Biochemical and Structural Characterization of β-Hydroxylases in Non-Ribosomal Peptide Biosynthesis

Hydroxylases are essential in the biosynthesis of natural products by functionalization of building blocks, which derive from primary metabolism. They provide hydrophily for specific interactions or enable further derivatisation, which ultimately results in a natural product with outstanding activity. Hydroxylases, which are associated with non-ribosomal peptide synthetases (NRPS) contribute to the generation of diverse compounds with great significance for human society such as antibiotics or tumour therapeutics. Their wide-ranging importance is in stark contrast to the scarce knowledge about their structure, functionality, and substrate recognition. Hence, the trans acting hydroxylases in the biosynthetases of the non-ribosomal peptides hypeptin, lysobactin and FR900359 were investigated in this work. Hypeptin is a cyclic octadepsipeptide from Lysobacter sp. with strong antibacterial activity against Gram-positive strains. The responsible biosynthetic gene cluster, (BGC) hyn encodes for two trans acting hydroxylases that are responsible for the β-hydroxylation of four amino acid residues. HynC belongs to the family of non-heme diiron monooxygenases (NHDM) and HynE is an iron- and α-ketoglutarate dependent hydroxylase. The characterization of HynC and HynE in vitro required the reconstitution of cognate NRPS modules, which were partly dependent on the MbtH-like protein, HynMLP that was not encoded within the hyn BGC. The successful reconstitution of HynC and HynE in vitro enabled determination of their substrate specificities, which were contradicting with the published stereoconfiguration of hypeptin. Finally, the reassigned configuration could be verified by in-depth NMR analyses. The cyclic depsipeptide lysobactin shows a similar activity compared to hypeptin. The respective lyb BGC encodes for the interesting NHDM LybC, which is predicted to hydroxylate three structurally diverse amino acids. The cognate NRPS modules were successfully reconstituted in vitro, but no activity of the hydroxylase could be detected. The selective and potent Gαq protein inhibitor FR9003559 (FR) is a cyclic octadepsipeptide with three β-hydroxylated leucine residues. During its biosynthesis, the monomodular NRPS FrsA, together with the NHDM synthesise the FR side chain N-propionylhydroxyleucine. Here, the biosynthesis of the side chain was reconstituted in vitro. To get a deeper insight into the mechanism of substrate recognition, the structure of FrsH was elucidated by crystallization trails and subsequent X-ray absorption spectroscopy. Additionally, the gene frsH was functionally substituted by hynC and lybC in the native producer Chromobacterium vaccinii, indicating a conserved structure of the NHDMs for substrate recognition. The gathered information were used for in silico docking studies to elucidate crucial residues for the interaction of FrsH and FrsA. Lastly, an assay was established to verify the outcome of the docking studies in vitro

Assessing the potential of perfect screw dislocations in SiC for solid-state quantum technologies

Although point defects in solids are one of the most promising physical systems to build functioning qubits, it remains challenging to position them in a deterministic array and to integrate them into large networks. By means of advanced ab initio calculations we show that undissociated screw dislocations in cubic 3C-SiC, and their associated strain fields, could be used to create a deterministic pattern of relevant point defects. Specifically, we present a detailed analysis of the formation energies and electronic structure of the divacancy in 3C-SiC when located in the vicinity of this type of dislocations. Our results show that the divacancy is strongly attracted towards specific and equivalent sites inside the core of the screw dislocations, and would form a one-dimensional arrays along them. Furthermore, we show that the same strain that attracts the divacancy allows the modulation of the position of its electronic states and of its charge transition levels. In the case of the neutral divacancy, we find that these modulations result in the loss of its potential as a qubit. However, these same modulations could transform defects with no potential as qubits when located in bulk, into promising defects when located inside the core of the screw dislocations. Since dislocations are still mostly perceived as harmful defects, our findings represent a technological leap as they show that dislocations can be used as active building blocks in future defect-based quantum computers

Rethinking Warning: Intelligence, Novel Events, and the COVID-19 Pandemic

17 USC 105 interim-entered record; under temporary embargo.U.S. Government affiliation is unstated in article text

Erste Beobachtungen mit dem abstimmbaren Infrarot-Heterodynsystem THIS

Gegenstand der vorliegenden Arbeit ist die technische Weiterentwicklung des durchstimmbaren Infrarot-Heterodynsystems THIS (Tuneable Heterodyne Infrared Spectrometer) sowie die Planung, Vorbereitung und Durchführung diverser Beobachtungseinsätze des Instruments an unterschiedlichen Teleskopen. Auf technischer Seite konnte der sehr erfolgreiche Einsatz von Quanten- Kaskadenlasern als Lokaloszillatoren, die Empfindlichkeit des Systems erstmals auf das Niveau von CO2-Laser gepumpten Instrumenten bringen. Frequenzstabilität und radiometrisches Verhalten des Instruments konnten für Beobachtungszeiträume von einigen Stunden nachgewiesen werden.Weiterhin wurde ein in den Empfänger integriertes, optisches Guide-System für den Einsatz an Teleskopen entwickelt und aufgebaut. Die neu entwickelte Optik macht THIS durch die Verwendung einer integrierten optischen Kamera sowie eines Scannerspiegels zur Auswahl von Beobachtungspositionen am Himmel weitgehend unabhängig vom Teleskoptyp und damit vielseitig einsetzbar. Die technischen Neuerungen und die mechanische Stabilität des neuen Aufbaus wurden u. a. während Messperioden am Observatorium Hoher List bei Daun/Eifel sowie am McMath-Pierce Solar Telescope auf dem Kitt Peak nahe Tucson/Arizona erstmalig außerhalb des Labors genutzt und getestet. Es konnten Molekül-Absorptionen aus Sonnenflecken aufgenommen und aus den vollständig aufgelösten Linienprofilen die Anregungstemperaturen des Materials bestimmt werden. In diesem Zusammenhang wurde eine theoretische Analyse der Kalibration von am Teleskop gewonnenen Spektren durchgeführt und auf die gemessenen Daten angewandt. Weiterhin konnten erstmals mit THIS nicht-terrestrische Signale in Form von schwachen, nicht-thermischen CO2- Emissionen aus der von der Sonne beleuchteten Venus- und Mars-Atmosphäre detektiert werden. Die exzellente Empfindlichkeit des Instruments konnte zusätzlich durch die Beobachtung von stratosphärischem Ozon gegen einen Stern als Hintergrundquelle demonstriert werden. Weitere Beobachtungseinsätze von THIS sind in Zusammenarbeit mit dem GSFC der NASA in Vorbereitung. Beobachtungsziele sind interessante molekulare Absorptions- und Emissionslinien in Planetenatmosphären, die nicht mit Gaslaser-Instrumenten beobachtbar sind. Dazu zählt die hochaufgelöste Vermessung der bereits erwähnten CO2-Laseremissionen oder die Bestimmung von Häufigkeiten von leichten Kohlenwasserstoffen wie C2H4 oder C2H6

A Methodology for Three-Dimensional Quantification of Anterior Tooth Width

The use of cone-beam computed tomography (CBCT) technology has been shown to be more accurate in measuring individual incisor tooth widths than the use of wax exemplars. There were fewer differences by investigators using CBCT than others using an F-test in a mixed model of the measurement differences of investigators, wax type, and which tooth was measured. In addition, the frequency of outliers was less in the CBCT method (a total of 5) as compared to the two-dimensional measurements in ether Aluwax (a total of 8) or Coprwax (a total of 12). Both results indicate that CBCT measurements accounted more precisely for tooth width and level of eruption

Quantification of the Individual Characteristics of the Human Dentition

The considerations for admissibility suggested by the Daubert trilogy challenge forensic experts to provide scientific support for opinion testimony. The defense bar has questioned the reliability of bitemark analysis. Under an award from the U. S. Department of Justice, via the Midwest Forensic Resource Center, a two-year feasibility study was undertaken to quantify six dental characteristics. Using two computer programs, the exemplars of 419 volunteers were digitally scanned, characteristics were measured, and frequency was calculated. The study demonstrates that there were outliers or rare dental characteristics in measurements. An analysis of the intra-observer and inter-observer consistency demonstrated a high degree of agreement. Expansion of the sample size through collaboration with other academic researchers will be necessary to be able to quantify the occurrence of these characteristics in the general population. The automated software application, Tom\u27s Toolbox, developed specifically for this research project, could also provide a template for precisely quantifying other pattern evidence

Plio-Pleistozängrenze und Günzeiszeit in Nordwestdeutschland

researc

Evaluating Return On Investment For Software Process Improvement Projects

This article presents a simple approach that will allow decision-makers to evaluate the return on investment of software process improvement prior to launching such an effort. Obviously, it will be easy to tell ten years up the road whether the right decision was made. But a CEO, or CIO contemplating laying out six, or seven figures for the additional personnel and resources to conduct SPI is not in a position to make that call and the wise ones will not be led into it by blind faith. The problem is assessing the risks and returns of such a project in terms and perspective that a non-technical decision-maker can understand. We believe our instrument serves that purpose

Assessing the potential of perfect screw dislocations in SiC for solid-state quantum technologies

editorial reviewedAlthough point defects in solids can be used as qubits, it remains challenging to position them in a deterministic array. By means of advanced ab initio calculations we show that perfect screw dislocations in 3C-SiC could be used to overcome this limitation. In addition, we show that such dislocations can change the spin configuration of a point defect located inside its core, without changing the localized nature of its defect states. Our findings represent a technological leap as they show that dislocations can be used as active building blocks in future defect-based quantum computers