scholarly journals High content Image Analysis to study phenotypic heterogeneity in endothelial cell monolayers

2022 ◽  
Author(s):  
Francois Chesnais ◽  
Jonas Hue ◽  
Errin Roy ◽  
Marco Branco ◽  
Ruby Stokes ◽  
...  

Endothelial cells (EC) are heterogeneous across and within tissues, reflecting distinct, specialised functions. EC heterogeneity has been proposed to underpin EC plasticity independently from vessel microenvironments. However, heterogeneity driven by contact-dependent or short-range cell-cell crosstalk cannot be evaluated with single cell transcriptomic approaches as spatial and contextual information is lost. Nonetheless, quantification of EC heterogeneity and understanding of its molecular drivers is key to developing novel therapeutics for cancer, cardiovascular diseases and for revascularisation in regenerative medicine. Here, we developed an EC profiling tool (ECPT) to examine individual cells within intact monolayers. We used ECPT to characterise different phenotypes in arterial, venous and microvascular EC populations. In line with other studies, we measured heterogeneity in terms of cell cycle, proliferation, and junction organisation. ECPT uncovered a previously under-appreciated single-cell heterogeneity in NOTCH activation. We correlated cell proliferation with different NOTCH activation states at the single cell and population levels. The positional and relational information extracted with our novel approach is key to elucidating the molecular mechanisms underpinning EC heterogeneity.

Author(s):  
Dan Song ◽  
Ming Guo ◽  
Shuai Xu ◽  
Xiaotian Song ◽  
Bin Bai ◽  
...  

Abstract Background Pseudouridine synthase (PUS) 7 is a member of the PUS family that catalyses pseudouridine formation. It has been shown to be involved in intellectual development and haematological malignancies. Nevertheless, the role and the underlying molecular mechanisms of PUS7 in solid tumours, such as colorectal cancer (CRC), remain unexplored. This study elucidated, for the first time, the role of PUS7 in CRC cell metastasis and the underlying mechanisms. Methods We conducted immunohistochemistry, qPCR, and western blotting to quantify the expression of PUS7 in CRC tissues as well as cell lines. Besides, diverse in vivo and in vitro functional tests were employed to establish the function of PUS7 in CRC. RNA-seq and proteome profiling analysis were also applied to identify the targets of PUS7. PUS7-interacting proteins were further uncovered using immunoprecipitation and mass spectrometry. Results Overexpression of PUS7 was observed in CRC tissues and was linked to advanced clinical stages and shorter overall survival. PUS7 silencing effectively repressed CRC cell metastasis, while its upregulation promoted metastasis, independently of the PUS7 catalytic activity. LASP1 was identified as a downstream effector of PUS7. Forced LASP1 expression abolished the metastasis suppression triggered by PUS7 silencing. Furthermore, HSP90 was identified as a client protein of PUS7, associated with the increased PUS7 abundance in CRC. NMS-E973, a specific HSP90 inhibitor, also showed higher anti-metastatic activity when combined with PUS7 repression. Importantly, in line with these results, in human CRC tissues, the expression of PUS7 was positively linked to the expression of HSP90 and LASP1, and patients co-expressing HSP90/PUS7/LASP1 showed a worse prognosis. Conclusions The HSP90-dependent PUS7 upregulation promotes CRC cell metastasis via the regulation of LASP1. Thus, targeting the HSP90/PUS7/LASP1 axis may be a novel approach for the treatment of CRC.


2020 ◽  
Vol 2020 ◽  
pp. 1-18
Author(s):  
Sonia Setia ◽  
Verma Jyoti ◽  
Neelam Duhan

The continuous growth of the World Wide Web has led to the problem of long access delays. To reduce this delay, prefetching techniques have been used to predict the users’ browsing behavior to fetch the web pages before the user explicitly demands that web page. To make near accurate predictions for users’ search behavior is a complex task faced by researchers for many years. For this, various web mining techniques have been used. However, it is observed that either of the methods has its own set of drawbacks. In this paper, a novel approach has been proposed to make a hybrid prediction model that integrates usage mining and content mining techniques to tackle the individual challenges of both these approaches. The proposed method uses N-gram parsing along with the click count of the queries to capture more contextual information as an effort to improve the prediction of web pages. Evaluation of the proposed hybrid approach has been done by using AOL search logs, which shows a 26% increase in precision of prediction and a 10% increase in hit ratio on average as compared to other mining techniques.


Bone Research ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yibo Gan ◽  
Jian He ◽  
Jun Zhu ◽  
Zhengyang Xu ◽  
Zhong Wang ◽  
...  

AbstractA comprehensive understanding of the cellular heterogeneity and molecular mechanisms underlying the development, homeostasis, and disease of human intervertebral disks (IVDs) remains challenging. Here, the transcriptomic landscape of 108 108 IVD cells was mapped using single-cell RNA sequencing of three main compartments from young and adult healthy IVDs, including the nucleus pulposus (NP), annulus fibrosus, and cartilage endplate (CEP). The chondrocyte subclusters were classified based on their potential regulatory, homeostatic, and effector functions in extracellular matrix (ECM) homeostasis. Notably, in the NP, a PROCR+ resident progenitor population showed enriched colony-forming unit-fibroblast (CFU-F) activity and trilineage differentiation capacity. Finally, intercellular crosstalk based on signaling network analysis uncovered that the PDGF and TGF-β cascades are important cues in the NP microenvironment. In conclusion, a single-cell transcriptomic atlas that resolves spatially regulated cellular heterogeneity together with the critical signaling that underlies homeostasis will help to establish new therapeutic strategies for IVD degeneration in the clinic.


2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii323-iii323
Author(s):  
Flavia W de Faria ◽  
Marta Interlandi ◽  
Natalia Moreno ◽  
Monika Graf ◽  
Viktoria Melcher ◽  
...  

Abstract Embryonal tumors with multilayered rosettes (ETMR) are deadly brain malignancies affecting young children. No standard treatment is available and the median survival is less than 12 months. Molecularly, the disease is characterized by the miRNA C19MC cluster amplification, with the expression of multiples miRNAs related to a stem cell program. The discoveries on the purely molecular mechanisms of the disease did not help to create a bridge for new treatment strategies so far and the cellular diversity of ETMR remains poorly understood. In this study, we used single-cell RNA sequencing of murine and human tumors to describe ETMR cellular heterogeneity. Our findings support that intra-tumoral heterogeneity is mainly characterized by 4 cellular programs defining a developmental hierarchy related to different metabolic states: 1) Early quiescent NSC-like cells supported by fatty-acid oxidation 2) Late NSC and NP-like proliferative cells fueled by glycolytic metabolism; 3) Post-mitotic neuroblast-like cells, relying on oxidative-phosphorylation; 4) NSC-like proliferative cells, with metabolic plasticity and capable of performing the three types of metabolism. Tumor-specific ligand-receptor interaction analysis revealed that ETMR exchange with microglia and vascular mural cells (MC) signals related to extracellular matrix (ECM) organization (Cxcl12-CxCr4), stem cell signaling (BMPs-BMP receptors), anti-apoptosis and survival (Ntf3-Ntrk), not seen in the control brain. In addition, the vascular MC showed a cancer-associated fibroblast (CAF) phenotype, with potential prognostic implications, as previously demonstrated for other tumors. This study provides new findings to build up a more robust understanding of ETMR biology and opens space for further studies in the field.


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Yong Zhong ◽  
Xiangcheng Xiao

Abstract Background and Aims The exact molecular mechanisms underlying IgA nephropathy (IgAN) remains incompletely defined. Therefore, it is necessary to further elucidate the mechanism of IgA nephropathy and find novel therapeutic targets. Method Single-cell RNA sequencing (scRNA-seq) was applied to kidney biopsies from 4 IgAN and 1 control subjects to define the transcriptomic landscape at the single-cell resolution. Unsupervised clustering analysis of kidney specimens was used to identify distinct cell clusters. Differentially expressed genes and potential signaling pathways involved in IgAN were also identified. Results Our analysis identified 14 cell subsets in kidney biopsies from IgAN patients, and analyzed changing gene expression in distinct renal cell types. We found increased mesangial expression of several novel genes including MALAT1, GADD45B, SOX4 and EDIL3, which were related to proliferation and matrix accumulation and have not been reported in IgAN previously. The overexpressed genes in tubule cells of IgAN were mainly enriched in inflammatory pathways including TNF signaling, IL-17 signaling and NOD-like receptor signaling. Moreover, the receptor-ligand crosstalk analysis revealed potential interactions between mesangial cells and other cells in IgAN. Specifically, IgAN with overt proteinuria displayed elevated genes participating in several signaling pathways which may be involved in pathogenesis of progression of IgAN. Conclusion The comprehensive analysis of kidney biopsy specimen demonstrated different gene expression profile, potential pathologic ligand-receptor crosstalk, signaling pathways in human IgAN. These results offer new insight into pathogenesis and identify new therapeutic targets for patients with IgA nephropathy.


Author(s):  
Tongbin Wu ◽  
Zhengyu Liang ◽  
Zengming Zhang ◽  
Canzhao Liu ◽  
Lunfeng Zhang ◽  
...  

Background: Left ventricular noncompaction cardiomyopathy (LVNC) was discovered half a century ago as a cardiomyopathy with excessive trabeculation and a thin ventricular wall. In the decades since, numerous studies have demonstrated that LVNC primarily impacts left ventricles (LVs), and is often associated with LV dilation and dysfunction. However, owing in part to the lack of suitable mouse models that faithfully mirror the selective LV vulnerability in patients, mechanisms underlying susceptibility of LV to dilation and dysfunction in LVNC remain unknown. Genetic studies have revealed that deletions and mutations in PRDM16 cause LVNC, but previous conditional Prdm16 knockout mouse models do not mirror the LVNC phenotype in patients, and importantly, the underlying molecular mechanisms by which PRDM16 deficiency causes LVNC are still unclear. Methods: Prdm16 cardiomyocyte (CM)-specific knockout ( Prdm16 cKO ) mice were generated and analyzed for cardiac phenotypes. RNA sequencing and ChIP sequencing were performed to identify direct transcriptional targets of PRDM16 in CMs. Single cell RNA sequencing in combination with Spatial Transcriptomics were employed to determine CM identity at single cell level. Results: CM-specific ablation of Prdm16 in mice caused LV-specific dilation and dysfunction, as well as biventricular noncompaction, which fully recapitulated LVNC in patients. Mechanistically, PRDM16 functioned as a compact myocardium-enriched transcription factor, which activated compact myocardial genes while repressing trabecular myocardial genes in LV compact myocardium. Consequently, Prdm16 cKO LV compact myocardial CMs shifted from their normal transcriptomic identity to a transcriptional signature resembling trabecular myocardial CMs and/or neurons. Chamber-specific transcriptional regulation by PRDM16 was in part due to its cooperation with LV-enriched transcription factors Tbx5 and Hand1. Conclusions: These results demonstrate that disruption of proper specification of compact CM may play a key role in the pathogenesis of LVNC. They also shed light on underlying mechanisms of LV-restricted transcriptional program governing LV chamber growth and maturation, providing a tangible explanation for the susceptibility of LV in a subset of LVNC cardiomyopathies.


2012 ◽  
Vol 68 (1) ◽  
pp. 40-56 ◽  
Author(s):  
Joachim Kusz ◽  
Maciej Zubko ◽  
Reinhard B. Neder ◽  
Phillipp Gütlich

In the spin-crossover compound [Fe(ptz)6](BF4)2 (where ptz=1-n-propyltetrazole) six different phases are observed. When a single crystal is slowly cooled from high temperatures to those below 125 K, the reflections broaden into diffuse maxima and split into two maxima along the c* direction [Kusz, Gütlich & Spiering (2004). Top. Curr. Chem. 234, 129–153]. As both maxima are broad along the c* direction, the short-range order exists only along the c direction and in the ab plane the structure remains long-range ordered. In this disordered phase additional satellite reflections appear. Upon heating above 135 K, the diffuse maxima return to their previous shape and this process is completely reversible. Rapidly cooled samples, on the other hand, do not show such splitting and the symmetry remains R\bar 3, despite a jump in lattice parameters. We use a special technique to analyse the disorder model of the slowly cooled samples, which consists of layered domains shifted in the hexagonal ab plane. The low-spin disordered phase was solved in a novel approach to accommodate the very unusual twinning and refined in the non-standard space group C\bar 1. In contrast to the ordered low-spin phase, the Fe ion is in a non-centrosymmetric coordination polyhedron and two of the six propyl groups change their conformation.


2021 ◽  
Author(s):  
Stella Belonwu ◽  
Yaqiao Li ◽  
Daniel Bunis ◽  
Arjun Arkal Rao ◽  
Caroline Warly Solsberg ◽  
...  

Abstract Alzheimer’s Disease (AD) is a complex neurodegenerative disease that gravely affects patients and imposes an immense burden on caregivers. Apolipoprotein E4 (APOE4) has been identified as the most common genetic risk factor for AD, yet the molecular mechanisms connecting APOE4 to AD are not well understood. Past transcriptomic analyses in AD have revealed APOE genotype-specific transcriptomic differences; however, these differences have not been explored at a single-cell level. Here, we leverage the first two single-nucleus RNA sequencing AD datasets from human brain samples, including nearly 55,000 cells from the prefrontal and entorhinal cortices. We observed more global transcriptomic changes in APOE4 positive AD cells and identified differences across APOE genotypes primarily in glial cell types. Our findings highlight the differential transcriptomic perturbations of APOE isoforms at a single-cell level in AD pathogenesis and have implications for precision medicine development in the diagnosis and treatment of AD.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Abha S. Bais ◽  
Débora M. Cerqueira ◽  
Andrew Clugston ◽  
Andrew J. Bodnar ◽  
Jacqueline Ho ◽  
...  

AbstractThe kidney is a complex organ composed of more than 30 terminally differentiated cell types that all are required to perform its numerous homeostatic functions. Defects in kidney development are a significant cause of chronic kidney disease in children, which can lead to kidney failure that can only be treated by transplant or dialysis. A better understanding of molecular mechanisms that drive kidney development is important for designing strategies to enhance renal repair and regeneration. In this study, we profiled gene expression in the developing mouse kidney at embryonic day 14.5 at single-cell resolution. Consistent with previous studies, clusters with distinct transcriptional signatures clearly identify major compartments and cell types of the developing kidney. Cell cycle activity distinguishes between the “primed” and “self-renewing” sub-populations of nephron progenitors, with increased expression of the cell cycle-related genes Birc5, Cdca3, Smc2 and Smc4 in “primed” nephron progenitors. In addition, augmented expression of cell cycle related genes Birc5, Cks2, Ccnb1, Ccnd1 and Tuba1a/b was detected in immature distal tubules, suggesting cell cycle regulation may be required for early events of nephron patterning and tubular fusion between the distal nephron and collecting duct epithelia.


2021 ◽  
Vol 12 ◽  
Author(s):  
Chunye Zhang ◽  
Ming Yang ◽  
Aaron C. Ericsson

Tissue-resident macrophages (TRMs) are heterogeneous populations originating either from monocytes or embryonic progenitors, and distribute in lymphoid and non-lymphoid tissues. TRMs play diverse roles in many physiological processes, including metabolic function, clearance of cellular debris, and tissue remodeling and defense. Macrophages can be polarized to different functional phenotypes depending on their origin and tissue microenvironment. Specific macrophage subpopulations are associated with disease progression. In studies of fate-mapping and single-cell RNA sequencing methodologies, several critical molecules have been identified to induce the change of macrophage function. These molecules are potential markers for diagnosis and selective targets for novel macrophage-mediated treatment. In this review, we discuss some of the recent findings regarding less-known molecules and new functions of well-known molecules. Understanding the mechanisms of these molecules in macrophages has the potential to yield new macrophage-mediated treatments or diagnostic approaches to disease.


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