Neue molekulare Targets von Boswelliasäuren und Charakterisierung bioaktiver Inhaltsstoffe aus Weihrauch

Lipophilic extracts from the gum resin of Boswellia spec. (BEs) have been traditionally used for the treatment of several inflammatory and cancer-related diseases. The extracts contain large amount of triterpenes, among them the prominent group of boswellic acids (BAs), as well as roburic acids (RAs) and lupanic acids (LAs). In search of the molecular mechanism of BEs, several targets of BAs were identified, which were mainly influenced by 3-O-acetyl-11-keto-beta-boswellic acid (AKBA). However, rather high concentrations were needed for efficient inhibition of these targets, while AKBA has a rather poor bioavailability. Therefore, it seems likely that additional targets or other ingredients participate in the beneficial outcome observed in several diseases treated with BEs. One aim of the present study was the identification of novel molecular targets of BAs. Several new targets were identified, among them LL-37, lipopolysaccharides (LPS), cathepsin G (catG), p21 Ras and Rap1B. A direct binding of BAs to LL-37 resulted in an inhibition of the biological activity of LL-37. This effect was most prominent for 3-O-acetyl-beta-boswellic acid (ABA, EC50 = 0.2 µM) and AKBA (EC50 = 0.8 µM). A 3-acetoxy or 3-hydroxy group was necessary for potent interference, while the 11-keto group was less important. Besides BAs, RAs and LAs were identified as LL-37-inhibiting compounds from frankincense. At higher concentrations (greater than or equal 10 µM), BAs stimulated LL-37-release from cytochalasin B/fMLP-stimulated neutrophils. Since inhibition of LL-37 activity occurs at much lower BA concentrations, the inhibitory effect seemingly dominates. BAs, RAs and LAs might play a role for the treatment of LL-37-related diseases as psoriasis. A second identified target was LPS, which was specifically bound in a direct manner by BAs without 11-keto moiety. This binding led to a potent inhibition of LPS activity (IC50 ~2 µM). Those BAs also influenced LPS signaling, as both LPS-induced iNOS expression and nitric oxide generation were inhibited. The obtained in vitro data suggests that BAs might become potentially valuable compounds for the treatment of severe LPS-mediated diseases as sepsis or septic shock. CatG was identified as a third functional target of BAs, which was bound through direct interaction. This binding led to an inhibition of the proteolytic activity, and the catG-induced Ca2+ influx in platelets was inhibited as well. In addition, cell migration into an extracellular matrix was inhibited by BAs. CatG activity of the plasma of stimulated blood from BE-treated patients was reduced, compared to the placebo-treated control group. Conclusively, catG inhibition might contribute to the anti-inflammatory effects of frankincense extracts. Besides LL-37, LPS and catG, two novel targets of BAs, small G-proteins belonging to the protein family Ras, were identified: p21 Ras and Rap1B. The direct interaction between p21 Ras and BAs did not led to a direct inhibition of p21 Ras activity, however. Similarly, the binding of BAs to Rap1B did not led to immediate functional effects. Therefore, a participation of these targets on the beneficial effects of frankincense seems questionable. An additional aim of the present study was the analysis of apotosis induction by ingredients of BEs. BAs without 11-keto moiety showed cytotoxic effects on Jurkat and PBMC cells (EC50 = 2.8-9.8 µM), while the related 11-keto BAs displayed less cytotoxicity. A prominent exception to this rule was ABA, which was cytotoxic for Jurkat cells but not for PBMCs. Such selectivity for cancer cells versus non-transformed cells might be of interest for the development of novel anti-cancer agents. Besides BAs, RAs and LAs were identified as apoptosis-inducing compounds from frankincense, which activated caspase-8 and caspase-3, cleaved PARP and finally led to DNA fragmentation. Taken together, LL-37, LPS and catG, all involved in inflammatory processes, were identified as novel targets of BAs. The BA concentrations needed for an efficient inhibition were in the range of BA plasma levels reached after treatment with BEs. The obtained data suggest that these molecular mechanisms might have physiological relevance. Moreover, novel triterpenes derived from frankincense were identified as potent apoptosis-inducing agents, encouraging for future studies of BE-derived compounds for their potential in the treatment of cancer and cancer-related diseases.Lipophile Extrakte des Harzes von Weihrauchbäumen (Boswellia spec.) werden in der Volksmedizin eingesetzt, um diverse Entzündungs- und Krebskrankheiten zu behandeln. Die Extrakte enthalten unter anderem die in großen Mengen vorkommenden Boswelliasäuren (BAs), sowie Robursäuren (RAs) und Lupansäuren (LAs). Die Suche nach den molekularen Wirkmechanismen der Extrakte führte zu einer Identifizierung verschiedener Targets, die hauptsächlich von der BA 3-O-acetyl-11-keto-beta-boswellic acid (AKBA) in höheren Konzentrationen gehemmt wurden. Demgegenüber steht eine niedrige Bioverfügbarkeit von AKBA, welche eine Beteiligung weiterer Targets oder anderer Inhaltsstoffe wahrscheinlich erscheinen lässt. Ein Ziel der vorliegenden Arbeit war die Identifikation neuer molekularer Targets für BAs. Dabei wurden LL-37, Lipopolysaccharide (LPS), Cathepsin G (catG), p21 Ras und Rap1B als molekulare Bindungspartner identifiziert. Die direkte Bindung von BAs an LL-37 führte zu einer Hemmung der biologischen Aktivität. Dieser Effekt war am stärksten bei 3-O-acetyl-beta-boswellic acid (ABA, EC50 = 0,2 µM) und AKBA (EC50 = 0,8 µM) ausgeprägt. Eine 3-acetoxy- oder eine 3-hydroxy-Gruppe war notwendig für eine Hemmung, während die 11-keto-Gruppe keinen großen Einfluss auf die Aktivität der BAs hatte. Neben BAs wurden mit RAs und LAs weitere Inhaltsstoffe des Weihrauchs als aktive, LL-37-hemmende Komponenten identifiziert. In höheren Konzentrationen (größer/gleich 10 µM) stimulierten BAs die LL-37-Freisetzung aus Cytochalasin B/fMLP-stimulierten Neutrophilen, ein Effekt, der allerdings wegen den relativ hohen benötigten Konzentrationen gegenüber dem hemmenden Effekt vermutlich eine geringere Rolle spielt. BAs, RAs und LAs könnten eine Rolle bei der Behandlung von LL-37-bedingten Krankheiten wie Psoriasis spielen. Ein zweites identifiziertes Target war LPS, welches direkt und spezifisch an BAs ohne 11-keto-Gruppe binden konnte. Diese Bindung führte zu einer Hemmung der LPS-Aktivität (IC50 ~2 µM). LPS-induzierte Signalwege wurden ebenfalls durch BAs beeinflusst. So konnte gezeigt werden, dass die LPS-induzierte iNOS-Expression und die Stickstoffmonoxid-Freisetzung durch BAs gehemmt wurde. Die gewonnenen in vitro-Daten deuten an, dass BAs eventuell das Potential dazu hätten, eine Grundlage für die Behandlung schwerwiegender LPS-abhängiger Krankheiten wie Sepsis oder Septischer Schock zu bieten. CatG wurde als drittes Target identifiziert, welches von BAs direkt gebunden wurde. Die proteolytische Aktivität von catG wurde durch BAs gehemmt, und der catG-induzierte Ca2+-Einstrom in Thrombozyten wurde ebenfalls durch BAs inhibiert. Zusätzlich wurde die Zellmigration in eine extrazelluläre Matrix durch BAs gehemmt. Im Vergleich zu einer Placebo-behandelten Kontrollgruppe war die catG-Aktivität des Plasmas von stimuliertem Blut von Weihrauchextrakt-behandelten Patienten reduziert. Dieses deutet auf eine mögliche pharmakologische Relevanz der catG-Hemmung hin, die an den entzündungshemmenden Effekten von Weihrauchextrakten beteiligt sein könnte. Mit p21 Ras und Rap1B wurden zwei weitere neue Targets von BAs gefunden, die zu den kleinen G-Proteinen der Proteinfamilie Ras gehören. Die direkte Bindung an p21 Ras führte allerdings nicht zu einer direkten Hemmung der p21 Ras-Aktivität. Auch konnten keine unmittelbaren funktionellen Effekte der direkten Bindung von BAs an Rap1B gezeigt werden. Es ist daher fraglich, ob diese beiden Targets eine Rolle bei den heilsamen Effekten von Weihrauchextrakten spielen. Ein weiteres Ziel der vorliegenden Arbeit war die Analyse der Apoptoseinduktion durch Weihrauchinhaltsstoffe. BAs ohne 11-keto-Gruppe wirkten cytotoxisch auf Jurkat- und PBMC-Zellen (EC50 = 2,8-9,8 µM), während die entsprechenden 11-keto-BAs weniger cytotoxisch waren. Eine bemerkenswerte Ausnahme von dieser Regel war ABA, welches cytotoxisch auf Jurkatzellen, nicht aber auf PBMC wirkte. Diese Selektivität (Krebszellen gegen nicht-transformierte Zellen) könnte eine Bedeutung für die Entwicklung neuer Krebsmedikamente haben. Zusätzlich zu BAs wurden RAs und LAs als Apoptoseinduktoren aus Weihrauch identifiziert, die Caspase-8 und Caspase-3 aktivierten, PARP spalteten und zu einer Fragmentierung der zellulären DNA führten. Zusammengefasst wurden in der vorliegenden Arbeit LL-37, LPS und catG als neue Targets von BAs identifiziert, die an Entzündungsvorgängen beteiligt sind. Die für eine Hemmung notwendigen BA-Konzentrationen lagen auf oder unter den erreichbaren BA-Plasmaspiegeln, welches auf eine mögliche physiologische Relevanz hindeutet. Darüber hinaus wurden bisher unbekannte Triterpene aus Weihrauch als wirksame Apoptoseinduktoren identifiziert, welche weitere Studien über einzelne Weihrauchbestandteile und ihrer Wirkung auf Krebs interessant werden lassen

Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G

Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging

Primitive Duplicate Hox Clusters in the European Eel's Genome

The enigmatic life cycle and elongated body of the European eel (Anguilla anguilla L., 1758) have long motivated scientific enquiry. Recently, eel research has gained in urgency, as the population has dwindled to the point of critical endangerment. We have assembled a draft genome in order to facilitate advances in all provinces of eel biology. Here, we use the genome to investigate the eel's complement of the Hox developmental transcription factors. We show that unlike any other teleost fish, the eel retains fully populated, duplicate Hox clusters, which originated at the teleost-specific genome duplication. Using mRNA-sequencing and in situ hybridizations, we demonstrate that all copies are expressed in early embryos. Theories of vertebrate evolution predict that the retention of functional, duplicate Hox genes can give rise to additional developmental complexity, which is not immediately apparent in the adult. However, the key morphological innovation elsewhere in the eel's life history coincides with the evolutionary origin of its Hox repertoire

Virtual Verification of Cause-Effect Chains in Automotive Cyber-Physical Systems

The technical complexity of automotive Cyber-Physical Systems (CPS) traditionally demands high development and validation efforts. Due to the new technologies entering the automotive market, such as Highly Automated Driving (HAD) (>= SAE L3) and connected infotainment, the overall system complexity is currently increasing significantly, challenging traditional system development methods and requiring new approaches for validation and verification (V&V). In parallel, new Electric/Electronic (E/E) architecture patterns are emerging in the automotive industry, distributing the functionalities across several multi-core Electrical Control Units (ECU) connected via Ethernet-based in-vehicle networks. This distributed approach leads to complex inter- and intra-ECU timing relations challenging the concept of freedom from interference according to the ISO 26262, and adding another dimension of effects analysis during V&V. This work enhances a cyber-physical functional simulation tool to include timing effects in distributed cause-effect chains and multi-technology-communication networks (incl. Ethernet and CAN). The resulting simulation allows the system designer to evaluate the impact of timing properties on a given distributed vehicle function, enabling an early validation of the system, avoiding rework during later stages of the development process resulting from wrong design choices

Using normalization to resolve RNA-seq biases caused by amplification from minimal input

RNA-Seq has become a widely used method to study transcriptomes, and it is now possible to perform RNA-Seq on almost any sample. Nevertheless, samples obtained from small cell populations are particularly challenging, as biases associated with low amounts of input RNA can have strong and detrimental effects on downstream analyses. Here we compare different methods to normalize RNA-Seq data obtained from minimal input material. Using RNA from isolated medaka pituitary cells, we have amplified material from six samples before sequencing. Both synthetic and real data are used to evaluate different normalization methods to obtain a robust and reliable pipeline for analysis of RNA-Seq data from samples with very limited input material. The analysis outlined here shows that quantile normalization outperforms other more commonly used normalization procedures when using amplified RNA as input and will benefit researchers employing low amounts of RNA in similar experiments

Using normalization to resolve RNA-seq biases caused by amplification from minimal input

RNA-Seq has become a widely used method to study transcriptomes, and it is now possible to perform RNA-Seq on almost any sample. Nevertheless, samples obtained from small cell populations are particularly challenging, as biases associated with low amounts of input RNA can have strong and detrimental effects on downstream analyses. Here we compare different methods to normalize RNA-Seq data obtained from minimal input material. Using RNA from isolated medaka pituitary cells, we have amplified material from six samples before sequencing. Both synthetic and real data are used to evaluate different normalization methods to obtain a robust and reliable pipeline for analysis of RNA-Seq data from samples with very limited input material. The analysis outlined here shows that quantile normalization outperforms other more commonly used normalization procedures when using amplified RNA as input and will benefit researchers employing low amounts of RNA in similar experiments

Delayed minimally invasive injection of allogenic bone marrow stromal cell sheets regenerates large bone defects in an ovine preclinical animal model

Cell-based tissue engineering approaches are promising strategies in the field of regenerative medicine. However, the mode of cell delivery is still a concern and needs to be significantly improved. Scaffolds and/or matrices loaded with cells are often transplanted into a bone defect immediately after the defect has been created. At this point, the nutrient and oxygen supply is low and the inflammatory cascade is incited, thus creating a highly unfavorable microenvironment for transplanted cells to survive and participate in the regeneration process. We therefore developed a unique treatment concept using the delayed injection of allogenic bone marrow stromal cell (BMSC) sheets to regenerate a critical-sized tibial defect in sheep to study the effect of the cells’ regeneration potential when introduced at a postinflammatory stage. Minimally invasive percutaneous injection of allogenic BMSCs into biodegradable composite scaffolds 4 weeks after the defect surgery led to significantly improved bone regeneration compared with preseeded scaffold/cell constructs and scaffold-only groups. Biomechanical testing and microcomputed tomography showed comparable results to the clinical reference standard (i.e., an autologous bone graft). To our knowledge, we are the first to show in a validated preclinical large animal model that delayed allogenic cell transplantation can provide applicable clinical treatment alternatives for challenging bone defects in the future

An RNA-seq time series of the medaka pituitary gland during sexual maturation

Abstract Directing both organismal homeostasis and physiological adaptation, the pituitary is a key endocrine gland in all vertebrates. One of its major tasks is to coordinate sexual maturation through the production and release of hormones stimulating gonad development. In order to study its developmental dynamics in the model fish medaka (Oryzias latipes), we sampled both the pituitary and the ovaries of 68 female fish. Of these, 55 spanned the entire course of sexual maturation from prepubertal juveniles to spawning adults. An additional 13 showed either considerably faster or slower growth and development than the majority of fish. We used histological examination of the ovaries to determine a histological maturation stage, and analyzed the pituitary glands using RNA-seq optimized for low input. Taken together, these data reveal the timing of hormone production priorities, and form a comprehensive resource for the study of their regulation