Equine oviduct explant culture: a basic model to decipher embryo–maternal communication

2014 ◽  
Vol 26 (7) ◽  
pp. 954 ◽  
Author(s):  
Hilde Nelis ◽  
Katharina D'Herde ◽  
Karen Goossens ◽  
Lynn Vandenberghe ◽  
Bart Leemans ◽  
...  
Keyword(s):  
Secretory Granules ◽  
Explant Culture ◽  
Marker Genes ◽  
Cell Degeneration ◽  
Dark Cell ◽  
Basal Nuclei ◽  
Basic Model ◽  
Set Up

Equine embryos remain for 6 days in the oviduct and thus there is a need for an in vitro model to study embryo–oviductal interactions in the horse, since this subtle way of communication is very difficult to analyse in vivo. Until now, no equine oviduct explant culture model has been characterised both morphologically and functionally. Therefore, we established a culture system for equine oviduct explants that maintained epithelial morphology during 6 days of culture, as revealed by light microscopy and transmission electron microscopy. We demonstrated the presence of highly differentiated, tall columnar, pseudostratified epithelium with basal nuclei, numerous nucleoli, secretory granules and apical cilia, which is very similar to the in vivo situation. Both epithelium and stromal cells originating from the lamina propria are represented in the explants. Moreover, at least 98% of the cells remained membrane intact and fewer than 2% of the cells were apoptotic after 6 days of culture. Although dark-cell degeneration, which is a hypoxia-related type of cell death, was observed in the centre of the explants, quantitative real-time PCR failed to detect upregulation of the hypoxia-related marker genes HIF1A, VEGFA, uPA, GLUT1 and PAI1. Since the explants remained morphologically and functionally intact and since the system is easy to set up, it appears to be an excellent tool for proteome, transcriptome and miRNome analysis in order to unravel embryo–maternal interactions in the horse.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

2021 ◽  
Vol 22 (13) ◽  
pp. 6663
Author(s):  
Maurycy Jankowski ◽  
Mariusz Kaczmarek ◽  
Grzegorz Wąsiatycz ◽  
Claudia Dompe ◽  
Paul Mozdziak ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Marker Genes ◽  
P Value ◽  
Illumina Platform

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.

scholarly journals Fabrication of 3D-Printed Interpenetrating Hydrogel Scaffolds for Promoting Chondrogenic Differentiation

Polymers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 2146
Author(s):  
Jian Guan ◽  
Fu-zhen Yuan ◽  
Zi-mu Mao ◽  
Hai-lin Zhu ◽  
Lin Lin ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Elastic Modulus ◽  
Chondrogenic Differentiation ◽  
Marker Genes ◽  
Self Healing ◽  
Polyethylene Glycol Diacrylate ◽  
3D Microenvironment ◽  
3D Printed

The limited self-healing ability of cartilage necessitates the application of alternative tissue engineering strategies for repairing the damaged tissue and restoring its normal function. Compared to conventional tissue engineering strategies, three-dimensional (3D) printing offers a greater potential for developing tissue-engineered scaffolds. Herein, we prepared a novel photocrosslinked printable cartilage ink comprising of polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GelMA), and chondroitin sulfate methacrylate (CSMA). The PEGDA-GelMA-CSMA scaffolds possessed favorable compressive elastic modulus and degradation rate. In vitro experiments showed good adhesion, proliferation, and F-actin and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) on the scaffolds. When the CSMA concentration was increased, the compressive elastic modulus, GAG production, and expression of F-actin and cartilage-specific genes (COL2, ACAN, SOX9, PRG4) were significantly improved while the osteogenic marker genes of COL1 and ALP were decreased. The findings of the study indicate that the 3D-printed PEGDA-GelMA-CSMA scaffolds possessed not only adequate mechanical strength but also maintained a suitable 3D microenvironment for differentiation, proliferation, and extracellular matrix production of BMSCs, which suggested this customizable 3D-printed PEGDA-GelMA-CSMA scaffold may have great potential for cartilage repair and regeneration in vivo.

scholarly journals Multiple screening approaches reveal HDAC6 as a novel regulator of glycolytic metabolism in triple-negative breast cancer

2021 ◽  
Vol 7 (3) ◽  
pp. eabc4897
Author(s):  
Catríona M. Dowling ◽  
Kate E. R. Hollinshead ◽  
Alessandra Di Grande ◽  
Justin Pritchard ◽  
Hua Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Histone Deacetylase Inhibitors ◽  
Triple Negative ◽  
Mitochondrial Apoptosis ◽  
Glycolytic Metabolism ◽  
Set Up ◽  
Screening Approaches

Triple-negative breast cancer (TNBC) is a subtype of breast cancer without a targeted form of therapy. Unfortunately, up to 70% of patients with TNBC develop resistance to treatment. A known contributor to chemoresistance is dysfunctional mitochondrial apoptosis signaling. We set up a phenotypic small-molecule screen to reveal vulnerabilities in TNBC cells that were independent of mitochondrial apoptosis. Using a functional genetic approach, we identified that a “hit” compound, BAS-2, had a potentially similar mechanism of action to histone deacetylase inhibitors (HDAC). An in vitro HDAC inhibitor assay confirmed that the compound selectively inhibited HDAC6. Using state-of-the-art acetylome mass spectrometry, we identified glycolytic substrates of HDAC6 in TNBC cells. We confirmed that inhibition or knockout of HDAC6 reduced glycolytic metabolism both in vitro and in vivo. Through a series of unbiased screening approaches, we have identified a previously unidentified role for HDAC6 in regulating glycolytic metabolism.

scholarly journals Targeting a Lipid Desaturation Enzyme, SCD1, Selectively Eliminates Colon Cancer Stem Cells through the Suppression of Wnt and NOTCH Signaling

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 106
Author(s):  
Yeongji Yu ◽  
Hyejin Kim ◽  
SeokGyeong Choi ◽  
JinSuh Yu ◽  
Joo Yeon Lee ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Notch Signaling ◽  
Cultured Cells ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Dependent Manner ◽  
Lipid Desaturation ◽  
Novel Target

The elimination of the cancer stem cell (CSC) population may be required to achieve better outcomes of cancer therapy. We evaluated stearoyl-CoA desaturase 1 (SCD1) as a novel target for CSC-selective elimination in colon cancer. CSCs expressed more SCD1 than bulk cultured cells (BCCs), and blocking SCD1 expression or function revealed an essential role for SCD1 in the survival of CSCs, but not BCCs. The CSC potential selectively decreased after treatment with the SCD1 inhibitor in vitro and in vivo. The CSC-selective suppression was mediated through the induction of apoptosis. The mechanism leading to selective CSC death was investigated by performing a quantitative RT-PCR analysis of 14 CSC-specific signaling and marker genes after 24 and 48 h of treatment with two concentrations of an inhibitor. The decrease in the expression of Notch1 and AXIN2 preceded changes in the expression of all other genes, at 24 h of treatment in a dose-dependent manner, followed by the downregulation of most Wnt- and NOTCH-signaling genes. Collectively, we showed that not only Wnt but also NOTCH signaling is a primary target of suppression by SCD1 inhibition in CSCs, suggesting the possibility of targeting SCD1 against colon cancer in clinical settings.

scholarly journals Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells

2006 ◽  
Vol 173 (2) ◽  
pp. 241-251 ◽  
Author(s):  
Malika Ahras ◽  
Grant P. Otto ◽  
Sharon A. Tooze
Keyword(s):  
Pc12 Cells ◽  
Secretory Granules ◽  
Granule Formation ◽  
Synaptotagmin Iv ◽  
Prohormone Convertase 2 ◽  
Granule Maturation ◽  
Golgi Network ◽  
Interfering Rna

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA–mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.

Opioids and testicular activity in the frog, Rana esculenta

1993 ◽  
Vol 137 (1) ◽  
pp. 49-NP ◽  
Author(s):  
F. Facchinetti ◽  
A. R. Genazzani ◽  
M. Vallarino ◽  
M. Pestarino ◽  
A. Polzonetti-Magni ◽  
...  
Keyword(s):  
Physiological Activity ◽  
Mammalian Species ◽  
Rana Esculenta ◽  
Cell Degeneration ◽  
Efferent System ◽  
Naltrexone Treatment ◽  
Nucleus Infundibularis ◽  
The Brain

ABSTRACT The presence and activity of brain, pituitary and testicular β-endorphin (β-EP)-like material have been studied in the frog, Rana esculenta, using reverse-phase high-pressure liquid chromatography, coupled with radioimmunoassay and immunocytochemistry. In-vivo and in-vitro treatments with naltrexone were carried out to assess the putative physiological activity of opioid peptides. β(1–31) and (1–27), together with their acetylated forms, have been identified in brain, pituitary and testis. In particular, β-EP(1–31) concentrations peaked during July in the brain and pituitary, whilst in testes maximum concentrations were found in April and November. β-EP immunoreactivity was present in the brain within the nucleus preopticus and nucleus infundibularis ventralis while positive fibres in the retrochiasmatic regions projected to the median eminence. In the testis, interstitial cells, canaliculi of the efferent system, spermatogonia and spermatocytes showed positive immunostaining for β-EP. In intact animals, naltrexone treatment increased plasma and testicular androgen levels and this effect was confirmed in in-vitro incubations of minced testes. Naltrexone also induced a significant increase in germ cell degeneration. Our results indicated that an opioid system modulates the hypothalamus-pituitary-gonadal axis in the frog, Rana esculenta and, for the first time, we have shown that the testicular activity of a non-mammalian species may be regulated by opiates locally. Journal of Endocrinology (1993) 137, 49–57

scholarly journals Amount of RNA secondary structure required to induce an alternative splice.

1987 ◽  
Vol 7 (9) ◽  
pp. 3194-3198 ◽  
Author(s):  
D Solnick ◽  
S I Lee
Keyword(s):  
Alternative Splicing ◽  
Secondary Structure ◽  
Alternative Splice ◽  
Rna Secondary Structure ◽  
Secondary Structures ◽  
Splice Sites ◽  
Perfect Complementarity ◽  
Set Up

We set up an alternative splicing system in vitro in which the relative amounts of two spliced RNAs, one containing and the other lacking a particular exon, were directly proportional to the length of an inverted repeat inserted into the flanking introns. We then used the system to measure the effect of intramolecular complementarity on alternative splicing in vivo. We found that an alternative splice was induced in vivo only when the introns contained more than approximately 50 nucleotides of perfect complementarity, that is, only when the secondary structure was much more stable than most if not all possible secondary structures in natural mRNA precursors. We showed further that intron insertions containing long complements to splice sites and a branch point inhibited splicing in vitro but not in vivo. These results raise the possibility that in cells most pre-mRNA secondary structures either are not maintained long enough to influence splicing choices, or never form at all.

scholarly journals Ultrasound-Mediated Drug Delivery With a Clinical Ultrasound System: In Vitro Evaluation

2021 ◽  
Vol 12 ◽  
Author(s):  
Josanne S. de Maar ◽  
Charis Rousou ◽  
Benjamin van Elburg ◽  
Hendrik J. Vos ◽  
Guillaume P.R. Lajoinie ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Clinical Translation ◽  
Drug Uptake ◽  
Single Element ◽  
Hydrophilic Drug ◽  
Ultrasound System ◽  
Fadu Cells ◽  
Set Up

Chemotherapy efficacy is often reduced by insufficient drug uptake in tumor cells. The combination of ultrasound and microbubbles (USMB) has been shown to improve drug delivery and to enhance the efficacy of several drugs in vitro and in vivo, through effects collectively known as sonopermeation. However, clinical translation of USMB therapy is hampered by the large variety of (non-clinical) US set-ups and US parameters that are used in these studies, which are not easily translated to clinical practice. In order to facilitate clinical translation, the aim of this study was to prove that USMB therapy using a clinical ultrasound system (Philips iU22) in combination with clinically approved microbubbles (SonoVue) leads to efficient in vitro sonopermeation. To this end, we measured the efficacy of USMB therapy for different US probes (S5-1, C5-1 and C9-4) and US parameters in FaDu cells. The US probe with the lowest central frequency (i.e. 1.6 MHz for S5-1) showed the highest USMB-induced intracellular uptake of the fluorescent dye SYTOX™ Green (SG). These SG uptake levels were comparable to or even higher than those obtained with a custom-built US system with optimized US parameters. Moreover, USMB therapy with both the clinical and the custom-built US system increased the cytotoxicity of the hydrophilic drug bleomycin. Our results demonstrate that a clinical US system can be used to perform USMB therapy as efficiently as a single-element transducer set-up with optimized US parameters. Therefore, future trials could be based on these clinical US systems, including validated US parameters, in order to accelerate successful translation of USMB therapy.

scholarly journals An in vitro ovarian explant culture system to examine sex change in a hermaphroditic fish

2020 ◽  
Author(s):  
Alexander Goikoetxea ◽  
Erin L Damsteegt ◽  
Erica V Todd ◽  
Andrew McNaughton ◽  
Neil J Gemmell ◽  
...  
Keyword(s):  
Culture System ◽  
Sex Change ◽  
Explant Culture ◽  
Culture Technique ◽  
Aromatase Activity ◽  
Histological Images ◽  
17Β Estradiol ◽  
Learning Software

AbstractMany teleost fishes undergo natural sex change, and elucidating the physiological and molecular controls of this process offers unique opportunities not only to develop methods of controlling sex in aquaculture settings, but to better understand vertebrate sexual development more broadly. Induction of sex change in some sequentially hermaphroditic or gonochoristic fish can be achieved in vivo through social manipulation, inhibition of aromatase activity, and steroid treatment. However, the induction of sex change in vitro has been largely unexplored. In this study, we established an in vitro culture system for ovarian explants in serum-free medium for a model sequential hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). This culture technique enabled evaluating the effect of various treatments with 17β-estradiol (E2), 11-ketotestosterone (11KT) or cortisol (CORT) on spotty wrasse ovarian architecture for 21 days. A quantitative approach to measuring the degree of ovarian atresia within histological images was also developed, using pixel-based machine learning software. Ovarian atresia likely due to culture was observed across all treatments including no-hormone controls, but was minimised with treatment of at least 10 ng/mL E2. Neither 11KT nor CORT administration induced proliferation of spermatogonia (i.e. sex change) in the cultured ovaries indicating culture beyond 21 days may be needed to induce sex change in vitro. The in vitro gonadal culture and analysis systems established here enable future studies investigating the paracrine role of sex steroids, glucocorticoids and a variety of other factors during gonadal sex change in fish.

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