Biology subjects

Song, G.

Publications and source records attributed to Song, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Internalized Components of Membrane Attack Complexes Disrupt Proteostasis and Acquire Alarmin-Like Properties

Immune effects of membrane attack complexes (MAC) have been widely attributed to their abilities to cause cell death. Here, we show that the MAC component, C9, forms non-cytolytic aggregates with pro-inflammatory effects. Intracellular aggregates of C9 are detected within inflamed tissues of patients in association with endothelial cell (EC) activation but not increased cell death. We identify NUMBL as a Rab35 effector that directly binds surface-bound C9 to promote C9 internalization and entry into the endolysosomal pathway. Within acidified endolysosomes, C9 forms insoluble aggregates that are targeted for degradative aggrephagy in a process that activates NF-{kappa}B. For C9 aggrephagy to occur, ZFYVE21, a Rab5 effector, complexes with RNF34 to bridge C9 aggregates to LC3B+ aggresome membranes. We detect C9 aggregates in vivo, and we show that a ZFYVE21-RNF34 signaling axis is required for C9 aggrephagy and NF-{kappa}B -dependent EC activation in three separate mouse models. Mice with conditional loss of ZFYVE21 in ECs show reduced aggregraphy, resulting in attenuated systemic inflammation and reduced tissue injury following skin transplantation. Our data show that the C9 component of MACs forms intracellular aggregates with alarmin-like properties.

biochemistry↗

bioRxiv2026

RNabel-A Standalone Software Tool for Annotating Tandem Mass Spectra of Modified Ribonucleic Acids

Ribonucleic acid (RNA) modifications, with over 170 identified types, play diverse roles in cellular processes. The past decade has witnessed surging demand for accurate identification and localization of RNA modifications in both endogenous and synthetic therapeutic RNAs. With accurate spectral annotation for RNA, tandem mass spectrometry (MS/MS) can meet this demand. Here we present RNabel, a user-friendly software tool for in-depth annotation of MS/MS spectra of RNA oligonucleotides. RNabel considers a full set of backbone-cleavage ions (a, b, c, d, a-B, w, x, y, z) in which the ribonucleotide unit could be A, U, C, G, Y (pseudouridine), or I (Inosine). Additionally, RNabel considers 196 modifications on the base, the phosphoribose linkage, the 5' or the 3' terminus, or detachment of a sub-nucleotide fragment as a neutral or charged group. Users can create new components if needed, including ribonucleotides, modifications, neutral or charged groups that could detach from a ribonucleotide. RNabel efficiently processes large datasets in four acceptable formats including .mgf, .raw, .txt from msConvert, and RNabel batch files. Multiple statistical metrics are provided for quality assessment of spectral annotation. To accelerate RNA modification analysis, RNabel is made freely available for Mac and Windows users at https://github.com/songge1111/RNabel/releases. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/733900v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@65af35org.highwire.dtl.DTLVardef@1d1c2d4org.highwire.dtl.DTLVardef@4e04d2org.highwire.dtl.DTLVardef@1eb2a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2026

Epigenetic activation of CHST2 by promoter hypomethylation promotes progression of triple-negative breast cancer

Epigenetic dysregulation is a hallmark of triple-negative breast cancer (TNBC), yet the oncogenic relevance of specific epigenetically activated genes remains poorly defined. In this study, we demonstrate that carbohydrate sulfotransferase 2 (CHST2) is aberrantly upregulated in TNBC across multiple transcriptomic analyses as a consequence of promoter hypomethylation. Elevated CHST2 expression is associated with aggressive clinicopathological characteristics and has been linked to unfavorable patient outcomes in independent cohorts. Functional analyses reveal that CHST2 primarily promotes TNBC cell migration and invasion, while genetic silencing of CHST2 markedly attenuates these malignant phenotypes. Importantly, mutational disruption of CHST2 catalytic activity abolishes its pro-migratory effects, indicating that sulfotransferase activity is essential for CHST2-driven invasiveness. Mechanistically, promoter hypomethylation is experimentally supported as a driver of CHST2 transcriptional activation, linking epigenetic deregulation to enhanced tumor aggressiveness. In addition, tumors with high CHST2 expression exhibit distinct immune-related transcriptional features within the tumor microenvironment. Collectively, these findings identify CHST2 as an epigenetically activated driver of invasive behavior in TNBC and highlight its potential value as a biomarker and therapeutic target for aggressive disease.

cancer biology↗

bioRxiv2026

Heterologous betacoronavirus spike immunization in nonhuman primates elicits cross-reactive antibodies that neutralize both sarbeco- and merbecoviruses

In anticipation of future coronavirus (CoV) pandemics, developing vaccines that elicit broadly neutralizing antibodies (bnAbs) against diverse CoVs is critical. Here, we vaccinated rhesus macaques with SARS-CoV-2 spike (S)-protein, then boosted with heterologous {beta}-CoV S-proteins to focus responses to common conserved S2 bnAb epitopes. Initial SARS-CoV-2 priming elicited receptor-binding domain (RBD)-focused responses, while MERS-CoV boosting redirected responses toward the S2 region, including the stem-helix bnAb site. Although S2-directed serum cross-neutralization was undetectable and most isolated cross-reactive monoclonal antibodies (mAbs) targeted non-neutralizing epitopes, two S2 stem-helix mAbs were identified from memory B cells. These bnAbs neutralized diverse sarbeco- and merbecoviruses, including MERS-CoV, and conferred robust in vivo protection against SARS-CoV-2 challenge. Structural studies revealed that these macaque bnAbs closely mimic human S2-stem bnAbs induced by infection. These findings provide proof-of-principle for vaccination strategies that elicit broadly protective {beta}-coronavirus responses and highlight non-human primates as a translational model for evaluating S2-targeted immunogens.

immunology↗

bioRxiv2026

Quantitative Full-length transcriptome analysis by nanopore sequencing with Error-Aware UMI mapping

Comprehensive transcriptome profiling is essential for understanding RNA diversity and regulation, yet accurate identification and quantification of full-length transcript isoforms remain challenging with short-read sequencing technologies. Nanopore sequencing enables direct sequencing of long cDNA molecules and thus offers a powerful solution for full-length transcriptome analysis, but its application to quantitative transcriptomics is limited by PCR amplification bias and the difficulty of unique molecular identifier (UMI) recognition under high sequencing error rates. Here, we developed UMImap, a dedicated pipeline for robust UMI identification, error correction, and deduplication in nanopore data. By integrating transcript-aware UMI correction with long-read isoform assembly, UMImap substantially improves UMI recognition accuracy compared with existing methods and effectively mitigates PCR-induced duplication bias. Using this framework, we identified tens of thousands of full-length transcript isoforms, including a large fraction of previously unannotated isoforms that are significantly longer than reference annotations. Quantitative analyses demonstrate the reliability of UMImap for transcript-level quantification. Functional and pathway enrichment analyses of highly expressed novel isoforms revealed coherent and biologically meaningful patterns, including strong enrichment in RNA processing, splicing, and translation pathways. Our results establish UMImap as an effective solution for UMI-based quantification in nanopore full-length transcriptome sequencing and highlight the potential of long-read sequencing to simultaneously achieve accurate isoform discovery and expression analysis in complex transcriptomes.

bioinformatics↗

bioRxiv2025

Panax notoginseng saponins protect the blood brain barrier against oxidative stress by activating the Akap12/PI3K/AKT/AQP1 signaling axis

Oxidative stress is a key pathological factor that contributes to blood-brain barrier (BBB) disruption during ischemic injury. Panax notoginseng saponins (PNS), the primary bioactive components of Panax notoginseng, possess potent antioxidative and vasoprotective activities. This study aimed to elucidate the molecular mechanisms by which PNS regulate endothelial integrity and BBB permeability under oxidative stress conditions. Mouse brain microvascular endothelial cells (bEnd.3) were treated with hydrogen peroxide to induce oxidative damage, followed by intervention with PNS. Transcriptomic and proteomic analyses revealed that Akap12 (A-kinase anchoring protein 12) and Aquaporin 1 (AQP1) were key molecules involved in PNS-mediated endothelial protection. H2O2 exposure markedly suppressed Akap12 expression and inhibited the PI3K/AKT signaling pathway, leading to elevated AQP1 expression and increased BBB permeability. PNS treatment reversed these effects by restoring Akap12 expression, reactivating PI3K/AKT signaling, and reducing AQP1 levels. Functional validation further showed that AQP1 dysregulation altered the expression balance of tight junction proteins, including Claudin-5 and ZO-1, thereby impairing barrier function. Collectively, these findings demonstrate that PNS protect endothelial cells from oxidative injury by activating Akap12-dependent PI3K/AKT signaling and modulating AQP1-associated tight junction remodeling. This study provides new mechanistic insights into the molecular basis of PNS-mediated BBB protection and suggests potential therapeutic targets for oxidative or ischemic cerebrovascular diseases.

pharmacology and toxicology↗

bioRxiv2025

Migration patterns and hybridization within the Asian stonechat complex in response to a major geographical barrier

Long-distance avian migration is thought to be under strong natural selection. Facing geographical barriers, migrants display various patterns considered to be adaptive. For example, they may detour along either side around the barrier or cross it, requiring specialized behavioral adaptations. Variations within closely related taxa are excellent sources for understanding the evolutionary background of migration and how barriers are shaping migration routes. In Asia, some species are assumed to have a migratory divide in response to the major geographical barrier, the Qinghai-Tibet Plateau (QTP), including the stonechat taxa (Siberian Stonechat Saxicola maurus maurus and Amur Stonechat S. stejnegeri). As they detour along either side of the QTP, these taxa are believed to disfavor a crossing over the highland. However, the more southernly distributed Tibetan Stonechat (S. m. przewalskii) breeds on the QTP, suggesting adaptation to high elevation. To investigate migration patterns and the potentially associated genetic differences, we studied migration routes and population genetics of four populations around the assumed migratory divide in Russia and Mongolia, and of one from the QTP in China. Our results confirmed the existence of a migratory divide between maurus and stejnegeri, albeit with extensive hybridization. We observed both the hypothesized western and eastern routes, but also found individuals employing intermediate routes crossing the QTP, of which two-thirds were clear hybrids. Meanwhile, przewalskii followed a highland-crossing route and was genetically differentiated from maurus and stejnegeri. The diverse migration routes among Asian stonechats show differential responses towards the geographical barrier. The intermediate route may be associated with hybridization, and its conditional viability may facilitate gene flow between maurus and stejnegeri. The Asian stonechat complex thus offers great opportunities for novel research of the genetics and evolution of migration. The specific evolutionary background associated with inhabiting and crossing the QTP can offer new perspectives in this field. Teaser textMigratory divides can arise in birds because alternative routes around migratory barriers would select for behaviors to restrict hybridization. Hybrids of parental types that employ alternative routes are hypothesized to embark on intermediate routes that would expose them to suboptimal conditions, resulting in post-zygotic reproductive isolation. However, this hypothesis is challenged when a sister taxon actually breeds on the geographical barrier. This is the case in the Asian stonechat complex that breeds near or on the Qinghai-Tibet Plateau (QTP), the roof of the world. We demonstrated a migratory divide in central Siberia to Mongolia for race maurus and stejnegeri, yet showed also evidence for extensive hybridization. Hybrids migrated along a newly discovered intermediate route, seemingly viable and overlaps with the migration trajectory of race przewalskii over the eastern part of the QTP. The Asian migratory divide relative to the QTP thus provided new insights to the evolution of landbird migration.

evolutionary biology↗

bioRxiv2025

Directed evolution of a stem-helix targeting antibody enables MERS-CoV cross-neutralization through enhanced binding affinity

Broadly neutralizing antibodies (bnAbs) targeting conserved regions of the betacoronavirus spike are important for pan-betacoronavirus protection and pandemic preparedness. Here, we report on the isolation of a human monoclonal antibody, CC65.1, from a SARS-CoV-2 convalescent donor that targets the conserved S2 stem helix region. CC65.1 neutralizes various sarbecoviruses, including SARS-CoV-2, and binds the MERS-CoV spike but lacks MERS-CoV-neutralizing activity due to insufficient binding affinity. We utilized directed evolution to enhance the binding affinity of CC65.1 for the MERS-CoV S2 stem helix, yielding engineered antibody variants with newly acquired MERS-CoV-neutralizing activity. High-resolution structural analysis reveals key paratope mutations that optimize binding and stabilize epitope engagement. Our findings demonstrate the potential of rational antibody engineering to expand bnAb breadth across divergent betacoronaviruses. This work supports the development of engineered bnAbs and S2-targeted vaccines for broad betacoronavirus countermeasures and highlights strategies to achieve cross-lineage immunity for future pandemic threats. Author SummaryThe persistent emergence of new SARS-CoV-2 variants of concern that evade neutralizing antibody responses and other zoonotic betacoronaviruses with pandemic potential have provided strong motivation to develop broadly neutralizing antibodies (bnAbs) that target more conserved regions of the spike protein in sarbecoviruses and other betacoronaviruses. Here, we employed a directed evolution strategy to engineer the sarbecovirus-neutralizing antibody CC65.1, which targets the conserved S2 stem helix, to enhance its binding affinity with the MERS-CoV stem helix region, thereby conferring MERS-CoV neutralization. High-resolution structural studies of engineered CC65.1 revealed that key mutations reshape the paratope to better accommodate and stabilize the MERS-CoV S2 stem helix, resulting in increased binding affinity and neutralization potency. This study emphasizes the critical role of affinity maturation in expanding neutralization breadth and provides valuable insights for design of bnAbs to prevent and treat pandemic threats by betacoronaviruses.

immunology↗

bioRxiv2025

Dual-mode intramolecular agonist-dependent mechanoactivation of the adhesion GPCR ADGRG1/GPR56

Mechanical stimuli instruct cardinal cellular decisions pertaining to cell fate, proliferation, morphology, and movement1,2. How adhesion G protein-coupled receptors (aGPCRs), a large family of mechanosensors with more than 30 members in humans3-5, transduce mechanical cues into metabotropic commands has been a matter of debate due to the lack of suitable approaches to analyze receptor activation during mechanotransduction in live cells6. A central question is whether aGPCR activation strictly requires mechanical dissociation of the autoproteolyzed N- and C-terminal fragment (NTF and CTF) complex to expose the tethered intramolecular agonist (TIA; 'Stachel'). Here, we investigated human ADGRG1/GPR56 (G1), an aGPCR with roles in brain development7, skeletal muscle8, and platelet function9, to study the events during aGPCR mechanotransduction by monitoring receptor dissociation and G protein recruitment in real time during cell migration and substrate deformation. Magnetic-tweezer measurements at a physiologically relevant loading rate showed that isolated G1 GAIN domains can partially unfold and refold at forces below those required for NTF-CTF dissociation. In cells, G1 dissociation occurred at retracting fibers during cell migration10 over an adhesive substrate through its native extracellular matrix ligand, tissue transglutaminase 2 (TG2), and was enhanced by cell stretching. Simultaneous live recording of G protein recruitment and pharmacological assays during mechanical stimulation of G1 reveal a dual-mode activation mechanism: mechanical force elicits robust metabotropic signaling without requiring NTF dissociation and generates an intracellular response commensurate with the fully dissociated receptor. Both signaling modes depend on an intact Stachel element11,12 of the receptor under our experimental conditions. Our findings determine mechanotransduction principles governing aGPCR activation at the cell-ECM interface. We reveal that G1 does not function as a simple binary switch, but rather as a highly sophisticated mechanosensor that dynamically encodes localized extracellular tension into distinct, highly scalable metabotropic outputs.

cell biology↗

bioRxiv2025

Pleistocene demographic histories dominate contemporary genomic diversity in a continental radiation of Himalayan-Hengduan songbirds

While population genetic theory expects genetic diversity to scale predictably with population size, mounting empirical evidence has challenged this fundamental prediction. This striking inconsistency has prompted a paradigm shift in our understanding of the determinants of genetic diversity, driving increased efforts to disentangle the relative contributions of Pleistocene demography and linked selection. Here, based on systematic sampling and a unified analytical pipeline, we de novo assembled genomes for 120 songbird species breeding in the Himalayas-Hengduan Mountains (HHMs) and conducted population genomic analysis to examine the drivers of their genomic diversity. We observed a 6.5-fold variation in genome-wide heterogeneity and a 16.4-fold variation nucleotide diversity and across species. Notably, these measures of genomic diversity showed no correlation with recent population dynamics, current population size, or other contemporary factors--such as natural selection, elevational distribution, or life-history traits. Instead, historical demography strongly predicted genetic diversity, with ancestral population size during the late Pleistocene emerging as the sole correlate: larger ancestral sizes consistently coincided with higher diversity. These findings underscore the critical influence of historical demography on contemporary genetic diversity in natural populations--an insight essential for designing effective conservation strategies.

genetics↗

bioRxiv2025

Impact of Inactivation Methods on Biosafety and Antigen Reactivity of Brucella melitensis from the Perspective of Astral-DIA Proteomics Based on Antibody Immunoprecipitation Mass Spectrometry

Effective Brucella inactivation is imperative for safe vaccine development, diagnostics, and sample handling, particularly in resource-limited regions lacking high-level containment facilities. This study investigated inactivation methods using the Rev.1 vaccine strain and three Brucella melitensis field isolates from Gansu (GS-XG, GS-SN, GS-MQ). Heat (80{degrees}C/95{degrees}C, 10-20 min) and formaldehyde (0.4%/0.6%, 48-72 h) inactivation were evaluated for biosafety and antigenicity. Rev.1 was completely inactivated by 80{degrees}C/95{degrees}C for 20 min or 0.6% formaldehyde for 48-72 h. Superior antigenicity, compared to phenol inactivation, was confirmed by ELISA and Western Blot. However, field isolates demonstrated greater resistance, surviving 80{degrees}C for 20 min and 0.4% formaldehyde for 72 h, necessitating stricter conditions (95{degrees}C, 20 min; 0.6% formaldehyde, 72 h) for their complete inactivation. Astral-DIA proteomics analyzed approximately 60% of the proteome ([~]2000/3300 proteins), revealing 256, 311, and 318 differentially expressed proteins between 80{degrees}C/95{degrees}C heat, 48-h/72-h formaldehyde, and heat/formaldehyde methods, respectively. Gene Ontology and KEGG analyses indicated heat inactivation upregulated cellular structure proteins but downregulated metabolic pathways, with 95{degrees}C potentially damaging conformational epitopes. Formaldehyde treatment (48 h) stabilized soluble antigens, preserving ribosomal and regulatory protein epitopes, while 72-h treatment induced organelle disintegration. Protein-Protein Interaction networks suggested heat inactivation enhanced immunogenicity of membrane and stress proteins, suitable for targeted studies, whereas formaldehyde preserved broader epitopes, beneficial for whole-cell vaccines and multi-epitope screening. Inactivation methods must be tailored to specific strain characteristics and applications. Astral-DIA provides molecular insights into antigenicity loss, guiding future research on protein functions and epitope dynamics for precise brucellosis control.

microbiology↗

bioRxiv2025

Structural insights into Measles virus RNA synthesis: C protein-mediated regulation of replication-transcription switch and allosteric inhibition by ERDRP-0519

Nonsegmented negative-sense RNA viruses (nsNSVs) rely on a multifunctional RNA-dependent RNA polymerase (RdRP) complex for transcription and replication. In measles virus (MeV), the nonstructural protein C has long been implicated in regulating RNA synthesis, yet its precise role remains unclear. Here, we show that the MeV C protein directly associates with the RdRP complex. Using cryo-electron microscopy, we determined atomic-resolution structures of the MeV polymerase with and without C, revealing that C binding stabilizes the accessory domains of L and locks the complex into a replication-competent elongation state. Biochemical data further show that C promotes N protein recruitment, enhancing polymerase processivity through facilitating encapsidation during replication. Additionally, we also resolved high-resolution structures of MeV and Nipah virus (NiV) polymerases bound to ERDRP-0519, an orally available morbillivirus inhibitor. Unexpectedly, the compound occupies a previously unrecognized allosteric pocket within the RdRp domain, rather than the predicted PRNTase domain, overlapping conserved resistance sites. This binding induces conformational changes in palm subdomain, blocking RNA template and nucleotide engagement, thereby halting RNA synthesis. These findings uncover distinct regulatory and inhibitory mechanisms in paramyxovirus polymerases and provide a structural framework for the rational design of broad-spectrum antivirals targeting MeV, NiV, and potentially other clinically relevant nsNSVs.

microbiology↗

bioRxiv2025

Spatial and temporal scale-dependent feedbacks govern dynamics of biocrusts in drylands

Biota could be ecosystem engineers in generating an intrinsic heterogeneous landscape through scale-dependent feedbacks. Thereby, they can form resource enriched patchiness or islands of fertility, comprising of self-organizing spatial patterns. Research so far has largely focused on the self-organized spatial patterns of plant communities in drylands. It, however, remains unclear whether and how biocrusts having distinct morphology and life history from plant communities could self-organize themselves and form unique spatial patterns. Here we conducted field observations of biocrusts across successional stages and employed a probabilistic Cellular Automaton (CA) model to investigate the distinct self-organized spatial patterns exhibited by mosaic patches of mosses and lichens with different patch size distributions (PSDs). Our study demonstrates that short-range positive feedbacks initially promote the development of patches, featured with a heavy-tailed PSD, while long-range negative feedbacks subsequently curtail further expansion of big patches, thereby establishing a characteristic patch scale with regular PSDs. Strikingly, only lichens reverted back to the heavy-tailed PSD in the late succession stage, presumably implying self-organized critical fragmentation of lichen patches. Field measurements of biocrust performance at the center and edge of patches of varying sizes along succession stages further support the classic scale-dependent feedback mechanism for Turing pattern formation. Collectively, our results clearly demonstrate the capability of the biocrust communities to self-organize themselves to form distinct spatial patterns governed by the spatial and temporal scale-dependent feedbacks, potentially impacting dryland ecosystem functions and resilience. Significance StatementAn intriguing phenomenon is the capability of biota to form striking self-organized spatial patterns. Biocrusts serve as the soils skin and have different morphology and life history as compared to plant communities. We investigated these distinct self-organized spatial patterns exhibited by mosaic patches of mosses and lichens across succession stages through field surveys and a probabilistic Cellular Automaton (CA) model. Our results provide empirical evidence that biocrusts act as ecosystem engineers, forming self-organized spatial patterns. Simulations and field measurements of biocrust performance elucidate the mechanism of spatial and temporal scale-dependent feedbacks in governing biocrust spatial pattern formation. This self-organizing ability of biocrusts holds significant implications for ecosystem functions and the resilience of dryland ecosystems.

ecology↗

bioRxiv2025

Rational Design and Optimization of a Potent IDO1 Proteolysis Targeting Chimera (PROTAC)

Indoleamine 2,3-dioxygenase 1 (IDO1) is a potently immunosuppressive protein that inhibits antitumor immunity through both tryptophan metabolism and non-enzymatic functions. Pharmacological therapies targeting IDO1 enzyme activity have generally failed to improve the overall survival of patients with cancer. Developing new therapeutic agents that are capable of neutralizing both enzyme-and non-enzyme-derived immunosuppressive IDO1 effects is therefore of high interest. We previously described the development of a novel Proteolysis Targeting Chimera (PROTAC), NU223612, that degrades IDO1 in cultured human glioblastoma (GBM) cells, as well as in well-established brain tumors, in vivo. In this study, we rationally optimized the composition, rigidity, and linker orientation of the PROTAC structure to create NU227326, which degrades IDO1 with a DC50 of 5 nM in human GBM cells. Mechanistic studies showed that IDO1 degradation occurred through the ubiquitin-proteasome system and was sustained for at least 2 days, supporting NU227326 as a highly potent IDO1 PROTAC suitable for further studies in GBM and other human cancers.

biochemistry↗

bioRxiv2024

Mechanical force regulates the inhibitory function of PD-1

The immune checkpoint molecule, programmed cell death 1 (PD-1), plays a pivotal role in regulating T-cell function. Upon binding to its ligands, PD-L1 or PD-L2, PD-1 suppresses T-cell receptor signaling, thereby preventing T-cell activation, making it a critical target in cancer immunotherapy. Although extensively studied, the molecular mechanism of PD-1s inhibitory function is still not fully understood, especially at the atomic level. Using the biomembrane force probe (BFP), we discovered that interactions between PD-1 and PD-L1/PD-L2 exhibit catch-slip bond behavior under force. At lower forces, catch bonds are observed, transitioning to slip bond as the force increases. Steered molecular dynamics (SMD) simulations revealed a force-induced bond state between PD-1 and its ligands, distinct from the force-free state observed in solved complex structures. Disrupting interactions that stabilize either state weakens the catch bond and diminishes PD-1s inhibitory function. Interestingly, soluble forms of PD-L1 and PD-L2 compete with their surface-bond counterparts, attenuating PD-1s suppression of T-cell activation. This suggests that soluble PD-1 ligands could potentially serve as anti-PD-1 drugs. Tumor growth studies in mice confirmed the anti-cancer activity of soluble PD-L1. Our findings highlight the critical role of mechanical force in PD-1s inhibitory function, suggesting that PD-1 may act as a mechanical sensor in suppressing T-cell activation. These insights indicate that mechanical regulation should be considered when designing PD-1 blocking inhibitors and other PD-1 related cancer immunotherapies. TeaserThis study shows that the interaction between PD-1 and its ligands is force-dependent, and soluble forms of PD-1 ligands can potentially serve as PD-1 blockers in cancer immunotherapy.

biophysics↗

bioRxiv2024

Engineered mitochondrial A-to-G editors with enhanced efficiency and targeting scope

Mitochondrial base editing is a powerful technology, but the current A-to-G mitochondrial DNA (mtDNA) base editors are reluctant to achieve high efficiency, which is critical for mtDNA engineering. Through laboratory evolution, TadA-8e variants were discovered with substantially increased activity and expanded targeting compatibility, especially for previously unfavorite sequence contexts, when applied to both nuclear and mitochondrial ABEs. Further engineering of the mtDNA editors (eTd-mtABEs) dramatically reduced both DNA and RNA off-targeting effects and enhanced strand-selective A-to-G editing with substitution of DddA to DNA nickases. Moreover, the eTd-mtABEs induced up to 145-fold editing frequencies compared to previous mtDNA adenine base editors in rat cells, and installed targeted mutations in all injected rat embryos with up to 74% efficiency in founders, resulting generation of SNHL and Leigh syndrome models with severe defects. It suggests that eTd-mtABEs are promising mtDNA engineering technology for basic research and for translational studies. HighlightsO_LIEngineered TadA variants enhance nuclear and mitochondrial ABE activity in all sequence contexts. C_LIO_LIAdditional mutations in eTd-mtABEs further minimize both DNA and RNA off-targeting effects. C_LIO_LIEngineered TadA variants are compatible to DNA nickase assisted strand-selective mtDNA editing. C_LIO_LIeTd-mtABEs enable highly efficient generation of rat mtDNA disease models with severe phenotypes. C_LI

bioengineering↗

bioRxiv2024

DEMINERS enables clinical metagenomics and comparative transcriptomic analysis by increasing throughput and accuracy of nanopore direct RNA sequencing

Nanopore direct RNA sequencing (DRS) advances RNA biology but is limited by relatively low basecalling accuracy, low throughput, yet high RNA input and costs. Here we introduce a novel DRS toolkit, DEMINERS, which integrates an RNA multiplexing experimental workflow, a machine-learning barcode classifier based on Random Forest and a novel basecaller built on an optimized convolutional neural network providing an additional species-specific training module. With the increased accuracy in barcode classification and basecalling, DEMINERS can demultiplex up to 24 samples and the required RNA input and running time are both substantially reduced. We demonstrated the applications of DEMINERS in clinical metagenomics, cancer transcriptomics and parallel comparison of transcriptomic features in different biological conditions, revealing altered airway microbial diversity in COVID-19 and a potential role of m6A in increasing transcriptomic diversity in glioma and the mature blood-stage of malaria parasites. Overall, DEMINERS is a simple, robust, high-throughput DRS method for accurately estimating transcript levels, poly(A) lengths, and mutation and RNA modification heterogeneity at single-read level, with minimal sequencing biases.

bioinformatics↗

bioRxiv2024

Regulatory architecture of disease resistance in maize revealed by multi-omic systems genetics

Complex traits such as disease resistance have been traditionally studied using quantitative genetics. Here, we use systems genetics to integrate disease severity and multi-omic quantitate trait loci (QTL) to uncover biological networks underlying interaction with northern leaf blight (NLB), a yield-limiting disease of corn. Specifically, we integrated transcriptome, (phospho)proteome, and metabolome measurements to map molecular QTL and build predictive regulatory networks following NLB infection. These inferred networks identified a critical signaling module that was genetically validated comprised of a kinase termed NLB SUSCEPTIBLE KINASE 1, a bHLH transcription factor, and the lignin biosynthesis enzyme BROWN MIDRIB 2. Our results demonstrate the feasibility of high-throughput mapping of genetic determinants of gene- product levels and demonstrates the power of systems genetics to identify upstream regulatory genes that confer resistance to NLB that can inform future strategies for crop protection.

plant biology↗