Biology subjects

Paul, A.

Publications and source records attributed to Paul, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Can We Really See the World in Three Dimensions? An Exploratory Study with Finger Alignment Task and Ponzo Illusion

Stereopsis is the perception of depth when two 2-dimensional (2D) images of an object from slightly different angles align with corresponding retinal positions in both eyes. However, when we consider the blind spot (BS), a new set of challenges arises. In binocular vision, one eye's higher visual areas compensate for the other's non-receptive region. However, our brain only receives information from one image patch in the BS region, and another visual patch does not contribute to constructing a comprehensive 3-dimensional (3D) representation of the scene. This suggests that the brain adeptly manages this dimensional transition. We aimed to determine the extent to which we perceive 3D across our entire field of view (FoV). We employed the Ponzo illusion, a stimulus relying on visual depth cues (pictorial depth cues), and an eye-tracking technique to assess participants' accurate fixation and eye movement patterns. The participants attempted to align their index fingers before a chart paper to create a Horopter-like setup, supporting the hypothesis about our capacity for stereoscopic perception. The results of our exploratory pilot study with two specific tasks revealed that our ability to perceive depth diminishes beyond approximately 8$^{\circ}$ of retinal eccentricity (in the horizontal meridian) in both halves of the visual field centered around the fovea. This limitation is not apparent in our day-to-day experience.

neuroscience↗

bioRxiv2026

DNA polymerase kappa supports repair-associated DNA synthesis and neuronal genome resilience

Postmitotic neurons must preserve genome integrity for decades despite continuous oxidative, transcription-associated, and environmental DNA damage. Although recent studies have established ongoing repair-associated DNA synthesis in mature neurons, the polymerases that execute this synthesis remain incompletely defined. DNA polymerase kappa (POLK), a Y-family translesion synthesis polymerase, is highly expressed in neurons and undergoes an age-associated redistribution from the nucleus to the cytoplasm, suggesting that loss of nuclear POLK may compromise neuronal genome maintenance. Here, we investigated the consequences of POLK loss and augmentation in primary mouse cortical neurons and human induced pluripotent stem cell-derived forebrain neurons, including APOE e4/e4 backgrounds. Partial POLK depletion reduced neuronal arbor complexity, enlarged neuronal nuclei, increased LAMIN A/C and nuclear-envelope abnormalities, and induced a multidimensional senescence-like stress phenotype characterized by increased senescence-associated {beta}-galactosidase, p21, nuclear p38 MAPK, inflammatory signaling, lysosomal remodeling, and reduced mitochondrial-associated staining. POLK localized in proximity to EdU-labeled DNA in mature neurons, and POLK depletion reproducibly reduced EdU incorporation across independent human neuronal backgrounds and maturation stages. Exploratory nuclear POLK immunoprecipitation-mass spectrometry, together with proximity ligation assays, placed POLK near proteins involved in nucleotide excision repair, base-excision/single-strand break repair, DNA-end repair, checkpoint signaling, and specialized polymerase regulation, including PCNA-Ub and RAD18. POLK loss also produced Alzheimer disease-relevant cellular phenotypes. In wild-type neurons, POLK depletion increased 6E10 immunoreactivity and was accompanied by remodeling of APP/endosomal, lipid/cholesterol, and lysosomal pathways. In APOE e4/e4 neurons, POLK depletion increased cytoplasmic dsDNA and phospho-tau. Conversely, neuron-directed POLK augmentation increased EdU incorporation while reducing 53BP1 and {gamma}H2AX signals, p38 MAPK, cytoplasmic dsDNA, 6E10 immunoreactivity, and phospho-tau. Together, these findings identify POLK as a component of repair-associated DNA synthesis in mature neurons and support a model in which neuronal POLK availability contributes to genome maintenance, stress resilience, and vulnerability in aging and Alzheimer disease-relevant states.

neuroscience↗

bioRxiv2026

Glutathione Protects Hematopoietic Stem Cells from Proteotoxic Stress

Glutathione is a redox buffer, best known for preventing oxidative stress and ferroptosis, but rarely studied in stem cells. We studied glutathione function in hematopoietic stem cells (HSCs) by deleting the glutamate-cysteine ligase catalytic subunit (GCLC), which synthesizes glutathione, or glutathione peroxidase 4 (GPX4), which employs glutathione to prevent ferroptosis. GCLC deficiency eliminated glutathione from hematopoietic cells, severely impairing HSC function and hematopoiesis. In contrast, GPX4 deficiency did not affect HSC function and had little effect on hematopoiesis, even after vitamin E depletion, suggesting glutathione has ferroptosis-independent functions in HSCs. Consistent with this, GCLC deficiency increased reactive oxygen species levels in hematopoietic cells. But treatment with antioxidants or ferroptosis inhibitors gave little or no rescue of HSC function despite reducing ROS levels. Rather, GCLC-deficient, but not GPX4-deficient, HSCs exhibited unfolded protein accumulation, proteostatic stress, and cell death marked by elevated p53 levels and Annexin V staining. Deficiency for p53 prolonged the function of GCLC-deficient HSCs after transplantation. Glutathione is thus necessary in HSCs for proteostasis but may be dispensable for ferroptosis inhibition in vivo.

cell biology↗

bioRxiv2026

Substrate binding reorganizes the energetic landscape of Plasmodium falciparum hexose transporter PfHT1

Malaria parasites depend on the Plasmodium falciparum hexose transporter PfHT1 for sugar uptake, yet how substrate binding reshapes transporter energetics and kinetics of sugar transport remains poorly understood. Here, we investigate how glucose reorganizes the conformational landscape, transition pathways, and residue interaction network underlying membrane transport. Using over 800 s of adaptive molecular dynamics simulations combined with Markov state models, transition-path theory, residue-contact analysis, and graph attention learning, we reconstruct the apo and glucose-bound conformational cycles. We show that glucose selectively stabilizes productive outward-facing, occluded, and inward-facing conformations, reshapes transition kinetics, and channels reactive flux through the occluded state. We identify TM7b helix cracking as a local structural transition coupled to extracellular-gate closure and substrate progression, providing a flexible connection between the binding pocket and global alternating access. Experimental testing of mechanistically critical residues validated their functional importance in PfHT1-dependent sugar utilization. Together, these results show how substrate binding reorganizes the energetic, kinetic, and interaction landscape of membrane transport and establish a transferable framework for studying transporter mechanisms.

biophysics↗

bioRxiv2026

Large-scale structural analysis of pre-mRNA structure using RADIS

Pre-mRNA secondary structure can modulate the regulatory function of intronic sequences by masking or exposing splice-site signals and altering the accessibility of other features. Yet experimentally supported structural models for human pre-mRNAs have been largely restricted to splice-site-proximal sequences, leaving the deep intronic regions where ~70% of intronic disease-causing variants reside almost entirely unmapped. We developed RADIS (Reactivity Analysis of Deep and Intergenic RNA Structure), a strategy that yields strand-resolved chemical probing reactivity profiles across entire intron-rich loci by creating tiled arrays of RNAs that comprehensively span long genomic distances. This approach removes the abundance and locus-specificity constraints that otherwise limit pre-mRNA probing approaches. RADIS recovers known E. coli ribosomal RNA architectures and yields high-correlation with in-cell dimethyl sulfate (DMS)-MaP reactivities at representative splice sites (r = 0.87-0.90). RADIS-constrained folding of 109 5' and 88 3' splice sites uncovers an inverse relationship between base-pairing within the spliceosome footprint and splice-site strength, and partitions 81 branchpoints into three structural classes. Across 233 full-length Alu elements, sense Alu RNAs are more structured than antisense elements, and both partition into strand- and lineage-dependent structural classes. RADIS enables experimentally grounded structure analysis at disease-associated intronic and intergenic loci, complementing sequence-based variant-effect predictors.

molecular biology↗

bioRxiv2026

Neuronal and vascular genome-maintenance states organize opposing multicellular architectures in the aging brain

Genome maintenance is usually treated as cell-intrinsic, yet brain cells age within multicellular neighborhoods. Whether a cell's DNA damage response (DDR) state is systematically embedded in native tissue architecture is unknown. We used MERFISH to map curated repair programs across 604,252 cells in matched sections from 2-3 and 22-month-old mice. Regional DDR expression and coordination formed an age-dependent mosaic, accompanied by immune and oligodendroglial shifts. Independent metacell and distance-resolved analyses identified a consistent anchor-dependent organization. Low-DDR neurons occupied immune- and OPC-oligodendroglial-rich niches. In contrast, DDR-high vascular anchors were associated with vascular-cell enrichment and, in cortex and cerebral nuclei, OPC-oligodendroglial enrichment, whereas immune cells were enriched around DDR-low vascular anchors. Thus, genome-maintenance state was associated with distinct anchor-cell-specific spatial context depending on anchor-cell identity and its DDR transcriptional state. These opposing neuronal and vascular patterns define anchor-dependent genome-maintenance niches as a spatial feature of brain aging, with implications for regional vulnerability.

neuroscience↗

bioRxiv2026

Scale-Aware Compositional Inference Improves Reproducibility and Uncovers Convergent Aging Programs in Spatial Transcriptomics

Spatial transcriptomics enables analysis of molecular organization with anatomical context. Existing spatial differential expression methods are restricted to within-sample inference, forcing between-sample comparisons to rely on approaches adapted from single-cell RNA-seq. Here, we establish a scale-aware inference framework for spatial differential expression by modeling compositional constraints and variation in total RNA abundance rather than removing them through normalization, enabling calibrated between-sample inference at cell-level resolution. Our method produces more reliable results in simulated data and different spatial platforms. When applied to aged mouse brains, the analysis reveals converging aging-associated programs involving cellular signaling, membrane homeostasis, and neurovasculature across independent datasets.

genomics↗

bioRxiv2026

Improved Calreticulin Nanobody by Framework Engineering

Calreticulin is an emerging cancer biomarker, but current detection methods rely on expensive monoclonal antibodies that suffer from inefficient protein production, pharmacokinetic challenges and poor tissue penetration. Cal3, a calreticulin-specific nanobody, was constructed by replacing the complimentary determining region 2 (CDR2) of a soluble, clinically validated nanobody with a calreticulin-specific CDR2 isolated from a phage display library. However, the poor solubility and low yield of Cal3 limit its usefulness. In this study, we engineered CALR-Nb02 by adapting the core of Cal3 to a partial consensus framework sequence of stable nanobodies. CALR-Nb02 was purified with a 240-fold higher yield as a predominantly monomeric, soluble protein that exhibits an increased thermal stability and a higher calreticulin binding affinity (KD: 25-50 nM) compared with Cal3. These results reveal a strategy for quickly altering the specificity of a stable nanobody, and provide an improved calreticulin-binding reagent for future diagnostic, imaging, and therapeutic applications.

bioengineering↗

bioRxiv2026

Improving splice site usage prediction with SPLAIRE

BackgroundAlternative splicing, the mechanism by which intronic sequences are excised from pre-mRNAs to produce mature mRNA, affects >95% of human protein-coding genes and is a major driver of human disease states. The spliceosome, a protein-RNA complex responsible for splicing pre-mRNA, identifies candidate splice sites partly through the recognition of characteristic sequence motifs at exon-intron junctions. Deep learning models that predict the presence of splice sites from pre-mRNA sequence have achieved breakthrough performance relative to previous machine-learning techniques, and these models have improved our ability to identify pathogenic genetic variants that alter splicing. ResultsWe show that, while overall performance measures from these models suggest near-perfect performance, substantial gaps in prediction remain, including the identification of splice sites with low usage rates and tissue-specific splice sites. We leverage one of the largest paired RNA and genotyping datasets used to date to train a novel splicing model optimized for a specific cell type, human airway epithelial cells. We trained a dilated convolutional neural network on data from cultured airway epithelial cells from 100 donors, and showed that this model outperforms current state-of-the-art models on splice site identification and splice site usage quantification, including on multiple tissues not included in the model training data. ConclusionsWe present the most comprehensive evaluation of state-of-the-art splicing models published to date, revealing reasonable performance across models for genetic variant effect prediction along with important performance gaps and insights into directions for future model development.

genomics↗

bioRxiv2026

Tau aggregate replication occurs at the pre-synapse of cultured human neurons and increases with application of TNFa

Tau aggregation at synapses is a key process driving Alzheimers disease but the mechanism(s) that cause this have not been established. We used a model system of forward-programming induced glutamatergic neurons (iNeurons) with three independent cell lines treated with TNF[a]. Using aggregate-specific SIMOA, STED microscopy, and SynPull to detect nanoscopic tau aggregates in bulk samples and at individual synapses, we found that TNF[a]-driven tau aggregation occurs preferentially at the pre-synapse, forming predominantly non-fibrillar aggregates that are larger than ones in the extra- and post-synaptic regions. Using mathematical models of aggregate formation, we fitted the frequency of AT8-positive tau aggregates in synaptosomes, which showed that aggregate replication is the dominant process and is much faster than de-novo aggregate formation, leading to rapid local amplification once one aggregate is formed. Our results provide direct evidence for tau aggregate replication at the pre-synapse, linking inflammation induced tau aggregation with synaptic pathology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/728997v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@169dacorg.highwire.dtl.DTLVardef@c9894corg.highwire.dtl.DTLVardef@1551253org.highwire.dtl.DTLVardef@260ae7_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

Explainable machine learning reveals an RBP regulatory logic of exon skipping

RNA binding proteins (RBPs) regulate the life cycle of an mRNA, often through RBP-RNA interactions. This life cycle includes splicing, whereby the intronic sequence of a pre-mRNA is removed and the exons are joined together. However, the patterns of RBP binding that lead to different splicing outcomes are still incompletely understood. Here, we build machine learning models from RBP-RNA binding and knockdown RNA-seq data for over 168 RBPs in two cell lines (HepG2 and K562) to better understand the binding patterns that predict exon skipping, the predominant form of alternative splicing in humans. We show that models trained exclusively on RBP binding patterns are indeed predictive and that a more sophisticated machine learning model (XGBoost) outperforms simpler linear models. In addition, we are able to extract a biologically interpretable logic embedded in these models. We show that SHAP, a machine learning explainability technique, captures activating and repressive behavior of RBP binding that is position-specific. In addition, we find that SHAP values are predictive of changes in unseen splicing events and that SHAP interactions between pairs of RBPs are predictive of protein-protein interactions. Our results demonstrate that using machine learning with interpretability techniques can reveal a regulatory logic of RBP binding. By estimating the impact of an RBP binding site on a splicing event, the SHAP values also provide a directly testable scientific hypothesis. We anticipate that models designed around biological processes and focused on interpretability will yield actionable biological insights both in splicing and genomics generally.

systems biology↗

bioRxiv2026

Tumor cell specific total mRNA expression informed neural networks predicts cancer progression

Inferring tumor molecular phenotypes from high-dimensional multi-omic data is a fundamental challenge in computational biology. Current methods for estimating tumor cell-specific total mRNA expression (TmS) require matched DNA and RNA sequencing data and rely on computationally intensive deconvolution pipelines. We present TmSNet, a deep learning framework that predicts TmS using mRNA, DNA methylation, miRNA, and immune cell proportions as input features. TmSNet integrates structured feature selection (gradient boosting, LASSO, elastic net) with specialized neural architectures to predict continuous TmS. Across 12 TCGA cancer types, TmSNet achieved cross-validated performance up to concordance correlation coefficient (CCC) = 0.93 and correlation R{superscript 2} = 0.88 and generalized to external cohorts with correlations of 0.54 (SCAN-B) and 0.43 (FUSCC). Predicted TmS values effectively stratify patients by risk and preserve known transcriptional profiles across tumor subtypes. These results demonstrate that TmSNet can infer biologically meaningful phenotypes from multi-omic data and provide a scalable framework for modeling tumor transcriptional activity in heterogeneous cohorts.

bioinformatics↗

bioRxiv2026

Quercetin and Fisetin activate circadian clock via RORα and inhibit adipocyte growth

The circadian clock maintains temporal control of metabolic processes and exerts a key role in adipocyte development. Discovery of clock-modulatory compounds may provide new avenues for metabolic disease therapy. Here we report the identification of flavonoid compounds, Quercetin and Fisetin, as clock-activating molecules with direct inhibitory action on adipogenesis and adipocyte lipid metabolism. Quercetin and Fisetin displayed robust ROR agonism that promoted clock oscillation with induction of clock genes. Treating preadipocytes with these compounds blocked their adipogenic differentiation. In mature adipocytes, Quercetin and Fisetin suppressed lipid accumulation by inhibiting lipogenic enzymes. Furthermore, activation of ROR by a synthetic agonist or ectopic expression were sufficient to inhibit adipogenesis. In mice treated with Quercetin or Fisetin, ROR was markedly induced in adipose depots with strong suppression of the adipogenic and lipogenic programs. While quercetin significantly attenuated lipid storage in adipose tissue in vivo accompanied with lowering of free fatty acids and improved insulin sensitivity, fisetin displayed a less robust effect with differential regulation of lipolytic pathway. Collectively, these findings uncovered the clock-activating properties of quercetin and fisetin that prevent adipocyte maturation and hypertrophy to limit adipose tissue expansion. These actions contribute, at least in part, to their beneficial effects on metabolic disorders.

cell biology↗

bioRxiv2026

ClonoScreen3D-CRISPRi Uncovers Genetic Modifiers of Radiation Response in Glioblastoma

BackgroundGlioblastoma (GBM) is the most aggressive primary brain tumor in adults. Radioresistance, partly mediated by glioma stem-like cells, represents a major clinical challenge which could be overcome by the identification of the modulators of radioresistance. Existing CRISPR screens in human GBM models have largely used two-dimensional cultures with short-term viability readouts, failing to capture the long-term clonogenic behaviour underlying tumour recurrence after radiotherapy. MethodWe developed ClonoScreen3D-CRISPRi, combining CRISPRi-mediated gene knockdown with three-dimensional clonogenic survival assays. Two GBM cell lines (G7 and GBML20), differing in MGMT promoter methylation status, were engineered to express the KRAB-dCas9 editor. Nine candidate radiosensitivity modifiers, selected through transcriptomic analysis, pharmacological studies, and literature review, were examined in both lines. Target validation was performed using full radiation dose-response assays and a pharmacological inhibitor. ResultsThe majority of candidate genes significantly altered survival fraction following irradiation in both cell lines. Knockdown of NFKB2, RELB, and CDK9 produced the most potent radiosensitization, with sensitizer enhancement ratios of 1.39-1.70 in validation studies -- exceeding those of established radiosensitizers including PARP and ATM inhibitors. Notably, knockdown of these genes induced no significant cytotoxicity in the absence of radiation. Pharmacological validation using an IKK inhibitor confirmed these findings, implicating non-canonical NF-{kappa}B signalling and CDK9-dependent transcriptional elongation as critical adaptive mechanisms in GBM radioresistance. ConclusionsClonoScreen3D-CRISPRi is a scalable, physiologically relevant platform for identifying genetic modifiers of radioresistance. The non-canonical NF-{kappa}B pathway and CDK9 represent promising radiosensitizing targets, and larger screens could enable systematic prioritisation of candidates for clinical translation. Key PointsO_LIClonoScreen3D-CRISPRi combines gene knockdown with 3D clonogenic survival assays C_LIO_LIWe identified NFKB2, RELB, and CDK9 as modifiers of radioresistance in two GBM cell lines C_LIO_LIValidation experiments show ClonoScreen3D-CRISPRi reliably identifies radiosensitizers in GBM C_LI Importance of the studyGlioblastoma (GBM) remains one of the most lethal human cancers, with radioresistance representing a central barrier to improved patient outcomes. While CRISPR-based screens have begun to illuminate genetic drivers of GBM biology, prior approaches using human models have largely relied on two-dimensional culture systems and short-term viability readouts that inadequately model the disease. This study introduces ClonoScreen3D-CRISPRi, a novel platform that integrates CRISPRi-mediated gene knockdown with three-dimensional clonogenic survival assays in patient-derived GBM cells -- more faithfully recapitulating the long-term clonogenic potential that underlies post-radiotherapy recurrence. Using this platform, we identified NFKB2, RELB, and CDK9 as potent genetic modifiers of radioresistance, with sensitizer enhancement ratios exceeding those of established clinical radiosensitizers such as PARP and ATM inhibitors. Pharmacological validation of the non-canonical NF-{kappa}B pathway demonstrates direct translational relevance, providing a rationale for targeting this axis in combination with radiotherapy to improve GBM treatment.

cancer biology↗

bioRxiv2026

Title: Multimodal profiling reveals Mycobacterium tuberculosis restricts lung mitochondrial immunometabolism to promote pathogenesis

Early events in the lung that shape protective immune responses to M. tuberculosis (Mtb) infection are not well understood but are critical for developing better vaccines and immunomodulatory therapies for tuberculosis. Here, we used high-dimensional flow cytometry, single-cell transcriptomics, and untargeted metabolomics to define the early lung immune environment that precedes the development of protective versus pathogenic outcomes following aerosolized Mtb infection of mice. We show that Mtb induced sustained glycolysis in the lung while restricting oxidative phosphorylation (OXPHOS) and impairing mitochondria, in part through the Mtb serine protease Hip1, leading to low energy output and suboptimal macrophage-T cell interactions that promoted pathogenic immunity. However, robust induction of mitochondrial OXPHOS, amino acid metabolism, and fatty acid oxidation in the early lung resulted in high ATP output and enhanced innate-T cell signaling networks that stimulated protective immune responses. Moreover, we identified a novel mitochondrial immunometabolic lung signature associated with protective outcomes to Mtb infection in animal models and humans. Our studies identify induction of mitochondrial dysfunction as a mechanism employed by Mtb to manipulate lung immunometabolism to its benefit and reveal that maintenance of intact mitochondrial metabolism in the early lung is pivotal for generating protective outcomes to Mtb infection.

immunology↗

bioRxiv2026

Overlapping MHC class I/II Epitopes Program cDC1-like Differentiation of Monocyte-Derived Dendritic Cells via mTORC1 Signaling Inhibition

Viral infection polarizes monocyte-derived dendritic cells (moDC) to initiate type 1 immunity. The availability of overlapping (homologous) MHC class I and II epitopes, an occurrence frequently and primarily associated with intracellular infection, significantly enhances this process; however, the underlying mechanism(s) are unclear. We demonstrate that moDC loaded with homologous MHC epitopes acquire a cDC1-like phenotype in a process governed by mTORC1. mTORC1 pathway inhibition leads to NF-{kappa}B-mediated expression of IL-12 and other type I immune polarizing genes. The observed cDC1-like gene signature was also significantly enhanced in clinical moDC vaccine products made through methodologies that enforced class I and II antigenic homology. Collectively, these findings reveal a novel and previously unrecognized mechanism of immune governance that might also be exploited in cancer immunotherapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/716309v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@15475borg.highwire.dtl.DTLVardef@ffe5fborg.highwire.dtl.DTLVardef@53c761org.highwire.dtl.DTLVardef@46c4ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Investigating the versatility of cytochalasan cytochrome P450 monooxygenases using combinatorial biosynthesis reveals stereochemical restrictions

BackgroundCytochalasans are a large family of fungal metabolites which inhibit actin polymerization and ultimately lead to a broad range of biological effects in different assays. Investigations into the biosynthesis of cytochalasans has revealed that the cytochrome P450 monooxygenase (P450s) tailoring enzymes possess a somewhat relaxed substrate-specificity and may accept structurally-related intermediates for oxidation, partly explaining the variety of structural variations observed in this family of molecules. In this study, we investigate a broad range of P450 enzymes via combinatorial biosynthesis to better understand their substrate scope and potential applications as biocatalysts. ResultsGenome mining enabled us to identify cryptic cytochalasan biosynthetic gene clusters (BGCs) in six different species of fungi, each with at least two P450 enzymes encoded. Comparative genomics identified a cryptic thioredoxin-like enzyme encoded in cytochalasan BGCs that co-occurs with the gene encoding a Baeyer-Villiger monooxygenase. Heterologous expression of seven P450s in Magnaporthe grisea mutant strains, lacking P450s required for pyrichalasin H biosynthesis, enabled functional characterization of three P450s, two of which were previously cryptic. The experimental results, combined with phylogenetic analysis of the P450 sequences, reveal subtle information regarding the structures of the associated cytochalasans and begins to explain why some P450s are inactive on the substrates available to them. ConclusionsThe P450 enzymes involved in cytochalasan biosynthesis are known to be site-selective in their native host but also possess intrinsic promiscuity due to being able to modify structurally-related analogues. By investigating a diverse set of P450s from characterized and cryptic BGCs, we were able to identify that the stereochemistry of functional groups around the cytochalasan backbone is more restrictive than the size of the macrocycle when introducing the P450 enzyme to non-native substrates.

biochemistry↗

bioRxiv2026

Surface Functionalized RBC Membrane-Derived Nanoparticles for Targeted Drug Delivery to Attenuate Fatty Liver Disease

Metabolic dysfunction-associated steatotic liver disease (MASLD) is marked by excessive hepatic lipid accumulation and is closely associated with hyperlipidemia. It poses significant health challenges and can progress to severe chronic liver disease if untreated. Several small-molecule pharmacological agents are either in clinical use (resmetirom) or advancing through preclinical development for the treatment of hepatic steatosis. However, some promising lead drug candidates have limited therapeutic potential due to poor solubility, low permeability, limited biocompatibility, and off-target effects. Cell membrane-derived nanoparticles (CMN), prepared from red blood cells, naturally exhibit immune-evasion properties and can overcome these limitations by encapsulating small molecules within their self-assembled structures. Further, CMN can be surface functionalized to enable precise targeting of liver hepatocytes. Here, we developed a hepatocyte-targeting CMN loaded with a model drug (resmetirom) for MASLD therapy. Using covalent bonds, we conjugated three different hepatocyte-targeting ligands to CMN and identified lactoferrin as the most effective ligand through comparative screening. We then confirmed the cellular internalization pathways of the selected ligand in both targeted CMN and non-functionalized CMN. Finally, in an in vitro hepatic steatosis model, the optimized targeted CMN demonstrated improved bioactivity, including significant reductions in lipid droplets, triglycerides, and liver enzyme levels. Altogether, this targeted CMN platform shows promising potential to enhance the therapeutic efficacy of small-molecule drugs for MASLD and may, overall, improve therapeutic outcomes in preclinical and clinical trials.

bioengineering↗