Neonatal lactocrine deficiency affects the adult porcine endometrial transcriptome at pregnancy day 13.

Reproductive performance of female pigs that do not receive sufficient colostrum from birth is permanently impaired. Whether lactocrine deficiency, reflected by low serum immunoglobulin immunocrit (iCrit), affects patterns of endometrial gene expression during the periattachment period of early pregnancy is unknown. Here, objectives were to determine effects of low iCrit at birth on the adult endometrial transcriptome on pregnancy day (PxD) 13. On the first day of postnatal life, gilts were assigned to high or low iCrit groups. Adult high (n = 8) and low (n = 7) iCrit gilts were bred (PxD 0), and humanely slaughtered on PxD 13 when tissues and fluids were collected. The endometrial transcriptome was defined for each group using mRNAseq and microRNAseq. Reads were mapped to the Sus scrofa 11.1 genome build. Mature microRNAs were annotated using miRBase 21. Differential expression was defined based on fold change (≥ ±1.5). Lactocrine deficiency did not affect corpora lutea number, uterine horn length, uterine wet weight, conceptus recovery, or uterine luminal fluid estrogen content on PxD 13. However, mRNAseq revealed 1157 differentially expressed endometrial mRNAs in high versus low iCrit gilts. Differentially expressed genes had functions related to solute transport, endometrial receptivity, and immune response. Six differentially expressed endometrial microRNAs included five predicted to target 62 differentially expressed mRNAs, affecting similar biological processes. Thus, lactocrine deficiency on the first day of postnatal life can alter uterine developmental trajectory with lasting effects on endometrial responses to pregnancy as reflected at the level of the transcriptome on PxD 13.

Enhanced Pulsatile Growth Hormone Secretion and Altered Metabolic Hormones by in Vivo Hexarelin Treatment in Streptozotocin-Induced Diabetic Rats.

Significant growth hormone (GH) reductions have been reported in diabetic animal models with disturbed metabolic balance coinciding with GH deficiency. Therefore, enhanced GH secretion may have beneficial effects in controlling diabetes. Thus, we aim to investigate the effect of hexarelin, a synthetic GH secretagogue (GHS), on GH secretion in streptozotocin (STZ, 65 mg/kg)-induced diabetic rats. Daily hexarelin (100 µg/kg) treatment was performed for two weeks in four-week-long STZ-diabetic and vehicle control rats. Pulsatile GH secretion in STZ-rats was significantly reduced in total, pulsatile, basal, and mass of GH secretion per burst. In addition, impaired GH secretion was followed by an increase in fasting-level free fatty acids (FFAs) and a decrease in insulin-like growth factor 1 (IGF-1) compared to control rats. After hexarelin treatment, pulsatile GH secretion in STZ-rats was significantly increased in total, pulsatile, and basal, but not in the mass GH secretion per burst, compared to STZ-rats without hexarelin treatment. However, there was no significant elevation in GH secretion in the hexarelin-treated control group. In addition, hexarelin-treated STZ-rats showed a significant decrease in fasting level FFAs, whereas suppression of fasting level for IGF-1 was maintained. These results suggest that STZ-induced diabetic rats have impaired pulsatile GH secretion, causing increased FFAs and decreased IGF-1 levels in circulation. Hexarelin injections for two weeks is able to normalize impaired pulsatile GH secretion with normal fasting levels of FFAs, but fails to recover IGF-1 levels.

Microparticles Produced by Activated Platelets Carry a Potent and Functionally Active Angiogenic Signal in Subjects with Crohn's Disease.

Microparticles (MPs) are submicron vesicles shed from various cell types upon activation, stimulation, and death. Activated platelets are an important source of circulating MPs in subjects with inflammatory diseases, including Crohn's disease (CD). Angiogenesis is a hallmark of inflammation in CD and plays an active role in sustaining disease progression, while targeting angiogenesis may be an effective approach to block colitis. In this study, we analyzed the angiogenic content of the MPs produced by activated platelets in subjects with CD. We also evaluated whether the angiogenic signal carried by these MPs was functionally active, or able to induce angiogenesis. We found that, in subjects with CD, MPs produced by activated platelets contain significantly higher levels of angiogenic mRNAs, such as epidermal growth factor (EGF), platelet-derived growth factor-α (PDGFα), fibroblast growth factor (FGF-2), and angiopoietin-1 (ANGPT1), compared to MPs isolated from control subjects. They also contain significantly higher levels of prototypical angiogenic proteins, including vascular endothelial growth factor (VEGF), angiopoietin-1, endoglin, endothelin-1, pentraxin 3, platelet factor-4, plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinases-1 (TIMP-1), and thrombospondin 1. The protein content of these MPs is functionally active, since it has the ability to induce a robust angiogenic process in an endothelial cell/interstitial cell co-culture in vitro assay. Our results reveal a potential novel mechanism through which the angiogenic signal is delivered in subjects with CD, with potentially important clinical and therapeutic implications.

[Effects of concentrated growth factors on proliferation and osteogenic differentiation in Beagle adipose-derived stem cells].

OBJECTIVE: To investigate the effect of concentrated growth factor (CGF) on proliferation and differentiation in Beagle adipose-derived stem cells (ADSCs).
 Methods: ADSCs were isolated from adipose tissue of healthy Beagles and cultured. The multi-directional differentiation potential of ADSCs was identified. The ADSCs were assigned to a CGF group and a control group. The rate of proliferation was analyzed by CCK-8 assay. The osteogenic differentiation capability was detected by ALP staining after the osteoinduction. Bone formation-related gene expression was detected by RT-PCR.
 Results: CGF promoted the proliferation of ADSCs in vitro. ADSCs in the CGF group showed higher level of ALP activity than that in the control group (P<0.05). CGF stimulated the expression of the genes associated with osteogenesis, such as Col-I and Runx2. 
 Conclusion: CGF can promote the proliferation and osteogenic differentiation in ADSCs in vitro.

Structural modulation of gut microbiota in Bama minipigs in response to treatment with a "growth-promoting agent", salbutamol.

Even though salbutamol (SAL) had remarkable effects on the enhancement of growth rate and carcass composition in different livestock species such as cattle, pigs, sheep and poultry, it was banned as a growth promoter because of its adverse effects on health. However, the specific mechanism by which salbutamol enhances growth efficiency remains unknown. In this study, Bama pigs were randomly allocated to receive salbutamol (5 mg/kg) for 30 or 60 days and were compared with untreated pigs. Pigs treated with salbutamol demonstrated enhanced growth rates and carcass composition; however, they showed deterioration in blood biochemical indices and organ development. We hypothesized that salbutamol exerts its effects by modulating the composition of the gut microbiota population. The faecal microbiome of pigs was characterized via pyrosequencing of the bacterial 16S rRNA gene. The gut microbiota population analysis showed that salbutamol caused shifts in the microbial composition of less abundant species. Redundancy analysis indicated an increase in abundance of the phylum Bacteroidetes, class Betaproteobacteria, family Christensenellaceae and genus Lactobacillus, and a decreased ratio of the phylum Firmicutes, class Clostridia and genera Ruminococcus, Blautia and Subdoligranulum. In conclusion, our study provided circumstantial evidence that the various effects of salbutamol are caused by gut microbiota modulation, and several potential candidates were identified for SAL detection via the gut microbiota. Our findings provided new insights into the roles of the gut microbiota during salbutamol treatment, and these findings will aid in the screening of alternative strategies for animal health improvement and production enhancement.

Actions of activin A, connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on the development of the bovine preimplantation embryo.

The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. In the present study we tested four growth factors expressed in the endometrium for their ability to modify the development of the bovine embryo to the blastocyst stage and alter the expression of genes found to be upregulated (bone morphogenetic protein 15 (BMP15) and keratin 8, type II (KRT8)) or downregulated (NADH dehydrogenase 1 (ND1) and S100 calcium binding protein A10 (S100A10)) in embryos competent to develop to term. Zygotes were treated at Day 5 with 0.01, 0.1 or 1.0nM growth factor. The highest concentration of activin A increased the percentage of putative zygotes that developed to the blastocyst stage. Connective tissue growth factor (CTGF) increased the number of cells in the inner cell mass (ICM), decreased the trophectoderm:ICM ratio and increased blastocyst expression of KRT8 and ND1. The lowest concentration of hepatocyte growth factor (HGF) reduced the percentage of putative zygotes becoming blastocysts. Teratocarcinoma-derived growth factor 1 increased total cell number at 0.01nM and expression of S100A10 at 1.0nM, but otherwise had no effects. Results confirm the prodevelopmental actions of activin A and indicate that CTGF may also function as an embryokine by regulating the number of ICM cells in the blastocyst and altering gene expression. Low concentrations of HGF were inhibitory to development.

The Growth Hormone Secretagogue Hexarelin Protects Rat Cardiomyocytes From in vivo Ischemia/Reperfusion Injury Through Interleukin-1 Signaling Pathway.

Hexarelin, a synthetic growth hormone-releasing peptide, has been proven to possess cardioprotective actions through its binding to the growth hormone secretagogue receptor (GHSR) 1a and the non-GHSR receptor CD36. However, its effect on myocardial ischemia/reperfusion (I/R) injury has not been fully clarified in vivo. We aimed to determine whether hexarelin treatment could protect cardiomyocytes from I/R injury and to examine the underlying mechanisms. In vivo hearts of male SD rats underwent 30 minutes of ischemia by left coronary artery ligation followed by reperfusion. The rats were then treated subcutaneously twice daily with hexarelin [100 µg/kg·day], ghrelin [400 µg/ kg·day], or saline for 7 days. Echocardiography, malondialdehyde detection, and histochemical staining were performed after treatment. In addition, Western blot was used to examine the expression levels of IL-1ß, IL-1Ra, and IL-1RI. Our study showed that hexarelin treatment improved cardiac systolic function, decreased malondialdehyde production, and increased the number of surviving cardiomyocytes. The beneficial effects of hexarelin treatment were slightly superior to those of equimolar ghrelin treatment. We meanwhile confirmed that hexarelin induced down-regulation of IL-1ß expression and up-regulation of IL-1Ra expression in I/R myocardium, which could be neutralized by the GHSR antagonist [D-Lys3]-growth hormone releasing peptide-6 ([D-Lys3]-GHRP-6). These findings suggest that hexarelin protects in vivo cardiomyocytes from I/R injury partly by modification of the IL-1 signaling pathway through the activation of cardiac GHSR1a receptors.

Radiographic Appearance of Transforaminal Lumbar Interbody Fusion Performed With and Without Recombinant Human Morphogenetic Protein-2.

OBJECTIVE: The purpose of this study is to determine whether recombinant human morphogenetic protein-2 (rhBMP-2) alters the findings on routine radiographs performed after transforaminal lumbar interbody fusion (TLIF). MATERIALS AND METHODS: A retrospective review of 256 TLIF procedures in 200 patients was performed over a 4-year period. The rhBMP-2 group included 204 TLIFs in 160 patients, and the control group included 52 TLIFs in 40 patients. Two musculoskeletal radiologists reviewed the postoperative radiographs for endplate resorption, resorption resolution, new bone formation, bridging bone, and allograft migration. Statistical analysis was performed using logistic regression. RESULTS: The median age was 53 years in the rhBMP-2 group and 54 years in the control group (p = 0.182). The groups were similar with regard to sex (p = 0.517), single or multilevel TLIF (p = 0.921), specific TLIF levels (p = 0.53), and median radiographic follow-up (373 vs 366 days; p = 0.34). Findings that were more common in the rhBMP-2 group than in the control group included endplate resorption (38% [78/204] vs 12% [6/52]; odds ratio [OR], 4.67; 95% CI, 1.99-12.54; p < 0.001), resorption resolution (59% [46/78] vs 0% [0/6]; OR, 8.09; 95% CI, 1.41 to ∞; p = 0.022), new bone formation (84% [171/204] vs 67% [35/52]; OR, 2.51; 95% CI, 1.24-4.99; p = 0.011), bridging bone (55% [112/204] vs 31% [16/52]; OR, 2.73; 95% CI, 1.43-5.34; p = 0.002), and allograft migration (17% [35/204] vs 2% [1/52]; OR, 6.30; 95% CI, 0.91-151.41; p = 0.065). CONCLUSION: A statistically significant higher frequency of endplate resorption, new bone formation, and bone bridging is present in TLIF augmented by rhBMP-2 compared with TLIF performed without rhBMP-2. Endplate resorption resolves without treatment in most cases after rhBMP-2 use.

Syndecan-4 regulates subcellular localization of mTOR Complex2 and Akt activation in a PKCalpha-dependent manner in endothelial cells.

Mammalian target of rapamycin (mTOR) activity is regulated by assembly of two functionally distinct complexes, mTORC1 and mTORC2. In syndecan-4 (S4) null endothelial cells, mTORC2 activity is reduced, resulting in decreased Akt activation, while mTORC1 activity is increased. Levels of rictor, mLST8, and mSin-1 are unchanged in total cell lysates but decreased in the rafts of S4(-/-) endothelial cells, as is the level of PKCalpha. Expression of myristoylated-PKCalpha in S4(-/-) cells restores rictor, mLST8, and mSin-1 presence in the rafts and rescues Akt phosphorylation. PKCalpha knockdown mimics the effect of S4 deletion on mTORC2 localization and Akt activation. Reduced mTORC2 activity in S4(-/-) endothelial cells results in decreased FoxO1/3a and eNOS phosphorylation, decreased endothelial cell size, and increased arterial blood pressure in S4(-/-) mice. Thus, S4-dependent targeting of PKCalpha to the plasma membrane is required for recruitment of mTORC2 components to the rafts and Akt activation.

Can the use of natural biostimulants be a potential means of phytoremediating contaminated soils from goldmines in South Africa?

Biostimulants offer great potential in improving phytoremediation of contaminated soils. In the current greenhouse-based study, Brassica juncea seedlings grown on soils collected from Krugersdorp Goldmine and the adjourning areas (a Game Reserve and private farmland) were supplemented with different biostimulants (Kelpak® = KEL, vermicompost leachate = VCL, smoke-water = SW). Indole-3-butyric acid (IBA) was included in the study for comparative purposes because these biostimulants are known to enhance rooting. Prior to the pot trial, concentrations of elements in the three soil types were determined using Inductively Coupled Plasma-Optical Emission Spectroscopy. Plants were harvested after 105 days and the growth and concentrations of elements in the various plant organs were determined. TheB. juncea seedlings with and without biostimulants did not survive when growing in soil from the Krugersdorp Goldmine. The Game Reserve and private farmland soils supplemented with KEL produced the highest plant biomass and the lowest accumulation of metals in the organs of B. juncea. High concentrations (>13 000 mg kg(-1)) of zinc and aluminium were quantified in the roots of IBA-supplemented soils from the Game Reserve. Generally, IBA and SW enhanced the phytoremediation of B. juncea due to elevated levels of elements that accumulated in their different organs.

Plasma rich in growth factors had limited effect on early bone formation in extraction sockets: Response to "Anitua, E., Alkhraisat, M.H. & Orive, G. (2013) Letter to the Editor: Rigorous methodology is the school of coherent conclusions in science. European Journal of Oral Implantology 6: 9-11.".

AIM: To address the criticisms raised by Anitua et al. (European Journal of Oral Implantology, 6, 2013, 9-11) to the article "Plasma Rich in Growth Factors (PRGF) in Human Post-Extraction Sockets: an Histological and Histomorphometric Study.", recently published by Farina and colleagues (Clinical Oral Implants Research 2012; doi: 10.1111/clr.12033). METHODS: All the methodological aspects criticized in the letter by Anitua et al. were thoroughly reconsidered and discussed in a structured short communication. When indicated, pertinent, additional material was included to reinforce our considerations. RESULTS: As most clinical studies in implant dentistry, including previous studies evaluating the efficacy/effectiveness of PRGF, the study by Farina et al. has some limitations. However, it is currently the only published controlled trial using quantitative parameters related to PRGF-induced early bone formation. CONCLUSIONS: Despite all limitations, the results of the study by Farina et al., which were based on different quantitative parameters (micro-CT scan, immunohistochemical markers of wound healing and bone deposition), indicated a limited effect of PRGF on early bone formation in extraction sockets.

Efficacy of plasma-rich growth factor in the healing of postextraction sockets in patients affected by insulin-dependent diabetes mellitus.

PURPOSE: To evaluate the efficacy of plasma-rich growth factor (PRGF) in improving socket healing after tooth extraction in diabetic patients. MATERIALS AND METHODS: This was a split-mouth study in which each patient also served as the control: the study socket was treated with PRGF, whereas the control socket underwent natural healing. The outcome variables were the Healing Index, residual socket volume, visual analog scale score, postsurgical complications, and outcome of a patient questionnaire. The investigation considered the impact of hyperglycemia, glycated hemoglobin, End Organ Disease Score, and smoking habits. Follow-up included 4 postextraction checkups over a 21-day period. Pairs of correlated continuous variables were analyzed with the Wilcoxon test, independent continuous variables with the Mann-Whitney test, and categorical variables with the χ(2) test or Fisher test. RESULTS: From January 2012 to December 2012, 34 patients affected by insulin-dependent diabetes mellitus underwent contemporary bilateral extractions of homologous teeth. The treatment-versus-control postoperative comparison showed that PRGF resulted in significantly smaller residual socket volumes and better Healing Indices from days 3 to 14. The patients' questionnaire outcomes were unanimously in favor of PRGF treatment. The small sample of patients with glycemia values of at least 240 mg/dL showed worse Healing Index and minor socket decreases. CONCLUSION: PRGF application after extraction improved the healing process in diabetic patients by accelerating socket closure (epithelialization) and tissue maturation, proving the association between PRGF use and improved wound healing in diabetic patients.

Angptl3 regulates lipid metabolism in mice.

The KK obese mouse is moderately obese and has abnormally high levels of plasma insulin (hyperinsulinemia), glucose (hyperglycemia) and lipids (hyperlipidemia). In one strain (KK/San), we observed abnormally low plasma lipid levels (hypolipidemia). This mutant phenotype is inherited recessively as a mendelian trait. Here we report the mapping of the hypolipidemia (hypl) locus to the middle of chromosome 4 and positional cloning of the autosomal recessive mutation responsible for the hypolipidemia. The hypl locus encodes a unique angiopoietin-like lipoprotein modulator, which we named Allm1. It is identical to angiopoietin-like protein 3, encoded by Angptl3, and has a highly conserved counterpart in humans. Overexpression of Angptl3 or intravenous injection of the purified protein in KK/San mice elicited an increase in circulating plasma lipid levels. This increase was also observed in C57BL/6J normal mice. Taken together, these data suggest that Angptl3 regulates lipid metabolism in animals.

Effects of replacing a dietary antibacterial agent (zinc bacitracin) with copper salts in Cherry Valley Pekin meat ducks.

1. A study was conducted to investigate the effectiveness of high dietary copper concentrations obtained from tribasic copper chloride (TBCC, 58% copper) and copper sulphate pentahydrate (CuSO4, 25% copper) in replacing antibiotic growth promoters (AGP) in duck diets. 2. A total of 960 one-day-old Cherry Valley meat-strain ducks were divided into 3 treatment groups, with 8 replicates per treatment, in a 6-week feeding trial. The ducks were fed a basal diet supplemented with AGP (40 mg zinc bacitracin/kg and 40 mg garlicin/kg of diet) or 150 mg of Cu/kg of diet, given as either CuSO4 or TBCC. 3. The body weight, average daily gain, average daily feed intake and mortality of ducks were not affected by the dietary treatments. However, the feed/gain ratio of ducks that were fed TBCC diets was significantly lower than those of ducks that were fed CuSO4 diets and were similar to those in the AGP group. 4. TBCC increased the Cu content in the liver tissue of ducks compared with the content in those that were fed the diet supplemented with AGP. TBCC also increased the Fe and Zn content in breast muscles compared with that in ducks that were fed the diet supplemented with CuSO4. 5. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were significantly higher in the serum of ducks that received the diet supplemented with TBCC than AGP or CuSO4. TBCC treatment decreased the malondialdehyde (MDA) content in serum of ducks compared with groups supplemented with CuSO4. 6. No significant difference was observed in liver or muscle fat content among the different dietary treatment groups. The serum low-density lipoprotein cholesterol concentration was lower in ducks fed AGP diets than those fed CuSO4 diets. 7. It was concluded that the replacement of AGP with 150 mg of Cu/kg of feed from TBCC improved the feed efficiency, trace mineral deposition and antioxidant status more than when the source of copper was CuSO4.

[Examination of cytoprotective and anti-inflammatory effect of PACAP-38 on small bowel autotransplantation]. / A PACAP-38 citoprotektív és antiinflammatórikus hatásának vizsgálata vékonybél-autotranszplantációs modellben.

INTRODUCTION: The small intestine is one of the most sensitive organs to ischemia-reperfusion injury during transplantation. Cytoprotective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) is well known. The aim of our study was to measure changes of PACAP-38-like immunoreactivities and cytokine levels in intestinal grafts stored PACAP-38 containing preservation solution. MATERIAL AND METHODS: Small-bowel autotransplantation was performed on male Wistar rats (n = 56). Grafts were stored in University of Wisconsin (UW) solution at 4 °C for 1 (GI), 3 (GII), and 6 hours (GIII); and in PACAP-38 containing UW solution for 1 (GIV), 3 (GV), and 6 hours (GVI). Reperfusion lasted 3 hours in each group. Intestinal PACAP-38 immunoreactivities were measured by radioimmunoassay. To measure cytokine from tissue homogenates we used rat cytokine array and Luminex Multiplex Immunoassay. RESULTS: Levels of PACAP-38-like and PACAP-27-like immunoreactivities decreased by preservation time compared to control. This decrease was significant following 6 hours cold storage (p < 0.05). Values remained significantly higher in grafts stored in PACAP-38 containing UW. Expressions of sICAM-1, L-selectin, tissue inhibitor of metalloproteinase-1 were increased in GIII and were decreased in GVI. CONCLUSION: PACAP-38 increased tissue levels of PACAP-38 and PACAP-27, and decreased cytokine expression. This indicates that PACAP-38 has anti-inflammatory and cytoprotective effects in intestinal autotransplantation model.

Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate.

Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004).

VEGF, FLT3 ligand, PlGF and HGF can substitute for M-CSF to induce human osteoclast formation: implications for giant cell tumour pathobiology.

Giant cell tumour of bone (GCTB) is a primary bone tumour that contains numerous very large, hyper-nucleated osteoclastic giant cells. Osteoclasts form from CD14+ monocytes and macrophages in the presence of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). GCTB contains numerous growth factors, some of which have been reported to influence osteoclastogenesis and resorption. We investigated whether these growth factors are capable of substituting for M-CSF to support osteoclast formation from cultured human monocytes and whether they influence osteoclast cytomorphology and resorption. Vascular endothelial growth factor-A (VEGF-A), VEGF-D, FLT3 ligand (FL), placental growth factor (PlGF) and hepatocyte growth factor (HGF) supported RANKL-induced osteoclastogenesis in the absence of M-CSF, resulting in the formation of numerous TRAP+ multinucleated cells capable of lacunar resorption. Monocytes cultured in the presence of M-CSF, HGF, VEGF-A and RANKL together resulted in the formation of very large, hyper-nucleated (GCTB-like) osteoclasts that were hyper-resorptive. M-CSF and M-CSF substitute growth factors were identified immunohistochemically in GCTB tissue sections and these factors stimulated the resorption of osteoclasts derived from a subset of GCTBs. Our findings indicate that there are growth factors that are capable of substituting for M-CSF to induce human osteoclast formation and that these factors are present in GCTB where they influence osteoclast cytomorphology and have a role in osteoclast formation and resorption activity.

Small GTPases and tyrosine kinases coregulate a molecular switch in the phosphoinositide 3-kinase regulatory subunit.

Phosphoinositide 3-kinase (PI3K) type IA is a heterodimer of a catalytic subunit, p110, and a regulatory subunit, p85. Here we show that p85 contains a GTPase-responsive domain and an inhibitory domain, which together form a molecular switch that regulates PI3K. H-Ras and Rac1 activate PI3K by targeting the GTPase-responsive domain. The stimulatory effect of these molecules, however, is blocked by the inhibitory domain, which functions by binding to tyrosine-phosphorylated molecules and is neutralized by tyrosine phosphorylation. The complementary effects of tyrosine kinases and small GTPases on the p85 molecular switch result in synergy between these two classes of molecules toward the activation of the PI3K/Akt pathway.

Transcriptomic markers meet the real world: finding diagnostic signatures of corticosteroid treatment in commercial beef samples.

BACKGROUND: The use of growth-promoters in beef cattle, despite the EU ban, remains a frequent practice. The use of transcriptomic markers has already proposed to identify indirect evidence of anabolic hormone treatment. So far, such approach has been tested in experimentally treated animals. Here, for the first time commercial samples were analyzed. RESULTS: Quantitative determination of Dexamethasone (DEX) residues in the urine collected at the slaughterhouse was performed by Liquid Chromatography-Mass Spectrometry (LC-MS). DNA-microarray technology was used to obtain transcriptomic profiles of skeletal muscle in commercial samples and negative controls. LC-MS confirmed the presence of low level of DEX residues in the urine of the commercial samples suspect for histological classification. Principal Component Analysis (PCA) on microarray data identified two clusters of samples. One cluster included negative controls and a subset of commercial samples, while a second cluster included part of the specimens collected at the slaughterhouse together with positives for corticosteroid treatment based on thymus histology and LC-MS. Functional analysis of the differentially expressed genes (3961) between the two groups provided further evidence that animals clustering with positive samples might have been treated with corticosteroids. These suspect samples could be reliably classified with a specific classification tool (Prediction Analysis of Microarray) using just two genes. CONCLUSIONS: Despite broad variation observed in gene expression profiles, the present study showed that DNA-microarrays can be used to find transcriptomic signatures of putative anabolic treatments and that gene expression markers could represent a useful screening tool.

ZEN and the art of breast health maintenance.

Zearalenone (ZEN) is a non-steroidal mycoestrogen that widely contaminates agricultural products. ZEN and its derivatives share similar molecular mechanisms and activity with estrogens and interact with ERα and ERß leading to changes in the reproductive system in both animals and humans. The reduced form of ZEN, α-ZEA ralenol, has been used as an anabolic agent for animals and also proposed as hormonal replacement therapy in postmenopausal women. Furthermore, both zearelanol ZEN and derivatives have been patented as oral contraceptives. ZEN has been widely used in the United States since 1969 to improve fattening rates in cattle by increasing growth rate and feed conversion efficiency. Evidence of human harm from this practice is provided by observations of central precocious puberty. As a result, this practice has been banned by the European Union. As ZEN has been associated with breast enlargement in humans, it has been included in many bust-enhancing dietary supplements but epidemiological evidence is lacking with regard to breast cancer risk. Extensive work with human breast cancer cell lines has shown estrogenic stimulation in those possessing ER but a reduction in DMBA-induced breast cancers in rodents given ZEN. Protein disulfide isomerase provides a molecular biomarker of dietary exposure to ZEN and its derivatives allowing the detection and control of harmful food intake. The interaction of ZEN with anti-estrogens, anticancer agents and antioxidants requires further investigation.