Biology subjects

You, H.

Publications and source records attributed to You, H..

At least 19 recordsLinked to original sources

bioRxiv2026

The secreted elastase SjCE2b drives host skin penetration by Schistosoma japonicum

Cercarial elastase is the most abundant protease secreted by Schistosoma mansoni and plays a critical role in cercarial invasion. Although Schistosoma japonicum encodes only a single elastase, SjCE2b, its secretion by cercariae and its specific function in skin penetration have remained elusive. Here, we report the first proteomic analysis of S. japonicum cercarial excretory-secretory products (ESPs) induced by linoleic acid or mouse skin, confirming the presence of SjCE2b in both ESPs preparations. Recombinant SjCE2b expressed in Pichia pastoris was characterized as a trypsin-like serine protease whose activity is entirely abolished by the elastase inhibitor MeoSuc-AAPF-CMK. Furthermore, SjCE2b expression was detected exclusively in cercarial extracts and localized specifically to the cercarial acetabular glands and ducts. Subsequent proteomic analysis indicates that SjCE2b can degrade numerous human epidermal proteins, including ten isoforms of type I and type II keratins. In vitro digestion assays further demonstrated that SjCE2b can digest key structural components of the dermis, including elastin, collogen, and fibronectin. Additionally, the cleavage of complement component C3 and immunoglobulins (IgA and IgG) suggests that SjCE2b may facilitate immune evasion by newly transformed schistosomula. Critically, the incubation of cercariae with anti-rSjCE2b antibody reduced the worm burden by 80.85%, confirming the essential role of SjCE2b in the skin penetration of S. japonicum cercariae and highlighting it as a compelling candidate for vaccine or therapeutic development. Author SummarySchistosomiasis constitutes a major global health burden caused by parasitic flatworms of the genus Schistosoma. Proteolytic and histolytic enzymes secreted by cercarial pre- and post-acetabular glands facilitate disruption of the host skin barrier and protect the parasite from localized dermal inflammatory response. Although cercarial elastase is a well-characterized invasion enzyme in Schistosoma mansoni, its role in Schistosoma japonicum remains poorly defined; consequently, S. japonicum cercariae have been hypothesized to rely on distinct repertoire of proteolytic enzymes during skin penetration. In the present study, we showed that SjCE2b was localized to the cercarial acetabular glands and ducts, and was secreted upon stimulation with linoleic acid or mouse skin. Functionally, SjCE2b can disrupt host skin integrity by degrading epidermal and dermal components, and may promote immune evasion through cleavage of complement component and immunoglobins. Furthermore, the antibody-mediated neutralization of secreted SjCE2b significantly impaired parasite penetration by greater than 80%. Together, these findings establish SjCE2b as a critical enzyme required for S. japonicum cercariae invasion and highlight its potential as a promising target for novel therapeutic interventions.

microbiology↗

bioRxiv2026

Mapping fibrotic microenvironments: single-cell and spatial profiling of Schistosoma mansoni-induced tissue fibrosis

Schistosoma-induced tissue fibrosis, driven by immune response to trapped schistosome eggs, is the principal cause of pathology and schistosomiasis-related morbidity. While praziquantel effectively clears adult parasites, no therapies exist to target eggs or egg-induced tissue fibrosis. Here, we utilise single-cell spatial transcriptomics to define the high-resolution molecular architecture of the fibrotic niche in Schistosoma mansoni-infected mice at 8 weeks post-infection. Our data reveal that the host executes divergent, organ-specific biological programs: constructing a rigid, concentric fibrotic granuloma for permanent egg sequestration in the liver while building a flexible, discontinuous collagen architecture in the intestine to facilitate egg transit. Spatially resolved cell-cell interaction analysis, unprecedented in schistosome-induced fibrosis, identifies the strongest cellular interactions between macrophages and collagen-producing cells, with TGF-{beta}-dominant ligand-receptor pairs in the hepatic niche and integrin-centered pairs in the intestine. Despite this architectural divergence, we detected a core signature of five ligand-receptor pairs shared across both tissues, suggesting shared signalling interactions that could be leveraged for pan-tissue therapeutics for treating schistosome-induced fibrosis. Together, these findings provide the first high-resolution spatial atlas of schistosomiasis-associated fibrosis and provide a foundational framework for discovering novel drug targets capable of reversing established tissue damage, addressing a critical clinical gap where current therapies fail.

molecular biology↗

bioRxiv2026

Membrane Curvature Activates Src kinase and Promotes Metastatic Cancer Cell Survival

Src family kinases (SFKs) play key roles in cancer metastasis. While SFKs are classically regulated by cell adhesions and transmembrane receptors, how they become activated following tumor cell detachment remains unclear. Here, we report curvature-induced kinase activation (CIKA), a distinct mechanism through which plasma membrane curvature directly promotes Src activation. Mechanistically, membrane curvature promotes the oligomerization of TOCA-family curvature-sensing proteins, inducing local biomolecular condensation. These condensates recruit Src, stabilize its open conformation, and exclude the negative regulator Csk, converting curved membrane domains into discrete kinase activation hubs. Disruption of CIKA using TOCA mutants inhibits curvature-induced Src activation and selectively impairs the viability of detached but not adherent cells. Functionally, curvature-induced Src activation promotes anchorage-independent survival, and its disruption suppresses metastatic colonization in xenograft mouse models. These findings reveal membrane curvature as a biophysical activator of Src and suggest CIKA inhibition as a potential therapeutic strategy to target metastatic cancer cells.

cell biology↗

bioRxiv2025

FCHo2, instead of talin, enables inside-out activation of integrin αvβ5 in curved adhesions

Inside-out activation of integrins is crucial for transducing mechanical forces through the extracellular ligand-integrin-talin-F-actin axis. Extensive studies have shown that talin is the essential player in this process by binding to the intracellular tail of {beta} integrins. Here, we show that, while talin binding is essential for inside-out integrin activation in focal adhesions, it is dispensable in curved adhesions - a distinct adhesion architecture that is exclusively mediated by integrin v{beta}5 and selectively formed at curved membranes. Instead, a curvature-sensing protein FCHo2 binds to the HDRRE motif in the cytoplasmic tail of integrin {beta}5 (ITG{beta}5) and inside-out activates integrin v{beta}5 in curved adhesions. Intriguingly, FCHo2 does not bind to a similar motif in the homologous integrin {beta}3 tail. Through truncations and mutations, we identified a pivotal tryptophan (W) in the {beta}3 tail, which is conserved in all homologous integrin {beta} isoforms except {beta}5, where it is replaced by a tyrosine (Y766). This tyrosine substitution is crucial for integrin {beta}5s unique capability in forming curved adhesions. A Y766W mutation abolishes integrin {beta}5s capacity to form curved adhesions, without affecting its ability to form focal adhesions. Furthermore, our studies suggest that the phosphorylation state of Y766 regulates whether integrin v{beta}5 forms curved adhesions or focal adhesions, providing a cellular mechanism governing different adhesion types. Overall, our work unveils distinct molecular interactions and regulatory mechanisms between curved adhesions and focal adhesions, and establishes a molecular basis for the formation of curved adhesions by integrin v{beta}5. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/691229v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@eb4addorg.highwire.dtl.DTLVardef@1802a01org.highwire.dtl.DTLVardef@145e805org.highwire.dtl.DTLVardef@83d641_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

ATP increases murine neuroblastoma cell size through a PANX1- and macropinocytosis-dependent mechanism

Macropinocytosis is an endocytic process that allows cells to respond to changes in their environment by internalizing nutrients and cell surface proteins, as well as modulating cell size. Here, we identify that adenosine triphosphate (ATP) triggers macropinocytosis in murine Neuro2a neuroblastoma cells, driving an increase in cell size, and internalizing the ATP release channel pannexin 1 (PANX1) to macropinosomes. Amiloride treatment and mutation of an extracellular tryptophan (W74) in PANX1 abolished ATP-evoked cell area enlargement, suggesting that PANX1 may itself regulate this form of macropinocytosis. Transient expression of the GTP-hydrolysis resistant ADP-ribosylation factor 6 GTPase (ARF6 Q67L) led to increased cell size, PANX1 internalization and localization to endosomal compartments, consistent with macropinocytosis. Inhibiting macropinocytosis-associated GTPases, phosphoinositide-3 kinase (PI3K), and disrupting actin polymerization abolished ATP-induced PANX1 internalization supporting a macropinocytic mechanism. Further, these inhibitors disrupted co-distribution of intracellular PANX1 with macropinosomal cargo. Several lipid-PANX1 interactions were identified with relevance to macropinocytic mechanisms. The role of PANX1 in ATP-mediated macropinocytosis could be particularly important for disease states implicating PANX1, such as cancer, where ATP can act as a purinergic regulator of cell growth/metastasis and as a supplementary energy source following internalization.

cell biology↗

bioRxiv2025

Label-Free Mapping of Subcellular Dynamics using Wide-field Interferometric Scattering Microscopy and Spectral Exponent Analysis

Interferometric scattering (iSCAT) microscopy combines label-free detection with nanometer-scale motion sensitivity. It captures temporal signal fluctuations from sub-diffraction movements of scatterers in live cells, including macromolecules and cell membranes. Here, we investigate label-free mapping of subcellular dynamics by calculating the power spectral density (PSD) of the iSCAT time series. Using a wide-field iSCAT, we observe an inverse-power-law relationship S(f) = {beta}f- over 30 - 1250 Hz in multiple cell types. We compute pixel-wise PSDs and obtain a color-coded spectral exponent map based on the fitted spectral exponent () and amplitude ({beta}), which indicates the characteristics and strength of subcellular movements. We also incorporate the goodness-of-fit as color saturation to enhance the contrast of the spectral exponent map. Following this approach, we demonstrate label-free imaging of live cells while effectively distinguishing between benign and malignant thyroid cancer subtypes, mitotic and interphase cells, as well as live and apoptotic cells. Dynamic imaging using wide-field iSCAT provides an intrinsic, label-free marker of cellular states and mechanical properties, which may benefit studies in mechanobiology, cancer diagnostics, and stem cell therapies.

bioengineering↗

bioRxiv2025

Embryonic Durotaxis: A Mechanical Framework for Understanding Cesarean Scar Pregnancy

Cesarean scar pregnancy (CSP) is an increasingly common complication associated with the rise in cesarean section rates, yet its underlying mechanisms remain poorly understood. In this study, we identified a stiffness gradient between healthy and scarred uterine tissues in both human cesarean samples and mouse uterine scar models. By employing gradient stiffness hydrogels, we demonstrated that mouse embryos and human trophoblast spheroids exhibit durotaxis, migrating preferentially toward stiffer areas. This migration occurs through three-dimensional complex behaviors--translation, swinging, and rolling--characterized by periodic patterns that align with cavity oscillations. Embryonic durotaxis is initiated from asymmetric adhesion forces and is driven by embryonic protrusive forces, generated from Marangoni-like tissue flows. The intrinsic cavity oscillations further amplify and sustain embryonic durotaxis. Finally, pharmacological inhibition of embryonic durotaxis significantly reduced CSP incidence in the mouse model. Our findings establish the concept of embryonic durotaxis, which may provide novel therapeutic avenues for CSP prevention.

developmental biology↗

bioRxiv2024

Stage-Specific Transcriptome Landscape of Hepatocellular Carcinoma: Insights from Super and Poor Survivors with Prognostic Signature Identification

BackgroundThe prognosis for hepatocellular carcinoma (HCC) patients can vary significantly even within the same clinical stage. This study aims to characterize the stage-specific transcriptomic landscape of super survivors with HCC and identify a prognostic gene signature. MethodsWe analyzed data from The Cancer Genome Atlas (TCGA) for 76 matched super survivors (alive at 5 years) and poor survivors (deceased within 1 year) with HCC. Gene set enrichment analysis stratified by stage was conducted and a key prognostic gene signature was developed. The signature was then applied to the full TCGA cohort and independently validated in International Cancer Genome Consortium (ICGC) data. FindingsStage-specific transcriptomic profiling revealed distinct patterns in super survivors. Stage I and II showed positive enrichment in immune response pathways, while stage III exhibited enhanced catabolic activities and reduced glycolysis. Across all stages, cell cycle processes were less active in super survivors. A 19-gene signature, incorporating immune, metabolic, and cell cycle genes, accurately distinguished super from poor survivors with 91% accuracy (69/76). The signature reliably predicted overall survival in both the verification cohort (1-year, 3-year, and 5-year AUROCs of 0.82, 0.80, and 0.78) and the independent validation cohort (1-year and 3-year AUROCs of 0.80 and 0.83). Consistent AUROCs were observed across all tumor stages. InterpretationOur findings reveal a dynamic shift in HCC progression, with immune dominance in early stages and metabolic dominance in later stages, alongside reduced cell cycle activity across all stages. Integrating stage-specific transcriptomic profiles offers a promising approach to enhancing individualized management for HCC patients.

bioinformatics↗

bioRxiv2024

A new stegosaur (Dinosauria: Ornithischia) from the Upper Jurassic Qigu Formation of Xinjiang, China and a revision on Chinese stegosaurs phylogeny

Stegosaurs are a small but iconic clade of ornithischian dinosaurs. They and their sister taxa, the ankylosaurs, formed the clade Eurypoda which means broad-footed. Here, we describe a stegosaur from the Upper Jurassic Qigu Formation of Xinjiang, China, based on an associated partial skeleton that includes axial, pectoral girdle, pelvic girdle, limb and armor elements. It can be diagnosed as a new taxon, Angustungui, based on numerous autapomorphies. Some morphologies of Angustungui are more similar to the taxa from Europe, Africa and North America than to those from Asia. Our phylogenetic analysis recovers it as the sister taxon of Loricatosaurus. More importantly, the narrow and claw-shaped ungual of Angustungui proves that Eurypoda, at least stegosaur, has claw-shaped unguals. Besides, we revised the character scores for Chinese stegosaurs based on observations of the specimens.

paleontology↗

bioRxiv2024

Identification of Growth Differentiation Factor-15 as An Early Predictive Biomarker for Metabolic Dysfunction-Associated Steatohepatitis: A Nested Case-control Study of UK Biobank Proteomic Data

Background & AimsThe lack of non-invasive biomarkers for the early prediction of patients with metabolic dysfunction-associated steatohepatitis (MASH) is a major challenge for timely intervention. This study aims to determine the predictive capability for MASH long before its diagnosis by using six previously identified diagnostic biomarkers for metabolic dysfunction-associated steatotic liver disease (MASLD) with proteomic data from the UK Biobank. MethodsA nested case-control study comprising of a MASH group and three age- and sex-matched controls groups (metabolic dysfunction-associated steatosis, viral hepatitis, and normal liver controls) were conducted. Olink proteomics, anthropometric and biochemical data at baseline levels were obtained from the UK Biobank. The baseline levels of CDCP1, FABP4, FGF21, GDF15, IL-6 and THBS2 were analyzed prospectively to determine their predictive accuracy for subsequent diagnosis with a mean lag time of over 10 years. ResultsAt baseline, GDF15 demonstrated the best performance for predicting MASH occurrence at 5 and 10 years later, with an AUC of 0.90 at 5 years and 0.86 at 10 years. A predictive model based on four biomarkers (GDF15, FGF21, IL-6, and THBS2) showed AUCs of 0.88 at both 5 and 10 years. Furthermore, a protein-clinical model that included these four circulating protein biomarkers along with three clinical factors (BMI, ALT and TC) yielded AUCs of 0.92 at 5 years and 0.89 at 10 years. ConclusionGDF15 at baseline levels outperformed other individual circulating protein biomarkers for the early prediction of MASH. Our data suggest that GDF15 and the GDF15-based model may be used as easy-to-implement tools to identify patients with high risk of developing MASH at a mean lag time of over 10 years.

molecular biology↗

bioRxiv2024

Force-Enhanced Sensitive Detection of New DNA-Interactive Agents from Microorganisms at the Single-Molecule Level

The discovery of microbial-derived DNA-interacting agents, which hold broad therapeutic potential, is inherently challenging due to the limited sensitivity and specificity of conventional methodologies. Our study introduces a pioneering application of single-molecule stretching assay (SMSA) in natural product chemistry to identify DNA-intercalating agents directly from microbial cultures or extracts. We demonstrate that mechanical force can enhance sensitivity by increasing both the binding affinity Ka and the quantity of ligands bound. The changes induced by intercalators in the counter length and overstretching transition of dsDNA yield a distinctive and highly specific signature indicative of DNA intercalative binding, thereby enabling straightforward detection of DNA intercalators even in trace amounts from microbial cultures. This methodology eliminates the need for extensive large-scale fermentation and purification processes, thus offering a more streamlined approach to DNA-intercalating natural product discovery. By applying SMSA to 17 microorganisms, we identified two DNA intercalator-producing strains: Streptomyces tanashiensis and Talaromyces funiculosus. Subsequently, three DNA intercalators, namely medermycin, kalafungin, and ligustrone B, were isolated and characterized. Among them, medermycin and kalafungin showed significant inhibitory effects against HCT-116 cancer cells, with IC50 values of 52 {+/-} 6 nM and 70 {+/-} 7 nM, respectively.

biophysics↗

bioRxiv2023

SCC3 acts as the cohesin and inhibits inter-sister chromatids repair during rice meiosis

Cohesin is a multi-subunit protein that plays a pivotal role in holding sister chromatids together during cell division. Sister chromatid cohesion 3 (SCC3), constituents of cohesin complex, is highly conserved from yeast to mammals. Since the deletion of individual cohesin subunit always causes lethality, it is difficult to dissect its biological function in both mitosis and meiosis. Here, we obtained scc3 weak mutants using CRISPR-Cas9 system to explore its function during rice mitosis and meiosis. The scc3 weak mutants displayed obvious vegetative defects and complete sterility, underscoring the essential roles of SCC3 in both mitosis and meiosis. SCC3 is localized on chromatin from interphase to prometaphase in mitosis. However, in meiosis, SCC3 acts as an axial element during early prophase I and subsequently situates onto centromeric regions following the disassembly of the synaptonemal complex. The loading of SCC3 onto meiotic chromosomes depends on REC8. scc3 shows severe defects in homologous pairing and synapsis. Consequently, SCC3 functions as an axial element that is essential for maintaining homologous chromosome pairing and synapsis during meiosis.

genetics↗

bioRxiv2023

Promotion of tumor angiogenesis and growth induced by low-dose antineoplastic agents via bone-marrow-derived cells

More research is needed to solidify the basis for reasonable metronomic chemotherapy regimens due to the inconsistent clinical outcomes from studies on metronomic chemotherapy with antineoplastic agents, along with signs of a nonlinear dose-response relationship at low doses. The present study therefore explored the dose-response relationships of representative antineoplastic agents in low dose ranges and their underlying mechanisms. Cyclophosphamide (CPA) and 5-fluorouracil (5-Fu) were employed to observe the effects of the frequent administration of low-dose antineoplastic agents on tumor growth, tumor angiogenesis, and bone-marrow-derived cell (BMDC) mobilization in mouse models. The effects of antineoplastic agents on tumor and endothelial cell functions with or without BMDCs were analyzed in vitro. Tumor growth and metastasis were significantly promoted after the administration of CPA or 5-Fu at certain low dose ranges, and were accompanied by enhanced tumor angiogenesis and proangiogenic factor expression in tumor tissues, increased proangiogenic BMDC release in the circulating blood, and augmented proangiogenic BMDC retention in tumor tissues. Low concentrations of CPA or 5-Fu were found to significantly promote tumor cell migration and invasion, and enhance BMDC adhesion to endothelial cells in vitro. These results suggest that there are risks in empirical metronomic chemotherapy using low-dose antineoplastic agents.

pharmacology and toxicology↗

bioRxiv2023

Lymphotoxin-β Promotes Bone Colonization and Osteolytic Outgrowth of Indolent Bone Metastatic cells of Breast Cancer

Bone metastatic relapse is a lethal consequence of breast cancer, occurring years after initial diagnosis. By analyzing single-cell transcriptomes of bone-seeding tumor cells and in vivo barcoded cDNA library screening, LT{beta} (lymphotoxin-{beta}) is identified as a key factor highly expressed in early-stage bone metastatic cells, associated with poor bone metastasis-free survival, and capable of promoting dormancy reactivation in multiple breast cancer models. Mechanistically, tumor-derived LT{beta} activates NF-{kappa}B2 signaling in osteoblasts to express CCL2/5, facilitating tumor cell seeding and accelerating osteoclastogenesis. Both processes contribute to the reactivation of dormancy and metastatic outgrowth. Blocking LT{beta} signaling with a decoy receptor significantly suppressed bone colonization and metastatic progression, whereas clinical sample analysis revealed significantly higher LT{beta} expression in bone metastases than in primary tumors. Our findings highlight LT{beta} as a bone niche-induced factor that promotes tumor cell seeding and dormancy reactivation, underscoring its potential as a therapeutic target for preventing bone metastatic relapse in patients with breast cancer.

cell biology↗

bioRxiv2023

Somatic Calcium Signals from Layer II/III Motor Cortex for Continuous Neural Decoding

The latest research shows that calcium signals can provide a new signal source for brain-machine interfaces (BMI). However, it remains a question whether the calcium signals from layer 2/3 motor cortex can be used for continuous neural decoding. And how they are involved in movement coding is also worth investigating. Here we collect the somatic signals in M1 layer 2/3 while mice performed a lever-press task under the one-photon imaging. We first present the potential of somatic calcium signals from layer 2/3 in continuous neural decoding through an improved recurrent neural network. Layer 2/3 neurons exhibit three types of calcium dynamics with distinct spatiotemporal coding patterns involved in the movement. Pre-pressing and pressing neurons enable sparse coding of movement through complementary spatiotemporal information. While post-pressing neurons predict the lever movement most accurately through the calcium dynamics with higher fidelity. These results demonstrate the capability of calcium signals from layer 2/3 neurons as a motor BMI driver and underscore their diversity in motor coding, opening a new avenue for studying the motor cortex and designing optical BMIs.

neuroscience↗

bioRxiv2022

CryoFIB milling large tissue samples for cryo-electron tomography

Cryo-electron tomography (cryoET), a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, locating and cutting out objects of interest from a large tissue sample is an important but difficult step. In this study, we report a sample thinning strategy and workflow for tissue samples based on cryo-focused ion beam (cryoFIB) milling. This workflow provides a full solution for isolating objects of interest by starting from a millimeter-sized tissue sample and ending with hundred-nanometer thin lamellae. The workflow involves sample fixation, pre-sectioning, a two-step milling strategy, and locating the object of interest using cellular secondary electron imaging (CSEI). The two-step milling strategy introduces a coarse milling method to solve the milling efficiency problem for samples as thick as tens of microns, followed by a fine milling method to create a furrow-ridge structure. The furrow-ridge structure guarantees the generation of large, thin lamellae with enhanced mechanical stability and charge-reducing design. CSEI is highlighted in the workflow, which provides conventional, on-the-fly locating during cryoFIB milling. Tests of the complete workflow were conducted to demonstrate the high efficiency and high feasibility of the proposed method.

biophysics↗

bioRxiv2022

SOGO-SOFI, light-modulated super-resolution optical fluctuation imaging using only 20 raw frames for high-fidelity reconstruction

Taking advantage of the stochastic photoswitching of genetically encodable reversibly photoswitchable fluorescent proteins (RSFPs), super-resolution optical fluctuation imaging (SOFI) and its variant photochromic stochastic optical fluctuation imaging (pcSOFI) are valuable tools for wide field super- resolution (SR) imaging. Live-cell (pc)SOFI, which requires a small number of original frames to reconstruct an SR image, is prone to structural discontinuity artifacts and low spatial resolution. Herein, we developed a repeated synchronized on- and gradually off-switching SOFI (SOGO-SOFI) that maximized the photoswitching frequency of RSFPs by light modulation and required only 20 frames for high-quality reconstruction. Live-cell SOGO-SOFI imaging of the endoplasmic reticulum (ER) exhibited 10 times higher temporal resolution (100 fps) and fewer artifacts than pcSOFI. Moreover, a combination of SOGO-SOFI with Airyscan further increased the image contrast and the resolution of Airyscan by a factor of 1.5 from 140 nm to 91 nm. The capabilities of SOGO-SOFI were further demonstrated by dual- color imaging of nucleolar proteins in mammalian cells and deep imaging of ER structures in thick brain slices (20.6 {micro}m).

biophysics↗

bioRxiv2022

CRISPR interference for Sequence-Specific Regulation of Fibroblast Growth Factor Receptor A in Schistosoma mansoni

Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A. SmFGFRA is essential for maintaining schistosome stem cells and critical in the schistosome-host interplay. The SmfgfrA gene was targeted in S. mansoni adult worms, eggs and schistosomula using a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor KRAB. We showed that SmfgfrA repression resulted in considerable phenotypic differences in the modulated parasites compared with controls, including reduced levels of SmfgfrA transcription and decreased protein expression of SmFGFRA, a decline in EdU (thymidine analog 5-ethynyl-2-deoxyuridine, which specifically stains schistosome stem cells) signal, and an increase in cell apoptosis. Notably, reduced SmfgfrA transcription was evident in miracidia hatched from SmfgfrA-repressed eggs, and resulted in a significant change in miracidial behavior, indicative of a durable repression effect caused by CRISPRi. Intravenous injection of mice with SmfgfrA-repressed eggs resulted in granulomas that were markedly reduced in size and a decline in the level of serum IgE, emphasizing the importance of SmFGFRA in regulating the host immune response induced during schistosome infection. Our findings show the feasibility of applying CRISPRi for effective, targeted transcriptional repression in schistosomes, and provide the basis for employing CRISPRi to selectively perturb gene expression in parasitic helminths on a genome-wide scale.

molecular biology↗