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bioRxiv2024

Genome-Wide Transcription Response of Staphylococcus epidermidis to Heat Shock and Medically Relevant Glucose Levels

Skin serves as both barrier and interface between body and environment. Skin microbes are intermediaries evolved to respond, transduce, or act in response to changing environmental or physiological conditions. Here, we quantify genome-wide changes in gene expression levels for one abundant skin commensal, Staphylococcus epidermidis, in response to an internal physiological signal, glucose levels, and an external environmental signal, temperature. We find 85 of 2354 genes change up to[~] 34-fold in response to medically relevant changes in glucose concentration (0 mM to 17 mM; adj P value [≤] 0.05). We observed carbon catabolite repression in response to a range of glucose spikes, as well as upregulation of genes involved in glucose utilization in response to persistent glucose. We observed 366 differentially expressed genes in response to a physiologically relevant change in temperature (37{degrees}C to 45{degrees}C; adj P value [≤] 0.05) and an S. epidermidis heat-shock response that mostly resembles the heat-shock response of related staphylococcal species. DNA motif analysis also revealed CtsR and CIRCE operator sequences arranged in tandem upstream of dnaK and groESL operons. We further identified 38 glucose-responsive genes as candidate ON or OFF switches for use in controlling synthetic genetic systems. Such systems might be used to instrument the in-situ skin microbiome or help control microbes bioengineered to serve as embedded diagnostics, monitoring, or treatment platforms.

microbiology↗

bioRxiv2024

How clinically relevant are prostate cancer cell lines? A comprehensive characterisation and multiomics comparison.

Cell line experiments arguably remain the most used tool in preclinical cancer research, despite their limitations. With almost 95% drugs entering human trials failing, and up to 90% preclinical research failing before even being tested in humans, we must shift the pre-clinical paradigm. A range of in silico, in vitro, in vivo and ex vivo approaches are gaining popularity, with the aim of potentially replacing cell line use. However, we cannot ignore the plethora of historical data from cell lines, nor write off their future use- especially within advanced bioengineered models. Therefore, we must question if and how cell lines hold clinical relevance. This study evaluates the clinical characteristics of 46 prostate cancer cell lines against worldwide data and investigates the biological features of seven cell lines in depth, comparing them to over 10,000 well characterised human cases from 24 studies in nine countries. Clinical features compared included age, ethnicity, Gleason grade, cancer type, treatment history and multiomics variables included mutations, copy number alterations, structural variants, microsatellite instability, mRNA and protein expression, and tumour mutational burden. We found that the most used cell lines accurately represent a minute proportion of prostate cancer patients. Furthermore, we recommend a pipeline for tailoring selection of clinically relevant cell lines with the ultimate aim of increasing the scientific methodology behind choosing a cell line.

cancer biology↗

bioRxiv2024

Conversion of natural cytokine receptors into orthogonal synthetic biosensors

Synthetic receptors enable bioengineers to build cell-based therapies that perform therapeutic functions in a targeted or conditional fashion to enhance specificity and efficacy. Although many synthetic receptors exist, it remains challenging to generate new receptors that sense soluble cues and relay that detection through orthogonal mechanisms independent of native pathways. Towards this goal, we investigated co-opting natural cytokine receptor ectodomains into Modular Extracellular Sensor Architecture receptors (yielding natural ectodomain, NatE MESA receptors). We generated multiple high-performing, orthogonal synthetic cytokine receptors, identified design principles and constraints, and propose guidance for extending this approach to other natural receptors. We demonstrate utility of NatE MESA by engineering T cells to sense an immunosuppressive cue and respond with customized transcriptional output to support CAR T-cell activity. Finally, we multiplex NatE MESA to logically evaluate multiple cues associated with the tumor microenvironment. These technologies and learnings will enable engineering cellular functions for new applications.

synthetic biology↗

bioRxiv2024

Plasmodium falciparum disruption of pericyte angiopoietin-1 secretion contributes to barrier breakdown in a 3D brain microvessel model

Disruption of the vascular protective angiopoietin-Tie axis is common in cerebral malaria (CM) patients, who display elevated angiopoietin-2 (Ang-2) and reduced angiopoietin-1 (Ang-1) blood concentrations. The role of pericytes in CM pathogenesis remains unexplored, despite being a major source of brain Ang-1 secretion and evidence of pericyte damage observed in CM postmortem samples. Here we engineered a human 3D microfluidics-based brain microvessel model containing the minimal cellular components to replicate the angiopoietin-Tie axis, human primary brain microvascular endothelial cells and pericytes. This model replicated pericyte vessel coverage and ultrastructural interactions present in the brain microvasculature. When exposed to P. falciparum-iRBC egress products, 3D brain microvessels presented decreased Ang-1 secretion, increased vascular permeability, and minor ultrastructural changes in pericyte morphology. Notably, P. falciparum-mediated barrier disruption was partially reversed after pre-treatment with recombinant Ang-1 and the Tie-2 activator, AKB-9778. Our approach suggests a novel mechanistic role of pericytes in CM pathogenesis and highlights the potential of therapeutics that target the angiopoietin-Tie axis to rapidly counteract vascular dysfunction caused by P. falciparum. The paper explainedO_ST_ABSProblemC_ST_ABSCerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, resulting in the majority of [~]600000 malarial deaths annually. Despite anti-malarial drug administration upon hospitalization, fatality rates still range from 15-25% and many survivors suffer long term neurological disabilities. A common dysregulated vascular pathway identified in CM patients is the angiopoietin-Tie axis. Treatments that restore this vascular homeostatic pathway appear as a potential avenue for adjunctive therapies in experimental rodent CM models. Nevertheless, the use of rodent CM models for therapeutic discovery is not ideal, given that P. falciparum pathogenesis is species-specific. Therefore, the development of novel and advanced human 3D microvascular models offers new avenues to study disease pathogenesis and explore potential adjunctive CM treatments. ResultsIn this study, we generate a 3D human brain microvasculature model that reproduces in vivo interactions between two key cell types necessary to reproduce the protective angiopoietin-Tie axis: human brain endothelial cells and pericytes. Addition of P. falciparum-infected red blood cell (iRBC) egress products causes vascular disruption and hampers the release of the vascular protective factor, angiopoietin-1, from brain pericytes. 18-hour pre-treatment of Ang-1 for 18h prevents iRBC egress product-induced vascular disruption. A short pre-treatment of the microvessels with AKB-9778, a downstream pharmaceutical inducer of angiopoietin-Tie axis activity currently in phase II clinical trial for diabetic retinopathy, partially restores vascular integrity. Our study highlights the role of pericytes in CM and the therapeutic potential of interventions that restore the angiopoietin-Tie2 axis as adjunctive CM treatments. ImpactOur study demonstrates the potential of bioengineered vascular models to recapitulate dysregulated pathways previously characterized in malaria patients, and in providing a physiologically-relevant platform to test adjunctive therapies. The use of the 3D brain microvascular model has enhanced our understanding of the mechanisms behind CM pathogenesis, uncovering a previously unappreciated effect of P. falciparum on brain pericytes, linking angiopoietin-Tie axis dysregulation and microvasculature disruption. These findings pave the way for the identification of novel, fast-acting therapeutics, such as AKB-9778, to restore vascular integrity in CM patients.

microbiology↗

bioRxiv2024

A Structural Proteome Screen Identifies Protein Mimicry in Host-Microbe Systems

Host-microbe systems are evolutionary niches that produce coevolved biological interactions and are a key component of global health. However, these systems have historically been a difficult field of biological research due to their experimental intractability. Impactful advances in global health will be obtained by leveraging in silico screens to identify genes involved in mediating interspecific interactions. These predictions will progress our understanding of these systems and lay the groundwork for future in vitro and in vivo experiments and bioengineering projects. A driver of host-manipulation and intracellular survival utilized by host-associated microbes is molecular mimicry, a critical mechanism that can occur at any level from DNA to protein structures. We applied protein structure prediction and alignment tools to explore host-associated bacterial structural proteomes for examples of protein structure mimicry. By leveraging the Legionella pneumophila proteome and its many known structural mimics, we developed and validated a screen that can be applied to virtually any host-microbe system to uncover signals of protein mimicry. These mimics represent candidate proteins that mediate host interactions in microbial proteomes. We successfully applied this screen to other microbes with demonstrated effects on global health, Helicobacter pylori and Wolbachia, identifying protein mimic candidates in each proteome. We discuss the roles these candidates may play in important Wolbachia-induced phenotypes and show that Wobachia infection can partially rescue the loss of one of these factors. This work demonstrates how a genome-wide screen for candidates of host-manipulation and intracellular survival offers an opportunity to identify functionally important genes in host-microbe systems.

bioinformatics↗

bioRxiv2024

Hydrogel-embedded precision-cut lung slices support ex vivo culture of in vivo-induced premalignant lung lesions

Lung cancer is the leading cause of global cancer death and prevention strategies are key to reducing mortality. Medical prevention may have a larger impact than treatment on mortality by targeting high-risk populations and reducing their lung cancer risk. Premalignant lesions (PMLs) that can be intercepted by prevention agents are difficult to study in humans but easily accessible in murine preclinical carcinogenesis studies. Precision-cut lung slices (PCLS) are underutilized as an ex vivo model for lung cancer studies due to limited culture time. Embedding PCLS within bioengineered hydrogels extends PCLS viability and functionality for up to six weeks. Here, we embedded PCLS generated from urethane-induced murine PMLs in cell-degradable and non-degradable hydrogels to study viability and activity of the tissues over six weeks. PMLs in hydrogel-embedded PCLS maintained viability, gene expression, and proliferation. Treatment of hydrogel-embedded PCLS containing urethane-induced PMLs with iloprost, a known lung cancer prevention agent, recapitulated in vivo gene expression and activity. These studies also showed that iloprost reduced proliferation and PML size in hydrogel-embedded PCLS, with some differences based on hydrogel formulation and suggested that hydrogel-embedded PCLS models may support long-term culture of in vivo generated PMLs to improve preclinical studies of lung cancer and prevention agents.

cancer biology↗

bioRxiv2024

Advancing thermostability of the key photorespiratory enzyme glycerate 3-kinase by structure-based recombination

As global temperatures rise, maintaining and improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalyzing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. Exploring inter-specific variation of the key photorespiratory enzyme glycerate kinase (GLYK) from various photosynthetic organisms, we found that the homolog from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand factors influencing thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold-predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, a series of hybrid proteins were produced and analyzed. These hybrid enzymes contained selected loop regions from CmGLYK replacing the most highly mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 {degrees}C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. While this hybrid enzyme exhibited enhanced thermostability and a similar Km for ATP compared to AtGLYK, its Km for glycerate increased threefold. This study demonstrates that molecular dynamics simulation-guided structure-based recombination offers a promising strategy for enhancing thermostability of other plant enzymes.

biochemistry↗

bioRxiv2024

Genetic modification of the shikimate pathway to reduce lignin content in switchgrass (Panicum virgatum L.) significantly impacts plant microbiomes

Switchgrass (Panicum virgatum L.) is considered a sustainable biofuel feedstock, given its fast-growth, low input requirements, and high biomass yields. Improvements in bioenergy conversion efficiency of switchgrass could be made by reducing its lignin content. Engineered switchgrass that expresses a bacterial 3-dehydroshikimate dehydratase (QsuB) has reduced lignin content and improved biomass saccharification due to the rerouting of the shikimate pathway towards the simple aromatic protocatechuate at the expense of lignin biosynthesis. However, the impacts of this QsuB trait on switchgrass microbiome structure and function remains unclear. To address this, wildtype and QsuB engineered switchgrass were grown in switchgrass field soils and samples were collected from inflorescences, leaves, roots, rhizospheres, and bulk soils for microbiome analysis. We investigated how QsuB expression influenced switchgrass-associated fungal and bacterial communities using high-throughput Illumina MiSeq amplicon sequencing of ITS and 16S rDNA. Compared to wildtype, QsuB engineered switchgrass hosted different microbial communities in roots, rhizosphere, and leaves. Specifically, QsuB engineered plants had a lower abundance of arbuscular mycorrhizal fungi (AMF). Additionally, QsuB engineered plants had fewer Actinobacteriota in root and rhizosphere samples. These findings may indicate that changes in the plant metabolism impact both organismal groups similarly, or potential interactions between AMF and the bacterial community. This study enhances understanding of plant-microbiome interactions by providing baseline microbial data for developing beneficial bioengineering strategies and by assessing non-target impacts of engineered plant traits on the plant microbiome.

microbiology↗

bioRxiv2024

AptaGPT: Advancing aptamer design with a generative pre-trained language model

Aptamers, synthetic oligonucleotide ligands, have shown significant promise for therapeutic and diagnostic applications owing to their high specificity and affinity for target molecules. However, the conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for aptamer selection is time-consuming and often yields limited candidates. To address these limitations, we introduce AptaGPT, a novel computational strategy that leverages a Generative Pre-trained Transformer (GPT) model to design and optimize aptamers. By training on SELEX data from early rounds, AptaGPT generated a diverse array of aptamer sequences, which were then computationally screened for binding using molecular docking. The results of this study demonstrated that AptaGPT is an effective tool for generating potential high-affinity aptamer sequences, significantly accelerating the discovery process and expanding the potential for aptamer research. This study showcases the application of generative language models in bioengineering and provides a new avenue for rapid aptamer development.

bioinformatics↗

bioRxiv2024

An expanded metabolic pathway for androgen production by host-associated bacteria

A growing body of literature implicates host-associated microbiota in the modulation of circulating androgen levels in the host, which could have far-reaching implications for androgen-mediated diseases. However, the microbial genetic pathways involved in androgen production remain unknown. Here, we report the first host-associated microbial gene (desF) encoding an enzyme that catalyzes conversion of androstenedione to epitestosterone (epiT) in the gut bacterium, Clostridium scindens. Despite current dogma that epiT is a nuclear androgen-receptor (AR) antagonist, we demonstrate that epiT is a potent androgen, as assessed by its ability to promote prostate cancer cell growth and expression of prostate specific antigen (PSA). We then quantified the desF gene in fecal samples collected from individuals with advanced prostate cancer (rising blood PSA) undergoing androgen deprivation therapy combined with abiraterone acetate and prednisone (AA/P). Strikingly, fecal desF levels were elevated in a subset of individuals progressing on AA/P versus samples taken during AA/P response (stable). Importantly, we observed that AA does not inhibit the bacterial desmolase enzyme that is analogous to the human drug target of AA. We then determined that bacterial isolates from urine or prostatectomy tissue are capable of androgen production. From these isolates we detected 17{beta}-hydroxysteroid dehydrogenase (17{beta}-HSDH) activity, which has not been previously reported in urinary tract bacteria, and discovered the desG gene in urinary isolates encoding 17{beta}-HSDH that catalyzed conversion of androstenedione to testosterone. Applying advanced artificial intelligence and molecular dynamics, we predict the structures and ligand binding to DesF and DesG. Using a novel bioengineered microencapsulation technique, we demonstrate that urinary androgen-producing bacterial strains can also promote prostate cancer cell growth through steroid metabolism. Taken together, our results are a significant advance for steroid microbiology in humans and suggest that these microbial biotransformations should be further studied in the context of androgen-mediated physiological processes and diseases.

microbiology↗

bioRxiv2024

Zinc-starved Brassicaceae Plants Secrete Peptides thatInduce Root Expansion

Zinc (Zn) deficiency is recognized as a global crisis as it is observed in half of all agricultural soils. However, the molecular mechanisms that drive plant physiological responses to soil Zn deficiency are not well understood. We used an untargeted metabolomics approach to search for metabolites exuded from roots during Zn deficiency stress, which led to the discovery of a collection of secreted small defensin-like peptides in Arabidopsis thaliana (named Zinc-Deficiency Responsive Peptides (ZDRPs)). Phylogenetic analysis and untargeted metabolomics revealed ZDRPs in at least eleven accessions of A. thaliana and nine members of the Brassicaceae family. Analysis of Arabidopsis gene mutants and overexpressing lines, in combination with chemical complementation experiments, unveiled a critical role of these peptides in plant root growth. We hypothesize that Brassicaceae secreted peptides enable plants to expand their root mass to reach Zn-rich soil layers and optimize Zn uptake. These data reveal a critical relationship between plant survival, Zn status, root morphology and peptide production. Taken together, our results expand our knowledge regarding micronutrient deficiency responses in plants and could enable in engineering approaches to make plants more resilient to low Zn conditions. SignificanceZinc deficiency is the most abundant micronutrient deficiency affecting about 50% of arable lands thus presenting a high burden for plant health and agriculture globally. In this study, we reveal a metabolic strategy by Brassicaceae to deal with low Zn concentrations. We characterize the role of peptides expressed upon zinc deficiency in a variety of important crop plants. The discovery of a cryptic class of peptides that are made by plant roots specifically suffering from Zn deficiency provides critical insight into the molecular mechanisms by which plants dynamically acclimate to nutrient-limited soils. The identification of peptides actively secreted by zinc-deprived plants has translational value for sustainable agriculture, human health, and bioengineering approaches to enable tolerance to low zinc.

plant biology↗

bioRxiv2024

A Spike Trimer Dimer-Inducing Nanobody with Anti-Sarbecovirus Activity

The continued emergence and zoonotic threat posed by coronaviruses highlight the urgent need for effective antiviral strategies with broad reactivity to counter new emerging strains. Nanobodies (or single-domain antibodies) are promising alternatives to traditional monoclonal antibodies, due to their small size, cost-effectiveness and ease of bioengineering. Here, we describe 7F, a llama-derived nanobody, targeting the spike receptor binding domain of sarbecoviruses and SARS-like coronaviruses. 7F demonstrates potent neutralization against SARS-CoV-2 and cross-neutralizing activity against SARS-CoV and SARS-like CoV WIV16 pseudoviruses. Structural analysis reveals 7Fs ability to induce the formation of spike trimer dimers by engaging with two SARS-CoV-2 spike RBDs, targeting the highly conserved class IV region. Bivalent 7F constructs substantially enhance neutralization potency and breadth, up to more recent SARS-CoV-2 variants of concern. Furthermore, we demonstrate the therapeutic potential of 7F against SARS-CoV-2 in the fully differentiated 3D tissue cultures mirroring the epithelium of the human airway ex vivo. The broad sarbecovirus activity and distinctive structural features of 7F underscore its potential as promising antiviral against emerging and evolving sarbecoviruses.

microbiology↗

bioRxiv2024

Substrate Rigidity Modulates Segmentation Clock Dynamics in Isolated Presomitic Mesoderm Cells

The segmentation clock, a genetic oscillator in the presomitic mesoderm (PSM), is known to be influenced by biochemical signals, yet its potential regulation by mechanical cues remains unclear. The complex PSM microenvironment has made it challenging to isolate the effects of mechanical signals on clock behavior. Here we investigated how mechanical stimuli affect clock oscillations by culturing zebrafish PSM cells on bioengineered elastic substrates (PDMS micropost arrays) with tunable rigidities ranging from 0.6 to 1,200 kPa. We observed an inverse sigmoidal relationship between substrate rigidity and the percentage of oscillating PSM cells, with a switching rigidity threshold between 3-6 kPa. The oscillation periods of oscillating PSM cells showed a consistently broad distribution across the substrate rigidity conditions tested. Moreover, these oscillatory PSM cells exhibited distinct biophysical properties, including reduced motility, contractility, and sustained circularity, compared to non-oscillating ones. These findings highlight a role of cell-substrate interactions in regulating segmentation clock behavior, providing insights into the mechanobiology of somitogenesis. HighlightsO_LIOscillatory behaviors of single PSM cells respond to substrate rigidity in a switch-like manner, transitioning from an oscillatory state to a quiescent state at a critical rigidity threshold between 2.9 kPa and 6 kPa. C_LIO_LIIncreased substrate rigidity significantly suppresses the percentage of oscillating PSM cells, while oscillation period and cycle number show no consistent rigidity-dependent trend. C_LIO_LINon-oscillating PSM cells exhibit distinct biophysical properties compared to oscillating ones, including reduced circularity, a polarized and elongated morphology, greater motility, and increased contractility. C_LIO_LIAggregates of PSM cells exhibit similar trends in response to substrate rigidity changes, except for increased oscillation percentages across different rigidity conditions, suggesting a potential interplay between cell-cell and cell-matrix communications in influencing clock oscillation behavior. C_LI

developmental biology↗

bioRxiv2024

Pressorum Sensing: Growth-induced Compression Activates cAMP Signaling in Pseudomonas aeruginosa

Bacteria employ various strategies to coordinate population-level behaviors, with quorum sensing being a well-established mechanism. Here, we report a novel population-level regulatory mechanism in Pseudomonas aeruginosa, which we term pressorum sensing. This mechanism allows bacteria to modulate their collective behavior in response to growth-induced mechanical compression in confined spaces. Using a highly sensitive cAMP biosensor in combination with microfluidics, we demonstrate that when compressive forces reach approximately 30 nN, P. aeruginosa cells rapidly increases intracellular cAMP levels via the Pil-Chp chemosensory system. This response leads to up-regulation of the Type III Secretion System, a key virulence factor. Unlike quorum sensing, which relies on diffusible chemical signals, pressorum sensing utilizes mechanical cues to gauge population density and spatial confinement. In bacterial colonies, this mechanism generates striking spatial patterns of cAMP signaling, including traveling rings that coincide with step-like structures in colony morphology. Our findings reveal a previously unknown link between mechanical compression and bacterial virulence, providing new insights into how P. aeruginosa coordinates population-level responses in confined environments. This work also expands our knowledge of mechanogenetics and opens up new possibilities in synthetic biology and bioengineering applications.

microbiology↗

bioRxiv2024

Molecular mimicry of a pathogen virulence target by a plant immune receptor

Plants and animals respond to pathogen attack by mounting innate immune responses that require intracellular nucleotide binding leucine-rich repeat (NLR) proteins. These immune receptors detect pathogen infection by sensing virulence effector proteins. However, the mechanisms by which receptors evolve new recognition specificities remain poorly understood. Here we report that a plant NLR has evolved the capacity to bind to a pathogen effector by acting as a molecular mimic of a virulence target of the effector, thereby triggering an immune response. The barley NLR Mildew Locus A 3 (MLA3) confers resistance to the blast fungus Magnaporthe oryzae by recognizing the effector Pwl2. Using structural analysis, we show that MLA3 has acquired the capacity to bind and respond to Pwl2 through molecular mimicry of the effector host target HIPP43. We demonstrate that the amino acids at the binding interface of MLA3 and Pwl2 are highly conserved in interface of HIPP43 with Pwl2, and are required to trigger an immune response. We used this discovery to bioengineer SR50--an MLA ortholog in rye that confers resistance to wheat stem rust--by introducing the Pwl2 binding interface of MLA3. This chimeric receptor has dual recognition activities, binding and responding to effectors from two major cereal pathogens. Collectively, these results provide evidence that plant immune receptors have evolved sophisticated mimicry strategies to counteract pathogen attack.

plant biology↗

bioRxiv2024

Silk-Ovarioids: Establishment and characterization of human ovarian primary cells 3D-model system

In vitro models that mimic ovaries are crucial for elucidating the biological mechanisms underlying follicle activation and growth. Three-dimensional (3D) systems are particularly relevant because they can replicate the heterogeneity and cell-cell communication between different ovarian cell types. However, complex models using human ovarian primary cells have not yet been established. In this study, we developed and characterized long-term cultured 3D models of primary ovarian somatic cells isolated from adult tissues, using Biosilk as a scaffold. We successfully established both ovarian cortex- and medulla-derived 3D systems, termed Silk-Ovarioids. The presence of key ovarian somatic cell types - including granulosa, stromal, endothelial, and perivascular cells - was confirmed by transcriptomics, proteomics, and immunostaining. Notably, Silk-Ovarioids exhibited the formation of a pro-angiogenic hypoxic core, as evidenced by the development of vessel-like structures after six weeks of culture. The Silk-Ovarioids demonstrated low intra-batch variability and long-term culture stability, underscoring their potential as a robust step towards creating a bioengineered, patient-specific artificial ovary.

cell biology↗

bioRxiv2024

A hollow fiber membrane-based liver organoid-on-a-chip model for examining drug metabolism and transport

Liver-on-a-chip models predictive for both metabolism as well as canalicular and blood transport of drug candidates in humans are lacking. Here, we established an advanced, bioengineered and animal component-free hepatocyte-like millifluidic system based on 3D hollow fiber membranes (HFMs), recombinant human laminin 332 coating and adult human stem cell-derived organoids. Organoid fragments formed polarized and tight monolayers on HFMs with improved hepatocyte-like maturation, as compared to standard 3D organoid cultures in Matrigel from matched donors. Gene expression profiling and immunofluorescence revealed that hepatocyte-like monolayers expressed a broad panel of phase I (e.g., CYP3A4, CYP2D6) and II (UGTs, SULTs) drug-metabolizing enzymes and drug transporters (e.g., OATP1B3, MDR1 and MRP3). Moreover, statically cultured monolayers displayed phase I and II metabolism of a cocktail of six relevant compounds, including midazolam and 7-hydroxycoumarin. We also demonstrated the disposition of midazolam in the basal/blood-like circulation and apical/canalicular-like compartment of the millifluidic chip. Finally, we connected the system to the other two PK/ADME-most relevant organ systems, i.e. small intestine- and kidney proximal tubule-like to study the bioavailability of midazolam and coumarin, and excretion of metformin. In conclusion, we generated a proof-of-concept liver organoid-on-a-chip model for examining metabolism and transport of drugs, which can be further developed to predict PK/ADME profiles in humans.

pharmacology and toxicology↗

bioRxiv2024

DeepSME: De Novo Nanopore Basecalling of Motif-insensitive DNA Methylation and Alignment-free Digital Information Decryptions at Single-Molecule Level

Hidden messages in DNA molecules by employing chemical modifications has been suggested for private data storage and transmission at high information density. However, rapidly decoding these "molecular keys" with corresponding basecallers remains challenging. We present DeepSME, a nanopore sequencing and deep-learning based framework towards single-molecule encryption, demonstrated by using 5-hydroxymethylcytosine (5hmC) substitution for individual nucleotide recognition rather than sequential interactions. This non-natural, motif-insensitive methylation disrupts ion current, resulting in a readout failure of 67.2%-100%, concealing the privacy within the DNAs. We further develop an alignment-free DeepSME basecaller as a key to reconstitute the digital information. Our three-stage training pipeline, expands k-mer size from 46 to 49, achieving over 92% precision and recall from scratch. DeepSME deciphers fully 5hmC concealed text and image within 16x coverage depth with an F1-score of 86.4%, surpassing all the state-of-the-art basecallers. Demonstrated on edge computing devices, DeepSME holds supreme potential for DNA-based private communications and broader bioengineering and medical applications.

bioinformatics↗