therapeutic nucleic acids. Sulfur modifications on RNA confer structural diversity, stability and enhance its functionality.

The H-cluster of [FeFe]-hydrogenase consists of a [4Fe–4S](H)-subcluster linked by a cysteinyl bridge to a unique organometallic [2Fe](H)-subcluster assigned as the site of interconversion between protons and molecular hydrogen. This [2Fe](H)-subcluster is assembled by a set of Fe–S maturase enzymes HydG, HydE and HydF. Here we show that the HydG product [Fe(II)(Cys)(CO)(2)(CN)] synthon is the substrate of the radical SAM enzyme HydE, with the generated 5′-deoxyadenosyl radical attacking the cysteine S to form a C5′–S bond concomitant with reduction of the central low-spin Fe(II) to the Fe(I) oxidation state. This leads to the cleavage of the cysteine C3–S bond, producing a mononuclear [Fe(I)(CO)(2)(CN)S] species that serves as the precursor to the dinuclear Fe(I)Fe(I) center of the [2Fe](H)-subcluster. This work unveils the role played by HydE in the enzymatic assembly of the H-cluster and expands the scope of radical SAM enzyme chemistry.

Cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) methyltransferases catalyze methylation reactions at non-nucleophilic centers in a wide range of substrates. CysS is a Cbl-dependent radical SAM methyltransferase involved in cystobactamid biosynthesis. This enzyme catalyzes the sequential methylation of a methoxy group to form ethoxy, i-propoxy, s-butoxy, and t-butoxy groups on a p-aminobenzoate peptidyl carrier protein thioester intermediate. This biosynthetic strategy enables the host myxobacterium to biosynthesize a combinatorial antibiotic library of 25 cystobactamid analogues. In this Article, we describe three experiments to elucidate how CysS uses Cbl, SAM, and a [4Fe-4S] cluster to catalyze iterative methylation reactions: a cyclopropylcarbinyl rearrangement was used to trap the substrate radical and to estimate the rate of the radical substitution reaction involved in the methyl transfer; a bromoethoxy analogue was used to explore the active site topography; and deuterium isotope effects on the hydrogen atom abstraction by the adenosyl radical were used to investigate the kinetic significance of the hydrogen atom abstraction. On the basis of these experiments, a revised mechanism for CysS is proposed.

Androgen-deprivation therapy (ADT) is only a palliative measure, and prostate cancer invariably recurs in a lethal, castration-resistant form (CRPC). Prostate cancer resists ADT by metabolizing weak, adrenal androgens to growth-promoting 5α-dihydrotestosterone (DHT), the preferred ligand for the androgen receptor (AR). Developing small-molecule inhibitors for the final steps in androgen metabolic pathways that utilize 17-oxidoreductases required probes that possess fluorescent groups at C-3 and intact, naturally occurring functionality at C-17. Application of the Pictet-Spengler condensation to substituted 4-(2-aminoethyl)coumarins and 5α-androstane-3-ones furnished spirocyclic, fluorescent androgens at the desired C-3 position. Condensations required the presence of activating C-7 amino or N,N-dialkylamino groups in the 4-(2-aminoethyl)coumarin component of these condensation reactions. Successful Pictet-Spengler condensation, for example, of DHT with 9-(2-aminoethyl)-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one led to a spirocyclic androgen, (3R,5S,10S,13S,17S)-17-hydroxy-10,13-dimethyl-1,2,2’,3’,4,5,6,7,8,8’,9,9’,10,11,12,12’,13,13’,14,15,16,17-docosahydro-7’H,11’H-spiro-[cyclopenta[a]phenanthrene-3,4’-pyrido[3,2,1-ij]pyrido[4’,3’:4,5]pyrano[2,3-f]quinolin]-5’(1’H)-one. Computational modeling supported the surrogacy of the C-3 fluorescent DHT analog as a tool to study 17-oxidoreductases for intracrine, androgen metabolism.

International audience; The Pdx1 enzyme catalyses condensation of two carbohydrates and ammonia to form pyridoxal 5-phosphate (PLP) via an imine relay mechanism of carbonyl intermediates. The I333 intermediate characterised here using structural, UV-vis absorption spectroscopy and mass spectrometry analyses rationalises stereoselective deprotonation and subsequent substrate assisted phosphate elimination, central to PLP biosynthesis.

RNA modifications and their corresponding epitranscriptomic writer and eraser enzymes regulate gene expression. Altered RNA modification levels, dysregulated writers, and sequence changes that disrupt epitranscriptomic marks have been linked to mitochondrial and neurological diseases, cancer, and multifactorial disorders. The detection of epitranscriptomics marks is challenging, but different next generation sequencing (NGS)-based and mass spectrometry-based approaches have been used to identify and quantitate the levels of individual and groups of RNA modifications. NGS and mass spectrometry-based approaches have been coupled with chemical, antibody or enzymatic methodologies to identify modifications in most RNA species, mapped sequence contexts and demonstrated the dynamics of specific RNA modifications, as well as the collective epitranscriptome. While epitranscriptomic analysis is currently limited to basic research applications, specific approaches for the detection of individual RNA modifications and the epitranscriptome have potential biomarker applications in detecting human conditions and diseases. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Processing > tRNA Processing RNA in Disease and Development > RNA in Disease.

Significance The circadian clock plays an essential role in energy metabolism and inflammation. In contrast, the importance of the clock in the pathogenesis of fibrosis remains poorly explored. This study describes a striking alteration in circadian biology during pulmonary fibrosis where the relatively arrhythmic alveolar structures gain circadian but asynchronous rhythmicity due to infiltration by fibroblasts. Disruption of the clock in these cells, which are not widely implicated in circadian pathophysiology, results in a profibrotic phenotype. Translation of these findings in humans revealed previously unrecognized important circadian risk factors for pulmonary fibrosis (sleep length, chronotype, and shift work). In addition, targeting REVERBα repressed collagen secretion from human fibrotic lung tissue, making this protein a promising therapeutic target., Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinβ1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.

The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes into two eventual daughter nuclei. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) anchor chromosomes within the spindle. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We sever k-fibers using laser ablation, thereby detaching them from poles and testing the contribution of pole-localized force generation to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often, but not always, splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced in response to ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the putative k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15 microtubule binding reduces k-fiber mechanical integrity. In contrast, inhibition of its motor activity but not its microtubule binding does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors.

The mammalian circadian clock exerts substantial control of daily gene expression through cycles of DNA binding. Understanding of mechanisms driving the circadian clock is hampered by lack of quantitative data, without which predictive mathematical models cannot be developed. Here we develop a quantitative understanding of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We have used fluorescent correlation spectroscopy (FCS) to track dynamic changes in CRISPR-modified fluorophore-tagged proteins in time and space in single cells across SCN and peripheral tissues. We determine the contribution of multiple rhythmic processes in coordinating BMAL1 DNA binding, including the roles of cycling molecular abundance, binding affinities and two repressive modes of action. We find that nuclear BMAL1 protein numbers determine corresponding nuclear CLOCK concentrations through heterodimerization and define a DNA residence time of 2.6 seconds for this complex. Repression of CLOCK:BMAL1 is in part achieved through rhythmic changes to BMAL1:CRY1 affinity as well as a high affinity interaction between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling of these data reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.

Epitranscriptomic marks, in the form of enzyme catalyzed RNA modifications, play important gene regulatory roles in response to environmental and physiological conditions. However, little is known with respect to how pharmaceuticals influence the epitranscriptome. Here we define how acetaminophen (APAP) induces epitranscriptomic reprogramming and how the writer Alkylation Repair Homolog 8 (Alkbh8) plays a key gene regulatory role in the response. Alkbh8 modifies tRNA selenocysteine (tRNASec) to translationally regulate the production of glutathione peroxidases (Gpx’s) and other selenoproteins, with Gpx enzymes known to play protective roles during APAP toxicity. We demonstrate that APAP increases toxicity and markers of damage, and decreases selenoprotein levels in Alkbh8 deficient mouse livers, when compared to wildtype. APAP also promotes large scale reprogramming of 31 RNA marks comprising the liver tRNA epitranscriptome including: 5-methoxycarbonylmethyluridine (mcm5U), isopentenyladenosine (i6A), pseudouridine (Ψ), and 1-methyladenosine (m1A) modifications linked to tRNASec and many others. Alkbh8 deficiency also leads to wide-spread epitranscriptomic dysregulation in response to APAP, demonstrating that a single writer defect can promote downstream changes to a large spectrum of RNA modifications. Our study highlights the importance of RNA modifications and translational responses to APAP, identifies writers as key modulators of stress responses in vivo and supports the idea that the epitranscriptome may play important roles in responses to pharmaceuticals.

A comprehensive analysis of the temporal evolution of tropospheric ozone in Antarctica using more than 25 years of surface ozone and ozonesonde measurements reveals significant changes in tropospheric ozone there. It shows a positive trend in ozone at the surface and lower and mid-troposphere, but a negative trend in the upper troposphere. We also find significant links between different climate modes and tropospheric ozone in Antarctica and observe that changes in residual overturning circulation, the strength of the polar vortex, and stratosphere-troposphere exchange make noticeable variability in tropospheric ozone. Therefore, this study alerts of increasing ozone concentration in Antarctica, which would have a profound impact on the future climate of the region as tropospheric ozone has warming feedback to the Earth's climate.

Trans-Pacific transport of enhanced ozone plumes has been mainly attributed to fossil fuel combustion in Asia in spring, but less attention has been paid to vegetation fires in Asia. Here we show that the El Niño-Southern Oscillation (ENSO)-modulated fires in Southeast Asia, rather than Asian fossil fuel plumes, dominate the interannual variability of springtime trans-Pacific transport of ozone across the entire North Pacific Ocean. During El Niño springs, the intensified fires from both the Indochinese Peninsula and Indonesia, together with large-scale circulation anomalies, result in enhanced ozone plumes that stretch over 15 000 km in both the lower-middle and upper troposphere. This enhancement is also observed in the in situ measurements of ozone concentration, with an almost 10% increase at Mauna Loa Observatory, Hawaii, a unique site to monitor the long-distance transport over the North Pacific. This study reports an unexpectedly strong influence of vegetation fires, linked with climate variability, on global tropospheric chemistry and proves once more how complex the interactions in the climate system are.

Here we highlight the role of epitranscriptomic systems in post-transcriptional regulation, with a specific focus on RNA modifying writers required for the incorporation of the 21(st) amino acid selenocysteine during translation, and the pathologies linked to epitranscriptomic and selenoprotein defects. Epitranscriptomic marks in the form of enzyme-catalyzed modifications to RNA have been shown to be important signals regulating translation, with defects linked to altered development, intellectual impairment, and cancer. Modifications to rRNA, mRNA and tRNA can affect their structure and function, while the levels of these dynamic tRNA-specific epitranscriptomic marks are stress-regulated to control translation. The tRNA for selenocysteine contains five distinct epitranscriptomic marks and the ALKBH8 writer for the wobble uridine (U) has been shown to be vital for the translation of the glutathione peroxidase (GPX) and thioredoxin reductase (TRXR) family of selenoproteins. The reactive oxygen species (ROS) detoxifying selenocysteine containing proteins are a prime examples of how specialized translation can be regulated by specific tRNA modifications working in conjunction with distinct codon usage patterns, RNA binding proteins and specific 3’ untranslated region (UTR) signals. We highlight the important role of selenoproteins in detoxifying ROS and provide details on how epitranscriptomic marks and selenoproteins can play key roles in and maintaining mitochondrial function and preventing disease.

AIMS The Australian Health Survey 2011 to 2013 indicated that Western Australian women had good iodine status, suggesting adequate consumption of iodine from food, however, little is known about pregnant women in this state. The aim was to investigate the iodine-containing food practices of Western Australian pregnant women. METHODS Pregnant women attending antenatal clinics at a public tertiary hospital in Perth, Western Australia, were invited to complete a questionnaire investigating consumption of key iodine food sources and knowledge of iodine-containing foods. Food frequency data were entered into FoodWorks based on the Australian Food and Nutrition Database 2007. RESULTS A total of 425 women took part in the study with a mean (SD) age of 29.4 (5.5) years. Sixty percent of women reported consuming bread at least daily. Only 37.6% of women used iodised salt, but the median (25, 75 percentile) iodine intake of these women was 183 (142, 267) μg/day compared to 148 (100, 228) μg/day of iodine from food only. Ethnicity was associated with iodised salt use: 76% of Asian women compared with 33% of Caucasian women. Three quarters of the women did not know if any foods are required to be fortified with iodine. CONCLUSION The iodine-containing food practices of pregnant women in this state suggest a risk of insufficiency. The present study is limited by the use of a semi-quantitative and non-validated food frequency questionnaire, thus assessment of the iodine intake and status of pregnant women representative of the ethnic mix of Western Australia is recommended.

Temperature-programmed native electrospray ionization mass spectrometry gives detailed insight into the assembly of model collagen triple helices., Collagen model peptides are useful for understanding the assembly and structure of collagen triple helices. The design of self-assembling heterotrimeric helices is particularly challenging and often affords mixtures of non-covalent assemblies that are difficult to characterize by conventional NMR and CD spectroscopic techniques. This can render a detailed understanding of the factors that control heterotrimer formation difficult and restrict rational design. Here, we present a novel method based on electrospray ionization mass spectrometry to investigate homo- and heterotrimeric collagen model peptides. Under native conditions, the high resolving power of mass spectrometry was used to access the stoichiometric composition of different triple helices in complex mixtures. A temperature-controlled electrospray ionization source was built to perform thermal denaturation experiments and provided melting temperatures of triple helices. These were found to be in good agreement with values obtained from CD spectroscopic measurements. Importantly, for mixtures of coexisting homo- and heterotrimers, which are difficult to analyze by conventional methods, our technique allowed for the identification and monitoring of the unfolding of each individual species. Their respective melting temperatures could easily be accessed in a single experiment, using small amounts of sample.

Concentrations of three monomers used in the manufacture of polyester resins for food cans, isophthalic acid (IPA), terephthalic acid (TA) and nadic acid (NA), and two oligomers were determined in canned food samples. All canned foods were purchased in local supermarkets in the United States. Concentrations of IPA and TA were determined to be from below the limit of detection in tomato sauce to 69 μg/kg in coconut milk, while the highest concentration of NA was found in pumpkin at 21 μg/kg. Estimated concentrations of oligomers in the food samples ranged from non-detect to 595 μg/kg. The influence of the storage time, lot type, and retail outlet on the migration concentrations was also investigated.

Radiolysis products from irradiated polyethylene resins were investigated using liquid chromatography coupled to photodiode array (PDA) and mass spectrometry (MS) detection. The resins were gamma-irradiated at applicable doses for food applications. The antioxidants and radiolysis products were extracted using accelerated solvent extraction (ASE) prior to analysis. Tris(2,4-di-tert-butylphenyl) phosphite and tris(nonylphenyl) phosphite (TNPP) were oxidized to form phosphate as the major product. Nonylphenol (from TNPP), and 2,4-di-tert-butyl phenol (from tris(2,4-di-tert-butylphenyl) phosphite) were also produced by the irradiation. Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate formed a dehydrogenated product (loss of two hydrogens). The remaining detectable peaks had low responses suggesting low concentrations. Some of the low concentration products were tentatively identified using information from their fragmentation patterns in the MS/MS spectrum, and high mass accuracy measurements.

Increases in free tropospheric ozone over the past two decades are mainly in the Northern Hemisphere that have been widely documented, while ozone trends in the Southern Hemisphere (SH) remain largely unexplained. Here we first show that in-situ and satellite observations document increases of tropospheric ozone in the SH over 1990–2015. We then use a global chemical transport model to diagnose drivers of these trends. We find that increases of anthropogenic emissions (including methane) are not the most significant contributors. Instead, we explain the trend as due to changes in meteorology, and particularly in transport patterns. We propose a possible linkage of the ozone increases to meridional transport pattern shifts driven by poleward expansion of the SH Hadley circulation (SHHC). The SHHC poleward expansion allows more downward transport of ozone from the stratosphere to the troposphere at higher latitudes, and also enhances tropospheric ozone production through stronger lifting of tropical ozone precursors to the upper troposphere. These together may lead to increasing tropospheric ozone in the extratropical SH, particularly in the middle/upper troposphere and in austral autumn. Poleward expansion of the Hadley circulation is partly driven by greenhouse warming, and the associated increase in tropospheric ozone potentially provides a positive climate feedback amplifying the warming that merits further quantification.

Aminofutalosine synthase (MqnE) catalyzes an important rearrangement reaction in menaquinone biosynthesis by the futalosine pathway. In this Letter, we report the identification of previously unreported inhibitors of MqnE using a mechanism-guided approach. The best inhibitor shows efficient inhibitory activity against H. pylori (IC50 = 1.8 ± 0.4 μM) and identifies MqnE as a promising target for antibiotic development.

Collagen model peptides are useful for understanding the assembly and structure of collagen triple helices. The design of self-assembling heterotrimeric helices is particularly challenging and often affords mixtures of non-covalent assemblies that are difficult to characterize by conventional NMR and CD spectroscopic techniques. This can render a detailed understanding of the factors that control heterotrimer formation difficult and restrict rational design. Here, we present a novel method based on electrospray ionization mass spectrometry to investigate homo- and heterotrimeric collagen model peptides. Under native conditions, the high resolving power of mass spectrometry was used to access the stoichiometric composition of different triple helices in complex mixtures. A temperature-controlled electrospray ionization source was built to perform thermal denaturation experiments and provided melting temperatures of triple helices. These were found to be in good agreement with values obtained from CD spectroscopic measurements. Importantly, for mixtures of coexisting homo- and heterotrimers, which are difficult to analyze by conventional methods, our technique allowed for the identification and monitoring of the unfolding of each individual species. Their respective melting temperatures could easily be accessed in a single experiment, using small amounts of sample., Chemical Science, 10 (42), ISSN:2041-6520, ISSN:2041-6539

Human exposure to per- and polyfluoroalkyl substances (PFAS) through the US diet has not been well-characterized. Highly consumed foods are routinely monitored through FDA's Total Diet Study program. Portions of these samples were used to develop and validate a method for PFAS in a wide variety of foods. The extraction of 16 PFAS was performed using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method and analyzed by liquid chromatography/mass spectrometry. Method optimizations are described including investigations into the QuEChERS sorbents, matrix effects, and solid-phase extraction (SPE) cartridges. The use of a custom push-through SPE cartridge showed promising results as a rapid cleanup option for food samples. Challenges in ion confirmation are discussed, and the use of enhanced product ion (EPI) full-scan MS/MS spectra is presented as a potential option for verifying false positives. The validated method was then used for the analysis of 179 total diet study samples, and positive detects for perfluorooctanesulfonic acid (PFOS) were found in two fish and one meat sample.

The addition of contaminated powdered spices and seasonings to finished products which do not undergo further processing represents a significant concern for food manufacturers. To reduce the incidence of bacterial contamination, seasoning ingredients should be subjected to a decontamination process. Ultraviolet light emitting diodes (UV-LEDs) have been suggested as an alternative to UV lamps for reducing the microbial load of foods, due to their increasing efficiency, robustness and decreasing cost. In this study, we investigated the efficacy of UV-LED devices for the inactivation of four bacteria (Listeria monocytogenes, Escherichia coli, Bacillus subtilis and Salmonella Typhimurium) on a plastic surface and in four powdered seasoning ingredients (onion powder, garlic powder, cheese and onion powder and chilli powder). Surface inactivation experiments with UV mercury lamps, UVC-LEDs and UVA-LEDs emitting at wavelengths of 254 nm, 270 nm and 365 nm, respectively, revealed that treatment with UVC-LEDs were comparable to, or better than those observed using the mercury lamp. Bacterial reductions in the seasoning powders with UVC-LEDs were less than in the surface inactivation experiments, but significant reductions of 0.75–3 log10 colony forming units (CFU) were obtained following longer (40 s) UVC-LED exposure times. Inactivation kinetics were generally nonlinear, and a comparison of the predictive models highlighted that microbial inactivation was dependent on the combination of powder and microorganism. This study is the first to report on the efficacy of UV-LEDs for the inactivation of several different bacterial species in a variety of powdered ingredients, highlighting the potential of the technology as an alternative to the traditional UV lamps used in the food industry.

Entamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota., Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His, and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here, we describe the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in tRNAGUCAsp. Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in the OS response, such as heat shock protein 70 (Hsp70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in tRNAGUCAsp, parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.

Phthalates are one of many chemical compounds that are used as plasticisers. Packaging can transfer plasticisers to the surfaces of foods or other materials. A recent study suggested a link between fast food consumption and increased urine phthalate metabolites even though phthalates are most commonly found in food contact materials made of PVC while fast food packaging is most commonly composed of paper and paper board. Phthalates in PVC are usually present in percent concentrations. In non-PVC food contact materials, such as paper or paperboard, the concentrations, if any, are expected to be significantly lower which can greatly impact the analytical method used for their determination. Due to the widespread use of plasticised PVC in many commercial applications, background concentrations of phthalates are a concern for trace concentration analyses and background contamination must be avoided when performing these analyses. A glassware cleaning method was developed and a solvent extraction with dichloromethane and hexane was used to extract phthalates from paper-based food packaging. The extracts were then analysed using a GC-MS/MS. The minimum reporting concentrations for the method were determined to be 0.10-0.40 µg/g depending on the phthalate investigated. Phthalate concentrations in several different non-PVC printed and unprinted packaging are presented. Of the 54 packaging samples tested, 10 samples contained no reportable concentrations of any of the 6 phthalates investigated. Of those that were reportable, all measured lower than 10 µg/g and in fact, most had concentrations less than 1 µg/g. These data demonstrate that phthalates from fast food packaging do not significantly contribute to overall consumer exposure.

DNA transcription by RNA polymerases has always interested the scientific community as it is one of the most important processes involved in genome expression. This has led scientists to come up with different protocols allowing analysis of this process in specific locations across the genome by quantitating the amount of RNA polymerases transcribing that genomic site in a cell population. This can be achieved by either detecting the total number of polymerases in contact with that region (i.e., by chromatin immunoprecipitation (ChIP) with anti-RNA polymerase antibodies) or by measuring the number of polymerases that are effectively engaged in transcription in that position. This latter strategy is followed using transcription run-on (TRO), also known as nuclear run-on (NRO), which was first developed in mammalian cells over 40 years ago and has since been adapted to many other different organisms and high-throughput methods. Here, we detail the procedure for performing TRO in Saccharomyces cerevisiae for single genomic regions to study active transcription on a single gene scale. To do so, we wash the cells in the detergent sarkosyl, which prevents new initiations at the promoter level, and then perform an in situ reaction, leading to the radiolabeling of transcripts by RNA polymerases that were already engaged in transcription at the moment of harvesting. By subsequently quantitating the signal of these transcripts, we can determine the level of active transcription in a single gene. This presents a major advantage over other forms of transcription quantitation such as RNA polymerase ChIP, since in the latter, both active and inactive polymerases are measured. By combining both ChIP and TRO, the amount of inactive or paused polymerases on a particular gene can be estimated. Graphic abstract: [Image: see text] Transcriptional run-on scheme

Bacteriocins are bacterially produced antimicrobial peptides. Although only two peptides have been approved for use as natural preservatives foods, current research is focusing on expanding their application as potential therapeutics against clinical pathogens. Our laboratory group has been working on bacteriocins for over 25 years, and during that time, we have isolated bacteriocin-producing microorganisms from a variety of sources including human skin, human faeces, and various foods. These bacteriocins were purified and characterised, and their potential applications were examined. We have also identified bioengineered derivatives of the prototype lantibiotic nisin which possess more desirable properties than the wild-type, such as enhanced antimicrobial activity. In the current communication, we discuss the main methods that were employed to identify such peptides. Furthermore, we provide a step-by-step guide to carrying out these methods that include accompanying diagrams. We hope that our recommendations and advice will be of use to others in their search for, and subsequent analysis of, novel bacteriocins, and derivatives thereof.

A collection of Listeria monocytogenes isolates from various food products, food processing environments and clinical sources (n = 153) were evaluated for their tolerance to acetic, lactic and propionic acids. A large variation in tolerance was observed amongst isolates under mildly acidic conditions (pH 5.3) for acetic (5-20 mM undissociated acid) and propionic acid (2-10 mM undissociated acid) but there was less variation for lactic acid (3-6 mM undissociated acid). Analysis of isolate genome sequences revealed thiT, gadT2, gadD2 and gadD3 genes were linked to higher acetic acid tolerance ( P

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5ʹ exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3ʹ was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation., This work has been supported by grants from the Ministerio de Economía, Industria y Competitividad – Agencia Estatal de Investigación, and European Union funds (FEDER) [BFU2016-77728-C3-1-P to S. C.],[BFU2016-77728-C3-3-P to J.E.P-O & P.A], [BFU2016-77728-C3-2-P to F.N.] and RED2018‐102467‐T to J.E.P-O, F.N. and S.C.; by FPI grants from the Spanish Government to V.B and A.C-B; by the Regional Andalusian Government [BIO271 and US-1256285 to S. C.], [BIO258 and FEDER-UJA 1260360 to F.N.] and from the Regional Valencian Government [AICO/2019/088 to J.E.P-O]. Funding for open access charge: [BFU2016-77728-C3-1-P].

Nature chemical biology xx, 1-20 (2020). doi:10.1038/s41589-020-0628-4, The natural antivitamin 2′-methoxy-thiamine (MTh) is implicated in the suppression of microbial growth. However, its mode of action and enzyme-selective inhibition mechanism have remained elusive. Intriguingly, MTh inhibits some thiamine diphosphate (ThDP) enzymes, while being coenzymatically active in others. Here we report the strong inhibition of Escherichia coli transketolase activity by MTh and unravel its mode of action and the structural basis thereof. The unique 2′-methoxy group of MTh diphosphate (MThDP) clashes with a canonical glutamate required for cofactor activation in ThDP-dependent enzymes. This glutamate is forced into a stable, anticatalytic low-barrier hydrogen bond with a neighboring glutamate, disrupting cofactor activation. Molecular dynamics simulations of transketolases and other ThDP enzymes identify active-site flexibility and the topology of the cofactor-binding locale as key determinants for enzyme-selective inhibition. Human enzymes either retain enzymatic activity with MThDP or preferentially bind authentic ThDP over MThDP, while core bacterial metabolic enzymes are inhibited, demonstrating therapeutic potential., Published by Nature Publishing Group, Basingstoke

N3-methylcytidine (m3C) is present in both eukaryotic tRNA and mRNA and plays critical roles in many biological processes. We report the synthesis of the m3C phosphoramidite building block and its containing RNA oligonucleotides. The base-pairing stability and specificity studies show that the m3C modification significantly disrupts the stability of the Watson-Crick C:G pair. Further m3C decreases the base pairing discrimination between C:G and the other mismatched C:A, C:U, and C:C pairs. Our molecular dynamic simulation study further reveals the detailed structural insights into the m3C:G base pairing pattern in an RNA duplex. More importantly, the biochemical investigation of m3C using reverse transcription shows thatN3-methylation specifies the C:A pair and induces a G to A mutation using HIV-1-RT, MMLV-RT and MutiScribe™-RT enzymes, all with relatively low replication fidelity. For other reverse transcriptases with higher fidelity like AMV-RT, the methylation could completely shut down DNA synthesis.

Staphylococcus epidermidis is frequently implicated in medical device-related infections. As a result of this, novel approaches for control of this opportunistic pathogen are required. We examined the ability of the natural peptide nisin A, produced by Lactococcus lactis, to inhibit S. epidermidis. In addition, a bank of 29 rationally selected bioengineered L. lactis strains were examined with the aim of identifying a nisin derivative with enhanced antimicrobial activity. Agar-based deferred antagonism assays revealed that wild type nisin A inhibited all 18 S. epidermidis strains tested. Larger zones of inhibition than those obtained from the nisin A producing L. lactis strain were observed for each derivative producer against at least one S. epidermidis strain tested. Six derivative producing strains, (VGA, VGT, SGK, M21A, M17Q, AAA), gave larger zones against all 18 strains compared to the wildtype producing strain. The enhanced bioactivity of M17Q was confirmed using well diffusion, minimum inhibitory concentration (MIC) and a broth-based survival assays. Biofilm assays were performed with plastic microtiter plates and medical device substrates (stainless-steel coupons and three catheter materials). The presence of nisin A significantly reduce the amount of biofilm formed on all surfaces. M17Q was significantly better at reducing biofilm production than nisin A on plastic and stainless-steel. Finally, M17Q was significantly better than nisin A at reducing bacterial numbers in a simulated wound fluid. The findings of this study suggest that nisin and bioengineered derivatives warrant further investigation as potential strategies for the control of S. epidermidis.

Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here we describe the effects of queuine on the physiology of the eukaryotic parasite, Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in tRNAAspGUC. Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in OS response, such as heat shock protein 70 (Hsp 70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in tRNAAspGUC, parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.ImportanceEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of the bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces a mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.

RATIONALE Polyamide (PA) is the generic name of polymers synthesized by linking monomers via amide bonds, and various types of PAs with different monomer compositions are known. Distinguishing PA polymers is useful in directing monomer residual testing, product testing, and reverse engineering, but is analytically challenging and cumbersome. To simplify this, we explored the applicability of direct analysis in real time mass spectrometry (DART-MS) for screening PA polymers. METHODS A DART ion source coupled to a quadrupole Orbitrap (high-resolution (HR) mass spectrometer) was employed for this study. Ten types of PA polymers and four retail samples were evaluated. The DART-HRMS data for these samples, as well as the DART-MS/MS (MS2 ) data for PA6 and PA66, were obtained, and their repeatability was assessed across days/calibrations, operators, and equipments. RESULTS Ions corresponding to the cyclic or linear monomers and oligomers of each PA polymer were detected in each DART-HR mass spectrum. Although similar DART-HR mass spectra were obtained for PA6, PA66, and PA6/PA66 (polymer blends of PA6 and PA66), their DART tandem mass spectra were completely different. The analysis was repeatable, and nearly identical DART tandem mass spectra were obtained on different days, by different operators, and with different equipment. This technique was successfully applied to commercially available samples. CONCLUSIONS Ten types of PA polymers were distinguished using DART-HRMS and DART-MS2 , and their identification using these techniques was straightforward as the characteristic ions for each PA polymer were identified and detected. Furthermore, the spectra were obtained rapidly. Therefore, DART-HRMS can be considered an efficient screening technique for the rapid identification and differentiation of PA polymers.

Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable despite the absence of both modifications, growth is however greatly impaired. The major observed consequence is a subsequent increase in protein aggregates and aberrant morphology. Proteomic analysis of the t6A-deficient strain (sua5 mutant) revealed a global mistranslation leading to protein aggregation without regard to physicochemical properties or t6A-dependent or biased codon usage in parent genes. However, loss of sua5 led to increased expression of soluble proteins for mitochondrial function, protein quality processing/trafficking, oxidative stress response, and energy homeostasis. These results point to a global function for t6A in protein homeostasis very similar to mcm5/s2U modifications.

Noradrenergic afferents to corticotropin releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) provide a major excitatory drive to the hypothalamic-pituitary-adrenal (HPA) axis via α1 adrenoreceptor activation. The ascending noradrenergic afferents are recruited preferentially by physiological, rather than psychological, stress modalities.Glucocorticoids secreted in response to HPA activation feed back onto the hypothalamus to negatively regulate the HPA axis, providing a critical autoregulatory constraint that prevents glucocorticoid overexposure. Whether differential negative feedback mechanisms target stress modality-specific HPA activation is not known. Here, we reveal a desensitization of the α1 adrenoreceptor activation of the HPA axis following acute stress that is mediated by rapid glucocorticoid regulation of adrenoreceptor trafficking. Prior stress desensitized the HPA axis to subsequent physiological, but not psychological, stress. Our findings demonstrate rapid glucocorticoid suppression of adrenoreceptor signaling in CRH neurons that is specific to physiological stress activation, and reveal, therefore, a rapid, modality-selective glucocorticoid feedback mechanism.

Extracting globally representative trend information from lower tropospheric ozone observations is extremely difficult due to the highly variable distribution and interannual variability of ozone, and the ongoing shift of ozone precursor emissions from high latitudes to low latitudes. Here we report surface ozone trends at 27 globally distributed remote locations (20 in the Northern Hemisphere, 7 in the Southern Hemisphere), focusing on continuous time series that extend from the present back to at least 1995. While these sites are only representative of less than 25% of the global surface area, this analysis provides a range of regional long-term ozone trends for the evaluation of global chemistry-climate models. Trends are based on monthly mean ozone anomalies, and all sites have at least 20 years of data, which improves the likelihood that a robust trend value is due to changes in ozone precursor emissions and/or forced climate change rather than naturally occurring climate variability. Since 1995, the Northern Hemisphere sites are nearly evenly split between positive and negative ozone trends, while 5 of 7 Southern Hemisphere sites have positive trends. Positive trends are in the range of 0.5–2 ppbv decade–1, with ozone increasing at Mauna Loa by roughly 50% since the late 1950s. Two high elevation Alpine sites, discussed by previous assessments, exhibit decreasing ozone trends in contrast to the positive trend observed by IAGOS commercial aircraft in the European lower free-troposphere. The Alpine sites frequently sample polluted European boundary layer air, especially in summer, and can only be representative of lower free tropospheric ozone if the data are carefully filtered to avoid boundary layer air. The highly variable ozone trends at these 27 surface sites are not necessarily indicative of free tropospheric trends, which have been overwhelmingly positive since the mid-1990s, as shown by recent studies of ozonesonde and aircraft observations. The IAGOS program acknowledges the European Commission for its support of the MOZAIC project (1994–2003) the preparatory phase of IAGOS (2005–2013) and IGAS (2013–2016).