3D exploration of gene expression in chicken embryos through combined RNA fluorescence in situ hybridization, immunofluorescence, and clearing.

BACKGROUND: Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity. RESULTS: We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons. CONCLUSIONS: We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.

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

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

The Effect of Short- and Long-Term Cryopreservation on Chicken Primordial Germ Cells.

Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14-16 Hamburger-Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers (SSEA1, CVH, and DAZL), as well as pluripotency markers (OCT4 and NANOG). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.

Thalamic activity-dependent specification of sensory input neurons in the developing chick entopallium.

During development, cell-intrinsic and cell-extrinsic factors play important roles in neuronal differentiation; however, the underlying mechanisms in nonmammalian species remain largely unknown. We here investigated the mechanisms responsible for the differentiation of sensory input neurons in the chick entopallium, which receives its primary visual input via the tectofugal pathway from the nucleus rotundus. The results obtained revealed that input neurons in the entopallium expressed Potassium Voltage-Gated Channel Subfamily H Member 5 (KCNH5/EAG2) mRNA from embryonic day (E) 11. On the other hand, the onset of protein expression was E20, which was 1 day before hatching. We confirm that entopallium input neurons in chicks were generated during early neurogenesis in the lateral and ventral ventricular zones. Notably, neurons derived from the lateral (LP) and ventral pallium (VP) exhibited a spatially distinct distribution along the rostro-caudal axis. We further demonstrated that the expression of EAG2 was directly regulated by input activity from thalamic axons. Collectively, the present results reveal that thalamic input activity is essential for specifying input neurons among LP- and VP-derived early-generated neurons in the developing chick entopallium.

Ontogeny of hepatic metabolism in two broiler lines divergently selected for the ultimate pH of the Pectoralis major muscle.

BACKGROUND: Nutrient availability during early stages of development (embryogenesis and the first week post-hatch) can have long-term effects on physiological functions and bird metabolism. The embryo develops in a closed structure and depends entirely on the nutrients and energy available in the egg. The aim of this study was to describe the ontogeny of pathways governing hepatic metabolism that mediates many physiological functions in the pHu + and pHu- chicken lines, which are divergently selected for the ultimate pH of meat, a proxy for muscle glycogen stores, and which differ in the nutrient content and composition of eggs. RESULTS: We identified eight clusters of genes showing a common pattern of expression between embryonic day 12 (E12) and day 8 (D8) post-hatch. These clusters were not representative of a specific metabolic pathway or function. On E12 and E14, the majority of genes differentially expressed between the pHu + and pHu- lines were overexpressed in the pHu + line. Conversely, the majority of genes differentially expressed from E18 were overexpressed in the pHu- line. During the metabolic shift at E18, there was a decrease in the expression of genes linked to several metabolic functions (e.g. protein synthesis, autophagy and mitochondrial activity). At hatching (D0), there were two distinct groups of pHu + chicks based on hierarchical clustering; these groups also differed in liver weight and serum parameters (e.g. triglyceride content and creatine kinase activity). At D0 and D8, there was a sex effect for several metabolic pathways. Metabolism appeared to be more active and oriented towards protein synthesis (RPS6) and fatty acid ß-oxidation (ACAA2, ACOX1) in males than in females. In comparison, the genes overexpressed in females were related to carbohydrate metabolism (SLC2A1, SLC2A12, FoxO1, PHKA2, PHKB, PRKAB2 and GYS2). CONCLUSIONS: Our study provides the first detailed description of the evolution of different hepatic metabolic pathways during the early development of embryos and post-hatching chicks. We found a metabolic orientation for the pHu + line towards proteolysis, glycogen degradation, ATP synthesis and autophagy, likely in response to a higher energy requirement compared with pHu- embryos. The metabolic orientations specific to the pHu + and pHu- lines are established very early, probably in relation with their different genetic background and available nutrients.

Coupling and uncoupling of midline morphogenesis and cell flow in amniote gastrulation.

Large-scale cell flow characterizes gastrulation in animal development. In amniote gastrulation, particularly in avian gastrula, a bilateral vortex-like counter-rotating cell flow, called 'polonaise movements', appears along the midline. Here, through experimental manipulations, we addressed relationships between the polonaise movements and morphogenesis of the primitive streak, the earliest midline structure in amniotes. Suppression of the Wnt/planar cell polarity (PCP) signaling pathway maintains the polonaise movements along a deformed primitive streak. Mitotic arrest leads to diminished extension and development of the primitive streak and maintains the early phase of the polonaise movements. Ectopically induced Vg1, an axis-inducing morphogen, generates the polonaise movements, aligned to the induced midline, but disturbs the stereotypical cell flow pattern at the authentic midline. Despite the altered cell flow, induction and extension of the primitive streak are preserved along both authentic and induced midlines. Finally, we show that ectopic axis-inducing morphogen, Vg1, is capable of initiating the polonaise movements without concomitant PS extension under mitotic arrest conditions. These results are consistent with a model wherein primitive streak morphogenesis is required for the maintenance of the polonaise movements, but the polonaise movements are not necessarily responsible for primitive streak morphogenesis. Our data describe a previously undefined relationship between the large-scale cell flow and midline morphogenesis in gastrulation.

Evaluating the effects of various ethanolic medicinal plant extracts on metastatic breast cancer proliferation, invasion, and expression of a novel potential drug target; CD82 metastatic suppressor protein, and on in vivo angiogenesis using the ex ovo yolk sac membrane (YSM) assay.

PURPOSE: Breast cancer metastasis relies on cellular invasion and angiogenesis facilitated by the downregulation of metastatic suppressor proteins like Cluster of Differentiation 82 (CD82). Currently, no medicines target multiple systems to prevent metastatic progression through CD82 upregulation. This study screened for plant extracts displaying effects on cell proliferation, invasion, and CD82 expression in breast cancer cells, and in vivo angiogenesis, and further correlated between the biological activities and effect on CD82 expression. METHODS: Seventeen ethanolic plant extracts were screened for their effect on cell proliferation (against MDA-MB-231 and MCF-7 breast cancer and Hek293 kidney cells), cell invasion and effect on CD82 expression in metastatic MDA-MB-231 cells. Selected extracts were further evaluated for in vivo anti-angiogenesis. RESULTS: Extracts displayed varying antiproliferative activity against the different cell lines, and those that showed selectivity indexes (SI) > 0.5 against MDA-MB-231 were selected for anti-invasion evaluation. Buddleja saligna Willd. (BS), Combretum apiculatum Sond. (CA), Foeniculum vulgare, Greyia radlkoferi, Gunnera perpensa and Persicaria senegalensis (Meisn.) Soják (PS) displayed 50% inhibitory concentration (IC50) values of 44.46 ± 3.46, 74.00 ± 4.48, 180.43 ± 4.51, 96.97 ± 2.29, 55.29 ± 9.88 and 243.60 ± 2.69 µg/mL, respectively against MDA-MB-231, and compared to Hek293 showed SI of 0.9, 0.7, 1.4, 1.1, 2.2 and 0.5. Significant invasion inhibition was observed at both 20 and 40 µg/mL for BS (94.10 ± 0.74 and 96.73 ± 0.95%) and CA (87.42 ± 6.54 and 98.24 ± 0.63%), whereas GR (14.91 ± 1.62 and 41 ± 1.78%) and PS (36.58 ± 0.54 and 51.51 ± 0.83%), only showed significant inhibition at 40 µg/mL, and FV (< 5% inhibition) and GP (10 ± 1.03 and 22 ± 1.31%) did not show significant inhibition at both concentrations. Due to the significant anti-invasive activity of BS, CA and PS at 40 µg/mL, these extracts were further evaluated for their potential to stimulate CD82. BS showed significant (p < 0.05) reduction in CD82 at 20 and 40 µg/mL (13.2 ± 2.2% and 20.3 ± 1.5% decrease, respectively), whereas both CA and PS at 20 µg/mL increased (p < 0.05) CD82 expression (16.4 ± 0.8% and 5.4 ± 0.6% increase, respectively), and at 40 µg/mL significantly reduced CD82 expression (23.4 ± 3.1% and 11.2 ± 2.9% decrease, respectively). Using the yolk sac membrane assay, BS (59.52 ± 4.12 and 56.72 ± 3.13% newly formed vessels) and CA (83.33 ± 3.17 and 74.00 ± 2.12%) at both 20 and 40 µg/egg showed significant (p < 0.001) angiogenesis inhibition, with BS showing statistical similar activity to the positive control, combretastatin A4 (10 nmol/egg), whereas PS only displayed significant (p < 0.001) angiogenesis stimulation at 40 µg/egg (120.81 ± 3.34% newly formed vessels). CONCLUSION: BS exhibits antiproliferative, anti-invasive, and anti-angiogenic activity despite inhibiting CD82, suggesting an alternative mode of action. CA at 20 µg/mL shows moderate anti-invasive and anti-angiogenic potential by stimulating CD82, while at 40 µg/mL it still displays these properties but inhibits CD82, suggesting an additional mode of action. PS, with the least antiproliferative activity, stimulates CD82 and inhibits angiogenesis at 20 µg/mL but inhibits CD82 and increases angiogenesis at 40 µg/mL, indicating CD82 targeting as a major mode of action. Future studies should explore breast cancer xenograft models to assess the extracts' impact on CD82 expression and angiogenesis in the tumor microenvironment, along with isolating bioactive compounds from the extracts.

Quantitative phosphoproteomic analysis of chicken DF-1 cells infected with Eimeria tenella, using tandem mass tag (TMT) and parallel reaction monitoring (PRM) mass spectrometry.

Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host cell-E. tenella interactions. However, no comprehensive phosphoproteomic analyses of host cells at various phases of E. tenella infection have been published. In this study, quantitative phosphoproteomic analysis of chicken embryo DF-1 fibroblasts that were uninfected (UI) or infected with E. tenella for 6 h (PI6, the early invasion phase) or 36 h (PI36, the trophozoite development phase) was conducted. A total of 10,122 phosphopeptides matched to 3,398 host cell phosphoproteins were identified and 13,437 phosphorylation sites were identified. Of these, 491, 1,253, and 275 differentially expressed phosphorylated proteins were identified in the PI6/UI, PI36/UI, and PI36/PI6 comparisons, respectively. KEGG pathway enrichment analysis showed that E. tenella modulated host cell processes through phosphorylation, including focal adhesion, regulation of the actin cytoskeleton, and FoxO signaling to support its early invasion phase, and modulating adherens junctions and the ErbB signaling pathway to favor its trophozoite development. These results enrich the data on the interaction between E. tenella and host cells and facilitate a better understanding of the molecular mechanisms underlying host-parasite relationships.

Title: Analyse phosphoprotéomique quantitative de cellules DF-1 de poulet infectées par Eimeria tenella, par spectrométrie de masse avec marqueur de masse en tandem (TMT) et surveillance des réactions parallèles (PRM). Abstract: Eimeria tenella est un parasite intracellulaire obligatoire qui cause de graves dommages à l'industrie de l'élevage de volailles. La phosphorylation des protéines joue un rôle essentiel dans les interactions entre la cellule hôte et E. tenella. Cependant, aucune analyse phosphoprotéomique complète des cellules hôtes à différentes phases de l'infection par E. tenella n'a été publiée. Dans cette étude, une analyse phosphoprotéomique quantitative de fibroblastes DF-1 d'embryon de poulet non infectés (NI) ou infectés par E. tenella pendant 6 h (PI6, la phase d'invasion précoce) ou 36 h (PI36, la phase de développement des trophozoïtes) a été réalisée. Un total de 10 122 phosphopeptides correspondant à 3 398 phosphoprotéines de cellules hôtes ont été identifiés et 13 437 sites de phosphorylation ont été identifiés. Parmi celles-ci, 491, 1 253 et 275 protéines différentiellement phosphorylées exprimées ont été identifiées respectivement dans les comparaisons PI6/NI, PI36/NI et PI36/PI6. L'analyse d'enrichissement de la voie KEGG a montré qu'E. tenella modulait les processus de la cellule hôte par phosphorylation, y compris l'adhésion focale, la régulation du cytosquelette d'actine et la signalisation FoxO, pour aider sa phase d'invasion précoce, et la modulation des jonctions adhérentes et de la voie de signalisation ErbB pour favoriser le développement de son trophozoïte. Ces résultats enrichissent les données sur l'interaction entre E. tenella et les cellules hôtes et facilitent une meilleure compréhension des mécanismes moléculaires sous-jacents aux relations hôtes­parasites.

Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine.

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Immunometric and functional measurement of endogenous vasoinhibin in human sera.

Introduction: Circulating levels of the antiangiogenic protein vasoinhibin, a fragment of prolactin, are of interest in vasoproliferative retinopathies, preeclampsia, and peripartum cardiomyopathy; however, it is difficult to determine the circulating levels of vasoinhibin due to the lack of quantitative assays. Methods: This study used human serum samples to assess the concentration and bioactivity of vasoinhibin using a novel enzyme-linked immunosorbent assay (ELISA) for human vasoinhibin, which employs an anti-vasoinhibin monoclonal antibody, a human umbilical vein endothelial cell (HUVEC) proliferation assay, and a chick chorioallantoic membrane (CAM) angiogenesis assay. Results: Serum samples from 17 pregnant women without (one group) and with preeclampsia and pregnancy induced hypertension (another group) demonstrated endogenous vasoinhibin concentrations in the range of 5-340 ng/ml. Immunoactive vasoinhibin levels were significantly higher in preeclampsia serum compared to healthy pregnancy serum (mean 63.09 ± 22.15 SD vs. 19.67 ± 13.34 ng/ml, p = 0.0003), as was the bioactive vasoinhibin level as determined by the HUVEC proliferation assay (56.12 ± 19.83 vs. 13.38 ± 4.88 ng/ml, p < 0.0001). There was a correlation between the concentration of vasoinhibin measured by ELISA and the HUVEC proliferation assay (Pearson r = 0.95, p < 0.0001). Healthy serum demonstrated a proangiogenic effect in the CAM assay (p < 0.05, compared to control), while serum from preeclamptic patients demonstrated an antiangiogenic effect (p < 0.05 vs. control), as did recombinant human vasoinhibin and a synthetic circular retro-inverse vasoinhibin analogue (CRIVi45-51). The antiangiogenic effects in the CAM assay and the inhibition of HUVEC proliferation were abolished by addition of the ELISA anti-vasoinhibin monoclonal antibody, but not by mouse IgG. Discussion: These results demonstrate the first quantitation of endogenous vasoinhibin in human sera and the elevation of it levels and antiangiogenic activity in sera from women with preeclampsia. The development and implementation of a quantitative assay for vasoinhibin overcomes a long-standing barrier and suggests the thorough clinical verification of vasoinhibin as a relevant biomarker.

Retinoic Acid-Mediated Control of Energy Metabolism Is Essential for Lung Branching Morphogenesis.

Lung branching morphogenesis relies on intricate epithelial-mesenchymal interactions and signaling networks. Still, the interplay between signaling and energy metabolism in shaping embryonic lung development remains unexplored. Retinoic acid (RA) signaling influences lung proximal-distal patterning and branching morphogenesis, but its role as a metabolic modulator is unknown. Hence, this study investigates how RA signaling affects the metabolic profile of lung branching. We performed ex vivo lung explant culture of embryonic chicken lungs treated with DMSO, 1 µM RA, or 10 µM BMS493. Extracellular metabolite consumption/production was evaluated by using 1H-NMR spectroscopy. Mitochondrial respiration and biogenesis were also analyzed. Proliferation was assessed using an EdU-based assay. The expression of crucial metabolic/signaling components was examined through Western blot, qPCR, and in situ hybridization. RA signaling stimulation redirects glucose towards pyruvate and succinate production rather than to alanine or lactate. Inhibition of RA signaling reduces lung branching, resulting in a cystic-like phenotype while promoting mitochondrial function. Here, RA signaling emerges as a regulator of tissue proliferation and lactate dehydrogenase expression. Furthermore, RA governs fatty acid metabolism through an AMPK-dependent mechanism. These findings underscore RA's pivotal role in shaping lung metabolism during branching morphogenesis, contributing to our understanding of lung development and cystic-related lung disorders.

Biosynthesized silver nanoparticles prevent bacterial infection in chicken egg model and mitigate biofilm formation on medical catheters.

Investigating the application of innovative antimicrobial surface coatings on medical devices is an important field of research. Many of these coatings have significant drawbacks, including biocompatibility, coating stability and the inability to effectively combat multiple drug-resistant bacteria. In this research, we developed an antibiofilm surface coating for medical catheters using biosynthesized silver nanoparticles (b-Cs-AgNPs) developed using leaves extract of Calliandra surinamensis. Various characterization techniques were employed to thoroughly characterize the synthesized b-Cs-AgNPs and c-AgNPs. b-Cs-AgNPs were compatible with human normal kidney cells and chicken embryos. It did not trigger any skin inflammatory response in in vivo rat model. b-Cs-AgNPs demonstrated potent zone of inhibition of 19.09 mm when subjected to the disc diffusion method in E. coli confirming strong antibacterial property. Different anti-bacterial assays including liquid growth curve, colony counting assay, biofilm formation assay supported the potent antimicrobial efficacy of b-Cs-AgNPs alone and when coated to medical grade catheters. Mechanistic studies reveal the presence of ferulic acid, that was important for the synthesis of b-AgNPs along with enhanced antibacterial effects of b-Cs-AgNPs compared to c-AgNPs, supported by molecular docking analysis. These results together demonstrated the effective role b-Cs-AgNPs in combating infections and mitigating biofilm formations, highlighting their need for further study in the field of biomedical applications.

Whole transcriptome profiling reveals a lncMDP1 that regulates myogenesis by adsorbing miR-301a-5p targeting CHAC1.

Myoblast proliferation and differentiation are essential for skeletal muscle development. In this study, we generated the expression profiles of mRNAs, long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) in different developmental stages of chicken primary myoblasts (CPMs) using RNA sequencing (RNA-seq) technology. The dual luciferase reporter system was performed using chicken embryonic fibroblast cells (DF-1), and functional studies quantitative real-time polymerase chain reaction (qPCR), cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry cycle, RNA fluorescence in situ hybridization (RNA-FISH), immunofluorescence, and western blotting assay. Our research demonstrated that miR-301a-5p had a targeted binding ability to lncMDP1 and ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1). The results revealed that lncMDP1 regulated the proliferation and differentiation of myoblasts via regulating the miR-301a-5p/CHAC1 axis, and CHAC1 promotes muscle regeneration. This study fulfilled the molecular regulatory network of skeletal muscle development and providing an important theoretical reference for the future improvement of chicken meat performance and meat quality.

Determination of the embryotoxic effects of propofol injected into eggs on the cerebellum and spinal cord using histologic methods: an animal study.

Background/aim: This study aims to determine the possible embryotoxic effects of propofol on the cerebellum and spinal cord using fertile chicken eggs. Materials and methods: A total of 430 fertile eggs were divided into 5 groups: control, saline, 2.5 mg.kg-1, 12.5 mg.kg-1, and 37.5 mg.kg-1 propofol. Injections were made immediately before incubation via the air chamber. On the 15th, 18th, and 21st day of incubation, 6 embryos from each group were evaluated. Serial paraffin sections taken from the cerebellum and spinal cord were stained with hematoxylin-eosin, Kluver-Barrera, toluidine blue, and periodic acid-Schiff's reaction. The outer granular layer and total cortex thickness were measured, and the linear density of the Purkinje cells was determined. The ratios of the substantia grisea surface area to the total surface area of the spinal cord were calculated. The transverse and longitudinal diameters of the canalis centralis were also assessed. Results: No structural malformation was observed in any embryos examined macroscopically. No significant difference was observed between the groups in terms of development and histologic organization of the cerebellum and spinal cord. However, on the 15th, 18th, and 21st day, the outer granular layer (p < 0.001 for all days) and the total cortex thickness (p < 0.01, p < 0.001, and p < 0.001, respectively) decreased significantly in different propofol dose groups in varying degrees in the cerebellum. Similarly, in the spinal cord, there were significant changes in the ratios of the substantia grisea surface area to the total surface area (p < 0.01 and p < 0.001, respectively). Conclusion: It was concluded that the in-ovo-administered propofol given immediately before incubation has adverse effects on the developing cerebellum and spinal cord. Therefore, it is important for anesthesiologists always to remain vigilant when treating female patients of childbearing age.

The haemagglutinin-neuraminidase protein of velogenic Newcastle disease virus enhances viral infection through NF-κB-mediated programmed cell death.

The haemagglutinin-neuraminidase (HN) protein, a vital membrane glycoprotein, plays a pivotal role in the pathogenesis of Newcastle disease virus (NDV). Previously, we demonstrated that a mutation in the HN protein is essential for the enhanced virulence of JS/7/05/Ch, a velogenic variant NDV strain originating from the mesogenic vaccine strain Mukteswar. Here, we explored the effects of the HN protein during viral infection in vitro using three viruses: JS/7/05/Ch, Mukteswar, and an HN-replacement chimeric NDV, JS/MukHN. Through microscopic observation, CCK-8, and LDH release assays, we demonstrated that compared with Mukteswar and JS/MukHN, JS/7/05/Ch intensified the cellular damage and mortality attributed to the mutant HN protein. Furthermore, JS/7/05/Ch induced greater levels of apoptosis, as evidenced by the activation of caspase-3/8/9. Moreover, JS/7/05/Ch promoted autophagy, leading to increased autophagosome formation and autophagic flux. Subsequent pharmacological experiments revealed that inhibition of apoptosis and autophagy significantly impacted virus replication and cell viability in the JS/7/05/Ch-infected group, whereas less significant effects were observed in the other two infected groups. Notably, the mutant HN protein enhanced JS/7/05/Ch-induced apoptosis and autophagy by suppressing NF-κB activation, while it mitigated the effects of NF-κB on NDV infection. Overall, our study offers novel insights into the mechanisms underlying the increased virulence of NDV and serves as a reference for the development of vaccines.

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.

Bone Regenerative Effect of Injectable Hypoxia Preconditioned Serum-Fibrin (HPS-F) in an Ex Vivo Bone Defect Model.

Biofunctionalized hydrogels are widely used in tissue engineering for bone repair. This study examines the bone regenerative effect of the blood-derived growth factor preparation of Hypoxia Preconditioned Serum (HPS) and its fibrin-hydrogel formulation (HPS-F) on drilled defects in embryonic day 19 chick femurs. Measurements of bone-related growth factors in HPS reveal significant elevations of Osteopontin, Osteoprotegerin, and soluble-RANKL compared with normal serum (NS) but no detection of BMP-2/7 or Osteocalcin. Growth factor releases from HPS-F are measurable for at least 7 days. Culturing drilled femurs organotypically on a liquid/gas interface with HPS media supplementation for 10 days demonstrates a 34.6% increase in bone volume and a 52.02% increase in bone mineral density (BMD) within the defect area, which are significantly higher than NS and a basal-media-control, as determined by microcomputed tomography. HPS-F-injected femur defects implanted on a chorioallantoic membrane (CAM) for 7 days exhibit an increase in bone mass of 123.5% and an increase in BMD of 215.2%, which are significantly higher than normal-serum-fibrin (NS-F) and no treatment. Histology reveals calcification, proteoglycan, and collagen fiber deposition in the defect area of HPS-F-treated femurs. Therefore, HPS-F may offer a promising and accessible therapeutic approach to accelerating bone regeneration by a single injection into the bone defect site.

Preparation and characterization of mouse-derived monoclonal antibodies against the hemagglutinin of the H1N1 influenza virus.

H1N1 influenza virus is a significant global public health concern. Monoclonal antibodies (mAbs) targeting specific viral proteins such as hemagglutinin (HA) have become an important therapeutic strategy, offering highly specific targeting to block viral transmission and infection. This study focused on the development of mAbs targeting HA of the A/Victoria/2570/2019 (H1N1pdm09, VIC-19) strain by utilizing hybridoma technology to produce two mAbs with high binding capacity. Notably, mAb 2B2 has demonstrated a strong affinity for HA proteins in recent H1N1 influenza vaccine strains. In vitro assessments showed that both mAbs exhibited broad-spectrum hemagglutination inhibition and potent neutralizing effects against various vaccine strains of H1N1pdm09 viruses. 2B2 was also effective in animal models, offering both preventive and therapeutic protection against infections caused by recent H1N1 strains, highlighting its potential for clinical application. By individually co-cultivating each of the aforementioned mAbs with the virus in chicken embryos, four amino acid substitution sites in HA (H138Q, G140R, A141E/V, and D187E) were identified in escape mutants, three in the antigenic site Ca2, and one in Sb. The identification of such mutations is pivotal, as it compels further investigation into how these alterations could undermine the binding efficacy and neutralization capacity of antibodies, thereby impacting the design and optimization of mAb therapies and influenza vaccines. This research highlights the necessity for continuous exploration into the dynamic interaction between viral evolution and antibody response, which is vital for the formulation of robust therapeutic and preventive strategies against influenza.

Impact of dichromatic lighted incubation on hatching result and post-hatch performance of broiler chickens.

This study was planned to evaluate the impact of dichromatic lights during incubation on the hatching and post-hatch performance of broiler chickens. A total of 500 eggs of broiler breeder (Ross 308; Age 44 weeks) were evenly divided according to a completely randomized design into 4 treatments having 5 replicates and 25 eggs each. Treatments consisted of dichromatic lights Blue + Red (BR), Green + Red (GR) and Green + Blue (GB) provided at an intensity of 250 lx for 12 h a day along with a Dark (D) environment. After hatching 200 chicks (50 from each respective light group) were divided into 4 treatments with 5 replicates each having 10 chicks. Results indicated a higher embryo index (13.12%) in the GR group on the 12th day of incubation; while an ideal hatch window was observed in GR and GB (98.18% and 96.00% hatched chicks) lighting groups. In hatching traits, higher hatchability (86.15) and hatch of fertile (93.85) percentages were observed in GR lighting followed by GB, BR and Dark treatment groups; while dead-in shell embryos were lowest in the GR group. In growth performance, higher feed intake (513.20 g) and body weight (479.20 g) were observed in the GB group followed by GR, BR and dark group; and feed conversion ratio (FCR) was better in the GR group (1.06). In welfare parameters, improved physical asymmetry (0.90 mm) and tonic immobility (54.40 s) were measured in the GR group followed by GB, BR and the dark group. It was concluded that under experimental conditions when broiler breeder eggs are provided with GR lighting during incubation, it can help to improve hatchability, growth performance and welfare traits in chicks.

A global gene regulatory program and its region-specific regulator partition neurons into commissural and ipsilateral projection types.

Understanding the genetic programs that drive neuronal diversification into classes and subclasses is key to understand nervous system development. All neurons can be classified into two types: commissural and ipsilateral, based on whether their axons cross the midline or not. However, the gene regulatory program underlying this binary division is poorly understood. We identified a pair of basic helix-loop-helix transcription factors, Nhlh1 and Nhlh2, as a global transcriptional mechanism that controls the laterality of all floor plate-crossing commissural axons in mice. Mechanistically, Nhlh1/2 play an essential role in the expression of Robo3, the key guidance molecule for commissural axon projections. This genetic program appears to be evolutionarily conserved in chick. We further discovered that Isl1, primarily expressed in ipsilateral neurons within neural tubes, negatively regulates the Robo3 induction by Nhlh1/2. Our findings elucidate a gene regulatory strategy where a conserved global mechanism intersects with neuron class-specific regulators to control the partitioning of neurons based on axon laterality.