Biology subjects

Fu, R.

Publications and source records attributed to Fu, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Mechanistic Insights into Proteomic Mutation-Phenotype Linkages from Tiling Mutagenesis Screens

High-throughput mutagenesis screens are powerful tools for mapping mutations to phenotypes. However, deciphering the molecular mechanisms that link mutations to phenotypic outcomes remains a significant challenge. Here, we present ProTiler-Mut, a versatile computational framework that harnesses tiling mutagenesis screens, which introduce variants across entire protein sequences, to facilitate investigation of mutation-to-phenotype associations at multiple levels, including individual residues, protein substructures, and protein-protein interactions (PPIs). As demonstrated through our analyses of base editing (BE) screens targeting DNA Damage Response (DDR) proteins and T cell regulators, ProTiler-Mut provides novel insights into the mutation-phenotype linkages, including: i) refined classification of mutation that reveals separation-of-function (SOF) category beyond the conventional binary classification of loss-of-function (LOF) and gain-of-function (GOF); ii) definition of phenotype-associated hotspot substructures that enable the inference of the function of unscreened pathogenic mutations; and iii) identification of phenotype-associated PPIs disrupted by functional mutations. Through ProTiler-Mut analyses, we identified a substructure harboring pathogenic GOF mutations that disrupt interactions between the kinases MAPK1 and RSK1, leading to MAPK1 activation and elevated expression of the immune checkpoint receptor PD-1. Furthermore, we demonstrate the applicability of ProTiler-Mut to various mutagenesis screening platforms, highlighting its broad utility and generalizability.

bioinformatics↗

bioRxiv2025

Proton-Detected Solid-State NMR for Deciphering Structural Polymorphism and Dynamic Heterogeneity of Cellular Carbohydrates in Pathogenic Fungi

Carbohydrate polymers in their cellular context display highly polymorphic structures and dynamics essential to their diverse functions, yet they are challenging to analyze biochemically. Proton-detection solid-state NMR spectroscopy offers high isotopic abundance and sensitivity, enabling rapid and high-resolution structural characterization of biomolecules. Here, an array of 2D/3D 1H-detection solid-state NMR techniques are tailored to investigate polysaccharides in fully protonated or partially deuterated cells of three prevalent pathogenic fungi: Rhizopus delemar, Aspergillus fumigatus, and Candida albicans, representing filamentous species and yeast forms. Selective detection of acetylated carbohydrates reveals fifteen forms of N-acetylglucosamine units in R. delemar chitin, which coexists with chitosan as separate domains or polymers and associates with proteins only at limited sites. This is supported by distinct order parameters and effective correlation times of their motions, analyzed through relaxation measurements and model-free analysis. Five forms of -1,3-glucan with distinct structural origins and dynamics were identified in A. fumigatus, important for this buffering polysaccharide to perform diverse roles of supporting wall mechanics and regenerating soft matrix under antifungal stress. Eight -1,2-mannan sidechain variants in C. albicans were resolved, highlighting the crucial role of mannan sidechains in maintaining interactions with other cell wall polymers to preserve structural integrity. These methodologies provide novel insights into the functional structures of key fungal polysaccharides and create new opportunities for exploring carbohydrate biosynthesis and modifications across diverse organisms.

biochemistry↗

bioRxiv2025

Anionic lipid catalyzes the generation of cytotoxic insulin oligomers

Misfolding and aggregation of proteins into amyloidogenic assemblies are key features of several metabolic and neurodegenerative diseases. Human insulin has long been known to form amyloid fibrils under various conditions, which affects its bioavailability and function. Clinically, insulin aggregation at recurrent injection sites poses a challenge for diabetic patients who rely on insulin therapy. Furthermore, decreased responsiveness to insulin in type 2 diabetic (T2D) patients may lead to its overproduction and accumulation as aggregates. Earlier reports have reported that various factors such as pH, temperature, agitation, and the presence of lipids or other proteins influence insulin aggregation. Our present study aims to elucidate the effects of non-micellar anionic DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) lipids on insulin aggregation. Distinct pathways of insulin aggregation and intermediate formation were observed in the presence of DMPG using a ThT fluorescence assay. The formation of soluble intermediates, alongside large insulin fibrils, was observed in insulin incubated with DMPG via TEM, DLS and NMR, as opposed to insulin aggregates generated without lipids. 13C magic angle spinning solid-state NMR and FTIR experiments indicated that lipids do not alter the conformation of insulin fibrils but do alter the time scale of motion of aromatic and aliphatic sidechains. Furthermore, the soluble intermediates were found to be more cytotoxic as compared to fibrils generated with or without lipids. Overall, our study elucidates the importance of anionic lipids in dictating the pathways and intermediates associated with insulin aggregation. These findings could be useful in determining various approaches to avoid toxicity and enhance the effectiveness of insulin in therapeutic applications.

biophysics↗

bioRxiv2024

Mycobacterium tuberculosis CrgA Forms a Dimeric Structure with Its Transmembrane Domain Sandwiched between Cytoplasmic and Periplasmic β-Sheets, Enabling Multiple Interactions with Other Divisome Proteins

CrgA is a key transmembrane (TM) protein in the cell division process of Mycobacterium tuberculosis (Mtb), the pathogen responsible for tuberculosis. While many of the Mtb divisome proteins have been identified, their structures and interactions remain largely unknown. Previous studies of CrgA using oriented-sample solid-state NMR have defined the tilt and rotation of the TM helices, but the cytoplasmic and periplasmic domains and even the oligomeric state were uncharacterized. Here, combining oriented-sample and magic-angle spinning solid-state NMR spectra, we solved the full-length structure of CrgA. The structure features a dimer with a TM domain sandwiched between a cytoplasmic {beta}-sheet and a periplasmic {beta}-sheet. The {beta}-sheets stabilize dimerization, which in turn increases CrgAs ability to participate in multiple protein interactions. Within the membrane, CrgA binds FtsQ, CwsA, PbpA, FtsI, and MmPL3 via its TM helices; in the cytoplasm, Lys23 and Lys25 project outward from the {beta}-sheet to interact with acidic residues of FtsQ and FtsZ. The structural determination of CrgA thus provides significant insights into its roles in recruiting other divisome proteins and stabilizing their complexes for Mtb cell wall synthesis and polar growth.

biophysics↗

bioRxiv2024

3D Printing of Multiscale Scaffolds with Microtopography for Guiding Tissue Organization and Regeneration

Engineering biomaterial scaffolds with multiscale structures, integrating anatomically accurate macroscale architecture (millimeters to centimeters) with microtopographic features (sub-microns to tens of microns), is critical for guiding cellular organization and tissue regeneration. However, fabricating such multiscale scaffolds remains challenging due to the limitations of conventional manufacturing techniques and the trade-off between speed and resolution in current 3D printing methods. Here, we present a multiscale micro-continuous liquid interface production (M{micro}CLIP) technique that enables rapid, one-step 3D printing of centimeter-scale scaffolds with spatially tunable microtopography of various sizes and geometries in just a few minutes (up to 1 mm/min). To showcase the versatility of our technique, we printed a one-centimeter-long tubular scaffold with dual microtopographic patterns, i.e. 20 {micro}m axially aligned grooves on the internal surface and 15 {micro}m circumferentially aligned rings on the external surface. These scaffolds induced the simultaneous orientation of vascular endothelial cells along the axial grooves and vascular smooth muscle cells along the circumferential rings, mimicking the orthogonally aligned bilayer architecture of natural arteries. Moreover, the groove patterns significantly accelerated endothelial cell migration, potentially enhancing endothelialization in vascular implants. This approach provides a versatile tool for designing advanced scaffolds and medical devices that harness microtopography to guide tissue organization and enhance regeneration.

bioengineering↗

bioRxiv2024

Dissecting cell-free DNA fragmentation variation in tumors using cell line-derived xenograft mouse

Cell-free DNA (cfDNA) is increasingly studied for its diverse applications in non-invasive detection. Non-randomly cleaved by nucleases and released into the bloodstream, cfDNA exhibits a variety of intrinsic fragmentation patterns indicative of cell status. Particularly, these fragmentation patterns have recently been demonstrated to be effective in predicting cancer and its tissue-of-origin, owing to increased variation of fragmentation features observed in tumor patients. However, there remains a lack of detailed exploration of altered cfDNA fragmentation profiles in tumors, which consist of a mixture of both non-tumor cfDNA and circulating tumor DNA (ctDNA). Hence, we leveraged the human tumor cell line-derived xenograft (CDX) mouse model, where different tumor cell lines were implanted into different anatomical sites, to isolate pure ctDNA and separately investigate the fragment properties of CDX-induced cfDNA and ctDNA. We found an enrichment of short cfDNA fragments in both CDX-induced cfDNA and ctDNA compared to normal plasma cfDNA, with more elevated short fragments in ctDNA. Moreover, the CDX-induced cfDNA fragmentation features distinguished between CDX models of different tumor cell lines, while the ctDNA fragmentation features conversely discriminate between CDX models of different anatomical sites. The results suggested that both non-tumor cfDNA and ctDNA contribute to the increased variation observed in tumors, and that cfDNA fragmentation may be highly variable and susceptible to regulations by both original cells and cells within the local niche.

cancer biology↗

bioRxiv2024

Multi-pronged analysis of pediatric low-grade glioma reveals a unique tumor microenvironment associated with BRAF alterations

Pediatric low-grade gliomas (pLGG) comprise 35% of all brain tumors. Despite favorable survival, patients experience significant morbidity from disease and treatments. A deeper understanding of pLGG biology is essential to identify novel, more effective, and less toxic therapies. We utilized single cell RNA sequencing (scRNA-seq), spatial transcriptomics, and cytokine analyses to characterize and understand tumor and immune cell heterogeneity across pLGG. scRNA-seq revealed tumor and immune cells within the tumor microenvironment (TME). Tumor cell subsets revealed a developmental hierarchy with progenitor and mature cell populations. Immune cells included myeloid and lymphocytic cells. There was a significant difference between the prevalence of two major myeloid subclusters between pilocytic astrocytoma (PA) and ganglioglioma (GG). Bulk and single-cell cytokine analyses evaluated the immune cell signaling cascade with distinct immune phenotypes among tumor samples. KIAA1549-BRAF tumors appeared more immunogenic, secreting higher levels of immune cell activators and chemokines, compared to BRAF V600E tumors. Spatial transcriptomics revealed the differential gene expression of these chemokines and their location within the TME. A multi-pronged analysis of pLGG demonstrated the complexity of the pLGG TME and differences between genetic drivers that may influence their response to immunotherapy. Further investigation of immune cell infiltration and tumor-immune interactions is warranted. Key pointsO_LIThere is a developmental hierarchy in neoplastic population comprising of both progenitor-like and mature cell types in both PA and GG. C_LIO_LIA more immunogenic, immune activating myeloid population is present in PA compared to GG. C_LIO_LIFunctional analysis and spatial transcriptomics show higher levels of immune mobilizing chemokines in KIAA1549-BRAF fusion PA tumor samples compared to BRAF V600E GG samples. C_LI Importance of the StudyWhile scRNA seq provides information on cellular heterogeneity within the tumor microenvironment (TME), it does not provide a complete picture of how these cells are interacting or where they are located. To expand on this, we used a three-pronged approach to better understand the biology of pediatric low-grade glioma (pLGG). By analyzing scRNA-seq, secreted cytokines and spatial orientation of cells within the TME, we strove to gain a more complete picture of the complex interplay between tumor and immune cells within pLGG. Our data revealed a complex heterogeneity in tumor and immune populations and identified an interesting difference in the immune phenotype among different subtypes.

cancer biology↗

bioRxiv2024

SKP2-mediated FBXO2 proteasomal degradation drives hepatocellular carcinoma progression via stabilizing Hsp47

Accumulating studies highlight that dysregulated E3 ubiquitin ligases are associated with the onset and advancement of cancers. Nevertheless, the impact and mechanism of most E3 ubiquitin ligases on tumorigenesis and tumor metastasis remain poorly understood. Here, we show that loss of FBXO2, an E3 ubiquitin ligase, accelerates hepatocellular carcinoma (HCC) tumor growth and metastasis to the lung through stabilizing heat shock protein 47 (Hsp47). Downregulation of FBXO2, caused by DNA-PKcs-medicated phosphorylation at serine 17 and E3 ligase SKP2-mediated ubiquitination at lysine 79 and subsequent proteasomal degradation, is observed in tumor tissues compared to their parallel non-tumor tissues resected from patients with HCC. Patients whose tumors are enriched for SKP2 or Hsp47 or express low levels of FBXO2 have poor median survival compared to those whose tumors have reversed levels of SKP2, FBXO2 and Hsp47. Together, FBXO2 acts as a tumor suppressor in HCC development. The components of the SKP2-FBXO2-Hsp47 axis provide newly prognostic and therapeutic factors for anti-HCC.

cancer biology↗

bioRxiv2024

Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay

Nonsense variants underlie many genetic diseases. The phenotypic impact of nonsense variants is determined by nonsense-mediated mRNA decay (NMD), which degrades transcripts with premature termination codons (PTCs). Despite its clinical importance, the factors controlling transcript-specific and context-dependent variation in NMD activity remain poorly understood. Through analysis of human genetic datasets, we discovered that the amino acid preceding the PTC strongly influences NMD activity. Notably, glycine codons promote robust NMD efficiency and show striking enrichment before PTCs but depletion before normal termination codons (NTCs). This glycine-PTC enrichment is particularly pronounced in genes tolerant to loss-of-function variants, suggesting evolutionary selection or neutrality conferred by efficient elimination of truncated proteins from non-essential genes. Using biochemical assays and massively parallel reporter analysis, we demonstrated that the peptide release rate during translation termination varies substantially with the identity of the preceding amino acid and serves as the primary determinant of NMD activity. We propose a "window of opportunity" model where translation termination kinetics modulate NMD efficiency. By revealing how sequence context shapes NMD activity through translation termination dynamics, our findings provide a mechanistic framework for improved clinical interpretation of nonsense variants.

molecular biology↗

bioRxiv2023

AP-1 regulates heterogeneous cellular dormancy in TNBC

BackgroundDormant or slow cycling cells (SSCs) pre-exist in tumor and responsible for chemo-resistant and tumor recurrence. Due to their low differentiation and dormancy characteristics, SCCs are resistant to standard chemotherapy and targeted therapy. Label-retaining is a common method used to identify and isolate live SCCs. However, it remains unclear whether different label-retaining methods yield distinct SCC subpopulations. In this study, we investigated that various label-retaining methods result in overlapping yet heterogeneous subpopulations of SCCs. Additionally, we explored the molecular mechanisms regulating dormancy in triple-negative breast cancer (TNBC). MethodsWe employed multiple label-retaining methods to simultaneously label MDA-MB-231 cells, thereby generating distinct subpopulations of SCCs. We subsequently analyzed these subpopulations for heterogeneity in cell cycle distribution, drug resistance, invasive capacity, and other characteristics using real-time PCR, flow cytometry, and Transwell assays. RNA-seq analysis was performed to characterize the gene expression profiles of the SCCs. Furthermore, we used real-time PCR, Western blotting, immunofluorescence, and luciferase assays to investigate the role of characteristic AP-1 expression in dormancy regulation. Finally, the therapeutic effects of targeting AP-1 in the treatment of TNBC were assessed using a cell-derived xenograft model. ResultsWe labeled and separated three overlapping but non-identical SCCs subpopulations. We found that all three SCCs subgroups are cell cycle arrested. Additionally, Violet enriched SCCs showed stronger drug resistance and more G1 phase arrest, while Claret enriched SCCs demonstrated enhanced migratory and invasive abilities, along with more G2/M phase arrest. Furthermore, we observed upregulation of AP-1 expression in SCCs, and the JunB subunit of AP-1 promoted the expression of CDKN1A and GADD45A, thereby maintaining cell cycle arrest. CC-930 can inhibit AP-1 transcriptional activity by suppressing JNK activity, ultimately improving the therapeutic efficacy and prognosis of TNBC when used in combination with chemotherapy drugs. ConclusionsWe obtained three subpopulations of SCCs with heterogeneous drug resistance. Our findings suggest that AP-1 plays a regulatory role in dormancy regulation in TNBC, and elucidated the molecular function of JunB subunit. Targeting AP-1 with CC-930 has the potential to improve the treatment and prognosis of TNBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/566980v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@129ce76org.highwire.dtl.DTLVardef@1b1cfa1org.highwire.dtl.DTLVardef@b68315org.highwire.dtl.DTLVardef@57faf1_HPS_FORMAT_FIGEXP M_FIG C_FIG TNBC harbors both fast-cycling cells (FCCs) and functionally overlapping slow-cycling cell (SCC) subpopulations that manifest differential drug sensitivities and motility (A) but are commonly regulated by the JunB-containing AP1 complex (B). (A). Slow-cycling (quiescent) TNBC cells in culture (a) identified by different label-retaining approaches phenotypically overlap (b), display differential drug sensitivities (c, d) and motility (e) but share common gene expression profiles (f). (B). Schematic depicting regulation of proliferation in FCCs by the c-Jun/c-Fos AP1 complex (left) and regulation of cellular dormancy in SCCs by c-Jun/JunB AP1 complex.

cancer biology↗

bioRxiv2023

3D-printed, Citrate-based Bioresorbable Vascular Scaffolds for Coronary Artery Angioplasty

Fully bioresorbable vascular scaffolds (BVSs) were designed to overcome the limitations of metallic drug-eluting stents (DESs). However, current polymer-based BVSs, such as Abbotts Absorb, the only US FDA-approved BVS, struggle with increased strut thickness (150 m for Absorb) and exacerbated tissue inflammation, leading to inferior clinical performance compared to metallic DESs. Here we develop a drug-eluting BVS (DE-BVS) through the innovative use of photopolymerizable, citrate-based materials and high-precision additive manufacturing process. Bare BVS with a clinically relevant strut thickness of 62 m can be produced in a high-throughput manner, i.e. one BVS per minute. By modulating the coating polymer and structure, we achieve a controlled release of anti-restenosis drug of everolimus from DE-BVSs. We show the mechanical competence of DE-BVS and the successful deployment in swine coronary arteries using a custom-built balloon catheter delivery system. We further demonstrate that BVS and DE-BVS remain safe and effective to keep the vessel patency, induce limited inflammation, and facilitate the recovery of smooth muscle and endothelial tissues over 28 days implantation in swine coronary arteries. All these evaluated pre-clinical performances are largely comparable to the commercial XIENCE DES (Abbott Vascular).

bioengineering↗

bioRxiv2023

Investigation of 3D Printed Bioresorbable Vascular Scaffold Crimping Behavior

The rise in additive manufacturing (AM) offers myriad opportunities for 3D-printed polymeric vascular scaffolds, such as customization and on-the-spot manufacturing, in vivo biodegradation, incorporation of drugs to prevent restenosis, and visibility under X-ray. To maximize these benefits, informed scaffold design is critical. Polymeric bioresorbable vascular scaffolds (BVS) must undergo significant deformation prior to implantation in a diameter-reduction process known as crimping which enables minimally invasive surgery. Understanding the behavior of vascular scaffolds in this step provides twofold benefits: first, it ensures the BVS is able to accommodate stresses occurring during this process to prevent failure, and further, it provides information on the radial strength of the BVS, a key metric to understanding its post-implant performance in the artery. To capitalize on the fast manufacturing speed AM provides, a low time cost solution for understanding scaffold performance during this step is necessary. Through simulation of the BVS crimping process in ABAQUS using experimentally obtained bulk material properties, we have developed a qualitative analysis tool which is capable of accurately comparing relative performance trends of varying BVS designs during crimping in a fraction of the time of experimental testing, thereby assisting in the integration of informed design into the additive manufacturing process.

bioengineering↗

bioRxiv2023

Co-regulator activity of Mediator of DNA Damage Checkpoint 1 (MDC1) is associated with DNA repair dysfunction and PARP inhibitor sensitivity in lobular carcinoma of the breast

Invasive lobular carcinoma of the breast (ILC) is typically estrogen receptor (ER)-positive and presents with biomarkers of anti-estrogen sensitive disease, yet patients with ILC face particularly poor long-term outcomes with increased recurrence risk, suggesting endocrine response and ER function are unique in ILC. ER is co- regulated by the DNA repair protein Mediator of DNA Damage Checkpoint 1 (MDC1) specifically in ILC cells, driving distinct ER activity. However, this novel MDC1 activity is associated with dysfunctional canonical DNA repair activity by MDC1, but without typical features of DNA repair deficiency. To understand reciprocal activities of MDC1, we profiled the MDC1 interactome and found MDC1-associated proteins in ILC cells mirror a "BRCA-like" state lacking key homologous recombination (HR) proteins, consistent with HR dysfunction but distinct from classic "BRCAness". HR dysfunction in ILC cells is supported by single-cell transcriptome and DNA repair activity analyses, with DNA repair signaling and functional data, showing dysfunctional induction and resolution of HR. In parallel, ILC tumor data are consistent with a distinct form of HR dysfunction via impaired HR resolution, lacking BRCA-like genomic scarring but showing elevated signatures of PARP inhibitor sensitivity. We demonstrate this HR dysfunction can be exploited using PARP inhibition, and found that talazoparib treatment produced a durable growth suppression both in vitro and in multiple ILC xenografts in vivo. ILC-specific ER:MDC1 activity creates a new context for ER and MDC1 function in ILC, at the cost of a DNA repair dysfunction, which may be therapeutically targetable. SignificanceILC is rarely associated with biomarkers of HR deficiency, and as such patients are rarely eligible for treatment with PARP inhibitors. Our work suggests ILC presents with a previously unappreciated form of HR dysfunction, linked to ILC-specific genomic activity of ER and MDC1, which imparts sensitivity to PARP inhibition.

cancer biology↗

bioRxiv2023

Global and local adaptation to aridity in a desert plant Gymnocarpos przewalskii

In order to thrive and survive plant species need to combine stability in the long term and rapid response to environmental challenges in the short term. The former would be reflected by global adaptation across species and the latter by pronounced local adaptation among populations of the same species. It remains unclear how much overlap is to be expected between the parts of the genome associated to these two contrasted adaptation processes. In the present study, we generated a high-quality genome and re-sequenced 177 individuals for Gymnocarpos przewalskii, an important desert plant species from North-West China, to detect local adaptation. To test for global adaptation to aridity at the molecular level we compared genomic data of 15 species that vary in their ability to withstand drought. A total of 118 genes were involved in global adaptation to aridity. Sixty-five G. przewalskii genes were shared across all xerophytic species, of which sixty-three were under stabilizing selection and two under directional selection. While 20% of G. przewalskii genome showed signatures of local adaptation to aridity during population divergence, only 13 of those genes were also under global adaptation. Hence, our results suggest that long-term stability is crucial for adaptation to extreme environmental stress but is only maintained in a small group of highly pleiotropic genes while a rapid response to recent changes elicits a genome-wide response, including gene family expansion. The overlap between the two evolutionary mechanisms appears limited.

evolutionary biology↗

bioRxiv2023

On-Pathway Oligomer of Human Islet Amyloid Polypeptide Induced and Stabilized by Mechanical Rotation During MAS NMR

Intermediates along the fibrillation pathway are generally considered to be the toxic species responsible for the pathologies of amyloid diseases. However, structural studies of these species have been hampered by heterogeneity and poor stability in standard aqueous conditions. Here, we report a novel methodology for producing stable, on-pathway oligomers of the human Type-2 Diabetes-associated islet amyloid polypeptide (hIAPP, or amylin) using the mechanical forces associated with magic angle spinning (MAS). The species were a heterogeneous mixture of globular and short rod-like species with significant {beta}-sheet content and the capability of seeding hIAPP fibrillation. We used MAS NMR to demonstrate that the nature of the species was sensitive to sample conditions including peptide concentration, ionic strength, and buffer. The methodology should be suitable for studies of other aggregating systems.

biophysics↗

bioRxiv2023

In situ spatial reconstruction of distinct normal and pathological cell populations within the human adrenal gland

The human adrenal gland consists of concentrically organized functionally distinct regions responsible for hormone production. Dysregulation of adrenocortical cell differentiation alters the proportion and organization of the functional zones of the adrenal cortex leading to disease. Current models of adrenocortical cell differentiation are based on mouse studies, but there are known organizational and functional differences between human and mouse adrenal glands. This study aimed to investigate the centripetal differentiation model in the human adrenal cortex and characterize aldosterone-producing micronodules (APMs) to better understand adrenal diseases such as primary aldosteronism. We applied spatially resolved in situ transcriptomics to human adrenal tissue sections from two individuals and identified distinct cell populations and their positional relationships. The results supported the centripetal differentiation model in humans, with cells progressing from the outer capsule to the zona glomerulosa, zona fasciculata, and zona reticularis. Additionally, we characterized two APMs in a 72-year-old female. Comparison with earlier APM transcriptomes indicated a subset of core genes, but also heterogeneity between APMs. The findings contribute to our understanding of normal and pathological cellular differentiation in the human adrenal cortex.

genomics↗

bioRxiv2023

Dimeric Transmembrane Structure of the SARS-CoV-2 E Protein

The SARS-CoV-2 E protein is a transmembrane (TM) protein with its N-terminus exposed on the external surface of the virus. Here, the TM structure of the E protein is characterized by oriented sample and magic angle spinning solid-state NMR in lipid bilayers and refined by molecular dynamics simulations. This protein has been found to be a pentamer, with a hydrophobic pore that appears to function as an ion channel. We identified only a symmetric helix-helix interface, leading to a dimeric structure that does not support channel activity. The two helices have a tilt angle of only 6{degrees}, resulting in an extended interface dominated by Leu and Val sidechains. While residues Val14-Thr35 are almost all buried in the hydrophobic region of the membrane, Asn15 lines a water-filled pocket that potentially serves as a drug-binding site. The E and other viral proteins may adopt different oligomeric states to help perform multiple functions.

biophysics↗

bioRxiv2023

Single cell analyses of cancer cells identified two regulatorily and functionally distinct categories in differentially expressed genes among tumor subclones

To explore the feature of cancer cells and tumor subclones, we analyzed 101,065 single-cell transcriptomes from 12 colorectal cancer (CRC) patients and 92 single cell genomes from one of these patients. We found cancer cells, endothelial cells and stromal cells in tumor tissue expressed much more genes and had stronger cell-cell interactions than their counterparts in normal tissue. We identified copy number variations (CNVs) in each cancer cell and found correlation between gene copy number and expression level in cancer cells at single cell resolution. Analysis of tumor subclones inferred by CNVs showed accumulation of mutations in each tumor subclone along lineage trajectories. We found differentially expressed genes (DEGs) between tumor subclones had two populations: DEGCNV and DEGreg. DEGCNV, showing high CNV-expression correlation and whose expression differences depend on the differences of CNV level, enriched in housekeeping genes and cell adhesion associated genes. DEGreg, showing low CNV-expression correlation and mainly in low CNV variation regions and regions without CNVs, enriched in cytokine signaling genes. Furthermore, cell-cell communication analyses showed that DEGCNV tends to involve in cell-cell contact while DEGreg tends to involve in secreted signaling, which further support that DEGCNV and DEGreg are two regulatorily and functionally distinct categories.

genomics↗