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Dong, G.

Publications and source records attributed to Dong, G..

At least 37 records · Page 2Linked to original sources
bioRxiv2025

Tracing the stemness and malignant transition in a heritable colorectal cancer Lynch Syndrome by single-cell RNA-seq analysis

BackgroundLynch Syndrome (LS) is an autosomal dominant disease characterized by germline heterozygous mutations in DNA mismatch repair (MMR) genes. High-risk LS patients may proceed to colorectal cancer (CRC). However, the drivers or biomarkers of LS benign colon tissue approaching malignant CRC are not completely understood. The similarity and difference between LS-related and nonLS-related CRC are also not well interrogated (LS-CRC vs. nonLS-CRC). This study aimed to understand the cellular changes during malignant transition in LS. MethodsSingle-cell RNA sequencing (scRNA-seq) was used to analyze paired biopsy samples from 3 patients with LS (cancer tissues vs. adjacent normal tissues, n=3). Single-nuclear RNA sequencing (snRNA-seq) was used to analyze a frozen biopsy sample from a patient with LS (n=1). scRNA-seq or snRNA-seq datasets from CRC were downloaded from the open source. Integrative computational analysis was performed to conclude the distinct pattern in the single-cell atlas. Immuno-histo-fluorescence staining (IHF) were also performed for three key markers. ResultsIn the single-cell atlas, we observed an increase in primitive cancer stem-cell-like cells with high expression of a general cancer biomarker CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 5 (CEACAM5) in the epithelium of the LS. Infiltration of immune cells and DNA repair biological activity are dramatically increased in LS carcinoma. The burden in LS is fundamentally elevated compared to that in CRC or healthy donors when the mutations are in the coding-sequence-wide range. Furthermore, T cell and macrophage-related tumor immunity is readily mobilized in carcinomas compared to paraCArcinomas. ConclusionsThis study provides single-cell transcriptomic resource using affected tissues from patients with Lynch Syndrome and describes an integrative profile covering the alterations (cancer stem cell markers, mutation burden, and tumor immunity) during the malignant transition from healthy to Lynch Syndrome and to colorectal cancer at the single-cell level. Highlights and Figure/Table Index (Take-Home Messages)O_LIColon tissues from HD (n=4), patients with LS (n=6) and with CRC (n=3) were collected for snRNA-seq or scRNA-seq analysis; Diagnosis of the 6 LS patients are clearly supported by our pedigree documentations (Fig. 1 and Supp. Fig. 1) and briefed in Table 1; C_LIO_LIAmong the 6 patients with LS, 1 for snRNA-seq analysis (a carcinoma tissue only), 3 for scRNA-seq analysis (3 paired para-carcinoma and carcinoma tissues) and 3 for experimental validations; (Fig. 1) C_LIO_LIFollowing studies are focused on the comparisons between HD, LS and CRC; In addition, comparisons between paired carcinoma and para-carcinoma tissues from patients with LS were also performed; (Fig. 2-7) C_LIO_LIWhen HD and LS are compared, a cancer stem cell marker CEACAM5 is readily detected by snRNA-seq analysis; (Fig. 5) C_LIO_LIWhen carcinoma and para-carcinoma from LS patients are compared, increased immune cell infiltration of and enhanced DNA repair activity in the malignant tissues is readily detected by scRNA-seq analysis; in addition, three upregulated markers in LS carcinoma (BACE2, GRPC5A and OLFM4) were identified and validated by immunohistoinfloresnce; (Fig. 3 and Fig. 4) C_LIO_LISComatic algorithm-based mutation calling analysis suggests a comparable mutation burden between carcinoma and para-carcinoma from LS patients, but such mutation burdens are grossly greater than that from HD or that from patients with CRC; (Fig. 6) C_LI O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@174af7dorg.highwire.dtl.DTLVardef@1ad3e71org.highwire.dtl.DTLVardef@dea1e4org.highwire.dtl.DTLVardef@568f91_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1C_FLOATNO Enrollment of LS patients and overall design of the study. (A) Three family pedigrees of the LS patients (LS-CRC) who donated the samples for single-cell transcriptomic analysis. Cropped pedigree information is provided in the Main text. See the Supp. Fig. 1 for the complete pedigree information. (B) Two different platforms were used in the study: snRNA-seq and scRNA-seq. (C) Various types of comparison were performed to characterize the malignant transition of LS. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@eb8e4eorg.highwire.dtl.DTLVardef@cbafaaorg.highwire.dtl.DTLVardef@79ca83org.highwire.dtl.DTLVardef@1de7544org.highwire.dtl.DTLVardef@1e4fcbc_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONClinical information of enrolled patients with LS. An Excel version of the table is provided in the Supp. Material C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=160 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig2.gif" ALT="Figure 2"> View larger version (56K): org.highwire.dtl.DTLVardef@8cd71corg.highwire.dtl.DTLVardef@7e75b3org.highwire.dtl.DTLVardef@61757eorg.highwire.dtl.DTLVardef@68ca5f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 2C_FLOATNO snRNA-seq analysis of a frozen colon biopsy sample from a LS patient reveals a cluster of cancer stem cells (A) Frozen colon biopsy samples were subjected for snRNA analysis (Healthy donors, N=4; Lynch Syndrome, N=1) and epithelial cells were filtered out for further study. Data were integrated by the Seurat and Harmony standard procedure. Clusters 0-9 were identified in a total of 11,353 cells (the Healthy Controls have 7,373 cells; the LS patient has 3,980 cells). The malignancy of the epithelial cells was inferred by the inferCNV and SCEVAN algorithm (two major compartments: tumor and no-tumor). The portion of tumor or non-tumor cells in each cluster was also illustrated (right panel). Note that the major contributor in Cluster 4, 6, and 7 is the tumor-like cells from the LS patient. (B) Each cluster of the epithelial cells was further annotated according to the combination of Seurat cluster identity and SCEVAN malignant identity. Mix_1 to 5 contain both tumor-like cells and non-tumor-like cells; Other clusters are marked as BEST4+-Enterocytes, Enterocytes and Goblet cells where much fewer tumor-like cells were assigned. Note that the epithelial cells from the LS patient are mainly classified into Mix_2, 4, and 5 tumor-like cells. (C) Three clones were inferred by SCEVAN algorithm. In the phylogenetic tree, Clone_1 and Clone_2 are closer and probably originated from Clone_3. (D) A Sankey plot showing overall belonging relationships in the epithelial snRNA-seq dataset. Note that the Mix_2 pool is dominated by cells from Lynch_01 and that in the Mix_2 pool Clone_3 is the most dominant clone. (E) The Mix_2 cells specifically express numerous colon epithelial stem cells signature markers such as SMOC2, RGBM, OLFM4, AXIN2 and LGR5. Of note, all these intestine stem cell signature genes were reported as marker genes for cancer stem cells, especially LGR5. (F) Pseudotime trajectory analysis of the Lynch Syndrome epithelium cells indicates the Mix_2 cells are the most primitive in development. (G) Top 36 differentially expressed genes (DEGs, both upregulated and downregulated) were highlighted when the non-tumor-like cells were compared with tumor-like cells in the Mix2. (H) The Mix_2, 4, 5 epithelial cells all have enriched signaling pathways involved in DNA repair response and MYC targets (fonts in red) while Mix_4 and 5 manifest Interferon alpha or gamma response (fonts in blue). C_FIG O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig7.gif" ALT="Figure 7"> View larger version (54K): org.highwire.dtl.DTLVardef@1be2c6aorg.highwire.dtl.DTLVardef@55f267org.highwire.dtl.DTLVardef@9d60fborg.highwire.dtl.DTLVardef@4a2d37_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 7C_FLOATNO Distinct tumor immunity microenvironment profiles in LS carcinoma (A) T cells from LS-CA and LS-paraCA patients were filtered for further annotation. The LS-paraCA group had 1410 T cells, whereas the Lynch CA group had 2125 T cells. Six major T cell subtypes were identified: Naive CD4-T, NKT, Treg, IL7R+CD8_T, GZMA+CD8_T, and GZMK+CD8_T. Note that GZMA+CD8_T cells appear to be more abundant in Lynch CA (arrows). (B) The pseudotime trajectory indicating the T cells developed towards two branches: the Naive CD4-T and Treg branches, and GZMA+CD8_T and GZMK+CD8_T branches. GZMA+CD8_T cells appear to be more mature in LS-CA than in LS-paraCA (arrows). (C-F) Biological function-instructed scoring of the paired single-cell datasets of T cells from the three LS patients, including T cell cytotoxicity (B), T cell exhaustion (C), T cell immune escape (D), and T cell surveillance (E). (G) Circle plots showing interaction profiles across indicated cells in the paired LS samples. The direction of the lines indicates the direction of the cell-cell communication, the width of the lines indicates the interaction strength, and the color of the lines indicates the cell types of the senders. (H) Summary of the Study. In this study, we profiled Lynch Syndrome biopsy samples using single-cell or single-nucleus RNA-seq analysis. Paired carcinoma and adjacent para-carcinoma samples were included to facilitate the analysis, including tracing tumor clones, calculating mutation burden, identifying biomarkers for cancer stem cells, and tumor immune microenvironment changes during the transition from pre-carcinoma to carcinoma in the inheritable cancer Lynch Syndrome. See the text in the Discussion section for further detail. C_FIG O_FIG O_LINKSMALLFIG WIDTH=148 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig5.gif" ALT="Figure 5"> View larger version (44K): org.highwire.dtl.DTLVardef@1de7f42org.highwire.dtl.DTLVardef@74e8ddorg.highwire.dtl.DTLVardef@1642847org.highwire.dtl.DTLVardef@907a23_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 5C_FLOATNO Distinct pre-cancer signs in epithelial and immune cells of LS para-carcinoma (A) The scRNA-seq datasets of LS-paraCA were integrated with those of the colons from healthy donors (HD) (N=3, respectively; the HD group had 7885 cells in total, whereas the LS-paraCA group had 4766 cells). A dominance of CEACAM5high epithelial cells were identified in LS-paraCA. The sample from the patient with relapsed LS is marked with an arrow in the bar plot (left panel). (B) The portion of CEACAM5high epithelial cells, infiltrated CD8_T cells and macrophages was quantified. The sample from the relapsed LS patient is marked with arrows. (C, E) Top marker genes co-expressed in CEACAM5high epithelial cells and the enriched pathways are illustrated in the gene expression heatmap. Cells from the Enterocytes, Tufts, and Goblet subtypes were used as controls. (D, F) Quantification of the expression of CEACAM5 in epithelial cells of HD and LS-paraCA cells. C_FIG O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig3.gif" ALT="Figure 3"> View larger version (48K): org.highwire.dtl.DTLVardef@142c991org.highwire.dtl.DTLVardef@1bf5d99org.highwire.dtl.DTLVardef@99edd5org.highwire.dtl.DTLVardef@1cd5c15_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 3C_FLOATNO Distinct infiltration of immune cells in carcinoma and para-carcinoma tissues of LS (A) Fresh paired carcinoma (LS-CA) and adjacent para-carcinoma (LS-paraCA) biopsies of colon tissue was subjected to scRNA-seq analysis (N=3). Colorectal cancer (nonLS-CRC, they are not diagnosed as LS) tumor samples from the public dataset were also included as controls (N=4). Cells were first grossly annotated as stromal, epithelial, or immune cells. Note that the LS-CA group showed increased infiltration of myeloid cells compared with LS-paraCA group, but T cell infiltration was comparable between the two groups. (B) Expression of classic markers for annotating the major 5 cell types. (C) Detailed annotations of 16 cell types in paired LS-CA and LS-paraCA samples. (D) Stacked bar plot showing the contribution of sample sources in each cell type. (E) Quantification of immune cell infiltration in LS-CA compared with LS-paraCA. Although no significant differences were observed, the fold-changes were approximately 2-10. Although the overall T cell infiltration was comparable, infiltration of GZMA+CD8_T and GZMK+CD8_T cells appeared to be higher in LS-CA than in LS-paraCA. Of the three LS patients enrolled in the study, one was a relapsed patient (gray dashed line, surgery treatment performed; whose son donated the frozen sample in Figure 2), while the other two were treatment-naive (gray solid lines). (F) Boxplots show DNA repair score quantification per sample (above) or per epi-cell. (bottom). C_FIG O_FIG O_LINKSMALLFIG WIDTH=152 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig4.gif" ALT="Figure 4"> View larger version (51K): org.highwire.dtl.DTLVardef@1588458org.highwire.dtl.DTLVardef@58db3borg.highwire.dtl.DTLVardef@1a12084org.highwire.dtl.DTLVardef@15de70e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 4C_FLOATNO Prioritizing novel epithelial biomarkers for tracing the transition between para-carcinoma and carcinoma in LS (LS-paraCA vs. LS-CA) (A) Epithelial cells were filtered out for further analysis and annotated as clusters 0-9 in the UMAP plot. (B, C) Bar and pie plots show the contribution of sources to epithelial clusters. Cluster 0 contained cells largely from Lynch CA_02, whereas Cluster 9 was composed of Lynch paraCA_02. The patient who donated Lynch CA_02 and paraCA_02 is identified as a relapsed LS. (D) KEGG enrichment analysis showing the enriched bioprocesses in the four epithelial cell clusters. (E) Five genes with upregulated expression in LS carcinoma epithelial cells were prioritized based on the scRNA-seq and snRNA-seq datasets. (F) Expression of BACE2, GPRC5A and OLFM4 in the paired LS-paraCA and LS-CA groups. Epithelial cells are circled by dashed lines. Note that the expression of genes in epithelial cells from nonLS-CRC samples was also very strong. (G) Verification of the low-to-high transition in the expression of BACE2, GPRC5A and OLFM4. Paraffin sections from paired LS-paraCA and LS-CA samples were prepared, and immunohistochemical (IHF) staining was performed. The expression of the indicated proteins is labeled in green, whereas the nuclei are stained with DAPI in blue. In total, biopsy samples from 4 patients with LS were used for IHF verification. C_FIG O_FIG O_LINKSMALLFIG WIDTH=148 HEIGHT=200 SRC="FIGDIR/small/647495v1_fig6.gif" ALT="Figure 6"> View larger version (28K): org.highwire.dtl.DTLVardef@1ec5210org.highwire.dtl.DTLVardef@1ffd6f9org.highwire.dtl.DTLVardef@17a7a8forg.highwire.dtl.DTLVardef@4819e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 6C_FLOATNO Increased mutation burden in the colon tissues from Lynch Syndrome (A) The schematic diagram for calculating mutation burden based on the scRNA-seq datasets using the SComatic algorithm. Only single-base substitutions (SBS) were called in this study. Once mutation calls were achieved, the mutation burden value was quantified by normalizing the total mutation calls to the total cell number in the samples or cell types (See Methods section for details). (B) The total SBS mutation burden in the four groups of samples was indicated. (C) The six different SBS mutation burdens in the four groups of samples as indicated. (D) The SBS mutation burden in the cell types of the four groups is indicated. Note that the The mutation burden appears to be higher in mast, epithelial, plasma, myeloid, and Treg cells in the LS-CA group than in that the LS-paraCA group. C_FIG

cancer biology↗
bioRxiv2025

Flagellar pocket collar biogenesis: Cytoskeletal organization and novel structures in a unicellular parasite

Understanding how cells build and organize their internal structures is a fundamental question in biology, with important implications for human health and disease. Trypanosomes are single-celled flagellated parasites that cause life-threatening diseases in human and animals. Their survival relies on a specialized compartment called the flagellar pocket (FP), which serves as a gateway for nutrient uptake, and immune evasion. The formation and function of the FP are supported by an intricate cytoskeletal structure known as the flagellar pocket collar (FPC). However, the mechanisms underlying its assembly remain poorly understood. In this study, we used cutting-edge ultrastructure expansion microscopy (U-ExM) to investigate FPC biogenesis in Trypanosoma brucei. We mapped the formation of the new microtubule quartet (nMtQ) alongside flagellum growth, providing new insights into its assembly. Additionally, we tracked the localization dynamics of key structural proteins - BILBO1, MORN1, and BILBO2 - during the biogenesis of the FPC and the hook complex (HC). Notably, we identified two previously undetected structures: the proFPC and the transient FPC-interconnecting fibre (FPC-IF), both of which appear to play crucial roles in linking and organizing cellular components during cell division. By uncovering these novel aspects of FPC biogenesis, our study significantly advances the understanding of cytoskeletal organization in trypanosomes and opens new avenues for exploring the functional significance of these structures.

microbiology↗
bioRxiv2025

Recurrent patterns of widespread neuronal genomic damage shared by major neurodegenerative disorders

Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimers disease (AD) represent two major categories of neurodegenerative disorders--TDP-43 and tau proteinopathies--for which the mechanisms driving neuronal death remain unclear. Single-cell whole-genome sequencing of 469 neurons from C9ORF72 ALS, C9ORF72 FTD, AD, and control brains revealed increased somatic single nucleotide variants (sSNVs) and insertions/deletions (sIndels) in all three diseases. Mutational signature analysis identified a disease-associated sSNV signature consistent with oxidative damage and an sIndel process affecting 22% of ALS, 76% of FTD, and 61% of AD neurons--but only 2% of control neurons--resembling signature ID4, previously linked to topoisomerase 1 (TOP1)-mediated mutagenesis. Rapid Approach to DNA Adduct Recovery (RADAR) assays confirmed increased TOP1-DNA covalent complexes and duplex sequencing confirmed the increased sIndels and identified single-strand events as likely precursor lesions. TOP1-associated sIndel mutagenesis and genome instability thus represents a mechanism shared by both TDP-43 and tau neurodegeneration.

genomics↗
bioRxiv2025

Diverse somatic genomic alterations in single neurons in chronic traumatic encephalopathy

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease that is linked to exposure to repetitive head impacts (RHI), yet little is known about its pathogenesis. Applying two single-cell whole-genome sequencing methods to hundreds of neurons from prefrontal cortex of 15 individuals with CTE, and 4 with RHI without CTE, revealed increased somatic single-nucleotide variants in CTE, resembling a pattern previously reported in Alzheimers disease (AD). Furthermore, we discovered remarkably high burdens of somatic small insertions and deletions in a subset of CTE individuals, resembling a known pattern, ID4, also found in AD. Our results suggest that neurons in CTE experience stereotyped mutational processes shared with AD; the absence of similar changes in RHI neurons without CTE suggests that CTE involves mechanisms beyond RHI alone.

genomics↗
bioRxiv2025

Depicting pathogenesis of osteomyelitis by single cell RNA-sequencing and an involvement of Morrbid in the autoinflammatory disease

Autoinflammatory diseases (AIDs) are defined as abnormal activation of the innate immune system leading to spontaneous and uncontrolled inflammation. The AIDs affect bone tissue and lead to chronic recurrent multifocal osteomyelitis (CRMO). However, the etiology and treatment of CRMO remain elusive. A mouse strain, Pstpip2cmo/cmo (cmo: chronic multifocal osteomyelitis), exhibits phenotypic characteristics similar to human CRMO. Morrbid is a long non-coding RNA gene and has been indicated in leukemogenesis in our previous studies. In this study, we demonstrated that Morrbid and Pstpip2 are co-expressed in mature myeloid cells and hypothesized a role of Morrbid in osteomyelitis. The Pstpip2-/- mice have the same phenotype as Pstpip2cmo/cmo, mimicking CRMO, while loss of Morrbid in Pstpip2-/-mice significantly inhibited the initiation and progression of CRMO symptoms, as well as the dysregulated activation of myeloid cells and the excessive release of inflammatory cytokines. Furthermore, single-cell RNA-sequencing (scRNA-seq) analysis from the Pstpip2-/- mice and the compound mutant mice supports that reduction of osteoclasts and inflammatory cells caused by Morrbid loss. The study systematically profiles the etiology of CRMO by scRNA-seq and warrants that inhibiting the lifespan of inflammatory myeloid cells by targeting Morrbid is an effective therapeutic strategy for osteomyelitis. HighlightsO_LIWe generated a frameshift mutant mouse strain Pstpip2-/-, which have a classic CRMO-like phenotype as same as Pstpip2cmo/cmo and could be used for testing various anti-inflammation perturbations. C_LIO_LILoss of Morrbid significantly inhibited the autoinflammatory symptoms in Pstpip2-/- mice, suggesting that Morrbid is a novel target for mitigating CRMO. C_LIO_LIscRNA-seq analysis of the affected bone marrow cells in Pstpip2-/-revealed the abnormalities of osteoclasts (OC), neutrophils (NE) and granulocyte macrophage progenitors (GMP) in both their fractions and inflammatory activities. C_LIO_LIUpon loss of Morrbid, we observed reduced composition and proliferation of OC and decreased activity of Nfkb2 and Rela in the compound mutants. C_LI

immunology↗
bioRxiv2025

Single-cell and spatial transcriptomics reveal the pathogenesis of chronic granulomatous disease in a natural model

Genetic defects in NOX2 can cause chronic granulomatous disease (CGD), characterized by increased susceptibility to infections and pronounced inflammatory responses that lead to granuloma formation. We developed a CGD model using Ncf2-/- mice through controlled environmental exposure. Unlike in specific pathogen-free environment, these mice spontaneously developed pulmonary granulomas under clean-grade conditions. Employing single-cell RNA sequencing, we observed an expansion of neutrophils and monocyte-derived macrophages (MDMs) within the lung tissue, identifying pro-inflammatory NOS2high-neutrophils and a distinct MDMs subset marked by NOS2 and ARG1. Spatial transcriptomics demonstrated that NOS2high-neutrophils were located at the core area, while the MDMs subset was positioned at the periphery, facilitating extracellular matrix remodeling. Pharmacological targeting of MIF, deletion of the pro-survival gene Morrbid, and elimination of Il1r1 all suppressed granuloma formation by mitigating inflammation. This study underscores how environmental control can establish a natural CGD model, elucidates the mechanisms of granuloma formation, and develops potent therapeutic interventions. HighlightsO_LIThrough installing a mutation in a NOX2 subunit gene Ncf2 and changing the husbandry environment, we generated a natural CGD mouse model with progressive pulmonary granuloma formation. C_LIO_LINOS2high neutrophils located in the core region of CGD lung granulomas, exhibit a pro-inflammatory transcriptional profile. C_LIO_LIMonocyte-derived macrophages marked by Nos2 and Arg1 are located at the periphery of granulomas and exhibit a profibrotic transcriptional profile. C_LIO_LIThe transcriptomic studies assist the development of three effective perturbations for treating the rare disease CGD via targeting Il1r1, Morrbid, and Mif respectively. C_LI

immunology↗
bioRxiv2024

Continual integration of single-cell multimodal data with MIRACLE

Single-cell sequencing technologies have revolutionized our understanding of cellular heterogeneity and facilitated the construction of multi-omics cell atlases via data integration. However, updating these atlases with new data conventionally requires reintegration of all data and is computationally intensive, hindering timely updates and dynamic adjustments in biological and medical research. To address this challenge, we present Multimodal Integration with Continual Learning (MIRACLE), a novel online learning framework for the adaptive and efficient integration of single-cell multimodal data. MIRACLE employs dynamic architectures and data rehearsal strategies to support continual learning, allowing diverse data to be integrated while minimizing information loss over time. Our evaluations demonstrate that MIRACLE achieves accurate online integration with reduced computational requirements, effectively updating and expanding atlases with new cross-tissue and cross-modal data, and precisely identifying novel cell types and transferring labels across datasets. MIRACLE provides an efficient and flexible tool for single-cell community to integrate, share and explore biological knowledge from single-cell multimodal data.

bioinformatics↗
bioRxiv2024

A regulatory module driving the recurrent evolution of irreducible molecular complexes.

To sustain life, molecular complexes require the concerted action of multiple proteins, each relying on one another to perform intricate tasks. However, how such interdependent protein interactions evolve in the first place is poorly understood. To address this, we investigated the origins of a group of fast-evolving genetic parasites--toxin-antidote elements--which boil down this dilemma to a simple question: what came first, the toxin or the antidote? By integrating quantitative genetics, biochemistry, and evolutionary genomics, we discovered that toxins and antidotes can arise simultaneously through the duplication of a regulatory module comprising an F-box protein in linkage to its substrate. Our findings provide one solution to the recurrent emergence of mutual dependence in protein complexes and illustrate in detail how complexity can swiftly arise from simplicity.

genomics↗
bioRxiv2024

Somatic mutation phasing and haplotype extension using linked-reads in multiple myeloma

Somatic mutation phasing informs our understanding of cancer-related events, like driver mutations. We generated linked-read whole genome sequencing data for 23 samples across disease stages from 14 multiple myeloma (MM) patients and systematically assigned somatic mutations to haplotypes using linked-reads. Here, we report the reconstructed cancer haplotypes and phase blocks from several MM samples and show how phase block length can be extended by integrating samples from the same individual. We also uncover phasing information in genes frequently mutated in MM, including DIS3, HIST1H1E, KRAS, NRAS, and TP53, phasing 79.4% of 20,705 high-confidence somatic mutations. In some cases, this enabled us to interpret clonal evolution models at higher resolution using pairs of phased somatic mutations. For example, our analysis of one patient suggested that two NRAS hotspot mutations occurred on the same haplotype but were independent events in different subclones. Given sufficient tumor purity and data quality, our framework illustrates how haplotype-aware analysis of somatic mutations in cancer can be beneficial for some cancer cases.

bioinformatics↗
bioRxiv2023

The C-terminus of CFAP410 forms a tetrameric helical bundle that is essential for its localization to the basal body

Cilia are antenna-like organelles protruding from the surface of many cell types in the human body. Defects in ciliary structure or function often lead to diseases that are collectively called ciliopathies. Cilia and flagella associated protein 410 (CFAP410) localizes at the basal body of cilia/flagella and plays essential roles in ciliogenesis, neuronal development, and DNA damage repair. It remains unknown how its specific basal body location is achieved. Multiple single amino acid mutations in CFAP410 have been identified in patients with various ciliopathies. One of the mutations, L224P, is located in the C-terminal domain (CTD) of human CFAP410 and causes severe spondylometaphyseal dysplasia, axial (SMDAX). However, the molecular mechanism for how the mutation causes the disorder remains unclear. Here, we report our structural studies on the CTD of CFAP410 from three distantly related organisms, Homo sapiens, Trypanosoma brucei, and Chlamydomonas reinhardtii. The crystal structures reveal that the three proteins all adopt the same conformation as a tetrameric helical bundle. Our work further demonstrates that the tetrameric assembly of the CTD is essential for the correct localization of CFAP410 in T. brucei, as the L224P mutation that disassembles the tetramer disrupts its basal body localization. Taken together, our studies reveal that the basal body localization of CFAP410 is controlled by the CTD and provide a mechanistic explanation for how the mutation L224P in CFAP410 causes ciliopathies in humans.

biochemistry↗
bioRxiv2023

A novel plasmid-based experimental system in Saccharomyces cerevisiae that enables the introduction of 10 different plasmids into cells

The budding yeast Saccharomyces cerevisiae is commonly used as an expression platform to produce valuable compounds. Yeast-based genetics research can uniquely utilize use auxotrophy in transformant selection: auxotrophic complementation by an auxotrophic marker gene on exogenous DNA (such as plasmids). However, the number of auxotrophic nutrients required restricts the number of plasmids maintained by the cells. We therefore developed novel {Delta}10 strains that are auxotrophic for 10 different nutrients, and new plasmids with two multicloning sites and auxotrophic markers for use in {Delta}10 strains. We confirmed that {Delta}10 strains could maintain 10 types of these plasmids. Because each plasmid can express two different genes, this {Delta}10 strain-based expression system has the potential to co-express a maximum of 20 different proteins.

bioengineering↗
bioRxiv2023

Chitosan oligosaccharide induces plant resistance gene expression in Pinus massoniana

Chitosan oligosaccharides are the main degradation products from chitosan or chitin and have been reported to induce resistance to diseases in herbaceous plants like cucumber and Arabidopsis. Concomitantly, pine wilt disease is a devastating disease of conifer tree species. Here, we hypothesized that chitosan oligosaccharides induce plant resistance gene (PRG) expression in the woody plant Masson pine, Pinus massoniana. Chitosan oligosaccharides were inoculated into P. massoniana seedlings and the BGISEQ-500 platform was used to generate transcriptomes from chitosan oligosaccharide-treated P. massoniana and control seedlings. A total of 501 differentially expressed genes (DEGs) were identified by comparing the treatment and control groups. A total of 251 (50.1%) DEGs were up-regulated in the treatment relative to the control seedlings and 250 (49.9%) were down-regulated. Inoculation of chitosan oligosaccharide induced the expression of 31 PRGs in P. massoniana seedlings and the relative expression levels of six of the PRGs were verified by RT-qPCR. This is the first study to demonstrate that chitosan oligosaccharide induces the expression of PRGs in a tree species. These results provide important insights into the function of chitosan oligosaccharides and further the prospects of developing a chitosan oligosaccharide-based immune inducer for controlling pine wilt disease.

genomics↗
bioRxiv2022

MIDAS: a deep generative model for mosaic integration and knowledge transfer of single-cell multimodal data

AO_SCPLOWBSTRACTC_SCPLOWRapidly developing single-cell multi-omics sequencing technologies generate increasingly large bodies of multimodal data. Integrating multimodal data from different sequencing technologies, i.e. mosaic data, permits larger-scale investigation with more modalities and can help to better reveal cellular heterogeneity. However, mosaic integration involves major challenges, particularly regarding modality alignment and batch effect removal. Here we present a deep probabilistic framework for the mosaic integration and knowledge transfer (MIDAS) of single-cell multimodal data. MIDAS simultaneously achieves dimensionality reduction, imputation, and batch correction of mosaic data by employing self-supervised modality alignment and information-theoretic latent disentanglement. We demonstrate its superiority to other methods and reliability by evaluating its performance in full trimodal integration and various mosaic tasks. We also constructed a single-cell trimodal atlas of human peripheral blood mononuclear cells (PBMCs), and tailored transfer learning and reciprocal reference mapping schemes to enable flexible and accurate knowledge transfer from the atlas to new data. Applications in mosaic integration, pseudotime analysis, and cross-tissue knowledge transfer on bone marrow mosaic datasets demonstrate the versatility and superiority of MIDAS.

bioinformatics↗
bioRxiv2022

Soft and stretchable organic bioelectronics for continuous intra-operative neurophysiological monitoring during microsurgery

Continuous intra-operative neurophysiological monitoring (CINM) that provides precise mapping of neural anatomy through the entire microsurgery process is essential to preserve the structural and functional integrity of the nerve. However, bulky and rigid electrodes used currently in clinics are usually unable to reliably maintain continuous and stable contacts with the vulnerable and complex nerve networks, thus often resulting in detrimental post-operative complications, such as hemiplegia and sensory disturbances. Here, we describe a biomechanically compatible, suture-free, and individually reconfigurable CINM based on soft and stretchable organic electronic materials. Due to both low impedance and modulus of our conducting polymer electrodes, we achieved for the first time continuous recording of near-field action potential with high signal-to-noise ratio and minimal invasiveness during microsurgeries. Utilizing this unprecedented CINM modality, in conjunction with localized neurostimulation, we further demonstrated our approach in enabling optimal post-operative prognosis in preclinical animal models by preserving normal neural functions after a variety of tumor resection surgeries.

bioengineering↗
bioRxiv2022

Structural studies of cilia and flagella associated protein 410 (CFAP410) reveal its bimodular organization with an N-terminal LRR motif and a C-terminal tetrameric helical bundle

Cilia and flagella associated protein 410 (CFAP410) is a protein localized at the basal body of cilia/flagella and plays essential roles in ciliogenesis. Multiple single amino acid mutations in CFAP410 have been identified in patients. However, the molecular mechanism for how the mutations cause these disorders remains poorly understood due to a lack of high-resolution structures of the protein. Our studies demonstrate that CFAP410 adopts a bimodular architecture. We have previously reported our structural studies on the C-terminal domain (CTD) of CFAP410 from various organisms. Here we report a 1.0-[A] resolution crystal structure of the N-terminal domain (NTD) of Trypanosoma brucei CFAP410. We further examined how the disease-causing mutations in this domain may affect the folding and structural stability of CFAP410. Our results suggest that the single-residue mutations in the CFAP410-NTD cause human diseases by destabilizing the structure that subsequently disrupts its interaction with other partners.

biochemistry↗
bioRxiv2022

Dictys: dynamic gene regulatory network dissects developmental continuum with single-cell multi-omics

Gene regulatory networks (GRNs) are key determinants of cell function and identity and are dynamically rewired during development and disease. Despite decades of advancement, challenges remain in GRN inference: dynamic rewiring, causal inference, feedback-loop modeling, and context specificity. To address them, we develop Dictys, a dynamic GRN inference and analysis method which leverages multi-omic single-cell assays of chromatin accessibility and gene expression, context specific transcription factor (TF) footprinting, stochastic process network, and efficient probabilistic modeling of scRNA-seq read counts. Dictys improves GRN reconstruction accuracy and reproducibility and enables the inference and comparative analysis of context specific and dynamic GRNs across developmental contexts. Dictys network analyses recover unique insights in human blood and mouse skin development with cell-type specific and dynamic GRNs. Its dynamic network visualizations enable time-resolved discovery and investigation of developmental driver TFs and their regulated targets. Dictys is available as a free, open source, and user-friendly Python package.

bioinformatics↗
bioRxiv2022

IGF-1 therapy improves muscle size and function in experimental peripheral arterial disease

CONDENSED ABSTRACTLower extremity peripheral arterial disease (PAD) has continued to increase in prevalence over the past several decades, yet therapeutic development has remained stagnant. Skeletal muscle health and function has been strongly linked to quality of life and medical outcomes in PAD patients. Using a rodent model of PAD, this study demonstrates that treatment of the ischemic limb with adeno-associated virus-mediated expression of insulin-like growth factor 1 (IGF1) significantly increases muscle size and strength, without improving limb hemodynamics. Interestingly, the effect size of IGF1 therapy was larger in female mice compared to their male counterparts, where substantial improvements in muscle specific force and a reduction in the progression of limb necrosis were observed. These findings indicate that clinical trials should carefully examine sex-dependent effects in experimental PAD therapies.

physiology↗
bioRxiv2022

Endocytosis is required for access of surface-bound cargo to the flagellar pocket of trypanosomes

All endo- and exocytosis in the African trypanosome Trypanosoma brucei occurs at a single subdomain of the plasma membrane. This subdomain, the flagellar pocket, is a small vase-shaped invagination containing the root of the cells single flagellum. Several cytoskeleton-associated multiprotein complexes are coiled around the neck of the flagellar pocket on its cytoplasmic face. One of these, the hook complex, was proposed to affect macromolecule entry into the flagellar pocket lumen. In previous work, knockdown of the hook complex component TbMORN1 resulted in larger cargo being unable to enter the flagellar pocket. In this study, the hook complex component TbSmee1 was characterised in bloodstream form Trypanosoma brucei and was found to be essential for cell viability. TbSmee1 knockdown resulted in flagellar pocket enlargement and impaired access to the flagellar pocket membrane by surface-bound cargo, similar to depletion of TbMORN1. Unexpectedly, inhibition of endocytosis by knockdown of clathrin phenocopied TbSmee1 knockdown, suggesting that endocytic activity itself is a prerequisite for the entry of surface-bound cargo into the flagellar pocket. SummaryCharacterisation of the essential trypanosome protein TbSmee1 suggests that endocytosis is required for flagellar pocket access of surface-bound cargo.

cell biology↗