A 3D micro-computed tomography study comparing embryonic skeletal development in layer versus broiler strains of the domestic chicken.

Our objective was to analyze the effect of selection for divergent traits in the domestic chicken on embryonic skeletal development, which could affect postnatal bird welfare. Development was compared between the Ross 308 broiler line (fast growth and muscle mass accrual) and Novoponte layers (high laying rate and egg quality). In Study 1 (Initial Conditions), we characterized egg composition prior to incubation and identified the onset of embryonic skeletal mineralization in the 2 strains. In Study 2 (Developmental Dynamics), we used 3D X-ray tomographic imaging (µCT) on incubation days ED11, ED13, ED15 and ED17 to track skeletal maturation trajectories as a pseudo-time series. Results showed that Ross 308 embryos, which are heavier than Novoponte embryos, possess higher levels of yolk nutrients including phosphorus and calcium, but lower eggshell mineral content, than Novoponte embryos. Skeletal mineralization started synchronously in both strains, on ED11. The higher mineral ion content in the larger yolk of Ross 308 eggs compared to Novoponte eggs may partly explain why skeletal mineralization in Ross 308 embryos advances faster: using µCT, we show that the mandible and tibiotarsi in Ross 308 embryos are larger at ED11 and ED13 compared with Novoponte embryos. However, Novoponte embryos catch up from this initial lag in mineralization by ED15. The timing of the Novoponte acceleration coincides with the functional activation of the chorioallantoic membrane in releasing calcium from the inner eggshell. This correlates with a decrease in eggshell thickness from ED11 to ED17 in Novoponte eggs, which was not observed during Ross 308 incubation. To conclude, while some temporal discrepancies exist in early skeletal development between the embryos of Ross 308 and Novoponte strains, overall prenatal skeletal maturation seems to be robustly regulated. Despite selection for antagonist production traits, layer and broiler prehatch skeletal morphology ultimately synchronizes. Practically, since the extent of skeletal maturity equalizes between strains towards the end of incubation, refinements of farming practices, postnatal environment, and diet should be considered for improving domestic fowl welfare.

Critical appraisal of the chorioallantoic membrane model for studying angiogenesis in preclinical research.

BACKGROUND: Angiogenesis, the biological mechanism by which new blood vessels are generated from existing ones, plays a vital role in growth and development. Effective preclinical screening is necessary for the development of medications that may enhance or inhibit angiogenesis in the setting of different disorders. Traditional in vitro and, in vivo models of angiogenesis are laborious and time-consuming, necessitating advanced infrastructure for embryo culture. MAIN BODY: A challenge encountered by researchers studying angiogenesis is the lack of appropriate techniques to evaluate the impact of regulators on the angiogenic response. An ideal test should possess reliability, technical simplicity, easy quantifiability, and, most importantly, physiological relevance. The CAM model, leveraging the extraembryonic membrane of the chicken embryo, offers a unique combination of accessibility, low cost, and rapid development, making it an attractive option for angiogenesis assays. This review evaluates the strengths and limitations of the CAM model in the context of its anatomical and physiological properties, and its relevance to human pathophysiological conditions. Its abundant capillary network makes it a common choice for studying angiogenesis. The CAM assay serves as a substitute for animal models and offers a natural setting for developing blood vessels and the many elements involved in the intricate interaction with the host. Despite its advantages, the CAM model's limitations are notable. These include species-specific responses that may not always extrapolate to humans and the ethical considerations of using avian embryos. We discuss methodological adaptations that can mitigate some of these limitations and propose future directions to enhance the translational relevance of this model. This review underscores the CAM model's valuable role in angiogenesis research and aims to guide researchers in optimizing its use for more predictive and robust preclinical studies. CONCLUSION: The highly vascularized chorioallantoic membrane (CAM) of fertilized chicken eggs is a cost-effective and easily available method for screening angiogenesis, in comparison to other animal models.

A one-platform comparison study of brinzolamide-loaded liposomes, niosomes, transfersomes, and transniosomes for better management of glaucoma.

Ocular drug delivery presents significant challenges due to various anatomical and physiological barriers. Ultradeformable vesicles have emerged as better vesicular systems for achieving deeper corneal penetration and enhanced ocular bioavailability. This research aims to develop a hybrid vesicular system with improved deformability and compare it to conventional vesicular carriers. The ultradeformable vesicle, termed "transniosomes," is a combination of niosomes, liposomes, and transfersomes, loaded with brinzolamide as model drug. The brinzolamide-loaded transniosomes (BRZ-TN) was formulated and compared with different vesicular systems through in vitro, ex vivo, and in vivo characterizations. The optimized BRZ-TN demonstrated a vesicle size of 112.06 ± 4.13 nm and an entrapment efficiency of 93.63 ± 0.30 %. With a deformability index of 6.405, the BRZ-TN exhibited a permeability of 86.68 ± 2.51 % over 10 h, which is approximately 1.3 times higher than other conventional vesicular systems. Additionally, the BRZ-TN showed a drug flux of 0.247 ± 0.01 mg/cm2/h and an apparent permeability of 0.09 ± 1.21 cm/s. Pre-clinical experiments confirmed the superiority of the optimized BRZ-TN, achieving a 37 % reduction in intraocular pressure (IOP), post 6hr of administration, indicating its prolonged therapeutic effect and improved ocular bioavailability. The findings of this study suggest that transniosomes are superior to other carriers and hold great promise as a nanocarrier for ocular drug delivery.

Exposure to a sublethal dose of technical grade flubendiamide hampers angiogenesis in the chicken chorioallantoic membrane.

Pesticides are commonly employed to enhance agricultural productivity to meet the demands of the expanding global populace. Their harmful impact on non-target organisms is a severe cause of concern, and hence, new, presumably safer variants are developed. Flubendiamide is one such insecticide that targets caterpillars of insect pests. To evaluate its safety, we exposed early chicken embryos to technical-grade flubendiamide. Based on a dose range analysis, a lowest observed effect concentration (LOEC) of 25 µg/50µL (500 ppm) was selected for further experiments. LC-MS/MS analysis confirmed the presence of flubendiamide in the treated embryos. Gross morphology of embryos on days 2, 3 and 4 revealed reduced vascular area in the chorioallantoic membrane (CAM). The CAM vessel analysis showed reduced vascular networks in treated group. Hence, we hypothesized that flubendiamide, at LOEC, alters the expression patterns of the essential signaling molecules involved in angiogenesis, leading to compromised blood vessel development in CAM. An initial in silico study of flubendiamide was conducted with proteins involved in the CAM angiogenesis pathway. The docking scores revealed flubendiamide's direct influence on the functionality of angiogenic proteins. Hence, the expression patterns of key regulators of angiogenesis were studied on days 2, 3 and 4 at the transcript and protein levels. The results revealed a significant reduction in VEGFα, AKT, KDR, PCNA, PI3K, BMP2, BMP6, SHH and WNT7A expression in treated embryos, while expression of CASPASE-3 and RHOB were upregulated. Immunolocalization of Cl. Caspase-3 reaffirmed heightened apoptosis in the CAM of day 2 embryos. The study thus confirms that flubendiamide at a sublethal dose can hamper CAM angiogenesis and reduce the vascular networks in developing chick embryos by targeting the VEGF signaling cascade. This finding points to the teratogenic potential of flubendiamide and prompts throughput screening for safety. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-024-00254-z.

Sulfamoylated Estradiol Analogs Targeting the Actin and Microtubule Cytoskeletons Demonstrate Anti-Cancer Properties In Vitro and In Ovo.

The microtubule-disrupting agent 2-methoxyestradiol (2-ME) displays anti-tumor and anti-angiogenic properties, but its clinical development is halted due to poor pharmacokinetics. We therefore designed two 2-ME analogs in silico-an ESE-15-one and an ESE-16 one-with improved pharmacological properties. We investigated the effects of these compounds on the cytoskeleton in vitro, and their anti-angiogenic and anti-metastatic properties in ovo. Time-lapse fluorescent microscopy revealed that sub-lethal doses of the compounds disrupted microtubule dynamics. Phalloidin fluorescent staining of treated cervical (HeLa), metastatic breast (MDA-MB-231) cancer, and human umbilical vein endothelial cells (HUVECs) displayed thickened, stabilized actin stress fibers after 2 h, which rearranged into a peripheral radial pattern by 24 h. Cofilin phosphorylation and phosphorylated ezrin/radixin/moesin complexes appeared to regulate this actin response. These signaling pathways overlap with anti-angiogenic, extra-cellular communication and adhesion pathways. Sub-lethal concentrations of the compounds retarded both cellular migration and invasion. Anti-angiogenic and extra-cellular matrix signaling was evident with TIMP2 and P-VEGF receptor-2 upregulation. ESE-15-one and ESE-16 exhibited anti-tumor and anti-metastatic properties in vivo, using the chick chorioallantoic membrane assay. In conclusion, the sulfamoylated 2-ME analogs displayed promising anti-tumor, anti-metastatic, and anti-angiogenic properties. Future studies will assess the compounds for myeloproliferative effects, as seen in clinical applications of other drugs in this class.

Raman spectroscopy for classification of neoplastic and non-neoplastic CAM colon tumors.

This paper demonstrates the potential of Raman spectroscopy for differentiating neoplastic from non-neoplastic colon tumors, obtained with the CAM (chicken chorioallantoic membrane) model. For the CAM model two human cell lines were used to generate two types of tumors, the RKO cell line for neoplastic colon tumors and the NCM460 cell line for non-neoplastic colon tumors. The Raman spectra were acquired with a 785 nm excitation laser. The measured Raman spectra from the CAM samples (n = 14) were processed with several methods for baseline correction and to remove artifacts. The corrected spectra were analyzed with PCA (principal component analysis). Additionally, machine learning based algorithms were used to create a model capable of classifying neoplastic and non-neoplastic tumors. The principal component scores showed a clear differentiation between neoplastic and non-neoplastic colon tumors. The classification model had an accuracy of 93 %. Thus, a complete methodology to process and analyze Raman spectra was validated, using a rapid, accessible, and well-established tumor model that mimics the human tumor pathology with minor ethical concerns.

Xenografting Human Musculoskeletal Sarcomas in Mice, Chick Embryo, and Zebrafish: How to Boost Translational Research.

Musculoskeletal sarcomas pose major challenges to researchers and clinicians due to their rarity and heterogeneity. Xenografting human cells or tumor fragments in rodents is a mainstay for the generation of cancer models and for the preclinical trial of novel drugs. Lately, though, technical, intrinsic and ethical concerns together with stricter regulations have significantly curbed the employment of murine patient-derived xenografts (mPDX). In alternatives to murine PDXs, researchers have focused on embryonal systems such as chorioallantoic membrane (CAM) and zebrafish embryos. These systems are time- and cost-effective hosts for tumor fragments and near-patient cells. The CAM of the chick embryo represents a unique vascularized environment to host xenografts with high engraftment rates, allowing for ease of visualization and molecular detection of metastatic cells. Thanks to the transparency of the larvae, zebrafish allow for the tracking of tumor development and metastatization, enabling high-throughput drug screening. This review will focus on xenograft models of musculoskeletal sarcomas to highlight the intrinsic and technically distinctive features of the different hosts, and how they can be exploited to elucidate biological mechanisms beneath the different phases of the tumor's natural history and in drug development. Ultimately, the review suggests the combination of different models as an advantageous approach to boost basic and translational research.

MOF-Modified Microrollers for Bioimaging and Sustained Antibiotic Delivery.

Central nervous system (CNS) infections caused by neurosurgery or intrathecal injection of contaminated cerebrospinal fluid are a common and difficult complication. Drug-delivery microrobots are among the latest solutions proposed for antibacterial applications. However, there is a lack of research into developing microrobots with the ability to sustain antibody delivery while can move efficiently in the CNS. Here, biocompatible antibacterial metal-organic framework (MOF)-modified microrollers (MMRs) to combat CNS infections are proposed. The MMRs are iron-based metal-organic framework (NH2-MIL-101(Fe)) modified for enhanced adsorption and Fe/Al coated for magnetic actuation and biocompatibility. The MMRs have demonstrated a faster and unhindered magnetically actuated motion on the uneven biological tissue surface in an organ-on-a-chip that mimicked the CNS compared to it on smooth surface. CFD results consistently align with the experimental findings. The MMRs can be loaded with rhodamine 6G for bioimaging, allowing them to be imaged through sections of the main human tissues by fluorescence microscopy, or tetracycline hydrochloride for antibiotic delivery, allowing them to inhibit the growth of Staphylococcus aureus biofilms by sustained release of antibiotics for 9 days. This study provides a strategy to integrate high-capacity adsorption material with magnetically actuated locomotion for long-term targeted antibacterial applications in biological environments.

Bibliometric study of the application of the chicken embryo chorioallantoic membrane model in cancer research: the top 100 most cited articles.

The chicken embryo chorioallantoic membrane (CAM) model has played a crucial role in various aspects of cancer research. The purpose of this study is to help researchers clarify the research direction and prospects of the CAM model. A bibliometric analysis was conducted on the top 100 most cited articles on use of the CAM model in tumour research, retrieved from the Web of Science Core Collection database. Tools such as Bibliometrix, VOSviewer, CiteSpace and Excel were utilized for the visualization network analysis. The 100 articles analysed were mainly from the USA, China and European countries such as Germany and France. Tumour research involving CAM model experiments demonstrated reliability and scientific rigor (average citation count = 156.2). The analysis of keywords, topics and subject areas revealed that the applications of this model ranged from the biological characteristics of tumours to molecular mechanisms and signaling pathways, to recent developments in nanotechnology and clinical applications. Additionally, nude mouse experiments have been more frequently performed in recent years. We conclude that the CAM model is efficient, simple and cost-effective, and has irreplaceable value in various aspects of cancer research. In the future, the CAM model can further contribute to nanotechnology research.

The Chorioallantoic Membrane (CAM) Assay for the Analysis of Starvation-Induced Autophagy.

During avian development, the chorioallantoic membrane (CAM) is generated around 4 days after fertilization following the fusion of the allantois and the chorion. The CAM develops rapidly over the next several days and gets heavily vascularized and therefore has been explored widely as a tool for the study of angiogenesis. Additionally, being immunodeficient, the CAM can be used for tumor growth of human origin and its metastasis. Of note, the CAM assay is minimally invasive for the chicken embryo and lacks innervation, which gives this in vivo model a low ethical burden. Here, we describe the protocol for the generation of microtumors from human colorectal cancer cell lines on the CAM, incubated in a nutrient-deficient medium for the activation of autophagy. We show that pre-inoculation markers of autophagy induced through nutrient deficiency are retained in the microtumors generated on the CAM.

Photoangiolysis with the 445-nm Blue Laser and the Potassium-Titanyl-Phosphate Laser: A Comparison.

OBJECTIVES: Photoangiolytic lasers have yielded significant innovation in laryngeal surgery in the last 25 years. After the discontinuation of the potassium titanyl phosphate (KTP) laser, a novel 445-nm blue laser was developed. The optimal balance between a laser's desired tissue effects and collateral tissue damage is a major determinant of laser selection in microlaryngeal surgery. The shell-less incubation system for the chick chorioallantoic membrane (CAM) simulates the microvasculature of the human vocal fold and is useful for testing effects of laser settings and in simulated surgery. The aim of this study is to compare the tissue effects of the KTP and blue lasers using the shell-less CAM model. METHODS: The shell-less incubation system contains: polymethylpentene film (used as a culture vessel), calcium lactate and distilled water supplementations. By using this system, the chick chorioallantoic membrane (CAM) can be fully exposed with a good field for surgery simulation. The effects of the 2 lasers (532 nm KTP and 445 nm blue) were quantified at clinically relevant energy settings and laser distances from target. Measures included imaging real-time vascular reactions in the CAM model, post-procedure histologic analysis of CAM tissue and temperature changes. RESULTS: Vessel coagulation and rupture rates were less common with the blue laser compared with the KTP laser. Histologic analysis demonstrated less tissue disruption with the blue laser. Temperature changes were less with the blue laser. CONCLUSION: In this CAM model with specific conditions, the blue laser reveals less tissue damage than the KTP laser. Suitable working distance and power setting of the laser are necessary for desired tissue effects.Level of Evidence: Level 3.

Liver cancer in ovo models for preclinical testing.

Immunotherapies have significantly improved the prognosis of patients with advanced hepatocellular carcinoma (HCC), although more than 70% of patients still do not respond to this first-line treatment. Many new combination strategies are currently being explored, which drastically increases the need for preclinical models that would allow large-scale testing of new immunotherapies and their combinations. We developed several in ovo (in the egg) human liver cancer models, based on human tumor xenografts of different liver cancer cell lines on the chicken embryo's chorioallantoic membrane. We characterized the angiogenesis, as well as the collagen accumulation and tumor immune microenvironment, and tested atezolizumab (anti-PD-L1) plus bevacizumab (anti-VEGF) treatment. Our results show the involvement of chicken immune cells in tumor growth, reproducing a classical non-inflamed "cold" as well as inflamed "hot" tumor status, depending on the in ovo liver cancer model. The treatment by atezolizumab and bevacizumab was highly efficient in the "hot" tumor model PLC/PRF/5 in ovo with the reduction of tumor size by 76% (p ≤ .0001) compared with the control, whereas the efficacy was limited in the "cold" Hep3B in ovo tumor. The contribution of the anti-PD-L1 blockade to the anti-tumoral effect in the PLC/PRF/5 in ovo model was demonstrated by the efficacy of atezolizumab monotherapy (p = .0080, compared with the control). To conclude, our study provides a detailed characterization and rational arguments that could help to partially replace conventional laboratory animals with a more ethical model, suited to the current needs of preclinical research of new immunotherapies for liver cancer.

Electroporation with Calcium or Bleomycin: First Application in an In Vivo Uveal Melanoma Patient-Derived Xenograft Model.

Uveal melanoma (UM) represents a rare tumor of the uveal tract and is associated with a poor prognosis due to the high risk of metastasis. Despite advances in the treatment of UM, the mortality rate remains high, dictating an urgent need for novel therapeutic strategies. The current study introduces the first in vivo analysis of the therapeutic potential of calcium electroporation (CaEP) compared with electrochemotherapy (ECT) with bleomycin in a patient-derived xenograft (PDX) model based on the chorioallantoic membrane (CAM) assay. The experiments were conducted as monotherapy with either 5 or 10 mM calcium chloride or 1 or 2.5 µg/mL bleomycin in combination with EP or EP alone. CaEP and ECT induced a similar reduction in proliferative activity, neovascularization, and melanocytic expansion. A dose-dependent effect of CaEP triggered a significant induction of necrosis, whereas ECT application of 1 µg/mL bleomycin resulted in a significantly increased apoptotic response compared with untreated tumor grafts. Our results outline the prospective use of CaEP and ECT with bleomycin as an adjuvant treatment of UM, facilitating adequate local tumor control and potentially an improvement in metastatic and overall survival rates.

Antioxidant Extracts from Greek and Spanish Olive Leaves: Antimicrobial, Anticancer and Antiangiogenic Effects.

Olea europaea L. is the most valuable species of the Olea type, and its products offer a wide range of therapeutical uses. The olive tree has been extensively studied for its nourishing qualities, and the "Mediterranean diet", which includes virgin olive oil as a key dietary component, is strongly associated with a reduced risk of cardiovascular disease and various malignancies. Olive leaves, a by-product in the olive harvesting process, are valued as a resource for developing novel phytomedicines. For this purpose, two ethanolic extracts obtained from Olivae folium from Spain (OFS) and Greece (OFG) were investigated. Our findings contribute to a wider characterization of olive leaves. Both extracts displayed important amounts of phenolic compounds and pentacyclic triterpenes, OFG having higher concentrations of both polyphenols, such as oleuropein and lutein, as well as triterpenes, such as oleanolic acid and maslinic acid. The antioxidant capacity is similar for the two extracts, albeit slightly higher for OFG, possibly due to metal polyphenol complexes with antioxidant activity. The extracts elicited an antimicrobial effect at higher doses, especially against Gram-positive bacteria, such as Streptococcus pyogenes. The extract with lower inorganic content and higher content of polyphenols and triterpenic acids induced a strong anti-radical capacity, a selective cytotoxic effect, as well as antimigratory potential on A375 melanoma cells and antiangiogenic potential on the CAM. No irritability and a good tolerability were noted after evaluating the extracts on the in vivo Hen's Egg Test-Chorioallantoic Membrane (HET-CAM). Therefore, the present data are suggestive for the possible use of the two types of olive leaf products as high-antioxidant extracts, potentially impacting the healthcare system through their use as antimicrobial agents and as anticancer and anti-invasion treatments for melanoma.

The Chick Chorioallantoic Membrane as a Xenograft Model for the Quantitative Analysis of Uveal Melanoma Metastasis in Multiple Organs.

Uveal melanoma (UM) is the most common intraocular tumor in adults, and nearly 50% of patients develop metastatic disease with a high mortality rate. Therefore, the development of relevant preclinical in vivo models that accurately recapitulate the metastatic cascade is crucial. We exploited the chick embryo chorioallantoic membrane (CAM) xenograft model to quantify both experimental and spontaneous metastasis by qPCR analysis. Our study found that the transplanted UM cells spread predominantly and early in the liver, reflecting the primary site of metastasis in patients. Visible signs of pigmented metastasis were observed in the eyes, liver, and distal CAM. Lung metastases occurred rarely and brain metastases progressed more slowly. However, UM cell types of different origins and genetic profiles caused an individual spectrum of organ metastases. Metastasis to multiple organs, including the liver, was often associated with risk factors such as high proliferation rate, hyperpigmentation, and epithelioid cell type. The severity of liver metastasis was related to the hepatic metastatic origin and chromosome 8 abnormalities rather than monosomy 3 and BAP1 deficiency. The presented CAM xenograft model may prove useful to study the metastatic potential of patients or to test individualized therapeutic options for metastasis in different organs.

In Vivo Xenograft Model to Study Tumor Dormancy, Tumor Cell Dissemination and Metastasis.

Metastasis is a complex, multistep process. To study the molecular steps of the metastatic cascade, it is important to use an in vivo system that recapitulates the complex tumor microenvironment. The chicken embryo chorioallantoic membrane (CAM) is an in vivo system suitable for the implantation of xenograft tumor models. It allows the study of different aspects of the metastatic process, including the dormancy-awakening transition. The main advantages of this system are its high reproducibility, cost-effectiveness, and versatility. Here, by using two dormancy tumor models, one of head and neck squamous cell carcinoma and one of breast cancer, we described a detailed protocol for the use of the CAM model in metastasis assays and for the study of tumor growth and dormancy.

A Comparative Analysis of the Cytotoxic and Vascular Activity Effects of Western Diamondback Rattlesnake (Crotalus atrox) and Eastern Diamondback Rattlesnake (Crotalus adamanteus) Venoms Using a Chick Embryo Model.

Crotalus snakebites induce various toxicological effects, encompassing neurological, myotoxic, and cytotoxic symptoms, with potentially fatal outcomes. Investigating venom toxicity is essential for public health, and developing new tools allows for these effects to be studied more comprehensively. The research goals include the elucidation of the physiological consequences of venom exposure and the assessment of toxicity using animal models. Chicken embryos serve as valuable models for assessing venom toxicity through the chick embryotoxicity screening test (CHEST) and the chick chorioallantoic membrane (CAM) assay, particularly useful for evaluating vascular impacts. C. adamanteus venom application resulted in higher embryotoxicity and morphological abnormalities, such as Siamese twins. The CAM assay demonstrated the hemorrhagic effects of venom, varying with venom type and concentration. The irritant potential of both venom types was classified as slight or moderate depending on their concentration. Additionally, acetylcholinesterase (AChE) activity was performed to receive information about organ toxicity. The results show that both venoms induced changes in the whole embryo, heart, and liver weights, but the C. adamanteus venom was identified as more toxic. Specific venom concentrations affected AChE activity in embryonic tissues. These findings underscore the embryotoxic and vasoactive properties of Crotalus venoms, providing valuable insights into their mechanisms of toxicity and potential applications in biomedicine.

Phytochemical profiling, cytotoxic, anti-migration, and anti-angiogenic potential of phenolic-rich fraction from Peganum harmala: in vitro and in ovo studies.

Peganum harmala has been extensively employed in Algerian traditional medicine practices. This study aimed to explore the impact of n-butanol (n-BuOH) extract sourced from Peganum harmala seeds on cell proliferation, cell migration, and angiogenesis inhibition. Cytotoxic potential of n-BuOH extract was evaluated using MTT (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide) assay against human breast adenocarcinoma MCF-7 cells, cell migration was determined using scratch assay, and anti-angiogenic effect was evaluated through macroscopic and histological examinations conducted on chick embryo chorioallantoic membrane. Additionally, this research estimated the phytochemical profile of n-BuOH extract. Fifteen phenolic compounds were identified using Ultra-performance liquid chromatography UPLC-ESI-MS-MS analysis. In addition, the n-BuOH extract of P. harmala exhibited potent antioxidant and free radical scavenging properties. The n-BuOH extract showed potent cytotoxicity against MCF-7 cell with an IC50 value of 8.68 ± 1.58 µg/mL. Furthermore, n-BuOH extract significantly reduced migration. A strong anti-angiogenic activity was observed in the groups treated with n-BuOH extract in comparison to the negative control. Histological analysis confirmed the anti-angiogenic effect of the n-BuOH extract. This activity is probably a result of the synergistic effects produced by different polyphenolic classes.

Mitigation of oxidative stress and inflammatory factors, along with the antibrowning and antimicrobial effects of cassia seed microbial fermentation solution.

Introduction: Cassia seeds, originating from the mature seeds of leguminous cassia species, possess pharmacological effects attributed to their rich composition of various active ingredients, notably anthraquinones. While current research predominantly focuses on pharmaceutical extractions, there has been limited progress in fermentation studies. Methods: Our study aimed to enhance the content of active compounds such as anthraquinones, flavonoids, and polyphenols using microbial fermentation techniques. We specifically optimized a fermentation system through a single-factor experimental design. Results: The antioxidant properties of the fermentation solution were validated through assays involving HaCaT cells and zebrafish. We observed effective suppression of inflammatory reactions in both RAW264.7 cells and transgenic zebrafish by the fermentation solution. Moreover, significant inhibition of tyrosinase activity and melanin production was evident in B16-F10 cells and zebrafish. Positive outcomes were also obtained in antibacterial assays and chick embryo experiments. Discussion: These findings highlight the potential of cassia seed fermentation solution as a safe and eco-friendly material in food chemistry and biomedical sciences.

The chicken chorioallantoic membrane assay revisited - A face-lifted approach for new perspectives in placenta research.

The study of very early human placentation is largely limited due to ethical restrictions on the use of embryonic tissue and the fact that the placental anatomy of common laboratory animal models varies considerably from that of humans. In recent years several promising models, including trophoblast stem cell-derived organoids, have been developed that have also proven useful for the study of important trophoblast differentiation processes. However, the consideration of maternal blood flow in trophoblast invasion models currently appears to be limited to animal models. An almost forgotten model to study the invasive behavior of trophoblasts is to culture them in vitro on the chicken chorioallantoic membrane (CAM), showing an extraembryonic vascular network in its mesenchymal stroma that is continuously perfused by the chicken embryonic blood circulation. Here, we present an extension of the previously described ex ovo CAM assay and describe the use of cavity-bearing trophoblast spheroids obtained from the first trimester cell line ACH-3P. We demonstrate how spheroids penetrated the CAM and that erosion of CAM vessels by trophoblasts led to filling of the spheroid cavities with chicken blood, mimicking initial steps of intervillous space blood perfusion. Moreover, we prove that this model is useful for state-of-the-art techniques including immunofluorescence and in situ padlock probe hybridization, making it a versatile tool to study aspects of trophoblast invasion in presence of blood flow.