TGF-ß-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-ß), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-ß-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-ß-induced EndMT. Using these techniques, we provide evidence that TGF-ß2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

Dual ß-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-ß and Hepatocyte Nuclear Factor 4α Signaling.

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of ß-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of ß-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-ß signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of ß-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by ß-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

Mechanism of threshold size assessment: Metamorphosis is triggered by the TGF-beta/Activin ligand Myoglianin.

Although the mechanisms that control growth are now well understood, the mechanism by which animals assess their body size remains one of the great puzzles in biology. The final larval instar of holometabolous insects, after which growth stops and metamorphosis begins, is specified by a threshold size. We investigated the mechanism of threshold size assessment in the tobacco hornworm, Manduca sexta. The threshold size was found to change depending on the amount of exposure to poor nutrient conditions whereas hypoxia treatment consistently led to a lower threshold size. Under these various conditions, the mass of the muscles plus integuments was correlated with the threshold size. Furthermore, the expression of myoglianin (myo) increased at the threshold size in both M. sexta and Tribolium castaneum. Knockdown of myo in T. castaneum led to larvae that underwent supernumerary larval molts and stayed in the larval stage permanently even after passing the threshold size. We propose that increasing levels of Myo produced by the growing tissues allow larvae to assess their body size and trigger metamorphosis at the threshold size.

Assessment of Hepatoprotective Effect of Chokeberry Juice in Rats Treated Chronically with Carbon Tetrachloride.

The aim of this study was to compare the protective effects of chokeberry juice and silymarin against chemical-induced liver fibrosis in rats. Liver fibrosis was induced by CCl4 administered two days a week for six weeks. Two groups of rats were co-treated with chokeberry juice, 10 mL/kg/day. or silymarin as a positive control, 100 mg/kg/day for six weeks. Hepatic lipid peroxidation was suppressed by 50% and the activity of hepatic antioxidant enzymes was increased by 19%-173% in rats co-treated with CCl4 and substances tested as compared to rats administered CCl4 alone. Hepatic hydroxyproline was decreased by 24% only in rats treated with silymarin. The messenger RNA (mRNA) expression levels of fibrosis-related molecules, procollagen I, α-SMA, TIMP-1, TGFß, and TNFα, which were significantly increased in the liver of CCl4-treated rats, were not modulated by substances tested. Histological evaluation revealed a slight protective effect of silymarin against fibrosis. However, in CCl4 + chokeberry-treated rats, the density of vacuolated hepatocytes was significantly lower than that in silymarin administered animals. Chokeberry juice did not demonstrate an antifibrotic effect in the applied experimental model of fibrosis, and the effect of the known antifibrotic agent, silymarin, was very limited.

Assessment of Bones Deficient in Fibrillin-1 Microfibrils Reveals Pronounced Sex Differences.

Defects in the extracellular matrix protein fibrillin-1 that perturb transforming growth factor beta (TGFß) bioavailability lead to Marfan syndrome (MFS). MFS is an autosomal-dominant disorder, which is associated with connective tissue and skeletal defects, among others. To date, it is unclear how biological sex impacts the structural and functional properties of bone in MFS. The aim of this study was to investigate the effects of sex on bone microarchitecture and mechanical properties in mice with deficient fibrillin-1, a model of human MFS. Bones of 11-week-old male and female Fbn1mgR/mgR mice were investigated. Three-dimensional micro-computed tomography of femora and vertebrae revealed a lower ratio of trabecular bone volume to tissue volume, reduced trabecular number and thickness, and greater trabecular separation in females vs. males. Three-point bending of femora revealed significantly lower post-yield displacement and work-to-fracture in females vs. males. Mechanistically, we found higher Smad2 and ERK1/2 phosphorylation in females vs. males, demonstrating a greater activation of TGFß signaling in females. In summary, the present findings show pronounced sex differences in the matrix and function of bones deficient in fibrillin-1 microfibrils. Consequently, sex-specific analysis of bone characteristics in patients with MFS may prove useful in improving the clinical management and life quality of these patients, through the development of sex-specific therapeutic approaches.

TGFß C-509T, TGFß T869C, XRCC1 Arg194Trp, IKBα C642T, IL4 C-590T Genetic polymorphisms combined with socio-economic, lifestyle, diet factors and gastric cancer risk: A case control study in South Indian population.

BACKGROUND: Gastric cancer is worldwide the third major cause of cancer related death. Risk factors for gastric cancer includes Helicobacter pylori infection, gastric ulcer, less hygienic condition, use of tobacco, alcohol consumption, use of salted, smoked food, genetic alterations etc. In order to identify the risk factors associated with gastric cancer in South Indian population a case-control study involving 200 proven gastric cancer cases and 400 controls was conducted. METHODS: A structured questionnaire was used to interview all the subjects who participated in our study. Genotyping assay was performed using Taqman allelic discrimination assay for 5 Single Nucleotide Polymorphisms (SNPs)-TGFß C-509T, TGFß T869C, XRCC1 Arg194Trp, IkBα C642T and IL4C-590T. RESULTS: Odds Ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression. Statistical analysis on socio-economic factors, lifestyle factors had showed that subjects from low socio economic status, use of tobacco and consumption of non-vegetarian food had increased risk of developing gastric cancer. Multi-factorial analysis for the SNPs adjusting for the risk factors obtained in this study showed that TGFΒ C-509T TT genotypes had four fold increased risk of gastric cancer (OR = 4.11, CI = 1.02-16.56) and TGFß T869C CC genotype had a decreased risk of gastric cancer (OR = 0.21, CI = 0.05-0.85). CONCLUSION: Economic status, tobacco use and food habits play a significant role in gastric cancer development. TT genotype for TGFß C-509T had an increased risk and CC genotype for TGFß T869C had a decreased risk of gastric cancer in south Indian population after adjusting for socio-economic factors and lifestyle factors.

Assessment of Topical Therapies for Improving the Optical Clarity Following Stromal Wounding in a Novel Ex Vivo Canine Cornea Model.

Purpose: To evaluate the effect of topical suberanilohydroxamic acid (SAHA) and 5-methyl-1-phenyl-2[1H]-pyridone (pirfenidone) on the degree of corneal haze in the stromal wounded ex vivo canine cornea. Methods: Twenty-four corneoscleral rims from normal dogs were uniformly wounded with an excimer laser and placed into culture medium with an air-liquid interface. The control group (n = 8) contained placebo-treated corneas. Treatment group 1 (n = 8) received SAHA topically every 6 hours. Treatment group 2 (n = 8) received pirfenidone topically every 6 hours. Each cornea was fluorescein stained and macrophotographed every 6 hours to assess epithelialization rate. All corneas were also macrophotographed weekly to assess optical clarity (haze). Images were analyzed for differences in pixel intensity between wounded (haze) and unwounded (nonhaze) regions, and haze surface area for each cornea was calculated. Results: The mean epithelialization time was 47.25 hours in the control group, 45.00 hours in the SAHA group, and 43.50 hours in the pirfenidone group, revealing no significant difference (P = 0.368). The median difference in pixel intensity between haze and nonhaze areas was 21.5 in the control group, 8.0 in the SAHA group, and 8.0 in the pirfenidone group, which is significant (P < 0.01). The median haze surface area was 12.96 mm2 in the control group, 5.70 mm2 in the SAHA group, and 5.92 mm2 in the pirfenidone group, which is significant (P < 0.01). Conclusions: Stromal-wounded ex vivo canine corneas exhibited greater optical clarity when treated with SAHA and pirfenidone than when placebo treated at 21 days. There was no significant difference in epithelialization rate between groups. Corneal contour was correlated with geographic haze distribution.

Computational Model of Secondary Palate Fusion and Disruption.

Morphogenetic events are driven by cell-generated physical forces and complex cellular dynamics. To improve our capacity to predict developmental effects from chemical-induced cellular alterations, we built a multicellular agent-based model in CompuCell3D that recapitulates the cellular networks and collective cell behavior underlying growth and fusion of the mammalian secondary palate. The model incorporated multiple signaling pathways (TGFß, BMP, FGF, EGF, and SHH) in a biological framework to recapitulate morphogenetic events from palatal outgrowth through midline fusion. It effectively simulated higher-level phenotypes (e.g., midline contact, medial edge seam (MES) breakdown, mesenchymal confluence, and fusion defects) in response to genetic or environmental perturbations. Perturbation analysis of various control features revealed model functionality with respect to cell signaling systems and feedback loops for growth and fusion, diverse individual cell behaviors and collective cellular behavior leading to physical contact and midline fusion, and quantitative analysis of the TGF/EGF switch that controls MES breakdown-a key event in morphogenetic fusion. The virtual palate model was then executed with theoretical chemical perturbation scenarios to simulate switch behavior leading to a disruption of fusion following chronic (e.g., dioxin) and acute (e.g., retinoic acid) chemical exposures. This computer model adds to similar systems models toward an integrative "virtual embryo" for simulation and quantitative prediction of adverse developmental outcomes following genetic perturbation and/or environmental disruption.

Assessment of 1,25-dihydroxyvitamin D3 effects on Treg cells in a mouse model of systemic lupus erythematosus.

CONTEXT: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease which is characterized by the presence of auto-reactive T cell and anti-ds DNA antibodies. Treg cells are crucial for maintaining immunologic self-tolerance and are shown to be reduced in SLE patients. 1,25-Dihydroxyvitamin D3 has immunomedulatory effects on the immune system and has recently received substantial attention. OBJECTIVE: In this study we evaluated the effects of 1,25-dihydroxyvitamin D3 on Treg cells and related cytokines in lupus-like induced mice model. MATERIALS AND METHODS: Female Balb/c mice were divided into four groups: Group one: injected with PBS and Freund's adjuvant; Group two: injected with non-activated chromatin; Group three: Lupus-like disease was induced with activated chromatin; Group four: Mice were initially treated for two weeks with 1,25-dihydroxyvitamin D3 and then lupus-like disease was induced. Group five: Four mice from group one were treated with 1,25-dihydroxyvitamin D3 for two weeks after disease establishment. Ten weeks after the last injection the mice were killed and spleens were studied for Treg percentages and expression of cytokine genes. RESULTS: We found that treatment with 1,25-dihydroxyvitamin D3 reduces IL-6 and IL-10 mRNA expression and increases TGF-ß and Foxp3 mRNA expression levels, and also enhances spleen Treg percentage. CONCLUSIONS: The remarkable reduction of IL-6 and IL-10 gene expressions, significant enhancement of TGF-ß and Foxp3 gene expressions, along with an increase in Treg cell population after oral 1,25-dihydroxyvitamin D3 administration suggest a possible role for this vitamin as a prophylactic supplement in SLE.

Assessment of the association between genetic polymorphisms in transforming growth factor beta, and its binding protein (LTBP), and the presence, and expansion, of Abdominal Aortic Aneurysm.

OBJECTIVES: Abdominal Aortic Aneurysm (AAA) has a strong genetic predisposition. Transforming growth factor beta 1 (TGF-beta1) is a causal factor in ascending aortic dilatation; however, a role in AAA pathology is unclear. The aim of the study was to determine whether genes coding TGF-beta and its binding protein are associated with the presence and expansion of AAA. METHODS: Four geographically distinct case control studies, totaling 1890 AAA cases and 3785 controls, were genotyped and compared to the presence, size and growth rate of AAA. 26 single nucleotide polymorphisms (SNPs) in 5 genes were genotyped in the UK cohort and the result was replicated in 3 independent cohorts. RESULTS: No associations between genotypes or haplotypes and the presence of AAA disease were confirmed. Five SNPs in Latent TGF-beta Binding Protein (LTBP4) and an allelic variant of TGFB3 were associated with a significant decrease in AAA growth (p< or =0.02), in the UK cohort. Altered growth was demonstrated in carriers of two common haplotypes of LTBP4 (+0.38 mm/year, p=0.003; -0.41 mm/year, p=0.02, per haplotype copy) and a single haplotype of TGFB3 (-0.53 mm/year, p=0.05). This association with AAA growth could not be demonstrated in two other independent cohorts. Meta-analysis of AAA size and growth rates in larger AAA (> or =45 mm), in all four cohorts, demonstrated a significant association with the LTBP4 21011A>T genotype (a 2% decrease in AAA diameter, or a 0.53 mm/year reduction in AAA growth rate, per T allele [p=0.03, p=0.01]). CONCLUSION: This study suggests that the LTBP4 gene may contribute to AAA progression.

Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children.

Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD(65) or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-gamma and TNF-alpha) and chemokines (IP-10, MCP-1, MIP-1alpha, MIP-1beta and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-beta mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-gamma and MCP-1, and mRNA expression of FOXP3 and TGF-beta, was detected after cryopreservation. Stimulation with GAD(65) induced higher levels of IL-6, IFN-gamma, TNF-alpha and MIP-1alpha, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCP-1 and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.

Safety assessment of intradiscal gene therapy II: effect of dosing and vector choice.

STUDY DESIGN: Clinical, biochemical, and histologic analysis was performed after in vivo delivery of cDNA encoding various anabolic cytokines and marker genes to the lumbar epidural space of New Zealand white rabbits, using both adenoviral and adeno-associated viral vectors. OBJECTIVE: To mimic errant or misplaced doses of gene therapy to better ascertain the potential risks associated with alternative vectors and transgene products with regard to their application to problems of the intervertebral disc. SUMMARY OF BACKGROUND DATA: Work done with several anabolic cytokines including bone morphogenic proteins and transforming growth factors, has demonstrated the potential of gene therapy. Recently, data has been published demonstrating that improperly dosed or delivered adenoviral-mediated gene therapy within the subarachnoid space can result in significant morbidity in rabbits. There are currently no studies examining the effect of these errors within the epidural space or using an adeno-associated viral (AAV) vector. METHODS: Using either adenoviral or AAV vectors, complementary DNA (cDNA) encoding anabolic cytokines bone morphogenic protein-2 (BMP-2) and transforming growth factor-beta 1 and marker proteins LacZ and green fluorescent protein were injected into the epidural space of 37 New Zealand white rabbits at the L5/6 level. Rabbits were then observed clinically for up to 6 weeks, after which the rabbits were sacrificed in order to perform a comprehensive biochemical and histologic analysis. RESULTS: Following adenoviral-mediated delivery of anabolic cytokine cDNA, up to eighty percent of rabbits demonstrated significant clinical, biochemical, and histologic morbidity. Conversely, AAV-mediated delivery of any cDNA and adenoviral-mediated delivery of marker protein cDNA resulted in no clinical, histologic, or biochemical morbidity. CONCLUSION: Properly dosed and directed gene therapy seems to be both safe and potentially efficacious. This study suggests that side effects of gene therapy may be due to a combination of dosing, transgene product, and vector choice, and that newer AAV vectors may reduce these side-effects and decrease the risk of this technology.

Genetic involvement in skin wound healing and scarring in domestic pigs: assessment of molecular expression patterns in (Yorkshire x Red Duroc) x Yorkshire backcross animals.

Yorkshire, red Duroc, and F1 (first-generation cross) pigs heal with normal, fibroproliferative/hypercontractile, and intermediate levels of scarring, respectively. The purpose of this study was to evaluate the healing phenotype of Yorkshire x F1 backcross animals, to address the molecular basis for genetic transmission of the red Duroc scarring phenotype. Macroscopically and histologically, full-thickness wounds on backcross animals followed the Yorkshire phenotype, with one exception; the backcross wounds exhibited contraction following re-epithelialization. The molecular expression patterns in the backcross animals generally correlated with the macroscopic and histologic findings. Compared to Yorkshire, red Duroc, and F1 wounds, the backcross wounds demonstrated a diminished initial inflammatory phase, followed by a prolonged expression of several relevant growth factors. Additionally, collagen expression was prolonged, expression of matrix metalloproteinases was increased, and alterations in tissue inhibitor of metalloproteinase expression were detected. Moreover, a subset of molecules still followed the red Duroc pattern of mRNA expression, a finding that allows for correlations between the scarring phenotype and the molecular expression patterns to be made in this model. The results indicate that a number of genes are likely involved in the red Duroc healing phenotype and that identification of the specific genes involved will require a more detailed genomic analysis.

Crk-associated substrate lymphocyte type regulates transforming growth factor-beta signaling by inhibiting Smad6 and Smad7.

Crk-associated substrate lymphocyte type (Cas-L) is a 105 kDa docking protein with diverse functional properties, including regulation of cell division, proliferation, migration and adhesion. Cas-L is also involved in beta1 integrin- or antigen receptor-mediated signaling in B and T cells. In the present study, we demonstrate that Cas-L potentiates transforming growth factor-beta (TGF-beta) signaling pathway by interacting with Smad6 and Smad7. Immunoprecipitation experiments reveal that single domain deletion of full-length Cas-L completely abolishes its docking function with Smad6 and Smad7, suggesting that the natural structure of Cas-L is necessary for its association with Smad6 and Smad7. On the other hand, both N-terminal and C-terminal deletion mutants of Smad6 and Smad7 still retain their docking ability to Cas-L, suggesting that Smad6 and Smad7 possess several binding motifs to Cas-L. Moreover, Cas-L interaction with Mad-homology (MH)2 domain, but not with MH1 domain of Smad6 or Smad7, ameliorates TGF-beta-induced signaling pathway. Finally, depletion of Cas-L by small-interfering RNA oligo attenuates TGF-beta-induced growth inhibition of Huh-7 cells, with a concomitant reduction in phosphorylation of Smad2 and Smad3. These results strongly suggest that Cas-L is a potential regulator of TGF-beta signaling pathway.

Assessment of linkage and association of 13 genetic loci with bone mineral density.

Bone mineral density (BMD), an important risk factor for osteoporosis, is a complex trait likely affected by multiple genes. The linkage and/or association of 13 polymorphic loci of seven candidate genes (estrogen receptor alpha [ERalpha] and beta [ERbeta], calcium-sensing receptor, vitamin D receptor, collagen type 1alpha1, low-density lipoprotein [LDL] receptor-related protein 5 [LRPS], and transforming growth factor beta1) were evaluated in 177 southern Chinese pedigrees of 674 subjects, with each pedigree identified through a proband having a BMD Z score of -1.28 or less at the hip or spine. A suggestive linkage was detected between the IVS1-351A/G polymorphism of ERalpha and spine BMD, and between the 1082G/A, 1730G/A, and D14S1026 polymorphisms of ERbeta and BMD at both spine and hip. The quantitative transmission disequilibrium test (QTDT) detected total family association between 1730G/A of ERbeta and BMD at spine and hip; between D14S1026 of ERbeta and hip BMD; and between the 266A/G and 2220C/T polymorphisms of LRP5 and hip BMD. Similar total family associations were detected when only the females were analyzed. In addition, the IVS1-397T/C polymorphism of ERalpha was associated with spine BMD, and the 266A/G and 2220C/T polymorphisms of LRP5 were associated with femoral neck BMD in the females. A within-family association was detected with the IVS1-397T/C polymorphism of ERalpha, and the 266A/G and 2220C/T polymorphisms of LRP5 in the females. The effect of each polymorphism on BMD variance ranged from 1% to 4%. In conclusion, ERalpha, ERbeta and LRP5 are important candidate genes determining BMD variation, especially in females.

Safety assessment of intradiscal gene transfer: a pilot study.

BACKGROUND: Several recent in vitro and in vivo studies have reported the beneficial properties of gene delivery of therapeutic factors to the intervertebral disc, as a potential treatment strategy for degenerative disc disease; however, to date, no studies have assessed the safety and toxicity of the practical application of this treatment modality. PURPOSE: To assess the safety of inappropriately dosed or misdirected gene delivery to the spinal column in an in vivo model. STUDY DESIGN: The potential toxicity of gene therapy to the spinal column was assessed in this pilot study by monitoring clinical and histological changes in the spinal cord after intradural injections of an adenoviral vector containing the complementary deoxyribonucleic acid (cDNA) for potentially therapeutic factors in the treatment of degenerative disc disease. METHODS: Fourteen New Zealand White rabbits were divided into experimental groups to receive an intradural injection (<10 microL) of saline alone or saline in combination with recombinant transforming growth factor beta1 (TGF-beta1) or an adenoviral vector containing the cDNA for either TGF-beta1 (at previously established therapeutic or elevated concentrations) or bone morphogenic protein-2 (BMP-2). Animals were monitored clinically and spinal cords were harvested for histological analysis. RESULTS: No neurological deficits developed in any of the animals receiving injections of saline alone or saline in combination with the therapeutic dose of Ad-TGF-beta1, Ad-BMP-2, or with recombinant TGF-beta1. However, animals receiving a higher concentration of Ad-TGF-beta1 developed bilateral lower extremity paralysis with significant histological changes. CONCLUSIONS: Inappropriately dosed or directed gene delivery to the spinal column may result in significant complications. However, with appropriate dosing, a therapeutic window may exist where the potential benefits of gene therapy in the treatment of degenerative disc disease outweigh its risks.

Combined genetic assessment of transforming growth factor-beta signaling pathway variants may predict breast cancer risk.

There is growing evidence that common variants of the transforming growth factor-beta (TGF-beta) signaling pathway may modify breast cancer risk. In vitro studies have shown that some variants increase TGF-beta signaling, whereas others have an opposite effect. We tested the hypothesis that a combined genetic assessment of two well-characterized variants may predict breast cancer risk. Consecutive patients (n = 660) with breast cancer from the Memorial Sloan-Kettering Cancer Center (New York, NY) and healthy females (n = 880) from New York City were genotyped for the hypomorphic TGFBR1*6A allele and for the TGFB1 T29C variant that results in increased TGF-beta circulating levels. Cases and controls were of similar ethnicity and geographic location. Thirty percent of cases were identified as high or low TGF-beta signalers based on TGFB1 and TGFBR1 genotypes. There was a significantly higher proportion of high signalers (TGFBR1/TGFBR1 and TGFB1*CC) among controls (21.6%) than cases (15.7%; P = 0.003). The odds ratio [OR; 95% confidence interval (95% CI)] for individuals with the lowest expected TGF-beta signaling level (TGFB1*TT or TGFB1*TC and TGFBR1*6A) was 1.69 (1.08-2.66) when compared with individuals with the highest expected TGF-signaling levels. Breast cancer risk incurred by low signalers was most pronounced among women after age 50 years (OR, 2.05; 95% CI, 1.01-4.16). TGFBR1*6A was associated with a significantly increased risk for breast cancer (OR, 1.46; 95% CI, 1.04-2.06), but the TGFB1*CC genotype was not associated with any appreciable risk (OR, 0.89; 95% CI, 0.63-1.21). TGFBR1*6A effect was most pronounced among women diagnosed after age 50 years (OR, 2.20; 95% CI, 1.25-3.87). This is the first study assessing the TGF-beta signaling pathway through two common and functionally relevant TGFBR1 and TGFB1 variants. This approach may predict breast cancer risk in a large subset of the population.

Myostatin and its implications on animal breeding: a review.

Myostatin, or growth and differentiation factor 8 (GDF8), has been identified as the factor causing a phenotype known as double muscling, in which a series of mutations render the gene inactive, and therefore, unable to regulate muscle fibre deposition. This phenotype occurs at a high frequency in some breeds of cattle such as Belgian Blue and Peidmontese. Phylogenetic analysis has shown that there has been positive selection pressure for non-synonymous mutations within the myostatin gene family, around the time of the divergence of cattle, sheep and goats, and these positive selective pressures on non-ancestral myostatin are relatively recent. To date, there have been reports of nine mutations in coding regions of myostatin that cause non-synonymous changes, of which three cause missense mutations, including two in exon 1 and one in exon 2. The remaining six mutations, located in exons 2 and 3, result in premature stop codons, which are the mutations responsible for the double-muscling phenotype. Unfortunately, breed management problems exist for double-muscled cattle, such as birthing difficulties, which can be overcome through genetically controlled breeding programmes, as shown in this review.

Assessment of mRNA levels for matrix molecules and TGF- beta1 in rabbit flexor and peroneus tendons reveals regional differences in steady-state expression.

This study analysed the differences on a molecular level between two segments of the deep flexor tendon, and compared the intrasynovial flexor tendon with the tendon sheath and the extrasynovial peroneus tendon in a rabbit model. The TRIspin method of RNA extraction was combined with the reverse transcription polymerase chain reaction to assess mRNA levels in the tissue segments. Significant differences were detected for all genes studied. mRNA levels for aggrecan, biglycan and collagen III were significantly higher in the fibrocartilaginous proximal segment of the flexor tendon. Collagen I was higher in the flexor tendon than the sheath and the peroneus tendon, and TGF-beta1 was significantly lower in the peroneus tendon. This study demonstrates differences at the mRNA level between different segments of tendon, indicating that the tendon tissue may be adapted to its environment.