Biology subjects

Wang, B.

Publications and source records attributed to Wang, B..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Structures of the human sodium-citrate cotransporter NaCT with and without substrates

The human sodium-citrate cotransporter NaCT imports various tri- and dicarboxylates into the cell as TCA cycle intermediates. This substrate uptake process is driven by an inward sodium gradient. The protein is a member of the Divalent Anion-Sodium Symporter (DASS) family. Whereas extensive biochemical and structural studies have been carried out for NaCT, how the substrate binding and translocation is coupled to the sodium gradient remains unclear. Here using single particle cryo-electron microscopy, we determined the structures of the human NaCT protein in three states: sodium-free, in the presence of sodium, and sodium- and substrate-bound. These structures suggest a simultaneous binding mechanism for sodium-substrate coupling, distinct from the sequential binding, conformational selection mechanism previously observed for the bacterial DASS protein VcINDY.

biophysics↗

bioRxiv2026

Bulk and single-cell transcriptomics reveal elevated endogenous retrovirus expression linked to immunopathology in severe COVID-19.

SARS-CoV-2 infection triggers expression of endogenous retroviruses (ERVs), but whether this persists during COVID-19 and contributes to disease severity has not been well characterized. In this study, we analyzed bulk and single-cell RNA-sequencing datasets from multiple cohorts of severe COVID-19 patients to investigate the role of ERVs in COVID-19 immunopathology. We identified 33 differentially expressed proviral ERV loci in severe COVID-19 compared with healthy individuals (severe COVID-19 signature ERVs). ERV activation was associated with dysregulation of ERV epigenetic regulators, and their expression strongly correlated with key inflammatory pathways implicated in severe COVID-19, including neutrophil degranulation, interleukin signaling, and inflammasome activation. Anakinra treatment reversed activation of signature ERVs. Six signature ERVs were specific to intensive care unit (ICU) admission and significantly correlated with hospital-free days at day 45 (HFD-45). At the single-cell level, signature ERVs were significantly upregulated in erythroid-like and erythroid precursor cells and macrophages of patients with severe disease. Within these cells, we found evidence of ERV-associated differences in inflammatory gene expression, whereby cells expressing signature ERVs showed heightened expression of innate immune genes compared with cells not expressing these ERVs. Together, our study unmasked specific ERV loci activated in severe COVID-19 that are linked to immunopathology.

immunology↗

bioRxiv2026

Azacitidine Response in Myelodysplastic Syndromes is Marked by NK-like CD8 T-Cell Expansion and CXCL12+ Reticular Cell Remodeling

Myelodysplastic syndromes (MDS) are driven by somatic mutations in hematopoietic stem and progenitor cells (HSPCs), leading to clonal expansion and ineffective hematopoiesis. Hypomethylating agents (HMAs; azacitidine or decitabine) are the standard of care for higher-risk MDS. However, their effects on the bone marrow (BM) microenvironment, and the extent to which these changes correlate with clinical response, remain poorly understood. We performed longitudinal analyses of BM aspirates, trephine biopsies, and peripheral blood samples from MDS patients treated with azacitidine in a clinical trial (NCT03493646), integrating CyTOF, 5' single-cell RNA and TCR sequencing, plasma proteomics, and multiplex immunofluorescence microscopy to characterize changes associated with azacitidine response. Clinical responders showed expansion of GzmBCD56CD8 T cells together with increased type I and type II interferon signaling within the T-cell compartment. Responders also exhibited marked alterations in circulating platelet- and myeloid-derived factors with the potential to remodel the BM niche. Spatial analyses revealed expansion of "neighborhoods" enriched for CXCL12-abundant reticular cells and CD8 T cells in responders, whereas HSPC-enriched neighborhoods were largely unchanged. In contrast, several HSPC-enriched neighborhoods expanded in non-responders. These microenvironmental changes were accompanied by evidence of enhanced myelopoiesis in clinical responders. Our findings support a model in which azacitidine response extends beyond direct effects on malignant hematopoietic cells to involve coordinated remodeling of the BM microenvironment which may be reinforced by platelet- and myeloid-derived signals that establish a feed-forward circuit promoting productive hematopoiesis. One Sentence SummaryLongitudinal bone marrow profiling reveals stromal and immune correlates of azacitidine response in myelodysplastic syndrome.

cancer biology↗

bioRxiv2026

In-Chip Volumetric Printing of Collagen-I Scaffolds for Perfusable and Stretchable Mammary Tissue Models

Engineered epithelial models require three-dimensional extracellular matrix environments that support organized cell growth and allow independent access to luminal and basal compartments. However, many organ-on-chip (OoC) fabrication strategies rely on planar geometries, non-native materials, or multi-step assembly workflows that limit architectural complexity and experimental control. Here, we report a direct in-chip volumetric printing strategy for fabricating stretchable and perfusable collagen-I scaffolds inside custom OoC devices. A vitamin C-regulated ruthenium/sodium persulfate photocrosslinking system enabled high-fidelity printing of collagen-I into open-lumen architectures with ductal- and alveolar-inspired features. By generating scaffolds directly within the final culture device, this workflow eliminates post-print transfer and integrates defined collagen architectures with compartmentalized fluidic access and a mechanically actuable chip format. To support chip-based culture, printed collagen constructs were stabilized after fabrication using EDC/NHS chemistry, which limited thermally induced collagen densification, improved shape retention, and maintained scaffold anchorage during perfusion. The chip design provided separate access to the printed lumen and surrounding basal compartment, which enabled compartment-specific fluid handling while preserving scaffold integrity during inflation, stretching, and perfusion of the printed construct. On the collagen-I scaffolds, human milk-derived mammary epithelial cells formed epithelial layers with tight junctions and lactation associated markers. The platform further supported perfusion culture, in situ staining, and whole-chip volumetric imaging. Together, this work establishes direct in-chip collagen-I volumetric printing as a biofabrication strategy for creating perfusable epithelial tissue chips with native matrix architecture and compartmentalized fluidic control.

bioengineering↗

bioRxiv2026

Transposon library anomaly reveals importance of cell wall teichoic acids for kin discrimination

Genome-wide transposon insertion sequencing (Tn-seq) is a powerful tool to measure the importance of genes for growth. In this study, we applied Tn-seq to the Gram-positive model system Bacillus subtilis, and found that after growth in liquid medium the transposon library lacked transposon insertions in several genes related to lipoteichoic acid biosynthesis and cell wall teichoic acid modification. This was unexpected since these genes are not essential for normal growth. By growing the transposon library as a confluent layer of cells, and as discrete colonies, we found that these genes are only important when the transposon library is grown as a confluent layer. Thus, growing the transposon library as a mixed population reduces the fitness of teichoic acid mutants. We provide further evidence showing that this anomaly is caused by the production of multiple autologous antimicrobials and/or toxins in the growth medium. These findings show that growing genome-wide mutant libraries as mixed cultures can influence the mutant library composition.

microbiology↗

bioRxiv2026

HLiCA: An integrated cell atlas of the healthy human liver

The human liver is composed of a heterogeneous mix of cell types. How these distinct populations contribute individually and collectively to liver function remains poorly understood. Although single-cell technologies have advanced our understanding of liver biology, individual studies have often been limited by small donor cohorts and inconsistent cell type annotations. Integrating multiple datasets can overcome these challenges and better capture biological variability. We present the Human Liver Cell Atlas (HLiCA), an integrated reference of non-disease liver cells assembled from eight datasets across six research centers, encompassing more than 525,000 cells from 110 donors. Developed in collaboration with the Human Cell Atlas Liver Bionetwork, the HLiCA incorporates expert-curated cell annotations refined through community feedback and dedicated cell type annotation meetings. The HLiCA classifies cells into six lineages and expands the cell type resolution to include 47 distinct cell types. Starting from raw sequencing reads, we realigned all data and performed rigorous benchmarking to ensure robust integration across technical and biological variables. Genetic ancestry was inferred for all samples to evaluate the range of ancestral backgrounds represented in the atlas. The expanded cell type annotation enabled identification of previously unrecognized liver cell types, including NRXN1+ stromal cells. Their presence was validated using spatial transcriptomics, which localized NRXN1+ stromal cells to periportal regions. With the number of donors included in the HLiCA we were able to examine cell type specific associations with demographic covariates. In hepatocytes, drug metabolism genes showed differential expression between sexes, and in cholangiocytes, mucus-production genes varied with age. As the largest and most genetically diverse human liver cell atlas to date, the HLiCA provides a comprehensive, well-annotated reference for the field, annotated by expert consensus. This resource will enable deeper interrogation of liver cellular diversity, architecture, and function in the healthy human liver and serve as a reference to understand changes that occur with disease.

genomics↗

bioRxiv2026

Environment-dependent landscapes of coding variant impacts on coproporphyrinogen oxidase

Hereditary coproporphyria (HCP) -- caused by variants in coproporphyrinogen oxidase (CPOX) -- can be diagnosed via genome sequencing. However, 74% of clinically-reported CPOX missense variants are classified as variants of uncertain significance (VUS) due to lack of evidence. CPOX variant classification is further complicated by environment-dependence: For example, the CPOX variant p.Asn272His (c.814A>C) is classified as benign yet has been associated with HCP-like symptoms in the context of mercury exposure. Here we measured the functional impact of nearly all possible CPOX amino acid substitutions in both the presence and absence of mercury. The resulting CPOX variant effect maps reflect known protein structure and mutational tolerance patterns while also offering new sequence-structure-function insights. Scores from this atlas not only distinguish pathogenic from benign variants but also identify mercury-dependent variant impacts, thus informing our clinical, structural, and functional understanding of CPOX deficiency and illustrating the value of systematic context-dependent multiplexed assays of genetic variant effects.

molecular biology↗

bioRxiv2026

A Glutamine antagonist-modulated tumor microenvironment unleashes enzalutamides immunotherapeutic effects

Androgen receptor (AR) antagonists, such as enzalutamide, suppress prostate cancer (PCa) cells to achieve temporary therapeutic effects. In addition to tumor cell-autonomous suppressive function, AR antagonists can also potentially exert anti-tumor effects via mitigating cytotoxic T cells exhaustion. However, strategies for effectively harnessing enzalutamides immunotherapeutic effects remain elusive. In studying a recently described glutamine antagonist prodrug (DRP-104) in PCa models, we found that despite the initial response, tumors ultimately became resistant. Intriguingly, compared to the untreated (DRP-104 treatment-naive) tumors, the resistant tumors became highly susceptible to enzalutamide in vivo. Additionally, treating tumors with DRP-104 and enzalutamide simultaneously also yielded superior therapeutic efficacy. We demonstrated that DRP-104 therapy promoted the infiltration of CD8+ T cells as well as regulatory T cells (Treg) in responsive tumors, and the tumor-infiltrating Treg were mostly depleted upon enzalutamide treatments. Mechanistically, we showed that Treg differentiation from mouse CD4+ T cells was attenuated by enzalutamide. We further demonstrated that Treg induction was accompanied by the interaction between AR and aryl hydrocarbon receptor (AhR), the nuclear receptor indispensable for Treg differentiation, in the nuclei of CD4+ T cells, and this interaction was diminished by enzalutamide. In further support of AR signaling in Treg biogenesis, analysis of available gene expression datasets found that AR expression was elevated in Treg when compared to CD4+ T cells in human peripheral blood mononuclear cells (PBMCs). In addition, it was positively correlated with Treg module scores in several human cancer types. Finally, using an anti-GPC3 (Glypican 3) vaccination model, we demonstrated that CD4+ T cells subjected to Treg induction in the presence of enzalutamide were less effective in protecting GPC3-expressing tumor cells from CD8+ T cells cytotoxic killing. Collectively, these results suggest that AR promotes Treg s differentiation and/or immunosuppressive functions, and nominate enzalutamide as a Treg-mitigating agent for potentiating immunotherapies. Our results also demonstrate that an otherwise unintended, Treg-promoting property of DRP-104 can be leveraged to unleash the immune-regulatory function of enzalutamide for the treatment of PCa.

cancer biology↗

bioRxiv2026

Critical period plasticity enables credit assignment

Synaptic plasticity is often guided by instructive inputs to neural circuits, but learning only succeeds when these instructions reach neurons that mediate relevant outputs. This creates the credit assignment problem: how does a neuron receive instructions suited to its own behavioral function? Here we report a developmental mechanism that enables credit assignment by using instructive inputs to organize the downstream architecture through which learning is expressed. In the olivocerebellar learning system, the inferior olive provides instructive inputs that guide plasticity within the cerebellum. During circuit assembly in zebrafish, we find these same inputs regulate long-range cerebellar projections during a highly plastic, two-day critical period. Developmental experience caused specific maturation of projections to targets that were coactivated with olivary inputs. Mathematical theory and computational modeling show how the resulting architecture constrains later learning, such that a single input can sculpt downstream connectivity and then leverage that circuit to assign credit. Simulated learning became paradoxically more robust if we reduced plasticity after a developmental critical period, protecting key architecture from corruption during learning. Thus, instructive inputs can first build the circuits they later teach, coordinating development and learning to enable effective credit assignment.

neuroscience↗

bioRxiv2026

Combining prior knowledge and transcriptomics data for logic models of patient subgroups

Computational modeling provides a powerful framework for in silico exploration of anti-cancer therapeutic targets and tumor response mechanisms. Oncogenic signaling pathways play a central role in tumor behavior and represent promising targets for personalized combination therapies. However, these pathways are complex, and although logic-based models are well suited for representing signaling dynamics, they are often constrained by model-specific data requirements, limited scalability, and time-consuming manual curation. Here, we introduce Functional Integration of Contextualized Omics for Unraveling regulatory dynamicS (FICUS), a framework that integrates omics-driven network contextualization with dynamic Boolean and logic-ODE modeling. FICUS enables automated, data-driven protein network inference and patient stratification, allowing shared signaling mechanisms to be identified across patient subgroups while preserving patient-specific dynamic responses. We applied FICUS to the SU2C-MARK lung cancer cohort and the The Cancer Genome Atlas kidney cancer cohort, demonstrating its utility for post-hoc analyses and downstream interrogation of dynamic tumor models. Overall, our results highlight the flexibility of FICUS in capturing heterogeneous signaling mechanisms across patient subgroups, addressing a key challenge in precision oncology.

Systems Biology↗

bioRxiv2026

A glycoRNA switch for malignancy: SNORA73B activates TIAR-dependent oncogenic signaling in lung adenocarcinoma

Although glycosylated small non-coding RNAs are emerging players in cancer, their functions in lung adenocarcinoma (LUAD) are largely unknown. We identify SNORA73B as a glycosylated small nucleolar RNA (glycol-snoRNA) that carries sialic acid-capped O-glycans in both normal lung epithelial and LUAD cells. SNORA73B is markedly elevated in LUAD, and its plasma levels distinguish early-stage LUAD from healthy controls with an area under the curve (AUC) of 0.7903. Subcellular fractionation reveals predominant nuclear localization. Functional assays demonstrate that SNORA73B depletion curbs LUAD cell proliferation, migration, and invasion, whereas its overexpression fosters these malignant phenotypes and accelerates tumor growth. Mechanistically, SNORA73B directly binds the T-cell-restricted intracellular antigen-related protein (TIAR), thereby enhancing TIAR protein abundance without affecting its mRNA levels. TIAR then recognizes the 3-untranslated region (3-UTR) of MYC mRNA to upregulate c-Myc, which subsequently augments AKT phosphorylation. Importantly, c-Myc knockdown largely rescues the oncogenic phenotypes and tumorigenesis induced by SNORA73B overexpression. Collectively, our data unveil a glycoRNA-dependent oncogenic axis SNORA73B-TIAR-c-Myc-AKT that drives LUAD progression. These findings position SNORA73B as a promising early diagnostic biomarker and a candidate therapeutic target in LUAD.

Cancer Biology↗

bioRxiv2026

A post-mitotic bistable switch actively safeguards mature neurons against reciprocal transdifferentiation in vivo

How mature cells actively defend their identity against alternative fates is poorly understood. Here we show that two types of fully differentiated Drosophila somatosensory neurons, derived from distinct lineages, are continuously prevented from transdifferentiating into one another by a post-mitotic bistable switch. Two homeodomain transcription factors, Hmx and Cut, are co-expressed in both neuronal types but adopt opposite stoichiometric dominances through mutual repression, each defining one identity. Disrupting this balance in mature neurons triggers deterministic, lineage-crossing reprogramming of transcriptome, dendritic and axonal architecture, and sensory behavior without reversion to a progenitor state. This regulatory logic is evolutionarily conserved: Drosophila sensory identities share transcriptomic signatures with distinct human A{delta} mechanoreceptors, and human CUX and HMX proteins functionally replace their fly counterparts. Mature neuronal identity is thus not a fixed endpoint but a poised equilibrium actively maintained by ongoing competition between opposing selectors. TEASERMature somatosensory identity is a poised equilibrium actively maintained by continuous competition between opposing terminal selectors.

Developmental Biology↗

bioRxiv2026

Sequence-to-function modeling uncovers the context-specific grammar of Drosophila chromatin insulation

Chromatin is organized into self-interacting topologically associating domains partitioned by boundary elements that insulate adjacent domains and restrict regulatory interactions. Yet, how sequence context and combinations of factors dictate boundary strength remains incompletely understood. Here we present Domino, a deep learning framework that maps genomic sequences to quantitative insulation scores defined directly from single-nucleosome resolution Drosophila melanogaster Micro-C data. Unlike traditional transcription factor motif scanning, Domino captures broad sequence context to resolve the functional contributions of individual sequence elements. We validate model predictions through experimental perturbations of insulator sequences. Model interpretation yields insulation-associated motifs genome-wide. Across 7,311 embryonic boundaries, Domino reveals a comprehensive insulation grammar defined by just 24 primary motifs that account for 59% of the boundaries, with an average of only two motifs per motif-containing boundary. Beyond known factors, we identify the zinc-finger proteins Trem, CG4854 and CG17385 as previously unreported insulation factors. We uncover distance- and orientation-dependent motif synergy, including a strict orientation preference of the prominent architectural factor M1BP. Finally, Domino traces tissue-specific shifts in the insulator landscape from the embryo to larval and adult brains, nominating new brain-specific insulation motifs. In sum, Domino provides a generalizable framework for decoding the regulatory logic of 3D genome architecture.

genomics↗

bioRxiv2026

NF1 deficiency induces metabolic reprogramming and epithelial-mesenchymal transition in glioblastoma

BackgroundMetabolic reprogramming is a common occurrence in tumor cells, where enhanced glycolysis promotes cell growth, invasion and migration. NF1 is tumor suppressor gene that downregulates the encoded neurofibromin protein. However, the effects of NF1 on energy metabolism and epithelial-mesenchymal transition (EMT) in glioblastoma multiforme (GBM), as well as the underlying molecular mechanisms, remain unclear. MethodsCRISPR/Cas9 gene editing technology was employed to construct GBM cell lines with NF1 gene mutations. Metabolomics was utilized to examine the impact of NF1 on metabolic remodeling in GBM. The Seahorse XF24 extracellular flux analyzer was used to detect the effect of NF1 knockdown on glycolysis and mitochondrial oxidative phosphorylation in GBM cells. Wound healing assay and Transwell chamber assay were utilized to detect the effect of NF1 on GBM cell invasion. Orthotopic tumor model in nude mice was established to explore the role of NF1 in vivo. In addition, Co-IP, western blotting, and immunofluorescence were used to explore the changes of key enzymes in glycolysis and mitochondrial oxidative phosphorylation and the relationship between NF1 and MFN1. ResultsThe expression of NF1 is decreased in glioma tissues and is significantly correlated with patient prognosis. NF1 knockdown may promote the invasion, migration, and EMT of GBM cells. At the same time, the activation of the AKT/mTOR signaling pathway promotes aerobic glycolysis in GBM cells, promotes mitochondrial division through targeted regulation of MFN1, and inhibits mitochondrial oxidative phosphorylation. NF1 deficiency promotes EMT in GBM cells by enhancing aerobic glycolysis and mitochondrial division. ConclusionNF1 deficiency promotes GBM glycolysis by activating the AKT/mTOR signaling pathway and inhibits the mitochondrial oxidative phosphorylation by regulating MFN1; NF1 deletion promotes GBM EMT by remodeling the pattern of energy metabolism.

Cancer Biology↗

bioRxiv2026

Chronic Intermittent Ethanol Exposure Produces Sex- and Tissue-Specific Metabolomic Signatures Across the Gut-Liver Axis in Adult Mice

Alcohol misuse leads to a range of health complications and induces various metabolic perturbations that impacts multiple physiological systems, including the cardiovascular system, liver, and gut microbiota. However, limited research has been reported on these metabolic profile changes, particularly using models of alcohol dependence such as after chronic intermittent ethanol (CIE) vapor exposure. This study investigated CIE-induced metabolomic alterations of CIE were investigated using fecal, liver, and serum samples of adult male and female C57BL/6J mice following 72 hr withdrawal. Significant metabolite changes were observed in both fecal and liver extracts and these changes were sex-specific. Both liver and fecal metabolites had systematic changes, while blood serum influences were limited after CIE. Female fecal samples showed higher metabolite perturbations than male samples according to PCA studies. The female samples showed significant butyrate downregulation and acetate upregulation, which are critical microbial products as beneficial microbe cell energy sources and influence intestinal absorption in the host. In addition, the female fecal samples showed significant downregulation of branched-chain amino acids including leucine, isoleucine, and valine, while male samples showed downregulation of glucose and taurine, with upregulated phenylalanine and tyrosine. In contrast, in the liver study, phenylalanine and tyrosine were upregulated while taurine was downregulated in females. Both sexes showed downregulation of liver glycine and glucose. These data indicate that CIE induces sex-specific metabolic perturbations in the mouse liver and fecal metabolome, and have implications for guy disturbances and liver damage observed following alcohol dependence. This study provides potential targets for future examination of mechanisms and treatment approaches for alcohol dependence.

neuroscience↗

bioRxiv2026

Histologically Informed Multiscale Modeling of the Neuronal Elements Activated by TMS

BackgroundThe primary neural site(s) at which action potentials are initiated by transcranial magnetic stimulation (TMS) remain poorly understood. Multiscale computational models provide biophysically based hypotheses, but model accuracy is constrained by limited histological knowledge of the microscopic organization of neural tissue. Recent high-resolution electron microscopy, in particular the petavoxel H01 dataset, provides novel, detailed axon morphologies and myelination patterns within the human cortex and superficial white matter. ObjectiveTo compare systematically multiple candidates for the neural elements activated by TMS using computer simulations informed by an extensive body of histological measurements, including neuron models directly reconstructed from the H01 dataset. MethodsWe developed a novel modeling pipeline to extract individual morphologically realistic multi-compartment models with exact myelination from serial section electron microscopic segmentations. To assess candidate excitation sites, we simulated the extracted neuron models together with parameterized models of a "ball-and-two-sticks", bifurcation, termination, and bend under uniform electric fields. In addition, smooth and sharply bending myelinated axons were embedded in a realistic human head model to evaluate activation thresholds under anatomically realistic electric field distributions. ResultsAxon terminations were only excitable by TMS when they were fully myelinated, which the histology suggested is unlikely. Partial myelination, even when separated by only 10 {micro}m from the terminal, increased activation thresholds by more than 100%. Reconstructed H01 neurons exhibited correspondingly high activation thresholds at axon terminals due to a lack of myelination. Further, most other candidate structures exhibited low thresholds only for histologically unrealistic parameter choices. In contrast, myelinated axonal bends of fibers transitioning from cortex to superficial white matter consistently showed low activation thresholds for both uniform electric fields and in realistic head model simulations. These thresholds fell within physiologically realistic ranges and, for larger diameter fibers, approached experimentally measured motor thresholds. ConclusionThese results identify myelinated axons bending from cortex into superficial white matter as possible neural targets for transcranial magnetic stimulation, and demonstrate the relevance of detailed histological and biophysical information to support robust modeling results.

neuroscience↗

bioRxiv2026

Miniaturized subcutaneous cellular implants for sustained therapeutic protein delivery in resource-limited settings

Cell encapsulation offers a promising strategy for sustained therapeutic protein delivery, obviating the need for repeated injections. Among potential implantation sites, the subcutaneous space is particularly attractive for its accessibility and amenability to minimally invasive procedures. However, performance of subcutaneous devices reported to date has been limited due to various challenges including foreign body response (FBR) and inadequate mass transfer. Moreover, typical encapsulation devices require surgeries for implantation and retrieval, limiting their potential use in resource-limited settings. Here we present a miniaturized cell encapsulation platform comprising cells engineered to produce therapeutic proteins and an FBR-mitigating zwitterionic polyurethane nanofibrous membrane, in a thin cylindrical form factor compatible with applicator-based minimally invasive implantation and retrieval. Clonal mesenchymal stromal cells engineered to produce PGT121, a broadly neutralizing anti-HIV-1 antibody, were encapsulated and inserted subcutaneously, achieving long-term cell survival and sustained serum PGT121 concentrations for up to 36 weeks across multiple murine models. Cell-loaded devices retained therapeutic function after cryopreservation, supporting their potential use as an off-the-shelf product that can be centrally manufactured and implanted on-site without specialized infrastructure. The custom-designed applicator-based implantation and minimally invasive retrieval procedures were demonstrated in a more clinically relevant minipig model. These mini-"cellular factories" represent a translatable strategy for sustained delivery of biologic drugs in resource-limited settings. One Sentence SummaryAn insertable and retrievable mini cellular construct enables sustained protein delivery, supporting its potential use in resource-limited settings.

bioengineering↗

bioRxiv2026

Deep learning-guided design of hydrolases for crystalline PET depolymerization

Poly(ethylene terephthalate) (PET), a ubiquitous polyester used in packaging and textiles, persists in the environment due to its high crystallinity and stability, contributing substantially to global plastic pollution. Enzymatic depolymerization by PET hydrolase (PETase) offers a chemically precise and environmentally sustainable route to convert PET into its monomeric building blocks, enabling recycling. However, practical implementation remains hindered by the rigid, crystalline architecture of PET, which restricts enzyme access and necessitates energy-intensive pretreatment to enable efficient depolymerization. In addition, most PETases achieve only partial depolymerization with low terephthalic acid (TPA) yields and accumulate inhibitory intermediates, while limited thermostability and slow surface kinetics further restrict efficiency. To overcome these barriers, we report VenusPETase, an engineered variant of KbPETase designed using PET-Flow, a state-of-the-art computational framework for PETase engineering. Compared with KbPETase, VenusPETase exhibits a 3.4-fold increase in hydrolytic activity, up to a 27-fold improvement after heat treatment of protein, and a 12 {degrees}C enhancement in thermostability. VenusPETase exhibits rapid degradation across a wide crystallinity range (8%-50%) at 50 {degrees}C, effectively spanning the entire spectrum of commercial PET products. Moreover, VenusPETase outperformed nine high-performance PETases under their respective optimal conditions and degraded untreated PET substrates across 8%-50% crystallinity, producing TPA as over 95% of the released products with minimal intermediate accumulation. X-ray crystallography and molecular dynamics simulations suggest that dynamic modulation, elevated surface electrostatic potential enhance the interaction of VenusPETase with crystalline PET, thereby lowering the hydrolytic energy barrier and improving catalytic performance. We also demonstrate that untreated, postconsumer-PET from nine different products can all be degraded by VenusPETase. The recovered monomers can be directly repolymerized into virgin-quality PET, demonstrating a closed-loop enzymatic recycling process. In a 100 L bioreactor, VenusPETase completely depolymerizes post-consumer crystalline PET (28% crystallinity) within 24 h under 50 {degrees}C. These results establish VenusPETase as a robust biocatalyst that enables efficient, closed-loop recycling of crystalline PET under mild conditions.

biochemistry↗