Concentrations Of Retinoic Acid Research Articles

Whole-transcriptome analysis reveals the effect of retinoic acid on small intestinal mucosal injury in cage-stressed young laying ducks

Retinoic acid (RA) is an active derivative of vitamin A and is involved in a variety of physiological processes, including cell growth, antioxidant, and inflammation. However, the role of RA in intestinal oxidative stress injury in caged-stressed laying ducks is unknown. In this study, we analyzed the effect and underlying mechanism of RA supplementation on intestinal damage in cage-stressed young laying ducks. One hundred and sixty laying ducks were divided into 5 treatment groups, including a control group (CR) and 4 treatment groups exposed to different RA concentrations (2,500, 5,000, 7,500 and 10,000 IU/kg, TG1 to TG4). The experimental period comprised a 7-d prefeeding period and a 10-d experimental feeding period, for a total of 17 d. Phenotypic analysis revealed that compared with the control group, RA addition increased the intestinal villus height and the villus-to-crypt ratio; decreased the crypt depth (P < 0.01); decreased the serum diamine oxidase and D-lactate concentrations (P < 0.05); increased the serum antioxidant capacity and intestinal antioxidant gene expression levels (P < 0.05); and increased the expression levels of tight junction-related genes, with the greatest effect observed in TG2 group. Our further whole-transcriptome analysis of duodenum tissues from CR and TG2 ducks revealed 706 differentially expressed mRNAs (DEmRNAs), 357 differentially expressed lncRNAs (DElncRNAs), 14 differentially expressed circRNAs (DEcircRNAs), and 4 differentially expressed miRNAs (DEmiRNAs). These DEGs are involved in calcium signaling, NOD-like receptor signaling, pyruvate metabolism, Jak-STAT signaling, Wnt signaling, riboflavin metabolism, and the adherens junction and tight junction pathways. The results of omics and marker gene expression analysis suggested that RA treatment may play a role in endoplasmic reticulum stress (ERS) and apoptosis. In conclusion, the addition of RA to the diet improved intestinal injury by improving the redox homeostasis of intestinal cells associated with ERS, enhancing the intestinal tight junction structure and alleviating the apoptosis of intestinal epithelial cells; moreover, 5,000 IU/kg RA was determined to be the most appropriate concentration for supplementation.

Lazy neutrophils - a lack of DGAT1 reduces the chemotactic activity of mouse neutrophils.

Neutrophils are key players in the innate immune system, actively migrating to sites of inflammation in the highly energetic process of chemotaxis. In this study, we focus on the role of acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1), an enzyme that catalyzes the synthesis of triglycerides, the major form of stored energy, in neutrophil chemotaxis. Using a mouse model of psoriasis, we show that DGAT1-deficiency reduces energy-demanding neutrophil infiltration to the site of inflammation, but this inhibition is not caused by decreased glycolysis and reduced ATP production by neutrophils lacking DGAT1. Flow cytometry and immunohistochemistry analysis demonstrate that DGAT1 also does not influence lipid accumulation in lipid droplets during inflammation. Interestingly, as has been shown previously, a lack of DGAT1 leads to an increase in the concentration of retinoic acid, and here, using real-time PCR and publicly-available next-generation RNA sequencing datasets, we show the upregulation of retinoic acid-responsive genes in Dgat1KO neutrophils. Furthermore, supplementation of WT neutrophils with exogenous retinoic acid mimics DGAT1-deficiency in the inhibition of neutrophil chemotaxis in in vitro transwell assay. These results suggest that impaired skin infiltration by neutrophils in Dgat1KO mice is a result of the inhibitory action of an increased concentration of retinoic acid, rather than impaired lipid metabolism in DGAT1-deficient mice.

Retinoic acid modulation guides human-induced pluripotent stem cell differentiation towards left or right ventricle-like cardiomyocytes

Background: Cardiomyocytes (CMs) induced from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and other non-cardiomyocytes. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart. Aim: Create engineered heart tissue (EHT) with left and right ventricular phenotype from hiPSCs by intervening with specific concentrations of RA during hiPSC differentiation towards CM. Methods: hiPSC were derived by reprogramming skin fibroblasts and erythroid progenitors. Different concentrations of RA (Control without RA, LowRA with 0.05 μM and HighRA with 0.1 μM) were administered during third to sixth days of the differentiation process. EHTs were generated by assembling CMs derived from hiPSC (hiPSC-CM) at high cell density in a low collagen hydrogel. EHTs were further matured and grown in a customized biomimetic tissue culture system, that provides continuous electrical stimulation, medium agitation and stretch. Finally, RNA extraction and tissue fixation were performed on CMs and EHTs for RT-qPCR and immunofluorescence staining analysis, while RNA sequencing was conducted on EHTs to examine how RA affects gene expression associated with function and structure of EHTs. Results: HighRA-hiPSC-CMs exhibited highest expression of maturity genes MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are transcription factors associated with left ventricular development, was also the highest in the HighRA-hiPSC-CM. The expression of TBX5 and NKX2.5 was also found to be highest in the HighRA-EHTs, while expression of right ventricular transcrition factors TBX20 and ISL1 was highest in control-EHTs. RNAseq of EHTs showed augmented extracellular matrix gene expression in HighRA-EHTs. The HighRA-EHTs were functionally most similar to left ventricle with the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline. Conclusion: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHT with a phenotype similar to that of the left ventricle or right ventricle. Our research paves the way for future studies on in vitro left ventricular or right ventricular function, personalized drug screening, and precision medicine. German Center for Cardiovascular Research (DZHK81Z0600207 to D.M. and P.S.) China Scholarship Council (CSC202108410141) and Henan Provincial Medical Science and Technology Research Project. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Systematic cryopreservation study of cardiac myoblasts in suspension.

H9c2 myoblasts are a cell line derived from embryonic rat heart tissue and demonstrate the ability to differentiate to cardiac myotubes upon reduction of the serum concentration (from 10% to 1%) and addition of all-trans retinoic acid in the growth medium. H9c2 cells are increasingly being used as an easy-to-culture proxy for some functions of cardiomyocytes. The cryobiology of cardiac cells including H9c2 myoblasts has not been studied as extensively as that of some cell types. Consequently, it is important to characterize the cryobiological response and systematically develop well-optimized cryopreservation protocols for H9c2 cells to have optimal and consistent viability and functionality after thaw for high quality studies with this cell type. In this work, an interrupted slow cooling protocol (graded freezing) was applied to characterize H9c2 response throughout the cooling profile. Important factors that affect the cell response were examined, and final protocols that provided the highest post-thaw viability are reported. One protocol uses the common cryoprotectant dimethyl sulfoxide combined with hydroxyethyl starch, which will be suitable for applications in which the presence of dimethyl sulfoxide is not an issue; and the other protocol uses glycerol as a substitute when there is a desire to avoid dimethyl sulfoxide. Both protocols achieved comparable post-thaw viabilities (higher than 80%) based on SYTO 13/GelRed flow cytometry results. H9c2 cells cryopreserved by either protocol showed ability to differentiate to cardiac myotubes comparable to fresh (unfrozen) H9c2 cells, and their differentiation to cardiac myotubes was confirmed with i) change in cell morphology, ii) expression of cardiac marker troponin I, and iii) increase in mitochondrial mass.

Abstract C031: Differential growth of pancreatic ductal adenocarcinoma cells in serum amyloid A (SAA) deficient mouse model through immune modulation

Abstract Objective: Pancreatic ductal adenocarcinoma (PDAC) is a formidable challenge to cure and expected to be the second leading cause of cancer-related deaths in the US by 2030. Its aggressive nature stems from late diagnosis and early metastasis. PDAC cells interact with the host system and impair the immune response. This communication leads to the production of mediators by host cells, creating an environment that supports PDAC cells growth and the formation of premetastatic niches. Despite progress, the specific factors and mechanisms underlying immune modulation in PDAC progression are not fully understood. Methods: PDAC mice models (WT-KPCL and KOSAA-KPCL) were developed by administering luciferase-expressing KPC cells (KPCL) intraperitoneally into eight weeks old male C57BL/6 mice. The control groups (WT and KOSAA) received PBS. Plasma proteomics was analyzed by using Mass Spectrometry (MS). PDAC cells growth was visualized by bioluminescence using Lago X in-vivo imaging system. Results: During tumor progression, KPCL mice exhibited significant weight loss, with the loss of adipose tissue and skeletal muscle, indicating a hallmark of pancreatic cancer. To identify potential circulating molecules involved in tumor progression, we conducted proteomics analysis on plasma samples using MS. Comparative analysis revealed that out of 734 circulating proteins, 86 were upregulated and 35 were downregulated in tumor mice compared to controls. Serum Amyloid A 1 and 2 were found to be the most abundantly expressed proteins in the plasma of tumor mice, with a remarkable 1024-fold increase compared to controls. To determine the major source of SAA in tumor mice, we measured SAA1, 2, and 3 expressions in various tissues, including adipose tissue, liver, muscle, and tumor. Interestingly, adipose tissue displayed a substantial contribution to SAA levels in WT-KPCL. However, we were unable to detect SAA expression in the tumor tissue from KOSAA-KPCL indicating negligible source of SAA from KPCL cells. Intrigued by these findings, we studied the role of SAA in PDAC cell proliferation in-vivo. Luciferase-expressing KPC cells were injected into SAA-KO mice (KOSAA-KPCL) and controls (WT-KPCL). Unexpectedly, bioluminescence imaging revealed a dramatic growth of KPC cells in the pancreas of SAA-deficient animals. This was supported by a significantly higher rate of metastasis to the liver, increased by 62.5% in KOSAA-KPCL compared to WT-KPCL. SAA is retinol-binding protein and retinoic acid plays a vital role in the immune system. We found a decreased concentration of retinoic acid in the plasma of KOSAA-KPCL mice, with a significant decrease in CD8 expression in tumor tissue isolated from KOSAA-KPCL. These results suggest that SAA modulates immune response during PDAC cells growth. Conclusion: Our study demonstrates that adipose tissue is likely a major source of SAA, where the absence of SAA leads to enhanced PDAC cells growth and metastasis through immune modulation. These findings suggest the therapeutic potential of SAA for pancreatic cancer. Citation Format: Yuki Takamuku, Mengistu Lemecha, Keiichi Itakura. Differential growth of pancreatic ductal adenocarcinoma cells in serum amyloid A (SAA) deficient mouse model through immune modulation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C031.

Dual transcriptomic and metabolomic analyses provide novel insights into the role of vitamins A and B metabolism in ectomycorrhizal symbiosis between Pinus yunnanensis and Lactarius deliciosus

AbstractEctomycorrhizal (EM) fungi play important roles in nutrient cycling and plant community establishment in forest ecosystems. Effects of EM formation on global alterations of the transcriptome and metabolome during plant‐fungal interaction and the key metabolites involved in EM development are largely unknown. Here, dual RNA‐Seq and untargeted metabolomic analyses were used to reveal stage‐specific and core responses of Pinus yunnanensis and Lactarius deliciosus during mycorrhizal colonization. We found that L. deliciosus colonization in P. yunnanensis roots induced different transcriptional changes across three interaction stages, with a small core of genes consistently regulated at all stages. Concentrations of retinol (vitamin A) and retinoic acid increased while that of B group vitamins decreased during EM formation, which was coordinately regulated by these two plant‐fungus partners, with L. deliciosus possibly playing a dominant role. Exogenous retinol altered the diameter and mantle thickness of P. yunnanensis ‐ L. deliciosus EM tips and affected host plant growth and phosphorus acquisition. In the absence of L. deliciosus, exogenous retinol increased the root diameter and the number of root tips of P. yunnanensis. Furthermore, the concentration of auxin increased, but that of abscisic acid decreased during EM formation, and the genes involved in plant hormone signal transduction were gradually activated, and auxin and cytokinin signal transduction potentially played a positive role in this EM symbiosis. In conclusion, we propose that the interaction of P. yunnanensis and L. deliciosus alters vitamin metabolism, which may further affect plant hormone biosynthesis and signal transduction, modulating root morphology and EM traits.

A critical role of retinoic acid concentration for the induction of a fully human-like atrial action potential phenotype in hiPSC-CM

Retinoic acid (RA) induces an atrial phenotype in human induced pluripotent stem cells (hiPSCs), but expression of atrium-selective currents such as the ultrarapid (IKur) and acetylcholine-stimulated K+ current is variable and less than in the adult human atrium. We suspected methodological issues and systematically investigated the concentration dependency of RA. RA treatment increased IKur concentration dependently from 1.1± 0.54pA/pF (0 RA) to 3.8± 1.1, 5.8± 2.5, and 12.2± 4.3 at 0.01, 0.1, and 1μM, respectively. Only 1μM RA induced enough IKur to fully reproduce human atrial action potential (AP) shape and a robust shortening of APs upon carbachol. We found that sterile filtration caused substantial loss of RA. We conclude that 1μM RA seems to be necessary and sufficient to induce a full atrial AP shape in hiPSC-CM in EHT format. RA concentrations are prone to methodological issues and may profoundly impact the success of atrial differentiation.

Optimisation of a Method for the Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Toward Renal Epithelial-like Cells.

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) can differentiate into multiple cell lineages, but few methods have been developed to generate kidney lineage cells. Due to their human origin, pluripotent nature and immunomodulatory properties, these stem cells are attractive candidates for clinical applications such as the repair or regeneration of damaged organs. This study evaluated the renal differentiation potential of hucMSCs, when exposed for 10 days to optimised concentrations of retinoic acid, activin-A and bone morphogenetic protein-7 (BMP-7) in various combinations, with and without the priming of the cells with a Wnt signalling pathway activator (CHIR99021). The hucMSCs were isolated and characterised according to surface marker expression (CD73, CD90, CD44, CD146 and CD8) and tri-lineage differentiation potential. The expression of key marker genes (OSR1, TBXT, HOXA13, SIX2, PAX2, KRT18 and ZO1) was examined by qRT-PCR. Specific marker protein expression (E-cadherin, cytokeratin-8 and cytokeratin-19) was analysed by immunocytochemistry. CHIR99021-primed cells treated with the retinoic acid, activin-A and BMP-7 cocktail showed epithelial cell-like differentiation - i.e. distinct phenotypic changes, as well as upregulated gene and protein expression, were observed that were consistent with an epithelial cell phenotype. Thus, our results showed that hucMSCs can efficiently differentiate into renal epithelial-like cells. This work may help in the development of focused therapeutic strategies, in which lineage-defined human stem cells can be used for renal regeneration.

Retinoic acid signaling pathway perturbation impacts mesodermal-tissue development in the zebrafish embryo: Biomarker candidate identification using transcriptomics.

The zebrafish embryo (ZE) model provides a developmental model well conserved throughout vertebrate embryogenesis, with relevance for early human embryo development. It was employed to search for gene expression biomarkers of compound-induced disruption of mesodermal development. We were particularly interested in the expression of genes related to the retinoic acid signaling pathway (RA-SP), as a major morphogenetic regulating mechanism. We exposed ZE to teratogenic concentrations of valproic acid (VPA) and all-trans retinoic acid (ATRA), using folic acid (FA) as a non-teratogenic control compound shortly after fertilization for 4 h, and performed gene expression analysis by RNA sequencing. We identified 248 genes specifically regulated by both teratogens but not by FA. Further analysis of this gene set revealed 54 GO-terms related to the development of mesodermal tissues, distributed along the paraxial, intermediate, and lateral plate sections of the mesoderm. Gene expression regulation was specific to tissues and was observed for somites, striated muscle, bone, kidney, circulatory system, and blood. Stitch analysis revealed 47 regulated genes related to the RA-SP, which were differentially expressed in the various mesodermal tissues. These genes provide potential molecular biomarkers of mesodermal tissue and organ (mal)formation in the early vertebrate embryo.

Zinc Treated Engineered Human Dendritic Cells Capable of Synthesizing High Active Vitamin D and Retinoic Acid Concentrations Enhance Gut-homing Regulatory T Cell Induction

Abstract Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the gastrointestinal tract (the Gut). Available IBD therapies can cause severe side effects, and many patients either fail to respond to the treatment or lose therapeutic effects with time. Hence, IBD therapy is still an unmet medical need. We recently proposed to enhance the induction of regulatory T cells (Tregs) expressing gut-homing molecules (gut-homing Tregs) to control chronic gut inflammation in IBD patients without compromising systemic immunity. We have shown that dendritic cells (DCs), engineered to de novo synthesize high concentrations of the active vitamin D (1,25-dihydroxy vitamin D or 1,25[OH]2D) and retinoic acid, increase the induction of gut-homing Tregs phenotypically and functionally in healthy mice and the mice induced for experimental colitis. This current study investigated whether the engineered DCs worked in humans. In addition, we also asked how zinc impacts the function of our engineered DCs because zinc is a biologically essential mineral widely included in daily supplements and has also been shown to tolerize DCs. Our data showed that the engineered DCs, derived from healthy control subjects and IBD patients, stimulated autologous naïve CD4+ T cells to express both regulatory and gut-homing molecules (gut-homing Tregs). In addition, the stimulated CD4+ T cells suppressed the proliferation of autologous effector T cells. Moreover, the addition of zinc enhanced the ability of the engineered human DCs to augment the induction of autologous gut-homing Tregs. Hence, our findings indicate zinc-treated engineered DCs are a promising novel IBD therapy. This study was partially supported by the AAI Careers in Immunology Fellowship; the US Army Medical Research and Materiel Command grant (W81XWH-15-0240); and the GI Foundation of Loma Linda University Innovation grant (RIG-GI 681207-2967).

Difenoconazole Exposure Induces Retinoic Acid Signaling Dysregulation and Testicular Injury in Mice Testes.

Difenoconazole (DFZ) is a broad-spectrum triazole fungicide that is widely utilized in agriculture. Although DFZ has been demonstrated to induce reproductive toxicity in aquatic species, its toxic effects on the mammalian reproductive system have yet to be fully elucidated. In vivo, male mice were administered 0, 20 or 40 mg/kg/d of DFZ via oral gavage for 35 days. Consequently, DFZ significantly decreased testicular organ coefficient, sperm count and testosterone levels, augmented sperm malformation rates, and elicited histopathological alterations in testes. TUNEL assay showed increased apoptosis in testis. Western blotting results suggested abnormally high expression of the sperm meiosis-associated proteins STRA8 and SCP3. The concentrations of retinoic acid (RA), retinaldehyde (RE), and retinol (ROL) were increased in the testicular tissues of DFZ-treated groups. The mRNA expression level of genes implicated in RA synthesis significantly increased while genes involved in RA catabolism significantly decreased. In vitro, DFZ reduced cell viability and increased RA, RE, and ROL levels in GC-2 cells. Transcriptome analysis revealed a significant enrichment of numerous terms associated with the RA pathway and apoptosis. The qPCR experiment verified the transcriptome results. In conclusion, our results indicate that DFZ exposure can disrupt RA signaling pathway homeostasis, and induce testicular injury in mice testes.

CRABP-I Expression Patterns in the Developing Chick Inner Ear.

The vertebrate inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions, regarded as an excellent system for analyzing events that occur during development, such as patterning, morphogenesis, and cell specification. Retinoic acid (RA) is involved in all these development processes. Cellular retinoic acid-binding proteins (CRABPs) bind RA with high affinity, buffering cellular free RA concentrations and consequently regulating the activation of precise specification programs mediated by particular regulatory genes. In the otic vesicle, strong CRABP-I expression was detected in the otic wall's dorsomedial aspect, where the endolymphatic apparatus develops, whereas this expression was lower in the ventrolateral aspect, where part of the auditory system forms. Thus, CRABP-I proteins may play a role in the specification of the dorsal-to-ventral and lateral-to-medial axe of the otic anlagen. Regarding the developing sensory patches, a process partly involving the subdivision of a ventromedial pro-sensory domain, the CRABP-I gene displayed different levels of expression in the presumptive territory of each sensory patch, which was maintained throughout development. CRABP-I was also relevant in the acoustic-vestibular ganglion and in the periotic mesenchyme. Therefore, CRABP-I could protect RA-sensitive cells in accordance with its dissimilar concentration in specific areas of the developing chick inner ear.

Altered Structure and Function of Murine Sclera in Form-Deprivation Myopia.

The sclera is believed to biomechanically influence eye size, facilitating the excessive axial elongation that occurs during myopigenesis. Here, we test the hypothesis that the sclera will be remodeled and exhibit altered biomechanics in the mouse model of form-deprivation (FD) myopia, accompanied by altered retinoid concentrations, a potential signaling molecule involved in the process. Male C57 Bl/6J mice were subjected to unilateral FD (n = 44 eyes), leaving the contralateral eye untreated (contra; n = 44). Refractive error and ocular biometry were measured in vivo prior to and after 1 or 3 weeks of FD. Ex vivo measurements were made of scleral biomechanical properties (unconfined compression: n = 24), scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 18, and immunohistochemistry: n = 22), and ocular all-trans retinoic acid (atRA) concentrations (retina and RPE+choroid+sclera, n = 24). Age-matched naïve controls were included for some outcomes (n = 32 eyes). Significant myopia developed after 1 (-2.4 ± 1.1 diopters [D], P < 0.001) and 3 weeks of FD (-4.1 ± 0.7 D, P=0.025; mean ± standard deviation). Scleral tensile stiffness and permeability were significantly altered during myopigenesis (stiffness = -31.4 ± 12.7%, P < 0.001, and permeability = 224.4 ± 205.5%, P < 0.001). Total scleral sGAG content was not measurably altered; however, immunohistochemistry indicated a sustained decrease in chondroitin-4-sulfate and a slower decline in dermatan sulfate. The atRA increased in the retinas of eyes form-deprived for 1 week. We report that biomechanics and GAG content of the mouse sclera are altered during myopigenesis. All scleral outcomes generally follow the trends found in other species and support a retina-to-sclera signaling cascade underlying mouse myopigenesis.

Retinoic acid released from self-assembling peptide activates cardiomyocyte proliferation and enhances repair of infarcted myocardium

The limited cardiomyocyte proliferation is insufficient for repair of the myocardium. Therefore, activating cardiomyocyte proliferation might be a reasonable option for myocardial regeneration. Here, we investigated effect of retinoic acid (RA) on inducing adult cardiomyocyte proliferation and assessed efficacy of self-assembling peptide (SAP)-released RA in activating regeneration of the infarcted myocardium. Effect of RA on inducing cardiomyocyte proliferation was examined with the isolated cardiomyocytes. Expression of the cell cycle-associated genes and paracrine factors in the infarcted myocardium was examined at one week after treatment with SAP-carried RA. Cardiomyocyte proliferation, myocardial regeneration and improvement of cardiac function were assessed at four weeks after treatment. In the adult rat myocardium, expression of RA synthetase gene Raldh2 and RA concentration were decreased significantly. After treatment with RA, the proliferated cardiomyocytes were increased. The formulated SAP could sustainedly release RA. After treatment with SAP-carried RA, expression of the pro-proliferative genes in cell cycle and paracrine factors in the infarcted myocardium were up-regulated. Myocardial regeneration was enhanced, and cardiac function was improved significantly. These results demonstrate that RA can induce adult cardiomyocytes to proliferate effectively. The sustained release of RA with SAP is a promise strategy to enhance repair of the infarcted myocardium.

Differentiation of human endometrial stem cells encapsulated in alginate hydrogel into oocyte-like cells.

Human endometrial mesenchymal stem cells (hEnMSCs) are a rich source of mesenchymal stem cells (MSCs) with multi-lineage differentiation potential, making them an intriguing tool in regenerative medicine, particularly for the treatment of reproductive and infertility issues. The specific process of germline cell-derived stem cell differentiation remains unknown, the aim is to study novel ways to achieve an effective differentiation method that produces adequate and functioning human gamete cells. We adjusted the optimum retinoic acid (RA) concentration for enhancement of germ cell-derived hEnSCs generation in 2D cell culture after 7 days in this study. Subsequently, we developed a suitable oocyte-like cell induction media including RA and bone morphogenetic protein 4 (BMP4), and studied their effects on oocyte-like cell differentiation in 2D and 3D cell culture media utilizing cells encapsulated in alginate hydrogel. Our results from microscopy analysis, real-time PCR, and immunofluorescence tests revealed that 10 µM RA concentration was the optimal dose for inducing germ-like cells after 7 days. We examined the alginate hydrogel structural characteristics and integrity by rheology analysis and SEM microscope. We also demonstrated encapsulated cell viability and adhesion in the manufactured hydrogel. We propose that in 3D cell cultures in alginate hydrogel, an induction medium containing 10 µM RA and 50 ng/mL BMP4 can enhance hEnSC differentiation into oocyte-like cells. The production of oocyte-like cells using 3D alginate hydrogel may be viable in vitro approach for replacing gonad tissues and cells.

The characteristics of neutrophil extracellular traps produced by all-trans retinoic acid-induced dHL-60 under PMA stimulation

Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.

All-Trans Retinoic Acid Effect on Candida albicans Growth and Biofilm Formation.

Candida albicans (C. albicans) is the most common fungal pathogen causing recurrent mucosal and life-threatening systemic infections. The ability to switch from yeast to hyphae and produce biofilm are the key virulence determinants of this fungus. In fact, Candida biofilms on medical devices represent the major risk factor for nosocomial bloodstream infections. Novel antifungal strategies are required given the severity of systemic candidiasis, especially in immunocompromised patients, and the lack of effective anti-biofilm treatments. Retinoids have gained attention recently due to their antifungal properties. Material and methods: The present study aimed at evaluating the in vitro effects of different concentrations (300 to 18.75 µg/mL) of All-trans Retinoic Acid (ATRA), a vitamin A metabolite, on Candida growth and biofilm formation. Results: ATRA completely inhibited the fungal growth, by acting as both fungicidal (at 300 µg/mL) and fungistatic (at 150 µg/mL) agent. Furthermore, ATRA was found to negatively affect Candida biofilm formation in terms of biomass, metabolic activity and morphology, in a dose-dependent manner, and intriguingly, its efficacy was as that of amphotericin B (AmB) (2–0.12 μg/mL). Additionally, transmission electron microscopy (TEM) analysis showed that at 300 μg/mL ATRA induced plasma membrane damage in Candida cells, confirming its direct toxic effect against the fungus. Conclusion: Altogether, the results suggest that ATRA has a potential for novel antifungal strategies aimed at preventing and controlling biofilm-associated Candida infections.

How the gut microbiota helps keep us vitaminized

Balanced intestinal retinoic acid concentrations are essential to maintain immune homeostasis in the gut. In this issue of Cell Host & Microbe, Bonakdar etal, sheds light on metabolic host-microbiota interactions and elegantly links diet, microbiota and host functions at the molecular level in the context of vitamin A metabolism.

In vitro and in vivo evaluation of porous alginate hydrogel containing retinoic acid for skin wound healing applications

The current study’s main aim was to fabricate and evaluate alginate (Alg) hydrogel containing retinoic acid (RA) as wound healing materials. Different RA concentrations (2, 10, and 50% w/w) were incorporated into the hydrogel. The results showed that the prepared hydrogels had a porous structure with a pore size of 69.69 ± 22.1 µm for pure Alg hydrogel and 78.44 ± 27.8 µm for Alg/RA hydrogel. The swelling measurement showed that the hydrogels swelled up to 65% and the incorporation of RA reduced the degree of swelling . The in vitro studies confirmed the hemo- and biocompatibility of the Alg/RA 2% and increasing the RA concentration induced hemolysis and toxic effects. The animal studies showed that the lowest RA concentration resulted in the best treatment outcome while increasing the RA concentration suppressed the healing process. In conclusion, these results showed that RA induced wound healing process at low concentration, and the prepared hydrogel could be used as the wound healing materials.

The synergistic effect of eucalyptus oil and retinoic acid on human esophagus cancer cell line SK-GT-4

BackgroundIn order to improve cancer patients' chances of survival, scientists have prioritized finding alternatives to chemotherapy, focusing their efforts on natural sources. The current study investigates the anti-cancer action of retinoic acid and Eucalyptus oil in esophageal cancer and studies their combined effect as well as the cellular pathways that each trigger as part of ongoing research in this field. As a model of esophageal cancer, the SK-GT-4 cancer cell line was treated with a series of concentrations of both materials.ResultsThe concentrations of Eucalyptus oil (10, 100, 1000, and 1500 g/mL) and Retinoic acid (5, 100, 150, and 200 M/mL) were used for treatment of cells. The MTT test was used to assess the anti-cancer activity of Eucalyptus oil and Retinoic acid, and qPCR was used to determine cellular pathways. Our findings show that both Eucalyptus oil and Retinoic acid inhibit cancer cell growth significantly. Our findings revealed that the IC50 values for eucalyptus oil were 63 g/mL and 111.3 M l/mL for retinoic acid. Furthermore, the impact was at the level that causes apoptosis. The findings suggested that any herbal substance could act as an inducer of the caspase-9-dependent pathway. The caspase-8-dependent pathway, on the other hand, was restricted to retinoic acid.ConclusionOur research discovered that the two chemicals worked together to create a synergistic effect. This synergistic effect could be attributed to a close connection between external and internal apoptotic pathways, which inhibits SK-GT-4 cell growth.Graphical