Biology subjects

Lin, H.

Publications and source records attributed to Lin, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Species-Specific Roles of RIPK1 and TRADD in TNF-Induced Cell Death Reveal a Translational Gap Between Mouse Models and Human Biology

Mouse models have historically been central to studies of TNF-induced cell death and guided pharmaceutical translation into clinic, based on the assumption that TNF signaling is conserved between human and mouse. Here, our work uncovers critical species-specific differences between the two. By systematically dissecting the roles of RIPK1, TRADD, and sensitivity to RIPK1 inhibitors in TNF signaling--including RIPK1 kinase-dependent and-independent apoptosis--we found that both apoptosis modalities diverge between human and mouse cells. In mouse cells, RIPK1 suppresses TRADD-mediated kinase-independent apoptosis, whereas in human cells, RIPK1 and TRADD act redundantly. Moreover, RIPK1 inhibitors block kinase-dependent apoptosis in mouse but not human cells, despite effectively inhibiting RIPK1 S166 phosphorylation. Cross-species complementation revealed that these discrepancies stem not from RIPK1 itself but from cell-context differences. These findings echo the clinical failures of RIPK1 inhibitors despite efficacy in mouse models and underscore the need for humanized models and therapeutics that more faithfully predict clinical outcomes.

Cell Biology↗

bioRxiv2026

mTORC1 Directs TNF-Induced Life-or-Death Decisions via Complex I Destabilization

The transition from tumor necrosis factor (TNF)-induced plasma membrane-bound complex I to cytosolic death-inducing complex II switches cells from survival to death. However, the precise regulation of this fatal decision is incompletely understood. Here, we show that mTORC1 promotes this transition by destabilizing later-stage complex I without affecting its initial assembly. Inhibition of mTORC1 unleashes ATG9A and FIP200 activity, thereby promoting the accumulation of CHUK (IKK) in complex I. CHUK scaffolds the kinase-active IKK{beta} to stabilize complex I and prevent complex II formation. Activation of this ATG9A/FIP200-CHUK/IKK{beta} axis protects against TNF-induced fulminant hepatitis while compromises antibacterial defense against Staphylococcus aureus. This mTORC1-governed life-or-death transition provides therapeutic insight into TNF-related pathologies--including cancer, metabolic tissue injury, and microbial infections--where mTORC1 activity is frequently suppressed.

Cell Biology↗

bioRxiv2026

Hydration and H/D exchange-dependent infrared signatures of the GCN4 leucine zipper

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

biochemistry↗

bioRxiv2026

Development of GS-441524 Derivatives as Potent SARS-CoV-2 Mac1 Inhibitors via a Direct-to-Biology Approach

Targeting viral macrodomains (Mac) has emerged as a promising strategy for antiviral drug development, especially after the outbreak of COVID-19 that claimed millions of lives worldwide. Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mac1 inhibitors have been reported in the past few years. In the present work, we converted GS-441524 (IC50 of [~]10 M for SARS-CoV-2 Mac1) to KP-S54 (18c), a potent inhibitor of both SARS-CoV-2 Mac1 (IC50: 44 nM) and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 (IC50: 91 nM) through an iterative direct-to-biology approach. This approach leverages efficient amide-coupling reaction and the mix-and-read fluorescence polarization (FP) assays where reaction mixtures could be screened directly without purification. Cocrystal structure of a selected derivative (12p) binding to SARS-CoV-2 Mac1 revealed the binding mode, which will guide future drug development against viral macrodomains.

pharmacology and toxicology↗

bioRxiv2026

Systematic benchmarking of zero-shot utility and robustness in single-cell transcriptomic foundation models

Single-cell foundation models (scFMs) have emerged as powerful representation learning approaches for single-cell transcriptomics. However, the utility and robustness of their pretrained representations across diverse analytical tasks and data conditions remain insufficiently characterized, particularly in zero-shot settings without task-specific fine-tuning. Here, we systematically analyze zero-shot performance of single-cell transcriptomic representations across 20 methods, 6 downstream tasks and 1,607 datasets comprising nearly 21.8 million cells. We evaluate model behavior along three complementary dimensions: utility on original datasets, robustness to controlled changes in dataset structure, and exploratory associations between dataset characteristics and performance variation. Our results show that scFM performance is strongly task dependent, with no single method consistently outperforming others across cell- and gene-level analyses. Notably, high utility on original datasets did not necessarily translate into robustness under structural perturbations, and several top-ranking methods were sensitive to changes in cell number, gene number, class composition, class imbalance, and batch complexity. Conventional statistical and task-specific methods remained competitive in several settings, while greater computational cost did not consistently correspond to better performance. Driver analyses further identified task-specific associations between performance and dataset characteristics, including cell-type complexity, train-test class overlap, batch number, and regulatory target-set size. Together, these findings show that the zero-shot utility and robustness of pretrained scFM representations depend jointly on analytical task and dataset structure. Our study provides a practical basis for context-aware representation selection and underscores the importance of evaluating structural robustness alongside utility when developing and applying scFMs.

bioinformatics↗

bioRxiv2026

A Brugada-related KCNT1 mutation unveils its conductance-independent activation of store-operated Ca 2+ entry

BackgroundPotassium sodium-activated channel subfamily T member 1 (KCNT1) is a Na+-activated K+ channel, associated with epilepsy and cardiac diseases. Besides K+ conductance, KCNT1 is also involved in Ca2+ handling. KCNT1:R1106Q was identified in a Brugada Syndrome (BrS) patient; however, its pathological characteristics regarding either K+ or Ca2+ homeostasis remain to be fully elucidated. MethodsFura-2-loaded HEK293T cells were used for intracellular Ca2+ investigations, and whole-cell patch-clamp technique were used as complementary tests. Stromal interaction molecule 1 (STIM1) aggregation and endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HeLa cells were visualized using spinning disc confocal microscope. Multielectrode array (MEA) assays were utilized to assess the field potential duration (FPD) and spontaneous beating rate of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). ResultsKCNT1 overexpression increased SOCE, which was further upregulated by the KCNT1R1106Q mutant. Furthermore, KCNT1 upregulated ER Ca2+ release and was found to localize at ER-PM junctions. The ER Ca2+ dynamics in KCNT1R1106-overexpressing cells was disrupted, while its SOCE remained intact. The KCNT1 cytoplasmic tail alone sufficiently modulated Ca2+ homeostasis. Given that KCNT1 possesses prominent charge-concentrated segments within its cytoplasmic domain, we substituted charged amino acids in either the 740-DDE-742 or 1114-RRLSR-1118 motifs with alanine; these substitutions abolished the KCNT1-mediated increases in ER Ca2+ release and ER-PM contact formation. MEA recordings of hiPSC-CMs revealed that the overexpression of KCNT1 shortened the FPD and accelerated the cardiomyocyte beating rate compared to baseline. However, the magnitude of beating rate acceleration induced by KCNT1R1106Q was significantly attenuated compared to that of KCNT1WT. ConclusionsOur findings indicate that, independent of KCNT1-mediated ion conductance, the charged motifs located at both ends of the KCNT1 cytoplasmic tail serve as structural anchors to facilitate ER-PM contact formation. This non-conducting role endows KCNT1 with a K+ current-independent mechanism to modulate intracellular Ca2+ homeostasis and cardiomyocyte physiology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/731813v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@104e234org.highwire.dtl.DTLVardef@19eceb3org.highwire.dtl.DTLVardef@1148512org.highwire.dtl.DTLVardef@2c8118_HPS_FORMAT_FIGEXP M_FIG C_FIG Novelty and SignificanceO_ST_ABSWhat is known?C_ST_ABSO_LIKCNT1 is a Na+-activated K+ channel, and mutations within this gene are often associated with epilepsy and epilepsy-related cardiac diseases. C_LIO_LIBesides functioning as a rectifier, KCNT1 is implicated in cellular Ca2+ handling, such as regulating Ca2+ oscillations in rat cortical neurons. C_LIO_LIKCNT1:R1106Q missense mutation has been identified in a patient with Brugada Syndrome (BrS); however, its pathophysiological characteristics and underlying mechanisms remain uncharacterized. C_LI What New Information Does This Article Contribute?O_LIKCNT1 overexpression upregulates both ER Ca2+ release and store-operated Ca2+ entry (SOCE). KCNT1R1106Q further increased SOCE intensity, however, its increase in ER Ca2+ release and [Ca2+]ER were attenuated. C_LIO_LIThe cytoplasmic tail of KCNT1 alone is suNicient to modulate Ca2+ closely mimicking the eNects of full-length KCNT1. C_LIO_LIThe charged clusters distributed along the KCNT1 cytoplasmic tail facilitate ER-PM contact. Alanine substitutions abolish the enhancements in ER Ca2+ release and ER-PM contact formation, while SOCE modulation remains intact. C_LIO_LIThe KCNT1 cytoplasmic tail alone shortens the field potential duration (FPD) and accelerates the spontaneous beating rate in hiPSC-CMs. However, the magnitude of beating rate acceleration induced by KCNT1R1106Q is significantly attenuated compared to KCNT1WT. C_LI This study uncovers a novel, conductance-independent role for the KCNT1 channel in structural organization, demonstrating that the charged motifs on its cytoplasmic tail serve as physical anchors facilitating ER-PM junctions. This structural mechanism regulates intracellular Ca2+ homeostasis and subsequently alters cardiomyocyte electrophysiology. By decoupling KCNT1s structural impact from its ion-conducting function, these findings provide new mechanistic insights into how specific KCNT1 variants, such as the BrS-associated KCNT1:R1106Q mutation, may contribute to arrhythmogenesis through imbalanced Ca2+ handling rather than classical K+ current alterations.

Molecular Biology↗

bioRxiv2026

Enhanced Target Binding by Leritrelvir Restores Dimerization of Mpro Mutants and Mitigates Drug Resistance

The SARS-CoV-2 main protease (Mpro) has been a major target of antiviral drug development, leading to the development of inhibitors such as nirmatrelvir, the antiviral component of the COVID-19 drug Paxlovid. However, resistance-associated mutations that reduce the efficacy of current Mpro inhibitors, particularly nirmatrelvir, have emerged. Here, we evaluated the inhibitory activity of leritrelvir (RAY1216), an Mpro inhibitor approved in China for COVID-19 monotherapy, against a panel of Mpro variants carrying mutations at 12 resistance-associated residues distributed across four catalytic subsites. Using integrated biochemical, biophysical, structural, and cellular analyses, we demonstrate that leritrelvir retains stronger inhibitory activity against most tested resistant mutants compared with nirmatrelvir. Most of the tested mutations promote Mpro dimer dissociation, with E166V showing a particularly pronounced effect and markedly compromising nirmatrelvir binding. In contrast, thermal shift and size-exclusion chromatography assays demonstrate that leritrelvir binding restores dimerization of these Mpro mutants. Sixteen high-resolution crystal structures reveal that leritrelvir binding re-instates key dimer-interface interactions disrupted by resistance mutations. Mini-replicon assays further confirm leritrelvir to possess enhanced cellular antiviral efficacy compared with nirmatrelvir. Our findings indicate that tighter leritrelvir binding enables more effective inhibition of dissociation-prone Mpro mutants than nirmatrelvir, supporting its use as a more resilient antiviral agent for SARS-CoV-2 treatment.

molecular biology↗

bioRxiv2026

ArchaicSeeker 3.0: A deep-learning framework for scalable, haplotype-resolved inference of archaic introgression

Archaic introgression has left a significant mark on human genetic diversity, but reliably identifying introgressed segments remains a major challenge, especially with complex demographic histories and limited sample sizes. Existing methods often rely on demographic assumptions or cohort-specific parameter fitting, which compromises robustness and scalability. We introduce ArchaicSeeker 3.0 (AS3), a deep-learning framework designed for haplotype-resolved detection of archaic introgression. AS3 integrates a tract-scale sequence model with an overlap-aware reassembly approach and boundary refinement, enabling accurate, boundary-coherent reconstruction of introgressed segments across diverse genomic contexts. By leveraging a simulation-trained model, AS3 avoids inference-time recalibration, offering stable performance across unrepresented demographic scenarios and small cohorts. In extensive simulations, AS3 outperforms existing methods in precision, recall, and F1 score, while providing more continuous segments with accurate boundary localization. It demonstrates robustness in small-target regimes and varying marker densities. Applied to 3,453 genomes from 209 populations, AS3 shows strong concordance with existing introgression callers and identifies additional introgressed regions, including high-frequency AS3-specific introgressed segments supported by locus-level haplotype and phylogenetic analyses. AS3 provides a scalable, robust solution for detecting archaic introgression from single individuals to large biobank datasets, marking a significant advancement in the field of local ancestry inference and opening new possibilities for the study of human evolutionary genetics. ArchaicSeeker 3.0 is available at https://github.com/Shuhua-Group/ArchaicSeeker3.0.

bioinformatics↗

bioRxiv2026

Coenzyme A is a redox sensing cofactor for malic enzyme 2 regulating oxidative stress and mitochondrial metabolism

Coenzyme A (CoA) is an essential cofactor required for numerous metabolic reactions, yet its ability to bind and regulate proteins remains poorly defined. Using a proteomic approach, we identified malic enzyme 2 (ME2) as a CoA-binding protein. ME2 uses NAD(P)+ to convert malate to pyruvate, generating NAD(P)H to support energy production and redox homeostasis. ME2 binds CoA at an allosteric site previously thought to bind NAD(P)+. Reduced CoA has minimal effect on ME2 activity, but the oxidized form, CoA disulfide, strongly activates ME2 by promoting ME2 tetramerization and a catalytically efficient closed conformation. Under oxidative stress, ME2 facilitates CoA disulfide formation, enhancing NADPH production and cellular defense against reactive oxygen species (ROS). Mice with ME2 mutation that cannot bind CoA show impaired muscle performance, elevated ROS, and mitochondrial dysfunction. These findings establish CoA as a redox-sensing cofactor, allowing cells to respond to ROS and promote mitochondrial metabolism, and expanding the function of this essential cofactor.

biochemistry↗

bioRxiv2026

CellChem: Cellular transcriptional responses reshape molecular representation space for efficient and multi-scale drug discovery

Despite decades of progress in computational drug discovery, deep learning-based molecular representation models remain largely structure-centric, assuming that chemical similarity approximates functional similarity. However, drug effects in cells are shaped not only by chemical similarity but also by molecular interactions in the cellular context. To capture this complexity, we introduce CellChem, a cellular-chemical representation-learning framework that learns cell-guided molecular representations of small-molecule actions within cells. By incorporating large-scale cellular transcriptional profiles during pretraining, CellChem reshapes molecular representation space from structure-centric to a balanced integration of structure and function. The learned CellChem molecular representations exhibit biologically meaningful geometric organization, such that distances between molecules encode not only structural similarity but also similarity in the cellular responses they elicit, independent of downstream tasks. Using downstream derivative models such as cell-guided compound-protein interaction prediction and drug-induced transcriptional response profile generation, CellChem supports highly efficient, multi-scale drug discovery, achieving significantly better performance than traditional models.

molecular biology↗

bioRxiv2026

SIRT5 acts in the tumor microenvironment via endothelial cell metabolism to support breast cancer growth

Triple-negative breast cancer (TNBC) is characterized by aggressive progression and poor prognosis, partly due to abnormal angiogenesis. While the metabolic reprogramming of tumor cells is well characterized, the metabolic regulation of tumor-associated endothelial cells (ECs) remains unclear. Here, we identified the mitochondrial deacylase SIRT5, which has established tumor-promoting roles in TNBC cells, as a key regulator of endothelial metabolic homeostasis and tumor angiogenesis. SIRT5-deficient host mice showed significant defects in supporting the growth of orthotopic SIRT5-proficient mammary tumor transplants, and the resulting neoplasms showed defects in tumor vascularization. In a 3D microfluidic vessel-on-chip model, SIRT5 loss compromised vascular barrier integrity and EC sprouting. Mechanistically, SIRT5-deficient ECs exhibited diminished mitochondrial respiratory capacity but apparently normal glycolysis. SIRT5 loss also caused increased mitochondrial reactive oxygen species levels, and a mitochondrial antioxidant rescued the endothelial cell defects following SIRT5 loss, indicating that SIRT5-mediated mitochondrial redox homeostasis in the tumor microenvironment is necessary to maintain vascular function. Orthotopic co-transplantation of TNBC and EC cells with or without SIRT5 knockdown demonstrated that endothelial SIRT5 promotes increased tumor growth in vivo. These results suggest that targeting SIRT5 offers a potential therapeutic strategy to disrupt tumor angiogenesis and suppress TNBC progression by targeting the metabolic vulnerabilities of the tumor endothelium.

cancer biology↗

bioRxiv2026

RNA Folding Nearest Neighbor Parameters Including the Modification 1-Methyl-Pseudouridine

Nearest neighbor analysis is commonly used to estimate RNA folding stabilities. In this contribution, we report a set of RNA folding nearest neighbor parameters for estimating free energy change for RNA sequences including 1-methyl-pseudouridine. Development of mRNA vaccines has identified 1-methyl-pseudouridine as a key nucleobase modification for suppressing innate immune responses. However, the contributions of these modifications to RNA folding stability were unclear. Our new parameters provide helical terms for 1-methyl-pseudouridine-adenine and 1-methyl-pseudouridine-guanine base pairs. The parameters also estimate loop stabilities for loops with 1-methyl-pseudouridine or a combination of 1-methyl-pseudouridine and uridine. These parameters are derived using 208 optical melting experiments and tested against an additional 16 optical melting experiments. On average, we find that substitution of uridine with 1-methyl-pseudouridine stabilizes RNA folding, with the extent of stabilization depending on adjacent sequence. The estimation of tRNA folding ensembles for tRNA sequences with 1-methyl-pseudouridine was significantly improved using the new nearest neighbor parameters. The new nearest neighbor parameters are provided as part of the RNAstructure software package. With these parameters, the secondary structures of natural sequences with 1-methyl-pseudouridine and mRNA therapeutics fully substituted with 1-methyl-pseudouridine can be modeled.

bioinformatics↗

bioRxiv2026

A High-throughput Fluorescence Polarization Assay for Screening Sirtuin Inhibitors

Sirtuins (SIRTs), which remove protein lysine acyl modifications, play crucial roles in diverse cellular processes, including metabolism, gene transcription, DNA damage repair, cell survival, and stress response. Several sirtuins are considered non-oncogene addiction of cancer cells and promising targets for anticancer drug development. High-throughput screening (HTS) methods for sirtuins are critical for the development of potent and isoform-selective sirtuin inhibitors, which are needed to validate the therapeutic potential. Herein, we designed and synthesized a fluorescent polarization (FP) tracer, KP-SC-1. Using this high-affinity tracer, we developed a robust, high-throughput FP competition assay for screening SIRT1-3 inhibitors. The assay was validated by testing known SIRT1-3 inhibitors. The assay can detect NAD+-independent SIRT1-3 inhibitors, as well as NAD+-dependent inhibitors, such as Ex-527 and TM. Finally, our assay showed satisfactory stability and outstanding performance in a pilot library screening. Compared to previous assays, the FP assay uses much less SIRT1-3 enzymes, a feature important for high-throughput library screening. We believe that the FP assay developed here will accelerate the discovery and development of SIRT1-3 inhibitors.

biochemistry↗

bioRxiv2026

SPEND-hSRS imaging of fumarate uncovers mitochondrial metabolic heterogeneity

Mitochondria, acting as the energy powerhouse, biosynthetic center, and reductive equivalent hub of the cell, participate in cellular metabolic activities. However directly imaging mitochondrial chemical content and quantifying metabolic activity in living cells remain challenging. Here, by Self-PErmutation Noise2noise Denoiser enhanced Hyperspectral Stimulated Raman Scattering (SPEND-hSRS) microscopy, we demonstrate fingerprint-region metabolic imaging of fumarate, a key intermediate in the tricarboxylic acid (TCA) cycle, with sub-millimolar sensitivity. In chemotherapy-stressed bladder cancer cells, fumarate imaging revealed two mitochondrial subpopulations with divergent TCA metabolic preferences quantified by ratio metric analysis. Pixel-wise least absolute shrinkage and selection operator (LASSO) spectral unmixing further reconstructs fumarate and lipid maps, uncovering localized fumarate enrichment in protrusions. Extending to CH-window hyperspectral SRS imaging, we uncover the interplay between mitochondria and lipid droplets (LDs) in protrusions, where fatty acid is found to be released from LDs, to fuel the TCA cycle. Together, our work establishes SPEND-hSRS as high-resolution platform for linking fumarate to mitochondrial heterogeneity. Our results provide new insights into how mitochondrial heterogeneity and interaction with LDs drive cancer cell adaptation to stress.

cancer biology↗

bioRxiv2026

High-speed 3D single-virus tracking reveals actin-aided viral trafficking of SARS-CoV-2 on the plasma membrane

Early interactions between viruses and live cells are difficult to resolve due to rapid extracellular motion, 3D nature of the cell membrane, and the fast, nanoscale interactions involved. While actin is a central regulator of viral entry, direct observations of actin-aided trafficking have been restricted to membrane protrusions on glass surfaces given the limitations of conventional methods. Here, high-speed 3D Tracking and Imaging microscopy (3D-TrIm) is integrated with highly photostable StayGold-labeled SARS-CoV-2 virus-like particles to capture long-term, high-resolution single-virus trajectories in live cells. This approach revealed distinct regimes of viral dynamics, including extracellular diffusion, protrusion-based surfing, and an unreported linear trafficking mode along the plasma membrane that precedes viral internalization. This work demonstrates that this membrane trafficking is actin-driven and positively correlated with ACE2 expression. These findings reveal new actin exploitation by viruses and demonstrate the utility of 3D-TrIm for dissecting dynamic virus-cell interactions at high spatiotemporal resolution.

biophysics↗

bioRxiv2026

DHHC7 palmitoylates KRAS4A and promotes mutant KRAS-driven pancreatic cancers

KRAS mutations underlie many human cancers. While inhibitors such as Sotorasib and Adagrasib targeting KRAS mutants have shown promise, additional strategies are required to address the broader spectrum of KRAS-driven cancers, particularly those displaying drug resistance. Thus, there is a need to better understand KRAS signaling and develop new therapeutic strategies. Here we show that KRAS4A is palmitoylated on Cys180 by a palmitoyl transferase, DHHC7 (gene name ZDHHC7). Palmitoylation promotes KRAS4A plasma membrane localization, and more importantly, nanoclustering. This in turn promotes the activation of ARAF and RAF1, but not BRAF. DHHC7 and KRAS4A Cys180 palmitoylation are important for the normal and anchorage independent growth of pancreatic cancer cell lines. Depletion of ZDHHC7 dramatically inhibits pancreatic tumor growth in mouse xenograft models. These studies provide new understandings about how palmitoylation regulates KRAS4A activity and suggest DHHC7 as a promising new target for KRAS mutant cancers.

cancer biology↗

bioRxiv2026

immunoPETE: A DNA-based integrated B-cell and T-cell receptor profiling platform

The vast diversity of B and T cell receptors generated through the recombination of Variable (V), Diversity (D), and Joining (J) gene segments plays a critical role in adaptive immunity. Profiling immune repertoires at the DNA level provides a robust and stable approach to capture the clonal composition of these receptors. immunoPETE is an assay designed to target recombined human T-cell Receptor Beta (TRB), T-cell Receptor Delta (TRD), and Immunoglobulin Heavy (IGH) chain genes directly from genomic DNA. Simultaneous profiling of B and T cell receptor chains in a single reaction provides internally normalized clone counts and facilitates the study of B-T cell interactions. Full-length amplicon consensus sequences representative of original template DNA molecules are accurately reconstructed using Unique Molecular Identifiers (UMIs). An in-house pipeline compiles VDJ rearrangements from the Complementarity-Determining Region 3 (CDR3) of TRB, TRD and IGH chains into comprehensive readouts at cell-level resolution. In this study, we describe the immunoPETE end-to-end workflow, followed by a comprehensive benchmarking of its performance in adaptive immune profiling. Where applicable, we used both natural and contrived samples and characterized the assays accuracy, linearity, and reproducibility across several metrics: retrieving CDR3 sequences, determining B and T cell ratios, total cell count, yield, fraction of functional rearrangements, clonal diversity, composition of dominant clones, pairwise similarity, and V/J gene usage frequencies. Furthermore, we assessed its quantitative limits concerning the total number of lymphocytes and the detection of rare clones. As an example of its applications, we show that adding immune biomarkers extracted from immunoPETE data to clinical factors improves prediction of progression-free survival in a cohort of non-muscle invasive bladder cancer (NMIBC) patients. Finally, we discuss the broad applications of immunoPETE in the study of aging, cancers, infections, and autoimmune disorders with reference to select published studies.

immunology↗

bioRxiv2026

HMA proteins produce 2',3'-cNMP signaling molecules and activate CNL-mediated immunity in rice

Plants deploy a sensor repertoire, usually NLRs, to perceive pathogen effectors and activate executor NLR-triggered immunity. Here we reveal that rice HMA proteins function as sensors detecting the Magnaporthe oryzae MAX effector AvrPigm to trigger broad-spectrum blast resistance mediated by the CNL PigmR. AvrPigm is conserved with multiple copies in blast genomes and targets HMA proteins, facilitating HMA translocation into the cytoplasm. Cryo-EM structure of the HPP04HMA reveals a filament-like oligomer with ssDNA/RNA bound inside the filament, which display cyclic nucleotide synthase activity to generate 2',3'-cNMP in a CNL-dependent manner. We further discovered that the LRR domains of the executor CNLs perceive 2',3'-cNMP to mount immunity. Our study thus establishes an unrecognized immunity mode with a distinct repertoire of sensor receptors that produce signaling molecules that are perceived by the LRR domains of executor NLRs to mediate immunity, shedding new light on the sensor-executor conception and immune activation in plants. HighlightsO_LIHMA proteins and CNL receptors form a new sensor-executor mode in plant immunity C_LIO_LICryo-EM reveals the filament-like structure of HMA-ssRNA/ssDNA C_LIO_LIHMA proteins are cyclic nucleotide synthetases to generate 2',3'-cNMP C_LIO_LILRR domains of CNLs perceive 2',3'-cNMP to mediate immunity C_LI

plant biology↗