Identification, characterization and molecular adaptation of class I redox systems for the production of hydroxylated diterpenoids

Background De novo production of multi-hydroxylated diterpenoids is challenging due to the lack of efficient redox systems. Results In this study a new reductase/ferredoxin system from Streptomyces afghaniensis (AfR·Afx) was identified, which allowed the Escherichia coli-based production of the trihydroxylated diterpene cyclooctatin, a potent inhibitor of human lysophospholipase. This production system provides a 43-fold increase in cyclooctatin yield (15 mg/L) compared to the native producer. AfR·Afx is superior in activating the cylcooctatin-specific class I P450s CotB3/CotB4 compared to the conventional Pseudomonas putida derived PdR·Pdx model. To enhance the activity of the PdR·Pdx system, the molecular basis for these activity differences, was examined by molecular engineering. Conclusion We demonstrate that redox system engineering can boost and harmonize the catalytic efficiency of class I hydroxylase enzyme cascades. Enhancing CotB3/CotB4 activities also provided for identification of CotB3 substrate promiscuity and sinularcasbane D production, a functionalized diterpenoid originally isolated from the soft coral Sinularia sp

Systematic drug screening reveals specific vulnerabilities and co-resistance patterns in endocrine-resistant breast cancer

Abstract Background The estrogen receptor (ER) inhibitor tamoxifen reduces breast cancer mortality by 31 % and has served as the standard treatment for ER-positive breast cancers for decades. However, 50 % of advanced ER-positive cancers display de novo resistance to tamoxifen, and acquired resistance evolves in 40 % of patients who initially respond. Mechanisms underlying resistance development remain poorly understood and new therapeutic opportunities are urgently needed. Here, we report the generation and characterization of seven tamoxifen-resistant breast cancer cell lines from four parental strains. Methods Using high throughput drug sensitivity and resistance testing (DSRT) with 279 approved and investigational oncology drugs, exome-sequencing and network analysis, we for the first time, systematically determine the drug response profiles specific to tamoxifen resistance. Results We discovered emerging vulnerabilities towards specific drugs, such as ERK1/2-, proteasome- and BCL-family inhibitors as the cells became tamoxifen-resistant. Co-resistance to other drugs such as the survivin inhibitor YM155 and the chemotherapeutic agent paclitaxel also occurred. Conclusion This study indicates that multiple molecular mechanisms dictate endocrine resistance, resulting in unexpected vulnerabilities to initially ineffective drugs, as well as in emerging co-resistances. Thus, combatting drug-resistant tumors will require patient-tailored strategies in order to identify new drug vulnerabilities, and to understand the associated co-resistance patterns

Fibroblast subsets in non-small cell lung cancer : Associations with survival, mutations, and immune features

Background Cancer-associated fibroblasts (CAFs) are molecularly heterogeneous mesenchymal cells that interact with malignant cells and immune cells and confer anti- and protumorigenic functions. Prior in situ profiling studies of human CAFs have largely relied on scoring single markers, thus presenting a limited view of their molecular complexity. Our objective was to study the complex spatial tumor microenvironment of non-small cell lung cancer (NSCLC) with multiple CAF biomarkers, identify novel CAF subsets, and explore their associations with patient outcome. Methods Multiplex fluorescence immunohistochemistry was employed to spatially profile the CAF landscape in 2 population-based NSCLC cohorts (n = 636) using antibodies against 4 fibroblast markers: platelet-derived growth factor receptor-alpha (PDGFRA) and -beta (PDGFRB), fibroblast activation protein (FAP), and alpha-smooth muscle actin (alpha SMA). The CAF subsets were analyzed for their correlations with mutations, immune characteristics, and clinical variables as well as overall survival. Results Two CAF subsets, CAF7 (PDGFRA-/PDGFRB+/FAP+/alpha SMA+) and CAF13 (PDGFRA+/PDGFRB+/FAP-/alpha SMA+), showed statistically significant but opposite associations with tumor histology, driver mutations (tumor protein p53 [TP53] and epidermal growth factor receptor [EGFR]), immune features (programmed death-ligand 1 and CD163), and prognosis. In patients with early stage tumors (pathological tumor-node-metastasis IA-IB), CAF7 and CAF13 acted as independent prognostic factors. Conclusions Multimarker-defined CAF subsets were identified through high-content spatial profiling. The robust associations of CAFs with driver mutations, immune features, and outcome suggest CAFs as essential factors in NSCLC progression and warrant further studies to explore their potential as biomarkers or therapeutic targets. This study also highlights multiplex fluorescence immunohistochemistry-based CAF profiling as a powerful tool for the discovery of clinically relevant CAF subsets.Peer reviewe

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology

KONECT: Implementation and Extension of a Method for the Development of Safety-Critical Human-Machine Interaction Interfaces

In safety-critical systems, monitoring can be risky, because overlooking or misinterpreting important information can lead to serious consequences. For this purpose, the KONECT method was developed. The method is capable of systematically deriving information visualizations for monitoring tasks in safety-critical systems. We have studied this method and describe planned extensions and implementations of the steps of the KONECT method. In this way, the method offers more possibilities and can be applied more easily by the user

Stereoselective chemo-enzymatic oxidation routes for (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene

The diterpene (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene from the marine brown alga Dilophus spiralis belongs to the dolabellanes natural product family and has antimicrobial activity against multi-drug resistant Staphylococcus aureus (MRSA). Recently, we generated a CotB2 diterpene synthase mutant (W288G), which instead of its native product cyclooctat-9-en-7-ol, generates (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene. In vivo CotB2 W288G reconstitution in an E. coli based terpene production system, allowed efficient production of this olefinic macrocycle. To diversify the 3,7,18-dolabellatriene bioactivity we evaluated chemical and enzymatic methods for selective oxidation. Epoxidation by acetic peracid, which was formed in situ by a lipase catalyzed reaction of acetic acid with H2O2, provided efficient access to two monooxidized dolabellanes and to a novel di-epoxidated dolabellane species. These compounds could act as synthons en-route to new dolabellanes with diversified bioactivities. Furthermore, we demonstrate the almost quantitative 3,7,18-dolabellatriene conversion into the new, non-natural compound (1R,3E,7E,11S,12S,18R)-dolabella-3,7-diene-20-ol by hydroboration-oxidation with an enantiomeric excess of 94 %, for the first time