Biology subjects

Ke, W.

Publications and source records attributed to Ke, W..

At least 19 recordsLinked to original sources

bioRxiv2026

The common evolution of fungicide persistence in cryptococcosis patients

Fungicide persistence, the ability of dormant fungal cells to survive lethal drug exposure, undermines treatment efficacy, yet its clinical evolution remains largely unexplored. We assembled a nationwide collection of clinical Cryptococcus neoformans isolates from China and revealed extensive inter strain variation in amphotericin B (AmB) persistence. This variation can arise from common evolutionary events in patients, generating previously undescribed high persistence cryptococcal variants refractory to AmB clearance. Genome wide fitness landscape analysis shows that persistence associated mutations generally impose minimal fitness costs, facilitating persistence evolution even in fungistatic resistant and hypervirulent genetic backgrounds. Machine learning identified deficient ACO2 expression as a key predictor of high persistence clinical isolates. ACO2 deficiency reproduces the high persistence phenotype by promoting POPC accumulation, which competes with AmB for its target. Moreover, the clinical stage antifungal T-2307 effectively eliminates high persistence strains across diverse genetic backgrounds. Collectively, our findings unveil common evolution of fungicide persistence in cryptococcosis patients, representing a previously overlooked clinical concern.

microbiology↗

bioRxiv2026

Sequence-to-function modeling uncovers the context-specific grammar of Drosophila chromatin insulation

Chromatin is organized into self-interacting topologically associating domains partitioned by boundary elements that insulate adjacent domains and restrict regulatory interactions. Yet, how sequence context and combinations of factors dictate boundary strength remains incompletely understood. Here we present Domino, a deep learning framework that maps genomic sequences to quantitative insulation scores defined directly from single-nucleosome resolution Drosophila melanogaster Micro-C data. Unlike traditional transcription factor motif scanning, Domino captures broad sequence context to resolve the functional contributions of individual sequence elements. We validate model predictions through experimental perturbations of insulator sequences. Model interpretation yields insulation-associated motifs genome-wide. Across 7,311 embryonic boundaries, Domino reveals a comprehensive insulation grammar defined by just 24 primary motifs that account for 59% of the boundaries, with an average of only two motifs per motif-containing boundary. Beyond known factors, we identify the zinc-finger proteins Trem, CG4854 and CG17385 as previously unreported insulation factors. We uncover distance- and orientation-dependent motif synergy, including a strict orientation preference of the prominent architectural factor M1BP. Finally, Domino traces tissue-specific shifts in the insulator landscape from the embryo to larval and adult brains, nominating new brain-specific insulation motifs. In sum, Domino provides a generalizable framework for decoding the regulatory logic of 3D genome architecture.

genomics↗

bioRxiv2026

Shared binding sites for the chromosomal architectural protein Su(Hw) mediate physical interactions between Drosophila TAD boundaries

The head-to-tail pairing of the nhomie and homie boundaries is responsible for the formation of a stem-loop TAD (topologically associated domain) that encompasses the even-skipped (eve) gene and its regulatory elements. TAD boundaries like homie and nhomie have partner preferences and one mechanism thought to be important in linking compatible boundaries is shared binding sites for the same architectural protein. Both eve boundaries have a single binding site for the polydactyl zinc finger protein Su(Hw). Here we have investigated the role of the shared Su(Hw) sites in the long-distance regulation of a dual reporter by the eve enhancers. Consistent with this mechanism, we find that when the transgene nhomie or homie boundary has a mutation in the Su(Hw) binding site, reporter activation by the eve enhancers is severely compromised. Using MicroC we show the mutating the single Su(Hw) substantially weakens the ability of the nhomie and homie boundaries in the transgene to physically pair with the two endogenous eve boundaries. Su(Hw) binding sites are not, however, sufficient as the gypsy su(Hw) insulator is unable to support long-distance regulation. In a less demanding transvection assay mutations in the nhomie and homie Su(hw) site have only a modest effect on regulatory interactions. The eve boundaries can mediate transvection with the gypsy insulator; however. they are not compatible with the dCTCF Fab-8 boundary even in the transvection assay. Finally, we show that the regulatory and physical interactions of the nhomie boundary in the transgene and the two endogenous eve boundaries are incompatible with the popular cohesin-loop extrusion model.

genetics↗

bioRxiv2026

iCNG99: a validated genome-scale metabolic model of Cryptococcus neoformans strain H99

Cryptococcus neoformans is a ubiquitous environmental fungus that can also cause life-threatening infections in immunocompromised individuals. As a competent pathogen, Cryptococcus needs to reprogram its metabolism to adapt to the drastic differences between environmental niches and host niches. A well-curated genome-scale metabolic model (GEM) is a powerful tool to facilitate the investigation of the metabolic resilience of an organism. Here we reconstructed and validated iCNG99, a GEM for C. neoformans reference strain H99, and evaluated its predictive performance across 43 growth conditions and gene essentiality benchmarks. The model achieved high confidence essential gene prediction (precision = 0.77) and recapitulated pathways targeted by clinically available antifungals. Integration with transcriptomic and metabolomic data enabled iCNG99 to capture condition-specific metabolic adaptations and to identify candidate vulnerabilities in drug tolerance, revealing metabolic adaptations associated with survival within host conditions and drug susceptibility. Together, iCNG99 provides a systems-level computational platform for investigating C. neoformans metabolism and for prioritizing antifungal vulnerabilities.

systems biology↗

bioRxiv2025

homie and nhomie insulators compete with each other for interaction with distant copies, affecting enhancer-promoter interactions and eve function

Chromatin insulators, a.k.a. boundaries, separate regions of the chromosome with distinct chromatin characteristics, including distinct histone modifications. This activity affects gene expression by allowing chromatin domains to be stably regulated and maintained. They also block enhancer-promoter interactions and, somewhat paradoxically, can facilitate other enhancer-promoter interactions, particularly when they stitch together distant regions of the chromosome by pairing with specific insulator partners. Here we explore how long-range interactions facilitated by insulator pairing are affected by the presence of two specifically interacting partners. Our results show that when two partners are present, they compete, reducing each others effects, suggesting that interactions tend to be limited to two interacting partners at any given time. In particular, when a distant transgenic copy of an eve insulator (homie or nhomie) is present, it can interact with either endogenous insulator. But when one endogenous insulator is removed, the remaining one interacts more strongly with the transgenic copy, biasing the induced enhancer-promoter interactions toward those nearest the remaining endogenous insulator. We also show that removing one or both endogenous eve insulators significantly reduces endogenous eve function at a critical early stage of development, and that the eve Polycomb domain expands in both directions when its insulator boundaries are removed.

genetics↗

bioRxiv2025

Metadomain and metaloop genome interactions in mammalian T cells

Recent studies have advanced our understanding of chromosomal organization and its principal role in gene regulation. However, most analyses have focused on short-range interactions (<2 Mb), limiting insight into broader regulatory architecture. In particular, the relationships between topologically associating domains (TADs), sub-TAD loops, long-range cross-TAD interactions, and higher-order chromosomal compartmentalization remain poorly understood. Here, we identify extensive multi-megabase and interchromosomal interactions (metaloops) in T lymphocytes, which organize into larger meta-TAD associations (metadomains). Metaloops bridge distal promoters and regulatory elements of key T cell-specific genes such as Ctla4, Ikzf2, Il2ra, Ets1, Lef1, Runx1, Bach2, Foxo1 and others, and are both shared and cell type-specific across functionally distinct T cell lineages. Reanalysis of published data confirms the reproducibility of these interactions in both mouse and human T cells and their dependence on superenhancers. Genome-wide clustering of metadomains reveals three interchromosomal hubs with distinct epigenomic profiles, including a superenhancer-enriched hub associated with T cell-specific gene activation. By integrating a compendium of new and public T cell epigenomic data, we infer distinct architectural factors associated with short-range loops and long-range metaloops. Altogether, our study reveals new features of T cell-specific 3D genome organization across scales, and our computational framework is broadly applicable to analyses of chromatin architecture across different cell types and experimental systems. Highlights- Ultra-long-range (>2 Mb) chromatin interactions linked to gene regulation in T cells - A new algorithm identifies distal and interchromosomal meta-TADs and metaloops - An interchromosomal T cell-specific active hub emerges from metadomain clustering - Epigenomic compendium implicates TFs associated with long-range interactions

genomics↗

bioRxiv2025

Macrophage-derived reactive oxygen species promote Salmonella aggresome formation contributing to bacterial antibiotic persistence

In this study, we reveal that macrophage-derived reactive oxygen species (ROS) can trigger the rapid formation of Salmonella aggresomes, which substantially contribute to the increased frequency of persisters induced by phagocytosis. Salmonella containing aggresomes exhibited a dormant phenotype characterized by reduced adenosine triphosphate (ATP) levels and decreased metabolic activity. Furthermore, these dormant bacteria showed upregulated expression of Salmonella pathogenicity island 1 (SPI-1)-encoded type III secretion system (T3SS)-related genes, followed by later expression of SPI-2 T3SS-related genes when macrophages ROS production declined. Our results demonstrate that Salmonella containing aggresomes can enter a dormant state to escape antibiotic attack, while crucially maintaining the ability to resuscitate when the stress environment is improved. Research on bacterial aggresomes could potentially provide therapeutic strategies to combat bacterial antibiotic persistence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/657582v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@7e5f1forg.highwire.dtl.DTLVardef@970a39org.highwire.dtl.DTLVardef@204449org.highwire.dtl.DTLVardef@632c47_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

FOSL2 Directly Regulates FSHR and CYP11A1 Transcription: An Essential Transcription Factor for Gonadotropin-Dependent Folliculogenesis

The meticulous orchestration of gonadotropin-dependent folliculogenesis constitutes the cornerstone of female reproductive cyclicity and fertility, with FSH/FSHR signaling recognized as the master regulator. Achieving the necessary amplification of this signaling is essential for successful GTH-dependent folliculogenesis, yet the mechanisms remain inadequately defined. Our study utilizes single-cell and spatial transcriptomics to identify FOSL2 as an FSH-inducible transcription factor, exhibiting precise spatiotemporal co-expression with FSHR. FOSL2 knockdown in vitro resulted in notable reductions in granulosa cell proliferation, induced apoptosis, and disrupted gonadotropin-dependent folliculogenesis. In vivo studies using conditional FOSL2 deletion in mouse granulosa cells corroborated these results, demonstrating a complete halt in GTH-dependent folliculogenesis and resultant infertility. Mechanistic exploration unveiled that FSH/FSHR initiates FOSL2 expression via the cAMP/PKA/CREB cascade, while FOSL2 in turn enhances FSHR transcription through direct promoter binding, thereby establishing a self-amplifying loop. This loop represents a molecular switch, modeled to the all-or-nothing dynamics of GTH-dependent folliculogenesis. The evolutionary conservation of this mechanism was confirmed through cross-species analyses in sheep, where FOSL2 deficiency similarly attenuated FSH/FSHR signaling and inhibited follicular growth. Our findings advance the understanding of folliculogenesis by revealing a novel FOSL2-centered amplification loop for FSH/FSHR signaling, highlighting the indispensable role of FOSL2 in reproductive biology.

cell biology↗

bioRxiv2025

Follicular mural granulosa cells stockpile glycogen to fuel corpus luteum pre-vascularization

The corpus luteum (CL) arises from the luteinization of follicular granulosa cells (GCs) and theca cells, marked by rapid progesterone elevation and angiogenesis. Intriguingly, angiogenesis lags behind progesterone elevation, creating an avascular phase during which luteal cells must fuel intensive steroidogenesis without perfusion. How the avascular CL meets this energetic demand remains a mystery. Here, we reveal a novel cellular adaptive mechanism-GC energy storage (GCES)-that resolves this enigma. We demonstrate that upon luteinization initiation, GCs enter a metabolically quiescent state yet enhance glucose uptake via SLC2A1, converting the glucose into glycogen through the hCG (LH)-MAPK-RUNX1-Insulin signaling axis. Catabolism of this glycogen reserve supplies the energy required for the avascular CL, ensuring normal luteogenesis. GCES is evolutionarily conserved across species. Genetic or pharmacological disruption of GCES or glycogenolysis induces luteal insufficiency, whereas timely glucose administration enhances GCES, improving luteal function and optimize reproductive outcome in both mouse and ovine models. In human study, orally intake of glucose post-hCG significantly augments GCES and enhances progesterone production in women. These results advance luteal physiology by uncovering a universal reproductive principle with direct clinical implications.

developmental biology↗

bioRxiv2024

Motor protein-assisted glycan translocation and sequencing using nanopores

The glycan sequencing remains a significant bottleneck in glycoscience. While nanopore platforms have achieved substantial progress in single-molecule nucleic acid sequencing, their application to glycan sequencing has faced considerable challenges, with limited advancements to date. In this study, we propose a novel strategy for controlling glycan translocation through the MspA nanopore as an initial step toward glycan sequencing. By conjugating the target glycan with a helicase-controlled single-stranded DNA, we successfully achieved sequencing reads of up to eleven glycans. For the first time, we isolated glycan-associated electrical signals, enabling the translocation, stretching, and controlled speed of neutral glycans through the nanopore. This method provides a platform for obtaining glycan read lengths and identifying different glycan modifications, demonstrating the capability to resolve monosaccharide composition and glycosidic linkages. To further improve resolution, we propose engineered M2-MspA to reduce the pore constriction size and enhance precision by minimizing the random thermal motion of the translocating glycan. These modifications are expected to increase the sequencing accuracy and reliability. This work represents the first proof-of-concept demonstration of glycan chain nanopore sequencing and lays a promising foundation for the development of single-molecule glycan fingerprinting and sequencing technologies. We anticipate that this approach will significantly advance the development and commercialization of nanopore-based glycan sequencing techniques.

bioengineering↗

bioRxiv2024

Combining Glycosidases and Nanopore Technology for Glycan Sequencing

The efficient characterization of glycan structures remains a critical challenge. Direct glycan sequencing technologies with improved sensitivity and throughput are still needed. Here, we propose a glycan sequencing strategy based on glycosidase -assisted nanopore sensing. We used engineered nanopore -hemolysin (M113R/T115A), which provided high-resolution discrimination of monosaccharide volume differences and sensitivity to variations in glycan chain length. By utilizing the specificity of glycosidases, we sequentially hydrolyzed the terminal residues of the glycan chains and detected the characteristic shifts in electrical signals generated by the translocation of hydrolysis products. The accuracy of recognition of hydrolysis fragments achieved over 90 % using machine learning. This allowed us to efficiently and conveniently determine the sequence of consecutive monosaccharide units in the glycan chains. Based on this principle, we conducted a proof-of-concept demonstration on actual samples and verified the accuracy via HPLC-MS. We achieved the sequencing of consecutive units in glycan chains, confirming the theoretical feasibility of glycosidase -assisted nanopore glycan sequencing. With future optimization, the development of arrayed nanopore glycan sequencing technology based on hydrolysis strategy will provide an innovative approach for rapid decoding of glycans, which could promote the progress of glycomics research and glycobiology.

biochemistry↗

bioRxiv2024

Cathelicidin antimicrobial peptides mediate immune protection of marsupial neonates

Marsupial neonates are born with immature immune systems, making them vulnerable to pathogens. While neonates receive maternal protection, they can also independently combat pathogens, though the mechanisms remain unknown. Using the sugar glider (Petaurus breviceps) as a model, we investigated immunological defense strategies of marsupial neonates. Cathelicidins - a family of antimicrobial peptides expanded in the genomes of marsupials - are highly expressed in developing neutrophils. Sugar glider cathelicidins reside in two genomic clusters and their coordinated expression is achieved by enhancer sharing within clusters and long-range physical interactions between clusters. These cathelicidins modulate immune responses and have potent antimicrobial effects, sufficient to provide protection in a mouse model of sepsis. Lastly, cathelicidins have a complex evolutionary history, where marsupials and monotremes are the only tetrapods that retained two cathelicidin clusters. Thus, cathelicidins are critical mediators of marsupial immunity, and their evolution reflects the life history-specific immunological needs of these animals.

evolutionary biology↗

bioRxiv2024

Granulosa cell-layer stiffening prevents the granulosa cells from escaping the post-ovulatory follicle

Ovulation is necessary for successful reproduction. After ovulation, cumulus cells and oocytes are released, while granulosa cells (GCs) remain trapped within the post-ovulatory follicle to form the corpus luteum. However, the mechanism underlying GC confinement has long been unclear. Here, we provide in vitro and in vivo evidence demonstrating that the stiffening of GC-layer as an evolutionarily conserved mechanism that hinders GCs from escaping the post-ovulatory follicles. Spatial transcriptome analysis reveals that the assembly of focal adhesions is primarily responsible for this stiffening. Disrupting focal adhesion assembly through RNA interference results in the release of GCs from the post-ovulatory follicle, leading to the formation of an aberrant corpus luteum with reduced cell density and cavities. We also uncover that the LH (hCG) -cAMP-PKA-CREB signaling axis stimulates focal adhesion assembly and induce GC-layer stiffening. Our findings introduce a novel concept of "GC-layer stiffening", which offers valuable insights into the factors that prevent GCs escape from the post-ovulatory follicle.

cell biology↗

bioRxiv2024

The homie insulator has sub-elements with different insulating and long-range pairing properties

Chromatin insulators are major determinants of chromosome architecture. Specific architectures induced by insulators profoundly influence nuclear processes, including how enhancers and promoters interact over long distances and between homologous chromosomes. Insulators can pair with copies of themselves in trans to facilitate homolog pairing. They can also pair with other insulators, sometimes with great specificity, inducing long-range chromosomal loops. Contrary to their canonical function of enhancer blocking, these loops can bring distant enhancers and promoters together to activate gene expression, while at the same time blocking other interactions in cis. The details of these effects depend on the choice of pairing partner, and on the orientation specificity of pairing, implicating the 3-dimensional architecture as a major functional determinant. Here we dissect the homie insulator from the Drosophila even skipped (eve) locus, to understand its substructure. We test pairing function based on homie-carrying transgenes interacting with endogenous eve. The assay is sensitive to both pairing strength and orientation. Using this assay, we found that a Su(Hw) binding site in homie is required for efficient long-range interaction, although some activity remains without it. This binding site also contributes to the canonical insulator activities of enhancer blocking and barrier function. Based on this and other results from our functional dissection, each of the canonical insulator activities, chromosomal loop formation, enhancer blocking, and barrier activity, are partially separable. Our results show the complexity inherent in insulator functions, which can be provided by an array of different proteins with both shared and distinct properties. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/578481v3_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@243157org.highwire.dtl.DTLVardef@234cfborg.highwire.dtl.DTLVardef@193012borg.highwire.dtl.DTLVardef@31d7e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Chromosome Structure II: Stem-loops and circle-loops

The chromosomes in multicellular eukaryotes are organized into a series of topologically independent loops called TADs. In flies, TADs are formed by physical interactions between neighboring boundaries. Fly boundaries exhibit distinct partner preferences, and pairing interactions between boundaries are typically orientation-dependent. Pairing can be head-to-tail or head-to-head. The former generates a stem-loop TAD, while the latter gives a circle-loop TAD. The TAD that encompasses the Drosophila even skipped (eve) gene is formed by the head-to-tail pairing of the nhomie and homie boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the nhomie boundary region with two attP sites. The attP sites were then used to generate four boundary replacements:{lambda} DNA, nhomie forward (WT orientation), nhomie reverse (opposite of WT orientation), and homie forward (same orientation as WT homie). The nhomie forward replacement restores the WT physical and regulatory landscape: In MicroC experiments, the eve TAD is a "volcano" triangle topped by a plume, and the eve gene and its regulatory elements are sequestered from interactions with neighbors. The{lambda} DNA replacement lacks boundary function: the endpoint of the "new" eve TAD on the nhomie side is ill-defined, and eve stripe enhancers activate a nearby gene, eIF3j. While nhomie reverse and homie forward restore the eve TAD, the topology is a circle-loop, and this changes the local physical and regulatory landscape. In MicroC experiments, the eve TAD interacts with its neighbors, and the plume at the top of the eve triangle peak is instead flanked by a pair of "clouds" of contacts with the next-door TADs. Consistent with the loss of isolation afforded by the stem-loop topology, the eve enhancers weakly activate genes in the neighboring TADs. Conversely, eve function is partially disrupted.

genetics↗

bioRxiv2023

Discrimination of Isomeric Glycans via High-sensitive Engineered Nanopore

Glycan isomers are the basis for forming the spatial structure of glycans, which poses great challenges to glycan structural analysis and hinders the study of the relationship between structure and function. An economical and convenient analytical method with high resolution is still in demand. Here, we designed an engineered -hemolysin nanopore with double-mutants (-HL M113R/T115A) to achieve sensing of glycans with different glycosidic linkages. By extracting three parameters to depict a 3D scatter plot, four glycans with different degrees of polymerization and glycosidic bonds could be distinguished. Through molecular dynamics (MD), we further elucidated the motion trajectories of two different glycosidic bonds in the pore region. In addition, the sensing ability was also applied in the direct identification of glycans in mixture systems. The obtained nanopore data permitted us to achieve a separation accuracy of over 90% independent of the assistance of machine learning algorithms. Designing biological nanopores with high discrimination and sensitivity for glycans with different lineages paves the way for nanopore glycan profiling and potentially sequencing.

bioengineering↗

bioRxiv2023

Chromosome Structure I: Loop extrusion or boundary:boundary pairing?

Two different models have been proposed to explain how the endpoints of chromatin looped domains ("TADs") in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the "loop-extrusion" and the "boundary-pairing" models. Our findings are incompatible with the loop-extrusion model and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion.

genetics↗

bioRxiv2023

Microglia facilitate and stabilize the response to general anesthesia

General anesthesia leads to a loss of consciousness and an unarousable state in patients. Although general anesthetics are widely used in clinical practice, their underlying mechanisms remain elusive. The potential involvement of nonneuronal cells is unknown. Microglia are important immune cells in the central nervous system (CNS) that play critical roles in CNS function and dysfunction. We unintentionally observed delayed anesthesia induction and early anesthesia emergence in microglia-depleted mice. We found that microglial depletion differentially regulates neuronal activities by suppressing the neuronal network of anesthesia-activated brain regions and activating emergence-activated brain regions. Thus, microglia facilitate and stabilize the anesthesia status. This influence is not mediated by dendritic spine plasticity. Instead, it relies on the activation of microglial P2Y12 and subsequent calcium influx, which facilitates the general anesthesia response. Together, we elucidate the regulatory role of microglia in general anesthesia, extending our knowledge of how nonneuronal cells modulate neuronal activities.

neuroscience↗