The drug-survival of low-dose thioguanine in patients with inflammatory bowel disease: a retrospective observational study.

Background: Thiopurines are commonly used to treat inflammatory bowel disease but withdrawal due to side effects are common. Thioguanine has been suggested to be better tolerated than conventional thiopurines. Objectives: We studied drug-survival of low dose of thioguanine in real-life clinical practice in comparison to conventional thiopurines. Design: Retrospective observational study. Methods: All patients born 1956 and later, and who at least once started thiopurine treatment between 2006 and 2022 were included. A medical chart review was performed that noted drug-survival for every thiopurine treatment attempt. The Mantel-Cox rank test was used to test differences in drug-survival for different thiopurines. Blood chemistry analysis and faecal calprotectin levels were registered for the first 5 years of treatment. Results: In the study population, there was 379 initiated thiopurine treatments (210 for Crohn's disease and 169 for ulcerative colitis) in 307 patients with inflammatory bowel disease (IBD). Low-dose thioguanine (median dose 11 mg; 25-75th percentile 7-19 mg) had been initiated in 31 patients. Overall, when including all thiopurine attempts, thioguanine had the longest drug-survival [Mantel-Cox rank test: thioguanine versus azathioprine p = 0.014; thioguanine versus 6-mercaptopurine (6-MP) p < 0.001]. For second-line thiopurine treatment thioguanine had longer drug-survival than 6-MP (Mantel-Cox rank test: p = 0.006). At 60 months, 86% of the patients who started low-dose thioguanine were still on treatment compared to 42% of the patients who started 6-MP (p = 0.022). The median 6-thioguanine nucleotide levels in patients treated with thioguanine was 364 pmol/8 × 108. Patients on thioguanine treatment showed significantly lower values of median mean corpuscular volume at follow-up than patients treated with azathioprine and 6-MP. Patients treated with 6-MP showed significantly lower levels of FC in the third year of treatment compared to patient treated with azathioprine (59 versus 109 µg/g; p = 0.023), but there was no significant difference in FC levels for thioguanine compared to azathioprine (50 versus 109 µg/g; p = 0.33). Conclusion: Treatment with a low dose of thioguanine is well-tolerated in patients with IBD and had a significantly higher drug-survival than conventional thiopurines.

Low-dose of the immunomodulator drug thioguanine are well tolerated by patients with inflammatory bowel disease Thiopurines are commonly used to treat inflammatory bowel disease but it is common that patients end treatment due to side-effects. The thiopurine thioguanine has been suggested to be better tolerated than other thiopurines. We aimed to study if a low-dose of thioguanine had been tolerated better and used longer than other thiopurines in patients with inflammatory bowel disease at our clinic. In the study population there was 379 initiated thiopurine treatments in 307 patients with inflammatory bowel disease. Among those patients a low-dose thioguanine had been initiated in 31 patients. Overall, when including all thiopurine attempts, thioguanine had longest drug-survival of all thiopurines. For second line thiopurine treatment thioguanine had longer drug-survival than the thiopurine 6-mercaptopurine that are usually used as second line thiopurine treatment. At 60 months, 86% of the patients who started low dose thioguanine was still on treatment compared to 42% of the patients who started 6-mercaptopurine.There was a similar response on inflammatory markers the first five years from starting treatment with thioguanines compared to conventional used thiopurines. We conclude that treatment with a low-dose of thioguanine is well tolerated in patients with inflammatory bowel disease and have a significantly higher drug survival than conventional thiopurines.

Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters.

Artificial dye-coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)-dependent XoxF-MDHs are able to incorporate different lanthanides (Lns) in their active site. Dye-coupled assays showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Despite widespread use, there are limitations: oftentimes a pH of 9 and activators are required for the assay. Moreover, Ln-MDH variants are not obtained by isolation from the cells grown with the respective Ln, but by incubation of an apo-MDH with the Ln. Herein, we report the cultivation of Ln-dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDHs and the assessment of the enzyme activity using the dye-coupled assay. We compare these results with a protein-coupled assay using its physiological electron acceptor cytochrome cGJ (cyt cGJ ). Depending on the assay, two distinct trends are observed among the Ln series. The specific enzyme activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds that measured by the dye-coupled assay. This suggests that early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt cGJ thereby increasing functional efficiency.

Learning from nature: recovery of rare earth elements by the extremophilic bacterium Methylacidiphilum fumariolicum.

We present the extremophilic bacterium Methylacidiphilum fumariolicum SolV as a platform for the recovery of rare earth elements (REE). Strain SolV is able to selectively extract the light REE from artificial industrial waste sources, natural REE-containing and post-mining waters. Upscaling, different media composition and accumulation over several cycles were successfully implemented, underlining the potential for bio-recovery of REE.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life.

Certain f-block elements-the lanthanides-have biological relevance in the context of methylotrophic bacteria. The respective strains incorporate these 4 f elements into the active site of one of their key metabolic enzymes, a lanthanide-dependent methanol dehydrogenase. In this study, we investigated whether actinides, the radioactive 5 f elements, can replace the essential 4 f elements in lanthanide-dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides when presented with a mixture of equal amounts of lanthanides together with americium and curium. Our combined in vivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one-carbon metabolism if they possess the correct size and a +III oxidation state.

Studies of pyrroloquinoline quinone species in solution and in lanthanide-dependent methanol dehydrogenases.

Pyrroloquinoline quinone (PQQ) is a redox cofactor in calcium- and lanthanide-dependent alcohol dehydrogenases that has been known and studied for over 40 years. Despite its long history, many questions regarding its fluorescence properties, speciation in solution and in the active site of alcohol dehydrogenase remain open. Here we investigate the effects of pH and temperature on the distribution of different PQQ species (H3PQQ to PQQ3- in addition to water adducts and in complex with lanthanides) with NMR and UV-Vis spectroscopy as well as time-resolved laser-induced fluorescence spectroscopy (TRLFS). Using a europium derivative from a new, recently-discovered class of lanthanide-dependent methanol dehydrogenase (MDH) enzymes, we utilized two techniques to monitor Ln binding to the active sites of these enzymes. Employing TRLFS, we were able to follow Eu(III) binding directly to the active site of MDH using its luminescence and could quantify three Eu(III) states: Eu(III) in the active site of MDH, but also in solution as PQQ-bound Eu(III) and in the aquo-ion form. Additionally, we used the antenna effect to study PQQ and simultaneously Eu(III) in the active site.

Neodymium as Metal Cofactor for Biological Methanol Oxidation: Structure and Kinetics of an XoxF1-Type Methanol Dehydrogenase.

The methane-oxidizing bacterium Methylacidimicrobium thermophilum AP8 thrives in acidic geothermal ecosystems that are characterized by high degassing of methane (CH4), H2, H2S, and by relatively high lanthanide concentrations. Lanthanides (atomic numbers 57 to 71) are essential in a variety of high-tech devices, including mobile phones. Remarkably, the same elements are actively taken up by methanotrophs/methylotrophs in a range of environments, since their XoxF-type methanol dehydrogenases require lanthanides as a metal cofactor. Lanthanide-dependent enzymes seem to prefer the lighter lanthanides (lanthanum, cerium, praseodymium, and neodymium), as slower methanotrophic/methylotrophic growth is observed in medium supplemented with only heavier lanthanides. Here, we purified XoxF1 from the thermoacidophilic methanotroph Methylacidimicrobium thermophilum AP8, which was grown in medium supplemented with neodymium as the sole lanthanide. The neodymium occupancy of the enzyme is 94.5% ± 2.0%, and through X-ray crystallography, we reveal that the structure of the active site shows interesting differences from the active sites of other methanol dehydrogenases, such as an additional aspartate residue in close proximity to the lanthanide. Nd-XoxF1 oxidizes methanol at a maximum rate of metabolism (Vmax) of 0.15 ± 0.01 µmol · min-1 · mg protein-1 and an affinity constant (Km) of 1.4 ± 0.6 µM. The structural analysis of this neodymium-containing XoxF1-type methanol dehydrogenase will expand our knowledge in the exciting new field of lanthanide biochemistry. IMPORTANCE Lanthanides comprise a group of 15 elements with atomic numbers 57 to 71 that are essential in a variety of high-tech devices, such as mobile phones, but were considered biologically inert for a long time. The biological relevance of lanthanides became evident when the acidophilic methanotroph Methylacidiphilum fumariolicum SolV, isolated from a volcanic mud pot, could only grow when lanthanides were supplied to the growth medium. We expanded knowledge in the exciting and rapidly developing field of lanthanide biochemistry by the purification and characterization of a neodymium-containing methanol dehydrogenase from a thermoacidophilic methanotroph.

Activity assays of methanol dehydrogenases.

The field of methanol dehydrogenases (MDHs) has experienced revival in the recent decade due to the observation of lanthanide-dependent MDH, in addition to widely known calcium-MDH. With the advent of lanthanide-dependent alcohol dehydrogenases, the need for reliable assays to evaluate and compare activities between different MDHs is obvious: from extremophilic to neutrophilic organisms, or with different lanthanide ions in the active site. Here we outline four assays that have been reported for Ln-MDH, discussing the advantages and disadvantages of the assays and their components. It should be noted, in 1990Day and Anthony produced a comprehensive summary in Methods in Enzymology on the available methods for Ca-MDH assays at the time (Day & Anthony, 1990). This chapter is an updated appraisal of the most important developments in the last 30years.

Americium preferred: lanmodulin, a natural lanthanide-binding protein favors an actinide over lanthanides.

The separation and recycling of lanthanides is an active area of research with a growing demand that calls for more environmentally friendly lanthanide sources. Likewise, the efficient and industrial separation of lanthanides from the minor actinides (Np, Am-Fm) is one of the key questions for closing the nuclear fuel cycle; reducing costs and increasing safety. With the advent of the field of lanthanide-dependent bacterial metabolism, bio-inspired applications are in reach. Here, we utilize the natural lanthanide chelator lanmodulin and the luminescent probes Eu3+ and Cm3+ to investigate the inter-metal competition behavior of all lanthanides (except Pm) and the major actinide plutonium as well as three minor actinides neptunium, americium and curium to lanmodulin. Using time-resolved laser-induced fluorescence spectroscopy we show that lanmodulin has the highest relative binding affinity to Nd3+ and Eu3+ among the lanthanide series. When equimolar mixtures of Cm3+ and Am3+ are added to lanmodulin, lanmodulin preferentially binds to Am3+ over Cm3+ whilst Nd3+ and Cm3+ bind with similar relative affinity. The results presented show that a natural lanthanide-binding protein can bind a major and various minor actinides with high relative affinity, paving the way to bio-inspired separation applications. In addition, an easy and versatile method was developed, using the fluorescence properties of only two elements, Eu and Cm, for inter-metal competition studies regarding lanthanides and selected actinides and their binding to biological molecules.

Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster's Blue in methanol dehydrogenase assays.

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

Sulfur mustard alkylates steroid hormones and impacts hormone function in vitro.

The chemical warfare agent sulfur mustard (SM) alkylates a multitude of biomacromolecules including DNA and proteins. Cysteine residues and nucleophilic nitrogen atoms in purine DNA bases are typical targets of SM but potentially every nucleophilic structure may be alkylated by SM. In the present study, we analyzed potential SM-induced alkylation of glucocorticoid (GC) hormones and functional consequences thereof. Hydrocortisone (HC), the synthetic betamethasone (BM) and dexamethasone (DEX) were chosen as representative GCs. Structural modifications were assessed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The hypothesized alkylation was verified and structurally allocated to the OH-group of the C21 atom. The biological function of SM-alkylated GCs was investigated using GC-regulated dual-luciferase reporter gene assays and an ex vivo GC responsiveness assay coupled with real-time quantitative polymerase chain reaction (RT-qPCR). For the reporter gene assays, HEK293-cells were transiently transfected with a dual-luciferase reporter gene that is transcriptional regulated by a GC-response element. These cells were then incubated either with untreated or SM-derivatized HC, BM or DEX. Firefly-luciferase (Fluc) activity was determined 24 h after stimulation. Fluc-activity significantly decreased after stimulation with SM-pre-exposed GC dependent on the SM concentration. The ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to DEX revealed a transcriptional dysregulation of GC-regulated genes (FKBP5, IL1R2, and GILZ) after stimulation with SM-alkylated DEX. Our results present GCs as new biological targets of SM associated with a disturbance of hormone function.