Biology subjects

Pan, D.

Publications and source records attributed to Pan, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Proteomic analysis of papillary thyroid carcinoma in the context of Hashimoto's thyroiditis

ObjectiveTo analyze the proteomic characteristics of papillary thyroid carcinoma in the context of Hashimotos thyroiditis by mass spectrometry and screen the corresponding differential proteins. MethodsPostoperative paraffin specimens of 3 patients with Hashimotos thyroiditis combined with papillary thyroid carcinoma and 3 patients with Hashimotos thyroiditis and benign nodules were collected. The differential proteins were obtained by qualitative and quantitative analysis using Q ExactiveTM mass spectrometer. Functional enrichment cluster analysis and protein interaction analysis were then performed on these differential proteins. Finally, key proteins were screened out and immunohistochemical verification was performed. Results72 up-regulated differentially expressed proteins and 21 down-regulated differentially expressed proteins were screened out. THBS2 and COL12A1 were further identified by bioinformatics analysis. After immunohistochemical verification, it was concluded that COL12A1 can be used as a specific tumor marker. ConclusionThere are differentially expressed proteins between the Hashimotos thyroiditis combined with papillary thyroid carcinoma group and the Hashimotos thyroiditis combined with benign nodules group. The up-regulated expression of COL12A1 can become an auxiliary diagnostic marker for papillary thyroid carcinoma with Hashimotos thyroiditis.

genomics↗

bioRxiv2024

Endophilin A1 facilitates organization of the GABAergic postsynaptic machinery to maintain excitation-inhibition balance

The assembly and operation of neural circuits in the brain rely on the coordination and balance of excitatory and inhibitory activities. Inhibitory synapses are key regulators of the functional balance of neural circuits. However, due to the diversity of inhibitory presynaptic neurons, the complex composition of postsynaptic receptor subunits and the lack of typical postsynaptic dense structure, there are relatively few studies on the regulatory mechanisms for inhibitory synaptic structure and function, and insufficient understanding of the cellular and molecular abnormalities of inhibitory synapses in neurological and neuropsychiatric disorders. Here, we report a crucial role for endophilin A1 in inhibitory synapses. We show that endophilin A1 directly interacts with the inhibitory postsynaptic scaffold protein gephyrin in excitatory neurons, and promotes organization of the inhibitory postsynaptic density and synaptic recruitment/stabilization of the {gamma}-aminobutyric acid type A receptors via its plasma membrane association and actin polymerization promoting activities. Loss of endophilin A1 by gene knockout in mouse hippocampal CA1 pyramidal cells weakens inhibitory synaptic transmission and causes imbalance in the excitatory/inhibitory function of neural circuits, leading to increased susceptibility to epilepsy. Our findings identify endophilin A1 as an iPSD component and provide new insights into the organization and stabilization of inhibitory postsynapses to maintain E/I balance as well as the pathogenesis of epilepsy.

neuroscience↗

bioRxiv2024

scCancerExplorer: a comprehensive database for interactively exploring single-cell multi-omics data of human pan-cancer

Genomic, epigenomic, and transcriptomic alterations are hallmarks of cancer cells, and are closely connected. Especially, epigenetic regulation plays a critical role in tumorigenesis and progression. The growing single-cell epigenome data in cancer research provide new opportunities for data mining from a more comprehensive perspective. However, there is still a lack of databases designed for interactively exploring the single-cell multi-omics data of human pan-cancer, especially for the single-cell epigenome data. To fill in the gap, we developed scCancerExplorer, a comprehensive and user-friendly database to facilitate the exploration of the single-cell genome, epigenome (chromatin accessibility and DNA methylation), and transcriptome data of 50 cancer types. Five major modules were provided to explore those data interactively, including "integrated multi-omics analysis", "single-cell transcriptome", "single-cell epigenome", "single-cell genome", and "TCGA analysis". By simple clicking, users can easily investigate gene expression features, chromatin accessibility patterns, transcription factor activities, DNA methylation states, copy number variations, and TCGA survival analysis results. Taken together, scCancerExplorer is distinguished from previous databases with rich and interactive functions for exploring the single-cell multi-omics data of human pan-cancer. It bridges the gap between single-cell multi-omics data and the end-users, and will facilitate progress in the field of cancer research. scCancerExplorer is freely accessible via https://bianlab.cn/scCancerExplorer.

cancer biology↗

bioRxiv2024

Adaptive optical third-harmonic generation microscopy for in vivo imaging of tissues

Third-harmonic generation microscopy is a powerful label-free nonlinear imaging technique, providing essential information about structural characteristics of cells and tissues without requiring external labelling agents. In this work, we integrated a recently developed compact adaptive optics module into a third-harmonic generation microscope, to measure and correct for optical aberrations in complex tissues. Taking advantage of the high sensitivity of the third-harmonic generation process to material interfaces and thin membranes, along with the 1,300-nm excitation wavelength used here, our adaptive optical third-harmonic generation microscope enabled high-resolution in vivo imaging within highly scattering biological model systems. Examples include imaging of myelinated axons and vascular structures within the mouse spinal cord and deep cortical layers of the mouse brain, along with imaging of key anatomical features in the roots of the model plant Brachypodium distachyon. In all instances, aberration correction led to significant enhancements in image quality.

biophysics↗

bioRxiv2024

Condensin II activation by M18BP1

Condensin complexes promote the drastic spatial rearrangement of the genome upon mitotic entry. Condensin II initiates chromosome condensation in early mitosis. To prevent chromosome condensation during interphase, condensin II is inhibited by MCPH1, but the mechanism is unknown. Through genetic and proteomic approaches, we identify M18BP1, a protein previously associated with centromere identity, as a factor required for condensin II localization to chromatin. M18BP1 directly binds condensin IIs CAP-G2 subunit and competes with MCPH1 for binding. Upon mitotic entry, CDK1 mediated phosphorylation may promote a switch from MCPH1 to M18BP1 binding to activate condensin II. Our results identify a fundamental and evolutionarily conserved mechanism of condensin II activation.

cell biology↗

bioRxiv2024

Label-free structural imaging of plant roots and microbes using third- harmonic generation microscopy

Root biology is pivotal in addressing global challenges including sustainable agriculture and climate change. However, roots have been relatively understudied among plant organs, partly due to the difficulties in imaging root structures in their natural environment. Here we used microfabricated ecosystems (EcoFABs) to establish growing environments with optical access and employed nonlinear multimodal microscopy of third-harmonic generation (THG) and three-photon fluorescence (3PF) to achieve label-free, in situ imaging of live roots and microbes at high spatiotemporal resolution. THG enabled us to observe key plant root structures including the vasculature, Casparian strips, dividing meristematic cells, and root cap cells, as well as subcellular features including nuclear envelopes, nucleoli, starch granules, and putative stress granules. THG from the cell walls of bacteria and fungi also provides label-free contrast for visualizing these microbes in the root rhizosphere. With simultaneously recorded 3PF fluorescence signal, we demonstrated our ability to investigate root-microbe interactions by achieving single-bacterium tracking and subcellular imaging of fungal spores and hyphae in the rhizosphere.

plant biology↗

bioRxiv2024

Role of PLK1 in the epigenetic maintenance of centromeres

The centromere, a chromosome locus defined by the histone H3-like protein CENP-A, seeds the kinetochore to bind microtubules during cell division. Centromere maintenance requires CENP-A to be actively replenished by dedicated protein machinery in the early G1 cell-cycle phase, compensating for its two-fold dilution following DNA replication. Cyclin-dependent kinases (CDKs) limit CENP-A deposition to once per cell cycle and function as negative regulators outside early G1. Antithetically, Polo-like kinase 1 (PLK1) promotes CENP-A deposition in early G1, but the molecular details are still unknown. We reveal a phosphorylation network that recruits PLK1 to the deposition machinery to control a conformational switch required for licensing the CENP-A deposition reaction. Our findings solve the long-standing question of how PLK1 contributes to the epigenetic maintenance of centromeres. One-Sentence SummaryPLK1 licenses epigenetic maintenance of centromeres by regulating a conformational switch on the MIS18 protein.

cell biology↗

bioRxiv2023

Tradeoff Between Speed and Robustness in Primordium Initiation Mediated by Auxin-CUC1 Interaction

Robustness is the reproducible development of a phenotype despite stochastic noise. It often involves tradeoffs with other performance metrics, but the mechanisms underlying such tradeoffs were largely unknown. An Arabidopsis flower robustly develops four sepals from four precisely positioned auxin maxima. The development related myb-like 1 (drmy1) mutant generates noise in auxin signaling that disrupts robustness in sepal initiation. Here, we found that increased expression of CUP-SHAPED COTYLEDON1 (CUC1), a boundary specification transcription factor, in drmy1 underlies this loss of robustness. CUC1 surrounds and amplifies stochastic auxin noise in drmy1 to form variably positioned auxin maxima and sepal primordia. Removing CUC1 from drmy1 provides time for noisy auxin signaling to resolve into four precisely positioned auxin maxima, restoring robust sepal initiation. However, removing CUC1 decreases auxin maxima intensity and slows down sepal initiation. Thus, CUC1 increases morphogenesis speed but impairs robustness against auxin noise. Further, using a computational model, we found that the observed phenotype can be explained by the effect of CUC1 in repolarizing PIN FORMED1 (PIN1), a polar auxin transporter. Lastly, our model predicts that reducing global growth rate improves developmental robustness, which we validated experimentally. Thus, our study illustrates a tradeoff between speed and robustness during development.

developmental biology↗

bioRxiv2023

VHL loss enables immune checkpoint blockade therapy by boosting type I interferon response.

Despite a moderate mutation burden, clear cell renal cell carcinoma (ccRCC) responds well to immune checkpoint blockade (ICB) therapy. Here we report that loss-of-function mutations in the von Hippel-Lindau (VHL) gene, the most frequent in ccRCC, underlies its responsiveness to ICB therapy. We demonstrate that genetic knockout of the VHL gene enhanced the efficacy of anti-PD-1 therapy in multiple murine tumor models in a T cell-dependent manner. Mechanistically, we discovered that upregulation of HIF1 and HIF2 induced by VHL gene loss decreased mitochondrial outer membrane potential and caused the cytoplasmic leakage of mitochondrial DNA (mtDNA), which triggered cGAS-STING activation and induced type I interferons. Our study thus provided novel mechanistic insights into the role of VHL gene loss in potentiating ccRCC immunotherapy.

cancer biology↗

bioRxiv2023

Molecular Regulation of Invasive Protrusion Formation at the Mammalian Fusogenic Synapse

Invasive membrane protrusions play a central role in a variety of cellular processes. Unlike filopodia, invasive protrusions are mechanically stiff and propelled by branched actin polymerization. However, how branched actin filaments are organized to create finger-like invasive protrusions remains a longstanding question in cell biology. Here, by examining the mammalian fusogenic synapse, where invasive protrusions are generated to promote cell membrane juxtaposition and fusion, we have uncovered the mechanism underlying invasive protrusion formation. We show that two Arp2/3 nucleation promoting factors (NPFs), WAVE and N-WASP, exhibit distinct and complementary localization patterns in the protrusions. While WAVE is at the leading edge, N-WASP is recruited by its interacting protein, WIP, to the shaft of the protrusion. During protrusion growth, new branched actin filaments are polymerized at the periphery of the shaft and crosslinked to preexisting actin bundles by the "pioneer" actin-bundling protein dynamin. The thickened actin bundles are further stabilized by WIP, which functions as a WH2 domain-mediated actin-bundling protein. Disrupting any of these components results in defective protrusions and myoblast fusion in cultured cells and/or in mouse embryos. Thus, our study has revealed the intricate spatiotemporal coordination between two NPFs and two actin-bundling proteins in creating invasive protrusions and has general implications in understanding protrusion formation in many cellular processes beyond cell-cell fusion.

cell biology↗

bioRxiv2023

Senescence of endplate osteoclasts induces sensory innervation and spinal pain

Spinal pain affects individuals of all ages and is the most common musculoskeletal problem globally. Its clinical management remains a challenge as the underlying mechanisms leading to it are still unclear. Here, we report that significantly increased numbers of senescent osteoclasts (SnOCs) are observed in mouse models of spinal hypersensitivity, like lumbar spine instability (LSI) or aging, compared to controls. The larger population of SnOCs is associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. We show that deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation and H-type vessel growth in the endplate. We also show that there is significantly increased SnOC-mediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. These findings suggest that pharmacological elimination of SnOCs may be a potent therapy to treat spinal pain.

cell biology↗

bioRxiv2023

KRAS inhibition activates an actionable CD24 do-not-eat-me signal in pancreas cancer

KRASG12C inhibitors (G12Ci) have produced encouraging, albeit modest and transient, clinical benefit in pancreatic ductal adenocarcinoma (PDAC). Identifying and targeting resistance mechanisms to G12Ci treatment is therefore crucial. To better understand the function of KRASG12C and possible G12Ci bypass mechanisms, we developed an autochthonous KRASG12C- driven PDAC model. Compared to the classical KRASG12D PDAC model, the G12C model exhibits slower tumor growth, yet similar histopathological and molecular features. Aligned with clinical experience, G12Ci treatment of KRASG12C tumors produced modest impact despite stimulating a hot tumor immune microenvironment. Immunoprofiling revealed that CD24, a dont eat me signal, is significantly upregulated on cancer cells upon G12Ci treatment. Blocking CD24 enhanced macrophage phagocytosis of cancer cells and significantly sensitized tumors to G12Ci treatment. Similar findings were observed in KRASG12D-driven PDAC. Together, this study reveals common and distinct oncogenic KRAS allele-specific biology and identifies a clinically actionable adaptive mechanism that may improve the efficacy of oncogenic KRAS inhibitor therapy in PDAC. SignificanceGeneration of an autochthonous KRASG12C-driven pancreatic cancer model enabled elucidation of specific effects of KRASG12C during tumor development, revealing CD24 as an actionable adaptive mechanism in cancer cells induced upon KRASG12C inhibition.

cancer biology↗

bioRxiv2023

maskNMF: A denoise-sparsen-detect approach for extracting neural signals from dense imaging data

A number of calcium imaging methods have been developed to monitor the activity of large populations of neurons. One particularly promising approach, Bessel imaging, captures neural activity from a volume by projecting within the imaged volume onto a single imaging plane, therefore effectively mixing signals and increasing the number of neurons imaged per pixel. These signals must then be computationally demixed to recover the desired neural activity. Unfortunately, currently-available demixing methods can perform poorly in the regime of high imaging density (i.e., many neurons per pixel). In this work we introduce a new pipeline (maskNMF) for demixing dense calcium imaging data. The main idea is to first denoise and temporally sparsen the observed video; this enhances signal strength and reduces spatial overlap significantly. Next we detect neurons in the sparsened video using a neural network trained on a library of neural shapes. These shapes are derived from segmented electron microscopy images input into a Bessel imaging model; therefore no manual selection of "good" neural shapes from the functional data is required here. After cells are detected, we use a constrained non-negative matrix factorization approach to demix the activity, using the detected cells shapes to initialize the factorization. We test the resulting pipeline on both simulated and real datasets and find that it is able to achieve accurate demixing on denser data than was previously feasible, therefore enabling faithful imaging of larger neural populations. The method also provides good results on more "standard" two-photon imaging data. Finally, because much of the pipeline operates on a significantly compressed version of the raw data and is highly parallelizable, the algorithm is fast, processing large datasets faster than real time.

neuroscience↗

bioRxiv2023

A brown fat-enriched adipokine, ASRA, is a leptin receptor antagonist that stimulates appetite

The endocrine control of food intake remains incompletely understood, and whether the leptin receptor (LepR)-mediated anorexigenic pathway in the hypothalamus is negatively regulated by a humoral factor is unknown. Here, we identify an appetite-stimulating factor - ASRA - that represents a peripheral signal of energy deficit and orthosterically antagonizes LepR signaling. Asra encodes an 8 kD protein that is abundantly and selectively expressed in adipose tissue and to a lesser extent, in liver. ASRA associates with autophagy vesicles and its secretion is enhanced by energy deficiency. In vivo, fasting and cold stimulate Asra expression and increase its protein concentration in cerebrospinal fluid. Asra overexpression attenuates LepR signaling, leading to elevated blood glucose and development of severe hyperphagic obesity. Conversely, either adipose- or liver-specific Asra knockout mice display increased leptin sensitivity, improved glucose homeostasis, reduced food intake, resistance to high-fat diet-induced obesity, and blunted cold-evoked feeding response. Mechanistically, ASRA acts as a high affinity antagonist of LepR. AlphaFold2-multimer prediction and mutational studies suggest that a core segment of ASRA binds to the immunoglobin-like domain of LepR, similar to the site 3 recognition of the A-B loop of leptin. While administration of recombinant wild-type ASRA protein promotes food intake and increases blood glucose in a LepR signaling-dependent manner, point mutation within ASRA that disrupts LepR-binding results in a loss of these effects. Our studies reveal a previously unknown endocrine mechanism in appetite regulation and have important implications for our understanding of leptin resistance.

molecular biology↗

bioRxiv2023

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

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

cell biology↗

bioRxiv2023

The Hippo kinase cascade regulates a contractile cell behavior and cell density in a close unicellular relative of animals

The genomes of close unicellular relatives of animals encode orthologs of many genes that regulate animal development. However, little is known about the function of such genes in unicellular organisms or the evolutionary process by which these genes came to function in multicellular development. The Hippo pathway, which regulates cell proliferation and tissue size in animals, is present in some of the closest unicellular relatives of animals, including the amoeboid organism Capsaspora owczarzaki. We previously showed that the Capsaspora ortholog of the Hippo pathway nuclear effector Yorkie/YAP/TAZ (coYki) regulates actin dynamics and the three-dimensional morphology of Capsaspora cell aggregates, but is dispensable for cell proliferation control (Phillips et al., 2022). However, the function of upstream Hippo pathway components, and whether and how they regulate coYki in Capsaspora, remained unknown. Here, we analyze the function of the upstream Hippo pathway kinases coHpo and coWts in Capsaspora by generating mutant lines for each gene. Loss of either kinase results in increased nuclear localization of coYki, indicating an ancient, premetazoan origin of this Hippo pathway regulatory mechanism. Strikingly, we find that loss of either kinase causes a contractile cell behavior and increased density of cell packing within Capsaspora aggregates. We further show that this increased cell density is not due to differences in proliferation, but rather actomyosin-dependent changes in the multicellular architecture of aggregates. Given its well-established role in cell density-regulated proliferation in animals, the increased density of cell packing in coHpo and coWts mutants suggests a shared and possibly ancient and conserved function of the Hippo pathway in cell density control. Together, these results implicate cytoskeletal regulation but not proliferation as an ancestral function of the Hippo pathway kinase cascade and uncover a novel role for Hippo signaling in regulating cell density in a proliferation-independent manner.

cell biology↗

bioRxiv2023

Structure and engineering of miniature Acidibacillus sulfuroxidans Cas12f1

The miniature CRISPR-Cas12f nucleases allow for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles, thereby showing promising potential for in vivo therapeutic applications. Acidibacillus sulfuroxidans Cas12f1 (AsCas12f1, 422 amino acids) is the most compact Cas12f nuclease identified to date, showing a moderate level of genome-editing activity in human cells compared to Cas9 and Cas12a. Understanding the mechanisms of why such a compact nuclease is active for genome editing would facilitate its rational engineering. Here, we report the cryo-EM structure of the AsCas12f1-sgRNA-dsDNA ternary complex, and reveal that AsCas12f1 functions as an asymmetric dimer for single-guide RNA (sgRNA) binding and DNA targeting. The detailed mechanisms for dimer formation, PAM recognition, and sgRNA accommodation are elucidated. Leading by the structural knowledge, we extensively engineer the AsCas12f1 nuclease and its corresponding sgRNA, resulting in an evolved AsCas12f1-sgRNA combination with drastically enhanced genome editing activity in human cells. These results provide further understanding of compact CRISPR systems and expand the mini CRISPR toolbox for therapeutic applications.

molecular biology↗

bioRxiv2023

Self-extinguishing relay waves enable homeostaticcontrol of human neutrophil swarming

Neutrophils exhibit self-amplified swarming to sites of injury and infection. How swarming is controlled to ensure the proper level of neutrophil recruitment is unknown. Using an ex vivo model of infection, we find that human neutrophils use active relay to generate multiple pulsatile waves of swarming signals. Unlike classic active relay systems such as action potentials, neutrophil swarming relay waves are self-extinguishing, limiting the spatial range of cell recruitment. We identify an NADPH-oxidase-based negative feedback loop that is needed for this self-extinguishing behavior. Through this circuit, neutrophils adjust the number and size of swarming waves for homeostatic levels of cell recruitment over a wide range of initial cell densities. We link a broken homeostat to neutrophil over-recruitment in the context of human chronic granulomatous disease.

cell biology↗