scholarly journals Microinjection to deliver protein, mRNA, and DNA into zygotes of the cnidarian endosymbiosis model Aiptasia sp.

2017 ◽  
Author(s):  
Victor A. S. Jones ◽  
Madeline Bucher ◽  
Elizabeth A. Hambleton ◽  
Annika Guse
Keyword(s):  
Transgene Expression ◽  
Molecular Mechanisms ◽  
Model System ◽  
Proof Of Concept ◽  
Coral Larvae ◽  
Functional Studies ◽  
Algal Symbiosis ◽  
Summary Statement ◽  
Exogenous Genes

AbstractReef-building corals depend on an intracellular symbiosis with photosynthetic dinoflagellates for their survival in nutrient-poor oceans. Symbionts are phagocytosed by coral larvae from the environment and transfer essential nutrients to their hosts. Aiptasia, a small tropical marine sea anemone, is emerging as a tractable model system for coral symbiosis; however, to date functional tools and genetic transformation are lacking. Here we have established an efficient workflow to collect Aiptasia eggs for in vitro fertilization and microinjection as the basis for experimental manipulations in the developing embryo and larvae. We demonstrate that protein, mRNA, and DNA can successfully be injected into live Aiptasia zygotes to label actin with recombinant Lifeact-eGFP protein; to label nuclei and cell membranes with NLS-eGFP and farnesylated mCherry translated from injected mRNA; and to transiently drive transgene expression from an Aiptasia-specific promoter, respectively, in embryos and larvae. These proof-of-concept approaches pave the way for future functional studies of development and symbiosis establishment in Aiptasia, a powerful model to unravel the molecular mechanisms underlying intracellular coral-algal symbiosis.Summary StatementToolkit extension: development of microinjection for cellular labelling, expression of exogenous genes and live imaging in Aiptasia, an emerging model for intracellular coral-algal symbiosis.

scholarly journals CSIG-30. miR-146a INHIBITS TUMORIGENESIS AND INDUCES TEMOZOLOMIDE SENSITIVITY VIA AFFECTING CANCER STEM CELL PROPERTIES IN GBM

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi50-vi50
Author(s):  
Tiantian Cui ◽  
Erica Hlavin Bell ◽  
Joseph McElroy ◽  
Kevin Liu ◽  
Pooja Manchanda Gulati ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cancer Stem Cell ◽  
Molecular Mechanisms ◽  
Functional Studies ◽  
Average Survival ◽  
Novel Biomarkers ◽  
The Ohio State University

Abstract BACKGROUND Glioblastomas (GBMs) are the most aggressive primary brain tumors, with an average survival time of less than 15 months. miRNAs are emerging as promising and novel biomarkers in GBM. The aims of this study are: 1) to investigate novel miRNAs biomarkers that affect tumorigenesis and therapeutic sensitivity, and 2) to study the underlying molecular mechanisms in GBM. METHODS Nanostring v3 was performed followed by univariable (UVA) and multivariable (MVA) analyses. Functional studies were conducted to define the role of miR-146a in GBM tumorigenesis and therapeutic response and the molecular mechanisms were investigated. RESULTS UVA analyses demonstrated that miR-146a is one of the top miRNAs that correlated with better prognosis in GBM patients (p=9.21E-05), which was independent of MGMT promoter methylation by MVA analyses (p< 0.001). miR-146a expression was significantly downregulated in recurrent GBM tumors compared with the paired primary GBM tumors (p=0.003). Overexpression of miR-146a significantly inhibited tumor cell growth and sensitized patient-derived primary GBM cells to temozolomide (TMZ) treatment in vitro, and showed statistically significant smaller tumor size (p< 0.01) and prolonged survival (p=0.001) in vivo. In addition, miR-146a is downregulated in glioma cancer stem cells, and overexpression of miR-146a significantly affected glioma cancer stem cell self-renewal. We also found that overexpression of miR-146a significantly inhibited the NF-κB, AKT, and ERK pathways. CONCLUSION Our data suggest, for the first time, that miR-146a predicts favorable prognosis for GBM patients and sensitizes primary GBM cells to TMZ treatment in vitro and in vivo through regulating glioma stem cells. Importantly, miR-146a may prove to be a master switch shutting off AKT, NF-κB, as well as other pathways and may overcome redundancies among these pathways leading to resistance. FUNDING: Bohnenn Fund (to PR), R01CA108633, R01CA169368, U10CA180850-01(NCI), Brain Tumor Funders Collaborative Grant, and The Ohio State University CCC (all to AC).

scholarly journals Functional analysis of synovial fluid from osteoarthritic knee and carpometacarpal joints unravels different molecular profiles

Rheumatology ◽  
2018 ◽  
Vol 58 (5) ◽  
pp. 897-907 ◽  
Author(s):  
Goncalo Barreto ◽  
Rabah Soliymani ◽  
Marc Baumann ◽  
Eero Waris ◽  
Kari K Eklund ◽  
...  
Keyword(s):  
Protein Expression ◽  
Molecular Mechanisms ◽  
Mrna Level ◽  
Digital Pcr ◽  
Label Free ◽  
Primary Chondrocytes ◽  
Functional Studies ◽  
Knee Oa ◽  
Carpometacarpal Joints

Abstract Objective In this work, we aimed to elucidate the molecular mechanisms driving primary OA. By studying the dynamics of protein expression in two different types of OA joints we searched for similarities and disparities to identify key molecular mechanisms driving OA. Methods For this purpose, human SF samples were obtained from CMC-I OA and knee joint of OA patients. SF samples were analysed by label-free quantitative liquid chromatography mass spectrometry. Disease-relevant proteins identified in proteomics studies, such as clusterin, paraoxonase/arylesterase 1 (PON1) and transthyretin were validated by enzyme-linked immunosorbent assays, and on the mRNA level by droplet digital PCR. Functional studies were performed in vitro using primary chondrocytes. Results Differential proteomic changes were observed in the concentration of 40 proteins including clusterin, PON1 and transthyretin. Immunoassay analyses of clusterin, PON1, transthyretin and other inflammatory cytokines confirmed significant differences in protein concentration in SF of CMC-I and knee OA patients, with primarily lower protein expression levels in CMC-I. Functional studies on chondrocytes unequivocally demonstrated that stimulation with SF obtained from knee OA, in contrast to CMC-I OA joint, caused a significant upregulation in pro-inflammatory response, cell death and hypertrophy. Conclusion This study demonstrates that differential expression of molecular players in SF from different OA joints evokes diverse effects on primary chondrocytes. The pathomolecular mechanisms of OA may significantly differ in various joints, a finding that brings a new dimension into the pathogenesis of primary OA.

scholarly journals Regulation of myogenic progenitor proliferation in human fetal skeletal muscle by BMP4 and its antagonist Gremlin

2006 ◽  
Vol 175 (1) ◽  
pp. 99-110 ◽  
Author(s):  
Natasha Y. Frank ◽  
Alvin T. Kho ◽  
Tobias Schatton ◽  
George F. Murphy ◽  
Michael J. Molloy ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Molecular Mechanisms ◽  
Side Population ◽  
Functional Studies ◽  
Morphogenetic Protein ◽  
Study Gene Expression ◽  
Main Population ◽  
Myogenic Progenitor

Skeletal muscle side population (SP) cells are thought to be “stem”-like cells. Despite reports confirming the ability of muscle SP cells to give rise to differentiated progeny in vitro and in vivo, the molecular mechanisms defining their phenotype remain unclear. In this study, gene expression analyses of human fetal skeletal muscle demonstrate that bone morphogenetic protein 4 (BMP4) is highly expressed in SP cells but not in main population (MP) mononuclear muscle-derived cells. Functional studies revealed that BMP4 specifically induces proliferation of BMP receptor 1a–positive MP cells but has no effect on SP cells, which are BMPR1a-negative. In contrast, the BMP4 antagonist Gremlin, specifically up-regulated in MP cells, counteracts the stimulatory effects of BMP4 and inhibits proliferation of BMPR1a-positive muscle cells. In vivo, BMP4-positive cells can be found in the proximity of BMPR1a-positive cells in the interstitial spaces between myofibers. Gremlin is expressed by mature myofibers and interstitial cells, which are separate from BMP4-expressing cells. Together, these studies propose that BMP4 and Gremlin, which are highly expressed by human fetal skeletal muscle SP and MP cells, respectively, are regulators of myogenic progenitor proliferation.

scholarly journals In Vitro Reconstitution of Yeast Translation System Capable of Synthesizing Long Polypeptide and Recapitulating Programmed Ribosome Stalling

2021 ◽  
Vol 4 (3) ◽  
pp. 45
Author(s):  
Riku Nagai ◽  
Yichen Xu ◽  
Chang Liu ◽  
Ayaka Shimabukuro ◽  
Nono Takeuchi-Tomita
Keyword(s):  
Unnatural Amino Acids ◽  
Molecular Mechanisms ◽  
In Vitro Translation ◽  
Critical Parameters ◽  
Translation System ◽  
Reaction Conditions ◽  
Translation Elongation ◽  
Functional Studies ◽  
Ribosome Stalling

The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors, utilizing CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing not only short oligopeptides but also long reporter proteins such as nanoluciferase. By setting appropriate translation reaction conditions, such as the Mg2+/polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) is properly recapitulated in this system. We describe protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention critical parameters for setting up the translation reaction conditions. This reconstituted translation system not only facilitates biochemical analyses of translation but is also useful for various applications, such as structural and functional studies with the aim of designing drugs that act on eukaryotic ribosomes, and the development of systems for producing novel functional proteins by incorporating unnatural amino acids by eukaryotic ribosomes.

scholarly journals The buds of Oscarella lobularis (Porifera): a new convenient model for sponge cell and developmental biology

2020 ◽  
Author(s):  
Rocher Caroline ◽  
Vernale Amélie ◽  
Fierro-Constaín Laura ◽  
Séjourné Nina ◽  
Chenesseau Sandrine ◽  
...  
Keyword(s):  
Cell Biology ◽  
Molecular Mechanisms ◽  
Full Description ◽  
Sponge Cell ◽  
Cell Architecture ◽  
Amphimedon Queenslandica ◽  
Summary Statement ◽  
The Comparative Study ◽  
Convenient Model

AbstractThe comparative study of the four non-bilaterian phyla (Cnidaria, Placozoa, Ctenophora, Porifera) should provide insights into the origin of bilaterian traits. Except for Cnidaria, present knowledge on the cell biology and development of these animals is so far limited. Non-bilaterian models are needed to get further into cell architecture and molecular mechanisms.Given the developmental, histological, ecological and genomic differences between the four sponge classes, we develop a new sponge model: the buds of the Oscarella lobularis (class Homoscleromorpha). This experimental model supplements the two other most famous sponge models Amphimedon queenslandica and Ephydatia muelleri, both belonging to the class Demospongiae.Budding is a natural and spontaneous asexual reproduction mean, but budding can be triggered in vitro ensuring availability of biological material all year long. We provide a full description of buds, from their formation to their development into juveniles. Their transparency enables fluorescent and live imaging, and their abundance allows for experimental replicates. Moreover, regeneration and cell reaggregation capabilities provide interesting experimental morphogenetic contexts. The numerous techniques now mastered on these buds make it a new suitable sponge model.Summary statementStudying sponge biology is needed to understand the evolution of metazoans. We developed a new model suitable for experimental biology that allows to study morphogenetic processes with modern tools.

scholarly journals Analysis of the Mouse CSF-1 Gene Promoter in a Transgenic Mouse Model1

2003 ◽  
Vol 51 (7) ◽  
pp. 941-949 ◽  
Author(s):  
Sherry L. Abboud ◽  
Maria Bunegin ◽  
Nandini Ghosh-Choudhury ◽  
Kathleen Woodruff
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Tissue Expression ◽  
Cellular Level ◽  
Liver And Kidney

CSF-1 stimulates monocyte and osteoclast populations. However, the molecular mechanisms involved in regulating CSF-1 gene expression are unclear. To identify regulatory regions that control normal CSF-1 gene expression, a −774/+183-bp fragment of the murine CSF-1 promoter was analyzed in vitro and in vivo. Transcriptional activity was high in cultured osteoblasts that express CSF-1 mRNA compared to ARH-77 B cells that lack CSF-1 gene expression. Transient transfection of osteoblasts with promoter deletion constructs showed that the −774-bp fragment conferred the highest transcriptional activity and contained activator and repressor sequences. To assess the ability of the CSF-1 promoter to confer normal tissue expression of CSF-1, transgenic mice containing the −774/+183-bp region driving the E. coli β-galactosidase (lacZ) reporter gene were generated. β-Gal analysis of whole tissue extracts showed transgene expression in all tissues tested except liver and kidney. At the cellular level, the pattern of β-gal expression in the spleen, thymus, bone, lung, and testes of adult transgenic mice mimicked normal endogenous CSF-1 mRNA expression in non-transgenic littermates detected by in situ hybridization. This region also directed appropriate transgene expression to sites in other tissues known to synthesize CSF-1, with the exception of the liver and kidney. These findings indicate that the −774-bp fragment contains cis-acting elements sufficient to direct CSF-1 gene expression in many tissues. CSF-1 promoter/lacZ mice may be useful for studying the transcriptional mechanisms involved in regulating CSF-1 gene expression in tissues throughout development.

scholarly journals EGR1 Modulated LncRNA HNF1A-AS1 Drives Glioblastoma Progression Via miR-22-3p/ENO1 Axis

2021 ◽  
Author(s):  
Chunchun Ma ◽  
Hongliang Wang ◽  
Gang Zong ◽  
Jie He ◽  
Yuyang Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Molecular Mechanisms ◽  
Bioinformatic Analysis ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Clinical Samples ◽  
Regulation Mechanism ◽  
Functional Studies

Abstract Background: Accumulating evidences revealed that long noncoding RNAs (lncRNAs) have been participated in cancer malignant progression, including glioblastoma multiforme (GBM). Despite much studies have found the precise biological role in the regulatory mechanisms of GBM，however the molecular mechanisms，particularly upstream mechanisms still need further elucidated. Methods: RT-QPCR, cell transfection, western blotting and bioinformatic analysis were executed to detect the expression of EGR1, HNF1A-AS1, miR-22-3p and ENO1 in GBM. Cell proliferation assay, colony formation assay, wound healing, migration and invasion assays were performed to detect the malignant characters of GBM cell. The molecular regulation mechanism was confirmed by luciferase reporter assay, ChIP and RIP. Finally, orthotopic mouse models were established to examine the effect of HNF1A-AS1 in vivo.Results: In the current study, we analyzed clinical samples to show that the long non-coding antisense transcript of HNF1A, HNF1A-AS1, is upregulated and associated with poor prognosis in GBM. Functional studies revealed that knockdown of HNF1A-AS1 markedly inhibits cell proliferation, migration and invasion both in vitro and in vivo, whereas overexpression of HNF1A-AS1 exerts opposite effect. Mechanistically, the transcription factor EGR1 forced the transcription of HNF1A-AS1 by directly binding the promoter region of HNF1A-AS1. Furthermore, combined bioinformatics analysis with our mechanistic work, using luciferase reporter assays and RIP, we first demonstrated that HNF1A-AS1 functions as a competing endogenous RNA (ceRNA) with miR-22-3p to regulate ENO1 expression in GBM cells. HNF1A-AS1 directly binds to miR-22-3p and significantly inhibits miR-22-3p expression, while ENO1 expression was increased. miR-22-3p inhibitor offsets the HNF1A-AS1 silencing induced suppression in proliferation, migration and invasion of GBM cells, as well as promotion effect on ENO1 expression. ENO1 was verified as a direct target of miR-22-3p and its expression levels was negatively with the prognosis in GBM patients. Conclusion: Taken together, our study illuminated the definite mechanism of HNF1A-AS1 in promoting GBM malignancy, and provided a novel therapeutic target for further clinical application.

In Vitro Models to Study Mechanisms of Lung Toxicity

1990 ◽  
Vol 18 (1_part_1) ◽  
pp. 267-281
Author(s):  
Åke Ryrfeldt ◽  
Ian A. Cotgreave ◽  
Peter Moldéus
Keyword(s):  
Molecular Mechanisms ◽  
In Vitro Models ◽  
Lung Toxicity ◽  
Lung Mechanics ◽  
Lung Cells ◽  
Suitable Model ◽  
Clara Cells ◽  
Functional Studies

Several functioning in vitro systems of varying complexity are currently in use for the study of mechanisms of lung toxicity. The isolated perfused lung is the model closest to the in vivo situation. It is a suitable model for combining metabolic and functional studies. It is, for instance, possible to relate changes in lung mechanics and lung perfusion flow to the release of various mediators during exposure of the lung to various agents. A simpler model may be constructed from lung slices which are less viable but suitable for uptake as well as metabolism studies. Specific lung cells such as Clara cells and type II pneumocytes have been isolated and cultured and are valuable tools for studies of the molecular mechanisms of lung toxicity, particularly in cases of cell-specific toxicity. There is, however, a great need to develop techniques for the isolation and culture of other types of lung cells and also to improve the culturing techniques for those already isolated.

scholarly journals Activation of the clock gene TIMELESS by H3k27 acetylation promotes colorectal cancer tumorigenesis by binding to Myosin-9

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Meng Cao ◽  
Yi Wang ◽  
Yijing Xiao ◽  
Dandan Zheng ◽  
Chunchun Zhi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clock Gene ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Clock Genes ◽  
Tumor Development ◽  
Creb Binding Protein ◽  
Functional Studies

Abstract Background Colorectal cancer (CRC) is a common tumor characterized by its high mortality. However, the underlying molecular mechanisms that drive CRC tumorigenesis are unclear. Clock genes have important roles in tumor development. In the present study, the expression and functions of clock gene TIMELESS (encoding the Timeless protein) in CRC were investigated. Methods Immunohistochemistry, cell proliferation, migration, invasion, EMT and xenograft tumor experiments were used to prove the function of Timeless in the tumorigenesis of CRC. Immunoprecipitation, mass spectrometry, Immunofluorescence and Chromatin immunoprecipitation (ChIP) were utilized to clarify the mechanism of Timeless in regulating CRC tumorigenesis. Results We found that Timeless was upregulated in CRC tissues compared with corresponding normal tissues and its expression was closely associated with the TNM stages and overall survival of CRC patients. Functional studies demonstrated that Timeless promoted the proliferation, invasion, and EMT of CRC cells in vitro and in vivo. Mechanistic investigations showed that Timeless activated the β-catenin signal pathway by binding to Myosin-9, which binds to β-catenin to induce its nuclear translocation. The upregulation of Timeless was attributed to CREB-binding protein (CBP)/p300-mediated H3K27 acetylation of the promoter region of Timeless. Conclusion Timeless regulates the tumorigenesis of CRC by binding to and regulating myosin-9, suggesting Timeless might be a potential prognostic biomarker and therapeutic target for CRC.

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