Variation in WNT7A is unlikely to be a cause of familial Congenital Talipes Equinovarus

<p>Abstract</p> <p>Background</p> <p>Genetic factors make an important contribution to the aetiology of congenital talipes equinovarus (CTEV), the most common developmental disorder of the lower limb. WNT7A was suggested as a candidate gene for CTEV on the basis of a genome-wide scan for linkage in a large multi-case family. WNT7A is a plausible candidate gene for CTEV as it provides a signal for pattern formation during limb development, and mutation in WNT7A has been reported in a number of limb malformation syndromes.</p> <p>Methods</p> <p>We investigated the role of WNT7A using a family-based linkage approach in our large series of European multi-case CTEV families. Three microsatellite markers were used, of which one (D3S2385) is intragenic, and the other two (D3S2403, D3S1252) are 700 kb 5' to the start and 20 kb from the 3' end of the gene, respectively. Ninety-one CTEV families, comprising 476 individuals of whom 211 were affected, were genotyped. LOD scores using recessive and incomplete-dominant inheritance models, and non-parametric linkage scores, excluded linkage.</p> <p>Results</p> <p>No significant evidence for linkage was observed using either parametric or non-parametric models. LOD scores for the parametric models remained strongly negative in the regions between the markers, and in the 0.5 cM intervals outside the marker map. No significant lod scores were obtained when the data were analysed allowing for heterogeneity.</p> <p>Conclusion</p> <p>Our evidence suggests that the WNT7A gene is unlikely to be a major contributor to the aetiology of familial CTEV.</p

Baicalin administration attenuates hyperglycemia-induced malformation of cardiovascular system

In this study, the effects of Baicalin on the hyperglycemia-induced cardiovascular malformation during embryo development were investigated. Using early chick embryos, an optimal concentration of Baicalin (6 μM), was identified which could prevent hyperglycemia-induced cardiovascular malformation of embryos. Hyperglycemia-enhanced cell apoptosis was reduced in embryos and HUVECs in the presence of Baicalin. Hyperglycemia-induced excessive ROS production was inhibited when Baicalin was administered. Analyses of SOD, GSH-Px, MAQE and GABAA suggested Baicalin plays an antioxidant role in chick embryos possibly through suppression of outwardly rectifying Cl(-) in the high-glucose microenvironment. In addition, hyperglycemia-enhanced autophagy fell in the presence of Baicalin, through affecting the ubiquitin of p62 and accelerating autophagy flux. Both Baicalin and Vitamin C could decrease apoptosis, but CQ did not, suggesting autophagy to be a protective function on the cell survival. In mice, Baicalin reduced the elevated blood glucose level caused by streptozotocin (STZ). Taken together, these data suggest that hyperglycemia-induced embryonic cardiovascular malformation can be attenuated by Baicalin administration through suppressing the excessive production of ROS and autophagy. Baicalin could be a potential candidate drug for women suffering from gestational diabetes mellitus

A novel COMP mutation in a pseudoachondroplasia family of Chinese origin

<p>Abstract</p> <p>Background</p> <p>Pseudoachondroplasia (PSACH) is caused exclusively by mutations in the gene for cartilage oligomeric matrix protein (<it>COMP</it>). Only a small number of studies have documented the clinical phenotype and genetic basis in Chinese PSACH patients.</p> <p>Case presentation</p> <p>We investigated a four-generation PSACH pedigree of Chinese Han origin. Two patients and two unaffected individuals were recruited for clinical evaluation and molecular genetic analysis. The genomic DNA was extracted from peripheral blood leukocytes. Polymerase chain reaction (PCR) was adopted to amplify the 8-19 exons of <it>COMP </it>gene. Then the products were sequenced bi-directionally for screening mutation. Clinical evaluation revealed that PSACH patients in this pedigree had a severe disproportionate short stature (-10SD). A heterozygous TGTCCCTGG insertion in exon 13, between nucleotide 1352T and 1353G, were identified in the patients except the unaffected individuals, which resulted in a three-amino-acid insertion (451V_452P ins VPG) in the sixth calmodulin-like repeat of the <it>COMP </it>protein.</p> <p>Conclusion</p> <p>This c. 1352_1353ins TGTCCCTGG is a novel mutation responsible for severe familial PSACH.</p

Cartilage oligomeric matrix protein in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5-3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-β1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-β1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded. © 2013 Vuga et al

STAT3 Activation in Skeletal Muscle Links Muscle Wasting and the Acute Phase Response in Cancer Cachexia

Cachexia, or weight loss despite adequate nutrition, significantly impairs quality of life and response to therapy in cancer patients. In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response. Acute phase proteins, including fibrinogen and serum amyloid A (SAA) are synthesized by hepatocytes in response to IL-6 as part of the innate immune response. To gain insight into the relationships among these observations, we studied mice with moderate and severe Colon-26 (C26)-carcinoma cachexia.Moderate and severe C26 cachexia was associated with high serum IL-6 and IL-6 family cytokines and highly similar patterns of skeletal muscle gene expression. The top canonical pathways up-regulated in both were the complement/coagulation cascade, proteasome, MAPK signaling, and the IL-6 and STAT3 pathways. Cachexia was associated with increased muscle pY705-STAT3 and increased STAT3 localization in myonuclei. STAT3 target genes, including SOCS3 mRNA and acute phase response proteins, were highly induced in cachectic muscle. IL-6 treatment and STAT3 activation both also induced fibrinogen in cultured C2C12 myotubes. Quantitation of muscle versus liver fibrinogen and SAA protein levels indicates that muscle contributes a large fraction of serum acute phase proteins in cancer.These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer. Through IL-6/STAT3 activation, skeletal muscle is induced to synthesize acute phase proteins, thus establishing a molecular link between the observations of high IL-6, increased acute phase response proteins and muscle wasting in cancer. These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such that amino acids liberated by increased proteolysis in cachexia are synthesized into acute phase proteins and exported into the blood

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

The chick embryo chorioallantoic membrane as a model system for the study of tumor angiogenesis, invasion and development of anti-angiogenic agents.

Angiogenesis, the formation of new blood vessels, is essential for tumor growth, progression and metastasis. The development of agents that target tumor vasculature is ultimately dependent on the availability of appropriate preclinical screening assays. The chorioallantoic membrane (CAM) assay is well established and widely used as a model to examine angiogenesis, and anti-angiogenesis. This review 1) summarizes the currently used angiogenesis assays and the importance of CAM model among them; 2) summarizes the current knowledge about the development and structure of the CAM's capillary bed; 3) reports findings regarding the role played by molecular signaling pathways in angiogenesis process; 4) discusses the use, advantages and limitations of the CAM as a model for studying tumor angiogenesis and invasiveness, as well as development of angiogenic and/or anti-angiogenic agents; 5) discusses the importance of standardization of the major methodologies for all aspects of the use of the CAM in angiogenesis-related studies; 6) and finally, summarizes major findings regarding the agents developed by the use of CAM model in the study of tumor angiogenesis, invasion and development of anti-angiogenic agents

the chick area vasculosa

The effect of ethanol (EtOH) exposure on extraembryonic vascular development was examined using the chick embryo area vasculosa (AV) in shell-less culture. Embryos were placed in cultures at Hamburger Hamilton (HH) stage 11/12 and a single dose of EtOH (10, 30 or 50%) was applied to the center of the blastodisc. Untreated/sodium-chloride-treated controls showed normal embryonic growth and well-developed extraembryonic vessels at 24/48 h of treatment. At doses of 30 and 50%, the mortality rate was significantly increased, and survivors demonstrated significant growth retardation and inhibition of normal vascular development in a dose-dependent manner. Immunostaining for vascular endothelial growth factor ( VEGF) showed that mesenchymal cells continued to differentiate into angioblasts to form blood islands, but their assembly into primitive vessels was perturbed in a dose-dependent manner. Northern blot analyses of basic fibroblast growth factor, VEGF, Flt-1 and Flk-1 mRNA expression supported these findings and showed a dose-dependent decrease in EtOH-treated cultures compared to controls. Co-treatment with alpha-tocopherol (0.05 M) or all-trans-retinoic acid (10(-8) M) significantly decreased the mortality rate and improved both embryonic growth and extraembryonic vascular development in the cultures. On the other hand, almost all embryos treated with 10% EtOH survived the first 48 h after treatment. However, the complexity of the vascular tree measured as the relative vasculogenesis index, the surface area of the AV and the mRNA expression of vasculogenic molecules were increased during the first 24 h. This acute effect disappeared 48 h after treatment and the vascular tree continued to develop parallel to the controls. No significant growth retardation was observed in this group. These results suggest that, in terms of extraembryonic vascular development, an early, single, low-dose EtOH exposure may have an acute, short-term positive effect, whereas moderate- or high-dose EtOH exposure may severely perturb this process disabling the necessary absorption of the nutrients for the embryo to develop properly. The mechanisms of action of EtOH on extraembryonic vascular development may involve the establishment of reactive oxygen species, resulting in the initiation of oxidative stress and perturbation of retinoic acid signaling and alterations in the expression of growth-regulatory vasculogenic factors and their receptors

Amelioration of ethanol-induced growth retardation by all-trans-retinoic acid and alpha-tocopherol in shell-less culture of the chick embryo.

The mechanisms of teratogenic action of ethanol (EtOH) were investigated by testing the hypothesis that all-trans-retinoic acid and/or alpha-tocopherol ameliorates ethanol-induced embryonic growth retardation. Chicken embryos were explanted in shell-less cultures and a single dose of EtOH (15, 30, or 50%) or 50% EtOH with either all-trans-retinoic acid (10(-8)M) or alpha-tocopherol (0.05 M) or a mix of all-trans-retinoic acid (10(-8)M) and alpha-tocopherol (0.05 M) was applied to the center of the blastodisc. EtOH significantly increased the mortality rate and induced growth retardation in a dose-dependent manner. In addition, EtOH increased malondialdehyde (MDA) levels, an indicator of oxidative stress and cell damage, in a dose dependent manner. All-trans-retinoic acid, the active form of Vitamin A, and/or alpha-tocopherol, an antioxidant, co-treatment with EtOH significantly diminished both the EtOH-induced mortality and growth retardation. However, only alpha-tocopherol co-treatment reduced the MDA levels. Thus, the mechanisms of teratogenic action of EtOH appear to involve initiation of oxidative stress as well as perturbation of retinoic acid (RA) signaling. It also appears likely that these mechanisms work independently of each other