Biology subjects

Li, Z.

Publications and source records attributed to Li, Z..

At least 541 records · Page 30Linked to original sources

bioRxiv2024

A comprehensive benchmark for multiple highly efficient base editors with broad targeting scope

As the toolbox of base editors (BEs) expands, selecting appropriate BE and guide RNA (gRNA) to achieve optimal editing efficiency and outcome for a given target becomes challenging. Here, we construct a set of 10 adenine and cytosine BEs with high activity and broad targeting scope, and comprehensively evaluate their editing profiles and properties head-to-head with 34,040 BE-gRNA-target combinations using genomically integrated long targets and tiling gRNA strategies. Interestingly, we observe widespread non-canonical protospacer adjacent motifs (PAMs) for these BEs. Using this large-scale benchmark data, we build a deep learning model, named BEEP (Base Editing Efficiency Predictor), for predicting the editing efficiency and outcome of these BEs. Guided by BEEP, we experimentally test and validate the installment of 3,558 disease-associated single nucleotide variants (SNVs) via BEs, including 20.1% of target sites that would be generally considered as "uneditable", due to the lack of canonical PAMs. We further predict candidate BE-gRNA-target combinations for modeling 1,752,651 ClinVar SNVs. We also identify several cancer-associated SNVs that drive the resistance to BRAF inhibitors in melanoma. These efforts benchmark the performance and illuminate the capabilities of multiple highly useful BEs for interrogating functional SNVs. A practical webserver (http://beep.weililab.org/) is freely accessible to guide the selection of optimal BEs and gRNAs for a given target.

molecular biology↗

bioRxiv2024

Bottom-up design of calcium channels from defined selectivity filter geometry

Native ion channels play key roles in biological systems, and engineered versions are widely used as chemogenetic tools and in sensing devices1,2. Protein design has been harnessed to generate pore-containing transmembrane proteins, but the capability to design ion selectivity based on the interactions between ions and selectivity filter residues, a crucial feature of native ion channels3, has been constrained by the lack of methods to place the metal-coordinating residues with atomic-level precision. Here we describe a bottom-up RFdiffusion-based approach to construct Ca2+ channels from defined selectivity filter residue geometries, and use this approach to design symmetric oligomeric channels with Ca2+ selectivity filters having different coordination numbers and different geometries at the entrance of a wide pore buttressed by multiple transmembrane helices. The designed channel proteins assemble into homogenous pore-containing particles, and for both tetrameric and hexameric ion-coordinating configurations, patch-clamp experiments show that the designed channels have higher conductances for Ca2+ than for Na+ and other divalent ions (Sr2+ and Mg2+). Cryo-electron microscopy indicates that the design method has high accuracy: the structure of the hexameric Ca2+ channel is nearly identical to the design model. Our bottom-up design approach now enables the testing of hypotheses relating filter geometry to ion selectivity by direct construction, and provides a roadmap for creating selective ion channels for a wide range of applications.

biochemistry↗

bioRxiv2024

Transposable elements drive regulatory and functional innovation of F-box genes

Protein domains of transposable elements (TEs) and viruses increase the protein diversity of host genomes by recombining with other protein domains. By screening 10 million eukaryotic proteins, we identified several domains that define multi-copy gene families and frequently co-occur with TE/viral domains. Among these, a Tc1/Mariner transposase helix-turn-helix (HTH) domain was captured by F-box genes in the Caenorhabditis genus, creating a new class of F-box genes. For specific members of this class, like fbxa-215, we found that the HTH domain is required for diverse processes including germ granule localisation, fertility, and thermotolerance. Furthermore, we provide evidence that HSF-1 mediates the transcriptional integration of fbxa-215 into the heat-shock response by binding to Helitron TEs directly upstream of the fbxa-215 locus. The interactome of HTH-bearing F-box factors suggests roles in post-translational regulation and proteostasis, consistent with established functions of F-box proteins. Based on AlphaFold2 multimer proteome-wide screens, we propose that the HTH domain may diversify the repertoire of protein substrates that F-box factors regulate post-translationally. We further demonstrate that F-box genes repeatedly and independently captured TE domains throughout eukaryotic evolution, and describe an additional instance in zebrafish. In conclusion, we identify recurrent TE domain captures by F-box genes in eukaryotes and provide insights into how these novel proteins are integrated within host gene regulatory networks.

evolutionary biology↗

bioRxiv2024

Adaptation to cystine limitation stress promotes PDAC tumor growth and metastasis through translational upregulation of OxPPP

Cystine/cysteine is critical for antioxidant response and sulfur metabolism in cancer cells and is one of the most depleted amino acids in the microenvironment of pancreatic ductal adenocarcinoma (PDAC). The effects of cystine limitation stress (CLS) on PDAC progression are poorly understood. Here we report that adaptation to CLS (CLSA) promotes PDAC cell proliferation and tumor growth through translational upregulation of the oxidative pentose phosphate pathway (OxPPP). OxPPP activates the de novo synthesis of nucleotides and fatty acids to support tumor growth. On the other hand, CLSA-mediated lipidomic reprogramming depends on triacylglycerides synthesis and lipid droplet formation to mitigate lipotoxicity. Through drug screening, we identify lomitapide as an inhibitor of CLSA PDAC tumor growth and a potent sensitizer of chemotherapy. Lomitapide inhibits triacylglycerides synthesis to interfere with CLSA and chemotherapy-induced lipidomic reprogramming. Taken together, we demonstrate that CLSA promotes PDAC tumor growth through metabolic reprogramming and lomitapide could be used to target the dysregulated lipid metabolism in PDAC.

cancer biology↗

bioRxiv2024

Feathered Icons: Drivers of Global Attention on Bird Species

Greater public support to conservation is needed to help address the global biodiversity crisis. However, human attention towards biodiversity is taxonomically biased, leaving many species neglected and their conservation needs unmet. Here we investigated the drivers of public and scientific interest for 10,509 bird species, measured by Wikipedia pageviews and articles in Web of Knowledge. We then used a quasi-experimental design to test the effect on public interest of published updates to the International Union for the Conservation of Nature (IUCN) Red List of threatened species (N = 479). Public and scientific interest were strongly predicted by species charisma, familiarity, utilization, and Red List category. While threatened species received significantly more attention, IUCN Red List category revisions produced asymmetric public response: uplistings (to categories of higher extinction risk) generated a significant but modest immediate increase in pageviews, while downlistings (to categories of lower risk) showed no detectable effect. The traits that predicted responsiveness, genuineness of change (real biological shifts rather than improved knowledge or revised taxonomy) and increasing population trend, differed from those driving underlying interest. These findings underscore the need to raise the profile of species lacking the charismatic or familiar traits that typically attract public and scientific interest.

ecology↗

bioRxiv2024

The 8-nm spaghetti: well-structured glycans coating linear tetrapeptide repeats discovered from freshwater with CryoSeek

We recently developed a research strategy, termed CryoSeek, to identify uncharacterized bio-entities from natural or endogenous resources using cryo-electron microscopy (cryo-EM). Here we report the discovery of a glycofibril whose primary molecular mass is attributed to a thick glycan shell. The 3.3-[A] resolution cryo-EM reconstruction reveals that the only protein component of the glycofibril, which is approximately 8 nm in diameter, is a linear chain of tetrapeptide repeats. Each tetrapeptide repeat consists of a 3,4-dihydroxyproline (diHyp), a Ser or Thr, and two less conserved residues. Two and one glycan chains are respectively O-linked to the diHyp and Ser/Thr residues. The protein sequence pattern of this glycofibril is similar to that of our recently observed TLP-4, although the glycan chains are different. We rename the previously characterized glycofibril as TLP-4a and designate this one as TLP-4b. Our discoveries reveal the critical role of glycans in structural folding of glycoconjugates and shed light on understanding the carbon/nitrogen ratio in biospheres.

biophysics↗

bioRxiv2024

Simulating big mechanically-active culture systems (BigMACS) using paired biomechanics-histology FEA modelling to derive mechanobiology design relationships.

Big mechanically-active culture systems (BigMACS) are promising to stimulate, control, and pattern cell and tissue behaviours with less soluble factor requirements, however, it remains challenging to predict if and how distributed mechanical forces impact single-cell behaviours to pattern tissue. In this study, we introduce a centimetre, tissue-scale, finite element analysis (FEA) framework able to correlate sub-cellular quantitative histology with centimetre-scale biomechanics. Our framework is relevant to diverse bigMACS; media perfusion, tensile-stress, magnetic, and pneumatic tissue culture platforms. We apply our framework to understand how the design and operation of a multi-axial soft robotic bioreactor can spatially control mesenchymal stem cell (MSC) proliferation, orientation, differentiation to smooth muscle, and extracellular vascular matrix deposition. We find MSC proliferation and matrix deposition correlate positively with mechanical stimulation but cannot be locally patterned by soft robot mechanical stimulation within a centimetre scale tissue. In contrast, local stress distribution was able to locally pattern MSC orientation and differentiation to smooth muscle phenotypes, where MSCs aligned perpendicular to principal stress direction and expressed increased -SMA with increasing 3D Von Mises Stresses from 0 to 15 kPa. Altogether, our new biomechanical-histological simulation framework is a promising technique to derive the future mechanical design equations to control cell behaviours and engineer patterned tissue generation.

bioengineering↗

bioRxiv2024

A Novel Bispecific Antibody CVL006 Superior to AK112 for Dual Targeting of PD-L1 and VEGF in Cancer Therapy

Both preclinical and clinical studies have shown that combining anti-VEGF/VEGFR drugs with immune checkpoint inhibitors (ICIs) significantly enhances anticancer efficacy. Currently, PDL-1/ VEGF bispecific antibodies demonstrate superior antitumor activity compared to monotherapy or even the combination of PD-L1 inhibitors with anti-VEGF antibodies. This enhanced efficacy results from the simultaneous blockade of the PD-1/PD-L1 pathway and the inhibition of VEGF-driven angiogenesis. In this study, we developed a novel bispecific antibody, CVL006, by fusing an anti-PDL1 VHH domain with a humanized IgG1 anti-VEGF monoclonal antibody while retaining antibody-dependent cellular cytotoxicity (ADCC) functionality. CVL006 demonstrated high affinity and specificity for both human PD-L1 and VEGF, effectively blocking both the VEGF/VEGFR signaling pathway and the PD-L1/PD-1 axis. This dual blockade not only suppressed VEGF-induced angiogenesis but also reactivated T cells, increasing the secretion of cytokines essential for immune response. In vivo studies further indicated that CVL006 achieved superior antitumor efficacy compared to the PD-L1 inhibitor atezolizumab in mouse models, with greater tumor growth inhibition and reduced angiogenesis. To compare with approved bispecific antibody PD-1/VEGF AK112 (ivonescimab), CVL006 showed superior antitumor efficacy in vivo. These findings underscore the therapeutic potential of CVL006, which integrates immune checkpoint inhibition with disruption of tumor vascularization, offering a robust and comprehensive anticancer strategy.

pharmacology and toxicology↗

bioRxiv2024

Chemical perturbations impacting histone acetylation governcolorectal cancer differentiation

Dysregulated epigenetic programs that restrict differentiation, reactivate fetal genes, and confer phenotypic plasticity are critical to colorectal cancer (CRC) development. By screening a small molecule library targeting epigenetic regulators using our dual reporter system, we found that inhibiting histone deacetylase (HDAC) 1/2 promotes CRC differentiation and anti-tumor activity. Comprehensive biochemical, chemical, and genetic experiments revealed that on-target blockade of the HDAC1/2 catalytic domain mediated the differentiated phenotype. Unbiased profiling of histone posttranslational modifications induced by HDAC1/2 inhibition nominated acetylation of specific histone lysine residues as potential regulators of differentiation. Genome-wide assessment of implicated marks indicated that H3K27ac gains at HDAC1/2-bound regions associated with open chromatin and upregulation of differentiation genes upon HDAC1/2 inhibition. Disrupting H3K27ac by degrading acetyltransferase EP300 rescued HDAC1/2 inhibitor-mediated differentiation of a patient-derived CRC model using single cell RNA-sequencing. Genetic screens revealed that DAPK3 contributes to CRC differentiation induced by HDAC1/2 inhibition. These results highlight the importance of specific chemically targetable histone modifications in governing cancer cell states and epigenetic reprogramming as a therapeutic strategy in CRC. BRIEF SUMMARYHDAC1/2 inhibition promotes colorectal cancer differentiation via gains in H3K27ac, which can be reversed by blocking its acetyltransferase EP300.

cancer biology↗

bioRxiv2024

SOX2-induced IL-1alpha-mediated immune suppression drives epithelial dysplasia malignant transformation

Squamous cell carcinomas (SCC) are often preceded by potentially malignant precursor lesions, most of which remain benign. The terminal exhaustion phenotypes of effector T-cells and the accumulation of myeloid-derived suppressor cells (MDSC) have been thoroughly characterized in established SCC. However, it is unclear what precancerous lesions harbor a bona fide high risk for malignant transformation and how precancerous epithelial dysplasia drives the immune system to the point of no return. Here we show that expression of SRY-box transcription factor 2 (SOX2) in precancerous lesions imparts an irreversible risk that recruits suppressive myeloid cells by promoting the release of CCL2. We developed a unique genetically engineered mouse model (GEMM) to recapitulate the malignant transformation of epithelial dysplasia to SCC in the oral mucosa with high histologic and phenotypic fidelity. Using a combination of longitudinal human specimens and the Sox2-GEMM, we found that the myeloid cells in precancerous epithelial dysplasia exhibit a distinctive dichotomous profile featuring high levels of IL-1-SLC2A1 and low levels of type-I interferon (IFN-I) signatures, which occurs before SCC emerges histologically. Brief priming of myeloid cells with IL-1 desensitizes them to IFN-I agonists and makes myeloid-derived suppressor cells (MDSC) even more suppressive of T-cell activation. Mechanistically, IL-1 activation represses the expression of DHHC3/7 enzymes, which are responsible for the palmitoylation of stimulator of interferon genes (STING). Early blockade of IL1 signaling using pharmacologic and genetic approaches similarly reduces MDSC and SLC2A1high myeloid cells, suppresses epithelial dysplasia transformation, and extends survival. This work establishes a previously unrecognized SOX2-CCL2-IL1 pathway that leads to irreversible immune escape when precancerous epithelial lesions transform.

cancer biology↗

bioRxiv2024

deepBlastoid: A Deep Learning-Based High-Throughput Classifier for Human Blastoids Using Brightfield Images with Confidence Assessment

Recent advances in human blastoids have opened new avenues for modeling early human development and implantation. Human blastoids can be generated in large numbers, making them suitable for high-throughput screening, which often involves analyzing vast numbers of images. However, automated methods for evaluating and characterizing blastoid morphology are still underdeveloped. We developed a deep-learning model capable of recognizing and classifying blastoid brightfield images into five distinct quality categories. The model processes 53.2 images per second with an average accuracy of 87%, without signs of overfitting or batch eHects. By integrating a Confidence Rate (CR) metric, the accuracy was further improved to 97%, with low-CR images flagged for human review. In a comparison with human experts, the model matched their accuracy while significantly outperforming them in throughput. We demonstrate the value of the model in two real-world applications: (1) systematic assessment of the eHect of lysophosphatidic acid (LPA) concentration on blastoid formation, and (2) evaluating the impact of dimethyl sulfoxide (DMSO) on blastoids for drug screening. In the applications involving over 10,000 images, the model identified significant eHects of LPA and DMSO, which may have been overlooked in manual assessments. The deepBlastoid model is publicly available and researchers can train their own model according to their imaging conditions and blastoid culture protocol. deepBlastoid thus oHers a precise, automated approach for blastoid classification, with significant potential for advancing mechanism research, drug screening, and clinical in vitro fertilization (IVF) applications.

developmental biology↗

bioRxiv2024

Structure of a zoonotic H5N1 hemagglutinin reveals a receptor-binding site occupied by an auto-glycan

Highly pathogenic avian influenza has spilled into many mammals, most notably U.S. dairy cows with several dozen human breakthrough infections. Zoonotic crossovers, with hemagglutinins mutated to enhance viral ability to use human 2-6-linked sialic acid receptors versus avian 2-3-linked ones, highlight the pandemic risk. To gain insight into these crossovers, we determined the cryo-EM structure of hemagglutinin from the zoonotic H5N1 A/Texas/37/2024 strain (clade 2.3.4.4b) in complex with a previously reported neutralizing antibody. Surprisingly, we found that the receptor-binding site of this H5N1 hemagglutinin was already occupied by an 2-3-linked sialic acid and that this glycan emanated from asparagine N169 of a neighboring protomer on hemagglutinin itself. This structure thus highlights recognition by influenza hemagglutinin of an "auto"-2-3-linked sialic acid from N169, an N-linked glycan conserved in 95% of H5 strains, and adds "auto-glycan recognition" to the complexities surrounding H5N1 zoonosis. HighlightsO_LIWe report the structure of hemagglutinin from the zoonotic H5N1 strain A/Texas/37/2024 from clade 2.3.4.4b responsible for the current H5N1 outbreak C_LIO_LIStructural analysis revealed that each receptor-binding site of H5N1 A/Texas/37/2024 HA is bound to an "auto" sialic acid originating from glycan N169 on an adjacent protomer C_LIO_LIThis structure provides new aspects of receptor binding for a highly pathogenic strain of avian influenza, and raises questions about the impact of auto-binding sialic acid, especially with respect to zoonosis C_LI

immunology↗

bioRxiv2024

Harmonising digitised herbarium data to enhance biodiversity knowledge: creating an updated checklist for the flora of Greenland.

International efforts to digitise herbarium specimens provide the building blocks for a global digital herbarium. However, taxonomic changes and errors can result in inconsistencies when amalgamating specimen metadata, that compromise the assignment of occurrence records to correct taxa, and the subsequent interpretation of patterns in biodiversity. We present a novel workflow to mass-curate digital specimens. By employing existing digital taxonomic backbones, we aggregate specimen names by their accepted name and flag remaining cases for manual review. We then validate names using site-specific floras, balancing automation with taxonomic expert-based curation. Applying our workflow to the vascular plants of Greenland, we harmonised 175,266 digitised herbarium specimens and observations from 92 data providers from the Global Biodiversity Information Facility (GBIF). The harmonised metacollection for the Greenland flora contains 780 plant species. Our workflow increases the number of species known from Greenland compared to other currently available species checklists and increases the mean number of occurrences per species by 42.6. Our workflow illustrates the integration required in order to create a global, universally accessible digital herbarium, and shows how previous obstacles to database curation can be overcome through a combination of automation and expert curation. From the specific perspective of the Greenland flora, our approach arrives at a new checklist of taxa, a new curated metacollection of occurrence data, and revised estimates of plant richness. The list of taxa and their prevalence allow a new basis for biodiversity assessment and conservation planning. Societal Impact StatementDigitising plant collections has allowed for data to be aggregated across multiple collections, forming a single harmonised resource of unprecedented scale. This resource is only accurate once the database names are assigned to one accepted name per species. We established a semi-automated workflow for processing plant name data, leveraging taxonomic backbones and employing taxonomic expertise at key stages. Applying our workflow to the flora of Greenland, we developed a curated checklist of 780 species, capturing greater species richness than previously published, while also curating 175,266 plant records. Our findings redefine our knowledge of Greenlandic plant diversity, while harmonising a vast digital collection for further research.

ecology↗

bioRxiv2024

A CRISPR/SpCas9M-reporting system for efficient and rapid genome editing in Caulobacter crescentus

As members of the -proteobacteria group, Caulobacter crescentus and its relatives are known for their asymmetric life cycle and comprehensive applications in gene delivery, agricultural biotechnology, and the production of high-value compounds. However, genetic manipulations of these bacteria are often time-consuming and labor-intensive due to the lack of efficient genome editing tools. Here, we report a practical CRISPR/SpCas9M-reporting system that overcomes the limitations of SpCas9 expression, enabling efficient, markerless, and rapid genome editing in C. crescentus. As a demonstration, we successfully knocked out two genes encoding the scaffold proteins, achieving apparent editing efficiencies up to 80%. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized to enhance the editing efficiency or decrease the cell lethality. A nearly zero off-target ratio was observed after the curing of the editor plasmid in editing strains. Furthermore, we applied the CRISPR/SpCas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti, establishing it as an efficient and reliable editing strategy. We anticipate that this system could be applied to other hard-to-edit organisms, accelerating both basic and applied research in -proteobacteria.

bioengineering↗

bioRxiv2024

Constructing a holistic map of cell fate decision by hyper solution landscape

The Waddington landscape metaphor has inspired extensive quantitative studies of cell fate decisions using dynamical systems. While these approaches provide valuable insights, the intrinsic nonlinear complexity and the parameter dependence limits systematic analysis of fate transitions. Here, we introduce the Hyper Solution Landscape (HSL), a minimally parameter-dependent methodology showing a comprehensive structure of all possible landscape configurations for gene regulatory networks. HSL connects different solution landscapes to reflect dynamic changes of the landscapes associated with bifurcations. Applied to the Cross-Inhibition with Self-activation motif, HSL analysis identifies key hyperparameters driving distinct directional changes in cell fate propensity. Different routes through the HSL between the same initial and final states can produce markedly different fate distributions. This enables rational design of transition strategies. We validate HSLs utility in the seesaw model of cellular reprogramming, establishing a powerful framework for understanding and engineering cell fate decisions. A record of this papers Transparent Peer Review process is included in the Supplemental Information.

systems biology↗

bioRxiv2024

LRRK2 G2019S mutation incites increased cell-intrinsic neutrophil effector functions and intestinal inflammation in a model of infectious colitis

Parkinsons Disease (PD) is a progressive, neurodegenerative disorder characterised by motor and non-motor symptoms. Emerging evidence suggests a link between PD and gastrointestinal dysfunction. Constipation is frequently observed years prior to development of motor dysfunction in PD, and people with inflammatory bowel disease (IBD) are more likely to develop PD. Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) account for approximately 1% of all PD cases and are associated with increased risk for IBD. Among them, LRRK2 Gly2019Ser (G2019S), located within the kinase domain, is the most common PD-associated mutation and increases kinase activity. It is unknown how LRRK2 mutation affects susceptibility to intestinal inflammation or pathogenesis of PD. Using single cell RNA sequencing (scRNAseq), we demonstrate that LRRK2 G2019S mutation promotes a dysregulated gene profile, especially within neutrophil, monocyte and {gamma}{delta} T cell populations, following Citrobacter rodentium infection in mice. Transcriptionally, LRRK2 G2019S neutrophils have a greater pro- inflammatory type I and II IFN response compared to those of WT mice. This is accompanied by an increase in neutrophil numbers in the lamina propria in LRRK2 G2019S mice. We also uncover cell-intrinsic functional defects in LRRK2 G2019S neutrophils, including increased chemotaxis, degranulation and neutrophil extracellular traps (NETosis) formation. Increased neutrophil infiltration is associated with an upregulation in Th17 immune responses, which may together contribute to the observed increase in colon pathology during infection. These findings increase our understanding of the role of PD-associated genes in immune cells and their contribution to immune dysregulation. Understanding the early perturbations driven by the LRRK2 G2019S mutation in gastrointestinal pathology may facilitate the development of biomarkers for early diagnosis and intervention in PD.

immunology↗

bioRxiv2024

Control of mitochondrial dynamics by dPGC1 limits Yorkie-induced oncogenic growth in Drosophila

Mitochondrial function and dynamics are essential for maintaining cellular homeostasis and overall health. Disruptions in these processes can contribute to various diseases, including cancer. The Hippo signaling pathway, a key regulator of tissue growth, plays a central role in cancer through its main effector, the Yes-associated protein (YAP), known as Yorkie (Yki) in Drosophila. In this model organism, Yki upregulation drives benign tissue overgrowth in imaginal discs. Our research shows that the conserved metabolic regulator dPGC1 restricts Yki-driven tissue hyperplasia and helps maintain epithelial integrity in vivo. Combined Yki upregulation and dPGC1 depletion results in tumors characterized by enlarged mitochondria and upregulation of genes promoting mitochondrial fusion, a condition that is both necessary and sufficient for Yki-driven oncogenic growth. We further demonstrate that mitochondrial enlargement is associated with increased levels of the cell cycle regulator Cyclin E, which is critical for tumor development. These findings identify dPGC1 as a context-dependent tumor suppressor that coordinates mitochondrial dynamics and cell cycle regulation in response to oncogene activation, with implications for understanding cancer development in humans.

developmental biology↗

bioRxiv2024

NaDyNet: A Toolbox for Dynamic Network Analysis of Naturalistic Stimuli

Experiments with naturalistic stimuli (e.g., listening to stories or watching movies) are emerging paradigms in brain function research. The content of naturalistic stimuli is rich and continuous. The fMRI signals of naturalistic stimuli are complex and include different components. A major challenge is isolate the stimuli-induced signals while simultaneously tracking the brains responses to these stimuli in real-time. To this end, we have developed a user-friendly graphical interface toolbox called NaDyNet (Naturalistic Dynamic Network Toolbox), which integrates existing dynamic brain network analysis methods and their enhanced versions. The main features of NaDyNet are: 1) extracting signals of interest from naturalistic fMRI signals; 2) incorporating six commonly used dynamic analysis methods and three static analysis methods; 3) enhanced versions of these dynamic methods by adopting inter-subject analysis to eliminate the effects of non-interest signals; 4) performing K-means clustering analysis to identify temporally reoccurring states along with their temporal and spatial attributes. We then introduced the rationale for incorporating inter-subject analysis to improve existing dynamic brain network analysis methods, and presented numerous examples. We also summarized research progress in comparing methodological efficacy, offered our recommendations for method selection in dynamic network analysis, and discussed the limitations of current approaches and directions for future research. We hope that this open source toolbox will promote the development of naturalistic neuroscience. The toolbox is available at https://github.com/yuanbinke/Naturalistic-Dynamic-Network-Toolbox.

neuroscience↗