Biology subjects

Qian, M.

Publications and source records attributed to Qian, M..

34 records · Page 2Linked to original sources

bioRxiv2022

Genetically engineered mesenchymal stem cells as a nitric oxide reservoir for acute kidney injury therapy

Nitric oxide (NO), as a gaseous therapeutic agent, shows great potential for the treatment of many kinds of diseases. Although various NO delivery systems have emerged, the immunogenicity and long-term toxicity of artificial carriers hinder the potential clinical translation of this gas therapeutics. Mesenchymal stem cells (MSCs), with the capacities of self-renewal, differentiation, and low immunogenicity, have been used as living carriers. However, MSCs as gaseous signaling molecule (GSM) carriers have not been reported. In this study, MSCs were genetically modified to produce mutant {beta}-galactosidase ({beta}-GALH363A). Furthermore, a new NO prodrug, 6-methyl-galactose-benzyl-oxy NONOate (MGP), was designed. MGP can entrance into cell and selectively trigger the NO release from genetically engineered MSCs (eMSCs) in the presence of {beta}-GALH363A. Moreover, our results revealed that eMSCs can release NO when MGP is systemically administered in a mouse model of acute kidney injury (AKI), which can achieve NO release in a precise spatiotemporal manner and augment the therapeutic efficiency of MSCs. This eMSC and NO prodrug system provides a unique and tunable platform for GSM delivery and holds promise for regenerative therapy by enhancing the therapeutic efficiency of stem cells. SignificanceGaseous signaling molecules (GSMs), such as nitric oxide (NO), hold tremendous potential in regenerative medicine and tissue engineering. However, the delivery of GSMs with stem cells to target tissues or organs in response to demand has never been reported. This study designed engineered mesenchymal stem cells (eMSCs) that produce mutant {beta}-galactosidase ({beta}-GALH363A) and trigger NO release when the NO prodrug is systemically administered, which can achieve NO release in a precise spatiotemporal manner and augment the therapeutic efficiency of MSCs in a mouse model of acute kidney injury (AKI). This eMSC and NO prodrug system provides a unique and tunable platform for GSM delivery and holds promise for regenerative therapy by enhancing the therapeutic efficiency of stem cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/519950v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1d8f4e4org.highwire.dtl.DTLVardef@b5e548org.highwire.dtl.DTLVardef@1dff1edorg.highwire.dtl.DTLVardef@184130e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss accompanied by cytoplasmic localization of TDP-43 proteins and their insoluble accumulations. Haploinsufficiency of TBK1 has been found to associate with or cause ALS. However, the cell-autonomous mechanisms by which reduced TBK1 activity contributes to human motor neuron pathology remain elusive. Here, we generated a human cellular model harboring loss-of-function mutations of TBK1 by gene editing and found that TBK1 deficiency was sufficient to cause TDP-43 pathology in human motor neurons. In addition to its functions in autophagy, we found that TBK1 interacted with endosomes and was required for normal endosomal maturation and subsequent lysosomal acidification. Surprisingly, TDP-43 pathology resulted more from the dysfunctional endo-lysosomal pathway than the previously recognized autophagy inhibition mechanism. Restoring TBK1 levels ameliorated lysosomal dysfunction and TDP-43 pathology and maintained normal motor neuron homeostasis. Notably, using patient-derived motor neurons, we found that haploinsufficiency of TBK1 sensitized neurons to lysosomal stress, and chemical regulators of endosomal maturation rescued the neurodegenerative process. Together, our results revealed the mechanism of TBK1 in maintaining TDP-43 and motor neuron homeostasis and suggested that modulating endosomal maturation was able to rescue neurodegenerative disease phenotypes caused by TBK1 deficiency.

neuroscience↗

bioRxiv2021

Sequencing uracil in DNA at single-nucleotide resolution

As an aberrant base in DNA, uracil is generated by dUMP misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Current methods for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase (UNG) and are limited in resolution or specificity. Here, we present a UNG-independent Single-Nucleotide resolution Uracil Sequencing (SNU-seq) method utilizing the UdgX protein which specifically excises the uracil and forms a covalent bond with the resulting deoxyribose. SNU-seq was validated on synthetic DNA and applied to mammalian genomes. We found that the uracil content of pemetrexed-treated cells fluctuated along with DNA replication timing. We also accurately detected uracil introduced through cytosine deamination by the cytosine base editor (nCas9-APOBEC) and verified uracil occurrence in "WRC" motif within Activation-Induced Cytidine Deaminase (AID) hotspot regions in CSR-activated UNG -/- B cells.

biochemistry↗

bioRxiv2021

Downsizing in plants - UV induces pronounced morphological changes in cucumber in the absence of stress

Ultraviolet (UV)-A- or UV-B-enrichment of growth light resulted in a stocky cucumber (Cucumis sativus L.) phenotype exhibiting decreased stem and petiole lengths and leaf area. Effects were larger in plants grown in UV-B- than in UV-A-enriched light. In plants grown in UV-A-enriched light, decreases in stem and petiole lengths were similar independently of tissue age. In the presence of UV-B radiation, stems and petioles were progressively shorter the younger the tissue. Also, plants grown under UV-A-enriched light significantly reallocated photosynthate from shoot to root and also had thicker leaves with decreased specific leaf area. Our data therefore imply different morphological plant regulatory mechanisms under UV-A and UV-B radiation. There was no evidence of stress in the UV-exposed plants, neither in photosynthetic parameters, total chlorophyll content, nor in accumulation of damaged DNA (cyclobutane pyrimidine dimers). The ABA content of the plants also was consistent with non-stress conditions. Parameters such as total leaf antioxidant activity, leaf adaxial epidermal flavonol content and foliar total UV-absorbing pigment levels revealed successful UV acclimation of the plants. Thus, the stocky UV-phenotype was displayed by healthy cucumber plants, implying a strong morphological response and regulatory adjustment as part of UV acclimation processes involving UV-A and/or UV-B photoreceptors.

plant biology↗

bioRxiv2020

MarkovHC: Markov hierarchical clustering for the topological structure of high-dimensional single-cell omics data

Distinguishing cell types and cell states is one of the fundamental questions in single-cell studies. Meanwhile, exploring the lineage relations among cells and finding the path and critical points in the cell fate transition are also of great importance. Existing unsupervised clustering methods and lineage trajectory reconstruction methods often face several challenges such as clustering data of arbitrary shapes, tracking precise trajectories and identifying critical points. Certain adaptive landscape approach1-3, which constructs a pseudo-energy landscape of the dynamical system, may be used to explore such problems. Thus, we propose Markov hierarchical clustering algorithm (MarkovHC), which reconstructs multi-scale pseudo-energy landscape by exploiting underlying metastability structure in an exponentially perturbed Markov chain4. A Markov process describes the random walk of a hypothetically traveling cell in the corresponding pseudo-energy landscape over possible gene expression states. Technically, MarkovHC integrates the tasks of cell classification, trajectory reconstruction, and critical point identification in a single theoretical framework consistent with topological data analysis (TDA)5. In addition to the algorithm development and simulation tests, we also applied MarkovHC to diverse types of real biological data: single-cell RNA-Seq data, cytometry data, and single-cell ATAC-Seq data. Remarkably, when applying to single-cell RNA-Seq data of human ESC derived progenitor cells6, MarkovHC not only could successfully identify known cell types, but also discover new cell types and stages. In addition, when using MarkovHC to analyze single-cell RNA-Seq data of human preimplantation embryos in early development7, the hierarchical structure of the lineage trajectories was faithfully reconstituted. Furthermore, the critical points representing important stage transitions had also been identified by MarkovHC from early gastric cancer data8. In summary, these results demonstrate that MarkovHC is a powerful tool based on rigorous metastability theory to explore hierarchical structures of biological data, to identify a cell sub-population (basin) and a critical point (stage transition), and to track a lineage trajectory (differentiation path). HighlightsO_LIMarkovHC explores the topology hierarchy in high-dimensional data. C_LIO_LIMarkovHC can find clusters (basins) and cores (attractors) of clusters in different scales. C_LIO_LIThe trajectory of state transition (transition paths) and critical points in the process of state transition (critical points) among clusters can be tracked. C_LIO_LIMarkovHC can be applied on diverse types of single-cell omics data. C_LI

bioinformatics↗

bioRxiv2020

Effects of NT5C2 germline variants on 6-mecaptopurine metabolism in children with acute lymphoblastic leukemia

6-mercaptopurine (6-MP) is widely used in the treatment of acute lymphoblastic leukemia (ALL), and its cytotoxicity is primarily mediated by thioguanine nucleotide metabolites (TGN). Recent genome-wide association study has identified germline polymorphisms (e.g., rs72846714) in the NT5C2 gene associated with 6-MP metabolism in patients with ALL. However, the full spectrum of genetic variation in NT5C2 is unclear and its impact on 6-MP drug activation has not been comprehensively examined. To this end, we performed targeted sequencing of NT5C2 in 588 children with ALL and identified 121 single nucleotide polymorphisms (SNPs) nominally associated with erythrocyte TGN during 6-MP treatment (P < 0.05). Of these, 61 variants were validated in a replication cohort of 372 children with ALL. After considering linkage disequilibrium and multivariate analysis, we confirmed two clusters of variants, represented by rs72846714 and rs58700372, that independently affected 6-MP metabolism. Functional studies showed that rs58700372 directly altered the activity of an intronic enhancer, with the variant allele linked to higher transcription activity and reduced 6-MP metabolism (lower TGN). By contrast, rs72846714 was not located in a regulatory element and instead its association signal was explained by linkage disequilibrium with a proximal functional variant rs12256506 that activated NT5C2 transcription in-cis. Our results indicated that NT5C2 germline variation significantly contributes to inter-patient variability in thiopurine drug disposition.

genetics↗

bioRxiv2020

4 β-hydroxycholesterol is a pro-lipogenic factor that promotes SREBP1c expression and activity through Liver X-receptor.

Oxysterols are oxidized derivatives of cholesterol that play signaling roles in lipid biosynthesis and homeostasis. Here we show that 4{beta}-hydroxycholesterol (4{beta}-HC), a liver and serum abundant oxysterol of poorly defined function, is a potent and selective inducer of the master lipogenic transcription factor, Sterol Regulatory Element Binding Protein 1c (SREBP1c), but not the related steroidogenic transcription factor SREBP2. Mechanistically, 4{beta}-HC acts as a putative agonist for Liver X receptor (LXR), a sterol sensor and transcriptional regulator previously linked to SREBP1c activation. Unique among the oxysterol agonists of LXR, 4{beta}-HC induced expression of the lipogenic program downstream of SREBP1c, and triggered de novo lipogenesis both in primary hepatocytes and in mouse liver. 4{beta}-HC-acted in parallel to insulin-PI3K-dependent signaling to stimulate triglyceride synthesis and lipid droplet accumulation. Thus, 4{beta}-HC is an endogenous regulator of de novo lipogenesis through the LXR-SREBP1c axis.

biochemistry↗

bioRxiv2020

KDM4 Orchestrates Epigenomic Remodeling of Senescent Cells and Potentiates the Senescence-Associated Secretory Phenotype

Cellular senescence restrains the expansion of neoplastic cells through several layers of regulation, including epigenetic decoration of chromatin structure and functional modulation of bioactive components. Here we report that expression of the histone H3-specific demethylase KDM4 is upregulated in human stromal cells upon cellular senescence. In clinical oncology, upregulated KDM4 and diminished H3K9/H3K36 methylation are correlated with adverse survival of cancer patients post-chemotherapy. Global chromatin accessibility mapping via ATAC-seq and expression profiling through RNA-seq reveal extensive reorganization of chromosomes and spatiotemporal reprogramming of the transcriptomic landscape, events responsible for development of the senescence-associated secretory phenotype (SASP). Selectively targeting KDM4 dampens the SASP of senescent stromal cells and enhances the apoptotic index of cancer cells in the treatment-damaged tumor microenvironment (TME), together prolonging overall survival of experimental animals. Our study supports the dynamic change of H3K9/H3K36 methylation marks during cellular senescence, identifies an unusually permissive chromatin state, unmasks KDM4 as a key modulator of the SASP, and presents a novel therapeutic avenue to manipulate cellular senescence and curtail age-related pathologies.

cell biology↗

bioRxiv2020

Senescent stromal cells promote cancer resistance through SIRT1 loss-potentiated overproduction of small extracellular vesicles

Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype (SASP) and implicated in age-related pathologies including cancer. Here we report that senescent cells actively synthesize and release small extracellular vesicles (sEVs) with a distinctive size distribution. Mechanistically, SIRT1 loss supports accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Once released, senescent stromal sEVs significantly alter the expression profile of recipient cancer cells and enhance their aggressiveness, specifically drug resistance mediated by expression of ATP binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with an agonist SRT2104 prevents development of cancer resistance through restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients are readily detectable by routine biotechniques, presenting a novel biomarker to monitor therapeutic efficacy and to predict long term outcome. Together, our study identifies a distinct mechanism supporting pathological activities of senescent cells, and provides a novel avenue to circumvent advanced human malignancies by co-targeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells.

cell biology↗

bioRxiv2020

A synergistic action of sirtuins directly recognizes DNA breaks and potentiates DNA damage response and repair

The DNA damage response (DDR) is a highly orchestrated process but how double-strand DNA breaks (DSBs) are initially recognized is unclear. Here, we show that polymerized SIRT6 deacetylase recognizes DSBs and potentiates the DDR. First, SIRT1 deacetylates SIRT6 at residue K33, which is important for SIRT6 polymerization and mobilization toward DSBs. Then, K33-deacetylated SIRT6 anchors to {gamma}H2AX, allowing its retention on and subsequent remodeling of local chromatin. We show that a K33R mutation that mimics hypoacetylated SIRT6 can rescue defective DNA repair as a result of SIRT1 deficiency in cultured cells. These data highlight the synergistic action between SIRTs in the spatiotemporal regulation of the DDR and DNA repair.

biochemistry↗

bioRxiv2020

25-Hydroxycholesterol amplifies microglial IL-1β production in an apoE isoform-dependent manner

Genome-wide association studies associated with Alzheimers disease (AD) have implicated pathways related to both lipid homeostasis and innate immunity in the pathophysiology of AD. However, the exact cellular and chemical mediators of neuroinflammation in AD remain poorly understood. The oxysterol 25-hydroxycholesterol (25-HC) is an important immunomodulator produced by peripheral macrophages with wide-ranging effects on cell signaling and innate immunity. Genetic variants of the enzyme responsible for 25-HC production, cholesterol 25-hydroxylase (CH25H), have been found to be associated with AD. In the present study, we found that the CH25H expression is upregulated in human AD brain tissue and in transgenic mouse brain tissue bearing amyloid-{beta} (A{beta}) plaques or tau pathology. Treatment with the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) markedly upregulates CH25H expression in the mouse brain in vivo. LPS also stimulates CH25H expression and 25-HC secretion in cultured mouse primary microglia. We also found that LPS-induced microglial production of the pro-inflammatory cytokine IL1{beta} is markedly potentiated by 25-HC and attenuated by genetic deletion of CH25H. Microglia expressing apolipoprotein E4 (apoE4), a genetic risk factor for AD, produce greater amounts of 25-HC than apoE3-expressing microglia following treatment with LPS. Remarkably, treatment of microglia with 25-HC results in a much greater level of IL1{beta} secretion in LPS-activated apoE4-expressing microglia than in apoE2- or apoE3-expressing microglia. Blocking potassium efflux or inhibiting caspase-1 prevents 25-HC-potentiated IL1{beta} release in apoE4-expressing microglia, indicating the involvement of caspase-1/NLRP3 inflammasome activity. 25-HC may function as a microglial secreted inflammatory mediator in brain, promoting IL1{beta}-mediated neuroinflammation in an apoE isoform-dependent manner (E4>>E2/E3) and thus may be an important mediator of neuroinflammation in AD.

neuroscience↗

bioRxiv2019

Exponential Growth of Glioblastoma In Vivo Driven by Rapidly Dividing and Outwardly Migrating Cancer Stem Cells

How overall tumor growth emerges from the properties of functionally heterogeneous tumor cell subpopulations is a fundamental question of cancer biology. Here we combined lineage tracing, continuous monitoring of tumor mass, proliferation assays and transcriptomics with mathematical modeling and statistical inference to dissect the growth of glioblastoma in mice. We found that tumors grow exponentially at the rate of symmetric divisions of brain tumor stem cells (BTSCs). Spatial modeling predicts, and data show, that BTSCs accumulate at the tumor rim rather than in the core. The physiological differentiation hierarchy downstream of BTSCs is preserved in mice and humans: transit amplifying progenitors give rise to terminally differentiated cells. Consistent with our quantification of the mechanisms underlying tumor growth, molecular data show elevated expression of cell cycle-and migration-related genes in BTSCs. Our systematic approach reveals fundamental properties of glioblastoma and may be transferable to the study of other animal models of cancer.

cancer biology↗

bioRxiv2019

SIRT6 as a transcriptional coactivator of GATA4 prevents doxorubicin cardiotoxicity independently of its deacylase activity

Activity dependent and independent functions for some enzymes are indispensable as significant biological regulators. Deacylase SIRT6 is well-known to improve stress resistance and promote lifespan extension through enzymatic activity-dependent gene silencing. However, whether and how SIRT6 non-enzymatically actives the transcriptional output hasnt been characterized. Here, we revealed SIRT6 as a coactivator of GATA4, an essential transcription factor for postnatal cardiomyocyte survival, promoting the expression of anti-apoptotic gene. Chemotherapeutic drug, doxorubicin (DOX), remarkably and rapidly decreased SIRT6 expression, leading to transcriptional repression of GATA4 and cardiomyocyte apoptosis. Interestingly, SIRT6 interacted with GATA4 yet enhanced GATA4 acetylation independent of its deacylase activity, by recruiting the acetyltransferase Tip60 to form a trimeric complex. Nonacyl-mimetic mutation of GATA4 thoroughly blocked its ability against DOX cardiotoxicity. Moreover, Sirt6 transgenic mice exhibited preserved cardiac function with attenuated GATA4 activity in response to DOX. Thus, our studies uncover a previously unrecognized role of SIRT6 in cardioprotection independently of deacylase activity, providing the molecular basis to prevent chemotherapeutic side effects.

molecular biology↗

bioRxiv2018

Novel 3,6-Dihydroxypicolinic Acid Decarboxylase Mediated Picolinic Acid Catabolism in Alcaligenes faecalis JQ135

Alcaligenesfaecalis strain JQ135 utilizes picolinic acid (PA) as sole carbon and nitrogen source for growth. In this study, we screened a 6-hydroxypicolinic acid (6HPA) degradation-deficient mutant through random transposon mutagenesis. The mutant hydroxylated 6HPA into an intermediate, identified as 3,6-dihydroxypicolinic acid (3,6DHPA) with no further degradation. A novel decarboxylase PicC was identified that was found to be responsible for the decarboxylation of 3,6DHPA to 2.5-dihydroxypyridine. Although, PicC belonged to amidohydrolase_2 family, it shows low similarity (<45%) when compared to other reported amidohydrolase_2 family decarboxylases. Moreover, PicC was found to form a monophyletic group in the phylogenetic tree constructed using PicC and related proteins. Further, the genetic deletion and complementation results demonstrated that picC was essential for PA degradation. The PicC was Zn2+-dependent non-oxidative decarboxylase that can specifically catalyze the irreversible decarboxylation of 3,6DHPA to 2.5-dihydroxypyridine. The Km and kcat towards 3,6DHPA were observed to be 13.44 M and 4.77 s-1, respectively. Site-directed mutagenesis showed that His163 and His216 were essential for PicC activity.\n\nImportancePicolinic acid is a natural toxic pyridine derived from L-tryptophan metabolism and some aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize picolinic acid for their growth, and thus, a microbial degradation pathway of picolinic acid has been proposed. Picolinic acid is converted into 6-hydroxypicolinic acid, 3,6-dihydroxypicolinic acid, and 2,5-dihydroxypyridine in turn. However, there was no physiological and genetic validation for this pathway. This study demonstrated that 3,6DHPA was an intermediate in PA catabolism process and further identified and characterized a novel amidohydrolase_2 family decarboxylase PicC. It was also shown that PicC could catalyze the decarboxylation process of 3,6-dihydroxypicolinic acid into 2,5-dihydroxypyridine. This study provides a basis for understanding PA degradation pathway and the underlying molecular mechanism.

microbiology↗

bioRxiv2018

Sirt7 regulates circadian phase coherence of hepatic circadian clock via a body temperature/Hsp70-Sirt7-Cry1 axis

The biological clock is generated in the hypothalamic suprachiasmatic nucleus (SCN), which synchronizes peripheral oscillators to coordinate physiological and behavioral activities throughout the body. Disturbance of circadian phase coherence between the central and peripheral could disrupt rhythms and thus cause diseases and aging. Here, we identified hepatic Sirt7 as an early element responsive to light, which ensures the phase coherence in mouse liver. Loss of Sirt7 leads to advanced liver circadian phase; restricted feeding in daytime entrains hepatic clock more rapidly in Sirt7-/- mice compared to wild-types. Molecularly, a light-driven body temperature (BT) oscillation induces rhythmic expression of Hsp70, which binds to and promotes the ubiquitination and proteasomal degradation of Sirt7. Sirt7 rhythmically deacetylates Cry1 on K565/579 and promotes Fbxl3-mediated degradation, thus coupling hepatic clock to the central pacemaker. Together, our data identify a novel BT/Hsp70-Sirt7-Cry1 axis, which transmits biological timing cues from the central to the peripheral and ensures circadian phase coherence in livers.

molecular biology↗

bioRxiv2017

Boosting ATM Activity Promotes Longevity in Nematodes and Mice

DNA damage accumulates with age1. However, whether and how robust DNA repair machinery promotes longevity is elusive. Here, we demonstrate that activation of ataxia-telangiectasia mutated (ATM) via low dose of chloroquine (CQ) promotes DNA damage clearance, rescues age-related metabolic shift, and extends lifespan in nematodes and mice. Molecularly, ATM phosphorylates SIRT6 deacetylase and thus prevents MDM2-mediated ubiquitination and proteasomal degradation. Extra copies of Sirt6 in Atm-/- mice extend lifespan, accompanied with restored metabolic homeostasis. In a progeria mouse model with low ATM protein level and DNA repair capacity, the treatment with CQ ameliorates premature aging features and extends lifespan. Thus, our data highlights a pro-longevity role of ATM, for the first time establishing direct causal links between robust DNA repair machinery and longevity, and providing therapeutic strategy for progeria and age-related metabolic diseases.

molecular biology↗