Chick Embryo Model Research Articles

Prenatal Maternal Stress Suppresses Embryonic Neurogenesis via Elevated Glucocorticoid Levels.

Although it is known that prenatal maternal stress (PNMS) has negative influence on nervous system development in offspring, there has been no solid evidence showing the effect of PNMS on early neurogenesis during development. In this study, we established a chick embryo model to investigate how PNMS affects early neurogenesis by mimicking an intrauterine environment with high dexamethasone (Dex) levels. The results showed that Dex-mimicked PNMS significantly suppressed the development of gastrula embryos and increased the risks of neural tube defects and cranial deformity. Using immunofluorescence staining and Western blots to determine the expression levels of pHIS3, PCNA/Sox2, we found that PNMS significantly inhibited the proliferation of neural progenitor cells, and the downregulation of TGFβ signaling pathway might be responsible for the inhibition. Furthermore, immunofluorescence staining and Western blots manifested that PNMS could suppress the differentiation of neural progenitor cells to neuronal lineages, but promote them to transform into neuroglial cells, which might be due to the restriction of expressions of key genes (BMP4, SHH, Wnt3a, Slug, and Msx1) related to neural differentiation. In summary, our data reveal that PNMS dramatically impacts the earliest stages of neural development, thereby greatly increasing the risk of physical and mental health problems in childhood or adulthood.

Relative fitness of wild-type and phage-resistant pyomelanogenic P. aeruginosa and effects of combinatorial therapy on resistant formation

Bacteriophages, the natural predators of bacteria, are incredibly potent candidates to counteract antimicrobial resistance (AMR). However, the rapid development of phage-resistant mutants challenges the potential of phage therapy. Understanding the mechanisms of bacterial adaptations to phage predation is crucial for phage-based prognostic applications. Phage cocktails and combinatorial therapy, using optimized dosage patterns of antibiotics, can negate the development of phage-resistant mutations and prolong therapeutic efficacy. In this study, we describe the characterization of a novel bacteriophage and the physiology of phage-resistant mutant developed during infection. M12PA is a P. aeruginosa-infecting bacteriophage with Myoviridae morphology. We observed that prolonged exposure of P. aeruginosa to M12PA resulted in the selection of phage-resistant mutants. Among the resistant mutants, pyomelanin-producing mutants, named PA-M, were developed at a frequency of 1 in 16. Compared to the wild-type, we show that PA-M mutant is severely defective in virulence properties, with altered motility, biofilm formation, growth rate, and antibiotic resistance profile. The PA-M mutant exhibited reduced pathogenesis in an allantoic-infected chick embryo model system compared to the wild-type. Finally, we provide evidence that combinatory therapy, combining M12PA with antibiotics or other phages, significantly delayed the emergence of resistant mutants. In conclusion, our study highlights the potential of combinatory phage therapy to delay the development of phage-resistant mutants and enhance the efficacy of phage-based treatments against P. aeruginosa.

A Novel’s Evidence of MSG-Induced Craniofacial Defects in Chick Embryo Models

Monosodium glutamate (MSG), commonly used as an additive food enhancer, has been reported to have teratogenic effects on the embryo during development. The present study aimed to investigate the effect of MSG-induced teratogenicity, focusing on craniofacial formation in the chick embryo model. One hundred eighty fertilized eggs were divided into control and MSG groups. The chemicals were administered, and the results were investigated in 3-day-old embryos (ED-3), ED-6, and ED-10. The morphology and histology of craniofacial structures were studied using a stereomicroscope and light microscopy. The neural crest cells (NCCs) were investigated by the immunostaining technique. The results showed that a dose of 3 mg of MSG induced anterior neuropore opening and eye malformation in ED-3, and this was clearly demonstrated in ED-6. MSG also caused craniofacial bone malformation and delayed calcification in ED-10. Moreover, MSG induced apoptosis of NCCs and reduced the proliferation of NCCs in the craniofacial structures. These findings are the 1st report to demonstrate the teratogenicity of high doses of MSG-induced craniofacial defects in chick embryos during organogenesis. HIGHLIGHTS Monosodium glutamate exposure causes craniofacial defects in chick embryos. The functions of the neural crest cells were abrogated due to monosodium glutamate-induced teratogenesis, which led to birth defects. The experimental protocols benefit teratogenicity studies, and the results can be applied to future studies to elucidate the causes of birth defects during organogenesis. GRAPHICAL ABSTRACT

In ovo toxico-pathological effects of medicinal plants used against coccidiosis on chicken embryos development and hatchability

TOXICO-pathological effects of herbal plants have always been a major concern, but little information is provided on the toxico-pathological effects of medicinal plants used against coccidiosis. This study aimed to assess the histopathological effects of Carica papaya seeds (CPS), Azadirachta indica leaves (AIL), and Sarcocephalus latifolius root (SLR) used as coccidiostat in traditional poultry farms with various doses using the chick embryo model. A total of 420 Sasso breeder eggs at ED4 of incubation were inoculated with the extract of these plants following a 3×4 factorial design with 3 plant extracts (CPS, AIL and SLR) and 4 inoculation doses (0, 25, 50 and 100 mg/kg egg-weight). From ED6 to ED19, the weights of the albumen and embryo were recorded, and the weights of ED19 embryo organs such as liver, heart, kidney, and lungs were measured. Additionally, histopathological lesions were examined. The results indicated that the presence of various phytoconstituents such as alkaloids, phenolics, flavonoids, tannins, saponins, coumarins, steroids, and triterpenes with statistically significant free-radical-scavenging ability differed among the plant extracts (p < 0.0001). Toxico-pathological examination revealed a dose-dependent slight toxicity (p < 0.0001) of the Azadirachta indica leaves extract compared to the other plants. Additionally, the relative organ weight showed kidney hypertrophy (p = 0.001) and liver hypertrophy (p = 0.0001), as well as dilation of hepatic and cardiac vessels. The conclusion drawn was that chicken embryos are more susceptible to in ovo inoculation with Azadirachta indica leaves compared to Carica papaya seeds and Sarcocephalus latifolius root.

A-143 The role of OX-40 in tumor microenvironment of a Cutaneous T-cell Lymphoma (CTCL) in vivo chick embryo model

A-143 The role of OX-40 in tumor microenvironment of a Cutaneous T-cell Lymphoma (CTCL) in vivo chick embryo model

Characterization and angiogenic potential of CD146+ endometrial stem cells

BackgroundThe human endometrium, lining the inner uterus, regenerates over 400 times uniquely during a woman’s reproductive life. Endometrial stem cells (eSCs) enrich the tissue, resulting in a dense vascular network, significant angiogenic potential, and effective regeneration power. Being of natural angiogenic properties and proven effective in the treatment of vascular disorders, eSCs can be considered safe, reliable, and superior to other post-natal stem cells. Cluster of Differentiation 146 (CD146) has emerged as a pivotal marker associated with pericytes and endothelial cells for promoting angiogenesis. Endometrial cells with high CD146 expression could proliferate and differentiate into multiple lineages. This study will explore the role of CD146 in eSCs, focusing on the potential to boost the angiogenic and regenerative functions of the cells. The novelty of this study lies in the investigation of CD146 on eSC function, which may open new possibilities for eSC-based therapy in regenerative medicine and vascular disorders.MethodsThe study involved obtaining endometrial biopsies from active reproducing women to isolate and cultivate eSCs. eSCs were assessed for growth factor secretion pattern, characterized for their mesenchymal properties. Finally, eSCs were tested for their angiogenic potential by angiogenic gene expression profile and in-ovo chick embryo model. As aimed, to check the role of CD146 in eSC angiogenesis, CD146+ cells were magnetically sorted and cultured. The sorted cells underwent various analyses, including flowcytometry to identify mesenchymal markers and human growth factor panel to analyze growth factor secretion profiles The study evaluated the angiogenic potential of CD146 + cells using functional assays, including ring formation, endothelial differentiation, and wound scratch assays, to evaluate cell migration and healing capabilities. Molecular insights were obtained through chemokine and cytokine investigations In-ovo Chick model assay was conducted to check the angiogenic potential and evaluated through macroscopic as well as through immunohistochemistry.ResultEndometrial stem cells (eSCs) were successfully isolated using a combination of mechanical and enzymatic digestion, followed by culturing in complete DMEM media. The secretion profile of eSCs revealed significant production of various angiogenic growth factors, including Granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Vascular endothelial growth factor (VEGF), Fibroblast growth factors (FGF), and Platelet derived growth factor AA (PDGF-AA). The angiogenic gene profile indicated upregulation of several angiogenic genes in eSCs. The mesenchymal nature of eSCs was demonstrated through surface marker analysis (Cluster of differentiation 73, Cluster of differentiation 90, Cluster of differentiation 105) and trilineage differentiation. The in-ovo chick model confirmed the angiogenic potential of eSCs. CD146+ cells, isolated via magnetic sorting, exhibited enhanced angiogenic potential. These cells secreted significant levels of angiogenic growth factors such as VEGF. In Matrigel assays, CD146+ cells formed endothelial ring structures more rapidly and persistently than unsorted eSCs. Semi-quantitative PCR confirmed their endothelial differentiation. CD146+ cells express various angiogenic chemokines such as CXCL5, CXCL8, CCL3, and CCL20 and cytokines such as GM-CSF, Interleukin-1β (IL-1β), Interleukin-6 (IL-6), PDGF AA/BB, Epidermal growth factor (EGF), Endothelin 1, Angiopoietin. In-ovo chick model assay showed that CD146+ cells had superior angiogenesis, with more nodes, junctions, and segments compared to eSCs and controls. Immunohistochemistry confirmed increased expression of endothelial markers (Cluster of differentiation 31, VEGF, Vascular associated protein (VAP), Von Willebrand factor (vWF) in CD146+ cells.ConclusionThe study highlights the angiogenic potential of endometrial stem cells, particularly the CD146+ cell population. These cells promote angiogenesis, secreting growth factors and forming stable blood vessel structures. CD146+ cells have higher expression levels of VEGF and TGF-α, key factors in angiogenesis. This suggests CD146+ eSCs may be promising for therapeutic applications in vascular diseases requiring angiogenesis. Further research is needed.

Antimicrobial Activity of Essential-Oil-Based Nanostructured Lipid Carriers against Campylobacter Spp. Isolated from Chicken Carcasses.

Campylobacter is a virulent Gram-negative bacterial genus mainly found in the intestines of poultry. The indiscriminate use of traditional antibiotics has led to drug resistance in these pathogens, necessitating the development of more efficient and less toxic therapies. Despite their complex biologically active structures, the clinical applications of essential oils (EOs) remain limited. Therefore, this study aimed to increase the bioavailability, stability, and biocompatibility and decrease the photodegradation and toxicity of EO using nanotechnology. The diffusion disk test revealed the potent anti-Campylobacter activity of cinnamon, lemongrass, clove, geranium, and oregano EOs (>50 mm). These were subsequently used to prepare nanostructured lipid carriers (NLCs). Formulations containing these EOs inhibited Campylobacter spp. growth at low concentrations (0.2 mg/mL). The particle size, polydispersity index, and zeta potential of these systems were monitored, confirming its physicochemical stability for 210 days at 25 °C. FTIR-ATR and DSC analyses confirmed excellent miscibility among the excipients, and FE-SEM elucidated a spherical shape with well-delimited contours of nanoparticles. The best NLCs were tested regarding nanotoxicity in a chicken embryo model. These results indicate that the NLC-based geranium EO is the most promising and safe system for the control and treatment of multidrug-resistant strains of Campylobacter spp.

Assessment of efficacy of chrysin in diabetes-associated cardiac complications in chick embryo and murine model.

Patients with type 2 diabetes or prolonged diabetic condition are webbed into cardiac complications. This study aimed to ascertain the utility of chick embryo as an alternative to the mammalian model for type 2 diabetes-induced cardiac complications and chrysin as a protective agent. Diabetes was activated in ovo model (chick embryo) using glucose along with β-hydroxybutyric acid. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Alamar, and Kenacid blue assay were used to compare with chrysin-administered group. Blood glucose level, total cholesterol, triglyceride, and high-density lipoprotein were considered as endpoints. Diabetes was induced in Wistar albino rats by administering a high-fat diet and a subdued dose of streptozotocin (35 mg/kg, b.w). Percentage of glycated hemoglobin, creatinine kinase-MB, tumor necrosis factor-α, and C-reactive protein were evaluated and compared with chrysin administered group. Chrysin treatment improved elevated blood glucose levels and dyslipidemia in a diabetic group of whole embryos. Condensed cellular growth and protein content as well as enhanced cytotoxicity in ovo were shielded by chrysin. Chrysin reduced cardiac and inflammatory markers in diabetic rats and provided cellular protection to damage the heart of diabetic rats. The protective action of chrysin in ovo model induced a secondary complication associated with diabetes, evidenced that the ovo model is an effective alternative in curtailing higher animal use in scientific research.

A new standardization for the use of chicken embryo: selection of target from the phage display library and infection

The filamentous bacteriophage M13KO7 (M13) is the most used in phage display (PD) technology and, like other phages, has been applied in several areas of medicine, agriculture, and in the food industry. One of the advantages is that they can modulate the immune response in the presence of pathogenic microorganisms, such as bacteria and viruses. This study evaluated the use of phage M13 in the chicken embryos model. We inoculated 13-day-old chicken embryos with Salmonella Pullorum (SP) and then evaluated survival for the presence of phage M13 or E. coli ER2738 (ECR) infected with M13. We found that the ECR bacterium inhibits SP multiplication in 0.32 (M13-infected ECR) or 0.44 log UFC/mL (M13-uninfected ECR) and that the ECR-free phage M13 from the PD library can be used in chicken embryo models. This work provides the use of the chicken embryo as a model to study systemic infection and can be employed as an analysis tool for various peptides that M13 can express from PD selection.Key points• SP-infected chicken embryo can be a helpful model of systemic infection for different tests.• Phage M13 does not lead to embryonic mortality or cause serious injury to embryos.• Phage M13 from the PD library can be used in chicken embryo model tests.

Epigenetic regulation by hypoxia, N-acetylcysteine and hydrogen sulphide of the fetal vasculature in growth restricted offspring: A study in humans and chicken embryos.

Fetal growth restriction (FGR) is a common outcome in human suboptimal gestation and is related to prenatal origins of cardiovascular dysfunction in offspring. Despite this, therapy of human translational potential has not been identified. Using human umbilical and placental vessels and the chicken embryo model, we combined cellular, molecular, and functional studies to determine whether N-acetylcysteine (NAC) and hydrogen sulphide (H2S) protect cardiovascular function in growth-restricted unborn offspring. In human umbilical and placental arteries from control or FGR pregnancy and in vessels from near-term chicken embryos incubated under normoxic or hypoxic conditions, we determined the expression of the H2S gene CTH (i.e. cystathionine γ-lyase) (via quantitative PCR), the production of H2S (enzymatic activity), the DNA methylation profile (pyrosequencing) and vasodilator reactivity (wire myography) in the presence and absence of NAC treatment. The data show that FGR and hypoxia increased CTH expression in the embryonic/fetal vasculature in both species. NAC treatment increased aortic CTH expression and H2S production and enhanced third-order femoral artery dilator responses to the H2S donor sodium hydrosulphide in chicken embryos. NAC treatment also restored impaired endothelial relaxation in human third-to-fourth order chorionic arteries from FGR pregnancies and in third-order femoral arteries from hypoxic chicken embryos. This NAC-induced protection against endothelial dysfunction in hypoxic chicken embryos was mediated via nitric oxide independent mechanisms. Both developmental hypoxia and NAC promoted vascular changes in CTH DNA and NOS3methylation patterns in chicken embryos. Combined, therefore, the data support that the effects of NAC and H2S offer a powerful mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy. KEY POINTS: Gestation complicated by chronic fetal hypoxia and fetal growth restriction (FGR) increases a prenatal origin of cardiovascular disease in offspring, increasing interest in antenatal therapy to prevent against a fetal origin of cardiovascular dysfunction. We investigated the effects between N-acetylcysteine (NAC) and hydrogen sulphide (H2S) in the vasculature in FGR human pregnancy and in chronically hypoxic chicken embryos. Combining cellular, molecular, epigenetic and functional studies, we show that the vascular expression and synthesis of H2S is enhanced in hypoxic and FGR unborn offspring in both species and this acts to protect their vasculature. Therefore, the NAC/H2S pathway offers a powerful therapeutic mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy.

Design, synthesis and biological evaluation of piperine derivatives as potent antitumor agents

A series of piperine derivatives were designed and successfully synthesized. The antitumor activities of these compounds against 293 T human normal cells, as well as MDA-MB-231 (breast) and Hela (cervical) cancer cell lines, were assessed through the MTT assay. Notably, compound H7 exhibited moderate activity, displaying reduced toxicity towards non-tumor 293 T cells while potently enhancing the antiproliferative effects in Hela and MDA-MB-231 cells. The IC50 values were determined to be 147.45 ± 6.05 μM, 11.86 ± 0.32 μM, and 10.50 ± 3.74 μM for the respective cell lines. In subsequent mechanistic investigations, compound H7 demonstrated a dose-dependent inhibition of clone formation, migration, and adhesion in Hela cells. At a concentration of 15 μM, its inhibitory effect on Hela cell function surpassed that of both piperine and 5-Fu. Furthermore, compound H7 exhibited promising antitumor activity in vivo, as evidenced by significant inhibition of tumor angiogenesis and reduction in tumor weight in a chicken embryo model. These findings provide a valuable scientific foundation for the development of novel and efficacious antitumor agents, particularly highlighting the potential of compound H7 as a therapeutic candidate for cervical cancer and breast cancer.

P-021 Development of recombinant Phospholipase C Zeta protein as a therapeutic intervention to overcome certain types of male infertility

Abstract Study question Developing recombinant Phospholipase C Zeta (PLCζ) protein using a bacterial expression system to be utilized for potential future replacement of Ca2+ ionophores during IVF treatments. Summary answer Our findings support the use of MBP-PLCζ recombinant protein as a therapeutic tool for treating certain types of male factor infertility associated with fertilization failure What is known already The long-term safety of calcium ionophores, which are exclusively used for treating cases of fertilization failure, is still questionable. PLCζ is an endogenous agent that may represent a better replacement for treating male infertility cases related to fertilization failure Study design, size, duration Maltose binding protein (MBP)-tagged PLCζ was produced by cloning wild-type PLCζ into a pET41MM vector and purified using affinity chromatography techniques. Circular dichroism (CD) was employed to evaluate the secondary structure and the thermal denaturation of PLCζ protein. Moreover, the PIP2 hydrolysis assay was used to test the enzymatic activity of PLCζ protein over time and under different storage conditions. Furthermore, the embryonic toxicity of the protein was tested using a chick embryo model. Participants/materials, setting, methods Several methods were used in this study including Molecular cloning, circular dichroism, and chick embryo model to investigate the embryonic viability. Main results and the role of chance Our findings demonstrated that PLCζ recombinant protein displayed a properly folded secondary structure mainly composed of alpha-helices. Moreover, the protein was effective in maintaining the enzymatic activity and Ca2+ sensitivity for 90 days when stored at -80oC. Moreover, exposing chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control group. Kaplan-Meier survival curve showed that the rate of survival did not differ between the treatment, control and sham groups until the endpoint of the study. Limitations, reasons for caution The toxicology assays used are not sufficient to confirm the safety of the protein. Further in vivo studies are required to investigate the effect of MBP-PLCζ protein on early embryonic development before being used as a replacement for calcium ionophores in cases of oocyte activation failure. Wider implications of the findings These findings can help develop a new therapeutic tool for treating cases of male infertility associated with oocyte activation failure. Trial registration number not applicable

Immobilized NRG1 Accelerates Neural Crest like Cell Differentiation Toward Functional Schwann Cells Through Sustained Erk1/2 Activation and YAP/TAZ Nuclear Translocation.

Neural Crest cells (NC) are a multipotent cell population that give rise to a multitude of cell types including Schwann cells (SC) in the peripheral nervous system (PNS). Immature SC interact with neuronal axons via the neuregulin 1 (NRG1) ligand present on the neuronal surface and ultimately form the myelin sheath. Multiple attempts to derive functional SC from pluripotent stem cells have met challenges with respect to expression of mature markers and axonal sorting. Here, they hypothesized that sustained signaling from immobilized NRG1 (iNRG1) might enhance the differentiation of NC derived from glabrous neonatal epidermis towards a SC phenotype. Using this strategy, NC derived SC expressed mature markers to similar levels as compared to explanted rat sciatic SC. Signaling studies revealed that sustained NRG1 signaling led to yes-associated protein 1 (YAP) activation and nuclear translocation. Furthermore, NC derived SC on iNRG1 exhibited mature SC function as they aligned with rat dorsal root ganglia (DRG) neurons in an in vitro coculture model; and most notably, aligned on neuronal axons upon implantation in a chick embryo model in vivo. Taken together their work demonstrated the importance of signaling dynamics in SC differentiation, aiming towards development of drug testing platforms for de-myelinating disorders.

Development of Recombinant PLC-Zeta Protein as a Therapeutic Intervention for the Clinical Treatment of Oocyte Activation Failure.

The sperm-specific phospholipase C zeta (PLCζ) protein is widely considered as the predominant physiological stimulus for initiating the Ca2+ release responsible for oocyte activation during mammalian fertilization. The increasing number of genetic and clinical reports that directly link PLCζ defects and/or deficiencies with oocyte activation failure (OAF) necessitates the use of a powerful therapeutic intervention to overcome such cases of male factor infertility. Currently, in vitro fertilization (IVF) clinics treat OAF cases after intracytoplasmic sperm injection (ICSI) with Ca2+ ionophores. Despite their successful use, such chemical agents are unable to trigger the physiological pattern of Ca2+ oscillations. Moreover, the safety of these ionophores is not yet fully established. We have previously demonstrated that recombinant PLCζ protein can be successfully used to rescue failed oocyte activation, resulting in efficient blastocyst formation. Herein, we produced a maltose binding protein (MBP)-tagged recombinant human PLCζ protein capable of inducing Ca2+ oscillations in mouse oocytes similar to those observed at fertilization. Circular dichroism (CD) experiments revealed a stable, well-folded protein with a high helical content. Moreover, the recombinant protein could retain its enzymatic properties for at least up to 90 days after storage at -80 °C. Finally, a chick embryo model was employed and revealed that exposure of fertilized chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control, giving a first indication of its safety. Our data support the potential use of the MBP-PLCζ recombinant protein as an effective therapeutic tool but further studies are required prior to its use in a clinical setting.

The biodistribution of polystyrene nanoparticles administered intravenously in the chicken embryo

Nanoplastics can cause severe malformations in chicken embryos. To improve our understanding of the toxicity of nanoplastics to embryos, we have studied their biodistribution in living chicken embryos. We injected the embryos in the vitelline vein at stages 18–19. We injected polystyrene nanoparticles (PS-NPs) tagged with europium- or fluorescence. Their biodistribution was tracked using inductively-coupled plasma mass spectrometry on tissue lysates, paraffin histology, and vibratome sections analysed by machine learning algorithms. PS-NPs were found at high levels in the heart, liver and kidneys. Furthermore, PS-NPs crossed the endocardium of the heart at sites of epithelial-mesenchymal transformation; they also crossed the liver endothelium. Finally, we detected PS-NPs in the allantoic fluid, consistent with their being excreted by the kidneys. Our study shows the power of the chicken embryo model for analysing the biodistribution of nanoplastics in embryos. Such experiments are difficult or impossible in mammalian embryos. These findings are a major advance in our understanding of the biodistribution and tissue-specific accumulation of PS-NPs in developing animals.

Atractylenolide-I Alleviates Hyperglycemia-Induced Heart Developmental Malformations through Direct and Indirect Modulation of the STAT3 Pathway

BackgroundGestational diabetes could elevate the risk of congenital heart defects (CHD) in infants, and effective preventive and therapeutic medications are currently lacking. Atractylenolide-I (AT-I) is the active ingredient of Atractylodes Macrocephala Koidz (known as Baizhu in China), which is a traditional pregnancy-supporting Chinese herb. PurposeIn this study, we investigated the protective effect of AT-I on the development of CHD in embryos exposed to high glucose (HG). Study design and MethodsFirst, systematic review search results revealed associations between gestational diabetes mellitus (GDM) and cardiovascular malformations. Subsequently, a second systematic review indicated that heart malformations were consistently associated with oxidative stress and cell apoptosis. We assessed the cytotoxic impacts of Atractylenolide compounds (AT-I, AT-II, and AT-III) on H9c2 cells and chick embryos, determining an optimal concentration of AT-I for further investigation. Second, immunofluorescence, western blot, Polymerase Chain Reaction (PCR), and flow cytometry were utilized to delve into the mechanisms through which AT-I mitigates oxidative stress and apoptosis in cardiac cells. Molecular docking was employed to investigate whether AT-I exerts cardioprotective effects via the STAT3 pathway. Then, we developed a streptozotocin-induced diabetes mellitus (PGDM) mouse model to evaluate AT-I's protective efficacy in mammals. Finally, we explored how AT-I protects hyperglycemia-induced abnormal fetal heart development through microbiota analysis and untargeted metabolomics analysis. ResultsThe study showed the protective effect of AT-I on embryonic development using a chick embryo model which rescued the increase in the reactive oxygen species (ROS) and decrease in cell survival induced by HG. We also provided evidence suggesting that AT-I might directly interact with STAT3, inhibiting its phosphorylation. Further, in the PGDM mouse model, we observed that AT-I not only partially alleviated PGDM-related blood glucose issues and complications but also mitigated hyperglycemia-induced abnormal fetal heart development in pregnant mice. This effect is hypothesized to be mediated through alterations in gut microbiota composition. We proposed that dysregulation in microbiota metabolism could influence the downstream STAT3 signaling pathway via EGFR, consequently impacting cardiac development and formation. ConclusionsThis study marks the first documented instance of AT-I's effectiveness in reducing the risk of early cardiac developmental anomalies in fetuses affected by gestational diabetes. AT-I achieves this by inhibiting the STAT3 pathway activated by ROS during gestational diabetes, significantly reducing the risk of fetal cardiac abnormalities. Notably, AT-I also indirectly safeguards normal fetal cardiac development by influencing the maternal gut microbiota and suppressing the EGFR/STAT3 pathway.

Effects of Resveratrol on In Vivo Ovarian Cancer Cells Implanted on the Chorioallantoic Membrane (CAM) of a Chicken Embryo Model.

Ovarian cancer poses a significant threat to patients in its advanced stages, often with limited treatment options available. In such cases, palliative management becomes the primary approach to maintaining a reasonable quality of life. Therefore, the administration of any medication that can benefit patients without a curative option holds potential. Resveratrol, a natural compound known for its in vitro anticancer activities, has generated contrasting results in vivo and human studies. In this study, we aimed to assess the anticancer effects of resveratrol on ovarian cancer cells grown on the chorioallantoic membrane (CAM) of chicken embryos. Two ovarian cancer cell lines, OVCAR-8 and SKOV-3, were cultured in collagen scaffolds for four days before being implanted on the CAM of chicken embryos on day 7. Different doses of resveratrol were applied to the CAM every two days for six days. Subsequently, CAM tissues were excised, fixed, and subjected to histological analysis. Some CAM tumours were extracted to analyse proteins through Western blotting. Our findings indicate that specific doses of resveratrol significantly reduce angiogenic activities, pNF-κB levels, and SLUG protein levels by using immunohistochemistry. These results suggest that resveratrol may have the potential to impact the behaviour of ovarian cancer CAM tumours, thereby warranting further consideration as a complementary treatment option for women with incurable ovarian cancer.

Pioneering a chick embryo model to explore the intrauterine etiology of developmental dysplasia of the hip in oligohydramnios conditions

ObjectiveTo explore the impact of oligohydramnios on fetal movement and hip development, given its association with Developmental Dysplasia of the Hip (DDH) but unclear mechanisms. MethodsChick embryos were divided into four groups based on the severity of oligohydramnios induced by amniotic fluid aspiration (control, 0.2 mL, 0.4 mL, 0.6 mL). Fetal movement was assessed by detection of movement and quantification of residual amniotic fluid volume. Hip joint development was assessed by gross anatomic analysis, micro-computed tomography (micro-CT) for cartilage assessment, and histologic observation at multiple time points. In addition, a subset of embryos from the 0.4 mL aspirated group underwent saline reinfusion and subsequent evaluation. ResultsIncreasing volumes of aspirated amniotic fluid resulted in worsening of fetal movement restrictions (e.g., 0.4mL aspirated and control group at E10: frequency difference -7.765 [95% CI: -9.125, -6.404]; amplitude difference -0.343 [95% CI: -0.588, -0.097]). The 0.4mL aspirated group had significantly smaller hip measurements compared to controls, with reduced acetabular length (-0.418 [95% CI: -0.575, -0.261]) and width (-0.304 [95% CI: -0.491, -0.117]) at day E14.5. Histological analysis revealed a smaller femoral head (1.084±0.264 cm) and shallower acetabulum (0.380±0.106 cm) in the 0.4mL group. Micro-CT showed cartilage matrix degeneration (13.6% [95% CI: 0.6%, 26.7%], P=0.043 on E14.5). Saline reinfusion resulted in significant improvements in the femoral head to greater trochanter (0.578 [95% CI: 0.323, 0.833], P=0.001). ConclusionsOligohydramnios can cause DDH by restricting fetal movement and disrupting hip morphogenesis in a time-dependent manner. Timely reversal of oligohydramnios during the fetal period may prevent DDH.

Unlocking the power of Libidibia ferrea extracts: antimicrobial, antioxidant, and protective properties for potential use in poultry production

Plant extracts are increasingly recognized as potential prophylactic agents in poultry production due to their diverse bioactive properties. This study investigated the phytochemical and biological properties of Libidibia ferrea (L. ferrea), a plant species native to the Caatinga region of northeastern Brazil. The aim of this study was to identify secondary metabolites and to demonstrate the antimicrobial, antioxidant and protective effects of the plant extract. Three extracts were produced: EHMV, a hydroalcoholic extract from the maceration of pods, and EEMC and EEMV ethanolic extracts from the maceration of peels and pods, respectively, from L. ferrea. High-performance liquid chromatography (HPLC-MS/MS) and atomic absorption spectroscopy (AAS) were used to characterize the metabolites and metals. The antimicrobial activity against Salmonella Galinarum (SG), Salmonella pullorum (SP), Salmonella Heidelberg (SH) and Avian pathogenic Escherichia coli (APEC) was evaluated alone and in combination with probiotic bacteria (Bacillus velenzensis) using agar diffusion and the bactericidal minimum concentration (CBM). The antioxidant potential of the extracts was evaluated in 5 in vitro assays and 6 assays in 3t3 cells. The toxicity of EHMV was tested, and its ability to combat SP infection was demonstrated using a chicken embryo model. The results showed that EHMV exhibited significant antimicrobial activity. The combination of EHMV with BV had synergistic effects, increased antimicrobial activity and induced bacterial sporulation. Composition analysis revealed the presence of 8 compounds, including tannins and phenolic compounds. In vitro antioxidant tests demonstrated that total antioxidant capacity(TAC) activity was increased, and the extract had strong reducing power and notable metal chelating effects. Analysis of 3T3 cells confirmed the protective effect of EHMV against oxidative stress. Toxicity assessments in chicken embryos confirmed the safety of EHMV and its protective effect against SP-induced mortality. EHMV from L. ferrea is rich in proteins and contains essential metabolites that contribute to its antimicrobial and antioxidant properties. When associated with probiotic bacteria such as B. velezensis, this extract increases the inhibition of SH, SG, SP, and APE. The nontoxic nature of EHMV and its protective effects on chicken embryos make it a potential supplement for poultry.

Repurposing the anti-angiogenic activity of homoeopathic medicines by employing Chick embryo model- An In-Ovo Study

Background: Homoeopathic medicines have the potential in treatment of patients suffering from cancers, Diabetic Retinopathy, and hemorrhagic disorders wherein anti- angiogenesis plays a vital role. Homoeopathic medicines have been used to treat haemorrhagic disorders. For example, Hamamelis is used in haemorrhages, varicose veins and in venous congestion. Thalaspi bursa is used in flabby condition of uterus, haemorrhagic conditions of uterine fibroid. This been said, their mechanism of action remains unknown. Therefore, this study was intended to establish angiogenic/anti-angiogenic effect of the selected homoeopathic medicines employing chick embryo model.&#x0D; Objective:&#x0D; &#x0D; To study the anti-angiogenic properties of selected homoeopathic medicines employing YSM assay in the chick embryo model.&#x0D; To test the toxicity of selected homoeopathic medicines through Chick embryo model.&#x0D; &#x0D; Materials and Method: Yolk Sac Membrane is widely utilized as an in vivo system to study angiogenesis and anti-angiogenesis. The images for all YSMs were captured and were analyzed for angiogenic/anti-angiogenic effect.&#x0D; Results: Significant anti-angiogenic effects were observed in 10% concentrations of Hamamelis200cH, Thalaspi bursa 200cH and Thalaspi bursa 1M. Reduction of vessel density and total vessel network length, total branching points, total nets and total segment symbolizes that these medicines have anti-angiogenic potential and so they help in treating menstrual disorders, which is followed with cramps and discharge of clots.&#x0D; Conclusion: The mechanism of action of selected homoeopathic medicines as anti- angiogenic agent is proved which can be used to combat various diseases like haemorrhages, varicose veins, in venous congestion and problems related to haemorrhagic disorders. This will further help to study the action of these Homoeopathic medicines as potential line of treatment in patients suffering from cancers, Diabetic Retinopathy, hemorrhagic disorders wherein anti- angiogenesis plays a vital role.