Biology subjects

Feng, M.

Publications and source records attributed to Feng, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Domain-resolved epitope mapping of B7-H3-targeting VHH-Fcs defines isoform-selective binders for antibody-drug conjugate development

The immune checkpoint molecule B7-H3 has emerged as one of the most actively pursued therapeutic targets in solid tumors, and multiple B7-H3-targeting antibody-drug conjugates have entered clinical evaluation. B7-H3 occurs as a 2Ig isoform carrying a single IgV-IgC domain pair and a predominant 4Ig isoform carrying two tandem pairs of near-identical sequence in their extracellular domains. Resolving which pair an antibody engages, or how antibodies distinguish the two isoforms, requires epitope mapping at residue level. Here we generated a panel of six B7-H3-targeting VHH-Fcs and characterized them by surface plasmon resonance, epitope binning, domain-resolved deep mutational scanning and receptor internalization, benchmarked against the clinically advanced antibodies ifinatamab, mirzotamab and vobramitamab. Most VHH-Fcs bound the membrane-proximal pair more tightly than the distal pair, and 4Ig 10- to 100-fold more tightly than 2Ig, whereas the three clinical antibodies showed no such discrimination. Displaying each domain pair independently on yeast identified the residues that account for this selectivity. Internalization varied several-fold among binders, including between VHH-Fc 620 and 636, which have near-identical footprints and comparable affinities. VHH-Fc 620 internalized most efficiently, also compared to ifinatamab, and was conjugated to exatecan through a protease-cleavable linker. The conjugate showed B7-H3-dependent cytotoxicity that scaled with B7-H3 cell surface density, though it was less potent than ifinatamab-exatecan. Affinity, epitope and internalization therefore behaved as independent characteristics rather than integrating to predict antibody-drug conjugate potency. Together, these results define the molecular basis for isoform-selective recognition of B7-H3 and identify VHH-Fc 620 as a starting point for isoform-selective targeting.

cancer biology↗

bioRxiv2026

Epithelial to mesenchymal transition and M2 macrophages drive aggressiveness in murine and human sarcomatoid urothelial carcinoma.

Patients with majority variant subtype histology including sarcomatoid urothelial carcinoma (SUC) have poor outcomes and limited treatment options, and there are few preclinical resources to investigate rare histologic subtypes. We developed three novel variant subtype histology bladder cancer (BC) cell lines by finding that basal BC tumor cells derived from mice treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) were initially not competent to grow in immune competent mice, but then acquired tumorigenicity following passaging (recycling) in mice lacking mature B and T cells. Three genitourinary oncology pathologists identified BBN966 as SUC, BBN964 as a hybrid between SUC and squamous, and BBN975 as squamous, and each had unique growth characteristics. Using a publicly available dataset and our spatial transcriptomics analysis of a cohort of human tumors with mixed SUC and UC as comparators, we found that consistent with human SUC, BBN966 lack expression of basal and luminal genes. Consistent with human SUC and spatial transcriptomics analysis of canine SUC, BBN966 had the highest epithelial to mesenchymal transition (EMT) upregulation, and all three recycled tumor cells had increased EMT pathway expression in recycled tumor cells. Moreover, increased expression of genes upregulated in all three recycled tumor cells associated with worse overall survival in patients with muscle invasive UC. Similar to human SUC, BBN966 tumors had higher proportion of M2 macrophage gene expression. Finally, we found in the largest cohort of PD-L1 IHC to date that 20 of 38 (52.6%) human SUC had CPS > 50, which was driven mostly by tumor proportion score. Consistent with this, we found anti-PD1 immune checkpoint inhibitor (ICI) significantly inhibited BBN966 tumor growth, while the hybrid BBN964 tumors had mixed response to ICI. Thus, the murine BBN966 tumors exhibited similarities to human SUC, and can be used to examine novel therapies for translation into future SUC clinical studies.

cancer biology↗

bioRxiv2026

Quantifying Cross-Modal Shared Information Between Histomorphology and Spatial Transcriptomics via Spatiotemporal Trajectory Correlation

Histopathological imaging and spatial transcriptomics (ST) provide synergistic morphological and molecular insights into tissue architecture. While conventional downstream analyses predominantly adopt a discrete paradigm, such as segmenting histological images or identifying spatial domains, emerging trajectory reconstruction methods offer a continuous perspective for analyzing these modalities. However, most current studies are confined to single-modality or single-organ analyses, lacking systematic integration across modalities and multiple organs. To address this limitation, we performed trajectory reconstruction on ST-derived gene expression data and on histopathological morphological features extracted using ten widely used pathology pretrained models across multiple cancer samples from six organs. The results demonstrate that trajectory reconstruction, as a continuous analytical framework, effectively bridges spatial transcriptomics and histopathological imaging. More importantly, we propose an innovative framework that uses trajectory pseudotime as a mediating variable to quantify the extent of information sharing between molecular and morphological features. This framework not only provides a new perspective for understanding the intrinsic links between modalities but also establishes a solid theoretical and methodological foundation for future cross-modal translation studies.

pathology↗

bioRxiv2026

Combined Checkpoint Inhibition Amplifies Post-Infarction Injury via T Cell-Mediated Macrophage Activation

BackgroundThis study aimed to investigate whether combined PD-1/CTLA-4 immune checkpoint inhibition predisposes the heart to a hyperinflammatory state, thereby exacerbating cardiac injury following acute myocardial infarction (MI), a critical unresolved question in cardio-oncology. MethodsMyocardial infarction was induced in Pd1-/-Ctla4+/- mice, a genetic model mimicking combined checkpoint inhibition. Key mechanistic insights were gained through in vivodepletion of CD8+ T cells (using anti-CD8a antibody) and pharmacological inhibition of the JAK-STAT1 pathway (using Tofacitinib). Cardiac function, structural injury, and immune responses were comprehensively assessed via echocardiography, flow cytometry, immunofluorescence, and molecular analyses. ResultsCompared to wild-type controls, Pd1-/-Ctla4+/- mice exhibited significantly increased post-MI mortality, worse cardiac function, and larger infarct size. Mechanistically, the aggravated injury was driven by an amplified infiltration of activated, IFN-{gamma}-producing CD8+ T cells, which activated the JAK-STAT1 pathway in macrophages, polarizing them towards a pro-inflammatory state. Depleting CD8+ T cells or inhibiting the JAK-STAT1 pathway effectively attenuated macrophage-driven inflammation and improved all aspects of post-MI injury. ConclusionsCombined PD-1/CTLA-4 blockade exacerbates post-infarction cardiac injury by promoting CD8+ T cell-mediated activation of macrophages via the JAK-STAT1 axis. This work elucidates MI as a context-dependent immune-related adverse event in ICI therapy and identifies CD8+ T cells and the JAK-STAT1 pathway as promising therapeutic targets for cardioprotection in these patients. RESEARCH PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABSO_LIThis study identifies acute myocardial infarction (MI) as a potential, context-dependent immune-related adverse event in the setting of combined PD-1/CTLA-4 checkpoint inhibition, shifting the paradigm beyond the classic focus on myocarditis. C_LIO_LIIt elucidates a novel pathogenic axis where combined checkpoint deficiency exacerbates post-MI injury specifically through CD8+ T cell-derived IFN-{gamma}, which activates macrophages via the JAK-STAT1 pathway. C_LI What Question Should Be Addressed Next?O_LIFuture studies should employ anti-PD-1/CTLA-4 monoclonal antibodies in wild-type or humanized mouse models to validate findings and better recapitulate the pharmacokinetics of clinical ICI therapy, strengthening translational relevance. C_LIO_LIThe long-term consequences of this primed inflammatory state on chronic cardiac remodeling, heart failure development, and the potential interplay with atherosclerosis warrant further investigation. C_LI

immunology↗

bioRxiv2026

Sphk1-S1P signaling drives blood-brain barrier breakdown after intracerebral hemorrhage via HIF-1α-dependent upregulation of Bsg-MMP-9

Blood-brain barrier (BBB) breakdown is a critical pathological event driving secondary brain injury and poor outcomes following intracerebral hemorrhage (ICH). However, the mechanisms governing acute BBB breakdown after ICH remain incompletely understood. Here we demonstrate that the Sphk1-S1P-S1PR3 signaling plays a pivotal role in this process. Sphk1 expression was significantly upregulated in the perihematomal endothelial cells of both ICH patients and mice, with levels positively correlating with BBB dysfunction severity and poor clinical outcomes. Using endothelial-specific genetic gain- and loss-of-function approaches, we found that Sphk1 knockdown attenuated BBB leakage, reduced hematoma volume and brain edema, preserved tight junction integrity, and improved neurological function at 1-day post-ICH, whereas Sphk1 overexpression exacerbated these pathological features. Mechanistically, transcriptomic profiling of perihematomal endothelial cells revealed that prior to its established role in Nlrp3-mediated pyroptosis, Sphk1 promotes early BBB breakdown by regulating the Bsg-MMP-9 axis. Endothelial-specific Bsg deletion completely abrogated the deleterious effects of Sphk1, confirming Bsg as an indispensable intermediary through which Sphk1 signals to MMP-9. ATAC-seq and dual-luciferase assays further demonstrated that Sphk1-generated S1P signals through S1PR3 to activate HIF-1, which directly binds the Bsg promoter to drive its transcription, ultimately promoting MMP-9-mediated tight junction degradation. These findings delineate a complete hierarchical signaling cascade from metabolic enzyme to transcriptional regulation and subsequent barrier injury, establishing the Sphk1-Bsg-MMP-9 axis as a promising therapeutic target for ICH. One sentence summaryThis work identifies an early and pivotal mechanism of blood-brain barrier breakdown after intracerebral hemorrhage, demonstrating that Sphk1-generated S1P signals through S1PR3 to activate HIF-1, which directly transactivates Bsg expression, leading to MMP-9-mediated tight junction degradation, thereby establishing a novel hierarchical axis with therapeutic potential.

cell biology↗

bioRxiv2026

Multispecific nanobody degraders co-deplete membrane receptors and enable targeted delivery of diverse payloads

Targeting membrane receptors underlies the success of antibody-drug conjugates (ADCs), yet single-receptor formats can be limited by heterogeneous expression, compensatory signaling, and variable internalization. Here we developed Multivalent Interchangeable Nanobody Degradation System (MINDS), a modular nanobody-Fc chassis that co-engages multiple membrane receptors, promotes their lysosomal co-depletion, and enables delivery of diverse intracellular payloads. As a proof of concept, we generated Tritazumab, a trispecific nanobody-Fc targeting three oncogenic receptors EGFR, cMET, and TfR1. Tritazumab incorporates a high-affinity, non-transferrin-competing anti-TfR1 nanobody that drives efficient uptake and lysosomal trafficking, enabling coordinated depletion of all three receptors. Across non-small cell lung cancer models, Tritazumab achieved rapid and sustained multi-receptor surface loss with picomolar degradation potency, reaching near-maximal depletion within approximately 1.5 hours. Conjugation of Tritazumab to MMAE preserved receptor binding and produced substantially greater antiproliferative activity and improved tumor selectivity relative to clinical ADCs in matched cell models, along with potent in vivo tumor growth inhibition and acceptable tolerability in a xenograft model. Extending the platform beyond cytotoxic payloads, a BRD4 molecular glue conjugate improved the selectivity window by > 100-fold and showed marked in vivo efficacy, while an EZH2-targeting PROTAC conjugate achieved an approximately 1,000-fold increase in intracellular degradation potency relative to the free PROTAC. These findings establish MINDS as a modular multispecific degrader-payload platform that integrates receptor co-depletion to enhance anticancer selectivity and efficacy.

cancer biology↗

bioRxiv2026

Mechanism of GPR84 allosteric modulation at a helix 8-proximate site

Allosteric modulators offer opportunities for pathway-selective GPCR signalling, but the structural mechanisms enabling biased allosteric modulation remain unclear. Here we identify a helix 8-proximate allosteric site in the immune-metabolic receptor GPR84 and define how it achieves Gi -biased signalling. Cryo-EM structures of the GPR84-Gi complexes bound to the orthosteric agonist OX04539 alone or in combination with the positive allosteric modulator (PAM) PSB-16671 reveal that PSB-16671 binds at the interface of TM1, TM7, and helix 8, a location distinct from previously characterized GPCR allosteric pockets. Molecular dynamics simulations and mutagenesis uncover a polar interaction network linking orthosteric and allosteric sites through conserved residues including Asp662.50, Asn1043.36, and Asn3627.45. Unexpectedly, disrupting this network enhances allosteric cooperativity, indicating that conformational flexibility within the network is essential for allosteric communication. PSB-16671 stabilizes a receptor conformation with pronounced TM6 displacement that favours Gi coupling while disfavouring {beta}-arrestin recruitment. This Gi-biased profile sustains macrophage phagocytosis of cancer cells without the desensitization induced by balanced agonists. Sequence analysis suggests that helix 8-proximate allosteric sites may be broadly targetable across class A GPCRs, while receptor-specific contacts enable selective modulation. These findings establish structural and mechanistic principles for biased allosteric modulation applicable beyond GPR84.

biochemistry↗

bioRxiv2025

Unraveling the archaeal virosphere: diversity, functional and virus-host interactions

Archaea, the third domain of life, play critical roles in global biogeochemical cycles. However, their virosphere, particularly the proviruses which integrated into host genomes, remains largely unexplored. To systematically reveal the landscape of archaeal proviruses, we conducted large-scale mining of public and in-house genomic datasets spanning all presently known archaeal phyla. We identified 9,697 archaeal proviruses across 19 archaeal phyla and 366 families, which clustered into 9,123 viral operational taxonomic units (vOTUs). Among these, 97.2% represent novel viruses, and 81.3% could not be classified at the family level, substantially expanding the known diversity of archaeal viruses. Host range analysis revealed that many proviruses exhibit broad infectivity across archaeal lineages, with some even capable of cross-domain infection. Genomic analysis identified 178 distinct types of antiviral systems in archaeal hosts, encompassing multiple CRISPR-Cas variants and restriction-modification (RM) systems. Meanwhile, we detected 747 anti-defense genes encoded by 710 proviruses, such as anti-CRISPR and anti-RM, directly corroborating the ongoing evolutionary arms race between archaeal hosts and their viruses. Additionally, we identified 532 auxiliary metabolic genes (AMGs) within archaeal proviruses that are involved in key processes including carbon, nitrogen, and sulfur metabolism, indicating their potential to reprogram host metabolic pathways and thereby influence biogeochemical cycling. This study establishes a systematic global genomic atlas of archaeal proviruses, advancing our understanding of their distribution and diversity while laying the groundwork for future investigations into how AMG-mediated processes influence archaeal metabolism and ecosystem functions.

ecology↗

bioRxiv2025

MCUR1-CCDC90B complex is a conserved regulator of metabolic homeostasis

The mitochondrial calcium uniporter regulator 1 (MCUR1) is an evolutionarily conserved protein of the inner mitochondrial membrane1, yet its physiological role has remained elusive. Although initially proposed to function as a subunit of the mitochondrial calcium uniporter complex (MCUC)2-4, emerging evidence suggests that MCUR1 has a broader functional spectrum5-7. Here, we identify a biallelic loss-of-function MCUR1 variant (c.802C>T; p.R268X) in a patient with a progressive neurological phenotype. This mutation leads to loss of MCUR1 protein and exerts a dominant-negative effect on its paralog, CCDC90B. We show that MCUR1 and CCDC90B form a hetero-oligomeric complex whose stability depends on MCUR1. Deletion of MCUR1 and CCDC90B in the fission yeast Schizosaccharomyces pombe, which lacks the MCUC, impairs lipid and amino acid metabolism and causes nitrogen source-dependent growth defects that are rescued by expression of human MCUR1. Patient serum metabolomics confirms an imbalance in the amino acid pool, while MCUR1 deficiency in patient-derived skin fibroblasts upregulates autophagy, perturbs non-essential amino acid metabolism, and limits biosynthetic capacity, resulting in delayed proliferation and migration. These findings redefine the MCUR1-CCDC90B coiled-coil complex as a transmembrane scaffold critical for the integrity of mitochondrial protein complexes and the maintenance of metabolic homeostasis, suggesting a potential link between MCUR1 deficiency and human neurometabolic disease.

cell biology↗

bioRxiv2025

Assembly of the complete horse genome reveals variants associated with reproduction and temperamental traits

A high-quality horse genome assembly is crucial for genetic analysis of equine. Benefiting from long-read sequencing techniques and trio-binning strategy, we assembled a complete telomere-to-telomere horse genome (T2T-horse 1.0) with a size of 2.80 Gb and a base accuracy of more than 99.9999%, comprising a 34.11-Mb entire Y chromosome. T2T-horse 1.0 filled the gaps and corrected errors in previous assemblies, and added 180.30 Mb of previously unresolved regions and 77 new genes compared with the previous reference genome TB-T2T. We identified five types of centromeric satellites and revealed the features of the horse centromeric regions. We characterized the structure of the Y chromosome, and determined the borders of PAR transposed region, and annotated 59 novel genes on Y chromosome of T2T-horse 1.0. Using SNPs in the X-degenerate region of the Y chromosome of T2T-horse 1.0, we performed selection signatures analysis between indigenous horses and horses from intensively selected breeds, and detected the selected genes ARSF and NLGN4Y, which are closely associated with reproduction and temperamental traits, respectively.

genomics↗

bioRxiv2025

piR-bmo-796514 Facilitates the Proliferation of Exogenous DNA Virus (Baculovirus) by Targeting the Host E3 Ubiquitin Ligase RNF181

PIWI-interacting RNAs (piRNAs), a class of 23-31 nucleotide non-coding RNAs, are known for silencing transposons and endogenous retroviruses that reside in animal genomes. However, the mechanisms by which host piRNAs affect exogenous viral infections, particularly those by DNA viruses, remain poorly understood. Here, we demonstrate that infection by Bombyx mori nucleopolyhedrovirus (BmNPV), a large DNA virus, induces significant upregulation of silkworm host piR-bmo-796514, which facilitates viral proliferation by suppressing the expression of E3 ubiquitin ligase RNF181. We further reveal that RNF181 exerts antiviral activity through ubiquitin-mediated degradation of Integrin 2b-like, a cellular membrane protein that interacts with viral GP64 protein to mediate BmNPV entry. This study unveils a previously unrecognized regulatory axis connecting host derived piRNAs with exogenous DNA virus infection, providing further mechanistic insights into the modulation of exogenous viral pathogenesis through the reprogramming of the piRNA pathway. Our findings not only advance the understanding of the immune escape mechanism of exogenous viruses but also provide new insights for the development of oligonucleotide antiviral drugs that target proviral piRNAs.

immunology↗

bioRxiv2025

Entamoeba histolytica Gal/GalNAc lectin intermediate subunit promotes inflammation and epithelial damage in intestinal amebiasis through its C3 region.

Entamoeba histolytica is a common enteric protozoan that sometimes transitions from a symbiont to a pathogen, causing intestinal amebiasis and extraintestinal abscesses in the host. The early-to-intermediate stages of intestinal amebiasis are characterized by an intense inflammatory response, during which host innate immune defenses play an important role. Here, using the cecal infection model of C3H/HeNCrl mice, we performed single-cell RNA sequencing of cecal tissues and identified the prominent pro-inflammatory function of host macrophages in intestinal amebiasis. On the amoebic cell surface, Igl is an intermediate subunit of galactose- and N-acetyl-D-galactosamine-inhibitable lectins, which contributes to parasite adherence. We proposed that E. histolytica Igl diffuses along with amoebic extracellular vesicles and contacts host macrophages, binding to the TLR4 co-receptor MD2 through its C3 region and initiating the TLR4/MyD88/NF-{kappa}B inflammatory signaling pathway in host cells. Moreover, by inducing macrophage inflammation and cytokine production, E. histolytica indirectly impairs intestinal epithelium integrity through the Igl protein. To our knowledge, this is the first report showing a host single-cell atlas in the field of amebiasis research. In response to the parasite invasion during intestinal amebiasis, our study provides new insights elucidating the inflammatory formation and epithelial damage of gut mucosal immune system, and also conduces to the identification of potential drug targets for these amoebas.

microbiology↗

bioRxiv2025

Acquired resistance to sotorasib in mutant NSCLC is vulnerable to PI3K-mTOR pathway inhibition mediated by 4E-BP1 regulator of cap-dependent translation

Sotorasib and adagrasib have shown significant efficacy in KRASG12C mutant NSCLC; however, acquired resistance (AR) occurs within 6-12 months. While some resistance arises from new mutations, over half of the resistant cases lack identifiable genomic alterations. We hypothesize that resistance is driven by signaling network rewiring, creating new therapeutic vulnerabilities. To investigate AR mechanisms, multiple AR models, including cell lines, PDXs, CDXs, and PDXOs, were developed. H23AR and H358AR cells displayed >600-fold and 200-fold; and PDXO303AR and PDXO314AR organoids exhibited >300-fold and >100-fold resistance to sotorasib, respectively; however, no additional mutations in KRAS or other potential genetic alterations were identified. The AR cells and PDXOs also showed comparable resistance to adagrasib. Distinct protein signatures associated with KRAS reactivation, mTORC1 signaling upregulation, and PI3K/AKT/mTOR pathway activation were identified in TC303AR & TC314AR PDXs. PI3K protein levels were significantly elevated in AR PDXs, H23AR, and H358AR cells. Pharmacological inhibition of PI3K with copanlisib or genetic knockout via CRISPR-Cas9 restored sotorasib sensitivity, suppressed colony formation, and inhibited downstream effectors, including p-AKT, p-mTOR, p-S6, p70S6K, p-GSK3{beta}, and p-PRAS40 in AR cells. Copanlisib also sensitized both acquired and primary resistant PDXOs and synergized with sotorasib in restoring drug sensitivity. p4E-BP1 was significantly upregulated in H23AR and H358AR cells, which is suppressed by copanlisib. The level of p4E-BP1 expression was correlated with Sotorasib sensitivity in PI3K knockout clones, where the most sensitive clone displayed reduced or no p4E-BP1 expression. CRISPR-Cas9-mediated knockout of 4E-BP1, either alone or in combination with PI3K knockout, dramatically restored sotorasib sensitivity to levels comparable to parental cells. Suppression of 4E-BP1 hyperphosphorylation required dual inhibition of mTORC1 and mTORC2, and treatment with AZD8055 or sapanisertib (mTORC1/2 dual inhibitors) significantly dephosphorylated 4E-BP1 and restored sotorasib sensitivity in resistant cells and PDXOs. In PDX, CDX, and xenograft models in vivo, the combination of sotorasib with either copanlisib or sapanisertib resulted in robust, synergistic, and durable tumor regression at well-tolerated doses. These findings showed the critical role of PI3K/mTOR signaling as a bypass mechanism of resistance to KRASG12C inhibitors. We conclude that mTORC1/2 mediated inhibition of p4E-BP1 and combination strategies targeting this pathway effectively overcome acquired resistance to KRASG12C inhibitors in NSCLC.

cancer biology↗

bioRxiv2025

Synthetic derivatives of vinpocetine as antiproliferative agents

Vincamine is an indole alkaloid initially isolated from plants of the Vinca genus and has previously been demonstrated to have antioxidant, hypoglycemic, and hypolipidemic activities. Vinpocetine, a synthetic derivative of vincamine with an enhanced pharmacological profile, has demonstrated promising antiproliferative properties. While previously reported vinpocetine derivatives have undergone extensive investigation for their pharmacological properties, the role of the E-ring ethyl ester in the anticancer properties of compounds with this scaffold has not yet been fully described. Here, we report the antiproliferative activity of two vinpocetine analogs with modifications at the E-ring. MTT assays revealed that reduction of the ethyl ester to an alcohol exhibited strong dose-dependent antiproliferative activity across five mammalian cell lines, but did not induce significant markers of apoptosis or necrotic death as determined by FITC/Annexin V and cell cycle flow cytometry, respectively. We further observe that vinpocetine and both analogs exhibit dose-dependent modulation of a TCF/LEF reporter cell, which appears to be decoupled from trends in antiproliferative activity Altogether, this work demonstrates the potential for E-ring modifications of vinpocetine as anticancer agents.

cancer biology↗

bioRxiv2025

Amoebicidal Action of Licorice-Derived Compounds: Mechanisms and Effects Against Acanthamoeba castellanii

BackgroundAcanthamoeba is a widely distributed unicellular eukaryotic organism, causing Acanthamoeba keratitis, granulomatous amoebic encephalitis and chronic infectious ulcers. At present, there are no effective drugs available for treating Acanthamoeba infections, highlighting the urgent need for the development of novel anti-Acanthamoeba therapies. PurposeThis study aims to investigate the inhibitory effect of licorice on A. castellanii trophozoites growth and elucidate its molecular mechanisms. MethodsThe effect of licorice on trophozoite viability was assessed employing the CellTiter-Glo assay, while the viability of cells was evaluated via CCK-8 assay. Apoptotic cells were identified through Hoechst 33,342/PI double staining and caspase-3 detection. ROS level was measured via flow cytometry utilising DCFH-DA. Mitochondrial dysfunction was analysed with JC-1 and mtSOX Deep Red staining. RNA sequencing and RT-qPCR analysis were conducted to investigate the potential anti-Acanthamoeba mechanism. ResultsOur results showed that isoliquiritigenin (ISL) and glabridin (GLA) effectively inhibited A. castellaniitrophozoite growth in vitro dose-and time-dependently. ISL and GLA induced several apoptosis features, including Hoechst/PI-positive staining and increased caspase-3 expression. ISL and GLA increased intracellular ROS production, decreased SOD expression and mitochondrial membrane potential and enhanced mitochondrial ROS generation. ISL and GLA effectively prevent host cells from A. castellanii trophozoites invasion. RNA sequencing indicated that ISL may modulate the NAD+ metabolic process, while GLA may influence the sterol metabolic process. ISL reduced the NAD+/NADH ratio, while GLA lowered the levels of 7-dehydrocholesterol in A. castellanii trophozoites. ConclusionThese findings suggest that ISL and GLA may serve as the active components of licorice for treating A. castellanii trophozoites. Our findings suggest that ISL suppresses trophozoites by regulating NAD+ metabolism, while GLA inhibits trophozoites through the regulation of sterol metabolism. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/638758v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1e3ec1borg.highwire.dtl.DTLVardef@a212d2org.highwire.dtl.DTLVardef@81b710org.highwire.dtl.DTLVardef@19b003_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

PSTP: Decoding Latent Sequence Grammar through Transfer Learning and Attention for Protein Phase Separation

Phase separation (PS) is essential in various biological processes, necessitating high-accuracy predictive algorithms for studying numerous uncharacterized sequences, and accelerating experimental validation. However, many recent prediction methods face challenges in generalizability due to their reliance on engineered features. Furthermore, accurately identifying protein regions involved in PS remains challenging. To address this, we propose PSTP, a model employing a dual-language model embedding strategy and a lightweight attention module. The attention layer enables reliable residue-level phase separation predictions, identifying 84% of PS regions in PhaSePro and substantially improving correlation coefficient compared to existing models. PSTP also demonstrates robust performance in predicting PS propensity across various types of PS proteins and shows potential for predicting artificial proteins. By analyzing 160,000+ variants, PSTP characterizes the link between the incidence of pathogenic variants and residue-level PS propensities. PSTPs predictive power and broad applicability make it a valuable tool for understanding biomolecular condensates and disease mechanisms.

bioinformatics↗

bioRxiv2024

High-throughput, multiplexed quantification, and sorting of single EVs at single-molecule level

We have developed a platform for the high-throughput, multiplexed, and ultra-sensitive profiling of individual extracellular vesicles (EVs) directly in plasma, which we call BDEVS - Agarose Bead-based Digital Single Molecule-Single EV Sorting. Unlike conventional approaches, BDEVS achieves single molecule sensitivity and moderate multiplexing (demonstrated 3-plex) without sacrificing the throughput (processing ten thousand of EVs per minute) necessary to resolve EVs directly in human plasma. Our platform integrates rolling circle amplification (RCA) of EV surface proteins, which are cleaved from single EVs, and amplified within agarose droplets, followed by flow cytometry-based readout and sorting, overcoming steric hindrance, non-specific binding, and the lack of quantitation of multiple proteins on EVs that have plagued earlier approaches. We evaluated the analytical capabilities of BDEVS through head-to-head comparison with gold-standard technologies, and demonstrated a [~]100x improvement in the limit of detection of EV subpopulations. We demonstrate the high throughput ([~]100k beads / minute) profiling of individual EVs for key immune markers PD-L1, CD155, and the melanoma tumor marker TYRP-1, and showed that BDEVS can precisely quantify and sort EVs, offering unprecedented resolution for analyzing tumor-immune interactions and detecting rare EV subpopulations in complex clinical specimens. We demonstrate BDEVSs potential as a transformative tool for EV-based diagnostics and therapeutic monitoring in the context of cancer immunology by analyzing plasma samples from patients with melanoma, where EV heterogeneity plays a critical role in disease progression and response to therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/621423v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1a8a542org.highwire.dtl.DTLVardef@f9b9f0org.highwire.dtl.DTLVardef@11e4758org.highwire.dtl.DTLVardef@de1645_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Identification of a series of pyrrolo-pyrimidine based SARS-CoV-2 Mac1 inhibitors that repress coronavirus replication

Coronaviruses (CoVs) can emerge from zoonotic sources and cause severe diseases in humans and animals. All CoVs encode for a macrodomain (Mac1) that binds to and removes ADP-ribose from target proteins. SARS-CoV-2 Mac1 promotes virus replication in the presence of interferon (IFN) and blocks the production of IFN, though the mechanisms by which it mediates these functions remain unknown. Mac1 inhibitors could help elucidate these mechanisms and serve as therapeutic agents against CoV-induced diseases. We previously identified compound 4a (a.k.a. MCD-628), a pyrrolo-pyrimidine that inhibited Mac1 activity in vitro at low micromolar levels. Here, we determined the binding mode of 4a by crystallography, further defining its interaction with Mac1. However, 4a did not reduce CoV replication, which we hypothesized was due to its acidic side chain limiting permeability. To test this hypothesis, we developed several hydrophobic derivatives of 4a. We identified four compounds that both inhibited Mac1 in vitro and inhibited murine hepatitis virus (MHV) replication: 5a, 5c, 6d, and 6e. Furthermore, 5c and 6e inhibited SARS-CoV-2 replication only in the presence of IFN{gamma}, similar to a Mac1 deletion virus. To confirm their specificity, we passaged MHV in the presence of 5a to identify drug-resistant mutations and identified an alanine-to-threonine and glycine-to-valine double mutation in Mac1. Recombinant virus with these mutations had enhanced replication compared to WT virus when treated with 5a, demonstrating the specificity of these compounds during infection. However, this virus is highly attenuated in vivo, indicating that drug-resistance emerged at the expense of viral fitness. IMPORTANCECoronaviruses (CoVs) present significant threats to human and animal health, as evidenced by recent outbreaks of MERS-CoV and SARS-CoV-2. All CoVs encode for a highly conserved macrodomain protein (Mac1) that binds to and removes ADP-ribose from proteins, which promotes virus replication and blocks IFN production, though the exact mechanisms remain unclear. Inhibiting Mac1 could provide valuable insights into these mechanisms and offer new therapeutic avenues for CoV-induced diseases. We have identified several unique pyrrolo-pyrimidine-based compounds as Mac1 inhibitors. Notably, at least two of these compounds inhibited both murine hepatitis virus (MHV) and SARS-CoV-2 replication. Furthermore, we identified a drug-resistant mutation in Mac1, confirming target specificity during infection. However, this mutant is highly attenuated in mice, indicating that drug-resistance appears to come at a fitness cost. These results emphasize the potential of Mac1 as a drug target and the promise of structure-based inhibitor design in combating coronavirus infections.

microbiology↗