Biology subjects

Lee, S.

Publications and source records attributed to Lee, S..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

Skin-derived G-CSF activates pathological granulopoiesis upon psoriasis

Psoriasis is an inflammatory skin disease initiated by environmental triggers and driven by disruption of T cell cytokine network in the cutaneous milieu. The fact that complete resolution of disease by targeting key inflammatory cytokines remains challenging indicates a contribution of other immune cells to the pathogenesis. Here, we study the role of neutrophils in psoriasis, the first-line innate immune defender that is short-lived but mobile and infiltrate into various tissues. We found that upon psoriasis induction, skin-resident endothelial cells are activated to produce G-CSF which activates emergency granulopoiesis in bone marrow and induces cutaneous infiltration and accumulation of neutrophil that are functionally overactive. Depletion of neutrophils or blockage of psoriasis-driven granulopoiesis by respective neutralizing antibodies results in reducing cutaneous neutrophil burden and mitigating psoriasis pathogenesis. This mechanism might be conserved in human psoriasis as confirmed by public RNA-seq database. Our findings uncovered and detailed the pathological crosstalk between skin and BM in psoriatic inflammation, proposing a potential therapeutic approach targeting cross-organ communication.

immunology↗

bioRxiv2025

A Cross-scale Causal Mapping Framework Pinpoints Macrophage Orchestrators of Balanced Arterial Development

Postnatal pulmonary arteries experience an abrupt surge in flow that demands tightly coordinated remodeling of vascular structure and mechanical compliance. However, the cellular programs orchestrating these multiscale adaptation remain poorly defined, partly due to the absence of analytical tools integrating gene-cell-tissue scales. Here, we present CausaLink, a cross-scale causal mapping framework that predicts how altered gene expression propagates through gene-cell-tissue networks by integrating time-course transcriptomic and tissue morpho-mechanical data. We constructed a single-cell transcriptomic atlas of 11,143 proximal pulmonary artery-derived cells from C57BL/6J mice across five developmental stages (P2, P10, P21, P42, and P84), revealing dynamic lineage transitions and transient mesenchymal population specifying into fibroblasts and smooth muscle cells from P2 to P21. Using this framework, we identified Mgl2 macrophages as central regulators promoting lumen expansion while preventing pathological wall thinning or thickening. To validate these predictions, we established a human induced pluripotent stem cell-derived arterial assembloid enriched in MGLhigh macrophages. In this model, MGLhigh macrophages supported lumen enlargement while maintaining overall wall thickness and induced formation of an adventitia-like fibroblast layer that recapitulated approximately 80% of the native adventitial fraction with balanced collagen-elastin remodeling. By linking predictive modeling to human organoid validation, this study establishes a cross-scale workflow for tracing how gene programs shape vascular architecture, offering mechanistic insights and a foundation for predictive regenerative medicine.

bioengineering↗

bioRxiv2025

Morphological plasticity in a reef-building coral is context-dependent and trades off with resistance to thermal stress

In sessile, long-lived organisms, such as reef-building corals, phenotypic plasticity may play a key role in mitigating the impacts of climate change. Plasticity is often context-dependent and has costs and limits, ultimately resulting in adaptive, neutral, or maladaptive effects. However, our understanding of the mechanisms that maintain variation in, and therefore drive the evolution of, plasticity is still limited. We use a field-based transplant experiment to show context-dependent tradeoffs to morphological plasticity in Acropora cervicornis under real-world conditions driven by climate change. We find that while morphological plasticity is neutral under some conditions, it trades off with thermal tolerance during moderate temperature stress during a marine heatwave. Additionally, we find that plasticity changes over time, underlining the importance of its temporal dynamics in long-lived organisms. Our results demonstrate a key role for environmental change in the eco-evolutionary dynamics of plasticity by demonstrating context-dependent costs under real-world thermal stress.

evolutionary biology↗

bioRxiv2025

The role of disulfide bonds in the GluN1 subunit in the early trafficking and functional properties of GluN1/GluN2 and GluN1/GluN3 NMDA receptors

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for excitatory neurotransmission. Previous studies proposed the existence of four disulfide bonds in the GluN1 subunit; however, their role in NMDAR trafficking remains unclear. Our study first confirmed the existence of four disulfide bonds in the GluN1 subunit using biochemistry in human embryonic kidney 293T (HEK293T) cells. Disrupting the individual disulfide bonds by serine replacements produced the following surface expression trend for GluN1/GluN2A, GluN1/GluN2B, and GluN1/GluN3A receptors: wild-type (WT) > GluN1-C744S-C798S > GluN1-C79S-C308S > GluN1-C420S-C454S > GluN1-C436S-C455S subunits. Electrophysiology revealed altered functional properties of NMDARs with disrupted disulfide bonds, specifically an increased probability of opening (Po) at the GluN1-C744S-C798S/GluN2 receptors. Synchronized release from the endoplasmic reticulum confirmed that disruption of disulfide bonds impaired early trafficking of NMDARs in HEK293T cells and primary hippocampal neurons prepared from Wistar rats of both sexes (embryonic day 18). The pathogenic GluN1-C744Y variant, associated with neurodevelopmental disorder and seizures, caused reduced surface expression and increased Po at GluN1/GluN2 receptors, consistent with findings for the GluN1-C744S-C798S subunit. The FDA-approved memantine inhibited GluN1-C744Y/GluN2 receptors more potently and with distinct kinetics compared to WT GluN1/GluN2 receptors. We also observed enhanced NMDA-induced excitotoxicity in hippocampal neurons expressing the GluN1-C744Y subunit, which memantine reduced more effectively compared to the WT GluN1 subunit. Lastly, we demonstrated that the presence of the hGluN1-1a-C744Y subunit counteracted the effect of the hGluN3A subunit on decreasing dendritic spine maturation, consistent with the reduced surface delivery of the NMDARs carrying this variant. Significance statementOur findings highlight the critical role of disulfide bonds in the GluN1 subunit in regulating trafficking and function of major conventional (GluN1/GluN2A, GluN1/GluN2B) and unconventional (GluN1/GluN3A) diheteromeric NMDAR subtypes in the postnatal forebrain. We further demonstrated that the pathogenic GluN1-C744Y variant reduces surface expression of all studied NMDARs, as well as increases the probability of opening (Po) of the GluN1/GluN2 receptors, leading to heightened NMDA-induced excitotoxicity in hippocampal neurons. Additionally, we introduced an ARIAD-based system for the synchronized release of NMDARs from the endoplasmic reticulum in hippocampal neurons. This system provides a powerful tool for studying pathogenic GRIN gene variants and addresses the current lack of molecular methods for analyzing the early trafficking of NMDARs.

neuroscience↗

bioRxiv2025

Few-Shot Classification of Cryo-EM Micrographs Using Triplet Loss Embeddings

Micrograph quality assessment in cryo-electron microscopy (cryo-EM) presents a significant challenge: users must either manually screen thousands of micrographs or expend substantial computational resources processing potentially low-quality data. While few-shot learning has been applied to particle picking and subtomogram classification in cryo-EM, its application to micrograph-level quality assessment remains largely unexplored. We present a framework combining few-shot learning with cryo-EM micrograph classification using triplet loss embeddings. By combining real-space and Fourier-space information in an embedding network trained with triplet loss, we achieve competitive performance across multiple EMPIAR datasets using as few as 1-5 labeled examples per class. Our approach demonstrates improvements over traditional cross-entropy training, which often collapses to predicting only the majority class in the few-shot regime. These results suggest a practical framework for rapid adaptation of automated micrograph screening to new experimental conditions with minimal manual labeling.

biophysics↗

bioRxiv2025

Succinate Dehydrogenase-Deficient Cancer Cells Have Increased Susceptibility to Ym155 Induced DNA Damage

The hereditary pheochromocytoma and paraganglioma (hPPGL) syndrome is caused by inherited mutations in Succinate Dehydrogenase genes (SDHx). Affected individuals are predisposed to developing pheochromocytomas (Pheo), paragangliomas (PGL), renal cell carcinoma (RCC) and gastrointestinal stromal tumors (GIST). Notably, tumors with succinate dehydrogenase subunit B (SDHB) deficiency demonstrate increased metastatic risk, for which treatments remain palliative. Hence, discovering novel therapeutic avenues to improve the prognosis for SDHB-cancer patients is an urgent need. Here we employed human SDHB-deficient UOK269 RCC cells (SDHB-KO) and isogenic SDHB-reconstituted control cells (SDHB-WT) to discover SDH-dependent mitochondria-directed cytotoxic agents. Given the reduced ATP-generating capacity of SDHB-KO cells, we hypothesized they would be uniquely sensitive to futile cycle induction with mitochondrial ionophores (2,4-Dinitrophenol (2-DNP), BAM15, Niclosamide, Nitazoxanide). Indeed, these compounds exhibited preferential cytotoxicity toward SDHB-KO cells. However, the chemotherapeutic compound Ym155 demonstrated the most potent and dramatic (five-fold) preferential cytotoxicity towards SDHB-KO cells. Importantly, the SDH-dependent cytotoxicity of Ym155 was validated in both primary human pheochromocytoma cells and mouse pheochromocytoma (MPC) cells. Furthermore, because few SDH-deficient cell lines are available, we buttressed our findings in additional relevant cell lines by modeling SDH-deficiency using chemical SDH enzyme inhibition with 3-nitropropionic acid (3-NPA). We observed a persistent cooperativity between SDH-deficiency and Ym155 cytotoxicity across multiple cell lineages and disease models. Mechanistically, Ym155-induced cytotoxicity was independent of its primary target, Survivin. Instead, SDH-deficiency sensitized cells to Ym155-induced DNA damage. Strikingly, the phenotype of SDH-deficient Ym155 sensitivity was recapitulated by inhibition of the histone demethylase KDM4, a downstream consequence of SDH deficiency. Thus, the accumulation of succinate in SDH-deficient tumors inhibits KDM4 activity, impairs DNA repair and yields enhanced susceptibility to Ym155-induced reactive oxygen species (ROS) generation. The identified intrinsic susceptibilities of SDHB-deficient cancers has the potential to be therapeutically leveraged.

cancer biology↗

bioRxiv2025

Is synuclein aggregation a derived or ancestral trait? Ancestral sequence reconstruction uncovers stepwise evolution of synuclein aggregation

Protein aggregation drives many neurodegenerative diseases, including Parkinsons disease, where misfolded -synuclein (Syn) forms fibrillar assemblies that accumulate as Lewy bodies. Although Syn aggregation has been extensively characterized, its evolutionary origins and sequence determinants remain unresolved. Here, we use ancestral sequence reconstruction (ASR) to trace the emergence of fibril-forming ability in the synuclein family. We inferred synuclein phylogeny and experimentally resurrected common ancestors, including ROOT synuclein, the last common ancestor of all synucleins, and key intermediates along the Syn lineage. Strikingly, ROOT synuclein is non-aggregating, demonstrating that fibril formation is an evolved, rather than ancestral property. Aggregation first emerges at the ancestral {beta} node, is retained in Syn, and suppressed in {beta}-synuclein. Biophysical analyses including mass spectrometry and NMR reveal that aggregation aligns with greater complexity and heterogeneity in the monomer conformational ensemble, suggesting that evolutionary sequence changes progressively remodel monomer landscapes to favor fibril formation. Complementing these insights, comparative sequence analysis reveals that the transition from ROOT to -WT is marked by the stepwise acquisition of residues critical for stabilizing the fibril core. Early mutations stabilized the {beta}-arch core, enabling the onset of fibril formation, followed by substitutions that reinforce protofilament-protofilament interactions. Together, ASR defines an evolutionary framework for synuclein aggregation linking progressive sequence evolution and conformational complexity to the molecular origins of Syn fibril formation.

biophysics↗

bioRxiv2025

E2F1 induces a G0-G1 reentry transcriptional program without changing chromatin accessibility

Quiescent cells actively repress cell-cycle genes via chromatin-based mechanisms to maintain a non-dividing state, yet remain poised to reenter upon stimulation. E2F1, a canonical activator of cell-cycle genes, is sufficient to induce reentry from quiescence, but how it overcomes chromatin-mediated repression remains unclear. Here, we show that inducible E2F1 expression triggers exit from quiescence and progression through the cycle without changes in chromatin accessibility, by harnessing regulatory elements with limited, pre-existing accessibility. Using time-resolved transcriptomics, we demonstrate that E2F1 induces an accelerated transcriptional program compared to serum. Unlike serum, which triggers broad chromatin remodeling, E2F1-induced activation occurs in a context of limited accessibility. ChIP-seq reveals that E2F1 directly binds target sites in quiescent cells to upregulate canonical genes. Biochemical reconstitution shows that E2F1 binds nucleosomes and accesses internal E2F sites within histone-wrapped DNA. These findings suggest that E2F1 can engage nucleosome-associated DNA and initiate transcription without major chromatin reorganization, redefining transcription factor-chromatin dynamics during cell fate transitions and establishing E2F1 as a potent regulator of cell-cycle reentry.

cell biology↗

bioRxiv2025

Non-integrating direct reprogramming generates therapeutic endothelial cells with sustained vascular regeneration capacity enhanced by nanomatrix delivery

RationaleDirect reprogramming of fibroblasts into endothelial cells (rECs) using ETV2 shows promise for vascular regeneration. However, current approaches using integrating viral vectors pose clinical translation barriers, and poor long-term cell survival limits therapeutic efficacy. ObjectiveTo develop a clinically compatible method for generating rECs using non-integrating adenoviral ETV2 (Ad-ETV2) and enhance their engraftment and therapeutic efficacy through peptide amphiphile (PA) nanomatrix encapsulation. Methods and ResultsHuman dermal fibroblasts were reprogrammed using Ad-ETV2 and characterized by flow cytometry, RNA sequencing, and functional assays. Therapeutic efficacy was evaluated in murine hindlimb ischemia with or without PA-RGDS encapsulation over 12 months. Ad-ETV2 induced robust endothelial gene expression (CDH5, KDR, PECAM1) within 6 days, with 40-50% reprogramming efficiency. KDR+ Ad-rECs demonstrated functional endothelial properties including Ac-LDL uptake, tube formation, and exceptional proangiogenic factor secretion (200-fold higher HGF than HUVECs). RNA sequencing revealed rapid transcriptional reprogramming with fibroblast gene suppression and endothelial/angiogenic gene activation. In hindlimb ischemia, Ad-rECs significantly enhanced blood flow recovery and capillary density versus controls. Long-term analysis revealed sustained vascular contribution through three mechanisms: direct incorporation, perivascular support, and vessel guidance, persisting throughout 12 months--the longest reported follow-up for reprogrammed cells. PA-RGDS encapsulation markedly improved cell retention; while 75% of cells were lost by 3 months, retention stabilized thereafter with minimal additional loss through 12 months. ConclusionsAdenoviral ETV2 delivery enables efficient generation of clinically compatible rECs without genomic integration. These cells demonstrate potent and sustained therapeutic efficacy through multiple vascular regeneration mechanisms. PA-RGDS encapsulation significantly enhances long-term engraftment, establishing this combined approach as a promising platform for treating ischemic cardiovascular diseases.

cell biology↗

bioRxiv2025

Proteolytic cleavage of G3BP1 by calpain 1 couples NMDAR activation to mTOR-dependent local translation

Ribonucleoprotein (RNP) granules are dynamic, membraneless organelles that sequester translationally repressed mRNAs and RNA-binding proteins, playing a pivotal role in the regulation of localized protein synthesis. While disassembly of RNP granules is essential for reactivating translation, the mechanisms by which neuronal activity regulates this process remain poorly understood. In this study, we show that stimulation of N-methyl-D-aspartate (NMDA) receptor (NMDAR) triggers calcium influx, leading to activation of calpain 1 and subsequent proteolytic cleavage of Ras-GTPase-activating protein binding protein 1 (G3BP1), a core component of stress granules. This cleavage results in the disassembly of G3BP1 granules in the neurites and promotes mTOR-dependent local translation, thereby linking synaptic activity to spatially restricted protein synthesis. Finally, we demonstrate that the NMDAR-calpain 1-G3BP1-mTOR signaling axis contributes to axonal regeneration, establishing proteolytic remodeling of RNP granules as a key mechanism of activity-dependent neural repair, with potential implications for therapeutic intervention in brain injury.

neuroscience↗

bioRxiv2025

Microbial oral-gut translocation in advanced chronic liver disease is linked to exacerbation of intestinal barrier dysfunction and hepatic fibrosis

While microbiome perturbations are associated with advanced chronic liver disease (ACLD), microbial disease mechanisms are poorly understood. Using multi-omics analyses of paired saliva and faecal samples from an ACLD cohort, we identified next-to-identical oral and gut bacterial strains (including Veillonella and Streptococcus spp.) which increased in absolute abundance in the gut of ACLD patients. These translocators uniquely encoded a collagenase-like proteinase (prtC) with the potential for gut barrier disruption and prtC faecal abundance was a robust ACLD biomarker (auPR=0.91). CCl4-treated mice inoculated with Veillonella and Streptococcus prtC-encoding patient isolates showed exacerbation of gut barrier impairment and hepatic fibrosis. Furthermore, faecal collagenase activity was increased in ACLD patients and experimentally confirmed for the prtC gene from translocating Veillonella parvula. Overall, our study establishes mechanistic links between oral-gut translocation and ACLD pathobiology, and identifies the oral microbiome as an important contributing factor with potential for microbial diagnostics and therapeutics.

microbiology↗

bioRxiv2025

Effective allogeneic natural killer cell therapy for pancreatic adenocarcinoma avails conserved activating receptors and evades HLA I-driven inhibition

BackgroundAt diagnosis, [~]80% of pancreatic ductal adenocarcinomas (PDAC) have metastasized. Relapse is thus common even among patients that undergo surgical resection, the only curative option. PDAC progresses rapidly, and existing immunotherapies have been ineffective. We hypothesized that natural killer (NK) cell immunotherapies could be effective against PDAC because they recognize conserved and heterogeneous features associated with cellular stress and transformation, and can seek out metastases distal to the primary tumour site. Here, we aim to define the key features of NK cells as effective agents for PDAC immunotherapy. MethodsWe used TCGA PDAC Firehose data and flow cytometry to predict and measure the most common activating or inhibitory ligands available on PDAC for NK cell activation. To ascertain how the tumour might alter expression of these ligands during treatment, inflammation or immune pressure, we measured expression of NK ligands at rest, or after exposure to immune cells or inflammation. To test and rank the functional importance of these dynamic ligands in the recognition, killing and control of PDAC< we used co-culture, antibody-blocking and an NK-competent humanized mouse model. ResultsLeveraging the known sequential acquisition of mutations as a surrogate for disease progression, we observed a progressive loss of transcript expression for activating NK cell ligands and chemoattractants. Exposure of PDAC to NK cells or IFN-{gamma}, an inflammatory stimulus, drove dynamic changes in expression of both activating and inhibitory ligands. In vitro co-culture assays revealed a redundancy in the activating receptors engaged in NK:PDAC interactions, but that HLA-KIR signalling dominantly interrupted anti-PDAC activity. In NK-competent humanized mice, adoptively transferred, unselected, unmodified NK cells slowed tumour growth in a dose-dependent manner, but NK cells selected to avoid HLA I-driven inhibition were the most competent effectors for PDAC control. ConclusionsAlthough there is redundancy among activating ligand:receptor pairs for recognizing PDAC tumours, but interactions between KIR and HLA define the extent to which anti-tumour activity can proceed. During tumour progression, and in response to immunotherapy. NK:tumour interactions drive upregulation of HLA I molecules. Thus, educated NK cells from HLA I-disparate donors may be the most effective allogeneic NK immunotherapy for PDAC. What is already known on this topicNK cells can be safely transferred across allogeneic barriers, so allogeneic therapy is possible. Since PDAC tumours progress very rapidly, there is insufficient time for engineered cell therapies. Although PDAC is typically considered to be an immunologically "cold" tumour, more recent studies have revealed that sub-tumour microenvironments can contain clusters of immune cells, and that the presence of immune cells in PDAC is associated with good prognosis. What this study addsWe explore how the naturally-occurring heterogeneity of activating and inhibitory receptor expression with and between individuals impacts recognition of PDAC tumour cells. We find that the natural cytotoxicity receptors and NKG2D are among the most likely activating receptors to be expressed on NK cells responding to PDAC. However, interactions between inhibitory killer immunoglobulin-like receptors (KIR) and class I human leukocyte antigens (HLA) dominantly inhibit NK cell killing. To enable NK cell reactivity without the liability of inhibition, we demonstrate that NK cells can be selected intentionally from allogeneic HLA-mismatched donors, where they retain programmed functionality, but are ignorant to the HLA-driven signals for inhibition present on the tumour cells. How this study might affect research, practice or policyDiversity among NK cell functions is driven by interactions between HLA I molecules and KIR, with the remaining receptor-ligand partnerships mostly conserved across people. KIR:HLA I interactions are predictable, and there is a limited range of potential combinations, so definitions of key functions would empower mass-production of NK cells from a limited range of healthy donors that could be used as off-the-shelf cellular immunotherapy. Our study provides key exclusion criteria that will inform these selections. Lee Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/677560v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@f6ed8forg.highwire.dtl.DTLVardef@1667904org.highwire.dtl.DTLVardef@9330ccorg.highwire.dtl.DTLVardef@168c85a_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Separable roles for Microprocessor and its cofactors ERH and SAFB1/2 during microRNA cluster assistance

While most conserved microRNA (miRNA) transcripts harbor a suite of features that mediate their efficient biogenesis into small RNAs, some loci bear suboptimal attributes that enable additional layers of processing regulation. A notable example is cluster assistance, whereby a miRNA hairpin with suboptimal nuclear biogenesis can be enhanced by an optimal neighbor. This process involves local transfer of the Microprocessor complex, composed of the RNase III enzyme Drosha and its partner DGCR8, in concert with cofactors such as ERH and SAFB1/2. However, the mechanism(s) that underlie miRNA cluster assistance remain largely unclear. Here, we gain insights into this process by integrating mutant cells of Microprocessor and its cofactors with analysis of miRNA structure-function variants, biochemical tests and genomewide profiling. We define features of suboptimal miRNAs that render them dependent on cluster assistance, and distinguish amongst a network of proposed interactions amongst Microprocessor and its cofactors, to reveal a subset that are critical for cluster assistance. Most importantly, we use epistatic tests to separate and order the functional requirements for ERH and SAFB1/2 into a pathway. Our data indicate that ERH may engage in the process of Microprocessor transfer between hairpins, while SAFB factors (especially SAFB2) mediate recognition and stable binding of a suboptimal miRNA hairpin after Microprocessor transfer. Finally, we show how cluster assistance integrates into a feedback regulatory loop on Microprocessor, via Drosha-mediated cleavage of a suboptimal miRNA hairpin in the DGCR8 transcript. Altogether, our findings reveal complex regulatory transactions during biogenesis of clustered miRNAs.

molecular biology↗

bioRxiv2025

Apical extracellular matrix regulates fold morphogenesis in the Drosophila wing disc

Tissue folding is a fundamental process occurring often in animal organ development. Here, we study the progression of fold shape and the underlying mechanics in the development of the Drosophila wing disc. We present a 3D segmentation of the apical surface of the wing disc proper from larval stages, when folds grow, to early pupariation, when the tissue unfolds and remodels into a bilayer. We establish morphological metrics to quantify and resolve fold shape in this dataset, introducing a definition of fold depth and width that can be used to characterize folds on a curved surface. Furthermore, we identify fibrous extracellular matrix on the apical side (aECM) that physically connects the two opposing sides of the folds. By modeling a tissue fold with a lateral vertex model endowed by an adhesive layer representing the aECM, we predict that unfolding in the wing disc is preceded by the removal of aECM. Using genetic perturbations, we confirm that aECM adhesion affects fold stability and mechanics: loss of aECM leads to abnormal fold shape and unfolding dynamics, whereas failure to remove aECM at pupariation inhibits unfolding. Finally, we show that these aECM perturbations in larval stages cause morphological phenotypes in the adult wing, demonstrating that the fold morphology of the wing disc helps to define adult wing shape. In total, our work establishes a key mechanical role for aECM in wing disc growth and morphogenesis and advances our general understanding of how epithelial tissue folds can be mechanically stabilized during development.

developmental biology↗

bioRxiv2025

A predictive framework for stop-loss variants with C-terminal extensions

Stop codons dictate translation termination, and variants occurring at these sites can result in stop-loss variants, leading to C-terminal extensions with potentially significant functional consequences. Despite their clinical relevance, existing prediction tools--primarily developed for missense variants--lack sufficient accuracy for assessing stop-loss variants, mainly due to their insufficiency in accounting for the sequence features of the extended peptide. To address this gap, we developed TAILVAR (Terminal codon Analysis and Improved prediction of Lengthened VARiants), a machine-learning classifier that integrates multi-omics features spanning transcript- and protein-level properties, along with variant effect annotations. Our analyses showed that transcripts lacking downstream stop codons in the 3 untranslated region exhibit lower evolutionary constraints. Additionally, we observed that deleterious variants exhibit greater C-terminal hydrophobicity, which is associated with reduced protein stability and increased degradation, as well as a higher aggregation propensity. TAILVAR outperformed existing benchmarks, demonstrating the highest correlation with functional experiments and establishing thresholds to classify variants as benign or pathogenic. This work offers a systematic framework for interpreting stop-loss variants, providing precise predictions of elongated protein effects that may aid genetic diagnosis and facilitate the discovery of novel disease-associated genes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/673407v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1d53baaorg.highwire.dtl.DTLVardef@403289org.highwire.dtl.DTLVardef@b5c8b4org.highwire.dtl.DTLVardef@8bd61a_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2025

Plant MutS2 proteins function in plastid ribosome quality control

Tight regulation of chloroplast translation is essential for plant growth, development and environmental response. Active translation can result in stalling and collision of ribosomes, which have negative fitness consequences. However, the ways in which chloroplasts respond to these types of translational stressors remain unknown. Here, we identify two MutS2 proteins that act as critical players in plastid ribosome-associated quality control (RQC) in Arabidopsis. We found that both MutS2A and MutS2B are required to overcome specific antibiotic-induced ribosome stalling and collisions. Further, these proteins appear to be essential for tissue greening during de-etiolation, potentially due to increased translational demand during the transition from etioplast to chloroplast. Although bacterial homologs of MutS2 have been widely recognized for their role in regulating homologous recombination, we found only weak support for this function in Arabidopsis plastids. Therefore, these proteins, which are widely conserved among photosynthetic eukaryotes, appear to be central in the resolution of ribosome collisions and may play a critical role during times of increased translational demand.

plant biology↗

bioRxiv2025

Enhanced Neutralization of Japanese Encephalitis Virus Using an Engineered VL Double-Domain

Japanese encephalitis virus (JEV) is an emerging virus responsible for thousands of deaths in Asia; however, an effective treatment has not yet been discovered. JEV envelope (E) proteins play important roles in viral survival and infection. In this study, we isolated scFv- and VL-targeting E proteins using phage display biopanning. The isolated VL was engineered to form VL double domains to increase its neutralization activity. Several candidates showed binding abilities, whereas only the DE2 VL double-domain treatment showed neutralizing activity against JEV. DE2 was also highly specific to JEV and did not bind to other flaviviruses. Immunocytochemistry results showed that DE2 was co-localized with the endosome 4 h post-infection. Thus, the mechanism of DE2 neutralization involves blocking the E protein from fusing with the endosomal membrane, resulting in the unrelease of the viral genome into the cytoplasm. Docking analysis showed that DE2 was bound to three domains, especially DIII, of the JEV E protein. In contrast, other scFv that were unable to bind to DIII showed no or low neutralizing activity. DE2 was also predicted to neutralize all the JEV genotypes. In conclusion, a combined in vitro and in silico study showed the high potential of DE2 to neutralize JEV infections, which may be applied in future therapeutic systems. ImportanceJapanese encephalitis virus (JEV) is a serious mosquito-borne disease that causes brain infections and deaths across Asia. However, there is still no specific treatment once infection occurs. In this study, we developed a new type of antibody fragment, called a VL double-domain, that can block the virus. Among several candidates, DE2 showed a strong ability to stop JEV from infecting cells, while others could bind to the virus but did not block infection. Our findings reveal that DE2 works by attaching to a key region of the virus, preventing it from releasing its genetic material. Moreover, DE2 is predicted to work against all known JEV strains, highlighting its potential as a universal treatment option. This research provides an important step toward developing new antiviral therapies for JEV and possibly for other viruses with similar infection mechanisms.

microbiology↗

bioRxiv2025

HEIP1 orchestrates pro-crossover protein activity during mammalian meiosis

Meiotic crossovers are needed to produce genetically balanced gametes. In mammals, crossover formation is mediated by a conserved set of pro-crossover proteins via mechanisms that remain unclear. Here, we characterize a mammalian pro-crossover factor HEIP1. In mouse HEIP1 is essential for crossing over and fertility of both sexes. HEIP1 promotes crossing over by orchestrating the recruitment of other pro-crossover proteins, including the MutS{gamma} complex (MSH4- MSH5) and E3 ligases (HEI10, RNF212, and RNF212B), that are required to mature crossover sites and recruit the crossover-specific resolution complex MutL{gamma}. Moreover, HEIP1 directly interacts with HEI10, suggesting a direct role in controlling the recruitment of pro-crossover E3 ligases. During early stages of meiotic prophase I, HEIP1 interacts with the chromosome axes, independently of recombination, before relocalizing to the central region of the synaptonemal complex. We propose that HEIP1 is a new conserved master regulator of crossover proteins that controls different crossover maturation steps. Significance StatementCrossovers are essential to produce gametes by promoting the proper segregation of the homologous chromosomes. But, if misregulated, they can lead to genetic disorders, miscarriage and infertility. Their formation depends on the conserved pro-crossover factors, which repertoire is expanding and mode of action tightly regulated. This study highlights how the HEIP1 protein organizes pro-crossover protein activities in the mouse. Our findings show that HEIP1, by interacting early with chromosomes independently of recombination initiation, and orchestrating the recruitment of pro-crossover factors, including the MutSg complex and the RING proteins HEI10, RNF212 and RNF212B, is pivotal in this regulation. Our work is of significance to unravel crossover control during meiotic recombination, a conserved mechanism essential for gametes formation.

genetics↗