Search Results (15)

Background: Routine use of the chorioallantoic membrane (CAM) tumor model in nuclear imaging studies is hampered by small tumors, embryonic movements and laborious volume-restricted intravenous tracer/drug administration. We sought a workaround by using fast-growing tumors, high-resolution autoradiography and non-intravenous tracer administration. Methods: Dekalb White chicken eggs were grafted with C3H mammary carcinoma fragments or MOC2 oral squamous cell carcinoma fragments from donor mice. The tumor uptake of ¹⁸F-fluorodeoxyglucose (FDG) following in-yolk-sac injection, dripping after CAM scoring or allantoic cavity injection was evaluated using positron emission tomography (PET) and autoradiography. Using in-yolk-sac injection, eggs were administered different tracer mixtures, namely (1) pimonidazole (hypoxia-marker), FDG and ¹⁴C-2-deoxyglucose (¹⁴C-2DG), (2) pimonidazole, FDG and ¹⁴C-acetate or (3) pimonidazole, the hypoxia-selective tracer ¹⁸F-fluoroazomycin-arabinoside (FAZA) and ¹⁴C-2DG. For comparison, tumor-bearing mice were administered FDG/¹⁴C-acetate/pimonidazole. Gross tumor uptake was evaluated using PET. Tumor cryosections were analyzed using dual-tracer autoradiography. Complementary autoradiograms were co-registered, covered by a square grid (0.5 × 0.5 mm). Pearson correlation coefficients (PCC) were calculated from scatterplots. Results: C3H tumors reached a mean weight (with 95% confidence interval) of 0.32 g (0.28–0.37 g), while for MOC2, it was 0.19 g (0.09–0.29 g). In-yolk-sac tracer injection was simple and effective, producing high tracer uptake and contrast 3 h post-administration. Spatial tracer overlap (PCC) was: FDG vs. ¹⁴C-2DG, 0.95–0.97; FAZA vs. ¹⁴C-2DG, 0.71–0.79 and FDG vs. ¹⁴C-acetate, 0.26–0.84 (0.15–0.76 in mice). Pimonidazole revealed tumor hypoxia. Conclusions: Direct-grafting from donor mice generated larger tumors than previously reported. In-yolk-sac tracer administration was practical and allowed larger injected volumes. Autoradiography revealed that: (1) FDG and ¹⁴C-2DG can be used interchangeably, (2) ¹⁴C-2DG was elevated in FAZA-positive areas, suggesting that in some tumors FDG-PET may provide information on the intratumoral distribution of hypoxic areas, and (3) FDG and ¹⁴C-acetate showed variable overlap. We conclude that in-yolk-sac tracer injection and autoradiography simplify and optimize CAM-based nuclear imaging research. Full article

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disorder characterized by persistent airflow limitation and chronic airway inflammation. Current therapeutic strategies primarily offer symptomatic relief and are often limited by systemic side effects, inadequate lung deposition, and poor patient compliance. Naringin (NAR), a natural flavonoid with strong antioxidant, anti-inflammatory, and anti-fibrotic activities, has demonstrated potential in mitigating COPD-associated pathophysiology. However, its therapeutic application is restricted by poor water solubility, low bioavailability, and rapid metabolism. Nanotechnology-based drug delivery systems, particularly poly(lactic-co-glycolic acid) (PLGA) nanoparticles, provide an effective approach for lung-targeted therapy. Their nanoscale size promotes deep lung deposition, enhanced cellular uptake, reduced lung clearance, improved therapeutic efficacy, and reduced systemic side effects. The present study aimed to develop NAR-loaded PLGA nanoparticles (NAR PLGA NP) for enhanced cell-targeting in inflammatory lung conditions. NAR PLGA NP were prepared using the emulsion solvent evaporation method, with PLGA in the organic phase and soya lecithin (SL) with poly(vinyl alcohol) (PVA) as surfactants in the aqueous phase. A face-centered central composite design was employed to optimize the formulation. The optimized nanoparticles were characterized for size distribution by dynamic light scattering, entrapment efficiency, Transmission Electron Microscopy (TEM), Fourier Transform Infrared (FTIR), Differential Scanning Calorimetry (DSC), X-Ray Diffraction (XRD), and in vitro drug release. The safety of PLGA and lecithin-coated PLGA nanoparticles (LC PLGA NP) was assessed using an MTT assay on lung epithelial cells, followed by cellular uptake studies, angiogenesis by chick Yolk Sac Membrane (YSM) assay, and in vitro evaluation of reactive oxidative stress (ROS) and anti-inflammatory activity. The optimized PLGA formulation showed a hydrodynamic diameter of 201 ± 1 nm with PDI 0.20 ± 0.03 and EE of 76.11 ± 2.1%, and 81.7 ± 4.9% drug release at 72 h, whereas LC PLGA NP showed a hydrodynamic diameter of 308 ± 3 nm, PDI of 0.21 ± 0.05, entrapment efficiency of 82.45 ± 4.8%, and 71.4 ± 3.2% drug release at 72 h. Both PLGA NP and LC PLGA NP demonstrated good cytocompatibility with lung epithelial cells, efficient cellular uptake, and a significant reduction in intracellular reactive oxygen species (ROS) levels (**** p value < 0.0001). Moreover, the formulations markedly suppressed pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, indicating anti-inflammatory activity. The angiogenesis assay further suggested their ability for lung tissue repair and remodeling. These findings support the potential of LC PLGA NP as a promising cell-specific targeting system for naringin in inflammatory lung conditions. Full article

The role of the yolk sac membrane (YSM) and digestive tract in the processing of egg yolk proteins during embryogenesis is unexplored in the duck Anas platyrhynchos domestica. Here, we investigated in the duck embryo the function of the YSM, proventriculus, and small intestine in protein digestion and uptake. We tested the expression of aminopeptidase N (APN) and the oligopeptide transporter PepT1 as well as the expression of cathepsin B (CTSB) and cathepsin D (CTSD) lysosomal genes in the YSM during incubation days 12, 14, 16–18, 20, 22, 24, 26, and 28 (the day of hatch). Also, we examined embryonic duck pepsinogen (EDPg) expression in the proventriculus and APN and PepT1 expression in the small intestine. In the YSM, CTSD expression was weak compared to that of CTSB, and the expression of CTSB, APN, and PepT1 reached its maximum on day 24 and decreased afterwards. In the proventriculus, EDPg expression peaked on days 17 to 20 and decreased thereafter. The APN and PepT1 expression levels were highest in the jejunum and ileum and reached their maximum on day 28. Our results suggest that the YSM plays a role in the degradation and uptake of the peptides that are digested by the activated yolk proteases, and it also functions in the lysosomal digestion of yolk lipoproteins. Furthermore, the proventriculus is possibly involved in the digestion of yolk proteins. Finally, the jejunum and ileum appear to be the primary sites for peptide digestion and absorption at the end of the incubation. Full article

Differences in cell count and growth factor expression between first- and second-generation autologous platelet concentrates (APCs) have been well described. The debate over which formula best supports wound healing in various surgical procedures is still ongoing. This study aims to assess the whole proteome assembly, cell content, immunological potential and pro-angiogenic potential of second-generation APC, Platelet-Rich Fibrin (PRF) vs. first-generation APC, Platelet-Rich Plasma (PRP). The global proteome of the APCs was analyzed using nano-liquid chromatography mass spectrometry. Blood cell concentrations were determined by an automated cell counter. The effect of APCs on macrophage polarization was analyzed by flow cytometry. A yolk sac membrane (YSM) assay was used to monitor the neo-vessel formation and capillary branching in vivo. Cell count analysis revealed a higher number/concentration of leukocytes in PRF vs. PRP. Incubation of macrophages with PRP or platelet-free plasma (PFP) did not induce a significant pro-inflammatory state but led to a shift to the M0/M2 phenotype as seen in wound healing for all tested formulas. Label-free proteomics analysis identified a total of 387 proteins from three biological replicates of the respective designated groups. PRF induced increased formation of neo-vessels and branching points in vivo in comparison to PRP and PFP (each p < 0.001), indicating the enhanced pro-angiogenic potential of PRF. Overall, PRF seems superior to PRP, an important representative of first-generation formulas. Inclusion of leucocytes in PRF compared to PRP suggested rather an anti-inflammatory effect on macrophages. These results are important to support the versatile clinical applications in regenerative medicine for second-generation autologous platelet concentrates to optimize wound healing. Full article

Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK’s origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4^CreRosa26^mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production. Full article

Cancerous cells are characterised by their ability to invade, metastasise, and induce angiogenesis. Tumour cells use various molecules that can be targeted to reverse these processes. Dasatinib, a potent Src inhibitor, has shown promising results in treating hepatocellular carcinoma (HCC) in vitro and in vivo. However, its effectiveness is limited by focal adhesion kinase (FAK) activation. Isothiocyanates, on the other hand, are phytochemicals with broad anticancer activity and FAK inhibition capabilities. This study evaluated the synergistic effect of dasatinib and phenethyl isothiocyanate (PEITC) on HCC. The combination was tested using various assays, including MTT, adhesion, scratch, Boyden chamber, chorioallantoic membrane (CAM), and yolk sac membrane (YSM) assays to evaluate the effect of the drug combination on HCC metastatic potential and angiogenesis in vitro and in vivo. The results showed that the combination inhibited the adhesion, migration, and invasion of HepG2 cells and reduced xenograft volume in the CAM assay. Additionally, the combination reduced angiogenesis in vitro, diminishing the growth of vessels in the tube formation assay. The inhibition of FAK/STAT3 signalling led to increased E-cadherin expression and reduced VEGF secretion, reducing HCC metastatic potential. Therefore, a combination of PEITC and dasatinib could be a potential therapeutic strategy for the treatment of HCC. Full article

Bone fracture healing is a multistep process, including early immunological reactions, osteogenesis, and as a key factor, angiogenesis. Molecules inducing osteogenesis as well as angiogenesis are rare, but hold promise to be employed in bone tissue engineering. It has been demonstrated that the bone sialoprotein (BSP) can induce bone formation when immobilized in collagen type I, but its effect on angiogenesis still has to be characterized in detail. Therefore, the aim of this study was to analyse the effects of BSP immobilized in a collagen type I gel on angiogenesis. First, in vitro analyses with endothelial cells (HUVECs) were performed detecting enhancing effects of BSP on proliferation and gene expression of endothelial markers. A spheroid model was employed confirming these results. Finally, the inducing impact of BSP-collagen on vascular density was proved in a yolk sac membrane assay. Our results demonstrate that BSP is capable of inducing angiogenesis and confirm that collagen type I is the optimal carrier for this protein. Taking into account former results, and literature showing that BSP also induces osteogenesis, one can hypothesize that BSP couples angiogenesis and osteogenesis, making it a promising molecule to be used in bone tissue regeneration. Full article

Y-27632 inhibits Rho-associated coiled-coil-containing protein kinase (ROCK) signaling, which is involved in various embryonic developmental processes, including angiogenesis, by controlling actin cytoskeleton assembly and cell contractility. Administration of Y-27632 impairs cytoskeletal arrangements in post-gastrulation chick embryos, leading to ventral body wall defects (VBWDs). Impaired angiogenesis has been hypothesized to contribute to VBWDs. ROCK is essential in transmitting signals downstream of vascular endothelial growth factor (VEGF). VEGF-mediated angiogenesis induces gene expressions and alterations of the actin cytoskeleton upon binding to VEGF receptors (VEGFRs). The aim of this study was to investigate effects of Y-27632 on angiogenesis in post-gastrulation chick embryos during early embryogenesis. After 60 h incubation, embryos in shell-less culture were treated with Y-27632 or vehicle for controls. Y-27632-treated embryos showed reduced extra-embryonic blood vessel formation with impaired circulation of the yolk sac, confirmed by fractal analysis. Western blot confirmed impaired ROCK downstream signaling by decreased expression of phosphorylated myosin light chain. Interestingly, RT-PCR demonstrated increased gene expression of VEGF and VEGFR-2 1 h post-treatment. Protein levels of VEGF were higher in Y-27632-treated embryos at 8 h following treatment, whereas no difference was seen in membranes. We hypothesize that administration of Y-27632 impairs vessel formation during angiogenesis, which may contribute to failure of VWB closure, causing VBWDs. Full article

This study aimed to isolate and freeze germ cells from the superior brown mealworm. Styrofoam diet changes were observed for 20 days to determine whether mealworms were useful insects for decomposing Styrofoam. The average weight of mealworms before the Styrofoam diet was 500 mg, which decreased to 336 mg at D20 after their diet. To preserve mealworms with excellent Styrofoam-degrading ability, we first isolated the reproductive organs of mealworms, testes, ovaries, sperms, and ovarioles. Morphologically, male and female adult brown mealworms were distinguished according to the presence or absence of a protrusion at the tip of the fifth segment of the abdomen. Sperms and ovarioles were observed in anatomically isolated testes and ovaries. We compared mechanical and enzymatic (collagenase I) methods to effectively isolate ovarioles from adult female brown mealworms. For the enzymatic method, most were torn and burst as the membrane of the ovarioles was damaged by collagenase I, unlike the mechanical method. To preserve the superior genetic resources of mealworms, we cryopreserved the ovaries of female brown mealworms using slow-freezing and vitrification. Histological analysis showed that the yolk sac was completely damaged in the ovaries after slow-freezing. However, only partial damage was achieved in the vitrification group compared to the control group (no freezing). The newly developed vitrification method with alginate-encapsulated ovarioles maintained the yolk sac in the ovarioles but was evenly distributed. These results provide basic data for reproductive studies of other useful insects and contribute to the biobanking and fertility preservation of superior mealworm germ cells and endangered insects. Full article

Melanoma cells secrete pro-angiogenic factors, which stimulates growth, proliferation and metastasis, and therefore are key therapeutic targets. Buddleja saligna (BS), and an isolated triterpenoid mixture (DT-BS-01) showed a fifty percent inhibitory concentration (IC₅₀) of 33.80 ± 1.02 and 5.45 ± 0.19 µg/mL, respectively, against melanoma cells (UCT-MEL-1) with selectivity index (SI) values of 1.64 and 5.06 compared to keratinocytes (HaCat). Cyclooxygenase-2 (COX-2) inhibition was observed with IC₅₀ values of 35.06 ± 2.96 (BS) and 26.40 ± 4.19 µg/mL (DT-BS-01). BS (30 µg/mL) significantly inhibited interleukin (IL)-6 (83.26 ± 17.60%) and IL-8 (100 ± 0.2%) production, whereas DT-BS-01 (5 µg/mL) showed 51.07 ± 2.83 (IL-6) and 0 ± 6.7% (IL-8) inhibition. Significant vascular endothelial growth factor (VEGF) inhibition, by 15.84 ± 4.54 and 12.21 ± 3.48%, respectively, was observed. In the ex ovo chick embryo yolk sac membrane assay (YSM), BS (15 µg/egg) significantly reduced new blood vessel formation, with 53.34 ± 11.64% newly formed vessels. Silver and palladium BS nanoparticles displayed noteworthy SI values. This is the first report on the significant anti-angiogenic activity of BS and DT-BS-01 and should be considered for preclinical trials as there are currently no US Food and Drug Administration (FDA) approved drugs to inhibit angiogenesis in melanoma. Full article

Parrot bornavirus (PaBV) might be transmitted vertically. Cockatiel embryonic brain cells and embryonated eggs of cockatiels (ECE) were infected with PaBV-2 and PaBV-4. In embryonic brain cells, PaBV-2 and PaBV-4 showed no differences in viral spread despite the slower growth of PaBV-2 compared with PaBV-4 in CEC-32 cells. ECE were inoculated with PaBV-4 and 13–14 dpi, organs were sampled for RT-PCR, immunohistochemistry/histology, and virus isolation. In 28.1% of the embryos PaBV-4-RNA and in 81.3% PaBV-4-antigen was detected in the brain. Virus isolation failed. Division of organ samples and uneven tissue distribution of the virus limited the results. Therefore, 25 ECE were inoculated with PaBV-4 (group 1) and 15 ECE with PaBV-2 (group 3) in the yolk sac, and 25 ECE were inoculated with PaBV-4 (group 2) and 15 eggs with PaBV-2 (group 4) in the chorioallantoic membrane to use the complete organs from each embryo for each examination method. PaBV-RNA was detected in the brain of 80% of the embryos in groups 1, 2, 3 and in 100% of the embryos in group 4. In 90% of the infected embryos of group 1, and 100% of group 2, 3 and 4, PaBV antigen was detected in the brain. PaBV antigen–positive brain cells were negative for anti-neuronal nuclear protein, anti-glial fibrillary acidic protein, and anti S-100 staining. Virus was not re-isolated. These results demonstrated a specific distribution pattern and spread of PaBV-4 and PaBV-2 in the brain when inoculated in ECE. These findings support a potential for vertical transmission. Full article

Background: The aim of this study was to analyze possible interactions of different xenogeneic collagen membranes (CM) and platelet-rich fibrin (PRF). PH values were evaluated in the CM rehydration process with PRF, and their influence on angiogenesis was analyzed in vivo. Materials and Methods: Porcine (Bio-Gide^®, Geistlich)- and bovine-derived collagen membranes (Symbios^®, Dentsply Sirona) were biofunctionalized with PRF by plotting process. PRF in comparison to blood, saline and a puffer pH7 solution was analysed for pH-value changes in CM rehydration process in vitro. The yolk sac membrane (YSM) model was used to investigate pro-angiogenic effects of the combination of PRF and the respective CM in comparison to native pendant by vessel in-growth and branching points after 24, 48 and 72 h evaluated light-microscopically and by immunohistochemical staining (CD105, αSMA) in vivo. Results: Significantly higher pH values were found at all points in time in PRF alone and its combined variants with Bio-Gide^® and Symbios^® compared with pure native saline solution and pH 7 solution, as well as saline with Symbios^® and Bio-Gide^® (each p < 0.01). In the YSM, vessel number and branching points showed no significant differences at 24 and 48 h between all groups (each p > 0.05). For PRF alone, a significantly increased vessel number and branching points between 24 and 48 h (each p < 0.05) and between 24 and 72 h (each p < 0.05) was shown. After 72 h, CM in combination with PRF induced a statistically significant addition to vessels and branching points in comparison with native YSM (p < 0.01) but not vs. its native pendants (p > 0.05). Summary: PRF represents a promising alternative for CM rehydration to enhance CM vascularization. Full article

Macrophage populations in most mammalian organs consist of cells of different origin. Resident macrophages originate from erythromyeloid precursors of the yolk sac wall; maintenance of the numbers of such macrophages in postnatal ontogenesis is practically independent of bone marrow haematopoiesis. The largest populations of the resident macrophages of embryonic origin are found in the central nervous system (microglia) and liver (Kupffer cells). In contrast, skin dermis and mucous membranes become predominantly colonized by bone marrow-derived monocytes that show pronounced functional and phenotypic plasticity. In the present study, we compared Kupffer cells and monocytes using the immunophenotype, gene expression profile, proteome, and pool of microRNA. The observed differences did not consider the resident liver macrophages as purely M2 macrophages or state that monocytes have purely M1 features. Monocytes show signs of high plasticity and sensitivity to pathogen-associated molecular patterns (e.g., high levels of transcription for Tlr 2, 4, 7, and 8). In contrast, the resident liver macrophages were clearly involved in the regulation of specific organ functions (nitrogen metabolism, complement system protein synthesis). Full article

Silver nanoparticles (AgNPs) are widely used in commercial applications as antimicrobial agents, but there have recently been increasing concerns raised about their possible environmental and health impacts. In this study, zebrafish embryos were exposed to two sizes of AgNP, 4 and 10 nm, through a continuous exposure from 4 to 96 h post-fertilisation (hpf), to study their uptake, impact and molecular defense responses. Results showed that zebrafish embryos were significantly impacted by 72 hpf when continuously exposed to 4 nm AgNPs. At concentrations above 0.963 mg/L, significant in vivo uptake and delayed yolk sac absorption was evident; at 1.925 mg/L, significantly reduced body length was recorded compared to control embryos. Additionally, 4 nm AgNP treatment at the same concentration resulted in significantly upregulated hypoxia inducible factor 4 (HIF4) and peroxisomal membrane protein 2 (Pxmp2) mRNA expression in exposed embryos 96 hpf. In contrast, no significant differences in terms of larvae body length, yolk sac absorption or gene expression levels were observed following exposure to 10 nm AgNPs. These results demonstrated that S4 AgNPs are available for uptake, inducing developmental (measured as body length and yolk sac area) and transcriptional (specifically HIF4 and Pxmp2) perturbations in developing embryos. This study suggests the importance of particle size as one possible factor in determining the developmental toxicity of AgNPs in fish embryos. Full article

Palythoa caribaeorum (class Anthozoa) is a zoantharian which, together with other cnidarians, like jellyfishes, hydra, and sea anemones, possesses specialized structures in its tissues, the cnidocytes, which deliver an array of toxins in order to capture prey and deter predators. The whole transcriptome of P. caribaeroum was deep sequenced, and a diversity of toxin-related peptide sequences were identified, and some retrieved for functional analysis. In this work, a peptide precursor containing a ShK domain, named PcShK3, was analyzed by means of computational processing, comprising structural phylogenetic analysis, model prediction, and dynamics simulation of peptide-receptor interaction. The combined data indicated that PcShK3 is a distinct peptide which is homologous to a cluster of peptides belonging to the ShK toxin family. In vivo, PcShK3 distributed across the vitelline membrane and accumulated in the yolk sac stripe of zebrafish larvae. Notably, it displayed a significant cardio-protective effect in zebrafish in concentrations inferior to the IC₅₀ (<43.53 ± 6.45 µM), while in high concentrations (>IC₅₀), it accumulated in the blood and caused pericardial edema, being cardiotoxic to zebrafish larvae. Remarkably, PcShK3 suppressed the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish. The present results indicated that PcShK3 is a novel member of ShK toxin family, and has the intrinsic ability to induce neuro- and cardio-protective effects or cause cardiac toxicity, according to its effective concentration. Full article