Biology subjects

Xu, J.

Publications and source records attributed to Xu, J..

At least 145 records · Page 8Linked to original sources

bioRxiv2025

Engineering the citrus phyllosphere microbiome for enhanced disease resistance to bacterial canker

Engineering phyllosphere microbiomes toward plant protection against pathogens on crops has shown promising effects. Here we tested the effect of application of beneficial bacteria individually and as a synthetic community (SynCom) on the phyllosphere microbiome and management of bacterial canker caused by Xanthomonas citri subsp. citri (Xcc). Foliar spray of a SynCom consisting of three citrus endophytic bacterial strains (Paenibacillus sp. ATY16, Bacillus megaterium PT6, and Bacillus subtilis PT26A, SynCom1) with mechanistically complemented plant-beneficial properties exerted a more profound effect on the leaf microbiota and suppression of citrus canker than single strains. Cultivation-dependent and bacterial 16S rRNA gene-sequence profiling analyses revealed that leaf endophytic Bradyrhizobium, Brevibacillus, Cellvibrio, Flavobacterium, and Pseudomonas were significantly enriched in the SynCom treated plants, in addition to Bacillus and Paenibacillus. The enriched Brevibacillus and Pseudomonas spp. were isolated, whole-genome sequenced, and confirmed to possess multiple beneficial traits including antimicrobial activity against Xcc, Moreover, the expression of citrus defense genes was induced by the SynCom1 inoculation. Foliar application of SynCom1 and a newly assembled SynCom (SynCom2; SynCom1 with representative Brevibacillus and Pseudomonas isolates) prior to Xcc inoculation significantly reduced the citrus canker disease severity in greenhouse assays, and SynCom2 had a better efficacy, comparable to a copper bactericide. Three-year field tests showed that both SynCom1 and SynCom2 effectively controlled citrus canker, with a 50% reduction of foliar and fruit canker incidence. Collectively, these findings provide novel insights and valuable clues for microbiome engineering to serve as a sustainable strategy for the control of phyllosphere pathogens on crops.

plant biology↗

bioRxiv2025

A Multi-omic Atlas of Human Choroid Plexus in Alzheimer's Disease

The choroid plexus (CP) regulates barrier integrity, cerebrospinal-fluid (CSF) dynamics, and immune surveillance, yet its role in Alzheimers disease (AD) remains poorly defined. We performed snRNA-seq on CP samples from 69 ROSMAP participants spanning normal cognition, mild cognitive impairment, and AD dementia, and integrated these data with spatial transcriptomics, snATAC-seq, and proteomics from CP tissue and CSF. We identified 17 CP cell states and uncovered widespread disease-associated transitions that converged into three major phenotypic axes. Along the inflammatory axis, epithelial cells and border-associated macrophages (BAMs) showed progressive immune activation, with BAMs shifting from inflammatory to stress-dominant states. In the barrier axis, epithelial cells, fibroblasts, and endothelial cells exhibited reduced junction-related gene expression and broad alterations in transport pathways. Epithelial cells also showed late-stage cilia loss and CSF-regulatory pathway impairment, indicating a breakdown in epithelial polarity and CSF sensing, consistent with abnormal CSF proteomic signatures. Along the remodeling axis, fibroblasts showed ECM alterations, while epithelial and stromal cells demonstrated aberrant cell-matrix adhesion pathways. Spatial neighborhood analysis revealed a multicellular signaling hub, with epithelial-rich niches showing the strongest dysregulation in AD. Together, these findings define a unified model of CP dysfunction in AD and position the CP as an active, multicellular contributor to AD pathophysiology.

genomics↗

bioRxiv2025

A Charge-reversal Point Mutation Completely Depletes Flavin Chromophore from European Robin Cryptochrome 4a Protein

Cryptochrome 4a (Cry4a) is a magnetically sensitive protein that could enable night-migratory birds to sense the geomagnetic field for navigation. The key to the protein magnetic sensitivity is the flavin adenine dinucleotide (FAD) cofactor, which initiates the electron transfer within the protein leading to a spin-correlated radical pair. De-spite its importance, the mechanism of FAD binding in avian Cry4a proteins remains unclear. Here we show that point mutagenesis of positively charged arginine residue at position 356 to negatively charged glutamic acid completely depletes FAD binding from European robin (Erithacus rubecula) Cry4a. The result indicates that electrostatic in-teractions constitute the primary driving force that enables FAD binding in European robin Cry4a. The finding provides new structural insight into the molecular basis of FAD binding in Cry4 and advances our understanding on the biophysical underpinnings of bird magnetoreception. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/690116v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@93344forg.highwire.dtl.DTLVardef@4f642corg.highwire.dtl.DTLVardef@39912corg.highwire.dtl.DTLVardef@169e1de_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Deep learning reveals a microprotein atlas in maize and uncovers novel regulators of seed amino acid metabolism

Microproteins represent a vast and functionally important class of genes that remain largely unexplored in plant genomes. Here, we developed DeepMp, a deep learning framework that integrated multi-omics evidence and built the most comprehensive plant microprotein atlas to date, identifying 18,338 high-confidence candidates in maize. The majority of these appear to have originated de novo from regions previously annotated as noncoding, and they show hallmarks of rapid, lineage-specific evolution and pronounced tissue specificity. These novel microproteins were found integrated into core regulatory networks, particularly in organ development and nutrient storage. Focusing on the maize kernel, population-scale analyses linked microprotein expression to natural variation in amino acid content. We functionally validated three grain-filling-specific candidates originating from noncoding regions by CRISPR-Cas9 knockouts, which confirmed their roles as precise modulators of arginine, aspartate, and methionine levels, without pleiotropic effects on kernel morphology. Our study provides a foundational resource and analytical framework, establishes microproteins as a new and functionally important coding layer in maize, and uncovers a previously untapped source of targets for crop improvement.

genomics↗

bioRxiv2025

Epigenetic silencing of transposable elements by IRF2BP2 is a selective dependency of myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous malignancy with limited curative treatment options. Transposable elements (TEs) are now recognized as key regulators of genome function, with aberrant activation implicated in cancer. However, their tumor-type-specific roles remain poorly characterized. Using single-cell Perturb-seq, we systematically screened for chromatin-associated regulators in primary AML patient cells to uncover dependencies required for leukemia cell viability. Our screen identified IRF2BP2 as an AML-selective dependency, functioning as a repressor of TE expression. Loss of IRF2BP2 induced differentiation, apoptosis, and impaired leukemic cell fitness, phenotypes linked to transcriptional activation of TEs, particularly evolutionarily young human endogenous retrovirus K (HERV-K). Mechanistically, IRF2BP2 cooperates with TRIM28 and DNMT1 to epigenetically silence TE expression. CRISPR-mediated activation of HERV-K/LTR5_Hs recapitulated the phenotypic effects of IRF2BP2 loss, while targeted re-silencing of HERV-K/LTR5_Hs partially rescued the effects, establishing a causal link between TE regulation and AML maintenance. Our findings highlight tumor-suppressive functions of TEs in leukemia and reveal IRF2BP2 as a key regulator of TE silencing in AML. Targeting the epigenetic machinery governing TE repression may represent a promising therapeutic avenue for differentiation-inducing and immunomodulatory strategies in AML.

cancer biology↗

bioRxiv2025

EMT activates ER-to-Golgi trafficking through upregulation of REEP2 to promote lung cancer progression

Membrane trafficking governs the transport of molecules to both intracellular and extracellular locations, thereby maintaining cell homeostasis. During cancer progression, alterations in membrane trafficking are frequently observed. However, the mechanisms underlying the dysregulation of membrane trafficking in cancer progression remain largely unresolved. Recent evidence has demonstrated that epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma (LUAD) employs a membrane trafficking program to coordinate cancer cell invasion and immunosuppression in the tumor microenvironment (TME). To further dissect the pro-tumorigenic membrane trafficking program, here we conducted a CRISPR interference (CRISPRi) in vivo screen for membrane trafficking regulators in a syngeneic mouse model with a complete immune system. This screen identified REEP2, an endoplasmic reticulum (ER) shaping protein, as a novel regulator of the EMT-dependent membrane trafficking program, which is associated with a poor prognosis in LUAD patients and is required for LUAD metastasis in a syngeneic orthotopic LUAD mouse model. Mechanistically, the EMT activator ZEB1 upregulates REEP2 expression through miR-183- and miR-193a-mediated regulation that promotes the transportation of secretory cargoes from the ER exit site (ERES) to the Golgi, thereby augmenting the secretion of pro-tumorigenic factors. The REEP2-driven secretion promotes cancer cell proliferation, migration, and the infiltration of myeloid-derived suppressor cells (MDSCs) in the TME. These findings identify REEP2 as a critical mediator of the EMT-driven pro-metastatic membrane trafficking program, revealing a specific vulnerability in mesenchymal LUAD.

cancer biology↗

bioRxiv2025

Protectin DX resolves fracture-induced postoperative pain in mice via neuronal signaling and GPR37-activated macrophage efferocytosis

Protectin DX (PDX) is a member of the superfamily of specialized pro-resolving mediators (SPMs) and exerts anti-inflammatory actions in animal models; but the specific signaling pathways involved in its actions have not been fully elucidated. Here we demonstrate the analgesic actions of PDX in a mouse model of tibial fracture-induced postoperative pain (fPOP) via activation of GPR37. Intravenous early-phase and late-phase treatment of PDX (100 ng/mouse) effectively alleviated fPOP. Compared to protectin D1/neuroprotectin D1, DHA, steroid, and meloxicam, PDX produced superior pain relief. While dexamethasone and meloxicam prolonged fPOP, PDX shortened the pain duration. The analgesic effects of PDX were abrogated in Gpr37-/- mice, which displayed deficits in fPOP resolution. PDX binds GPR37 and induces calcium responses in peritoneal macrophages. LC-MS-MS-based lipidomic analysis revealed that endogenous PDX levels were [~]10-fold higher than PD1 in muscle at the fracture site. PDX promotes macrophage polarization via GPR37-dependent phagocytosis and efferocytosis through calcium signaling in vitro, and it further enhances macrophage viability and efferocytosis in vivo via GPR37. Finally, PDX rapidly modulates nociceptor neuron responses by suppressing C-fiber-induced muscle reflex in vivo and calcium responses in DRG neurons ex vivo and reducing TRPA1/TRPV1-induced acute pain and neurogenic inflammation in vivo. Our findings highlight multiple benefits of PDX to manage postoperative pain and promote perioperative recovery.

neuroscience↗

bioRxiv2025

Overglycosylation introduces local changes in triple helix alignment in collagen type I fibril structure

Collagen fibrils constitute the structural scaffold of bone, and their hierarchical organization is central to biomineralization. While hydroxylation and glycosylation of lysine residues are well-known collagen post-translational modifications, their structural consequences remain poorly understood. Here we show that excess of glycosylation of hydroxylysine residues leads to significant alterations in the local packing of collagen molecules within the fibrils. By prolonging the enzymatic modification window during helix folding through the use of cyclosporin A, an increase in double glycosylation was observed at specific sites. These overglycosylated residues were pinpointed by mass spectrometry, while cryogenic TEM revealed fibrils with reduced diameters and distinct displacements of defined sub-bands within the D-period, without altering the overall periodicity. By mapping the modified residues onto the quarter-staggered model, we have been able to correlate site-specific glycosylation with sub-band shifts, linking chemical modification to supramolecular order. These results provide molecular-level evidence that collagen glycosylation is an active determinant of fibril structure. Such insights not only advance fundamental understanding of collagen assembly but could also illuminate mechanisms underlying bone fragility disorders, including osteogenesis imperfecta, that feature altered glycosylation.

biochemistry↗

bioRxiv2025

Synergistic therapeutic effects of endolysin BNT331 and a Lactobacillus crispatus consortium in a human vagina chip model of bacterial vaginosis

Bacterial vaginosis (BV) is a vaginal infection caused by an imbalance in the vaginal microbiome characterized by a decrease in healthy bacteria dominated by Lactobacillus crispatus along with a concomitant increase in dysbiotic bacteria, such as Gardnerella. Current treatments commonly fail to fully eradicate BV, and the lack of more effective therapies is due in part to the absence of relevant human models. Here, we used a human vagina-on-a-chip (Vagina Chip) microfluidic culture device that has been previously shown to faithfully recapitulate the human vaginal microenvironment as well as the inflammatory and injurious effects of G. vaginalis to test the therapeutic efficacy of a consortium of L. crispatus alone or in combination with endolysin BNT331, which specifically targets and lyses Gardnerella. These studies revealed that when L. crispatus was added alone, it suppressed inflammation even though it failed to engraft or displace the G. vaginalis bacteria. In contrast, BNT331 effectively killed Gardnerella in dysbiotic Vagina Chip. Importantly, the combined administration of both treatments resulted in restoration of a healthier vaginal microenvironment, as indicated by higher engraftment of L. crispatus on-chip, inhibition of G. vaginalis, and a reduction in inflammation. Similar effects of this combined treatment were observed when administered to Vagina Chips infected with vaginal swab samples from BV patients. These data suggest that combination of a live biotherapeutic product composed of a L. crispatus consortium with a potent antimicrobial agent that targets G. vaginalis, such as BNT331, may offer an effective therapeutic strategy for patients with BV. One Sentence SummaryEndolysin BNT331 combined with Lactobacillus crispatus reduces bacterial load and suppresses inflammation in a human vagina chip model of bacterial vaginosis.

pathology↗

bioRxiv2025

Carbohydrate adaptation drives liver-brain axis maturation

Mammalian postnatal life requires adaptation to a carbohydrate-rich diet, yet how metabolic programs are coordinated within and across organs is unclear. Using time-resolved transcriptomic and metabolomic analyses from the neonatal period through adulthood, we show that mouse liver rapidly acquires oxidative and detoxification capacity after weaning. This transition enables the brain to establish energy-sufficient, low-toxicity metabolic environment for neuronal development. This maturation process is marked by progressive activation of the hepatic electron transport chain (ETC), with the mitochondrial RNA endoribonuclease LACTB2 acting as a key regulator. LACTB2 prevents the accumulation of mitochondrial RNAs and sustains expression of mtDNA-encoded ETC subunits, thereby preserving mitochondrial competence for oxidative metabolism. LACTB2 is postnatally induced in hepatocytes, and its loss causes defective glucose utilization, systemic metabolic toxicity, and impaired brain metabolism and myelination, leading to prepubertal lethality, particularly in males. Restoring ETC function through liver-targeted expression of yeast NADH dehydrogenase NDI1, inhibiting the integrated stress response or ammonia scavenging improved survival. Our findings identify LACTB2-dependent hepatic mitochondrial maturation as a central mechanism that aligns carbohydrate adaptation with the liver-brain metabolic coordination to support early-life development.

developmental biology↗

bioRxiv2025

Captamer: A Novel Quantitative Protein Detecting Method Depending on Aptamer-activated Molecular Switches and RPA Signal Amplification

There are various protein assays for specific and quantitative detection and widely used for laboratory and clinic purposes, but current methods still have limitations. Immunoassays based on antibodies, like ELISA, suffer from slow response and a long antibody-screening period, while physical or electrochemical methods are generally restricted by high cost or the stringent requirement of equipment or operating skills. In this study, we developed an in vitro sensitive protein quantification method: Captamer. The Captamer system comprises a molecular switch derived from aptamer sequence and an exponential fluorescence signal amplification pathway based on recombinase polymerase amplification (RPA). We demonstrated the Captamer for SARS-CoV-2 nucleocapsid protein detection and obtained results from samples within 30 min, displaying a wide detection window from 0.2 pg/mL to 200 pg/mL with high specificity. Furthermore, we tested the Captamer for Tau441 protein (a potential Alzheimers disease biomarker) and thrombin (a classic aptamer-protein interaction model), showing the limit of detection as low as 1 ng/mL and 0.02pg/mL respectively, which suggested the capacity of Captamer to be applied to various aptamer-protein pairs. Compared with the most commonly used and recently reported protein quantification methods, Captamer stands out for its high sensitivity, short response time, low cost, and simplicity, indicating its great potential to be widely used in protein quantification. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/686180v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1e38ffaorg.highwire.dtl.DTLVardef@1019e72org.highwire.dtl.DTLVardef@1498b68org.highwire.dtl.DTLVardef@15f5ea2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Circuit-specific gene editing for precision modulation of neuronal activity with CRISPR-rabies virus

CRISPR gene editing has revolutionized our ability to study and manipulate specific genes, enabling novel insights into gene function and potential therapies for brain disorders. Recent advances in cell-type-specific regulatory elements and viral delivery systems have made precise in vivo gene editing possible. However, neurons with similar molecular profiles can belong to different circuits, complicating efforts to manipulate circuit function and behavior. To address this, we developed CRISPR-rabies virus (CRV), which leverages the trans-synaptic spread of rabies virus to enable gene editing within anatomically defined neural circuits. By pairing CRV with cell type-specific Cas9 expression, we achieved targeted gene modifications in specific circuits. We demonstrate that CRV can modulate sodium and potassium channel expression in parvalbumin interneurons, thereby effectively regulating synaptic transmission of pyramidal neurons in the CA3 region of the hippocampus. Its compatibility with 3'-capture single-cell RNA-seq allows simultaneous circuit perturbation and molecular profiling. In summary, CRV allows precise circuit-level gene modulation, providing a platform for studying gene function in neural circuits and developing novel gene therapies for brain disorders.

neuroscience↗

bioRxiv2025

In-Cell Synthesis of Nε-acetyl-L-lysine for Facile Protein Acetylation

N{varepsilon}-acetylation of proteins is a crucial post-translational modification (PTM) that occurs across all kingdoms of life and plays key roles in nearly every cellular process. Due to its broad significance, there is a strong demand for chemical biology tools that enable efficient, site-specific acetylation of target proteins in live cells. Genetic code expansion (GCE) has emerged as a powerful tool for introducing N{varepsilon}-acetylation at specific lysine residues in proteins. However, achieving adequate expression levels typically requires adding 2-10 mM of chemically synthesized N{varepsilon}-Acetyl-L-lysine (AcK) to the culture medium, which can be cumbersome and costly. To overcome this limitation, we present the first proof-of-concept for a one-pot acetylation platform that simplifies protein acetylation in both E. coli and mammalian cells. Our approach begins with the discovery of 10 novel lysine acetyltransferases (KATs) capable of biosynthesizing AcK from basic carbon sources. When these enzymes are co-expressed with the genetic incorporation machinery for AcK, they facilitate streamlined, site-specific acetylation of any target protein without compromising E. coli viability. This innovative platform not only broadens the range of unnatural amino acids (UAAs) that can be biosynthesized and incorporated but also provides a powerful tool for probing the histone and non-histone acetylation events in live cells. In addition, this technique offers an eco-friendly and scalable method to produce synthetic acetylated proteins, which will provide practical value in acetylation/deacetylation-related research such as chemical biology, biotechnology, and drug development.

synthetic biology↗

bioRxiv2025

A Unified Protein Embedding Model with Local and Global Structural Sensitivity

Structural comparison between proteins is key to many research tasks, including evolutionary analysis, peptidomimetics, and functional annotation. Traditional structure alignment tools based on three-dimensional protein structures, such as TM-Align, DALI, or ProBiS, are accurate, but they are computationally expensive and impractical at scale. Existing protein language models (PLMs), such as TM-Vec, improve computational efficiency but only capture global structural similarity, overlooking important motif-level structural details. In this paper, we propose a novel PLM consisting of a Siamese neural network, enabling efficient embedding-based structural comparison while also capturing both global and local structural similarity. Our model was trained on a dual loss function combining TM-score, a global similarity metric, and a variation of lDDT scores, a per-residue similarity metric. We tested against two datasets: a varied TM-score dataset from TM-Vec, and a high TM-score mutant dataset from VIPUR. Against these sets, our model achieved a TM-score MAE of 0.0741 and 0.0583, respectively, and a lDDT-score MAE of 0.0788 and 0.0038, respectively. Our model fulfills two key roles: first, it rapidly detects global structural differences. Second, it supports fine-grained structural assessments, improving sensitivity to subtle but functionally important structural changes.

bioinformatics↗

bioRxiv2025

A biased allosteric modulator functions as a molecular glue to induce β2AR dimerization

Family A G-protein coupled receptors (GPCRs) are typically described as monomers, yet growing evidence suggests they can form dimers with distinct signaling properties1-3. The mechanisms and therapeutic potential of such dimerization, however, remain poorly understood. Here, we show that AP-7-168, an optimized derivative of a {beta}-arrestin-biased negative allosteric modulator of the {beta}2-adrenergic receptor ({beta}2AR) that sustains bronchorelaxation in cell and tissue models4, functions as a molecular glue to promote {beta}2AR homodimerization. Cryo-EM structures reveal a unique binding mode in which two AP-7-168 molecules pack within a pocket formed by transmembrane helices 3, 4, and 5 of two protomers, stabilizing a dimeric conformation that selectively prevents {beta}-arrestin coupling. In cells, AP-7-168 robustly induces {beta}2AR dimerization and drives enlarged nanocluster formation. Combined with extensive functional studies, our findings unveil a novel allosteric mechanism by which a small molecule biases {beta}2AR signaling through dimerization, highlighting ligand-induced dimerization as a strategy for GPCR modulation.

biochemistry↗

bioRxiv2025

Aberrant splicing of MBD1 reshapes the epigenome to drive convergent myeloerythroid defects in MDS

Myelodysplastic neoplasms (MDS) feature hematopoietic deficits driven in part by transcript splicing abnormalities. Thus far, such disease-driving transcripts have been identified in association with specific splicing factor mutations. However, it remains unclear whether there also exists a set of disease-wide conserved pathological transcripts, which drive MDS independently of mutational status. Here, we characterize an MDS-associated long isoform of MBD1 (MBD1-L) as the first described member of this class of transcripts. Overexpression of MBD1-L in healthy human HSPCs recapitulates archetypal defects of MDS including deficits in erythroid differentiation and reconstitution capacity. These defects arise from an isoform-specific switching of MBD1s binding behavior, refocusing its heterochromatin-promoting activity from methylated to unmethylated CpGs and enacting broad downregulation of CpG-rich promoters as well as secondary epigenetic effects mediated by its downstream target BCOR. Remarkably, we also find that directly reversing abnormal MBD1 splicing in primary human MDS using nanoparticle-encapsulated ASOs enhances erythroid differentiation. Key pointsO_LIGlobal mis-splicing of MBD1 represents a novel gain-of-function epigenetic axis driving erythropoietic and proliferative defects in MDS. C_LIO_LIASO based depletion of pathogenic MBD1 transcripts restores erythroid differentiation, advancing RNA-based therapies for MDS. C_LI

cancer biology↗

bioRxiv2025

A line attractor maintains aggressiveness during feeding in hangry mice

Aggression evolved to protect resources such as food from competitors, but animals must balance fighting and feeding so that they facilitate rather than hinder re-establishment of energy homeostasis. How this balancing is computed is not well understood. We have approached this problem at the level of neural population-coding by examining the effect of progressive starvation on a hypothalamic line attractor that encodes an internal state of aggressiveness. Moderate fasting yielded "hangry" mice, decreasing attack latency and increasing attack frequency. In parallel, line attractor ramping rate and stability were increased, suggesting that hunger enhances aggressiveness by modifying neural dynamics. In contrast, prolonged starvation inhibited aggression and eliminated the line attractor. In satiated mice, titrated acute chemogenetic activation of arcuate AgRP neurons recapitulated the biphasic effects of progressive starvation, suggesting that a continuous increase in hunger exerts bi-directional influences at different intensities. When confronted with food and an intruder, hangry mice alternated between feeding and fighting. During eating, population neural activity moved out of the line attractor while activity in the attractor dimension remained unchanged. Following feeding, activity rapidly relaxed back into the attractor and aggression resumed. Thus, the line attractor may serve to keep hungry animals primed for aggression during intermittent feeding bouts.

animal behavior and cognition↗

bioRxiv2025

An ultra-long acting insulin enables glucose-synchronised release

Delayed and weak glucose-responsive kinetics, coupled with short plasma exposure and complicated formulation, remain major obstacles to the clinical translation of glucose-responsive insulin formulations. Here we report a rapidly absorbable, ultra-long acting, and glucose-responsive insulin analogue via engineering recombinant human insulin with two phenylboronic acids. The insulin analogue has a long circulating half-life (t1/2{beta} [~]150 h) due to its unique, unprecedented, glucose-responsive binding to blood circulation-associated proteins. This interaction drives the formation of a unique systemic reservoir that allows synchronised dynamic response in insulin levels to glucose fluctuations. In type 1 diabetic mice and minipigs, a single subcutaneous dose can maintain normoglycaemia for over one week (47.5-fold compared to insulin glargine). After the last injection of four consecutive weekly administrations in mice, the glucose-lowering effect can last 800 hours. Administering this glucose-responsive insulin analogue with a fully automated insulin delivery system can leverage dual closed loops and achieve a glucose time in range of over 85% and a coefficient of variation of approximately 30%, better than the recommended targets for human therapy. Importantly, no toxicity was identified in the study.

bioengineering↗