Fertilized Chicken Eggs Research Articles

Effects of histidin-2-ylidene vs. imidazol-2-ylidene ligands on the anticancer and antivascular activity of complexes of ruthenium, iridium, platinum, and gold

Couples of N-heterocyclic carbene complexes of ruthenium, iridium, platinum, and gold, each differing only in the carbene ligand being either 1,3-dimethylimidazol-2-ylidene (IM) or 1,3-dimethyl-N-boc-O-methylhistidin-2-ylidene (HIS), were assessed for their antiproliferative effect on seven cancer cell lines, their interaction with DNA, their cell cycle interference, and their vascular disrupting properties. In MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays only the platinum complexes were cytotoxic at single-digit micromolar IC50 concentrations with the (HIS)Pt complex being on average twice as active as the (IM)Pt complex. The former was highly efficacious against cisplatin-resistant HT-29 colon carcinoma cells where the latter had no effect. Both Pt complexes were accumulated by cancer cells and bound to double-helical DNA equally well. Only the (HIS)Pt complex modified the electrophoretic mobility of circular DNA in vitro due to the HIS ligand causing greater morphological changes to the DNA. Both platinum complexes induced accumulation of 518A2 melanoma cells in G2/M and S phase of the cell cycle. A disruption of blood vessels in the chorioallantoic membrane of fertilized chicken eggs was observed for both platinum complexes and the (IM)gold complex. The (HIS)platinum complex was as active as cisplatin in tumor xenografted mice while being tolerated better. We found that the HIS ligand may augment the cytotoxicity of certain antitumoral metal fragments in two ways: by acting as a transmembrane carrier increasing the cellular accumulation of the complex, and by initiating a pronounced distortion and unwinding of DNA. We identified a new (HIS)platinum complex which was highly cytotoxic against cancer cells including cisplatin-resistant ones.

Toxicity of different forms of graphene in a chicken embryo model.

In the present work, the toxicity of three forms of graphene: pristine graphene (pG), graphene oxide (GO), and reduced graphene oxide (rGO) was investigated using a chicken embryo model. Fertilized chicken eggs were divided into the control group and groups administered with pG, GO, and rGO, in concentrations of 50, 500, and 5000μg/ml. The experimental solutions were injected in ovo into the eggs, and at day 18 of incubation, the embryo survival, body and organ weights, the ultrastructure of liver samples, and the concentration of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the livers were measured. Survival of embryos decreased significantly after treatment with all types of graphene, but not in a dose-dependent manner. The body weights were only slightly affected by the highest doses of graphene, while the organ weights were not different among treatment groups. In all experimental groups, atypical hepatocyte ultrastructure and mitochondrial damage were observed. The concentration of the marker of DNA damage 8-OHdG in the liver significantly decreased after pG and rGO treatments. Further in vivo studies with different animal models are necessary to clarify the level of toxicity of different types of graphene and to estimate the concentrations appropriate to evaluate their biomedical applications and environmental hazard.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System.

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

Combretastatin A-4 derived 5-(1-methyl-4-phenyl-imidazol-5-yl)indoles with superior cytotoxic and anti-vascular effects on chemoresistant cancer cells and tumors

5-(1-Methyl-4-phenyl-imidazol-5-yl)indoles 5 were prepared and tested as analogs of the natural vascular-disrupting agent combretastatin A-4 (CA-4). The 3-bromo-4,5-dimethoxyphenyl derivative 5c was far more active than CA-4 with low nanomolar IC50 concentrations against multidrug-resistant KB-V1/Vbl cervix and MCF-7/Topo mamma carcinoma cells, and also against CA-4-resistant HT-29 colon carcinoma cells. While not interfering markedly with the polymerization of tubulin in vitro, indole 5c completely disrupted the microtubule cytoskeleton of cancer cells at low concentrations. It also destroyed real blood vessels, both in the chorioallantoic membrane (CAM) of fertilized chicken eggs and within tumor xenografts in mice, without harming embryo or mouse, respectively. Indole 5c was less toxic than CA-4 to endothelial cells, fibroblasts, and cardiomyocytes. In highly vascularized xenograft tumors 5c induced distinct discolorations and histological features typical of vascular-disrupting agents, such as disrupted vessel structures, hemorrhages, and extensive necrosis. In a first preliminary therapy trial, indole 5c retarded the growth of resistant xenograft tumors in mice. © 2016 Elsevier Science Ltd. All rights reserved.

Biochemical and molecular investigation of thermal manipulation protocols during broiler embryogenesis and subsequent thermal challenge.

BackgroundThe aim of the current study was to evaluate the effect of different thermal manipulation (TM) protocols during embryogenesis on thermotolerance acquisition parameters during subsequent thermal challenge (TC) at posthatch day 28. A total of 1500 fertile chicken eggs were divided randomly into five treatments (300 eggs each): control was maintained at 37.8 °C and 56 % relative humidity (RH) whereas, TM1, TM2, TM3 and TM4 were subjected to 38.5, 39, 39.5 and 40 °C for 18 h and 65 % RH daily during embryonic days ED 12–18. Hatched chicks from each treatment group allocated randomly into two sub-treatment groups (thermo-neutral, naïve (TN) and thermal challenge (TC). At day 28 of age, chicks subjected to TC by adjusting room temperature to 42 °C for 6 h while naïve chicks kept under regular conditions (22 ± 1 °C and 50–60 % RH). Chick’s response to TC evaluated by determination of plasma T3, T4, corticosterone, total proteins, albumin, selected enzymes and some electrolytes at the beginning (0 h) and after 1, 3 and 5 h of TC in TM and TN chicks. Furthermore, pectoral and thigh muscles mRNA expression of Atrogin-1, CK, avUCP, DIO3, DIO2 were evaluated in TC and TN sub-treatment groups.ResultsTM induced a significant reduction in free T3 and elevation in total proteins and albumin in plasma with significant down-regulation of Atrogin-1 and DIO2 and significant up-regulation of DIO3 mRNA expression in muscle of TM chicks compare to control. During TC at day 28, decrease in the concentrations of plasma free T3, total proteins and albumin with increase in T4 have been detected in control and TM chicks. TC induced up-regulation of Atrogin-1 and DIO3 with down-regulation of DIO2 gene expression in muscles of all TC chicks.ConclusionThe present study indicated that, TM improved thermotolerance acquisition by decreasing basal metabolic rate and muscle injury during thermal stress. Basal metabolic rate decreased via reduction of plasma T3 concentration with up and down regulation of expression of DIO3 and DIO2, respectively in muscles. Muscle injury protected by stimulation of protein biosynthesis and down-regulation of Atrogin-1 expression.

The cooling time of fertile chicken eggs at different stages of incubation

We asked whether or not the thermal characteristics of fertile avian eggs changed throughout incubation. The cooling and warming times, expressed by the time constant τ of the egg temperature response to a rapid change in ambient temperature, were measured in fertile chicken eggs at early (E7), intermediate (E11) and late (E20) stages of embryonic development. Same measurements were conducted on eggs emptied of their content and refilled with water by various amounts. The results indicated that (1) the τ of a freshly laid egg was ~50min; (2) τ decreased linearly with the drop in egg water volume; (3) the dry eggshell had almost no thermal resistance but its wet inner membrane contributed about one-third to the stability of egg temperature; (4) the egg constituents (yolk, albumen and embryonic tissues) and the chorioallantoic circulation had no measurable effect on τ; (5) the presence of an air pocket equivalent in volume to the air cell of fertile eggs reduced τ by about 3min (E7), 5min (E11) and 11min (E20). Hence, in response to warming the egg τ at E20 was slightly shorter than at E7. In response to cooling, the egg τ at E20 was similar to, or longer than, E7 because embryonic thermogenesis (evaluated by measurements of oxygen consumption during cold) offset the reduction in τ introduced by the air cell. In conclusion, until the onset of thermogenesis the thermal behavior of a fertile egg is closely approximated by that of a water-filled egg with an air volume equivalent to the air cell. It is possible to estimate the cooling τ of avian eggs of different species from their weight and incubation time.

Transcription Factor SomA Is Required for Adhesion, Development and Virulence of the Human Pathogen Aspergillus fumigatus.

The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ) in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus.

Thermal manipulation during broiler chicken embryogenesis: Effect on mRNA expressions of Hsp108, Hsp70, Hsp47 and Hsf-3 during subsequent post-hatch thermal challenge

Effects of thermal manipulation during broiler chicken embryonic days 12–18 on body temperature (Tb) and mRNA expressions of Hsp108, Hsp70, Hsp47 and Hsf-3 in muscle, heart and brain tissues during subsequent thermal challenge (TC) were investigated. Fertile chicken eggs were divided randomly into four groups (n=375): eggs in the control group were maintained at 37.8°C and 56% (RH). Eggs in TM1 group were subjected to TM at 39°C for 9h during ED 12–18. Eggs in the TM2 and TM3 groups were subjected to the same protocol of TM1 except for increasing the period of exposure to 12h and 18h, respectively. During TC (43°C for 6h) at days 10 and 28, Tb of TM chicks was significantly lower compared to controls. Furthermore, significant changes in mRNA expressions of Hsp108, Hsp70 and Hsp47 in muscle, heart and brain tissues were observed.

Development of collateral vessels: A new paradigm in CAM angiogenesis model

The chorioallantoic membrane (CAM) assay is one of the most widely used models to study angiogenesis. In this study, collateral vessel development is reported in CAM assay useful in analysis of angiogenesis. Four days old white Leghorn fertilized chicken eggs were inoculated with vehicle, standard or test angiogenesis inhibitor using standard protocol. Central vessel growth was seen tapering down and collateral vessels were developed from the lower side of the chorioallantoic membrane moving upward in 12days old standard or test treated CAMs. In the absence of the central vessel, collateral blood supply helped in survival of embryos. Hence, development of collateral vessels was used for ranking of blood vessels and angiogenesis in addition to well-known standard parameters related to central vessel. The finding could differentiate molecules inhibiting angiogenesis with or without collateralization which is crucial in anti-angiogenic therapy used for cardiovascular diseases and cancer.This study proposes a new avenue to distinguish pro-angiogenic molecules from anti-angiogenic ones as well as anti-angiogenic molecules which may or may not support alternative vascularization pathway that would have great impact on future angiogenic and anti-angiogenic therapy.

Angiogenic activity of latex from Euphorbia tirucalliLinnaeus 1753 (Plantae, Euphorbiaceae)

To assess the pro-angiogenic activity of Euphorbia tirucalli, commonly known as "avelós" plant, we performed a series of tests by applying an aqueous E. tirucalli latex solution (10 mg/mL) to the chorioallantoic membranes (CAMs) of 80 fertilized chicken eggs incubated in a temperature- and humidity-controlled automatic incubator. The results indicated that the aqueous latex solution increased vascular network formation compared to that with the negative control (p < 0.05) and the inhibitor control (p < 0.05). This suggests that under the experimental conditions tested, the aqueous latex solution induced an inflammatory response leading to neoangiogenesis.

Adjusting the DNA Interaction and Anticancer Activity of Pt(II) N-Heterocyclic Carbene Complexes by Steric Shielding of the Trans Leaving Group.

Five platinum(II) complexes bearing a (1,3-dibenzyl)imidazol-2-ylidene ligand but different leaving groups trans to it were examined for cytotoxicity, DNA and cell cycle interference, vascular disrupting properties, and nephrotoxicity. The cytotoxicity of complexes 3a-c increased with the steric shielding of their leaving chloride ligand, and complex 3c, featuring two triphenylphosphanes, was the most efficacious, with submicromolar IC50 concentrations. Complexes 3a-c interacted with DNA in electrophoretic mobility shift and ethidium bromide binding assays. The cationic complex 3c did not bind coordinatively to DNA but led to its aggregation, damage that is not amenable to the usual repair mechanisms. Accordingly, it arrested the cell cycle of melanoma cells in G1 phase, whereas cis-dichlorido[(1,3-dibenzyl)imidazol-2-ylidene](dimethyl sulfoxide) platinum(II) 3a induced G2/M phase arrest. Complex 3c also disrupted the blood vessels in the chorioallantoic membrane of fertilized chicken eggs. Ex vivo studies using precision-cut tissue slices suggested the nephrotoxicities of 3a-c to be clinically manageable.

LED measurement for development of a non-destructive detector of unsuitable chicken eggs in influenza vaccine production

In Japan, fertilised chicken eggs are used to produce influenza vaccines; however, some eggs die and then contaminate the vaccine stock solution, which causes large economic losses and raises health concerns. Therefore, a non-destructive test that allows distinction of normal and unsuitable chicken eggs was developed, first using visible and near-infrared (VNIR) spectroscopy and subsequently a light-emitting diode (LED) light source. Eggs were sampled from an egg farm, and each of the eggs categorised as either normal or unsuitable using candling. Linear discriminant analysis was applied using wavelength absorbance data to distinguish between normal and unsuitable eggs. All of the optical absorbance values of the normal eggs were found to be higher than those of unsuitable eggs. To reduce the production cost, LED lights and photodiodes were used as the light source and light receptors, respectively. A discrimination rate of 92.9% was obtained when LED was used as the light source.

The Effects of Fertilized Egg Yolk Isolates on Anabolic and Catabolic Signaling in Skeletal Muscle

Fortetropin, a bioactive ingredient derived from fertilized chicken egg yolk, augmented gains in muscle in trained humans compared to exercise alone. This study investigated the impact of Fortetropin on protein synthesis and protein degradation in rats after acute exercise. Male wistar rats were fed for 8 days with either 1.2 ml of tap water (control) or 0.39 g Fortetropin (MYOS Corp). Rats underwent an electrically-stimulated lower body unilateral exercise regimen. Muscles in the non-exercised and exercised limbs were harvested at 180 minutes following exercise and analyzed for gene and protein expression in myostatin, mTOR and ubiquitin signaling pathways. Activin IIB receptor mRNA was significantly lowered in the exercise + Fortetropin group, but not the exercise + control group indicating depression of the myostatin pathway via decreasing receptor expression (p<0.05). An increase in both Ubiquitin monomer protein expression and polyubiquitination were greater in the control + exercise, but not Fortetropin + exercise (p<0.05). The mRNA expression of Atrogin-1 was depressed in the Fortetropin + exercise compared to control + exercise (p<0.05). Phosphorylation status of 4EBP1, rp6, and p70S6K were increased for both groups. The Fortetropin + exercise resulted in elevations of these markers compared to the control (p<0.05). Fortetropin supplementation reduced mRNA expression of the myostatin receptor, Activin IIB. Fortetropin also appears to promote muscle growth via increasing anabolic (mTOR) and decreasing catabolic (ubiquitin) signaling.

The synergic effect of glycyrrhizic acid and low frequency electromagnetic field on angiogenesis in chick chorioallantoic membrane.

Much attention is paid to angiogenesis due to its mutual role in health and disease. Therefore, the effect of various chemical and physical agents on inhibition of this process has been recently studied. This study was conducted to investigate the synergic effect of glycyrrhizic acid and electromagnetic field on angiogenesis. In this experimental study, 44 Ross fertilized chicken eggs were randomly divided into four groups, one control and three experimental. Control group was kept with dimethyl sulfoxide on the eighth day, experimental group 1 treated with 200 gauss, 50 Hz electromagnetic field on the 10th day, experimental group 2 treated with 1 mg/ml glycyrrhizic acid on the eighth day, and experimental group 3 simultaneously treated with glycyrrhizic acid on the eighth day and electromagnetic field on the 10th day. On the 12th day, the images of chorioallantoic membrane samples were prepared using photostreomicroscope and the number and length of vessels were measured. The mean number of vessels in the experimental groups 1 and 3 (29.31±3.60 and 27.43±4.61, respectively) was not significantly different from that in the control group (29.11±4.76) (p>0.05). The length of vessels in the experimental groups 1 and 3 (52.35±3.25 mm and 54.94±4.70 mm, respectively) decreased significantly (p<0.05) compared with the control group (61.79±6.46 mm). In experimental group 2, both length and number of vessels (54.53±5.85 mm and 23.96±3.94) decreased significantly compared with the control group (p<0.05). Electromagnetic field and glycyrrhizic acid separately led to inhibition of angiogenesis. However, use of electromagnetic field accompanied with glycyrrhizic acid not only did not increase but also decreased the inhibitory effect.

PD33-11 METABOLIC MODULATION WITH DICHLOROACETATE REVERSES THE WARBURG EFFECT AND REDUCES THE GROWTH OF CLEAR CELL RENAL CELL CARCINOMA IN VITRO AND IN VIVO

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2015PD33-11 METABOLIC MODULATION WITH DICHLOROACETATE REVERSES THE WARBURG EFFECT AND REDUCES THE GROWTH OF CLEAR CELL RENAL CELL CARCINOMA IN VITRO AND IN VIVO Adam Kinnaird, Peter Dromparis, Roxane Paulin, Bruno Saleme, Trevor Stenson, Desmond Pink, John Lewis, and Evangelos Michelakis Adam KinnairdAdam Kinnaird More articles by this author , Peter DromparisPeter Dromparis More articles by this author , Roxane PaulinRoxane Paulin More articles by this author , Bruno SalemeBruno Saleme More articles by this author , Trevor StensonTrevor Stenson More articles by this author , Desmond PinkDesmond Pink More articles by this author , John LewisJohn Lewis More articles by this author , and Evangelos MichelakisEvangelos Michelakis More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2109AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clear Cell Renal Cell Carcinoma (ccRCC) exhibits a distinct metabolic phenotype using cytoplasmic glycolysis over a suppressed glucose oxidation (GO) even in normoxia (Warburg effect; WE). This offers growth advantages including suppressed apoptosis and increased proliferation and angiogenesis. Most ccRCC have constitutively active Hypoxia Inducible Factor (HIF) (due to mutation or silencing of the von Hippel Lindau tumor factor), which induces the WE by upregulating Pyruvate Dehydrogenase kinase (PDK) and thus inhibiting Pyruvate Dehydrogenase (PDH), the gate keeping enzyme in GO. PDK inhibitors like the clinically available small molecule Dichloroacetate (DCA) have been shown to increase GO and reverse the WE, decreasing growth of solid tumors. The resulting increase in mitochondrial signals (mitochondrial-derived reactive oxygen species and α-ketoglutarate) can inhibit the redox-sensitive HIF transcriptional activity (PMID: 22614004). We hypothesized that DCA will decrease ccRCC growth even in the presence of constitutively active HIF. METHODS The 786-O ccRCC cell line was used in vitro, in ovo (cells injected into the chorioallantoic membrane of fertilized chicken eggs) and in vivo in Nu/Nu mice, following both a prevention and reversal protocol and at doses similar to those achieved in the serum of DCA treated patients. RESULTS In vitro DCA increased PDH activity and GO, increased apoptosis, decreased proliferation and decreased HIF activity, including decreasing VEGF levels. siRNA inhibition of FIH prevented most of the DCA-induced decrease in HIF activity in an α-ketoglutarate dependent manner. DCA both prevented tumor growth and decreased tumor size in the reversal protocol: in ovo mean tumor weight 74mg control, 18mg DCA, n=20 and 29 eggs respectively, p<0.05; in vivo mean tumor volume 271mm3 control, 136mm3 DCA, n=10 and 20 respectively, p<0.01. DCA significantly decreased angiogenesis in vivo, measured by lectin imaging. CONCLUSIONS DCA can reverse the WE in ccRCC and decrease tumor growth and angiogenesis even though HIF is constitutively expressed. The DCA-induced inhibition of angiogenesis may be superior to VEGF inhibitors since it suppresses the HIF axis proximal to VEGF. We suggest that DCA should be tested in early phase clinical trials in ccRCC. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e713 Peer Review Report Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Adam Kinnaird More articles by this author Peter Dromparis More articles by this author Roxane Paulin More articles by this author Bruno Saleme More articles by this author Trevor Stenson More articles by this author Desmond Pink More articles by this author John Lewis More articles by this author Evangelos Michelakis More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Influenza virus propagation in embryonated chicken eggs.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 °C and 55-60% humidity for 10-11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 °C, the eggs are chilled for at least 4 hr at 4 °C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 °C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Nanoparticles of copper stimulate angiogenesis at systemic and molecular level.

Copper is a key element affecting blood vessel growth and muscle development. However, the ions released from Cu salts are toxic. Given their specific physicochemical properties, nanoparticles of Cu (NanoCu) may have different bioactivity and affect the development of blood vessel and muscles in a different manner than Cu salts. The objective of the study was to evaluate the influence of NanoCu on embryo development and angiogenesis at the systemic and molecular level, in experiments using a chick embryo model. Fertilized chicken eggs were divided into a control group, and groups injected with a placebo, CuSO4 or NanoCu. Embryo development at the whole body level and molecular indices using an embryo chorioallantoic membrane model were measured during embryogenesis. The present study indicated for the first time that NanoCu have pro-angiogenic properties at the systemic level, to a greater degree than CuSO4 salt. The properties of NanoCu were confirmed at the molecular level, demonstrating significant effects on mRNA concentration and on mRNA gene expression of all pro-angiogenic and pro-proliferative genes measured herein.

Anti-angiogenic effect of Nelumbo nucifera leaf extracts in human umbilical vein endothelial cells with antioxidant potential.

Nelumbo nucifera Gaertn (Nymphaeaceae) has long been used as a traditional herb in Chinese, Japanese, Indian, and Korean medicinal practices since prehistoric times and flourishes today as the primary form of medicine. This study reports for the first time the potent ability of N. nucifera leaf extracts to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro and in vivo, as well as their antioxidant efficacy in various scavenging models and an analysis of their chemical composition. In vivo anti-angiogenic activity was evaluated in a chick chorioallantoic membrane (CAM) model using fertilized chicken eggs, in human umbilical vein endothelial cells (HUVECs) by using cell viability, cell proliferation and tube formation assays, and by determining intracellular reactive oxygen species (ROS) in vitro. The antioxidant efficacy of N. nucifera leaf extracts was determined in various scavenging models, including total phenolic and flavonoid content. The chemical composition of N. nucifera leaf extracts was determined by GC-MS analysis, which revealed the presence of different phytochemicals. The IC50 values for the DPPH radical scavenging activities of water and methanol extracts were found to be 1699.47 and 514.36 μg ml−1, and their total phenolic and flavonoid contents were 85.01 ± 2.32 and 147.63 ± 2.23 mg GAE g dry mass−1 and 35.38 ± 1.32 and 41.86 ± 1.07 mg QA g dry mass−1, respectively. N. nucifera leaf extracts (10–100 μg ml−1) exhibited significant dose-dependent inhibition of VEGF-induced angiogenesis, as well as VEGF-induced proliferation and tube formation in HUVECs. In this study, N. nucifera leaf extracts displayed potent antioxidant and inhibitory effects on VEGF-induced angiogenesis. N. nucifera exerted an inhibitory effect on VEGF-induced proliferation and tube formation, as well as CAM angiogenesis in vivo. Moreover, N. nucifera leaf extracts significantly blocked VEGF-induced ROS production in HUVECs, confirming their possible anti-angiogenic mechanism.

Development and efficacy of a novel live-attenuated QX-like nephropathogenic infectious bronchitis virus vaccine in China

In this study, we attenuated a Chinese QX-like nephropathogenic infectious bronchitis virus (IBV) strain, YX10, by passaging through fertilized chicken eggs. The 90th passage strain (YX10p90) was selected as the live-attenuated vaccine candidate strain. YX10p90 was found to be safe in 7-day-old specific pathogen free chickens without induction of morbidity or mortality. YX10p90 provided nearly complete protection against QX-like (CH I genotype) strains and partial protection against other two major Chinese genotype strains. YX10p90 also showed no reversion to virulence after five back passages in chickens. An IBV polyvalent vaccine containing YX10p90 was developed and showed that it could provide better protection against major Chinese IBV virulent strains than commercial polyvalent vaccines. In addition, the complete genome sequence of YX10p90 was sequenced. Multiple-sequence alignments identified 38 nucleotide substitutions in the whole genome which resulted in 26 amino acid substitutions and a 110-bp deletion in the 3′ untranslated region. In conclusion, the attenuated YX10p90 strain exhibited a fine balance between attenuation and immunogenicity, and should be considered as a candidate vaccine to prevent infection of Chinese QX-like nephropathogenic IBV.

Ethical euthanasia and short-term anesthesia of the chick embryo.

Fertilized chicken eggs are suggested as an alternative to mammalian models. The chorioallantoic membrane (CAM) of the chick embryo is widely used for examination of angiogenesis, xenotransplants and for virus production. Unfortunately, it is mostly not taken into account, that the chick embryo's ability to experience pain starts to develop at day 7 of breeding. In our view, this model is only in accordance with the 3 R principles, if an appropriate anesthesia of the chick embryo in potentially painful procedures is provided. Although many experimental approaches are performed on the none-innervated CAM, the euthanasia of the embryo strongly requires a more human technique than the usually used freezing at -20°C, decapitation or in ovo fixation with paraformaldehyde without prior anesthesia. However, protocols regarding feasible and ethical methods for anesthesia and euthanasia of avian embryos are currently not available. Therefore, we established an easy and reliable method for the euthanasia and short-term anesthesia of the chick embryo.