Single-cell RNA-seq analysis revealed long-lasting adverse effects of tamoxifen on neurogenesis in prenatal and adult brains

The CreER/LoxP system is widely accepted to track neural lineages and study gene functions upon tamoxifen (TAM) administration. We have observed that prenatal TAM treatment caused high rates of delayed delivery and fetal mortality. This substance could produce undesired results, leading to data misinterpretation. Here, we report that administration of TAM during early stages of cortical neurogenesis promoted precocious neural differentiation, while it inhibited neural progenitor cell (NPC) proliferation. The TAM-induced inhibition of NPC proliferation led to deficits in cortical neurogenesis, dendritic morphogenesis, synaptic formation, and cortical patterning in neonatal and postnatal offspring. Mechanistically, by employing single-cell RNA-sequencing (scRNA-seq) analysis combined with in vivo and in vitro assays, we show TAM could exert these drastic effects mainly through dysregulating the Wnt-Dmrta2 signaling pathway. In adult mice, administration of TAM significantly attenuated NPC proliferation in both the subventricular zone and the dentate gyrus. This study revealed the cellular and molecular mechanisms for the adverse effects of TAM on corticogenesis, suggesting that care must be taken when using the TAM-induced CreER/LoxP system for neural lineage tracing and genetic manipulation studies in both embryonic and adult brains.

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Single cell transcriptomic analysis revealed long-lasting adverse effects of prenatal tamoxifen administration on neurogenesis in prenatal and adult brains

10.1101/811893 ◽

2019 ◽

Author(s):

Chia-Ming Lee ◽

Liqiang Zhou ◽

Jiping Liu ◽

Jiayu Shi ◽

Yanan Geng ◽

...

Keyword(s):

Adverse Effects ◽

Single Cell ◽

Molecular Mechanisms ◽

Genetic Manipulation ◽

Lineage Tracing ◽

High Rate ◽

Neural Lineage ◽

Cortical Neurogenesis

SummaryCreER/LoxP system has enabled precise gene manipulation in distinct cell subpopulations at any specific time point upon tamoxifen (TAM) administration. This system is widely accepted to track neural lineages and study gene functions. We have observed prenatal TAM treatment caused high rate of delayed delivery and mortality of pups. These substances could promote undesired results, leading to data misinterpretation. Here, we report that TAM administration during early stages of cortical neurogenesis promoted precocious neural differentiation, while inhibited neural progenitor cell (NPC) proliferation. The TAM-induced inhibition of NPC proliferation led to deficits in cortical neurogenesis, dendritic morphogenesis, and cortical patterning in neonatal and postnatal offspring. Mechanistically, single cell RNA sequencing (scRNA-seq) analysis combined with in vivo and in vitro assays showed TAM could exert these drastic effects mainly through dysregulating the expression of Dmrta2 and Wnt8b. In adult mice, administration of TAM significantly attenuated NPC proliferation in both the subventricular zone and the dentate gyrus. This study revealed the cellular and molecular mechanisms for the adverse effects of prenatal tamoxifen administration on corticogenesis, suggesting that tamoxifen-induced CreER/LoxP system may not be suitable for neural lineage tracing and genetic manipulation studies in both embryonic and adult brains.SignificantFor the first time, our study revealed the molecular mechanisms underlying tamoxifen activities on cortical development. This study also clearly showed that care must be taken when using tamoxifen-induced CreER/LoxP system for neural lineage tracing and genetic manipulation studies.

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L1CAM promotes ovarian cancer stemness and tumor initiation via FGFR1/SRC/STAT3 signaling

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02117-z ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Marco Giordano ◽

Alessandra Decio ◽

Chiara Battistini ◽

Micol Baronio ◽

Fabrizio Bianchi ◽

...

Keyword(s):

Molecular Mechanisms ◽

Genetic Manipulation ◽

Tumor Formation ◽

In Vitro Assays ◽

Tumor Initiation ◽

Loss Of Function ◽

Stat3 Signaling

Abstract Background Cancer stem cells (CSC) have been implicated in tumor progression. In ovarian carcinoma (OC), CSC drive tumor formation, dissemination and recurrence, as well as drug resistance, thus contributing to the high death-to-incidence ratio of this disease. However, the molecular basis of such a pathogenic role of ovarian CSC (OCSC) has been elucidated only to a limited extent. In this context, the functional contribution of the L1 cell adhesion molecule (L1CAM) to OC stemness remains elusive. Methods The expression of L1CAM was investigated in patient-derived OCSC. The genetic manipulation of L1CAM in OC cells provided gain and loss-of-function models that were then employed in cell biological assays as well as in vivo tumorigenesis experiments to assess the role of L1CAM in OC cell stemness and in OCSC-driven tumor initiation. We applied antibody-mediated neutralization to investigate L1CAM druggability. Biochemical approaches were then combined with functional in vitro assays to study the molecular mechanisms underlying the functional role of L1CAM in OCSC. Results We report that L1CAM is upregulated in patient-derived OCSC. Functional studies showed that L1CAM promotes several stemness-related properties in OC cells, including sphere formation, tumor initiation and chemoresistance. These activities were repressed by an L1CAM-neutralizing antibody, pointing to L1CAM as a druggable target. Mechanistically, L1CAM interacted with and activated fibroblast growth factor receptor-1 (FGFR1), which in turn induced the SRC-mediated activation of STAT3. The inhibition of STAT3 prevented L1CAM-dependent OC stemness and tumor initiation. Conclusions Our study implicate L1CAM in the tumorigenic function of OCSC and point to the L1CAM/FGFR1/SRC/STAT3 signaling pathway as a novel driver of OC stemness. We also provide evidence that targeting this pathway can contribute to OC eradication.

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Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18073332 ◽

2021 ◽

Vol 18 (7) ◽

pp. 3332

Author(s):

Olga V. Naidenko ◽

David Q. Andrews ◽

Alexis M. Temkin ◽

Tasha Stoiber ◽

Uloma Igara Uche ◽

...

Keyword(s):

Immune System ◽

High Throughput ◽

Environmental Protection Agency ◽

High Throughput Screening ◽

Molecular Mechanisms ◽

Specific Activity ◽

Laboratory Animals ◽

In Vitro Assays ◽

Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

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Fishing for Targets of Alien Metabolites: A Novel Peroxisome Proliferator-Activated Receptor (PPAR) Agonist from a Marine Pest

Marine Drugs ◽

10.3390/md16110431 ◽

2018 ◽

Vol 16 (11) ◽

pp. 431 ◽

Cited By ~ 12

Author(s):

Rosa Vitale ◽

Enrico D'Aniello ◽

Stefania Gorbi ◽

Andrea Martella ◽

Cristoforo Silvestri ◽

...

Keyword(s):

Native Species ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Invasion Biology ◽

In Vitro Assays ◽

Bioactive Metabolites ◽

Peroxisome Proliferator ◽

Invasive Pests

Although the chemical warfare between invasive and native species has become a central problem in invasion biology, the molecular mechanisms by which bioactive metabolites from invasive pests influence local communities remain poorly characterized. This study demonstrates that the alkaloid caulerpin (CAU)—a bioactive component of the green alga Caulerpa cylindracea that has invaded the entire Mediterranean basin—is an agonist of peroxisome proliferator-activated receptors (PPARs). Our interdisciplinary study started with the in silico prediction of the ligand-protein interaction, which was then validated by in vivo, ex vivo and in vitro assays. On the basis of these results, we candidate CAU as a causal factor of the metabolic and behavioural disorders observed in Diplodus sargus, a native edible fish of high ecological and commercial relevance, feeding on C. cylindracea. Moreover, given the considerable interest in PPAR activators for the treatment of relevant human diseases, our findings are also discussed in terms of a possible nutraceutical/pharmacological valorisation of the invasive algal biomasses, supporting an innovative strategy for conserving biodiversity as an alternative to unrealistic campaigns for the eradication of invasive pests.

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Single-cell analysis revealed that IL4I1 promoted ovarian cancer progression

Journal of Translational Medicine ◽

10.1186/s12967-021-03123-7 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Hongyu Zhao ◽

Yu Teng ◽

Wende Hao ◽

Jie Li ◽

Zhefeng Li ◽

...

Keyword(s):

Ovarian Cancer ◽

Single Cell ◽

Cancer Progression ◽

Cox Regression ◽

Single Cell Analysis ◽

Cell Types ◽

In Vitro Assays ◽

Migration And Invasion ◽

Dominant Type

Abstract Background Ovarian cancer was one of the leading causes of female deaths. Patients with OC were essentially incurable and portends a poor prognosis, presumably because of profound genetic heterogeneity limiting reproducible prognostic classifications. Methods We comprehensively analyzed an ovarian cancer single-cell RNA sequencing dataset, GSE118828, and identified nine major cell types. Relationship between the clusters was explored with CellPhoneDB. A malignant epithelial cluster was confirmed using pseudotime analysis, CNV and GSVA. Furthermore, we constructed the prediction model (i.e., RiskScore) consisted of 10 prognosis-specific genes from 2397 malignant epithelial genes using the LASSO Cox regression algorithm based on public datasets. Then, the prognostic value of Riskscore was assessed with Kaplan–Meier survival analysis and time-dependent ROC curves. At last, a series of in-vitro assays were conducted to explore the roles of IL4I1, an important gene in Riskscore, in OC progression. Results We found that macrophages possessed the most interaction pairs with other clusters, and M2-like TAMs were the dominant type of macrophages. C0 was identified as the malignant epithelial cluster. Patients with a lower RiskScore had a greater OS (log-rank P < 0.01). In training set, the AUC of RiskScore was 0.666, 0.743 and 0.809 in 1-year, 3-year and 5-year survival, respectively. This was also validated in another two cohorts. Moreover, downregulation of IL4I1 inhibited OC cells proliferation, migration and invasion. Conclusions Our work provide novel insights into our understanding of the heterogeneity among OCs, and would help elucidate the biology of OC and provide clinical guidance in prognosis for OC patients.

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Long noncoding RNA FAM225A promotes the malignant progression of gastric cancer through the miR-326/PADI2 axis

Cell Death Discovery ◽

10.1038/s41420-021-00809-1 ◽

2022 ◽

Vol 8 (1) ◽

Author(s):

Xiang Ma ◽

Gang Wang ◽

Hao Fan ◽

Zengliang Li ◽

Wangwang Chen ◽

...

Keyword(s):

Gastric Cancer ◽

Noncoding Rna ◽

Molecular Mechanisms ◽

Therapeutic Targets ◽

In Vitro Assays ◽

Advanced Tumor Stage ◽

Advanced Tumor ◽

Mechanistic Investigation

AbstractGastric cancer (GC) is a global health problem and further studies of its molecular mechanisms are needed to identify effective therapeutic targets. Although some long noncoding RNAs (lncRNAs) have been found to be involved in the progression of GC, the molecular mechanisms of many GC-related lncRNAs remain unclear. In this study, a series of in vivo and in vitro assays were performed to study the relationship between FAM225A and GC, which showed that FAM225A levels were correlated with poor prognosis in GC. Higher FAM225A expression tended to be correlated with a more profound lymphatic metastasis rate, larger tumor size, and more advanced tumor stage. FAM225A also promoted gastric cell proliferation, invasion, and migration. Further mechanistic investigation showed that FAM225A acted as a miR-326 sponge to upregulate its direct target PADI2 in GC. Overall, our findings indicated that FAM225A promoted GC development and progression via a competitive endogenous RNA network of FAM225A/miR-326/PADI2 in GC, providing insight into possible therapeutic targets and prognosis of GC.

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Abstract MP175: Disruption of the Genome Architecture at the PRRX1 Locus is Associated With the Pathogenesis of LMNA -related Dilated Cardiomyopathy

Circulation Research ◽

10.1161/res.127.suppl_1.mp175 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Yuan Zhang ◽

Mohamed Ameen ◽

Isaac Perea Gil ◽

Jennifer Arthur ◽

Alexandra A Gavidia ◽

...

Keyword(s):

Dilated Cardiomyopathy ◽

Single Cell ◽

Genome Organization ◽

Molecular Mechanisms ◽

Critical Role ◽

Lamin A ◽

Candidate Locus ◽

Gene Encoding ◽

Gene Dysregulation

Background: LMNA , a gene encoding A-type lamin proteins (abbreviated as lamin A), is one of the most frequently mutated genes in dilated cardiomyopathy (DCM). The molecular mechanisms underlying cardiomyocyte dysfunction in LMNA -related DCM remain elusive, translating to the lack of disease-specific therapies. Lamin A has been shown to play a critical role in genome organization via interactions with the chromatin at specific regions called lamina-associated domains (LADs). However, little is known about whether DCM-causing LMNA mutations rearrange the genome conformation and chromosome accessibility. The overarching goal of this study is to define the role of genome organization in LMNA -related DCM. Methods: LMNA -related DCM was modeled in vitro using cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) from DCM patients carrying a frameshift mutation in the LMNA gene (c. 348_349insG; p. K117fs) and isogenic controls. We combined genome-wide single cell functional genomic and epigenomic mapping analyses to define the gene regulation and cis-regulatory interactions in isogenic iPSC-CMs. Results: Single-cell RNA-seq revealed global gene dysregulation in LMNA mutant compared to isogenic control iPSC-CMs. The homeodomain transcription factor PRRX1 was significantly upregulated in mutant cells. We showed that LAD integrity is disrupted at the PRRX1 locus in mutant iPSC-CMs. In agreement, DNA fluorescence in situ hybridization (FISH) revealed that the PRRX1 locus loses peripheral association and relocates towards the transcriptionally active nuclear interior in mutant iPSC-CMs. Correspondingly, single-cell assay for transposase accessible chromatin (ATAC)-seq showed increased chromatin co-accessibility at the PRRX1 locus, providing a plausible explanation for ectopic activation of PRRX1 in LMNA mutant iPSC-CMs. Conclusion: Our data suggest that LMNA haploinsufficiency disrupts the structure of LADs, leading to ectopic promoter interactions and altered gene expression in LMNA -related DCM iPSC-CMs. We identified PRRX1 as a promising candidate locus linking changes in LAD organization with gene dysregulation in LMNA -related DCM.

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Differentiation of Mesenchymal Stem Cells to Neuroglia: in the Context of Cell Signalling

Stem Cell Reviews and Reports ◽

10.1007/s12015-019-09917-z ◽

2019 ◽

Vol 15 (6) ◽

pp. 814-826 ◽

Cited By ~ 10

Author(s):

Sajan George ◽

Michael R. Hamblin ◽

Heidi Abrahamse

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adult Stem Cells ◽

Molecular Mechanisms ◽

Culture Media ◽

Cell Signalling ◽

Specific Cell ◽

Ongoing Research ◽

Neural Lineage

Abstract The promise of engineering specific cell types from stem cells and rebuilding damaged or diseased tissues has fascinated stem cell researchers and clinicians over last few decades. Mesenchymal Stem Cells (MSCs) have the potential to differentiate into non-mesodermal cells, particularly neural-lineage, consisting of neurons and glia. These multipotent adult stem cells can be used for implementing clinical trials in neural repair. Ongoing research identifies several molecular mechanisms involved in the speciation of neuroglia, which are tightly regulated and interconnected by various components of cell signalling machinery. Growing MSCs with multiple inducers in culture media will initiate changes on intricately interlinked cell signalling pathways and processes. Net result of these signal flow on cellular architecture is also dependent on the type of ligands and stem cells investigated in vitro. However, our understanding about this dynamic signalling machinery is limited and confounding, especially with spheroid structures, neurospheres and organoids. Therefore, the results for differentiating neurons and glia in vitro have been inconclusive, so far. Added to this complication, we have no convincing evidence about the electrical conductivity and functionality status generated in differentiating neurons and glia. This review has taken a step forward to tailor the information on differentiating neuroglia with the common methodologies, in practice.

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Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons

eLife ◽

10.7554/elife.10841 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 36

Author(s):

Sebastian Poliak ◽

Daniel Morales ◽

Louis-Philippe Croteau ◽

Dayana Krawchuk ◽

Elena Palmesino ◽

...

Keyword(s):

Motor Neurons ◽

Molecular Mechanisms ◽

Neural Circuit ◽

Growth Cones ◽

In Vitro Assays ◽

Dependent Manner ◽

Motor Axons ◽

Spinal Motor Neurons ◽

Spinal Motor

During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin–ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

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Evaluation of the Cytopathicity (Fusion/Hemifusion) of Patient-Derived HIV-1 Envelope Glycoproteins Comparing Two Effector Cell Lines

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057112439890 ◽

2012 ◽

Vol 17 (6) ◽

pp. 727-737 ◽

Cited By ~ 6

Author(s):

Francesc Cunyat ◽

Marta Curriu ◽

Silvia Marfil ◽

Elisabet García ◽

Bonaventura Clotet ◽

...

Keyword(s):

Cell Death ◽

Single Cell ◽

Cell Lines ◽

Effector Cell ◽

Cell Loss ◽

Biological Properties ◽

In Vitro Assays ◽

Envelope Glycoproteins ◽

Hiv 1

HIV-1 envelope glycoprotein (Env) is a major determinant of viral pathogenicity. The evaluation of the biological properties of patient-derived envelopes by comparing two effector cell lines (293T and HeLa) is reported. A standard cell-to-cell fusion assay was used to evaluate fusogenicity, whereas a coculture with CD4+ cells was used to evaluate absolute cell loss, single cell death, and hemifusion events. Fusion and absolute cell loss assays showed that Env-expressing 293T and HeLa cells had different fusion efficiencies; fusion was magnified in 293T cells despite a significantly lower cell-surface Env expression. Conversely, gp41-mediated single cell death and hemifusion induced in CD4+ cells by 293T-Env-positive cells were significantly lower than that induced by HeLa-Env-positive cells. These data showed that the effector cell line used in the in vitro assays is crucial, and a combination of assays is recommended to evaluate the biological properties of patient-derived envelope glycoproteins: preferentially, 293T-Env-positive cells for the evaluation of fusogenicity and HeLa-Env-positive cells for the evaluation of cell death parameters. The combination of assays described in our work could be a valuable tool for dual screenings of large collections of primary Envs or Env mutants and drugs acting on these Envs.

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