An artificial metalloenzyme for a bimolecular Diels–Alder reaction

The Diels–Alder reaction, one of the most important in organic chemistry, forms functionalized six-membered cycloadducts in a single step. While widely used to construct complex biologically active molecules in the laboratory, [4+2] cycloadditions are rarely employed for natural product biosynthesis in cells owing to the lack of appropriate enzymes. Creating artificial metalloenzymes able to exploit Lewis acid catalysis for substrate activation could change this situation. Embedding a metal ion in a chiral protein binding pocket potentially combines the best aspects of two worlds – transition metal and enzymatic catalysis – to achieve both high activity and selectivity. Here we report the transformation of a zinc-binding helical bundle into an artificial metalloenzyme that efficiently catalyzes a hetero-Diels–Alder reaction between 3-vinyl indole and an azachalcone derivative by a process of design and laboratory evolution. The best enzyme, DA7, performed \u3e15,000 turnovers per active site and produced only a single product stereoisomer (\u3e99% ee). Detailed kinetic analysis showed that this catalyst is more than two orders of magnitude more proficient than other known Diels–Alderases, including many designed catalysts and natural enzymes involved in polyketide natural products biosynthesis. The remarkable activity of DA7 can be ascribed to the Zn(II) ion, which activates the heterodiene for reaction, and a shape complementary binding pocket that preorganized the reactants for efficient reaction and exacting control over chemo-, diastereo-, and enantioselectivity. These results establish the feasibility of combining design and evolution to harness the structural and functional properties of metal ions to produce remarkably active enzymes for an important abiological reaction. Extending this approach to metal ions other than zinc, and to scaffolds beyond helical bundles, can be expected to produce proficient custom-metalloenzymes for a wide spectrum of unnatural chemical transformations

Environmental harm assessment of a wastewater discharge from Hammerfest LNG: A study with biomarkers in mussels (Mytilus sp.) and Atlantic cod (Gadus morhua)

Biologically treated wastewater (WW) from the Hammerfest LNG (liquefied natural gas) plant is discharged to the sea. A study using biomarkers in mussels and Atlantic cod was performed to examine whether this discharge meets a zero harmful emission requirement. Caging of mussels close to the outfall and exposure of mussels and fish to WW in the laboratory were conducted, and a suite of contaminant responsive markers was assessed in exposed animals. In mussels the markers included chemical contaminant levels, haemocyte lysosomal instability and nucleus integrity, cellular energy allocation, digestive gland and gonad histopathology and shell-opening behaviour. In fish, biliary PAH metabolites and gill histopathology biomarkers were measured. A consistent cause-effect relationship between WW treatments and markers measured in test animals was not found. The results therefore indicate that the WW emission is unlikely to represent a significant stress factor for the local marine environment under the conditions studied.acceptedVersio

Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE), or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML). However, shared CD123 expression on healthy hematopoietic stem and progenitor cells (HSPCs) bears the risk for myelotoxicity. We demonstrate that epitope-engineered HSPCs were shielded from CD123-targeted immunotherapy but remained functional, while CD123-deficient HSPCs displayed a competitive disadvantage. Transplantation of genome-edited HSPCs could enable tumor-selective targeted immunotherapy while rebuilding a fully functional hematopoietic system. We envision that this approach is broadly applicable to other targets and cells, could render hitherto undruggable targets accessible to immunotherapy, and will allow continued posttransplant therapy, for instance, to treat minimal residual disease (MRD)

What can inactivity (in its various forms) reveal about affective states in non-human animals? A review

Captive/domestic animals are often described as inactive, with the implicit or explicit implication that this high level of inactivity is a welfare problem. Conversely, not being inactive enough may also indicate or cause poor welfare. In humans, too much inactivity can certainly be associated with either negative or positive affective states. In non-human animals, however, the affective states associated with elevated or suppressed levels of inactivity are still not well understood. Part of the complexity is due to the fact that there are many different forms of inactivity, each likely associated with very different affective states. This paper has two aims. One is to identify specific forms of inactivity that can be used as indicators of specific affective states in animals. The other is to identify issues that need to be resolved before we could validly use the remaining, not yet validated forms of inactivity as indicators of affective state. We briefly discuss how inactivity is defined and assessed in the literature, and then how inactivity in its various forms relates to affective (either negative or positive) states in animals, basing our reasoning on linguistic reports of affective states collected from humans displaying inactivity phenotypically similar to that displayed by animals in similar situations, and, when possible, on pharmacological validation. Specific forms of inactivity expressed in response to perceived threats (freezing, tonic immobility, and hiding) appear to be, to date, the best-validated indicators of specific affective states in animals. We also identify a number of specific forms of inactivity likely to reflect either negative (associated with ill-heath, boredom-like, and depression-like conditions), or positive states (e.g. ‘sun-basking’, post-consummatory inactivity), although further research is warranted before we could use those forms as indicators of the affective states. We further discuss the relationship between increased inactivity and affective states by presenting misleading situations likely to yield wrong conclusions. We conclude that more attention should be paid to inactivity in animal welfare studies: specific forms of inactivity identified in this paper are, or have the potential to be, useful indicators of affective (welfare) states in animals

The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis.

Background: Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: In this systematic analysis, we used a candidate gene approach to identify mutations associated with resistance or consistent with susceptibility for 13 WHO-endorsed antituberculosis drugs. We collected existing worldwide MTBC whole-genome sequencing data and phenotypic data from academic groups and consortia, reference laboratories, public health organisations, and published literature. We categorised phenotypes as follows: methods and critical concentrations currently endorsed by WHO (category 1); critical concentrations previously endorsed by WHO for those methods (category 2); methods or critical concentrations not currently endorsed by WHO (category 3). For each mutation, we used a contingency table of binary phenotypes and presence or absence of the mutation to compute positive predictive value, and we used Fisher's exact tests to generate odds ratios and Benjamini-Hochberg corrected p values. Mutations were graded as associated with resistance if present in at least five isolates, if the odds ratio was more than 1 with a statistically significant corrected p value, and if the lower bound of the 95% CI on the positive predictive value for phenotypic resistance was greater than 25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: We analysed 41 137 MTBC isolates with phenotypic and whole-genome sequencing data from 45 countries. 38 215 MTBC isolates passed quality control steps and were included in the final analysis. 15 667 associations were computed for 13 211 unique mutations linked to one or more drugs. 1149 (7·3%) of 15 667 mutations were classified as associated with phenotypic resistance and 107 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was more than 80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were identified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: We present the first WHO-endorsed catalogue of molecular targets for MTBC drug susceptibility testing, which is intended to provide a global standard for resistance interpretation. The existence of this catalogue should encourage the implementation of molecular diagnostics by national tuberculosis programmes. Funding: Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation

High-Throughput Engineering of Nonribosomal Extension Modules

Nonribosomal peptides constitute an important class of natural products that display a wide range of bioactivities. They are biosynthesized by large assembly lines called nonribosomal peptide synthetases (NRPSs). Engineering NRPS modules represents an attractive strategy for generating customized synthetases for the production of peptide variants with improved properties. Here, we explored the yeast display of NRPS elongation and termination modules as a high-throughput screening platform for assaying adenylation domain activity and altering substrate specificity. Depending on the module, display of A-T bidomains or C-A-T tridomains, which also include an upstream condensation domain, proved to be most effective. Reprograming a tyrocidine synthetase elongation module to accept 4-propargyloxy-phenylalanine, a noncanonical amino acid that is not activated by the native protein, illustrates the utility of this approach for altering NRPS specificity at internal sites.ISSN:1554-8929ISSN:1554-893

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