Structures of the Apo and FAD-Bound Forms of 2-Hydroxybiphenyl 3-monooxygenase (HbpA) Locate Activity Hotspots Identified by Using Directed Evolution

The FAD-dependent monooxygenase HbpA from Pseudomonas azelaica HBP1 catalyses the hydroxylation of 2-hydroxybiphenyl (2HBP) to 2,3-dihydroxybiphenyl (23DHBP). HbpA has been used extensively as a model for studying flavoprotein hydroxylases under process conditions, and has also been subjected to directed-evolution experiments that altered its catalytic properties. The structure of HbpA has been determined in its apo and FAD-complex forms to resolutions of 2.76 and 2.03 Å, respectively. Comparisons of the HbpA structure with those of homologues, in conjunction with a model of the reaction product in the active site, reveal His48 as the most likely acid/base residue to be involved in the hydroxylation mechanism. Mutation of His48 to Ala resulted in an inactive enzyme. The structures of HbpA also provide evidence that mutants achieved by directed evolution that altered activity are comparatively remote from the substrate-binding site

Preparative Scale Achmatowicz and aza-Achmatowicz Rearrangements Catalyzed by Agrocybe aegerita Unspecific Peroxygenase

The unspecific peroxygenase (UPO) from Agrocybe aegerita (rAaeUPO-PaDa-I-H) is an effective and practical biocatalyst for the oxidative expansion of furfuryl alcohols/amines on a preparative scale, using the Achmatowicz and aza-Achmatowicz reaction. The high activity and stability of the enzyme, which can be produced on large scale as an air-stable lyophilised powder, renders it a versatile and scalable biocatalyst for the preparation of synthetically valuable 6-hydroxypyranones and dihydropiperidinones. In several cases, the biotransformation out-performed the analogous chemo-catalysed process, and operates under milder and greener reaction conditions

Complementary Specificity of Unspecific Peroxygenases (UPOs) Enables Access to Diverse Products from Terpene Oxygenation

Unspecific Peroxygenases (UPOs) have emerged as attractive biocatalysts for selective oxygenations as, unlike cytochromes P450, they can be employed as easy-to-use lyophilised powders and depend only upon hydrogen peroxide as the external oxidant. The application of UPOs to a range of synthetic challenges relies on the characterisation of activity and specificity of complementary enzymes. Here we show that two UPOs, artUPO and rAaeUPO-PaDa-I-H, which are representative members of the ‘short’ Family I and ‘long’ Family II UPOs, display complementary activity in a series of scalable, preparative biotransformations of a diverse array of terpenes. The UPOs were also applied to the biotransformation of chrysanthemic acid derived fragments relevant in the agrichemical industry, culminating in the highly diastereoselective and enantioselective oxidation of a racemic synthetic pyrethroid derivative via kinetic resolution

The Right Light – De Novo Design of a Robust Modular Photochemical Reactor for Optimum Batch and Flow Chemistry

Having identified inconsistencies when repeating literature examples of photochemical transformations and difficulties recreating experimental setups, we devised several criteria that an ideal labscale reactor should achieve. Herein, we introduce a versatile photoreactor for high throughput screening, preparative scale batch reactions and continuous processing, all with a single light source. The reactor utilizes interchangeable arrays of pseudo-monochromatic high-power LEDs in a range of synthetically useful wavelengths, combined with excellent temperature control. Moreover, light intensity can be modulated in an accurate and straightforward manner. This system has subsequently been tested on a range of literature methodologies

Chromoselective Photocatalysis Enables Stereocomplementary Biocatalytic Pathways

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photochemo-enzymatic cascades that give either the (S)- or the (R)- enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CNOA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99% e.e.). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93% e.e.)

Loss of Metabolic Fitness Drives Tumor Resistance After CAR-NK Cell Therapy and Can Be Overcome by Cytokine Engineering

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells is promising, with early-phase clinical studies showing encouraging responses. However, the transcriptional signatures that control the fate of CAR-NK cells after infusion and factors that influence tumor control remain poorly understood. We performed single-cell RNA sequencing and mass cytometry to study the heterogeneity of CAR-NK cells and their in vivo evolution after adoptive transfer, from the phase of tumor control to relapse. Using a preclinical model of noncurative lymphoma and samples from a responder and a nonresponder patient treated with CAR19/IL-15 NK cells, we observed the emergence of NK cell clusters with distinct patterns of activation, function, and metabolic signature associated with different phases of in vivo evolution and tumor control. Interaction with the highly metabolically active tumor resulted in loss of metabolic fitness in NK cells that could be partly overcome by incorporation of IL-15 in the CAR construct

Functional parameters indicative of mild cognitive impairment: a systematic review using instrumented kinematic assessment

Background: Patients with mild cognitive impairment (MCI) experience alterations of functional parameters, such as an impaired balance or gait. The current systematic review set out to investigate whether functional objective performance may predict a future risk of MCI; to compare functional objective parameters in patients with MCI and a control group; and to assess changes in these parameters after different physical activity interventions. Methods: Electronic databases, including PubMed, AMED, CINAHL, EMBASE, PEDro and Web of Science as well as grey literature databases, were searched from inception to February 2020. Cohort studies and Randomized Controlled Trials (RCTs) were included. The risk of bias of the included studies was assessed independently by reviewers using quality assessment checklists. The level of evidence per outcome was assessed using the GRADE criteria. Results: Seventeen studies met inclusion criteria including patients with MCI. Results from RCTs suggested that gait speed, gait variability and balance may be improved by different physical activity interventions. Cohort studies showed that slower gait speed, above all, under Dual Task (DT) conditions, was the main impaired parameter in patients with MCI in comparison with a Control Gorup. Furthermore, cohort studies suggested that gait variability could predict an incident MCI. Although most of included cohort studies reported low risk of bias, RCTs showed an unclear risk of bias. Conclusions: Studies suggest that gait variability may predict an incident MCI. Moreover, different gait parameters, above all under DT conditions, could be impaired in patients with MCI. These parameters could be improved by some physical activity interventions. Although cohort studies reported low risk of bias, RCTs showed an unclear risk of bias and GRADE criteria showed a low level of evidence per outcome, so further studies are required to refute our findings

Heterologous Expression and Characterisation of Unspecific Peroxygenases

In 2004, unspecific peroxygenases (UPOs) from fungi were first identified and showed to catalyse selective oxygenation reactions, with high turnovers, good stability and a broad reaction scope, providing a valuable alternative to established biocatalytic hydroxylation systems such as cytochrome P450s. At the start of this project, access to UPOs was limited to expression in their native fungi, hence studies were performed looking into heterologous expression of the enzymes from Agrocybe aegerita and Agaricus bisporus, followed by characterisation, and application of the enzymes

Juristische Lehrcomics – Anforderungen und Möglichkeiten

Ziel unseres Beitrags ist die Untersuchung, inwiefern sich Comics als Darstellungsform für Lehrbücher im juristischen Bereich eignen. Dazu erläutern wir zunächst die Begriffe Comic, Cartoon und Graphic Novel und grenzen sie voneinander ab. Es folgt ein Überblick zum bisherigen Einsatz von Comics oder Cartoons in den Bereichen Sachbuch und wissenschaftliches Lehrbuch auch im Vergleich zu anderen Visualisierungsformen sowie den möglichen Vorteilen einer comicartigen Darstellung. Am Ende wird ein Problem aus dem Erbrecht beispielhaft als Comic sowie als Cartoon dargestellt

Selective Oxidations of Toluenes and Benzyl Alcohols by an Unspecific Peroxygenase (UPO)

Unspecific Peroxygenases (UPOs) have emerged as robust biocatalysts for selective oxygenation reactions, as they are easily produced at scale and require only hydrogen peroxide as the external oxidant. UPOs can catalyze the oxygenation of the primary benzylic carbons of toluenes to give alcohol, aldehyde and carboxylic acid products. They can also catalyze hydroxylation at the benzylic position of ethylbenzenes, and the subsequent oxidation of the secondary alcohols to ketones. In this study, we have investigated factors that affect the balance of products in UPO-catalyzed benzylic oxygenations using a range of functionalised toluenes and ethyl benzenes, and a UPO from Agrocybe aegerita (rAaeUPO-PaDa-I-H variant). The product distribution is dependent upon a mixture of steric and electronic effects and, in selected cases, controlling the reaction conditions permits products from each product series to be generated chemoselectively. In this way, electron poor toluenes were converted directly into carboxylic acids in isolated yields of 36–99% on preparative scale