Hettich, Timm
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Hettich, Timm
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- PublikationWhole-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, Adrian [in: Genome Medicine]Background 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 Zeitschrift
- PublikationGABAA 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, Mouhssin [in: Journal of Chromatography B]In 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 Zeitschrift
- PublikationAltered (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, Francis [in: Scientific Reports]The (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 Zeitschrift
- PublikationEpigenetic activation of MGAT3 and corresponding bisecting GlcNAc shortens the survival of cancer patients(Impact Journals LLC, 12.07.2016) Kohler, Reto; Anugraham, Merrina; Nunez Lopez, Monica; Xiao, Christina; Schoetzau, Andreas; Hettich, Timm; Schlotterbeck, Götz; Fedier, André; Jacob, Francis; Heinzelmann-Schwarz, Viola [in: Oncotarget]Bisecting GlcNAc on N-glycoproteins is described in E-cadherin-, EGF-, Wnt- and integrin- cancer-associated signaling pathways. However, the mechanisms regulating bisecting GlcNAc expression are not clear. Bisecting GlcNAc is attached to N-glycans through beta 1-4 N-acetylglucosaminyl transferase III (MGAT3), which is encoded by two exons flanked by high-density CpG islands. Despite a recently described correlation of MGAT3 and bisecting GlcNAc in ovarian cancer cells, it remains unknown whether DNA methylation is causative for the presence of bisecting GlcNAc. Here, we narrow down the regulatory genomic region and show that reconstitution of MGAT3 expression with 5-Aza coincides with reduced DNA methylation at the MGAT3 transcription start site. The presence of bisecting GlcNAc on released N-glycans was detected by mass spectrometry (LC-ESI-qTOF-MS/MS) in serous ovarian cancer cells upon DNA methyltransferase inhibition. The regulatory impact of DNA methylation on MGAT3 was further evaluated in 18 TCGA cancer types (n = 6118 samples) and the results indicate an improved overall survival in patients with reduced MGAT3 expression, thereby identifying long-term survivors of high-grade serous ovarian cancers (HGSOC). Epigenetic activation of MGAT3 was also confirmed in basal-like breast cancers sharing similar molecular and genetic features with HGSOC. These results provide novel insights into the epigenetic regulation of MGAT3/bisecting01A - Beitrag in wissenschaftlicher Zeitschrift
- PublikationCaco-2 Permeability Studies and In Vitro hERG Liability Assessment of Tryptanthrin and Indolinone(Thieme, 2016) Jähne, Evelyn A.; Eigenmann, Daniela E.; Moradi-Afrapoli, Fahimeh; Verjee, Sheela; Butterweck, Veronika; Hebeisen, Simon; Hettich, Timm; Schlotterbeck, Götz; Smiesko, Martin; Hamburger, Matthias; Oufir, Mouhssin [in: Planta Medica]Tryptanthrin and (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolinone (indolinone) were recently isolated from Isatis tinctoria as potent anti-inflammatory and antiallergic alkaloids, and shown to inhibit COX-2, 5-LOX catalyzed leukotriene synthesis, and mast cell degranulation at low μM to nM concns. To assess their suitability for oral administration, we screened the compds. in an in vitro intestinal permeability assay using human colonic adenocarcinoma cells. For exact quantification of the compds., validated UPLC-MS/MS methods were used. Tryptanthrin displayed high permeability (apparent permeability coeff. > 32.0 × 10-6 cm/s) across the cell monolayer. The efflux ratio below 2 (< 1.12) and unchanged apparent permeability coeff. values in the presence of the P-glycoprotein inhibitor verapamil (50 μM) indicated that tryptanthrin was not involved in P-glycoprotein interactions. For indolinone, a low recovery was found in the human colon adenocarcinoma cell assay. High-resoln. mass spectrometry pointed to extensive phase II metab. of indolinone (sulfation and glucuronidation). Possible cardiotoxic liability of the compds. was assessed in vitro by measurement of an inhibitory effect on human ether-a-go-go-related gene tail currents in stably transfected HEK 293 cells using the patch clamp technique. Low human ether-a-go-go-related gene inhibition was found for tryptanthrin (IC50 > 10 μM) and indolinone (IC50 of 24.96 μM). The anal. of compds. using various in silico methods confirmed favorable pharmacokinetic properties, as well as a slight inhibition of the human ether-a-go-go-related gene potassium channel at micromolar concns.01A - Beitrag in wissenschaftlicher Zeitschrift
- PublikationMetabolic Profiling(2015) Berchtold, Christian; Hettich, Timm; Schlotterbeck, Götz; Senner, Frank [in: GIT: Labor-Fachzeitschrift]01 - Zeitschriftenartikel, Journalartikel oder Magazin
- PublikationNovel Analytical Workflow for Comprehensive(Schweizerische Chemische Gesellschaft, 2015) Hettich, Timm; Schlotterbeck, Götz [in: Chimia]01A - Beitrag in wissenschaftlicher Zeitschrift