scholarly journals Mutant MAPK7-Induced Idiopathic Scoliosis is Linked to Impaired Osteogenesis

2018 ◽  
Vol 48 (3) ◽  
pp. 880-890 ◽  
Author(s):  
Taifeng Zhou ◽  
Chong Chen ◽  
Caixia Xu ◽  
Hang Zhou ◽  
Bo Gao ◽  
...  
Keyword(s):  
Idiopathic Scoliosis ◽  
Western Blotting ◽  
Messenger Rna ◽  
Human Mesenchymal Stem Cells ◽  
Control Group ◽  
Zebrafish Embryos ◽  
Small Interfering Rnas ◽  
Cell Stage ◽  
Alizarin Red ◽  
The One

Background/Aims: Three rare MAPK7 variants that predispose individuals to adolescent idiopathic scoliosis have previously been identified. However, the mechanism underlying the effects of the mutations remain unknown. Methods: Human mesenchymal stem cells (hMSCs) were isolated from both patients and healthy volunteer donors, and MAPK7 expression was detected by western blotting and real-time quantitative PCR (RT-qPCR). Zebrafish embryos were injected with mapk7 morpholinos or co-injected with morpholinos and wild-type (WT) MAPK7 messenger RNA (mRNA) at the one-cell stage, followed by calcein staining to evaluate bone formation. hMSCs were transfected with MAPK7 small interfering RNAs and osteogenesis was induced for 14 days. Alizarin red staining was performed and osteoblast markers were detected by western blotting and RT-qPCR. Since RPS6KA3 is a downstream target of MAPK7 and plays an important role in the osteogenesis, zebrafish embryos were then injected with rps6ka3 morpholinos, or co-injected with rps6ka3 or mapk7 morpholinos and WT RPS6KA3 mRNA at the one-cell stage. Results: MAPK7 expression in the patient group was much lower than in the control group. Morpholino-induced mapk7 knockdown in zebrafish embryos led to body curvature, which was significantly reversed by WT MAPK7 mRNA. Calcein staining revealed that mapk7-knockdown delayed the ossification of the vertebrae. MAPK7 silencing in hMSCs impaired osteogenesis and downregulated osteoblast marker expression. Morpholino-induced rps6ka3-knockdown in zebrafish embryos led to body curvature, which was reversed by WT RPS6KA3 mRNA. Interestingly, RPS6KA3 mRNA also partially reversed the phenotype induced by mapk7 morpholinos. Conclusion: Impaired osteogenesis is linked to mutant MAPK7-induced idiopathic scoliosis , and RPS6KA3 may play an important role in this process.

scholarly journals Direct Visualization of Live Zebrafish Glycan via Single-step Metabolic Labeling with Fluorophore-tagged Nucleotide Sugars

2019 ◽  
Author(s):  
Senlian Hong ◽  
Pankaj Sahai-Hernandez ◽  
David Traver ◽  
Peng Wu
Keyword(s):  
Developmental Stages ◽  
Single Step ◽  
Zebrafish Embryos ◽  
Metabolic Labeling ◽  
Cell Stage ◽  
Imaging Probes ◽  
Nucleotide Sugars ◽  
Living Organisms ◽  
The One

ABSTRACTDynamic turnover of cell-surface glycans is involved in a myriad of biological events, making this process an attractive target for in vivo molecular imaging. The metabolic glycan labeling coupled with ‘bioorthogonal chemistry’ has paved the way for visulizing glycans in living organisms. However, a two-step labeling sequence is required, which is prone to tissue penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we developed a single-step fluorescent glycan labeling strategy by using fluorophore-tagged analogs of nucleotide sugars directly. Injecting the fluorophore-tagged sialic acid and fucose into the yolk of zebrafish embryos at the one-cell stage enables a systematic imaging of sialylation and fucosylation in live zebrafish embryos at various developmental stages. From these studies, we obtained insights into the role of sialylated and fucosylated glycans in zebrafish hematopoiesis.

103 BIRTH OF CLONED PIGLETS DERIVED FROM AN OPTIMIZED IN VITRO BLASTOCYST PRODUCTION SYSTEM BY TRICHOSTATIN A TREATMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
Y.-H. Zhang ◽  
Y.-T. Du ◽  
K. Zhang ◽  
J. Li ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Culture Media ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Deacetylase Inhibitor ◽  
The One ◽  
Effective Group

The present study was designed to examine the effect of trichostatin A (TSA, a histone deacetylase inhibitor) treatment on in vitro developmental ability of pig cloned embryos and to evaluate the feasibility of producing piglets from these embryos. Cell lines were established from 40-day-old fetuses, and adult ear skin was used as nuclear donor. In vitro-matured oocytes from abattoir-derived sow ovaries were used as cytoplast recipients for micromanipulator-assisted somatic cell nuclear transfer (SCNT). Data were analyzed by using SPSS (11.0) with one-way ANOVA, and each experiment was replicated at least 3 times. In Experiment 1, immediately after simultaneous fusion and activation, the reconstructed couplets were randomly cultured in porcine zygote medium 3 (PZM3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) with 10 �g mL-1 cytochalasin B (CB), 10 �g mL-1 cycloheximide (CHX), and 0 nM, 5 nM, or 50 nM TSA for the first 4 h. Cloned embryos (fused reconstructed couplets) were moved to the same culture media but without CB and CHX and further cultured at 38.5�C, under 5% CO2, 5% O2, 90% N2 and 100% humidity. After incubation for a total of 8–14 h in 50 nM, 19–24 h in 50 nM or 5 nM, and 31–36 h in 50 nM TSA in PZM3 (0 nM TSA serves as control for each group), the embryos were further cultured in vitro without TSA in PZM3 for up to 168 h. Cleavage and blastocyst development rates, based on embryos cultured, were recorded at 48 and 168 h of IVC, respectively. Results showed that 50 nM TSA treatment for 19-24 h supported a higher blastocyst development rate than the control group [No. blastocysts/No. embryos cultured (mean � SEM): 107/258, 47.4 � 5.9% vs. 65/324, 20.0 � 2.3%, respectively; P < 0.05], whereas similar pre-implantation development was obtained between the other 3 test groups and the control. In Experiment 2, TSA-treated cloned embryos at the one-cell stage or blastocyst stage were transferred to recipients to examine the possibility of producing piglets. Ten cloned piglets (2 are healthy and 8 died shortly after birth) and one ongoing pregnancy were obtained from 3 recipients who received an average of 110 one-cell stage embryos, whereas 4 piglets originating from traditional cloning were produced from one recipient which received 28 traditional cloned blastocysts (produced from the effective group in Experiment 1) and 30 handmade but non-TSA-treated ones. Our data demonstrate that TSA treatment after SCNT in porcine can significantly improve the in vitro blastocyst production, and embryos treated with TSA could support full-term development and result in healthy offspring.

scholarly journals Developmental exposure window influences silver toxicity but does not affect the susceptibility to subsequent exposures in zebrafish embryos

2020 ◽  
Vol 154 (5) ◽  
pp. 579-595
Author(s):  
Paige C. Robinson ◽  
Hannah R. Littler ◽  
Anke Lange ◽  
Eduarda M. Santos
Keyword(s):  
Early Development ◽  
Toxic Metal ◽  
Positive Control ◽  
Zebrafish Embryos ◽  
Chemical Toxicity ◽  
Experimental Conditions ◽  
Cell Stage ◽  
Developmental Exposure ◽  
Methylation Inhibitor ◽  
The One

AbstractSilver is a non-essential, toxic metal widespread in freshwaters and capable of causing adverse effects to wildlife. Its toxic effects have been studied in detail but less is known about how sensitivity varies during development and whether pre-exposures affect tolerance upon re-exposure. We address these knowledge gaps using the zebrafish embryo (Danio rerio) model to investigate whether exposures encompassing stages of development prior to mid-blastula transition, when chorion hardening and epigenetic reprogramming occur, result in greater toxicity compared to those initiated after this period. We conducted exposures to silver initiated at 0.5 h post fertilisation (hpf) and 4 hpf to determine if toxicity differed. In parallel, we exposed embryos to the methylation inhibitor 5-azacytidine as a positive control. Toxicity increased when exposures started from 0.5 hpf compared to 4 hpf and LC50 were significantly lower by 1.2 and 7.6 times for silver and 5-azacyitidine, respectively. We then investigated whether pre-exposure to silver during early development (from 0.5 or 4 hpf) affected the outcome of subsequent exposures during the larvae stage, and found no alterations in toxicity compared to naïve larvae. Together, these data demonstrate that during early development zebrafish embryos are more sensitive to silver when experiments are initiated at the one-cell stage, but that pre-exposures do not influence the outcome of subsequent exposures, suggesting that no long-lasting memory capable of influencing future susceptibility was maintained under our experimental conditions. The finding that toxicity is greater for exposures initiated at the one-cell stage has implications for designing testing systems to assess chemical toxicity.

The embryonic nucleologenesis during inhibition of major transcriptional activity in bovine preimplantation embryos

Biologia ◽  
2012 ◽  
Vol 67 (4) ◽  
Author(s):  
Mária Kovalská ◽  
Marián Hruška-Plocháň ◽  
Oľga Østrup ◽  
Marian Adamkov ◽  
Ján Lehotský ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Actinomycin D ◽  
De Novo ◽  
Specific Inhibitor ◽  
Preimplantation Embryos ◽  
Control Group ◽  
Cell Stage ◽  
Initial Development ◽  
Embryonic Genome ◽  
Autoradiographic Labeling

AbstractCommon features of embryonic genome activation in mammalian and non-mammalian embryos are the colocalization of pre-assembled complexes of maternally inherited nucleolar proteins, the so-called nucleolus precursor bodies and de novo synthesized transcripts with ribosomal DNA. The de novo transcription of messenger RNA and ribosomal RNA proteins is required for the development of functional nuclei during the major activation of the embryonic genome. The aim of our work was to investigate to what extent. Autoradiography and transmission electron microscopy has been applied in in vitro produced bovine embryos. The embryos were cultured to the late 8-cell stage with: α-amanitin; a specific inhibitor of RNA-polymerases II and III transcription; actinomycin D; a specific inhibitor of RNA polymerase I transcription; and without inhibitors (control group). Nucleoplasm and nucleolar structures displayed strong autoradiographic labeling and showed the initial development of fibrillo-granular nucleoli in the control group. In α-amanitin groups, however, in both inhibited groups of embryos, lack of autoradiographic labeling and disintegrated nucleolus precursor bodies stage were observed. Our study of α-amanitin as well as in actinomycin D groups proves inhibition of transformation nucleolus precursor bodies to active nucleoli. From our results follows, actinomycin D is able to penetrate through zona pellucida, what was shown for the first time.

scholarly journals Advanced platelet-rich fibrin plus gold nanoparticles enhanced the osteogenic capacity of human mesenchymal stem cells

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Dara Ghaznavi ◽  
Amirreza Babaloo ◽  
Adileh Shirmohammadi ◽  
Arezoo Rezaie Nezhad Zamani ◽  
Mehdi Azizi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gold Nanoparticles ◽  
Mesenchymal Stem Cells ◽  
Bone Mineralization ◽  
Human Mesenchymal Stem Cells ◽  
Conditioned Media ◽  
Calcium Deposition ◽  
Control Group ◽  
Alizarin Red ◽  
Platelet Rich Fibrin

Abstract Objectives There is still insufficient clinical evidence of platelet-rich fibrin beneficial effects on bone regeneration. Gold nanoparticles have been shown to enhance osteogenic differentiation and bone mineralization. The purpose of this study was to investigate the effect of advanced-platelet-rich fibrin modified by gold nanoparticles on the osteoblastic differentiation of human mesenchymal stem cells. Results MTT assay revealed 0.0125 mM gold nanoparticles had no cytotoxic effects on stem cells after 7 days. The addition of 0.0125 mM gold nanoparticle to advanced-platelet-rich fibrin clot increased cell viability compared to the non-treated control group (p < 0.05). 7-day incubation of stem cells with advanced-platelet-rich fibrin modified by gold nanoparticles conditioned media was shown to promote alkaline phosphatase activity compared to the control cells and group treated with advanced-platelet-rich fibrin condition media (p < 0.05). By using Alizarin Red S staining, red-colored calcium deposits were observed in the group treated with advanced-platelet-rich fibrin and gold nanoparticles conditioned media in comparison with non-treated cells (p < 0.05). Advanced-platelet-rich fibrin conditioned medium was unable to promote calcium deposition compared to the combination of advanced-platelet-rich fibrin and gold nanoparticles (p < 0.05). Adding gold nanoparticles to advanced-platelet-rich fibrin and fibrin and platelet byproducts could be an alternative strategy to improve osteogenic capacity of stem cells.

Gene silencing in bovine zygotes: siRNA transfection versus microinjection

2011 ◽  
Vol 23 (4) ◽  
pp. 534 ◽  
Author(s):  
Ciara M. O'Meara ◽  
James D. Murray ◽  
Solomon Mamo ◽  
Emma Gallagher ◽  
James Roche ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Relative Abundance ◽  
Messenger Rna ◽  
Blastocyst Stage ◽  
Small Interfering Rnas ◽  
Sirna Transfection ◽  
Cell Stage ◽  
Transfection Medium ◽  
E Cadherin

The aim of this study was to compare gene silencing in bovine zygotes when small interfering RNAs (siRNAs) were introduced into bovine zygotes by microinjection or lipid-based transfection. In Experiment 1, E-cadherin siRNA was injected at 100 or 375 µM and compared with PBS-injected and non-injected controls. Embryos were then cultured in vitro for 7 days and periodically assessed for development. For transfection, zona-free zygotes were incubated in transfection medium with siRNA for 1 h at 39°C and then cultured to Day 7. Injection of PBS or 375 µM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5% and 45.5%) or the blastocyst stage (39.0 and 32.5%) compared with non-injected controls (62.9 and 45.0%, respectively; P < 0.05). Messenger RNA abundance was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 and 375 µM, respectively, compared with controls (P < 0.05). Transfection with 100 nM E-cadherin siRNA decreased development to the 8-cell stage (20.3 versus 53.0%) and blastocyst stage (7.2 versus 18.2%) compared with controls (P < 0.05). Messenger RNA relative abundance was not different between controls (non-transfected or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100 and 200 nM E-cadherin siRNA led to a 72 and 38% reduction, respectively, in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P < 0.05).

126 NUCLEAR TRANSLOCATION OF NUCLEAR FACTOR KAPPA B AND ITS ROLE IN MOUSE PRE-IMPLANTATION DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 172
Author(s):  
Y. Fumiiwa ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
Treatment Group ◽  
Nuclear Factor Kappa B ◽  
Nuclear Translocation ◽  
Blastocyst Stage ◽  
Control Group ◽  
Nuclear Factor ◽  
Cell Stage ◽  
Nuclear Factor Kappa ◽  
Morphological Aberration ◽  
The One

In mouse pre-implantation development, it has been reported that RelA, one of the subunits of nuclear factor kappa B (NF-�B), is expressed in eggs and embryos from the Metaphase II oocyte to the blastocyst stage. However, the role of NF-�B in the pre-implantation development has not yet been elucidated in detail. In this study, we examined (1) the activation of NF-�B during mouse pre-implantation development and (2) the effect of a synthetic peptide inhibitor of NF-�B, SN-50, which inhibits nuclear translocation of NF-�B on the pre-implantation development. Fertilized one-cell embryos were collected 17 h post-hCG from the ampullae of oviducts of superovulated ICR mouse females that had been mated with the same strain of males and then were cultured in KSOM medium at 37�C under 5% CO2 in air for 4 d. To elucidate the timing of NF-�B activation, we examined the localization of NF-�B in the nucleus by an immunofluorescence approach using RelA antibody with a laser confocal microscope. RelA was distributed mainly in the cytoplasm of embryos from the one-cell stage through the blastocyst stages. The presence of RelA in the nucleus, evidence for NF-�B activation, was observed in embryos from the one-cell to the compacted 8-cell stages. Moreover, we observed RelA punctate localization in nucleoplasm of embryos from the one-cell to the 4-cell stages, and nuclear dots were enriched conspicuously in the one-cell embryos and the late 2-cell embryos. These results suggest that NF-�B is activated in embryos from the one-cell to the compacted 8-cell stages and that its activation seems to be particularly strong at the developmental stage when RelA appeared to be concentrated in nuclear dots, as it has been reported that NF-�B and other transcription factors and co-activators form punctate structures called 'enhanceosom' in association with particular promoters in the nucleus. Next, we examined the effect of SN-50 on the pre-implantation development of mouse embryos. When embryos were treated with SN-50 at 20 �g/mL from the 2-cell stage, 63% (33 of 52) of the embryos developed to blastocysts, but 55% (18 of 33) of the blastocysts showed abnormal morphology, such as poor cavitation, and many degenerating cells extruded into the perivitelline space. The percentages of 2-cell embryos that formed morphologically normal blastocysts were significantly lower in the SN-50 treatment group (29%; 15 of 52) than in the untreated control group (76%; 35 of 46) and in the SN-50M (inactive analogue of SN-50, 20 �g/mL) treatment group (72%; 38 of 53). These experiments were done in 4 replicates, and the statistical analyses of the data were done by ANOVA and Fisher's PLSD test. Nuclear location of RelA was not observed in the embryos at the 4-cell stage when treated with SN-50 from the 2-cell stage, although observed in control and SN-50M-treated embryos. Furthermore, it was found that most of embryos (23 of 37) treated with SN-50 from the compacted 8-cell or morula stages developed normally to the blastocyst stage as control embryos (25 of 36). These results suggest that morphological aberration at the blastocyst stage is elicited by inhibiting NF-�B activation.

Human Mesenchymal Stem Cells Behavior on Synthetic Coral Scaffold

2016 ◽  
Vol 696 ◽  
pp. 205-211 ◽  
Author(s):  
Erlina Sih Mahanani ◽  
Indra Bachtiar ◽  
Ika Dewi Ana
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Attachment ◽  
Platelet Rich Plasma ◽  
Human Mesenchymal Stem Cells ◽  
Control Group ◽  
Freeze Dried ◽  
Initial Support ◽  
The One ◽  
Coral Scaffold

For bone tissue engineering, corals have long history to be used as scaffold to promote bone regeneration. However the use of a lot of corals may damage their habitates. For this reason, a strategy to mimic coral in a synthetic form is needed. As an ideal scaffold, synthetic coral must provide structure and initial support for cell attachment and proliferation. The aim of this study was to investigate the attachment and proliferation of human Mesenchymal Stem Cells (h-MSC) on synthetic coral scaffold, to provide information on the behavior of h-MSC on the designated scaffold. Synthetic coral scaffolds were prepared from bovine gelatine and CaCO3 with 5:5 in 10% w/v concentration in aquadest. Sodium citrate was used as dispersant in the suspension. Gelatin-CaCO3 suspension was moulded in a plastic cover of 24 well plate, then freezed at -20°C for 24 hours, freeze dried for 24 hours and continued by dehydrothermal crosslinking for 72 hours. After the fabrication, synthetic coral scaffolds were subjects to cover the bottom of the well for cell culture. Human Mesenchymal Stem Cells (h-MSC) were seeded and divided into 2 groups, control group without scaffold and the one with scaffold. All groups were incubated for 3, 6, and 24 hours. Cells attatchment were determined by deduction of the cells unattached from total cells seeding. Proliferation of h-MSC were done in 3 groups ie., control group without scaffold, scaffold only and scaffold incorporated Platelet Rich Plasma (PRP) in the bottom of well. All groups were incubated for 24, 48 and 72 hours. Human Mesenchymal Stem Cells attached faster to synthetic coral scaffold than the control. Its proliferation behavior was faster in the scaffold incorporated PRP, showing better interaction of scaffold and cells with the incorporation of morphogenetic factor.

scholarly journals MEK1/2 Inhibitor (GDC0623) Promotes Osteogenic Differentiation of Primary Osteoblasts Inhibited by IL-1β through the MEK-Erk1/2 and Jak/Stat3 Pathways

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Zeng-Qiao Zhang ◽  
Xiao-Shen Hu ◽  
Ye-Chen Lu ◽  
Jun-Peng Zhang ◽  
Wen-Yao Li ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Western Blotting ◽  
Cell Counting ◽  
Nuclear Antigen ◽  
Control Group ◽  
Induction Medium ◽  
Alizarin Red ◽  
Real Time Quantitative Pcr ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction

Objective. We evaluated the effects and mechanisms of GDC0623 on osteogenic differentiation of osteoblasts induced by IL-1β. Methodology. Osteoblasts were treated with 20 ng/ml IL-1β and 0.1 µM GDC0623. Cell proliferation levels were evaluated by the cell counting kit 8 (CCK8), EdU assay, and western blotting [proliferating cell nuclear antigen (PCNA) and Cyclin D1]. Osteoblasts were cultured in an osteogenic induction medium for 1–3 weeks after which their differentiations were assessed by alkaline phosphatase (ALP) staining, Alizarin Red staining, calcium concentration, immunocytochemistry staining, real-time quantitative PCR (RT-qPCR), and immunofluorescence staining. The osteogenesis-associated mechanisms were further evaluated by western blotting using appropriate antibodies. Results. Relative to the control group, IL-1β induced the rapid proliferation of osteoblasts and suppressed their osteogenic differentiations by upregulating the activities of MEK-Erk1/2 as well as Jak-Stat3 pathways and by elevating MMP13 and MMP9 levels. However, blocking of the MEK-Erk1/2 signaling pathway by GDC0623 treatment reversed these effects. Conclusion. Inhibition of Jak-Stat3 pathway by C188-9 downregulated the expression levels of MMP9 and MMP13, activated MEK-Erk1/2 pathway, and inhibited osteogenic differentiation.

The Effect of The Using of Computer Games On Academic Success In Teaching English To 8th Grade Students

2019 ◽  
Author(s):  
Bulent Dos ◽  
Zeynep Sinem Balıkçıoğlu ◽  
Semih Şengel
Keyword(s):  
Academic Success ◽  
Computer Games ◽  
Experimental Information ◽  
Control Group ◽  
Traditional Teaching ◽  
National Education ◽  
8Th Grade ◽  
Post Test ◽  
Education Measurement ◽  
The One

<p>In this study, the effect of using computer games in an English teaching classof the 8th grade students in secondary school is investigated. A total of 112 8th grade students, 57 in the experimental group and 55 in the control group, participated in the study. Academic Achievement Tests prepared by Ministry of National Education, Measurement, Evaluation and Exam Services Department were used as pre-test and post-test. Eight of the games, which were prepared specifically for Grade 8 students, were used in the Experimental Information Network (EBA). Preliminary tests as covariant, final tests as dependent variables and applied to groups of teaching and traditional teaching were discussed as independent variables. According to the one-way covariance analysis (COVARIANCE) results, it was found that the game was more effective than traditional teaching and this effect was moderate. In addition, it was determined that 36% of the final test scores of the students learning the game were explained by the game.</p>

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