Biology subjects

Feng, L.

Publications and source records attributed to Feng, L..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Effect of Caenorhabditis elegans U6 Promoters on CRISPR/Cas9-Mediated Gene Editing Efficiency

ObjectiveThis study investigated the effects of endogenous C. elegans U6 promoters on dpy-10 gene editing efficiency. MethodsWe screened endogenous U6 snRNA genes from the WormBase database and constructed 14 editing plasmids targeting dpy-10 by replacing the U6r07e5.16 promoter in pSX524 (Peft-3::cas9::tbb-2 terminator::U6 r07e5.16::dpy-10 sgRNA) through molecular cloning. Following standardized microinjection protocols, we quantified gene editing efficiency and high-efficiency gene editing index based on dpy-10 mutant phenotypes in F1 progeny. ResultsFifteen U6 snRNA genes (r07e5.16, f35c11.9, t20d3.13, k09b11.15, k09b11.16, w05b2.8, c28a5.7, f54c8.9, k09b11.11, k09b11.12, k09b11.14, t20d3.12, f54c8.8, f54c8.10, k09b11.13) were identified from WormBase database. Comparative analysis revealed four U6 promoters (w05b2.8, c28a5.7, f54c8.9, and k09b11.11) significantly enhanced gene editing compared to other U6 promoters, including commonly used U6r07e5.16 and U6k09b11.12 promoters in C. elegans community. Notably, the gRNAF+E scaffold showed no improvement over the gRNA scaffold when paired with the optimal U6w05b2.8 promoter. ConclusionThis study identifies superior U6 promoters for C. elegans gene editing and demonstrates the critical role of promoter optimization in CRISPR systems, providing novel insights for technical refinement.

molecular biology↗

bioRxiv2025

Development and application of a genotyping by target sequencing SNP array panel in Salix suchowensis

Salix suchowensis is an important species of Salix, known for its rapid growth property and wide application in environmental construction, ecological restoration, wicker production, and biomass energy production. Due to its significance as a sustainable biological resource, S. suchowensis has been the centre of intensive breeding. However, rapid improvement of growth and biomass has been hindered by a lack of genomic resources. To address this limitation, we designed a genotyping by target sequencing SNP array panel to facilitate efficient genotyping. Using whole-genome resequencing data, a total of 39,076 SNPs were selected for the array panel, consisting of trait-associated SNPs, intragenic SNPs, and intergenic SNPs. This panel was validated by genotyping 550 new samples, demonstrating high call rates and effective capture of the population structure. Genome-wide association analysis identified 72 SNPs associated with plant height and ground diameter. Additionally, the array panel shows a high potential for genomic selection, with high prediction accuracy for various traits. These results highlight the efficiency of this panel in capturing genomic variations that are highly valuable for future genetic research and breeding applications.

genetics↗

bioRxiv2025

Robust Prediction of Multiple Protein Conformations with Entropy Guidance

Deep learning approaches, exemplified by AlphaFold, have made enormous progress in addressing the grand challenge of protein structure prediction. However, these methods typically predict only a single conformation, while accurately predicting multiple conformations, which are essential for the functions and regulations of many protein classes, remains challenging. Here, we demonstrate that AlphaFold2 has internal knowledge of alternative protein conformations, which can be effectively extracted by learning modifications to its intermediate hidden states using a negative entropy loss. Our resulting method, Entropy Guided Fold (EGF), achieves a success rate of over 80% in accurately predicting two conformations of 37 tested membrane proteins within a 2.0 [A] RMSD threshold, with similar performance for proteins both within and outside AlphaFold2s training dataset. In addition, our method successfully reveals three or more conformational states in several cases. We have also developed a post-processing strategy to effectively select accurate predictions. Overall, our method provides an efficient and robust approach for predicting multiple conformations of target proteins, with broad potential applications in mechanistic studies and translational applications.

bioinformatics↗

bioRxiv2025

Inferring non-coding RNA regulatory network from transcriptomic data and curated databases

Non-coding RNAs (ncRNAs) have played an indispensable role in regulating gene expression and cellular processes. However, the complex regulatory circuits between ncRNAs and coding genes remain understudied. Major challenges for inferring ncRNA regulatory network (NRN) on a transcriptome-wide scale include high dimensionality of both ncRNA and coding gene expression data, their context-dependent interaction, and lack of validation. We hereby propose a comprehensive analytical framework, namely Construction and Analysis of non-coding RNA regulatory NETwork (CAR-NET), specifically designed to infer NRN from transcriptomic data and curated databases on experimentally validated ncRNA-gene interactions and disease-related ncRNAs. At its core is a novel computationally efficient Bayesian network structure learning method that leverages the semi-bipartite graph structure of NRN for dimension reduction, edge orientation and global network search. We also developed and applied algorithms to perform differential network, subnetwork and pathway analysis of the inferred network. We showed the strength of CAR-NET in simulations and three case studies, encompassing lncRNA regulation in brain development, miRNA differential regulation in renal cell carcinoma and regulation by different classes of ncRNAs in different cell lines using bulk or single cell RNA-seq data. An R-shiny app (https://github.com/kehongjie/CAR-NET) was developed to implement the method(s) with interactive GUI.

bioinformatics↗

bioRxiv2025

Conjugated GLP-1 and Estradiol Treatment as a Novel Treatment for Age-related Cognitive Decline in Males and Females

Middle age represents a critical window for metabolic and cognitive health, particularly in the context of rising obesity and diabetes rates. Glucagon-like peptide-1 (GLP-1)-based therapies, which regulate blood glucose and body weight, show sex-specific effects, with estradiol potentiating their metabolic benefits. However, research on GLP-1s cognitive and neuroprotective roles has largely been conducted in males. Here, we investigated the effects of GLP-1 conjugated to estradiol (GE2) on metabolism, cognition, cytokine levels and neurogenesis in the dentate gyrus of middle-aged male and female rats fed a standard (SD) or Western (WD) diet. In both sexes, WD increased body weight and plasma leptin levels, regardless of sex. GE2 treatment led to weight loss, enhanced cued and contextual fear memory, reduced cytokine levels in the hippocampus in SD rats, and increased neurogenesis in the dorsal dentate gyrus (DG), regardless of sex. Sex-specific differences were observed in fat distribution, glucose regulation, central cytokine levels, and neuroplasticity after WD and GE2 treatment. In females only, GE2 reduced visceral (gonadal) fat, reduced cytokines in the dorsal hippocampus, and improved basal blood glucose in response to a WD. In males only, GE2 restored neurogenesis in the DG after WD exposure, and reduced cytokine levels in the amygdala. These findings suggest that although WD increased body weight and GE2 improved associative learning in both sexes, both WD and GE2 had differential affects on metabolic hormones, insulin regulation, cytokine levels and neuroplasticity. Our findings underscore the importance of sex-specific approaches in metabolic and neuroprotective therapeutics in middle age.

neuroscience↗

bioRxiv2025

Palmdelphin facilitates R-spondin2 secretion to activate Wnt signaling and promote colorectal cancer stemness and tumorigenesis

The Wnt signaling pathway is a key driver of stemness and progression which contributes to mortality in colorectal cancer (CRC). R-spondins bind to LGR receptors to inhibit ubiquitin E3 ligases, thus protecting Frizzled from degradation and activating downstream Wnt signaling. Herein, we identify Palmdelphin (PALMD) as a functional marker of CRC stem cells, interspersed between intestinal and colonic crypt base epithelial cells, and predictive of aggressive CMS4 CRC and poor survival. Gene knockdown and overexpression studies revealed that PALMD initiates paracrine activation of Wnt/{beta}-catenin signaling via interacting with and facilitating the secretion of R-spondin2 (RSPO2), resulting in enhanced stemness and tumor growth in vitro and in vivo. Physiologic or pharmacologic inhibition of the PALMD-RSPO2 axis using R-spondin2-specific antibody or 6-methyl-1,3,8-trihydroxyanthraquinone (emodin), respectively, attenuates PALMD- mediated Wnt reporter activation, self-renewal, and tumorigenesis in cell and patient-derived organoid models. Together, these findings identify PALMD as a previously unknown player in Wnt signaling in CRC, and underscore the pro-tumorigenic role of R-spondin2 in this context.

cancer biology↗

bioRxiv2025

METTL1/WDR4-mediated m7G Hypermethylation of SCLT1 mRNA Promotes Gefitinib Resistance in Non-small Cell Lung Cancer

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have produced durable complete responses, but the eventual development of acquired resistance presents a huge challenge in the treatment of non-small cell lung cancer (NSCLC). N7-methylguanosine (m7G), a prevalent post-transcriptional modification within RNA, play regulatory roles in RNA stability, expression dynamics, and functional diversity. Despite these insights, the contribution of m7G methylation to EGFR-TKIs resistance remains poorly characterized. Here, we identified substantial upregulation of mRNA internal m7G modifications and their associated methyltransferase complex composed of methyltransferase-like 1 (METTL1) /WD repeat domain 4 (WDR4) were significantly elevated in NSCLC specimens, correlating with therapeutic resistance. Functional assays confirmed that METTL1/WDR4 enhances gefitinib resistance in both cellular and animal models via the internal RNA m7G methyltransferase activity in NSCLC. Mechanistically, m7G MeRIP-seq coupled with RNA-seq identified sodium channel and clathrin linker 1 (SCLT1) as the m7G target of METTL1/WDR4. METTL1/WDR4 knockdown may result in a decrease in both the demethylation level and mRNA stability of the SCLT1 transcript, while overexpression of wild-type METTL1, but not its catalytically inactive mutant, rescues mRNA stability. METTL1/WDR4-mediated m7G modification of SCLT1 regulates gefitinib resistance by activating the NF-{kappa}B signaling. These results establish aberrant mRNA internal m7G modification as a novel resistance mechanism and propose therapeutic targeting of the METTL1/WDR4-SCLT1-NF-{kappa}B axis cascade to overcome EGFR-TKIs resistance.

molecular biology↗

bioRxiv2025

PIEZO1 Drives Trophoblast Fusion and Placental Development

PIEZO1, a mechanosensor1,2 in endothelial cells, plays a critical role in fetal vascular development during embryogenesis3,4. However, its expression and function in placental trophoblasts remain unexplored. Here, we demonstrate that PIEZO1 is expressed in placental villus trophoblasts, where it is essential for trophoblast fusion and placental development. Mice with trophoblast-specific PIEZO1 knockout exhibit embryonic lethality without obvious vascular defects. Instead, PIEZO1 deficiency disrupts the formation of the syncytiotrophoblast layer in the placenta. Mechanistically, PIEZO1-mediated calcium influx activates TMEM16F lipid scramblase, facilitating the externalization of phosphatidylserine, a key "fuse-me" signal for trophoblast fusion5,6. These findings reveal PIEZO1 as a crucial mechanosensor in trophoblasts and highlight its indispensable role in trophoblast fusion and placental development, expanding our understanding of PIEZO1s functions beyond endothelial cells during pregnancy.

cell biology↗

bioRxiv2025

Posttranscriptional activity of the eukaryotic translation initiation factoreIF4E contributes to HoxA9-driven leukemogenesis

HoxA9, a homeodomain-containing transcription factor, is mis-expressed in over half of acute myeloid leukemia (AML) cases, and is associated with poor prognosis. Previous studies indicated that HoxA9 binds to the eukaryotic translation initiation factor eIF4E in primary specimens and that HoxA9 stimulated the RNA export and translation efficiency of selected RNAs via eIF4E. However, the relevance of this to its leukemogenic transformation capacity was unknown. Here, we used a double point mutation (HoxA9AA) to disrupt the physical and functional interaction between eIF4E and HoxA9 while retaining the HoxA9 transcriptional signature. Surprisingly, the mutation dramatically increased AML latency from a median of 90 to 280 days and resulted in incomplete penetrance. Re-transplantation of bone marrow cells from leukemic animals demonstrated even more pronounced differences in disease kinetics and penetrance with all animals succumbing to disease by day 60 in the wildtype group, while some HoxA9AA mice never developed leukemia. Collectively, these findings uncover a novel, transcription-independent mechanism of HoxA9-driven leukemogenesis through eIF4E and positions eIF4E as a potential therapeutic target AML patients expressing high levels of HoxA9. Key PointsO_LIA double point mutation in HoxA9 disrupted the physical and functional interaction between eIF4E and HoxA9 while retaining the HoxA9 transcriptional signature. C_LIO_LIEukaryotic translation initiation factor eIF4E contributes to HoxA9-driven leukemogenesis and is important for the maintenance of acute myeloid leukemia. C_LI

molecular biology↗

bioRxiv2025

The metabolic enzyme GYS1 condenses with NONO/p54nrb in the nucleus to spatiotemporally regulate glycogenesis and myogenic differentiation

Accumulating evidence indicates that metabolic enzymes can directly couple metabolic signals to transcriptional adaptation and cell differentiation. Glycogen synthase 1 (GYS1), the key metabolic enzyme for glycogenesis, is a nucleocytoplasmic shuttling protein compartmentalized in the cytosol and nucleus. However, the spatiotemporal regulation and biological function of nuclear GYS1 (nGYS1) microcompartments remain unclear. Here, we show that GYS1 dynamically reorganizes into nuclear condensates under conditions of glycogen depletion or transcription inhibition. nGYS1 complexes with the transcription factor NONO/p54nrb and undergoes liquid-liquid phase separation to form biomolecular condensates, leading to its nuclear retention and inhibition of glycogen biosynthesis. Compared to their wild-type littermates, Nono-deficient mice exhibit exercise intolerance, higher muscle glycogen content, and smaller myofibers. Additionally, Gys1 or Nono deficiency prevents C2C12 differentiation and cardiotoxin-induced muscle regeneration in mice. Mechanistically, nGYS1 and NONO co-condense with the myogenic transcription factor MyoD and preinitiation complex (PIC) proteins to form transcriptional condensates, driving myogenic gene expression during myoblast differentiation. These results reveal the spatiotemporal regulation and subcellular function of nuclear GYS1 condensates in glycogenesis and myogenesis, providing mechanistic insights into glycogenoses and muscular dystrophy.

cell biology↗

bioRxiv2025

Harnessing landscape genomics to evaluate genomic vulnerability and future climate resilience in an East Asia perennial

In this era of rapid climate change, understanding the adaptive potential of organisms is imperative for buffering biodiversity loss. Genomic forecasting provides invaluable insights into population vulnerability and adaptive potential under diverse climatic conditions, thereby facilitating management interventions, bolstering population resilience, and shaping germplasm conservation strategies tailored to specific species. Here we integrated population genomics and landscape genomics approaches, leveraging single-nucleotide polymorphisms obtained through whole-genome resequencing of 43 Rheum palmatum complex populations, to pinpoint adaptive variation and its significance in the context of future climates, delineate seed zones, and establish guidelines for ex situ germplasm conservation to capture the majority of existing adaptive diversity. Our analysis unveiled that the species complex comprised two distinct genetic clusters, exhibiting differential climate adaptation and genomic vulnerabilities across its distribution range. We also determined that the species range could be subdivided into three distinct seed zones, with varying sample requirements per seed zone corresponding to differed conservation efforts. Overall, our findings provide a genome-wide perspective on climate adaptation and valuable insights into germplasm conservation strategies aimed at enhancing population resilience in future climates, serving as a blueprint for restoration plans of other vulnerable species.

evolutionary biology↗

bioRxiv2025

Unveiling the Evolution of Antimicrobial Peptides in Gut Microbes via Foundation Model-Powered Framework

Antimicrobial resistance poses a growing threat to public health, emphasizing the urgent need for novel therapeutic strategies. Antimicrobial peptides (AMPs), short peptide sequences with diverse mechanisms of action, offer a promising alternative due to their broad-spectrum activity against pathogens. Recent advances in protein language models (PLMs) have revolutionized protein structure prediction and functional annotation, highlighting their potential for AMP discovery and therapeutic development. In this context, we present AMP-SEMiner (Antimicrobial Peptide Structural Evolution Miner), an AI-driven framework designed to identify AMPs from metagenome-assembled genomes (MAGs). By integrating PLMs, structural clustering and evolutionary analysis into the framework, AMP-SEMiner can identify AMPs encoded by small open reading frames (smORFs) and encrypted peptides (EPs), significantly expanding the discovery space. Using this approach, we identified 1,670,600 AMP candidates from diverse habitats. Experimental validation of 29 candidates revealed antimicrobial activity in 18, with 13 surpassing antibiotics in effectiveness. Further analysis of AMPs from human gut microbiomes demonstrated both conserved and adaptive evolutionary strategies, ensuring their functional efficacy in the dynamic gut environment. These findings position AMP-SEMiner as a powerful tool for the discovery and characterization of novel AMPs, with significant potential to drive the development of new antimicrobial therapies.

microbiology↗

bioRxiv2025

C. elegans interprets dietary quality through context-dependent serotonergic modulation

Animals sense their metabolic needs to guide foraging decisions using neuronal pathways that are only partly understood. Here, we systematically investigate how foraging in the nematode Caenorhabditis elegans is influenced by its bacterial diet, E. coli. By screening C. elegans behavior on 3983 E. coli knockout strains, we identified 22 E. coli metabolic mutants that are aversive to C. elegans in a long-term foraging assay. These include the global metabolic regulator CRP and genes affecting cysteine synthesis, vitamin B6 synthesis, and iron uptake. Serotonin, a neurotransmitter associated with feeding in many animals, allows C. elegans to distinguish wild-type E. coli from these "mediocre" diets through bidirectional signaling. Serotonin produced by the ADF serotonergic neurons supports attraction to wild-type E. coli with the serotonin receptor genes ser-4 and ser-5, whereas serotonin produced by the NSM serotonergic neurons differentially drives aversion to two mediocre diets through four serotonin receptor genes, ser-1, ser-7, mod-1, and lgc-50. Serotonin receptors act in multiple target neurons, including octopamine-producing neurons that suppress aversion across all diets. In addition, dopamine promotes aversion, in part by inhibiting octopaminergic neurons. These results reveal interactions between neuromodulatory circuits in the context-dependent evaluation of dietary quality.

neuroscience↗

bioRxiv2024

CD40 ligation-induced ERK activation promotes autophagy which leads to enhanced radiosensitivity in cervical carcinoma cells

CD40, a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role not only in the immune system, but also in tumor progression. CD40 ligation reportedly promotes autophagy in immune cells. However, the effects of CD40 ligation on autophagy and its mechanism in solid tumor cells are still unclear. In this study, we find that CD40 ligation promotes autophagosome formation, and consequently promotes autophagic flux in cervical cancer cells. Mechanically, this effect relies on that ERK contributes to CD40 ligation-induced ATG13 upregulation by p53. Furthermore, we demonstrates that CD40 ligation-induced autophagy increases the radiosensitivity of cervical cancer cells. Taken together, our results provide new evidence for involvement of the CD40 pathway in autophagy and radiotherapy in cervical cancer cells.

cell biology↗

bioRxiv2024

Biomolecular condensation of ERC1 recruits ATG8 and NBR1 to drive autophagosome formation for plant heat tolerance

Macroautophagy (hereafter autophagy) is essential for cells to respond to nutrient stress by delivering cytosolic contents to vacuoles for degradation via the formation of a multi-layer vesicle named autophagosome. A set of autophagy-related (ATG) regulators are recruited to the phagophore assembly site for the initiation of phagophore, as well as its expansion and closure and subsequent delivery into the vacuole. However, it remains elusive that how the phagophore assembly is regulated under different stress conditions. Here, we described an unknown Arabidopsis (Arabidopsis thaliana) cytosolic ATG8-interaction protein family (ERC1/2), that binds ATG8 and NBR1 to promote autophagy. ERC1 proteins translocate to the phagophore membrane and develop into classical ring-like autophagosomes upon autophagic induction. However, ERC1 proteins form large droplets together with ATG8e proteins when in the absence of ATG8 lipidation activity. We described the property of these structures as phase-separated membraneless condensates by solving the in vivo organization with spatial and temporal resolution. Moreover, ERC1 condensates elicits a strong recruitment of the autophagic receptor NBR1. Loss of ERC1 suppressed NBR1 turnover and attenuated plant tolerance to heat stress condition. This work provides novel insights into the mechanical principle of phagophore initiation via an unreported ERC1-mediated biomolecular condensation for heat tolerance in Arabidopsis.

plant biology↗

bioRxiv2024

Structural and molecular basis of the epistasis effect in enhanced affinity between SARS-CoV-2 KP.3 and ACE2

The recent emergence of SARS-CoV-2 variants KP.2 and KP.3 has been marked by mutations F456L/R346T and F456L/Q493E, respectively, which significantly impact the viruss interaction with human ACE2 and its resistance to neutralizing antibodies. KP.3, featuring F456L and Q493E, exhibits a markedly enhanced ACE2 binding affinity compared to KP.2 and the JN.1 variant due to synergistic effects between these mutations. This study elucidated the structures of KP.2 and KP.3 RBD in complex with ACE2 using cryogenic electron microscopy (Cryo-EM) and decipher the structural and thermodynamic implications of these mutations on receptor binding by molecular dynamics (MD) simulations, revealing that F456L mutation facilitates a more favorable binding environment for Q493E, leading to stronger receptor interactions which consequently enhance the potential for incorporating additional evasive mutations. These results underscore the importance of understanding mutational epistatic interactions in predicting SARS-CoV-2 evolution and optimizing vaccine updates. Continued monitoring of such epistatic effects is crucial for anticipating new dominant strains and preparing appropriate public health responses.

molecular biology↗

bioRxiv2024

Non-destructive in situ monitoring of structural changes of 3D tumor spheroids during the formation, migration, and fusion process

For traditional laboratory microscopy observation, the multi-dimensional, real-time, in situ observation of three-dimensional (3D) tumor spheroids has always been the pain point in cell spheroid observation. In this study, we designed a side-view observation petri dish/device that reflects light, enabling in situ observation of the 3D morphology of cell spheroids using conventional inverted laboratory microscopes. We used a 3D-printed handle and frame to support a first surface mirror, positioning the device within a cell culture petri dish to image cell spheroid samples. The imaging conditions, such as the distance between the mirror and the 3D spheroids, the light source, and the impact of the culture medium, were systematically studied to validate the in-situ side-view observation. The results proved that placing the surface mirror adjacent to the spheroids enables non-destructive in situ real-time tracking of tumor spheroid formation, migration, and fusion dynamics. The correlation between spheroid thickness and dark core appearance under light microscopy and the therapeutic effects of chemotherapy doxorubicin and Natural Killer cells on spheroidss 3D structure was investigated.

cancer biology↗

bioRxiv2024

eLemur: A cellular-resolution 3D atlas of the mouse lemur brain

The gray mouse lemur (Microcebus murinus), one of the smallest living primates, emerges as a promising model organism for neuroscience research. This is due to its genetic similarity to humans, its evolutionary position between rodents and humans, and its primate-like features encapsulated within a rodent-sized brain. Despite its potential, the absence of a comprehensive reference brain atlas impedes the progress of research endeavors in this species, particularly at the microscopic level. Existing references have largely been confined to the macroscopic scale, lacking detailed anatomical information. Here, we present eLemur, a new resource, comprising a repository of high-resolution brain-wide images immunostained with multiple cell type and structural markers, elucidating the cyto- and chemoarchitecture of the mouse lemur brain. Additionally, it encompasses a segmented two-dimensional (2D) reference and 3D anatomical brain atlas delineated into cortical, subcortical, and other vital regions. Furthermore, eLemur includes a comprehensive 3D cell atlas, providing densities and spatial distributions of non-neuronal and neuronal cells across the mouse lemur brain. Accessible via a web-based viewer (https://eeum-brain.com/#/lemurdatasets), the eLemur resource streamlines data sharing and integration, fostering the exploration of new hypotheses and experimental designs using the mouse lemur as a model organism. Moreover, in conjunction with the growing 3D datasets for rodents, non-human primates, and humans, our eLemur 3D digital framework enhances the potential for comparative analysis and translation research, facilitating the integration of extensive rodent study data into human studies. Significance StatementThe gray mouse lemur (Microcebus murinus) represents a promising model for neuroscience research, offering insights into brain structure and function due to its genetic and evolutionary proximity to humans and rodents. Our development of eLemur, a comprehensive 3D digital brain atlas, fills critical gaps in microscopic data availability, enabling nuanced investigations at cellular resolutions. This resource not only enhances our understanding of the mouse lemur brain but also catalyzes broader neuroscience endeavors. By facilitating data sharing and integration, eLemur empowers researchers to explore novel hypotheses and experimental designs. Moreover, its compatibility with existing neuroanatomy frameworks and the growing repository of 3D datasets positions eLemur as a pivotal tool for advancing basic and translational neuroscience studies.

neuroscience↗