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Genes May 2024The low survival rate of transplanted plantlets, which has limited the utility of tissue-culture-based methods for the rapid propagation of tree peonies, is due to...
The low survival rate of transplanted plantlets, which has limited the utility of tissue-culture-based methods for the rapid propagation of tree peonies, is due to plantlet dormancy after rooting. We previously determined that the auxin response factor PsARF may be a key regulator of tree peony dormancy. To clarify the mechanism mediating tree peony plantlet dormancy, genes were systematically identified and analyzed. Additionally, was transiently expressed in the leaves of tree peony plantlets to examine its regulatory effects on a downstream gene network. Nineteen genes were identified and divided into four classes. All genes encoded proteins with conserved B3 and ARF domains. The number of motifs, exons, and introns varied between genes in different classes. The overexpression of altered the expression of , , , , , and other key genes in abscisic acid (ABA) and gibberellin (GA) signal transduction pathways, thereby promoting ABA synthesis and decreasing GA synthesis. Significant changes to the expression of some key genes contributing to starch and sugar metabolism (e.g., , , , , and ) may be associated with the gradual conversion of sugar into starch. This study provides important insights into functions in tree peonies.
Topics: Gene Expression Regulation, Plant; Plant Proteins; Plant Dormancy; Paeonia; Abscisic Acid; Gibberellins; Plant Growth Regulators; Trees; Transcription Factors; Signal Transduction
PubMed: 38927602
DOI: 10.3390/genes15060666 -
Reproductive Biology and Endocrinology... Jun 2024Testis is an immune privileged organ, which prevents the immune response against sperm antigens and inflammation. Testicular cells responsible for immune tolerance are...
BACKGROUND
Testis is an immune privileged organ, which prevents the immune response against sperm antigens and inflammation. Testicular cells responsible for immune tolerance are mainly Sertoli cells, which form the blood-testis barrier and produce immunosuppressive factors. Sertoli cells prevent inflammation in the testis and maintain immune tolerance by inhibiting proliferation and inducing lymphocyte apoptosis. It has been shown that 9-cis-retinoic acid (9cRA) blocks ex vivo apoptosis of peripheral blood lymphocytes and promotes the differentiation of Treg cells in the gut. However, the role of retinoid signaling in regulating the immune privilege of the testes remains unknown.
OBJECTIVE
The aim of this study was to determine whether 9cRA, acting via the retinoic acid receptors (RAR) and the retinoic X receptors (RXR), controls the immunomodulatory functions of Sertoli cells by influencing the secretion of anti-inflammatory/pro-inflammatory factors, lymphocyte physiology and Treg cell differentiation.
METHODS
Experiments were performed using in vitro model of co-cultures of murine Sertoli cells and T lymphocytes. Agonists and antagonists of retinoic acid receptors were used to inhibit/stimulate retinoid signaling in Sertoli cells.
RESULTS
Our results have demonstrated that 9cRA inhibits the expression of immunosuppressive genes and enhances the expression of pro-inflammatory factors in Sertoli cells and lymphocytes, increases lymphocyte viability and decreases apoptosis rate. Moreover, we have found that 9cRA blocks lymphocyte apoptosis acting through both RAR and RXR and inhibiting FasL/Fas/Caspase 8 and Bax/Bcl-2/Caspase 9 pathways. Finally, we have shown that 9cRA signaling in Sertoli cells inhibits Treg differentiation.
CONCLUSION
Collectively, our results indicate that retinoid signaling negatively regulates immunologically privileged functions of Sertoli cells, crucial for ensuring male fertility. 9cRA inhibits lymphocyte apoptosis, which can be related to the development of autoimmunity, inflammation, and, in consequence, infertility.
Topics: Male; Animals; Sertoli Cells; T-Lymphocytes, Regulatory; Signal Transduction; Mice; Tretinoin; Cell Differentiation; Alitretinoin; Receptors, Retinoic Acid; Apoptosis; Coculture Techniques; Mice, Inbred C57BL; Cells, Cultured; Immunomodulation
PubMed: 38926848
DOI: 10.1186/s12958-024-01246-2 -
Microbial Cell Factories Jun 2024Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of...
BACKGROUND
Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application.
RESULTS
In this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1',4'-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1',4'-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1',4'-trans-ABA-diol synthesis. To further increase the yield of 1',4'-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1',4'-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1',4'-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain.
CONCLUSIONS
We utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1',4'-trans-ABA-diol. By overexpressing the crucial genes involved in the 1',4'-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1',4'-trans-ABA-diol.
Topics: Botrytis; Abscisic Acid; Fermentation; Metabolic Engineering; Fungal Proteins
PubMed: 38926702
DOI: 10.1186/s12934-024-02460-8 -
Methods in Molecular Biology (Clifton,... 2024CHO cell pools with desirable characteristics of high titer and consistent product quality are useful for rapid production of recombinant proteins. Here, we describe the...
CHO cell pools with desirable characteristics of high titer and consistent product quality are useful for rapid production of recombinant proteins. Here, we describe the generation of CHO cell pools using the piggyBac transposon system for mediating gene integration. The method describes the co-transfection of cells with the donor plasmid (coding for the gene of interest) and the helper plasmid (coding for the transposase) using polyethyleneimine (PEI). This is followed by a genetic selection for the generation of a cell pool. The resulting cell pool can be used to start a batch or fed-batch culture. Alternatively, it can be used for generation of clonal cell lines or generation of cell banks for future use.
Topics: Animals; CHO Cells; Cricetulus; DNA Transposable Elements; Transfection; Plasmids; Recombinant Proteins; Polyethyleneimine; Transposases; Genetic Vectors
PubMed: 38926277
DOI: 10.1007/978-1-0716-3878-1_9 -
Methods in Molecular Biology (Clifton,... 2024The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or...
The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or functional studies as well as for their characterization as biotherapeutics. While transient gene expression (TGE) in mammalian cells constitutes a rapid and well-established approach, non-clonal stably transfected cells, or "pools," represent another option, which is especially attractive when recurring productions of the same protein are required. From a culture volume of just a few liters, stable pools can provide hundreds of milligrams to gram quantities of high-quality secreted recombinant proteins.In this chapter, we describe a highly efficient and cost-effective procedure for the generation of Chinese Hamster Ovary cell stable pools expressing secreted recombinant proteins using commercially available serum-free media and polyethylenimine (PEI) as the transfection reagent. As a specific example of how this protocol can be applied, the production and downstream purification of recombinant His-tagged trimeric SARS-CoV-2 spike protein ectodomain (SmT1) are described.
Topics: CHO Cells; Cricetulus; Animals; Recombinant Proteins; Transfection; Polyethyleneimine; Spike Glycoprotein, Coronavirus; SARS-CoV-2; Cricetinae; Culture Media, Serum-Free
PubMed: 38926275
DOI: 10.1007/978-1-0716-3878-1_7 -
Methods in Molecular Biology (Clifton,... 2024Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital...
Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.
Topics: Humans; Dependovirus; HEK293 Cells; Genetic Vectors; Transfection; Plasmids; Polyethyleneimine; Bioreactors; Chromatography, Ion Exchange; Virion
PubMed: 38926272
DOI: 10.1007/978-1-0716-3878-1_4 -
Methods in Molecular Biology (Clifton,... 2024We describe a method for polyethyleneimine (PEI)-mediated transient transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells for protein expression...
We describe a method for polyethyleneimine (PEI)-mediated transient transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells for protein expression applicable at scales from 2 mL to 2 L. The method involves transfection at a high cell density (5 × 10 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31 °C with agitation by orbital shaking. This method requires 0.3 mg/L of coding pDNA, 2.7 mg/L of nonspecific (filler) DNA, and 15 mg/L of PEI. The production phase is performed at 31 °C in the presence of 0.25% N,N-dimethylacetamide (DMA). If desired, the method can be modified to avoid use of DMA by increasing the amount of coding DNA. We also provide information on culture vessel options, recommended working volumes, and recommended shaking speeds for transfections at scales from 2 mL to 2 L.
Topics: Animals; CHO Cells; Cricetulus; Polyethyleneimine; Transfection; Plasmids; Gene Expression; Cricetinae; DNA
PubMed: 38926269
DOI: 10.1007/978-1-0716-3878-1_1 -
Discovery Medicine Jun 2024Facilitating the healing process of skin post-trauma is crucial for minimizing infection risks and reinstating normal tissue functionality. While past studies have...
BACKGROUND
Facilitating the healing process of skin post-trauma is crucial for minimizing infection risks and reinstating normal tissue functionality. While past studies have established astaxanthin (ASX) as an effective compound in promoting wound healing, the precise mechanism of its action remains unclear. Consequently, the objective of this study was to explore the impact of ASX on the acute wound healing of rat skin by modulating macrophage polarization.
METHODS
Eighteen male SD rats were randomly assigned to control, dimethylsulfoxide (DMSO), and ASX groups. Acute skin wounds were induced in the rats, and the effects of different treatments on wound area and healing were assessed. Hematoxylin-eosin (H&E) staining was employed to detect histopathological changes in the skin, while Masson staining was utilized to observe collagen expression. Immunohistochemistry was conducted to identify clusters of differentiation (CD) 206 macrophages in the tissues. Furthermore, enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, IL-10, IL-4, and IL-13. The expression of inducible nitric oxide synthase (iNOS), arginase (Arg)-1, and mannose receptor C-type 1 (Mrc1) proteins in the injured skin of rats was assessed through Western blot analysis.
RESULTS
On postoperative days 7 and 14, the ASX treatment demonstrated notable reductions in inflammatory cell infiltration and inflammatory cytokine expression when compared to the Control and DMSO groups. This was accompanied by evident improvements in the pathological changes in skin tissue, characterized by the regeneration of new epidermis, dermal repair, and increased thickness of granulation, contributing to enhanced scar formation. Furthermore, ASX therapy exhibited an upregulation in the expression levels of collagen I and collagen III, along with markers indicative of M2 macrophages. These findings collectively signify the accelerated progression of wound healing attributed to ASX intervention.
CONCLUSIONS
In summary, these findings collectively indicate that ASX facilitates the healing of rat skin wounds by suppressing inflammatory responses and fostering M2 macrophage polarization. Consequently, ASX holds promise as a potentially effective drug for the treatment of skin wounds.
Topics: Animals; Wound Healing; Male; Macrophages; Rats; Rats, Sprague-Dawley; Xanthophylls; Collagen; Skin; Cytokines; Macrophage Activation
PubMed: 38926104
DOI: 10.24976/Discov.Med.202436185.108 -
PloS One 2024This study investigated the mitigating effects of spermidine on salinity-stressed yarrow plants (Achillea millefolium L.), an economically important medicinal crop....
This study investigated the mitigating effects of spermidine on salinity-stressed yarrow plants (Achillea millefolium L.), an economically important medicinal crop. Plants were treated with four salinity levels (0, 30, 60, 90 mM NaCl) and three spermidine concentrations (0, 1.5, 3 μM). Salinity induced electrolyte leakage in a dose-dependent manner, increasing from 22% at 30 mM to 56% at 90 mM NaCl without spermidine. However, 1.5 μM spermidine significantly reduced leakage across salinities by 1.35-11.2% relative to untreated stressed plants. Photosynthetic pigments (chlorophyll a, b, carotenoids) also exhibited salinity- and spermidine-modulated responses. While salinity decreased chlorophyll a, both spermidine concentrations increased chlorophyll b and carotenoids under most saline conditions. Salinity and spermidine synergistically elevated osmoprotectants proline and total carbohydrates, with 3 μM spermidine augmenting proline and carbohydrates up to 14.4% and 13.1% at 90 mM NaCl, respectively. Antioxidant enzymes CAT, POD and APX displayed complex regulation influenced by treatment factors. Moreover, salinity stress and spermidine also influenced the expression of linalool and pinene synthetase genes, with the highest expression levels observed under 90 mM salt stress and the application of 3 μM spermidine. The findings provide valuable insights into the responses of yarrow plants to salinity stress and highlight the potential of spermidine in mitigating the adverse effects of salinity stress.
Topics: Spermidine; Achillea; Salt Stress; Chlorophyll; Photosynthesis; Carotenoids; Proline; Gene Expression Regulation, Plant; Salinity; Antioxidants; Sodium Chloride; Chlorophyll A
PubMed: 38923971
DOI: 10.1371/journal.pone.0304831 -
Marine Drugs May 2024The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in...
The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in carotenoid biosynthesis, which has been extensively investigated in bacteria, land plants and microalgae. However, this step in macroalgae remains unknown. In the present study, a gene encoding putative phytoene synthase was cloned from the economic red alga -a species that has long been used in food and pharmaceuticals. The conservative motifs/domains and the tertiary structure predicted using bioinformatic tools suggested that the cloned should encode a phytoene synthase; this was empirically confirmed by pigment complementation in . This phytoene synthase was encoded by a single copy gene, whose expression was presumably regulated by many factors. The phylogenetic relationship of PSYs from different organisms suggested that red algae are probably the progeny of primary endosymbiosis and plastid donors of secondary endosymbiosis.
Topics: Rhodophyta; Phylogeny; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Carotenoids; Escherichia coli; Cloning, Molecular; Edible Seaweeds; Porphyra
PubMed: 38921568
DOI: 10.3390/md22060257