Long-Term Protein Synthesis with PURE in a Mesoscale Dialysis System.

Cell-free systems are powerful tools in synthetic biology with versatile and wide-ranging applications. However, a significant bottleneck for these systems, particularly the PURE cell-free system, is their limited reaction lifespan and yield. Dialysis offers a promising approach to prolong reaction lifetimes and increase yields, yet most custom dialysis systems require access to sophisticated equipment like 3D printers or microfabrication tools.

RNA Order Regulates Its Interactions with Zwitterionic Lipid Bilayers.

RNA-lipid interactions directly influence RNA activity, which plays a crucial role in the development of new applications in medicine and biotechnology. However, while specific preferential behaviors between RNA and lipid bilayers have been identified experimentally, their molecular origin remains unexplored. Here we use molecular dynamics simulations to investigate the interaction between RNA and membranes composed of zwitterionic lipids at the atomistic level.

Structure of the yeast ceramide synthase.

Ceramides are essential lipids involved in forming complex sphingolipids and acting as signaling molecules. They result from the N-acylation of a sphingoid base and a CoA-activated fatty acid, a reaction catalyzed by the ceramide synthase (CerS) family of enzymes. Yet, the precise structural details and catalytic mechanisms of CerSs have remained elusive.

Yop1 stability and membrane curvature generation propensity are controlled by its oligomerisation interface.

The DP1 family of integral membrane proteins stabilize high membrane curvature in the endoplasmic reticulum and phagophores. Mutations in the human DP1 gene REEP1 are associated with Hereditary Spastic Paraplegia type 31 and distal hereditary motor neuropathy. Four missense mutations map to a putative dimerization interface but the impact of these mutations on DP1 structure and tubule formation are unknown.

Bio-Informed Porous Mineral-Based Composites.

Certain biominerals, such as sea sponges and echinoderm skeletons, display a fascinating combination of mechanical properties and adaptability due to the well-defined structures spanning various length scales. These materials often possess high density normalized mechanical properties because they contain well-defined pores. The density-normalized mechanical properties of synthetic minerals are often inferior because the pores are stochastically distributed, resulting in an inhomogeneous stress distribution.

Rescaling protein-protein interactions improves Martini 3 for flexible proteins in solution.

Multidomain proteins with flexible linkers and disordered regions play important roles in many cellular processes, but characterizing their conformational ensembles is difficult. We have previously shown that the coarse-grained model, Martini 3, produces too compact ensembles in solution, that may in part be remedied by strengthening protein-water interactions. Here, we show that decreasing the strength of protein-protein interactions leads to improved agreement with experimental data on a wide set of systems.

Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain.

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors.

Universal Click-Chemistry Approach for the DNA Functionalization of Nanoparticles.

Nanotechnology has revolutionized the fabrication of hybrid species with tailored functionalities. A milestone in this field is the deoxyribonucleic acid (DNA) conjugation of nanoparticles, introduced almost 30 years ago, which typically exploits the affinity between thiol groups and metallic surfaces. Over the last decades, developments in colloidal research have enabled the synthesis of an assortment of nonmetallic structures, such as high-index dielectric nanoparticles, with unique properties not previously accessible with traditional metallic nanoparticles.

Large-area, self-healing block copolymer membranes for energy conversion.

Membranes are widely used for separation processes in applications such as water desalination, batteries and dialysis, and are crucial in key sectors of our economy and society. The majority of technologically exploited membranes are based on solid polymers and function as passive barriers, whose transport characteristics are governed by their chemical composition and nanostructure. Although such membranes are ubiquitous, it has proved challenging to maximize selectivity and permeability independently, leading to trade-offs between these pertinent characteristics.

Molecular dynamics simulations of intracellular lipid droplets: a new tool in the toolbox.

Lipid droplets (LDs) are ubiquitous intracellular organelles with a central role in multiple lipid metabolic pathways. However, identifying correlations between their structural properties and their biological activity has proved challenging, owing to their unique physicochemical properties as compared with other cellular membranes. In recent years, molecular dynamics (MD) simulations, a computational methodology allowing the accurate description of molecular assemblies down to their individual components, have been demonstrated to be a useful and powerful approach for studying LD structural and dynamical properties.

Disulfide-Cross-Linked Tetra-PEG Gels.

The preparation of polymer gels via cross-linking of four-arm star-shaped poly(ethylene glycol) (Tetra-PEG) precursors is an attractive strategy to prepare networks with relatively well-defined topologies. Typically, Tetra-PEG gels are obtained by cross-linking heterocomplementary reactive Tetra-PEG precursors. This study, in contrast, explores the cross-linking of self-reactive, thiol-end functional Tetra-PEG macromers to form disulfide-cross-linked gels.

Engineering an artificial catch bond using mechanical anisotropy.

Catch bonds are a rare class of protein-protein interactions where the bond lifetime increases under an external pulling force. Here, we report how modification of anchor geometry generates catch bonding behavior for the mechanostable Dockerin G:Cohesin E (DocG:CohE) adhesion complex found on human gut bacteria. Using AFM single-molecule force spectroscopy in combination with bioorthogonal click chemistry, we mechanically dissociate the complex using five precisely controlled anchor geometries.

screening of potential agonists of a glucagon-like peptide-1 receptor among female sex hormone derivatives.

Glucagon-like peptide-1 (GLP-1) is an intestinal hormone that exerts its pleiotropic effects through a specific GLP-1 receptor (GLP-1R). The hormone-receptor complex might regulate glucose-dependent insulin secretion, and energy homeostasis; moreover, it could decrease inflammation and provide cardio- and neuroprotection. Additionally, the beneficial influence of GLP-1 on obesity in women might lead to improvement of their ovarian function.

Understanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing.

Understanding the complex relationships between enzyme sequence, folding stability and catalytic activity is crucial for applications in industry and biomedicine. However, current enzyme assay technologies are limited by an inability to simultaneously resolve both stability and activity phenotypes and to couple these to gene sequences at large scale. Here we present the development of enzyme proximity sequencing, a deep mutational scanning method that leverages peroxidase-mediated radical labeling with single cell fidelity to dissect the effects of thousands of mutations on stability and catalytic activity of oxidoreductase enzymes in a single experiment.

Enzyme Cascade with Horseradish Peroxidase Readout for High-Throughput Screening and Engineering of Human Arginase-1.

We report an enzyme cascade with horseradish peroxidase-based readout for screening human arginase-1 (hArg1) activity. We combined the four enzymes hArg1, ornithine decarboxylase, putrescine oxidase, and horseradish peroxidase in a reaction cascade that generated colorimetric or fluorescent signals in response to hArg1 activity and used this cascade to assay wild-type and variant hArg1 sequences as soluble enzymes and displayed on the surface of . We screened a curated 13-member hArg1 library covering mutations that modified the electrostatic environment surrounding catalytic residues D128 and H141, and identified the R21E variant with a 13% enhanced catalytic turnover rate compared to wild type.

Connexins orchestrate progression of breast cancer metastasis to the brain by promoting FAK activation.

Brain metastasis is a complication of increasing incidence in patients with breast cancer at advanced disease stage. It is a severe condition characterized by a rapid decline in quality of life and poor prognosis. There is a critical clinical need to develop effective therapies to prevent and treat brain metastases.

Mapping Mechanostable Pulling Geometries of a Therapeutic Anticalin/CTLA-4 Protein Complex.

We used single-molecule AFM force spectroscopy (AFM-SMFS) in combination with click chemistry to mechanically dissociate anticalin, a non-antibody protein binding scaffold, from its target (CTLA-4), by pulling from eight different anchor residues. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in by 2-3 orders of magnitude over a force range of 50-200 pN. Five of the six internal anchor points gave rise to complexes significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.

Phosphate and Kidney Healthy Aging.

Background: The aging population is increasing rapidly, much faster than our understanding on how to promote healthy aging free of multimorbidities. The aging kidney shows a decline in its function. Whether this decline is preventable or physiological is still debated.

Exploring Chemical Space with Machine Learning.

Chemical space is a concept to organize molecular diversity by postulating that different molecules occupy different regions of a mathematical space where the position of each molecule is defined by its properties. Our aim is to develop methods to explicitly explore chemical space in the area of drug discovery. Here we review our implementations of machine learning in this project, including our use of deep neural networks to enumerate the GDB13 database from a small sample set, to generate analogs of drugs and natural products after training with fragment-size molecules, and to predict the polypharmacology of molecules after training with known bioactive compounds from ChEMBL.

Mapping the Azolog Space Enables the Optical Control of New Biological Targets.

Photopharmacology relies on molecules that change their biological activity upon irradiation. Many of these are derived from known drugs by replacing their core with an isosteric azobenzene photoswitch (azologization). The question is how many of the known bioactive ligands could be addressed in such a way.

Optimizing TRPM4 inhibitors in the MHFP6 chemical space.

We recently reported 4-chloro-2-(2-chlorophenoxy)acetamido)benzoic acid (CBA) as the first potent inhibitor of TRPM4, a cation channel implicated in cardiac diseases and prostate cancer. Herein we report a structure-activity relationship (SAR) study of CBA resulting in two new potent analogs. To design and interpret our SAR we used interactive color-coded 3D-maps representing similarities between compounds calculated with MHFP6 (MinHash fingerprint up to six bonds), a new molecular fingerprint outperforming other fingerprints in benchmarking virtual screening studies.

A probabilistic molecular fingerprint for big data settings.

Background: Among the various molecular fingerprints available to describe small organic molecules, extended connectivity fingerprint, up to four bonds (ECFP4) performs best in benchmarking drug analog recovery studies as it encodes substructures with a high level of detail. Unfortunately, ECFP4 requires high dimensional representations (≥ 1024D) to perform well, resulting in ECFP4 nearest neighbor searches in very large databases such as GDB, PubChem or ZINC to perform very slowly due to the curse of dimensionality.

Results: Herein we report a new fingerprint, called MinHash fingerprint, up to six bonds (MHFP6), which encodes detailed substructures using the extended connectivity principle of ECFP in a fundamentally different manner, increasing the performance of exact nearest neighbor searches in benchmarking studies and enabling the application of locality sensitive hashing (LSH) approximate nearest neighbor search algorithms.

Type I interferon/IRF7 axis instigates chemotherapy-induced immunological dormancy in breast cancer.

Neoadjuvant and adjuvant chemotherapies provide survival benefits to breast cancer patients, in particular in estrogen receptor negative (ER) cancers, by reducing rates of recurrences. It is assumed that the benefits of (neo)adjuvant chemotherapy are due to the killing of disseminated, residual cancer cells, however, there is no formal evidence for it. Here, we provide experimental evidence that ER breast cancer cells that survived high-dose Doxorubicin and Methotrexate based chemotherapies elicit a state of immunological dormancy.

Exploring DrugBank in Virtual Reality Chemical Space.

The recent general availability of low-cost virtual reality headsets and accompanying three-dimensional (3D) engine support presents an opportunity to bring the concept of chemical space into virtual environments. While virtual reality applications represent a category of widespread tools in other fields, their use in the visualization and exploration of abstract data such as chemical spaces has been experimental. In our previous work, we established the concept of interactive two-dimensional (2D) maps of chemical spaces followed by interactive web-based 3D visualizations, culminating in the interactive web-based 3D visualization of extremely large chemical spaces.