Sleeping Beauty mRNA-LNP enables stable rAAV transgene expression in mouse and NHP hepatocytes and improves vector potency.

Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel in vivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spfash mouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types.

A phenotypic screening platform for chronic pain therapeutics using all-optical electrophysiology.

ABSTRACT: Chronic pain associated with osteoarthritis (OA) remains an intractable problem with few effective treatment options. New approaches are needed to model the disease biology and to drive discovery of therapeutics. We present an in vitro model of OA pain, where dorsal root ganglion (DRG) sensory neurons were sensitized by a defined mixture of disease-relevant inflammatory mediators, here called Sensitizing PAin Reagent Composition or SPARC. Osteoarthritis-SPARC components showed synergistic or additive effects when applied in combination and induced pain phenotypes in vivo. To measure the effect of OA-SPARC on neural firing in a scalable format, we used a custom system for high throughput all-optical electrophysiology. This system enabled light-based membrane voltage recordings from hundreds of neurons in parallel with single cell and single action potential resolution and a throughput of up to 500,000 neurons per day. A computational framework was developed to construct a multiparameter OA-SPARC neuronal phenotype and to quantitatively assess phenotype reversal by candidate pharmacology. We screened ∼3000 approved drugs and mechanistically focused compounds, yielding data from over 1.2 million individual neurons with detailed assessment of functional OA-SPARC phenotype rescue and orthogonal "off-target" effects. Analysis of confirmed hits revealed diverse potential analgesic mechanisms including ion channel modulators and other mechanisms including MEK inhibitors and tyrosine kinase modulators. Our results suggest that the Raf-MEK-ERK axis in DRG neurons may integrate the inputs from multiple upstream inflammatory mediators found in osteoarthritis patient joints, and MAPK pathway activation in DRG neurons may contribute to chronic pain in patients with osteoarthritis.

Oxidative stress and DNA damage in earthworms induced by methyl tertiary-butyl ether in natural soils.

Adverse effects of methyl tertiary-butyl ether (MTBE) have been noticed at different trophic levels by international researchers. However, there was unclear evidence about its effects on oxidative stress and DNA damage in earthworms. In this study, earthworms were cultivated in various doses of MTBE (0.0 mg/kg, 10.0 mg/kg, 30.0 mg/kg, and 60.0 mg/kg) contaminated agricultural soil for 7 days, 14 days, 21 days, and 28 days, respectively. The result showed that the reactive oxygen species (ROS) content of earthworms significantly increased in MTBE treatment groups compared to the control group. In MTBE treatment groups, the activities of superoxide dismutase, catalase, peroxidase, and glutathione S-transferase were significantly activated at the exposure of 7 days, which increased by 36.3-78.9%, 51.8-97.3%, 36.5-61.9%, and 12.0-54.8%, respectively. Then, the activities of these defense enzymes showed various changes following the changes in exposure times and MTBE concentrations. Especially in the 60.0 mg kg-1 group, both antioxidant enzymes and GST were still significantly activated at the exposure of 14 days and then significantly inhibited at the exposure of 28 days. The analysis of olive tail moment showed significant DNA damage in the 10.0 mg kg-1 group at the exposure of 28 days, and this damage in 30.0 mg/kg and 60.0 mg/kg groups was found at the exposure of 7 days. This result was consistent with the malondialdehyde accumulation in earthworms. Additionally, the analysis of IBRv2 showed the effects of MTBE treatments on earthworms in dose- and time-dependent manners. This study helps better to understand the effects of MTBE on soil invertebrate animals and provide theoretical support for soil protection in governing MTBE application.

Sub-chronic ecotoxicity of triphenyl phosphate to earthworms (Eisenia fetida) in artificial soil: Oxidative stress and DNA damage.

As a flame retardant, triphenyl phosphate (TPHP) is commonly added to various daily products. Due to its easy diffusion, TPHP pollution has become a global concern. Despite the wide focus on environmental risk, the sub-chronic ecotoxicity of TPHP in soil organisms remains unclear. In this study, the artificial soil exposure method was used to analyze the oxidative stress and DNA damage in earthworms with 0, 20, 40, 60 and 80 mg/kg TPHP treatments through the response of reactive oxygen species (ROS), antioxidant and detoxifying enzymes, malondialdehyde (MDA) and olive tail moment (OTM) at 7, 14, 21 and 28 days. Throughout the experimental period, the results showed that the ROS content in earthworms treated with 20, 40, 60 and 80 mg/kg TPHP treatments increased by 9.43-18.37 %, 6.07-25.73 %, 7.71-42.61 % and 8.22-46.70 %, respectively, compared to the control treatment. Meanwhile, the activities of antioxidant and detoxification enzymes in earthworms with all TPHP treatments were significantly activated after exposure for 7 and 14 days, and then inhibited at 21 and 28 days. Despite the protection of antioxidant enzymes and detoxification enzymes, MDA content in earthworms with the 20 mg/kg treatment still significantly increased at 7 and 14 days of exposure, as well as in the other three treatments. Compared to the control treatment, the obviously higher OTM values in earthworms with TPHP treatments possibly indicated a genotoxicity of TPHP in earthworms. Furthermore, the integrated biomarker response index (IBRv2) revealed that earthworms showed an obvious biochemical response TPHP-contaminated soil, which was strongly correlated with TPHP concentrations and exposure time. This study provides insights into the TPHP hazard in the soil environment and offers a reference to assess its environmental risk to soil ecosystems.

[Quantitative Comparison of Methods to Assess the Airborne Particulate Matter Retention Capacity of Leaves].

The objective of this study was to explore the differences of five methods for evaluating the PM retention capacity of leaves based on the same experimental materials and leaf area measurement method and to summarize the advantages and disadvantages of each method. In this study, four tree species (Pinus tabuliformis, Platycladus orientalis, Ginkgo biloba, and Platanus occidentalis), which are common in Beijing and have greatly different leaf characteristics, were selected as the research objects. The mass subtraction method (MS), the membrane filter method (MF), the aerosol regenerator method(AR), the scanning electron method (SEM), and the elution weighing method coupled with a particle size analysis based on ultrasonic cleaning (ultrasonic-EWPA) were used to evaluate the PM retention capabilities and characteristics of the leaves of the four tree species. The total time needed and the total cost were measured simultaneously during the evaluation process. The results showed that although the values of PM retention efficiency obtained by different methods were quite different, the ranks of the efficiency of four tree species obtained by different methods were the same or partially the same. Additionally, the results obtained by the methods with the same or similar principles were more overlapped (AR and SEM had the most overlapped results). In addition, 89% of the species ranks of the same index obtained by each method were P. orientalis > P. tabuliformis > P. occidentalis > G. biloba, and the remaining 11% were P. occidentalis > G. biloba > P. orientalis > P. tabuliformis. Among the five methods, ultrasonic-EWPA was the one with the largest number of indexes and the highest cost, and MS was the one with the least number of indexes and the lowest cost. The one that needed most time was MF, while the one that needed the least time was AR. ultrasonic-EWPA and SEM are high input and high output methods. That is to say, although they needed more time and cost, they can prove more information; however, MS was opposite, which resulted in less information but lower time and cost needed. So, it is suitable for roughly evaluating the total PM retention capacities of trees; MF had a medium amount of information, low cost, but required too much time, which needs to be weighed and balanced before selecting this method. The AR method had strict requirements for equipment and parameters and should be used with caution. The results of this study can provide a comprehensive and detailed scientific basis for researchers to choose specific methods in the future.

Use of integrated biomarker response for evaluating antioxidant stress and DNA damage of earthworms (Eisenia fetida) in decabromodiphenyl ethane-contaminated soil.

Decabromodiphenyl ethane (DBDPE) is a new and popular type of brominated flame retardant (BFR) with high bromine content, strong thermal stability, and ultraviolet resistance. To evaluated the potential toxicity of this new BFR to soil ecosystem, different concentrations of DBDPE were used to observe effects on earthworms (Eisenia fetida) in artificial soil. The reactive oxygen species (ROS) contents, activities of antioxidase system and detoxify enzyme, levels of malondialdehyde (MDA), as well as DNA damage in earthworms were measured after exposure to 0, 2.5, 5, 10, and 20 mg/kg DBDPE in artificial soil for 7, 14, 21, and 28 days. The results showed that ROS and MDA content significantly increased for all treatments from days 7-21, followed by a decrease. Throughout the experimental period, SOD, POD, and CAT activities increased. The GST activity was stimulated significantly from days 14-28. Besides, the olive tail moment (OTM) value in all treated groups was significantly higher than that in the control and exhibited a concentration-related and exposure time-related response. This is the first study evaluating the biological toxicity of BFR at different concentrations using an integrated biomarker response index. Our results show that DBDPE has biochemical toxicity on earthworms, which sheds some light on the potential risks of DBDPE in the soil environment and provides a basis for the monitoring and diagnosis of soils contaminated with DBDPE.

Biochemical responses of the Protaetia brevitarsis Lewis larvae to subchronic copper exposure.

Copper (Cu) is one of the most commonly detected heavy metals in livestock manure pollution. Protaetia brevitarsis Lewis larvae are widely used in the decomposition of livestock manure. During decomposition, heavy metals in livestock manure can accumulate in P. brevitarsis larvae and affect normal growth and reproduction. Therefore, this research focused on characterizing the toxic effects of Cu to P. brevitarsis Lewis larvae. Larvae were exposed to Cu concentrations of 0, 100, 200, 400, and 800 mg kg-1 in edible fungi residue for 7, 14, 21, and 28 days. Results showed that the soluble protein content was markedly increased in 200 and 400 mg kg-1 treatment groups on day 28. Compared to the control group, superoxide dismutase (SOD) and glutathione S-transferase (GST) activities were significantly stimulated on day 7 and then decreased as exposure length was increased (e.g., after 14, 21, and 28 days). Catalase (CAT) activity was also significantly increased after 7 days of exposure. Malondialdehyde (MDA) levels were markedly increased in the 100, 200, and 400 mg kg-1 treatment groups on day 7. However, as time and Cu concentration were increased, MDA levels gradually decreased. These results indicate that Cu has biochemical effects on P. brevitarsis Lewis larvae, and both time and dose affect this biochemical response.

Biomarker responses of earthworms (Eisenia fetida) to soils contaminated with perfluorooctanoic acid.

Perfluorooctanoic acid (PFOA) is considered a persistent environmental pollutant. The aim of this study was to assess the potential toxicity of PFOA to earthworms (Eisenia fetida) in artificial soil. The activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione S-transferase (GST) as well as the contents of malondialdehyde (MDA) were measured after exposure to 0, 5, 10, 20, and 40 mg kg-1 PFOA in soils for 7, 14, 21, and 28 days. The results showed that SOD activity increased at 14 days and decreased from 21 to 28 days; MDA levels were highest in the treatment with 40 mg kg-1 PFOA after 28 days of exposure. In contrast, CAT and POD activities increased after 14-21 days of exposure and significantly decreased with long-term exposure (28 days). GST activity increased significantly from 14 to 28 days. Our results indicate that PFOA has biochemical effects on E. fetida, thereby contributing to our understanding of the ecological toxicity of PFOA on soil invertebrates.

Gene activation of SMN by selective disruption of lncRNA-mediated recruitment of PRC2 for the treatment of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.

Evaluation of DNA damage and antioxidant system induced by di-n-butyl phthalates exposure in earthworms (Eisenia fetida).

Di-n-butyl phthalates (DBP) are recognized as ubiquitous contaminants in soil and adversely impact the health of organisms. The effect of DBP on the activity of antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT), malondialdehyde (MDA) content and DNA damage were used as biomarkers to analyze the relationship between DNA damage and oxidative stress and to evaluate the genotoxic effect of DBP on earthworms (Eisenia fetida). DBP was added to artificial soil in the amounts of 0, 5, 10, 50 and 100mg per kg of soil. Earthworm tissues exposed to each treatment were collected on the 7th, 14th, 21st, and 28th day of the treatment. The results showed that SOD and CAT levels were significantly inhibited in the 100mgkg(-1) treatment group on day 28. MDA content in treatment groups was higher than in the control group throughout the exposure time, suggesting that DBP may lead to oxidative stress in cells. A dose-response relationship existed between DNA damage and total soil DBP levels. The comet assay showed that increasing concentrations of DBP resulted in a gradual increase in the OTM, Comet Tail Length and Tail DNA %. The degree of DNA damage was increased with increasing concentration of DBP. These results suggested that DBP induced serious oxidative damage on earthworms and induced the formation of reactive oxygen species (ROS) in earthworms. The excessive generation of ROS caused damage to vital macromolecules including lipids and DNA. DBP in the soils were responsible for the exerting genotoxic effects on earthworms.

Biomarker responses in earthworms (Eisenia fetida) to soils contaminated with di-n-butyl phthalates.

Di-n-butyl phthalates (DBP) are recognized as ubiquitous contaminants in soil and adversely impact the health of organisms. Changes in the activity of antioxidant enzymes and levels of glutathione-S-transferase (GST), glutathione (GSH), and malondialdehyde (MDA) were used as biomarkers to evaluate the impact of DBP on earthworms (Eisenia fetida) after exposure to DBP for 28 days. DBP was added to artificial soil in the amounts of 0, 5, 10, 50, and 100 mg kg(-1) of soil. Earthworm tissues exposed to each treatment were collected on the 7th, 14th, 21st, and 28th day of the treatment. We found that superoxide dismutase (SOD) and catalase (CAT) levels were significantly inhibited in the 100 mg kg(-1) treatment group on day 28. After 21 days of treatment, GST activity in 10-50 mg kg(-1) treatment groups was markedly stimulated compared to the control group. MDA content in treatment groups was higher than in the control group throughout the exposure time, suggesting that DBP may lead to lipid peroxidation (LPO) in cells. GSH content increased in the treatment group that received 50 mg kg(-1) DBP from 7 days of exposure to 28 days. These results suggest that DBP induces serious oxidative damage on earthworms and induce the formation of reactive oxygen species (ROS) in earthworms. However, DBP concentration in current agricultural soil in China will not constitute any threat to the earthworm or other animals in the soil.

Overexpression of TaLEA gene from Tamarix androssowii improves salt and drought tolerance in transgenic poplar (Populus simonii × P. nigra).

Late embryogenesis abundant (LEA) genes were confirmed to confer resistance to drought and water deficiency. An LEA gene from Tamarixandrossowii (named TaLEA) was transformed into Xiaohei poplar (Populussimonii × P. nigra) via Agrobacterium. Twenty-five independent transgenic lines were obtained that were resistant to kanamycin, and 11 transgenic lines were randomly selected for further analysis. The polymerase chain reaction (PCR) and ribonucleic acid (RNA) gel blot indicated that the TaLEA gene had been integrated into the poplar genome. The height growth rate, malondialdehyde (MDA) content, relative electrolyte leakage and damages due to salt or drought to transgenic and non-transgenic plants were compared under salt and drought stress conditions. The results showed that the constitutive expression of the TaLEA gene in transgenic poplars could induce an increase in height growth rate and a decrease in number and severity of wilted leaves under the salt and drought stresses. The MDA content and relative electrolyte leakage in transgenic lines under salt and drought stresses were significantly lower compared to those in non-transgenic plants, indicating that the TaLEA gene may enhance salt and drought tolerance by protecting cell membranes from damage. Moreover, amongst the lines analyzed for stress tolerance, the transgenic line 11 (T11) showed the highest tolerance levels under both salinity and drought stress conditions. These results indicated that the TaLEA gene could be a salt and drought tolerance candidate gene and could confer a broad spectrum of tolerance under abiotic stresses in poplars.

[Evaluation of nitrogen loss way in summer maize system under different fertilizer N managements].

The objective of this study was to investigate nitrogen (N) loss from soil-crop systems under different fertilizer N managements, and to provide some suggestions on optimizing fertilizer management practices. The experiment was carried in high yield production area of Huantai county in Shandong province in 2009. Four kinds of fertilizer N application practices were designed, including CK, farmer practice (FP), optimizing fertilizer application (OPT) and controlled release fertilizer (CRT) for studying the fate of N during the maize growth season in 2009. The water and nitrogen management model (WNMM) was used to simulate the dynamics of soil water and N fate. The results indicated that the ratio of nitrate leaching and NH3 volatilization accounting of fertilizer N ranged from 6% to 18% and 5% to 34%, and their means were 12.7% and 20.7%, respectively. The amount of N leaching under OPT was 14.5 kg x hm(-2), was the lowest in all treatments. The amount of NH3 volatilization under CRT was 7.6 kg x hm(-2), respectively, was the lowest in all treatments. The order of total N loss under four treatments followed as: FP > OPT > CRF approximately CK. Both OPT and CRT treatments are the best management practices considering their high grain yield, water and nitrogen use efficiencies, and environmental protection.

[Simulation of soil water dynamics in triploid Populus tomentosa root zone under subsurface drip irrigation].

Based on the observed data of triploid Populus tomentosa root distribution, a one-dimensional root water uptake model was proposed. Taking the root water uptake into account, the soil water dynamics in triploid P. tomentosa root zone under subsurface drip irrigation was simulated by using HYDRUS model, and the results were validated with field experiment. Besides, the HYDRUS model was used to study the effects of various irrigation technique parameters on soil wetting patterns. The RMAE for the simulated soil water content by the end of irrigation and approximately 24 h later was 7.8% and 6.0%, and the RMSE was 0.036 and 0.026 cm3 x cm(-3), respectively, illustrating that the HYDRUS model performed well in simulating the short-term soil water dynamics in triploid P. tomentosa root zone under drip irrigation, and the root water uptake model was reasonable. Comparing with 2 and 4 L x h(-1) of drip discharge and continuous irrigation, both the 1 L x h(-1) of drip discharge and the pulsed irrigation with water applied intermittently in 30 min periods could increase the volume of wetted soil and reduce deep percolation. It was concluded that the combination of 1 L x h(-1) of drip discharge and pulsed irrigation should be the first choice when applying drip irrigation to triploid P. tomentosa root zone at the experiment site.

[Heavy metals pollution and its stability assessment of sediments in flowing rivers around lake Taihu].

16 main inflow and outflow rivers around Lake Taihu were chosen as the research object, and the concentrations and distribution of four heavy metals (Pb, Cd, Cu and Zn) in the surface sediments of these river estuaries were detected. The pollution extent and stability were analyzed by using three-step sequential extraction method (BCR method). Aim of this study is to control heavy metal pollution of Lake Taihu and provide the basic information. Based on the results, the monitored river estuaries all had been contaminated by different degrees, and four heavy metals' concentrations all exceeded the threshold effect level (TEL) at most sampling sites. A distinct spatial heterogeneity was found in extracted fractions of these heavy metals distribution: northern rivers > southern rivers, inflow rivers > outflow rivers. The Stability Assessment Code (SAC) for different metals varied in the descending order of Cd, Zn, Pb and Cu. Compared with Pb and Cu, Cd and Zn had a higher second release potential and ecological risk.

Identification and characterization of 4-[[4-(2-butynyloxy)phenyl]sulfonyl]-N-hydroxy-2,2-dimethyl-(3S)thiomorpholinecarboxamide (TMI-1), a novel dual tumor necrosis factor-alpha-converting enzyme/matrix metalloprotease inhibitor for the treatment of rheumatoid arthritis.

Tumor necrosis factor (TNF)-alpha is a well validated therapeutic target for the treatment of rheumatoid arthritis. TNF-alpha is initially synthesized as a 26-kDa membrane-bound form (pro-TNF) that is cleaved by a Zn-metalloprotease named TNF-alpha-converting enzyme (TACE) to generate the 17-kDa, soluble, mature TNF-alpha. TACE inhibitors that prevent the secretion of soluble TNF-alpha may be effective in treating rheumatoid arthritis (RA) patients. Using a structure-based design approach, we have identified a novel dual TACE/matrix metalloprotease (MMP) inhibitor 4-[[4-(2-butynyloxy)phenyl]sulfonyl]-N-hydroxy-2,2-dimethyl-(3S)thiomorpholinecarboxamide (TMI-1). This molecule inhibits TACE and several MMPs with nanomolar IC(50) values in vitro. In cell-based assays such as monocyte cell lines, human primary monocytes, and human whole blood, it inhibits lipopolysaccharide (LPS)-induced TNF-alpha secretion at submicromolar concentrations, whereas there is no effect on the TNF-alpha mRNA level as judged by RNase protection assay. The inhibition of LPS-induced TNF-alpha secretion is selective because TMI-1 has no effect on the secretion of other proinflammatory cytokines such as interleukin (IL)-1beta, IL-6, and IL-8. Importantly, TMI-1 potently inhibits TNF-alpha secretion by human synovium tissue explants of RA patients. In vivo, TMI-1 is highly effective in reducing clinical severity scores in mouse prophylactic collagen-induced arthritis (CIA) at 5, 10, and 20 mg/kg p.o. b.i.d. and therapeutic CIA model at 100 mg/kg p.o. b.i.d. In summary, TMI-1, a dual TACE/MMP inhibitor, represents a unique class of orally bioavailable small molecule TNF inhibitors that may be effective and beneficial for treating RA.

TGFbeta regulates the expression of G alpha(i2) via an effect on the localization of ras.

The negative chronotropic response of the heart to parasympathetic stimulation is mediated via the interaction of M(2) muscarinic receptors, Galpha(i2) and the G-protein coupled inward rectifying K(+) channel, GIRK1. Here TGFbeta(1) is shown to decrease the expression of Galpha(i2) in cultured chick atrial cells in parallel with attenuation of the negative chronotropic response to parasympathetic stimulation. The response to the acetylcholine analogue, carbamylcholine, decreased from a 95+/-2% (+/-SEM, n=8) inhibition of beat rate in control cells to 18+/-2% (+/-SEM,n =8) in TGFbeta(1) treated cells. Data support the conclusion that TGFbeta regulation of Galpha(i2) expression was mediated via an effect on Ras. TGFbeta(1) inhibited Galpha(i2) promoter activity by 56+/-6% (+/-SEM, n=4) compared to control. A dominant activating Ras mutant reversed the effect of TGFbeta on Galpha(i2) expression and stimulated Galpha(i2) promoter activity 1.7 fold above control. A dominant negative Ras mutant mimicked the effect of TGFbeta(1) on Galpha(i2) promoter activity. TGFbeta had no effect on the ratio of GDP/GTP bound Ras, but markedly decreased the level of membrane associated Ras and increased the level of cytoplasmic Ras compared to control. Furthermore, farnesol, a precursor to farnesylpyrophosphate, the substrate for the farnesylation of Ras, not only reversed TGFbeta(1) inhibition of Ras localization to the membrane, but also reversed TGFbeta(1) inhibition of Galpha(i2)promoter activity. FTI-277, a specific inhibitor of the farnesylation of Ras, mimicked the effect of TGFbeta(1) on Ras localization and Galpha(i2) promoter activity. These data suggest a novel relationship between TGFbeta signaling, regulation of Ras function and the autonomic response of the heart.