DNA surface exploration and operator bypassing during target search.

Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI hops one or two grooves (10-20 bp) every 200-700 µs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein-DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT-FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.

A frame-shift mutation in COMTD1 is associated with impaired pheomelanin pigmentation in chicken.

The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5th exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin.

Rapid and Bifunctional Chemoselective Metabolome Analysis of Liver Disease Plasma Using the Reagent 4-Nitrophenyl-2H-azirine.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts that has been associated with diverse metabolic carboxylic acids. Mass spectrometric techniques are the method of choice for their analysis. However, the broad investigation of this metabolite class remains challenging. Derivatization of carboxylic acids represents a strategy to overcome these limitations but available methods suffer from diverse analytical challenges. Herein, we have designed a novel strategy introducing 4-nitrophenyl-2H-azirine as a new chemoselective moiety for the first time for carboxylic acid metabolites. This moiety was selected as it rapidly forms a stable amide bond and also generates a new ketone, which can be analyzed by our recently developed quant-SCHEMA method specific for carbonyl metabolites. Optimization of this new method revealed a high reproducibility and robustness, which was utilized to validate 102 metabolic carboxylic acids using authentic synthetic standard conjugates in human plasma samples including nine metabolites that were newly detected. Using this sequential analysis of the carbonyl- and carboxylic acid-metabolomes revealed alterations of the ketogenesis pathway, which demonstrates the vast benefit of our unique methodology. We anticipate that the developed azirine moiety with rapid functional group transformation will find broad application in diverse chemical biology research fields.

Immobilized Enzymes on Magnetic Beads for Separate Mass Spectrometric Investigation of Human Phase II Metabolite Classes.

The human body has evolved to remove xenobiotics through a multistep clearance process. Non-endogenous metabolites are converted through a series of phase I and different phase II enzymes into compounds with higher hydrophilicity. These compounds are important for diverse research fields such as toxicology, nutrition, biomarker discovery, doping control, and microbiome metabolism. One of the challenges in these research fields has been the investigation of the two major phase II modifications, sulfation and glucuronidation, and the corresponding unconjugated aglycon independently. We have now developed a new methodology utilizing an immobilized arylsulfatase and an immobilized ß-glucuronidase to magnetic beads for treatment of human urine samples. The enzyme activities remained the same compared to the enzyme in solution. The separate mass spectrometric investigation of each metabolite class in a single sample was successfully applied to obtain the dietary glucuronidation and sulfation profile of 116 compounds. Our new chemical biology strategy provides a new tool for the investigation of metabolites in biological samples with the potential for broad-scale application in metabolomics, nutrition, and microbiome studies.

Chemoselective and Highly Sensitive Quantification of Gut Microbiome and Human Metabolites.

The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.

Amino Acid Modified RNA Bases as Building Blocks of an Early Earth RNA-Peptide World.

Fossils of extinct species allow us to reconstruct the process of Darwinian evolution that led to the species diversity we see on Earth today. The origin of the first functional molecules able to undergo molecular evolution and thus eventually able to create life, are largely unknown. The most prominent idea in the field posits that biology was preceded by an era of molecular evolution, in which RNA molecules encoded information and catalysed their own replication. This RNA world concept stands against other hypotheses, that argue for example that life may have begun with catalytic peptides and primitive metabolic cycles. The question whether RNA or peptides were first is addressed by the RNA-peptide world concept, which postulates a parallel existence of both molecular species. A plausible experimental model of how such an RNA-peptide world may have looked like, however, is absent. Here we report the synthesis and physicochemical evaluation of amino acid containing adenosine bases, which are closely related to molecules that are found today in the anticodon stem-loop of tRNAs from all three kingdoms of life. We show that these adenosines lose their base pairing properties, which allow them to equip RNA with amino acids independent of the sequence context. As such we may consider them to be living molecular fossils of an extinct molecular RNA-peptide world.

Sensitive mass spectrometric analysis of carbonyl metabolites in human urine and fecal samples using chemoselective modification.

Metabolites with ketone or aldehyde functionalities comprise a large proportion of the human metabolome, most notably in the form of sugars. However, these reactive molecules are also generated through oxidative stress or gut microbiota metabolism and have been linked to disease development. The discovery and structural validation of this class of metabolites over the large concentration range found in human samples is crucial to identify their links to pathogenesis. Herein, we have utilized an advanced chemoselective probe methodology alongside bioinformatic analysis to identify carbonyl-metabolites in urine and fecal samples. In total, 99 metabolites were identified in urine samples and the chemical structure for 40 metabolites were unambiguously validated using a co-injection procedure. We also describe the preparation of a metabolite-conjugate library of 94 compounds utilized to efficiently validate these ketones and aldehydes. This method was used to validate 33 metabolites in a pooled fecal sample extract to demonstrate the potential for rapid and efficient metabolite detection over a wide metabolite concentration range. This analysis revealed the presence of six metabolites that have not previously been detected in either sample type. The constructed library can be utilized for straightforward, large-scale, and expeditious analysis of carbonyls in any sample type.

Unexpected Acetylation of Endogenous Aliphatic Amines by Arylamine N-Acetyltransferase NAT2.

N-Acetyltransferases play critical roles in the deactivation and clearance of xenobiotics, including clinical drugs. NAT2 has been classified as an arylamine N-acetyltransferase that mainly converts aromatic amines, hydroxylamines, and hydrazines. Herein, we demonstrate that the human arylamine N-acetyltransferase NAT2 also acetylates aliphatic endogenous amines. Metabolomic analysis and chemical synthesis revealed increased intracellular concentrations of mono- and diacetylated spermidine in human cell lines expressing the rapid compared to the slow acetylator NAT2 phenotype. The regioselective N8 -acetylation of monoacetylated spermidine by NAT2 answers the long-standing question of the source of diacetylspermidine. We also identified selective acetylation of structurally diverse alkylamine-containing drugs by NAT2, which may contribute to variations in patient responses. The results demonstrate a previously unknown functionality and potential regulatory role for NAT2, and we suggest that this enzyme should be considered for re-classification.

Coupled Enzymatic Treatment and Mass Spectrometric Analysis for Identification of Glucuronidated Metabolites in Human Samples.

Glucuronidation is the most common phase II modification and plays an important role in human clearance metabolism. Glucuronidated metabolites have also been linked to disease development and microbiota-host co-metabolism. Although many of these compounds have been identified, the total number of unknown glucuronides and their impact on the human host's physiology can only be estimated. Herein, we describe the combination of an untargeted metabolomics analysis and enzymatic metabolic conversion for the selective detection of glucuronide conjugates by using UPLC-MS/MS in human urine samples. Our study demonstrates that this powerful strategy can be used for the selective identification of glucuronidated molecules and to discover unknown natural metabolites. In total, we identified 191 metabolites in a single sample including microbiota-derived compounds as well as previously unidentified molecules.

Comprehensive kinetic and substrate specificity analysis of an arylsulfatase from Helix pomatia using mass spectrometry.

Sulfatases hydrolyze sulfated metabolites to their corresponding alcohols and are present in all domains of life. These enzymes have found major application in metabolic investigation of drugs, doping control analysis and recently in metabolomics. Interest in sulfatases has increased due to a link between metabolic processes involving sulfated metabolites and pathophysiological conditions in humans. Herein, we present the first comprehensive substrate specificity and kinetic analysis of the most commonly used arylsulfatase extracted from the snail Helix pomatia. In the past, this enzyme has been used in the form of a crude mixture of enzymes, however, recently we have purified this sulfatase for a new application in metabolomics-driven discovery of sulfated metabolites. To evaluate the substrate specificity of this promiscuous sulfatase, we have synthesized a series of new sulfated metabolites of diverse structure and employed a mass spectrometric assay for kinetic substrate hydrolysis evaluation. Our analysis of the purified enzyme revealed that the sulfatase has a strong preference for metabolites with a bi- or tricyclic aromatic scaffold and to a lesser extent for monocyclic aromatic phenols. This metabolite library and mass spectrometric method can be applied for the characterization of other sulfatases from humans and gut microbiota to investigate their involvement in disease development.

Structural elucidation of major selective androgen receptor modulator (SARM) metabolites for doping control.

Selective androgen receptor modulators (SARMs) are a class of androgen receptor drugs, which have a high potential to be performance enhancers in human and animal sports. Arylpropionamides are one of the major SARM classes and get rapidly metabolized significantly complicating simple detection of misconduct in blood or urine sample analysis. Specific drug-derived metabolites are required as references due to a short half-life of the parent compound but are generally lacking. The difficulty in metabolism studies is the determination of the correct regio and stereoselectivity during metabolic conversion processes. In this study, we have elucidated and verified the chemical structure of two major equine arylpropionamide-based SARM metabolites using a combination of chemical synthesis and liquid chromatography-mass spectrometry (LC-MS) analysis. These synthesized SARM-derived metabolites can readily be utilized as reference standards for routine mass spectrometry-based doping control analysis of at least three commonly used performance-enhancing drugs to unambigously identify misconduct.

Chemoselective Probe Containing a Unique Bioorthogonal Cleavage Site for Investigation of Gut Microbiota Metabolism.

While metabolites derived from gut microbiota metabolism have been linked to disease development in the human host, the chemical tools required for their detailed analysis and the discovery of biomarkers are limited. A unique and multifunctional chemical probe for mass spectrometric analysis, which contains p-nitrocinnamyloxycarbonyl as a new bioorthogonal cleavage site has been designed and synthesized. Coupled to magnetic beads, this chemical probe allows for straightforward extraction of metabolites from human samples and release under mild conditions. This isolation from the sample matrix results in significantly reduced ion suppression, an increased mass spectrometric sensitivity, and facilitates the detection of metabolites in femtomole quantities. The chemoselective probe was applied to the analysis of human fecal samples, resulting in the discovery of four metabolites previously unreported in this sample type and confirmation of the presence of medically relevant gut microbiota-derived metabolites.

Validation of onchocerciasis biomarker N-acetyltyramine-O-glucuronide (NATOG).

The Neglected Tropical Disease onchocerciasis is a parasitic disease. Despite many control programmes by the World Health Organization (WHO), large communities in West and Central Africa are still affected. Besides logistic challenges during biannual mass drug administration, the lack of a robust, point-of-care diagnostic is limiting successful eradication of onchocerciasis. Towards the implementation of a non-invasive and point-of-care diagnostic, we have recently reported the discovery of the biomarker N-acetyltyramine-O-glucuronide (NATOG) in human urine samples using a metabolomics-mining approach. NATOG's biomarker value was enhanced during an investigation in a rodent model. Herein, we further detail the specificity of NATOG in active onchocerciasis infections as well as the co-infecting parasites Loa loa and Mansonella perstans. Our results measured by liquid chromatography coupled with mass spectrometry (LC-MS) reveal elevated NATOG values in mono- and co-infection samples only in the presence of the nematode Onchocerca volvulus. Metabolic pathway investigation of l-tyrosine/tyramine in all investigated nematodes uncovered an important link between the endosymbiotic bacterium Wolbachia and O. volvulus for the biosynthesis of NATOG. Based on these extended studies, we suggest NATOG as a biomarker for tracking active onchocerciasis infections and provide a threshold concentration value of NATOG for future diagnostic tool development.

Onchocerca volvulus-neurotransmitter tyramine is a biomarker for river blindness.

Onchocerciasis, also known as "river blindness", is a neglected tropical disease infecting millions of people mainly in Africa and the Middle East but also in South America and Central America. Disease infectivity initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious third-stage larvae. Ivermectin has controlled transmission of microfilariae, with an African Program elimination target date of 2025. However, there is currently no point-of-care diagnostic that can distinguish the burden of infection--including active and/or past infection--and enable the elimination program to be effectively monitored. Here, we describe how liquid chromatography-MS-based urine metabolome analysis can be exploited for the identification of a unique biomarker, N-acetyltyramine-O,ß-glucuronide (NATOG), a neurotransmitter-derived secretion metabolite from O. volvulus. The regulation of this tyramine neurotransmitter was found to be linked to patient disease infection, including the controversial antibiotic doxycycline treatment that has been shown to both sterilize and kill adult female worms. Further clues to its regulation have been elucidated through biosynthetic pathway determination within the nematode and its human host. Our results demonstrate that NATOG tracks O. volvulus metabolism in both worms and humans, and thus can be considered a host-specific biomarker for onchocerciasis progression. Liquid chromatography-MS-based urine metabolome analysis discovery of NATOG not only has broad implications for a noninvasive host-specific onchocerciasis diagnostic but provides a basis for the metabolome mining of other neglected tropical diseases for the discovery of distinct biomarkers and monitoring of disease progression.

Glycation Reactivity of a Quorum-Sensing Signaling Molecule.

Reported herein is that (4S)-4,5-dihydroxy-2,3-pentanedione (DPD) can undergo a previously undocumented non-enzymatic glycation reaction. Incubation of DPD with viral DNA or the antibiotic gramicidin S resulted in significant biochemical alterations. A protein-labeling method was consequently developed that facilitated the identification of unrecognized glycation targets of DPD in a prokaryotic system. These results open new avenues toward tracking and understanding the fate and function of the elusive quorum-sensing signaling molecule.

Flagellin as carrier and adjuvant in cocaine vaccine development.

Cocaine abuse is problematic, directly and indirectly impacting the lives of millions, and yet existing therapies are inadequate and usually ineffective. A cocaine vaccine would be a promising alternative therapeutic option, but efficacy is hampered by variable production of anticocaine antibodies. Thus, new tactics and strategies for boosting cocaine vaccine immunogenicity must be explored. Flagellin is a bacterial protein that stimulates the innate immune response via binding to extracellular Toll-like receptor 5 (TLR5) and also via interaction with intracellular NOD-like receptor C4 (NLRC4), leading to production of pro-inflammatory cytokines. Reasoning that flagellin could serve as both carrier and adjuvant, we modified recombinant flagellin protein to display a cocaine hapten termed GNE. The resulting conjugates exhibited dose-dependent stimulation of anti-GNE antibody production. Moreover, when adjuvanted with alum, but not with liposomal MPLA, GNE-FliC was found to be better than our benchmark GNE-KLH. This work represents a new avenue for exploration in the use of hapten-flagellin conjugates to elicit antihapten immune responses.

Litomosoides sigmodontis: a jird urine metabolome study.

The neglected tropical disease onchocerciasis affects more than 35 million people worldwide with over 95% in Africa. Disease infection initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious L3 larvae. New treatments and diagnostics are required to eradicate this parasitic disease. Herein, we describe that a previously discovered biomarker for onchocerciasis, N-acetyltyramine-O-glucuronide (NATOG) is also present in urine samples of jirds infected with the onchocerciasis model nematode Litomosoides sigmodontis. Increased NATOG values paralleled a progressing infection and demonstrated that quantification of NATOG in this rodent model can be utilized to track its infectivity. Moreover, our findings suggest how NATOG monitoring may be used for evaluating potential drug candidates.

The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA.

The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.

Identification of New Ketamine Metabolites and Their Detailed Distribution in the Mammalian Brain.

Ketamine is a common anesthetic used in human and veterinary medicine. This drug has recently received increased medical and scientific attention due to its indications for neurological diseases. Despite being applied for decades, ketamine's entire metabolism and pharmacological profile have not been elucidated yet. Therefore, insights into the metabolism and brain distribution are important toward identification of neurological effects. Herein, we have investigated ketamine and its metabolites in the pig brain, cerebrospinal fluid, and plasma using mass spectrometric and metabolomics analysis. We discovered previously unknown metabolites and validated their chemical structures. Our comprehensive analysis of the brain distribution of ketamine and 30 metabolites describes significant regional differences detected mainly for phase II metabolites. Elevated levels of these metabolites were identified in brain regions linked to clearance through the cerebrospinal fluid. This study provides the foundation for multidisciplinary studies of ketamine metabolism and the elucidation of neurological effects by ketamine.

Characterising and preventing the gut microbiota's inactivation of trifluridine, a colorectal cancer drug.

The gut microbiome can metabolise hundreds of drugs, potentially affecting their bioavailability and pharmacological effect. As most gut bacteria reside in the colon, drugs that reach the colon in significant proportions may be most impacted by microbiome metabolism. In this study the anti-colorectal cancer drug trifluridine was used as a model drug for characterising metabolism by the colonic microbiota, identifying correlations between bacterial species and individuals' rates of microbiome drug inactivation, and developing strategies to prevent drug inactivation following targeted colonic delivery. High performance liquid chromatography and ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry demonstrated trifluridine's variable and multi-route metabolism by the faecal microbiota sourced from six healthy humans. Here, four drug metabolites were linked to the microbiome for the first time. Metagenomic sequencing of the human microbiota samples revealed their composition, which facilitated prediction of individual donors' microbial trifluridine inactivation. Notably, the abundance of Clostridium perfringens strongly correlated with the extent of trifluridine inactivation by microbiota samples after 2 hours (R2 = 0.8966). Finally, several strategies were trialled for the prevention of microbial trifluridine metabolism. It was shown that uridine, a safe and well-tolerated molecule, significantly reduced the microbiota's metabolism of trifluridine by acting as a competitive enzyme inhibitor. Further, uridine was found to provide prebiotic effects. The findings in this study greatly expand knowledge on trifluridine's interactions with the gut microbiome and provide valuable insights for investigating the microbiome metabolism of other drugs. The results demonstrate how protection strategies could enhance the colonic stability of microbiome-sensitive drugs.