Propofol Impairs Specification of Retinal Cell Types in Zebrafish by Inhibiting Zisp-mediated Noggin-1 Palmitoylation and Trafficking

2020 ◽  
Author(s):  
Xiaoqing Fan ◽  
Haoran Yang ◽  
Lizhu Hu ◽  
Delong Wang ◽  
Ruiting Wang ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Cell Formation ◽  
Cognitive Disorders ◽  
Cell Types ◽  
Retinal Cell ◽  
Soluble Form ◽  
In Vivo Experiments ◽  
Developing Neurons

Abstract Background: Propofol can have adverse effects on developing neurons, leading to cognitive disorders. However, the mechanism remains elusive. Here, we aimed to investigate the effect and molecular mechanism of propofol on neuronal development in zebrafish. Methods: The effect of propofol on neuronal development was demonstrated by a series of in vitro and in vivo experiments. mRNA injections, Whole-mount in situ hybridization and immunohistochemistry, quantitative real-time PCR, TUNEL, EdU, Co-Immunoprecipitation and acyl–biotin exchange (ABE) labeling method were carried out to demonstrate the potential mechanisms of propofol-mediated zisp expression and specification of retinal cell types.Results: Propofol impaired the specification of retinal cell types, thereby inhibiting neuronal and glial cell formation in retinas, mainly through inhibition of Zisp expression. Furthermore, Zisp promoted the secretion of a soluble form and the stabilization of a membrane-associated form of Noggin-1, a specific palmitoylation substrate.Conclusions: Propofol caused a severe phenotype during neuronal development in zebrafish. Our findings established a direct link between an anesthetic agent and protein palmitoylation in the regulation of neuronal development. This could be used to investigate the mechanisms via which the improper use of propofol might result in neuronal defects.

scholarly journals Propofol impairs specification of retinal cell types in zebrafish by inhibiting Zisp-mediated Noggin-1 palmitoylation and trafficking

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaoqing Fan ◽  
Haoran Yang ◽  
Lizhu Hu ◽  
Delong Wang ◽  
Ruiting Wang ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Cell Formation ◽  
Cognitive Disorders ◽  
Cell Types ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Retinal Cell ◽  
Soluble Form ◽  
In Vivo Experiments

Abstract Background Propofol can have adverse effects on developing neurons, leading to cognitive disorders, but the mechanism of such an effect remains elusive. Here, we aimed to investigate the effect of propofol on neuronal development in zebrafish and to identify the molecular mechanism(s) involved in this pathway. Methods The effect of propofol on neuronal development was demonstrated by a series of in vitro and in vivo experiments. mRNA injections, whole-mount in situ hybridization and immunohistochemistry, quantitative real-time polymerase chain reaction, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, 5-ethynyl-2′-deoxyuridine labeling, co-immunoprecipitation, and acyl–biotin exchange labeling were used to identify the potential mechanisms of propofol-mediated zisp expression and determine its effect on the specification of retinal cell types. Results Propofol impaired the specification of retinal cell types, thereby inhibiting neuronal and glial cell formation in retinas, mainly through the inhibition of Zisp expression. Furthermore, Zisp promoted the stabilization and secretion of a soluble form of the membrane-associated protein Noggin-1, a specific palmitoylation substrate. Conclusions Propofol caused a severe phenotype during neuronal development in zebrafish. Our findings established a direct link between an anesthetic agent and protein palmitoylation in the regulation of neuronal development. This could be used to investigate the mechanisms via which the improper use of propofol might result in neuronal defects.

scholarly journals Postprandial transfer of colostral extracellular vesicles and their protein and miRNA cargo in neonatal calves

2019 ◽  
Author(s):  
Benedikt Kirchner ◽  
Dominik Buschmann ◽  
Vijay Paul ◽  
Michael W. Pfaffl
Keyword(s):  
Extracellular Vesicles ◽  
Expression Profiles ◽  
Bovine Milk ◽  
Cell Types ◽  
Specific Protein ◽  
In Vivo Experiments ◽  
Neonatal Calves ◽  
Almost All

Abstract Background Extracellular vesicles (EVs) such as exosomes are key regulators of intercellular communication that can be found in almost all bio fluids. Although studies in the last decade have made great headway in discerning the role of EVs in many physiological and pathophysiological processes, the bioavailability and impact of dietary EVs and their cargo still remain to be elucidated. Due to its widespread consumption and high content of EV-associated microRNAs and proteins, a major focus in this field has been set on EVs in bovine milk and colostrum. Despite promising in vitro studies in recent years that show high resiliency of milk EVs to degradation and uptake of milk EV cargo in a variety of intestinal and blood cell types, in vivo experiments continue to be inconclusive and sometimes outright contradictive. Results To resolve this discrepancy, we assessed the potential postprandial transfer of colostral EVs to the circulation of newborn calves by analysing colostrum-specific protein and miRNAs, including specific isoforms (isomiRs) in cells, EV isolations and unfractionated samples from blood and colostrum. Our findings reveal distinct populations of EVs in colostrum and blood from cows that can be clearly separated by density, particle concentration and protein content (BTN1A1, MFGE8). Postprandial blood samples of calves show a time-dependent increase in EVs that share morphological and protein characteristics of colostral EVs. Analysis of miRNA expression profiles by Next-Generation Sequencing gave a different picture however. Although significant postprandial expression changes could only be detected for calf EV samples, expression profiles show very limited overlap with highly expressed miRNAs in colostral EVs or colostrum in general. Conclusions Taken together our results indicate a selective uptake of membrane-associated protein cargo but not luminal miRNAs from colostral EVs into the circulation of neonatal calves.

scholarly journals The Rise of Retinal Organoids for Vision Research

2020 ◽  
Vol 21 (22) ◽  
pp. 8484 ◽  
Author(s):  
Kritika Sharma ◽  
Tim U. Krohne ◽  
Volker Busskamp
Keyword(s):  
Molecular Mechanisms ◽  
Cell Types ◽  
Therapeutic Interventions ◽  
Retinal Cell ◽  
Retinal Diseases ◽  
Organ Systems ◽  
Cell Sources ◽  
Retinal Degenerative Diseases

Retinal degenerative diseases lead to irreversible blindness. Decades of research into the cellular and molecular mechanisms of retinal diseases, using either animal models or human cell-derived 2D systems, facilitated the development of several therapeutic interventions. Recently, human stem cell-derived 3D retinal organoids have been developed. These self-organizing 3D organ systems have shown to recapitulate the in vivo human retinogenesis resulting in morphological and functionally similar retinal cell types in vitro. In less than a decade, retinal organoids have assisted in modeling several retinal diseases that were rather difficult to mimic in rodent models. Retinal organoids are also considered as a photoreceptor source for cell transplantation therapies to counteract blindness. Here, we highlight the development and field’s improvements of retinal organoids and discuss their application aspects as human disease models, pharmaceutical testbeds, and cell sources for transplantations.

scholarly journals Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening

2020 ◽  
Vol 21 (13) ◽  
pp. 4804
Author(s):  
Vincent van Duinen ◽  
Wendy Stam ◽  
Eva Mulder ◽  
Farbod Famili ◽  
Arie Reijerkerk ◽  
...  
Keyword(s):  
Drug Screening ◽  
Cell Formation ◽  
Cell Types ◽  
Culture Conditions ◽  
In Vitro Assays ◽  
Vegf Receptor ◽  
Screening Assay ◽  
Angiogenesis Assay

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

scholarly journals Glucocorticoid agonists enhance retinal stem cell self-renewal and proliferation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kenneth N. Grisé ◽  
Nelson X. Bautista ◽  
Krystal Jacques ◽  
Brenda L. K. Coles ◽  
Derek van der Kooy
Keyword(s):  
Cell Fate ◽  
Progenitor Cell ◽  
Cell Types ◽  
Retinal Cell ◽  
Ciliary Epithelium ◽  
Cell Transplant ◽  
Self Renewal ◽  
Synthetic Glucocorticoid

Abstract Background Adult mammalian retinal stem cells (RSCs) readily proliferate, self-renew, and generate progeny that differentiate into all retinal cell types in vitro. RSC-derived progeny can be induced to differentiate into photoreceptors, making them a potential source for retinal cell transplant therapies. Despite their proliferative propensity in vitro, RSCs in the adult mammalian eye do not proliferate and do not have a regenerative response to injury. Thus, identifying and modulating the mechanisms that regulate RSC proliferation may enhance the capacity to produce RSC-derived progeny in vitro and enable RSC activation in vivo. Methods Here, we used medium-throughput screening to identify small molecules that can expand the number of RSCs and their progeny in culture. In vitro differentiation assays were used to assess the effects of synthetic glucocorticoid agonist dexamethasone on RSC-derived progenitor cell fate. Intravitreal injections of dexamethasone into adult mouse eyes were used to investigate the effects on endogenous RSCs. Results We discovered that high-affinity synthetic glucocorticoid agonists increase RSC self-renewal and increase retinal progenitor proliferation up to 6-fold without influencing their differentiation in vitro. Intravitreal injection of synthetic glucocorticoid agonist dexamethasone induced in vivo proliferation in the ciliary epithelium—the niche in which adult RSCs reside. Conclusions Together, our results identify glucocorticoids as novel regulators of retinal stem and progenitor cell proliferation in culture and provide evidence that GCs may activate endogenous RSCs.

scholarly journals Adipose-tissue derived signals control bone remodelling

2020 ◽  
Author(s):  
Maria-Bernadette Madel ◽  
He Fu ◽  
Dominique D. Pierroz ◽  
Mariano Schiffrin ◽  
Carine Winkler ◽  
...  
Keyword(s):  
Bone Erosion ◽  
Cell Formation ◽  
Cell Types ◽  
Whole Body ◽  
Long Bone ◽  
Bone Homeostasis ◽  
Multiple Cell ◽  
The One

SummaryLong bones from mammals host blood cell formation and contain multiple cell types, including adipocytes. Physiological functions of bone marrow adipocytes are poorly documented. Herein, we used adipocyte-deficient PPARγ-whole body null mice to investigate the consequence of total adipocyte deficiency on bone homeostasis in mice. We first highlight the dual bone phenotype of PPARγ null mice: on the one hand the increase bone formation and subsequent trabecularization extending in the long bone diaphysis, due to the well-known impact of PPARγ deficiency on osteoblasts formation and activity; on the other hand, an increased osteoclastogenesis in the cortical bone. We then further explore the cause of this unexpected increased osteoclastogenesis using two independent models of lipoatrophy, which recapitulated this phenotype. This demonstrates that hyperosteoclastogenesis is not intrinsically linked to PPARγ deficiency, but is a consequence of the total lipodystrophy. We further showed that adiponectin, a cytokine produced by adipocytes and mesenchymal stromal cells is a potent inhibitor of osteoclastogenesis in vitro and in vivo. Moreover, pharmacological activation of adiponectin receptors by the synthetic agonist AdipoRon inhibits mature osteoclast activity both in mouse and human cells by blocking podosome formation through AMPK activation. Finally, we demonstrated that AdipoRon treatment blocks bone erosion in vivo in a murine model of inflammatory bone loss, providing potential new approaches to treat osteoporosis.

scholarly journals An alternative approach to produce versatile retinal organoids with accelerated ganglion cell development

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Philip E. Wagstaff ◽  
Anneloor L. M. A. ten Asbroek ◽  
Jacoline B. ten Brink ◽  
Nomdo M. Jansonius ◽  
Arthur A. B. Bergen
Keyword(s):  
Ganglion Cells ◽  
Cell Types ◽  
Retinal Cell ◽  
Specific Cell ◽  
Retinal Ganglion ◽  
In Vivo Models ◽  
Alternative Approach

AbstractGenetically complex ocular neuropathies, such as glaucoma, are a major cause of visual impairment worldwide. There is a growing need to generate suitable human representative in vitro and in vivo models, as there is no effective treatment available once damage has occured. Retinal organoids are increasingly being used for experimental gene therapy, stem cell replacement therapy and small molecule therapy. There are multiple protocols for the development of retinal organoids available, however, one potential drawback of the current methods is that the organoids can take between 6 weeks and 12 months on average to develop and mature, depending on the specific cell type wanted. Here, we describe and characterise a protocol focused on the generation of retinal ganglion cells within an accelerated four week timeframe without any external small molecules or growth factors. Subsequent long term cultures yield fully differentiated organoids displaying all major retinal cell types. RPE, Horizontal, Amacrine and Photoreceptors cells were generated using external factors to maintain lamination.

scholarly journals Novel use of a chemically modified siRNA for robust and sustainable in vivo gene silencing in the retina

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Takazumi Taniguchi ◽  
Ken-ichi Endo ◽  
Hidetoshi Tanioka ◽  
Masaaki Sasaoka ◽  
Kei Tashiro ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Intravitreal Injection ◽  
Target Genes ◽  
Vitreous Body ◽  
Cell Types ◽  
Retinal Cell ◽  
Mrna Levels ◽  
Chemically Modified

AbstractDespite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40–70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60–90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.

Characterization of soluble CD39 (SolCD39/NTPDase1) from PiggyBac nonviral system as a tool to control the nucleotides level

2019 ◽  
Vol 476 (11) ◽  
pp. 1637-1651
Author(s):  
Liziane Raquel Beckenkamp ◽  
Isabele Cristiana Iser ◽  
Giovana Ravizzoni Onzi ◽  
Dieine Maira Soares da Fontoura ◽  
Ana Paula Santin Bertoni ◽  
...  
Keyword(s):  
Purinergic Signaling ◽  
Hydrolytic Activity ◽  
Soluble Form ◽  
Purinergic System ◽  
In Vivo Experiments ◽  
High Doses ◽  
And Migration

Abstract Extracellular ATP (eATP) and its metabolites have emerged as key modulators of different diseases and comprise a complex pathway called purinergic signaling. An increased number of tools have been developed to study the role of nucleotides and nucleosides in cell proliferation and migration, influence on the immune system and tumor progression. These tools include receptor agonists/antagonists, engineered ectonucleotidases, interference RNAs and ectonucleotidase inhibitors that allow the control and quantification of nucleotide levels. NTPDase1 (also called apyrase, ecto-ATPase and CD39) is one of the main enzymes responsible for the hydrolysis of eATP, and purified enzymes, such as apyrase purified from potato, or engineered as soluble CD39 (SolCD39), have been widely used in in vitro and in vivo experiments. However, the commercial apyrase had its effects recently questioned and SolCD39 exhibits limitations, such as short half-life and need of high doses to reach the expected enzymatic activity. Therefore, this study investigated a non-viral method to improve the overexpression of SolCD39 and evaluated its impact on other enzymes of the purinergic system. Our data demonstrated that PiggyBac transposon system proved to be a fast and efficient method to generate cells stably expressing SolCD39, producing high amounts of the enzyme from a limited number of cells and with high hydrolytic activity. In addition, the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other enzymes from the purinergic system. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.

scholarly journals In vitro Cytotoxicity Studies of Industrially Used Common Nanomaterials on L929 and 3T3 Fibroblast Cells

2020 ◽  
Vol 1 (5) ◽  
pp. 192-200
Author(s):  
Madhulika Srikanth ◽  
Waseem S Khan ◽  
Ramazan Asmatulu ◽  
Heath E Misak ◽  
Shang-You Yang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
In Vitro Cytotoxicity ◽  
In Vitro Tests ◽  
Fibroblast Cells ◽  
In Vivo Experiments ◽  
Cytotoxicity Studies ◽  
Biomedical Electronics

The unique structures and properties of nanomaterials have attracted many engineers and scientists to these resources for different applications, including biomedical, electronics, manufacturing, transportation, energy, and defense. The increasing applications of nanomaterials have also caused some concern among the scientific community about their safety and cytotoxicity. To successfully use nanomaterials in different fields, their interaction with mammalian cells in vitro must be addressed before in vivo experiments can be carried out successfully. In this study, the cytotoxicity values of commonly known nanomaterials, such as 100-ply Carbon Nanotube (CNT) wires, graphene, CNTs, nanoclay, and fullerene, were investigated through in vitro tests on human L929 and mice 3T3 fibroblast cells and compared with each other. The effects of cytotoxicity on both cell types were similar in many ways, but not closely identical due to structural and morphological differences. Compared to mice fibroblast cells, human fibroblast cells have a larger surface area; therefore, the viability values of L929 cells at different dilutions and time durations vary over a larger range. Pristine 100-ply CNT wires were found to be the least cytotoxic, with an average viability of 86.9%, whereas materials with high aspect ratio (e.g., CNTs and graphene) had higher cytotoxicity values due to their potential to pierce through cell membranes.

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