Metrics details A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering such as the Alzheimer’s medication galantamine are complex plant secondary metabolites with recognized therapeutic value Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus we propose an efficient biosensor-machine learning technology stack for biocatalyst development which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli Directed evolution is used to develop a highly sensitive (EC50 = 20 μM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4’-O-methylnorbelladine A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase which are rapidly screened with the biosensor Functional enzyme variants are identified that yield a 60% improvement in product titer and 3-fold lower off-product regioisomer formation A solved crystal structure elucidates the mechanism behind key beneficial mutations yielding enzymes and pathways with improved stability and activity we synergize the development of custom biosensors with machine learning(ML)-guided protein design to improve microbial fermentation of the branchpoint AA 4′-O-methylnorbelladine (4NB) is evolved into a highly sensitive biosensor for 4NB that precisely discriminates against the non-methylated precursor norbelladine and the biosensor is then used to monitor the activity of norbelladine 4’-O-methyltransferase (Nb4OMT) from the daffodil Narcissus pseudonarcissus in Escherichia coli We then develop MutComputeX: a structure-based self-supervised residual neural network (3DResNet) trained to generalize at protein:non-protein interfaces which is used to generate activity-enriched Nb4OMT designs from an ML-generated protein-cofactor-substrate structure The evolved biosensor is used to rapidly screen a panel of MutComputeX-guided Nb4OMT designs leading to the identification of one variant that yields a 60% improvement in product titer A newly solved crystal structure of this engineered enzyme helps elucidate the mechanism behind key beneficial mutations and highlight important discrepancies with the AlphaFold2 model a. Abbreviated biosynthetic plant pathways for therapeutic amaryllidaceae alkaloids. b Response of the RamR transcription factor to amaryllidaceae alkaloid pathway intermediates norbelladine and 4′-O-methylnorbelladine. Error bars represent the S.D. +/− the mean. Experiments were conducted in biological triplicate. c Structure of RamR (PDB: 3VVX_A) docked with 4′-O-methylnorbelladine using GNINA (see “Methods”) Predicted ligand-interacting residues are highlighted green this method involves a growth-based selection to first filter out biosensor variants that are incapable of repressing transcription from their cognate promoter followed by a fluorescence-based screen to isolate sensor variants highly responsive to the target analyte a Schematic illustrating the mutations that resulted after round one (4NB1) and round two (4NB2) of RamR evolution towards 4′-O-methylnorbelladine and 4NB2 mutants with 4′-O-methylnorbelladine and 4NB2 mutants to norbelladine and 4′-O-methylnorbelladine e AlphaFold2 structural model of 4NB2 docked with 4′-O-methylnorbelladine Predicted ligand interactions with WT residues and mutations that arose in 4NB2 are colored gray (f) Correlation between fluorescent response measured with the 4NB2 sensor and 4′-O-methylnorbelladine measured with high-performance liquid chromatography (g) The distribution of fluorescent cell populations in response to 4′-O-methylnorbelladine concentration All data was performed in biological triplicate Cells were cultured for 4 h with the ligand in (b) and (c) and for 18 h with the ligand in (f) and (g) a Schematic representation of the biosensor-monitored enzymatic reaction within E Blue and purple hexagons denote norbelladine and 4′-O-methylnorbelladine The schematic was designed using a vector graphics editor b Correlation between cell population fluorescence and biosynthesized 4′-O-methylnorbelladine measured by high-performance liquid chromatography c Chromatographic traces of supernatant collected from E coli cells expressing an active Nb4OMT enzyme (green) or an empty plasmid (red) Traces for norbelladine and 4′-O-methylnorbelladine standards are shown in blue and purple These observations all suggested that Nb4OMT activity and specificity could be improved by enzyme engineering To improve Nb4OMT activity in a microbial host we initially carried out directed evolution starting from randomly mutagenized libraries which generated an average of three mutations per gene The library of enzyme variants was transformed into cells containing the pSens4NB2.1 plasmid plated on solid media containing norbelladine and highly fluorescent colonies were isolated and then individually phenotyped in a secondary quantitative liquid-based fluorescence screen where they were compared to the wild-type enzyme this approach was not able to identify variants that outperformed the wild-type enzyme in the liquid-based screen d Workflow using MutComputeX for enzyme engineering In the A53 masked microenvironment that is shown the light blue spheres represent the masked alanine and S-adenosyl-homocysteine (SAH) is shown in pink b Time-dependent fluorescent signals produced by E coli cells containing the 4′-O-methylnorbelladine reporter plasmid (pSens-4NB2) and expressing Nb4OMT (WT) or ML-designed mutants Data was performed in biological triplicate and shaded error bands represent the S.D c Ion-extracted chromatograms of chemical standards (blue) or the supernatant of cells expressing Nb4OMT or ML-designed mutants (purple) cultured with norbelladine a Global structure of Nb4OMTE36P/G40E/A53M solved in 2.4 A resolution One dimer is colored blue while the other dimer is transparent b Comparison of N-termini of Nb4OMT crystal structure (orange) and wild-type Nb4OMT AlphaFold2 structure (blue) c Local context of the A53M mutant residue d Local context of the E36P and G40E mutant residues Black arrow indicates the shift of glutamate from position 36 to position 40 e Active site context of Nb4OMTE36P/G40E/A53M in complex with S-Adenosyl-L-homocysteine (SAH) and docked with norbelladine the color coding is as follows—calcium ions: green A sulfur-π interaction between the catechol group of norbelladine and Methionine 53 may also restrict the rotation of the catechol group thereby reducing the cross-methylation of the 3’ position and improving specificity Herein we report the use of directed evolution and machine learning-guided design for the development of custom microbial biosensors that could be used to monitor substantive improvements in amaryllidaceae alkaloid pathway activity The RamR transcription factor was evolved to respond to low micromolar levels of the pathway branchpoint 4NB After only four substitutions exquisite specificity emerges for the methylated oxygen moiety in 4NB with a barely detectable response to the non-methylated precursor norbelladine The high specificity was also essential for measuring the real-time activity of the plant-derived Nb4OMT enzyme in E these results highlight the powerful capability of using evolved biosensors for precisely reporting on pathway intermediates while avoiding cross-reactivity with closely related precursor molecules The RamR protein is now well positioned as an ideal starting point for the generation of biosensors for not only benzylisoquinoline alkaloids Together with the 4NB2 sensor and MutComputeX model the Nb4OMT variant structure should accelerate progress towards further engineering of the AA pathway 4′-O-methylnorbelladine was purchased from Toronto Research Chemicals (Toronto Research Chemicals and NaBH4 were purchased from Sigma Aldrich CD3OD) were purchased from Cambridge isotope laboratories NMR spectra were taken on the 500 MHz Bruker prodigy at University of Texas at Austin strains were made competent for chemical transformation Five milliliters of an overnight culture of DH10B cells was subcultured into 500 mL LB medium and grown at 37 °C and 250 r.p.m until an optical density of 0.7 was reached (~3 h) and pellets were washed with 70 mL chemical competence buffer (10% glycerol 100 mM CaCl2) and centrifuged again (3500 × g The resulting pellets were resuspended in 20 mL chemical competence buffer cells were divided into 250-μL aliquots and flash-frozen in liquid nitrogen Competent cells were stored at −80 °C until use where cultures were grown for an additional 18 h at 37 °C and 250 r.p.m in order to compare sensor response in fermentation-relevant conditions and cell pellets were resuspended in 1 mL PBS (137 mM NaCl One hundred microliters of the cell resuspension for each condition was transferred to a 96-well microtiter plate (Corning 509 nm) and absorbance (600 nm) were measured using the Tecan Infinite M1000 plate reader Three semi-rational libraries were designed, each targeting three inward-facing residues within the RamR ligand-binding pocket (K63, L66, M71; E120, A123, D124; L133, C134, S137) (Supplementary Fig. 1) Libraries were generated using overlap PCR with redundant NNS codons using AccuPrime Pfx (Thermo Fisher coli DH10B bearing pSELIS-RamR was transformed with the resulting library Transformation efficiency always exceeded 106 for each round of selection indicating several fold coverage of the library Transformed cells were grown in LB medium overnight at 37 °C with carbenicillin and chloramphenicol Cell culture (20 μl) bearing the sensor library was seeded into 5 ml fresh LB containing appropriate antibiotics R25001) and 100 μM of norbelladine (for round two) and grown at 37 °C for 7 h 0.5 μl of culture was diluted into 1 ml LB medium from which 100 μl was further diluted into 900 μl LB medium Three hundred microliters of this mixture was then plated across three LB agar plates (100 μL per plate) containing carbenicillin the brightest colonies were picked and grown overnight in 1 ml LB medium containing appropriate antibiotics in a 96-deep-well plate sealed with an AeraSeal film at 37 °C A glycerol stock of cells containing pSELIS-RamR and pReg-RamR encoding the template RamR variant was also inoculated into 5 ml LB for overnight growth 20 μl of each culture was used to inoculate two separate wells in a new 96-deep-well plate containing 900 μl LB medium eight separate wells containing 1 ml LB medium were inoculated with 20 μl of the overnight culture expressing the parental RamR variant the top half of the 96-well plate was induced with 100 μl LB medium containing 10 µl DMSO whereas the bottom half of the plate was induced with 100 μl LB medium containing 4NB dissolved in 10 μl DMSO The concentration of 4NB used for induction is typically the same concentration used in the LB agar plate for screening during that particular round of evolution Cultures were grown for an additional 4 h at 37 °C and 250 r.p.m and cell pellets were resuspended in 1 ml PBS One hundred microliters of the cell resuspension for each condition was transferred to a 96-well microtiter plate Clones with the highest signal-to-noise ratio (generally the top 5–10% of the screened clones) were then sequenced and subcloned into a fresh pReg-RamR vector the subcloned pReg-RamR vectors expressing the sensor variants were transformed into DH10B cells expressing Pramr-GFP as described in the section ‘Biosensor response assay’ using eight different concentrations of the 4NB The sensor variant that displayed a combination of low background a reduced EC50 for 4NB and a high signal-to-noise ratio was then used as the template for the next round of evolution Glycerol stocks (20% glycerol) of strains containing the plasmids of interest were inoculated into 1 ml LB medium and grown overnight at 37 °C Twenty microliters of overnight culture was seeded into 900 μl LB medium containing ampicillin and chloramphenicol in a 2-ml 96-deep-well plate sealed with an AeraSeal film cultures were induced with 100 μl of an LB medium solution containing appropriate antibiotics and the inducer molecule dissolved in 10 μl DMSO The cell resuspension (100 μl) for each condition was transferred to a 96-well microtiter plate Nb4OMT was expressed with the P150-RBS(riboJ) promoter–RBS on the pReg-RamR plasmid backbone (no regulator present) Cells were co-transformed with both the Nb4OMT plasmid and the 4NB reporter plasmid and plated on an LB agar plate containing appropriate antibiotics Three individual colonies from each transformation were picked into LB and grown overnight Resulting cultures were diluted 50-fold into 1 mL LB medium containing the indicated concentration of norbelladine in a 96-deep-well plate and were grown at the indicated temperature for 24 h the fluorescence of cultures was measured in the same manner as previously described in ‘Dose–response measurements’ above To generate a voxelized molecular representation of a microenvironment a 20 Å cube of atoms was filtered from the structure centered on the Calpha and oriented with respect to the backbone where the side chain was along the +z axis All atoms in the center residue are then removed prior to insertion into a voxelized grid with 1 Å resolution Each atom is placed into a corresponding element channel except halogen atoms (which are placed into a multi-atom channel that consist of F resulting in the following atomic channels: C Each atom’s partial charge and SASA value are placed into the partial charge and SASA channels atom values are gaussian blurred according to their Van-der-Waals radii The P and Halogen channels were added to the original MutCompute framework in order to generalize to ligands and nucleic acids The 3D residual neural network was built in Tensorflow 2.7. The architecture is provided in Supplementary Fig. 7 Each model run was parallelized over 4 AMD Radeon Instinct MI50s with a batch size of 200 Models were trained for up to 8 epochs where each epoch was saved as a checkpoint with a variety of hyperparameters We used a scheduled learning rate that began at 0.001 and had an exponential decay constant of either 0.3 or 0.5 and an adaptive learning rate that would lower the learning rate by 0.25 if the training accuracy did not improve by 0.1% after either 30 K Weights were updated with the Adam optimizer and all convolutional layers had weight decay regularization of 0.001 Assay samples were filtered using a 0.2-μm PTFE syringe filter prior to running the HPLC The measurement of Norbelladine and 4′-O-methylnorbelladine was performed using a Vanquish HPLC system (Thermo Fisher Scientific) equipped with a BDS Hypersil TM C18 (3.0 × 150 mm2 3 μm) (Thermo Fisher Scientific) with detection wavelength 277 nm The mobile phase consisted of 0.1% formic acid in water or 0.1% formic acid in acetonitrile over the course of 28 min under the following conditions: 10% organic (vol/vol) for 2 min A standard curve for norbelladine was prepared using synthesized norbelladine (see ‘Chemical synthesis and NMR analysis of norbelladine’) A standard curve for 4′-O-methylnorbelladine was prepared using commercially available 4′-O-methylnorbelladine Reactions for kinetics measurements were performed in triplicate for all enzyme variants 1.5 ml reactions containing 3.5 nM of enzyme or 500 µM norbelladine in PBS pH 7.5 were incubated at 37 °C for 4 h Every hour a 200 µl aliquot of each reaction was quenched by pipetting it into a 1.5 ml microcentrifuge tube with 20 µl of 2 M HCl The concentration of 4′-O-methylnorbelladine was then determined using HPLC as described Cells containing the plasmid expressing each Nb4OMT variant with the P150-RBS(RiboJ) promoter were transformed and plated onto an LB agar plate containing appropriate antibiotics three colonies from each plate (n = 3) were cultured overnight in LB and subsequently diluted 50-fold into 1 ml LB containing 1 mM norbelladine These cultures were grown for 24 h at 37 °C and centrifuged at 16,000 × g for 1 min and the resulting supernatant was filtered using a 0.2-μm filter signal counts from the EIC within a window ±0.05 min relative to the retention time of the substrate and products were extracted for each scan (m/z ratios 260.1281 and 274.1438) Kinetic data were fit in KinTek Explorer simulation and data fitting software v1156 The following minimal model was used as an input Each line represents a step in the model and the forward reaction goes from left to right while the reverse reaction goes from right to left as written Starting concentrations were entered into the software just as the reactions were performed: this threshold was 0.85 to estimate the upper and lower limits for each parameter While the model described above is the simplest model that could describe the data and gave reasonable estimates for kcat and kcat/Km there was evidence for substrate inhibition at the highest norbelladine concentration for the two variants (A53M and triple mutant) that this model did not account for We then fit the data for these two variants to the model shown below For bacterial overexpression of Nb4OMT wild type and its variants (A53M and E36P + G40E + A53M) coli BL21 (DE3) was used as the expression host and its competent cell was transformed with the corresponding constructed plasmids coli BL21 (DE3) strain harboring one of the constructed plasmids was inoculated into 2 mL of Luria Bertani broth (LB) medium with 100 µg/mL ampicillin and grown overnight at 37 °C/225 rpm The overnight-grown culture (using 1 mL) was scaled up into a 500-mL autoinduction media at 37 °C/225 rpm Protein expression was automatically induced and cells were cultured for 24 h at 25 °C/225 rpm The induced cell culture was harvested by centrifugation at 4000 × g and 4 °C for 20 min Cell pellets were then resuspended in 200 mL of lysis buffer (50 mM TRIS pH 8.0 Cells were lysed by sonication and the resulting cell lysate was centrifuged at 15,000 × g and 4 °C for 20 min to obtain the supernatant that contains soluble proteins The supernatant was equilibrated with HisPur™ Ni-NTA Resin (Thermo Fisher Scientific MA) and washed with 10x bed volumes of wash buffer (50 mM TRIS pH 8.0 Then protein was eluted by using a 10 mL elution buffer (50 mM TRIS pH 8.0 The eluate was dialyzed with 3 C protease added to the dialysis cassette into the appropriate buffer (20 mM TRIS pH 7.5 10 mM β-mercaptoethanol) followed by size-exclusion fast protein liquid chromatography All Nb4OMT variants were stored in 20 mM Tris (pH 7.5) To identify crystallization conditions of the Nb4OMT variant with triple mutations (E36P + G40E + A53M) 20 mg/ml purified enzyme samples were directly used in sparse matrix screening Rod-shaped crystals formed after incubating screening plates at room temperature for 3 days A crystallization condition with the best crystal morphology (0.1 M Calcium Acetate and 20% PEG3350) was chosen and further optimized by manually setting sitting-drop vapor diffusion experiments by varying pH and precipitant concentration resulting diffraction-quality single crystals in 0.1 M Calcium Acetate Purified Nb4OMT variants in the concentration of 5 µM were prepared in 96-well low-profile PCR plates (ABgene 10X SYPRO® Orange (Molecular Probes) was added into each well and mixed prior to measurement in an RT-PCR machine (LightCycler 480 The protein melting experiments were carried out with a continuous temperature acquisition mode using 10 acquisitions per 1 °C in each cycle from 20 °C to 95 °C The melting curves of the Nb4OMT variants were monophasic and Tm values were derived using Boltzmann equation All data in the text are displayed as mean ± S.D dose–response functions were all plotted in Python 3.6.9 using Matplotlib Dose–response curves and EC50 values were estimated by fitting to the Hill equation y  =  d  +  (a  −  d)xb(cb + xb)−1 (where y = output signal with the scipy.optimize.curve_fit library in Python Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article in The Alkaloids: Chemistry and Biology (ed Biological evaluation of structurally diverse amaryllidaceae alkaloids and their synthetic derivatives: discovery of novel leads for anticancer drug design The Amaryllidaceae alkaloids haemanthamine haemanthidine and their semisynthetic derivatives as potential drugs Lycorine: a prospective natural lead for anticancer drug discovery Nasal application of the galantamine pro-drug memogain slows down plaque deposition and ameliorates behavior in 5X familial Alzheimer’s disease mice The case of galantamine: repurposing and late blooming of a cholinergic drug Effect of bulb age on alkaloid contents of narcissus pseudonarcissus bulbs Integrating Narcissus-derived galanthamine production into traditional upland farming systems Effect of fertilizers on galanthamine and metabolite profiles in Narcissus bulbs by 1H NMR A microbial biomanufacturing platform for natural and semisynthetic opioids Complete biosynthesis of the bisbenzylisoquinoline alkaloids guattegaumerine and berbamunine in yeast Biosynthesis of medicinal tropane alkaloids in yeast A microbial supply chain for production of the anti-cancer drug vinblastine The Amaryllidaceae alkaloids: biosynthesis and methods for enzyme discovery Accelerating the semisynthesis of alkaloid-based drugs through metabolic engineering Using fungible biosensors to evolve improved alkaloid biosyntheses Taking control over control: use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways Combining mechanistic and machine learning models for predictive engineering and optimization of tryptophan metabolism Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter Machine learning-aided engineering of hydrolases for PET depolymerization Adaptive machine learning for protein engineering Machine learning-guided acyl-ACP reductase engineering for improved in vivo fatty alcohol production Machine learning-assisted directed protein evolution with combinatorial libraries GNINA 1.0: molecular docking with deep learning Highly accurate protein structure prediction with AlphaFold Cloning and characterization of a norbelladine 4′-O-methyltransferase involved in the biosynthesis of the Alzheimer’s drug galanthamine in Narcissus sp Synthetic biology strategies for microbial biosynthesis of plant natural products Discovery of novel gain-of-function mutations guided by structure-based deep learning Improved Bst DNA polymerase variants derived via a machine learning approach Learning the local landscape of protein structures with convolutional neural networks He, K., Zhang, X., Ren, S. & Sun, J. Deep residual learning for image recognition. Preprint at https://doi.org/10.48550/arXiv.1512.03385 (2015) He, K., Zhang, X., Ren, S. & Sun, J. Identity mappings in deep residual networks. Preprint at https://doi.org/10.48550/arXiv.1603.05027 (2016) FireProtDB: database of manually curated protein stability data Temporal patterns of seed germination in early spring-flowering temperate woodland geophytes are modified by warming Crystal structures of alfalfa caffeoyl coenzyme A 3-O-methyltransferase Characterization of two O-methyltransferases involved in the biosynthesis of O-methylated catechins in tea plant Accurate prediction of protein structures and interactions using a three-track neural network Evolutionary-scale prediction of atomic-level protein structure with a language model Wu, R. et al. High-resolution de novo structure prediction from primary sequence. Preprint at https://doi.org/10.1101/2022.07.21.500999 (2022) De novo design of protein structure and function with RFdiffusion Ig-VAE: generative modeling of protein structure by direct 3D coordinate generation AlphaFill: enriching AlphaFold models with ligands and cofactors Cytochrome P450s in plant terpenoid biosynthesis: discovery characterization and metabolic engineering Evaluation of Brachypodium distachyon L-tyrosine decarboxylase using L-tyrosine over-producing Saccharomyces cerevisiae Metabolic engineering of muconic acid production in Saccharomyces cerevisiae RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes Identification of a noroxomaritidine reductase with amaryllidaceae alkaloid biosynthesis related activities Cloning and characterization of norbelladine synthase catalyzing the first committed reaction in Amaryllidaceae alkaloid biosynthesis Transcriptome analysis of Leucojum aestivum and identification of genes involved in norbelladine biosynthesis pseudonarcissus catalyzes formation of the Para-Para’ C-C phenol couple in the amaryllidaceae alkaloids Mehta, N., Meng, Y., Zare, R., Kamenetsky-Goldstein, R. & Sattely, E. A developmental gradient reveals biosynthetic pathways to eukaryotic toxins in monocot geophytes. Preprint at https://doi.org/10.1101/2023.05.12.540595 (2023) Atomic Charge Calculator II: web-based tool for the calculation of partial atomic charges Mitternacht, S. FreeSASA: an open source C library for solvent accessible surface area calculations. Preprint at https://doi.org/10.12688/f1000research.7931.1 (2016) Global Kinetic Explorer: a new computer program for dynamic simulation and fitting of kinetic data FitSpace Explorer: an algorithm to evaluate multidimensional parameter space in fitting kinetic data d’Oelsnitz, S. SBplot: a plotting library for synthetic biology. Zenodo https://doi.org/10.5281/zenodo.10655833 (2024) Diaz, D. & Loy, J. danny305/MutComputeX: v1.0.0. Zenodo https://doi.org/10.5281/zenodo.10655415 (2024) Download references Funding from National Institute of Standards and Technology (70NANB21H100 to A.D.E.) and the Air Force Office of Scientific Research (FA9550-14-1-0089 to A.D.E.) is acknowledged This work was partially supported by grants from the National Institutes of Health (R35GM148356 to Y.Z We would like to thank NSF AI Institute for Foundations of Machine Learning (IFML) for their support and AMD for the donation of hardware and support resources from its HPC fund Crystallographic data collections were conducted at Advanced Light Source (BCSB - BL 8.2.2) Department of Energy scientific user facility at Lawrence Berkeley National Laboratory Institute for Foundations of Machine Learning McKetta Department of Chemical Engineering and performed biosensor evolution and characterization with support from M.W.S developed MutComputeX with support from J.M.L and enzyme designs were generated by D.J.D performed enzyme engineering with support from H.D performed HPLC measurements with support from S.d’O performed enzyme kinetic assays and analysis purified protein and conducted X-ray crystallography with support from Y.J.Z The authors declare the following competing interests Patent applicant: The Board of Trustees of the University of Texas at Austin Title: METHODS AND COMPOSITIONS RELATED TO MODIFIED METHYLTRANSFERASES AND ENGINEERED BIOSENSORS has financial relationships with Retna Bio LLC has financial relationships with Intelligent Proteins LLC All other authors declare no competing interests Nature Communications thanks Michael Jensen reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-024-46356-y Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Students wearing garments from SOLACE Collective’s new line of clothing for neurodivergent individuals Carolyn Fernandes ’25 remembers walking out of an interview for a design internship that she ended up not getting She wondered if fidgeting with her nails—a common repetitive expression of her nervous energy every day—was misconstrued as a sign of not paying attention and a reason for her not getting the position Fernandes is part of the 20% of people worldwide who are neurodivergent and live with neurological differences like autism attention-deficit hyperactivity disorder (ADHD) Stories like the one above motivated Fernandes and fellow students Lucas d’Oelsnitz ’25, Aphrodite Gioulekas ’25 and Aidan Turner ’25 to create SOLACE Collective with comfortable garments that alleviate anxiety increase confidence and reduce distractions by providing opportunities for fidgeting discreetly or openly “People like me need to fidget to cope and feel comfortable so I started researching ways clothing could be made to help neurodiverse people like myself feel more comfortable,” Fernandes says After an inspiring conversation with Linda Dickerson Hartsock—the retired founder and executive director of Blackstone LaunchPad at Syracuse University Libraries—and after taking an Intelligence++ class focused on inclusive product design Turner came up with the idea for SOLACE “to meet the needs of a huge portion of the population that is currently underserved,” says Turner After sharing their personal experiences with uncomfortable clothing with each other Turner pitched creating the neurodivergent clothing line The project’s potential impact immediately resonated with d’Oelsnitz d’Oelsnitz does a lot of idea pitching but always struggles to control his fidgeting After one of his first pitches before his peers unable to rely on his usual coping mechanisms like playing with his rings “Realizing this is something I would wear motivated me to go all-in on this project because this is not just a clothing brand; it’s solving an important social issue,” says d’Oelsnitz The team set out to collect data on how SOLACE could better meet the needs of neurodiverse individuals beginning with a wardrobe analysis and personal interviews to determine what types of clothing neurodivergent people liked or didn’t like—including textures and how a garment feels Lucas d’Oelsnitz (right) discusses SOLACE Collective’s line of clothing with a student during one of 10 on-campus events where students could try on the clothes and provide feedback Fernandes conducted a survey of 430 people ages 5 to 91 and discovered that 95% of respondents experienced discomfort from their clothing Incorporating the feedback and relying on sketches Turner created a visual design brief that served as the blueprint for the first line of clothing and Fernandes and others on the team used sewing machines to create prototypes from fabric samples collected from their closets and local thrift shops Currently there are four prototypes consisting of a matching sweatshirt or hoodie and sweatpants of assorted colors The clothes are tagless (product details and cleaning instructions are screen-printed on the outside) with many of the items providing its wearer a sense of grounded-ness thanks to the use of heavy fabric while others rely on light and airy fabrics “Our product line serves to confirm to a lot of neurodivergent people that what they’re doing is okay and that they can be normal,” says Fernandes who serves as SOLACE’s industrial designer “And it’s not just neurodivergent people that want this clothing then no one is going to feel like they’re sticking out like a sore thumb while they fidget.” Aidan Turner (far right) talks with students during one of SOLACE Collective’s on-campus feedback events “We try to achieve a grounded feeling while providing warmth and the reassurance that you’re safe We’re trying to find a sustainable way to produce items of clothing for everyone no matter how your neurodiversity presents,” says d’Oelsnitz The group has received more than $32,000 in startup investment money through entrepreneurial competitions held on campus and through grants, including the 2025 School of Information Studies’ Raymond von Dran Fund for Student Entrepreneurship iPrize competition they held 10 events on campus for students to try on the clothes and provide feedback including the launch of a website and social media channels they hope to start accepting pre-orders in late June with items being distributed to customers by the end of the summer Knowing how well-received their casual prototypes have been Fernandes and d’Oelsnitz next want to expand into business casual clothing and I was introduced to the lack of solutions that exist for neurodivergent individuals,” says Turner “There are a lot of opportunities for us to give this overlooked community reassurance and warmth through our clothing and I’m proud of what this wonderful team has accomplished so far.” Members of the campus community learn about the SOLACE Collective’s line of clothing for neurodivergent individuals Service members injured in active duty are entitled to receive disability benefits and associated medical care from the U.S But navigating the process can be intimidating Thanks to the efforts of the Betty and Michael… Those were the principles behind the creation of the Mary Ann Shaw Center for Public and Community Service in 1994 under the direction of then-Syracuse University Chancellor Kenneth Shaw and his… The cutting-edge weight loss and diabetes research developed by medicinal chemist Robert Doyle has offered significant and consistent weight loss and glucose control to its recipients through peptide-based treatments Doyle and his fellow collaborators reported that two new peptide compounds­­—GEP44… Jonida Konjufca was not sure what she wanted to do professionally before she enrolled in the Department of Human Development and Family Science (HDFS) but her professors and coursework soon opened her eyes the Syracuse University Volunteer Organization (SUVO) took on its first bed build event on campus The organization teamed up with the local chapter of Sleep in Heavenly Peace (SHP) to build 44 beds for children in the community… If you need help with your subscription, contact sunews@syr.edu Metrics details Some of these approaches do not actually delimit the relative importance of each base within the operator sequence possibly leading to further dissection of the binding site a generalizable tool for predicting binding sequences would enable biotechnologists to identify and repurpose many more transcription factors A simple input (one protein accession ID) and interactive data visualization interface should facilitate the adoption of Snowprint among synthetic biologists and biotechnologists A RefSeq or GenBank accession ID is used to fetch the protein sequence of the regulator and the DNA sequence of the local genetic context (1) predicted to contain the regulator’s corresponding operator is then scanned for inverted repeat sequences (2) BLAST is then used to collect regulator homologs (3) which are used to collect homologous inter-operon regions (4) that are also scanned for inverted repeats similar to that found for the original regulator (5) The homologous inverted repeat sequences are then used to create a consensus sequence The Snowprint logo was designed using a vector graphics editor this algorithm design is encoded in a program we call Snowprint a lightweight computational method to determine a regulator’s DNA “footprint” Separate phyla are color coded and labeled d Representative examples of predicted operator motifs generated by Snowprint Regulators were further filtered on the basis that their annotation contained the word “regulator” and that they were adjacent to genes annotated as multidrug efflux pumps Snowprint was then used to predict operators for the resulting regulators Upon testing all 33 predicted operators with their cognate regulators, 28 were able to reduce the level of expressed GFP by at least 50%, and 24 were able to reduce signal by over 20-fold (Fig. 3c, Supplementary Fig. 6) This ~85% success rate is quite surprising given that the regulators were sourced from wildly divergent microbial hosts and that synthetic promoters were crafted in accord with simple rules in vivo repression could fail for reasons other than correct regulator:operator matching such as issues relating to heterologous protein production natural biosensors have not been identified for any of these ligands and in the future it is unlikely that it will immediately prove possible to regularly identify natural regulators for any biomanufacturing-relevant compound of interest b–e Dose response measurements for the BAK71752.1 and SMC09139 regulators with tetrahydropapaverine (THP) The ligand concentration was chosen based on the compound’s solubility limit in 1% DMSO Assays were performed in biological triplicate and individual data points are shown Snowprint was able to accurately predict operator sequences for 67 of 147 experimentally-validated regulator:operator pairs across the TetR these data suggest that Snowprint may be able to completely bypass traditional laborious methods for determining a regulator’s operator sequence rapidly accelerating the pace of regulator discovery The consensus score for an operator prediction was made using the following formula \(c=\frac{{\sum }_{o=0}^{x}{b}^{2}}{x}\) where \(c\) = the consensus score o = the position along the length of the operator \(b\) = the relative frequency of the dominant base within the predicted operator and \(x\) = the length of the predicted operator The inverted repeat score for an operator sequence was made using the following formula \(x\) = the number of matches within an inverted repeat \(y\) = the number of mismatches within an inverted repeat which adds or removes from the score based on the distance between repeat half sites as follows (0–4 bases = +4; 5–6 = +2; 7–8 = 0; 9–10 = −2; 11–12 = −4; 13–14 = −6; 15–16 = −8; 17–18 = −10) The pairwise2 algorithm of Biopython was used to generate alignment scores for all other sequence comparisons As outlined in Supplementary Fig. 3 the UniRef50 database was used to fetch TetR-family regulators using the search query “tetr regulator” CD-HIT was subsequently used to cluster the resulting sequences into 30% identity groups The Galaxy software suite was then used to filter out regulators with sequences above 260 amino acids and below 140 amino acids a python script was used to filter out sequences that did not contain the word “regulator” in their annotation name and the Entrez API was then used to extract only the regulators that were located adjacent to proteins annotated as multidrug efflux pumps Snowprint predictions were generated for the remaining regulators and the top 33 with the best consensus scores were used for in vivo experimental validation Tetrahydropapaverine was purchased from Tokyo Chemical Industry Co Ursodiol was purchased from MP Bio (0215825201) Geraniol was purchased from Tokyo Chemical Industry (G0027) Sitagliptin was purchased from Ambeed (654671-77-9) Artemisinic acid was purchased from Cayman Chemical (25059) Olivetolic acid was purchased from Cayman Chemical (26282) All data in the text are displayed as mean ± standard deviation unless specifically indicated All experimental assays were performed in biological triplicate which represent three individual bacterial colonies picked from an agar plate and dose–response functions were all plotted in Python 3.10.6 using Matplotlib and Seaborn Dose–response curves and EC50 values were estimated by fitting to the Hill equation y = d + (a − d)xb(cb + xb)−1 (where y = output signal The only difference between the command line tool and the web application is that the former uses NCBI’s BLAST to collect protein homologs while the latter uses DIAMOND While results returned from the command line tool and web application are similar Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article The GenBank ID for the CamR protein used as a control regulator is BAA03510.1 Cell-free biosensors for rapid detection of water contaminants Dynamic upregulation of the rate-limiting enzyme for valerolactam biosynthesis in Corynebacterium glutamicum Effective use of biosensors for high-throughput library screening for metabolite production Synthetic addiction extends the productive life time of engineered Escherichia coli populations Development and application of whole-cell biosensors for the detection of gallic acid A new biosensor for stilbenes and a cannabinoid enabled by genome mining of a transcriptional regulator Evolving a generalist biosensor for bicyclic monoterpenes Stockley, P. G. & Persson, B. Surface plasmon resonance assays of DNA-protein interactions. in DNA-Protein Interactions: Principles and Protocols, Third Edition (eds. Leblanc, B. & Moss, T.) 653–669 (Humana Press, 2009). https://doi.org/10.1007/978-1-60327-015-1_38 DNAase footprinting a simple method for the detection of protein-DNA binding specificity A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system+ Mapping genome-wide transcription-factor binding sites using DAP-seq Embryonic ε and γ globin genes of a prosimian primate (Galago crassicaudatus): nucleotide and amino acid sequences developmental regulation and phylogenetic footprints An integrative and applicable phylogenetic footprinting framework for cis-regulatory motifs identification in prokaryotic genomes An integrated toolkit for accurate prediction and analysis of cis-regulatory motifs at a genome scale MicroFootPrinter: a tool for phylogenetic footprinting in prokaryotic genomes DMINDA 2.0: integrated and systematic views of regulatory DNA motif identification and analyses Integrating genome sequence and structural data for statistical learning to predict transcription factor binding sites P2TF: a comprehensive resource for analysis of prokaryotic transcription factors Design of gene circuits: lessons from bacteria Local and global regulation of transcription initiation in bacteria Phylogenetic footprinting: a boost for microbial regulatory genomics Phenylacetic acid catabolism and its transcriptional regulation in corynebacterium glutamicum Jungle express is a versatile repressor system for tight transcriptional control A perfectly symmetric lac operator binds the lac repressor very tightly Genomic mining of prokaryotic repressors for orthogonal logic gates Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor d’Oelsnitz, S. et al. Synthetic microbial sensing and biosynthesis of amaryllidaceae alkaloids. bioRxiv 2023.04.05.535710 https://doi.org/10.1101/2023.04.05.535710 (2023) Programming gene expression with combinatorial promoters Production of the antimalarial drug precursor artemisinic acid in engineered yeast Engineering Escherichia coli for production of geraniol by systematic synthetic biology approaches and laboratory-evolved fusion tags Synthetic pathway for the production of olivetolic acid in escherichia coli Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture Latest development in the synthesis of ursodeoxycholic acid (UDCA): a critical review Regulation mechanism of nicotine catabolism in sphingomonas melonis TY by a dual role transcriptional regulator NdpR Crystal structure of the transcription activator BmrR bound to DNA and a drug Structural basis of direct and inverted DNA sequence repeat recognition by helix–turn–helix transcription factors Fast and sensitive protein alignment using DIAMOND Hanko, E. K. R., Joosab Noor Mahomed, T. A., Stoney, R. A. & Breitling, R. TFBMiner: a user-friendly command line tool for the rapid mining of transcription factor-based biosensors. ACS Synth. Biol. https://doi.org/10.1021/acssynbio.2c00679 (2023) Deglycosylation as a mechanism of inducible antibiotic resistance revealed using a global relational tree for one-component regulators Protein engineers turned evolutionists—the quest for the optimal starting point SensiPath: computer-aided design of sensing-enabling metabolic pathways GroovDB: a database of ligand-inducible transcription factors Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors Protein–DNA/RNA interactions: machine intelligence tools and approaches in the era of artificial intelligence and big data LogoJS: a Javascript package for creating sequence logos and embedding them in web applications The Snowprint repository. https://doi.org/10.5281/zenodo.10442767 NGPhylogeny.fr: new generation phylogenetic services for non-specialists Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation Download references Funding from National Institute of Standards and Technology (70NANB21H100) the Air Force Office of Scientific Research (FA9550-14-1-0089) and the National Institutes of Health (R01EB026533) is acknowledged The Snowprint command line tool was developed and benchmarked by S.D The Snowprint web application was developed by J.D.L The authors declare no competing interests Communications Biology thanks Jean-Loup Faulon and the other Primary Handling Editors: Cesar de la Fuente and David Favero Download citation DOI: https://doi.org/10.1038/s42003-024-05849-8 image: An Alzheimer's drug usually harvested from daffodils is one step closer to being fermented inside bacteria thanks to some help from artificial intelligence and biosensors.  Credit: Photo-Illustration: Martha Morales/The University of Texas at Austin Galantamine is a common medication used by people with Alzheimer’s disease and other forms of dementia around the world to treat their symptoms synthesizing the active compounds in a lab at the scale needed isn’t commercially viable The active ingredient is extracted from daffodils through a time-consuming process researchers at The University of Texas at Austin have developed tools — including an artificial intelligence system and glowing biosensors — to harness microbes one day to do all the work instead In a paper in Nature Communications researchers outline a process using genetically modified bacteria to create a chemical precursor of galantamine as a byproduct of the microbe’s normal cellular metabolism the bacteria are programmed to convert food into medicinal compounds “The goal is to eventually ferment medicines like this in large quantities,” said Andrew Ellington a professor of molecular biosciences and author of the study “This method creates a reliable supply that is much less expensive to produce and it can’t be impacted by drought or floods.” a postdoctoral fellow with the Deep Proteins research group in UT’s Institute for Foundations of Machine Learning (IFML) developed an AI system called MutComputeX that is key to the process It identifies how to mutate proteins inside the bacteria to improve their efficiency and operating temperature in order to maximize production of a needed medicinal chemical “This system helped identify mutations that would make the bacteria more efficient at producing the target molecule,” Diaz said it was up to three times as efficient as the natural system found in daffodils.” The process of harnessing microbes to produce useful byproducts is nothing new and bacteria help create cheese and yogurt Microbial fermentation is currently used to make certain types of insulin for diabetes treatment hormones and recombinant proteins used in several drugs such as autoimmune treatments But applying AI in the process is relatively new and expands what is possible with microbial fermentation coli to produce 4-O’Methyl-norbelladine The complex molecule is in a family of compounds extracted from daffodils that have medicinal uses in treating conditions such as cancer but using microbial fermentation to create a chemical in this family is new The scientists also created a fluorescent biosensor to quickly detect and analyze which bacteria were producing the desired chemicals and how much comes into contact with the chemical researchers wanted to create “The biosensor allows us to test and analyze samples in seconds when it used to take something like five minutes each,” said Simon d’Oelsnitz a postdoctoral researcher formerly at UT Austin and now at Harvard University “And the machine learning program allows us to easily narrow candidates from tens of thousands to tens Jessie Zhang of UT and Matthew Minus of Prairie View A&M University were also authors of the paper The research was supported by the National Institute of Standards and Technology the Air Force Office of Scientific Research and the National Institutes of Health and the National Science Foundation supports IFML Computing resources were provided by Advanced Micro Devices Those involved in this research have submitted required financial disclosure forms with the University Diaz and d’Oelsnitz have filed a patent application on materials described in this text Diaz and d’Oelsnitz are each involved with startups related to this research 10.1038/s41467-024-46356-y Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme Diaz and d’Oelsnitz have filed a patent application on materials described in this text Diaz and d’Oelsnitz are each involved with startups related to this research are not responsible for the accuracy of news releases posted to EurekAlert by contributing institutions or for the use of any information through the EurekAlert system Copyright © 2025 by the American Association for the Advancement of Science (AAAS) sustainable drug options than we have today the medication we take to treat high blood pressure pain or memory loss may one day come from engineered bacteria And thanks to a new bacterial tool developed by scientists at The University of Texas at Austin the process of improving drug manufacturing in bacterial cells may be coming sooner than we thought the UT Austin team introduces a biosensor system that can be adapted to detect all kinds of therapeutic compounds accurately and in mere hours “We’re figuring out how to give bacteria ‘senses,’ similar to olfactory receptors or taste receptors and use them for detection of the various compounds they might make,” said Andrew Ellington a professor of molecular biosciences and corresponding author on the paper Many of the medicines we take are made with ingredients extracted from plants (think the narcotic painkiller that comes from poppies a drug treatment for dementia that comes from daffodils) Extracting drugs from these plants is complicated and resource-intensive requiring water and acreage to grow the crops Deriving similar therapeutic components using synthetic chemistry brings problems since the process depends on petroleum and petroleum-based products linked to waste and expense The genetic code of bacteria can be easily manipulated to become factories for drug production the bacteria’s biological systems are harnessed to produce specific molecules as part of the natural cellular process manufacturers have not had a way to quickly analyze different strains of engineered bacteria to identify the ones capable of producing quantities of a desired drug at commercial volumes — until now Accurately analyzing the thousands of engineered strains on the way to a good producer can take weeks or months with current technology “There are currently no biosensors for most plant metabolites,” said Simon d’Oelsnitz a research scientist in the Department of Molecular Biosciences and first author on the paper it should be possible to create biosensors for a wide range of medicines.” Ellington and colleagues quickly and accurately determine the amount of a given molecule that a strain of bacteria is producing The team developed the biosensors for several types of common drugs such as cough suppressants and vasodilators Molecular images of the biosensors taken by X-ray crystallographers Wantae Kim and Yan Jessie Zhang show exactly how they tightly grab onto their partner drug When the drug is detected by the biosensor the team engineered their own bacteria to produce a compound found in several FDA-approved drugs and used the biosensors to analyze product output in essence showing how industry might adopt biosensors to quickly optimize chemical manufacturing “While this is not the first biosensor,” d’Oelsnitz said “this technique allows them to be developed faster and more efficiently that opens the door to more medicines being produced using biosynthesis.” Yan Jessie Zhang and Hal Alper of UT Austin and Ross Thyer previously of UT Austin and now at Rice University The research was funded by the Defense Advanced Research Projects Agency The University of Texas at Austin is committed to transparency and disclosure of all potential conflicts of interest University investigators involved in this research have submitted required financial disclosure forms with the university UT Austin and its researchers filed patent applications on the technology described here Get all the latest news and stories with Frontline See the latest vacancies and find out about working for MSF UK MSF staff will happily speak about their work at a fundraising or educational event Find a job working overseas with Médecins Sans Frontières raise funds and help our expert teams save lives around the world Amadeus is a nurse from Hamburg and is also on the board of MSF Germany Having completed multiple assignments with MSF worldwide he recently returned from Haiti where he worked as a medical team leader has seen waves of clashes between armed groups The violence has driven a stark need for emergency trauma care and surgery and cut people off from the everyday healthcare services they need Nurse Amadeus von der Oelsnitz explains how the Médecins Sans Frontières / Doctors Without Borders (MSF) principles of neutrality, impartiality and independence help our teams provide vital healthcare in a city torn apart by insecurity Port-au-Prince in the early hours of the morning I can feel the tension in our off-road vehicle - as always when we approach a roadblock An important basic rule that all MSF staff know.  at the transition from one neighbourhood to the next the balance of power in Haiti's capital is changing A few young men are standing behind a barricade made of car tyres They carry rapid-fire rifles and raise their arms to signal us to stop The men are checking who wants to come into their territory “Let's be clear: the security of our staff and the patients who come to our services is always our top priority We keep the dangers for them as low as possible.” Get the latest news on war and conflict with our monthly email newsletter critical updates and unique ways to get involved They know that we are on our way to our mobile clinic consultations bon boulot!" one of them shouts out - thank you And it’s because of exactly this – the joy about us and the acceptance of our work These are essential parts of our approach to security people interested in working with us have asked me:  "How can you actually work in conflict crisis areas Let's be clear: the security of our staff and the patients who come to our services is always our top priority We keep the dangers for them as low as possible The greatest risk for accidents and assaults for our teams is when we move from A to B – for example on the way to a clinic which in Haiti we always did in convoy with several vehicles.  The most important thing is to communicate with all those in power this meant that we met regularly with all the armed groups our project coordinators talk to the group leaders They explain to them when we would like to visit their area and where we would like to offer medical consultations.  We inform them that it is our task as humanitarian aid workers to treat them and the people in their neighbourhood but that we will also be at their opponents’ neighbouring quarter the following day to hold a consultation there When the groups have understood and accepted this principle of neutrality those in power ask us why we are providing aid – who our donors are Then we explain that we receive donations from many private individuals worldwide who simply want to help And that there are no political or economic interests behind our work.  This is an extremely important aspect of acceptance and security So a thank you goes out to all those who support us with donations Help us save lives in conflict zones The impact of conflict goes far beyond the frontlines We have been active in Haiti for more than 30 years The people in the country know us and they trust us.  MSF provided life-saving emergency aid after the earthquake in 2010 and during the following years when a devastating cholera epidemic broke out I heard again and again that they had personally received help from us or that family members had been saved by our teams.  Even those in power and their relatives need medical care I experienced this first-hand in Haiti.  several men came to our clinic with their pistols in their waistbands I approached them and asked them to leave their weapons outside This is also a basic rule for our security: no weapons are allowed in our clinics.  The men were initially indignant and wanted to take their turn immediately I had to explain to them that our rules mean we decide who gets treated next based solely on medical necessity mothers with seriously ill children were waiting in the room I was aware that I now had to appear confident One of the men had a chronic gunshot wound I managed to calm the situation down – and the man was later treated by our team that you're in the wrong place at the wrong time – and get caught in the crossfire of fights that have nothing to do with you” MSF's mission is to be there for the most vulnerable groups in crisis areas.  During my time in Haiti, we treated up to 100 patients a day during our mobile clinics. Many of our patients were pregnant women, elderly people with high blood pressure and diabetes, children with severe respiratory infections and diarrhoea, some acutely malnourished due to persistent infections Most of these illnesses are direct consequences of the living conditions Thousands live in displacement camps in Port-au-Prince Skin diseases such as scabies are widespread.  We offer some of our mobile clinics directly in the camps for displaced people others in empty warehouses or former schools.  we visit an abandoned health centre and practically put it back into operation for that short time The building was quite close to the border between two rival neighbourhoods which is why there were frequent gunshots in the area we secured the windows and balconies with sandbags to be protected from stray bullets that you're in the wrong place at the wrong time – and get caught in the crossfire of fights that have nothing to do with you.  That's why we minimise this risk by never being out in the dark poor visibility increases the risk for our drivers and our logo on the car can be harder to recognise.  There were also rare days when we could not hold our medical consultations We had received warnings from the armed groups or from the community that there could be fighting in our area of operation there is also a greatly increased risk of kidnapping Some criminal gangs earn their money through kidnappings As we are in constant contact with the armed groups we are well placed to prevent such situations All groups know that MSF does not pay ransom the best protection is that our medical aid is important to everyone and no one wants to harm us.  we immediately set up a specially trained team whose only concern is to ensure that the colleagues concerned are released as quickly as possible The risk of kidnapping is particularly high for our Haitian staff who don’t live in MSF accommodation and have to travel to and from work we don't have a good solution for these situations and it makes me angry that here it shows in miniature how unfair the world often is We generally moved around as little as necessary in Haiti This meant that me and my teammates who lived on-site spent a lot of time in our accommodation which was half flat and half office.  This is quite common when you’re working with MSF going jogging or even shopping in the supermarket are always much more intense for me after such an assignment I then always realise how much it means that in Germany That's why I think it's all the more important that we also look at what's happening elsewhere and that we get involved We provide medical care based on needs alone and try to reach the people who need help most If warring parties see aid organisations as being on one side of a conflict we are less likely to gain access to those in need and more likely to be attacked you'll get the best stories and urgent updates from our expert staff worldwide Metrics details A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can improve yield The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production We have developed a combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors; using RamR as a starting point we evolve highly specific (>100-fold preference) and sensitive (half-maximum effective concentration (EC50) < 30 μM) biosensors for the alkaloids tetrahydropapaverine High-resolution structures reveal multiple evolutionary avenues for the malleable effector-binding site and the creation of new pockets for different chemical moieties These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine a precursor to four modern pharmaceuticals collapsing multiple methylation steps into a single evolved enzyme Our methods for evolving biosensors enable the rapid engineering of pathways for therapeutic alkaloids Prices may be subject to local taxes which are calculated during checkout Code used to generate bar plots, dose–response functions and orthogonality matrices presented in this text is accessible at https://github.com/simonsnitz/plotting Complete biosynthesis of cannabinoids and their unnatural analogues in yeast Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli Cell-free synthetic biology: thinking outside the cell High-throughput screening for high-efficiency small-molecule biosynthesis Transcription factor-based biosensors: a molecular-guided approach for natural product engineering Genetic biosensor design for natural product biosynthesis in microorganisms Engineering and application of a biosensor with focused ligand specificity Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps Evolution-guided engineering of small-molecule biosensors Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes Complete biosynthesis of noscapine and halogenated alkaloids in yeast Structure-guided engineering of a scoulerine 9-O-methyltransferase enables the biosynthesis of tetrahydropalmatrubine and tetrahydropalmatine in yeast nalD encodes a second repressor of the mexAB–oprM multidrug efflux operon of Pseudomonas aeruginosa a repressor of the Stenotrophomonas maltophilia multidrug efflux pump SmeDEF The crystal structure of multidrug-resistance regulator RamR with multiple drugs Escherichia coli ‘Marionette’ strains with 12 highly optimized small-molecule sensors Robust and flexible platform for directed evolution of yeast genetic switches Bleomycin resistance conferred by a drug-binding protein from opium poppy: involvement in papaverine biosynthesis Isolation and characterization of O-methyltransferases involved in the biosynthesis of glaucine in Glaucium flavum Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution Directed enzyme evolution: beyond the low-hanging fruit Design and application of a lactulose biosensor A bacterial platform for fermentative production of plant alkaloids Genome engineering Escherichia coli for l-DOPA overproduction from glucose Progress in the microbial production of S-adenosyl-l-methionine Bohand, X., Jordan, D. & Dubois, F. Managing the risk of shortages and medication errors with curares during the COVID-19 pandemic: a hospital pharmacy experience. Eur. J. Hosp. Pharm. https://doi.org/10.1136/ejhpharm-2020-002605 (2021) Comparing the outcomes of different agents to treat vasospasm at microsurgical anastomosis during the papaverine shortage Privileged scaffolds for library design and drug discovery Design of an in vitro biocatalytic cascade for the manufacture of islatravir Dynamic control in metabolic engineering: theories Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals A suppressor tRNA-mediated feedforward loop eliminates leaky gene expression in bacteria Regulation by tetracycline of gene expression in Saccharomyces cerevisiae Tn10-encoded tet repressor can regulate an operator-containing plant promoter Tight control of gene expression in mammalian cells by tetracycline-responsive promoters Macromolecular structure determination using X-rays neutrons and electrons: recent developments in Phenix Coot: model-building tools for molecular graphics MolProbity: more and better reference data for improved all-atom structure validation Download references Funding from DARPA Soils (HR00111920019 to A.D.E.) Welch (F-1654 to A.D.E.) and AFSOR (FA9550-14-1-0089 to H.S.A This work is partially supported by grants from the National Institutes of Health (R01GM104896 and R01GM125882 to Y.Z.) Blake in the Chemistry Department at the University of Texas at Austin for performing LC–MS analysis and S Kar for his thoughtful advice on selection circuit design Department of Chemical and Biomolecular Engineering designed the experiments and performed biosensor evolution and characterization and X-ray crystallography was conducted by W.K have filed two patent applications on materials described in this text The other authors declare no conflict of interest Nature Chemical Biology thanks Michael Jensen and the other The same promoter was used to express variants from each evolutionary trajectory (see methods) Individual fluorescent measurements for each condition performed in biological triplicate are shown alongside error bars that represent the SE ± the mean Source data Fold-response is shown for all BIAs for the native RamR protein 100 μM of the indicated BIA was applied in all conditions Measurements for each condition represent the average of three biological replicates Source data Contains .XLSX sheets with statistical source data Download citation DOI: https://doi.org/10.1038/s41589-022-01072-w This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page Metrics details Prostate cancer (PCa) is defined by dysregulated lipid signaling and is characterized by upregulation of lipid metabolism-related genes including fatty acid binding protein 5 (FABP5) FASN and MAGL are enzymes that generate cellular fatty acid pools while FABP5 is an intracellular chaperone that delivers fatty acids to nuclear receptors to enhance PCa metastasis and MAGL have been independently implicated in PCa progression we hypothesized that FABP5 represents a central mechanism linking cytosolic lipid metabolism to pro-metastatic nuclear receptor signaling we show that the abilities of FASN and MAGL to promote nuclear receptor activation and PCa metastasis are critically dependent upon co-expression of FABP5 in vitro and in vivo Our findings position FABP5 as a key driver of lipid-mediated metastasis and suggest that disruption of lipid signaling via FABP5 inhibition may constitute a new avenue to treat metastatic PCa FABP5 thus represents a key transport protein delivering cytosolic lipids to nuclear receptors to promote a metastatic PCa phenotype Given the robust increase in fatty acid metabolism and upregulation of FABP5 in metastatic PCa we hypothesized that FABP5 may represent a central mechanism linking cytosolic lipid biosynthesis to pro-metastatic nuclear signaling using FASN and MAGL as prototypical examples we show that the ability of these lipid-metabolizing enzymes to enhance PCa metastasis in vitro and in vivo is critically dependent upon FABP5 thus positioning FABP5 as a key node in a lipid signaling network that promotes PCa metastasis FASN enhances the metastatic potential of LNCaP cells only in the presence of FABP5 (A) Migration and invasion of LNCaP cells expressing FABP5 (grey bars) or vector control (white bars) (B) Effect of FASN knockdown upon migration and invasion of FABP5 expressing LNCaP cells (C) Overexpression of FASN in LNCaP cells (red bars) increases their migration and invasion only in cells co-expressing FABP5 (red and grey bars) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; (n ≥ 5) MAGL enhances the metastatic potential of LNCaP cells only in the presence of FABP5 Overexpression of MAGL in LNCaP cells (blue bars) increases migration and invasion only when FABP5 is co-expressed (blue and grey bars) Cells overexpressing FABP5 and MAGL were also treated with vehicle *p < 0.05; **p < 0.01; ****p < 0.0001; (n ≥ 5) FABP5 knockdown suppresses metastatic potential in PC3 cells that express FASN and/or MAGL (A) Migration and invasion of PC3 cells bearing an FABP5 knockdown (white bars) or vector control (grey bars) as well as migration and invasion of PC3 cells treated with vehicle (B) Overexpression of FASN (red bars) or MAGL (blue bars) increases the metastatic potential of PC3 cells in vector-expressing but not FABP5 shRNA-expressing cells (red/blue and white bars) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; (n ≥ 5) Knockdown of FABP5 lowers PPARγ activation regardless of FASN and/or MAGL expression while increased interaction of PPARγ and FABP5 is observed when FASN/MAGL expression is elevated (A) Relative PPARγ-luciferase activity (luciferase/β-galactosidase) in vector-expressing PC3 cells and PC3 cells bearing an FABP5 knockdown and/or overexpressing FASN or MAGL (B) PPARγ immunoprecipitations (IP) were performed using PC3 vector control cells and PC3 cells overexpressing FASN or MAGL IP samples contained antibody directed against PPARγ The PPARγ IPs were western blotted using antibodies directed against FABP5 and PPARγ The control antibody IPs (IgG1) were negative for FABP5 Samples were derived from the same experiment and blots were processed in parallel (C) Following co-immunoprecipitation with PPARγ FABP5 expression in vector-expressing PC3 cells and PC3 cells overexpressing FASN or MAGL was quantified using densitometry analysis of western blots (D) Western blot of vector-expressing PC3 cells and PC3 cells overexpressing FASN or MAGL following extraction of cytosolic and nuclear protein fractions The purity of the collected fractions was corroborated using GAPDH and histone H3 as cytosolic and nuclear markers Western blot of FABP5 expression in the extracted nuclear fractions of PC3 vector control cells and PC3 cells overexpressing FASN or MAGL FABP5 expression was quantified using densitometry analysis of western blots Ex vivo analysis of metastasis to femurs and lungs of mice orthotopically implanted with PC3-Luc cells *p < 0.05; **p < 0.01; ***p < 0.001; (n = 4) (C) Representative images of femurs (left) and primary tumor (bottom right) taken from each cohort at the conclusion of week 7 modulation of lipid metabolism holds therapeutic promise in the treatment of PCa although the redundancy in lipid-metabolizing enzymes and nuclear receptors likely necessitates targeting multiple proteins in parallel Our study provides evidence that FABP5 links cytosolic lipid metabolism and nuclear signaling thus positioning FABP5 as a critical node in lipid signaling networks that drive PCa metastasis Using FASN and MAGL as prototypical examples of fatty acid metabolizing enzymes commonly overexpressed in PCa our study demonstrates that the pro-metastatic capabilities of these enzymes are critically dependent upon FABP5 expression in vitro and in vivo and MAGL extended to the in vivo setting as evidenced by suppression of metastasis in FABP5 knockdown cells despite the concomitant overexpression of FASN or MAGL our study demonstrates that FABP5 plays a critical role in gating lipid-mediated metastasis and may represent a druggable node in a PCa lipid signaling network that drives metastasis Male BALB/c nude mice (BALB/cOlaHsd-Foxn1nu IN; RRID: MGI:2161064) were used for all in vivo experiments (20–30 g Animals were housed at room temperature and kept on a 12:12-hour light:dark cycle with access to food and water ad libitum Euthanasia was performed utilizing CO2 asphyxiation followed by decapitation All of the experiments were approved by the Stony Brook University Institutional Animal Care and Use Committee (#850980) Male LNCaP and PC3 cells were purchased from ATCC (ATCC respectively) and authenticated by the ATCC human short-tandem repeat profiling cell authentication service (ATCC Male PC3-Red-FLuc cells (annotated as PC3-Luc) were purchased from PerkinElmer (PerkinElmer and PC3-Luc) were grown in RPMI 1640 (Gibco – Thermo Fisher Scientific MD) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products CA) and 100 units/mL of penicillin/streptomycin (Gibco – Thermo Fisher Scientific MD) in a humidified incubator set to 37 °C Female HEK293T cells were purchased from ATCC (ATCC HEK293T cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco – Thermo Fisher Scientific MD) supplemented with 10% FBS and 100 units/mL of penicillin/streptomycin in a humidified incubator set to 37 °C a lentiviral plasmid containing shRNA corresponding to human FASN was purchased (shRNA clone V3LHS_332906; GE Dharmacon A non-silencing shRNA control for human FASN expression was also purchased (shRNA clone V3LHS_173006; GE Dharmacon All lentiviral plasmids utilized to alter protein expression levels in LNCaP and V3LHS_173006) were packaged into functional lentiviruses and used to infect host cells HEK293T cells were sub-cultivated in a 10 cm cell culture plate to reach a confluency of 70–80% in complete DMEM (containing 10% FBS and 100 units/mL of penicillin/streptomycin) the HEK293T cells were co-transfected with the desired lentiviral plasmid and the third-generation lentiviral packaging plasmids p-RSV-Rev (Addgene at a 3:1:1:1 ratio using GenJet Plus transfection reagent (SignaGen MD) according to manufacturer’s instructions the media of transfected HEK293T cells was refreshed and functional packaged lentiviruses were harvested from the media at 48 hours LNCaP cells were infected at a multiplicity of infection (MOI) of 10 and PC3/PC3-Luc cells were infected at a MOI of 50 cells were subjected to puromycin-selection (1 µg/mL) and/or blasticidin-selection (5 µg/mL) (MilliporeSigma Appropriate overexpression/knockdown of proteins was assayed and confirmed via Western blotting Infected cells were kept under selective pressure for at least one week and passaged at least once in the absence of selection prior to experimentation PC3-Luc cells were propagated in culture and implanted orthotopically into the ventral lobe of the prostate gland of male BALB/c nude mice Mice were anesthesized under 2.0% isoflurane anesthesia (Henry Schein NY) and received continuous anesthesia throughout the entirety of the procedure mice received a subcutaneous injection of buprenorphine (0.1 mg/kg) (Henry Schein The surgical area (lower abdomen) was alternately swabbed 3 times with 70% ethanol and betadine A low midline abdominal incision of 3–4 mm was then made utilizing sterile surgical scissors the bladder of the mice was lifted (without disturbing other organs or musculature) to expose the ventral lobe of the prostate gland found directly beneath the bladder (if necessary fat was moved away using a sterile cotton swab) 2.5 × 105 cells were resuspended in 20 µL of sterile PBS and implanted directly into the ventral lobe of the prostate gland the bladder was replaced and the muscle layer closed using 4–0 absorbable vicryl monofilament sutures in an uninterrupted pattern The skin layer was closed using sterile 9 mm staples The animal was then removed from isoflurane anesthesia placed on a heating pad during the recovery period Mice were administered buprenorphine 4 hours post-surgery followed by an additional administration every 12–24 hours for the next 48 hours Staples were removed from the mice 7–10 days post-surgery once the incision wound had healed Mice were continually monitored for weight and food consumption Humane endpoints for all animals that underwent surgery were as follows: body weight decreasing by >15% Sample size analysis was carried out using Russ Lenth’s Java Applets for Power and Sample Size computer software which determined 8 animals/group (one-way ANOVA with Tukey multiple comparisons test; SD = 20%; Power = 80%; Contrast difference detection = 30%) Measurement of PC3-Luc cellular growth and metastasis was carried out utilizing a Caliper IVIS Spectrum imaging system (PerkinElmer Beginning at 24 hours after orthotopic implantation mice were imaged weekly for 7 weeks (when mice began to display morbidity) mice received an intraperotineal injection of luciferin (150 mg/kg) (PerkinElmer MA) and were imaged 10 minutes later under 2.0% isoflurane anesthesia extant mice received a luciferin injection and femurs and primary tumors were expediently excised Luminescence was quantified as total flux (photons/second) using LivingImage software (PerkinElmer 20 µg of protein (or 50 µg of protein derived from co-immunoprecipitations) were run on a 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) gel at 90 V for 75 minutes The protein was then transferred to a 0.45 µm nitrocellulose membrane (Bio-Rad followed by blocking in 5% non-fat dry milk in PBST the blots were trimmed and probed with mouse anti-β-actin (1:10,000) (Abcam rabbit anti-GAPDH (1:5000) (Cell Signaling Technology rabbit anti-histone H3 (1:2000) (Cell Signaling Technology mouse anti-PPARγ (1:1000) (Cell Signaling Technology or mouse anti-IgG1 (1:1000) (Cell Signaling Technology RRID: AB_10829607) overnight with shaking at 4 °C The blots were then washed at least three times with PBST followed by probing with one of the three following HRP-conjugated secondary antibodies for 1 hour at room temperature: goat anti-rabbit IgG (1:5000) (Life Technologies - Thermo Fisher Scientific goat anti-mouse IgG (1:5000) (Life Technologies - Thermo Fisher Scientific or donkey anti-sheep IgG (1:15,000) (Jackson ImmunoResearch Laboratories Inc. followed by development using the Immun-star HRP substrate (Bio-Rad Densitometry analysis of western blots was carried out using ImageJ software (NIH Cells were pre-treated for 2 hours before the initiation of the experiment with C75 (40 µM) and/or JZL184 (10 µM) 210 µL of cell media (±the appropriate pharmacological agent(s)/vehicle) was added to the bottom well of a Neuro Probe blind well chemotaxis chamber (Neuro Probe Inc. and a porous hydrophilic polycarbonate membrane (8.0 µm pore size 13 mm diameter) was placed on top (MilliporeSigma 100 µL of Matrigel (300 ng/µL) (Corning Inc. NY) was added to the top of the polycarbonate membrane and allowed to solidify in a humidified incubator set to 37 °C for 2 hours Cells to be utilized in the assay were then harvested resuspended in the appropriate culture media and added to the top well of the chemotaxis chamber (50,000 cells in 200 µL of media per chamber (±the pharmacological agent(s)/vehicle)) The completed chemotaxis chamber was then placed in a humidified incubator set to 37 °C containing 95% air and 5% CO2 for 20 hours sterile cottons swabs were used on the top of the polycarbonate membrane to mechanically remove excess media the membrane was then removed from the chemotaxis chamber and the bottom fixed in 4% paraformaldehyde for 20 minutes at room temperature (Ted Pella Inc. and fixed cells were stained using Hoechst 33342 (1:1000) for 1 hour in the absence of light (Thermo Fisher Scientific the membrane was then mounted onto a slide and all cells that had successfully migrated/invaded through the membrane were quantified using a Zeiss LSM 510 META NLO Two-Photon Laser Scanning Microscope (DAPI channel; 5x field of view) (Carl Zeiss Vision Inc. PC3 cells were seeded into 24-well plates to reach a confluency of 60–70% cells were co-transfected with plasmids encoding the PPARγ ligand binding domain fused to a GAL4 DNA binding domain (PPARγ LBD-GAL4-DBD) 4x upstream activation sequence for GAL4 upstream of luciferase (UAS 4x-TK Luc) and β-galactosidase as a transfection efficiency control (Kaczocha et al. the cells were lysed and processed using the Bright-Glo Luciferase Assay System (Promega Corporation WI) and β-galactosidase Assay System (Promega Corporation PPARγ activation (measured via luciferase luminescence at 595 nm) and β-galactosidase activity (measured via absorbance at 405 nm following hydrolysis of ο-nitrophenyl-β-d-galactopyranoside to ο-nitrophenyl) were quantified using an F5 Filtermax Multi-Mode Microplate Reader (Molecular Devices Background luminescence from non-transfected PC3 cells was subtracted from all samples and PPARγ activity was reported as relative luciferase activity (luciferase/β-galactosidase) Twenty-four hours prior to the initiation of extraction PC3 vector control cells and PC3 cells overexpressing FASN or MAGL were sub-cultivated in 10 cm cell culture plates to reach a confluency of 70–80% and the cytosolic and nuclear protein fractions were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Life Technologies - Thermo Fisher Scientific the purity of the collected fractions was assessed via western blot using antibodies directed against GAPDH and histone H3 as cytosolic and nuclear markers Vector expressing LNCaP or PC3 cells were seeded into 24-well plates (50,000 cells/well) JZL184 (10 µM) or C75 (40 µM) or JZL184 and C75 were added to the cells and subsequently incubated for 20 hours cells were harvested and counted after the addition of trypan blue using a hemocytometer PC3 vector control cells and PC3 cells overexpressing FASN or MAGL were harvested and lysed using ice-cold lysis buffer (20 mM Tris-HCl pH 7.4) containing both protease and phosphatase inhibitors (MilliporeSigma The cells were then scraped and centrifuged (13,000 g) for 15 minutes at 4 °C Lysates were collected and incubated with 20 µL of 50% Protein G Agarose beads (Cell Signaling Technology MA; Cat# 37478) for 30 minutes at 4 °C on an end-over-end rocker Lysates/beads were then centrifuged (13,000 g) for 10 minutes at 4 °C and incubated with mouse anti-PPARγ (1:50) (Cell Signaling Technology or mouse anti-IgG1 (1:50) (Cell Signaling Technology RRID: AB_10829607) antibodies overnight at 4 °C on an end-over-end rocker the lysates were incubated with 20 µL of 50% Protein G Agarose beads for 2 hours at 4 °C with gentle rocking The samples were then centrifuged (13,000 g) for 30 seconds at 4 °C The beads were then washed 3 times with 1 mL of sterile PBS before being boiled for 5 minutes and subjected to western blotting with antibodies directed against FABP5 After LNCaP cells were transduced (as previously described) with either the hFABP5-eGFP-N1 or the NES-FABP5-eGFP-N1 plasmid the cells were fixed using 4% paraformaldehyde (Ted Pella Inc. CA) for 15 minutes at room temperature and subsequently mounted onto slides using ProLong Gold Antifade Mountant (Thermo Fisher Scientific The images were captured using a Zeiss LSM 510 META NLO Two-Photon Laser Scanning Microscope (Carl Zeiss Vision Inc. CA) using both the GFP-channel and DAPI-channel and subsequent images were merged using AxioVision Software (Carl Zeiss Vision Inc. purified FABP5 (3 µM) was incubated with 0.5 µM NBD-stearate (Avanti Polar Lipids 100 mM NaCl buffer (pH 7.4) in the presence of positive control arachidonic acid (10 µM) (Cayman Chemical MI) or increasing concentrations of the FASN inhibitor C75 (5–500 µM) (Cayman Chemical Loss of fluorescence intensity (attributable to NBD-stearate ligand displacement) was measured at the respective excitation and emission wavelengths of 466 nm and 520–566 nm using an F5 Filtermax Multi-Mode Microplate Reader (Molecular Devices Fluorescence of wells lacking FABP5 was subtracted from all samples and stearate (SA) levels were quantified using liquid chromatography/mass spectrometry LNCaP and PC3 cells (5.0 × 105) were harvested and homogenized in 4 mL of 2:1:1 chloroform:methanol:Tris (50 mM pH 8) in the presence of 50 ng d2-PA and 50 ng d17-OA (Cayman Chemical Following centrifugation at 2800 RPM for 10 minutes at room temperature the organic layer was dried down with argon and resuspended in 120 µL of 40% acetonitrile in water and subsequently injected into a Thermo TSQ Quantum Access Max mass spectrometer (Thermo Fisher Scientific LC separation was achieved on a Gemini C18 (50 × 2 mm) (Phenomenex Mobile phase A consisted of H2O while mobile phase B was composed of acetonitrile and quantification was performed in the negative ion mode with select ion monitoring and the voltage set to 4 kV The sheath pressure was 30 and the capillary was set to 270 °C The gradient started at 40% B for 5 minutes and was equilibrated for 35 minutes at 40% B Excised tumor tissues from all animal cohorts were immersion fixed in 4% paraformaldehyde (Ted Pella Inc. CA) in a 0.1 M phosphate buffer (pH 7.4) at room temperature for 2 hours and then transferred to 4 °C overnight tissues were transferred to 30% sucrose in a 0.1 M phosphate buffer for cryoprotection for 48 hours Tissues were then embedded in OCT (Sakura Finetek USA Inc. CA; Cat# 4583) and frozen using a liquid nitrogen chilled isopentane bath Cryostat sections (16 µM thick) were then thaw-mounted on slides immersed in 4% paraformaldehyde for 3 minutes (to firmly fix the sections to the slide) and then rinsed in PBS three times for 10 minutes each Following a 30 minute wash in 5% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories Inc. RRID: AB_2337258) in PBS (to block non-specific labeling) the slides were incubated for 24–48 hours at 4 °C in rabbit anti-Ki67 antibody (1:500) (Abcam RRID: AB_302459) diluted in PBS containing 0.3% Triton-X 100 and 5% NDS Slides were then washed with PBS and incubated for 1 hour at room temperature with biotinylated donkey anti-rabbit antibody (1:500) (Jackson ImmunoResearch Laboratories Inc. RRID: AB_2340593) diluted in PBS with 0.3% Triton X-100 and 5% NDS incubated in ImmPACT DAB (Vector Laboratories Inc. Slides were counterstained with hematoxylin and eosin Images were captured using an Infinity 2 color digital camera Images were only adjusted for brightness and contrast All data were obtained from greater than/equal to three independent experiments and the n values described in each figure legend represent each independent trial or animal Data for all experiments were analyzed using a Mann-Whitney U test or repeated measures one-way ANOVA with Bonferroni post-hoc test using Prism software (GraphPad Prism All data are represented as means ± SEM and p < 0.05 was considered statistically significant and the degree of significance is indicated in each figure legend All experiments were performed in accordance with relevant guidelines and regulations including all experimentation using recombinant or synthetic nucleic acid molecules as approved by the Institutional Biosafety Committee of Stony Brook University (#850844) The animal experiments conducted were approved by the Stony Brook University Institutional Animal Care and Use Committee (#850980) The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request Ahmad, I. et al. 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AAPS J 12, 197–201, https://doi.org/10.1208/s12248-010-9180-6 (2010) Download references We would like to thank Dale Deutsch for the use of his equipment Robert Rieger and the Stony Brook Cancer Center Biological Mass Spectrometry core facility for expert assistance with fatty acid quantification Boris Kantor and the Viral Vector Core at Duke University and members of the Kaczocha group for helpful discussions Funding for this study was provided by the Translational Research Opportunities grant from the Stony Brook University School of Medicine and the Department of Anesthesiology The funding bodies did not have any role in the design of the study and collection and interpretation of data and in writing the manuscript Department of Biochemistry and Cell Biology Graduate Program in Molecular and Cellular Biology Institute of Chemical Biology and Drug Discovery All authors have read and approved the final version of the manuscript Download citation DOI: https://doi.org/10.1038/s41598-019-55418-x Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research The TimesA rare “Führer Quartets” card game featuring Adolf Hitler Heinrich Himmler and other senior Nazi leaders has been stolen from a palace in eastern Germany prompting the museum to put up a €1,000 reward for information leading to its return The game is said to be in excellent condition It was stolen from a display case in the Voigtsberg Palace museum in Oelsnitz last week and was on loan from a private collector Designed by the Munich publisher Anton Lehner it was first sold in 1934 and featured Hitler in two categories: “The Führer as role model” and “The Führer’s development” players must collect four cards of the top-ranking Nazi leaders The property is located at 700 Union Landing Road in Cinnaminson, New Jersey. Sale price is $4,700,000.  AGC Flat Glass North America, Inc. is a leading North American flat glass manufacturer. The building was formerly used by AGC for the manufacture of glass for residential windows.  Read more here. Log in or register to post comments The extensive refurbishment of the Oelsnitz Mining Museum is entering the home straight but will be far more expensive than originally planned the "Coal World" will reopen to visitors as an experience in an authentic location the costs amount to almost 30 million euros - originally around 18 million euros were estimated this was due to increased construction costs and unexpected difficulties with renovations to the historic building structure The museum was opened in 1986 in the listed building of a former coal mine The establishment of the new exhibition is being supported by the state with around 2.9 million euros A vivid and entertaining presentation is planned In addition to the history of coal mining in Saxony information will also be provided on how coal was created in the first place a forest will be staged as it existed here around 300 million years ago a replica of a pub for miners and a memorial for miners who died in accidents The museum can draw on its collection of around 50,000 exhibits for the exhibition "I am sure that the future coal world with its winding tower visible from afar will generate a great deal of interest," said Barbara Meyer State Secretary in the Ministry for Regional Development Coal mining was once important in driving forward the industrialization of Saxony at least 30,000 to 40,000 people are expected to visit the museum every year art lovers will also be able to get their money's worth in future: An impressive light installation by US artist James Turrell is planned there one of the flagships for Chemnitz as European Capital of Culture 2025 © 2009 - 2025 DieSachsen.de | Alle Rechte vorbehalten | Entwickelt mit publizer in Sachsen um Ihr unglaubliches Erlebniss auf DieSachsen.de weiter verbessern zu können.