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[Nachruf] Willy Hilgenberg
(2013)
Plastic products leach chemicals that induce in vitro toxicity under realistic use conditions
(2021)
Plastic products contain complex mixtures of extractable chemicals that can be toxic. However, humans and wildlife will only be exposed to plastic chemicals that are released under realistic conditions. Thus, we investigated the toxicological and chemical profiles leaching into water from 24 everyday plastic products covering eight polymer types. We performed migration experiments over 10 days at 40 °C and analyzed the migrates using four in vitro bioassays and nontarget high-resolution mass spectrometry (UPLC-QTOF-MSE). All migrates induced baseline toxicity, 22 an oxidative stress response, 13 antiandrogenicity, and one estrogenicity. Overall, between 17 and 8681 relevant chemical features were present in the migrates. In other words, between 1 and 88% of the plastic chemicals associated with one product were migrating. Further, we tentatively identified ∼8% of all detected features implying that most plastic chemicals remain unknown. While low-density polyethylene, polyvinyl chloride, and polyurethane induced most toxicological endpoints, a generalization for other materials is not possible. Our results demonstrate that plastic products readily leach many more chemicals than previously known, some of which are toxic in vitro. This highlights that humans are exposed to many more plastic chemicals than currently considered in public health science and policies.
1. Das Wachstum und die Fähigkeit zur Butyratproduktion von E. callanderi KIST612 wurde in geschlossenen Batch-Kulturen mit den Substraten Glukose, Methanol, Formiat, H2 + CO2 und CO untersucht. E. callanderi KIST612 zeigte sich nur bei Wachstum auf 20 mM Glukose oder 20 mM Methanol in der Lage, Butyrat in größeren Mengen (3,7 – 4,3 mM) zu produzieren. Das Hauptprodukt bei allen untersuchten Wachstumssubstraten war jedoch Acetat.
2. In bioinformatischen Analysen des Genoms von E. callanderi KIST612 konnte nur eine A1AO-ATP-Synthase gefunden werden, welche eine V-typ c-Untereinheit bestehend aus 4 TMH‘s mit nur einer Na+-Bindestelle aufweist. Diese konnte aus gewaschenen Membranen von E. callanderi durch Saccharose-Dichtegradientenzentrifugation, Anionenaustausch-Chromatographie (DEAE) sowie einer Größenausschluss-Chromatographie (Superose 6) bis zur apparenten Homogenität gereinigt werden. Nach Produktion einzelner Untereinheiten (A, B, C, D, E, F und H) in E. coli und Generierung von Antikörpern, konnten alle Untereinheiten (A, B, C, D, E, F, H, a sowie c) in der gereinigten Enzympräparation immunologisch oder mittels „Peptide-Mass-Fingerprinting“ nachgewiesen werden. Es konnte somit erstmals eine A1AO-ATP-Synthase aus einem mesophilen Organismus ohne Verlust von Untereinheiten gereinigt werden.
3. Der Gesamtkomplex wies unter nativen Bedingungen eine molekulare Masse von ca. 670 kDa auf. In elektronenmikroskopischen Aufnahmen zeigte sich anhand der hantelförmigen Strukturen, dass die A1AO-ATP-Synthase als intakter Gesamtkomplex gereinigt werden konnte.
4. Die gereinigte A1AO-ATP-Synthase wurde zunächst anhand ihrer ATP-Hydrolyse-Aktivität biochemisch charakterisiert. Die ATP-Hydrolyse-Aktivität hatte ein pH-Optimum von 7 – 7,5 und ein Temperaturoptimum bei 37 °C. Durch Messung der ATPase-Aktivität in Abhängigkeit von verschiedenen Mengen an Na+ konnte die vorhergesagte Na+-Abhängigkeit des Enzyms nachgewiesen werden. Zudem zeigten Hemmstoffexperimente mit DCCD, dass dieser Inhibitor mit Na+ um die gemeinsame Bindestelle in der c-Untereinheit konkurriert. Dies bestätigte nochmals, dass das Enzym funktionell gekoppelt gereinigt werden konnte.
5. Zur weiteren Untersuchung der Ionenspezifität wurde der an die ATP-Hydrolyse gekoppelte Ionentransport durch Rekonstitution des Enzyms in Liposomen und anschließender Messung des Na+- oder H+-Transports gemessen. In den Proteoliposomen konnte mit Hilfe von 22Na+ gezeigt werden, dass das Enzym Natriumionen translozieren kann. Während in Anwesenheit des Natriumionophors ETH 2120 kein 22Na+-Transport beobachtet werden konnte, führte die Anwesenheit des Protonophors TCS zu einer geringfügigen Stimulation der 22Na+-Translokation. Insgesamt konnte ein primärer Na+-Transport nachgewiesen werden, welcher von der A1AO-ATP-Synthase aus E. callanderi katalysiert wird.
6. Durch Rekonstitution der A1AO-ATP-Synthase aus E. callanderi in Liposomen konnte erstmals biochemisch nachgewiesen werden, dass ein solches Enzym trotz seiner V-Typ c-Untereinheit in der Lage ist, ATP zu synthetisieren. Durch die Zugabe von Ionophoren (ETH 2120 und TCS) konnte der elektrochemische Ionengradient aufgehoben werden, wodurch keine ATP-Synthese beobachtet werden konnte. Der erstmalige Nachweis der ATP-Synthese wurde bei einem ΔµNa+ von 270 mV erbracht.
7. Die ATP-Synthese zeigte sich ebenfalls abhängig von der Na+-Konzentration. Der KM-Wert lag bei 1,1 ± 0,4 mM und war vergleichbar mit dem für die ATP-Hydrolyse ermittelten Wert. Ebenso konnte für die ATP-Synthese-Richtung gezeigt werden, dass DCCD mit Na+ um die gemeinsame Bindestelle in der c-Untereinheit konkurriert.
8. Um den biochemischen Nachweis zu erbringen, dass die A1AO-ATP-Synthase auch unter physiologisch relevanten Potentialen zur ATP-Synthese befähigt ist, wurde der energetische Schwellenwert der ATP-Synthese bestimmt. Dieser betrug 87 mV als Triebkraft für ΔpNa, 94 mV als Triebkraft für Δψ und 90 mV als Triebkraft für ΔµNa+. Erstaunlicherweise konnte die ATP-Synthese der A1AO-ATP-Synthase aus E. callanderi KIST612 sowohl durch Δψ als auch ΔpNa angetrieben werden. Unterschiedliche Kombinationen von Δψ und ΔpNa führten zu dem gleichen energetischen Schwellenwert; Δψ und ΔpNa waren im Enzym aus E. callanderi KIST612 äquivalente Triebkräfte.
9. Der energetische Schwellenwert der A1AO-ATP-Synthase aus E. callanderi KIST612 wurde mit dem der F1FO-ATP-Synthasen aus A. woodii, E. coli und P. modestum verglichen. Dazu wurden die Enzyme im ATP-Synthase-defizienten E. coli-Stamm DK8 produziert und anschließend durch Ni2+-NTA-Affinitätschromatographie gereinigt. Nach Einbau der Enzyme in Liposomen waren alle Enzyme in der Lage, ATP als Reaktion auf ΔµNa+ (A. woodii und P. modestum) oder ΔµH+ (E. coli) zu synthetisieren. Im Vergleich zum Enzym aus E. callanderi zeigten sich zwei auffällige Unterschiede. Erstens war keine der F1FO-ATP-Synthasen in der Lage, ΔpNa/ΔpH als alleinige Triebkraft zu nutzen. Während die ATP-Synthese in den Enzymen aus E. coli und P. modestum nur durch ΔµH+ bzw. ΔµNa+ angetrieben werden konnte, konnte das Enzym aus A. woodii zusätzlich auch durch Δψ als einzige Triebkraft angetrieben werden.
...
Nature's non-material contributions to people are difficult to quantify and one aspect in particular, nature's contributions to communication (NCC), has so far been neglected. Recent advances in automated language processing tools enable us to quantify diversity patterns underlying the distribution of plant and animal taxon labels in creative literature, which we term BiL (biodiversity in literature). We assume BiL to provide a proxy for people's openness to nature's non-material contributions enhancing our understanding of NCC. We assembled a comprehensive list of 240,000 English biological taxon labels. We pre-processed and searched a subcorpus of digitised literature on Project Gutenberg for these labels. We quantified changes in biodiversity indices commonly used in ecological studies for 16,000 books, encompassing 4,000 authors, as proxies for BiL between 1705 and 1969. We observed hump-shape patterns for taxon label richness, abundance and Shannon diversity indicating a peak of BiL in the middle of the 19th century. This is also true for the ratio of biological to general lexical richness. The variation in label use between different sections within books, quantified as β-diversity, declined until the 1830s and recovered little, indicating a less specialised use of taxon labels over time. This pattern corroborates our hypothesis that before the onset of industrialisation BiL may have increased, reflecting several concomitant influences such as the general broadening of literary content, improved education and possibly an intensified awareness of the starting loss of biodiversity during the period of romanticism. Given that these positive trends continued and that we do not find support for alternative processes reducing BiL, such as language streamlining, we suggest that this pronounced trend reversal and subsequent decline of BiL over more than 100 years may be the consequence of humans’ increasing alienation from nature owing to major societal changes in the wake of industrialisation. We conclude that our computational approach of analysing literary communication using biodiversity indices has a high potential for understanding aspects of non-material contributions of biodiversity to people. Our approach can be applied to other corpora and would benefit from additional metadata on taxa, works and authors.
This work comprises the investigation of four different biosynthesis gene clusters from Xenorhabdus. Xenorhabdus is an entomopathogenic bacterium that lives in mutualistic symbiosis with its Steinernema nematode host and together they infect and kill insect larvae. Xenorhabdus is well known for the production of so-called specialised metabolites and many of these compounds are synthesised by non-ribosomal peptide synthetases (NRPSs) or NRPS-polyketide synthase (PKS)-hybrids. These enzymes are organised in a modular manner and produce structurally very diverse molecules, often with the help of modifying domains and tailoring enzymes. In general, the genes involved in the biosynthesis are organised in so-called biosynthetic gene clusters (BGCs) in the genome of the producing strain. Exchanging the native promoter with an inducible promoter, e.g. PBAD, allows the targeted activation of the BGC and in turn the analysis of the biosynthesis product via LC-MS analysis.
The first BGC investigated in this work is responsible for the biosynthesis of xenofuranones. Based on gene deletions, this work shows that the NRPS-like enzyme XfsA produces a carboxylated furanone intermediate which is subsequently decarboxylated by XfsB to yield xenofuranone B. The next step in xenofuranone biosynthesis is the O-methylation of xenofuranone B to yield xenofuranone A. A comparative proteomics approach allowed the identification of four methyltransferase candidates and subsequent gene deletions confirmed one of the candidates to be responsible for methylation of xenofuranone B. The proteome analysis was based on the comparison of X. szentirmaii WT and X. szentirmaii Δhfq because distinct levels of the methylated xenofuranone A were observed when the xfs BGC was activated in either WT or Δhfq strain. Hfq is a global transcriptional regulator whose deletion is associated with the down regulation of natural product biosynthesis in Xenorhabdus. The strong PBAD activation of the xfs BGC also allowed the detection of two novel xenofuranone derivatives which arise from incorporation of one 4-hydroxyphenylpyruvic acid as first or second building block, respectively.
PBAD based activation of the second BGC addressed in this work lead to the detection of a novel metabolite and compound purification allowed NMR-based structure elucidation. The molecule exhibits two pyrrolizidine moieties and was named pyrrolizwilline (pyrrolizidine + twin (German: “Zwilling”)). The BGC comprises seven genes and single gene deletions as well as heterologous expression in E. coli and NRPS engineering were conducted to investigate the biosynthesis. The first two genes xhpA and xhpB encode a bimodular NRPS and a monooxygenase which synthesise a pyrrolizixenamide-like structure, similar to PxaA and PxaB in pyrrolizixenamide biosynthesis. It is suggested that the acyl side chain incorporated by XhpA is removed by the α,β-hydrolase XhpG. The keto function is then reduced by two subsequent two electron reductions catalysed by XhpC and XhpD. One of these two reduced pyrrolizidine units most likely is extended with glyoxalate prior to non-enzymatic dimerisation with the second pyrrolizidine moiety. To finally yield pyrrolizwilline, L-valine is incorporated, probably by the free-standing condensation domain XhpF.
The third BGC investigated is responsible for the production of a tripeptide composed of β-D-homoserine, α-hydroxyglycine and L-valine and is referred to as glyoxpeptide. This work demonstrates that the previously observed glyoxpeptide derivative is derived from glycerol present in the culture medium. Furthermore, this work shows that the monooxygenase domain, which is found in an unusual position between motifs A8 and A9 within the adenylation domain, is responsible for the α-hydroxylation of glycine. It is suggested that the α-hydroxylation of glycine renders the tripeptide prone to hydrolysis via hemiacetal formation. Hence, the XgsC_MonoOx domain might be an interesting candidate for further NRPS engineering.
The fourth BGC addressed is responsible for the production of xildivalines and this work describes two additional derivatives which are detected only when the promoter is exchanged and activated in the X. hominickii WT strain but not in X. hominickii Δhfq. Deletion of the methyltransferase encoding gene xisE results in the production of non-methylated xildivalines. It remains to be determined when the N-methylation of L-valine takes place. It is discussed that the methyltransferase could act on the NRPS released product but also during the assembly. The peptide deformylase is not involved in the proposed biosynthesis as xildivaline production is detected in a ΔxisD strain. The PKS XisB features two adjacent, so-called tandem T domains. The inactivation of the first or the second T domain by point mutation causes decreased production titres of detected xildivalines in the respective mutant strain when compared to the wild type.
Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.
Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.