Modulation of the oxidative damage, inflammation, and apoptosis-related genes by dicinnamoyl-L-tartaric acid in liver cancer.

Cancer cells can become resistant to existing treatments over time, so it is important to develop new treatments that target different pathways to stay ahead of this resistance. Many cancer treatments have severe side effects that can be debilitating and even life-threatening. Developing drugs that can effectively treat cancer while minimizing the risks of these side effects is essential for improving the quality of life of cancer patients. The study was designed to explore whether the combination of dicinnamoyl-L-tartaric (CLT) and sorafenib ((SOR), an anti-cancer drug)) could be used to treat hepatocellular carcinoma (HCC) in the animal model and to assess whether this combination would lead to changes in certain biomarkers associated with the tumour. In this study, 120 male mice were divided into 8 groups of 15 mice each. A number of biochemical parameters were measured, including liver functions, oxidative stress (malondialdehyde, (MDA); nitric oxide (NO)), and antioxidative activity (superoxide dismutase (SOD), and glutathione peroxidase (GPx)). Furthermore, the hepatic expressions of Bax, Beclin1, TNF-α, IL1ß, and BCl-2 genes were evaluated by qRT-PCR. The combination of SOR and CLT was found to reduce the levels of liver enzymes, such as AST, ALT, ALP, and GGT, and reduce the pathological changes caused by DAB and PB. The upregulation of TNF-α, IL1ß, and Bcl-2 genes suggests that the CLT was able to initiate an inflammatory response to combat the tumor, while the downregulation of the Bax and Beclin1 genes indicates that the CLT was able to reduce the risk of apoptosis in the liver. Furthermore, the combination therapy led to increased expression of cytokines, resulting in an enhanced anti-tumor effect.

Amygdalin potentiates the anti-cancer effect of Sorafenib on Ehrlich ascites carcinoma and ameliorates the associated liver damage.

The burden of cancer diseases is increasing every year, therefore, the demands to figure out novel drugs that can retain antitumor properties have been raised. This study aimed to investigate the anti-tumor properties of amygdalin (Amy) against Ehrlich ascites carcinoma (EAC) bearing mice and its protective properties against liver damage. Amy and the standard anticancer drug Sorafenib (Sor) were given alone or in combination to Swiss albino female mice that had been injected with EAC cells. Biochemical parameters of liver function (AST, ALT, GGT, total protein, albumin), tumor volume, oxidative stress [malondialdehyde, (MDA)] and antioxidative [superoxide dismutase (SOD), and reduced glutathione (GSH)] markers were measured. The hepatic expression of the antioxidant-related gene [nuclear factor erythroid-2-related factor 2 (Nrf2)], the migration-related gene [matrix metalloprotease 9 (MMP9)], and the angiogenesis-related gene [vascular endothelial growth factor (VEGF)] were evaluated by qPCR. The results revealed that EAC-bearing mice treated with Amy and/or Sor showed a decrease in the tumor burden and hepatic damage as evidenced by (1) decreased tumor volume, number of viable tumor cells; (2) increased number of dead tumor cells; (3) restored the liver function parameters; (4) reduced hepatic MDA levels; (5) enhanced hepatic GSH and SOD levels; (6) upregulated expression of Nrf2; (7) downregulated expression of MMP9 and VEGF, and (8) improved hepatic structure. Among all treatments, mice co-treated with Amy (orally) and Sor (intraperitoneally) showed the best effect. With these results, we concluded that the Amy improved the antitumor effect of Sor and had a protective role on liver damage induced by EAC in mice.

Ruthenium(II)/(III) DMSO-Based Complexes of 2-Aminophenyl Benzimidazole with In Vitro and In Vivo Anticancer Activity.

New anticancer ruthenium(II/III) complexes [RuCl2(DMSO)2(Hapbim)] (1) and [RuCl3(DMSO) (Hapbim)] (2) (Hapbim = 2-aminophenyl benzimidazole) have been synthesized and characterized, and their chemotherapeutic potential evaluated. The interaction of the compounds with DNA was studied by both UV-Visible and fluorescence spectroscopies, revealing intercalation of both the Hapbim ligand and the Ru complexes. The in vitro cytotoxicity of the compounds was tested on human breast cancer (MCF7), human colorectal cancer (Caco2), and normal human liver cell lines (THLE-2), with compound (2) the most potent against cancer cells. The cytotoxic effect of (2) is shown to correlate with the ability of the Ru(III) complex to induce apoptosis and to cause cell-cycle arrest in the G2/M phase. Notably, both compounds were inactive in the noncancerous cell line. The anticancer effect of (2) has also been studied in an EAC (Ehrlich Ascites Carcinoma) mouse model. Significantly, the activity of the complex was more pronounced in vivo, with removal of the cancer burden at doses that resulted in only low levels of hepatotoxicity and nephrotoxicity. An apoptosis mechanism was determined by the observation of increased Bax and caspase 3 and decreased Bcl2 expression. Furthermore, (2) decreased oxidative stress and increased the levels of antioxidant enzymes, especially SOD, suggesting the enhancement of normal cell repair. Overall, compound (2) shows great potential as a chemotherapeutic candidate, with promising activity and low levels of side effects.

Combination therapy with melatonin, stem cells and extracellular vesicles is effective in limiting renal ischemia-reperfusion injury in a rat model.

OBJECTIVE: To evaluate the therapeutic value of melatonin, mesenchymal stem cells and their extracellular vesicles, exosomes, on renal ischemia-reperfusion. METHODS: Female albino rats (n = 64) were divided into eight groups (n = 8 per group): control, sham (only laparotomy), renal ischemia-reperfusion (renal ischemia-reperfusion + phosphate-buffered saline), melatonin (renal ischemia-reperfusion + melatonin), mesenchymal stem cells (renal ischemia-reperfusion + mesenchymal stem cells), exosomes (renal ischemia-reperfusion + exosomes), melatonin + mesenchymal stem cells (renal ischemia-reperfusion + melatonin + mesenchymal stem cells) and melatonin + exosomes (renal ischemia-reperfusion + melatonin + exosomes). After the establishment of the renal ischemia-reperfusion model, rats in each group were bilaterally injected once with either mesenchymal stem cells or exosomes in both renal arteries during reperfusion. RESULTS: Notable improvement of renal ischemia-reperfusion was obtained after different treatments, as evidenced by a lower histopathological score of kidney injury; decreased serum levels of urea, creatinine and retinol-binding protein; reduced lipid peroxidation marker malondialdehyde; increased superoxide dismutase and catalase activities; reduced apoptosis (lower DNA damage and B-cell lymphoma 2-associated X protein, and higher B-cell lymphoma 2 genes/proteins); and inhibition of kidney inflammatory and damage markers (tumor necrosis alpha, interleukin-1ß, nuclear factor kappa B, kidney injury molecule-1, IL-18, matrix metalloproteinase 9, neutrophil gelatinase-associated lipocalin). The improvement order was (highest to lowest): melatonin + exosomes, melatonin + mesenchymal stem cells, exosomes, mesenchymal stem cells and melatonin group. CONCLUSIONS: Our data suggest a potential therapeutic effect of combined therapy with melatonin, mesenchymal stem cells and their exosomes to minimize renal ischemia-reperfusion injury in rats.

Effect of pristine and functionalized multiwalled carbon nanotubes on rat renal cortex.

The increasing application of carbon nanotubes (CNTs) within environmental, occupational and consumer settings has raised concerns regarding their biosafety and adverse effects on human health. The present study was designed to investigate the possible adverse effect of pristine and functionalized (amylated and polyethelene glycol coated) multi-walled (MW) CNTs on rat kidney with special concern to the histological alterations and the associated oxidative stress, apoptosis and inflammation. Healthy male albino rats (n = 40) were randomly divided into 4 groups: group I (control), group II (pristine MWCNTs), group III (amylated MWCNTs) and group IV [polyethelene glycol (PEG)-coated MWCNTs]. Animals of groups II, III and IV received a single dose of 1 mg/kg body weight of MWCNTs via intra-tracheal (IT) instillation at the beginning of the experiment and all rats were sacrificed after 30 days. Rats in groups II and III showed, nearly similar, renal tissue damage (evidenced by thin collapsed glomeruli, packed mesangial and endothelial cells as well as edematous hemorrhagic glomeruli with apoptotic changes) and functional disruptions (indicated by high serum levels of urea and creatinine) probably through induction of oxidative stress [revealed by high level of the lipid peroxidation marker malondialdehyde (MDA) and lower levels of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx)], apoptosis (indicated by high caspase 3 activity), and inflammation (evidenced by high level of IL1ß). However, PEG-coated MWCNTs-treated group (group IV) showed nearly normal renal structure and function. It could be concluded that pristine and functionalized amylated MWCNTs have nephrotoxic effect, while PEG-coated MWCNTs had lowest, or none, toxic effects making them safer for therapy and diagnosis of a variety of diseases.

Dietary propionic acid enhances antibacterial and immunomodulatory effects of oxytetracycline on Nile tilapia, Oreochromis niloticus.

This study was carried out to evaluate the potential antibacterial and immunomodulatory effects of the dietary acidifier propionic acid (PA) when given alone or in combination with oxytetracycline (OTC) on Nile tilapia (Oreochromis niloticus). Apparently healthy O. niloticus (n = 240; 52 ± 3.75 g) were randomly allocated into four equal groups (n = 60/group): control group fed a basal diet alone and the other three groups fed basal diets supplemented with either PA (200 mg /kg of diet, PA group) or OTC (500 mg/kg of diet, OTC group) alone or in combination (PA + OTC group). Each group was subdivided into two subgroups (n = 30/subgroup, each subgroup had triplicate of 10 fish); subgroup (A) was used to evaluate the antibacterial effects with the aforementioned 2 weeks feeding regime, and subgroup (B) was used to evaluate the immunomodulatory effects against Aeromonas hydrophila infection with similar 2 weeks feeding regime. Among the four groups, PA + OTC group showed the highest significant (p < 0.0001) antibacterial activity as indicated by widest inhibition zones against A. hydrophila and lowest total gastrointestinal bacterial counts. Additionally, this group had the best immunomodulatory effect as noticed by a significant (p < 0.05) increase in total serum protein, globulin, IgM, phagocytic activity and index, lysosome activity, and significant (p < 0.05) upregulation in the expression levels of immunity-related genes (MHC I, MHC IIA, MHC IIB, Tlr7, IgM heavy chain, TNFα, and IL1ß) in head-kidney. Notably, the combined dietary PA and OTC improved the hematological parameters and reduced the oxidative damage of hepatopancreas and head-kidney induced by OTC. This data suggests dietary PA as potential adjuvant to OTC in O. niloticus diets to get maximal antibacterial and immunomodulatory effects.

Hesperidin, piperine and bee venom synergistically potentiate the anticancer effect of tamoxifen against breast cancer cells.

Despite advances in cancer treatment, breast cancer remains one of the main life threatening diseases in women. Most anti-breast cancer drugs cause severe health complications and multidrug resistance. Although, some natural products, such as hesperidin (Hes), piperine (Pip) and bee venom (BV), showed anti-breast cancer effect when used separately, their combined effect together or with the anti-cancer drug tamoxifen (Tam) has not yet been studied. Herein, we hypothesized that these three natural products could potentiate the therapeutic effect of Tam when used together. First, we studied the cytotoxic effect of Hes, Pip, and BV on MCF7 and T47D cells using MTT assay and found reasonable IC50 comparable to that of Tam. Second, we checked the effect of all combinations (n = 67 for each cell line, prepared as non-constant ratio from fractions of IC50 of the four compounds) and found enhanced anti-proliferative effects on MCF7 and T47D and synergistic effect, revealed by combination index (CI) values below one. Next, the best 5 combinations with lowest Tam doses and CI but with highest cell death were selected for further molecular analysis in comparison to single-drug treatment. All single- and combined-treated groups showed a significant increase in apoptosis (indicated by upregulated mRNA level of the pro-apoptotic marker Bax and downregulated mRNA level of the anti-apoptotic marker Bcl2) and a significant decrease in mRNA level of the two breast cancer related receptors EGFR and ERα, with the best effect in combined groups especially that contained the 4 compounds, as compared to vehicle-treated group. Moreover, Pip, BV and all combinations, except Tam + Hes group, arrested MCF7 and T47D in G2/M phase of cell cycle, while Tam and/or Hes caused G0/G1 phase arrest. These results indicate that Hes, Pip and BV synergistically enhance the anti-cancer effect of Tam and could be used as safe adjuvant/vehicle to Tam in treatment of breast cancer after further confirmatory in vivo investigations.

Therapeutic potential effect of bone marrow-derived mesenchymal stem cells on chronic liver disease in murine Schistosomiasis Mansoni.

Some reports have shown that mesenchymal stem cells (MSCs) therapy could ameliorate chemically-induced hepatic fibrosis. This research assesses the therapeutic action of bone marrow mesenchymal stem cells (BM-MSCs) on chronic diseased liver in Schistosoma mansoni infected mice. All infected female mice divided into three groups, one group (15 mice) treated with oral praziquantel (PZQ), second group (15 mice) received intravenous injection of BM-MSCs and third group (15 mice) treated with both MSCs + PZQ. Two control groups (15 mice each) subdivided into one infected and second healthy one. BM-MSCs were obtained from bones of both femur and tibia of male mice (30 mice), then cultured and characterized morphologically by detection of CD105 by flow cytometer. Liver tissues for all groups were examined histopathologically. Measuring of the collagen 1 gene expression was done by real-time PCR and immunohistochemical study to detect stem cells differentiation for detection of MSCs engraftments in liver tissue. MSCs treatment caused marked improvement and regression of fibrosis, and prevents deposition of collagen and reduced the expression of collagen 1 gene in infected mice on their liver tissues, especially when used with PZQ in mice treatment. It can be concluded that, MSCs is a good therapeutic method for liver fibrosis caused by S. mansoni infection.

Ameliorative Effect of Cardamom Aqueous Extract on Doxorubicin-Induced Cardiotoxicity in Rats.

This study was conducted to evaluate the potential cardioprotective effect of cardamom (CAR) against myocardial injuries induced by doxorubicin (DOX) in rats through investigation of histological alterations and the associated oxidative stress, apoptosis, inflammation, and angiogenesis. This study included 30 adult male albino rats that were randomized to 3 groups (n = 10/group): group I (control), group II (DOX) rats injected with DOX (2.5 mg/kg body weight [BW] i.p.) every other day for 2 weeks, and group III (CAR+DOX) received CAR extract (200 mg/kg BW) orally for 3 weeks, and 1 week later (starting from the 2nd week) they were injected with DOX (2.5 mg/kg BW i.p.) every other day for 2 weeks. Rats treated with DOX alone exhibited notable myocardial damage (discontinuity and disorganization of cardiac muscle fibers, mononuclear cell infiltration, and apparent increases in collagen fiber deposition) accompanied by loss of function (revealed by elevated serum levels of lactate dehydrogenase, creatine kinase, and cardiac troponin), induction of oxidative stress (indicated by higher levels of nitric oxide and malon-dialdehyde, and lower levels of superoxide dismutase, catalase, and glutathione peroxidase), apoptosis (evidenced by high caspase 3 activity and immunostaining), and inflammation (marked by high cardiac NFκB level). However, administration of CAR not only ameliorated all deleterious effects of DOX but also induced angiogenesis, as indicated by a significant increase in VEGF immunoreactivity. These data indicate that CAR could relieve DOX-induced cardiotoxicity, at least in part, via reductions in oxidative stress, apoptosis, and inflammation and increased tissue regeneration via induction of angiogenesis. Therefore, CAR could be a promising cytoprotective agent against DOX cardiotoxicity.

Evolutionary insights into the fusion protein of Newcastle disease virus isolated from vaccinated chickens in 2016 in Egypt.

Newcastle disease virus (NDV) infections are one of the most devastating causes of economic losses in the poultry industry and despite extensive vaccination, outbreaks are being reported around the globe especially from developing and tropical countries. Analysis of NDV field strains from vaccinated flocks would highlight essential areas of consideration not only to design effective immunization strategies but also to devise vaccines that provide sterile immunity. For this purpose, 91 NDV suspected outbreaks were investigated and screened for NDV genetic material. A total of 16 NDV-positive isolates were examined using biological, genetics and bioinformatics analysis to assess the epidemiological association and to identify motifs that are under vaccine-induced immune pressures. In line with the clinical outcomes, all isolates showed the 112RRQKR|F117 cleavage motif and phylogenetic analysis revealed grouping of isolates into the genotype VII, and specifically sub-genotype VIId. Further analysis of the putative fusion protein sequence showed a number of substitutions (n=10) in functionally important domains and based on these differences, the studied isolates could be categorized into four distinct groups (A-D). Importantly, two residues (N30 and K71) were conserved in the commercial live vaccine and Egyptian field strains that are present in class II, genotype II. Collectively, these data enhance our knowledge of the evolution of genotype VIId NDV under the vaccine-induced immune pressures. In addition, our findings suggest that the use of genotype II-type vaccines in Egypt may be implicated in the emergence of new variants rather than providing benefits against NDV infections.

Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression.

Despite widespread use of bromuconazole as a pesticide for food crops and fruits, limited studies have been done to evaluate its toxic effects. Here, we evaluated the hepatotoxic effect of bromuconazole using classical toxicological (biochemical analysis and histopathological examination) and gene-based molecular methods. Male rats were treated either orally or topically with bromuconazole at doses equal to no observed adverse effect level (NOAEL) and 1/10 LD50 for 90 d. Bromuconazole increased activities of liver enzymes (ALT, AST, ALP, and ACP), and levels of bilirubin. It also induced hepatic oxidative stress as evidenced by significant decrease in the activities of superoxide dismutase (SOD), and significant increase in levels of malondialdehyde (MDA) in liver. In addition, bromuconazole caused an increase in liver weights and necrobiotic changes (vacuolation and hepatocellular hypertrophy). It also strongly induced the expression of PXR and its downstream target CYP3A1 gene as well as the activity of CYP3A1. However, it inhibited the expression of CAR and its downstream target CYP2B1 gene without significant changing in CYP2B1 activity. Overall, the oral route showed higher hepatotoxic effect and molecular changes than the dermal route and all changes were dose dependent. This is the first investigation to report that bromuconazole-induced liver oxidative damage is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1.

Is really endogenous ghrelin a hunger signal in chickens? Association of GHSR SNPs with increase appetite, growth traits, expression and serum level of GHRL, and GH.

Chicken growth hormone secretagogue receptor (GHSR) is a receptor for ghrelin (GHRL), a peptide hormone produced by chicken proventriculus, which stimulates growth hormone (GH) release and food intake. The purpose of this study was to search for single nucleotide polymorphisms (SNPs) in exon 2 of GHSR gene and to analyze their effect on the appetite, growth traits and expression levels of GHSR, GHRL, and GH genes as well as serum levels of GH and GHRL in Mandara chicken. Two adjacent SNPs, A239G and G244A, were detected in exon 2 of GHSR gene. G244A SNP was non-synonymous mutation and led to replacement of lysine amino acid (aa) by arginine aa, while A239G SNP was synonymous mutation. The combined genotypes of A239G and G244A SNPs produced three haplotypes; GG/GG, GG/AG, AG/AG, which associated significantly (P<0.05) with growth traits (body weight, average daily gain, shank length, keel length, chest circumference) at age from >4 to 16w. Chickens with the homozygous GG/GG haplotype showed higher growth performance than other chickens. The two SNPs were also correlated with mRNA levels of GHSR and GH (in pituitary gland), and GHRL (in proventriculus and hypothalamus) as well as with serum level of GH and GHRL. Also, chickens with GG/GG haplotype showed higher mRNA and serum levels. This is the first study to demonstrate that SNPs in GHSR can increase appetite, growth traits, expression and level of GHRL, suggesting a hunger signal role for endogenous GHRL.

Pineal gland plays a role in gonadal development after eyelids separation in puppies / La glándula pineal juega un rol en el desarrollo gonadal luego de la separación de los párpados en cachorros

Several functional and morphological studies have been conducted on the pineal gland in many mammalian species; however, no published reports are available on the role of pineal gland on the gonadal development before and after eyelids separation in puppies. Therefore, this study aimed to trace the postnatal histo-morphological changes in the pineal gland and gonads of puppies before (2, 10 and 11 days old) and after (25, 35 and 40 days old) eyelids separation in an attempt to investigate the possible role of pineal gland on the gonadal development. In general, the differentiation of pineal cells, interstitial endocrine cells of testes and stromal ovarian cells coincides with the start of eyelids separation in puppies. Histological examination of stained pineal and gonadal slices of puppies after eyelids separation revealed a remarkable differentiation of pinealocytes and testicular interstitial endocrine cells, as well as presence of some evidence of folliculogenesis in ovary. Surprisingly, melatonin receptor (MT1) protein expression levels were significantly increased in the ovaries and testes of puppies after eyelids separation. Moreover, the mRNA and protein expression of AANAT, a rate-limiting enzyme in melatonin biosynthesis, was notably increased in the pineal gland of opened eyes puppies. Our results suggest an increase of melatonin production from the pineal gland of opened eyes puppies and this could play a vital role in the developmental changes observed in the gonads of these puppies.

Diversos estudios morfológicos y funcionales han sido realizados sobre la glándula pineal en distintas especies de mamíferos. Sin embargo, no hay informes publicados acerca del rol de la glándula pineal en el desarrollo gonadal antes y después de la separación de los párpados en cachorros. Este estudio tuvo como objetivo trazar los cambios histo-morfológicos postnatales en la glándula pineal y las gónadas de los cachorros antes (2, 10 y 11 días de edad) y después (25, 35 y 40 días de edad) de la separación de los párpados, en un intento por investigar el posible rol de la glándula pineal en el desarrollo gonadal. En general, la diferenciación de los pinealocitos, células intersticiales endocrinas de los testículos y las células estromales del ovario coincide con el inicio de la separación de los párpados en cachorros. El examen histológico de glándula pineal y los cortes gonadales de los cachorros, después de la separación de los párpados, reveló una notable diferenciación de los pinealocitos y las células intersticiales endocrinas testiculares, así como la posible evidencia de foliculogénesis en el ovario. Sorprendentemente, en el receptor de melatonina (MT1) los niveles de expresión de proteínas fueron significativamente superiores en los ovarios y los testículos de los cachorros después de la separación de los párpados. Además, el ARNm y la expresión de la proteína AANAT, una enzima limitante de la velocidad en la biosíntesis de la melatonina, aumentaron notablemente en la glándula pineal de los cachorros con los ojos abiertos. Nuestros resultados sugieren que existe un aumento de la producción de melatonina por parte de la glándula pineal en los cachorros con los ojos abiertos, lo que podría jugar un rol vital en los cambios evolutivos observados enlas gónadas de estos cachorros.

Human peripheral blood CD34+ cells attenuate oleic acid-induced acute lung injury in rats.

BACKGROUND AIMS: Adult stem cell-based therapy is a promising novel approach for treatment of acute lung injury (ALI). In this study, we evaluated the therapeutic effect of isolated human peripheral blood CD34+ progenitor cells in an ALI rat model, induced by oleic acid (OA) injection. METHODS: Seventy-five adult female rats were used in this study. Group A, control without treatment, and group B, control injected with phosphate-buffered saline, comprised 15 rats each; the remaining 45 rats were injected with OA to induce ALI and were further subdivided into 3 groups: group C (ALI group, 15 rats), group D (ALI and fibroblast group, 15 rats) and group E (ALI and CD34+ cell group, 15 rats). RESULTS: CD34+ cells transplantation in rats with OA-induced lung injury improves the arterial PaO(2) and wet/dry ratio, reduces infiltration of inflammatory cells and decreases lung vascular permeability as determined by reduced intra-alveolar and interstitial patchy congestion and hemorrhage as well as decreased interstitial edema. Additionally, lung inflammation determined by expression of the pro-inflammatory mediators intercellular adhesion molecule 1 and tumor necrosis factor-α was attenuated in CD34+ cell-treated rats at 6, 24 and 48 h post-OA challenge compared with non-treated rats. Moreover, the expression of anti-inflammatory molecule interleukin-10 was up-regulated in the lung of OA-induced ALI rats after administration of CD34+ cells. The important finding was that human TNF-α-induced protein 6 (TSG-6) gene expression was significantly up-regulated in rats treated with CD34+ cells. CONCLUSIONS: The freshly isolated human peripheral blood-derived CD34+ cells may be used as an important source of stem cells that improve ALI. The anti-inflammatory properties of CD34+ cells in the lung are explained, at least in part, by activation of CD34+ cells to express TSG-6.

Shh regulates chick Ebf1 gene expression in somite development.

The chick early B-cell factor 1 (cEbf1) is a member of EBF family of helix loop helix transcription factors. Recently, we have proved that cEbf1 expression in feather is regulated by Shh. It is therefore possible that the somitic expression of cEbf1 is controlled by Shh signals from the notochord. To assess this hypothesis, the expression profile of cEbf1 was first detailed in somites of chick embryos (from HH8 to HH28). cEbf1 expression was mainly localised in the medial sclerotome and later around the vertebral cartilage anlagen of body and pedicles. Tissue manipulations (notochord ablation) and Shh gain and loss of function experiments were then performed to analyse whether the notochord and/or Shh regulate cEbf1 expression. Results from these experiments confirmed our hypothesis that the medial somitic expression of cEbf1 is regulated by Shh from the notochord. In conclusion, cEbf1 gene is considered as a medial sclerotome marker, downstream to and regulated by the notochord derived Shh, which may be functionally involved in somitogenesis.

The effect of RA on the chick Ebf1-3 genes expression in somites and pharyngeal arches.

Expression of chick early B cell factor 1-3 (cEbf1-3) genes in regions of high retinoic acid (RA) activity, such as somites and pharyngeal arches (PAs), and regulation of other EBF members by RA raise the possibility that the internal cue RA may regulate cEbf1-3 expression in these tissues. To check this possibility, RA gain and loss of function experiments were conducted. Ectopic expression of RA led to up-regulation of cEbf2, 3 but did not change cEbf1 expression in somites. Expectedly, inhibition of RA by disulfiram resulted in downregulation of cEbf2, 3, but did not change cEbf1 expression in somites. The same RA gain and loss of function experiments did not change cEbf1-3 expression in PAs. However, ectopic expression of RA in the cranial neural tube before migration of neural crest cells downregulated cEbf1, 3 and up-regulated cEbf2 expression in the PAs. The same experiment, but with application of disulfiram, resulted in downregulation of cEbf2, but did not alter the expression of the other two genes. We conclude that the three cEbf genes act differently in response to RA signals in somitic mesoderm. cEbf1 may be not RA dependant in somites; however, the other two cEbf genes positively respond to RA signalling in somites. Additionally, only the migratory cEbf-expressing cells into the PAs are affected by RA signals.

Association of a novel SNP in exon 10 of the IGF2 gene with growth traits in Egyptian water buffalo (Bubalus bubalis).

Insulin-like growth factor 2 (IGF2) plays an important role in muscle growth and it might be used as a marker for the growth traits selection strategies in farm animals. The objectives of this study were to detect polymorphisms in exon 10 of IGF2 and to determine associations between these polymorphisms and growth traits in Egyptian water buffalo. PCR-single-strand conformation polymorphism (SSCP) and DNA sequencing methods were used to detect any prospective polymorphism. A novel single nucleotide polymorphism (SNP), C287A, was detected. It was a non-synonymous mutation and led to replacement of glutamine (Q) amino acid (aa) by histidine (H) aa. Three different SSCP patterns were observed: AA, AC, and CC, with frequencies of 0.540, 0.325, and 0.135, respectively. Association analyses revealed that the AA individuals had a higher average daily gain (ADG) than other individuals (CC and AC) from birth to 9 months of age. We conclude that the AA genotype in C287A SNP in the exon 10 of the IGF2 gene is associated with the ADG during the age from birth to 9 months and could be used as a potential genetic marker for selection of growth traits in Egyptian buffalo.

Effects of a novel SNP of IGF2R gene on growth traits and expression rate of IGF2R and IGF2 genes in gluteus medius muscle of Egyptian buffalo.

Insulin-like growth factor 2 receptor (IGF2R) is responsible for degradation of the muscle development initiator, IGF2, and thus it can be used as a marker for selection strategies in the farm animals. The aim of this study was to search for polymorphisms in three coding loci of IGF2R, and to analyze their effect on the growth traits and on the expression levels of IGF2R and IGF2 genes in the gluteus medius muscle of Egyptian buffaloes. A novel A266C SNP was detected in the coding sequences of the third IGF2R locus (at nucleotide number 51 of exon 23) among Egyptian water buffaloes. This SNP was non-synonymous mutation and led to replacement of Y (tyrosine) amino acid (aa) by D (aspartic acid) aa. Three different single-strand conformation polymorphism patterns were observed in the third IGF2R locus: AA, AC, and CC with frequencies of 0.555, 0.195, and 0.250, respectively. Statistical analysis showed that the homozygous AA genotype significantly associated with the average daily gain than AC and CC genotypes from birth to 9 mo of age. Expression analysis showed that the A266C SNP was correlated with IGF2, but not with IGF2R, mRNA levels in the gluteus medius muscle of Egyptian buffaloes. The highest IGF2 mRNA level was estimated in the muscle of animals with the AA homozygous genotype as compared to the AC heterozygotes and CC homozygotes. We conclude that A266C SNP at nucleotide number 51 of exon 23 of the IGF2R gene is associated with the ADG during the early stages of life (from birth to 9 mo of age) and this effect is accompanied by, and may be caused by, increased expression levels of the IGF2 gene.

Regulation of chick early B-cell factor-1 gene expression in feather development.

The chick Ebf1 (early B-cell factor-1) gene is a member of a novel family of helix loop helix transcription factors. The expression profile, regulation and significance of this gene have been extensively studied in lymphatic, nervous, adipose and muscular tissues. However, cEbf1 expression, regulation and function in the feather of chick embryo have not yet been investigated. cEbf1 expression was first detected throughout the mesenchymal core of some few feather placodes (D7-D7.5). After feathers became mature and grew distally (D9 and D10), the mesenchymal expression of cEbf1 became confined to the caudal margin of the proximal half of all formed feather buds. Because this dynamic pattern of expression resembles that of Sonic Hedgehog (Shh) protein and bone morphogenetic protein (Bmp4) plus the crucial role of these two major signals in feather development, we hypothesized that cEbf1 expression in the feather may be regulated by Shh and Bmp4. In a feather explant culture system, Shh signals are necessary to initiate and maintain cEbf1 expression in the posterior half of the feather bud, while Bmp4 is crucial for the initial cEbf1 expression in the anterior half of the feather bud. Inhibition of Shh, not only down-regulates cEbf1, but also changes the morphology of feather buds, which become irregular and fused. This is the first study to demonstrate that cEbf1 expression in the feather bud is under the control of Shh and Bmp4 signals and that expression may play a role in the normal development of feathers.

Regulation of chick Ebf1-3 gene expression in the pharyngeal arches, cranial sensory ganglia and placodes.

This study was conducted to identify the regulation of the expression of the cEbf1-3 (chick early B-cell factor 1-3) genes in the pharyngeal arches (PAs), cranial sensory ganglia and placodes. cEbf1 and cEbf3 were mainly expressed in the cranial neural crest cells (NCCs) occupying the PAs, but cEbf2 was expressed in the mesenchymal core. cEbf1-3 were prominently expressed in the olfactory placodes, but cEbf1 and cEbf3 were only expressed in the otic vesicle. cEbf1 was expressed in all cranial sensory ganglia, cEbf2 (only) in the dorsolateral ganglia and cEbf3 in the trigeminal and vestibular ganglia. The removal of the source (the cranial neural tube) of the cranial NCCs before their migration to the PAs led to downregulation of cEbf1 and cEbf3 and upregulation of cEbf2 expression. Gain- and loss-of-function experiments showed that sonic hedgehog did not regulate cEbf1-3 expression in the PAs or associated ganglia. Bone morphogenetic protein 2 (Bmp2) can, however, directly and indirectly regulate cEbf1 and cEbf3 expression in the PAs and the proximal (NCC-derived) portion, but not the distal (placodal-derived) portion of the cranial sensory ganglia. Conversely, cEbf2 expression was upregulated following injection of Noggin before the migration of NCCs, but did not change after the overexpression of either Noggin or Bmp2 in the arch after NCC migration. In conclusion, Bmp2 regulates cEbf1 and cEbf3 expression in PAs and cranial sensory ganglia both directly and indirectly, via the migration of cranial NCCs. However, cEbf2 expression in the mesenchymal core of PAs is controlled by other undetermined signals.