Biology subjects

Wang, P.

Publications and source records attributed to Wang, P..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

Early developmental placental defects and pregnancy loss in lupus mice expressing human TLR8

Anti-phospholipid (APL) autoantibodies confer a high risk for adverse pregnancy outcomes, especially in Systemic Lupus Erythematosus (SLE). While human TLR8 (huTLR8) has been implicated in APL antibody-mediated placental injury in vitro, its in vivo role in pregnancy is unexplored. We report a novel mouse model of pregnancy loss in SLE-prone mice expressing huTLR8. Placental analysis revealed early developmental defects starting post-implantation, including a thin junctional zone, impaired vascularization, infarcts, and inflammation. Profound immune dysregulation was evident at E8.5 including increased myeloid cells and CD8 T cells and decreased uterine natural killer (uNK) cells. RNA sequencing revealed downregulated pregnancy-specific glycoproteins, reduced uNK cell-associated genes, and an upregulated myeloid cell signature. Bone marrow chimera studies demonstrated preferential activation of huTLR8-expressing placental Ly6C+ monocytes. Spatial transcriptomics at E9.5 confirmed uNK cell loss, decreased IL15 expression by both stromal and myeloid cells, and discrete inflammatory aggregates in the maternal layers containing myeloid cells and IFN{gamma}-expressing CD8 T cells. We propose that huTLR8, likely through myeloid cell activation and cytolytic T cell recruitment, drives placental injury in the context of SLE and APL autoantibodies. This model provides a valuable platform to dissect early pathogenic events in APL-associated pregnancy loss and identify new therapeutic targets.

immunology↗

bioRxiv2026

Positional grammar of transcription factor binding partitions developmental and stress-response regulation in plants

Understanding how transcription factor binding site (TFBS) position influences gene regulation remains a fundamental challenge in plants. Here, we integrate conserved multiDAP TFBS maps for 244 transcription factors (TFs) with single-nucleus chromatin accessibility, cell type-resolved gene expression, and hormone-response datasets across Brassicaceae species to determine how TFBS position relates to regulatory function. Although conserved TFBSs are enriched near transcription start sites (TSSs), TSS-proximal accessibility poorly predicts cell type-specific expression. Instead, cell type-specific expression correlates best with conserved TFBSs embedded in cell type-restricted chromatin, with TF family-specific distributions across distal promoters and introns. In contrast, TSS-proximal TFBSs in broadly accessible chromatin are associated with rapid transcriptional responses to abiotic and biotic stress hormones. Coding sequence TFBSs mark a distinct regulatory context in which the same DNA sequence encodes both amino acid sequence and TF motifs, including evidence that CDS-localized ABR1 binding may contribute to repression during hormone response. Finally, distal upstream regions contain conserved multi-family TF clusters with enhancer-like features overlapping rare cell type-specific accessible chromatin and enriched near genes controlling embryonic, meristematic, and hormone-dependent developmental patterning. Together, these results support a positional grammar in which TFBS position and chromatin context jointly partition developmental, stress-responsive, and repressive regulatory output in plants.

genomics↗

bioRxiv2026

Precision CRISPR annotation of the functional enhancer landscape in primary human T cells

Precise modulation of T cell function through engineering the non-coding genome holds great promise for advancing next-generation immunotherapies. However, robust high-throughput approaches to annotate functional cis-regulatory elements (CRE) in human T cells remain limited. Here, we developed a simple and highly efficient CRISPR interference (CRISPRi) perturbation platform to systematically annotate CREs in human primary T cells. Using this platform, we identified novel CREs controlling PDCD1, HAVCR2, and TBX21 expression. Combinatorial CRE perturbations revealed synergistic CRE pairs that fine-tune PDCD1 and HAVCR2 expression, while Cas9-indel-based mutagenesis pinpointed the critical nucleotides within each enhancer that are essential for their activity. Functional experiments demonstrated that CRE-edited HAVCR2 outperformed conventional total gene knockout in enhancing CAR T cell anti-tumor efficacy. Moreover, CRE editing of PDCD1 and HAVCR2 repressed PD-1 and TIM-3 expression in human tumor-infiltrating lymphocyte CD8 T cells, highlighting regulatory role of these CREs in disease relevant exhausted T cells. Together, this approach offers a compact CRISPRi platform that enables high-throughput dissection of functionally relevant non-coding genomic regions in T cells, providing insights for mechanistic studies and precision genome engineering of advanced cellular therapies.

immunology↗

bioRxiv2026

Defects in tissue-resident macrophages lead to smaller eardrums and abnormal neurovascular networks with increased middle-ear infection

The tympanic membrane (TM), or eardrum, is essential for hearing. Macrophages, the primary innate immune cells, are densely distributed in the eardrum after birth, especially near blood vessels and nerve fibers. During postnatal development, neonatal tissue-resident macrophages (TRMs) gradually polarize, and their population declines. These dynamic changes closely parallel the maturation of neurovascular networks. What are the precursors of the early wave of TRMs? Are they critical for postnatal TM development? Using fate mapping, single-cell RNA sequencing, and macrophage depletion, this study reveals for the first time that postnatal eardrum TRMs are heterogeneous, originating mainly from embryonic myeloid lineages, with increasing input from postnatal monocyte progenitor-derived cells. Single-cell RNA sequencing identifies gene signatures vital for vascular and neuronal development. Depleting TRMs results in smaller eardrums, disrupted vascular-neuronal networks, and increased risk of middle-ear infection. This study offers new insight into how the innate immune system supports TM maturation and protects against middle-ear infection during a critical postnatal window. TeaserTissue-resident macrophages guide eardrum neurovascular maturation and help prevent middle-ear abnormalities.

immunology↗

bioRxiv2026

Rapid sensing and relaying of cellular hyperosmotic-stress signals via RAF-SnRK2 core condensates

Hyperosmolarity caused by drought, high salinity, or cold stress inhibits plant growth and crop productivity1,2. A conserved protein-kinase cascade of cytosolic B-RAFs and SnRK2s is rapidly activated upon osmotic stresses to initiate downstream adaptive responses, which represents one of the fastest known responses to osmotic stress in plants3-8. How the kinase cascade is activated by osmotic stress is unknown. Here, we show that Arabidopsis B4 subgroup RAFs have intrinsically disordered regions and directly sense both ionic and non-ionic hyperosmolarity by reversible condensation. B4-RAFs recruit and co-condense with subclass-I SnRK2s to phosphorylate and turn on SnRK2s, evading the non-condensable inhibitory A-clade PP2C phosphatases. This straightforward osmosensing and relaying module can be fully reconstituted in E. coli by co-expressing three components or in solution in a test tube using recombinant proteins. Our findings identify B-RAFs as the chief cellular osmosensors that detect low water potential by co-condensation, forming a signal hub with SnRK2s to orchestrate adaptive responses in plants, and represent an evolutionarily conserved osmosensing mechanism across kingdoms.

plant biology↗

bioRxiv2025

3'to 5'translation of linear mRNAs?

3[->]5 translation (termed "backward translation") mediated by eukaryotic circular RNAs has been reported recently. Whether linear mRNA can produce proteins through backward translation remained elusive. Here, we demonstrate that linear mRNAs can mediate backward translation in vitro. Backward translation was detected in human cells transfected with expression vectors used to produce linear mRNAs. Importantly, synthetic linear mRNAs could produce backward translation signal in multiple in vitro translation systems, with a level comparable to that of conventional 5 to 3 forward translation. Furthermore, we demonstrated that a single translation initiating sequence (TIS) was able to drive backward and forward translation in an in vitro experimental setup, with the efficiency of backward translation found to be significant lower than that of forward translation. In summary, this study further reinforces the notion of translation flexibility, demonstrates a broader applicability of backward translation and heralds a new strategy in synthetic biology.

cell biology↗

bioRxiv2025

ZIKV NS5-mediated STAT2 degradation as a determinant of immune evasion and pathogenicity

Type I interferon (IFN) signaling is a central antiviral defense, with STAT2 driving the expression of IFN-stimulated genes (ISGs) to restrict viral infection. Many flaviviruses, including Zika virus (ZIKV), evade this pathway through non-structural protein 5 (NS5)-mediated ubiquitination and proteasomal degradation of STAT2, yet the contribution of this immune evasion strategy to viral fitness and pathogenesis remains incompletely defined. Here, using a multi-step mutational scanning strategy that jointly assessed STAT2 degradation and viral RNA replication, we identified a single NS5 residue, L162, as uniquely permissive to substitution that abolishes STAT2 degradation without compromising intrinsic replication functions. Substitution of L162 with alanine or glycine (L162A or L162G) selectively disrupted NS5 recruitment of the ZSWIM8-CUL3 E3 ubiquitin ligase, thereby preserving STAT2 stability and restoring IFN signaling. Recombinant ZIKV carrying these mutations exhibited reduced replication and enhanced ISG induction in human cells, defects fully rescued by STAT2 knockout. In A129 mice (type I IFN receptor deficient), mutant and WT viruses replicated comparably, but in human STAT2 knock-in mice, ZIKV-NS5L162G exhibited markedly reduced viral loads and disease. Notably, infection with ZIKV-NS5L162G elicited robust neutralizing antibody and T cell responses that conferred protection against WT challenge. Together, these findings establish NS5-mediated STAT2 degradation as a central determinant of ZIKV immune evasion, viral fitness and pathogenesis, and highlight disruption of NS5-STAT2 antagonism as a promising strategy for antiviral intervention and rational attenuation.

microbiology↗

bioRxiv2025

Ultrahigh-Throughput Forster Resonance Energy Transfer-Activated Droplet Sorting for Terminator Polymerase Engineering

Droplet microfluidic screening systems enable high-throughput, labor-saving enzyme directed evolution by employing fluorescence, absorbance, and Raman-activated sorting strategies for library screening. Forster resonance energy transfer (FRET) - a nanoscale technique for monitoring intramolecular/intermolecular conformational changes - is yet to be integrated into this process. We upgraded a single-channel sorter to a dual-channel one without redesigning the microscopy setup, which can monitor FRET signals during enzyme reactions in droplets at kilohertz rates. We applied this upgraded sorter to improve the incorporation efficiency of KOD DNA polymerase towards reversible terminator deoxyribonucleotides, a property crucial for its application in next-generation sequencing (NGS). Our data show that a single-round sorting can achieve 30-fold enrichment of active variants. Five KOD variants enabling 100-cycle single-end runs of DNA sequencing were identified using a novel cyclic reversible termination (CRT) substrate featuring a terminator group {approx} 5-fold bulkier than the classical azidomethyl moiety. We also engineered a metagenome-derived novel polymerase, and a variant achieving 90% terminator incorporation efficiency within 2 minutes was identified after two rounds of enrichment. In sum, we provide a practical setup for dual-channel FRET-based droplet sorting, and demonstrate its ability in terminator polymerases engineering, thereby broaden the scope of microfluidic applications.

biochemistry↗

bioRxiv2025

Dopamine biosynthesis in plants

Dopamine, an essential human neurotransmitter, also plays important roles in plant-microbe interactions and abiotic stress tolerance. While dopamine biosynthesis is well-characterized in mammals, it remains largely unclear in plants. Through metabolite-based genome-wide association studies, we identified two putative polyphenol oxidase genes for dopamine biosynthesis in Brachypodium distachyon, located in the same chromosomal region as two tyrosine decarboxylase genes. Biochemical and knockout mutant analyses demonstrated that dopamine biosynthesis proceeds primarily via tyramine as an intermediate and contributes to plant tissue browning. Homology searches and functional assays indicate that this dopamine biosynthetic pathway is conserved across diverse plant species. This discovery provides valuable insights into plant dopamine biosynthesis with potential applications in reducing food browning, improving stress resilience, and bioengineering valuable compounds.

plant biology↗

bioRxiv2025

Occipital tACS does not modulate manual performance but fixation variability under visuo-proprioceptive conflict

The modulation of oscillations at alpha and beta frequency ranges recorded over sensory cortices has been linked to the top-down weighting of sensory information relative to (competing) information from other modalities. Here, using a virtual reality-based hand-target matching task under visuo-proprioceptive conflict, we tested whether alpha-/beta-tACS over the occipital cortex would improve action performance when conflicting visual movement feedback needed to be ignored in favor of proprioception. Participants had to match a target rhythm with either their unseen real hand (RH) grasping movements, or with the movements of a virtual hand (VH) that displayed their actual movements after a constant time delay. Visual movement feedback was, consequently, either task-relevant (VH task) or a distractor (RH task). In a cross-over, double-blind, within-subject design, we applied either low-intensity (2 mA peak-to-peak) transcranial alternating current stimulation (tACS) at 10 Hz ("alpha") or 20 Hz ("beta"), or sham, over the occipital cortex during this task. Neither alpha- nor beta-tACS significantly affected hand-target matching performance, but fixation variability in the RH condition significantly decreased during beta-tACS relative to sham stimulation. Thus, despite not directly modulating manual performance, occipital beta-tACS could have facilitated ignoring the mismatching (distracting) visual movement feedback when needed.

animal behavior and cognition↗

bioRxiv2025

3' to 5' Translation of Circular RNAs?

For decades, the 5[->]3 direction of translation has been a central dogma of molecular biology(1). Here we present evidence that eukaryotic circular RNAs (circRNAs) can serve as templates for 3[->]5 backward translation (BT), yielding polypeptides with distinct sequence and structural features not found in canonical proteomes. Mining of more than 6,000 multi-omics datasets identified [~]1 million candidate BT proteins across eukaryotes, including 59,000 high-confidence human BT proteins supported by mass spectrometry. Genetic combinatorial experiments (KO and KI) and cell-free translation of synthetic circRNAs establish BT as a conserved mechanism. Loss-of-function studies of hs.circCAPN15 in human and dm.circROLS in Drosophila underscore the functional importance of BT-derived proteins. Our work challenges the long-standing unidirectional translation paradigm, expands the functional landscape of the genetic code, and reveals a hitherto hidden layer of proteomic complexity with broad implications for biology and therapeutics.

molecular biology↗

bioRxiv2025

Harmine Selectively Drives Human Beta Cell Differentiation and Function Via Protein Kinase A Pathways

Harmine and other small molecule inhibitors of the kinase DYRK1A induce human beta cells to replicate and regenerate in vitro and in vivo, and are effective at reversing diabetes in animal models. In addition to its beta cell proliferative and regenerative effects, harmine also induces expression of transcription factors and other genes involved in beta cell differentiation and function, exemplified by PDX1, MAFA, NKX6.1, GLP1R, PCSK1 and many others. Harmine also rapidly enhances glucose-stimulated insulin secretion in vitro and in vivo, reversing diabetes within days in diabetic mice transplanted with a marginal mass of human islets. We had assumed that this pro-differentiation or pro-function effect was a common feature of all DYRK1A inhibitors, and was mediated by DYRK1A inhibition. Thus, DYRK1A is a primary target (we refer to it as Target 1) for harmine. Here, to our surprise, we report that the pro-differentiation effect is not a generalized action for all small molecule DYRK1A inhibitors and does not result from DYRK1A interference or genetic silencing. Instead, the prodifferentiation effect is restricted to a small select subset of DYRK1A inhibitors (harmine, 2-2c and 5-IT). Remarkably, the pro-differentiation effect results from the unique ability of these drugs to activate protein kinase A (PKA), a dual mechanism that distinguishes this class from other DYRK1A inhibitors. The beneficial effects of harmine on PKA appear to be indirect, driven by an as yet unidentified, Target 2 in the PKA pathway. These findings make it clear that all DYRK1A inhibitors are not interchangeable, and that those that drive both proliferation and differentiation/function will likely be preferable in human clinical therapeutic settings. They also provide a novel target or roadmap for enhancing human beta cell differentiation in Type 1 and Type 2 diabetes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/692818v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@bd015corg.highwire.dtl.DTLVardef@a46f9borg.highwire.dtl.DTLVardef@19b4f65org.highwire.dtl.DTLVardef@179aa28_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Tissue-specific tolerance mechanisms and lymph node co-drainage converge to shape T cell immunity in the upper digestive system and regulate pancreatic cancer progression

The liver, pancreas, and duodenum share lymph nodes (LNs), providing a unique system to examine how tissue origin of self-antigens shapes T cell fate. Comparing mice expressing ovalbumin (OVA) from distinct subcellular compartments, we found that cytosolic OVA from liver or pancreas, but not gut, was immunologically ignored. High-dose hepatic secreted OVA triggered antigen-specific T cell deletion, whereas secreted pancreatic and intestinal OVA induced regulatory T (Treg) cells, revealing immunological ignorance, clonal deletion and Treg cell generation as tissue-specific tolerance mechanisms. Of these, LN co-drainage only influenced Treg cell induction, establishing gut-pancreas-liver axes: Intestinal viral infection rendered hepatocyte- and exocrine pancreas-specific T cells inflammatory; liver injury promoted pancreas- and gut-directed responses. These self-reactive T cells caused tissue destruction but enhanced pancreatic tumor control when neoantigen OVA was secreted but not cytosolic. Thus, LN co-drainage and tissue-specific tolerance mechanisms jointly shape immune homeostasis and disease susceptibility in the upper digestive system.

immunology↗

bioRxiv2025

Constraining inference of across-region interactions using neural activity perturbations

Functional interactions between brain regions are often inferred from multi-region models fit to neural activity recorded in behaving animals. Here, we show that inference of acrossregion interactions is hindered by the wide breadth of model fits consistent with naturally occurring neural activity. In contrast, models fit to activity that includes region-wide activity perturbations provide well-constrained estimates of across-region interactions; simulations suggest these estimates can be accurate.

neuroscience↗

bioRxiv2025

SpaTranslator: A deep generative framework for universal spatial multi-omics cross-modality translation

Recent advances in spatial omics technologies have enabled the simultaneous analysis of multiple molecular patterns in tissue sections, offering unprecedented insights into cellular microenvironments. However, the high cost of measurements and the sparsity of data restrict the availability of paired spatial multi-omics datasets. Here, we present SpaTranslator, a deep generative framework that integrates graph neural networks with an adversarial variational generative model to fully capture spatial characteristics and enable effective cross-modality translation of spatial omics data, enabling simulation of paired spatial multi-omics data from single-omics measurements. Extensive experiments demonstrate that SpaTranslator consistently outperforms baseline methods in both clustering accuracy and biological coherence across various real-world scenarios, including spatial transcriptomics-epigenomics and spatial transcriptomics-proteomics translation tasks. Furthermore, SpaTranslator provides biologically meaningful insights through marker genes and proteins recovery, motif enrichment analysis, and gene regulation inference. Our work offers an effective and adaptable solution for spatial multi-omics cross-modality translation, supporting a broad range of biological and biomedical research.

bioinformatics↗

bioRxiv2025

Tumor cell-specific loss of GPX4 reprograms triacylglycerol metabolism to escape ferroptosis and impair antitumor immunity in NSCLC

Glutathione peroxidase 4 (GPX4) is a master regulator of ferroptosis, a process that has been proposed as a potential therapeutic strategy for cancer. Here we have unexpectedly found that inducible knockout of GPX4 in tumor cells significantly promotes non-small cell lung cancer (NSCLC) progression in the autochthonous KrasLSL-G12D/+Lkb1fl/fl (KL) and KrasLSL-G12D/+Tp53fl/fl (KP) mouse models, whereas inducible overexpression of GPX4 in tumor cells exerts the opposite effect. GPX4-deficient tumor cells evade ferroptosis by upregulating the expression of DGAT1/2 to promote the synthesis of triacylglycerol (TAG) and oxidized TAG (oxTAG) and the formation of lipid droplets in cells. In addition, GPX4-deficient tumor cells secrete TAG and oxTAG into the extracellular space to induce dysfunction of antitumor CD8+ T cells, thereby coordinating an immunoinhibitory tumor microenvironment (TME). Consistently, treatment with DGAT1/2 inhibitors or inducible overexpression of GPX4 in tumor cells significantly resensitizes tumor cells to ferroptosis and ignites the activation of T cells in the TME to inhibit NSCLC progression. These findings highlight a previously uncharacterized role of tumor cell-specific GPX4 in NSCLC progression and provide potential therapeutic strategies for NSCLC.

cell biology↗

bioRxiv2025

The O-glycosyltransferase SECRET AGENT Participates in Abscisic Acid-Induced Microtubule Remodeling and Stomatal Closure in Arabidopsis thaliana

Secret Agent (SEC) encodes the major O-GlcNAc transferase in Arabidopsis thaliana and has been implicated in stress and signaling pathways, but its role in stomatal regulation remains unknown. In this study, we investigated the function of SEC in abscisic acid (ABA)-induced stomatal closure and drought responses. Loss of SEC function in the sec-5 mutant delayed stomatal closure and increased water loss under drought, whereas SEC overexpression enhanced stomatal responsiveness and drought tolerance. Pharmacological assays revealed that sec-5 was less sensitive to microtubule-stabilizing and -disrupting drugs during ABA-induced stomatal closing, consistent with a defect in cytoskeletal dynamics. Confocal imaging and quantitative analysis of microtubule rearrangements showed that SEC loss-of-function mutation retarded microtubule reorganization in response to ABA, with abnormal microtubule bundling and density. Mechanistically, SEC physically interacted with -tubulin 4 (TUA4) in both yeast two-hybrid and bimolecular fluorescence complementation assays, suggesting tubulin as a potential substrate of SEC-modified O-GlcNAcylation. Together, these findings establish SEC as a key regulator of ABA-induced microtubule remodeling in guard cells, thereby linking O-GlcNAcylation to stomatal regulation and drought tolerance.

plant biology↗

bioRxiv2025

Correlative 4D-STEM Ptychography and EELS for Cryogenic Single-Particle Analysis

Cryogenic electron microscopy has enabled atomic resolution structural determination of biological macromolecules. However, phase contrast imaging alone does not reveal elemental composition or other valuable chemical information. Electron energy loss spectroscopy (EELS) provides rich vibrational and chemical details, integrating it with high-resolution structural imaging for correlative analysis of biological specimens remains a major challenge, as conventional acquisition modes are often mutually exclusive. Here we propose two optical geometries that enable correlative 4D-STEM with EELS: (1) a sequential acquisition with a post-specimen beam deflector, and (2) a simultaneous acquisition with a hollow detector. We compared the two setups for biological specimens, and focused on the hollow detector geometry which allows simultaneous acquisition of diffraction patterns and EELS spectra in a single dose budget. Using an apoferritin dataset, we demonstrate sub-nanometre 3D resolution in single particle analysis, even with a hollow angle as of 55% of the convergence angle and approximately 30% of the total dose allocated to EELS. Finally, we propose a workflow that integrates ptychography with EELS for correlative, energy-resolved 3D mapping of biological macromolecules, paving the way toward unified structural, chemical, and vibrational analysis of biological specimens.

biophysics↗