Strukturelle Charakterisierung von Genprodukten aus uncharakterisierten Operons aus Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen. It is considered as one of the most persistent infectious bacteria in human disease, leading to a high rate of morbidity. In most cases, P. aeruginosa infections strike hospitalized people and/or cystic fibrosis patients, where it is the main reason for low life expectancy. The genome of P. aeruginosa covers 6.3 million base pairs. Common with other sequenced organisms, a high percentage (approx. 40%) of the identified genes are functionally not annotated. In order to unveil new opportunities for drug discovery, it is important to shed light onto this “white space” of the genome map. Here the results of the pilot-phase of new structure based approach with follow up metabolomic analysis are reported. In this approach it is made use of the fact that in prokaryotes functionally linked genes are usually clustered in operons that can be predicted by bioinformatics methods. Regarding these operons loss- and gain-of-function mutants have been generated to be used for follow up metabolomic analysis. Further, x-ray crystallography was employed to gain detailed structural insight and to derive additional functional hypothesis. Genes of two selected operons, PA1621-PA1624 and PA5506-PA5509, both comprising previously uncharacterized genes were structurally elucidated in the study, leading to five new crystals structures determined, employing x-ray crystallographic methods. Amongst them, a transcription regulator which is involved in regulation of bacterial virulence and is therefore an interesting putative drug target. Furthermore, a new protein fold was discovered with no similarity to any previously reported structures deposited to the Protein Data Bank (PDB).Pseudomonas aeruginosa ist ein gram-negatives opportunistisches human-pathogenes Bakterium, welches in den letzten Jahren auf Grund seiner weiten Verbreitung und rapiden Resistenzentwicklung immer mehr in den Focus der wissenschaftlichen Forschung rückt. Vor allem in Krankenhäusern infiziert es immunsupprimierte Patienten, oder Patienten, deren natürliches Abwehrsystem bereits durch eine andere Krankheit geschwächt ist, z.B. Mukoviszidose Patienten, deren zähflüssiger Schleim in den Bronchien einen idealen Nährboden bietet, sind besonders schlimm betroffen im Falle einer Pseudomonas Infektion. Das Genom von P. aeruginosa wurde vor mehr als zehn Jahren sequenziert und umfasst ungefähr 6,5 Millionen Basenpaare, die sich in auf 5600 Gene aufteilen. Neu sequenzierte Organismen weisen generell eine Vielzahl unbekannter Gene auf, deren Zahl auf ungefähr 40% der Genomgröße beziffert wird. Allerdings ist es von entscheidender Wichtigkeit um einen Organismus charakterisieren können, nicht nur die Sequenz des Genoms zu kennen, sondern auch die Funktion der translatierten Genprodukte. In der hier vorliegenden Arbeit werden die Ergebnisse aus der Pilot-Phase eines neuartigen Ansatzes zur Annotierung zuvor unbekannter Gene berichtet, der sich von anderen dieser Art vor allem darin unterscheidet, dass nicht Proteine alleine charakterisiert werden, sondern diese zunächst im ihrem physiologischen Kontext annotiert werden bevor die individuelle Betrachtung folgt. In dieser Arbeit wurden zwei Operons unbekannter Funktion betrachtet, PA1621-PA1624 und PA5506-PA5509. Mittels Methoden der Röntgenkristallographie konnten fünf Protein Strukturen bestimmt werden; unter ihnen ein interessanter Transkriptionsfaktor, der indirekt in die Regulation bakterieller Virulenz involviert scheint und ein neuartige Form der Proteinfaltung, die keine Homologien zu bekannten Strukturen aufweist. Weiterhin wurden Pseudomonas Mutanten erzeugt, in denen eines der selektierten Operons deletiert wurde, um diese in späteren vergleichenden Metabolomic Studien zur Identifikation von Unterschieden in der Metabolom-Zusammensetzung zu nutzen

Reconstructing the infrared spectrum of a peptide from representative conformers of the full canonical ensemble

Leucine enkephalin (LeuEnk), a biologically active endogenous opioid pentapeptide, has been under intense investigation because it is small enough to allow efficient use of sophisticated computational methods and large enough to provide insights into low-lying minima of its conformational space. Here, we reproduce and interpret experimental infrared (IR) spectra of this model peptide in gas phase using a combination of replica-exchange molecular dynamics simulations, machine learning, and ab initio calculations. In particular, we evaluate the possibility of averaging representative structural contributions to obtain an accurate computed spectrum that accounts for the corresponding canonical ensemble of the real experimental situation. Representative conformers are identified by partitioning the conformational phase space into subensembles of similar conformers. The IR contribution of each representative conformer is calculated from ab initio and weighted according to the population of each cluster. Convergence of the averaged IR signal is rationalized by merging contributions in a hierarchical clustering and the comparison to IR multiple photon dissociation experiments. The improvements achieved by decomposing clusters containing similar conformations into even smaller subensembles is strong evidence that a thorough assessment of the conformational landscape and the associated hydrogen bonding is a prerequisite for deciphering important fingerprints in experimental spectroscopic data.</p

DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis

RNA上の遺伝情報を書き換える酵素であるDYWドメインの構造を解明 --植物オルガネラRNA編集のユニークな活性制御--. 京都大学プレスリリース. 2021-06-23.RNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts—mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated. DYW domains harbour a cytidine deaminase fold and a C-terminal DYW motif, with catalytic and structural zinc atoms, respectively. A conserved gating domain within the deaminase fold regulates the active site sterically and mechanistically in a process that we termed gated zinc shutter. Based on the structures, an autoinhibited ground state and its activation are cross-validated by RNA editing assays and differential scanning fluorimetry. We anticipate that, in vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated cytidine deamination playing a key role in mitochondrial and chloroplast homeostasis

CORDATA: an open data management web application to select corrosion inhibitors

The large amount of corrosion inhibition efficiencies in literature, calls for a more efficient way to organize, access and compare this information. The CORDATA open data management application (https://datacor.shinyapps.io/cordata/) can help select appropriate corrosion inhibitors for application specific challenges.publishe

Inhaled Nitric Oxide Treatment for Aneurysmal SAH Patients With Delayed Cerebral Ischemia

BACKGROUND: We demonstrated experimentally that inhaled nitric oxide (iNO) dilates hypoperfused arterioles, increases tissue perfusion, and improves neurological outcome following subarachnoid hemorrhage (SAH) in mice. We performed a prospective pilot study to evaluate iNO in patients with delayed cerebral ischemia after SAH. METHODS: SAH patients with delayed cerebral ischemia and hypoperfusion despite conservative treatment were included. iNO was administered at a maximum dose of 40 ppm. The response to iNO was considered positive if: cerebral artery diameter increased by 10% in digital subtraction angiography (DSA), or tissue oxygen partial pressure (PtiO(2)) increased by > 5 mmHg, or transcranial doppler (TCD) values decreased more than 30 cm/sec, or mean transit time (MTT) decreased below 6.5 secs in CT perfusion (CTP). Patient outcome was assessed at 6 months with the modified Rankin Scale (mRS). RESULTS: Seven patients were enrolled between February 2013 and September 2016. Median duration of iNO administration was 23 h. The primary endpoint was reached in all patients (five out of 17 DSA examinations, 19 out of 29 PtiO(2) time points, nine out of 26 TCD examinations, three out of five CTP examinations). No adverse events necessitating the cessation of iNO were observed. At 6 months, three patients presented with a mRS score of 0, one patient each with an mRS score of 2 and 3, and two patients had died. CONCLUSION: Administration of iNO in SAH patients is safe. These results call for a larger prospective evaluation

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

Automation of the UNICARagil Vehicles

The German research project UNICARagil is a collaboration between eight universities and six industrial partners funded by the Federal Ministry of Education and Research. It aims to develop innovative modular architectures and methods for new agile, automated vehicle concepts. This paper summarizes the automation approach of the driverless vehicle concept and its modular realization within the four demonstration vehicles to be built by the consortium. On-board each vehicle, this comprises sensor modules for environment perception and modelling, motion planning for normal driving and safe halts, as well as the respective control algorithms and base functionalities like precise localization. A control room and cloud functionalities provide off-board support to the vehicles, which are additionally addressed in this paper

The Effect of Corporate Earnings Announcements on the Volatility of Equity Prices: A New Methodology for More Accurate and Reliable Predictions

This study examines the effect of corporate earnings announcements on volatility of equity prices. It examines the dynamics of why stock prices of certain publicly listed companies are very sensitive to new information and others seemingly not at all. While the economic literature is rich in the study of the asymmetrical nature of information and its effect on the actions of market participants, there is gap in the literature about how information has a differential effect on the actions of investors based on the endogenous characteristics of individual companies. Using multiple regression analysis with panel data, this study examines the historical stock price movements of all the companies listed on the NASDAQ stock exchange from December 31, 2012 to December 31, 2018 and identifies the characteristics of those companies that reveal a disproportionate sensitivity in stock price movements to new information. The findings suggest that the relationship between a firm’s characteristics, new information, and stock price volatility are statistically significant. It concludes that the elasticities in stock prices to future quarterly earnings announcements can be predicted and used to develop a more reliable and profitable options-based, earnings announcement driven trading strategy