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In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. We identified two groups of exonucleases from the Herpes Virus and the bacteriophage T7 families that conferred an up to 38-fold increase in HDR frequencies when fused to Cas9/Cas12a in a Tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrate stable and heritable knock-ins of in wheat in 1% of the primary transformants. Our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

A bottleneck in the development of new anti‐cancer drugs is the recognition of their mode of action (MoA). Metabolomics combined with machine learning allowed to predict MoAs of novel anti‐proliferative drug candidates, focusing on human prostate cancer cells (PC‐3). As proof of concept, 38 drugs are studied with known effects on 16 key processes of cancer metabolism, profiling low molecular weight intermediates of the central carbon and cellular energy metabolism (CCEM) by LC‐MS/MS. These metabolic patterns unveiled distinct MoAs, enabling accurate MoA predictions for novel agents by machine learning. The transferability of MoA predictions based on PC‐3 cell treatments is validated with two other cancer cell models, i.e., breast cancer and Ewing\'s sarcoma, and show that correct MoA predictions for alternative cancer cells are possible, but still at some expense of prediction quality. Furthermore, metabolic profiles of treated cells yield insights into intracellular processes, exemplified for drugs inducing different types of mitochondrial dysfunction. Specifically, it is predicted that pentacyclic triterpenes inhibit oxidative phosphorylation and affect phospholipid biosynthesis, as confirmed by respiration parameters, lipidomics, and molecular docking. Using biochemical insights from individual drug treatments, this approach offers new opportunities, including the optimization of combinatorial drug applications.

Histamine is a biogenic amine that can cause food poisoning in an increasing fraction of the population. Histamine detection and quantification are crucial for evaluating the freshness of food products and informing histamine-sensitive consumers regarding histamine concentration in fermented or processed food products. Several analytical methods exist for quantifying histamine from food samples, most based on chromatographic analysis. This review summarizes the current knowledge of analytical methodologies for detecting and quantifying histamine. We highlight the importance of using timely detection tools for biogenic amines to indicate the degree of freshness or deterioration of food. A multidisciplinary approach based on molecular and enzymatic methods for detecting and quantifying histamine and other biogenic amines is presented, where histamine dehydrogenase and histamine oxidase enzymes from microbial sources stand out as potential molecular tools for histamine detection, and with which rapid, scalable, and user-friendly assay platforms can be used. In addition to typical enzyme technology concerns, the enzymatic detection of histamine faces substrate specificity and substrate inhibition challenges that affect the specific identification of histamine and the detection limit of the enzymatic assay. These challenges can be overcome by enzyme engineering, immobilization, or their simultaneous integration to obtain biocatalysts with increased histamine detection, quantification, or performance.

Tubulysins are among the most recent antimitotic compounds to enter into antibody/peptide‐drug conjugate (ADC/PDC) development. Thus far, the design of the most promising tubulysin payloads relied on simplifying their structures, e.g., by using small tertiary amide N‐substituents (Me, Et, Pr) on tubuvaline residue. Cumbersome solution‐phase approaches are typically used for both syntheses and functionalization with cleavable linkers. p‐Aminobenzyl quaternary ammonium (PABQ) linkers were a remarkable advancement for targeted delivery, but the procedures to incorporate them into tubulysins are only of moderate efficiency. Here we describe a novel all‐on‐resin strategy permitting a loss‐free resin linkage and an improved access to super potent tubulysin analogs showing close resemblance to the natural compounds. For the first time, a protocol enables the integration of on‐resin tubulysin derivatization with, e.g., a maleimido‐Val‐Cit‐PABQ linker, which is a notable progress for the payload‐PABQ‐linker technology. The strategy also allows tubulysin diversification of the internal amide N‐substituent, thus enabling to screen a tubulysin library for the discovery of new potent analogs. This work provides ADC/PDC developers with new tools for both rapid access to new derivatives and easier linker‐attachment and functionalization.

Surfactants are molecules derived primarily from petroleum that can reduce the surface tension at interfaces. Their slow degradation is a characteristic that could cause environmental issues. This and other factors contribute to the allure of biosurfactants today. Progress has been made in this area of research, which aims to satisfy the need for effective surfactants that are not harmful to the environment. In previous studies, we demonstrated the surface tension activity of the Escherichia coli transmembrane protein OmpA. Here, we carried out DNA shuffling on ompA to improve its interfacial activity. We evaluated changes in interfacial tension when exposing mutants to a water-oil interface to identify the most promising candidates. Two mutants reached an interfacial tension value lower (9.10 mN/m and 4.24 mN/m) than the original protein OmpA (14.98 mN/m). Since predicted isoelectric point values are far from neutral pH, the charge of the protein was a crucial factor in explaining the migration of proteins towards the interface. Low molecular weight mutants did not exhibit a significant difference in their migration to the interface.

Introduction: The genus Clusia L. is mostly recognised for the production of prenylated benzophenones and tocotrienol derivatives.Objectives: The objective of this study was to map metabolome variation within Clusia minor organs at different developmental stages.Material and Methods: In total 15 organs/stages (leaf, flower, fruit, and seed) were analysed by UPLC‐MS and 1H‐ and heteronuclear multiple‐bond correlation (HMBC)‐NMR‐based metabolomics.Results: This work led to the assignment of 46 metabolites, belonging to organic acids(1), sugars(2) phenolic acids(1), flavonoids(3) prenylated xanthones(1) benzophenones(4) and tocotrienols(2). Multivariate data analyses explained the variability and classification of samples, highlighting chemical markers that discriminate each organ/stage. Leaves were found to be rich in 5‐hydroxy‐8‐methyltocotrienol (8.5 μg/mg f.w.), while flowers were abundant in the polyprenylated benzophenone nemorosone with maximum level detected in the fully mature flower bud (43 μg/mg f.w.). Nemorosone and 5‐hydroxy tocotrienoloic acid were isolated from FL6 for full structural characterisation. This is the first report of the NMR assignments of 5‐hydroxy tocotrienoloic acid, and its maximum level was detected in the mature fruit at 50 μg/mg f.w. Seeds as typical storage organ were rich in sugars and omega‐6 fatty acids.Conclusion: To the best of our knowledge, this is the first report on a comparative 1D‐/2D‐NMR approach to assess compositional differences in ontogeny studies compared with LC‐MS exemplified by Clusia organs. Results derived from this study provide better understanding of the stages at which maximal production of natural compounds occur and elucidate in which developmental stages the enzymes responsible for the production of such metabolites are preferentially expressed.

Indole-3-carbinol (I3C) is a major dietary component produced in Brassica vegetables from glucosinolates (GLS) upon herbivores’ attack. The compound is gaining increasing interest due to its anticancer activity. However, reports about improving its level in plants or other sources are still rare. Unfortunately, I3C is unstable in acidic media and tends to polymerize rendering its extraction and detection challenging. This review presents a multifaceted overview of I3C regarding its natural occurrence, biosynthesis, isolation, and extraction procedure from dietary sources, and optimization for the best recovery yield. Further, an overview is presented on its metabolism and biotransformation inside the body to account for its health benefits and factors to ensure the best metabolic yield. Compile of the different analytical approaches for I3C analysis in dietary sources is presented for the first time, together with approaches for its detection and its metabolism in body fluids for proof of efficacy. Lastly, the chemopreventive effects of I3C and the underlying action mechanisms are summarized. Optimizing the yield and methods for the detection of I3C will assist for its incorporation as a nutraceutical or adjuvant in cancer treatment programs. Highlighting the complete biosynthetic pathway and factors involved in I3C production will aid for its future biotechnological production.