Biology subjects

van den Brink, M.

Publications and source records attributed to van den Brink, M..

7 recordsLinked to original sources

bioRxiv2026

Combinatorial optimization of protein systems in synthetic cells

In vitro reconstitution of protein systems - e.g., metabolic pathways, genetic circuits or biosensors - often requires optimization to enhance their activity. Combinatorial DNA libraries that simultaneously target multiple genes allow for a holistic optimization strategy by studying the interplay between the systems components, which may reveal DNA variants that would be hidden when testing each element in isolation. Here, we screen large populations of synthetic vesicles that express combinatorial DNA variants of a DNA self-replicator or a phospholipid synthesis pathway. We simultaneously vary the strengths of multiple RBSs or synonymously mutate the first codons of multiple genes to explore the effects of the protein translation rates directly on the functionality of the two core synthetic cell modules. We isolated high performers through DNA self-selection or functional screening by fluorescence-activated cell sorting. Long-read sequencing of the fittest variants informed on the optimal RBS strengths and base substitutions in the first codons and indicated which genes were most impactful in regulating the functionality of the protein systems. Single-mutation data were used to predict the fitness of combinatorial variants, which was compared with the experimental fitness observed. The theoretical fitness of combinatorial variants was extremely predictive for the two-gene library of the DNA replicator but less for the larger pathway library. Altogether, our approach exemplifies how combinatorial testing can be expanded from single proteins to multiprotein systems, which can in the future be extended to the evolutionary engineering of even larger genetic and metabolic networks, and eventually an entire artificial cell. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/707944v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@7029e4org.highwire.dtl.DTLVardef@1ce778forg.highwire.dtl.DTLVardef@b61b3org.highwire.dtl.DTLVardef@13b6215_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Image-based phenotypic sorting of synthetic cells

Understanding the relationships between genotype and phenotype is key to many areas of biological research and to the development of synthetic cells. We describe an image-based screening and sorting workflow that explores the phenotypes of gene-expressing vesicles within nonclonal populations and selects the desired variants. Using automated confocal microscopy and real-time, neural-network-assisted image analysis, we demonstrate that liposomes can be selected for fluorescence intensity, protein localization, membrane morphology, and dynamic behaviors, and their phenotype can be linked to genetic content. This approach could substantially accelerate the evolution of cellular functions in a minimal synthetic context.

synthetic biology↗

bioRxiv2024

Damage-induced IL-18 stimulates thymic NK Cells limiting endogenous tissue regeneration

Interleukin-18 is an acute phase pro-inflammatory molecule crucial for mediating viral clearance by activating Th1 CD4+, cytotoxic CD8+ T, and NK cells. Here, we show that mature IL-18 is generated in the thymus following numerous distinct forms of tissue damage, all of which cause caspase-1-mediated immunogenic cell death. We report that IL-18 stimulated cytotoxic NK cells limit endogenous thymic regeneration, a critical process that ensures restoration of immune competence after acute insults like stress, infection, chemotherapy, and radiation. NK cells suppressed thymus recovery by aberrantly targeting thymic epithelial cells (TECs), which act as the master regulators of organ function and regeneration. Together these studies reveal a novel pathway regulating tissue regeneration in the thymus and offer IL-18 as a potential therapeutic target to boost thymic function. Moreover, given the enthusiasm for IL-18 as a cancer immunotherapy for its capacity to elicit a type-1 immune response, these findings also offer insight into potential off-target effects.

immunology↗

bioRxiv2024

Microbial metabolite-guided CAR T cell engineering enhances anti-tumor immunity via epigenetic-metabolic crosstalk

The microbiome is a complex host factor and key determinant of the outcome of antibody-based and cellular immunotherapy. Its postbiotics are a blend of soluble commensal byproducts that are released into the host environment and have been associated with the regulation of immune homeostasis, particularly through impacts on epigenetics and cell signaling. In this study, we show that the postbiotic pentanoate is metabolized to citrate within the TCA cycle via both the acetyl- and succinyl-CoA entry points, a feature uniquely enabled by the chemical structure of the C5 aliphatic chain. We identified ATP-citrate lyase as the crucial factor that redirects pentanoate-derived citrate from the succinyl-CoA route to the nucleus, thereby linking metabolic output and histone acetylation. This epigenetic-metabolic crosstalk mitigated T cell exhaustion and promoted naive-like differentiation in pentanoate-programmed chimeric antigen receptor (CAR) T cells. The predictive and therapeutic potential of pentanoate was corroborated in two independent patient cohorts and three syngeneic models of CAR T adoptive therapy. Our data demonstrate that postbiotics are integrated into mitochondrial metabolism and subsequently incorporated as epigenetic imprints. This bridge between microbial and mammalian interspecies communication can ultimately impact T cell differentiation and efficacy.

immunology↗

bioRxiv2024

MOSAIC: a highly efficient, one-step recombineering approach to plasmid editing and diversification

The editing of plasmids and construction of plasmid libraries is paramount to the engineering of desired functionalities in synthetic biology. Typically, plasmids with targeted mutations are produced through time- and resource-consuming DNA amplification and/or cloning steps. In this study, we establish MOSAIC, a highly efficient protocol for the editing of plasmids and generation of combinatorial plasmid libraries. This quick protocol employs the efficient single-stranded DNA annealing protein (SSAP) CspRecT to incorporate (libraries of) DNA oligos harboring the desired mutations into a target plasmid in E. coli. In addition to up to 90% single-target plasmid editing efficiency, we demonstrate that MOSAIC enables the generation of a combinatorial plasmid library spanning four different target regions on a plasmid, in a single transformation. Lastly, we integrated a user-friendly validation pipeline using Nanopore sequencing reads, requiring minimal computational experience. We anticipate that MOSAIC will provide researchers with a simple, rapid and resource-effective method to edit plasmids or generate large, diverse plasmid libraries for a wide range of in vivo or in vitro applications in molecular and synthetic biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/586135v3_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@929537org.highwire.dtl.DTLVardef@1825039org.highwire.dtl.DTLVardef@60e89aorg.highwire.dtl.DTLVardef@46ef46_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

synthetic biology↗

bioRxiv2022

A novel lineage of RORγt+Aire+ antigen presenting cells promotes peripheral generation of intestinal regulatory T cells and tolerance during early life

Establishing and maintaining tolerance to self- or innocuous foreign antigens is vital for preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing AutoImmune Regulator, Aire, play a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development. Within weeks of birth, a separate wave of Treg cell differentiation occurs in the periphery, upon exposure to dietary and commensal microbiota derived antigens, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells are not known. Here we identified a new class of ROR{gamma}t+ antigen-presenting cells (APC), dubbed Thetis cells (TCs), with transcriptional features of both mTECs and dendritic cells (DCs), comprising 4 major sub-groups (TC I-IV). We uncovered a developmental wave of TCs within intestinal lymph nodes during a critical early life window, coincident with the wave of pTreg cell differentiation. While TC I and III expressed the signature mTEC nuclear factor Aire, TC IV lacked Aire expression and were enriched for molecules required for pTreg generation, including the TGF-{beta} activating integrin v{beta}8. Loss of either MHCII or Itgb8 expression by TCs led to a profound impairment in intestinal pTreg differentiation, with onset of intestinal inflammation. In contrast, MHCII expression by ROR{gamma}t+ group 3 innate lymphoid cells (ILC3) and classical DCs was neither sufficient nor required for pTreg generation, further implicating TCs as the tolerogenic ROR{gamma}t+ APC with an essential early life function. Our studies reveal parallel pathways for establishment of tolerance to self and foreign antigen within the thymus and periphery, marked by involvement of shared cellular and transcriptional programs.

immunology↗

bioRxiv2020

Rapidemic, a versatile and label-free DNAzyme-based platform for visual nucleic acid detection

In the last three decades, there have been recurring outbreaks of infectious diseases, brought to light with the recent outbreak of coronavirus disease 2019 (COVID-19). Attempts to effectively contain the spread of infectious diseases have been hampered by the lack of rapidly adaptable, accurate, and accessible point-of-care diagnostic testing. In this study, we present a novel design of a label-free DNAzyme-based detection method called Rapidemic. This assay combines recombinase polymerase amplification (RPA) with linear strand-displacement amplification (LSDA) and guanine-quadruplex (GQ) DNAzyme-catalysed colour-changing reaction. The colorimetry basis of the signal readout omits the need for extensive instrumentation. Moreover, the primer-based sequence detection of RPA gives Rapidemic a potential to be rapidly adapted to target a new sequence. As a proof of concept, we developed the assay to detect isolated genomic DNA of Saccharomyces cerevisiae. The use of low-pH buffers and the optimization of the dilution rates from each preceding reaction to the next showed to be successful strategies to enable visible detection with this method. These findings demonstrate for the first time that a label-free DNAzyme-based detection method can be coupled to RPA and LSDA for nucleic acid detection.

molecular biology↗