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Skeletal muscle formation involves tight interactions between muscle cells and associated connective tissue fibroblasts. Every muscle displays the same type of organisation, they are innervated in the middle and attached at both extremities to tendons. Myonuclei are heterogeneous along myotubes and regionalised according to these middle and tip domains. During development, as soon as myotubes are formed, myonuclei at muscle tips facing developing tendons display their own molecular program. In addition to molecular heterogeneity, a subset of tip myonuclei has a fibroblastic origin different to the classical somitic origin, highlighting a cellular heterogeneity of myonuclei in foetal myotubes. To gain insights on the functional relevance of myonucleus heterogeneity during limb development, we used 2D culture and co-culture systems to dissociate autonomous processes (occurring in 2D-cultures) from 3D-environment of tissue development. We also assessed the role of muscle contraction in myonucleus heterogeneity in paralysed limb muscles. The regionalisation of cellular heterogeneity was not observed in 2D cell culture systems and paralyzed muscles. The molecular signature of MTJ myonuclei was lost in a dish and paralysed muscles indicating a requirement of 3D-enviroment and muscle contraction for MTJ formation. Tip genes that maintain a regionalized expression at myotube tips in cultures are linked to sarcomeres. The behaviour of regionalized markers in cultured myotubes and paralyzed muscles allows us to speculate whether the genes intervene in myogenesis, myotube attachment or MTJ formation. Highlights: • The molecular signature of MTJ myonuclei is lost in cultured myotubes and paralysed muscles • Genes expressed in muscle tips that maintain their regionalised expression in cultured myotubes are linked to sarcomeric proteins • Cellular heterogeneity of myonuclei is observed in cultured myotubes but with no regionalisation • BMP signalling regulates fibroblast nucleus incorporation into cultured myotubes [ABSTRACT FROM AUTHOR]

Introduction: Safflower, a traditional Chinese medicine, is rich in chemical components including flavonoids, polysaccharides, and alkaloids. It exhibits pharmacological effects such as antioxidant, anti-inflammatory, anti-tumor, and anti-thrombosis properties, making it a valuable resource in the medical field. Furthermore, due to its antioxidant and anti-inflammatory effects, safflower is increasingly being utilized in the cosmetics industry. Methods: In this study, yeast was employed to ferment safflower, and the optimal fermentation conditions were established through single-factor experiments and response surface methodology. Subsequently, the antioxidant and anti-inflammatory efficacy of the safflower fermentation solution was assessed using both cellular and zebrafish models. Finally, the safety of the safflower fermentation solution was evaluated through a cosmetic eye irritation test. Results: From a total of 20 yeast strains, YF-5 was identified as the dominant strain for safflower fermentation. By optimizing the fermentation conditions, it was established that the optimal parameters for YF-5 fermentation of safflower are as follows: a fermentation temperature of 36.55°C, a material-to-liquid ratio of 1:20.46, a fructose concentration of 6.20%, a fermentation duration of 72 h, and an inoculum volume of 4%. The biological activities of safflower, including its antioxidant and anti-inflammatory properties, were enhanced through yeast fermentation. In HaCaT cell and zebrafish oxidative damage assays, safflower fermentation solution inhibits the production of malondialdehyde (MDA) and increases superoxide dismutase (SOD) activity as well as total antioxidant capacity (T-AOC). In the RAW264.7 cell inflammatory damage assays, a 20% safflower fermentation solution was found to inhibit the release of TNF-α and NO in the inflammatory model, with inhibition rates of 30.94 and 28.86%, respectively. In the zebrafish inflammatory damage assays, the quantity of fluorescent neutral proteins in the 5% safflower fermentation solution was 0.7 times that observed in the dexamethasone (0.1 mg/mL) positive control group, indicating that its anti-inflammatory activity is comparable to that of dexamethasone (0.1 mg/mL). In the chicken embryo chorionic membrane experiment, it was observed that the safflower fermentation solution did not cause significant damage to the blood vessels of the chorionic allantoic membrane (CAM). This finding demonstrates that the safflower fermentation solution possesses a certain degree of safety. Discussion: Safflower fermentation solution has antioxidant and anti-inflammatory bioactivities, and it has passed cosmetic safety evaluations. It can be used as a new natural cosmetic ingredient added to cosmetic products. [ABSTRACT FROM AUTHOR]

Genetic modification in vivo could provide direct functions of genes that could potentially contribute to diverse areas of research including genetics, developmental biology, and physiology. It has been reported that genes of interest could be introduced via recombinant adenovirus type 5 (Ad5) in poultry. Successful gene delivery to mammal fetuses in utero promises substantial progress in clinical and developmental biology, but it is limited because of difficulties in injecting specific sites and invasiveness. On the other hand, developing avian embryos are easily accessible by making a window on the eggshell. Therefore, the objective of this study is to determine permissive embryonic stages for gene transfer into specific avian tissue/organs by injection of Ad5 containing the green fluorescent protein (GFP) gene into blood vessels. At 2 d of post-injection, a strong GFP signal was predominantly identified in the heart of chicken embryos injected at Hamilton–Hamburger (HH) 14, 15, 16 and17 stages with the percentages (44%, 53%, 25%, and 14%, respectively) of GFP positive embryos. In quail embryos, the injection at the HH 15 resulted in heart-specific expression of GFP. Western blot analysis revealed that GFP was exclusively expressed in the avian hearts. These results suggest that the GFP gene is specifically delivered to the avian embryonic hearts when Ad5 is injected through the blood vessel at HH 14–17. This adenoviral transduction of genes of interest in avian embryonic hearts can provide new models for understanding functions of genetic factors on embryonic heart development and unravel genetic etiology of congenital heart diseases. [ABSTRACT FROM AUTHOR]

Background: Osteocalcin is a small protein abundant in the bone extracellular-matrix, that serves as a marker for mature osteoblasts. To become activated, osteocalcin undergoes a specific post-translational carboxylation. Osteocalcin is expressed at advanced stages of embryogenesis and after birth, when bone formation takes place. Neural crest cells (NCCs) are a unique cell population that evolves during early stages of development. While initially NCCs populate the dorsal neural-tube, later they undergo epithelial-to-mesenchymal-transition and migrate throughout the embryo in highly-regulated manner. NCCs give rise to multiple cell types including neurons and glia of the peripheral nervous system, chromaffin cells and skin melanocytes. Remarkably, in the head region, NCCs give rise to cartilage and bone. Finding: Here we report that osteocalcin is detected in cranial NCCs. Analysis of chick embryos at stages of cranial NCC migration revealed that osteocalcin mRNA and protein is expressed in pre-migratory and migratory NCCs in-vivo and ex-vivo. Addition of warfarin, an inhibitor of osteocalcin carboxylation, onto neural-tube explants, reduced the amount of NCC migration. These results provide the first evidence of osteocalcin presence in cranial NCCs, much before they give rise to craniofacial skeleton, and propose its possible involvement in the regulation of NCC migration. [ABSTRACT FROM AUTHOR]

The avian bursa of Fabricius (BF) is a primary lymphoid organ, where B-cell development occurs within bursal follicles of epithelial origin. During embryogenesis the epithelial anlage of the BF emerges as a diverticulum of the cloaca surrounded by undifferentiated tail bud mesenchyme. While it is believed that the epithelial-mesenchymal BF primordium provides a selective microenvironment for developing B cells, the initial events inducing lymphoid follicle formation are not fully elucidated. Using wild type and CSF1R- eGFP transgenic chick embryos, we find that separate B cell, macrophage and dendritic cell precursors enter the BF mesenchyme, migrate to the surface epithelium, and colonize the lymphoid follicle buds. Detailed immunocytochemical characterization revealed a novel EIV-E12+ blood-borne cell type, colonizing the surface epithelium of the BF rudiment before the entry of myeloid and lymphoid lineages and the appearance of this cell type coincides with the onset of follicle bud formation. Chick-duck chimeras and chick-quail tissue recombination experiments suggest that EIV-E12+ cells represent a transient lymphoid inducer cell population. They are not dendritic or B cells precursors, and they are capable of follicle bud induction in both dendritic cell- and B cell-depleted bursae. [ABSTRACT FROM AUTHOR]

Background: The utilization of titanium dioxide nanoparticles (TiO2 NPs) has significantly increased across various industries. Objectives: This study rigorously explored the impact of TiO2 NPs exposure on chicken embryos, focusing particularly on alterations in the immune system, liver functionality and key biochemical markers. Methods: The study involved three groups of 30 eggs each, subjected to increasing doses of TiO2 NPs: Group C (control), Group T1 (150 µg/mL) and Group T2 (300 µg/mL). After 48 h of incubation, the eggs in Groups T1 and T2 each received an injection of 0.3 mL of the TiO2 NPs solution. In contrast, the eggs in the control group (Group C) were injected with 0.3 mL of saline solution. Histopathological changes were analysed using haematoxylin and eosin (H&E) staining, whereas amniotic fluid's biochemical properties were examined photometrically. The study also assessed the expression of immune genes (AvBD9, IL6 and IL8L2) through quantitative PCR. The evaluations included growth metrics, amniotic fluid biochemistry and histological analysis of the liver, caecal tonsil and bursa of Fabricius. Results: The results revealed subcutaneous haemorrhage, significant reductions in total body weight and marked changes in biochemical markers, including urea, creatinine, alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), in the amniotic fluid of the groups treated with TiO2 NPs, compared to the control. Histological examinations indicated noticeable alterations in the liver, caecal tonsil and bursa of Fabricius following TiO2 NP exposure. These alterations were characterized by disruptions in cellular structures and variations in lymphocyte counts. Furthermore, a notable decrease in the expression of immunity genes, namely, AvBD9, IL8L2 and IL6, was observed in the TiO2 NP‐treated groups compared to the control. Conclusion: The findings underscore the need for risk assessments of TiO2 NPs exposure due to its impact on development and immunity. Future research should explore its impact on neurodevelopment and degeneration. [ABSTRACT FROM AUTHOR]

In ovo sexing identifies chicken embryo sex before or during incubation to avoid euthanising male chicks after hatching, enhancing animal welfare in the laying hen industry. Recently, researchers demonstrated the potential for non-invasive and early in ovo sexing through the analysis of volatile organic compounds (VOCs) emitted by eggs. However, a knowledge gap remains in understanding prediction model robustness, the efficacy of faster acquisition techniques, and day-to-day performance. In this study, two experiments were performed to fill these gaps. In Experiment 1, passive VOC extractions were performed on 110 eggs on incubation day 10 using sampling bags employing headspace sorptive extraction-gas chromatography-mass spectrometry (HSSE-GC-MS), proton transfer reaction-time-of-flight-mass spectrometry (PTR-TOF-MS), and selected ion flow tube-mass spectrometry (SIFT-MS). Prediction models were built using partial least squares-discriminant analysis (PLS-DA) and variable selection methods. As a result, prediction accuracies ranged from 57.6 % to 61.4 %, indicating no significant difference between the devices and highlighting the need for further optimisations. In Experiment 2, passive VOC samplings were performed on 42 eggs in glass jars during the initial 12 days of incubation using HSSE-GC-MS. Consequently, the optimised setup yielded higher accuracies ranging from 63.1 % (on day 0) to 71.4 % (on days 4, 6, and 12), revealing VOCs consistently elevated in relative abundance for a specific sex, and overall VOC abundance was higher in male embryos. Suggestions for future experiments to increase the accuracy of VOC in ovo sexing include active sampling with inert materials, expanding sample sets, and targeting consistent compounds. [Display omitted] • VOCs allow early, non-invasive in ovo sexing, yet with currently a low accuracy. • Up to 71.4 % accuracy was obtained within 12 days of incubation using HSSE-GC-MS. • Certain VOCs consistently had higher relative abundances for a specific sex. • Male embryos showed a significantly higher total VOC abundance. [ABSTRACT FROM AUTHOR]

Stress in birds disrupts the homeostasis of the organism, leading to an inability to neutralize reactive oxygen species. Taurine, an effective antioxidant, affects various cellular mechanisms, including cation modulation, protein phosphorylation, and cell proliferation. The aim of the study was to evaluate the effect of colloid with taurine applied in ovo to Albumin on embryonic development, oxidative stress indicators and the content of bioactive peptides—carnosine and anserine—in the pectoral muscle. The research materials were eggs of the parent flock (Ross 308) divided into four groups (K (without injection), T50-concentration of taurine hydrocolloid 50 ppm (mg/L); T100-colloid concentration 100 ppm (mg/L) taurine; T500-colloid concentration of 500 ppm (mg/L) taurine). The experimental solutions were injected in an amount of 0.3 mL into egg white. Eggs were incubated under standard incubation conditions. The addition of 100 and 500 ppm taurine had a highly significant (p = 0.001) effect on the plasma antioxidant potential in chicks. The level of anserine increased with increasing concentrations of taurine. These changes were highly significant (p = 0.007). The level of anserine in the T2 and T3 groups was determined to be 2.5 times higher than in the pectoral muscles of embryos not treated with taurine colloid. An analysis of the results showed that the administration of an increased dose of hydrocolloid with taurine increased the content of carnosine and anserine in the pectoral muscle. Colloid with taurine applied in ovo to chicken white egg reduces oxidative stress and increases homeostasis of the organism. [ABSTRACT FROM AUTHOR]

Birds, especially the chick and hen, have been important biomedical research models for centuries due to the accessibility of the avian embryo and the early discovery of avian viruses. Comprehension of avian tumor virology was a milestone in basic cancer research, as was that of non-viral genesis, as it enabled the discovery of oncogenes. Furthermore, studies on avian viruses provided initial insights into Kaposi's sarcoma and EBV-induced diseases. However, the role of birds in human carcinogenesis extends beyond the realm of virology research. Utilization of CAM, the chorioallantoic membrane, an easily accessible extraembryonic tissue with rich vasculature, has enabled studies on tumor-induced angiogenesis and metastasis and the efficient screening of potential anti-cancer compounds. Also, the chick embryo alone is an effective preclinical in vivo patient-derived xenograft model, which is important for the development of personalized therapies. Furthermore, adult birds may also closely resemble human oncogenesis, as evidenced by the laying hen, which is the only animal model of a spontaneous form of ovarian cancer. Avian models may create an interesting alternative compared with mammalian models, enabling the creation of a relatively cost-effective and easy-to-maintain platform to address key questions in cancer biology. [ABSTRACT FROM AUTHOR]

The trigeminal ganglion, the largest of the vertebrate cranial ganglia, is comprised of sensory neurons that relay sensations of pain, touch, and temperature to the brain. These neurons are derived from two embryonic cell types, the neural crest and ectodermal placodes, whose interactions are critical for proper ganglion formation. While the T-cell leukemia homeobox 3 (Tlx3) gene is known to be expressed in placodally-derived sensory neurons and necessary for their differentiation, little was known about Tlx3 expression and/or function in the neural crest-derived component of the developing trigeminal ganglion. By combining lineage labeling with in situ hybridization in the chick embryo, we show that neural crest-derived cells that contribute to the cranial trigeminal ganglion express Tlx3 at a time point that coincides with the onset of ganglion condensation. Importantly, loss of Tlx3 function in vivo diminishes the overall size and abundance of neurons within the trigeminal ganglion. Conversely, ectopic expression of Tlx3 in migrating cranial neural crest results in their premature neuronal differentiation. Taken together, our results demonstrate a critical role for Tlx3 in neural crest-derived cells during chick trigeminal gangliogenesis. [Display omitted] • Tlx3 is expressed in both neural crest and placode cells within the condensing chick trigeminal ganglion • Ectopic expression of Tlx3 delays neural crest migration and causes premature neuronal differentiation • Loss of Tlx3 disrupts trigeminal ganglion formation [ABSTRACT FROM AUTHOR]

The toxicity of methylmercury (MeHg) during embryonic development is a relevant issue that remains unclear and deserves investigation. In this sense, there is evidence that links the intake of contaminated food with cardiovascular pathologies in human adults and children. Thus, this study aimed to verify the impact of MeHg on the structure and integrity of extraembryonic and cardiac blood vessels and the contractile function of cardiomyocytes, also evaluating embryonic weight and the cardiosomatic index (CSI). Thus, chicken embryos, used as an experimental model, were exposed to a single dose of 0.1 μg MeHg/50 μl saline at E1.5 and analyzed at E10. After exposure, an increase in the number of extraembryonic blood vessels and the veins of the cardiac tissue was observed. These increases were accompanied by a reduction in the content of VEGF and VCAM proteins related to vessel growth and adhesiveness. Together, these results were related to reduced nitrite (NOx) levels. Furthermore, MeHg reduces the number of sarcomeres and increases the content of cardiac troponin I (cTnI), a protein that regulates contraction. In general, exposure to MeHg affected the integrity of extraembryonic and cardiac vessels and the contractile function of cardiomyocytes, which had a systemic impact evidenced by the reduction in embryonic weight gain and CSI. Ingestion of MeHg through contaminated food is associated with cardiovascular problems. This study assessed MeHg effects on extraembryonic and cardiac blood vessels, cardiomyocyte function, embryonic weight, and cardiosomatic index (CSI). Gallus domesticus embryos received a single dose 0.1 μg MeHg/50 μl saline at E1.5, analyzed at E10. MeHg exposure increased extraembryonic and cardiac vessel numbers, reduced VEGF and VCAM proteins, and lowered NOx levels. MeHg also decreased sarcomeres and increased cTnI, impacting cardiomyocyte function, embryonic weight, and CSI. [ABSTRACT FROM AUTHOR]

Background: Gliomas are the most common malignant brain tumors characterized by angiogenesis and invasive growth. A detailed understanding of its molecular characteristics could provide potential therapeutic targets. In the present study, we sought to explore the key gene CXCL8 in methionine metabolism in gliomas and its potential role in angiogenesis. Methods: U251 glioma cells were divided into control and methionine-restriction tolerant (constructed with 1/4 of the standard level of methionine in the culture medium) groups for transcriptome and metabolome analysis. To confirm the functions and mechanism of CXCL8 in glioma, heat map, volcano map, Go enrichment, gene set enrichment analysis (GSEA), protein–protein interaction network analysis, RT-PCR, western blotting assays, chicken embryo chorioallantoic membrane (CAM) test, chicken embryo yolk sac membrane (YSM) test and transplantation tumor nude mice model were performed. The TCGA database, CGGA database and clinical tissue samples were used to analyze CXCL8's significance on prognosis for patients with glioma. Results: CXCL8 expression was significantly up-regulated in methionine-restricted tolerance cells, it also activated vascular system development and triggered angiogenesis. CXCL8 expression is negatively correlated with survival prognosis in gliomas. Conclusions: Glioma cells promote angiogenesis in methionine-restricted environments through the activation of CXCL8, compensating for nutrient deprivation, and possibly contributing to the failure of antiangiogenic therapy. [ABSTRACT FROM AUTHOR]

Addressing a critical challenge in current tissue-engineering practices, this study aims to enhance vascularization in 3D porous scaffolds by incorporating bioceramics laden with pro-angiogenic ions. Specifically, freeze-dried gelatin-based scaffolds were infused with sol-gel-derived powders of Cu-doped akermanite (Ca 2 MgSi2O 7) and bredigite (Ca 7 MgSi 4 O 16) at various concentrations (10, 20, and 30 wt%). The scaffolds were initially characterized for their structural integrity, biodegradability, swelling behavior, impact on physiological pH, and cytocompatibility with human umbilical vein endothelial cells (HUVECs). The silicate incorporation effectiveness in promoting vascularity was then assessed through HUVEC attachment, capillary tube formation, and ex-ovo chick embryo chorioallantoic membrane assays. The findings revealed significant improvements in both in-vitro and ex-ovo vascularity of the gelatin scaffolds upon the addition of Cu-doped akermanite. The most effective concentrations were determined to be 10 and 20 %, which led to notable HUVEC metabolic activity, a well-spread morphology with extensive peripheral filopodia and lamellipodia at 10 % and a cobblestone phenotype indicative of in-vivo endothelium at 20 % during cell attachment, the formation of complex networks of tubular structures, and robust vascularization in chick embryo development. Moving forward, the incorporation of Cu-doped akermanite into tissue-engineering scaffolds shows great potential for addressing the limitations of vascularization, especially for critical-sized bone defects, by facilitating the controlled release of pro-angiogenic and pro-osteogenic ions. [ABSTRACT FROM AUTHOR]

This study was conducted to analyze the viability of primary chicken embryo fibroblasts and the efficiency of adipogenic trans-differentiation for cultured meat production. In isolating chicken embryo fibroblasts (CEFs) from a heterogeneous cell pool containing chicken satellite cells (CSCs), over 90% of CEFs expressed CD29 and vimentin. The analysis of the proliferative capabilities of CEFs revealed no significant differences in EdU-positive cells (%), cumulative cell number, doubling time, and growth rate from passage 1 to passage 9 (p > 0.05). This indicates that CEFs can be isolated by 2 h of pre-plating and survive stably up to passage 9, and that primary fibroblasts can serve as a valuable cell source for the cultured meat industry. Adipogenic trans-differentiation was induced up to passage 9 of CEFs. As passages increased, lipid accumulation and adipocyte size significantly decreased (p < 0.05). The reduced differentiation rate of primary CEFs with increasing passages poses a major challenge to the cost and efficiency of cultured meat production. Thus, effective cell management and the maintenance of cellular characteristics for a long time are crucial for ensuring stable and efficient cultured fat production in the cultured meat industry. [ABSTRACT FROM AUTHOR]

Meat goose can represent one of the most significant economic waterfowl in China. An excessively high stocking density can reduce the production performance and welfare of geese. Moreover, the light-emitting diode (LED) green light can play a pivotal role in regulating the growth and development of embryos and offspring in chickens. This study aims to determine the optimal regimen of LED green light in the high-density rearing mode of goslings. According to the actual production, a two-factor experiment was designed with the incubation light (dark and LED green) and growth light (LED white and LED green). A systematic analysis was made to explore the effects of LED green light on growth performance, intestinal development, blood hormones, feather quality, and pecking behaviors of geese in high-stocking density. The results show that the light intensities were 30–40 and 30–35 lx during the incubation and brooding period, respectively, with a light cycle of L:D = 24:0. The growth performance of geese was significantly improved by LED green light during the gosling brooding period. The LED green light during the growing period was found to significantly increase the body weight of geese at 14 days, as well as the daily gain and feed intake of geese for 1 to 14 days (P < 0.05). Furthermore, there was a significant decrease in the ratio of feed to gain of geese for 1 to 14 days (P < 0.05). A significant reduction was found in the concentration of serotonin and corticosterone in the blood, as well as the duodenal crypt depth of the geese at 14 days in the green light group during the growth period (P < 0.05), compared with the white light group. Furthermore, the LED green light irradiation during the growing period was observed to significantly increase the length of the chest and abdomen feathers of geese at 14 days post-hatching (P < 0.05), whereas, the back bare score of geese was reduced at 14 days post-hatching (P < 0.01), and the average pecking time and frequency of geese was reduced for 1 to 14 days post-hatching (P < 0.001). Thus, the green light environment during the growing period was significantly beneficial to the quality of the feathers and the development of geese plumage. The LED green light during the embryonic period was found to significantly reduce the average pecking time and frequency (P < 0.05) for better feather pecking behavior in geese. Moreover, the hatching procedure of green light outstandingly reduced the duodenal crypt depth (P < 0.01), where there was an increase in the jejunal villus height of geese at 14 days (P < 0.05), compared with the control group. The adverse effects of intensive farming were reduced on the intestinal development of geese. Moreover, there was no significant interaction to identify between the incubation and brood lights, in terms of the regulation of growth performance, intestinal development, blood hormones, feather quality (feather length, cleanliness, and damage degree), and feather pecking behavior of geese. Therefore, the LED green light shared the positive regulations on the negative effects of high-density rearing of goslings. Moreover, the LED green light during the growing period of goslings yielded superior outcomes. It is thus recommended that green light irradiation can be employed at the brood stage of goslings in actual production, in order to mitigate the adverse effects of modern intensive farming on goslings. The findings can provide a theoretical basis for the efficient production of geese. [ABSTRACT FROM AUTHOR]

Genetic modification in vivo could provide direct functions of genes that could potentially contribute to diverse areas of research including genetics, developmental biology, and physiology. It has been reported that genes of interest could be introduced via recombinant adenovirus type 5 (Ad5) in poultry. Successful gene delivery to mammal fetuses in utero promises substantial progress in clinical and developmental biology, but it is limited because of difficulties in injecting specific sites and invasiveness. On the other hand, developing avian embryos are easily accessible by making a window on the eggshell. Therefore, the objective of this study is to determine permissive embryonic stages for gene transfer into specific avian tissue/organs by injection of Ad5 containing the green fluorescent protein (GFP) gene into blood vessels. At 2 d of post-injection, a strong GFP signal was predominantly identified in the heart of chicken embryos injected at Hamilton-Hamburger (HH) 14, 15, 16 and17 stages with the percentages (44%, 53%, 25%, and 14%, respectively) of GFP positive embryos. In quail embryos, the injection at the HH 15 resulted in heart-specific expression of GFP. Western blot analysis revealed that GFP was exclusively expressed in the avian hearts. These results suggest that the GFP gene is specifically delivered to the avian embryonic hearts when Ad5 is injected through the blood vessel at HH 14-17. This adenoviral transduction of genes of interest in avian embryonic hearts can provide new models for understanding functions of genetic factors on embryonic heart development and unravel genetic etiology of congenital heart diseases. [ABSTRACT FROM AUTHOR]

Chicken embryos have many advantages in the study of amniote embryonic development. In particular, culture techniques developed for early‐stage embryos have promoted the advancement of modern developmental studies using chicken embryos. However, the standard technique involves placing chicken embryos in the ventral‐upward (ventral‐up) orientation, limiting manipulation of the epiblast at the dorsal surface, which is the primary source of ectodermal and mesodermal tissues. To circumvent this limitation, we developed chicken embryo cultures in the dorsal‐up orientation and exploited this technique to address diverse issues. In this article, we first review the history of chicken embryo culture techniques to evaluate the advantages and limitations of the current standard technique. Then, the dorsal‐up technique is discussed. These technological discussions are followed by three different examples of experimental analyses using dorsal‐up cultures to illustrate their advantages: (1) EdU labeling of epiblast cells to assess potential variation in the cell proliferation rate; (2) migration behaviors of N1 enhancer‐active epiblast cells revealed by tracking cells with focal fluorescent dye labeling in dorsal‐up embryo culture; and (3) neural crest development of mouse neural stem cells in chicken embryos. [ABSTRACT FROM AUTHOR]

This study presents an innovative method for producing thermosensitive bioink from chitosan hydrogels saturated with carbon dioxide and agarose. It focuses on a detailed characterisation of their physicochemical properties and potential applications in biomedicine and tissue engineering. The ORO test approved the rapid regeneration of the three-dimensional structure of chitosan–agarose composites in a unidirectional bench press simulation test. The diffusion of dyes through the chitosan–agarose hydrogel membranes strongly depended on the share of both polymers in the composite and the molecular weight of the dyes. Glucose, as a nutrient marker, also diffused through all membranes regardless of composition. Biocompatibility assessment using MTT tests on 46BR.1N fibroblasts and HaCaT keratinocytes confirmed the safety of the bioink. The regenerative potential of the bioink was confirmed by efficient cell migration, especially HaCaT. Long-term viability studies showed that chitosan–agarose scaffolds, unlike the agarose ones, support cell proliferation and survival, especially 14 days after bioink extrusion. Experiments in a skin wound model in mice confirmed the biocompatibility of the tested dressing and the beneficial action of chitosan on healing. Studies on vessel formation in chicken embryos highlight the potential of the chitosan–agarose composition to enhance proangiogenic effects. This composition meets all entry criteria and possesses excellent biological properties. [ABSTRACT FROM AUTHOR]

Agricultural areas can provide sources of food and hiding and nesting places for wild birds. Thus, the chemical load of potentially toxic elements (Cd, Cu, Pb) due to industrial and agricultural activities can affect not only the adult birds but also the embryos developing in the egg. The toxic effects of heavy metals applied alone were investigated on chicken embryos in the early and late stages of embryonic development using injection and immersion treatment methods. On day 3 of incubation, permanent preparations were made from the embryos to study the early development stage. There were no significant differences observed in embryo deaths and developmental abnormalities in this stage. On day 19 of incubation, the number of embryonic deaths, the body weight of the embryos, and the type of developmental abnormalities were examined. The embryonic mortality was statistically higher in the groups treated with cadmium and lead in the case of the injection treatment. A significant increase in developmental disorders was observed in the copper-treated group using the immersion application. The body weight significantly decreased in the cadmium- and lead-treated group using both treatment methods. However, a significant change in the body weight in the copper-treated group was only realized due to the injection method. [ABSTRACT FROM AUTHOR]

The BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS), a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits, ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes, little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A +/− induced pluripotent stem cells to model CNCC specification, we discovered that ARID1A -haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT), a process necessary for CNCC delamination and migration from the neural tube. Furthermore, wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level, these EMT enhancers contain binding motifs for ZIC2, and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers, ZIC2 relocates to neuronal enhancers, triggering aberrant neuronal gene activation. In mice, deletion of Zic2 impairs NCC delamination, while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination. [Display omitted] ARID1A modulates chromatin accessibility at enhancers of genes required for epithelial-to-mesenchymal transition, a process essential for cranial neural crest cell (CNCC) specification. Haploinsufficiency of ARID1A attenuates ZIC2 binding at these enhancers, resulting in impaired CNCC formation with an aberrant neuronal trajectory. This study reveals an ARID1A-ZIC2 axis essential for CNCC specification. [ABSTRACT FROM AUTHOR]

The optimal cellular reservoir for the conservation and propagation of endangered poultry is acknowledged to be primordial germ cells (PGCs). This study aimed to evaluate and compare the efficacy of cryopreserving PGCs derived from purified sources and blood samples. The experimental design encompassed two treatment groups, each iterated six replication. Blood samples that had been filled with 500 µL of phosphate-buffered saline without Ca2+ and Mg2+ (PBS) underwent centrifugation at 1200 rpm for six minutes. The supernatant was discarded and the pellet was mixed with 500 µL 10% FBS-PBS and was then subdivided for cryopreservation and PGCs purification. Cryopreservation included the controlled freezing of blood and purified PGCs samples in Nalgene cryogenic vials with the incorporation of banker cells. Following thawing, statistical analyses revealed no significant variance in the mean PGCs count between purified PGCs and blood samples (p > 0.05). The percentages of PGCs mortality and the recovery rate demonstrated statistical significance (p < 0.05). Further investigations indicated that cryopreserving blood samples significantly increased the recovery rate of PGCs while decreasing post-thaw mortality. Morphology of PGCs from blood cryopreservation showed similar morphology to PGCs in general. The potential application of cell-based cryobanking will contribute to the preservation of poultry genetic diversity for sustainability and adaptation to future poultry demands. [ABSTRACT FROM AUTHOR]

BACKGROUND RESULTS CONCLUSION Carnosine, a natural bioactive dipeptide derived from meat muscle, possesses strong antioxidant properties. Dexamethasone, widely employed for treating various inflammatory diseases, raises concerns regarding its detrimental effects on bone health. This study aimed to investigate the protective effects of carnosine against dexamethasone‐induced oxidative stress and bone impairment, along with its underlying mechanisms, utilizing chick embryos and a zebrafish model in vivo, as well as MC3T3‐E1 cells in vitro.Our findings revealed that carnosine effectively mitigated bone injury in dexamethasone‐exposed chick embryos, accompanied by reduced oxidative stress. Further investigation demonstrated that carnosine alleviated impaired osteoblastic differentiation in MC3T3‐E1 cells and zebrafish by suppressing the excessive production of reactive oxygen species (ROS) and enhancing the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPX). Moreover, mechanistic studies elucidated that carnosine promoted the expression and nuclear translocation of nuclear factor erythroid 2‐related factor 2 (NRF2), thereby facilitating the transcription of its downstream antioxidant response elements, including heme oxyense‐1 (HO‐1), glutamate cysteine ligase modifier (GCLM), and glutamate cysteine ligase catalytic (GCLC) to counteract dexamethasone‐induced oxidative stress.Overall, this study underscores the potential therapeutic efficacy of carnosine in mitigating oxidative stress and bone damage induced by dexamethasone exposure, shedding light on its underlying mechanism of action by activating the NRF2 signaling pathway. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: Fetal calf serum (FCS), an existing cell culture supplement, is effective but has several drawbacks, including being expensive, requiring a lengthy process of production, and requiring a hard currency. With this in mind, we planned to evaluate chick embryo extract and egg yolk extracts in cell culture as alternatives to fetal calf serum (FCS). Methods: Specific pathogen-free eggs were purchased from the National Veterinary Institute, Bishoftu, Ethiopia, and incubated in a humidified incubator at 37 °C for 11 days. Egg yolk extract (EYE) and chick embryo extract (CEE) were collected after the egg was opened with caution not to destroy the yolk sack or the chick embryo itself. Chick fibroblasts and Vero cells were cultured in minimum essential medium (MEM) supplemented with egg yolk extract or chick embryo extract at ratios of 0:10, 1:9, 2.5:7.5, and 5:5% fetal calf serum. Results: Fibroblast cell attachment was better in media supplemented with 5% CEE and 5% FCS. The confluency was also greater than 50% at this concentration. Vero cells cultured with 5% CEE and 5% FCS also exhibited very good cell attachment and a confluency of up to 70%. Viability and confluency were also observed at 5%:5% ratios of 50 and 70%, respectively. Conclusion: This investigation evaluated these two extracts as cell culture supplements and revealed promising results as alternatives to fetal calf serum. The limitation of this study is that it only used two cell types and additional cell lines, and different ratios should be tested. With the above findings, further research using different cell lines, ratios and conditions is warranted. [ABSTRACT FROM AUTHOR]

Assisted reproduction is a key aspect of modern animal breeding, providing valuable assistance in improving breeding programs. In this field, the administration of exogenous hormones, such as follicle-stimulating hormone (FSH), plays a crucial role in the induction of multiple ovulations. However, commercial FSH used in veterinary practice has been derived primarily from pituitary glands, obtained mostly from pigs for nearly four decades. Although these hormones have contributed significantly to the advancement of assisted reproductive techniques, they have certain limitations that warrant further improvements. These limitations include contamination with luteinizing hormone (LH), the potential risk of pathogen contamination, the potential to trigger an immune response in non-pig species, and the short half-life in circulation, requiring the implementation of complex 8-dose superovulation schedules. Our research team has developed and characterized a new variant of bovine follicle-stimulating hormone (bscrFSH) to address these limitations. The new hormone is produced recombinantly in CHO cell cultures, with a specific productivity of about 30 pg/cell/day. The bscrFSH can be purified to a high purity of 97 % using a single step of immobilized metal affinity chromatography (IMAC). N-glycan analysis of bscrFSH showed that approximately 74 % of the glycans corresponded to charged structures, including mono-, di-, tri-, and tetra-sialylated glycans. Superovulation trials conducted in cattle revealed that bscrFSH, administered at a total dose of about 0.5 μg per kg of body weight, using a decrescent schedule of 4 doses with 24-h intervals, resulted in an average yield of 8–12 transferable embryos per animal. Further research is required; however, the preliminary findings indicate that bscrFSH, currently packaged under the provisional brand name of Cebitropin B, holds potential as a commercial product for assisted reproduction in ruminants. • The bovine FSH variant (brscFSH) designed by the team is functional in inducing superovulation in cattle. • brscFSH can be produced at 30 pg/cell/day and purified to >95% purity using immobilized-metal affinity chromatography (IMAC). • brscFSH yields 8–12 transferable embryos on average at 0.5 μg/kg in a four-dose schedule over 12 hours. • Further studies are needed to assess brscFSH in two/single-dose schedules and in other species like sheep, goats, camelids, and mares. • brscFSH is now available for research use only on the Centro de Biotecnología y Biomedicina SpA website. [ABSTRACT FROM AUTHOR]

Due to the growing safety and environmental concerns associated with biocides, phenolic-soy branched chain fatty acids (phenolic-soy BCFAs) are synthesized as new bio-based antimicrobial agents. Safety evaluation is essential before the wide adoption of these new antimicrobial products. This study was initiated to evaluate the safety of four phenolic-soy BCFAs (with phenol, thymol, carvacrol, or creosote branches). Methyl-branched iso-oleic acid, phenol, and creosote were included in the study as controls. In silico toxicity simulation tools predicted that the phenolic BCFAs had much higher toxicities to aquatic organisms than free phenolics did, while the opposite was predicted for rats. The developmental toxicity of four phenolic-soy BCFAs was assessed using an in vivo chicken embryonic assay. Results showed that creosote-soy BCFA had much lower mortality rates than creosote at the same dosages. Additionally, creosotesoy BCFA and methyl-branched iso-oleic acid induced minimal estrogenic activity in the concentration range of 10 nM - 1 μM. Carvacrol-soy BCFA treatments significantly increased (p < 0.05) oxidative stress levels with higher thiobarbituric acid reactive substances in the livers of chicken embryos. Altogether, the phenolic-soy BCFAs, especially creosote-soy BCFA, reported in this study are potentially promising and safer bio-based antimicrobial products. [ABSTRACT FROM AUTHOR]

The pharyngeal arch (PA) is a transient structure of vertebrate embryos. Their number varies from species to species; nine in lampreys, seven in teleosts, and six in amniotes. Each pharyngeal arch contains a nerve and an artery as well as skeletomuscular elements. There has been a long-standing controversy about the formation of the fifth PA. Here, we investigated the formation of the fifth PA by tracking the distribution of neural crest cells in pharyngeal arches in chick embryos. Fertilized chick and quail eggs were used. Neural crest cells were detected by using quail-chick cell tracing technique and in ovo electroporation of EGFP constructs. Our results showed that neural crest cells from the post-otic level contributed to all post-otic pharyngeal arches (PA3-6). In contrast, neural crest cells from the somite 1-3 level avoided to invade the third pharyngeal arch, although they gave rise post-otic pharyngeal arches. Based on the trajectory of neural crest cells, the fifth pharyngeal arch was visible between the fourth and sixth pharyngeal arch, although it was considerably smaller compared to other arches. The fifth PA forms during early development at least in avian species. It is closer to the sixth PA than the fourth PA. This is in line with the formation of the fifth PAA, which can form a bypass and even fuse with the sixth PAA. [ABSTRACT FROM AUTHOR]

Objectives: 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. Methods: 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. Key findings: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Thyroid hormone (TH) is essential for growth and development, yet its specific role during embryogenesis remains incompletely understood. This study investigates the impact of TH deficiency, induced by thiourea, a known inhibitor of thyroid peroxidase (TPO), on the development of domestic chicks. Thiourea was administered before thyroid gland formation, and its presence in treated embryos was confirmed through liquid chromatography‐mass spectrometry. In silico docking revealed a strong interaction between thiourea and the CCP‐like domain of TPO, which was corroborated by TPO activity assays showing reduced enzyme function. This reduction in enzyme activity led to lower embryonic TH levels and increased thyroid‐stimulating hormone (TSH) secretion. Morphological analysis of newly hatched chicks revealed significant structural anomalies, particularly in lateral plate mesoderm‐derived structures, including omphalocele, limb deformities, anophthalmia and craniofacial defects. Alcian blue and Alizarin red staining demonstrated reduced ossification in ribs and forelimbs, while histological analysis showed incomplete abdominal wall closure and abnormal vertebral column development. Haematological profiling of TH‐deficient newly hatched chicks revealed significantly lower blood cell counts, highlighting TH's critical role in haematopoiesis. These findings emphasise the multifaceted role of TH in embryonic development, with potential implications for understanding congenital hypothyroidism and its developmental impacts, especially in regions with limited healthcare access. [ABSTRACT FROM AUTHOR]

Drug use during pregnancy is an important issue that must be investigated due to its adverse effects on maternal and foetal health. This study aimed to determine the embryotoxic and teratogenic effects of in‐ovo administered metamizole (dipyrone), which can be used when needed during pregnancy and has potent analgesic, antipyretic, anti‐inflammatory, and long bone (tibia and femur) effects. This study used 240 fertile eggs from Atak S breed chickens, divided into eight equal groups: control, vehicle control, and 15.62, 31.25, 62.5, 125, 250 and 500 mg/kg metamizole. The eggs were hatched on the 21st day of incubation, and the chicks' body weights and mortality rates were determined. The right and left femur and tibia bones were resected from the chicks. Anatomical reference points were determined after removing the soft tissues of the bones, and necessary morphometric measures were taken from these points with a 0.01 mm precision using digital callipers. The 100% lethal dose (LD100) was identified in the highest examined dose (500 mg/kg) in the Chicken Embryotoxicity Screening Test (CHEST)‐I stage. The CHEST‐II stage determined the 50% lethal dose (LD50). High‐dose metamizole affected skeletal development, significantly decreasing tibia and femur lengths and corpus thicknesses and increasing mortality. [ABSTRACT FROM AUTHOR]

For most quadrupeds, locomotion involves alternating movements of the fore- and hindlimbs. In birds, however, while walking generally involves alternating movements of the legs, to generate lift and thrust, the wings are moved synchronously with each other. Neural circuits in the spinal cord, referred to as central pattern generators (CPGs), are the source of the basic locomotor rhythms and patterns. Given the differences in the patterns of movement of the wings and legs, it is likely that the neuronal components and connectivity of the CPG that coordinates wing movements differ from those that coordinate leg movements. In this study, we used in vitro preparations of embryonic chicken spinal cords (E11–E14) to compare the neural responses of spinal CPGs that control and coordinate wing flapping with those that control alternating leg movements. We found that in response to N-methyl-d-aspartate (NMDA) or a combination of NMDA and serotonin (5-HT), the intact chicken spinal cord produced rhythmic outputs that were synchronous both bilaterally and between the wing and leg segments. Despite this, we found that this rhythmic output was disrupted by an antagonist of glycine receptors in the lumbosacral (legs), but not the brachial (wing) segments. Thus, our results provide evidence of differences between CPGs that control the wings and legs in the spinal cord of birds. [ABSTRACT FROM AUTHOR]

The chicken infectious anemia virus (CIAV) has been reported in major poultry-producing countries and poses a significant threat to the poultry industry worldwide. In this study, two Marek's disease virus (MDV) recombinants, rMDV-CIAV-1 and rMDV-CIAV-2, were generated by inserting the CIAV VP1 and VP2 genes into the MDV vaccine strain 814 at the US2 site using the fosmid-based rescue system. For rMDV-CIAV-1, an internal ribosome entry site was inserted between VP1 and VP2, so that both proteins were produced from a single open reading frame. In rMDV-CIAV-2, VP1 and VP2 were cloned into different open reading frames and inserted into the MDV genome. The recombinant viruses simultaneously expressed VP1 and VP2 in infected chicken embryo fibroblasts and exhibited growth kinetics similar to those of the parent MDV. The two recombinant viruses induced antibodies against CIAV in chickens. A single dose of the recombinant viruses provided strong protection against CIAV-induced anemia in chickens. These recombinant VP1- and VP2-expressing MDVs are potential vaccines against CIAV in chickens. [ABSTRACT FROM AUTHOR]

Objective: The methodology employed in this research was designed to identify and characterize the infectious bursal disease virus (IBDV) at the molecular level, originating from recent outbreaks in Bangladesh. Materials and Methods: The IBDV outbreak farm was investigated, and bursa of fabricius (BF) specimens were acquired from infected chickens. Initially, viruses in the processed samples were detected in chicken embryo fibroblast (CEF) cells, and the RT-PCR method was used to confirm IBDV. The positive samples were injected through chorioallantoic membrane route into the embryo of a 10-day-old specific pathogen-free (SPF) egg for virus isolation and pathogenicity testing. Finally, we sequenced the VP2 gene to identify phylogenetic relationships and detect mutations. Results: From the 77 collected samples, 42.85% (33/77) were found positive for cytopathic effects in CEF cells, and IBDV was detected in 31.16% (24/77) of the samples by RT-PCR. IBDV was isolated in SPF chicken embryos. In the pathogenicity test, infectious bursal disease was evident in seronegative chickens with visible signs of disease. Sequence analysis shows that the broiler-isolated viruses clustered with genotype A3B2 and backyard chickens with genotype A1B1. The presence of amino acid motifs for virulence markers was revealed in the partially sequenced VP2 gene with a mutation at S254G in four IBDV isolates from broilers. However, amino acids for virulence markers were absent in two isolates from backyard chickens, which shows sequence homology with IBDV classic strains. Conclusion: In this study, we identified and characterized circulating reassorted IBDV from vaccinated broilers, which may be one of the major causes of vaccination failure in broilers. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The functioning of the cardiovascular system is critical for embryo survival. Cardiac contractions depend on the sequential activation of different classes of voltage-gated ion channels. Understanding the fundamental features of these interactions is important for identifying the mechanisms of pathologies development in the myocardium. However, at present there is no consensus on which ion channels are involved in the formation of automaticity in the early embryonic stages. The aim of this study was to elucidate the expression of genes encoding various types of ion channels that are involved in the generation of electrical activity chicken heart at different stages of ontogenesis. We analyzed the expression of 14 genes from different families of ion channels. It was revealed that the expression profiles of ion channel genes change depending on the stages of ontogenesis. The HCN4 , CACNA1D , SCN1A , SCN5A , KCNA1 genes have maximum expression at the tubular heart stage. In adult, a switch occurs to the higher expression of CACNA1C , KCNH6 , RYR and SLC8A1 genes. This data correlated with the results obtained by the microelectrode method. It can be assumed that the automaticity of the tubular heart is mainly due to the mechanism of the «membrane–clock» (hyperpolarization-activated current (I f), Ca2+–current L–type (I CaL), Na+–current (I Na) and the slow component of the delayed rectifier K+–current (I Ks)). Whereas in adult birds, the mechanism for generating electrical impulses is determined by both « membrane– clock» and «Ca2+–clock». [Display omitted] • At different stages of ontogenesis, the electrophysiology of the developing heart was studied. • These data coincide with changes in gene expression of voltage-gated ion channels. • The HCN4, CACNA1D, SCN1A, SCN5A, KCNA1 genes have maximum expression at the tubular heart. • In adult birds, a switch occurs to the expression of CACNA1C, KCNH6, RYR, SLC8A1 genes. • Thus, the embryonic heart automatism is due to M-clock, in adults – M-clock and Ca-clock. [ABSTRACT FROM AUTHOR]

MS‐275, a histone deacetylase inhibitor, has proven anticancer activities against various malignancies. However, its clinical application has been constrained by dose‐limiting toxicity, off‐target effects, and variable clinical outcomes. Clinical data suggest that sustained low MS‐275 doses could achieve a more selective and consistent effect. This study aimed at enhancing the anticancer activity of MS‐275 by encapsulating it in D‐α‐tocopheryl polyethylene glycol 1000 succinate (TPGS) micelles. The produced nanoformulations were characterized by their low polydispersity (0.201), negative zeta potential (−0.397 mV), and high entrapment efficiency (98.8%). Experimental evaluation of the formulation showed a significant reduction in HepG2, HCT116, and MCF7 cells' viability, associated with enhanced apoptosis at a lower IC50 compared to MS‐275 alone. The formulation was further examined on cancer cells xenografted on the chorioallantoic membrane (CAM) of chick embryos. The results showed a substantial reduction in tumor size. TPGS micelles alone induced an accumulation in G1 and slightly reduced the cellular viability of the examined cell lines. Our results suggest that encapsulating MS‐275 in TPGS micelles represents a promising strategy to enhance MS‐275 therapeutic impact while minimizing its pharmacological dosage. [ABSTRACT FROM AUTHOR]

In the avian species, genetic modification by cell nuclear transfer is infeasible due to its unique reproductive system. The in vitro primordial germ cell modification approach is difficult and cumbersome, although it is the main method of genetic modification in chickens. In the present study, the adenoviral CRISPR/Cas9 vector was directly microinjected into the dorsal aorta of chicken embryos to achieve in vivo genetic modification. The results demonstrated that keratin 75-like 4 (KRT75L4), a candidate gene crucial for feather development, was widely knocked out, and an 8bp deletion was the predominant mutation that occurred in multiple tissues in chimeras, particularly in the gonad (2.63–11.57%). As we expected, significant modification was detected in the sperm of G0 (0.16–4.85%), confirming the potential to generate homozygous chickens and establishing this vector as a simple and effective method for genetic modification in avian species. [ABSTRACT FROM AUTHOR]

37/67 kDa laminin receptor (LamR)/ribosomal protein SA exhibits dual function as both a ribosomal protein and cell surface receptor for laminin. LamR influences critical cellular processes such as invasion, adhesion, and migration when acting as a receptor. Despite the acknowledged importance of LamR/67LR in various cellular processes, its contribution to the peripheral nervous system development is obscure. Thus, this study investigated the biological activity of LamR in peripheral axonal outgrowth in the presence of laminin-1 or Ile-Lys-Val-Ala- Val (IKVAV) peptide, whose important role in dorsal root ganglia (DRG) axonal outgrowth we recently showed. Unexpectedly, we did not observe LamR on the surface of DRG cells or in a conditioned medium, suggesting its intracellular action in the negative regulation of DRG axonal outgrowth. Using C-terminus LamR-targeting IgG, we demonstrated the role of LamR in that process, which is independent of the presence of Schwann cell precursors (SCPs) and is mediated by extracellular signal-regulated kinase (Erk) and Protein kinase B (Akt1/2/3) signaling pathways. Additionally, we show that the action of LamR towards laminin-1-dependent axonal outgrowth is unmasked only when the activity of integrin β1 is perturbed. We believe that modulation of LamR activity provides the basis for its use for inhibiting axon growth as a potential therapeutic agent for regulating abnormal or excessive neurite growth during neurodevelopmental diseases or pathological nerve regeneration. [ABSTRACT FROM AUTHOR]

Background:Pancreatic cancer is a leading cause of cancer-related mortality worldwide with increasing global incidence. We previously reported the anticancer effect of Rhus coriaria ethanolic extract (RCE) in triple negative breast and colon cancer cells. Herein, we investigated the anticancer effect of RCE on human pancreatic cancer cells. Methods: Cell viability was measured using Cell Titer-Glo and staining of viable and dead cells based on differential permeability to two DNA binding dyes. Cell cycle distribution and annexin V staining was carried out in Muse cell analyzer. Protein level was determined by Western blot. Tumor growth was assessed by in ovo chick embryo chorioallantoic membrane assay. Results: We found that RCE significantly inhibited the viability and colony growth of pancreatic cancer cells (Panc-1, Mia-PaCa-2, S2-013, AsPC-1). The antiproliferative effects of RCE in pancreatic cancer cells (Panc-1 and Mia-PaCa-2) were mediated through induction of G1 cell cycle arrest, Beclin-1-independent autophagy, and apoptosis. RCE activated both the extrinsic and intrinsic pathways of apoptosis and regulated the Bax/Bcl-2 apoptotic switch. Mechanistically, we found that RCE inhibited the AKT/mTOR pathway, downstream of which, inactivation of the cell cycle regulator p70S6K and downregulation of the antiapoptotic protein survivin was observed. Additionally, we found that RCE-induced autophagy preceded apoptosis. Further, we confirmed the anticancer effect of RCE in a chick embryo xenograft model and found that RCE inhibited the growth of pancreatic cancer xenografts without affecting embryo survival. Conclusion: Collectively, our findings demonstrate that Rhus coriaria exerts potent anti-pancreatic cancer activity though cell cycle impairment, autophagy, and apoptosis, and is hence a promising source of anticancer phytochemicals. [ABSTRACT FROM AUTHOR]

Introduction: The therapeutic effect of different doses of the traditional aqueous extract of dried leaves of yerba mate (Ilex paraguariensis A. St.-Hil.) was investigated in an experimental cataract model in chicken embryos. Methods and Results: LC-MS/MS analysis allowed the identification and quantification of 53 metabolites. In the hydrocortisone-induced cataract model, lenses were examined morphologically after treatment and parameters related to oxidative stress (total antioxidant/oxidant status (TAS/TOS), glutathione (GSH), and malondialdehyde (MDA)) were evaluated. Antiproliferative cell nuclear antigen (PCNA) and caspase-3 H-scores were determined and crystallin alpha A (CRYAA) gene expression in the lenses was measured by RT-PCR. The degree of cataract decreased in all treatment groups. While there was no significant difference in TAS levels compared to the negative control, TOS, GSH, and MDA levels were dose-dependently regulated. Treatment groups other than the high-dose group regulated the decrease in PCNA and the increase in caspase-3. CRYAA gene expression increased significantly only at the lowest dose. Conclusion: YM, which is becoming increasingly popular as a traditional tea, showed a therapeutic effect on hydrocortisone-induced cataracts in chicken embryos at relatively low doses. [ABSTRACT FROM AUTHOR]

Nanofibers can be utilized to overcome the challenges faced by conventional ophthalmic formulations. This study aimed to develop and characterize cysteamine (Cys)-loaded nanofiber-based ophthalmic inserts (OIs) as a potential candidate for the treatment of ophthalmic cystinosis using water-soluble polyvinyl alcohol (PVA)/poloxamer 407 (PO-407) and water-insoluble tetraethoxysilane (TEOS)/PVA nanofibers. Plain and Cys-loaded fibers in different proportions were prepared by the electrospinning method and studied for their morphological, physicochemical, release study, cytocompatibility effects, and stability study. The fiber formation was confirmed by scanning electron microscopy, while Fourier transform infrared spectra showed the most critical peaks for the Cys and the excipients. The release of the Cys was fast from the two polymeric matrices (≤20 min). The release from TEOS/PVA nanofibers is characterized by Case II transport (0.75 < β < 1), while the release from PVA/PO-407 nanofibers follows Fickian diffusion (β < 0.75). The cytocompatibility of compositions was confirmed by hen eggs tested on the chorioallantoic membrane (HET-CAM) of chick embryos. All formulations remained stable under stress conditions (40 ± 2 °C, 75 ± 5% relative humidity) regarding morphology and physicochemical characteristics. The developed nanofibrous mats could be an excellent alternative to available Cys drops, with better stability and convenience of self-administration as OIs. [ABSTRACT FROM AUTHOR]

Simple Summary: In commercial hatcheries, chicken embryos are incubated in complete darkness. However, in a natural environment, chicken embryos receive light stimulation during the incubation process as hens need to leave the nest to feed or turn the eggs. Therefore, providing light during incubation is closer to the natural growth environment of chicken embryos, which may have a positive effect on their growth and development. This study investigated the effects of providing different wavelengths of light during incubation on the behaviour, growth, immunity, and stress responses of post-hatch chicks. The results indicated that providing white light during incubation may enhance the growth performance of post-hatch chicks and potentially improve their ability to cope with environmental changes. This study aims to determine the effects of light exposure during incubation on the growth and development of post-hatch chicks and to provide a basis for the development of effective lighting strategies during the incubation period. Manipulation of light during incubation may have an effect on post-hatch chicks through the role of prenatal stage. The effects of providing different wavelengths of light (white, blue, and green lights, dark as control) during incubation on the growth performance, organ development, immune response, stress related hormones, digestive enzymes and behaviour of post-hatch chicks were investigated for 1–42 days. A total of 60 chicks per light treatment in three batches were used in this study. The results showed that the percentage of chicks accessing to feed and water resources appeared not to be affected by incubation light. Chicks hatched under white light were found to have a growth advantage (p < 0.05). The weight of organs (except thymus), IgA, IgY, IgM and heterophil to lymphocyte (H/L) ratio for post-hatch chicks were not affected by incubation light (p > 0.05). Thymus weight was reduced in chicks incubated under blue light compared to dark incubation (p < 0.05). The jejunum amylase and ileum lipase activities were significantly affected by the light treatments (p < 0.01). All light incubation chicks had stable plasma corticosterone levels and may have better ability to cope with environmental changes. Hence, white light photoperiod incubation may have potential to improve post-hatch chicks' growth performance and environmental adaptability. [ABSTRACT FROM AUTHOR]

Simple Summary: Establishing a cellular steatosis model is crucial for studying liver lipid deposition in poultry. This study used leghorn male hepatoma (LMH) cells to investigate the effects of oleic acid (OA), sodium oleate (SO), palmitic acid (PA), sodium palmitate (SP), and their pairwise combinations on steatosis development. Subsequently, RNA-seq was employed to comprehensively analyze alterations in gene expression within cells under various steatosis-inducing conditions. In primary chicken embryonic liver cells and liver tissue, the optimal conditions for inducing steatosis identified in this study can also induce steatosis effectively. Overall, this study found that a combination of SO and PA efficiently induces steatosis in various chicken liver cell types and chicken embryonic liver tissues. Research on hepatic steatosis in animal husbandry has been a prominent area of study. Developing an appropriate in vitro cellular steatosis model is crucial for comprehensively investigating the mechanisms involved in liver lipid deposition in poultry and for identifying potential interventions to address abnormalities in lipid metabolism. The research on the methods of in vitro liver steatosis in chickens, particularly the effects of different fat mixtures, is still lacking. In this study, LMH cells were utilized to investigate the effects of OA, SO, PA, SP, and their pairwise combinations on steatosis development, with the aim of identifying the optimal conditions for inducing steatosis. Analysis of triglyceride (TG) content in LMH cells revealed that OA and SP had limited efficacy in increasing TG content, while a combination of SO and PA in a 1:2 ratio exhibited the highest TG content. Moreover, Oil Red O staining results in LMH cells demonstrated that the combination treatment had a more pronounced induction effect compared to 0.375 mM SO. Additionally, RNA-seq analysis showed that 0.375 mM SO significantly influenced the expression of genes associated with fatty acid metabolism compared to the control group, whereas the combination of SO and PA led to an enrichment of key GO terms associated with programmed cell death. These findings suggest that varying conditions of cellular steatosis could lead to distinct disruptions in gene expression. The optimal conditions for inducing steatosis in LMH cells were also tested on chicken embryonic liver cells and embryos. TG detection and Oil Red O staining assays showed that the combination of SO and PA successfully induced steatosis. However, the gene expression pattern differed from that of LMH cells. This study lays the foundations for further investigations into avian hepatic steatosis. [ABSTRACT FROM AUTHOR]

Melanoma is one of the most aggressive and fatal skin cancers owing to its ability to metastasize and develop resistance to chemotherapy. Photodynamic therapy (PDT) is a minimally noninvasive treatment modality comprising photosensitizers (PSs), light sources, and endogenous molecular oxygen that exert a localized cytotoxic effect on cancer cells. The current study explores the therapeutic potential of sodium copper chlorophyllin‐loaded chitosan nanoparticles (CH‐SCC NPs) along with handheld laser‐based PDT on B16 cancer cells. A modified chlorophyll derivative identified as sodium copper chlorophyllin (SCC) is a dietary supplement that has anticancer properties. Herein, we have synthesized CH‐SCC NPs using the ionic gelation method to enhance the PS's bioavailability and efficiency. Chitosan nanoparticles exhibited high biocompatibility in a normal cell line L929, zebrafish, and chick embryos, and were successfully employed to deliver the SCC to cancer cells. CH‐SCC NPs showed an enhanced PDT effect that killed approximately 80%–85% of B16 cells. CH‐SCC NPs in combination with a handheld portable laser source showed significant therapeutic potential against the B16 skin cancer cell line. The experimental findings further strengthen our device‐repurposing strategy, which suggests that SCC nanoformulations along with handheld laser can be a suitable treatment for skin cancer even in remote areas where power source and treatment cost can be a limitation. [ABSTRACT FROM AUTHOR]

The determination of sex in mammals is established and controlled by various complex mechanisms. In contrast, sex control in poultry remains an unresolved issue. In this study, RNA-sequencing was conducted for male gonads and ovarian tissues in chicken embryos of up to 18.5 days to identify metabolic factors influencing male and female sex differentiation, as well as gonadal development. Our results reveal that PKM2, a critical glycolysis-related protein, plays a significant role in chicken sex differentiation via PPARG, a crucial hormone gene. We propose that our discoveries bolster the notion that glycolysis and oxidative phosphorylation function as antecedent contributors to sexual phenotypic development and preservation. [ABSTRACT FROM AUTHOR]

1. Skeletal muscle is an important component of chicken carcass. In chickens, the number of muscle fibres is fixed during the embryonic period, and muscle development during the embryonic period determines the muscle development potential after hatching. 2. Beijing-You (BY) and Cornish (CN) chickens show completely different growth rates and body types, and two breeds were used in this study to explore the role of lncRNAs in muscle development during different chicken embryonic periods. A systematic analysis of lncRNAs and mRNAs were conducted in the pectoral muscle tissues of BY and CN chickens at embryonic days 11 (ED11), 13 (ED13), 15 (ED15), 17 (ED17), and 1-day-old (D1) using RNA-seq. A total of 4,104 differentially expressed transcripts (DETs) were identified among the five stages, including 2,359 lncRNAs and 1,745 mRNAs. 3. The number of DETs between the two breeds at ED17 (1,658 lncRNAs and 1,016 mRNAs) was much higher than the total number of DET at all the other stages (692 lncRNAs and 729 mRNAs), indicating that the two breeds show the largest difference in gene regulation at ED17. 4. Correlation analysis was performed for all differentially expressed lncRNAs and mRNAs during the five periods. Forty-three, cis interaction pairs of lncRNA-mRNA related to chicken muscle development were predicted. The expression of four pairs was verified, and the results showed MSTRG.12395.2-FGFBP2 and MSTRG.18590.6-FMOD were significantly up-regulated in CN at ED11 compared to BY and might be important candidate genes for embryonic muscle development. [ABSTRACT FROM AUTHOR]

1. The accumulation of excessive fat plays a role in the development of non-alcoholic fatty liver disease (NAFLD) and phytogenic feed additives have the potential to ameliorate this. This study involved the isolation and culture of primary hepatocytes from chicken embryos to establish a model of hepatic steatosis induced by oleic acid/dexamethasone (OA/DEX). Lipid accumulation and cell viability were assessed using Nile Red staining, Oil Red O staining and cell count Kit −8 (CCK8) following treatment with varying concentrations of quercetin (Que). The potential mechanism by which Que exerts its effects was preliminarily investigated. 2. The results indicated that OA effectively treated lipid accumulation in hepatocytes. There was no notable variance in cell proliferation between the normal and OA/DEX groups when subjected to Que treatment at concentrations of 1000 ng/ml and 10 000 ng/ml. Triglycerides and cholesterol (low and high density) decreased with Que treatment, with the most substantial reduction observed at 10 000 ng/ml. 3. Gene expression levels decreased to levels similar to those in the control groups. Western blot data demonstrated that sterol regulatory element-binding protein 1 (SREBP-1) protein expression correlated with its mRNA expression level. Que mitigated lipid accumulation through the alpha serine/threonine protein kinase (AKT) and extracellular signal-regulated kinase (ERK) pathways. Expression levels of lipid-related genes (APOB, PPARα, CYP3A5 and SREBP-1) decreased to levels similar to the control groups. Western blot data demonstrated that the SREBP-1 protein expression correlated with its mRNA expression level. 4. Supplementation with Que ameliorated lipid accumulation through AKT and ERK signalling pathway in OA/DEX-induced high-fat hepatocytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study was undertaken to evaluate and compare the efficacy of different multi-antigen vaccines, including heat-inactivated, whole lysate, and subunit (outer membrane proteins [OMPs]) C. jejuni vaccines along with the immunostimulant CpG ODN in controlling Campylobacter colonization in chickens. In the first trial, 125 μg of C. jejuni OMPs and 50 μg of CpG ODN were administered individually or in combination, either in ovo to chick embryos or subcutaneously (SC) to one-day-old chicks. In the second trial, different concentrations of C. jejuni antigens (heat-killed, whole lysate, and OMPs) were administered SC to one-day-old chicks. The results of the first trial revealed that SC immunization with the combination of CpG ODN and C. jejuni OMPs elevated interferon (IFN)-γ, interleukin (IL)-1β, and IL-13 gene expression in the spleen, significantly increased serum IgM and IgY antibody levels, and reduced cecal C. jejuni counts by approximately 1.2 log10. In contrast, in ovo immunization did not elicit immune responses or confer protection against Campylobacter. The results of the second trial showed that SC immunization with C. jejuni whole lysate or 200 μg OMPs reduced C. jejuni counts by approximately 1.4 and 1.1 log10, respectively. In conclusion, C. jejuni lysate and OMPs are promising vaccine antigens for reducing Campylobacter colonization in chickens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Like all coronaviruses, infectious bronchitis virus, the causative agent of infectious bronchitis in chickens, exhibits a high mutation rate. Adaptive mutations that arise during the production of live attenuated vaccines against IBV often decrease virulence. The specific impact of these mutations on viral pathogenicity, however, has not been fully elucidated. In this study, we identified a mutation at the 3' end of the S gene in an IBV strain that was serially passaged in chicken embryos, and showed that this mutation resulted in a 9-aa truncation of the cytoplasmic tail (CT) of the S protein. This phenomenon of CT truncation has previously been observed in the production of attenuated vaccines against other coronaviruses such as the porcine epidemic diarrhea virus. We next discovered that the 9-aa truncation in the S protein CT resulted in the loss of the endoplasmic-reticulum-retention signal (KKSV). Rescue experiments with recombinant viruses confirmed that the deletion of the KKSV motif impaired the localization of the S protein to the endoplasmic-reticulum-Golgi intermediate compartment (ERGIC) and increased its expression on the cell surface. This significantly reduced the incorporation of the S protein into viral particles, impaired early subgenomic RNA and protein synthesis, and ultimately reduced viral invasion efficiency in CEK cells. In vivo experiments in chickens confirmed the reduced pathogenicity of the mutant IBV strains. Additionally, we showed that the adaptive mutation altered the TRS-B of ORF3 and impacted the transcriptional regulation of this gene. Our findings underscore the significance of this adaptive mutation in the attenuation of IBV infection and provide a novel strategy for the development of live attenuated IBV vaccines. Author summary: The administration of live attenuated vaccines is currently recognized as the most effective strategy for preventing infections caused by IBV. Understanding how mutations arising during vaccine production influence IBV virulence is crucial for developing more effective vaccines. In this study, we focused on a specific mutation in the S gene of IBV, which arose during viral propagation in chicken embryos. This mutation resulted in the truncation of the CT region of the S protein and concurrently eliminated a critical motif required for the retention of the protein at the viral assembly site. Similar S protein truncation has been observed in the production of attenuated vaccines against other coronaviruses, suggesting that the mutation identified in IBV in the present study was an adaptive mutation driven by natural selection pressure. Our findings underscore the value of targeting this adaptive mutation site as a strategy for reducing IBV virulence, and for the development of effective live attenuated vaccines against IBV and other coronaviruses. [ABSTRACT FROM AUTHOR]