Studies on the biosynthesis and heterologous expression of complex secondary metabolites from streptomycetes

The heterologous expression of natural product biosynthetic pathways is of increasing interest in biotechnology and drug discovery. This approach enables the production of complex metabolites in more amenable host organisms and provides the basis for the generation of novel analogs through genetic engineering. In this thesis, we describe a straightforward strategy for the heterologous expression of the highly complex phenalinolactone biosynthetic pathway, which was recently cloned from Streptomyces sp. Tü6071. Using Red/ET recombineering, the phenalinolactone pathway was reconstituted from two cosmids and heterologously expressed in several Streptomyces strains. The established expression system now provides a convenient platform for functional investigations of the biosynthetic genes and the generation of novel analogs, by genetic engineering of the pathway in Escherichia coli. The second part of this thesis describes work on a distinct class of secondary metabolites, the GE81112 tetrapeptide family. We developed a strategy for the cloning and identification of the GE81112 biosynthetic gene cluster, in order to investigate the biosynthetic pathway in detail. Generation of a cosmid library enabled us to identify the corresponding biosynthetic gene cluster on two overlapping cosmids. In parallel, we established methods to manipulate the strain genetically, allowing us to verify the identity of the GE81112 gene cluster by gene inactivation experiments. In addition, we characterized several proteins from the pathway using enzymatic assays in vitro. Taken together, these data have enabled us to propose a preliminary model for GE81112 biosynthesis. The results also open the door to developing new derivatives of these promising compounds by genetic engineering.Die heterologe Expression komplexer Naturstoff-Biosynthesewege spielt eine immer größer werdende Rolle bei der Entdeckung und Modifikation von Wirkstoffen aus der Natur und gewinnt somit zunehmend an Bedeutung in der biotechnologischen Forschung. Diese Methode ermöglicht die Produktion komplexer Metabolite in leicht handhabbaren Wirtsorganismen und bildet so die Grundlage für die Entwicklung neuer Naturstoffderivate durch genetische Manipulation der Biosynthesewege. Die vorliegende Arbeit beschreibt eine innovative Strategie für die heterologe Expression des komplexen Phenalinolacton- Biosynthesewegs aus dem Streptomyceten Tü6071. Mit Hilfe der Red/ET Rekombination wurde dieses Gencluster, das auf 2 Cosmiden vorlag, kloniert und in verschiedenen Streptomyceten-Stämmen heterolog exprimiert. Das somit etablierte Expressionssystem bietet nun die Möglichkeit, durch gezielte genetische Manipulation der Biosynthesewege in Escherichia coli und anschließende heterologe Expression neue, möglicherweise wirksamere Naturstoffe und Naturstoffderivate zu entwickeln. Der zweite Teil dieser Arbeit beschäftigt sich mit Untersuchungen zur Biosynthese einer ungewöhnlichen Klasse von Sekundärstoffen: den GE81112 Tetrapeptiden. Um die zugrundeliegende Biosynthese dieser Naturstoffe nun detailliert zu untersuchen, wurde eine Strategie für die Klonierung und Identifizierung des entsprechenden Genclusters entwickelt, das schließlich auf zwei überlappenden Cosmiden identifiziert wurde. Gleichzeitig konnten Methoden zur genetischen Manipulation des Stammes entwickelt werden, die es ermöglichten das Gencluster durch Geninaktivierungsexperimente zu identifizieren. Mit Hilfe der erhaltenen Ergebnisse konnte schließlich ein erstes Model für die GE81112 Biosynthese erarbeitet werden. Die in dieser Arbeit erhaltenen Einblicke in die Biosynthese können in Zukunft einen wichtigen Beitrag zur Entwicklung neuer GE81112 Derivate leisten

Advances in testing for sample manipulation in clinical and forensic toxicology—part B: hair samples

As a continuation of part A, focusing on advances in testing for sample manipulation of urine samples in clinical and forensic toxicology, part B of the review article relates to hair, another commonly used matrix for abstinence control testing. Similar to urine manipulation, relevant strategies to manipulate a hair test are lowering drug concentrations in hair to undercut the limits of detection/cut-offs, for instance, by forced washout effects or adulteration. However, distinguishing between usual, common cosmetic hair treatment and deliberate manipulation to circumvent a positive drug test is often impossible. Nevertheless, the identification of cosmetic hair treatment is very relevant in the context of hair testing and interpretation of hair analysis results. Newly evaluated techniques or elucidation of specific biomarkers to unravel adulteration or cosmetic treatment often focused on specific structures of the hair matrix with promising strategies recently proposed for daily routine work. Identification of other approaches, e.g., forced hair-washing procedures, still remains a challenge in clinical and forensic toxicology

Single sample preparation for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids in hair

A combined sample preparation for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids was developed based on three independent fully validated analytical methods. Recently, we published a multi-analyte method for the simultaneous analysis of 116 drugs and pharmaceuticals including different substance groups like opioids, stimulants, benzodiazepines, z-drugs, antidepressants and neuroleptics based on a single sample workup followed by a single analytical measurement with LC-MS/MS. However, in some cases, additional analysis of further substance groups, such as cannabinoids and endogenous steroids, is required, which are analyzed in our laboratory using separate sample preparation and separate analytical methods. The goal of this study was to use the knowledge from the different sample preparations and combine them into a single sample preparation and extraction workflow for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids, and endogenous steroids to be analyzed with the appropriate analytical methods. A partial validation of selected parameters such as selectivity, linearity, limit of quantification (LOQ), accuracy, precision and robustness for the different analytical methods was carried out and revalidated. In addition, comparative measurements of quality controls and authentic pools were performed and statistically evaluated using the unpaired t-test or the non-parametric Mann-Whitney test. The results using the newly established sample preparation and extraction were in good agreement with the original data. In conclusion, the newly established sample preparation is suitable for the combined extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids, and gives reliable results for quantification of various substances

Analytical description of adolescent binge drinking patients

Background Binge drinking is a widespread health compromising behavior among adolescents and young adults, leading to significant health problems, injuries and mortality. However, data on alcohol consumption is often unreliable, as it is mainly based on self-reporting surveys. In this five-year study (2014–2019) at the University Children’s Hospital Zurich, we analyzed blood samples from adolescent binge drinking patients to investigate blood alcohol concentrations (BACs), co-ingestion of drugs, assess compliance between self-reported and measured substance use, and test for genetic components of innate alcohol tolerance. Furthermore, hair analysis was performed to retrospectively access drug exposure and to evaluate the potential of hair analysis to assess binge drinking. Methods In a prospective, single-center study, patients with alcohol intoxications aged 16 years and younger were included. Blood and hair samples were analyzed by sensitive liquid chromatography – tandem mass spectrometry drug analysis. HTTLPR genotyping was performed with PCR and fragment analysis. Results Among 72 cases, 72 blood and 13 hair samples were analyzed. BACs ranged from 0.08–3.20‰ (mean 1.63‰, median 1.60‰), while a mean concentration of 3.64 pg/mg hair (median 3.0 pg/mg) of the alcohol marker ethyl glucuronide (EtG) was detected in eleven hair samples, providing no evidence of chronic excessive drinking. In 47% of the cases, co-ingested drugs were qualitatively detected next to ethanol, but only 9% of the detected drugs had blood concentrations classified as pharmacologically active. Cannabis consumption (22%) and stimulant intake (16%) were the most frequently observed drugs. Compliance between patients’ statements and measured substances matched well. Although we investigated the genetic contribution to innate alcohol tolerance via the 5-HTTLPR polymorphism, the diverse genetic background of the cohort and small sample size did not allow any conclusions to be drawn. Conclusion Almost half of our binge drinking patients tested positive for other substances, primarily cannabis. We anticipate that our study enhances understanding of consumption behavior of young people and encourage continued efforts to address the harmful effects of binge drinking and co-occurring substance use

Stereoselective Syntheses of Deuterated Pipecolic Acids as Tools to Investigate the Stereoselectivity of the Hydroxylase GetF

Members of the GE81112 family are interesting candidates for the development of antibiotics. The configuration of the OH group on the pipecolic acid moiety plays a pivotal role in antibiotic activity. To investigate the stereoselectivity of the corresponding hydroxylase GetF, involved in the biosynthetic pathway, we synthesized the two deuterium-labeled pipecolic acid diastereomers in a highly stereoselective fashion via chelate-enolate Claisen rearrangement. The stereochemical outcome of the enzymatic hydroxylation step could easily be determined by analysis of mass differences between the products

How Do Road Traffic Noise and Residential Greenness Correlate with Noise Annoyance and Long-Term Stress? Protocol and Pilot Study for a Large Field Survey with a Cross-Sectional Design

Urban areas are continuously growing, and densification is a frequent strategy to limit urban expansion. This generally entails a loss of green spaces (GSs) and an increase in noise pollution, which has negative effects on health. Within the research project RESTORE (Restorative potential of green spaces in noise-polluted environments), an extended cross-sectional field study in the city of Zurich, Switzerland, is conducted. The aim is to assess the relationship between noise annoyance and stress (self-perceived and physiological) as well as their association with road traffic noise and GSs. A representative stratified sample of participants from more than 5000 inhabitants will be contacted to complete an online survey. In addition to the self-reported stress identified by the questionnaire, hair cortisol and cortisone probes from a subsample of participants will be obtained to determine physiological stress. Participants are selected according to their dwelling location using a spatial analysis to determine exposure to different road traffic noise levels and access to GSs. Further, characteristics of individuals as well as acoustical and non-acoustical attributes of GSs are accounted for. This paper presents the study protocol and reports the first results of a pilot study to test the feasibility of the protocol

Higher paracetamol levels are associated with elevated glucocorticoid concentrations in hair: findings from a large cohort of young adults

Paracetamol is one of the most commonly used over-the-counter medications. Experimental studies suggest a possible stress-suppressing effect of paracetamol in humans facing experimental stress-inducing paradigms. However, no study has investigated whether paracetamol and steroid hormones covary over longer time frames and under real-life conditions. This study addresses this gap by investigating associations between steroid hormones (cortisol, cortisone, and testosterone) and paracetamol concentrations measured in human hair, indexing a timeframe of approximately three months. The data came from a large community sample of young adults (N = 1002). Hair data were assayed using liquid chromatography–tandem mass spectrometry. Multiple regression models tested associations between paracetamol and  steroid hormones, while adjusting for a wide range of potential confounders, such as sex, stressful live events, psychoactive substance use, hair colour, and body mass index. Almost one in four young adults from the community had detectable paracetamol in their hair (23%). Higher paracetamol hair concentrations were robustly associated with more cortisol (β = 0.13, η$_{p}$ = 0.016, p < 0.001) and cortisone (β = 0.16, η$_{p}$ = 0.025, p < 0.001) in hair. Paracetamol and testosterone hair concentrations were not associated. Paracetamol use intensity positively correlated with corticosteroid functioning across several months. However, a potential corticosteroid-inducing effect of chronic paracetamol use has yet to be tested in future experimental designs

Endocannabinoid and steroid analysis in infant and adult nails by LC-MS/MS

A common method to quantify chronic stress is the analysis of stress markers in keratinized matrices such as hair or nail. In this study, we aimed to validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the combined quantification of steroid hormones and endocannabinoids (eCBs) in the keratinized matrix nail. Furthermore, we aimed to investigate the suitability of the nail matrix for the detection of these stress markers in a pilot study. An LC-MS/MS method was used for the simultaneous identification and quantification of four eCBs (2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA)) and five steroid hormones (cortisol, cortisone, androstenedione, progesterone, testosterone) in human nails using a surrogate analyte method for each analyte. The method was validated in terms of selectivity, response factor, linearity, limit of quantification (LOQ), precision, accuracy, matrix effect, recovery, robustness, and autosampler stability. Nail samples were extracted for 1 h with methanol following a clean-up with a fully automated supported liquid extraction (SLE). The influence of nail weight on the quantification was investigated by using 0.5-20 mg of nail sample. As a proof of concept, nail samples (N = 57) were analyzed from a cohort representing newborns (1 month old), children (between 1 and 10 years), and adults (up to 43 years). It could be shown that the established workflow using a 1 hour extraction and clean-up by SLE was very robust and resulted in a short sample preparation time. The LC-MS/MS method was successfully validated. Matrix effects with ion enhancement occurred mainly for 2-AG. Sample weights below 5 mg showed variations in quantification for some analytes. Certain analytes such as PEA and progesterone could be accurately quantified at a sample weight lower than 5 mg. This is the first study where steroids and eCBs could be simultaneously detected and quantified in infant and adult nails. These results show that nails may serve as an alternative keratinized matrix (compared to hair) for the retrospective monitoring of cumulative eCB and steroid hormone levels. The combined assessment of eCBs and steroids from nails could provide a new approach to gain new insights into stress exposure in newborns and adults

Associations of psychoactive substances and steroid hormones in hair: Findings relevant to stress research from a large cohort of young adults

Objective: Epidemiological studies increasingly use hair samples to assess people’s cumulative exposure to steroid hormones, but how the use of different psychoactive substances may affect steroid hormone levels in hair is, so far, largely unknown. The current study addresses this gap by establishing the substance exposure correlates of cortisol, cortisone, and testosterone in hair, while also accounting for a number of relevant covariates. Method: Data came from a large urban community-sample of young adults with a high prevalence of substance use (N = 1002, mean age=20.6 years, 50.2% female), who provided 3 cm of hair samples. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantified cortisol, cortisone, and testosterone, as well as delta-9-tetrahydrocannabinol (THC), 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”), cocaine, several opioids, and their respective metabolites. Multiple linear regression models with covariates were used to predict steroid hormone levels from substance exposure in a four-step approach: In the full sample, low and high substance hair concentrations (median split) were first tested against no use for each substance individually (step 1) and for all substances together (step 2). Then, within the participants with any substance in hair only, the continuous hair concentration of each substance in pg/mg (step 3) and finally of all substances together, were regressed (step 4). Results: Low, high, and continuous levels of THC in hair were robustly associated with higher levels of cortisol (sig. in step 1 low THC: β = 0.29, p = .021; high THC: β = 0.42, p = .001; step 2: low THC: β = 0.27, p = 0.036, and high THC: β = 0.40, p = .004, and step 4: β = 0.12, p = .041). Participants with high MDMA levels had higher levels of cortisone without adjusting for other substances (step 1: β = 0.34, p = .026), but this effect was not significant in the other models. While high THC levels were associated with lower levels of testosterone in step 2 (β = -0.35, p = .018), MDMA concentration was positively related to testosterone concentration with and without adjusting for other substances (step 3: β = 0.24, p = .041; step 4: β = 0.17, 95%, p = .015) in male participants. Conclusion: The use of psychoactive substances, especially of cannabis and ecstasy, should be considered in studies investigating steroid hormones in hair

Molecular Blocking of CD23 Supports Its Role in the Pathogenesis of Arthritis

BACKGROUND: CD23 is a differentiation/activation antigen expressed by a variety of hematopoietic and epithelial cells. It can also be detected in soluble forms in biological fluids. Initially known as the low-affinity receptor for immunoglobulin E (Fc epsilonRII), CD23 displays various other physiologic ligands such as CD21, CD11b/c, CD47-vitronectin, and mannose-containing proteins. CD23 mediates numerous immune responses by enhancing IgE-specific antigen presentation, regulating IgE synthesis, influencing cell differentiation and growth of both B- and T-cells. CD23-crosslinking promotes the secretion of pro-inflammatory mediators from human monocytes/macrophages, eosinophils and epithelial cells. Increased CD23 expression is found in patients during allergic reactions and rheumatoid arthritis while its physiopathologic role in these diseases remains to be clarified. METHODOLOGY/PRINCIPAL FINDINGS: We previously generated heptapeptidic countrestructures of human CD23. Based on in vitro studies on healthy and arthritic patients' cells, we showed that CD23-specific peptide addition to human macrophages greatly diminished the transcription of genes encoding inflammatory cytokines. This was also confirmed by significant reduction of mediator levels in cell supernatants. We also show that CD23 peptide decreased IgE-mediated activation of both human and rat CD23(+) macrophages. In vivo studies in rat model of arthritis showed that CD23-blocking peptide ameliorates clinical scores and prevent bone destruction in a dose dependent manner. Ex-vivo analysis of rat macrophages further confirmed the inhibitory effect of peptides on their activation. Taken together our results support the role of CD23 activation and subsequent inflammatory response in arthritis. CONCLUSION: CD23-blocking peptide (p30A) prevents the activation of monocytes/macrophages without cell toxicity. Thus, targeting CD23 by antagonistic peptide decreases inflammatory markers and may have clinical value in the treatment of human arthritis and allergic reactions involving CD23