Hettich, Timm
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Hettich, Timm
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Publikation Validation of a commercial enzyme-linked immunosorbent assay for allopregnanolone in the saliva of healthy pregnant women(MDPI, 27.09.2022) Grötsch, Maria Katharina; Wietor, Denise Margret; Hettich, Timm; Ehlert, UlrikeEnzyme-linked immunosorbent assays (ELISAs) for saliva are simple, non-invasive methods for hormone detection. Allopregnanolone (ALLO) is a neuroactive steroid hormone that plays a crucial role in the aetiology of reproductive mood disorders. To better understand the relationship between ALLO and mood, a validated method to measure peripheral hormone levels is required. Currently, there is no commercially available ELISA with which to measure ALLO in saliva. We validated two ELISAs, developed for use with blood, with the saliva samples of 25 pregnant women, examining the range and sensitivity, intra- and inter-assay precision, parallelism, linearity of dilution, and recovery. The samples were simultaneously analysed using the liquid-chromatography–mass-spectrometry (LC-MS) method. The kits differed in range (31.2–2000 pg/mL vs. 1.6–100 ng/mL) and sensitivity (<9.5 pg/mL vs. 0.9 ng/mL), with the latter showing significant matrix effects and the former fulfilling the acceptance criteria of all the parameters. The concentrations measured with LC–MS were below the lower limit of quantification (<1.0 ng/mL) and no signal was detected. One of the tested ELISAs is a valid method for detecting ALLO in the saliva of pregnant women. It has a suitable measurement range and higher sensitivity than the conventional LC–MS method.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Tradeoff between micropollutant abatement and bromate formation during ozonation of concentrates from nanofiltration and reverse osmosis processes(Elsevier, 2022) Wünsch, Robin; Hettich, Timm; Prahtel, Marlies; Thomann, Michael; Wintgens, Thomas; Von Gunten, UrsWater treatment with nanofiltration (NF) or reverse osmosis (RO) membranes results in a purified permeate and a retentate, where solutes are concentrated and have to be properly managed and discharged. To date, little is known on how the selection of a semi-permeable dense membrane impacts the dissolved organic matter in the concentrate and what the consequences are for micropollutant (MP) abatement and bromate formation during concentrate treatment with ozone. Laboratory ozonation experiments were performed with standardized concentrates produced by three membranes (two NFs and one low-pressure reverse osmosis (LPRO) membrane) from three water sources (two river waters and one lake water). The concentrates were standardized by adjustment of pH and concentrations of dissolved organic carbon, total inorganic carbon, selected micropollutants (MP) with a low to high ozone reactivity and bromide to exclude factors which are known to impact ozonation. NF membranes had a lower retention of bromide and MPs than the LPRO membrane, and if the permeate quality of the NF membrane meets the requirements, the selection of this membrane type is beneficial due to the lower bromate formation risks upon concentrate ozonation. The bromate formation was typically higher in standardized concentrates of LPRO than of NF membranes, but the tradeoff between MP abatement and bromate formation upon ozonation of the standardized concentrates was not affected by the membrane type. Furthermore, there was no difference for the different source waters. Overall, ozonation of concentrates is only feasible for abatement of MPs with a high to moderate ozone reactivity with limited bromate formation. Differences in the DOM composition between NF and LPRO membrane concentrates are less relevant than retention of MPs and bromide by the membrane and the required ozone dose to meet a treatment target.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Whole-genome sequence-informed MALDI-TOF MS diagnostics reveal importance of Klebsiella oxytoca group in invasive infections: a retrospective clinical study(Springer, 2021) Cuenod, Aline; Wüthrich, Daniel; Seth-Smith, Helena; Ott, Chantal; Gehringer, Christian; Foucaul, Frederic; Mouchet, Roxanne; Kassim, Ali; Revathi, Gunturu; Vogt, Deborah; von Felten, Stefanie; Bassetti, Stefano; Tschudin-Sutter, Sarah; Hettich, Timm; Schlotterbeck, Götz; Homberger, Christina; Casanova, Carlo; Moran-Gilad, Jakob; Sagi, Orli; Rodriguez-Sanchez, Belen; Müller, Franco; Aerni, Martina; Gaia, Valeria; van Dessel, Helke; Kampinga, Greetje; Müller, Claudia; Daubenberger, Claudia; Pflüger, Valentin; Egli, AdrianBackground Klebsiella spp. are opportunistic pathogens which can cause severe infections, are often multi-drug resistant and are a common cause of hospital-acquired infections. Multiple new Klebsiella species have recently been described, yet their clinical impact and antibiotic resistance profiles are largely unknown. We aimed to explore Klebsiella group- and species-specific clinical impact, antimicrobial resistance (AMR) and virulence. Methods We analysed whole-genome sequence data of a diverse selection of Klebsiella spp. isolates and identified resistance and virulence factors. Using the genomes of 3594 Klebsiella isolates, we predicted the masses of 56 ribosomal subunit proteins and identified species-specific marker masses. We then re-analysed over 22,000 Matrix-Assisted Laser Desorption Ionization - Time Of Flight (MALDI-TOF) mass spectra routinely acquired at eight healthcare institutions in four countries looking for these species-specific markers. Analyses of clinical and microbiological endpoints from a subset of 957 patients with infections from Klebsiella species were performed using generalized linear mixed-effects models. Results Our comparative genomic analysis shows group- and species-specific trends in accessory genome composition. With the identified species-specific marker masses, eight Klebsiella species can be distinguished using MALDI-TOF MS. We identified K. pneumoniae (71.2%; n = 12,523), K. quasipneumoniae (3.3%; n = 575), K. variicola (9.8%; n = 1717), “K. quasivariicola” (0.3%; n = 52), K. oxytoca (8.2%; n = 1445), K. michiganensis (4.8%; n = 836), K. grimontii (2.4%; n = 425) and K. huaxensis (0.1%; n = 12). Isolates belonging to the K. oxytoca group, which includes the species K. oxytoca, K. michiganensis and K. grimontii, were less often resistant to 4th-generation cephalosporins than isolates of the K. pneumoniae group, which includes the species K. pneumoniae, K. quasipneumoniae, K. variicola and “K. quasivariicola” (odds ratio = 0.17, p < 0.001, 95% confidence interval [0.09,0.28]). Within the K. pneumoniae group, isolates identified as K. pneumoniae were more often resistant to 4th-generation cephalosporins than K. variicola isolates (odds ratio = 2.61, p = 0.003, 95% confidence interval [1.38,5.06]). K. oxytoca group isolates were found to be more likely associated with invasive infection to primary sterile sites than K. pneumoniae group isolates (odds ratio = 2.39, p = 0.0044, 95% confidence interval [1.05,5.53]). Conclusions Currently misdiagnosed Klebsiella spp. can be distinguished using a ribosomal marker-based approach for MALDI-TOF MS. Klebsiella groups and species differed in AMR profiles, and in their association with invasive infection, highlighting the importance for species identification to enable effective treatment options.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Environmental glucocorticoids corticosterone, betamethasone and flumethasone induce more potent physiological than transcriptional effects in zebrafish embryos(Elsevier, 01.07.2019) Willi, Raffael Alois; Faltermann, Susanne; Hettich, Timm; Fent, KarlMany glucocorticoids occur in the aquatic environments but their adverse effects to fish are poorly known. Here we investigate effects of the natural glucocorticoid corticosterone and the synthetic glucocorticoids betamethasone and flumethasone in zebrafish embryos. Besides studying the effects of each steroid, we compared effects of natural with synthetic glucocorticoids, used as drugs. Exposure at concentrations of 1 μg/L and higher led to concentration-related decrease in spontaneous muscle contractions at 24 h post fertilization (hpf) and increase in heart rate at 48 hpf. Betamethasone showed a significant increase at 0.11 μg/L in heart rate. Corticosterone also accelerated hatching at 60 hpf at 0.085 μg/L. Transcription of up to 24 genes associated with different pathways showed alterations at 96 and 120 hpf for all glucocorticoids, although with low potency. Corticosterone caused transcriptional induction of interleukin-17, while betamethasone caused transcriptional down-regulation of the androgen receptor, aromatase and hsd11b2, indicating an effect on the sex hormone system. Furthermore, transcripts encoding proteins related to immune system regulation (irg1l, gilz) and fkbp5 were differentially expressed by corticosterone and betamethasone, while flumethasone caused only little effects, mainly alteration of the irg1l transcript. Our study shows that these glucocorticoids caused more potent physiological effects in early embryos than transcriptional alterations in hatched embryos, likely due to increased metabolism in later developmental stages. Thus, these glucocorticoids may be of concern for early stages of fish embryos in contaminated aquatic environments.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation A Versatile Biocompatible Antibiotic Delivery System Based on Self‐Assembling Peptides with Antimicrobial and Regenerative Potential(Wiley, 15.04.2019) Koch, Franziska; Kilian, David; Hettich, Timm; Germershaus, OliverPeriodontitis is a chronic inflammatory and tissue‐destructive disease. Since the polymicrobiome in the oral cavity makes it difficult to treat, novel therapeutic strategies are required. Hydrogels based on self‐assembling peptides (SAP) can be suitable candidates for periodontal therapy due to their injectability, biocompatibility, cargo‐loading capacity, and tunable physicochemical and mechanical properties. In this study, two SAP hydrogels (P11‐4 and P11‐28/29) are examined for their intrinsic antimicrobial activity, regenerative potential, and antibiotic delivery capacity. A significant antibacterial effect of P11‐28/29 hydrogels on the periodontal pathogen Porphyromonas gingivalis and a less pronounced effect for P11‐4 hydrogels is demonstrated. The metabolic activity rates of human dental follicle stem cells (DFSCs), which reflect cell viability and may thus indicate the regenerative capacity, are similar on tissue culture polystyrene (TCPS) and on P11‐4 hydrogels after 14 days of culture. Noticeably, both SAP hydrogels strengthen the osteogenic differentiation of DFSCs compared with TCPS. The incorporation of tetracycline, ciprofloxacin, and doxycycline does not affect fibril formation of either SAP hydrogel and results in favorable release kinetics up to 120 h. In summary, this study reveals that P11‐SAP hydrogels combine many favorable properties required to make them applicable as prospective novel treatment strategy for periodontal therapy.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation A vitellogenin antibody in honey bees (Apis mellifera ): Characterization and application as potential biomarker for insecticide exposure(Wiley, 02/2019) Christen, Verena; Fent, Karl; Hettich, TimmThe insect yolk precursor vitellogenin is a lipoglycoprotein synthesized and stored in the fat body and secreted into the hemolymph. In honey bees, vitellogenin displays crucial functions in hormone signaling, behavioral transition of nurse bees to foragers, stress resistance, and longevity in workers. Plant protection products such as neonicotinoids, pyrethroids, and organophosphates alter the transcriptional expression of vitellogenin. To assess plant protection product‐induced alterations on the protein level, we developed a rabbit polyclonal vitellogenin antibody. After characterization, we assessed its specificity and vitellogenin levels in different tissues of worker bees. The vitellogenin antibody recognized full‐length 180‐kDa vitellogenin and the lighter fragment of 150 kDa in fat body, hemolymph, and brain. In hemolymph, a band of approximately 75 kDa was detected. Subsequent mass spectrometric analysis (liquid chromatography–mass spectrometry) confirmed the 180‐ and 150‐kDa bands as vitellogenin. Subsequently, we evaluated vitellogenin expression in brain, fat body, and hemolymph on 24‐h exposure of bees to 3 ng/bee to the neonicotinoid clothianidin. Full‐length vitellogenin was upregulated 3‐fold in the fat body, and the 150‐kDa fragment was upregulated in the brain of exposed honey bees, whereas no alteration occurred in the hemolymph. Upregulation of the vitellogenin protein by the neonicotinoid clothianidin is in line with the previously shown induction of its transcript. We conclude that vitellogenin might serve as a potential biomarker for neonicotinoid and other pesticide exposure in bees. Environ Toxicol Chem 2019;00:1–10. © 2019 SETAC01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Biodegradation of sulfamethoxazole by a bacterial consortium of Achromobacter denitrificans PR1 and Leucobacter sp. GP(Springer, 12/2018) Reis, Ana C.; Cvancarova Småstuen, M.; Liu, Ying; Lenz, Markus; Hettich, Timm; Kolvenbach, Boris; Corvini, Philippe; Nunes, Olga C.In the last decade, biological degradation and mineralization of antibiotics have been increasingly reported feats of environmental bacteria. The most extensively described example is that of sulfonamides that can be degraded by several members of Actinobacteria and Proteobacteria. Previously, we reported sulfamethoxazole (SMX) degradation and partial mineralization by Achromobacter denitrificans strain PR1, isolated from activated sludge. However, further studies revealed an apparent instability of this metabolic trait in this strain. Here, we investigated this instability and describe the finding of a low-abundance and slow-growing actinobacterium, thriving only in co-culture with strain PR1. This organism, named GP, shared highest 16S rRNA gene sequence similarity (94.6–96.9%) with the type strains of validly described species of the genus Leucobacter. This microbial consortium was found to harbor a homolog to the sulfonamide monooxygenase gene (sadA) also found in other sulfonamide-degrading bacteria. This gene is overexpressed in the presence of the antibiotic, and evidence suggests that it codes for a group D flavin monooxygenase responsible for the ipso-hydroxylation of SMX. Additional side reactions were also detected comprising an NIH shift and a Baeyer–Villiger rearrangement, which indicate an inefficient biological transformation of these antibiotics in the environment. This work contributes to further our knowledge in the degradation of this ubiquitous micropollutant by environmental bacteria.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation GABAA receptor activity modulating piperine analogs: In vitro metabolic stability, metabolite identification, CYP450 reaction phenotyping, and protein binding(Elsevier, 12/2017) Zabela, Volha; Hettich, Timm; Schlotterbeck, Götz; Wimmer, Laurin; Mihovilovic D., Marko; Guillet, Fabrice; Belkacem, Bouaita; Shevchenko, Bénédicte; Hamburger, Matthias; Oufir, MouhssinIn a screening of natural products for allosteric modulators of GABAA receptors (γ-aminobutyric acid type A receptor), piperine was identified as a compound targeting a benzodiazepine-independent binding site. Given that piperine is also an activator of TRPV1 (transient receptor potential vanilloid type 1) receptors involved in pain signaling and thermoregulation, a series of piperine analogs were prepared in several cycles of structural optimization, with the aim of separating GABAA and TRPV1 activating properties. We here investigated the metabolism of piperine and selected analogs in view of further cycles of lead optimization. Metabolic stability of the compounds was evaluated by incubation with pooled human liver microsomes, and metabolites were analyzed by UHPLC-Q-TOF-MS. CYP450 isoenzymes involved in metabolism of compounds were identified by reaction phenotyping with Silensomes™. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. Piperine was the metabolically most stable compound. Aliphatic hydroxylation, and N- and O-dealkylation were the major routes of oxidative metabolism. Piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Extensive binding to blood constituents was observed for all compounds.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Altered (neo-) lacto series glycolipid biosynthesis impairs α2-6 sialylation on N-glycoproteins in ovarian cancer cells(Nature, 30.03.2017) Alam, Shahidul; Anugraham, Merrina; Huang, Yen-Lin; Kohler, Reto; Hettich, Timm; Winkelbach, Katharina; Grether, Yasmin; Nunez Lopez, Monica; Khasbiullina, Nailia; Bovin, Nicolai V.; Schlotterbeck, Götz; Jacob, FrancisThe (neo-) lacto series glycosphingolipids (nsGSLs) comprise of glycan epitopes that are present as blood group antigens, act as primary receptors for human pathogens and are also increasingly associated with malignant diseases. Beta-1, 3-N-acetyl-glucosaminyl-transferase 5 (B3GNT5) is suggested as the key glycosyltransferase for the biosynthesis of nsGSLs. In this study, we investigated the impact of CRISPR-Cas9 -mediated gene disruption of B3GNT5 (∆B3GNT5) on the expression of glycosphingolipids and N-glycoproteins by utilizing immunostaining and glycomics-based PGC-UHPLC-ESI-QTOF-MS/MS profiling. ∆B3GNT5 cells lost nsGSL expression coinciding with reduction of α2-6 sialylation on N-glycoproteins. In contrast, disruption of B4GALNT1, a glycosyltransferase for ganglio series GSLs did not affect α2-6 sialylation on N-glycoproteins. We further profiled all known α2-6 sialyltransferase-encoding genes and showed that the loss of α2-6 sialylation is due to silencing of ST6GAL1 expression in ∆B3GNT5 cells. These results demonstrate that nsGSLs are part of a complex network affecting N-glycosylation in ovarian cancer cells.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Anti-Inflammatory Activity of Cyanobacterial Serine Protease Inhibitors Aeruginosin 828A and Cyanopeptolin 1020 in Human Hepatoma Cell Line Huh7 and Effects in Zebrafish (Danio rerio)(MDPI, 14.07.2016) Faltermann, Susanne; Hutter, Simon; Christen, Verena; Hettich, Timm; Fent, KarlIntensive growth of cyanobacteria in freshwater promoted by eutrophication can lead to release of toxic secondary metabolites that may harm aquatic organisms and humans. The serine protease inhibitor aeruginosin 828A was isolated from a microcystin-deficient Planktothrix strain. We assessed potential molecular effects of aeruginosin 828A in comparison to another cyanobacterial serine protease inhibitor, cyanopeptolin 1020, in human hepatoma cell line Huh7, in zebrafish embryos and liver organ cultures. Aeruginosin 828A and cyanopeptolin 1020 promoted anti-inflammatory activity, as indicated by transcriptional down-regulation of interleukin 8 and tumor necrosis factor α in stimulated cells at concentrations of 50 and 100 µmol·L−1 aeruginosin 828A, and 100 µmol·L−1 cyanopeptolin 1020. Aeruginosin 828A induced the expression of CYP1A in Huh7 cells but did not affect enzyme activity. Furthermore, hatched zebrafish embryos and zebrafish liver organ cultures were exposed to aeruginosin 828A. The transcriptional responses were compared to those of cyanopeptolin 1020 and microcystin-LR. Aeruginosin 828A had only minimal effects on endoplasmic reticulum stress. In comparison to cyanopeptolin 1020 our data indicate that transcriptional effects of aeruginosin 828A in zebrafish are very minor. The data further demonstrate that pathways that are influenced by microcystin-LR are not affected by aeruginosin 828A.01A - Beitrag in wissenschaftlicher Zeitschrift