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Peroxygenases are heme-dependent enzymes that use peroxide-borne oxygen to catalyze a wide range of oxyfunctionalization reactions. Herein, we report the engineering of an unusual cofactor-independent peroxygenase based on a promiscuous tautomerase that accepts different hydroperoxides (t-BuOOH and H 2 O 2 ) to accomplish enantiocomplementary epoxidations of various α,β-unsaturated aldehydes (citral and substituted cinnamaldehydes), providing access to both enantiomers of the corresponding α,β-epoxy-aldehydes. High conversions (up to 98 %), high enantioselectivity (up to 98 % ee), and good product yields (50-80 %) were achieved. The reactions likely proceed via a reactive enzyme-bound iminium ion intermediate, allowing tweaking of the enzyme's activity and selectivity by protein engineering. Our results underscore the potential of catalytic promiscuity for the engineering of new cofactor-independent oxidative enzymes., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been associated with many diseases. Most studies found in literature describe MIF as a proinflammatory cytokine involved in chronic inflammatory conditions, but evidence from last years suggests that many of its key effects are not directly related to inflammation. In fact, MIF is constitutively expressed in most human tissues and in some cases in high levels, which does not reflect the pattern of expression of a classic proinflammatory cytokine. Moreover, MIF is highly expressed during embryonic development and decreases during adulthood, which point toward a more likely role as growth factor. Accordingly, MIF knockout mice develop age-related spontaneous emphysema, suggesting that MIF presence (e.g., in younger individuals and wild-type animals) is part of a healthy lung. In view of this new line of evidence, we aimed to review data on the role of MIF in the pathogenesis of chronic lung diseases.

Objectives: In this review, we focus on the application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome editing system, in the identification of resistance mechanisms and in overcoming drug resistance in the most frequent solid tumors., Data Acquisition: Data were collected by conducting systematic searching of scientific English literature using specific keywords such as "cancer", "CRISPR" and related combinations., Results: The review findings revealed the importance of CRISPR/Cas9 system in understanding drug resistance mechanisms and identification of resistance-related genes such as PBRM1, SLFN11 and ATPE1 in different cancers. We also provided an overview of genes, including RSF1, CDK5, and SGOL1, whose disruption can synergize with the currently available drugs such as paclitaxel and sorafenib., Conclusion: The data suggest CRISPR/Cas9 system as a useful tool in elucidating the molecular basis of drug resistance and improving clinical outcomes. Graphical abstract The mechanisms of CRISPR/Cas9-mediated genome editing and double-strand breaks (DSBs) repair.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is considered to be a promising antitumor drug because of its selective proapoptotic properties on tumor cells. However, the clinical application of TRAIL is until now limited because of the resistance of several cancer cells, which can occur at various levels in the TRAIL signaling pathway. The role of decoy receptors that can side-track TRAIL, thereby preventing the formation of an activated death receptor, has been extensively studied. In this study, we have focused on extracellular vesicles (EVs) that are known to play a role in cell-to-cell communication and that can be released by donor cells into the medium transferring their components to recipient cells. TRAIL-induced apoptotic signaling is triggered upon the binding of two death receptors, DR4 and DR5. Here, we found that DR5 but not DR4 is present in the conditioned medium (CM)-derived from various cancer cells. Moreover, we observed that DR5 was exposed on EVs and can act as "decoy receptor" for binding to TRAIL. This results in a strongly reduced number of apoptotic cells upon treatment with DR5-specific TRAIL variant DHER in CM. This reduction happened with EVs containing either the long or short isoform of DR5. Taken together, we demonstrated that colon rectal tumor cells can secrete DR5-coated EVs, and this can cause TRAIL resistance. This is to our knowledge a novel finding and provides new insights into understanding TRAIL sensitivity., (Copyright © 2020 Setroikromo, Zhang, Reis, Mistry and Quax.)

Enzymes have evolved to function under aqueous conditions and may not exhibit features essential for biocatalytic application, such as the ability to function in high concentrations of an organic solvent. Consequently, protein engineering is often required to tune an enzyme for catalysis in non-aqueous solvents. In this study, we have used a collection of nearly all single mutants of 4-oxalocrotonate tautomerase, which promiscuously catalyzes synthetically useful Michael-type additions of acetaldehyde to various nitroolefins, to investigate the effect of each mutation on the ability of this enzyme to retain its "Michaelase" activity in elevated concentrations of ethanol. Examination of this mutability landscape allowed the identification of two hotspot positions, Ser30 and Ala33, at which mutations are beneficial for catalysis in high ethanol concentrations. The "hotspot" position Ala33 was then randomized in a highly enantioselective, but ethanol-sensitive 4-OT variant (L8F/M45Y/F50A) to generate an improved enzyme variant (L8F/A33I/M45Y/F50A) that showed great ethanol stability and efficiently catalyzes the enantioselective addition of acetaldehyde to nitrostyrene in 40 % ethanol (permitting high substrate loading) to give the desired γ-nitroaldehyde product in excellent isolated yield (89 %) and enantiopurity (ee=98 %). The presented work demonstrates the power of mutability-landscape-guided enzyme engineering for efficient biocatalysis in non-aqueous solvents., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Receptor tyrosine kinases, such as VEGFR, PDGFR and EGFR, play important roles in renal cancer. In this study, we investigated EGFR knockout as a therapeutic approach in renal cell carcinoma (RCC). We showed that a renal cell carcinoma cell line (RC21) has higher expression of EGFR as compared to other frequently used cell lines such as HEK293, A549, Hela and DLD1. Ablation of EGFR by CRISPR/Cas9 significantly restrained tumor cell growth and activated the MAPK (pERK1/2) pathway. The VEGFR and PDGFR inhibitor, sunitinib, attenuated the expression of MAPK (pERK1/2) and pAKT induced by EGFR loss and further inhibited EGFR-/- cell proliferation. We showed that loss of EGFR eventually leads to resistance to SAHA and cisplatin. Furthermore, EGFR loss induced G2/M phase arrest and resulted in an increased resistance to TNF-related apoptosis-inducing ligand (TRAIL) in renal cell carcinoma. Thus, ablation of overexpressed EGFR by CRISPR/Cas9 alone or in combination with sunitinib may be a new treatment option for renal cell carcinoma., Competing Interests: The authors have declared that no competing interests exist.

The enzyme 4-oxalocrotonate tautomerase (4-OT) can promiscuously catalyze various carboligation reactions using acetaldehyde as a nucleophile. However, the highly reactive nature of acetaldehyde requires intricate handling, which can impede its usage in practical synthesis. Therefore, we investigated three enzymatic routes to synthesize acetaldehyde in situ in one-pot cascade reactions with 4-OT. Two routes afforded practical acetaldehyde concentrations, using an environmental pollutant, trans-3-chloroacrylic acid, or a bio-renewable, ethanol, as starting substrate. These routes can be combined with 4-OT catalyzed Michael-type additions and aldol condensations in one pot. This modular systems biocatalysis methodology provides a stepping stone towards the development of larger artificial metabolic networks for the practical synthesis of important chemical synthons., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Much effort within the nanosafety field is currently focused on the use of advanced in vitro models to reduce the gap between in vitro and in vivo studies. Within this context, precision-cut tissue slices are a unique ex vivo model to investigate nanoparticle impact using live tissue from laboratory animals and even humans. However, several aspects of the basic mechanisms of nanoparticle interactions with tissue have not yet been elucidated. To this end, liver slices are exposed to carboxylated and amino-modified polystyrene known to have a different impact on cells. As observed in standard cell cultures, amino-modified polystyrene nanoparticles induce apoptosis, and their impact is affected by the corona forming on their surface in biological fluids. Subsequently, a detailed time-resolved study of nanoparticle uptake and distribution in the tissue is performed, combining fluorescence imaging and flow cytometry on cells recovered after tissue digestion. As observed in vivo, the Kupffer cells accumulate high nanoparticle amounts and, interestingly, they move within the tissue towards the slice borders. Similar observations are reproduced in liver slices from human tissue. Thus, tissue slices can be used to reproduce ex vivo important features of nanoparticle outcomes in the liver and study nanoparticle impact on real tissue., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transcriptional coactivators p300 and CBP catalyze the acetylation of lysine residues in histone proteins. Upregulation of p300 and CBP has been associated with lung, colorectal and hepatocellular cancer, indicating an important role of p300 and CBP in tumorigenesis. Recently, the novel p300 and CBP-selective inhibitor A485 became available, which was shown to inhibit proliferation of 124 different cancer cell lines. Here, we found that downregulation of EP300 or CREBBP enhances apoptosis upon TRAIL stimulation in non-small-cell lung cancer (NSCLC) cells. A485 upregulates pro- and anti-apoptotic genes at the mRNA level, implying an apoptosis-modulating effect in NSCLC cells. However, A485 alone does not induce apoptosis. Interestingly, we observed that the number of apoptotic cells increases upon combined treatment with A485 and TRAIL. Therefore, A485, as a TRAIL-sensitizer, was used in combination with TRAIL in wild type of NSCLC cell lines (HCC827 and H1650) and cells with acquired erlotinib resistance (HCC827-ER and H1650-ER). Our results show that the combination of A485 and TRAIL synergistically increases cell death and inhibits long-term cell proliferation. Furthermore, this combination inhibits the growth of 3D spheroids of EGFR-TKI-resistant cells. Taken together, we demonstrate a successful combination of A485 and TRAIL in EGFR-TKI-sensitive and resistant NSCLC cells., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Stem cell therapy is an emerging treatment for tendon disorders., Purpose: To systematically review the efficacy of stem cell therapy for patients with tendon disorders., Study Design: Systematic review; Level of evidence, 4., Methods: MEDLINE/PubMed, EMBASE, CINAHL, CENTRAL, PEDro, and SPORTDiscus; trial registers; and gray literature were searched to identify randomized controlled trials (RCTs) and non-RCTs, cohort studies, and case series with 5 or more cases. Studies investigating any type of stem cell therapy for patients with tendon disorders were eligible if they included patient-reported outcome measures or assessed tendon healing. Risk of bias was assessed through use of the Cochrane risk of bias tools., Results: This review included 8 trials (289 patients). All trials had moderate to high risk of bias (level 3 or 4 evidence). In Achilles tendon disorders, 1 trial found that allogenic-derived stem cells led to a faster recovery compared with platelet-rich plasma. Another study found no retears after bone marrow-derived stem cell therapy was used in addition to surgical treatment. There were 4 trials that studied the efficacy of bone marrow-derived stem cell therapy for rotator cuff tears. The controlled trials reported superior patient-reported outcomes and better tendon healing. A further 2 case series found that stem cell therapy improved patient-reported outcomes in patients with patellar tendinopathy and elbow tendinopathy., Conclusion: Level 3 evidence is available to support the efficacy of stem cell therapy for tendon disorders. The findings of available studies are at considerable risk of bias, and evidence-based recommendations for the use of stem cell therapy for tendon disorders in clinical practice cannot be made at this time. Stem cell injections should not be used in clinical practice given the lack of knowledge about potentially serious adverse effects., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: This systematic review was supported by the Dutch National Olympic Committee. However, this funding had no influence on the collection, analysis, interpretation, and writing of the current manuscript. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto., (© The Author(s) 2020.)

Metabolic flexibility is an essential characteristic of eukaryotic cells in order to adapt to physiological and environmental changes. Especially in mammalian cells, the metabolic switch from mitochondrial respiration to aerobic glycolysis provides flexibility to sustain cellular energy in pathophysiological conditions. For example, attenuation of mitochondrial respiration and/or metabolic shifts to glycolysis result in a metabolic rewiring that provide beneficial effects in neurodegenerative processes. Ferroptosis, a non-apoptotic form of cell death triggered by an impaired redox balance is gaining attention in the field of neurodegeneration. We showed recently that activation of small-conductance calcium-activated K + (SK) channels modulated mitochondrial respiration and protected neuronal cells from oxidative death. Here, we investigated whether SK channel activation with CyPPA induces a glycolytic shift thereby increasing resilience of neuronal cells against ferroptosis, induced by erastin in vitro and in the nematode C. elegans exposed to mitochondrial poisons in vivo. High-resolution respirometry and extracellular flux analysis revealed that CyPPA, a positive modulator of SK channels, slightly reduced mitochondrial complex I activity, while increasing glycolysis and lactate production. Concomitantly, CyPPA rescued the neuronal cells from ferroptosis, while scavenging mitochondrial ROS and inhibiting glycolysis reduced its protection. Furthermore, SK channel activation increased survival of C. elegans challenged with mitochondrial toxins. Our findings shed light on metabolic mechanisms promoted through SK channel activation through mitohormesis, which enhances neuronal resilience against ferroptosis in vitro and promotes longevity in vivo.

Squalene synthase (SQS) catalyzes the conversion of two farnesyl pyrophosphates to squalene, an important intermediate in between isoprene and valuable triterpenoids. In this study, we have constructed a novel biosynthesis pathway for squalene in Bacillus subtilis and performed metabolic engineering aiming at facilitating further exploitation and production of squalene-derived triterpenoids. Therefore, systematic studies and analysis were performed including selection of multiple SQS candidates from various organisms, comparison of expression vectors, optimization of cultivation temperatures, and examination of rate-limiting factors within the synthetic pathway. We were, for the first time, able to obtain squalene synthesis in B. subtilis . Furthermore, we achieved a 29-fold increase of squalene yield (0.26-7.5 mg/L) by expressing SQS from Bacillus megaterium and eliminating bottlenecks within the upstream methylerythritol-phosphate pathway. Moreover, our findings showed that also ispA could positively affect the production of squalene.

High-throughput construction of multivalent binders and subsequent screening for biological activity represent a fundamental challenge: A linear increase of monovalent components translates to the square of possible bivalent combinations. Even high-efficiency cloning and expression methods become limiting when thousands of bispecific binders need to be screened for activity. In this study, we present an in vitro method for the efficient production of flexibly linked bispecific binding agents from individually expressed and purified monovalent binders. We established a sortase A-mediated coupling reaction to generate bispecific designed ankyrin repeat proteins (DARPins), with an optimized reaction maximizing the bivalent coupling product with low levels of monovalent side-products. These one-pot reaction mixtures could be used directly, without further purification, in cell-based assays. We generated a matrix of 441 different bispecific DARPins against the extracellular domains of the cancer-associated receptors EGFR, ErbB2, ErbB3, ErbB4, EpCAM, and c-MET and screened on two different ErbB2-positive cancer cells lines for growth-inhibitory effects. We identified not only known but also novel biologically active biparatopic DARPins. Furthermore, we found that the cancer cell lines respond in a highly reproducible and defined manner to the treatment with the 441 different bivalent binding agents. The generated response profiles can thus be used for functional characterization of cell lines because they are strongly related to the cell line-specific surface receptor landscape. Thus, our method not only represents a robust tool for screening and lead identification of bispecific binding agents, but additionally offers an orthogonal approach for the functional characterization of cancer cell lines., (©2019 American Association for Cancer Research.)

A series of aminocarboxylic acid analogues of aspergillomarasmine A (AMA) and ethylenediamine-N,N'-disuccinic acid (EDDS) were chemoenzymatically synthesized via the addition of various mono- and diamine substrates to fumaric acid catalyzed by the enzyme EDDS lyase. Many of these novel AMA and EDDS analogues demonstrate potent inhibition of the bacterial metallo-β-lactamase NDM-1. Isothermal titration calorimetry assays revealed a strong correlation between the inhibitory potency of the compounds and their ability to bind zinc. Compounds 1a (AMA), 1b (AMB), 5 (EDDS), followed by 1d and 8a, demonstrate the highest synergy with meropenem resensitizing an NDM-1 producing strain of E. coli to this important carbapenem of last resort.

The biosynthesis of pyoverdine, the major siderophore of Pseudomonas aeruginosa, is a well-organized process involving a discrete number of enzyme-catalyzed steps. The final step of this process involves the PvdP tyrosinase, which converts ferribactin into pyoverdine. Thus, inhibition of the PvdP tyrosinase activity provides an attractive strategy to interfere with siderophore synthesis to manage P. aeruginosa infections. Here, we report phenylthiourea as a non-competitive inhibitor of PvdP for which we solved a crystal structure in complex with PvdP. The crystal structure indicates that phenylthiourea binds to an allosteric binding site and thereby interferes with its tyrosinase activity. We further provide proofs that PvdP tyrosinase inhibition by phenylthiourea requires the C-terminal lid region. This provides opportunities to develop inhibitors that target the allosteric site, which seems to be confined to fluorescent pseudomonads, and not the tyrosinase active site. Furthermore, increases the chances to identify PvdP inhibitors that selectively interfere with siderophore synthesis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Aromatic β-hydroxyaldehydes, 1,3-diols, and α,β-unsaturated aldehydes are valuable precursors to biologically active natural products and drug molecules. Herein we report the biocatalytic aldol condensation of acetaldehyde with various aromatic aldehydes to give a number of aromatic α,β-unsaturated aldehydes using a previously engineered variant of 4-oxalocrotonate tautomerase [4-OT(M45T/F50A)] as carboligase. Moreover, an efficient one-pot two-step chemoenzymatic route toward chiral aromatic 1,3-diols has been developed. This one-pot chemoenzymatic strategy successfully combined a highly enantioselective aldol addition step catalyzed by a proline-based carboligase [4-OT(M45T/F50A) or TAUT015] with a chemical reduction step to convert enzymatically prepared aromatic β-hydroxyaldehydes into the corresponding 1,3-diols with high optical purity (e.r. up to >99:1) and in good isolated yield (51-92%). These developed (chemo)enzymatic methodologies offer alternative synthetic choices to prepare a variety of important drug precursors., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Dysregulated epidermal growth factor receptor (EGFR) is an oncogenic driver of many human cancers, promoting aberrant cell proliferation, migration, and survival. Pharmacological targeting of EGFR is often challenged by acquired mechanisms of resistance. Ligand-dependent mechanisms in EGFR wild-type cells rely on ligand or receptor overexpression, allowing cells to outcompete inhibitors and perpetuate signaling in an autocrine manner. Importantly, EGFR ligands are synthesized as membrane-bound precursors that must be solubilized to enable receptor-ligand interactions. The A disintegrin and metalloproteinase 17 (ADAM17) is considered the main sheddase of several EGFR ligands, and a potential pharmacological target. However, its broad substrate range and ubiquitous expression complicate its therapeutic targeting. Here, we present a novel bispecific fusion protein construct consisting of the inhibitory prodomain of ADAM17 (TPD), fused to an EGFR-targeting designed ankyrin repeat protein (DARPin). TPD is a natural inhibitor of ADAM17, maintaining the protease in a zymogen-like form. Meanwhile, the high affinity anti-EGFR DARPin E01 binds to EGFR and inhibits ligand binding. The resulting fusion protein E01-GS-TPD retained binding ability to both molecular targets EGFR and ADAM17. The large difference in affinity for each target resulted in enrichment of the fusion protein in EGFR-positive cells compared to EGFR-negative cells, suggesting a possible application in autocrine signaling inhibition. Accordingly, E01-GS-TPD decreased migration and proliferation of EGFR-dependent cell lines with no significant increase in apoptotic cell death. Finally, inhibition of proliferation was observed through EGFR ligand-dependent mechanisms as growth inhibition was not observed in EGFR mutant or KRAS mutant cell lines. The use of bispecific proteins targeting the EGFR/ADAM17 axis could be an innovative strategy for the treatment of EGFR-dependent cancers., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The bacterial biofilm plays a key role in nosocomial infections, especially those related to medical devices in sustained contact with patients. The active dispersion of bacterial cells out of biofilms acts as a reservoir for infectious diseases. The formation of such biofilms is a highly complex process, which is coordinated by many regulatory mechanisms of the pathogen including quorum sensing (QS). Many bacteria coordinate the expression of key virulence factors dependent on their population density through QS. The inhibition of this system is called quorum quenching (QQ). Thus, preventing the development of biofilms is considered a promising approach to prevent the development of hard to treat infections. Enzymatic QQ is the concept of interfering with the QS system of bacteria outside the cell. PvdQ is an acylase with an N-terminal nucleophile (Ntn-hydrolase) that is a part of the pyoverdine gene cluster ( pvd ). It is able to cleave irreversibly the amide bond of long chain N-acyl homoserine lactones (AHL) rendering them inactive. Long chain AHLs are the main signaling molecule in the QS system of the gram-negative pathogen Pseudomonas aeruginosa PA01, which is known for surface-associated biofilms on indwelling catheters and is also the cause of catheter-associated urinary tract infections. Furthermore, PA01 is a well characterized pathogen with respect to QS as well as QQ. In this study, we immobilized the acylase PvdQ on polydimethylsiloxane silicone (PDMS), creating a surface with quorum quenching properties. The goal is to control infections by minimizing the colonization of indwelling medical devices such as urinary catheters or intravascular catheters. The enzyme activity was confirmed by testing the degradation of the main auto-inducer that mediates QS in P. aeruginosa . In this article we report for the first time a successful immobilization of the quorum quenching acylase PvdQ on PDMS silicone. We could show that immobilized PvdQ retained its activity after the coating procedure and showed a 6-fold reduction of the auto-inducer 3-oxo-C12 in a biosensor setup. Further we report significant reduction of a P. aeruginosa PA01 biofilm on a coated PDMS surface compared to the same untreated material., (Copyright © 2020 Vogel, Wakker-Havinga, Setroikromo and Quax.)

The creation of microbial cell factories for sustainable production of natural products is important for medical and industrial applications. This requires stable expression of biosynthetic pathways in a host organism with favorable fermentation properties such as Bacillus subtilis. The aim of this study is to construct B. subtilis strains that produce valuable terpenoid compounds by overexpressing the innate methylerythritol phosphate (MEP) pathway. A synthetic operon allowing the concerted and regulated expression of multiple genes was developed. Up to 8 genes have been combined in this operon and a stably inherited plasmid-based vector was constructed resulting in a high production of C 30 carotenoids. For this, two vectors were examined, one with rolling circle replication and another with theta replication. Theta-replication constructs were clearly superior in structural and segregational stability compared to rolling circle constructs. A strain overexpressing all eight genes of the MEP pathway on a theta-replicating plasmid clearly produced the highest level of carotenoids. The level of transcription for each gene in the operon was similar as RT-qPCR analysis indicated. Hence, that corresponding strain can be used as a stable cell factory for production of terpenoids. This is the first report of merging and stably expressing this large-size operon (eight genes) from a plasmid-based system in B. subtilis enabling high C 30 carotenoid production.

The efficient engineering of iminium biocatalysis has drawn considerable attention, with many applications in pharmaceutical synthesis. Here, we report a tailor-made iminium-activated colorimetric "turn-on" probe, specifically designed as a prescreening tool to facilitate engineering of iminium biocatalysis. Upon complexation of the probe with the catalytic Pro-1 residue of the model enzyme 4-oxalocrotonate tautomerase (4-OT), a brightly colored merocyanine-dye-type structure is formed. 4-OT mutants that formed this brightly colored species upon incubation with the probe proved to have a substantial activity for the iminium-based Michael-type addition of nitromethane to cinnamaldehyde, whereas mutants that showed no staining by the probe exhibited no or very low-level "Michaelase" activity. This system was exploited in a solid-phase prescreening assay termed as activated iminium colony staining (AICS) to enrich libraries for active mutants. AICS prescreening reduced the screening effort up to 20-fold. After two rounds of directed evolution, two artificial Michaelases were identified with up to 39-fold improvement in the activity for the addition of nitromethane to cinnamaldehyde, yielding the target γ-nitroaldehyde product with excellent isolated yield (up to 95%) and enantiopurity (up to >99% ee). The colorimetric activation of the turn-on probe could be extended to the class I aldolase 2-deoxy-d-ribose 5-phosphate aldolase, implicating a broader application of AICS in engineering iminium biocatalysis., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1-9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human macrophage migration-inhibitory factor (MIF) is an evolutionarily-conserved protein that has both extracellular immune-modulating and intracellular cell-regulatory functions. MIF plays a role in various diseases, including inflammatory diseases, atherosclerosis, autoimmunity, and cancer. It serves as an inflammatory cytokine and chemokine, but also exhibits enzymatic activity. Secreted MIF binds to cell-surface immune receptors such as CD74 and CXCR4. Plants possess MIF orthologs but lack the associated receptors, suggesting functional diversification across kingdoms. Here, we characterized three MIF orthologs (termed MIF/d-dopachrome tautomerase-like proteins or MDLs) of the model plant Arabidopsis thaliana Recombinant Arabidopsis MDLs ( At MDLs) share similar secondary structure characteristics with human MIF, yet only have minimal residual tautomerase activity using either p -hydroxyphenylpyruvate or dopachrome methyl ester as substrate. Site-specific mutagenesis suggests that this is due to a distinct amino acid difference at the catalytic cavity-defining residue Asn-98. Surprisingly, At MDLs bind to the human MIF receptors CD74 and CXCR4. Moreover, they activate CXCR4-dependent signaling in a receptor-specific yeast reporter system and in CXCR4-expressing human HEK293 transfectants. Notably, plant MDLs exert dose-dependent chemotactic activity toward human monocytes and T cells. A small molecule MIF inhibitor and an allosteric CXCR4 inhibitor counteract this function, revealing its specificity. Our results indicate cross-kingdom conservation of the receptor signaling and leukocyte recruitment capacities of human MIF by its plant orthologs. This may point toward a previously unrecognized interplay between plant proteins and the human innate immune system., (© 2020 Sinitski et al.)

Macrophage migration inhibitory factor (MIF) is a versatile protein that plays a role in inflammation, autoimmune diseases and cancers. Development of novel inhibitors will enable further exploration of MIF as a drug target. In this study, we investigated structure-activity relationships of MIF inhibitors using a MIF tautomerase activity assay to measure binding. Importantly, we notified that transition metals such as copper (II) and zinc (II) interfere with the MIF tautomerase activity under the assay conditions applied. EDTA was added to the assay buffer to avoid interference of residual heavy metals with tautomerase activity measurements. Using these assay conditions the structure-activity relationships for MIF binding of a series of triazole-phenols was explored. The most potent inhibitors in this series provided activities in the low micromolar range. Enzyme kinetic analysis indicates competitive binding that proved reversible. Binding to the enzyme was confirmed using a microscale thermophoresis (MST) assay. Molecular modelling was used to rationalize the observed structure-activity relationships. The most potent inhibitor 2d inhibited proliferation of A549 cells in a clonogenic assay. In addition, 2d attenuated MIF induced ERK phosphorylation in A549 cells. Altogether, this study provides insights in the structure-activity relationships for MIF binding of triazole-phenols and further validates this class of compounds as MIF binding agents in cell-based studies., (Copyright © 2019 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Ethnopharmacological Relevance: Cymbopogon citratus (lemongrass) essential oil has been widely used as a traditional medicine and is well known for antimicrobial properties. Therefore, it might be a potent anti-infective and biofilm inhibitive against Candida tropicalis infections. Until now, no ideal coating or cleaning method based on an essential oil has been described to prevent biofilm formation of Candida strains on silicone rubber maxillofacial prostheses, voice prostheses and medical devices susceptible to C. tropicalis infections., Aim of the Study: To investigate the antifungal and biofilm inhibitory effects of Cymbopogon citratus oil. Clinical isolates of C. tropicalis biofilms on different biomaterials were used to study the inhibitory effect., Materials and Methods: The efficacy of Cymbopogon citratus, Cuminum cyminum, Citrus limon and Cinnamomum verum essential oils were compared on biofilm formation of three C. tropicalis isolates on 24 well polystyrene plates. C. citratus oil coated silicone rubber surfaces were prepared using hypromellose ointment as a vehicle. The antifungal tests to determine minimum inhibitory and minimum fungicidal concentrations were assessed by a microbroth dilution method and biofilm formation was determined by a crystal violet binding assay., Results: C. tropicalis strains formed more biofilm on hydrophobic materials than on hydrophilic glass. C. citratus oil showed a high antifungal effect against all C. tropicalis strains. For comparison, C. limon oil and C. cyminum oil showed minor to no killing effect against the C. tropicalis strains. C. citratus oil had the lowest minimal inhibitory concentration of all essential oils tested and inhibited biofilm formation of all C. tropicalis strains. C. citratus oil coating on silicone rubber resulted in a 45-76% reduction in biofilm formation of all C. tropicalis strains., Conclusion: Cymbopogon citratus oil has good potential to be used as an antifungal and antibiofilm agent on silicone rubber prostheses and medical devices on which C. tropicalis biofilms pose a serious risk for skin infections and may cause a shorter lifespan of the prosthesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aspartic acid derivatives with branched N-alkyl or N-arylalkyl substituents are valuable precursors to artificial dipeptide sweeteners such as neotame and advantame. The development of a biocatalyst to synthesize these compounds in a single asymmetric step is an as yet unmet challenge. Reported here is an enantioselective biocatalytic synthesis of various difficult N-substituted aspartic acids, including N-(3,3-dimethylbutyl)-l-aspartic acid and N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-l-aspartic acid, precursors to neotame and advantame, respectively, using an engineered variant of ethylenediamine-N,N'-disuccinic acid (EDDS) lyase from Chelativorans sp. BNC1. This engineered C-N lyase (mutant D290M/Y320M) displayed a remarkable 1140-fold increase in activity for the selective hydroamination of fumarate compared to that of the wild-type enzyme. These results present new opportunities to develop practical multienzymatic processes for the more sustainable and step-economic synthesis of an important class of food additives., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Brain tumours are among the deadliest tumours being highly resistant to currently available therapies. The proliferative behaviour of gliomas is strongly influenced by ion channel activity. Small-conductance calcium-activated potassium (SK/K Ca ) channels are a family of ion channels that are associated with cell proliferation and cell survival. A combined treatment of classical anti-cancer agents and pharmacological SK channel modulators has not been addressed yet. We used the gold-derivative auranofin to induce cancer cell death by targeting thioredoxin reductases in combination with CyPPA to activate SK channels in neuro- and glioblastoma cells. Combined treatment with auranofin and CyPPA induced massive mitochondrial damage and potentiated auranofin-induced toxicity in neuroblastoma cells in vitro. In particular, mitochondrial integrity, respiration and associated energy generation were impaired. These findings were recapitulated in patient-derived glioblastoma neurospheres yet not observed in non-cancerous HT22 cells. Taken together, integrating auranofin and SK channel openers to affect mitochondrial health was identified as a promising strategy to increase the effectiveness of anti-cancer agents and potentially overcome resistance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme's stability while at the same time enhancing the enzyme's activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

There is increasing evidence that histone deacetylase (HDAC) inhibitors can (re)sensitize cancer cells for chemotherapeutics via "epigenetic priming". In this work, we describe the synthesis of a series of class I-selective HDAC inhibitors with 2-aminoanilides as zinc-binding groups. Several of the synthesized compounds revealed potent inhibition of the class I HDAC isoforms HDAC1, HDAC2, and/or HDAC3 and promising antiproliferative effects in the human ovarian cancer cell line A2780 and the human squamous carcinoma cell line Cal27. Selected compounds were investigated in a cellular model of platinum resistance. In particular, compound 2a revealed potent chemosensitizing properties and full reversal of cisplatin resistance in Cal27CisR cells. This effect is related to a synergistic increase in caspase 3/7 activation and induction of apoptosis. Thus, this work demonstrates that pan-HDAC inhibition or dual class I/class IIb inhibition is not required for full reversal of cisplatin resistance.

L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Metastatic melanoma is amongst the most difficult types of cancer to treat, with current therapies mainly relying on the inhibition of the BRAF V600E mutant kinase. However, systemic inhibition of BRAF by small molecule drugs in cancer patients results - paradoxically - in increased wild-type BRAF activity in healthy tissue, causing side-effects and even the formation of new tumors. Here we show the development of BRAF V600E kinase inhibitors of which the activity can be switched on and off reversibly with light, offering the possibility to overcome problems of systemic drug activity by selectively activating the drug at the desired site of action. Based on a known inhibitor, eight photoswitchable effectors containing an azobenzene photoswitch were designed, synthesized and evaluated. The most promising inhibitor showed an approximately 10-fold increase in activity upon light-activation. This research offers inspiration for the development of therapies for metastatic melanoma in which tumor tissue is treated with an active BRAF V600E inhibitor with high spatial and temporal resolution, thus limiting the damage to other tissues., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Around three million patients die due to airway inflammatory diseases each year. The most notable of these diseases are asthma and chronic obstructive pulmonary disease (COPD). Therefore, new therapies are urgently needed. Promising targets are histone deacetylases (HDACs), since they regulate posttranslational protein acetylation. Over a thousand proteins are reversibly acetylated, and acetylation critically influences aberrant intracellular signaling pathways in asthma and COPD. The diverse set of selective and non-selective HDAC inhibitors used in pre-clinical models of airway inflammation show promising results, but several challenges still need to be overcome. One such challenge is the design of HDAC inhibitors with unique selectivity profiles, such as selectivity towards specific HDAC complexes. Novel strategies to disrupt HDAC complexes should be developed to validate HDACs further as targets for new anti-inflammatory pulmonary treatments.

Fibrosis is characterized by the progressive alteration of the tissue structure due to the excessive production of extracellular matrix (ECM). The signaling system encompassing Receptor Activator of Nuclear factor NF-κB Ligand (RANKL)/RANK/Osteoprotegerin (OPG) was discovered to play an important role in the regulation of ECM formation and degradation in bone tissue. However, whether and how this signaling pathway plays a role in liver or pulmonary ECM degradation is unclear up to now. Interestingly, increased decoy receptor OPG levels are found in fibrotic tissues. We hypothesize that RANKL can stimulate RANK on macrophages and initiate the process of ECM degradation. This process may be inhibited by highly expressed OPG in fibrotic conditions. In this case, RANKL mutants that can bind to RANK without binding to OPG might become promising therapeutic candidates. In this study, we built a structure-based library containing 44 RANKL mutants and found that the Q236 residue of RANKL is important for OPG binding. We show that RANKL_Q236D can activate RAW cells to initiate the process of ECM degradation and is able to escape from the obstruction by exogenous OPG. We propose that the generation of RANKL mutants with reduced affinity for OPG is a promising strategy for the exploration of new therapeutics against fibrosis., (© 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Histone deacetylases (HDACs) play an important role in cancer, degenerative diseases and inflammation. The currently applied HDAC inhibitors in the clinic lack selectivity among HDAC isoforms, which limits their application for novel indications such as inflammatory diseases. Recent, literature indicates that HDAC 3 plays an important role among class I HDACs in gene expression in inflammation. In this perspective, the development and understanding of inhibitory selectivity among HDACs 1, 2 and 3 and their respective influence on gene expression need to be characterized to facilitate drug discovery. Towards this aim, we synthesized nine structural analogues of the class I HDAC inhibitor Entinostat and investigated their selectivity profile among HDACs 1, 2 and 3. We found that we can explain the observed structure activity relationships by small structural and conformational differences between HDAC 1 and HDAC 3 in the 'lid' interacting region. Cell-based studies indicated, however, that application of inhibitors with improved HDAC 3 selectivity did not provide an anti-inflammatory response in contrast to expectations from biochemical evidence in literature. Altogether, in this study, we identified structure activity relationships among class I HDACs and we connected isoform selectivity among class I HDACs with pro- and anti-inflammatory gene transcription in macrophages., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The enzyme vascular non-inflammatory molecule-1 (vanin 1) is highly expressed at gene and protein level in many organs, such as the liver, intestine, and kidney. Its major function is related to its pantetheinase activity; vanin 1 breaks down pantetheine in cysteamine and pantothenic acid, a precursor of coenzyme A. Indeed, its physiological role seems strictly related to coenzyme A metabolism, lipid metabolism, and energy production. In recent years, many studies have elucidated the role of vanin 1 under physiological conditions in relation to oxidative stress and inflammation. Vanin's enzymatic activity was found to be of key importance in certain diseases, either for its protective effect or as a sensitizer, depending on the diseased organ. In this review, we discuss the role of vanin 1 in the liver, kidney, intestine, and lung under physiological as well as pathophysiological conditions. Thus, we provide a more complete understanding and overview of its complex function and contribution to some specific pathologies.

Gene doping confers health risks for athletes and is a threat to fair competition in sports. Therefore the anti-doping community has given attention on its detection. Previously published polymerase chain reaction-based methodologies for gene doping detection are targeting exon-exon junctions in the intron-less transgene. However, because these junctions are known, it would be relatively easy to evade detection by tampering with the copyDNA sequences. We have developed a targeted next-generation sequencing based assay for the detection of all exon-exon junctions of the potential doping genes, EPO, IGF1, IGF2, GH1, and GH2, which is resistant to tampering. Using this assay, all exon-exon junctions of copyDNA of doping genes could be detected with a sensitivity of 1296 copyDNA copies in 1000 ng of genomic DNA. In addition, promotor regions and plasmid-derived sequences are readily detectable in our sequence data. While we show the reliability of our method for a selection of genes, expanding the panel to detect other genes would be straightforward. As we were able to detect plasmid-derived sequences, we expect that genes with manipulated junctions, promotor regions, and plasmid or virus-derived sequences will also be readily detected.

Human 15-lipoxygenase-1 (15-LOX-1) is a mammalian lipoxygenase which plays an important regulatory role in several CNS and inflammatory lung diseases. To further explore the role of this enzyme in drug discovery, novel potent inhibitors with favorable physicochemical properties are required. In order to identify such new inhibitors, we established a combinatorial screening method based on acylhydrazone chemistry. This represents a novel application of combinatorial chemistry focusing on the improvement of physicochemical properties, rather than on potency. This strategy allowed us to efficiently screen 44 reaction mixtures of different hydrazides and our previously reported indole aldehyde core structure, without the need for individual synthesis of all possible combinations of building blocks. Our approach afforded three new inhibitors with IC 50 values in the nanomolar range and improved lipophilic ligand efficiency., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Cellular senescence is a state of stable cell cycle arrest arising in response to DNA and mitochondrial damages. Senescent cells undergo morphological, structural and functional changes that are influenced by a number of variables, including time, stress, tissue, and cell type. The heterogeneity of the senescent phenotype is exemplified by the many biological properties that senescent cells can cover. The advent of innovative model organisms has demonstrated a functional role of senescent cells during embryogenesis, tissue remodeling, tumorigenesis and aging. Importantly, prolonged and aberrant persistence of senescent cells is often associated with tissue dysfunction and pathology, and is partially the consequence of mechanisms that enhance survival and resistance to cell death. Here, we describe the main molecular players involved in promoting survival of senescent cells, with particular emphasis on the regulation of senescence-associated anti-apoptotic pathways. We discuss the consequences these pathways have in providing resistance to intrinsic and extrinsic pro-apoptotic signals. Finally, we highlight the importance of these pathways in the development of targets for senolytic interventions., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Staphylococcus aureus with spa-type t437 has been identified as a predominant community-associated methicillin-resistant S. aureus clone from Asia, which is also encountered in Europe. Molecular typing has previously shown that t437 isolates are highly similar regardless of geographical regions or host environments. The present study was aimed at assessing to what extent this high similarity is actually reflected in the production of secreted virulence factors. We therefore profiled the extracellular proteome, representing the main reservoir of virulence factors, of 20 representative clinical isolates by mass spectrometry. The results show that these isolates can be divided into three groups and nine subgroups based on exoproteome abundance signatures. This implies that S. aureus t437 isolates show substantial exoproteome heterogeneity. Nonetheless, 30 highly conserved extracellular proteins, of which about 50% have a predicted role in pathogenesis, were dominantly identified. To approximate the virulence of the 20 investigated isolates, we employed infection models based on Galleria mellonella and HeLa cells. The results show that the grouping of clinical isolates based on their exoproteome profile can be related to virulence. We consider this outcome important as our approach provides a tool to pinpoint differences in virulence among seemingly highly similar clinical isolates of S. aureus.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis that may be a promising agent in cancer therapy due to its selectivity toward tumor cells. However, many cancer cells are resistant to TRAIL due to defects in apoptosis signaling or activation of survival pathways. We hypothesized that a disruption of pro-survival signaling cascades with the multi-tyrosine kinase inhibitor sunitinib and would be an effective strategy to enhance TRAIL-mediated apoptosis. Here we demonstrate that sunitinib significantly augments the anticancer activity of TRAIL in models of colon cancer. The therapeutic benefit of the TRAIL/sunitinib combination was associated with increased apoptosis marked by enhanced caspase-3 cleavage and DNA fragmentation. Overexpression of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2) in HCT116 cells reduced TRAIL/sunitinib-mediated apoptosis, further supporting that sunitinib enhances the anticancer activity of TRAIL via augmented apoptosis. Analysis of pro-survival factors identified that the combination of TRAIL and sunitinib significantly downregulated the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP) through a c-Jun N-terminal kinase (JNK)-mediated mechanism. Short hairpin RNA (shRNA)-mediated knockdown of JNK confirmed its key role in the regulation of sensitivity to this combination as cells with suppressed JNK expression exhibited significantly reduced TRAIL/sunitinib-mediated apoptosis. Importantly, the therapeutic benefit of the TRAIL/sunitinib combination was validated in the HCT116-Luc and HCT15 colon cancer xenograft models, which both demonstrated significant anti-tumor activity in response to combination treatment. Collectively, our data demonstrate that sunitinib enhances TRAIL-mediated apoptosis by heightened JNK activation, diminished XIAP levels, and augmented apoptosis., Competing Interests: The authors declare no conflict of interest.

For more effectiveness and overcoming the drug resistance Chemotherapy agents, the combination treatment is raised. Flavonoids with different anti-cancer effects are an appropriate choice as lead compounds. Over expressed MiR-21 in prostate cancer is associated with metastasis and drug resistance to chemotherapy with Docetaxel. In this study, the anticancer effect of 4', 5, 7-Trihydroxy-3', 5'-dimethoxyflavone (Tricin) was investigated with Docetaxel on PC3 cell line. Tricin was initially isolated from the Allium atroviolaceum by column chromatography and recrystallization method. The chemical structure of isolate was elucidated by spectroscopic techniques. IC 50 of Tricin and Docetaxel were assessed 117.5 ± 4.4 μM and 0.1 ± 0.02 nM by MTT assay, respectively. Analysis of results indicates the synergistic effect of combination therapy on decreased proliferation. MiR-21 in treated cells with Tricin significantly decreased compared to control cells. So, Tricin can be effective in the reduction of metastasis and drug resistance of Docetaxel.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as a promising anti-cancer therapeutic. However, many cancers have been found to be or to become inherently resistant to TRAIL. A combination of epigenetic modifiers, such as histone deacetylase inhibitors (HDACi's), with TRAIL was effective to overcome TRAIL resistance in some cancers. Broad spectrum HDACi's, however, show considerable toxicity constraining clinical use. Since overexpression of class I histone deacetylase (HDAC) has been found in colon tumors relative to normal mucosa, we have focused on small spectrum HDACi's. We have now tested agonistic receptor-specific TRAIL variants rhTRAIL 4C7 and DHER in combination with several class I specific HDACi's on TRAIL-resistant colon cancer cells DLD-1 and WiDr. Our data show that TRAIL-mediated apoptosis is largely improved in WiDr cells by pre-incubation with Entinostat-a HDAC1, 2, and 3 inhibitor- and in DLD-1 cells by RGFP966-a HDAC3-specific inhibitor- or PCI34051-a HDAC8-specific inhibitor. We are the first to report that using RGFP966 or PCI34051 in combination with rhTRAIL 4C7 or DHER represents an effective cancer therapy. The intricate relation of HDACs and TRAIL-induced apoptosis was confirmed in cells by knockdown of HDAC1 , 2 , or 3 gene expression, which showed more early apoptotic cells upon adding rhTRAIL 4C7 or DHER. We observed that RGFP966 and PCI34051 increased DR4 expression after incubation on DLD-1 cells, while RGFP966 induced more DR5 expression on WiDr cells, indicating a different role for DR4 or DR5 in these combinations. At last, we show that combined treatment of RGFP966 with TRAIL variants (rhTRAIL 4C7/DHER) increases apoptosis on 3D tumor spheroid models.

The enzyme 4-oxalocrotonate tautomerase (4-OT) exploits an N-terminal proline as main catalytic residue to facilitate several promiscuous C-C bond-forming reactions via enzyme-bound enamine intermediates. Here we show that the active site of this enzyme can give rise to further synthetically useful catalytic promiscuity. Specifically, the F50A mutant of 4-OT was found to efficiently promote asymmetric Michael additions of nitromethane to various α,β-unsaturated aldehydes to give γ-nitroaldehydes, important precursors to biologically active γ-aminobutyric acids. High conversions, high enantiocontrol, and good isolated product yields were achieved. The reactions likely proceed via iminium ion intermediates formed between the catalytic Pro-1 residue and the α,β-unsaturated aldehydes. In addition, a cascade of three 4-OT(F50A)-catalyzed reactions followed by an enzymatic oxidation step enables assembly of γ-nitrocarboxylic acids from three simple building blocks in one pot. Our results bridge organo- and biocatalysis, and they emphasize the potential of enzyme promiscuity for the preparation of important chiral synthons., Competing Interests: The authors declare no competing financial interest.

Human 15-lipoxygenase-1 (15-LOX-1) belongs to the class of lipoxygenases, which catalyze oxygenation of polyunsaturated fatty acids, such as arachidonic and linoleic acid. Recent studies have shown that 15-LOX-1 plays an important role in physiological processes linked to several diseases such as airway inflammation disease, coronary artery disease, and several types of cancer such as rectal, colon, breast and prostate cancer. In this study, we aimed to extend the structural diversity of 15-LOX-1 inhibitors, starting from the recently identified indolyl core. In order to find new scaffolds, we employed a combinatorial approach using various aromatic aldehydes and an aliphatic hydrazide tail. This scaffold-hopping study resulted in the identification of the 3-pyridylring as a suitable replacement of the indolyl core with an inhibitory activity in the micromolar range ( IC =16±6 μm) and a rapid and efficient structure-activity relationship investigation.50 =16±6 μm) and a rapid and efficient structure-activity relationship investigation.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome-editing tool, has revolutionized genetic engineering. It is widely used to investigate the molecular basis of different cancer types. In this review, we present an overview of recent studies in which CRISPR/Cas9 has been used for the identification of potential molecular targets. Based on the collected data, we suggest here that CRISPR/Cas9 is an effective system to distinguish between mutant and wild-type alleles in cancer. We show that several new potential therapeutic targets, such as CD38, CXCR2, MASTL, and RBX2, as well as several noncoding (nc)RNAs have been identified using CRISPR/Cas9 technology. We also discuss the obstacles and challenges that we face for using CRISPR/Cas9 as a therapeutic., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

KRAS-driven non-small cell lung cancer (NSCLC) patients have no effective targeted treatment. In this study, we aimed to investigate targeting epidermal growth factor receptor ( EGFR ) as a therapeutic approach in KRAS-driven lung cancer cells. We show that ablation of EGFR significantly suppressed tumor growth in KRAS-dependent cells and induced significantly higher expression of CX chemokine receptor 7 (CXCR7) and activation of MAPK (ERK1/2). Conversely, rescue of EGFR led to CXCR7 downregulation in EGFR cells. Dual EGFR and CXCR7 inhibition led to substantial reduction of MAPK (pERK) and synergistic inhibition of cell growth. Analysis of two additional -/- cells. Dual EGFR and CXCR7 inhibition led to substantial reduction of MAPK (pERK) and synergistic inhibition of cell growth. Analysis of two additional EGFR knockout NSCLC cell lines using CRISPR/Cas9 revealed genotype dependency of CXCR7 expression. In addition, treatment of different cells with gefitinib increased CXCR7 expression in EGFR wt cells. CXCR7 protein expression was detected in all NSCLC patient samples, with higher levels in adenocarcinoma as compared to squamous cell lung carcinoma and healthy control cases. In conclusion, EGFR and CXCR7 have a crucial interaction in NSCLC, and dual inhibition may be a potential therapeutic option for NSCLC patients.EGFR mut cells. CXCR7 protein expression was detected in all NSCLC patient samples, with higher levels in adenocarcinoma as compared to squamous cell lung carcinoma and healthy control cases. In conclusion, EGFR and CXCR7 have a crucial interaction in NSCLC, and dual inhibition may be a potential therapeutic option for NSCLC patients.

Non-alcoholic fatty liver disease (NAFLD) is a common liver disorder closely related to metabolic syndrome. NAFLD can progress to an inflammatory state called non-alcoholic steatohepatitis (NASH), which may result in the development of fibrosis and hepatocellular carcinoma. To develop therapeutic strategies against NAFLD, a better understanding of the molecular mechanism is needed. Current in vitro NAFLD models fail to capture the essential interactions between liver cell types and often do not reflect the pathophysiological status of patients. To overcome limitations of commonly used in vitro and in vivo models, precision-cut liver slices (PCLSs) were used in this study. PCLSs, prepared from liver tissue obtained from male Wistar rats, were cultured in supraphysiological concentrations of glucose, fructose, insulin, and palmitic acid to mimic metabolic syndrome. Accumulation of lipid droplets was visible and measurable after 24 h in PCLSs incubated with glucose, fructose, and insulin, both in the presence and absence of palmitic acid. Upregulation of acetyl-CoA carboxylase 1 and 2, and of sterol responsive element binding protein 1c, suggests increased de novo lipogenesis in PCLSs cultured under these conditions. Additionally, carnitine palmitoyltransferase 1 expression was reduced, which indicates impaired fatty acid transport and disrupted mitochondrial β-oxidation. Thus, steatosis was successfully induced in PCLSs with modified culture medium. This novel ex vivo NAFLD model could be used to investigate the multicellular and molecular mechanisms that drive NAFLD development and progression, and to study potential anti-steatotic drugs.

Terpenoids are natural products known for their medicinal and commercial applications. Metabolic engineering of microbial hosts for the production of valuable compounds, such as artemisinin and Taxol, has gained vast interest in the last few decades. The Generally Regarded As Safe (GRAS) Bacillus subtilis 168 with its broad metabolic potential is considered one of these interesting microbial hosts. In the effort toward engineering B. subtilis as a cell factory for the production of the chemotherapeutic Taxol, we expressed the plant-derived taxadiene synthase (TXS) enzyme. TXS is responsible for the conversion of the precursor geranylgeranyl pyrophosphate (GGPP) to taxa-4,11-diene, which is the first committed intermediate in Taxol biosynthesis. Furthermore, overexpression of eight enzymes in the biosynthesis pathway was performed to increase the flux of the GGPP precursor. This was achieved by creating a synthetic operon harboring the B. subtilis genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway ( dxs , ispD , ispF , ispH , ispC , ispE , ispG ) together with ispA (encoding geranyl and farnesyl pyrophosphate synthases) responsible for providing farnesyl pyrophosphate (FPP). In addition, a vector harboring the crtE gene (encoding geranylgeranyl pyrophosphate synthase, GGPPS, of Pantoea ananatis ) to increase the supply of GGPP was introduced. The overexpression of the MEP pathway enzymes along with IspA and GGPPS caused an 83-fold increase in the amount of taxadiene produced compared to the strain only expressing TXS and relying on the innate pathway of B. subtilis . The total amount of taxadiene produced by that strain was 17.8 mg/l. This is the first account of the successful expression of taxadiene synthase in B. subtilis . We determined that the expression of GGPPS through the crtE gene is essential for the formation of sufficient precursor, GGPP, in B. subtilis as its innate metabolism is not efficient in producing it. Finally, the extracellular localization of taxadiene production by overexpressing the complete MEP pathway along with IspA and GGPPS presents the prospect for further engineering aiming for semisynthesis of Taxol.

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, as antidepressants, and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from simple precursors (acetaldehyde and nitroalkenes), using a tailor-made highly enantioselective artificial "Michaelase" (4-oxalocrotonate tautomerase mutant L8Y/M45Y/F50A), an aldehyde dehydrogenase with a broad non-natural substrate scope, and a cofactor recycling system. We also report a three-step chemoenzymatic cascade route for the efficient chemical reduction of enzymatically prepared γ-nitrobutyric acids into GABA analogues in one pot, achieving high enantiopurity (e.r. up to 99:1) and high overall yields (up to 70%). This chemoenzymatic methodology offers a step-economic alternative route to important pharmaceutically active GABA analogues, and highlights the exciting opportunities available for combining chemocatalysts, natural enzymes, and designed artificial biocatalysts in multistep syntheses., Competing Interests: The authors declare no competing financial interest.

The remarkable pro-apoptotic properties of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) have led to considerable interest in this protein as a potential anticancer therapeutic. However, TRAIL is largely ineffective in inducing apoptosis in certain cancer cells, and the mechanisms underlying this selectivity are unknown. In colon adenocarcinomas, posttranslational modifications including O- and N- glycosylation of death receptors were found to correlate with TRAIL-induced apoptosis. Additionally, mRNA levels of fucosyltransferase 3 (FUT3) and 6 (FUT6) were found to be high in the TRAIL-sensitive colon adenocarcinoma cell line COLO 205. In this study, we use agonistic receptor-specific TRAIL variants to dissect the contribution of FUT3 and FUT6-mediated fucosylation to TRAIL-induced apoptosis via its two death receptors, DR4 and DR5. Triggering of apoptosis by TRAIL revealed that the low FUT3/6-expressing cells DLD-1 and HCT 116 are insensitive to DR5 but not to DR4-mediated apoptosis. By contrast, efficient apoptosis is mediated via both receptors in high FUT3/6-expressing COLO 205 cells. The reconstitution of FUT3/6 expression in DR5-resistant cells completely restored TRAIL sensitivity via this receptor, while only marginally enhancing apoptosis via DR4 at lower TRAIL concentrations. Interestingly, we observed that induction of the salvage pathway by external administration of l-fucose restores DR5-mediated apoptosis in both DLD-1 and HCT 116 cells. Finally, we show that fucosylation influences the ligand-independent receptor association that leads to increased death inducing signalling complex (DISC) formation and caspase-8 activation. Taken together, these results provide evidence for the differential impact of fucosylation on signalling via DR4 or DR5. These findings provide novel opportunities to enhance TRAIL sensitivity in colon adenocarcinoma cells that are highly resistant to DR5-mediated apoptosis., (© 2018 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Antimicrobial resistance resulting in ineffective treatment of infectious diseases is an increasing global problem, particularly in infections with pathogenic bacteria. In some bacteria, such as Streptococcus pyogenes, the pathogenicity is strongly linked to the attachment of virulence factors. Their attachment to the cellular membrane is a transpeptidation reaction, catalyzed by sortase enzymes. As such, sortases pose an interesting target for the development of new antivirulence strategies that could yield novel antimicrobial drugs. Using the substitution-oriented fragment screening (SOS) approach, we discovered a potent and specific inhibitor (C10) of sortase A from S. pyogenes. The inhibitor C10 showed high specificity towards S. pyogenes sortase A, with an IC 50 value of 10 μM and a K d of 60 μM. We envision that this inhibitor could be employed as a starting point for further exploration of sortase's potential as therapeutic target for antimicrobial drug development., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)