RESUMO
With increases in life expectancy, the number of patients requiring joint replacement therapy and experiencing periprosthetic osteolysis, the most common complication leading to implant failure, is growing or underestimated. In this study, we found that osteolysis progression and osteoclast differentiation in the surface of the skull bone of adult mice were accompanied by significant expansion of lymphatic vessels within bones. Using recombinant VEGF-C protein to activate VEGFR3 and promote proliferation of lymphatic vessels in bone, we counteracted excessive differentiation of osteoclasts and osteolysis caused by titanium alloy particles or inflammatory cytokines LPS/TNF-α. However, this effect was not observed in aged mice because adipogenically differentiated mesenchymal stem cells (MSCs) inhibited the response of lymphatic endothelial cells to agonist proteins. The addition of the JAK inhibitor ruxolitinib restored the response of lymphatic vessels to external stimuli in aged mice to protect against osteolysis progression. These findings suggest that inhibiting SASP secretion by adipogenically differentiated MSCs while activating lymphatic vessels in bone offers a new method to prevent periprosthetic osteolysis during joint replacement follow-up.
Assuntos
Vasos Linfáticos , Células-Tronco Mesenquimais , Osteólise , Animais , Osteólise/prevenção & controle , Camundongos , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Envelhecimento , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Osteoclastos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Masculino , Fenótipo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Crânio/patologia , Crânio/efeitos dos fármacos , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , TitânioRESUMO
The three major anatomic regions of the human kidney include the cortex, medulla and papilla, with different functions and vulnerabilities to kidney diseases. Epigenetic mechanisms underlying these anatomic structures are incompletely understood. Here, we performed chromatin conformation capture with Hi-C and histone modification H3K4me3/H3K27me3 Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing on the kidney cortex, medulla and papilla dissected from one individual donor. Nuclear suspensions were generated from each region and split subjected to paired Hi-C and CUT&RUN sequencing. We evaluated the quality of next-generation sequencing data, Hi-C chromatin contact matrices and CUT&RUN peak calling. H3K4me3 and H3K27me3 histone modifications represent active and repressive gene transcription, respectively, and differences in chromatin conformation between kidney regions can be analyzed with this dataset. All raw and processed data files are publicly available, allowing researchers to survey the epigenetic landscape across regional human kidney anatomy.
Assuntos
Cromatina , Histonas , Rim , Humanos , Cromatina/metabolismo , Histonas/metabolismo , Rim/metabolismo , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Código das HistonasRESUMO
Simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq) profiles transcriptomics and chromatin accessibility in the same cells at high throughput. Here, we present a protocol for multimodal profiling of human kidneys with SHARE-seq. We describe steps for processing fixed nuclei for SHARE-seq split-pool barcoding and library preparation. We also detail how to determine the optimal working concentration of Tn5 transposase for transposition and tagmentation. This protocol allows researchers to generate large-scale single-cell multiomics data at low reagent cost. For complete details on the use and execution of this protocol, please refer to Li et al.1.
RESUMO
Cortex, medulla and papilla are three major human kidney anatomic structures and they harbour unique metabolic functions, but the underlying metabolomic profiles are largely unknown at spatial resolution. Here, we generated a spatially resolved metabolomics dataset on human kidney cortex, medulla and papilla tissues dissected from the same donor. Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry (MALDI-IMS) was used to detect metabolite species over mass-to-charge ratios of 50 -1500 for each section at a resolution of 10 × 10 µm2 pixel size. We present raw data matrix of each sample, feature annotations, raw AnnData merged from three samples and processed AnnData files after quality control, dimensional reduction and data integration, which contains a total of 170,459 spatially resolved metabolomes with 562 features detected. This dataset can be either visualized through an interactive browser or further analyzed to study metabolomic heterogeneity across regional human kidney anatomy.
RESUMO
A large-scale multimodal atlas that includes major kidney regions is lacking. Here, we employed simultaneous high-throughput single-cell ATAC/RNA sequencing (SHARE-seq) and spatially resolved metabolomics to profile 54 human samples from distinct kidney anatomical regions. We generated transcriptomes of 446,267 cells and chromatin accessibility profiles of 401,875 cells and developed a package to analyze 408,218 spatially resolved metabolomes. We find that the same cell type, including thin limb, thick ascending limb loop of Henle and principal cells, display distinct transcriptomic, chromatin accessibility, and metabolomic signatures, depending on anatomic location. Surveying metabolism-associated gene profiles revealed non-overlapping metabolic signatures between nephron segments and dysregulated lipid metabolism in diseased proximal tubule (PT) cells. Integrating multimodal omics with clinical data identified PLEKHA1 as a disease marker, and its in vitro knockdown increased gene expression in PT differentiation, suggesting possible pathogenic roles. This study highlights previously underrepresented cellular heterogeneity underlying the human kidney anatomy.
Assuntos
Epigenômica , Rim , Metabolômica , Transcriptoma , Humanos , Rim/metabolismo , Masculino , Perfilação da Expressão Gênica , FemininoRESUMO
Ten percent of the population worldwide suffers from chronic kidney disease (CKD), but the mechanisms driving CKD pathology are incompletely understood. While dysregulated lipid metabolism is one hallmark of CKD, the pathogenesis of cellular lipid accumulation remains unclear. In this issue of the JCI, Mukhi et al. Identify acyl-CoA synthetase short-chain family 2 (ACSS2) as a disease risk gene and demonstrate a role for ACSS2 in de novo lipogenesis (DNL). Notably, genetic or pharmacological inhibition of DNL protected against kidney disease progression in mice. These findings warrant evaluation of DNL inhibition with respect to efficacy and safety in people with CKD.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Insuficiência Renal Crônica , Humanos , Animais , Camundongos , Lipogênese/fisiologia , Insuficiência Renal Crônica/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
BACKGROUND: Mosaic loss of Y chromosome (LOY) is the most common chromosomal alteration in aging men. Here, we use single-cell RNA and ATAC sequencing to show that LOY is present in the kidney and increases with age and chronic kidney disease. RESULTS: The likelihood of a cell having LOY varies depending on its location in the nephron. Cortical epithelial cell types have a greater proportion of LOY than medullary or glomerular cell types, which may reflect their proliferative history. Proximal tubule cells are the most abundant cell type in the cortex and are susceptible to hypoxic injury. A subset of these cells acquires a pro-inflammatory transcription and chromatin accessibility profile associated with expression of HAVCR1, VCAM1, and PROM1. These injured epithelial cells have the greatest proportion of LOY and their presence predicts future kidney function decline. Moreover, proximal tubule cells with LOY are more likely to harbor additional large chromosomal gains and express pro-survival pathways. Spatial transcriptomics localizes injured proximal tubule cells to a pro-fibrotic microenvironment where they adopt a secretory phenotype and likely communicate with infiltrating immune cells. CONCLUSIONS: We hypothesize that LOY is an indicator of increased DNA damage and potential marker of cellular senescence that can be applied to single-cell datasets in other tissues.
Assuntos
Cromossomos Humanos Y , Insuficiência Renal Crônica , Humanos , Masculino , Mosaicismo , Envelhecimento/genética , Fenótipo , Insuficiência Renal Crônica/genéticaRESUMO
An altered proteome in lymph nodes often suggests abnormal signaling pathways that may be associated with diverse lymphatic disorders. Current clinical biomarkers for histological classification of lymphomas have encountered many discrepancies, particularly for borderline cases. Therefore, we launched a comprehensive proteomic study aimed to establish a proteomic landscape of patients with various lymphatic disorders and identify proteomic variations associated with different disease subgroups. In this study, 109 fresh-frozen lymph node tissues from patients with various lymphatic disorders (with a focus on Non-Hodgkin's Lymphoma) were analyzed by data-independent acquisition mass spectrometry. A quantitative proteomic landscape was comprehensively characterized, leading to the identification of featured protein profiles for each subgroup. Potential correlations between clinical outcomes and expression profiles of signature proteins were also probed. Two representative signature proteins, phospholipid-binding proteins Annexin A6 (ANXA6) and Phospholipase C Gamma 2 (PLCG2), were successfully validated via immunohistochemistry. We also evaluated the capability of acquired proteomic signatures to segregate multiple lymphatic abnormalities and identified several core signature proteins, such as Sialic Acid Binding Ig Like Lectin 1 (SIGLEC1) and GTPase of immunity-associated protein 5 (GIMAP5). In summary, the established lympho-specific data resource provides a comprehensive map of protein expression in lymph nodes during multiple disease states, thus extending the existing human tissue proteome atlas. Our findings will be of great value in exploring protein expression and regulation underlying lymphatic malignancies, while also providing novel protein candidates to classify various lymphomas for more precise medical practice. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-022-00075-w.
RESUMO
Conjugated polymers featuring thermally activated delayed fluorescence (TADF) attract tremendous attention in both academic and industry communities due to their easy solution processing for fabricating large-area and low-cost high-performance polymer light-emitting diodes (PLEDs). However, current nondoped solution-processed PLEDs frequently encounter significant efficiency roll-offs and unreasonably high operating voltages at high brightness, especially for red-emitting polymers. Herein, we design hyperbranched conjugated polymers (HCPs) with D-A-D type TADF characteristics for high-performance red-emitting PLEDs. Multiple intramolecular charge transfer (ICT) channels induced by quasi-equivalent donors of the TADF core strongly boost the reverse intersystem crossing (RISC) process and singlet excitons radiative transition. Coupling with the efficient energy transfer process generated by structure advantages of HCPs, the strongly electron-withdrawing oxygen atoms located on the TADF cores further accelerate hole transportation from the host chains to the TADF cores. Under a rational regulation of the TADF core ratio, the related nondoped red-emitting device performs an outstanding performance with an EQEmax of 8.39% and exhibits no roll-off while the luminance is less than 100 cd/m2 and only 3.3% decrease at 500 cd/m2. Simultaneously, the EQE can maintain 7.4% under 1000 cd/m2. Furthermore, the corresponding nondoped device exhibits a low turn-on voltage of around 2.5 V and achieves a luminance of 500 cd/m2 at 3.5 V and even 1000 cd/m2 at 3.9 V. To our knowledge, this is the best performance among all nondoped red PLEDs with high brightness obtained at low operating voltage.
RESUMO
The underlying cellular events driving kidney fibrogenesis and metabolic dysfunction are incompletely understood. Here, we employed single-cell combinatorial indexing RNA sequencing to analyze 24 mouse kidneys from two fibrosis models. We profiled 309,666 cells in one experiment, representing 50 cell types/states encompassing epithelial, endothelial, immune, and stromal populations. Single-cell analysis identified diverse injury states of the proximal tubule, including two distinct early-phase populations with dysregulated lipid and amino acid metabolism, respectively. Lipid metabolism was defective in the chronic phase but was transiently activated in the very early stages of ischemia-induced injury, where we discovered increased lipid deposition and increased fatty acid ß-oxidation. Perilipin 2 was identified as a surface marker of intracellular lipid droplets, and its knockdown in vitro disrupted cell energy state maintenance during lipid accumulation. Surveying epithelial cells across nephron segments identified shared and unique injury responses. Stromal cells exhibited high heterogeneity and contributed to fibrogenesis by epithelial-stromal crosstalk.
Assuntos
Rim , Lipólise , Animais , Camundongos , Fibrose , LipídeosRESUMO
Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).1.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , RNA , Animais , Camundongos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biblioteca Gênica , Análise de Sequência de RNA/métodos , RimRESUMO
Single-cell RNA-sequencing (scRNA-seq) has been widely adopted in recent years due to standardized protocols and automation, reliability, and standardized bioinformatic pipelines. The most widely adopted platform is the 10× Genomics solution. Although powerful, this system is limited by its high cost, moderate throughput, and the inability to customize due to fixed kit components. This study will cover new approaches that do not rely on microfluidics and thus have low entry costs, are highly customizable, and are within the reach of any laboratory possessing molecular biology expertise.
Assuntos
Microfluídica , Análise de Célula Única , Genômica/métodos , Reprodutibilidade dos Testes , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodosRESUMO
The quantitative analysis of trace water in organic solvents has always been a research hotspot, and it is still in the development stage and needs to be continuously developed. In this study, a facile and rapid approach was developed for the preparation of carbon quantum dots (CQDs) with yellow fluorescence emission and ultrahigh absolute fluorescence quantum yields (92.6%). Compared to traditional organic fluorescent molecules, the preparation of CQDs is simpler, faster and more environmentally friendly. It is found that the fluorescent properties of CQDs are excellent in organic solvents and could be quenched by trace water, which makes them a promising material used without any modification for the detection of water in organic solvents. As a result, the as-prepared CQDs were adopted as fluorescent probes for the detection of water in organic solvents (ethanol, tetrahydrofuran, and 1,4-dioxane). The limit of detection was as low as 0.01%. To the best of our knowledge, this is the first time that CQDs have been used as water sensing fluorescent probes in organic solvents. The possible mechanism for trace water detection of the as-prepared CQDs in organic solvents is attributed to the specific water-fluorophore interaction and partially to the increase in polarity of the solvent caused by an increase in water concentration.
RESUMO
A great challenge in developing nanotechnologies for cancer diagnosis and therapy has been the combined functionalities required for complicated clinical procedures. Among all requirements, toxicity has been the major hurdle that has prevented most of the nano-carriers from clinical use. Here, we extracted chlorophyll (Chl) from vegetable and encapsulated it into polymer (pluronic F68, Plu) micelles for cancer imaging and therapy. The results showed that the Chl-containing nanocomposites were capable of mouse tumor targeting, and the nanocomposite fluorescence within the tumor sites remained at high intensity more than two days after tail-vein injection. It is interesting that oral administration with the nanocomposites was also successful for tumor target imaging. Furthermore, the dietary Chl was found to be able to efficiently convert near-infrared laser irradiation to heat. The growths of melanoma cells and mouse tumors were effectively inhibited after being treated with the nanocomposites and irradiation. The suppression of the tumors was achieved by laser-triggered photothermal and photodynamic synergistic effects of Chl. As a natural substance from vegetable, Chl is non-toxic, making it an ideal nano-carrier for cancer diagnosis and treatment. Based on the results of this research, the Plu-Chl nanocomposites have shown promise for future clinical applications.
Assuntos
Clorofila/uso terapêutico , Melanoma/diagnóstico , Melanoma/terapia , Nanocompostos/química , Fármacos Fotossensibilizantes/uso terapêutico , Animais , Linhagem Celular , Clorofila/administração & dosagem , Sistemas de Liberação de Medicamentos , Humanos , Hipertermia Induzida , Lasers , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Micelas , Fotoquimioterapia , Fármacos Fotossensibilizantes/administração & dosagem , Poloxâmero/químicaRESUMO
A combination of abâ initio calculations, circular dichroism, nuclear magnetic resonance, and X-ray photoelectron spectroscopy has shown that aluminum ions can induce the formation of backbone ring structures in a wide range of peptides, including neurodegenerative disease related motifs. These ring structures greatly destabilize the protein and result in irreversible denaturation. This behavior benefits from the ability of aluminum ions to form chemical bonds simultaneously with the amide nitrogen and carbonyl oxygen atoms on the peptide backbone.
Assuntos
Alumínio/química , Proteínas/química , Teoria Quântica , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Conformação Proteica , Desnaturação ProteicaRESUMO
The photothermal effect of Fe3O4 magnetic nanoparticles is investigated for cancer therapy both in vitro and in vivo experiments. Heat is found to be rapidly generated by red and near-infrared (NIR) range laser irradiation of Fe3O4 nanoparticles with spherical, hexagonal and wire-like shapes. These Fe3O4 nanoparticles are coated with carboxyl-terminated poly (ethylene glycol)-phospholipid for enhanced dispersion in water. The surface-functionalized Fe3O4 nanoparticles can be taken up by esophageal cancer cells and do not obviously affect the cell structure and viability. Upon irradiation at 808 nm however, the esophageal cancer cell viability is effectively suppressed, and the cellular organelles are obviously damaged when incubated with the NIR laser activated Fe3O4 nanoparticles. Mouse esophageal tumor growth was found to be significantly inhibited by the photothermal effect of Fe3O4 nanoparticles, resulting in effective tumor reduction. A morphological examination revealed that after a photothermal therapy, the tumor tissue structure exhibited discontinuation, the cells were significantly shriveled and some cells have finally disintegrated.