Biology subjects

Mao, R.

Publications and source records attributed to Mao, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Enhancing cortical electrophysiological signatures of sleep overrides ER stress responses in sleep-deprived mice

Sleep deprivation (SD) triggers characteristic changes in the cortical electroencephalogram (EEG) during waking and sleep, as well as upregulating molecular signatures of neuronal activity, synaptic plasticity, and endoplasmic reticulum (ER) stress. This includes the induction of the unfolded protein response (UPR), the primary marker and adaptive mechanism of ER stress. Yet how cortical neuronal activity during SD contributes to the induction of these molecular signatures remains unknown. Here we investigate whether manipulating cortical activity during SD with a physiological stimulation paradigm (flickering light stimulation, FLS) affects the induction of the UPR. As expected, SD alone upregulated activity-dependent and UPR-related genes in cortex and led to increased EEG slow-wave activity (SWA) during subsequent NREM sleep, an electrophysiological signature of sleep need. Delivering FLS during SD, however, prevented the upregulation of activity- and UPR-related genes while inducing an additional increase in SWA during subsequent sleep. Further analysis of neuronal firing revealed that FLS induced brief neuronal silent (OFF) periods in awake animals during stimulation, thereby shifting cortical activity towards a brain state resembling sleep. These findings suggest that physiological manipulation of cortical activity during waking is sufficient to dissociate molecular signatures of ER stress from electrophysiological markers of sleep pressure. We propose that global sleep wake history and cortical network dynamics differentially regulate activity-dependent transcription and the accumulation of sleep pressure.

neuroscience↗

bioRxiv2026

Fast receptor-antigen dissociation kinetics promote CAR-T cell tumor infiltration, functional fitness, and antitumor efficacy in solid tumors

Background: CAR-T cells have shown limited efficacy against solid tumors, where they face two major challenges: restricted tumor infiltration and sustained intensive engagement that drive them to exhaustion. Conventional CAR design has largely emphasized high-affinity interactions with slow dissociation, further limiting tumor infiltration and exacerbating exhaustion and dysfunction. We hypothesize that most current CARs are kinetically over-stabilized for solid tumors and that a fast-on/fast-off, cheek-kiss mode of engagement permits transient CAR-antigen disengagement, thereby facilitating tumor infiltration and preserving CAR-T cell fitness in tumor mass. Methods: We identified two monoclonal antibodies targeting the same epitope of epidermal growth factor receptor with comparable kon but distinct koff rates: cetuximab (Cet) and its humanized derivative H1. CAR-T cells incorporating these binders were evaluated in vitro and in vivo. Large, established human tumor xenografts were treated with low-dose CAR-T cells in NSG mice to model conditions of limited T cell availability and sustained tumor antigen exposure. Results: Cet and H1 exhibited comparable kon but markedly different koff rates, with H1 displaying fast-off kinetics with approximately 18-fold shorter binding dwell time. As CARs, H1 showed reduced antigen occupancy and less stable tumor-T cell engagement than Cet. Despite comparable in vitro cytotoxicity, H1 CAR-T cells accumulated more efficiently following tumor stimulation. At high doses, both CAR-T cell products eradicated established tumors. Under low-dose conditions, however, H1 CAR-T cells exhibited superior expansion, persistence, tumor infiltration, and antitumor efficacy, whereas Cet CAR-T cells showed limited expansion and only transient tumor control. Cet CAR-T cells progressively downregulated surface CAR expression, increased PD-1 expression, and exhibited reduced tumor infiltration and impaired functional recovery. In contrast, H1 CAR-T cells preserved surface CAR expression, exhibited greater tumor infiltration, maintained a less exhausted, more central memory-like phenotype, and retained effector function. Conclusion: Fast-on/fast-off binding kinetics enhance CAR-T tumor infiltration, expansion, persistence, and functional fitness, resulting in improved control of established solid tumors. The data provides a rationale for shifting CAR design from affinity optimization toward kinetic optimization to improve the efficacy and safety of CAR-T therapy for solid tumors.

immunology↗

bioRxiv2026

Plasma membrane PI4P recruits SNAP47 to mediate SNARE-dependent AMPAR exocytosis during synaptic potentiation

The synaptic delivery of the AMPA-type glutamate receptors (AMPARs) is crucial for longterm potentiation (LTP) of excitatory synapses, yet the mechanisms underlying neuronal activity-dependent AMPAR exocytosis at the plasma membrane (PM) remain unclear. We previously demonstrated that the PM synthesis of the phosphoinositide PI4P is enhanced upon LTP induction and that PM PI4P, not PI(4,5)P2, is required for activity-induced AMPAR exocytic trafficking. Here, we show that AMPARs are exocytosed at PI4P-enriched dendritic PM microdomains in potentiated hippocampal neurons. The Q-SNARE SNAP47 binds PI4P via its pleckstrin homology (PH)-like domain. This interaction recruits SNAP47 to the PM, promoting the exocytic fusion of AMPAR vesicles through the SNAP47-Syntaxin-3-VAMP2 SNARE complex. In the hippocampus, the SNAP47-PI4P interaction is necessary for both LTP and long-term memory. Our findings reveal a mechanistic role for PI4P in mediating activity-dependent, SNARE-driven fusion of AMPAR exocytic vesicles with the PM.

neuroscience↗

bioRxiv2026

The cellular and genetic basis of inflorescence divergence between maize and teosinte

The domestication of maize from teosinte involved dramatic remodeling of the ear, yet the cellular and genetic bases of this transformation remain unclear. Here, we generate a single-nucleus and spatial transcriptome atlas of developing maize and teosinte ears. Comparative analysis reveals divergence in cob-associated cell types, with enhanced cytokinin signaling and reduced growth-inhibitory signals collectively driving cob thickening and enlargement in maize. We further demonstrate that domestication expanded the spatial expression domain of key transcription factors in maize meristem cells, enhancing the potential for increasing kernel number. Additionally, we verified a major domestication gene, ZmSPD1, in which two nonsynonymous SNPs differentiate maize from teosinte and alter jasmonic acid (JA) levels in the ear, thereby suppressing spikelet abortion to effectively double kernel production. These findings provide a cell-resolved mechanistic framework for how cob architecture and kernel number were shaped during maize domestication, offering new insights into the formation of key agronomic traits.

plant biology↗

bioRxiv2026

Distal conformational steering by N-terminal pyroglutamylation enables subtype-selective GPCR activation across Aplysia PRXamide and human Neuromedin U signaling

Post-translational modifications (PTMs) diversify neuropeptide function, yet how minimal modifications encode receptor specificity without direct contact remains a fundamental challenge in chemical biology. Pyroglutamylation (pQ), a prevalent N-terminal PTM in bioactive peptides, introduces a rigid cyclic constraint, yet its mechanistic role in receptor signaling is unclear. Here, using two newly identified endogenous Aplysia PRXamide receptors (ApPRXa R1 and ApPRXa-R2) as a model system, we find that the same ligand, MMG2-pDPb (pQPPLPRYamide), produces opposite functional outcomes: pQ suppresses ApPRXa-R1 activation while enhancing ApPRXa-R2 activation. In vitro and in silico analyses demonstrate that the N-terminal pQ/Q remains solvent-exposed and does not directly contact receptor residues. Instead, pQ reshapes the ligand conformational ensemble and redistributes interaction networks across shared receptor contact sites. Strikingly, this molecular logic extends to mammalian Neuromedin U (NmU) receptors, as canine NmU (pQFLFRPRNamide) similarly biases subtype preference of human NmU receptors. Both static and dynamic analyses further reveal that receptor pocket mechanics determine the direction of this modulation: a loose and permissive pocket better accommodates the rigid pQ-constrained ligand, whereas a more compact pocket favors the non-pyroglutamylated ligand. These findings define a "PTM distal steering" mechanism that bridges "lock-key" and "induced-fit" paradigms and establish a general principle by which a minimal, non-contacting modification encodes receptor preference through ligand conformational biasing and pocket-dependent permissiveness, providing a chemical framework for optimizing stable, conformation-biased neuropeptide analogs.

biochemistry↗

bioRxiv2026

Lineage-Specific Neofunctionalization of Polyacetylene-Directing UGT76 Glycosyltransferases in Campanulaceae

Polyacetylene glycosides exhibit notable pharmacological activities, yet the glycosyltransferases acting on their polyacetylene scaffolds remain unknown. Here we report a telomere-to-telomere genome assembly of Codonopsis pilosula and, guided by spatial metabolomics, characterize three UDP-glycosyltransferases: CpUGT76BG1 and CpUGT76BG2 catalyze the direct glycosylation of lobetyol to lobetyolin, while CpUGT94BY2 performs subsequent sugar-sugar coupling to produce lobetyolinin, with each activity confirmed by in planta overexpression. Structural modeling reveals that CpUGT76BG1 and CpUGT76BG2 employ a deep hydrophobic tunnel to fully encase the linear polyacetylene chain, a binding architecture distinct from the shallow pockets used by canonical plant UGTs for planar aromatic substrates. Ancestral sequence reconstruction across eleven nodes partitions the UGT76 lineage into three functionally distinct evolutionary stages, tracing the trajectory from an ancestral shallow pocket to this specialized deep architecture. These findings establish the key glycosylation steps of polyacetylene glycoside biosynthesis, define a tunnel-based paradigm for non-planar substrate recognition, and reveal how tandem duplication-driven active site remodeling generates metabolic novelty.

evolutionary biology↗

bioRxiv2026

PanKbase Integrated Single-Cell Map: A Comprehensive Atlas of Human Pancreatic Islets

Single-cell RNA sequencing (scRNA-seq) of human pancreatic islet tissue is a powerful tool for investigating type 1 diabetes (T1D). However, individual datasets are limited in size and fragmented across donors, laboratories, and experimental conditions. To address this, we constructed a comprehensive, integrated scRNA-seq atlas of isolated human pancreatic islets by collating publicly available data generated from tissue provided by resources including the Human Pancreas Analysis Program, the Integrated Islet Distribution Program, and Prodo Labs. Systematic quality controls were implemented to select high-quality samples, reads, and cells. During integration, we accounted for important variables such as age, sex, body mass index, origin study, treatments, islet distribution resources, and sequencing chemistry. Our single-cell atlas comprises 191 high-quality samples from 140 donors (59 female, 81 male) across five phenotypic groups: no diabetes (controls, n=69), autoantibody positivity without diabetes (n=12), pre-diabetes (n=11), T1D (n=12), and type 2 diabetes (T2D) (n=36). In total, the atlas contains 448,935 cells, capturing 13 distinct populations, including alpha cells (43.3%) and beta cells (26.8%), as well as groups such as immune cells (0.6%). Publicly available at www.pankbase.org, this atlas provides a platform for hypothesis-driven investigation of diabetes pathophysiology and, given rigorous quality control, is well-suited for downstream machine-learning applications. Article HighlightsO_LICurrent scRNA-seq datasets of pancreatic islet tissue are limited in size and scattered across donors, laboratories, and experimental conditions, underscoring the need for a consolidated resource. C_LIO_LIWe harmonized datasets from multiple sources to build a comprehensive single-cell map of isolated human pancreatic islets. C_LIO_LIOur atlas captures 448,935 cells from 191 high-quality samples across 140 donors and multiple phenotypic groups, identifying 13 distinct cell populations. C_LIO_LIAvailable at www.pankbase.org, the atlas provides a scalable, rigorously curated platform to support hypothesis-driven diabetes research and can enable a broad range of downstream computational applications. C_LI

genomics↗

bioRxiv2026

An Essential Role of the AMPK-Related Kinase SNRK in Regulating Drosophila Sleep

Protein kinases are important regulators of sleep in animals, but their loss-of-function mutations usually show moderate phenotypes. We report here that sucrose non-fermenting related kinase (SNRK), a member of the AMP-activated protein kinase-related kinase (ARK) family, plays an essential role in sleep regulation: Snrk gene knockout resulted in a profound loss of night-time sleep phenotype, much stronger than that of the well-known ARK member Sik3. This phenotype was completely rescued by neuronal SNRK expression. SNRK in adulthood was required for sleep. Catalytic and regulatory sites in SNRK were important for sleep. A chemoconnectomic (CCT) screen revealed SNRK functioning in cholinergic neurons whose activation is known to inhibit sleep. Sleep loss in SNRK mutants was rescued by pharmacological blockade of nicotinic acetylcholine receptors. Our findings have uncovered a fundamental role for SNRK in cholinergic neurons to regulate sleep.

neuroscience↗

bioRxiv2026

Synthetic Immunological Niche Reveals Early Immune Dysregulationand Stratifies Therapeutic Response in Type 1 Diabetes

In Type 1 Diabetes (T1D), disease onset and response to immunotherapy vary widely among individuals, reflecting heterogeneous stage-specific immune dysregulation that remains undefined. To investigate this heterogeneity, we engineered a microporous polycaprolactone scaffold that forms a synthetic immunological niche (IN) upon subcutaneous implantation, enabling in vivo capture of systemic immune dysregulation. In non-obese diabetic (NOD) mice, longitudinal transcriptomic profiling of IN-infiltrating cells identified early-stage genes relatively enriched for myeloid cells, followed by progressive increases in T cell-associated dysregulation at later stages that distinguished T1D progressors from non-progressors. We derived an early-stage IN-based T1D gene signature capturing immune alterations. The signature stratified NOD progressors from non-progressors as early as 6 weeks of age and was conserved across human T1D datasets, distinguishing T1D from non-diabetic individuals in spleen and pancreatic lymph node samples, but not peripheral blood. Signature-based stratification further revealed enrichment of macrophage-associated TNF- pathways in NOD progressors, validated in human T1D islets. Given heterogeneous response to anti-TNF- therapy, IN profiling identified resistance-associated mechanisms and enabled derivation of a pathway score that prospectively distinguished treatment-sensitive from resistant mice prior to therapy, establishing the IN as a minimally invasive platform for detecting stage-wise immune dysregulation and stratifying immunotherapy response in T1D.

bioengineering↗

bioRxiv2026

Fast and Luminous: CLIP-tag2

CLIP-tag is a self-labeling protein tag used for the specific fluorescence labeling of proteins. However, its low labeling speed and the poor cell permeability of its substrates result in low labeling efficiencies in live-cell applications. Here, we introduce a substrate optimized for live-cell applications, as well as an engineered CLIP-tag variant, CLIP-tag2, which reacts with the new substrate almost 1000-fold faster than the original CLIP-tag-substrate pair. CLIP-tag2 fusion proteins can be specifically and efficiently fluorescently labeled in cells within minutes at nanomolar substrate concentrations, and can be multiplexed with other self-labeling tags such as SNAP-tag2 and HaloTag7. These advances establish CLIP-tag2 as a powerful tagging platform for high-performance live-cell bioimaging.

biochemistry↗

bioRxiv2026

Is torpor a quiescent state? Periodic motility and transient brain activation during daily torpor in Djungarian hamsters

Torpor is a hypometabolic state employed by many mammalian and non-mammalian species to cope with harsh environments. When exposed to a short photoperiod, Djungarian hamsters (Phodopus sungorus) enter daily torpor with body temperatures dropping to as low as 15{degrees}C. Despite the widely-held notion that torpor is a form of deep sleep, torpid animals are not completely inactive but exhibit occasional movements reflected in an increase in EMG tone. Little is known about these EMG events during torpor and whether they have a functional role during the torpid state. We here analysed EEG, EMG, and brain temperature data from Djungarian hamsters, and used an automatic detection algorithm to identify periods of EMG activation during spontaneous daily torpor. The hamsters exhibited regular periods of motility that were invariably initiated during a decline in brain temperature and were followed by a brain temperature increase. The frequency of EMG events exhibited a negative correlation with brain temperature, such that lower brain temperature was associated with a higher frequency of EMG events. In addition, EMG events were associated with a pronounced increase in EEG power, especially between 9.5-15.5 Hz, which often started with an EEG pattern similar to an evoked potential preceding the increase in the EMG activity. On the contrary, micro-arousals during normothermic NREM sleep were associated with a decrease in EEG power, a decrease in brain temperature and were of shorter duration than torpor EMG events, indicating that the two phenomena may serve different purposes. We speculate that periodic motility associated with increased brain activity during torpor may play a role in thermoregulation, and help retain vigilance to potentially mitigate predation risk during this hypometabolic state.

neuroscience↗

bioRxiv2025

Pocket-based molecule generation with an SE(3)-equivariant language model leads to a potent and selective HPK1 inhibitor with in vivo efficacy

Deep learning shows promise in structure-based drug discovery, yet challenges persist in generating pharmacologically plausible molecules with valid 3D conformation and decent binding mode in the pocket. We introduce SE3-BiLingoMol, an SE(3)-equivariant Transformer for pocket-based 3D molecule generation, addressing two key limitations of existing language-model approaches. First, it uses Geometric Algebra Transformers for SE(3)-equivariant handling of continuous 3D coordinates. Second, a bidirectional attention mechanism mitigates conformational errors accumulated during autoregressive sampling. These innovations enable SE3-BiLingoMol to generate 2D drug-like, 3D geometrically valid molecules with superior binding modes. Validated on DUD-E dataset containing over 100 targets, the model achieved state-of-the-art performance in de novo design and optimization. We applied SE3-BiLingoMol to design potent and selective inhibitors for HPK1, a promising immunotherapy target. Through an iterative human-AI workflow, integrating AI generation with experimental validations (X-ray crystallography, bioassays), we identified Cmpd. 6. This novel tetracyclic compound demonstrates potent HPK1 inhibition, excellent cellular activity, favorable pharmacokinetics, and robust anti-tumor in vivo efficacy as monotherapy and with PD-1 blockade. Our work establishes a sophisticated generative AI framework for 3D molecule design and demonstrates its application in developing cancer immunotherapy. Highlights[bullet] SE(3)-Equivariant Language Model for Molecule Generation: We introduce a dual-channel SE(3)-equivariant language model that supports de novo molecule design and optimization, addressing key challenges in prior language model-based 3D molecule generation approaches. [bullet]Discovery of Potent HPK1 Inhibitors: Application of this model led to the discovery of highly selective HPK1 inhibitors with robust in vivo efficacy, resulting in a promising lead compound. [bullet]AI-Guided Rational Drug Development Paradigm: This work establishes a paradigm for AI-guided rational drug development, showcasing AI as a "co-pilot" that inspires drug designers with novel ideas in an "evidence-based context".

bioinformatics↗

bioRxiv2025

Fast, Bright and Reversible Rhodamine Tags for Live-Cell Imaging

We present Rho-tag and SiR-tag, engineered protein tags derived from bacterial multidrug-resistance proteins that bind unsubstituted (silicon-) rhodamines with nanomolar affinity, enabling fast, reversible, and fluorogenic protein labeling. In live cells, Rho-tag labeling occurs within seconds -- faster than HaloTag7 -- and the tags are compatible with super-resolution methods like STED, SMLM, and MINFLUX. The high specificity of Rho-tag and SiR-tag for unsubstituted rhodamines allows their use alongside HaloTag7 and SNAP-tag. In vivo applications are demonstrated by efficient neuronal labeling in zebrafish larvae.

bioengineering↗

bioRxiv2025

A high-affinity split-HaloTag for live-cell protein labeling

We introduce a high-affinity split-HaloTag comprised of a short peptide tag (Hpep, 14 residues) and a large, inactive fragment (cpHalo{Delta}3). Hpep binds to cpHalo{Delta}3 spontaneously with nanomolar affinity, enabling subsequent labeling with fluorescent HaloTag ligands. The small size of Hpep facilitates cloning-free endogenous protein tagging using CRISPR/Cas9 and the complementation of Hpep-tagged proteins can be achieved in live cells through co-expression with cpHalo{Delta}3 and in fixed cells through incubation with cpHalo{Delta}3. The approach is compatible with advanced microscopy techniques such as expansion microscopy and live-cell STED imaging. Additionally, variants of Hpep that modulate the spectral properties of labeled fluorophores enable simultaneous imaging of two different Hpep-tagged proteins via fluorescence lifetime microscopy. In summary, our high-affinity split-HaloTag is a robust and versatile tool for live-cell imaging and diverse applications in chemical biology.

biochemistry↗

bioRxiv2025

Ancient DNA Reveals Hominoid Evolution: Intermediate DNA Sequences and Advances in Molecular Paleontology

AbstractFossils and ancient crude pottery vessels function as physical "DNA containers," preserving oriDNA (original, in situ DNA) and accumulating eDNA (environmental DNA) over time. Ancient DNA (aDNA) serves as molecular fossils, chronicling evolutionaryhistory. Using "nano-affinitybead technology", we extracted and sequenced DNAfrom Lycoptera fossils in the Jehol region and a "round-bellied jar (Jar)" from the Erlitou period in Guangwu Town, yielding 236,545 primate sequences from the fossils and 86,908 from the pottery. We observed that the AFF value of DNA sequences negatively corre lates with species divergence time1, offering a quantitative measure of DNA preservation and host divergence. Some fragments distinct from modern genomic sequences, termed "intermediate DNAsequences" (IDS), have been identified. Many IDS exhibit an upper age limit, preserving characteristics of the last common ancestor (LCA) of the Hominidae and offering molecular insights into "Darwins puzzle". Among IDS, the SRRA subtype (Sequence Reversal and Rearrangement), identified in mRNA-coding exonic sequences, arises from the incorporation of a complementary antisense strand upstream of the sense strand, either adjacent to it or separated bya sequence interval. This introduces a novel post-transcriptional regulatory mechanism at the mRNA level, driven by SRRA, which presets hairpin structures and Indels in the UTR or CDS of exonic sequences, modulating gene variation. We propose: "SRRAs played a critical trial-and-error role in early Hominidae evolution, facilitating adaptive genomic changes, with some SRRA sequences later excised from exons", positioning SRRAas an evolutionary "genetic switch". Additionally, seven species -specific fragments (SSFs) of non-human primates (NHPs) linked to Asian Homo erectus were identified in the fossils, and pottery DNA reveale d sequences from tropical species (e.g., zebrass, oil palms), providing evidence of climate-driven local extinction and supporting paleo-ecological and paleo-environmental reconstruction. This method of analyzing aDNA from non-skeletal materials opens new avenues in paleontology, archaeology, and geology, guiding the tracing of ancient migration patterns and fossil searches. DNAfragments preserved within "DNA containers " exhibit an "old-few, new-many" turnover pattern, with many aDNA fragments displaying non-deamination. This evidence challenges prevailing perspectives, the authenticity criteria for aDNA, and the capabilities and scope of the traditional research method, necessitating a thorough reevaluation of the relevant knowledge framework. Furthermore, this study opens new opportunities for frontier research.

paleontology↗

bioRxiv2024

Bergeyella cardium variant induces unique cytoplasmic vacuolization cell death

Bacterial pathogens have evolved multiple mechanisms to modulate host cell death, evade host immunity, and establish persistent infection. Here, we show that an emerging pathogen Bergeyella cardium variant (BCV) survives serum killing, inducing unique cytoplasmic vacuolization cell death in the majority of cells and apoptosis in a small number of bystanders. The cytoplasmic vacuolization cell death triggered by BCV is characterized by fused lysosome-associated termination and is inhibited by the sodium channel inhibitor amiloride. Endosomal solute carrier family 9 member A9 (SLC9A9) was identified as a critical regulator in the process of BCV-triggered cytoplasmic vacuolization cell death. Moreover, outer membrane vesicles (OMVs) or transfection of barrel-like membrane proteins lipocalin, {beta}-barrel, and PorV dramatically induced cytoplasmic vacuolization. BCV hijacked SLC9A9 by maintaining its high expression, increasing cytoplasmic vacuolization, and enhancing pathogenicity. These findings contribute to the development of novel approaches to modulate cytoplasmic vacuolization cell death and treat infectious diseases. HighlightsO_LIBCV triggers unique cytoplasmic vacuolization cell death. C_LIO_LISLC9A9 is essential for BCV-induced cytoplasmic vacuolization cell death. C_LIO_LIOMVs and the transfection of barrel-like proteins derived from BCV induce cytoplasmic vacuolization cell death. C_LIO_LIBCV increases pathogenicity via cytoplasmic vacuolization cell death. C_LI

cell biology↗

bioRxiv2024

Yeast-MetaTwin for Systematically Exploring Yeast Metabolism through Retrobiosynthesis and Deep Learning

Underground metabolism plays a crucial role in understanding enzyme promiscuity, cellular metabolism, and biological evolution, yet experimental exploration of underground metabolism is often sparse. Even though yeast genome-scale metabolic models have been reconstructed and curated for over 20 years, more than 90% of the yeast metabolome is still not covered by these models. To address this gap, we have developed a workflow based on retrobiosynthesis and deep learning methods to comprehensively explore yeast underground metabolism. We integrated the predicted underground network into the yeast consensus genome-scale model, Yeast8, to reconstruct the yeast metabolic twin model, Yeast-MetaTwin, covering 16,244 metabolites (92% of the total yeast metabolome), 2,057 metabolic genes and 59,914 reactions. We revealed that Km parameters differ between the known and underground network, identified hub molecules connecting the underground network and pinpointed the underground percentages for yeast metabolic pathways. Moreover, the Yeast-MetaTwin can predict the by-products of chemicals produced in yeast, offering valuable insights to guide metabolic engineering designs.

synthetic biology↗

bioRxiv2024

Bio-macromolecular assembly machine for human limb lengthening

Growth plate (GP) is the critical cartilaginous structure for longitudinal bone growth. Herein, employing high-resolution analytical techniques, we explore the intricate mechanisms that govern the polarized mineralization patterns within GP. The GP-epiphysis interface displays a sharp transition in tissue modulus, acting as a "protective shell" for the underlying GP, whereas the GP-metaphysis interface exhibits a gradual modulus increase, enabling efficient load redistribution to metaphysis. The unique mechanical environments at these two interfaces contribute to polarized CaP mineralization patterns, which are regulated by a complex protein-based molecular machinery. The mineralization inhibitors SPP1 and AHSG enriched at the GP-epiphysis interface could act as a line of defence against mineralization. When these two proteins coexisted with the mineralization-promoting ENPP1 and ALPL at the GP-metaphysis interface, a sequential event of precise nucleation and programmable assembly of CaP minerals occur, forming "mineralization waves" to guide bone elongation. By replicating such specific macromolecular environment at GP-metaphysis interface, a hypertable amorphous calcium phosphate (ACP) phase is well-retained in vitro, demonstrating the possibility for precise and gentle control of ACP-hydroxyapatite (HAp) transformation under physiological conditions. Our study defines a novel concept of "mineralization waves" that govern the velocity and amplitude of GP-guided mineralization process.

bioengineering↗