Biology subjects

Xiang, L.

Publications and source records attributed to Xiang, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids

Studies of cultured embryos have provided insights into human peri-implantation development. However, detailed knowledge of peri-implantation lineage developments as well as underlying mechanisms remain obscure. Using 3D-cultured human embryos, herein we report a complete cell atlas of the early post-implantation lineages and decipher cellular composition and gene signatures of the epiblast and hypoblast derivatives. In addition, we develop an embryo-like assembloid (E-assembloid) by assembling naive hESCs and extraembryonic cells. Using human embryos and E-assembloids, we reveal that WNT, BMP and Nodal signaling synergistically, but functionally differently, orchestrate human peri-implantation lineage development. Specially, we dissect mechanisms underlying extraembryonic mesoderm and extraembryonic endoderm specifications. Finally, an improved E-assembloid is developed to recapitulate the epiblast and hypoblast developments and tissue architectures in the pre-gastrulation human embryo. Our findings provide insights of the human peri-implantation development, and the E-assembloid offers a useful model to disentangle cellular behaviors and signaling interactions that drive human embryogenesis.

developmental biology↗

bioRxiv2023

DNA 5-methylcytosine regulates genome-wide formation of G-quadruplex structures

G-quadruplex structures (G4s) have been identified in genomes of multiple organisms and proven to play important epigenetic regulatory roles in various cellular functions. However, the G4 formation mechanism remains largely unknown. Here, we found a negative correlation between DNA 5mC methylation and G4 abundance. The abundance of genomic G4s significantly increased when the whole-genome methylation level was reduced in DNMT1-knockout cells. This increase was then suppressed by DNMT1 over-expression. And more G4s were detected in the hypomethylated cancer cell line HepG2 and rectal cancer tissues. Besides, 5mC modification significantly inhibited G4 formation of the potential G-quadruplex sequences (PQSs). The transcription of genes with 5mC modification sites in their promoter PQSs was affected after treatment with G4 stabilizer pyridostatin or methylation inhibitor 5-aza-dC. The global reduction of genomic methylation elevates gene transcription levels through increased G4s. Taken together, DNA 5mC methylation prevents PQSs from folding into G4s in genomes.

molecular biology↗

bioRxiv2023

Single-molecule displacement mapping indicates unhindered intracellular diffusion of small (<~1 kDa) solutes

While fundamentally important, the intracellular diffusion of small (<~1 kDa) solutes has been difficult to elucidate due to challenges in both labeling and measurement. Here we quantify and spatially map the translational diffusion patterns of small solutes in mammalian cells by integrating several recent advances. In particular, by executing tandem stroboscopic illumination pulses down to 400-s separation, we extend single-molecule displacement/diffusivity mapping (SMdM), a super-resolution diffusion quantification tool, to small solutes with high diffusion coefficients D of >300 m2/s. We thus show that for multiple water-soluble dyes and dye-tagged nucleotides, intracellular diffusion is dominated by vast regions of high diffusivity ~60-70% of that in vitro, up to ~250 m2/s in the fastest cases. Meanwhile, we also visualize sub-micrometer foci of substantial slowdowns in diffusion, thus underscoring the importance of spatially resolving the local diffusion behavior. Together, these results suggest that the intracellular diffusion of small solutes is only modestly scaled down by the slightly higher viscosity of the cytosol over water, but otherwise not further hindered by macromolecular crowding. We thus lift a paradoxically low speed limit for intracellular diffusion suggested by previous experiments. Abstract Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2023

Single-molecule displacement mapping unveils sign-asymmetric protein charge effects on intraorganellar diffusion

Using single-molecule displacement/diffusivity mapping (SMdM), an emerging super-resolution microscopy method, here we quantify, at nanoscale resolution, the diffusion of a typical fluorescent protein (FP) in the endoplasmic reticulum (ER) and mitochondrion of living mammalian cells. We thus show that the diffusion coefficients D in both organelles are ~40% of that in the cytoplasm, with the latter exhibiting higher spatial inhomogeneities. Moreover, we unveil that diffusions in the ER lumen and the mitochondrial matrix are markedly impeded when the FP is given positive, but not negative, net charges. Calculation shows most intraorganellar proteins as negatively charged, thus a mechanism to impede the diffusion of positively charged proteins. However, we further identify the ER protein PPIB as an exception with a positive net charge, and experimentally show that the removal of this positive charge elevates its intra-ER diffusivity. We thus unveil a sign-asymmetric protein charge effect on the nanoscale intraorganellar diffusion.

biophysics↗

bioRxiv2022

Enrichment of breast cancer stem cells following cytotoxic chemotherapy is mediated by hypoxia-inducible factors

Breast cancers (BCs) that do not express the estrogen or progesterone receptor or human epidermal growth factor receptor 2 are known as triple negative breast cancers (TNBCs). Women with TNBC receive non-targeted chemotherapy with a durable response rate of less than 20%. BC stem cells (BCSCs) are a small subpopulation of BC cells that are characterized by the capacity for infinite self-renewal; are the only BC cells capable of forming a secondary (recurrent or metastatic) BC; and must be eliminated in order to eradicate BC. Hypoxia-inducible factors (HIFs) activate hundreds of genes in TNBCs and HIF-1 expression in the diagnostic tumor biopsy is associated with patient mortality. In this paper, we report that treatment of TNBC cells with cytotoxic chemotherapy increased HIF-1 and HIF-2 protein levels and HIF target gene expression. Chemotherapy also increased the percentage of BCSCs through pathways involving interleukin-6 (IL-6), IL-8, and multidrug resistance 1. HIF inhibitors blocked increased BCSC specification in response to cytotoxic chemotherapy and combination therapy led to tumor eradication. Increased HIF target gene expression in BC biopsies was correlated with increased mortality, especially in those patients treated with chemotherapy alone. Our results suggest that HIF-dependent BCSC enrichment provides a molecular and cellular basis for the high incidence of relapse in women with TNBC.

cancer biology↗

bioRxiv2022

Benzopyrene biodegradation by multiple and individual mesophilic bacteria in axenic conditions and in soil samples

Thus far, only a handful of bacterial strains that can independently degrade and utilize benzo[a]pyrene (BaP) as the sole carbon source have been isolated and characterized. Here, three new bacterial strains, JBZ1A, JBZ2B, and JBZ5E, were isolated from contaminated soil and, using 16S rRNA sequencing, were identified as Bradyrhizobium japonicum, Micrococcus luteus, and Bacillus cereus, respectively. The growth ability of each individual strain and a consortium of all strains in the presence of BaP (4-400 {micro}mol{middle dot}l-1, pH 7, 37{degrees}C) was identified by the doubling time (dt). The results illustrated that dt decreased with increasing BaP concentrations for individual strain and the consortium. The optimum growth conditions of the consortium were 37{degrees}C, 0.5% NaCl (w/v), and pH 7. Under these conditions, the degradation rate was 1.06 {micro}mol{middle dot}l-1{middle dot}day-1, whereas that of individual strains ranged 0.9-0.38 {micro}mol{middle dot}l-1{middle dot}day-1. B. cereus had the strongest contribution to the consortiums activity, with a degradation rate of 0.9 {micro}mol{middle dot}l-1{middle dot}day-1. The consortium could also remove BaP spiked with soil, but at a lower rate (0.01 {micro}mol.l-1.day-1). High-performance liquid chromatography-high-resolution tandem mass spectrometry permitted the detection of the metabolites of these strains, and a biodegradation pathway has been proposed.

microbiology↗

bioRxiv2022

Locus c{oelig}ruleus noradrenergic neurons phase-lock to prefrontal cortical and hippocampal infra-slow rhythms which synchronize with behavioral events

The locus cruleus (LC) is the primary source of noradrenergic projections to the forebrain, and, in prefrontal cortex, is implicated in decision-making and executive function. LC neurons phase-lock to cortical infra-slow wave oscillations during sleep. Such infra-slow rhythms are rarely reported in awake states, despite their interest, since they correspond to the time scale of behavior. Thus, we investigated LC neuronal synchrony with infra-slow rhythms in awake rats performing an attentional set-shifting task. Local field potential (LFP) oscillation cycles in prefrontal cortex and hippocampus on the order of 0.4 Hz phase-locked to task events at crucial maze locations. Indeed, successive cycles of the infra-slow rhythms showed different wavelengths, as if they are periodic oscillations that can reset phase relative to salient events. Simultaneously recorded infra-slow rhythms in prefrontal cortex and hippocampus could show different cycle durations as well suggesting independent control. Most LC neurons (including optogenetically identified noradrenergic neurons) recorded here were phase-locked to these infra-slow rhythms, as were hippocampal and prefrontal units recorded on the LFP probes. The infra-slow oscillations also phase-modulated gamma amplitude, linking these rhythms at the time scale of behavior to those coordinating neuronal synchrony. This would provide a potential mechanism where noradrenaline, released by LC neurons in concert with the infra-slow rhythm, would facilitate synchronization or reset of these brain networks, underlying behavioral adaptation.

neuroscience↗

bioRxiv2021

Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter (AOTF), up to 6 subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low ([~]1%) crosstalks and high temporal resolutions (down to [~]10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and FRET (Forster resonance energy transfer) fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.

cell biology↗

bioRxiv2021

Apoptotic Extracellular Vesicles (ApoEVs) Safeguard Liver Homeostasis and Regeneration via Assembling an ApoEV-Golgi Organelle

Apoptosis is an integral physiological cell death process that occurs frequently and generates a huge number of apoptotic extracellular vesicles (apoEVs). However, whether apoEVs are necessary for maintaining organ homeostasis remains unclear. Here, we show that circulatory apoEVs engraft in liver and undergo specialized internalization by hepatocytes (HCs) based on surface signature of galactose and N-acetylgalactosamine. Furthermore, apoEVs rescue liver injury in apoptotic-deficient Fas mutant and Caspase-3 knockout mice, which is exerted by restoring the featured hepatic ploidy homeostasis. Surprisingly, apoEVs form a chimeric organelle complex with recipient Golgi apparatus via SNARE-mediated membrane interaction, which consequently facilitates microtubule organization and HC cytokinesis. Notably, through Golgi recovery and ploidy transition, apoEVs contribute to liver regeneration and protect against acute hepatic failure. Collectively, these results identify a previously unrecognized role for apoEVs and the specific mechanisms by which they safeguard liver homeostasis, and suggest the potential of apoEV-based therapy for liver disorders.

cell biology↗

bioRxiv2020

Modulation of Transient receptor potential melastatin 3 by protons through its intracellular binding sites

Transient receptor potential melastatin 3 channel (TRPM3) is a calcium-permeable nonselective cation channel that plays an important role in modulating glucose homeostasis in the pancreatic beta cells. However, how TRPM3 is regulated under physiological and pathological conditions is poorly understood. In this study, we found that both intracellular and extracellular protons block TRPM3 through its intracellular binding sites. We demonstrated that external protons indirectly block TRPM3, whereas internal protons inhibit TRPM3 directly with an inhibitory pH50 of 6.9 {+/-} 0.11. We identified three titratable residues, D1059, D1062, and D1073, at the inner vestibule of the channel pore that contribute to pH sensitivity. The mutation of D1073Q reduces TRPM3 current intensity and pH sensitivity; Replacement of Asp 1073 by Gln 1073 changes the reduction of TRPM3 outward current by low external pH 5.5, from 62 {+/-} 3 % in WT to 25 {+/-} 6.0 % in D1073Q. These results indicate that D1073 is not only essential for intracellular pH sensitivity, but it is also crucial for TRPM3 channel gating. In addition, a single mutation of D1059 or D1062 enhances pH sensitivity. In summary, our findings provide a novel molecular determinant for pH regulation of TRPM3. The inhibition of TRPM3 by protons may indicate an endogenous mechanism governing TRPM3 gating and its physiological/ pathological functions.

pharmacology and toxicology↗

bioRxiv2020

Transferrin receptor (Tfr1) ablation in satellite cells impacts skeletal muscle regeneration through the activation of ferroptosis

Satellite cells (SCs) are critical to the postnatal development and skeletal muscle regeneration. Inactivation of SCs is linked with the skeletal muscle loss. Leveraging on the RNAseq screening, transferrin receptor (Tfr1) is identified to be associated with muscle/SC ageing and the declined regeneration potential. Muscle-specific deletion of Tfr1 results in the growth retardation, metabolic disorder and lethality, shedding light on the importance of Tfr1 in skeletal muscle physiology. Here, our investigation reported that conditional SC-ablation of Tfr1 leads to the SCs inactivation and skeletal muscle regeneration defects, followed by the labile iron accumulation, de novo lipogenesis via fibroadipogenic progenitors (FAPs) and Gpx4/Nrf2-mediated ROS-scavenger defects. These abnormal phenomena, such as Hmox1-mediated myoglobin degradation, Tfr1-Slc39a14 functional switch and the activation of unsaturated fatty acid biosynthesis pathway are orchestrated with the occurrence of ferroptosis in skeletal muscle. Ferroptosis may further prevent SC proliferation and skeletal muscle regeneration. Ferrostatin-1, a ferroptosis inhibitor could not rescue Tfr1-ablation induced ferroptosis. However, intramuscular administration of lentivirus expressing Tfr1 could partially reduce labile iron accumulation, decrease de novo lipogenesis and promote skeletal muscle regeneration. Most importantly, Tfr1/Slc39a14 functional switch, labile iron accumulation and fatty acid biosynthesis are recapitulated in aged skeletal muscle of rodents, indicating that ferroptosis occurs in the skeletal muscles of aged rodents. The present study also bridges the gap between pathogenesis of iron and functional defects in the skeletal muscle, providing mechanistic information to develop anti-aging strategies. One Sentence SummaryConditional ablation of Tfr1 in satellite cells (SCs) results in the SC inactivation, skeletal muscle regeneration defects, labile iron accumulation, and unsaturated fatty acid biosynthesis, leading to the activation of ferroptosis, which is recapitulated in skeletal muscles of aged rodents to be a new cell death form identified in skeletal muscle and sheds light on the development of novel anti-ageing strategies.

cell biology↗

bioRxiv2020

Robotic Assay for Drought (RoAD): An Automated Phenotyping System for Brassinosteroid and Drought Response

Brassinosteroids (BRs) are a group of plant steroid hormones involved in regulating growth, development, and stress responses. Many components of the BR pathway have previously been identified and characterized. However, BR phenotyping experiments are typically performed on petri plates and/or in a low-throughput manner. Additionally, the BR pathway has extensive crosstalk with drought responses, but drought experiments are time-consuming and difficult to control. Thus, we developed Robotic Assay for Drought (RoAD) to perform BR and drought response experiments in soil-grown Arabidopsis plants. RoAD is equipped with a bench scale, a precisely controlled watering system, an RGB camera, and a laser profilometer. It performs daily weighing, watering, and imaging tasks and is capable of administering BR response assays by watering plants with Propiconazole (PCZ), a BR biosynthesis inhibitor. We developed image processing algorithms for both plant segmentation and phenotypic trait extraction in order to accurately measure traits in 2-dimensional (2D) and 3-dimensional (3D) spaces including plant surface area, leaf length, and leaf width. We then applied machine learning algorithms that utilized the extracted phenotypic parameters to identify image-derived traits that can distinguish control, drought, and PCZ-treated plants. We carried out PCZ and drought experiments on a set of BR mutants and Arabidopsis accessions with altered BR responses. Finally, we extended the RoAD assays to perform BR response assays using PCZ in Zea mays (maize) plants. This study establishes an automated and non-invasive robotic imaging system as a tool to accurately measure morphological and growth-related traits of Arabidopsis and maize plants, providing insights into the BR-mediated control of plant growth and stress responses.

plant biology↗

bioRxiv2020

Reconstruction and dynamics of human intestinal microbiome observed in situ

Gut microbiome are studied primarily using fecal samples in humans and we gained vital knowledge of compositional and functional capacities of gastro-intestinal microbial communities. Yet, fecal materials limit our ability to investigate microbial dynamics in different locations along GI-tract (in situ), nor in finer temporal scales as they are infrequent. With a technology developed originally for fecal material transplantation, colonic transendoscopic enteral tubing, we were able to sample ileocecal microbiome twice daily, and carried out metagenomic as well as metatranscriptomic analyses. Ileocecal and fecal microbiome are similar in metagenomic profiling, yet their active genes (in metatranscriptomes) are highly distinct. Both were perturbed after laxatives and then became more similar to microbiome prior to treatment, demonstrating resilience as an innate property of gut microbiome. Ileocecal microbiome transcriptomes sampled during day and night revealed diurnal rhythmes exist in certain bacterial species and functional pathways, in particular those related to short-chain fatty acid production. Lastly, metabolomic analysis in fecal and urine samples mirrored the perturbance and recovery in gut microbiome, indicating crucial contribution of gut microbiome to many of the key metabolites involved in host health. Our study provides interesting novel insights into human gut microbiome, and demonstrates the inner resilience, diurnal rhythmes and potential consequences to the host.

microbiology↗

bioRxiv2020

A novel fibrinogen gamma-chain mutation, p. γAla327Val, causes structural abnormality of D region and ultimately leads to congenital dysfibrinogenemia

Congenital dysfibrinogenemia (CD) is a coagulation disorder caused by mutations in the fibrinogen gene, which result in abnormal fibrinogen function. Many studies have confirmed that over half of dysfibrinogenemia cases are asymptomatic. In this study, we aimed to investigate the pathogenesis of CD caused by {gamma} Ala327Val heterozygous mutation, a new mutation, by studying fibrinogen function. Blood samples of patients were collected and the coagulation function, fibrinogen (Fg) aggregation test, fibrin clot lysis test, and SDS-PAGE were performed. Coagulation was monitored using a thromboelastometer, and the fibrin clot network structure was observed by scanning electron microscopy. The effect of the mutation on fibrinogen structure and function was predicted by molecular modeling. The fibrinogen activity concentration in patients with CD was significantly lower than that in healthy individuals. Thromboelastography showed that the K value of patients with CD was higher than that for healthy individuals. The Angle values were also decreased. The function of fibrinogen in patients with CD was low. Compared to fibrinogen from healthy individuals, fibrin size was different, the fiber network structure was loose, the pore size was increased, and the fiber branch nodes were increased for fibrinogen isolated from the proband. The {gamma} Ala327Val mutation led to changes in the structure of fibrinogen D region, affecting its structural stability. Ala327Val heterozygous missense mutation in exon 8 of FGG gene {gamma}-chain thus leads to abnormal fibrinogen structure and impairs the aggregation function of fibrinogen. This mutation is reported here for the first time.

molecular biology↗

bioRxiv2019

Crif1 Promotes Osteoporosis in Mice after Radiation

Radiation induces rapid bone loss and enhances bone resorption and RANKL expression. RANKL provides the crucial signal to induce osteoclast differentiation and plays an important role in bone resorption. However, the mechanisms of radiation-induced osteoporosis are not fully understood. Here, we show that Crif1 expression increases in bone marrow cells after radiation. Conditional Crif1 deletion in bone marrow cells causes decreases in RANKL expression and the RANKL/OPG ratio, and relieves bone loss after radiation in mice. We further demonstrated in vitro that Crif1 promotes RANKL secretion via the cAMP/PKA pathway. Moreover, protein-protein docking screening identified five compounds as Crif1 inhibitors; these compounds dramatically suppressed RANKL secretion and CREB phosphorylation when cells were exposed to forskolin. This study enriches current knowledge of the pathogenesis of osteoporosis and provides insights into potential therapeutic strategies for osteoporosis treatment.

cell biology↗

bioRxiv2019

Super-resolution displacement mapping of unbound single molecules reveals nanoscale heterogeneities in intracellular diffusivity

Intracellular diffusion underlies vital processes of the cell. However, it remains difficult to elucidate how an average-sized protein diffuses in the cell with good spatial resolution and sensitivity. Here we report single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanoscale mapping of intracellular diffusivity through the local statistics of instantaneous displacements of freely diffusing single molecules. We thus show that diffusion in the cytoplasm and in the nucleus to both be spatially heterogeneous at the nanoscale, and such variations in local diffusivity correlate strongly with the ultrastructure of the actin cytoskeleton and the chromosome, respectively. Moreover, we identify the net charge of the diffuser as a key determinant of diffusion rate: intriguingly, the possession of positive, but not negative, net charges significantly impedes diffusion, and the exact degree of slowdown is determined by the specific subcellular environments.

biophysics↗

bioRxiv2019

Delta/Notch signaling controls neuroepithelial morphogenesis in the zebrafish spinal cord

The morphogenesis of the nervous system requires coordinating the specification and differentiation of neural precursor cells, the establishment of neuroepithelial tissue architecture and the execution of specific cellular movements. How these aspects of neural development are linked is incompletely understood. Here we inactivate a major regulator of embryonic neurogenesis - the Delta/Notch pathway - and analyze the effect on zebrafish central nervous system morphogenesis. While some parts of the nervous system can establish neuroepithelial tissue architecture independently of Notch, Notch signaling is essential for spinal cord morphogenesis. In this tissue, Notch signaling is required to repress neuronal differentiation and promote neuroepithelial apico-basal polarity. Concomitant with a loss of their neuroepithelial properties, Notch signaling deficient cells also alter their morphogenetic behavior. In the wild-type zebrafish neural tube, cells divide at the organ midline to contribute one daughter cell to each organ half. Notch deficient animals fail to display this behavior and therefore form a misproportioned spinal cord. Taken together, our findings show that Notch signaling governs not only the cellular composition but also the morphogenetic shaping of the zebrafish spinal cord.

developmental biology↗

bioRxiv2018

Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates, and humans

Targeting genes to specific neuronal or glial cell types is valuable both for understanding and for repairing brain circuits. Adeno-associated viral vectors (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is a challenge. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that ~11% of these AAVs specifically target expression to neuronal and glial cell types in the mouse retina, mouse brain, non-human primate retina in vivo, and in the human retina in vitro. We demonstrate applications for recording, stimulation, and molecular characterization, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast, and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

neuroscience↗